Improvement of a commercial calcium phosphate bone cement by means of drug delivery and increased injectability

Table of contents :
Improvement of a commercial calcium phosphate bone cement by means of drug delivery and increased injectability
1 Introduction
2 Materials and methods
2.1 Preparation of unloaded bone cement samples
2.2 Preparation of drug loaded bone cement samples
2.3 Handling properties
2.3.1 Setting time
2.3.2 Injectability
2.4 Compressive strength
2.5 In vitro drug release
2.6 Antimicrobial activity
2.7 In vitro cytotoxicity
2.7.1 Cytotoxicity of bone cement extracts
2.7.1.1 Cell culture
2.7.1.2 Preparation of cement extracts
2.7.1.3 Cytotoxicity assay
2.7.2 Cellular adhesion to the optimal CPC
2.7.2.1 Sample ageing and cell seeding
2.7.2.2 Scanning electron microscopy (SEM)
2.7.2.3 Alkaline phosphatase activity
2.8 XRD
2.9 Chemical analysis: fourier transform infrared (FTIR)
2.10 Surface morphology: SEM
2.11 Human trabecular bone
2.11.1 Bone harvesting and CPC injection
2.11.2 Compressive strength tests
2.11.3 Micro-computed tomography (Micro-CT) analysis
2.12 Statistical analysis
3 Results and discussion
3.1 Optimization of Neocement® basic properties: effect of liquid phase content and polymeric additive
3.2 In vitro drug release
3.3 Cellular response to the materials
3.3.1 Cytotoxicity of bone cement extracts
3.3.2 Cellular adhesion to the optimal CPC
3.4 Characterization of the optimal drug-loaded CPC
3.4.1 Physical properties
3.4.2 Chemical structure
3.4.3 Morphology
3.5 Application of the optimal CPC in human trabecular bone
4 Conclusions
Data availability
Declaration of competing interest
Acknowledgements
Appendix A Supplementary data
References

Citation preview

Ceramics International 48 (2022) 33361–33372

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Ceramics International journal homepage: www.elsevier.com/locate/ceramint

Improvement of a commercial calcium phosphate bone cement by means of drug delivery and increased injectability N. Ribeiro a, M. Reis b, c, L. Figueiredo d, A. Pimenta d, L.F. Santos b, A.C. Branco b, e, A.P. Alves de Matos e, M. Salema-Oom e, A. Almeida f, M.F.C. Pereira g, R. Colaço c, A.P. Serro b, e, * a

Departamento de Ortopedia, Hospital Lusíadas Lisboa, Lisboa, Portugal CQE e Departamento de Engenharia Química, Instituto Superior T´ecnico, Universidade de Lisboa, Lisboa, Portugal IDMEC e Departamento de Engenharia Mecˆ anica, Instituto Superior T´ecnico, Universidade de Lisboa, Lisboa, Portugal d Bioceramed, S˜ ao Juli˜ ao do Tojal, Portugal e CiiEM, Instituto Universit´ ario Egas Moniz, Monte de Caparica, Portugal f CeFEMA e Departamento de Engenharia Química, Instituto Superior T´ecnico, Universidade de Lisboa, Lisboa, Portugal g CERENA e Departamento de Engenharia Civil, Instituto Superior T´ecnico, Universidade de Lisboa, Lisboa, Portugal b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Calcium phosphate bone cement Liquid phase Polymeric additive Injectability Drug release Gentamicin sulphate

Calcium phosphate cements (CPCs) have been increasingly used as synthetic bone substitutes for repair and regeneration of bone defects given their biocompatibility, resemblance to bone and malleability. Moreover, their use as local antibiotic delivery systems is of main interest against bone infections, avoiding the adverse effects of high dosages of conventional therapy. The main goals of this work were to improve the properties of a com­ mercial CPC (Neocement®), turning it injectable, and to provide it with a new functionality as a drug delivery system able to ensure a sustained release of an antibiotic commonly used in orthopaedics (gentamicin sulphate, GS). For this, the influence of the liquid phase amount (%LP) and type of polymer contained in the formulation (chitosan, Chi, or hydroxypropyl methylcellulose, HPMC) on the basic properties of the material was evaluated. It was found that the formulation containing 42%LP + HPMC+1.87% wt GS was the best one. It showed suitable setting and mechanical properties, and injectability around 87% (much superior to the original Neocement®, with 31%). It ensured a sustained release of GS for at least 14 days, at antibacterial levels. The antibiotic released is highly effective against S. epidermidis, but also presents some antibacterial activity against S. aureus. The CPC revealed to be non-cytotoxic. Moreover, it demonstrated good flowability and connectivity with human cadaveric trabecular bone.

