Chronic Wound Management: The Significance of Evidence and Technology 3031261097, 9783031261091

Table of contents :
Contents
1 Chronic Wound Management—A Continuing Challenge
Wound Aetiology
A Complex Profile
Economic Impact
Human Impact
Healing Outcomes
Future Opportunities
References
2 The Role of Technology in Managing Vascular Wounds
Abstract
Introduction
Background
Venous Leg Ulcers (VLU)
The Physics of Compression Delivered by Hosiery/Bandaging Systems
Healing Rates of VLU Treated with Compression Are Variable
Other Devices to Deliver Compression
Mixed Arterio-Venous Ulcers
Surgical Treatments of Venous Insufficiency in Conjunction with Venous Ulcers
Ischemic Leg Ulcers
Discussion
References
3 The Diabetic Foot, Its Complications, Role of Technology in Evidence-Based Management
Abstract
Introduction
Diagnosis
Assessment of Neuropathy as a Predisposing Factor to Ulceration
Use of Technology to Assess Large Fibre Neuropathy
Monofilament
Vibration Perception Threshold (VPT)
Use of Technology to Assess Small Fibre Neuropathy
Screening for Neuropathy
The Value of Screening to Predict Outcome
The Value of Screening to Affect Outcome
Measurement of Pressure Abnormalities of the Foot as Precipitants of Ulceration
Detection of Skin Temperature Rise as an Immediate Prelude to Ulceration
Characterisation of Infection, Which Often Complicates Ulceration
Wound Management
Wound Debridement
Innovative Technologies in Wound Debridement
Microwater Jet Technology
Ultrasound Assisted Wound Debridement
Near Infrared (NIR) Optical Imaging
Innovative Technology in Wound Applications to Accelerate Healing
Innovative Technology in Cellular and Molecular Therapies to Accelerate Healing
Stem Cell Therapy
Gene Therapy
Nanotechnology
Evidence for the Use of Technology on Offloading
Covid 19 Pandemic and Management of the Diabetic Foot
Conclusion
References
4 Role of Technology for wound Care in Diabetic Foot
Abstract
Introduction
Burden of Diabetic Foot Ulcers in India
Newer Technology as an Early Diagnostic Tool For DFU
Foot Evaluation
Recent Advances in the Management of DFU
Impact of Covid 19 Among People With DFU
Future in the Prevention of DFU
Conclusion
References
5 Biologic Transducers in Wound Healing
Abstract
Introduction
Effectiveness of Enriched Platelet for Skin Chronic Wounds
Effectiveness of Topical Recombinant Growth Factors/Cytokines for Chronic Skin Wounds
Cytokines
Growth Factors
PDGF
FGF
EGF
Combination Application of Growth Factors in Chronic Wound
Effectiveness of (Stem) Cells for Skin Chronic Wounds
Outlook
References
6 Physical, Electromagnetic, Biologic Devices
Abstract
Introduction
Physical/Delivery Systems
Materials
Skin Substitutes
Bone Substitutes
Vascular-Related Technologies
Conclusions
References
7 Medicinal Plants and Products from Traditional Medicine Systems Contribute to Clinical Wound Management
Abstract
Introduction
What are Ideal Properties to Benefit Wound Healing?
Randomized Clinical Trial on Wound Dressing Agents from Plant Origin
Objectives
Azadirachta Indica as Dressing Agent
Ayurvedic Polyherbal Formulation as Dressing Agent
Banana Leaves Dressings (BLD)
Randomised Controlled Trial on Wound Healing Plant Medicine
Topical Application
Aloe Vera
Hypericum Perforatum
Honey
Systemic Uses of Curcumin
Curcuma Longa
Centella Asiatica (L.)
Taking a Wound Healing Agent from Bench to Bedside: Validating a Natural Wonder with Advanced Technology
Discussion
Acknowledgements
Conflict of interest
References
8 Innovation in Laboratory Evaluations of the Performance of Treatment and Prophylactic Dressings Under Clinically-Relevant Usage Conditions
Abstract
Introduction
Robotic Wound Systems Designed and Built for the Evaluation of Treatment Dressings
Computational Modelling Reveals the Efficacy of Wound Dressings in Prophylactic Use
Summary and Conclusions
Acknowledgements
References
9 Atypical Wounds and Wounds Resulting from Infection
Abstract
Introduction
Neoplastic Ulcers
Haematologic Ulcers
Haemoglobinopathies-Associated Ulcers
Polycythaemia Vera and Leg Ulcers
Metabolic Anomalies
Calcific Uremic Arteriolopathy (Calciphylaxis)
Inflammatory and Immune Disorders-Correlated
Vasculitis
Pyoderma Gangrenosum
Occlusive Non Vasculitic Vasculopathies
Livedoid Vasculopathy
Antiphospholipid Antibodies-Associated Leg Ulcers
Drug-Induced
Hydroxyurea
Drug-Related Cutis Embolism (Nicolau’s Disease)
Warfarin-Induced Skin Necrosis
Drug Abuse
Ulcer Resulting from Arterial Hypertension (Martorell Hypertensive Ischemic Ulcer)
Atypical Ulcers Associated to Infections
Mycobacterial-Induced Ulcers
Buruli Ulcer
Lehismania
Deep Fungal Infection-Related Atypical Wounds
Sporothricosis
Mycetoma
Ecthyma Gangrenosum
Microbiological Investigations: Methods and Drawing Techniques
Mainly Qualitative Methods
Qualitative and Quantitative Methods
Laboratory Markers
The Future
References
10 Biofilms and Impaired Wound Healing: How Do We Detect the Presence of Biofilms in Chronic Wounds Non-invasively
Abstract
How Biofilms Impair Wound Healing
Disruption of Immune and Skin Cells
Disruption of Microenvironment
Detection Methods
Biofilm Detection Issues
Conventional Detection Methods
Novel Sensor-Based Detection Methods
Novel Imaging-Based Detection Methods
Discussion
References
11 Update on Technology and Evidence-Based Management of Scars
Abstract
Introduction
Biological Resume of Events Occurring After a Skin Injury
Factors Impacting Scars
The Young Age
The Elderly
The Reappearance of a Wound on a Scar
Degenerescence/Marjolin’s Ulcer
Hyperkeratosis
Scar Assessment Scales
Non-surgical Technologies
Intra-Lesional Steroid Injections
Silicone Gel/Sheet
Radiotherapy
Photodynamic Therapy (PDT)
Electrical Stimulation
Surgical Strategies
Scar Prevention
Conclusion
References
12 Surgical Flaps in Wound Healing—An Update on Evidence-Based Management
Abstract
Introduction
Patient Selection, Multidisciplinary Approach and the Wound Preparation
Surgical Techniques
Case Examples
Outcomes
Conclusion
References
13 Wound Measurement is an Essential Part of Wound Management
Abstract
Introduction
Skin Assessment
Wound Size Analysis
Hyperspectral Imaging
Laser Doppler Imaging (LDI)
Laser Speckle Imaging (LSI)
Near-Infrared Spectroscopy (NIRS)
Thermography
Videomicroscopy
Optical Coherence Tomography
Fluorescence Imaging
Confocal Microscopy
Ultrasound
Discussion
References
14 Translation of Wound Devices into Practice—A Myth? Translation is the Process of Taking an Invention Through to Clinical Practice—How Successful Are We?
Abstract
Gaining Approval for Clinical Use of an Innovation
Gaining Adoption and Usage of a Translated Product or Therapy
Case Studies
Discussion
References
15 Pain in Chronic Wounds: Mechanism and Management
Abstract
Introduction
The Burden of the Problem
Pathophysiology of Wound-Related Pain
Pain Pathways
Effects of Pain in Patients with Chronic Wounds
Diagnosis and Assessment
Pain Assessment
Management
WHO Pain Management Ladder
Management of Breakthrough Pain
Wound Pain Management
Topical Treatment
Management of Infection
Moisture Balance
Management of Specific Wounds
Diabetic Foot Ulcer
Venous Ulcers
Ischaemic Leg Ulcers
Conclusion
References

Citation preview

Chronic Wound Management The Significance of Evidence and Technology Raj Mani Editor

123

Chronic Wound Management

Raj Mani Editor

Chronic Wound Management The Significance of Evidence and Technology

123

Editor Raj Mani Shanghai Jiao Tong University Shanghai, China

ISBN 978-3-031-26109-1 ISBN 978-3-031-26110-7 https://doi.org/10.1007/978-3-031-26110-7

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

Chronic Wound Management—A Continuing Challenge . . . . . . . . . . . . Georgina Gethin and Raj Mani

1

The Role of Technology in Managing Vascular Wounds . . . . . . . . . . . . Bodo Erhardt Günther and Raj Mani

7

The Diabetic Foot, Its Complications, Role of Technology in Evidence-Based Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Danielle Dixon and Michael Edmonds

45

Role of Technology for wound Care in Diabetic Foot . . . . . . . . . . . . . . V. Viswanathan and R. Mirshad

67

Biologic Transducers in Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . Biao Cheng and Xiaobing Fu

77

Physical, Electromagnetic, Biologic Devices . . . . . . . . . . . . . . . . . . . . . . 107 Alberto Piaggesi Medicinal Plants and Products from Traditional Medicine Systems Contribute to Clinical Wound Management . . . . . . . . . . . . . . . . . . . . . . 117 Tuhin Kanti Biswas, Shrabana Chakrabarti, Srikanta Pandit, and Raj Mani Innovation in Laboratory Evaluations of the Performance of Treatment and Prophylactic Dressings Under Clinically-Relevant Usage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Amit Gefen Atypical Wounds and Wounds Resulting from Infection . . . . . . . . . . . . 153 Massimo Papi and Ersilia Fiscarelli Biofilms and Impaired Wound Healing: How Do We Detect the Presence of Biofilms in Chronic Wounds Non-invasively . . . . . . . . . 195 Ida C. Thaarup and Thomas Bjarnsholt Update on Technology and Evidence-Based Management of Scars . . . . 229 Luc Téot, Hester Colboc, and Sylvie Meaume

v

vi

Contents

Surgical Flaps in Wound Healing—An Update on Evidence-Based Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Joon Pio Hong and Asli Datli Wound Measurement is an Essential Part of Wound Management . . . . 263 Valentina Dini, Giammarco Granieri, Alessandra Michelucci, and Marco Romanelli Translation of Wound Devices into Practice—A Myth? Translation is the Process of Taking an Invention Through to Clinical Practice—How Successful Are We? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Mark Richardson and Raj Mani Pain in Chronic Wounds: Mechanism and Management . . . . . . . . . . . . 297 Aakansha Giri Goswami, Praveen Talawar, Somprakas Basu, and Vijay Kumar Shukla

Chronic Wound Management—A Continuing Challenge Georgina Gethin

and Raj Mani

This book “Chronic Wound Management—the significance of evidence and technology” examines the benefits of innovation to manage an enormous clinical and therapeutic challenge. Chronic wounds are those cutaneous wounds which fail to follow a predictable course of healing. Since the use of Doppler ultrasound to measure ankle brachial pressure index (ABI)—a reliable measure of the blood flow into the ankle and foot—in the early 1980s, evidence emerged to the effect that a great proportion of such wounds present on the lower extremities or legs and feet and are of vascular and/or diabetic in origin. A range of innovative technology to diagnose as well to manage wounds appeared on the scene, some were widely accepted and available and led to objective evidence. Study designs in the form of randomised controlled studies emerged and penetrative data analysis using meta-analysis appeared, pioneered by the Cochrane Wounds Group, became available. It was possible to define the concept of standardised care. Journals dedicated to the clinical science and management of wounds were launched innervating communications that were indexed in the Web of Science, Pub Med, G. Gethin Alliance for Research and Innovation in Wounds, Galway, Ireland e-mail: [email protected] CURAM (SFI Centre for Research in Medical Devices), Galway, Ireland School of Nursing and Midwifery, University of Galway, Galway, Ireland HES-SO University of Applied Sciences and Arts, Delémont, Switzerland Journal of Wound Management—Official Journal of EWMA, Frederiksberg, Denmark School of Nursing and Midwifery, Aras Moyola, University of Galway, Galway, Ireland R. Mani (&) Shanghai Jiao Tong University School of Medicine, Shanghai, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_1

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G. Gethin and R. Mani

and such other data bases. Numerous books were also published as learned societies also grew, initially in Europe and USA, later in many countries. All these developments raised expectations of improving healing outcomes. What is the current order of the problem? It is difficult to be precise about the prevalence of wounds of all aetiologies. In the United States 3% of the population >65 years have open wounds and an estimated 2% of the total population are affected by wounds (Sen 2019). In the United Kingdom (UK) it was estimated that in the year 2017/2018, 3.8 million adults had a wound, equivalent to 7% of the adult population (Guest et al. 2020). In Spain, an incidence and prevalence study over the years 2010–2014 among people over 40 years of age, reported prevalence and incidence increase over the years, doubling in those aged over 65 years (Berenguer Perez et al. 2019). Incidence increased from 0.5 new cases per 1,000 people/year in 2010 to 1 new case for every 1,000 people/year in 2014. A meta-analysis of eleven studies representing 15,748,658 people reported an estimated total prevalence of chronic wounds of 1.67 per 1,000 population (Martinengo et al. 2019). Subgroup analysis based on the type of wounds revealed a combined prevalence of 2.21 per 1000 population for studies reporting ulcers of more than one aetiology (Martinengo et al. 2019). The average age of patients with chronic wounds is around 65–70 years with 67% of those in one study being or = 85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed < 85% during treatment (P < 0.05) Complete resolution of the ulcers was seen

Ulcers treated with cytokines had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Patients treated with GM-CSF or bFGF had higher levels of their respective cytokine after treatment

Results/Conclusions

Biologic Transducers in Wound Healing 89

1995

Arnold et al. (1995)

Malik et al. 1998 (1998)

Cianfarani et al. (2006)

Vascular leg ulcers

Chronic wounds

Chronic venous ulcers

2006

Particular year

Author

Disease species

Table 1 (continued)

Comparison before and after treatment

Comparison before and after treatment

Results/Conclusions

Fifty-four of 61 patients completed the follow-up period with 68.5% of the patients (37 of 54) being healed after 1 year. Of patients healing > or = 85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed < 85% during treatment (P < 0.05) GM-CSF 10 microg/cm2 was injected subcutaneous injection of a single dose of subcutaneously along the edges and base of the GM-CSF may induce healing in refractory wound. The treatment was given only once and chronic wounds. Trials are necessary to patients were followed weekly for a minimum validate these initial observations and to decide of six weeks the optimal dose and route, and whether any additional benefit may be derived from repeated injections Patients with nonhealing venous leg ulcers Blood vessel density was significantly were treated with intradermal injection of increased in the ulcer bed following GM-CSF recombinant human GM-CSF treatment. VEGF transcripts were localized in keratinocytes at the ulcer margin both before and after GM-CSF treatment, whereas a VEGF hybridization signal was evident within the ulcer bed only following administration. PlGF mRNA was barely detectable in keratinocytes at the ulcer margin and was not visibly increased after treatment. Unlike VEGF, a specific PlGF hybridization signal could not be detected in cells within the ulcer following GM-CSF administration. Monocytes/macrophages were the main cell (continued)

Therapeutic method

Comparison before Subcutaneous injection of GM-CSF around and after treatment ulcer

Experimental method

90 B. Cheng and X. Fu

A systematic review

2011

Hu et al. (2011)

The wound healing

To evaluate the effect of the rhGM-CSF on wound healing, 8 RCT studies and 23 clinical studies and case reports are collected for analysis of the evidence

Comparison before One patient had a neuropathic-diabetic ulcer, and after treatment and four had long-standing vascular leg ulcers. Ulcers were cleansed with 0.9% sodium chloride solution and sprinkled with 1–2 mL of the rHuGM-CSF working solution. The working solution was applied three times a day for the first week then daily for the remainder of the treatment period. The wounds were covered with non-adhesive simple dressing changed every day

2002

Bianchi et al. (2002)

Chronic cutaneous leg ulcers

Therapeutic method

Experimental method

Particular year

Author

Disease species

Table 1 (continued)

population transcribing VEGF after GM-CSF treatment. In vitro analysis demonstrated that VEGF transcription can be directly stimulated by GM-CSF in a differentiated monocytic cell line, but not in keratinocytes. Data show that increased vascularization is associated with GM-CSF treatment of chronic venous ulcers and indicate that inflammatory cell-derived VEGF may act as an angiogenic mediator of the healing effect of GM-CSF in chronic ulcers Pathogenesis, size and duration of the ulcers seemed to be the most important parameters regarding wound repairing capability of rHuGM-CSF. None of the ulcers increased in size and none of the patients developed clinical side-effects or peripheral blood cell count abnormalities during the treatment. All the results described were stable after 6 months of follow up. The absence of peripheral leucocyte count variation and the size-dependent therapeutic effect indicate that the drug exercises local rather than systemic actions The overall effects of rhGM-CSF on the healing of wound are diverse. Topically applied rhGM-CSF is beneficial for deep partial-thickness burn wounds, chronic leg ulcers, and leprosy ulcers. rhGM-CSF may (continued)

Results/Conclusions

Biologic Transducers in Wound Healing 91

Disease species

Author

Table 1 (continued)

Particular year

Experimental method

Therapeutic method

have a positive effect on other type of chronic ulcers such as pressure ulcers and cancer related ulcers, but the evidence is not sufficient for generalised use at present. rhGM-CSF is suggested have no accelerating effect on the healing of healthy wounds or surgical incisions

Results/Conclusions

92 B. Cheng and X. Fu

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important role in the physiological and pathological processes of tissue repair. They include chemotaxis of inflammatory cells (such as neutrophils and monocytes) and recruitment repaired cells to the wound, promoting the mitosis of VECs, fibroblasts, SMCs and keratinocytes, regulating the synthesis and remodeling of extracellular matrix (ECM), contribute to angiogenesis, granulation tissue forming, and re-epithelialization of the wound site. The grades of evidence and expert recommendations for PDGF to promote wound healing in different types of wounds, especially chronic ulcers. In the 1980s, PDGF became the first growth factor that was isolated and purified to homogeneity. The first human experience of PDGF using in chronic wound, published as a preliminary report in the Lancet in 1992 and based on preclinical results, reported the use of exogenous PDGF-BB in the treatment of pressure ulcers (Robson et al. 1992a). Soon afterwards, a more detailed report of that phase I/II trial was published in the Annals of Plastic Surgery. Total three dose levels of PDGF (1 lg/ml, 10 lg/ml, and 100 lg/ml) were used and compared with a placebo. rPDGF-BB topically used in chronic pressure ulcers, which can promote wound closure compared with that in a similarly managed placebo control group. In addition, this article indicated the effective doses of wound-healing with PDGF are about 10–20 pg/cm2, similar to the total dose delivered in the patients who received 100 µg/ml. In 1997, rhPDGF-BB was approved by the American FDA for the therapy of chronic lower extremity diabetic neuropathic ulcers, it became the first pharmacological agent approved for treatment of a chronic ulcer condition. The recombinant growth factor is loaded in a aqueous-based sodium carboxymethylcellulose (NaCMC) gel and is marketed as Regranex®. The local used for diabetic foot ulcers that could arrived to the subcutaneous tissue and supplied adequate blood flow. Steed reported the therapeutic efficacy of rhPDGF-BB gel accelerating healing of lower extremity diabetic ulcers, he used a multi-center, randomized, prospective, double-blind, parallel-group, placebo-controlled clinical trial in 1995. This Phase II trial was the first to investigate PDGF in human diabetic ulcers and recruited 118 patients. The results illustrate that rhPDGF-BB gel is safety profile of repeated, once-daily, topical application, and could easily be employed. And there was a statistically significant difference in both the number of patients healed and the healing rates in diabetic patients with chronic ulcers treated topically with rhPDGF-BB gel (Steed 1995). In 2006, Steed again evaluated the safety and efficacy of rhPDGF therapy for the treatment of diabetic foot ulcers. It was effective of rhPDGF applied once daily on healing chronic diabetic foot ulcers, but should be used in conjunction with good wound care. There was a trend toward more recurrences in the placebo control group than in the rhPDGF treatment group, it was not statistically significant, no difference between these two groups in the incidence of adverse events (Steed 2006). A multicenter, double-blind, placebo-controlled, Phase III trial assigned 382 patients to becaplermin gel (30 and 100 lg/g) and placebo gel treatment groups (the control. The becaplermin treated wounds had higher incidences of complete healing over the study period, but only the 100 lg/g becaplermin dose yielded statistically

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significant results when compared to placebo gel (50% vs. 35%, p = 0.01). In order to ensure efficacy of topical becaplermin gel, the treatment process must be administered along with a standardized regimen of good wound care, which consisted of twice-daily dressing changes, debridement to remove nonviable tissue, systemic control of infection (Wieman et al. 1998). Therefore, the utility of using becaplermin therapy earlier in those patients with wounds at high risk for failure, such as large foot ulcers (Wieman 2005). In a word, rhPDGF is a safe and effective treatment for lower-extremity diabetic neuropathic ulcers. Robson et al. also performed a meta-analysis after the Phase IV trial to integrate those results with the previous RCTs. In spite of the phase IV study itself did not showing statistical significance results, the overall meta-analysis results did not deviate remarkably from that of the RCTs (Robson et al. 2005). A few authorities recommend becaplermin as an adjuvant treatment for diabetic foot ulcers that do not respond acceptably to optimized standard strategies. The scholars suggest that ulcer wound therapy would be more readily add becaplermin in the case of aged patients. As there are likely more senescent cells and a relative growth factor deficiency that may derive more benefit from exogenous growth factor supplementation (Fang and Galiano 2008). In 2014, Zhao and his colleagues systematically reviewed and meta analyzed about topical recombinant human platelet-derived growth factor treating diabetic lower-extremity ulcers. A total of 6 randomized controlled trials including 992 patients were selected from 173 identified studies. The studies compared rhPDGF treatment in the context of standard of care (SOC) to placebo or SOC alone. In the absence of study heterogeneity, a fixed-effects model was performed, and the combined odds ratio (OR) indicated a significantly greater complete healing rate in patients treated with rhPDGF compared to placebo or SOC alone. The ORs ranged from 0.58 to 2.77, with a combined OR of 1.53 (95% CI = 1.14– 2.04, p = 0.004). A sensitivity analysis (leave-one-out method) indicated good study reliability, and a funnel plot with Egger test showed no publication bias. The results showed that rhPDGF is useful for treating diabetic lower-extremity ulcers (Zhao et al. 2014). Another systematic reviews has reported that employing of rhPDGF (becaplermin) combined with good wound management was cost effective in many developed country, such as United States, Canada, the United Kingdom, Switzerland, and Sweden, etc. (Zhao et al. 2014; Langer and Rogowski 2009).

FGF FGF is a very important of significant growth factors in body. It has a wide range of biological effects on tissues and cells (fibroblasts, vascular endothelial cells, epithelial cells, etc.) derived from mesoderm and neuroectoderm, and is involved in wound healing. In the family of FGFs, topical bFGF and aFGF have been used widely. The possible biological functions of exogenous FGF for promoting wound healing are as follows. (1) FGF can observably promote angiogenesis, exert chemotaxis of various cells involved in angiogenesis, and promote their proliferation and migration, which is one of the main angiogenetic factors. (2) Injury-induced FGF promotes the aggregation of monocytes, neutrophils,

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95

macrophages and fibroblasts via chemotaxis to injured tissue sites. It can also promote mitogenic activity, which mainly demonstrates could be promote cell proliferation and division. The evidence grades and wound expert recommendations for FGF to promote the healing of kinds of wounds (Fu et al. 1998; Ohura et al. 2011; Hong et al. 2004). Robson’s group (Robson et al. 1992b) firstly reported the randomized, blinded, placebo-controlled phase I/II clinical trials for recombinant bFGF. All patients with stage III/IV pressure ulcers were treated with eight different dosage regimens of three different bFGF concentrations (1.0 lg/cm2, 5.0 lg/cm2, 10 lg/cm2). There was a significantly tendency to accelerate healing in six of eight groups treated with topical bFGF, compared with the vehicle-treated groups. All patients receiving bFGF at the two institutional sites were combined as a group, the difference between the slopes of the treated and placebo curves was significant (p < 0.05). When the data were analyzed in terms of the number of patients achieving a 69% volume reduction, compared with 59% for the control group. This outcome was significantly different when analyzed by the Fisher's exact test (p = 0.047). This first human trial suggests that topically applied recombinant bFGF is safe, and may be effective in the treatment of chronic wounds, especially pressure sores. In 1995, Richard et al. (1995) assessed the efficacy and safety of topical recombinant human basic fibroblast growth factor (rh-bFGF) on the healing of diabetic foot neurotrophic ulcers. Cases inclusion criteria were a typical neuropathic ulcer of Wagner grade I-III, more than 0.5 cm in the largest diameter, with an abnormally high vibration perception threshold in the absence of significant peripheral vascular disease or wound infection. Rh-bFGF or placebo (normal saline) was applied once a day during the first 6 weeks, then twice a week last 12 weeks. Changes of ulcer size was assessed through weekly clinical examination and computerized photographs. This pilot (phase I and II), randomized, double-blind, placebo-controlled study showed there was not significantly different, repartition in Wagner's classification was similar in both groups at the end of the clinical trails. The weekly reduction in ulcer perimeter and area was identical in both groups, as was the rate of linear advance from entry to the 6th week of treatment (bFGF: 0.053 ± 0.048 mm vs. normal saline: 0.116 ± 1.129 mm): the same result was obtained at the 11th week. Moreover, percent healed area at the end of the study did not differ significantly. In addition, this study indicate that topical rh-bFGF application was well tolerated; the clinical studies observed no clinical drug-related adverse events or abnormalities in hematological or biochemical data. In a word, topical application recombinant human bFGF has no advantage over placebo for healing chronic neuropathic ulcer of the diabetic foot. The authors inferenced that using a single recombinant growth factor might be insufficient to accelerate wound closure of diabetic ulcers.

EGF EGF is found in almost all kinds of body fluids, secretions and most tissues. EGF receptor (EGFR) is mainly expressed by keratinocytes. Other EGFR-expressing cells include normal fibroblasts, Vascular endothelial cells, smooth muscle cells

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(SMCs), glial cells, Mucosal epithelium and chondrocytes. EGF exerts chemotaxis and mitogenic effects mainly by binding to receptors on the cell membrane and forming a complex signal pathway during its intracellular transmission, resulting in the regulation of cell metabolism, differentiation and other biological activities. Based on literature literature, specialist of wound healing inferred and verified that EGF can promote the healing of various kinds of skin and soft tissue defects. In 1992, a double-blind randomized study conducted at a single center was used observed that recombinant human epidermal growth factor (rh-EGF) treat 44 patients with venous ulceration of the lower extremities. An aqueous solution (10 lg/mh) of rh-EGF was applied topically to the ulcers, twice a day until healing. On the basis of this study findings, the authors thought using rh-EGF for chronic wound with in the dose and manner was safe. Althought failed to significantly enhance reepithelialization of venous ulcers. A greater reduction in ulcer size and a larger number of healed ulcers with the use of rh-EGF are be worth looking forward to results (Falanga et al. 1992). In 2014, Gomez-Villa (2014) investigated the efficacy and safety of rhEGF in patients with diabetic foot ulcer (DFU). A randomized, two-center, double-blinded, placebo-controlled study was conducted comparing a thrice-per-week intralesional application of rhEGF (75 lg) or vehicle (placebo) in patients with DFU for 8 weeks, three times per week. Most of DFU were stage IIIA, B and stage IIB. All patients received standard care, which included a complete physical examination and consultation by an internist. The number of completely healed ulcers, size, and wound bed characteristics were evaluated to determine the efficacy of rhEGF with Student’s t test. Intralesional rhEGF could promoted the epithelialization of the wound bed, and significantly reduced the area of the DFU treated. Therefore, rhEGF resulted in better outcomes for patients suffering from DFU. The only adverse event that we observed more frequently in the rhEGF group was dizziness, not identified a mechanism to explain why dizziness occurred. Yang et al. (2016) topically used recombinant human epidermal growth factor (rhEGF) on the ulcers of diabetic foot. This systematic review and meta-analysis was designed to evaluate if rhEGF increased the complete healing rate of diabetic foot ulcers compared with the control. This searched the MEDLINE, Cochrane Library, EMBASE, and Web of Knowledge databases (up to December 22, 2015). Studies were identified and selected, and data were extracted by 2 independent reviewers. The initial search identified 399 articles. they also hand-searched 12 articles from reference lists of relevant review articles.. All statistical analyses were performed using Review Manager Meta-Analysis software, version 5.1 (The Nordic Cochrane Centre, The Cochrane Collaboration; http://ims.cochrane.org/revman). The studies evaluated the rate of healing of diabetic foot that were treated with rhEGF or controls (placebo). On account of study heterogeneity, a random-effects model was performed, and the combined odds ratio (OR) indicated a significantly greater complete healing rate in patients treated with rhEGF compared to placebo. These results indicate that rhEGF is beneficial in the treatment of diabetic foot ulcers by increasing the rate of wound healing. These findings provides evidence for a significant effect of rhEGF in treating diabetic foot ulcers. In future, the studies

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should be assess the effects of rhEGF alone or in combination with other growth factors or wound dressing agents. In 2019, Bui et al. (2019) published a meta-analysis (registration number CRD42019126404) that was performed to synthesize the evidence of rhEGF treatment in DFUs in comparison to placebo. Databases included for the search were PubMed, EMBASE, the Cochrane Library, Web of Science, EBSCOhost, ScienceDirect, and Scopus (up to 10 January 2019). A randomized, placebo-controlled trials evaluating the effects of rhEGF administration, for example intralesional injection, topical-gel, cream using for patients with DFUs. The outcome of interest was the complete healing rate of DFUs. Quality of evidence on the complete healing rate was estimated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. The Stata/IC software (version 15.1, StataCorp LL, College Station, Texas, USA) was used to make a funnel plot and Egger’s regression test. The results showed that combined OR (intralesional injection and topical apply) was 4.005 (95% CI: (2.248; 7.135), p < 0.001). The ORs for intralesional injection and topical application were 3.599 (95% CI: (1.213; 10.677), p = 0.021) and 4.176 (95% CI: (2.112; 8.256), p < 0.001), respectively. Statistical heterogeneity might not be important in overall treatment and both of the subgroups. They results support the application of rhEGF using for DFUs. However, the GRADE approach on rhEGF treatment is low evidence. Hence, the effect estimate of rhEGF may be limited, and the true effect may be substantially different from the estimate effect. At the same time, the amount of data on adverse effects was also limited. The study emphatically point out wound care is a key step throughout the treatment process, but it is not enough. Many other factors, for example control of infection, ischemia, and blood glucose, offloading, must also be seriously consideration, along with local wound management. In 2020, Yang et al. (2020) finished a meta-analysis of the efficacy and safety of topical recombinant human epidermal growth factor (rhEGF) on the treatment of diabetic foot ulcers. The authors conducted a comprehensive review of PubMed, EMBASE, Cochrane Library databases, and Web of Science (up to November 30, 2018). Seven randomized controlled trials (RCTs) that involved 610 participants were included in this review. These results were analysized with RevMan 5.3 software. The pooled results showed that topical rhEGF could significantly promote the healing of diabetic foot ulcers. Topical application of rhEGF could promote ulceration healing of diabetic feet of Wagner grade 1 or 2 significantly, and intralesional injection of rhEGF appeared to promote the healing of more severe ulcers. The quality of the evidence was low due to unclear risk of bias in the original trial and moderate statistical heterogeneity. The statistical result displayed that there is no significant difference in the incidence of adverse events between the rhEGF treatment group and the control group. Oliveira and his colleagues (2021) investigated whether the addition of recombinant human epidermal growth factor (rh-EGF) to 2% carboxymethyl cellulose gel is more effective in diabetic wound healing than standard treatment (the best standard of usual care available either alone or in addition to the intervention), a pilot, double-blind (patient and statistician), randomized (1:1 ratio) and controlled

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clinical trial (NCT12616798, Brazilian) with therapeutic intervention was performed at a university hospital. All sample contained 25 patients (14 in the intervention group with using rh-EGF (4 ppm) and 11 in the control group with using 2% carboxymethyl cellulose gel) with diabetic wound or a chronic venous ulcer, from 2 cm2 to 100 cm2, time longer than 12 weeks. Data were tabulated in SPSS and analyzed by intention-to-treat approach, without loss or exclusion of participants. Twenty-five subjects participated with a mean age of 60.6 years, a predominance of males in both groups and 100% prevalence of type-2 diabetes. Within 12 weeks, complete wound healing occurred in three ulcers in the intervention group versus one ulcer in the control group. The percent reduction in the wound area was significantly higher in the intervention group than in the control group (p = 0.049). About the types of tissue, increase of granulation and epithelialization, and a reduction of exudation were observed in both groups. Decreased slough occurred only in the intervention group. No participant experienced serious or local adverse events during the study period. This study showed that rh-EGF is effective and safe treatment for chronic wounds, with a statistically significant reduction in wound area, improvement of tissue quality.

