Orthopedic biomaterials : from materials science to clinical applications 9783035700374, 3035700370

Table of contents :
Content: ""Orthopedic Biomaterials --
From Materials Science to Clinical Applications""
""Preface""
""Table of Contents""
""Chapter 1: Current Strategies and Advanced Biomaterials for Bone Repair and Regeneration""
""Advanced Microstructural Characterizations of Some Biomaterials and Scaffolds for Regenerative Orthopaedics""
""Magnetic Nanoparticles Inclusion into Scaffolds Based on Calcium Phosphates and Biopolymers for Bone Regeneration""
""Current Strategies and Advances Materials for the Treatment of Injured Meniscus""
""Acrylic Bone Cements: New Insight and Future Perspective"" ""Results of In Vivo Biological Tests Performed on a Mg-0.8Ca Alloy""""Fractographic Evaluation of the Metallic Materials for Medical Applications""
""Chapter 2: Evaluation of the Clinical Performance of Different Options and Methods of Orthopedic Treatment and Implantation""
""Metallosis --
Literature Review and Particular Cases Presentation""
""Use of Collagen Scaffolds in Conjunction with NPWT for the Care of Complex Wounds: Clinical Report""
""Bioabsorbable Anchors for Medial Patellofemoral Ligament Reconstruction"" ""The Use of the Ligament Augmentation and Reconstruction System (LARS) in Clinical Practice""""Functional Outcomes after Surgical Treatment of Hand Fractures --
ORIF vs. CRIF Analysis""
""Short Term Behaviour of Fixed-Loop Cortical Suspension System Used for Surgical Treatment of Hallux valgus""
""Advantages of Modular Radial Head Prosthesis Use in Highly Comminuted Fractures""
""Keyword Index""

Citation preview

Orthopedic Biomaterials - From Materials Science to Clinical Applications

Edited by Iulian Antoniac Cirstoiu Catalin

Orthopedic Biomaterials - From Materials Science to Clinical Applications

Special topic volume with invited peer reviewed papers only

Edited by

Iulian Antoniac and Cirstoiu Catalin

Copyright  2017 Trans Tech Publications Ltd, Switzerland All rights reserved. No part of the contents of this publication may be reproduced or transmitted in any form or by any means without the written permission of the publisher. Trans Tech Publications Ltd Reinhardstrasse 18 8008 Zurich Switzerland http://www.scientific.net

Volume 745 of Key Engineering Materials ISSN print 1013-9826 ISSN cd 1662-9809 ISSN web 1662-9795

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Preface The goal of this special issue is to provide material scientists and orthopedic surgeons with an appreciation of the fundamental aspects of orthopedic biomaterials from materials science point of view, as well as their clinical applications in orthopedic surgery. There is a wide range of biomaterials used for different implants and prostheses used in orthopedic surgery, from metallic alloys to ceramics and from polymers to hybrid composites. Functional properties play an important role in discussions about their clinical applications and have a significant role in the evaluation of the clinical performance for different orthopedic implants and articular prostheses. This special issue is focused also on the current strategies and advanced biomaterials for bone repair and regeneration. Based on the requirements of the modern biomedical technology, the novel research strategies in orthopedic biomaterials field are nowadays directed toward biomaterials endowed with surface properties, controlled adhesion, and for the controlled release of active principles, especially against infections. In this view, several research groups and clinicians have been invited to contribute to this special issue with their original research papers that could stimulate efforts of comprehensive knowledge of orthopedic biomaterials-from materials science to clinical applications. This special issue is divided into two categories based on the following keywords: advanced orthopedic biomaterials and clinical performance for different orthopedic methods and implants. In the category of orthopedic biomaterials, metallic biomaterials have attracted a significant attention being widely used in manufacturing orthopedic implants. Biodegradable metals like magnesium-based alloys appear to be a new paradigm in orthopedic materials and this subject are discussed into a very interesting paper about the in vivo testing of these novel metallic biomaterials. Also, fractographic evaluation of the metallic materials for medical applications was a theme discussed into a paper from the first part of this special issue. Advanced microstructural characterizations of some biomaterials and scaffolds for regenerative orthopaedics represent another new hot topic for development new biomaterials and applications for orthopedic surgery. Different biomaterials are used now for manufacturing the scaffolds for bone tissue regeneration. According to this hot topic worldwide, this issue presents not just a general view about scaffolds requirements but also some experimental results about the magnetic nanoparticles inclusion into scaffolds based on calcium phosphates and biopolymers for bone regeneration. New insight and future perspective about acrylic bone cements are still interesting topic both for clinicians and for material scientists because new challenges appear today resulted from clinical evaluation of these materials. And specialists in materials science must develop and propose new bone cements, with improved properties like antibacterial effect. In the part dedicated to the evaluation of the clinical performance for different orthopedic implants, different authors present their perspective and clinical results for a large variety of the orthopedic implants from total hip prosthesis and modular radial head prosthesis, to the bioresorbable anchor for medial patellofemural ligament reconstruction and Ligament Augmentation and Reconstruction System in clinical practice. Also, the surgical treatment of hand fractures or the systems used for surgical treatment of hallux valgus was evaluated. Anyway, the continuous analysis and monitoring of the clinical results obtained after the use of the orthopedic implants and prosthesis represent clearly a good way to establish new research directions and develop new solutions for the future orthopedic biomaterials. Authors prove that the orthopedic biomaterials play not just an important role in the functionality of the orthopedic implants and prostheses but also in their effects on the human tissues. By collecting these papers, we hope to enrich our readers and researchers in the field of

orthopedic biomaterials and their clinical applications. We believe that novel orthopedic biomaterials and scaffolds for bone tissue regeneration, together with the improved understanding of the interface between biomaterials and adjacent tissue, will be an important part of future orthopedic biomaterials and implants with better functionality in orthopedic surgery.

Acknowledgment I would like to thank all the authors participating in the present Special Issue: Orthopedic Biomaterials: from Materials Science to Clinical Applications. Iulian Antoniac Materials Science and Engineering Faculty, Biomaterials Group, University Politehnica of Bucharest, Bucharest, Romania [email protected]

Table of Contents Preface

Chapter 1: Current Strategies and Advanced Biomaterials for Bone Repair and Regeneration Advanced Microstructural Characterizations of Some Biomaterials and Scaffolds for Regenerative Orthopaedics F. Fiori, E. Girardin, V.S. Komlev, A. Manescu and F. Rustichelli Magnetic Nanoparticles Inclusion into Scaffolds Based on Calcium Phosphates and Biopolymers for Bone Regeneration F.D. Ivan, V. Balan, M. Butnaru, I.M. Popa and L. Verestiuc Current Strategies and Advances Materials for the Treatment of Injured Meniscus I.B. Codorean, S. Tanase, E. Cernat, F. Diaconu, D. Popescu, A. Cirlan and S. Mitulescu Acrylic Bone Cements: New Insight and Future Perspective S. Cavalu Results of In Vivo Biological Tests Performed on a Mg-0.8Ca Alloy R. Adam, H. Orban, E. Plopeanu, D. Voinescu and A. Barbilian Fractographic Evaluation of the Metallic Materials for Medical Applications B. Ghiban, F.C. Varlan, M. Niculescu and D. Voinescu