Combination Application of Growth Factors in Chronic Wound Wound healing is a complicated process that comprises of various cytokines and growth factors which play roles in a repair signal pathway, it is not surprising that single growth factor therapy outcome in not remarkable benefits. To move forward in optimising growth factor delivery to wounds, especially ulcer, some research investigated the ways for delivering individual or combinations of growth factors. In 2000, Robson compared the healing response of sequential topically applied cytokines to that of each growth factor or cytokine alone and to a placebo-treated patients with pressure ulcers, and to evaluated the molecular and cellular responses. Because of a deficiency of growth factors and cytokines in chronic wounds and the reversal of impaired healing in animal models, pressure ulcer trials have been performed with several exogenous growth factors/cytokines application. Because single growth factor therapy has not been uniformly successful in therapy pressure sore, combination or sequential cytokine or/and growth factor treatment has been proposed. Laboratory data have suggested that sequential treatment with granulocyte–macrophage/colony-stimulating factor (GM-CSF)/basic fibroblast growth factor (bFGF) might augment the previously reported effect of bFGF alone. A double-blind, randomized, placebo-controlled pressure ulcer trial was performed comparing sequential GM-CSF/bFGF therapy with that of each cytokine alone and with placebo during a 35-day period. The primary measure was wound volume decrease over time. Level of cytokine/growth factor in wound were serially determined. Ulcers treated with cytokines and growth factor had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Although more than 85% wound closure appeared to distinguish cytokine/growth factor-treated wounds from those

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treated with placebos, and a statistically greater number of bFGF-treated patients achieved more than 85% closure, only quarter of all the patients had more than 85% wound closure. The reason may be due to the only 35-day treatment period (Robson et al. 2000). In future, combination application of growth factors/cytokines for chronic wound should be focus their temporal and spatial characteristics in the process of wound (e.g. waterfall versus slow release), protect the growth factor from degradation and provide tissue concentrations consistent with wound repair and regeneration.

Effectiveness of (Stem) Cells for Skin Chronic Wounds Wound healing has been greatly challenging in different chronic refractory ulceration. Among them, nonrevascularizable critical limb ischemic ulcers, venous leg ulcers, and diabetic neurovascular ulcers are well-known refractory skin defects that are difficult toheal within reasonable periods. Cellular therapy, characterized by using cells from diverse sources, with self-renewing potential and multi-differentiation ability, has shown promise in the management of chronic wounds. Partly differentiated, progenitor cell-based graft transplantation or direct injection of autologous stem cells might promote the wound healing. In 2021, Dong’s studies aiming to comprehensively analyzed the effects of cell therapy on skin wound healing could provide clinical evidence for skin defects treatment. Different databases (Science Direct, Springer Link, Web of Science, ProQuest, and Network Digital Library, Cochrane Central Register of Controlled Trials, etc.) were searched for full-text publications on the comparison between (stem) cell therapy and regular therapy, from January 2000 to March 2020. After a long-term follow-up, fewer patients underwent major amputation in the cell-therapy group, compared with the standard therapy group, and those in the cell therapy group were characterized by a smaller ulcer area. Moreover, there was a significant difference in the wound healing rate between the intervention and control groups. However, pain caused by skin wounds was hardly mitigated by cell therapy in patients with critical limb ischemia. In this study, cell therapy proved effective in decreasing the size of ulcers and improving the wound closure rate. Additionally, the rate of major amputation was significantly decreased in the cell therapy group, compared with the normal treatment. However, the symptoms of pain were hardly alleviated by cell therapy in patients with cutaneous ulcers caused by peripheral artery disease-related critical limb ischemia (Dong et al. 2021). Autologous stem cell therapy (ASCT) has emerged as a promising alternative treatment for those who suffered from Lower extremity chronic wounds (LECWs). Chiang and his colleagues (Chiang et al. 2021) assessed the effects of ASCT on LECWs with the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines and PROSPERO CRD42021248746. The authors searched three core databases (PubMed, Embase, and Cochrane Controlled

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Trials Register), and independently identified evidence according to predefined criteria. They also individually assessed the quality of the included randomized controlled trials (RCTs) (up to December 2020), and extracted data on complete healing rate, amputation rate, and outcomes regarding peripheral circulation. The extracted data were pooled using a random-effects model due to clinical heterogeneity among the included RCTs. A subgroup analysis was further performed according to etiology, source of stem cells, follow-up time, and cell markers. This systematic review and meta-analysis of RCTs were conducted following the recommendations of the Cochrane Collaboration, and were reported according to the Preferred Reporting Items for Systematic Review and Meta-Analyses Protocols (PRISMA-P) guidelines. ASCT through intramuscular injection can significantly improve wound healing in patients with LECWs caused by either DM or critical limb ischemia. The results showed significantly increased values of Ankle brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) levels (indicating better perfusion and higher levels of tissue oxygen available for healing), which were meaningful to confirm the improvements of total and major amputation rates and wound healing rate. ASCT significantly and similarly increased the complete wound healing rates in patients with either DM. The intramuscular subgroup had a significantly higher complete wound healing rate than the intra-arterial subgroup. Moreover, intramuscular injection seems to be a rather safe choice for administration. However, more extensive scale and well-designed studies are necessary to explore the details of ASCT and chronic wound healing. Shu assessed the current evidence regarding the efficiency and potential advantages of stem cell-based therapy compared with conventional standard treatment and/or placebo in the treatment of diabetic foot ulcer. A comprehensive search in PubMed, EmBase, Cochrane Central and Web of Science databases was conducted during December 2016 and a systematic review and meta-analysis of all relevant studies were performed. A total of 7 studies that involved 224 diabetic foot patients, classified as Wagner grades 1–5, were analyzed. The pooled results confirmed the benefits of using the stem cell treatment. Partial and/or complete healing were significantly faster in the stem cell group compared with the control group. The present meta-analysis indicates that stem cell-based therapy can enhance the healing of diabetic foot ulcers and is associated with lesser pain, lower amputation rate and improved prognosis compared with normal treatment. Larger sample sizes and thorough clinical research are required in order to determine whether the source, the dose and/or the delivery method of stem cells are key factors for the successful accomplish of the process. In future, well-designed randomized controlled trials are required to confirm and update these findings (Shu et al. 2018). Although primary studies in animal models and humans have showed the therapeutic potential of autologous stem cell help to accelerate healing of chronic wounds. The results of pilot randomized controlled trials (RCTs) in humans have been inconsistent. A systematic review and meta-analysis of RCTs was performed to evaluate the role of autologous stem cell-based therapy (derived either from bone marrow or from peripheral blood) for lower extremity ulcers. Studies were identified during a systematic search of Pubmed, Embase, Cochrane's library,

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and references cited in related reviews and studies. Studies were included if they were RCTs published in English, recruited patients with lower extremity ulcers who were assigned to either a group for the topical therapy with autologous stem cells, a control group (with no treatment or placebo), and reported data regarding the healing of the chronic wounds. Overall, autologous stem cell-based therapy was associated with better healing of lower extremity ulcers with little heterogeneity. Moreover, autologous stem cell-based therapy was associated with a greater reduction in ulcer size. Subgroup analyses indicated that stem cells from peripheral blood and bone marrow seemed to exert similar beneficial effects on the healing of ulcers. Stem cell therapy was not associated with any increased risks for procedure-related adverse events. The optimized sources, amounts, and delivery methods of stem cell-based therapy for patients with chronic lower extremity ulcers need to be determined, and the long-term effects of stem cell-based therapy on clinical outcomes need further investigate. Autologous stem cell-based therapy is effective and safe for improving the healing of chronic lower extremity ulcers, and large-scale RCTs are needed to confirm the findings (Jiang et al. 2016). Accumulating evidence from basic science studies and clinical trials have pointed out that cellular therapy could focus on multiple facets during chronic wounds (like diabetic foot ulcer) healing disorder through dysfunction of cell proliferation, vascularization, neuro-restoration, inflammation regulation, exosomes synthesis, and others. A meta-analysis of randomized controlled clinical trials evaluated and synthesized clinical evidence, its aims to estimate the therapeutic efficacy of cellular therapy for diabetic foot ulcer compared to standard therapy. The results showed cellular therapy seemed to be safe and effective treatment of diabetic foot ulcer, associated with a higher ABI, TcPO2, more reduction in pain, decreased amputation risk, and with no serious complications and low risk of short-term slight complications (Zhang et al. 2017b).

Outlook The aim of this chapter was to review the value that biological transducers bring to chronic wound management. Although many aspects of the healing process are influenced by many factors, the obstruction of cytokines and growth factors formation may be a major factor of chronic wound healing disorder. Even with cytokines/growth factors present, wound cells might be unresponsive due to a lack of cell surface receptors. Therefore, if chronic ulcer is to healing, we must be to supply more cytokines/growth factors (Cheng and Fu 2018) together with mainstay treatment as in standardized care. Mainstay treatment for venous leg ulcers is compression, for neuropathic diabetic foot ulcers is offloading and for ischemic diabetic foot ulcers, it is revascularization. Highlighted mainstay is an essential aspect of chronic wound treatment.

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To overcome this unmet need, therapeutic growth factors has evolved in attempt to increase repaired cells proliferation, migration, differentiation in injury sites. One unavoidable and important problem involves the local protease-rich environment of a chronic wound, which has been shown to actively degrade and inactivate most growth factors. This finding may explain in part the altered levels of growth factors in chronic wounds, but it also suggests that topical therapeutic applications probably allow only a brief suboptimal exposure of the wound to the growth factor unless the protease-rich, pro-inflammatory wound environment is first addressed. Drug delivery strategies suffer from the inherent loss of drug activity due to the combined effects of physical inhibition and biological degradation. In order to overcome the limitations of topical using recombinant growth factors or peptides, a molecular genetic approach in which genetically modified cells synthesize and deliver the desired growth factor in a time-regulated manner is a powerful means. Recently, two multicenter, double-blind, placebo-controlled clinical trials in Japan (phase III) and US (phase II) demonstrated that hepatocyte growth factor (HGF) gene therapy for CLI significant improved primary end points and tissue oxygenation up to two years in comparison to placebo. These clinical results implicate a distinct action of HGF on cellular processes involved in vascular remodeling under pathological condition. This review presents data from phase I-III clinical trials of therapeutic angiogenesis by gene therapy in patients (Sanada et al. 2014). In addition, current investigations are studying the combination of growth factors, the improvement of growth factor vehicles, the utilization of tissue engineering constructs with growth factors, the interaction of stem cells in conjunction with growth factors, and gene therapy modalities. Future successful therapeutics for chronic ulcers will likely parallel the development of effective therapies for nonhealing wounds as a whole. A new generation of growth factor therapy may very well be on the horizon for these wounds, which the therapeutics will likely need to be integrated into a combination therapy to be most effective.

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Physical, Electromagnetic, Biologic Devices Alberto Piaggesi

Abstract

The inception of Physical, Electromagnetic and Biologic devices into the field of wound management has greatly changed the standard approach to wound healing—only think of negative pressure wound therapy. This and other innovations introduced a completely different approach both on diagnostic and therapeutic aspects of this multi-specialistic area, in a variety of conditions all characterized by chronic ulceration. The number and variety of medical devices focused on wound management would deserve a thorough evaluation and description, which is beyond the scope of this report, in which a synthesis of the more relevant among the new technologies applied so far in the clinical field, reporting also their level of evidence, addressing the reader to the relevant literature. Keywords

Bio-Physic Evidence


Electromagnetism
Biologic Devices
Wound management


A. Piaggesi (&) Diabetic Foot Section, Department of Medicine, University of Pisa, Pisa, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_6

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Introduction The ways and the extent at which new technologies influenced the practice of wound management progressively increased in the last years, changing both diagnosis and therapy of a number of different pathologies, all characterized by the presence of chronic wounds (Mani et al. 2016). The impact of this reevolution has been so relevant that the European Wound of Management Association (EWMA), a scientific society focused on wound management, decide to edit two documents dedicated to this topic; the first, entitled Advanced Therapies in Wound Management, was published in 2018 (Piaggesi et al. 2018), while the second, entitled New Technologies for Tissue Replacement, will be published in September 2022 as a supplement of the Journal of Wound Management (Piaggesi et al. 2022). In these two documents, both edited by the author of this chapter, a panel of experienced and extremely qualified authors synthetized both the technical and clinical features of many classes of devices and technologies related to wound management, from physical –related technologies to new materials, to dermal and bone substitutes to nanotechnologies to internet-related technologies. The importance of this contribution is evident when one thinks of the increasing number of patients with chronic wounds and complex cases, which deserve effective solutions, to the cost-effectiveness of the interventions, to the new medical technology rules, which have drastically changed the scenario of medical devices in Europe (Rayman et al. 2019; Lindholm and Searle 2016). To adequately address the new technologies in wound management an exhaustive research on the most important databases has been carried out, in order to sort out the evidence behind the technologies and evidence tables for any section were produced as a support for the readers, alongside the reference tables, in which all the papers included in the analysis were synthetically summarized. A similar approach will be followed in this display, in which the most important chapters of the documents will be synthetized, providing the relative evidence tables.

Physical/Delivery Systems The inception of physical means into the management of chronic ulceration was actually a game changer, since it opened to a brand-new philosophy behind diagnosis and treatment of these complex conditions, based on the interaction between physical forces and the biology of the lesions, rather than on chemical and /or biochemical reactions. This was in a way a revolution, because the easiness of supplying, the re-usability, the lack of direct contact and the wide range of solutions, from electric and electro-magnetic fields to light and lasers, ionic plasma to fluorescence, made it possible to re-shape the diagnostic and therapeutic strategies

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Fig. 1 Auto fluorescence (left to right). In a chronic infected lesion, where it is difficult to identify biofilm and infected site to be debrided, the illumination with Infrared polarized light activate auto-fluorescence in eucariotic cells and bacteria, which react producing light at different wavelengths, thus separating the infected areas (cyan green) from healthy granulating tissue (dark green) (Anghel et al. 2016)

in many different complex situations. This has improved our possibility of curing the patients. Either the diagnostic approach, with new ecographic-based technologies and point-of care detectors (Fig. 1) (Pieruzzi et al. 2020; Anghel et al. 2016) and therapy, with a wide range of new possibilities (Ennis et al. 2016) has been deeply affected. More recently, besides the physical technologies strictu sensu, also delivery systems and materials came into play, opening new possibilities for patients suffering from chronic wounds. For the sake of the exposition, the different technologies have been grouped according to their basic physical principles; In Table 1 the most important physical technologies and delivery systems are reported together with their level of evidence. according to SORT system, ranging from 1A (= more than 1 randomized controlled trial) to 4 (= expert opinion).

Materials Tissue replacement relies on natural or artificial tri-dimensional (3D) matrices that provide a temporary template for the invasion of host cells that gradually deposit their own matrix and neo tissue. Naturally, a successful interaction with host cells is expected to be reached if they encounter a support that resembles their own extracellular matrix (ECM) maximizing their response. In fact, ECM-derived

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Table 1 Evaluation of evidence levels: physical technologies for tissue replacement (VLU = venous leg ulcers; PU = pressure ulcers; DFU = diabetic foot ulcers) Technology

Indication

Level of evidence

Comments

Autofluorescence

VLU, PU, DFU VLU, PU, DFU

1C

Cold atmospheric plasma Blue light

VLU, PU, DFU VLU, DFU

1B

Light activated nanofiber textile Electric stimulation

VLU

2B

PU

1B

Topical oxygen, magnetic stimulation and low-energy light Injectable hydrogels

VLU

1C

Preliminary positive results, both in vitro and in clinical trials Positive results both in vitro and in vivo, some initial clinical evidence no RCT at present Positive findings in clinical trials, good evidence in pre-clinical models Good evidence in vitro, preliminary positive results in observational studies Good preliminary results in a pivotal clinical experience Solid evidence in vitro and in animal models, positive results in one RCT Positive results in one RCT and in one observational trial

–

–

Hyperspectral imaging

1B

1C

Too early to be proposed for clinical applications, but extremely promising

structures to which cells were removed while preserving (not completely) native structure and composition, can be considered the gold standard of dermal templates. Additionally, ECM has been the source of components that are combined in various formulations and then processed/manufactured as 3D porous structures to form scaffolds that tend to provide the elements that stand out in the native tissue, to get improved clinical performance (Casey 2002). ECM has been the font of inspiration for the development of artificial (bio)materials, but it is not evident if these have superior performance than the ECM-derived ones or if that depends on the application/tissue to be healed (Luo and Wu 2020). The properties of artificial materials are highly controlled in opposition to the variability associated to natural sources, allowing the use of a greater number of processing methodologies to generate 3D structures that can act as tissue templates. Nonetheless, this is also directly linked to their bioactivity since the coupling of biomolecules/cues to those materials narrows that window. Therefore, a well-balanced compromise between bioactivity/ECM resemblance and processing conditions, is required in the development of tissue templates with a maximized potential for tissue replacement (Rudge 1999). In Table 2 the most important technologies on biomaterials are reported, together with their level of evidence.

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Table 2 Evaluation of evidence levels of biomaterials-based technologies for tissue replacement Technology

Indication

Level of evidence

Comments

Acellular Dermal or non-dermal Matrices Acellular Dermal Matrix Acellular Dermal Matrix Artificial Matrices

DFU

1B

VLU

2C

Burns

2C

Burns

2C

Complex DFU Wounds –

2B

–

–

–

–

High-quality studies and good evidence of effectiveness despite variabilities among trials Few RCT with weak evidence; likely to perform equal to other technologies Few RCT with weak evidence; likely to perform equal to other technologies Few RCT with weak evidence; likely to perform equal to other technologies Positive results non-RCT studies and case series regarding the healing of bone and/or tendon tissues during wound closure Promising in vivo indications regarding the importance of biomaterials responding to specific wound features (vascularization; inflammation) Promising in vivo indications in promoting wound closure and neovascularization, potentially reducing scarring Relevant results of customised prosthesis (Bone and device for NPWT) in feasibility clinical studies but limited use as regenerative templates for wounds

Non-living tissue derived matrices Extracellular Matrix-derived Biomaterials Bioprinting (cellular skin substitutes) 3D printing

–

Skin Substitutes Since the last three decades the acellular dermal substitutes have changed the concept of skin reconstruction. The neo-dermal component forming the dermal substitute limits the secondary retraction of the thin autologous skin graft used to cover it. Many products have been proposed and they can be with or without elastin, their collagen can come from different animals like cows, fish, or pigs with different combination with elastin, and they can be covered by a protective film in silicone and secondarily skin grafted after 3 weeks. This period is fundamental in order to permit scaffold’s degradation and consequently the formation of a new functional tissue which can avoid scar formation (Dai et al. 2020). The heterogeneity of the different dermal substitutes and their different indications make the global perception of these medical devices somehow confusing starting from their classification. Skin substitutes can be classified as epidermal, dermal, and composite, and further split into different categories depending on their composition and source of material (xenograft, acellular allograft, cellular allograft, autograft, synthetic skin substitutes), contraction capacity, pores size, and shape

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Table 3 Level of evidence of skin substitute based on the last 5 years of references Dermal substitute

Level of Evidence

Comments

Integra

3b

Matriderm Nevelia Pelnac

3b 4 3b

Kerecis Dehidrated amniotic Membrane Apligraf Oasis DeNovo skin

4 3b

Positive results from case series and retrospective studies. Few prospective and/or randomized control trials Positive results from case series and retrospective studies Positive results from case series Positive results from retrospective studies. Few prospective or randomized clinical trials Positive results from case series Positive results from case series retrospective studies. Few randomized control trials

5 N/A N/A

Narrative reviews, only one RCT Positive results from case reports, only 1 RCT Phase I trial

(Goodarzi et al. 2018). Because there is no ideal option for skin substitutes there are many researches evaluating and developing different skin substitute options (Límová 2010). In Table 3 the different options in the field of skin substitutes, with the relative evidence are reported.

Bone Substitutes It has been estimated that 60% of diabetic foot ulcerations (DFU) are infected at the time of initial evaluation (59). In the setting of osteomyelitis and/or soft tissue infection, antibiotic therapy most often in conjunction with surgical debridement of infected, non-viable tissue and bone is the usual initial course of treatment. In addition to debridement and systemic antibiotic therapy, local antibiotic delivery via non-absorbable/non-resorbable bone cement polymethyl-methacrylate (PMMA) and absorbable/resorbable bone graft substitutes may be a beneficial adjunct to surgical treatment of osteomyelitis in patients with infected diabetic foot ulceration. Antibiotic impregnated cement has been used for many years, and recently resorbable bone graft substitutes have been utilized in the treatment of diabetic foot osteomyelitis (DFO). The addition of this method for local delivery of antibiotics during the surgical treatment of DFO may help improve outcomes and reduce amputation rates (Fillingham and Jacobs 2016). There are several challenges that can occur with surgical resection of infected tissue/bone and systemic antibiotic therapy. The use of local antibiotic delivery via non-resorbable and resorbable carriers may help mitigate these potential issues. The decision to surgically resect infected bone is dependent on several variables to

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include location of osteomyelitis, specialty of the provider and available resources. The extent of debridement is also an area of debate as some advocate for total resection and clean margins, while others perform limited bone resections. Surgical excision of infected bone can result in dead space and/or bone defects, which can impact skeletal stability. In addition, despite surgical debridement, residual microorganisms may remain at the site of infection (i.e. positive margins). It is also thought that biofilm related to long standing diabetic foot ulceration may have a role in the development of chronic DFO, serving as a barrier to systemic antibiotic (Campana et al. 2014). Systemic antibiotics can lead to complications such as renal toxicity, bacterial resistance and gastrointestinal dysfunction. In theory, local delivery of antibiotics may provide several benefits compared to oral and intravenous antibiotic therapy. Local delivery can lower the risk for systemic complications and can result in increased concentration of antibiotic at the infection site which is especially beneficial in the setting of peripheral arterial disease. Local antibiotic delivery systems can elude a higher concentration of local antibiotics to a site of infection, as much as 10 to 100 times greater than the minimum inhibitory concentration. When used as a bone substitute, they can also fill a void or dead space left by resection of bone and tissue (Busch et al. 2021). Local antibiotic delivery systems in the form of cement and bone graft substitutes have been widely used and described in the orthopedic literature. Few studies have been published regarding their role in the surgical treatment of DFO. Recently a new class of bone substitutes, called bio-glasses because of their derivation from medical glasses for human use, have proven to be effective to treat osteomyelitis (OM) in DFU, without the need for using local antibiotics (Fig. 2) (Iacopi et al. 2022). In Table 4 the recent evidence for local antibiotic delivery systems, with a focus on the role of the newer resorbable bone graft substitutes and their potential benefits in the surgical management of DFO is reported.

Fig. 2 Bioglass (left to right). In an acutely infected calcaneal bone in a DFU, after drastic debridement and elimination of all the infected bone, the inception of bioglass granules leads to the sterilization of the osteomyelitic focus and to the formation of new healthy bone, with complete restitutio ad integrum (Iacopi et al. 2022)

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Table 4 Levels of Evidence for non-resorbable bone cement (PMMA) and resorbable bone graft substitutes for local antibiotic delivery used for the surgical treatment of diabetic foot osteomyelitis Number

Therapy

Indication for use

Level of evidence

Comments

1

Non-resorbable bone cement for local antibiotic delivery (PMMA) Resorbable bone graft substitutes for local antibiotic delivery Bioactive Glass

Surgical treatment of diabetic foot osteomyelitis Surgical treatment of diabetic foot osteomyelitis Surgical treatment of diabetic foot osteomyelitis

4

Small numbers, case series, retrospective Small numbers, case series, retrospective Small numbers, case series, retrospective

2

3

4

4

Vascular-Related Technologies In 2015, occlusive peripheral arterial disease (PAD) was diagnosed in 236,62 million adults older than 25 year of age world-wide. The prevalence, estimated between 4 et 20%, varies according to, age, smoking habits, diabetes, high blood pressure, hypercholesterolemia and social status. Of these patients 5–10% will develop chronic limb-threatening ischemia (CLTI) within five years. CLTI patients are at high risk of amputation and death. PAD is often underdiagnosed. In a retrospective German study based on statutory health scheme 81% of patients received a vascular diagnostic measure and only 50% had a vascular procedure before amputation. To improve limb salvage and life there is a consensus to proceed to a revascularization whenever feasible, which has been proved to be effective in short and long term (Beckman et al. 2021). The strategies of CLTI management have been reviewed thoroughly by international societies which issued recommendations concerning the grading of the diseases, prognosis, explorations, treatments and follow-up (Rogers and Laird 2007). In Table 5 the evidence of vascular-related technologies is reported.

Conclusions Despite the extreme interest and variety of new technologies in wound management field, still evidence is scarce and of poor quality and their implementation in clinical practice, despite being potentially useful, is negatively conditioned by the lack of solid data in support. New, more solid and prospective randomized trials are expected to finalize the inception of many of these technologies in many different pathologies characterized by chronic wounds.

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Table 5 Vascular-related technologies and their evidence Technology

Indication

Level of evidence

Comments

Conservative treatment

Frail or Non-revascularisable patients Long lesions, saphenous vein available Short lesions

1C

High Mortality and/or amputation

1A

More durable option, Incisional Wound healing problems

2C

Used as a primary option

2B

Used as a bailout procedure

1A

Improved patency But safety uncertainty Improved patency But safety uncertainty More data required

Bypass

Balloon angioplasty Bare stent Drug coated balloon Drug eluting stent Covered stents Atherotom Cellular therapies

Short and medium length lesions Short and medium length lesions Short and medium length lesions Long lesions Calcified lesions Non-revascularizable CLTI patients

1A 2B 2C 1C

More data required Few long-term adequately dimensioned studies Too short follow up Lack of comparative studies

References Anghel EL, Falola RA, Kim PJ. Fluorescence technology for point of care wound management. Surg Technol Int. 2016;28:58–64. PMID: 27175815. Beckman JA, Schneider PA, Conte MS. Advances in revascularization for peripheral artery disease: revascularization in PAD. Circ Res. 2021;128(12):1885–912. Busch A, Jäger M, Mayer C, Sowislok A. Functionalization of synthetic bone substitutes. Int J Mol Sci. 2021;22(9):4412. Campana V, Milano G, Pagano E, Barba M, Cicione C, Salonna G, Lattanzi W, Logroscino G. Bone substitutes in orthopaedic surgery: from basic science to clinical practice. J Mater Sci Mater Med. 2014;25(10):2445–61. Casey G. Wound repair: advanced dressing materials. Nurs Stand. 2002;17(4):49–53; quiz 54, 56. Dai C, Shih S, Khachemoune A. Skin substitutes for acute and chronic wound healing: an updated review. J Dermatolog Treat. 2020;31(6):639–48. Ennis WJ, Lee C, Gellada K, Corbiere TF, Koh TJ. Advanced technologies to improve wound healing: electrical stimulation, vibration therapy, and ultrasound-what is the evidence? Plast Reconstr Surg. 2016;138(3 Suppl):94S–104S. Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Joint J. 2016;98-B(1 Suppl A):6–9. Goodarzi P, Falahzadeh K, Nematizadeh M, Farazandeh P, Payab M, Larijani B, Tayanloo Beik A, Arjmand B. Tissue engineered skin substitutes. Adv Exp Med Biol. 2018;1107:143–88. Iacopi E, Pieruzzi L, Goretti C, Piaggesi A. Pilot experience on the use of S53P4 bioactive glass in the surgical management of diabetic foot osteomyelitis. Int J Low Extrem Wounds. 2022;21 (1):57–64.

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Límová M. Active wound coverings: bioengineered skin and dermal substitutes. Surg Clin North Am. 2010;90(6):1237–55. Lindholm C, Searle R. Wound management for the 21st century: combining effectiveness and efficiency. Int Wound J. 2016;13 Suppl 2(Suppl 2):5–15. Luo GX, Wu J. Modern functional materials for promoting cutaneous wound healing. Zhonghua Shao Shang Za Zhi. 2020;36(12):1113–6. Mani R, Margolis DJ, Shukla V, Akita S, Lazarides M, Piaggesi A, Falanga V, Teot L, Xie T, Bing FX, Romanelli M, Attinger C, Han CM, Lu S, Meaume S, Xu Z, Viswanathan V. Optimizing technology use for chronic lower-extremity wound healing: a consensus document. Int J Low Extrem Wounds. 2016;15(2):102–19. Piaggesi A, Låuchli S, Bassetto F, Biedermann T, Marques A, Najafi B, Palla I, Scarpa C, Seimetz D, Triulzi I, Turchetti G, Vaggelas A. Advanced therapies in wound management: cell and tissue based therapies, physical and bio-physical therapies smart and IT based technologies. J Wound Care. 2018;27(Sup6a):S1–137. Piaggesi A, Bassetto F, Becquemin JP, Dalla Paola L, Den Braber E, Marques A, Raspovic K, Scarpa C, Turchetti G, Teot L. New technologies for tissue replacement. J Wound Manag (JOWM). 2022; In press. Pieruzzi L, Napoli V, Goretti C, Adami D, Iacopi E, Cicorelli A, Piaggesi A. Ultrasound in the modern management of the diabetic foot syndrome: a multipurpose versatile toolkit. Int J Low Extrem Wounds. 2020;19(4):315–33. https://doi.org/10.1177/1534734620948351. Epub 2020 Aug 21. PMID: 32820699. Rayman G, Vas P, Dhatariya K, Driver V, Hartemann A, Londahl M, Piaggesi A, Apelqvist J, Attinger C, Game F. International Working Group on the Diabetic Foot (IWGDF). Guidelines on use of interventions to enhance healing of chronic foot ulcers in diabetes (IWGDF 2019 update). Diab Metab Res Rev. 2020;36 Suppl 1:e3283. Rogers JH, Laird JR. Overview of new technologies for lower extremity revascularization. Circulation. 2007;116(18):2072–85. Rudge T. Situating wound management: technoscience, dressings and “other” skins. Nurs Inq. 1999;6(3):167–77.

Medicinal Plants and Products from Traditional Medicine Systems Contribute to Clinical Wound Management Tuhin Kanti Biswas , Shrabana Chakrabarti, Srikanta Pandit, and Raj Mani

Abstract

Medicinal plants and plant products used in traditional medicine systems play potent roles in the management of many pathological conditions by accelerating the process of healing. The properties of plants and products are derived from basic laboratory studies on animal models, data from clinical studies in man are lacking. Some notable exceptions are single medicinal plants such as Aloe vera, Azadirachta indica, Curcuma longa, Hypericum perforatum, Centella asiatica and Pterocarpus santalinus and products such as honey and leaves of banana plant. Some data from clinical studies of the value of a polyherbal preparation used in Ayurvedic medicine to irrigate wounds also exist. These are examined in this chapter to support the argument that the medicinal plant systems valued by Traditional Medicine systems may have untapped potential to benefit clinical wound management. There are data driven offers from Traditional Medicine systems from which allopathic or western medicine may benefit. Keywords

Wound management Medicine




Wound dressings




Ayurveda




Traditional Chinese

T. K. Biswas (&)
S. Pandit Department of Kayachikitsa (Medicine), J. B. Roy State Ayurvedic Medical College and Hospital, Kolkata 700004, India e-mail: [email protected] S. Chakrabarti Chigurapati Pharmaceuticals, Hyderabad, India R. Mani Shanghai Jiao Tong University School of Medicine, Shanghai, China © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_7

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Abbreviations

AYUSH BLD BPPB DFU FGF FPG HOMA-IR MMP PUSH PWAR PWSR QUICKI REEDA TCM TGF-a VAS VEGF

Ayurveda, Yoga, Unani, Siddha, and Homeopathy Banana leaves dressings Boiled potato peel bandage Diabetic foot ulcer Fibroblast growth factor Fasting plasma glucose Homeostasis model assessment-estimated insulin resistance Matrix metalloproteinase Pressure ulcer scale for healing Percentage wound area reduction Percentage wound score reduction Quantitative insulin sensitivity check index Redness, edema, ecchymosis, discharge, and approximation Traditional Chinese Medicine Transforming growth factor–alpha Visual analogue scale Vascular endothelial growth factor

Introduction Wounds are the outcome of injuries or following surgery. Healing of wounds follows a pathological process rarely needing any clinical intervention unless it becomes complicated and/or non-healing. In most of the conditions, wounds may heal through natural process, but it may lack quality of healing in terms of days required, formation of scar and overgrowth of granulation tissues. Therapeutic application of healing agents varies according to the variations of stages and condition of diverse types of wounds. The process of wound healing can be divided broadly into two types as procedural and therapeutic. Even though there are tremendous developments of diverse surgical procedures to manage the healing process there are very few drugs developed from any source for their specific role in acceleration of healing. Besides drugs of synthetic origin, there are number of natural resources, medicinal plants, which are mentioned for their wound healing activity in various traditional systems of medicine including Ayurveda, Yoga, Unani, Siddha, Homeopathy, Chinese Medicine, and others in most countries. The wound healing potential of more than one hundred and fifty medicinal plants are mentioned in Ayurveda (Biswas and Mukherjee 2003). In the African continent about 80% of the population depends upon the medicinal plants for primary source of medication using forty five thousand medicinal plants under major five hundred species. This

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wide spectrum of biodiversity includes twenty-five plants of African origin as reported in the literature for possessing wound healing properties. Equally interestingly twenty medicinal plants of African origin have been screened in the laboratory for the development of biogenic nanoparticles in combination with silver, selenium, gold, zinc oxide and zinc sulphide with anti-inflammatory and antioxidant properties which promote wound healing (Tyavambiza et al. 2021). In Ghana, evidence exists of the use of one hundred and four medicinal plants with potential to benefit wound healing activity. Of these, historically seventeen have been used to treat leg ulcers caused by an infestation by the guinea worm (Dracunculus medinensis) (Freiesleben et al. 2017). In the Persian system of traditional medicine sixty-six plants are reported for their wound healing property but nine among them have been scientifically screened for such activity which is modulated owing to the antimicrobial, radical scavenging and anti-inflammatory properties (Hosseinkhani et al. 2017). Much research into traditional and complementary medicine has been conducted in China under the name of traditional Chinese medicine (TCM). Medicinal plants in TCM are broadly classified into four categories which are Ministerial or Principal herbs that act directly to alleviate disease, Deputy or Alternate herbs that assist Ministerial herbs, Assistant herbs that reduce adverse effects and Envoy herbs which are used to detoxify and have a direct role in the therapy. Most TCM products used in wound management are polyherbal in origin and among these two are Yunnan Paiyao and Shiunko. The evidence of the properties of these two polyherbals were based on laboratory studies using a rat model to study fibroblast cell regeneration and angiogenesis (Chak et al. 2013).Yunnan Paiyaois comprised Radix notoginseng (40%), Ajugaforrestiidiels (17%), Rhizomadioscoreae (13%), Rhizomadioscoreaenipponicae(10%), Herbageranli & Herbaerodii (7.2%), Dioscoreaeparviflora ting (6%), Herbainulaecappae (5%), Borneol (1.5%) and Musk (0.3%) to control bleeding and oedema. The formulation behaves as Deputy herbs do. Shiunko, on the other hand is a formulation composed with sesame oil (500 ml), Lithospermi radix (10 g), A. sinensis (10 g), lard (3 g) and Bees wax (45 g), that acts as Principal herbs since it aids information granulation tissue, speeding wound healing, re-epithelialization and angiogenesis (Chak et al. 2013). The Leguminosae family which comprise great many of medicinal plants used in Ayurveda have no direct specificity of family or constituent compounds (Biswas et al. 2016). Active constituents of various plants of this family may alleviate healing of wound by different mechanisms, for example, saponins may enhance the synthesis of pro-collagen, tannins and flavonoids benefit anti-bacterial and antiseptic activities which in turn help healing process (Ibrahim et al. 2018). Medicinal plants/products may be tested for efficacy using cell culture lines to study specific constituents, animal models or in clinical studies. Animal model studies have in previous years, demonstrated efficacy based on controlled studies. Investigation of such databases in English language as SCOPUS, Web of Science, PubMed, Science Direct led to a few clinical randomized controlled studies on specific plants and products used in Ayurveda. In this chapter, these studies are analysed to better comprehend their potential for use in clinical wound healing.

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What are Ideal Properties to Benefit Wound Healing? Research with plant medicine for their wound healing efficacy gained momentum about 3 to 4 decades ago and recent advancements included investigations of their mechanisms of actions at molecular level. The fundamental cellular aspect of acute wound healing involves four steps as haemostasis, inflammation, proliferation, and matrix remodelling of wound tissues. During haemostasis, there is plugging of platelets and formation of fibrin matrix, which stops bleeding. The process of inflammation has beneficial roles in removal of debris as well as in the prevention of secondary infection. The wound is closed by keratinocytes in the stage of proliferation and finally the closure of wound occurs in matrix remodelling stage when there is formation of fibroblasts and myofibroblasts. The acute stage of wound healing is dynamic that leads rapid healing but in the chronic stage, process of healing is delayed due to ageing as well as absence of certain molecular factors like matrix metalloproteinase (MMPs), FGF fibroblast growth factor (FGF), transforming growth factor−alpha (TGF-a), vascular endothelial growth factor (VEGF), and so on, due to excessive inflammation followed by scarring (Wilkinson and Hardman 2022). An ideal drug of plant origin should overcome all these phenomena.

Randomized Clinical Trial on Wound Dressing Agents from Plant Origin Objectives A high quality of healing can only occur if the wound bed is found healthy. Dressing of wounds appropriately may provide debridement of wound, non-adherence of wound surface, free from toxins and allergens, if any as well as absorb excess exudates and foul odour. Primarily the dressing of wounds is done by conventional method with Spunbond−meltblown−spunbond (SMS) fabric (Dhinakaran et al. 2017) which is placed over a layer of adhesives. Occlusive dressings are also in use for providing moisture in wound area followed by acceleration of collagen synthesis, epithelialization, maintains wound pH and minimizes chances of infection (Ghomi et al. 2019). Herbal dressing, in the form of paste or fresh juice in a specialised process of preparation, plays a crucial role in the healing process which may be non-toxic and having capacity of debridement of wounds.

Azadirachta Indica as Dressing Agent Azadirachta indica L. (Family Meliaceae), commonly known in the name of Neem, is a tall tree distributed particularly in Southeast Asian countries like India, Pakistan, Bangladesh, Myanmar, and Sri Lanka. This plant was introduced in Australia during 1940s where it flourishes (Nishteswar and Hemadri 2013). In Ayurveda, all

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five parts of the plant viz. leaves, seed, stem bark, root and fruits are used for various therapeutic purposes. The plant contains multiple phytochemicals such as nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin and quercetin. The leaves of the plant are widely used for different therapeutic purposes in different forms and contain a specific alkaloids nimbin, nimbanene, nimbandiol, nimbolide and nimbiol (Alzohairy 2016). The plant has antibacterial and antifungal properties favouring it to be an ideal wound dressing agent as described in Ayurveda. The antimicrobial and antifungal activities of the aqueous extract of leaves, bark and seed of A. Indica were screened separately in three doses 500, 1000 and 2000 µg/ml for each compared to Ciprofloxacin (5 µg/ml) and Amphotericin B (100 µg/ml) and both these were tested against specific bacteria such as Staphylococcus aureus, Pseudomonas eruginosa, Proteus mirabilis, Enterococcus foecalis and specific fungal infections like Aspergillus fumigates as well as Candida albicans. It was observed that leaf extract of A. Indica at the concentration of 2000 µg/ml has potent antifungal activity while bark extract exhibited significant antimicrobial activity at the concentration of 500 µg/ml (Reddy et al. 2013). The antimicrobial activity of A. indica in comparison with antibiotics was determined based on the minimum inhibitory concentration (MIC) at the dilution of 500, 1000 and 2000 µg/ml in micro-tire wells. In another study it was observed that the isolated chemical constituent of A. Indica nimonol has no antifungal activity though the 5% aqueous extract of leaves of the plant has significant antifungal activity against Aspergillus niger and A. flavus (Mahmoud et al. 2011). A clinical trial was done to study with the extracts of neem (A. indica) leaves to examine its wound irrigation properties using normal saline as a control or standardized agent in patients with diabetic foot ulcer (DFU). Patients enrolled had DFU grade 1−3 i.e. (Localized superficial ulcer to deep ulcer with abscess, osteomyelitis, or joint sepsis) defined using the Wagner classification (Mehraj 2018). DFU had wound areas greater than or equal to 1sq. cm. A total 246 patients of DFU, in the age group 20 and 80 years, uncomplicated by clinical signs of ischemia, attending in a diabetic foot ulcer care clinic in Assam, India, were screened of which N = 46 were excluded using the inclusion/exclusion criteria and unwillingness to participate in the study. The patients with DFU of grade 4 or 5 as per Wagner classification, had drug or alcohol addiction, any cognitive or mental illness, history of prior hospitalization for DFU and were under treatment on immunosuppressive drugs or on treatment for end stage renal disease were excluded from the study. The remaining two hundred patients were randomized into two groups. Each group was treated with wound dressings of either neem leaves aqueous extract or with normal saline as irrigation twice a week for period of four weeks. Forty patients dropped out and were lost to analysis. One hundred and sixty patients continued the treatment with eighty patients in each group in which average distribution of patients in two treatment groups in age groups between 41 and 60 years was maximum (62.5%) followed by age group between 61 and 80 years (25 to 31.25%). Patients of DFU in age group between 20 and 40 years were least (5 to 11.25%). Number of male patients in normal saline and Neem leave extract

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irrigation treated group were 68 and 64 respectively while females were 12 and 16, respectively. Effects of both treatments were assessed using wound PUSH (Pressure Ulcer Score on Healing) score (Neola Satish and Srinivasan 2021) where wound contraction size, exudation and type of tissue involved in the healing process were assessed. Assessment of PUSH was done based on percentage wound area reduction (PWAR) and percentage wound score reduction (PWSR). The result of the study shows that of the wound mean score in the normal saline group reduced from 12.93 to 9.14, while this reduction in neem extract group was from 11.97 to 6.7 which was highly statistically significant (p < 0.001). Mean wound area decreased from 19.84 cm2 to 14.31 cm2 in the normal saline group and 17.14 cm2 to 7.04 cm2 in the neem extract group (Jayalakshmi and Thenmozhi 2021).The number of individuals in whom ulcers achieved  50% PWAR was more in the neem leaves group (64%) compared to 35% in the normal saline group during the four-week study period. On the other hand, PWSR achieved  50% in 80.6% individuals of neem group while 55.8% individuals in the saline group (Jayalakshmi and Thenmozhi 2021).The results showed the neem leaves extract have a promising role in wound dressing.

Ayurvedic Polyherbal Formulation as Dressing Agent In Ayurveda, detailed descriptions exist for the management of wounds. These include procedures of cleaning of wounds, dressing, healing, maintenance of wound area hygiene, special dietary advice for patients having wounds and so on. There are multiple formulations of plant origin narrated in Ayurveda for their wound dressing properties. Jatyadi Tailam is a polyherbal preparation prepared with sesame oil, which is mentioned in Ayurvedic text Sharangadhar Samhita (Sarngadhar 2017) as a wound dressing as well as wound healing agent. The formulation is composed of various parts of Ayurvedic plants like Myristica fragrans, Azadirachta indica, Tricosanthes dioica, Pongamia pinnata, Glycyrrhiza glabra, Saussure alappa, Curcuma longa, Berberis aristata, Rubia cordifolia, Picrorhiza kurroa, Prunus cerasoides, Symplocos racemosa, Terminalia chebula, Nelumbo nucifera, Hemidesmus indicus, Sesame indicum along with bee wax and purified copper sulphate in different proportions. Quantitative phytochemical analyses showed presence of phenols, proteins, tannins, sterols and terpenoids with specifically high amount of Octadecanoic acid, a type of stearic acid. Besides, the formulation showed potent antibacterial effect against Gram (+) bacteria like Staphylococcus aureus, Staphylococcus epidermidis and Entercoccus faecalis and Gram (−) bacteria like Escherichia coli, Pseudomonas aeruginosa, Pseudomonas mirabilis and Klebsiella pneumonia (Mandrika et al. 2021). The formulation is widely used in Ayurveda as dressing agent for cleaning purpose before application of any healing drug or as a healing drug as well for the management of wounds like fissure-in ano, haemorrhoids and non-healing wounds.

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A randomized clinical trial was undertaken with this polyherbal formulation on N = 30 patients with clean wounds for its wound cleaning and healing properties. Patients were equally divided into two groups and randomly treated either with Jatyadi tailam (an oil) or Jatyadi ointment. Randomization of the patients was done by using special computerized software. Treatments were continued for seven consecutive days and the effects were compared using the values of selected parameters of Bates-Jensen Wound Assessment Tool i.e.1,2,3,7,9,10,11,12 and 13 (size, depth, edge, exudates type, skin colour surrounding wound, peripheral tissue oedema, peripheral tissue in duration, granulation tissue, epithelialization) on baseline, and the 8th day. It was observed that there was significant improvement within and between the groups in all the selected parameters of the Bates Jensen wound assessment tool. It was predicted from the study that dressing and healing properties of oil and ointment respectively of this formulation (Jatyadi) were promising thus giving a scientific basis to textual descriptions of Ayurveda (Shindhe et al. 2020). In another clinical study, for the management of filarial lymphoedema using an integrated approach following the guidelines of morbidity control agenda of the global alliance for the elimination of lymphatic filariasis (GAELF) Jatyadi tailam was one of the components successfully used to treat the unhealthy granulation tissues and slough at the base of the ulcer. It was reported that the Ayurvedic component in the treatment costs was $32.5 to $107 for six months (Narahari et al. 2007).

Banana Leaves Dressings (BLD) Banana (Musa paradisiacal Linn, family Musaceae) leaves (Fig. 1) and skin are being used traditionally in certain countries as wound dressing agent (Hoetzenecker et al. 2013). An experimental study was carried out with the aqueous and methanolic extracts of the peeled skin of the unripe banana and was applied over the excision, incision and dead space wounds in rats using an excision model. The study revealed that there was statistically significant reduction of wound contraction size (p < 0.001) between 4th day to 12th days with the aqueous extract of the test sample supported by significant generation of hydroxyproline, hexosamine as well as antioxidative materials (Agarwal et al. 2009). In another study, a water soluble substance extracted from leaves, skin and peels of unripe banana named as super green (SG) was studied in streptozototin (STZ) induced diabetic excision wounds on rats in different ratio under the ointment form and it was observed that there was significant reduction (p < 0.05) of wound contraction size within 15 days with concomitant potent expression of collagen I, collagen III, IL-1b, IL-6 and TNF-a in wound tissues treated with SG ointment prepared with 1:6000 dilution (Cheng et al. 2020). However, reports of animal experimental study with banana leaf alone for wound healing activity are limited. The banana leaf dressing (BLD) was reported to

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Fig. 1 Banana tree (musa paradisiaca) and its leaves

be a valuable dressing for superficial burns from an RCT; also, useful to treat head and neck injuries (Chendake et al. 2021). Banana leaves are widely available in over hundred countries throughout the world. Wounds treated with banana leaves healed in the same duration as wounds treated with Vaseline gauze dressing. The most advantage of banana leaves is the smooth and waxy surfaces which do not stick on the wound surface. Moreover, it was estimated that BLD are 1500 to 5000-times less expensive than collagen or biosynthetic dressings, respectively (Hoetzenecker et al. 2013). There are some interesting clinical studies with banana leaves in diverse types of wounds with special reference to burn wounds. In a study on N = 60 patients with contused, lacerated, and sutured wounds over the head, neck and face regions, comparative effects of autoclaved BLD with petroleum jelly gauze dressing were done by assigning equal number of patients to the two groups. Effects were observed by scoring or grading pain, status of wound bed (dry, moist, wet, saturated, and leaking). The result showed BLD treated wound fared better that the petroleum jelly gauze treated group, the significant results being statistically significant (p < 0.01) (Chendake et al. 2021). Burn injuries are a global problem on intense pain, long term morbidity and sometimes even loss of life. The World Health Organization reported in 2008 that burden of burn injury falls on the world’s poor: the vast majority (over 95%) of fire-related burns occurred in low- and middle-income countries (Mock 2008a, 2008b). Successful management of burns with low-cost dressing is unbelievably valuable the, BLD is a tried and tested alternative. A pilot study on N = 38 cases of burn wounds, divided into two groups,

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were treated with banana leaf; this was prepared by cutting the midrib of the leaf, carefully washed and dried, and then cut into pieces with average size 40  60 cm for Group-I: Group II received a simple dressing (antimicrobial ointment, then layers of gauze). The rate of healing in terms of outcome criteria viz. discomfort score: it was in the group BLD 2.1 ± 1.3 compared to the 6.0 ± 1.5 in Group II; dressing removal pain scores were 2.3 ± 1.2 and 5.9 ± 1.2 in the BLD and Group II respectively; ease of dressing removal score was significantly lower in BLD treated group when compared to the simple dressing group (3.5 ± 1.9 and 7.4 ± 1.2) respectively. Finally, time to complete healing in the BLD group was 8.4 ± 1.4 days compared to 13.4 ± 1.9 days in the simple dressing group. Control of infection was favourably comparable in BLD: as evidence of signs of wound infection (defined the presence of unpleasant odor, pus) was not found in 81.6 and 68.4% patients in the BLD and simple dressing groups. Signs of infection were noted in 7% (BLD group) and 12% patients simple dressing or Group II) respectively. Moreover, the scoring of visual analogue scale (VAS) on the criteria like discomfort, dressing removal pain and ease of dressing removal was favourable to the BLD groups (Ali and Eazaym 2015). An open controlled clinical trial of BLD versus povidone iodine ointment on superficial partial thickness burns was done. Burns on both upper and lower extremities treated with gauze impregnated with boiled potato peel bandage (BPPB) on N = 30 patients and BLD. BLD and BPPB were applied in same patients on different sites simultaneously. It was found that pain with dressing change, comfort score and dressing handling score were parallel in both the groups. The BLD is reported to be eleven times cheaper than BPPB. Interestingly, the boiled potato skin used in this study was donated to the surgeon conducting the study by families in the city. Donations of potato peels were bagged and transported free of cost to the hospital where the potato skins were boiled before use in the study (Gore and Akoleka 2003). The duration of area of epithelialization with number of days and mode were also found to be better in the BLD treated group. It was claimed that BLD is non-adherent, non-toxic, non-antigenic, cheap, simple to prepare and easily available. It is an effective and acceptable alternative for management of partial thickness burn wounds (Gore and Akoleka 2003). The estimated cost of BLD for a complete treatment was estimated at INR 2000−3000 (13−38 USD).

Randomised Controlled Trial on Wound Healing Plant Medicine Topical Application Most of the wound healing drugs of any origin are for topical use. The main aim being to regenerate epithelial and granulation tissues composed of extracellular matrix, proteoglycans, hyaluronic acid, collagen, and elastin. There are several

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pre-clinical trial reports of many drugs of plant origin which can perform these biochemical and pharmacological activities though reports from randomized controlled clinical trial are rare. However, stimulating reports from RCT using Aloe Vera exist and follow.

Aloe Vera Aloe vera (L) Burm. f. (family Asphodelaceae) is a plant of cactus species, widely available throughout the world. The plant is being used for various therapeutic purposes since 1500 BC in Egypt, India, Greece, Rome and China (Oliveira et al. 2016). The fleshy mucilaginous part of the centre of leaves of the plant is used for different therapeutic purpose including wound healing (Fig. 2). The plant is often confused with its other variety Aloe barbadensis is but these two are separate plants and have differences of therapeutic activities. It is reported that about seventy-five varieties of chemical components are available in Aloe vera gel like vitamins, minerals, sugar, lignin, saponins, phenolic compounds, etc. (Hamman 2008). There are several scientific reports of the gel of the plant for its wound healing potential. A randomized, double blind clinical trial was conducted on N = 64 patients with 2nd degree burns. Patients were divided into two equal groups of thirty-two each. A group was topically treated with 1% silver sulphadiazine while other group was

Fig. 2 Aloe vera herb and its fleshy juice

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treated with1% Aloe vera gel. Wound healing effects were assessed using the Bates-Jenson scale. There was a significant difference between groups on the fifteenth day (P = 0.000, F = 467.602) and it was argued that the Aloe vera gel is an effective treatment for burn wounds (Hosseini et al. 2013). In another clinical trial Aloe vera gel was applied on N = 27 patients of burn wounds from flames, scalding, and electric injuries, ranging of area of involvement between 5 and 45.7%, over different body parts. Complete healing of wounds within 14th day was observed. Efficacy of the plant for wound healing potential was confirmed by relevant histological screening (Visuthikosol et al. 1995). In another randomized split body controlled clinical trial, Aloe vera gel was used in N = 30 patients on superficial partial thickness burns having wounds in less than 20% of involvement and occurring in more than one site of the body with wound diameters within 16 cm. In the trial, wounds of one side of the body were treated with Aloe vera gel while the other side received 2% Nitrofurazone ointment, treatments being randomly assigned. Burn area was evaluated according to the criteria of American Burn Association Consensus Conferences and healing of wounds was assessed using the Baes-Jensen Wound Assessment tool (BWAT). The study revealed no differences in BWAT scores between these two groups the 2nd and 3rd weeks. Wound closure in both groups was statistically significantly less than at the start of the study (p = 0.037) although there were no differences between the groups (Varaei et al. n.d). Another randomized double blind clinical trial was done on N = 90 mothers aged 18 to 36 who underwent Caesarean section. They were divided into two equal groups of forty-five and were treated with either fresh Aloe vera gel or conservative dressing (control) respectively. Treatments were randomly assigned. The inclusion criteria were term pregnancy (37–42 weeks) Body Mass Index (BMI) > 29, not having a history more than two caesarean sections, and willingness to participate in the study. Pregnant mothers with a history of placenta previa, placental abruption, chorioamnionitis, meconium discharge, polyhydramnios, dysfunctional uterine bleeding, hysterectomy, myomectomy, suffering with any serious dreadful diseases were excluded from the study. Aloe vera gel was applied on the wound edges of caesarean section area along with systemic anti-inflammatory and antibiotic drugs. The other group received simple dressing with same group of anti-inflammatory and antibiotic drugs. Healing effects of these two treatments were observed using the REEDA scale. It is an instrument containing five factors, namely redness, oedema, ecchymosis, discharge, and approximation of the two edges of the wound. A significant (P = 0.003) results of wound healing effect were found in Aloe vera gel treated group within 24 h with REEDA scale value zero. Mean REEDA scale value in other group was 0.11 after 8 days of caesarean section (Molazem et al. 2014). The results showed that Aloe Vera is useful adjuvant to reduce post operative inflammation. Aloe vera-olive oil (AVO) combination therapy compared with phenytoin ointment was tried on N = 60 patients, 30 per group to treat such chronic wounds as pressure ulcers, diabetic foot ulcer and venous ulcer. Treatment effects were assessed based on VAS (visual analogue scale). The total score of wound healing

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showed significant improvement within both AVO (p < 0.001) and phenytoin (p < 0.01) groups: between groups, the AVO group fared marginally better (p < 0.001). Although both treatments had reduced VAS score, the efficacy of AVO significantly at a higher level (p < 0.001) (Panahi et al. 2015). It was argued that the due to presence of various chemical components, Aloe vera is able retain skin moisture and integrity. Further it was argued that the healing of wounds was accelerated in these studies to presence of mucopolysaccharides, amino acids, zinc, and water in Aloe Vera (Hekmatpou et al. 2019).

Hypericum Perforatum The plant belongs to the family Hypericaceae, commonly known as St. John’s Wort and is traditionally used across the Europe for the treatment of wounds (Scotti et al. 2019). It is a leafy herb that grows in open space (see Fig. 3) and whole part is used to treat various diseases, as well as cuts and burns. Recent research suggests that the plant has multiple therapeutic effects against cancer, inflammation-related disorders, and act as antioxidant, anti-bacterial and anti-viral as well as neuroprotective agents (Klemow et al. 2011). Neuroprotective effect of the standard extract of the plant on rat pheochromocytoma cell line PC12 as well as in Parkinsonism are also reported (Oliveira et al. 2016). Study reveals that the plant contains two major chemical components such as avicularin and guaiaverin (Scotti et al. 2019). An experimental study was conducted at the Department of Plastic, Reconstructive and Aesthetic Surgery, MuğlaSıtkıKoçman University, Turkey in Spraque-Dawley rats on incised wounds with combination of extract of H. perforatum and olive oil. Effect of the preparation showed that tensile strength of the wound as well as angiogenesis and epithelialization improved significantly (p < 0.05), which was corroborated by histological study of the wound tissues (Altıparmak and Kule 2019). A randomized clinical trial was conducted with this plant for evaluating wound healing activity on N = 125 women who underwent having Caesarean childbirth. Oily extract of the flowering top of the plant was prepared and mixed with grape seed oil in a ratio of 1:3 and then a 20% ointment was prepared with petroleum jelly. Patients were randomly divided into three groups twenty-four (24) hours after caesarean. First group consisting of N = 47 patients were treated with H. perforatum ointment, second group (N = 44 patients) received placebo ointment (petroleum jelly) and third or control group (N = 34 patients) remained without any intervention. Patients of first two treatment groups treated with the ointment three times a day for a period of 16 days. The randomization was based on consecutive numbering and ointments were coded by a staff member of the team and none of the members of the research team knew about the intervention group. Assessment of healing was observed at the 10 and 40th days using the REEDA scale (REEDA stands for redness, oedema, ecchymosis, discharge, and approximation) (Hill 1990). A total REEDA score ranges from 0 to 15 and score of 0 reflects normal skin. The study showed that there was significant reduction (p < 0.4–0.008) of all the characteristics

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Fig. 3 Hypericum perforatum Linn. (Hypericaceae) [Courtesy: flagstaffotos @gmail.com]

features of REED except ecchymosis in H. Perforatum treated group with respect to placebo and untreated control groups (Samadi et al. 2010). The chemical analysis of the plant by HPLC methods showed reveals that the plant is enriched with phenolics and flavonoids. Hypericin was found to be main active principle which was isolated from the plant during flowering. The plant also possesses comparable property of antioxidant activity as observed based on DPPH∙ scavenging assay (Kladar et al. 2017). The antioxidant property plays a key role for healing of wound by activation of pro-healing and anti-inflammatory genes pathways (Comino-Sanz et al. 2021). Hypericin is reported that it significantly helps in proliferation phase of the wound-healing cascade, contributing to increased fibroblast proliferation, maturation, and subsequent collagen deposition (Ozturk et al. 2007). The plant is, therefore, found to be useful for healing of post Cesarean incision wounds.