3 16 26 39 50 62

Chapter 2: Evaluation of the Clinical Performance of Different Options and Methods of Orthopedic Treatment and Implantation Metallosis - Literature Review and Particular Cases Presentation R. Marinescu, C. Socoliuc, I. Botezatu, D. Laptoiu and D. Voinescu Use of Collagen Scaffolds in Conjunction with NPWT for the Care of Complex Wounds: Clinical Report I. Cristescu, D. Vilcioiu, F. Safta, M. Istodorescu, C. Milea, F. Fiori, I. Mates and I. Lascar Bioabsorbable Anchors for Medial Patellofemoral Ligament Reconstruction R. Ene, Z. Panti, M. Nica, M. Pleniceanu, P. Ene, M. Cîrstoiu and C. Cirstoiu The Use of the Ligament Augmentation and Reconstruction System (LARS) in Clinical Practice I.B. Codorean, S. Tanase, E. Cernat, F. Diaconu and D. Popescu Functional Outcomes after Surgical Treatment of Hand Fractures - ORIF vs. CRIF Analysis I. Cristescu, D. Vilcioiu, L. Mirea, C. Milea, I. Mates and A. Mohan Short Term Behaviour of Fixed-Loop Cortical Suspension System Used for Surgical Treatment of Hallux valgus R. Ene, M. Nica, Z. Panti, M. Pleniceanu, P. Ene, M. Cîrstoiu and C. Cirstoiu Advantages of Modular Radial Head Prosthesis Use in Highly Comminuted Fractures M. Stănescu, A.B. Popescu and Z. Radu

77 91 101 111 124 134 145

CHAPTER 1: Current Strategies and Advanced Biomaterials for Bone Repair and Regeneration

Key Engineering Materials ISSN: 1662-9795, Vol. 745, pp 3-15 doi:10.4028/www.scientific.net/KEM.745.3 © 2017 Trans Tech Publications, Switzerland

Submitted: 2017-03-10 Revised: 2017-03-17 Accepted: 2017-03-17

Advanced Microstructural Characterizations of Some Biomaterials and Scaffolds for Regenerative Orthopaedics Fabrizio Fiori1,2,*, Emmanuelle Girardin1, Vladimir S. Komlev3, Adrian Manescu1, Franco Rustichelli1,2 1

Università Politecnica delle Marche, DiSCO - Sezione di Biochimica, Biologia e Fisica, 60131 Ancona, Italy 2 3

INBB - Istituto Nazionale Biostrutture e Biosistemi, Italy

Baikov Institute of Metallurgy and Materials Science, Moscow, Russia *corresponding author: [email protected]

Keywords: scaffolds, hydroxyapatite, bioglasses, synchrotron radiation, microtomography.

Abstract. In the last decades, very significant advances have been made for what concerns bone and joint substitution and in the repair and regeneration of bone defects. Though some strong requirements are still to be met, biomaterials for these purposes have known an impressive evolution, for what concerns their mechanical behaviour, their bioresorbability and finally their capability to generate new bone tissue in a stable way in long periods. The validation of such materials necessarily depends on a suitable characterization of their properties. In this article a brief review of some works in this field, carried out by the authors’ research group, is presented. It was shown in particular how advanced experimental methods, such as synchrotron radiation µCT and synchrotron radiation diffraction can offer very important information, can be not only complementary methods to more standard techniques (electron microscopy, X-ray diffraction), but can also offer the possibility to measure parameters that cannot be obtained otherwise. Introduction Orthopaedic biomaterials are meant to be implanted in the human body as constituents of devices that are designed to perform certain biological functions, by substituting or repairing different tissues such as bone, cartilage or ligaments and tendons, and even by guiding bone repair when necessary. Since the human body consists of a highly corrosive environment, very stringent requirements are imposed on the material properties. In particular, they are required to be as inert as possible in order to reduce their corrosion and their release of ions and particles after implantation. Mechanical properties also play a leading role in the selection of candidate materials for implant manufacture. Furthermore the concept of biocompatibility, associated with a set of in vitro kland in vivo standardized tests, was introduced in order to assess the biological behaviour of synthetic materials. Hydroxyapatite (HA) coating on medical implants has been used in commercial applications for several decades [1–3]. The coatings, commercially made by plasma-sprayed method, function as an intermediate layer between human tissues and the metal implant. However, the long-term stability of the plasma-sprayed HA on metal substrate has been questioned [4]. There are two major shortcomings associated with plasma-sprayed HA coatings. First, due to the high temperature plasma, a large fraction of crystalline HA turns amorphous. This phase is soluble in body fluids and results in subsequent dissolution of the material. Second, the poor mechanical property between the coating layer and titanium implant has been the point of potential weakness in prosthesis [5]. Mechanical evaluation of plasma-sprayed bio-active HA coatings on bio-inert metallic substrate have brought worldwide attention in both orthopedic and dental applications where the demands of operational stresses of the coatings are stringently required. To bridge the gap in HA coatings having poor mechanical properties, bio-ceramic composite coatings are being developed [6–8]. However, poor adhesion strength may lead to wear debris accumulation, which results in an adverse cellular response leading to inflammation, release of damaged enzymes, bone cells lysis, osteolysis

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and implant loosening [9]. On the other hand, a suitable method of improving HA coating properties is based on the nanostructure fabrication in thermal sprayed coatings, retaining nanostructures from starting nanophase/nanostructured feedstock and spray forming nanostructures owing to the rapid cooling of the droplets upon their impingement [1]. In order to best mimic bone tissue in bone replacement/repair, materials exhibiting high porosity (scaffolds) are being developed. The scaffolds used in tissue engineering, also in combination with mesenchymal stem/progenitor cells (MSC), should have 3D interconnected porosity and appropriate pore size and morphology to allow cell migration, fluid circulation and cell proliferation. Moreover, the scaffold surface should favour cell attachment, growth and differentiation [10]. Other requirements concern osteoconductivity, biocompatibility and appropriate mechanical properties to allow optimal integration with the surrounding tissue [11]. If the scaffolds are biodegradable, their degradation products should not be toxic and should not provoke an adverse immune response [12]. Among the many biomaterials developed for bone tissue scaffolds, composite materials containing both inorganic (glass, ceramic or glass–ceramic) and organic (biocompatible polymer) phases are considered an optimal approach [12,13]. The bioactive ceramic phase is brittle but has good compressive strength as well as good bone-bonding ability. The polymeric phase confers the toughness and plasticity. Therefore, by combining a bioactive ceramic phase with a polymeric one, composite materials with high mechanical properties and good biological behaviour can be obtained [12-14]. The ultimate efficiency of an artificial bone construct also depends on exchange of oxygen and nutrients from blood vessels to the MSC, and removal of waste products. Therefore, the control of angiogenesis as a microvascular network with properly structured spatial organization is crucial to any attempts to obtain bone regeneration/repair by tissue engineering. In this article a brief review of characterization studies on selected biomaterials is presented. A hydroxyapatite coating from nanostructured granules was investigated with different techniques, in order to determine the cristallinity grade and the mean size of original granules (X-ray diffraction – XRD and Scanning Electron Microscopy – SEM) and the residual stresses at the interface between the coating and the Ti alloy substrate (synchrotron radiation XRD) [15]. Another work concerned the use of synchrotron radiation X-ray microtomography (µCT) to investigate bioactive glass–ceramic scaffolds [16]. In particular, µCT was used to study the new phase 3-D distribution in the bulk material and its evolution as a function of the soaking time in a simulated body fluid (SBF) and Tris–HCl media. µCT was also used, together with other techniques, to asses the in vitro biocompatibility of glass–ceramic scaffolds based on 45S5 Bioglass©, using a human osteosarcoma cell line (HOS-TE85) [17]. Two different types of scaffolds with different porosities were analysed, coated with a biodegradable polymer, poly(3-hydroxybutyrate) (P(3HB)). The scaffold bioactivity was evaluated by soaking in a SBF for different durations. Finally, the microvascularization in bioceramic scaffold, implanted with MSC and subsequently transferred into the animal model, was evaluated 24 days after transplantation using a holotomography-like technique based on phasecontrast µCT [18]. The results showed that this method is able to visualize the new vessels formed inside the scaffold, and to give quantitative information about them, which is not achievable by standard absorption µCT. Plasma sprayed hydroxyapatite coatings from nanostructured granules Nanostructured HA powders were synthetized in a gelatine solution at 10 °C, as described in previous paper [15]. Subsequently the nanostructured HA particles were formed into porous granules by a method based on a liquid immiscibility effect using HA/gelatin suspension and oil as the liquid [19]. The SEM image of the spherical granules obtained from the HA powder are shown in Fig. 1.