Honey Honey is used as food and medicine for the management of varieties of diseases since a long time in various parts of the world. Honey is derived from flowers of diverse types of plants. Therapeutic use of honey was first mentioned in India, in Ayurveda and honey from the Sundarbans (Worlds’ largest mangrove forest lies on the delta of Ganges, Meghna and Brahmaputra on the Bay of Bengal) is claimed to

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be the best among all. Honey can be produced by a synthetic process though that offers opportunities for adulteration. Natural processing of honey is important for its therapeutic application, particularly in wounds of various origins. Traditionally there is anecdotal evidence of the use of honey to treat the chronic non-healing wounds like diabetic foot ulcers (DFU), burn wounds, pressure ulcers and so on (Fig. 4) but reports of RCT are limited. Natural honey contains many essential amino acids like arginine, proline, alanine, isoleucine, serine, valine and glycine (Biswas and Honey 2019). In a meta-analysis done from 1966 to 2008 covering PubMed, Medline, Embase, Cochrane database for topical application of honey in the treatment of wound healing, which revealed that in ten randomized controlled trials, covering nine hundred and sixty three patients, N = 511 were treated with honey and rest treated with silver sulfadizine, antiseptic, soframycin and acriflavin, etc., considered as

Fig. 4 Application of honey a in a male uncontrolled diabetic patient, aged 45 years, with a non-healing leg ulcer for more than 30 days and progress of healing b after 7 days. Intervention done at J. B. Roy State Ayurvedic Medical College and Hospital, Kolkata, India

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control group. In treated group modest improvement was there (56%) and duration of healing ranged from 5 to 30 days while in control group duration of healing ranged from 7 days to 17 months (Medhi et al. 2008). Evidence of a prospective, randomized, double blind, controlled study on eighty-seven patients among workers from a gold mine: the study was done on shallow wounds and abrasions not deeper than 2 cm and not larger than one hundred sq.cm. with honey in comparison with hydrogel (Intrasite gel®, Smith & Nephew). Honey was applied topically on forty-two patients and hydrogel to forty patients. The study showed that the healing time and reduction of wound sizes with honey treated group was faster than Hydrogel (Ingle et al. 2006). There is also evidence of prospective randomized controlled clinical study of honey with respect to silver sulfadiazine in superficial burn wound. In this study a total fifty patients with superficial burns involving 40% of body surface were randomly divided into two groups comprising of twenty-five patients in each group and were topically treated with honey and silver sulfadiazine (SSD) respectively. Topical applications of intervention (honey) or standard control (silver sulfadiazine) were done once a day for twenty one days. In honey treated wounds, there was no eschar, the margins of the wounds were free of oedema. On day twenty-one, in the honey treated group only one had fluid exudate as compared to four in SSD treated wounds. In honey treated patients, all the wounds healed by day 21 (100%) and in the SSD treated group in N = 21 patients (84%) (p < 0.001). On the other hand, honey treated group showed epithelialization and granulation tissue formation to all the patients on day twenty one as compared to the SSD treated group where N = 13 wounds showed reparative changes and granulation formation (52%), no evidence of granulation and reactive changes in one (4%) and in N = 11 wounds evidence of infection was present on the 7th day. The acceleration of epithelialization in the honey treated group appeared to occur between 6 and 9 days clinically as well as histologically (Subhramanayam 1998). In comparison to silver sulfadiazine, honey is better as a topical treatment for superficial burns because it promotes fast re-epithelialization and decreased inflammatory reaction. Most of the FDA approved honey-based wound agents are prepared with medical grade Manuka honey (Albaridi 2019) which is reported to have potent anti-bacterial activity against anti-biotic resistance bacteria resistant. The mechanism of honey in wound and burn healing involves several biochemical and pharmacological pathways. Honey itself act as antibacterial agent due to its high osmolarity, low water activity, and acidity as well as some compounds, such as hydrogen peroxide, phenolic compounds, methylglyoxal, or bee defensin-1 peptide, directly affect the bacterial growth and survival (Albaridi 2019; Proaño et al. 2021; Peršuri´and Paveli´ 2021). Stimulation of angiogenesis by honey was demonstrated in an in vitro study with analogues of angiogenesis and an endothelial proliferation assay (Rossiter et al. 2010).Wound debridement is a principal issue for healing and honey facilitates the wounds’ autolytic debridement process due to its high osmotic pressure which pulls out lymphatic fluid from the deeper zones followed by automatic removal of dead, damaged or infected scar tissue (ScepankovaH 2021). Other two important biological functions that honey performed are anti-inflammatory by reducing COX-1 and COX-2 (Silva et al. 2021) and

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anti-oxidant (Yadav et al. 2018) activities that help in promotion of healing process. Honey was recommended for topical use as a potent wound dressing agent (Molan 1999). Besides, honey is cost effective than any other natural or synthetic agents used for the healing of wounds. These are widely available, easily affordable alternatives. The processing of synthetic honey involves several technologies.

Systemic Uses of Curcumin A meta-analysis of plant drugs used as healing agent was done and only one plant Curcuma longa so far been reported as potent healing agent for systemic use.

Curcuma Longa The plant, commonly known as ‘Haridra’ or ‘Haldi’ in India and adjacent countries as well as Turmeric, is described in different traditional classical texts for various therapeutic purposes like anti-inflammatory, analgesic, antifungal, anti-viral, antibacterial and antioxidant activities (Bhat et al. 2015). Reports are also available of the paste of turmeric for its wound healing activity in the rabbit model in which it was found quite comparable with honey (Kundu et al. 2005). In another study wound healing potential of the plant was assayed using nanostructure porous curcumin loaded electrospun polyhydroxybutrate (PHB) mat (Fig. 5) and its was observed that the natural process of formation of extracellular matrix (ECM) scaffold as wound bed was formed through in vitro study in cell culture. Moreover, it was also observed that rate of release of 1 and 3% PHB-curcumin composition from nanofibrils are 37 and 46% respectively within a time of 6 h. Almost half of the drug is released from the nanofibril coated with curcumin within 72 h which is important from the point of view of its pharmacodynamics and pharmacokinetics (Ghavami et al. 2020). However, a prospective, randomized, double blind, placebo controlled clinical trial was undertaken on final fifty patients aged between 45 and 85 years of grade 3 DFU having features like deep wounds with abscess or osteomyelitis according to “Wagner-Meggitt’s” scores. The test drug C. longa was administered in the form of nanocurcumin tablets 80 mg per day for a period of 12 weeks and compared with placebo control with equal number of patients distribution (25) in each group in a randomized technique. All the patients were conventionally treated with antibiotic Ciprofloxacin and relevant antidiabetic therapy. Assessment was made based on the physical parameters like ulcer length, width, and depth (cms). Besides, diabetic profile like FPG, plasma insulin levels, HOMA-IR, QUICKI and some lipid profiles including total-and LDL-cholesterol were also estimated periodically. The study showed that in-spite there were no significant improvement of change of ulcer size with curcumin nanoparticle treated group with respect to placebo but there was significant change (p < 0.01−0.001) of glycaemic and lipid profiles (Mokhtari et al. 2020). In most of the DFU there are insulin resistance resulting delayed and poor healing progress. Improvement of glycaemic and lipid profile by oral administration of curcumin may indirectly help in acceleration of healing or may resist the deterioration of healing process. Simultaneous oral and topical application of C. longa

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Fig. 5 SEM images of curcumin-loaded nanofibers. a and b Unloaded PHB nanofibers; c and d loaded PHB nanofibers with curcumin 1%; and e and f loaded PHB nanofibers with curcumin 3%. (Magnification: 1000 and 2000  ) (Courtesy Dr. Esmaeil Biazar Department of Biomaterials Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran]

may have promising results in this direction, specifically to prevent wounds in diabetic patients. Though no direct evidence for wound healing of this plant is observed but it may prompt a future study to explore its preventive benefit in DFU as it can control diabetes. Chances of bacterial and/or fungal infections in DFU are very much common and the healing in this condition can be modulated by any drugs which have protective role against these noxious agents. The crude extract of curcuminoids and essential oil of C. longa has most sensitive effect against Bacillus subtilis with a minimum zone of inhibition of 20.6 mm diameter (Nazi et al. 2010). The aqueous extract from the rhizome powder of Curcuma longa is reported to have most sensitive effect against the filamentous fungi Cladosporium exospore and Cladosporium subliforme with inhibitory concentrations of 3.12 mg/ml and 6.25 mg/ml respectively (Marchia et al. 2019). This evidence lends support to the role of C. longa in wound healing.

Centella Asiatica (L.) The plant is commonly known as Gotu kola or Mandukaparni in India (family Apiaceae) (Fig. 6) and therapeutically used for the treatment of various diseases in Southeast Asian countries such as India, Sri Lanka, China, Indonesia, and Malaysia as well as South Africa and Madagascar (Jamil et al. 2007). The most important clinical use in various traditional system of medicine is mood elevation and anti-depressant activity and this folklore evidence was scientifically screened as neuroprotective potentiality. In the study it was observed that ethanol extract of C. asiatica could inhibit acetylcholinesterase (AchE), butyrylcholinesterase (BChE) and tyrosine (TYRO) through in vitro study. Study reveals that it can prevent

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Fig. 6 Centella asiatica (L) creeper

neurological diseases like Alzheimer’s disease (AD) or Parkinson’s disease (PD) (Orhan 2012). Wound healing potential of the plant was tested in fundamental research on several aspects. In a study, effect of the aqueous extract of C. asiatica was evaluated at different concentrations like 7.8, 15.6, 31.2, 62.5, 125, 250, 500 and 1000 ppm on scratched isolated rabbit corneal epithelium (RCE) and it was observed that number of viable RCE was promisingly increased with 1000 ppm of C. asiatica based on the MTT assay and gene expression analysis (Idrusa et al. 2012). The plant contains many chemical components of which major are Asiatic acid, madecassic acid, asiaticoside, madecassoside, madasiatic acid, etc. (Pan et al. 2007). Fundamental study also carried out with isolated compound madecassoside for its facilitating effect on burn wound in mice. In the study aqueous solution of madecassoside was applied in a dose of 24 mg/kg b.w. on artificial induced 30% burn in mice. Result showed that there were remarkable proliferation of fibroblast cells and neoangiogenesis on histological examination as well as antioxidant activities (Liu et al. 2008). A study also conducted towards the genetic expression of C. asiatica triterpenoids on normal human fibroblasts (ATCC CRL-2450) where proliferation was observed in terms of cDNA microarray sequence indicating it potent wound healing profile (Coldren et al. 2003). Study on isolated compound of 0.2% solution of asiaticoside from C. asiatica also carried out in delayed type of wounds in guinea pig model and it was observed that there were 56% increase in hydroxyproline, 57% increase in tensile strength, increased collagen content, angiogenesis and better epithelisation (Shukla et al. 1999). A trial was specifically carried out with the plant C. asiatica for their wound healing activity. A detailed RCT on this aspect on two hundred diabetic patients with chronic wounds were done dividing patients into two groups distributed with equal number of patients. Finally, N = 170 patients were included: eighty-four patients in group-I and eighty-six in group-II were included. In group–I the isolated chemical component asiaticoside at a dose of 300 mg in three divided doses after meal were administered orally for 21 days and group-II were treated with placebo. Effects were evaluated

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based on the physical parameters like wound contraction and granulation tissue formation on open eye observation. Effects was found significantly higher (p < 0.001) than the placebo group (Paocharoen 2010). However, more extensive RCTs are needed to establish its role in wound healing.

Taking a Wound Healing Agent from Bench to Bedside: Validating a Natural Wonder with Advanced Technology There are numbers of medicinal plants which have been screened primarily in lower animal models and screened with sophisticated molecular markers for establishing wound healing efficacy. However, the efficacy of few of these are rigorously tested using RCT. Pterocarpus santalinus or red sandal wood is an exception. The plant is found in the southern India and had been screened by preparation of 15% w/w crude powder ointment with petroleum jelly in 8 mm diameter punch wound model on Charles foster rats. There was significant (p < 0.05) reduction of wound size within a time of nine days. Tissue molecular markers like DNA, RNA, Protein and Hydroxyproline generation were also found to be significantly increased (p < 0.001). The healed tissue showed remarkable generation of biological factors like collagenesis, epithelial tissue formation and neovascularisation on histological investigation indicating its potent role in wound healing (Biswas et al. 2004a). Thereafter, the ointment was applied on selected N = 6 patients with non-healing wounds where significant reduction of wound size was observed within a period of fifteen days and was comparable with Racketing® ointment (Biswas et al. 2004b). Owing to the results of crude powder of the plant, further studies were carried out with 5% ointment of the ethanol extract of the plant in ointment base of petroleum jelly. In the same experimental model, the extracted plant ointment with low concentration (5%w/w) showed more potent reduction of wound size (p < 0.05) within a period of 8 days which was significantly better than vehicle control (petroleum jelly) or Framycetin® ointment treated group. Genesis of hydroxyproline, one of the three components of collagen, was also found to be significantly (p < 0.001) better than the previous experiment. Formation of tissue markers responsible for healing process like ECM, collagen, new blood vessel and epithelial tissues were also synthesized faster than the crude powder ointment. A pilot clinical study was carried out where the ointment (5% w/w) of ethanol extracted P. santalinus was applied on six (6) patients of non-healing chronic wounds like DFU and pressure ulcer and it was observed that wounds could healed up within 08.25 days while it required about 21.26 days in vehicle control (soft paraffin) group. The formation of granulation and epithelia tissues were also faster than the vehicle control group. A detail toxicity study was carried out with the ethanol extract of P. santalinus, and no toxic effect was observed. A special test for assessment of DNA damage by comet assay was performed with the extract and found to be safe. More interestingly the crude extract also showed potent DPPH radical scavenging activity in vitro, indicating its’ possible good antioxidant activity (Biswas et al. 2021). However, an RCT remain to be reported.

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Traditional Chinese Medicine (TCM) is presently playing a leading role for the management of different conditions: in China doctors in TCM and Western systems work closely together. A most important achievement of TCM is the discovery of drug Artemisinin from the plant Artemisia annua (Compositae) for the management of malaria caused by Plasmodium falciparum (Valavanidis 2019). Artemisinin is key in the combined therapy used to treat malaria today and this success is very encouraging to wound healers. There are some selective medicinal plants scientifically screened as wound healing and wound dressing drugs in TCM though data from controlled studies was not found in English language databases. It has been argued that TCM interventions are complex which makes it difficult to test its efficacy using standardised RCT models (Sun et al. 2021). In this way, it differs from the products used in Western or Allopathic medicine. The possibilities of publications in Chinese language were not checked by the authors of this chapter on account of language difficulties.

Discussion The aim of this chapter was to review data from controlled studies to better understand the wound healing potential of some medicinal plants and products simply because plants and related products have evolved over the millennia. During this period, plants have survived harsh climatic changes and other environmental insults: it is likely the survivors have had encoded means of combating inflammation and infection, and this could be expressed in the bark, leaves, fruits, or pods. Many plants have mention in Ayurveda, TCM and other Traditional Systems of Medicine. The purpose of this book is to examine the benefits of evidence and technology in chronic wound management: this chapter was focused on published data from controlled studies: a select few plants and products’ efficacy was studied from publications. The results of RCT show the potential of Neem extracts as a wound irrigant, the banana leaf dressing as a valuable wound cover for superficial burn wounds, honey as a valuable dressing material, among others. From basic studies on animal models, the intrinsic properties of Pterocarpus santalinus to control inflammation and support better tissue growth are evident. This begs the question–how to complete the steps from the bench to the bedside with such a product? Existing products such as the Banana Leaf Dressing are effective, safe, and commercially low cost. There is a marked absence of the evidence of adverse events also. It also needs mention that where efficacy has been demonstrated controlled studies, specific component or components acting favourably have not been identified. Is this a limitation? Only carefully designed studies with sample sizes large enough would be able to address this question.

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This chapter (and indeed this book) were planned as we emerged from the shadow of COVID-19. One of the lessons learnt from COVID-19 has surely got to be an increased willingness to change: the offer of potentially valuable dressings, oils, ointments, and medication for systemic use with origins in plants have been described and are offered to the wound healing community. Acknowledgements Authors are thankful to Dr. Sayan Halder, BAMS for providing original photograph of Banana tree (Musa paradisiaca), Aloe vera and Centella asiatica. Conflict of interest None

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Innovation in Laboratory Evaluations of the Performance of Treatment and Prophylactic Dressings Under Clinically-Relevant Usage Conditions Amit Gefen

Abstract

The effectiveness of wound dressing performance in exudate management when applied to treatment, and in redistribution of skin loads when used prophylactically, are commonly and typically gauged in simple, non-realistic laboratory setups, such as where dressing specimens are submersed in vessels containing aqueous solutions to evaluate their absorbency, or by means of interface pressure measurements in the context of pressure ulcer/injury prevention. In the last several years, we have developed a portfolio of clinically-relevant laboratory test configurations for dressings used in treatment and preventative applications. In the context of treatment, we developed laboratory test methods and robotic wound systems for evaluating two key fluid–structure interaction concepts: Sorptivity−the ability of wound dressings to transfer exudate, including viscous fluids, away from the wound-bed by capillary action; and Durability−the capacity of dressings to maintain their structural integrity over time and particularly, at removal events. In the prevention arena, we developed sophisticated, anatomically-accurate computational models of parts of the human body to evaluate the biomechanical protective efficacy of dressings in redistributing and alleviating skin and subdermal tissue loads due to bodyweight or medical device-related forces. This chapter reviews our recent published research concerning the development of these testing methods for wound dressings, focusing on the clinical relevance of the tests as well as on the standardization and automation of the laboratory measurements of dressing performance. The chapter further demonstrates differences across product

A. Gefen (&) Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv-Yafo, Israel e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_8

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performance metrics detected by means of the above advanced test methods for products that supposedly belong to the same families, and how these differences relate to the materials, structure and composition of the tested dressings. Keywords







Wound dressings Chronic and acute wounds Bioengineering laboratory test methods Exudate management Performance metrics







Introduction Wounds of all types affect millions of people globally and are a financial burden on healthcare systems, costing tens of billions of dollars annually (Guest et al., 2020). The prevalence of chronic wounds, for example, is relatively high and similar to that of heart failure, affecting 6.5 million people in the United States, which equates to 2% of the US population (Fife et al., 2012). In addition, chronic wounds account for 3−6% of total healthcare expenditure in developed countries, and conservative estimates for the US point to an associated cost of $28 billion per year to the American Medicare system (Nussbaum et al., 2018). Wound dressings remain the primary means for treating wounds and are the oldest medical device in history, and since ancient times have been used for protecting the wound and absorbing wound fluids. However, the ability of dressings to effectively protect a wound−not only mechanically but also from biological hazards, and to not merely absorb exudates but retain them so that they are not returned into the wound and potentially deteriorate it−only developed after the 2nd World War with the invention of polyurethane foams and later on, silicone-foam composites and superabsorbent materials (Gefen, 2020). One of the primary roles of a modern wound dressing is to manage exudate, a serum-based fluid that is secreted from a wound as part of the inflammatory process. Exudate contains proteins, nutrients, inflammatory mediators, digestive enzymes, growth factors, waste products, cells (e.g., neutrophils and macrophages) and platelets, and sometimes also bacteria. The exact composition of exudate and its biochemical and biophysical properties (such as the pH, viscosity etc.) depend on the wound aetiology, the health and infection status of the patient and the stage of wound healing (Gefen and Ousey, 2020; Gefen and Santamaria, 2021). In general, wounds must be kept moist,1 i.e., not too wet and not too dry at all times, as a standard of care delivered by any modern wound care device (Gefen, 2020). The well-established theory and practice of moist wound healing states that

1

It should be noted that some wounds will benefit from being kept dry, e.g., chronic ulcers with necrotic tissue.

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moisture in wounds is critical for preventing the wound from drying out; for supporting migration of tissue-repairing cells; for diffusing nutrients to cells and tissues; for diffusing inflammatory mediators such as histamine; for diffusing growth factors (e.g., for angiogenesis); for transporting signalling molecules between cells for cell−cell communication; for allowing immune cell migration to reduce bacterial burden; and for allowing tissue-repairing cell migration, i.e., of fibroblasts which synthesise collagen for wound closure. Excess exudate is known to be harmful to both the wound and the peri-wound skin (Gefen and Ousey, 2020; Gefen and Santamaria, 2021). Excessive exudate can degrade the wound via several different damage pathways and multiple damage routes can apply concurrently. For example, if the wound is infected, the exudate is a carrier of pathogens within and outside the wound. If a non-healing wound secrets excess exudate, nearby skin may be exposed to a high concentration of proteolytic enzymes (e.g., matrix metalloproteinases) which compromise forming granulation tissue. Excess exudate can also cause softening and weakening of the peri-wound stratum corneum and dissolving dermal collagen crosslinks. Decelerated migration of tissue-repairing cells from the wound edges (e.g., fibroblasts and keratinocytes) can also be caused by excessive exudate and the hostile biochemical environment that it induces for these cells (Tompan et al., 2012). Overall, these issues slow the rates of wound healing and decrease the extent and rate of wound closure, or may even enlarge the wound. In addition to ensuring appropriate moisture balance in the wound by absorbing and retaining excess exudate, dressings should not disintegrate and must not leave any microscopic or macroscopic (i.e., visually recognised) debris in the wound-bed, particularly during dressing changes when the dressing is subjected to pull-out forces, as this will likely cause chronic inflammation, thereby critically delaying tissue repair and healing (Gefen and Ousey, 2020; Lustig et al., 2021b; Gefen et al., 2022a). In materials science, the structure−function principle is the concept that microstructure determines properties. For wound dressings, “function” encompasses mechanical, thermal, fluid transport and retention properties, which altogether form a metrics of physical and engineering quantitative performance parameters. It is important to remember that physical and engineering characteristics of wound dressings belonging to the same family of products, such as foam-based dressings or gelling fibre dressings, may differ considerably across manufacturers, and this micro-structure affects the structural, mechanical and thermal properties and ultimately, the functions and clinical performance of the dressings. As noted above, evaluating the ability of wound dressings to manage exudate is of critical importance. However, the effectiveness of wound dressing performance in exudate management is commonly gauged in simple, non-realistic laboratory setups rather than by means of clinically-relevant test configurations. Two key fluid–structure interaction concepts, sorptivity and durability, should be specifically highlighted in this context. Sorptivity is the ability of wound dressings to transfer exudate away from the wound-bed, by means of capillary action, even if

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the exudate is viscous. Good sorptivity is required for transferring exudate from a primary to a secondary dressing, or from the wound-facing surface of a dressing to its external surface, from which fluids can evaporate to the environment, and thereby, clear the dressing reservoir for additional inflowing exudate regardless of the orientation of the wound and dressing with respect to the gravity vector. Durability is the capacity of wound dressings to maintain their structural integrity over time, after exposure to usage conditions, and during removal when pull-out forces are applied. Both factors, sorptivity and durability, are often ignored in existing test protocols. In the prophylaxis of wounds, exudate management is not relevant as the skin is intact, however, many of the mechanical features that are required for a wound dressing in a treatment application are also highly relevant and needed in the context of prevention (Gefen, 2021a; 2022b). Focusing now on the prevention of medical device-related pressure ulcers/injuries (MDRPUs) as a common example, the most frequently used dressing materials for the prevention of facial MDRPUs associated with non-invasive ventilation (continuous positive airway pressure, CPAP) masks are currently hydrocolloid-based and foam-based dressings. The alleviation of localised and sustained tissue loads is the most fundamental requirement from any type of dressing in prophylactic use under a CPAP mask, and avoiding sharp stiffness gradients between the skin and the protecting dressing serves this purpose well (Lustig et al., 2021a). The compressive stiffness of a dressing used for prophylaxis and the compressive stiffness of the skin region covered by the dressing are therefore the most important and relevant properties to consider in this regard, given the common techniques of the CPAP device attachment to skin which apply localised, intense compressive forces to the skin while strapping the mask to the head (Gefen, 2021a). Based on the above criterion, hydrocolloid-based dressings which are relatively stiff exhibit poor biomechanical prophylactic efficacy in protecting healthy skin, and more so, in preventing injuries in fragile or aged skin. Foam-based dressings, on the other hand, typically have stiffness properties that closely resemble those of human skin, and, though foam dressings by different manufacturers vary in their specific stiffness properties, some low-stiffness foams provide a near-ideal stiffness matching with skin (Gefen, 2021b). This chapter reviews our recent published research concerning the development of testing methods for wound dressings used in both treatment and prevention applications, focusing on the clinical relevance of the tests as well as on the standardisation and automation of the laboratory measurements of dressing performance. The chapter further demonstrates differences across product performance metrics detected by means of the above advanced test methods, for products that supposedly belong to the same families, and explains how these differences relate to the materials, structure and composition of the tested dressings.

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Robotic Wound Systems Designed and Built for the Evaluation of Treatment Dressings Robotic, computerised phantoms of a sacral pressure ulcer/injury (PU/PI), a non-offloaded diabetic foot ulcer (DFU) and other complex wounds simulating, e.g., venous leg ulcers (VLU), were developed and are described in detail in our published work (Lustig and Gefen, 2022a, 2022b; Orlov et al., 2022) (Fig. 1). These novel experimental platforms, which robustly simulate common wound aetiologies, facilitate methodological studies of wound dressing performance metrics under clinically-relevant scenarios, including with regards to patient positioning and the practice of application and removal of the dressings under investigation. These tests further allow evaluations of the function of primary and secondary dressing combinations, according to typical usage practice.

Fig. 1 Different robotic wound systems representing a variety of wound aetiologies, namely, a sacral pressure ulcer/injury (PU/PI), a diabetic foot ulcer (DFU) and a venous leg ulcer (VLU). The latter simulated wound system was built in replicates to allow simultaneous testing of the same wound dressing type or of different dressing products for high statistical power

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For example, we have designed, developed and produced a laboratory phantom of an exuding sacral PU/PI which mimics an active wound environment in an anatomically−and pathophysiologically-realistic form (Fig. 1a) (Lustig et al., 2021b; Lustig and Gefen, 2022b). The robotic PU/PI includes a plastic replica of the pelvis bones and soft tissue substitutes made of silicone casted to the shape of an adult male buttocks. A cylindrical wound geometry has been carved at the sacral region, into which disposable sponge components are inserted to simulate different wound-beds, with either a crater shape or undermining. To simulate the secretion of exudate, we embedded a hierarchal tubing system within the silicone volume which is connected to an electromechanical syringe pump. This flow system allows the release of exudate substitutes at controlled, pre-determined flow volumes and rates. Replica fluids are synthetic, containing, e.g., a xanthan gum-based thickener, and can be produced at a range of viscosities and pH levels which resemble those of real exudates (Lustig et al., 2021b; Lustig and Gefen, 2022b). Five thermocouples are further embedded around the simulated wound to monitor spatial temperatures during testing, while an adjustable-distance infrared lamp stationed above the phantom acts as a heat source (Lustig et al., 2021b; Lustig and Gefen, 2022b). This PU/PI robotic wound system facilitated, for the first time, experiments that expose treatment dressings to exudate-like fluids at the mechanical, thermodynamic and use conditions which duplicate real-world settings, as opposed to simple immersion and weighing tests that are commonly accepted in the wound dressing industry for testing fluid handling (Lustig et al., 2021b; Lustig and Gefen, 2022b). Moreover, pre-use and post-use physical and mechanical studies of dressing products and simulated wound-beds, such as measurements of the ratio of fluid mass returned to the wound-bed versus the mass retained in the dressing, or tensile testing of the used dressings, generate fundamental new efficacy data that shed light on the expected performance of the relevant dressing products in a clinical setting, under real-world conditions (Figs. 1, 2). These robotic-based testing methods for advanced wound dressings, shown in Fig. 1, focus on clinical relevance, as well as on the standardisation and automation of contemporary laboratory measurements of dressing performance. Utilisation of these novel robotic-based test methods for characterising the performance of gelling fibre dressings is demonstrated in Fig. 2, and facilitates the identification of key performance differences between products offered for similar clinical indications, particularly sorptivity and durability. Specifically, the clinically-relevant testing using robotic wounds revealed differences across products that apparently belong to the same “gelling fibre” family, but differ remarkably in materials, structure and composition, and thereby, in laboratory and clinical performance (Fig. 2) (Lustig et al., 2021b; Lustig and Gefen, 2022a, 2022b; Orlov et al., 2022). For example, a robotic phantom system containing six identical wound simulant units has been developed and employed to determine the synergy in fluid handling of two commercially available silver-containing gelling fibre primary dressings when used with a secondary foam-based dressing, as per clinical practice. The durability of the primary dressings post simulated use was further investigated, through tensile mechanical testing. The silver-containing gelling fibre primary dressing

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Fig. 2 Example results obtained by means of the robotic wound systems: a Absorbency and retention of a viscous exudate simulant and sharing of the exudate simulant between primary and secondary wound dressings after 5 h of simulated use, in a diabetic foot ulcer (DFU) and a sacral pressure ulcer/injury (PU/PI) robotic systems. The primary dressing was the Exufiber® gelling fibre dressing (Mölnlycke Health Care, Gothenburg, Sweden) versus a comparator commercial gelling fibre dressing, and the secondary dressing was Mepilex® Border Flex or Mepilex® Border Sacrum for the DFU and PU/PI robotic wound systems, respectively. b The durability of the tested primary gelling fibre dressing after 5 h of simulated use in the DFU robotic wound, quantified as the strain energy density to failure (Lustig et al., 2021b; Lustig and Gefen, 2022a, 2022b; Orlov et al., 2022)

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incorporating polyvinyl alcohol (PVA) fibres (Exufiber® Ag + manufactured by Mölnlycke Health Care, Gothenburg, Sweden) delivered greater fluid amounts for absorbency and retention by the secondary foam dressing (sorptivity), approximately twofold and 1.5-fold more than the comparator silver-containing primary dressing incorporating sodium carboxymethyl cellulose CMC) fibres, after 10 and 15 h of simulated use, respectively (Orlov et al., 2022). The above PVA fibre-based primary dressing type further demonstrated greater post-use mechanical strength that was *4-times and *6-times greater than that of the comparator primary dressing, when the latter dressing was tested out-of-alignment with its seams, after 10 and 15 h of usage, respectively (Orlov et al., 2022). The PVA fibre-based primary dressing type thus exhibited better sorptivity and durability than the comparator product, but this could only be detected through the robotic wound-based, clinically-relevant testing approach (Orlov et al., 2022). The above results exemplify that gelling fibre dressings, belonging to the same product category but made of different base materials (i.e., PVA versus CMC) and constructed differently yield remarkably distinct performance metrics when tested against each other. Accordingly, the robotic wound systems (Fig. 1) contribute towards the development of clinically-relevant testing methods for wound dressings and importantly, progress the standardisation and automation of the performance measurements of dressings. These innovative robotic phantom studies reported in our aforementioned published work are pivotal for improving the decision-making process of clinicians and regulatory personnel, by basing their choices of wound dressings on quantitative efficacy research. This should ultimately improve patient safety, the effectiveness of treatments and the overall quality of the delivered wound care.

Computational Modelling Reveals the Efficacy of Wound Dressings in Prophylactic Use Non-invasive CPAP ventilation masks are commonly used for respiratory support where intubation or surgical airway procedure can be avoided. These masks were massively used during the COVID-19 pandemic as a first line of respiratory support, in an attempt to avoid invasive ventilation in patients who responded to the non-invasive respiration. However, prolonged use of CPAP masks involves risk to the integrity and viability of facial tissues, which are subjected to sustained deformations caused by tightening the mask and microclimate conditions (Gefen et al., 2022b). The risk of developing such MDRPUs can be reduced by providing additional cushioning at the mask-face contact areas. We determined differences in facial skin and underlying soft tissue stresses while a CPAP mask is being used, with or without cushioning using cuts of the Mepilex® Lite (Mölnlycke Health Care, Gothenburg, Sweden) dressings (Peko Cohen et al., 2019). First, we developed a force measurement system consisting of five force sensors connected to a microcontroller board. The aforementioned system was used to experimentally

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determine local forces applied to facial skin at the bridge of the nose, cheeks and chin of healthy subjects while using a medium-size CPAP mask. Each subject was tested with or without Mepilex® Lite dressings cut to the shape of their individual face. Next, we used the Scan-IP module of Simpleware® to generate a three-dimensional computational head model using the visible human project® image database, and segmented and meshed the tissues, mask and dressing cuts (Peko Cohen et al., 2019) (Fig. 3). Using the finite element (FE) method (FEBio

Fig. 3 The intensity of facial stress concentrations at the nasal bridge, cheeks and chin, visualised by means of a computational (finite element) model of an adult male head with simulated continuous positive airway pressure (CPAP) mask mounted and tightened to the face (the mask is not shown here to visualise the facial stress concentrations). This modelling framework facilitates quantitative investigations of the protective efficacy delivered by dressing cuts applied to avoid a CPAP-related injury (e.g., using the foam-based Mepilex® Lite dressing type manufactured by Mölnlycke Health Care, Gothenburg, Sweden) on the facial stress concentration levels (Peko Cohen et al., 2019)

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software suite), we delivered the measured compressive forces per site of the face to the respective skin sites in the model. We compared maximal effective, shear and compressive tissue stresses, as well as strain energy densities (SED) in facial skin and subdermally, with or without the dressing cuts applied as cushioning. Application of the Mepilex® Lite dressing cuts substantially alleviated the exposure of facial skin and subdermal tissues to elevated stresses with respect to the no-dressing case, as reported in Peko Cohen et al., (2019). The Mepilex® Lite dressings have shown substantial biomechanical effectiveness in alleviating facial skin and underlying tissue deformations, by providing localised cushioning to the tissues at-risk which is only possible if there is adequate stiffness matching between the dressing and skin (Gefen, 2021b). Of note, use of the FE method for this purpose (Fig. 3) not only identifies and visualises the tissue stress concentrations in compression, tension and shear, but also allows to determine their diffusion into subdermal tissues (Peko Cohen et al., 2019), which traditional interface pressure measurements are not capable of achieving. The ability of the aforementioned foam-based dressings to alleviate localised and sustained facial soft tissue loads (Fig. 3) is achieved by avoiding sharp stiffness gradients between the skin and the protecting dressing cuts, because the stiffness of the foam material in the dressing is relatively close to that of native skin (Gefen, 2021b). In fact, the ratio of the compressive stiffness between the material of the dressing intended for prophylaxis and that of native skin, termed the compressive stiffness matching ratio (CSMR) is a highly useful, intuitive and easy-to-implement biomechanical performance measure in this regard. Based on this CSMR criterion, hydrocolloid-based wound dressings which are popular for facial skin protection from MDRPUs, probably due to historical reasons and availability, exhibit poor biomechanical prophylactic efficacy in protecting facial skin from MDRPUs associated with use of CPAP masks (Gefen, 2021b). Foam-based dressings such as the Mepilex® Lite dressing, which have substantially lower stiffness than hydrocolloids, are much more suitable for prevention of MDRPUs due to their good stiffness matching with skin, i.e., foam-based dressings have a CSMR value which is typically much closer to unity than that of hydrocolloid-based dressings (Gefen, 2021b).

Summary and Conclusions The effectiveness of wound dressing performance in exudate management when applied to treatment, and in the redistribution of skin and subdermal tissue loads when used prophylactically, are commonly and typically gauged in oversimplified, non-realistic setups. Examples of these are where wound dressing specimens are submersed in vessels containing watery solutions to evaluate their absorbency, or by means of interface pressure measurements in the context of PU/PI prevention. We have developed a portfolio of clinically-relevant, experimental and

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computationally-based laboratory test configurations for dressings used in treatment and preventative applications. In the context of application of wound dressings for treatment, we developed advanced test methods and robotic wound systems for evaluating two key fluid– structure interaction concepts, that is, sorptivity−the ability of wound dressings to transfer exudate including viscous wound fluids away from the wound-bed by capillary action; and durability−the capacity of dressings to maintain their structural integrity over time and particularly, at removal events (Figs. 1, 2). In the PU/PI prevention arena, we developed sophisticated, anatomically−accurate computational models of parts of the human body to evaluate the biomechanical protective efficacy of dressings in redistributing and alleviating subdermal tissue loads due to bodyweight or medical device-related forces (Fig. 3). Our published work referenced here details the specific materials and methods for each laboratory test type. The purpose of this chapter is to provide a high-level review, with some illustrative examples, regarding our recent published research concerning the development of these testing methods for wound dressings, focusing on the clinical relevance of the tests as well as on the standardisation and automation of the laboratory measurements of dressing performance. The current chapter further demonstrates that differences across product performance metrics can be detected by means of the above advanced test methods, including for products that supposedly belong to the same families, or products that are used for the same clinical purpose. According to the fundamental structure–function principle in engineering, these differences in dressing performance always relate to the materials, structure and composition of the tested dressings, and our currently reported test methods are able to make these connections between the structure and the performance metrics of wound dressings, in either treatment or preventative clinical applications. Acknowledgements The research work reviewed in this chapter was supported by Mölnlycke Health Care (Gothenburg, Sweden).

References Fife CE, Carter MJ, Walker D, Thomson B. Wound care outcomes and associated cost among patients treated in US out-patient wound centers: data from the US wound registry. Wounds. 2012;24:10–7. Gefen A. Innovations and emerging technologies in wound care. Edited by Amit Gefen. Academic Press (Elsevier);2020. ISBN 9780128150283. Gefen A, Ousey K. Safe and effective wound care during the COVID-19 pandemic. J Wound Care. 2020;29(11):622–3. Gefen A. The aetiology of medical device-related pressure ulcers and how to prevent them. Br J Nurs. 2021;30(15):S24-30. https://doi.org/10.12968/bjon.2021.30.15.S24. Gefen A. The selection of cushioning and padding materials for effective prophylaxis of medical device-related pressure ulcers: clinical intuition does not always work. Wounds Int. 2021;13 (1):10–9. Gefen A, Santamaria N. Saturation of a dressing applied to an exuding wound: the gap between clinical judgment and laboratory testing. Wounds Int. 2021;12(2):20–6.

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Gefen A, Alves P, Beeckman D, Cullen B, Lázaro-Martínez JL, Lev-Tov H, Najafi B, Santamaria N, Sharpe A, Swanson T, Woo K. How should clinical wound care and management translate to effective engineering standard testing requirements from foam dressings? mapping the existing gaps and needs. Adv Wound Care (new Rochelle). 2022. https://doi.org/10.1089/wound.2021.0173. Gefen A, Brienza DM, Cuddigan J, Haesler E, Kottner J. Our contemporary understanding of the aetiology of pressure ulcers/pressure injuries. Int Wound J. 2022;19(3):692–704. https://doi. org/10.1111/iwj.13667. Guest JF, Fuller GW, Vowden P. Cohort study evaluating the burden of wounds to the UK’s national health service in 2017/2018: update from 2012/2013. BMJ Open. 2020;10(12): e045253. https://doi.org/10.1136/bmjopen-2020-045253. Lustig A, Margi R, Orlov A, Orlova D, Azaria L, Gefen A. The mechanobiology theory of the development of medical device-related pressure ulcers revealed through a cell-scale computational modeling framework. Biomech Model Mechanobiol. 2021;20(3):851–60. https://doi.org/10.1007/s10237-021-01432-w. Lustig A, Alves P, Call E, Santamaria N, Gefen A. The sorptivity and durability of gelling fibre dressings tested in a simulated sacral pressure ulcer system. Int Wound J. 2021;18(2):194–208. https://doi.org/10.1111/iwj.13515. Lustig A, Gefen A. Fluid management and strength post-simulated use of primary and secondary dressings for treating diabetic foot ulcers: robotic phantom studies. Int Wound J. 2022;19 (2):305–15. https://doi.org/10.1111/iwj.13631. Lustig A, Gefen A. The performance of gelling fibre wound dressings under clinically relevant robotic laboratory tests. Int Wound J. 2022. https://doi.org/10.1111/iwj.13761. Nussbaum SR, Carter MJ, Fife CE, DaVanzo J, Haught R, Nusgart M, Cartwright D. An economic evaluation of the impact, cost, and medicare policy implications of chronic nonhealing wounds. Value Health. 2018;21(1):27–32. Orlov A, Lustig A, Grigatti A, Gefen A. Fluid handling dynamics and durability of silver-containing gelling fiber dressings tested in a robotic wound system. Adv Skin Wound Care. 2022;35(6):326–34. Peko Cohen L, Ovadia-Blechman Z, Hoffer O, Gefen A. Dressings cut to shape alleviate facial tissue loads while using an oxygen mask. Int Wound J. 2019;16(3):813–26. https://doi.org/10. 1111/iwj.13101. Topman G, Lin FH, Gefen A. The influence of ischemic factors on the migration rates of cell types involved in cutaneous and subcutaneous pressure ulcers. Ann Biomed Eng. 2012;40(9):1929– 39. https://doi.org/10.1007/s10439-012-0545-0.

Atypical Wounds and Wounds Resulting from Infection Massimo Papi and Ersilia Fiscarelli

Abstract

Atypical cutaneous ulcers are caused by inflammatory, neoplastic, vasculopathic, haematological, drug-induced and infectious etiologies.They present in unusual sites and have abnormal clinical aspects. They are underestimated. They may be clinically difficult to recognize and complicated in their features by the possible concurrent presence of multiple pathogenetic processes. The absence of response of a chronic ulcer to standard therapies requires a more specific diagnostic investigation. Diagnosis involves obtaining an accurate history and performing clinical examination and additional tests. A skin biopsy is fundamental to have a basic information on the type of atypical ulcer. Keywords




Atypical ulcers Inflammatory ulcers ulcers Infectious ulcers





Neoplastic ulcers
Micro-thrombotic

M. Papi (&) Chair ADOI (National Study Group Vascular Dermatology and Vulnology), Rome, Italy e-mail: [email protected] E. Fiscarelli Clinical Management and Technological Innovations, Research Center S. Paolo, Bambin Gesù Hospital, IRCCS, Rome, Italy © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_9

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Introduction Chronic skin ulcers are mainly diagnosed as venous, arterial, mixed (venous-arterial), pressure on bone prominences-related or neuro-ischemic ulcers. They represent most skin wounds. Atypical wounds (AW) of the skin are a daily challenge for clinicians. They need accurate diagnosis, management, and specific treatment. Even if atypical ulcers for their unusual aspect are likely difficult to obtain a standard care, the aim of our present and future work will be to inform us about this delicate topic and to standardize diagnostic strategies and treatments (Mani et al. 2016). AW are chronic skin ulcers which do not show clinical-histological aspects, localization and response to therapies that are usually seen in most common ulcers (Hoffman 2013). They may present features that the clinician has not previously encountered. They are increasingly and properly diagnosed with the improvement of investigative and diagnostic skills (Janowska et al. 2019). AW result from several different pathogenetic processes. They can be summarized as: neoplastic, haematologic (i.e. haemoglobin disorders, polycythaemia vera), metabolic anomalies (i.e. calciphylaxis), inflammatory (i.e. vasculitis, pyoderma gangrenosum), occlusive small vessel vasculopathies (i.e. coagulation anomalies, micro-thrombosis, livedo vasculopathies), drug assumption (i.e. hydroxyurea) or addiction (i.e. heroin-cocaine injection). Many other biological conditions may determine difficult-to-diagnose atypical chronic skin ulcers above all in the case of concurrent presence of venous-arterial insufficiency (double-hit effect). A special chapter of atypical ulcers is primary cutaneous infection (Tang and Kirsner 2012). Mycobacteria, Leishmania, Sporotrichum and skin parasites may be the causative agents of many chronic and often disabling wounds all over the world. Gram + and gram-bacteria are responsible for severe complication of many skin wounds but they may be the cause of primary-induced ulcers in immunocompromised, diabetic and elderly patients (i.e. ecthyma gangrenosum). Clinical examination, specific diagnostic investigation, and additional tests are required to diagnose AW. Despite recent emerging cutting-edge technologies provide innovative pathways to make diagnose and to improve AW treatments skin biopsy remains the first diagnostic step to obtain information about an undetermined chronic-cutaneous ulcer (Tottoli et al. 2020; Miteva and Romanelli 2012).

Neoplastic Ulcers Chronic skin ulcers can be caused by cutaneous neoplasms. They are mainly non-melanoma skin cancer and, in a limited number of cases, cutaneous lymphoma (Fig. 1).

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Fig. 1 Cutaneous B-cell lymphoma initial presentation in a 45 yrs-old female

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Sacchelli and co-workers recently examined 866 consecutive patients with chronic non-healing leg ulcers who underwent skin biopsy of the wound. They found 7% of neoplasms, more commonly basal and squamous cell carcinomas in elderly patients (Sacchelli et al. 2018). Chronic leg ulcers can be misdiagnosed as chronic venous ulcers and masquerade a malignant complication (Gil et al. 2015). The neoplastic lesions can become chronically ulcerated, as the well-known Marjolin ulcer, or present as ulceration from their early appearance (Khan et al. 2016; Pranteda et al. 2014). Marjolin ulcer occurs in burned, constantly injured or chronically inflamed skin. It is mainly a squamous cell carcinoma (Figs. 2 and 3) but it may be histologically defined by many other pathological types of neoplasms. Malignant transformation of a chronic ulcer is preferentially toward a well-differentiated form of squamous cell carcinoma. The clinical verrucous aspect creates difficulties in histologically distinguishing from a benign pseudoepitheliomatous hyperplasia (Pranteda et al. 2014; Senet et al. 2012) (Fig. 4). The clinical suspicion of a skin cancer may rise from: the development of an exophytic mass, irregularities of the ulcer bed or thickening, abnormal and extensive granulation, unusual pain and abnormal bleeding (Figs. 5 and 6).

Fig. 2 Marjolin ulcer. A vegetating squamous cell carcinoma developed in a osteomyelitis sinus tract

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Fig. 3 Marjolin ulcer. Squamous cell carcinoma complicating a chronic skin ulcer

Fig. 4 Basal cell carcinoma. Hyperplastic nodules are difficult to differentiate from benign pseudoepithelioumatous hyperplasia

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Fig. 5 Chronic ulcers caused by a squamous cell carcinomas

Fig. 6 Basal cell carcinoma recurrently bleeding

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The risk of a tumoral nature of a chronic ulcer increases over time. The necessity of a skin biopsy is commonly delayed in clinical decision. The opportunity to obtain multiple samples from a suspected chronic wound, may be encouraged by the rapid healing of the wound bed edge after punch biopsy (Panuncialman et al. 2010).

Haematologic Ulcers Haemoglobinopathies-Associated Ulcers Major haemoglobinopathies complicated by chronic ulcers are sickle cell syndromes and thalassemia disorders. Leg ulcers are the most common cutaneous complication of sickle cell disease (SCD). They may be disabling and are often misdiagnosed (Alavi and Kirsner 2015). They may be caused by various and concurrent pathogenetic processes: mechanical obstruction due to dense sickle red cells, venous incompetence, bacterial infections, abnormal autonomic control with excessive vasoconstriction when in the dependent position, in situ thrombosis, anaemia with decreased oxygen carrying capacity, and decreased nitric oxide bioavailability leading to impaired endothelial function. In brief, the increased susceptibility to leg ulcers in people with SCD is due to a chronic ischaemia and defective immunity. Chronic ischaemia may be explained by a blood hypercoagulability, venous incompetence, or postural vasoconstriction, with these mechanisms conditioned by a low nitric oxide that may itself be genetically influenced. Skin wounds are prevalent in areas with low subcutaneous fat and which are frequently traumatized. Medial malleolus of the leg is the preferential site (Fig. 7). The role of hydroxyurea in the treatment of SCD’s ulcers is still unclear, though it is the first drug to be approved in the treatment of SCD (Lanzkron et al. 2008). Pentoxifylline has been administered with success in the vessel-occlusive phases of the disease. It may be useful in healing ulcers and preventing their recurrence (Monfort and Senet 2019). However, allogeneic bone marrow transplantation or peripheral blood stem cell transplantation are presently considered the best curative therapies for patients with sickle cell disease (Connor et al. 2017). An updated Cochrane Review of interventions for treating leg ulcers in people with sickle SCD provided a limited evidence of very low quality that a systemic pharmaceutical interventions (arginine butyrate) may reduce ulcer size in treated participants compared to controls (MartíCarvajal et al. 2021). Diagnosis: blood count, detection of haemoglobin anomalies, exclusion of primary venous refluxes. Leg ulcers are less often seen in b thalassemia in comparison to SCD.

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Fig. 7 Chronic bilateral ulcer in a young man (31) with sickle cell anaemia

Polycythaemia Vera and Leg Ulcers Polycythaemia vera is characterized by an abnormal proliferation of bone marrow elements, erythrocytes, leukocytes, and platelets. Arterial thrombosis, superficial thrombophlebitis, leg ulcers, and livedo reticularis may be a complication of the disorder. Skin ulcers are extremely painful and prevalently located in the acral areas of the lower limbs and feet (Figs. 8 and 9). They may be improved with the decrease of the number of platelets or with the treatment with modern biological molecules (e.g. Jak-1 and 2 inhibitors) (Wirth et al. 1998; Tremblay et al. 2021; Shanmugam et al. 2013). Diagnosis: blood count, skin biopsy, arterial circulation assessment, coagulation screening.

Metabolic Anomalies Calcific Uremic Arteriolopathy (Calciphylaxis) Calcific uremic arteriolopathy (CUA) also known as calciphylaxis is the term which indicates a tissue deposit of calcium frequently secondary to chronic renal insufficiency (CRI). However, the frontier between uremic calciphylaxis and non-uremic calciphylaxis is difficult to define. CUA seems to be the result of multiple

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Fig. 8 Polycythaemia vera. Acral necrotizing and ulcerative lesions in a 35 years-old male

Fig. 9 Polycythaemia vera. Chronic painful ulcer of the foot and blue toe (40 years old male)

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conditions (obesity, bone mineral disease abnormalities, uraemia, inflammation) with a broad spectrum of variations (Gaisne et al. 2020). It is clinically characterized by distal cutaneous necrosis induced by ischemia following calcium deposition in the media and intimal hyperplasia of the sub-cutaneous arterioles or by acral gangrene due to extensive calcification of the media and thickening of the arterial intima of hands and feet. Hafner et co-workers debated the pathogenetic role of the secondary hyperparathyroidism noticed in the course of CRI and in dialyzed patients (Hafner et al. 1995). In CUA skin ulcers are severe, often multiple, painful and prevalently localized in the lower limbs (Fig. 10). On the dorsum of the hands the ulcers can be multiple, symmetrical and present a typically crater-like aspect (Fig. 11). Hands Rx shows complex thin arteriolar calcifications. Therapeutic guidelines and evidence-based recommendations for CUA are only partially defined (Kodumudi et al. 2020). Calcium-based phosphate binders, Vitamin D and calcium supplements should be avoided. For patients with secondary hyperparathyroidism and hyperphosphatemia, cinacalcet may be used to correct the excess of phospho-calcic products. In several cases, parathyroidectomy has allowed a quick resolution of the pain and the ulcerative lesions by correcting the condition of secondary hyper-parathyroidism. Treatment of pain is central in this condition. Diagnosis: kidney function screening (including glomerular filtration rate), calcium phosphate products detection, parathyroid function anomaly, Rx examination of the lower limbs and hands showing calcium deposit in the arterioles.

Inflammatory and Immune Disorders-Correlated Vasculitis Cutaneous vasculitis (CV) is a spectrum of conditions characterized by an angiocentric inflammation diffusely involving the vessels of the skin (Shavit et al. 2018). They are caused by the deposition of circulating immunocomplexes in the vessel wall. A smaller group of CV is caused by anti-neutrophils antibodies: they include mainly cutaneous-systemic vasculitis. The inflammatory infiltration and the severe vessel damage commonly result in purpuric palpable lesions of the lower half of the legs. The type, location and calibre of the involved vessel district influence the localization and severity of the clinical features which may cause cutaneous ulcers. The vasculitic etiology of a chronic skin ulcer can be usually only suspected. We often can’t see the early clinical aspects (palpable purpura) of the CV which commonly tend to heal after some days or weeks (Papi and Papi 2016). The histological confirmation of the diagnosis is mandatory for a vasculitic ulcer. Tissue culture is useful if a suspicion exists of an infective etiology (Papi and Didona 1999).

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Fig. 10 Calciphylaxis of the lower limb. Severe necrotic-ulcerative lesions in an elderly female patient

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Fig. 11 Calciphylaxis. Multiple painful non-healing crateriform ulcers of the hand. 71 years old men under dialytic treatment

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Table 1 Investigation protocol in case of suspected cutaneous ulcerative vasculitis First level investigation Confirm Histological examination Assessment of extracutaneous involvement - Direct immunofluorescence - Haemochrome, ESR, PCR, fibrinogen - Proteic electrophoresis - Creatinine, blood urea - GOT, GPT, alkaline phosphatases, cGT - PT, PPT - C3, C4 - Antinuclear antibody (ANA) - Extractable nuclear antigens antibodies (anti-ENA) - Antineutrophil cytoplasmic antibodies (c, p-ANCA) - Cryoglobulins - Rheuma-test - Antibodies anti Hepatitis B virus (anti-HBV), antibodies anti Hepatitis C (anti-HCV) - Urine test Second level investigation - Tumour markers - Virological research (cytomegalovirus, Epstein-Barr, Chlamydia) - Rx thorax - Tuberculosis tests - Electromyography, muscular biopsy - Selective angiographic tests

Theoretically, any type of CV can cause skin ulcerations due to a focal ischemia which develops in a small cutaneous area as result of vessel-function loss or a severe decrease in microvascular blood flow (Papi 2008). CV are idiopathic in 50% of cases and associated with drug intake, infections, connective tissue diseases or malignancies in other cases. A laboratory investigation protocol is necessary to identify skin-limited forms from extracutaneous visceral involvement (Table 1). Kidney, lung, and gut are the most commonly affected internal organs. Panarteritis nodosa, cryoglobulinemic vasculitis and granulomatosis with polyangiitis (Wegener disease) are commonly associated with cutaneous necrotizing lesions and possibly chronic atypical ulcers (Figs. 12 and 13). Corticosteroids and other immunosuppressant drugs (most commonly cyclophosphamide and methotrexate) can be useful in non-healing vasculitic ulcers caused by cutaneous-systemic vasculitis. In recent years, rituximab has been used with very good results in many systemic vasculitis (Terrier and Durel 2020). Topical lidocaine and prilocaine lidocaine help to reduce pain during local treatments.

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Fig. 12 Multiple painful leg and feet ulcers in 73 years old female with cryoglobulinaemic vasculitis

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Fig. 13 Necrotic and ulcerative lesions in a livedoid leg of a patient with panarteritis nodosa

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Pyoderma Gangrenosum It is a cutaneous inflammatory disease of unknown etiology at ulcerative evolution, included in the group of “neutrophilic dermatoses”. It has also been considered as a cutaneous marker for some autoinflammatory diseases (Marzano et al. 2016). The initial lesion is characterized by a small purplish papule-pustule that increases in the peripheral area with the formation of a violaceous skin ulcer (Papi et al. 1997). It typically affects adults and tends to recur. The ulcer is very painful and, sometimes, grows on one side and decreases on the opposite. The active area shows an undermined purplish margin. It tends to heal leaving a characteristic atrophic-scar outcomes (cribriform) (Fig.14). The lesion can be single but, more often, is multiple and localized at the lower limbs (Maverakis et al. 2020). No skin area may be excluded. The localization on previous surgical scars is common. Several clinical forms are known: superficial, granulomatous, panniculiticsuppurative (Fig. 15). About 50% of patients present an associated disease (intestinal inflammatory pathologies, seronegative arthritis, rheumatoid arthritis, chronic hepatitis, monoclonal gammopathy) (Table 2). In the atypical cases (multifocality) or severe, haematological diseases (myelofibrosis, leukaemia) and solid tumours should be excluded (Croitoru et al. 2020; Janowska et al. 2020). The non-specific histological exam shows a diffuse dermal infiltration of neutrophil leukocytes responsible, sometimes, of non-specific signs of leukocytoclastic vasculitis. There is no specific diagnostic test and the diagnosis is usually obtained from exclusion. Its early recognition and proper management with prompt initiation of immunosuppressive therapy are essential to improve the quality of life and the prognosis of patients. In the forms without concomitant pathologies, therapy is based on corticosteroids (1 mg/prednisone/kg/die), cyclosporine (3–5 mg/kg/die), clofazimine and dapsone. Good results have been reported using anti-TNFalpha drugs (etanercept and infliximab) and new biological molecules, (Papi and PapiC. 2018). Diagnosis: it is an exclusion diagnosis. A biopsy and the histological examination may help in many cases in which a clinical suspicion is present.

Occlusive Non Vasculitic Vasculopathies Occlusive cutaneous small vessel vasculopathies cause several cutaneous lesions which may result in AW, which are difficult to distinguish from inflammatory ulcerative disorders and clinically atypical. Embolization due to cholesterol and oxalate emboli, cutaneous intravascular metastasis from visceral malignancies, atrial myxomas, intravascular angiosarcoma, intralymphatic histiocytosis, intravascular lymphomas, endocarditis, crystal globulin vasculopathy, hypereosinophilic syndrome, and foreign material have been described to have a pathogenetic role in these disturbances. (Velasco et al. 2017). When platelet

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Fig. 14 Typical lesion of PG tending to extend with purplish edges and ulcerated central area cribriform like

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Fig. 15 Suppurative PG in a 72 year old male with recurrent septic arthritis

Table 2 Systemic diseases associated with PG (50% of patients)

Seronegative arthritis Rheumatoid arthritis Chron’s disease Ulcerative colitis

Myelodysplastic syndrome Myelocytic leukaemia Lymphoproliferative disorders Autoimmune hepatitis

pugging is involved, heparin necrosis, thrombocytosis secondary to myeloproliferative disorders, paroxysmal nocturnal haemoglobinuria, and thrombotic thrombocytopenic purpura may be responsible for the occlusion. A special role may be played by cryoproteins (cryofibrinogenemia, cryoglobulinemia) which may cause multiple and extremely painful atypical ulcers. Systemic coagulopathies due to defects in C and S proteins, coumarin/warfarin-induced skin necrosis, disseminated intravascular coagulation, and antiphospholipid antibody/lupus anticoagulant syndrome may also result in occlusive non vasculitic vasculopathy. Finally, livedoid vasculopathy is a distinct entity which may also cause occlusion of the vessels and result in recurrent skin ulcers on the legs with ambiguous etiology and clinical interpretation (Jorizzo 1998).Though many of the above conditions can cause skin ulcers which may become chronic and atypical, we will limit our description to livedoid vasculopathy and antiphospholipid antibodies-associated leg ulcers.

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Livedoid Vasculopathy Livedoid vasculopathy (LV) is a chronic, painful, thrombo-occlusive cutaneous vasculopathy that involves the distal lower extremities and feet. Typical clinical features include livedoid skin changes (linear or angular, erythematous nodules), atrophie blanche (smooth, ivory-white plaques), and painful atypical ulceration. The diagnosis needs to be confirmed by a histological exam which shows characteristic vascular abnormalities, including intraluminal thrombosis, endothelial proliferation, and subintimal hyaline degeneration and no signs of vasculitis, (Papi et al. 1998) For several years the confusing term “livedo vasculitis” has been used to identify a characteristic and an autonomous clinical entity. It presented focal livedoid lesions, purplish ridge-like (livedo reticularis) and a treelike aspect (livedo racemosa), often associated to painful ulcerations and starred scars (Figs. 16 and 17.) which are located in the lower third of the legs and in the dorsum of the feet. It preferentially affects young women and has a chronic course with frequent worsening in winter (livedo reticularis with winter ulcerations) or in summer (livedo with summer ulcerations) (Bilgic et al. 2021) (Table 3). The histological feature, also very specific, includes thrombosis of the “candelabrum artery”, hyalinization of the vessel walls, swelling of the endothelial cells and poor infiltration of lymphocytes. These aspects suggest a thrombotic pathogenesis of LV, as it is also confirmed by several studies which indicate the presence of various thrombotic defects associated to LV patterns and the clear presence of platelet activation (Papi et al. 1998; Alavi et al. 2013). An incisional surgical skin biopsy and the histological confirm of the diagnosis is mandatory. An extensive investigation for inherited or acquired thrombophilic disorders, immune-related ulcerating diseases and metabolic disturbances must be performed (Table 4). Therapeutic options: antiplatelet agents have been extensively used for a long period as a first-line therapy. They did not demonstrate to be effective in many cases. Anticoagulation by heparin was the most successful treatment as reported in some case-series study (Gardette et al. 2018). Recent studies reported good results with the use of rivaroxaban, a direct factor Xa inhibitor that prevents thrombus formation. (Weishaupt et al. 2016; Lee and Kim 2016). It may be increasingly recommended in the near future. In some cases with recurrences the therapy is a real challenge and the clinical lesions are respectively worsened by hot or cold environmental temperatures or other physical variants (Papi 2006). Diagnosis: histological examination, clinical aspects, young age, pain.