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Fig. 1 SEM micrograph of the fabricated HA granules. The grain size after heat treatment at 900 °C was about 100 nm (Fig. 2A). When the green compacts were sintered at 1200 °C (Fig. 2B), the grain size was increased to a value close to 1 µm.

Fig. 2 SEM micrographs of the HA granules sintered at (A) 900°C and (B) 1200°C. XRD analysis (Fig. 3) did not show any decomposition up to 1200 °C. The granules with diameter 80 µm, synthesized at the gelatine concentration 2.8 g/L and heat treated at 900 °C, were used for coating experiments. The powder was sprayed onto a commercial Ti-6Al-4V alloy substrate, using a plasma spray system, up to a coating thickness was in the range 50-100 µm. XRD also showed that all the HA coating is fully crystalline phase. The influence of the coating thickness on the residual stress at the coating-substrate interface was also evaluated. Measurements were performed by synchrotron radiation XRD at the BESSY laboratory in Berlin, using the “sin2 ψ” method [20].

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Fig. 3 XRD of the HA granules sintered at various temperatures. As shown in Fig. 4, the residual stresses for thinner HA coatings are higher than those in the thicker coatings, probably due to a higher level of porosity and microcracks in the investigated samples.

Fig. 4 Residual stresses in the coating for three different HA thicknesses: (A) 100 µm, (B) 80 µm and (C) 50 µm. 3-D bioactive glass–ceramic scaffolds The aim of this study was to characterize bioactive and bioresorbable glass–ceramic scaffolds [16]. In particular, microarchitectural parameters were evaluated by µCT. µCT is similar to conventional CT systems usually employed in medical diagnostics. Unlike these systems, which typically have a maximum spatial resolution of about 0.5 mm, advanced µCT can achieve a spatial resolution up to 0.3 µm. Such a high spatial resolution can be obtained only for samples of reduced size, in the range of a few cubic millimeters. Synchrotron radiation allows quantitative high spatial resolution images to be generated with high signal-to-noise ratio [21,22]. Use of synchrotron X-rays has several advantages compared to laboratory or industrial X-ray sources. These include:

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a high photon flux, which permits measurements with a high spatial resolution in short acquisition times; a tunable X-ray source, allowing measurements at different energies; a monochromatic X-ray radiation, which eliminates beam hardening effects; a parallel beam geometry, allowing the use of exact tomographic reconstruction algorithms; the possibility of using different sample-to-detector distances, thus exploiting phase-contrast and holotomography options [18].

The experimental set-up of a typical micro-CT instrument is shown in Fig. 5.

Fig. 5 Schematic set-up of a micro-CT system. According to the description made in a previous paper [16], an accurate analysis of the scaffold 3-D structure was performed on CEL2 glass–ceramic, belonging to the SiO2-P2O5-CaOMgO-Na2O-K2O system, as effective biomaterial for scaffolding. In particular, µCT analysis was used to study the new phase 3-D distribution in the bulk material and its evolution as a function of the soaking time in a SBF and Tris–HCl media. Specifically, the study in SBF is useful to analyse the in vitro bioactivity of the CEL2 scaffolds, whereas the study in Tris solution gives information about the sample bioresorbability. Fig.6 and 7 show the scaffold morphology reconstructed by µCT, performed at ELETTRA, Trieste. The different X-ray absorption properties of the scaffold and the new HA layers grown on the sample surface permits their visualization in different colours.

Fig. 6 Three-dimensional reconstruction of scaffolds subvolume (a) before treatment and after treatment in a SBF for (b) 1 week and (c) 4 weeks. The scaffold is in green and the new phase is in blue.

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Fig. 7 Three-dimensional reconstruction of scaffolds subvolume (a) before treatment and after treatment in a buffered medium (Tris) for (b) 1 week and (c) 4 weeks The scaffold is in green and the new phase is in blue. It is also possible to extract some significant quantitative parameters from the µCT images, such as the scaffold porosity: both before and after soaking in Tris/SBF the scaffolds exhibit a bimodal porous structure (Fig. 8), where both macropores, (100 - 500 µm), necessary for the growth of new bone and the vascularisation of the implant, and micropores (1–100 µm), important for cell adhesion and proliferation.

Fig. 8 3-D pore network showing the bimodal porous structure of the scaffold. The mean thickness of the newly formed HA in SBF is shown in Fig. 9, while the bioresorption of the scaffold material soaked in Tris is evaluated through the observation of the reduction of the scaffold wall mean thickness (Fig. 10).

Fig. 9 Evolution of the new HA thickness as a function of soaking time in SBF.

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Fig. 10 Evolution of the scaffold wall thickness as a function of soaking time in Tris. In vitro biocompatibility of 45S5 Bioglass©-derived glass–ceramic scaffolds coated with poly(3-hydroxybutyrate) Among the many biomaterials developed for bone tissue scaffolds, composite materials containing both inorganic (glass, ceramic or glass–ceramic) and organic (biocompatible polymer) phases are considered an optimal approach [12,13]. The bioactive ceramic phase is brittle but has good compressive strength as well as good bone-bonding ability. The polymeric phase confers the toughness and plasticity. Therefore, by combining a bioactive ceramic phase with a polymeric one, composite materials with high mechanical properties and good biological behaviour can be obtained. 45S5 Bioglass© is a commercially available material that has been used as bone replacement for more than 15 years. P(3HB) is a natural thermoplastic polymer produced by many types of microrganisms. It is highly biocompatible, biodegradable and has good processability. In previous published paper [17], the macrostructure and pore morphology of the scaffolds before and after P(3HB) coating were investigated, while the bioactivity of the P(3HB)–Bioglass© composites was analysed by immersion in SBF. Moreover, the compressive strength of the scaffold was measured before and after soaking in SBF. In vitro biocompatibility was studied by using the HOS-TE85 human osteosarcoma cell line. Two types of fully reticulated polyester-based polyurethane foams with 60 pores/inch (p.p.i.) (labelled B) and 45 p.p.i. (labelled W) were used as templates for the replication method. µCT images of B-scaffolds before and after coating with P(3HB) solution are illustrated in Fig. 11.

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Fig. 11 µCT images of B-type scaffolds, (a) before and (b) after coating with P(3HB). The images show the scaffold material (green) and the coating (blue). As can be observed, the coating seems to be homogeneously distributed inside the scaffold. The P(3HB) coating did not change the pore structure significantly: the porosity is still open and interconnected. The porosity of the W-scaffolds is approximately 85 % (mean pore size, 470 μm), while the porosity of B-scaffolds (derived from the 60 p.p.i. PU foam) is 80 % (mean pore size, 350 μm). The B-foam, having a smaller pore size in comparison with the W-foam, also displays a lower porosity. After soaking for 28 days in SBF, the scaffold surface is homogeneously coated by a HA layer (Fig. 12). In Fig. 12a, the cracks present on the glass–ceramic struts correspond to the dried silica-rich layer. The ionic exchange will initially take place between the uncoated glass–ceramic and SBF, leading subsequently to the formation of the HA-rich layer. Therefore, the presence of these small uncoated regions of the scaffold struts favours the precipitation of HA and hence the bioactivity of the scaffolds is not impaired by the P(3HB) coating.

Fig. 12 SEM micrographs of B-type glass–ceramic scaffolds after 28 days in SBF, (a) before and (b) after P(3HB) coating (arrows indicate areas coated by the polymer). SEM micrographs of the inner part of uncoated and P(3HB)-coated composite scaffolds (W-type) after 1, 3 and 7 days of cell culture are illustrated in Figure 13a–f. The HOS cells are seen to adhere and spread on the scaffold surfaces. With longer culture periods, the cells grow steadily on the scaffold. It seems also that cells spread more on the coated samples (see Figure 13f).