Antiphospholipid Antibodies-Associated Leg Ulcers Antiphospholipid syndrome (APS) is an acquired thrombophilic disorder in which autoantibodies are produced against a variety of phospholipids and phospholipidbinding proteins. They are called antiphospholipid antibodies (APLAs). APL share

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Fig. 16 Multiple ulcerative lesions with branch morphology in a 35 years old woman

anticoagulant properties in vitro, while associate to a higher incidence of thrombotic phenomena in vivo (Knight and Kanthi 2022). In fact, the venous and arterial thromboses and the repeated abortions are the main clinical features of the syndrome that sometimes precedes or is connected to systemic lupus erythematosus. The skin is often site of necrotizing or gangrenous lesions at ulcerative evolution

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Fig. 17 Livedoid aspects and racemose necrotizing lesion in a 23 years-old female

Table 3 Diagnostic screening in occlusive non-vasculitic vasculopathies

Immunologic exams ANA, antibodies anti ENA, anticardiolipin and antiß2 glycoprotein 1, C3, C4, CH50, CIC, cryoglobulinemia, cryo-blood fibrinogen Screening for thrombophilia Haplotypes of MTHFR, PAI-1, factor II, Factor V Leiden, LAC, antithrombin III, factors C and S of the coagulation, PT, PTT; fibrinogen Metabolic screening Homocystinemia, vit B12, folic acid, homocysteine dosing and oxalic acid in urines Instrumental exams Ecocolordoppler of lower limbs, ecocardiodoppler

and, sometimes, of less evident manifestations characterized by small irregular and branched ulcerations that result in atrophic-cicatricial areas resembling “white atrophy” (Fig. 18) (Flores et al. 2021). In these cases the histological exam shows a picture of thrombotic microangiopathy of the dermal and subcutaneous vessels, lacking real signs of vasculitis. However, severity of deep venous-arterial thrombotic events often causes serious systemic complications. The relation between

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Fig. 18 Antiphospholipid antibodies chronic atypical ulcer and residual scars of previous ulcers in a 45 years-old female

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APL immunity and the associated thrombotic events is not clear, yet. From time to time APLs have been considered responsible for a reduced production of prostacyclin, thus interfering with the anticoagulant proteins of protein C and inhibiting the fibrinolytic process. Lifelong anticoagulation with vitamin K antagonists remains the cornerstone of the therapy for thrombotic APS and its complication (warfarin) (Cohen et al. 2020). Rivaroxaban and antiplatelets (acetylsalicylic acid) are considered second line drugs. However, contradictory guidelines have been published in the last decade. Immunosuppressive (corticosteroids, rituximab) and antimalarial synthesis treatments (hydroxyquinolines), if associated to lupus erythematosus, may be advised. Diagnosis: positivity for APS, no histological aspects of vasculitis, pain.

Drug-Induced Hydroxyurea Hydroxyurea (HU) is an antiproliferative molecule mainly used by haematologists to treat chronic myeloid leukaemia and polycythaemia vera. In the case of prolonged therapies, dermatomyositis-like lesions, cutaneous atrophy, spread alopecia (baldness), linear hyperpigmentation of the nails, dyskeratosis and multiple epitheliomas of the light-exposed areas can appear on the skin (Papi et al. 1993). The possible onset of ulcers on the legs has been known for several years. They are mainly supra-malleolar ulcers, almost always very painful, roundish shape, covered with a constant yellowish fibrinous type material (Fig. 19). The preferred location is an area that covers a bone protuberance and that is often the site of ulcers which may be confused with other type of wounds (Sirieix et al. 1999). Patients sometimes refer to a trauma as a triggering factor. The drug antiproliferative action makes difficult it to repair the ulcer damage. The cutaneous atrophy and the thinning out of the capillary bed responsible for ischemia condition are other reasons that confirm HU direct role. The increase in the absolute number of platelets and/or their increased clustering in some of the haematological diseases treated with HU can constitute a favouring factor. Recent studies have demonstrated a significant volume increase of blood cells and the reduction of their deformability in patients treated with HU. The largest and most rigid erythrocyte can induce microcirculatory obstructions and, therefore, tissue hypoxia. HU interruption or substitution is generally followed by a significant improvement of the ulcer. Good results have been obtained by using vasodilators, pentoxifylline and elastic compression (Bulte et al. 2021). Diagnosis: HU therapy, lower extremities, over bone prominences location, pain.

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Fig. 19 Hydroxyurea ulcer in the supra-malleolar area. Typical linear hyperpigmentation of the nail

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Drug-Related Cutis Embolism (Nicolau’s Disease) Drug related cutaneous embolism (DICE) called also “Nicolau livedoid dermatitis” is an adverse reaction to drugs that arises in the seat of intra vascular injection of various drugs whose pathogenesis is uncertain. It has been reported as being caused by penicillin injection, ant-inflammatory non steroidal drugs, local anaesthetics, antihistamines, steroids and vitamin B complex. Less frequently, it has been observed after administration of pyradolon, chlorpromazine, interferon alfa and beta, vaccines and anodynes (Sarifakioglu 2007). Clinically, it is characterized by sharp pain that arises immediately after intramuscular injection with quick appearance of paleness due to local vasospasm. In the following 24 h a livedoid reticulum appears that, in some patients, can present with a haemorrhagic aspect with cutaneous and subcutaneous necrosis that might involve muscles and develop deep ulcers difficult to heal (Fig. 20). It has been reported more frequently on the glutei but other localizations, such as shoulder, thigh and knee have been reported. Generally, there is not supra-infection; if the glutei are involved, livedoid dermatitis can be associated to rectal haemorrhage up to the transitory or permanent ischemia of the homolateral limb. Neurological disorders, such as hypoesthesia and paraplegia, are possible; an increase of hepatic enzymes and creatine-phosphokinase can occur.

Fig. 20 Drug-related cutis embolism (Nicolau’s disease) Initial necrotic-livedoid lesion soon after a penicillin injection. A severe-chronic ulcer developed after 2 weeks

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It involves mainly adult patients, although some cases have been reported also at paediatric age. Presumably, MCE pathogenesis is of vascular origin, as proven by the histological aspects characterized in some cases by thrombotic phenomena in the medium and small size vessels at the reticular dermis level: a periarterial or perivenous injection causes the stimulation of the sympathetic nerve endings, thus causing sharp pain, vasospasm with consequent ischemia and cutaneous necrosis. Diagnosis: intramuscular injection, glutei most common area, pain.

Warfarin-Induced Skin Necrosis Warfarin-induced skin necrosis (WISN) is a rare but well-known complication of warfarin treatment with literature estimating WISN to cause complications in between 0.01 and 0.1% of patients on warfarin (Murad et al. 2014). WISN is thought to be caused by the paradoxical prothrombotic state that arises from warfarin therapy as a result of an initial relative decrease in vitamin K-dependent clotting factors (e.g. protein C). This imbalance can cause microthrombi which interrupt blood flow to the skin and cause necrosis. Protein C, S and antithrombin III deficiencies are, in fact, considered risk factors for WISN.

Drug Abuse Chronic skin ulcers are rare among healthy young adults. Local injection of cocaine and heroin has been identified as a cause of chronic skin ulcers in young adults abusing intravenous drugs (Sönmez Ergün et al. 2012). Chronic skin ulcers in young adults should be an indication of intravenous drug abuse and should be considered in the differential diagnosis of nonhealing AW (Fig. 21). Abscesses are common in those who use heroin because the substance is not sterile and is often mixed with citric acid. Citric acid can also cause acid burns in the vessels or subcutaneous tissues, leading to necrosis. When an user cannot find a good site (skin popping), the drug can build up under the skin and be absorbed into subcutaneous tissues and result in clinically atypical skin ulcers (Onesti et al. 2014).

Ulcer Resulting from Arterial Hypertension (Martorell Hypertensive Ischemic Ulcer) Martorell hypertensive ischemic leg ulcer (HYTILU) is an uncommon but probably underestimated lower extremity wound characterized by progressive, painful unique or multiple necrotic ulceration. “Necrotic angiodermitis” is the descriptive term used in French literature but it is also indicated with the term “ulcer with cyanotic and purpuric edges”.

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Fig. 21 Drug abuser atypical leg ulcer

It is included in the group of organic microangiopathies due to its characteristic clinic-histological aspects. It prevalently affects females. The local micro trauma can be a triggering factor. It is characterized by purplish plaques with central necrosis that tend to extend to the periphery with livedoid-inflamed edges that progressively necrotize (Fig. 22). During its evolution we can observe one or

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Fig. 22 Hypertensive ulcer in a 71 years old male with arterial hypertension. Livedoid and inflamed aspects of the periulcerative area

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multiple necrotic, blackish plaques that, although superficial, are strongly attached to the deep dermis, are very painful with surrounding erythematosus and/or purpuric skin. It is a less rare pathology than thought and generally associated to badly controlled arterial hypertension (90% of cases). The most frequent location is on the legs and the lesions are often multiple or bilateral. It tends to relaps (Alavi et al. 2012; Lima Pinto et al. 2015). The necrosis that is observed at the beginning can be removed quite easily and, when it is superficial, the ulcer may rapidly heal. The histological picture is characterized by arteriosclerosis of the deep dermis arteries associated with sub-endothelial non-specific hyalinosis, intima thickening, concentric hyperplasia of the muscle smooth media muscle fibres and substantial reduction of the vessel lumen. Such alterations sometimes are also present on areas of healthy skin of the same patient. The distal wrists are palpable and the exams confirm the absence of occlusive arteriopathies of the larger vessels and in anomalies in the venous circulation. These ulcers are typical of young people with severe hypertension, according to Martorell’s initial description. The functional and structural alterations on hypertensive basis firstly involve the cutaneous arterioles narrowing and might be responsible for an increased “vascular resistance” and a reduced “perfusion pressure”. The therapy must first aim at reducing the hypertension and mitigating the pain. Early surgical management is the most valuable definitive treatment for Martorell HYTILU. Ulcers > 3 cm in diameter benefit from surgical debridement of necrotic tissue followed by split-thickness grafting (Conde Montero et al. 2018) Prostanoids, pentoxifylline, and other vasodilators have been used with variable results. Diagnosis: arterial hypertension out of control, presence of peripheral arterial pulses, pain.

Atypical Ulcers Associated to Infections Chronic wounds are always contaminated by microorganisms originating from the surrounding skin. The initial bacterial burden, virulence and capacity of invading pathogens to grow within biofilms, together with the ability of the host to create protective immune responses may cause the development of an ulcer infection. Chronic wounds can also occur as a result of a primary infection with microorganisms. Some of wounds may be clinically unusual and do not respond to standard care treatments.

Mycobacterial-Induced Ulcers Tuberculosis (TB) is still prevalent in many developing countries and can pose a new potential threat to global health due to international migration. In 2020, the 30 high TB burden countries accounted for 86% of new TB cases. Eight countries

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account for two thirds of the total, with India leading the count, followed by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa (data from World Health Organization 2021). As an uncommon form of extrapulmonary TB, cutaneous TB is complicated in its clinical manifestation, pathogenesis, and classification (Kaul et al. 2022a, b). Cutaneous TB can be divided into two major categories, true cutaneous TB and tuberculid, depending on the source of infection, the route of transmission, the amount of bacteria, and the immune state of the host. Clinical manifestations may include patches and plaques (lupus vulgaris, TB verrucosa cutis), macules and papules (acute miliary TB, papulonecrotid tuberculid, lichen scrofulosorum), nodules, and abscesses (erythema induratum of Bazin, tuberculous gumma), erosions, and ulcers (tuberculous chancre, orificial TB (Fig. 23), scrofuloderma), mimicking diverse skin diseases. Cutaneous mycobacterial infections may cause a wide range of clinical manifestations, which are divided into four main disease categories: (i) cutaneous manifestations of Mycobacterium tuberculosis infection, (ii) Buruli ulcer caused by Mycobacterium ulcerans and other related slowly growing mycobacteria, (iii) leprosy caused by Mycobacterium leprae and Mycobacterium lepromatosis, and (iv) cutaneous infections caused by rapidly growing mycobacteria. Clinically, cutaneous mycobacterial infections present with widely different clinical presentations, including cellulitis, nonhealing ulcers, subacute or chronic nodular lesions, abscesses, superficial lymphadenitis, verrucous lesions, and other types of findings. Mycobacterial infections of the skin and subcutaneous tissue are associated with

Fig. 23 TBC ulcerative lesion in periorificial area

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severe stigma, deformity, and disability. Geography-based environmental exposures influence the epidemiology of cutaneous mycobacterial infections. Cutaneous tuberculosis exhibits different clinical phenotypes acquired through different routes, including via extrinsic inoculation of the tuberculous bacilli and dissemination to the skin from other sites, or represents hypersensitivity reactions to M. tuberculosis infection.

Buruli Ulcer Buruli ulcer is a chronic skin wound due to the infection of Micobacterium ulcerans (Clancey 1964; Kumar et al. 2015). It is the third most common mycobacterial disease worldwide, The identification of a microdeletion on chromosome 8 in a familial form of severe Buruli ulcer suggested a monogenic basis of susceptibility (Manry 2020) It prevalently occurs in tropical developing countries. It has been reported in several countries in Africa, the Americas, Asia and the Western Pacific. Most cases occur in tropical and subtropical regions (Guarner 2018). The higher concentration of cases has been reported West Africa. Buruli ulcer has been widely described also in Australia (Johnson and Roltgen 2019). The mycobacteria produce mycolactones that cause tissue necrosis. The disease presents as a painless skin nodule that ulcerates as necrosis expands (Fig. 24). Finding acid-fast bacilli in smears or histopathology, culturing the mycobacteria, and performing M. ulcerans PCR in presumed cases confirm the diagnosis. The skin and the bone are the favourite target organs. The chronicity of the lesion may lead to disfigurement and disability. M. ulcerans is environmental but the exact mechanism of transmission is still unclear. Rifampicin (10 mg/kg once daily) and clarithromycin (7.5 mg/kg twice daily) for 8 weeks is now the first-line treatment (Yotsu et al. 2018).

Lehismania The protozoan parasite Lehismania (L) is another major cause of primary infectious ulcers. Cutaneous Leishmaniasis (CL) is endemic in 88 countries. Ninety percent of cases present as CL, but the infection may also affect internal organs (visceral leishmaniasis). Nodular lesions on exposed skin with a tendency to ulcerate over time in combination with a travel history should therefore do a prompt workup for leishmaniasis (Fig. 25). The diagnosis is made through histology, parasite culture, and PCR using biopsy material (Handler et al. 2015). It is caused by the protozoa of the genus L. The disease is transmitted by phlebotomes: Phlebotomus (P) sp. in the Old World and Lutzomyia in the New World. The carriers are represented by dogs, mice, rats, wild rodents and, more rarely, by humans. In the Mediterranean basin, CL is commonly observed: L infantum, transmitted by P. Perniciosus and P. Perfiliewi, is responsible for most cases. On the clinical side, the leishmaniasis of the Mediterranean basin is

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Fig. 24 Buruli’s ulcer in a 32 years-old male from Sudan

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Fig. 25 Ulcerated cutaneous leishmaniasis in 18 years old Italian boy

characterized by a single polymorphic lesion, localized to the uncovered areas, especially the face, followed by the upper limbs (Fig. 24). Atypical lesions may include the following forms: erythematous volcanic ulcer, lupoid, eczematous, erysipeloid, verrucous, dry, zosteriform, paronychial, sporotrichoid, chancriform and annular (Meireles et al. 2017). In recent years, various cases of cutaneous L caused by tropical and sub-tropical species of leishmaniasis, such as L. tropica, L. mexicana, L. panamensis and L. brasiliensis, have been observed in the Western world, especially in tourists returning from trips to exotic countries: the latter may cause ulcers localized on the wrist and back of the hands, often atypical and very painful.

Deep Fungal Infection-Related Atypical Wounds Sporothricosis Cutaneous sporotrichosis is the most common form of this fungal infection. It usually occurs on a person’s hand or the arm after touching contaminated plant matter. Sporotrichosis is caused by scratches or bites from animals, above all cats. Sporothrix scenckii is the fungus saprophyte which causes the subcutaneous mycoses. It tends to involve lymphatics and develops lymphangitis. The common clinical aspect is multiple inflammatory nodules that are connected by lymphangitic linear inflamed lesions. Single nodules can evolve into a chronic ulcer (Roldan-Mari et al. 2009). A biopsy is mandatory (often not specific), but the diagnosis is usually

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obtained with a fungal culture. In immunocompromised patients, the inhalation of conidia may lead to pulmonary infection and multi-organ involvement.

Mycetoma The disease is characterised by numerous deformations and disabilities, high morbidity, and in its late stage it is potentially fatal (Sehgal 1990). Mycetoma is endemic in the so-called “mycetoma belt” that includes various countries across the world, but it is reported extensively in Sudan, Mexico, and India. Mycetoma can be caused by bacteria (actinomycete mycetoma) or by soil and plant saprophytic fungi (eufungal mycetoma). Firstly and most frequent in Central and South America, secondly in Africa. Mycetomas have in common some characteristics: (a) the penetration of etiological agents through continuous skin swounds caused by thorns, shrubs, wood chips, that is why mycetoma is considered an occupational disease among farmers, (b) the long latency time (months or years), (c) the characteristic, although not exclusive, location to the ankles and feet (Madura’s foot) (Sehgal 1990; Verma and Jha 2019) (Fig. 26), (d) the formation of nodular and plaque lesions, poorly inflammatory followed by the development of abscesses, fistulas and ulcerations: the latter drain a purulent serum exudate containing granules, that are, the colonies of etiological agents and may help the histological diagnosis, (e) the not uncommon involvement of muscles tendons ligaments joints and bones, with consequent deformities and functional impotence such as alterations in walking, (f) the chronic clinical course and the frequent

Fig. 26 Madura’s foot (with kind permission of Prof. Stefano Veraldi)

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absence of local symptoms and systemic clinical manifestations, (g) the histopathological picture characterized by granulomas and the frequent resistance to systemic antifungals drugs. Diagnosis in suspected lesions is made with the help of grain examination, microscopy, imaging (radiography, ultrasonography, magnetic resonance imaging) and culture, and more recently by molecular methods such as PCR and molecular sequencing (Ahmed et al. 2017).

Ecthyma Gangrenosum Ecthyma gangrenosum is usually seen in immunocompromised patients with leukaemia, lymphoma, other malignant diseases, severe burns or organ transplant, or in people receiving immunosuppressive therapy (Singh et al. 2005). It is one of the major dermatologic manifestations of severe, systemic pseudomonas aeruginosa infection (Burnett et al. 2022). It has ben also reported as the consequence of staphylococcus aureus (Santhaseelan and Muralidhar 2017), group A streptococcus, serratia marcescens, escherichia coli and other bacterial species. It is characterized by unique or multiple necrotic lesions which tend to enlarge, ulcerate and to be covered by sloughy-purulent material (Fig. 27). If a bacteriemia

Fig. 27 Ecthyma gangrenosum (multiple ulcers of the lower limb) in a patient with leukemia

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is excluded antibiotic therapy should be started in accordance with bacteriological exams. The patient should be investigated for the immunological condition and potential viral infections.

Microbiological Investigations: Methods and Drawing Techniques Mainly Qualitative Methods Swab: it is performed by moving the swab in a rotary manner inside the deepest ulcer area or margins and, afterwards, inside the culture medium. It can also be quantitative if performed on a specific lesion area and subsequently shaken in a certain quantity of growth medium for a specific period of time. Curettage: aspiration with needle: to remove the material from the ulcer surface a disposable curette or a syringe to aspirate material of a purulent sac can be used. It is advisable that the described techniques are preceded by the ulcer lavage with physiologic salt solution to reduce the risk of developing occasional contaminants.

Qualitative and Quantitative Methods Biopsy: it is performed after ulcer lavage with physiologic salt solution, local anaesthesia (lidocaine injection, xylocaine), rotary movement and light pressure performed with a disposable 4 mm diameter punch. The tissue fragment obtained can be homogenized and then weighed before being placed in the culture medium. It allows to evaluate the presence of microbes in depth and to monitor the extent of microbic proliferation. Sometimes, it requires a light compressive haemostasis. Deep-tissue biopsy is a qualitative and quantitative culture of wound tissue. Irrigation—aspiration: it is mainly used in presence of deep ulcers or ulcers caused by pressure. It allows taking material from anfractuous areas or in sites where a biopsy cannot easily be performed. It consists in irrigation, through 1 ml sterile syringe of 0.9% sterile saline solution, followed by a light massage of the ulcer margins. Immediately after, 1 ml of saline solution is again injected through a new syringe and, after a light massage, 0.25 ml of fluid is inspired. It is advisable that the microbiological laboratory carries out culture tests for aerobic and anaerobic germs, especially in the presence of a real infection. The most often isolated microbial species in the cutaneous ulcers are reported on Table 4. Particular attention should be given to the ulcers caused by pressure and in diabetic patients where anaerobic germs are frequently present (Clostridium perfrigens, Bacteroides fragilis, Peptostreptococcus spp). They often cause sepsis and, occasionally, gangrene.

Atypical Wounds and Wounds Resulting from Infection Table 4 Pathogenic microbial species more commonly found in the cutaneous ulcers

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Gram+ • Staphylococcus aureus • Staphylococcus coagulase neg. • Streptococcus group A, D • Pepto-streptococcus spp Mycetes • Candida albicans Gram– • Pseudomonas aeruginosa • Acinetobacter • Escherichia coli • Proteus spp • Bacteroides spp

As an adjunct to wound cultures, culture-independent investigation of the microbial DNA applying pyrosequencing, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis, and quantitative real-time PCR have identified a greater range of bacteria than traditional culture techniques (Renick and Tang 2021; Rerkasem and Mani 2015).

Laboratory Markers In addition to the wound culture techniques, laboratory markers can also be measured to aid the diagnosis of wound infection. These markers include C-reactive protein (CRP), procalcitonin, presepsin, microbial DNA, and bacterial protease activity. In response to inflammation and infection, CRP, a peptide produced in the liver, is stimulated by cytokines, primarily interleukin-6, and employed in complement binding and phagocytosis by macrophages.

The Future Many new technologies have been created in recent years to diagnose infected wounds. Traditional imaging modalities such as radiography and magnetic resonance imaging have still a role in the diagnostics, and new hybrid imaging techniques, including single-photon emission computed tomography, computed tomography (CT) and positron emission tomography (PET)/MRI, are being utilized as wound infection diagnostic tools, mainly in research at present.

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Biofilms and Impaired Wound Healing: How Do We Detect the Presence of Biofilms in Chronic Wounds Non-invasively Ida C. Thaarup and Thomas Bjarnsholt

Abstract

The pervasive presence of biofilms in chronic wounds is a well-acknowledged phenomenon. A recent meta-review estimated that 80% of all chronic wounds contain biofilms and this number is thought to be an underestimation, as the presence of a biofilm can be difficult to establish. The presence of a biofilm is believed to prevent a wound from progressing through the normal phases of healing, which are haemostasis, inflammation, proliferation, and remodelling. Biofilms are thought to arrest the wound in the inflammatory state, causing an exaggerated immune response with continuous tissue damage and harmful change to the wound microenvironment. Therefore, a key to successful wound healing is to focus on biofilm detection and subsequent biofilm eradication. Non-invasive biofilm detection methods present a new and challenging field of study. One of the main challenges lies within the random spatial distribution of wound biofilms. Despite the limited number of studies in this particular area, biofilms are currently thought to exist in an unstructured, heterogeneous fashion within the wound, occupying different spatial regions and both shallow and deeper layers of the wound. Several distinct species are thought to be present within a single wound creating many different microenvironments. Regardless of these challenges, a handful of non-invasive techniques have been developed that I. C. Thaarup
T. Bjarnsholt (&) Costerton Biofilm Centre, Department of Immunology and Microbiology, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark e-mail: [email protected] I. C. Thaarup e-mail: [email protected] T. Bjarnsholt Department of Clinical Microbiology, Copenhagen University Hospital, Juliane Maries Vej 22, 2100 Copenhagen, Denmark © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_10

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attempt to survey and locate biofilms in wounds using both direct and indirect methods. In this chapter, we begin by introducing the reader to the devastating effect biofilms have in a wound environment. This will include some of the complex interactions between the microbes, their host, and the surrounding microenvironment. We then cover the conventional methods used today to detect wound microbes and subsequently we introduce a number of new, non-invasive methods to detect the presence of wound biofilms. Some methods are still in their infancy and have yet to be tested in human subjects, while others are further along in their development. Finally, we discuss the importance of biofilm detection in wounds and other concerns which might be just as important to consider. Keywords

Biofilms


Detection methods
Biological markers
Imaging techniques

How Biofilms Impair Wound Healing Disruption of Immune and Skin Cells Normal wound healing is often described to progress through four overlapping phases of healing: haemostasis, inflammation, proliferation including tissue regeneration and finally, remodelling (Zhao et al. 2016). In wounds that are progressing normally, the inflammatory phase usually only lasts a few days. However, in the case of an infection, the presence of bacteria is thought to cause a deviant and exaggerated immune response, stalling the wound in the inflammatory phase of healing (Grice and Segre 2012). A meta-review has estimated that up to 80% of all chronic wounds contain bacterial biofilms (Malone et al. 2017a); however, these numbers are often thought to be underestimated since the presence of a biofilm can be difficult to prove due to the heterogeneous distribution in the wounds. While biofilms have already been shown to slow the healing of trauma-induced wounds (Schierle et al. 2009; Zhao et al. 2010; Seth et al. 2012), the specific methods by which biofilms slow the healing of chronic wounds, and the conclusive proof that they do so, is still being investigated. However, many in vivo and in vitro investigations have been performed, and in combination with clinical observations, they have uncovered several mechanisms by microbial biofilms that might lead to delayed healing. Excessive neutrophil numbers have been observed in many chronic biofilm infections, furthering the belief that the presence of a biofilm exaggerates the immune response (Bjarnsholt et al. 2008). When present in excessive numbers, the neutrophils produce compounds such as reactive oxygen species and proteolytic enzymes in such amounts that they cause damage to neighbouring tissue. The

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neutrophils also produce cytokines, which recruit and activate even more neutrophils, enhancing the inflammatory response. Cytokines such as TNF-a, interleukin-1 (IL-1) and interleukin-6 (IL-6) have been found to be significantly increased in wound fluid obtained from non-healing ulcers compared to healing ulcers (Wallace and Stacey 1998; Trengove et al. 2000). Moreover, neutrophils recruit macrophages from the bloodstream, which are also affected by the presence of a biofilm. Macrophages are functionally divided into two groups: the M1 and the M2 macrophages. M1 macrophages are pro-inflammatory and produce cytokines, reactive oxygen species and phagocytize debris and microbes. M1 macrophages are therefore sometimes termed ‘microbicidal’ macrophages. M2 macrophages are reparative and produce polyamines which promote skin cell proliferation and induce collagen production (Zhao et al. 2013; Motz et al. 2021; Sindrilaru and Scharffetter-Kochanek 2013). In normal wound healing, there is a regulated balance between M1 and M2 macrophages, but in biofilm-infected wounds, this balance seems disrupted. Initially, the presence of bacteria causes a large infiltration of M1 macrophages into the wound, intensifying the inflammatory cytokine production (Raziyeva et al. 2021). Yet, some in vitro studies have found that S. aureus biofilms specifically can alter the ratio of M1 to M2 macrophages, by altering the macrophage gene expression away from the M1 phenotype towards the less microbicidal M2 phenotype (Hanke et al. 2012; Thurlow et al. 2012). Moreover, one of these studies observed that S. aureus biofilms caused a decreased macrophage migration towards the biofilm and induced macrophage death by unknown mechanisms in an in vivo mouse model (Thurlow et al. 2012). Lipopolysaccharides of bacterial origin can also prevent macrophages from recognising apoptotic neutrophils destined for engulfing. When the neutrophils are not properly cleared by the macrophages, this leads to necrotic disintegration and further wound damage (Khandaker et al. 1998; Wolcott et al. 2008). Studies have also found that microbial biofilms might disrupt healing by interfering with the normal behaviour of human skin cells. In one study, S. aureus biofilms were found to promote altered gene expressions in human skin keratinocytes causing an upregulation of inflammatory genes and inducing the production of several interleukins (Secor et al. 2011). In another study, an extracellular fibronectin-binding protein produced by S. aureus was found to slow the migration of keratinocytes in vitro, thereby presenting another mechanism by which microbes might restrict epithelialization (Kintarak et al. 2004). Besides changing phenotypic responses and migration, biofilms may cause direct damage to the skin and immune cells (Gajula et al. 2020). Rhamnolipids produced by P. aeruginosa have been found to cause necrosis of neutrophils in vitro (Jensen et al. 2007), as well as disruption of macrophage membranes, leading to decreased phagocytosis (McClure and Schiller 1992). In high amounts, rhamnolipids have also been correlated to increased microbial killing of macrophages (Chua et al. 2017). Furthermore, P. aeruginosa has been found to produce an exotoxin named pyocyanin which drastically increases neutrophil apoptosis both in vitro and in a murine animal model (Prince et al. 2008). Other unknown compounds in biofilm conditioned media produced by S. aureus and P. aeruginosa have been found to be

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highly cytotoxic against keratinocytes (Kirker et al. 2009; Jeffery Marano et al. 2015). Transcriptome analyses of isolates obtained from chronic wounds and joint infections have found that many virulence factors are upregulated in microbes isolated from infectious environments (Xu et al. 2016; Cornforth et al. 2018). Such analyses have also shown that wound isolates express altered metabolic pathways and upregulation of resistance genes. In general, biofilm in wounds survives well due to several survival mechanisms, but that is outside the scope of this chapter and has recently been reviewed elsewhere (Thaarup et al. 2022).