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Fig. 13 SEM images of HOS cells on uncoated and P(3HB)-coated [W-P(3HB)] scaffolds after 1, 3 and 7 days of cell culture. A histogram representing cell proliferation and growth on and inside the scaffolds is shown in Fig. 14. The cells proliferated and grew on all samples. In contrast with the control surface (tissue culture plastic), the number of cells that proliferate on the scaffolds in all cases, e.g. W- and B-type, with and without coating, is lower. This fact can be partly explained in terms of pH. The scaffolds were not pre-conditioned in SBF before the cells were seeded and the ionic exchange between the scaffolds surface and the medium may have induced a pH increase, which could have affected cell proliferation and viability.

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Fig. 14 Cell proliferation for 1, 3 and 7 days, on P(3HB) coated and -uncoated samples B and W samples. µCT study of vascularisation in tissue engineered constructs X-ray attenuation coefficients of soft tissues are generaly low, and thus, in standard absorption µCT images, these structures have little difference in contrast from each other, unless a contrast agent is adopted. This is the case of vascularization, which usually cannot be displayed in a standard absorption µCT experiment. Nevertheless, in previous published paper [18] this result could be achieved, by exploiting a technique based on the phase contrast principle. In fact the interaction of a X-ray with the sample can be generally described in terms of the refractive index n: n = 1 - δ + iβ

(1)

where the imaginary part β describes the attenuation of the wave due to absorption and the δ describes the phase variation with respect to propagation in vacuum. In standard µCT the detector, set directly behind the sample, measures the attenuation, which allows calculation of the integral of β along the transmitted path. By repeating this measurement for a large number of angular positions of the sample and by using a tomographic reconstruction algorithm, it is possible to reconstruct the 3-D β map. On the other hand, holotomography allows reconstructing the δ map by measuring the phase distribution for each angular setting of the sample. In fact, with a coherent X-ray beam, phase contrast may be simply obtained by free space propagation (i.e., by positioning the detector at some distance from the sample), while a bidimensional projection of the phase map can be obtained from three or four series of images, each series being recorded at different distance from the object at each of the different angles of rotation. Then, the 3-D δ map map is reconstructed with the same algorithm as in standard µCT. Unfortunately, although a number of details can be observed with holotomography, it is still hard to distinguish very soft tissues such as vessels. In contrast, a suitable weighted superposition of both β (x,y,z) and δ (x,y,z) maps, called pseudoholotomography in [18], was found to generate more useful images. Marrow aspirates were obtained from iliac crest of sheep, and bone-marrow derived MSC were extracted and expanded in vitro according to standard protocols, to be used for implants on bioceramic scaffolds made of 100% synthetic calcium phosphate multiphase biomaterial containing 67% silicon stabilized TCP (Si-TCP; SkeliteTM), exhibiting 65% porosity. Subsequently, bioceramic/MSC composites were implanted subcutaneously on the back of immounocompromised mice and finally extracted after 24 weeks for the µCT observations. The measurements were carried out at ESRF, Grenoble. A standard absorption µCT image of a bone tissue-engineered construct 24 weeks after implantation in a mouse are shown in Fig. 15. Scaffold material and newly formed bone are clearly visible, but the organic tissue is poorly detectable due to its low contrast. On the

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contrary, in Fig.16 a reconstruction of a bone tissue-engineered construct 24 weeks after the implantation is shown, as obtained by pseudoholotomography. Three phases are clearly distinguishable: the scaffold (white), the engineered bone (light brown), and the vessel networks within the pores (green).

Fig. 15 Absorption µCT image of the tissue-engineered construct after 24 weeks of implantation in an immunocompromised mouse.

Fig. 16 3D Pseudo-holotomographic images of the tissue-engineered construct after 24 weeks of implantation in an immunocompromised mouse. The images show the vessel ingrowth inside the scaffold: vessel growth occurred both in the presence (A, green) and in the absence of newly formed bone (B, brown/pink). Details of 3D spatial distribution of the phases into scaffolds within one single pore (C, D). From these images a quantitative analysis could be also performed, obtaining the distribution of vessel size, which turned out to be in good agreement with the results of histological observations (Fig. 17).

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Fig. 17 Vessel diameter distribution obtained from pseudo-holotomography µCT and histology. Conclusion A few examples of microstructural characterizations of biomaterials and scaffolds for regenerative orthopaedics have been presented. It was shown in particular how advanced experimental methods, such as synchrotron radiation µCT and synchrotron radiation diffraction can offer very important information, can be not only complementary methods to more standard techniques (electron microscopy, X-ray diffraction), but can also offer the possibility to measure parameters that cannot be obtained otherwise. This is for what concerns the biomaterial mechanical properties, as well as other fundamental parameters such as bioactivity, bioresorption and vascularization. References [1] H. Li, K.A. Khor, Characteristics of the nanostructures in thermal sprayed hydroxyapatite coatings and their influence on coating properties, Surface and Coatings Technology, 201 (2006) 2147-2154. [2] M.F. Morks, A. Kobayashi, Influence of spray parameters on the microstructure and mechanical properties of gas-tunnel plasma sprayed hydroxyapatite coatings, Materials Science and Engineering B, 139 (2007) 209-215. [3] M. Inagaki, T. Kameyama, Phase transformation of plasma-sprayed hydroxyapatite coating with preferred crystalline orientation, Biomaterials, 28 (2007) 2923-2931. [4] Y. Yang, J. Kyo-Han Kim, L. Ong, A review on calcium phosphate coatings produced using a sputtering process - An alternative to plasma spraying, Biomaterials, 26 (2005) 327-337. [5] H. Li, K.A. Khor, P. Cheang, Adhesive and bending failure of thermal sprayed hydroxyapatite coatings: Effect of nanostructures at interface and crack propagation phenomenon during bending, Engineering Fracture Mechanics, 74 (2007) 1894-1903. [6] P.L. Silva, J.D. Santos, F.J. Monterio, J.C. Knowles, Adhesion and microstructural characterization of plasma-sprayed hydroxyapatite/glass ceramic coatings onto Ti-6A1-4V substrates, Surface and Coatings Technology, 102 (1998) 191-196. [7] M. Gaona, R.S. Lima, B.R. Marple, Nanostructured titania/hydroxyapatite composite coatings deposited by high velocity oxy-fuel (HVOF) spraying, Materials Science and Engineering: A, 458 (2007) 141-146.

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[8] A. Balamurugan, G. Balossier, S. Kannan, J. Michael, J. Faure, S. Rajeswari, Electrochemical and structural characterisation of zirconia reinforced hydroxyapatite bioceramic sol–gel coatingson surgical grade 316L SS for biomedical applications, Ceramic International, 33 (2007) 605-614. [9] M. Long, H.T. Rack, Titanium alloys in total joint replacement - a materials science perspective, Biomaterials, 19 (1998) 1621-1639. [10] R.P. Lanza, R. Langer, J. Vacanti, Principles of Tissue Engineering, (2000) Academic Press, New York. [11] V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials, 26 (2005) 5474-5471. [12] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering, Biomaterials, 27 (2006) 34133431. [13] V. Guarino, F. Causa, L. Ambrosio, Bioactive scaffolds for bone and ligament tissue, Expert Review of Medical Devices, 4 (2007) 405-418. [14] K. Zhang, Y. Wang, M.A. Hillmayer, L.F Francis, Processing and properties of porous poly(Llactide)/bioactive glass composites, Biomaterials, 25 (2004) 2489-2500. [15] C. Renchini, E. Girardin, A.S. Fomin, A. Manescu, A. Sabbioni, S.M. Barinov, V.S. Komlev, G. Albertini, F. Fiori, Plasma sprayed hydroxyapatite coatings from nanostructured granules, Materials Science and Engineering: B, 152 (2008) 86-90. [16] C. Renghini, V. Komlev, F. Fiori, E. Vernè, F. Baino, C. Vitale-Brovarone, Micro-CT studies on 3-D bioactive glass-ceramic scaffolds for bone regeneration, Acta Biomaterialia, 5 (2009) 13281337. [17] O. Bretcanu, S. Misra, I. Roy, C. Renghini, F. Fiori, A.R. Boccaccini, V. Salih, In vitro biocompatibility of 45S5 Bioglass®‐derived glass–ceramic scaffolds coated with poly (3‐hydroxybutyrate), Journal of Tissue Engineering and Regenerative Medicine, 3 (2009) 139-148. [18] V. Komlev, M. Mastrogiacomo, F. Peyrin, R. Cancedda, F. Rustichelli, X Ray Synchrotron Radiation Pseudo-Holotomography as a New Imaging Technique to Investigate Angio- and Microvasculogenesis with No Usage of Contrast Agents, Tissue Engineering Part C, 15 (2009) 425430. [19] V.S. Komlev, S.M. Barinov, E.V. Koplik, A method to fabricate porous spherical hydroxyapatite granules intended for time-controlled drug release, Biomaterials, 23 (2002) 34493454. [20] V. Hauk, Structural and Residual Stress Analysis by Nondestructive Methods, (1997) Elsevier, Amsterdam. [21] S. Nuzzo, F. Peyrin, P. Cloetens, J. Baruchel, G. Boivin, Quantification of the degree of mineralization of bone in three dimensions using synchrotron radiation microtomography, Medical Physics, 19 (2002) 2672–268. [22] M. Salomé, F. Peyrin, P. Cloetens, C. Odet, A.M. Laval-Jeantet, J. Baruchel, P. Spanne, A synchrotron radiation microtomography system for the analysis of trabecular bone samples, Medical Physics, 26 (1999) 2194-2204.

Key Engineering Materials ISSN: 1662-9795, Vol. 745, pp 16-25 doi:10.4028/www.scientific.net/KEM.745.16 © 2017 Trans Tech Publications, Switzerland

Submitted: 2017-01-15 Revised: 2017-01-16 Accepted: 2017-01-16

Magnetic Nanoparticles Inclusion into Scaffolds Based on Calcium Phosphates and Biopolymers for Bone Regeneration Florina D. Ivan1,2, Vera Balan2, Maria Butnaru2, Ionel M. Popa1, Liliana Verestiuc2a * 1

Gheorghe Asachi Technical University, Faculty of Chemical Engineering and Environmental Protection, Department of Chemical Engineering, Iasi 2

Grigore T. Popa University of Medicine and Pharmacy, Faculty of Medical Bioengineering, Department of Biomedical Sciences, Iasi, Romania a

[email protected]

Keywords:bone regeneration, scaffold, magnetic nanoparticles, biopolymers, calcium phosphates

Abstract. Considering its functions (support, protection, assisting in movement and storage of minerals), the bone is an essential organ for the human body and the bone trauma/damages have a great impact on the human body functionality. For that reason a variety of biomaterials are studied for potential applications in bone regeneration or substitution. Bone substitution materials, with similar chemical composition to that of natural bone, and specifically those obtained by processes which mimic the natural bone formation in vivo, has been shown to be among the best. In this study, using a process of co-precipitation of calcium phosphate precursors on a mixture of biopolymers (chitosan, collagen, hialuronic acid) and magnetic nanoparticles (magnetite functionalized with chitosan), biodegradable biomimetic scaffolds have been obtained. In order to study their chemical structure, the biodegradable scaffolds have been characterized by Fourier Transform Infrared Spectroscopy (FTIR). The morphology of the biodegradable scaffolds, studied using scanning electron microscopy (SEM) indicated a macroporous morphology, which influenced the retention of simulated biological fluids. A direct relationship between the scaffolds’ degradation rate and the concentration of the polymeric phase has been observed. The in vitro cytocompatibility tests indicate that the prepared scaffolds are biocompatible and assure and adequate mediums for osteoblasts. 1. Introduction Bone tumor is one of the major bone diseases and the treatment of such a bone disease typically requires the removal of bone tumor and regeneration of tumor-initiated bone defect [1]. The conventional therapies for bone defects (autografts, allografts, metals, ceramics and so on) have some drawbacks. Autografts serve as the gold standard for bone grafts because they are histocompatible and non-immunogenic, osteoinductive (bone morphogenetic proteins (BMPs) and other growth factors), osteogenetic and osteoconductive. However, autografts involve harvesting bone from the patient’s donor site, and thus, requires a second operation at the site of tissue harvest. Autografts are not an option in cases where the defect site requires larger volumes of bone than is available. Autologous bone transplants are very expensive procedures, and they may result in significant donor site injury. Allografts involve transplanting donor bone tissue, often from a cadaver. Allografts are associated with risks of immunoreactions and transmission of infections and reduced osteoinductive properties [2]. Tissue engineering and regenerative medicine contribute significantly to the achievement of scaffolds for bone repair and regeneration [3]. The scaffolds, which act as an artificial matrix, must have, first of all, properties like biocompatibility, osteoconductivity, osteoinductivity, osteogenecity and osteointegration, in order to regenerate the osseous tissue. Also, they must have suitable surface chemistry to facilitate cell attachment, proliferation and differentiation and should have a three dimensional structure with interconnected pores for cell growth and transport of nutrients and metabolic waste [4].

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Biodegradability is another essential requirement for the scaffolds with potential applications in tissue engineering and regenerative medicine; the scaffolds degradation rate should be similar to the neo tissue formation rate [5]. The enzymatic degradation is the most used method for characterizing the biodegradability of scaffolds. The enzymes used for the study of biodegradability are selected considering the scaffolds composition. For example, for collagen, one of the most used biopolymers in tissue engineering and regenerative medicine, degradation studies are carried out using collagenase, and in the case of chitosan, other biopolymer widely used in the field, the degradation studies are carried out using lysozyme. In vivo, collagenase cleaves collagen fibrils at the Gly775– Leu/Ile776 site, located in the area of loose triple helical structure domain [6]. Lysozyme is an enzyme normally present in the human serum, saliva, and other fluids. It is involved in hydrolyzing process of the β-(1-4) glycosides linkages between N-acetyl glucosamine and N-acetyl muramic acid residues that occur in the cell walls of bacteria. More precisely, lysozyme active site binds six sugar rings, three consecutive N-acetyl-D-glucosamine residues being necessary for lysozyme catalytic activity [7]. Various materials have been tested in the aim to substitute the natural bone. Synthetic and natural inorganic ceramic materials (hydroxyapatite, tricalcium phosphate, bicalcium phosphates) as candidate scaffold material have been tested bone tissue engineering [8-10] because these ceramics resemble the natural inorganic component of bone and have osteoconductive properties. However, these ceramics are brittle and cannot match the mechanical properties of the bone. This seriously limits their clinical relevance as synthetic bone scaffolds. Natural polymers (collagen, chitosan, hyaluronic acid) and their composites with calcium phosphates (Fig. 1) are an attractive and versatile alternative in bone tissue applications [11] and several techniques have been developed to process synthetic and natural scaffold materials into porous structures. Several systems have been successfully adapted to produce synthetic and natural biodegradable polymer, bioceramic and hydrogel scaffolds [12,13].

Fig. 1 Collagen, chitosan and sodium hyaluronate interactions with calcium phosphates The study presents the preparation and characterization of biodegradable scaffolds using a precipitation process of calcium phosphates precursors on a polymeric matrix based on collagen, chitosan, hyaluronic acid and magnetic nanoparticles. The morphology and the structure of the scaffolds have been analyzed, as well as their interaction with simulated biological fluids containing enzymes that in human body are involved in the degradation process of the polymers used for scaffold preparation.