Disruption of Microenvironment Microbial biofilms impair wound healing not only by affecting the skin and immune cells but also by altering the microenvironment to an unfavourable state which does not promote tissue regeneration. This is problematic, as a favourable wound microenvironment has been described as the most important supporting factor in achieving successful healing (Uluer et al. 2018). Healthy skin is usually found to be slightly acidic with a pH in the range of 4.0– 6.0 (Schneider et al. 2007). Acidic skin environment deters the growth of many pathogens and fungi, as they often need pH above 6.0 to grow. Unfortunately, the pH of chronic wounds has often been found to be alkaline with a pH within the range of 7.3–8.9 (Schneider et al. 2007). Moreover, healing has been reported to progress at a reduced speed in wounds with an alkaline microenvironment (Leveen et al. 1973; Tsukada et al. 1992). The alkalinity observed in chronic wounds is thought to be partially due to tissue necrosis and bacterial activity. Many bacterial wound species are able to produce ammonia, which in itself is a toxic compound causing tissue damage but it also increases the wound pH (Leveen et al. 1973; Percival et al. 2014). One study reported that the normal acidic environment of skin became alkaline following the colonisation of microbes (Schneider et al. 2007). Several in vitro experiments have corroborated that microbial growth in various media types, even acidic media, often results in an alkaline microenvironment and some studies have noted that co-cultures of bacteria increases the pH to a higher degree than monocultures do (Kadam et al. 2021; Cendra et al. 2019). A study by Hostacka et al. (2010) found that both P. aeruginosa and K. pneumoniae exhibited increased biofilm production in vitro when exposed to an alkaline pH of 8.5. A different study found that biofilms of P. aeruginosa produced larger amounts of both alginate and proteases when grown at a pH of 8.0 (Harjai et al. 2005). In general, alkaline wound environments support the continued degradation of skin tissue while potentially promoting bacterial biofilm formation. In vitro and ex vivo skin models have been used to investigate the effect of pH on the behaviour of human immune cells. Such investigations generally find that immune cells function best at neutral pH levels. According to one study, leukocyte motility peaks at a pH of 7.5 (Percival et al. 2014), while another study found that polymorphonuclear leukocytes (PMNs) showed the highest motility at pH levels between 6.7 and 7.2 (Leblebicioglu et al. 1996). That same study also found that

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phagocytosis of bacteria functioned best at a pH of 7.7. A later study by the same group found that PMNs undergo apoptosis to a larger degree under alkaline conditions (Leblebicioglu and Walters 1999). Moreover, it has been observed that macrophage production of TNF-a is increased at alkaline pH (Heming et al. 2001). Unfortunately, tissue-destroying enzymes such as matrix metalloproteases and collagenases also function best at alkaline pH, which further delays healing (Schneider et al. 2007; Percival et al. 2014). Oxygen limitation is another microenvironmental factor that decreases the healing potential of a wound. The hypoxic microenvironment observed in chronic wounds is most likely due to the combined oxygen consumption of both immune cells and microbes (Wu et al. 2018). Oxygen is a necessity for most cellular functions, as it is involved in the creation of biological energy equivalents (Schreml et al. 2010). It is also particularly vital for wound healing as it is needed for forming new blood vessels, synthesising new collagen, cell proliferation and other reparative processes (Schreml et al. 2010; Gottrup 2004). Oxygen is also used in the defence mechanisms employed by the immune cells. Both neutrophils and macrophages produce reactive oxygen species that are meant to defer bacterial contamination (Trostrup et al. 2013). PMNs in particular are known for their ability to mount a ‘respiratory burst’, an essential component of the innate immune response, which is the accelerated production of superoxide anions (Kolpen et al. 2010). Unfortunately, when produced in large amounts, reactive oxygen species cause oxidative damage to both tissue and cells. They also function as important signalling molecules and increased levels promote a continuous infiltration of immune cells into the wound (Zhao et al. 2016). Oxygen is also needed for the creation of nitric oxide (NO), one of the most effective antioxidants found in wounds (Zhao et al. 2016). M1 macrophages produce this antioxidant in an oxygen-dependant manner, so in the hypoxic environment of chronic wounds, this antioxidant is often lacking, and the balanced level of reactive oxygen species is disrupted. Finally, the alkaline pH and anoxic environment of wounds are closely associated, as less oxygen is available at high pH levels due to the Bohr effect, which states that oxygen is released more readily from haemoglobin at low pH (Schneider et al. 2007). Attention is often paid to wound temperatures and how they correlate with healing. It is important to include bacterial biofilms in these considerations, as bacterial behaviour has been found to be temperature-dependent in several instances. One study measured biofilm production at two different temperatures and found that 3 out of 4 tested V. cholera strains, that were isolated from a hospital setting, produced more biofilm at 30 °C compared to 37 °C (Hoštacká et al. 2010). The same study found that this was also true for 3 out of 4 tested P. aeruginosa strains. Other studies have found that the optimal temperature for P. aeruginosa biofilm production is highly strain-dependent, with some strains producing the most robust biofilms at 20 °C (Kim et al. 2020). Gene expression patterns and microbial virulence factors have also been found to be temperature regulated (Bisht et al. 2021). While the temperature of chronic wounds or the adjacent skin is often measured, an unequivocal answer to an optimum temperature for healing is hard to come by. On one hand, hypothermic wounds are often reported to heal slower than

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normothermic wounds, and colder wounds have been reported to have a worse wound bed score (Kurz et al. 1996; Lin et al. 2021; Dini et al. 2015). Hypothermia leads to thermoregulatory vasoconstriction, which limits the supply of oxygen to the wound site (Kurz et al. 1996). Moreover, neutrophil killing of microbes has been found to be reduced when the temperature was lowered from 37 to 30 °C (Allen et al. 1997). PMN locomotion is also significantly decreased in cold temperatures (Nahas et al. 1971). When the effects of cold wound temperatures are considered together with the observations that some bacterial strains produce larger amounts of biofilm in a colder microenvironment, it seems that warm wound temperatures would always be favourable. Yet, several studies interpret local increased wound temperature as a negative sign that signifies the presence of an infection. Armstrong et al. (2006) found that patients with a significant temperature increase above 10 °F (5 °C) of the affected limb compared to the unaffected limb had a less favourable clinical outcome. Another study that included data from 112 patients found that wounds with elevated temperature compared to the opposing limb were far more likely to have moderate or heavy bacterial growth (Woo and Sibbald 2009). Finally, Chanmugam et al. (2017) found that infected wounds showed higher wound bed and periwound temperatures relative to the opposing limb, compared to wounds which were inflamed but not infected. The same study also observed how efficient antibiotic treatment caused a notable lowering of the temperature. These studies suggest that a moderate wound temperature increase might signify inflammation and progression of healing whereas a high wound temperature increase could signify infection. This view is supported by the study of Lin et al. (2021) who found that non-infected pressure ulcers with higher temperatures healed faster than those with lower temperatures.

Detection Methods Biofilm Detection Issues There are several inherent issues with detecting biofilms in wounds. These issues are connected to the intrinsic nature of microbial biofilms in the chronic wound environment. The first challenge, which is also the largest issue, is the random and heterogeneous spatial distribution of biofilms in wounds. This has been observed in several studies of chronic wounds, in which the spatial distribution was investigated (Thomsen et al. 2010; Fazli et al. 2009; Travis et al. 2020). Such studies have shown that not only is the abundance of a single species substantially different from one surface area to another, but the number of species between different locations may also vary significantly. These studies exemplify the randomness and heterogenic distribution of wound biofilms, which in turn imposes complications on their detection. Fazli et al. (2009) investigated the depth distribution of biofilm aggregates of S. aureus and P. aeruginosa within chronic venous leg ulcers by using confocal microscopy in combination with peptide nucleic acid-based fluorescence

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in situ hybridization (PNA-FISH). By measuring the distance from the wound exterior to the observed bacterial aggregates, they could determine the mean distance from the wound surface for each species. They found that S. aureus was located 20–30 µm from the surface of the wound, whilst P. aeruginosa was located deeper within the tissue, around 50–60 µm from the wound surface. This study illustrates the problem with detection methods that rely on routinely used surface swabbing, as biofilm aggregates may be absent from the surface of a wound while still inhabiting deeper regions in the wound tissue. Dunyach-Remy et al. (2014) investigated the bacterial species found in 20 diabetic ulcers. They took both surface swabs and deep-tissue biopsies and analysed the samples using PCR coupled to denaturing gradient gel electrophoreses (DGGE). They found that a significantly larger number of species were present in the deeper tissues than in the surface swab samples, once again emphasizing the importance of considering the depth distribution of wound biofilms. Another challenge is the large variety of species. No two wounds are going to be alike in terms of species amount and composition. Thomsen et al. (2010) investigated 14 venous ulcers using molecular detection methods and found an average of 5.4 species per wound, while Price et al. (2011) found an average of 20.9 bacterial genera per wound when investigating 13 chronic wounds of different aetiology. The situation is further complicated as both viruses and fungi have been isolated from chronic wounds and their role in the infectious microenvironment has yet to be elucidated (Wolcott et al. 2009; Xu and Hsia 2018). A small number of biofilms obtained from wound samples have previously been found to be multispecies, which causes further complications (James et al. 2008; Johani et al. 2017; Malone et al. 2017b; Choi et al. 2019). Detection methods targeted toward specific species or compounds produced solely by a limited group of microbes will fall short under these circumstances. Yet, the detection of specific species is not without merit, as the presence of certain microbes has been found to significantly affect healing outcomes. Madsen et al. (1996) analysed 59 venous leg ulcers and found that ulcers containing P. aeruginosa increased in size compared to those that did not. They also observed that ulcers containing S. aureus or haemolytic streptococci healed more slowly than ulcers without. More recent studies have confirmed this trend that the presence of either P. aeruginosa or S. aureus leads to a worse healing outcome (Gjødsbøl et al. 2006; Kalan et al. in press). The large variety of wound microbes also complicates methods based on the detection of extracellular polymeric substances (EPS) produced by the microbes. In general terms, EPS is described to consist of proteins, lipids, exopolysaccharides and extracellular DNA (eDNA), although this description might only be true for in vitro biofilms (Koo and Yamada 2016). To our knowledge, no thorough examination has been performed that analyses the EPS produced in chronic wounds and the actual content of the in vivo wound EPS might differ. Some researchers suggest that wound microbes are likely to incorporate host materials such as fibrin and collagen into their biofilm, which could alter the function and structure (Trivedi et al. 2017). It is important to note that there is no one specific EPS composition, as different species and even different strains of the same species have been found to

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secrete EPS components of vastly different types and amounts (Hobley et al. 2015). One study analysed several strains of Streptococcus thermophilus all grown in the same medium and found that EPS production, proteolytic activity and monosaccharide composition varied widely between strains (Aslim et al. 2006). One final difficulty in detecting biofilms in wounds is that the number of pathogens present might be minuscule and therefore below various detection limits (Bjarnsholt et al. 2009). However, the presence of even a small number of bacteria in a wound can cause an infection, as the bacteria are able to proliferate, resulting in a large biofilm and subsequently causing a delayed healing response. Detection methods that depend on large quantities of microbes being present should thus be careful with ruling out the possibility of infection.

Conventional Detection Methods Current conventional detection methods can be divided into three groups: direct culturing methods, molecular analyses, and imaging methods. Direct culturing techniques assess the viability of the microbes found in a wound. When performed non-invasively, the sample is taken with a surface swab that is run across either part of a wound or sometimes the whole wound surface. While this method is cheap, fast to perform and easy to do in most hospital settings, the results are usually incomplete and cultures often take 24 h or more before they are visible. The bacterial numbers tend to be underestimated and the species found often do not match the ones found using molecular methods (Davies et al. 2004; Xu et al. 2020; Malone et al. 2017c). Results vary greatly depending on the type of swab, the media used for incubation, the incubation conditions including oxygen and temperature, and the duration of incubation (Jakobsen et al. 2021). All these factors need to be considered when comparing results between institutions or different hospitals, as standard practices might differ. As mentioned earlier, wound biofilms exist in deeper layers of the tissue, and superficial swabs are therefore unlikely to reach these. Moreover, superficial swabs may by accident sample commensal skin microbes. Finally, and perhaps the most important point related to traditional culturing techniques is the presence of a wound biofilm cannot be proven using these methods. Molecular methods are often deemed more accurate than culturing methods since molecular methods have the possibility to detect microbes, such as anaerobes or slow-growing variants that often can not be detected using traditional culturing (Dowd et al. 2008). Molecular techniques can be performed on both non-invasive surface swab samples or on wound biopsies that are invasive to acquire. Molecular methods used for detecting wound pathogens include 16S rRNA sequencing, Bacterial Tag-Encoded FLX Amplicon Pyrosequencing (bTEFAP) Partial Ribosomal Amplification and Pyrosequencing (PRAPS), Full Ribosomal Amplification, Cloning and Sanger sequencing (FRACS), Partial Ribosomal Amplification, DGGE and Sanger sequencing (PRADS), Whole Genome Sequencing (WGS) and mRNA sequencing (Cornforth et al. 2018; Malone et al. 2017c). Major concerns when

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using molecular methods include the possibility of DNA contamination from the environment, the amplification of molecular material from dead microbes and finally: the presence of a biofilm can also not be proven using the majority of these methods. mRNA sequencing is the one exception to most of these limitations. mRNA degrades rather fast and will therefore only be found from alive and metabolically active microbes, while dormant microbes might not be detected using mRNA sequencing. Moreover, mRNA sequencing might be able to determine whether the isolated strains were growing in a biofilm or not. In vitro studies have been performed comparing planktonic cells to biofilm-growing cells and specific expression patterns have been observed (Dötsch et al. 2012; Rumbo-Feal et al. 2013). In the future, perhaps mRNA sequencing of wound microbes could be used to determine whether the isolates were growing in a biofilm or not. Yet, some studies have observed how chronic wounds with a high clinical infection score contain surprisingly small amounts of bacterial mRNA (Fritz et al. 2022). Finally, imaging techniques such as confocal laser scanning microscopy (CLSM) or scanning electron microscopy (SEM) can be performed. However, for both of these methods, wound biopsies will need to be acquired making them more invasive than detection methods that can use surface swab samples. Using SEM, microcolonies can be observed and the presence of EPS be confirmed (Johani et al. 2017). Using CLSM, universal probes or species-specific probes can be utilized to confirm microbial aggregates of both unknown and particular species. Specific stains have been developed that target different components of the biofilm EPS such as polysaccharides, glycoproteins or eDNA (Neut et al. 2011; Oates et al. 2014; Schlafer and Meyer 2017). Although direct visualization of biofilms is currently seen as the gold standard for proving biofilm presence in a wound, these methods also have several limitations (Kvich et al. 2020). Imaging techniques carry the risk of false-negative results, as the investigated biopsy samples might not contain any biofilm aggregates due to the random and patchy distribution of biofilms in wounds. These imaging techniques also require expensive and specialized equipment making them unsuitable to use in routine diagnostics at hospitals.

Novel Sensor-Based Detection Methods Novel detection methods are often based on wearable sensors. Sensors have been reported to cause minimal patient inconvenience and wearable sensors incorporated into dressings can limit the need for bandage removal every time a measurement needs to be taken (Pusta et al. 2022). These sensors are able to detect various biological markers which broadly can be divided into three categories (see Fig. 1). Indirect infection markers that monitor the microenvironment and correlate this to the infection status and wound healing prospects. Microbe markers that directly detect microbes or pathogens within the wound. This can also include the detection of a secreted product of microbial origin. Finally, biofilm markers, including EPS matrix components, as well as microbe secreted products that are only produced by biofilm residing bacteria. There is an overlap between microbe markers and biofilm

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Fig. 1 Novel detection methods based on the measurement of infection markers. These markers can broadly be divided into three categories: indirect infection markers, microbe markers and biofilm markers. Some overlap can occur between the different markers. Illustrated wound is shown to contain both biofilm and single cells of the rod-morphology, but all morphological cell types can be found in wounds, in addition to fungi and viruses. Figure created using BioRender.com

markers in regards to density regulated compound production, which is sometimes inferred to be equal to biofilm production (see Table 1 for an overview of sensor-based detection methods). Alternatively, instead of using biological sensors, non-invasive imaging techniques are being developed, which can detect the presence of microbes and microbial compounds within a wound, without the need to take samples. Monitoring microenvironmental changes in the wound is a way to indirectly determine if there is an infection present. As described above, the presence of an infection cause changes to both wound temperature, pH and oxygen levels. Moreover, the immune response will be altered in the presence of pathogens and by observing the actions of the immune cells it can be determined if an infection is present. Antibodies produced by the body have been investigated as they might function as suitable detection markers. The antimicrobial peptide alpha defensin has already been shown to be a sensitive and specific marker for discovering infection in periprosthetic joint infections, however, while this antibody is produced against a wide spectrum of microbes it is not biofilm specific (Deirmengian et al. 2015). Gao et al. (2021) developed a multiplexed immunosensor which was able to detect several immune system signalling molecules in the collected wound exudate. Among the measured signalling molecules were TNF-a, IL-6 and IL-8 which have previously been observed to be increased in non-healing ulcers (Wallace and Stacey 1998; Trengove et al. 2000; Edsberg et al. 2012). The immunosensor was tested in situ in a wounded mouse model and subsequently on wound exudate retrieved

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Table 1 Table presenting the sensor-based detection methods mentioned in this chapter Type of marker

References

Markers measured

Indirect infection

Vu et al. (2020) pH

Indirect infection

Tamayol et al. (2016)

pH

Indirect infection

Pan et al. (2019)

pH

Indirect infection

Shukla et al. (2014)

pH

Indirect infection

Dini et al. (2015)

Temperature

Indirect infection

Woo and Sibbald (2009)

Temperature

Indirect infection

Fierheller and Sibbald (2010)

Temperature

Indirect infection

He et al. (2020) Oxygen

Indirect infection and microbe Indirect infection and microbe

Gao et al. (2021)

Sharifuzzaman et al. (2020)

TNFa, IL-6, IL-8, TGF, S. aureus cell wall epitopes, temperature and pH pH, uric acid, temperature

Indirect infection and microbe

Simoska et al. (2020)

Pyocyanin, uric acid, nitric oxide

Details

Tested in humans or on human samples?

A multilayered wound alkalinity monitoring system was developed that changed colour based on the detected pH pH-responsive hydrogel fibres were developed which changed colour based on the pH. The measurement could be read using a smartphone Curcumin was loaded into a fibrous material and exhibited colour changes based on the pH. Measurements could be read using a smartphone app Wound pH measured by litmus paper was correlated to the type of organism present Using an infrared camera, wound temperatures were measured and correlated to wound bed score A handheld infrared thermometer was used to assess wounds together with bacterial culturing methods A handheld thermometer was used to measure wound temperature Wound dressings with incorporated methylene blue were developed. The dressings turned yellow due to oxygen depletion Measurements were performed using multiplex biosensors in a mouse wound model and in human wound exudate samples A smart bandage with laser-guided graphene electrode sensors were used to measure pH, uric acid and temperature. The bandage was tested on wound exudate samples Flexible carbon ultramicroelectrode arrays were used to detect infection markers

No

No

No

Yes

Yes

Yes

Yes

No

Yes

Yes

No

(continued)

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Table 1 (continued) Type of marker

References

Markers measured

Details

Tested in humans or on human samples?

Indirect infection and microbe

Ashley et al. (2019)

Lactic acid, oxygen

No

Microbe

Sharp et al. (2008)

Uric acid

Microbe

Kassal et al. (2015)

Uric acid

Microbe

Roy et al. (2021)

S. aureus DNA

Microbe

Jarošová et al. (2019)

Uric acid, pyocyanin

Microbe

Zhou et al. (2018)

Toxins

Microbe

Thet et al. (2016)

Toxins

Microbe

Thet et al. (2020)

Toxins

Biofilm

Li et al. (2014) Uncharacterised EPS components

A flexible electrochemical biosensor was developed, which could detect both lactate and oxygen. It was designed to be able to be integrated into wound bandages A sensor added to carbon fibre mesh was used to measure uric acid in whole blood, serum and wound fluid A smart bandage was created by screen printing an amperometric biosensor directly on a wound dressing A dressing was developed which was based on a composite of zeolitic imidazolate framework and carbon nitride conjugated with S.aureus probe-DNA Electrodes were developed which could monitor changes in uric acid and pyocyanin levels A wound dressing that changed colour and released antimicrobials in response to bacterial toxins was developed and tested on a mouse wound model Presents a wound dressing with a lipid-encapsulated fluorescent dye, which was released when in contact with bacterial toxins. Tested on an ex vivo porcine model Swab from a wound was added to a liposome-encapsulated fluorescent dye, which was released when in contact with bacterial toxins A gold-particle based multichannel nanosensor was developed, which detected various EPS components, creating different patterns depending on the specific species that produced the EPS

Yes

No

No

No

No

No

Yes

No

(continued)

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Table 1 (continued) Type of marker

References

Markers measured

Details

Tested in humans or on human samples?

Biofilm

Ngernpimai et al. (2017)

Uncharacterised EPS components

No

Biofilm

Wu et al. (2020)

EPS polysaccharides

Biofilm

Nakagami et al. EPS (2017) mucopolysaccharides

Biofilm

Ashrafi et al. (2018)

Multichannel polymer sensors in combination with inter-polymer FRET between channels detected various EPS components, creating different patterns depending on the species that produced the EPS Wound blotting using alcian blue that stained EPS polysaccharides, was found to correlate well with microbiological results Wound blotting using ruthenium red that stained EPS mucopolysaccharides, was found to correlate to wound size and slough production Gas chromatography-MS was used to detect volatile organic compounds. VOC profiles were found to be specific for biofilm growth and even metabolic activity and biomass. The method was tested on human ex vivo skin samples

Volatile organic compounds specific to biofilms

Yes

Yes

Yes

from patients with chronic venous ulcers over the course of 5 weeks (Gao et al. 2021). Although temporal fluctuations amongst the measured markers occurred as well as conflicting interpatient differences, some common features were evident amongst all 5 patients, suggesting that the developed multiplexed immunosensor may serve as a beneficial tool in the future. On top of immune signalling molecules, the authors also measured temporal changes in temperature and pH. Both of these markers are commonly measured as they can be quantified using rather cheap and simple instruments. Handheld infrared thermometers are often utilized to measure wound temperatures and there is an increasing amount of documentation which shows that wound temperature and wound infection is closely linked (Dini et al. 2015; Woo and Sibbald 2009; Fierheller and Sibbald 2010). pH is often measured using colour gradients of different pH-sensitive stains. These can be incorporated into fibrous dressings in which a visible colour change can be observed in response to the pH dynamics of the wound (Pan et al. 2019; Tamayol et al. 2016; Vu et al. 2020). Shukla et al. (2014) measured the pH of 50 patients using simple litmus paper strips. They also performed microbial culturing of the wounds and found an association between the detected species and the wound pH. An additional

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microenvironmental change that can be monitored is the presence or absence of oxygen. He et al. (2020) created a smart wound dressing with incorporated methylene blue that turned yellow due to bacterial oxygen depletion. As this colour change is visible to the naked eye, the authors argued that this made real-time monitoring of wound infection possible (He et al. 2020). While all of these indirect markers may indicate whether an infection is present or not, none of them can prove the establishment of a biofilm. Measuring microbial markers is a complicated task due to the large variety of species found in wounds. Very often, a single biological marker will not be present for all species, and although some commonalities exist, sensors that detect microbes or microbial produced compounds are often limited by this fact. Yet attempts are still being made to create sensors that can report on the microbial presence in wounds. Smart wound dressings are being developed that responds to the presence of bacterial toxins, which are commonly utilized as microbial markers. This is often done by encapsulating dyes in lipid vesicles which are broken down in the presence of most bacterial toxins or bacterially derived enzymes, thus releasing the dye and causing a colour change (Thet et al. 2016, 2020; Zhou et al. 2018). It has been argued that these systems only react to pathogens, as commensal bacteria are not known to produce toxins to the same degree. However, some commensal skin bacteria cause extensive infections despite their limited production of toxins, as is the case for S. epidermidis (Otto 2009). Some studies maintain that the production of toxins for certain species is a density regulated action, and can therefore be correlated to the presence of biofilms (Thet et al. 2016). Other species, for example E. faecalis, are known to produce toxins in the presence of target cells, no matter the microbial amount (Coburn et al. 2004). For some species, the production of toxins is believed to be regulated by quorum sensing (QS). However, the role of QS in wounds is not yet established. In fact, one study that analysed the transcriptome of P. aeruginosa obtained from clinical samples found a down-regulation of QS genes (Cornforth et al. 2018). Some sensors are developed to detect specific toxins, as was done in the study by Simoska et al. (2020). In this study, a flexible carbon ultramicroelectrode array was developed that could perform quantitative electrochemical detection of pyocyanin, a toxin produced solely by P. aeruginosa strains. Pyocyanin has previously been found in varying quantities in wound exudate, and in this study pyocyanin amounts in the range of 1–250 lM could be detected. In addition to pyocyanin, the developed sensor was also able to detect uric acid and NO. Uric acid is another microbial marker, which is discussed later on, while NO is a common immune cell signalling molecule, secreted by both PMNs and macrophages in response to wounding. However, in the infectious anoxic environment of chronic wounds, NO is often lacking, and its absence can therefore be used as an infection marker. Jarosova et al. (2019) also created a sensor for detecting pyocyanin but did so using polyacrylamide-coated carbon nanotube electrodes, managing to detect pyocyanin in concentrations as small as 0.1 lM. Their sensor was also developed to detect uric acid.

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Uric acid (UA) is a commonly studied wound marker and high levels of UA have been found in uninfected chronic wounds (Fernandez et al. 2012). The precursor for UA is ATP, which is released in the wound microenvironment when cells rupture. Cell damage caused by inadequate oxygen supply leads to ATP release and subsequent build-up of purine metabolites (Fernandez et al. 2014). The conversion of UA also causes a release of ROS, further sustaining inflammation. Chronic wounds are therefore expected to contain an intrinsic high level of UA, however, in the presence of infectious microbes UA is rapidly metabolised and local levels of UA are decreased. This decrease in UA is therefore used as a biological marker signifying the presence of metabolising microbes and several different types of bandages have been developed to detect UA levels (Sharp et al. 2008; Kassal et al. 2015; Sharifuzzaman et al. 2020). Another microbial marker is lactic acid, which has also been found in increased amounts in infected chronic ulcers (Löffler et al. 2011). Lactate is thought to be produced not only by PMNs during their respiratory burst but also by several microbial species during fermentation (Löffler et al. 2011). Sensors which detect the levels of lactate within a wound have been developed. Ashley et al. (2019) developed a flexible electrochemical biosensor which could detect both lactate and oxygen. The sensor was designed to be able to integrate into wound bandages and in vivo tests of the sensor were reportedly underway. On top of several immune signalling molecules, the multiplexed immunosensor developed by Gao et al. (2021) also contained a channel for detecting S. aureus specifically, as S. aureus is one of the most common wound pathogens. The aptamers in the device were designed to bind to specific epitopes on the cell wall of S. aureus (Ranjbar and Shahrokhian 2018). However, due to inherent strain differences, it can be speculated if the specific aptamer only shows an affinity for certain similar strains of S. aureus as the one used in the experiment in the paper. A different study presented a wound dressing designed to detect the presence of S. aureus DNA (Roy et al. 2021). The dressing was based on a composite of zeolitic imidazolate framework and carbon nitride conjugated with S.aureus probe-DNA. The sensor was tested against human serum and non-complimentary DNA, and based on this the authors concluded that the chosen probe was selective only towards S. aureus DNA. Lastly, sensors that detect biofilm markers such as EPS components are being developed and tested. In 2014, Li et al. developed a gold-particle based multichannel nanosensor that could detect various EPS components, creating different patterns depending on the species which produced the EPS (Li et al. 2014). A multichannel output was created via reversible adsorption and subsequently partial displacement of three distinct fluorescent proteins. The three fluorescent proteins all contained negative surface charges allowing for electrostatic interactions with two cationic functional groups on gold nanoparticles. When presented with negatively charged EPS components, competitive interactions occurred, and distinct patterns were generated based on the specific EPS composition. The multichannel sensor was tested against biofilm produced by five different species and for each species a distinct fluorescent pattern was observed. The sensor was also tested against two different strains of E. coli and once again produced distinct

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fluorescent patterns. Ngernpimai et al. (2017) created a similar multichannel sensor containing three fluorescent poly(oxanorborneneimide) polymers which each contained a cationic recognition element and an environmentally sensitive transducer, selected to create two Förster Resonance Energy Transfer (FRET) partners. When exposed to different biofilms of various species, distinct fluorescent patterns were observed. In the two studies by Li et al. and Ngernpimai et al., both author groups recognized that creating sensors for specific EPS components would be too extensive due to the large variety and combination of EPS produced by different species. Instead, creating multichannel sensors which allow for unspecific EPS detection increases the likelihood that any encountered biofilm could be detected. However, to believe that such multichannel sensors will be able to recognise the specific microbial species responsible for a biofilm infection seems unlikely, as this would require an immense amount of sensor training. Nakagami et al. (2017) used a different and more simple approach to detect wound biofilms, termed wound blotting. A nitrocellulose membrane was pressed to a wound surface to collect biofilm components. The membrane was subsequently stained using ruthenium red which detects the presence of mucopolysaccharides, a component commonly found in biofilm EPS. This wound blotting technique was tested on 23 pressure ulcers and a biofilm-positive wound blotting outcome was found to correlate to increased or unchanged slough production. Wu et al. (2020) performed similar wound blotting but utilised alcian blue staining instead which also stains EPS polysaccharides. Wound blotting has previously been used to show the distribution of TNF-a on pressure wound surfaces and the distinct patterns were found to correlate to the healing process of the wounds (Minematsu et al. 2013). A common issue for all of the EPS sensors and detection methods presented above is their ability to only detect biofilms on the surface of wounds. This issue was averted using the detection method presented in Ashrafi et al. (2018). This method was based on the detection of volatile organic compounds (VOCs) produced by microbial pathogens. The VOCs were measured using gas chromatography coupled to mass spectrometry on ex vivo samples of human skin which had been incubated with bacteria. The authors found that the VOC profiles differed between planktonic and biofilm growing cells, which means that the method could arguably be used to detect biofilm infections specifically. For some species, it was also found that the VOC profiles correlated with metabolic activity and biofilm biomass. VOC profiles have previously been found to differ between chronic wounds and healthy skin in individual patients and have been speculated to relate to the infecting pathogens (Thomas et al. 2010). The VOC profiles in the study by Ashrafi et al. (2018) were also found to differ between different species, and although the authors argued that the profiles could be used to distinguish between infections caused by different species, it remains to be seen if this is clinically feasible.

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Novel Imaging-Based Detection Methods An alternative to sensors is non-invasive, non-destructive imaging techniques (see Table 2 for an overview). Imaging techniques cause little to no interference with the wound and have a high precision when used for repeated and continuous measurements over longer intervals (Li et al. 2020). Particularly approaches that use both photography and digital tracings to create a multidimensional image that not only assesses surface conditions are gaining popularity. One such technique is Table 2 Table presenting the imaging-based detection methods mentioned in this chapter Imaging method

References

Details

Tested in humans or on human samples?

Near-infrared imaging

Dinjaski et al. (2014)

No

Near-infrared imaging

LópezÁlvarez et al. (2022)

Raman Spectroscopy

Bullock et al. (2020)

Surface enhanced Raman spectroscopy Surface enhanced Raman spectroscopy MSI/HSI methods

Bodelón et al. (2016)

Detection of luciferase produced by bioengineered bacteria and ROS. The bacteria were added to implants, placed into mice and then followed using an IVIS platform Detection of a fluorescent tracer composed of vancomycin coupled to a fluorophore. The method was tested on plates and screws extracted from human patients who needed revision surgery following orthopaedic trauma Raman microscopy was used to measure microbe-induced pH changes in tissue-engineered skin Surface Enhanced Raman Spectroscopy was used to detect pyocyanin in vivo in a mouse model

MSI/HSI methods

No

No

No

Nguyen et al. (2018)

Pyocyanin produced by P. aeruginosa in aqueous media was detected using surface enhanced Raman spectroscopy

No

Nouvong et al. (2009)

HSI was used to measure superficial tissue oxyhemoglobin and deoxyhemoglobin at the edge of diabetic ulcers. Healing was found to correlate strongly to tissue oxygenation status HSI was used to investigate infected diabetic ulcers and by comparing the reflectance spectrum of pure bacterial cultures, they managed to discriminate between infections caused by S. aureus and E. coli

Yes

Poosapadi Arjunan et al. (2018)

Yes

(continued)

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Table 2 (continued) Imaging method

References

Details

Tested in humans or on human samples?