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2. Materials and methods 2.1. Chemicals Chitosan - Cs (Mn=755900, DD 79.2 %), Vascon Chemical Co, hyaluronic acid, sodium salt Hya (solubility in H2O of 5mg/mL, BioChemika-Sigma Aldrich), collagen- Col(type I, kindly donated by Lohmann&Rauscher GmbH, Germany), calcium phosphates – CP precursors: calcium chloride CaCl2 and monosodium phosphate NaH2PO4·H2O (Sigma-Aldrich) and magnetic nanoparticles – MNPs (magnetite coated with chitosan) have been used for the preparation of the scaffolds. Phosphate buffered solutions (PBS), pH=7.2 0.01 M, have been used for thein vitro degradation studies and for the retention of simulated biological fluids studies. For the in vitro degradation studies, lysozyme (Fulka-Switzerland), potassium ferricyanide - K3Fe(CN)6 (Sigma Aldrich), collagenase clostridium histolyticum (Sigma Aldrich) and ninhidrin reagent have been also used. Hank's Balanced Salt Solution - HBSS, Dulbecco’s Modified Eagle’s Medium, high glucose with L-glutamate and pyruvate - DMEM, fetal bovine serum - FBS, PenicillinStreptomycin-Neomycin – P/N/S, Thiazolyl Blue Tetrazolium Bromide - MTT, all from SigmaAldrich, have been used for the cytocompatibility studies. 2.2. Scaffolds preparation In first stage, biopolymers solutions, Cs (1%), Col (1%), Hya (1%), and MNPs (5%) have been combined with calcium phosphates precursors (CaCl2 (40%), NaH2PO4 (25%)). In second stage, thepH of the obtained mixture was adjusted to 7.2-7.4 using NH4OH (25%). In third stage the final mixture has been freezedried for 24 hours. Four biodegradable scaffolds have been obtained combiningCs, Col solutionsin a different percent and a diffrent Ca/P ratio. The Hya solution has been 5% reported to the whole content of biopolymers for all the scaffolds. The obtained scaffolds have been noted as follows: S1 -Cs 65%, Col 35%, Ca/P=1.6; S2 - Cs 35%, Col 65%, Ca/P=1.6; S3 - Cs 65%, Col 35%, Ca/P=1.7 and S4 - Cs 35%, Col 65%, Ca/P=1.7.

Fig. 2 Biodegradable composite scaffolds preparation 2.3. Scaffolds characterization 2.3.1. Scanning electron microscopy (SEM) Tescan-Vega scanning electron microscope has been used to analyze the scaffolds morphology. The samples have been coated with gold before analyzing.

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2.3.2. Fourier transform infrared spectroscopy (FTIR) analysis FTIR analysis has been used to determinate the structure of the scaffolds and the interactions between the components of the scaffolds. Before the FTIR analysis, in a first step, samples from the scaffolds have been grounded and in a second step they have been mixed with an alkali powder, like the potassium bromide (KBr). 2.3.3. Retention of simulated biological fluids In order to study the retention of simulated biological fluids (PBS) a volumetric method was used. The volume retained by each sample has been determinated using micro-columns(QIA quickVR Spin Column 50, Ø 10 mm) and the retention degree was calculated using the equation: RD (%)=

× 100

(1)

w0 is the initial weight of the scaffold and we, the equilibrium weight. 2.3.4. In vitro degradation studies Particular attention has been given to the study of in vitro enzymatic degradation. The degradation of the scaffolds has been studied using collagenase and lysozyme, enzymes involved in the in vivodegradation of the major component of the polymeric matrix. Samples of each scaffold have beenimmersed in a PBS solution with lysozyme (1200 mg/mL) and collagenase (0.01%), in a dialysis membrane, thathas been submerged in 10 mL PBS and in a last stage incubated at 37°C. From time to time 1 mL PBS has been extracted and evaluated by mesuring the amount of Cs reducing ends using potassium ferricyanide K3Fe(CN)6 and the amount of degraded collagen using ninhydrin reagent. 2.3.5. In vitro cytocompatibility studies Sterilized scaffolds have been teste for their cytocompatibility, by using the indirect extraction method. In order to obtain the extract, sterilized fragments of the scaffolds have been immersed in DMEM medium at 37°C for 24 hours. The cytocompatibility has been studied using preosteoblasts (cell line MC-3T3) that have been cultured in DMEM + 10% fetal bovine serum + 1% P/S/N, removed after 24 hours and replaced with medium containing scaffolds extracts. Cells were seeded at a density of 3×103cells per well in 96 well plates. At 24 hours, 48 hours and 72 hours MTT assays have been performed. This assay is based on the ability of viable cells (with active metabolism) to reduce MTT into a colored formazan product (purple), which can be spectrometrically detected at 570 nm. The formazan has been solubilized with iso-propanol and then the absorbance was measured using a Tecan Sunrise Plate Reader. The percentage cell viability was calculated as: %Cell viability=Abs samples/Abs control × 100.

(2)

where Abs samples is the absorbance of cells tested with various formulation and Abscontrol is the absorbance of control cells (incubated with cell culture media only). 3. Results and discussions 3.1. Scaffolds morphology The scaffolds have been analysed by scanning electron microscopy (SEM) in order to study their morphology (pore size and pores connectivity and distribution). It has been observed in Fig.3 that all scaffold samples displayed a highly porous structure. Porosity, characterized as the percentage of void space in a solid, allows vascularization and, very important, migration and proliferation of osteoblasts and mesenchymal cells, and is fundamental for bone tissue formation. Furthermore, the mechanical interlocking between the implant biomaterial and the surrounding natural bone is improved by a porous surface [14].

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SEM micrographs of the scaffolds revealed a three-dimensional structure with interconnected pores, structure that include both calcium phosphate crystals and MNPs, homogenously distributed within the polymeric matrix. 3.2. Fourier transform infrared spectroscopy (FTIR) analysis The FTIR data are presented in Fig. 4. For Cs were mainly observed the following characteristic absorption bands: 3428 cm-1 (S1) for the hydroxyl group absorption, 1556 cm-1 (S1 and S2) for amide II and 1318 cm-1 (S1) for amide III [15]. The FTIR spectrum for sodium hyaluronate (Hya) has been shown peaks such as 1652 cm-1 (S1 and S2) for amide I, 1032 cm-1 (S1) and 1031 cm-1 (S2) for the ester group, 1408 cm-1 (S1) and 1404 cm-1 (S2) can be attributed to the asymmetric (C=O) and symmetric (C−O) stretching modes of the planar carboxyl groups [16,17].

Fig.3 SEM images of the scaffolds S1, S2, S3 and S4 For collagen (Col), peak 1237 cm-1 (S2) can be attributed to amide III, and for CP have been observed typical peaks of phosphate bands at the positions of 603 cm-1 (S1), 604 cm-1 (S2) and 561 cm-1 (S1 and S2) [18].

Fig. 4 FTIR data of the scaffolds

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3.3.Retention of simulated biological fluids The results of the retention of simulated biological fluids study are shown in Fig. 5. The values have been correlated with the porosity of the scaffolds highlighted by Fig. 3. The highest value of the retention degree (RD) was obtained for the scaffold S1. The quantity of simulated body fluid included into composite materials has two components: water retained into materials porosity - as higher with material porosity increasing, and water interacted by hydrogen bonds with hydrophilic groups of the biopolymers. The materials porosity is considered to be the most significant contributor to the values of fluid retention, and S1 scaffold is characterized of non-uniform pores, some large which are able to include higher quantities of fluids.

Retention degree, RD (%)

400 350 300 250 200 150 100 50 0 S1

S2

S3

S4

Fig. 5 PBS Retention Degree (RD %) of the scaffolds 3.4. In vitro degradation studies Fig. 6 and Fig.7 exhibits the in vitro degradation study results. Regarding the collagen degradation (Fig. 6) has been seen a close link between the concentrations of degraded collagen and polymer composition of the scaffolds. Collagenase-catalysed hydrolysis of material with collagenic component is produced in two steps: in a first step the enzyme is fixed on substrate and the second one involve the specific active sites breaking. Environmental conditions (pH, ionic strength, enzyme inhibitor presence) and material characteristics (composition, supramolecular structure) are parameters which are influencing both the enzyme access to the aminoacids and obtaining of enzyme – substrate complex with sensitivity to hydrolysis. For the scaffolds with 35% collagen (S1 and S3), the values have been considerably lower than for those who have 65% collagen (S2 and S4). Also, it has been observed that the Ca/P ratio influence the collagen degradation, lower values being observed for the scaffolds with higher Ca/P ratio (1.7).

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4 hours

24 hours

48 hours

7 days

14 days

Degraded collagen, mmols/L

1.4 1.2 1 0.8 0.6 0.4 0.2 0

S1

S2

S3

S4

Fig. 6 In vitro degradation of the scaffold. Degraded collagen In terms of the degradation of chitosan (Fig. 7), an increase in time of the concentration of degraded chitosan has been observed. It appears that the degradation rate of the polymeric matrix is directly proportional with the increase of Ca content in materials. The polymeric matrix is strongly bonded with CP, which makes it difficult for lysozyme to destroy these bonds and, as result, the degradation of matrix is slower. Through the hydrophilic nature of the biopolymers, the diffusion of PBS into composites is faster than the degradation. Blank experiments in PBS without lysozyme indicated no contribution of the composite dissolution on the degradation data. The results of this study allow us to name the obtained scaffolds, biodegradable scaffolds.

Degraded chitosan * 100, mmols/L

4 hours

24 hours

48 hours

7 days

14 days

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 S1

S2

S3

S4

Fig. 7 In vitro degradation of the scaffold: Degraded chitosan. 3.5. In vitro cytocompatibility studies MTT colorimetric assay is mainly used for the evaluation of cytotoxicity, cell viability, and proliferation studies. The yellow MTT solution, on reduction by dehydrogenases and reducing agents present in metabolically active cells, turnout into violet-blue formazan [19]. A MTT viability assay has been performed for the scaffolds S1 and S2 and the results are shown in Fig. 8. and Fig. 9. Each result represents the mean viability ± standard deviation (SD) of four

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independent experiments. It was observed that the cell viability is greater than 94 % for the two analyzed scaffolds and also, that the viability slowly increase in time, meaning that the scaffolds do not exhibit any significant adverse effects on cells, so they are cytocompatible. Control

S1

S2

Cell viability (%)

120 100 80 60 40 20 0 24 hours

48 hours

72 hours

Fig. 8 The cell viability, measured by the MTT assay The microscopy analysis of the 24 hours 72 hours cultured osteoblasts revealed a normal cell spreading and shape (Fig.9).

Fig.9 Optical microscopy images of the cells in contact with the composite scaffolds (x200)

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4. Conclusions Biodegradable scaffolds were obtained using a co-precipitation process of calcium phosphates precursors on a polymeric matrix based on collagen, chitosan and hyaluronic acid, with inclusion of magnetic nanoparticles (magnetite coated with chitosan). The morphology and the structure of the scaffolds are suitable for their future use in bone tissue engineering applications. Regarding the scaffolds biodegradability, a direct relationship between the scaffolds degradation rate and their composition has been observed, the concentration of polymeric phase having a remarkable influence. In vitro cell culture tests confirmed of the biodegradable scaffolds are citocompatible. These results indicate that the prepared scaffolds are promising materials for bone tissue engineering Acknowledgement This work was financially supported by the Romanian Ministry of Education and Scientific Research; grant PN-IIPTPCCA-2013-4-2287- MAGBIOTISS. References [1] H. Ma, C. Jiang, D. Zhai, Y. Luo, Y. Chen, F. Ly, Z. Yi, Y. Deng, J. Wang, J. Chang, C. Wu, A bifunctional biomaterial with photothermal effect for tumor therapy and bone regeneration. Adv. Funct. Mater, 26 (2016), 1197-1208. [2] A.R. Amini, C.T. Laurencin, S.P. Nukavarapu, Bone tissue engineering: recent advances and challenges, Crit Rev Biomed Eng, 40 (2012), 363-408. [3]N. Bock, A. Riminucci, C. Dionigi, A. Russo, A. Tampieri, E. Landi, V.A. Goranov, M. Marcacci, V. Dediu, A novel route in bone tissue engineering: Magnetic biomimetic scaffolds, Acta Biomater. 6 (2010), 786-796. [4] S. Naznin, Biodegradable polymer-based scaffolds for bone tissue engineering, SpringerBriefs in Applied Sciences and Technology, 2013. [5] P.X. Ma, Biomimetic materials for tissue engineering, Adv. Drug Deliv. Rev. 60 (2008), 184– 198. [6] H.M. Wang, Y.T. Chou, Z.H. Wen, Z.R. Wang, C.H. Chen, M.L. Ho, Novel biodegradable porous scaffold applied to skin regeneration. PLoS ONE 8(2013), e56330. [7] M.A. Barbosa, A.P. Pego, I.F. Amaral, Chitosan, Materials of Biological Origin, Elsevier Ltd (2011), 221-237. [8] M.Azami, A.I. Jafar, S. Ebrahimi-Barougha, M. Farokhic, S.E. Fard, In vitro evaluation of biomimetic nanocomposite scaffold using endometrial stem cell derived osteoblast-like cells, Tissue and Cell 45 (2013), 328– 329. [9] S.V. Dorozhkin, Bioceramics of calcium orthophosphates, Biomaterials 6 (2010),2773-86. [10] W.W. Thein-Han, R.D.K. Misra, Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone tissue engineering, Acta Biomaterials 5 (2009), 1182-1197. [11] J. Wang, C. Liu, Biomimetic collagen/hydroxyapatite composite scaffolds: fabrication and characterizations, Journal of Bionic Engineering 11(2014), 600-601. [12] M. Kikuchi, T. Ikoma, S. Itoh, H. N. Matsumoto, Y. Koyama, K. Takakuda, K.Shinomiya, J. Tanaka, Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen, Comp. Sci. and Tech 64 (2004), 819-820.

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[13] H.Fatemeh, M.E.Bahrololoom, In situ preparation of iron oxide nanoparticles in natural hydroxyapatite/chitosan matrix for bone tissue engineering application, Ceramics International 4 (2015), 3094–3100. [14] V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials, 26 (2005), 5474-5491. [15] N. V. Afanaseva, V. A. Petrova, E. N. Vlasova, S. V. Gladchenko, A. R. Khayrullin, B. Z. Volchek, A. M. Bochek, Molecular mobility of chitosan and its interaction with montmorillonite in composite films:dielectric spectroscopy and FTIR studies, Polym. Sci. Ser. A Chem. Phys. 55 (2013), 738-748. [16] K. Jagadeeswara Reddy, K.T. Karunakaran, Purification and characterization of hyaluronic acid produced by Streptococcus zooepidemicus strain 3523-7, J. BioSci.Biotech. 2 (2013), 173-179. [17] Y. Wu, Preparation of low-molecular-weight hyaluronic acid by ozone treatment, Carbohydr. Polym. 89 (2012), 709–712. [18] Y. Zhai, F.Z. Cui, Recombinant human-like collagen directed growth of hydroxyapatite nanocrystals, J. Cryst. Growth, 291 (2006), 202–206. [19] J. C. Stockert, A. Blázquez-Castro, M.Cañete, R. W. Horobin, Á.Villanueva, MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets, Acta Histochemica,114 (2012), 785–796.

Key Engineering Materials ISSN: 1662-9795, Vol. 745, pp 26-38 doi:10.4028/www.scientific.net/KEM.745.26 © 2017 Trans Tech Publications, Switzerland

Submitted: 2016-02-28 Revised: 2017-01-16 Accepted: 2017-01-16

Current Strategies and Advances Materials for the Treatment of Injured Meniscus Codorean Ion Bogdan1, a *, Tanase Stefania1,b, Cernat Eduard1, c, Diaconu Florin1,d, Popescu Dragos2,e, Cirlan Alexandru1,f, Mitulescu Stefan1,g 1

Central Military Hospital Dr Carol Davila, Department of Orthopedics and Traumatology, Calea Plevnei street, 134, Sector 1, Bucharest,

2

National Aeronautical and Space Medicine Institute “Gen. Dr. Aviator Victor Anastasiu”, Mircea Vulcanescu Street, 88, Sector 1, Bucharest a

[email protected], b [email protected], [email protected], [email protected], [email protected], [email protected], g [email protected]

d

Keywords: artificial scaffold, meniscal implant, osteoarthritis.