MSI/HSI methods

Chang et al. (2018)

Yes

MSI/HSI methods

Herrmann et al. (2020)

MSI/HSI related methods

Raizman et al. (2021)

MSI/HSI related methods Spatial frequency domain imaging

Rennie et al. (2017)

HSI was combined with 3D wound size measurements and thermal profiling. This was employed on 23 patients suffering pressure ulcers HSI was combined with UV excitation and fluorescence profiling from infected diabetic ulcers was recorded MolecuLight i:X technology that detected pyoverdine fluorescence in chronic wounds was linked to biofilm formation MolecuLight i:X technology was used to detect red porphyrin production of bacteria An expansion of an MSI/HSI technique termed spatial frequency domain imaging was used to determine the infection status of rodent burn wounds in situ. The authors could follow blood flow, oxygenation and tissue changes over time X-ray computed tomography was performed in combination with the use of iron sulphate as a contrast agent. The authors were able to distinguish biofilm biomass from the surroundings Positron emission tomography imaging was performed using a radio-labelled antibiotic as the contrast agent and enabled the visualisation of infections in rodents Method not specifically developed for wounds. Ultrasound and ultrasound contrast agents, which bound to biofilm specific ligands were developed. Only tested on S. aureus

Nguyen et al. (2013)

X-ray CT

Carrel et al. (2017)

X-ray PET

Sellmyer et al. (2017)

Ultrasound

Anastasiadis et al. (2014)

Yes

Yes

Yes

No

No

No

No

near-infrared imaging. In Vivo Imaging Systems (IVIS) that use near-infrared imaging in combination with optical imaging are able to visualize bioluminescence and fluorescence signals in live tissue. Near-infrared imaging has the advantage that it can provide high-resolution images deep within a tissue, up to several centimetres (Dang et al. 2019). One study utilised an IVIS system in combination with a bioluminescent strain in a mouse model to follow the establishment of an implant-related infection in vivo (Dinjaski et al. 2014). A different study used an

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IVIS system to detect fracture-related infections following orthopaedic trauma surgery. The authors used vancomycin coupled to a near-infrared fluorophore as an optical tracer, which was found to bind well to biofilms of gram-positive microbes, although the visualisation was only performed on plates and screws that had already been removed from the patients (López-Álvarez et al. 2022). In the future, using biofilm-specific fluorescent compounds in combination with this method could provide a novel non-invasive detection method to visualise biofilms within wounds and other infections, although universal biofilm-specific compounds have yet to be found. Another popular imaging technique is Raman Spectroscopy which detects the energy change of photons when scattering off a material. Raman spectroscopy is highly sensitive and can detect biomolecules in low abundances (Xu et al. 2020). Bullock et al. (2020) used Raman Spectroscopy to detect bacteria-induced pH changes in tissue-engineered human skin. They managed to measure the pH to a depth of 600 um into the skin. Specific microenvironments were observed within the skin in a patchy distribution when the skin had been infected with S. aureus and P. aeruginosa. Interestingly, the authors noted that when they averaged all pH measurements across the infected skin model, the net pH value was not significantly different to a control model. This finding emphasizes how single point pH measurements or average measurements across a wound might overlook pockets of alkalinity occurring due to microbial actions. Surface-Enhanced Raman Scattering spectroscopy (SERS) is an enhanced method of Raman Spectroscopy and can detect even smaller amounts of biomolecules. Nguyen et al. (2018) used SERS to detect pyocyanin produced by P. aeruginosa in aqueous media and Bodelon et al. (2016) detected pyocyanin in vivo in a mouse model. The authors argued that SERS could be used to detect P. aeruginosa infections in very early stages. Multispectral and Hyperspectral Imaging (MSI/HSI) are other imaging techniques which have been used to monitor wound progression. The imaging systems contain the ability to split light into multiple narrow bands, and thereby recognise and differentiate spectrally distinctive materials (Saiko et al. 2020). The method is often used to quantify oxygenation levels in wound tissue, and a few commercial systems developed for this purpose already exist, although more complex HSI systems are also being developed. Nouvong et al. (2009) used HSI to measure superficial tissue oxyhemoglobin and deoxyhemoglobin at the edge of diabetic ulcers from 54 patients. After 24 weeks all wounds were re-evaluated and the rate of healing was found to correlate strongly to tissue oxygenation status. Poosapadi Arjunan et al. (2018) performed HSI on infected diabetic ulcers and by comparing the reflectance spectrum of pure bacterial cultures, they managed to discriminate between infections caused by S. aureus and E. coli. Some studies integrate multispectral and hyperspectral imaging with other imaging technologies. Chang et al. (2018) combined hyperspectral imaging with 3D wound size measurements and thermal profiling and employed this clinically on 23 patients suffering pressure ulcers. Finally, in a study by Herrmann et al. (2020) a hyperspectral imaging system was combined with UV excitation and fluorescence profiling from infected diabetic ulcers was recorded. The authors argued that fluorescent substances naturally

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produced by bacteria would be visible on the wound surface. A commercial device termed MolecuLight i:X has been developed which is based on the same concept as the one presented in Herrmann et al. (2020). This handheld imaging device has been tested to measure different fluorescent biomolecules such as porphyrin and pyoverdine (Raizman et al. 2021; Jones et al. 2020). The creators of the device argue that the amount of fluorescent biomolecules increases when the microbes are in the biofilm mode of growth, hence functioning as a specific biofilm detection device. Yet, care should be taken when measuring fluorescent signals in human tissue, as autofluorescence from tendons, slough and other human tissues can interfere with the bacterial fluorescent signals (Rennie et al. 2019). Moreover, the maximum depth of excitation for UV light has been estimated to be 1 mm in human tissue, hence any bacteria found deeper than this will not be visualised (Jones et al. 2020). Finally, the device has a limit of detection of around 104 CFU/g, as lower amounts than this do not produce enough fluorescent signal to be detected (Rennie et al. 2017). Another expansion of MSI/HSI techniques is Spatial Frequency Domain Imaging (SFDI), which can measure the optical properties over a large field of view with increased depth sensitivity and resolution. SFDI separates and quantifies absorbed and scattered light by imparting a structural pattern to the tissue illumination (Li et al. 2020; Thatcher et al. 2016). Nguyen et al. (2013) used SFDI to determine the infection status of rodent burn wounds in situ. The wounds were imaged daily over a period of 10 days, and by using SFDI the authors could follow blood flow, oxygenation and tissue changes. In the future, a combination of fluorescence profiling together with the depth resolution of SFDI might allow for greater biofilm detection in wounds. This combination of methods is already being investigated for use in cancer surgery (Sibai et al. 2019). Recently, novel types of fluorescent molecules that attempt to target some of the most ubiquitous EPS components have gained a lot of traction. The method is known as optotracing and it is based on conformation-sensitive fluorescent tracer molecules that bind to amyloids, polysaccharides and cell wall glycan strands (Choong et al. 2016; Butina et al. 2020). Optotracers are small anionic fluorescent tracer molecules that interact with their target via electrostatic interactions. For now, the fluorescent molecules have been visualized using standard fluorescent microscopy and spectrophotometric methods, but if the optotracers were to be used in combination with some of the newer visualisation techniques presented here, their use in infection diagnosis could prove very relevant. However, their usefulness relies on the matrix components being expressed in the wounds. While standard X-ray computed tomography scans can determine the presence of infections, specific biofilm detection will require a contrast agent (Xu et al. 2020). Carrel et al. (2017) performed X-ray computed tomography in combination with iron sulphate as the contrast agent and were able to distinguish biofilm biomass from the surrounding porous media, despite the high water content observed in biofilms. An expansion to this is to use X-ray microforce computed tomography instead, which has a higher resolution. Sellmyer et al. (2017) performed positron emission tomography (PET) imaging using a radio-labelled antibiotic as the

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contrast agent and were able to visualise rodent infections caused by several different species. They did however observe a decreased and delayed uptake of the contrast agent in resistant bacterial strains and while labelled antibiotics are often shown to be very specific in targeting microbes, the spread of antibiotic resistance can diminish the use of such tracers. The biofilm detection method presented in Anastasiadis et al. (2014) was not developed for wounds specifically, yet it could easily be adapted to fit this situation. The detection method uses high-frequency acoustic microscopy in conjunction with ultrasound contrast agents (UCAs) developed to target biofilm specific ligands of S. aureus. The authors argue that since ultrasound imaging devices are already readily available in most clinical settings, implementing this detection method should prove fairly easy. Gas microbubbles in combination with ultrasound are already being investigated extensively for their use in the controlled delivery of drugs to infectious biofilms (LuTheryn et al. 2020). While this detection method seems promising, to our knowledge, tests have yet to be performed on humans. Moreover, similar to other detection methods developed, this method focuses solely on S. aureus infections. The development of universal UCAs against biofilm components would surely be valuable in the detection of infectious biofilms.

Discussion The aim of this chapter is to elucidate methods used to detect biofilms in wounds (see Table 3 for a summary). Thus, it is important to address the question: is it relevant to know whether or not there is a biofilm present in a wound? Or rather: could it not just be assumed that a non-healing wound always contains a certain amount of biofilm? The purpose of this book is to address the topic of significance of evidence and technology in the context of wound management. The burden of chronic wounds is enormous as discussed widely in this book: technology is used to generate evidence which, appropriately applied, should permit better wound healing in the context of standardised care, and generate better evidence. Most recent studies find that 80% of all chronic wounds contain biofilms, but this number is often thought to be an underestimation (Malone et al. 2017a). Perhaps many of these detection methods could be used in combination with a biofilm-based wound care treatment in carefully designed studies using a presumptive hypothesis which is that a chronic wound with impaired healing will contain a biofilm: on the contrary a freshly cleansed and debrided wound should be free of biofilms. In this situation, it would be useful to have a fast, non-invasive method to detect if the debridement left the wound free of biofilm. At any rate, sharp debridement guided by an initial biofilm detection could lead to more accurate and efficient biofilm removal without collateral damage to nearby granulation tissue. The same is true for other biofilm-based wound care treatments, as detection methods may have their merits both before and after such treatments.

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Table 3 Summary of the biofilm detection methods presented in this chapter Method based on

References

Sensor-based detection methods pH Gao et al. (2021), Pan et al. (2019), Indirect Tamayol et al. (2016), Vu et al. infection (2020), Shukla et al. (2014), markers Sharifuzzaman et al. (2020) Oxygen (He et al. (2020), Ashley et al. (2019) Temperature Dini et al. (2015), Woo and Sibbald (2009), Gao et al. (2021), Fierheller and Sibbald (2010), Sharifuzzaman et al. (2020) Immune signals Gao et al. (2021), Simoska et al. (2020) Microbe Uric acid Simoska et al. (2020), Jarošová et al. markers (2019), Sharp et al. (2008), Kassal et al. (2015), Sharifuzzaman et al. (2020) Lactic acid Ashley et al. (2019) Cell wall epitopes Gao et al. (2021) DNA Roy et al. (2021) Pyocyanin Simoska et al. (2020), Jarošová et al. (2019) Uncharacterised Thet et al. (2016), (2020), Zhou et al. toxins (2018) Biofilm Uncharacterised EPS Li et al. (2014), Ngernpimai et al. markers components (2017) Polysaccharides and Nakagami et al. (2017), Wu et al. mucopolysaccharides (2020) Volatile Organic Ashrafi et al. (2018) Compounds Imaging-based detection methods Near-infrared imaging and Dinjaski et al. (2014), López-Álvarez related methods et al. (2022) Bullock et al. (2020), Bodelón et al. Raman Spectroscopy and (2016), Nguyen et al. (2018) Surface Enhanced Raman Spectroscopy MSI/HSI and related methods Nouvong et al. (2009), Poosapadi Arjunan et al. (2018), Chang et al. (2018), Herrmann et al. (2020), Raizman et al. (2021), Rennie et al. (2017), Nguyen et al. (2013) X-ray CT and X-ray PET Carrel et al. (2017), Sellmyer et al. (2017) Ultrasound Anastasiadis et al. (2014)

Was the method tested in humans or on human samples? Yes

No Yes

Yes Yes

No Yes No No No No Yes Yes

No No

Yes

No No

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The feasibility of some of the detection methods presented here might prove impractical as they require large and expensive equipment, which can only be operated by specially trained personnel. To expect this to occur at any common hospital is unrealistic, so using detection methods which utilize equipment that is already present, such as ultrasound devices or PET imagers, is more likely to become widespread. Alternatively, cheap and easily created equipment such as paper-based sensors might be more viable for use everywhere. Simple diagnostic tools such as thermometers and litmus paper for measuring wound temperature and alkalinity should also be encouraged as these might help to show early signs of infection. Many sensor-based detection methods including several imaging techniques have limitations with respect to detecting very small biofilms. Yet this is an important feature, as wound aggregates may only be a few micrometres across in diameter (Bjarnsholt et al. 2013). The volume-sensitivity of detection techniques and the spatial resolution of imaging techniques limits their use to detect small aggregates. This issue extends into our next discussion point: even small amounts of bacteria can lead to an infection. This is particularly true in patients who are already immunocompromised or suffer from poor vascularisation and therefore, tissue oxygenation, as is often the case of chronic wound patients (Sen 2019). Detection methods that rely on large quantities of bacteria, or the by-product of large microbial masses, including QS controlled secretions, might fail to diagnose infections in their early stages. Many detection methods rely on the EPS products of biofilms, yet actual investigations of EPS components in vivo are incredibly scarce. Some studies have used imaging techniques to show the presence of EPS and certain EPS compounds in wound samples, but no large studies have been performed which have analysed the EPS components of chronic wound biofilms (Johani et al. 2017; Neut et al. 2011; Oates et al. 2014). Hence, we need to be careful when making assumptions about chronic wounds based on data obtained from in vitro experiments. EPS components produced in abundance in vitro might not be found to the same extent in vivo. There is an ongoing search for universal biofilm markers, yet such markers might not exist, as we have modestly suggested here. It is often cited that only chronic wounds contain biofilms while acute wounds do not (Attinger and Wolcott 2012). Yet, newer studies show that this assumption might not be true. In 2018, Bay et al. found biofilm formations in 67% of acute wounds (Bay et al. 2018), while Schaber et al. (2007) found biofilm formations in 91% of acute wounds in an animal model, and a recent study by Kolpen et al., that compared chronic and acute lung infections, found both biofilm aggregates and single cells in all samples (Kolpen et al. 2022). Based on their finding, Kolpen et al. analysed the growth rates of the aggregates and found that the aggregates isolated from acute lung infections had a higher metabolism than the ones from chronic infections. This finding may cultivate speculations about whether it is not the presence of a biofilm, but rather the growth rate of the bacteria in the biofilm, which causes the main differences between acute and chronic infections. If this is the case, detection methods that infer details in regards to the metabolic status of the

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surveyed microbes might prove more valuable than initially thought. Further speculations can be made in regards to the microenvironment of the wound infection, as it is not yet known whether the presence of a biofilm leads to an unfavourable wound microenvironment or if the unfavourable wound microenvironment promotes the establishment of said biofilm. Hence, detection methods that continuously survey the microenvironmental conditions of the wound, (e.g. temperature, oxygenation and pH) could help prevent the early establishment of an infection, if these conditions are corrected in a timely manner. Several of the detection methods presented here specializes in the detection of a single species. Pyocyanin and pyoverdine, which are detected in the methods presented by Simoska et al. (2020), Jarosova et al. (2019) and Raizman et al. (2021), are only produced by P. aeruginosa. The detection method presented by Gao et al. (2021) contained an aptamer specific to S. aureus and the method in Roy et al. (2021) used an S. aureus specific DNA probe. Arguments can be made that the detection of these two species is highly relevant, as their presence in chronic wounds has previously been linked to a worse healing outcome and even wound enlargement (Madsen et al. 1996; Gjødsbøl et al. 2006; Kalan et al. in press). Yet, their absence does not automatically result in a positive healing outcome, as other species have been found to dominate in some chronic ulcers (Redkar et al. 2000). Hence, detection methods which solely look for a single species, or a certain range of species, must be supplemented with a different detection method in case of a negative result. Yet, specific pathogen identification has its value, especially when a medical physician needs to choose a suitable antibiotic for treatment. The identification of certain resistance genes is also very relevant in this regard, and consequently, molecular techniques, which are already used today, are still incredibly valuable. The same holds true for transcriptional profiles, as they might make implications in regards to the metabolic status of the infectious pathogens. It has previously been shown how certain antibiotics may have a limited effect on metabolically inactive microbes, thus knowing the activity status of the infectious microbes might be equally relevant to the treating physician (Liu et al. 2020). Some studies make distinctions between commensals and pathogens, yet this distinction seems dangerous when it is known that commensals in a new environment may act unpredictably (Otto 2009). This is particularly important to consider when using detection methods that rely on the production of toxins, as toxin production is not a guarantee in all infections. In conclusion, when a patient develops a chronic wound it should be investigated using a well-rounded, holistic approach which includes immune cell signals, microenvironmental factors such as pH, temperature and oxygenation, but also various microbial factors such as species, microbial amount, EPS components, metabolic rates, toxins, transcriptomes and resistance profiles. A single detection method can not be expected to fulfil all these requirements, hence the development of a range of different detection methods with different angles, based on a variety of biological markers is likely to be highly beneficial, though it may be costly and complicated. More importantly, before we continue the development of different detection methods, it would be more useful to first expand on our knowledge in

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regards to the chronic wound microbiome. Once we understand the role of both biofilms and single cells in the wound, and once we have established the presence and function of various EPS components, and determined how and if these components affect the healing process, we can then turn to develop adequate methods to help us detect the most important factors for wound healing. Improved understanding of microbial infections in chronic wounds is essential.

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Update on Technology and Evidence-Based Management of Scars Luc Téot, Hester Colboc, and Sylvie Meaume

Abstract

Scars form a medical problem still considered as a cosmetic issue by most of the societies and poorly covered except for burns and trauma. The classification established in 2002 (Mustoe et al. 2002) separates 6 classes of scars, from immature scar to large spreading keloidas. POSAS scale is now considered as one of the most valuable tool concerning objective assessment, taking in account the perception and the psychological consequences observed by the patient him (her)self and the surrounding actors. Amo ng the 30 Millions of surgical procedures realized every year in Europe, 1–6% lead to postoperative complications like surgical site infection, largely impacting the scar quality. The development of hypertrophic scars depends on the age, the anatomical location and its specific mechanical properties (the face is not submitted to the same mechanical forces than the back), the origin of the lesion (burns, post-op local infection,…) and the compliance of the patient to the proposed treatments. Scar management drastically changed during the last decade. Thanks to a large transdisciplinary approach, new technologies emerged. These technologies are mainly based on early mechanotherapy, antiproliferative drugs and lasers. Keywords

Pathological scarring


Mechanotherapy
Lasers
Prevention

L. Téot (&)
H. Colboc
S. Meaume Montpellier University Hospital, Montpellier, France e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. Mani (ed.), Chronic Wound Management, https://doi.org/10.1007/978-3-031-26110-7_11

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Introduction Scars form a medical problem still considered as a cosmetic issue by most of the societies and poorly covered except for burns and trauma. The classification established in 2002 (Mustoe et al. 2002) separates 6 classes of scars (Table 1), from immature scar to large spreading keloids. POSAS scale is now considered as one of the most valuable tool concerning objective assessment, taking in account the perception and the psychological consequences observed by the patient him(her)self and the surrounding actors. More than 30 Millions of surgical procedures are realized every year in Europe, leading to a variable rate of postoperative complications (between 1 and 6%) impacting the scar quality. the development of hypertrophic scars is variable, depending on the age, the anatomical location and the compliance of the patient to the proposed treatments. Scar management drastically changed during the last decade. Thanks to a large transdisciplinary approach, some important principles are now confirmed and new technologies emerged. These Table 1 Classification of scars (Mustoe et al. 2002)

A Normal scar is flat and pale without itching and not enlarged An Immature scar, frequently observed in children is a mild transitory rise in height of the scar, becoming red, sometimes itching, presenting a slight augmentation of the density. This scar will normalize after a variable period of time, reaching 18 months–2 years in some patients Atrophic scars can develop by a combined separation of the dermal edge with preservation of epidermis continuity. These scars are often observed in adolescents after skin resection for benign tumors on the back An Hypertrophic scar is a scar proliferation staying into the limits of the edges of the initial scar, with a progression starting one month after the complete healing, growing for five to six months and presenting a plateau with a slow decrease of the inflammatory signs, issuing after one year (sometimes more) to a stable scar having lost its inflammatory signs. It may be linear after a surgical suture or wide spreading after burns. They are usually red, itching and raised in height Keloids are characterized by a pseudo-tumor proliferation extending over the edges of the initial wound and keep growing along time, some of them reaching high volumes. They are more observed over the thorax, on the ear lobes and the neck Skin contractures are frequent in burns, with bands of hypertrophic retractile scars limitating the joint movement. Their mechanical force is strong enough, when not correctly managed, to impact the growth plates of diaphyseal bones, issuing to permanent deformities of limbs along the children growth Self inflicted scars are usually linear and located on the anterior part of the forearms. These situations are observed in psychologically affected adolescent patients

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technologies are mainly based on mecanotherapy, antiproliferative drugs and lasers. Most of these therapies are used with a lack of firm randomized controlled clinical trials to support their efficacy, and there has been often a lack of appropriate labelling or classification of scars to allow optimal evaluation of existing literature. Scars change over time so that improvements may not necessarily be due to the treatment intervention but simply to scar maturation.

Biological Resume of Events Occurring After a Skin Injury After injury the sequence of events associate platelet aggregation, provisional fibrin matrix followed by influx of inflammatory cells, and subsequent cell proliferation including fibroplasia and angiogenesis. After the third day, matrix collagen deposition begins. Maximal collagen deposition occurs in the first few weeks with a combination of Type 1 and Type 3, followed by many months of collagen breakdown and synthesis with increasing type 1 collagen with increased organization and scar strength. Once the wound is covered by keratinocytes a cellular apoptosis occurs, with a resolution of inflammation. These phases of inflammation, cell proliferation, and collagen remodeling results, for a surgical incision, to a fine line scar or “normal” scar, and in case of broader injury a flat scar. Hypertrophic scars may be a consequence of bad transmission of signaling between keratinotcytes and the underlying layer of proliferating fibroblasts (Andrews et al. 2016). Keloids occur with a genetic predisposition, and are formed of anarchic layers of collagen deposition.

Factors Impacting Scars Several factors may impact on the scar occurrence and evolution, the age of onset the extent on the body and the anatomical location.

The Young Age Scarring will follow the hormonal sequences accompanying the child growth, with peaks occurring between 3 and 7 years, and a second period of intense activity around the puberty. Scars will stay inflammatory for long periods of time, more than 2 years, imposing long periods of care to prevent hypertrophic scarring. Between 70 and 85% of burns scars become hypertrophic in children. In the first years of life the psychological consequences are more on the surrounding parents, and during puberty the consequences may be devastating, especially if visible areas are concerned, like the face or the upper limbs.

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Technologies should be adapted to the child with less painful treatments and more delayed surgical scar revisions when possible.

The Elderly Traumatic and surgical acute wounds healing is rather good even in very old patients and hypertrophic or keloid scars are rare, at least in the Caucasian population (Monarca et al. 2012). The poor quality of the scar is usually not a major problem (appearance, color, shape) (Brands-Appeldoorn et al. 2018) except in exposed regions (face) or when impacting the function (heel, eyelid and peri-orificial areas). Pruritus and/or pain may appear very lately after the trauma/surgery and occur after 20 or 30 years. They are linked to dermatological problems (dry skin) or neurogenic disorders. These situations may be treated symptomatically with appropriate cosmetics (Humbert et al. 2016). A few of them need surgery, but war, post-traumatic post surgical scars are not usually reasons to go to see a doctor. Atrophic and adherent cars can be improved by injecting fat under the scar, a recent technique presenting the advantage of being minimally invasive when anticoagulation is not needed or should be stopped.

The Reappearance of a Wound on a Scar The recurrence of a tumor may be at the origin of the scar. A biopsy or the recurrence of a chronic ulcer (arterial or venous, pressure, or diabetic foot ulcers) is needed to diagnose a malignant transformation. It can also lead to the reassessment of the patient and indication of preventive treatment of compression or discharge (cushion, shoe, soles). Unstable scars may appear because of their location, sometimes due to a poor quality of the dermal component (absent or fibrotic) or insufficient preventive measures taken against aggressive external agents (shoes, stockings, bandages, prosthesis ...) (Figs. 1 and 2). Post irradiation scars in cancer treatments (breasts for example) poses the problem of radiodermatitis and radionecrosis which evolves and worsens over time, in particular in the elderly who were irradiated at a time when the administered doses were high. Some scars from childhood link to operated orthopedic malformations are associated with joint deformations of osteoarthritis because of mechanical forces pressure or friction exerted on those scars or because of underlying medical problems in the region: arterial disease or neuropathy.

Update on Technology and Evidence-Based Management of Scars Fig. 1 Pressure ulcer

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Fig. 2 Post surgical wound

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Consequence of poor quality of dermal component (fibrotic) Succession of closure and reoppening phase More and more complicated to treat and to obtain total wound closure Should be biopsied if persistance

Location : On the back beetween the two shoulders • • •

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Melanoma excision Excessive tension Poor quality of dermal component Poor preventive measure taken against external agressive agent (taxi driver) Succession of healed and evolutive stage (biopsy to detect melanoma recurrence)

Degenerescence/Marjolin’s Ulcer A rare and aggressive skin cancer, develops late on scars from burns, or from delayed wound healing problems: chronic osteitis, burns, pressure ulcers, lupus scar, skin graft, radiodermatitis. Squamous cell carcinoma are more frequently observed than basal cell carcinoma, melanoma or sarcoma. The occurrence of a wound on a scar aged of more than 20 years always requires a biopsy (Cruickshank and Gaskele 1963; Yu et al. 2013).

Hyperkeratosis Hyperkeratosis is common especially on scars on plantar aspect of the foot, the heel, next to the Achilles tendon or on the toes and the lateral aspects of the foot. Due to a thickening of the stratum corneum reaction to friction or to mechanical conflict, especially in case of loss of the sensibility (diabetes, nerve damage). The cracks can appear and constitute entry doors exposing to the risk of deep infection. These wounds finally closed after a succession of closure and multiple re-openings, source of discomfort and risk of cancer transformation. The treatment of these unstable scars become more and more complex due to underlying diseases and comorbidities. Biopsies often need to be done to rule out a malignant degenerescence.

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Adherence to the depth is a problem that worsens with age. A long term healing occurring on a wound with a loss of deep substance (dermis, fat, gliding capacities) and not correctly repaired thanks to negative pressure therapy, skin substitute or flap may lead to an atrophic scar adherent to the deep plans, creating of a fixed point, disrupting normal skin movements, in particular at the junction between the edges of the skin flap and normal skin. These adherences expose to skin reopening due to the loss of elasticity of age-related skin. Generic principles of scar management depending on the time of onset after healing: • At one month local signs are minor and formed by a scar redness, itching and a mild elevation. These signs should be better defined and included in the training program of any nurse and doctor, especially surgeons (any kind) for a precise assessment of the scar during their first post op clinic after suturing or skin grafting. Silicone can be applied locally either as a sheet to be maintained locally by clothes or bandages, or using gels to regularly apply over exposed scars or even sprays easier to use and reapplied during the day. Some authors recommend a systematic use of silicone sprays after trauma or surgery. Starting early the silicone application may limit redness and mild elevation. The mechanism of action of silicone therapy on scar maturation has not been completely elucidated but a growing body of evidence indicates that the beneficial effects of silicone gel is decreased water evaporation of skin and therefore increased hydration of the stratum corneum. After application of silicone gel or cream in combination with an occlusive dressing, a silicone film forms which may explain a comparable effect on water loss and hydration of the upper layers of the epidermis (Mustoe 2008). Limit or suppress sun exposure should be the rule, even anti UV creams cannot prevent the local inflammation induced by sun exposure. The use of hydrocolloid dressings has been proposed by some Burns teams, their self adhesion capacity and suppleness during movements making them comfortable and easy to wear for the child. They prevent sun exposure and maintain a local adapted level of humidity, prone to decrease local inflammation. • At three months the scar is red, itching and mildlyelevated silicone application is recommended and, depending on the extent of the scar and the degree of inflammation, complementary techniques can be proposed. Local compression using compressive garments are used and realized at fashion. Compression forces should not exceed 25 mm Hg, measurements of the body segments (skull, upper arm, trunk, lower arms) being taken by specialists on special paper sheets and sent to the fabric. Different companies may provide this service around the world. These garments are coated with silicone. They should be redone 4 times a year during the child growth. They are considered as mandatory in all post

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burns extended scars in children and reimbursed in some countries (Anzarut et al. 2009). Plastic Orlen based may be needed in case of severe retraction, imposing uncomfortable positions to the children. Corticosteroid injections are not recommended in children, even if the cortisone is injected and stays locally, the pain induced by successive picking being intolerable for most of the children even if nitrous oxide may be used in children over the age. The prolonged use of hydrocolloid dressings can be proposed for a long period of time, even if some local allergic reactions were reported. • At six months the scar evolution may be stabilised, or not. Depending on the local signs of persisting inflammation, the local treatment is maintained, more or less well accepted by the child. In adolescent burn patients, wearing compressive garments is difficult. In some patients refusing any active local treatment a psychological management may help to find a compromise helping them to temporarily hide the scar zone. • At one year, when local inflammation signs slowed down or completely disappeared, a surgical treatment can be proposed. In children who do not accept compressive garments or painful injections, a surgical approach can be proposed, either reducing the scar surface by serial excision or replacing the scarred area by a flap. Other techniques like the use of dermal substitutes, or overgrafting has been proposed (Kim et al. 2019; Hori et al. 2016).

Scar Assessment Scales Several scar assessment scales were developed since the first description called the Vancouver scale in 1990 (Baryza and Baryza 1995). The POSAS scale (Draaijers et al. 2004) introduced a new method of analysis taking care of the perception of the patient, and other scales were further proposed (Lee et al. 2020; Busche et al. 2018; Simons et al. 2019).

Non-surgical Technologies Silicone Compressive garments Injectables cortisone 5-FU and Bleomycin

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Lasers Led and photobiomodulation Depigmentation lasers Dye lasers Non-surgical Technologies

Intra-Lesional Steroid Injections Intra-lesional injection of corticosteroid (alone or with other agents) is one of the commonest treatment methods for hypertrophic scars and keloids. They are injected each four to six weeks as insoluble triamcinolone acetonide (0–40 mg/mL) until pain, scar and pruritis subside. The response of patients to the treatment ranges from 50 to 100% while the recurrence rate ranges from 9 to 50%. However, 63% presents with complications like localized ulceration, dermal atrophy and telangiectasia or hypopigmentation. Noteworthy, pain might be managed through applying a local anaesthetic. Corticosteroids alone represent the most efficient for young keloids, while older keloids are further resistant (Oliveira and Gold 2020).

Silicone Gel/Sheet Using silicone gel/sheets is assumed to diminish mobility and decrease the scar tension. How silicone gel works is uncertain, however, it may serve as an impermeable membrane to preserve hydration of the skin. De Oliveira and colleagues, (O’Brien 2006) found no statistically significant difference in the symptoms or size of scar by taking any of the treatments; however, both scar types were combined in their analysis, and the follow up period was