Abstract. The lack of meniscal tissue increases the risk of early cartilage degeneration. Classic treatment includes suturing and partial menscectomies, total meniscectomies being abandoned. Modern treatments are based on the implantation of special scaffolds that replace some of the lost meniscal tissue. This paper reviews the basic principles of modern treatment of the menisci and it includes a retrospective study, in which a total of 10 patients (7 men and 3 women, mean age: 28.28 (21-38)) were enrolled. All patients had previous surgery and were subjected to arthroscopic treatment with a biodegradable scaffold (Actifit®). They received KOOS (Knee Injury and Ostheoarthritis Outcome Score), Lysholm and Tegner score. The Tegner score was not very useful in determining the success or failure of the surgery. The Lysholm and KOOS score results improved at the 1-year follow-up. The results of the scores that the patients filled out, showed an improvement in their preoperatively knee related problems. The Actifit® scaffold is safe and effective in treating meniscal defects. 1. Introduction The meniscal lesion is one of the most frequent injury seen and treated by orthopedic surgeons. The basic rule of treating such an injury is preserving as much meniscal tissue as possible through meniscal suturing, but when that is not possible the elected treatment is meniscectomy. After meniscectomy the pressures and forces that appear at the chondral level is higher than normal, leading to early chondral lesions which require more surgery. Chondral lesions combined with meniscus loss lead to osteoarthritis. Different therapeutical strategies have been employed to replace the lost meniscal tissue, starting with meniscal transplant leading up to the development of artificial scaffolds for implantation. Artificial scaffolds are based either on natural or synthetic polymers. Many such polymers have been and are studied, but to this day only two scaffolds have been approved for worldwide use: Actifit® and Menaflex® and they can only be used as implants in patients that have undergone partial meniscectomies. The development of an ideal replacement not only for partial meniscectomy patients but for patients who require a total meniscectomy, is crucial. Osteoarthritis is the leading cause of chronic disability in today’s world, requiring costly medical attention. Even patients with early signs of osteoarthritis require medication for symptoms such as pain, their daily and work activities being altered. As the disease evolves, patients require more drugs, more medical advice, even multiple surgical intervention, leading to an overall higher cost; patients requiring long periods of time off work. The artificial scaffold, although expensive at first, is a good alternative to the high costs that appear in treating osteoarthritis.

Key Engineering Materials Vol. 745

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Anatomy and histology The menisci are 2 fibrocartilaginous structures, medial and lateral, that are situated between the femur and the tibial plateau, and play an important role in the knee joint. They were first described as “functionless remnants of leg muscle origin” [1]; nowadays it has been widely accepted that they are crucial structures of the knee stability, with an important role in shock absorption, in direct load transmission and in the restraint mechanism of the human knee. Due to their location and structural layout, they are susceptible to injury and possess a small chance of healing [2]. This type of injury often appears in young patients in contact-sport activities, but it also appears in elderly patients due to the aging process. There are different types of meniscal lesions: radial lesion, degenerative lesion, root lesion, horizontal lesion or bucket lesion. Treatment of the lesions of the menisci has evolved during the last period of time, numerous techniques have been established, and the procedure of total meniscectomy has been abandoned due to long term results of studies, that have shown development of early osteoarthritis. Todays’ guidelines indicate partial meniscectomy or special sutures, but great potential is hold by regenerative medicine, by inserting an artificial meniscus, for restoring function and form of the meniscal fibrocartilage. Special consideration must be given to the type of scaffold, to the types of cells needed and to the microenvironment in which the menisci are located for the ideal development of engineered meniscus tissue [3]. The menisci are anchored to other intra-articular structures, including medial collateral ligament, the transverse ligament, and the meniscofemural ligaments. The menisci are divided into three parts, from a vascular point of view, the red zone or the external part, rich in vascular supply and it possesses the highest chance of healing, the red-white zone or the middle part, with a poor healing potential, and the white-white zone or inner part, with a minimal healing potential [4]. The predominant cell type that makes up the menisci, is the fibrochondrocite, and creates in a microscopic view two main zones: superficial and deep. This cell type is similar phenotipically to the chondrocyte, but it also possesses the capability to establish and develop a fibrous territorial matrix [5]. Depending on their location in the menisci, the fibrochondrocites differ in form, from an oval or fusiform shape with sparse cytoplasm, in the superficial layer to round or polygonal shape with endoplasmatic reticulum and other cytoplasmic organelles in the deep layer [6]. The extracellular matrix that the fibrochondrocites secrete is composed out of collagen and elastin fibers. The percentage that the collagen accounts for is about 60-70% of the extracellular dry weight, but with age the content decreases. The types of collagen present in the meniscus are I, II, III, V, the predominant type being type I collagen [7]. From a structural point of view type I collagen forms three layers: the outer layer with a random orientation of the collagen fibers, a middle or lamellar layer with the fibers having a parallel orientation and radial fibers at the peripheral ends and the deep layer that contains circumferentially oriented type I fibers and small amounts of radial oriented fibers. The main roles of the proteoglycans that exist in the menisci are compression and maintain tissue hydration. The quantity of proteoglicans produced is higher in the avascular two-thirds, but the glycosaminoglycan composition of proteoglycans remains the same throughout the meniscal tissue. The types of proteoglycans that exist in normal menisci are: 40% chondroitin-4-sulfate, 1020% chondroitin-6-sulfate, 20-30% dermatan-sulfate and 15% keratin sulfate [8]. The menisci have a unique structure that can withstand a complex combination of movements: flexion, extension, rotation and translation, that appear in the knee joint during normal motion. Modern testing of the biomechanical properties of the meniscus has revealed that in the moment of compression of the tibiofemural joint, a vertical force appears at the surface of the meniscus and a radial oriented component, that outwardly displace the menisci. The outward force that displaces the menisci is limited by the rigid meniscal attachment of the horns, that in turn produce a circumferentially directed force and tensile hoop stress that dissipates throughout the meniscal tissue. All these forces that appear during normal conditions are distributed within the tissue by the circumferentially collagen fibers located in the deep layer of the menisci [9].

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Orthopedic Biomaterials - From Materials Science to Clinical Applications

The circumferential modulus and the radial modulus have been extensively tested. The circumferential modulus ranges between 48-198MPa and the radial modulus ranges between 370MPa [10]. It should be noted that the values are subject to many biological and experimental variables, including age, specimen orientation, strain rate, handling and storage. The difference in the strength may explain the frequency of circumferential splitting of the menisci versus radial tears [11]. A basic classification of the meniscal lesion is into: horizontal, longitudinal, radial, root lesion or circumferential. Another type of classification divides meniscal lesions in acute and chronic [12]. Most often acute or traumatic lesions appear after a force movement of the knee joint, although they can also appear without any trauma. Chronic or degenerative lesions appear as a result of the aging process or as a result of tissue deterioration. The most frequent causes of mensical lesions in children are due to trauma, but more frequently they are due to congenital meniscal variants, like discoid meniscus or meniscal cysts and in adults, meniscal lesions are mostly due to trauma and or the degenerative process that appears with aging or a combination of the two. More frequently in adults the meniscal lesions are accompanied by other lesions such as concomitant ligament and cartilage lesions, also the lesions in adults are more complex [3]. The healing capability of the human meniscus is greater in the vascularized area, the third external region, the red-red zone, making repairing techniques necessary in this region. Surgical treatment Some patients with traumatic or generative meniscal lesions can be treated without surgery. Thus, adequate treatment can be applied in patients (i) without a blocked knee joint, (ii) without meniscal lesion that appear to be biomechanically unstable and (iii) without pain, classified as nonresponsive to pharmacological treatment. Meniscal lesions could be neglected when these types of lesions include partial thickness tears (