1. Introduction The gradual increase in the life expectancy of the world population in the last years resulted in a higher incidence of bone diseases like oste­ oporosis, osteomalacia, tumours and also of traumatic accidents with bone injuries [1]. In many cases, these conditions give rise to large bone defects that exceed the biological repair capabilities and bone grafting procedures are required. Every year, over 2 million surgeries worldwide involve bone grafting [2]. Although autograft is considered the “gold standard” method, the low availability of autologous bone material and the need for additional surgical procedures constitute strong limitations. Allografts and xenografts have also been used, but the risks of

immunogenic responses and infective agents’ transmission remain a major concern [2]. Since they were first proposed by Brown and Chow in the early 1980s [3], calcium phosphate cements (CPCs) revealed to be good alternatives to natural grafts and other synthetic materials (e.g., metals, ceramics, polymers) due to their similarity with the mineral phase of bone and excellent biological behaviour, i.e. good biocompatibility, bioactivity and osteoconductivity, as well as good handling properties [4]. CPCs are produced by a chemical reaction between a solid phase that contains one or more calcium phosphate powders, and a liquid phase, usually water or an aqueous solution with additives. Upon mixing of the two phases, a mouldable paste is formed. When oversaturation is

* Corresponding author. CQE e Departamento de Engenharia Química, Instituto Superior T´ ecnico, Universidade de Lisboa, Lisboa, Portugal. E-mail address: [email protected] (A.P. Serro). https://doi.org/10.1016/j.ceramint.2022.07.279 Received 8 December 2021; Received in revised form 25 July 2022; Accepted 26 July 2022 Available online 7 August 2022 0272-8842/© 2022 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

N. Ribeiro et al.

reached, precipitation of calcium phosphates creates an entangled network of crystals, leading to the setting of the paste [4,5]. According to the literature [6], the setting reaction products depend on the solu­ bility of calcium phosphates and the pH. It is generally accepted that two main end-products are formed: (i) brushite, also called dicalcium phosphate dihydrate or (ii) a poorly crystalline calcium-deficient hy­ droxyapatite. Hydroxyapatite is the most stable (least soluble) calcium phosphate at pH > 4.2, while brushite is the most stable one at pH < 4.2. Contrarily to polymethyl methacrylate (PMMA)-based cements, quite common in clinical practice, CPCs can self-set at body temperature, and, depending on their composition, biodegrade and be replaced by new bone material [5,7]. Some CPCs can be injected, allowing the repair of intricate/narrow defect sites through minimally invasive surgical pro­ cedures. Nonetheless, for several CPC formulations this is not possible due to liquid-solid phase separation phenomena (filter-pressing effect) during extrusion, in which the liquid phase drains at a faster rate than the powder particles, resulting in an extruded with a significantly higher liquid content than the paste initially loaded into the syringe and leading to syringe/needle blockage [8]. Tailoring of injectability can be done by changing CPCs liquid phase amounts, introducing additives like poly­ mers or changing calcium phosphates particle size [4,8]. One major concern in orthopaedics is the high incidence of bone infections (e.g., osteomyelitis), usually caused by bacteria and some­ times fungi, that leads to necrosis and destruction of bone. Although bone infections may be associated with other health problems (e.g., diabetes, tuberculosis, removal of the spleen), it is oftentimes a complication of bone surgery [9]. The standard treatment for osteo­ myelitis involves debridement of the necrosed tissue and systemic administration of antibiotics in high dosages for at least 6 weeks to successfully reach the site of infection, usually with impaired vascular supply [9]. Due to the risk of secondary effects and toxicity, local de­ livery of antibiotics to the site of infection for adequate periods, in sufficiently high amounts is strongly desired [10]. PMMA based cements have been clinically used with this purpose, but they present limited release capacity and since they are not resorbable, a second surgery must be done for remotion after antibiotics release [11]. CPCs have raised particular interest as alternative antibiotic delivery systems, either for prophylaxis or treatment of established infections, due to their intrinsic porosity, degradability and low temperature of setting [12]. Still, their clinical use for antibiotic delivery has only been performed on an off-label basis since further studies are required to fully establish their potential [13]. Neocement® is a CPC commercialized in the European market that is used to repair small bone defects in craniofacial and trauma surgeries, being manually placed within the trabecular bone. Follow up of different cases several months after surgical procedure, show that when implan­ ted it is radiopaque, does not migrate and does not loss contact with native bone [14]. This work intended to develop a CPC-based drug delivery system, starting from the original composition of Neocement®, with improved injectability, suitable handling and mechanical performance for clinical application and sustained release of an antibiotic, gentamicin sulphate (GS), an antibiotic commonly used in orthopaedics. The effect of changing the liquid phase (LP) content and using hydroxypropyl methylcellulose (HPMC) instead of chitosan (Chi), on the main proper­ ties of the cement was evaluated. HPMC is a biocompatible watersoluble polymer derived from cellulose, that has gelling properties and therefore can promote higher liquid phase viscosity of CPCs. Besides, it has been recently used for its ability to greatly improve injectability, cohesion and anti-washout properties of CPC pastes and also for controlled drug release systems [15]. The most promising formulations were loaded with GS and their drug release profiles and antimicrobial activity were studied aiming to find the ones that combined the best material properties and a sustained drug release. The optimal formula­ tion was selected after performing cytotoxicity studies and was further characterized regarding other relevant properties and chemical

Ceramics International 48 (2022) 33361–33372

structure. Finally, the material’s ability to be injected into cadaveric human bone and fill voids was evaluated ex vivo. 2. Materials and methods 2.1. Preparation of unloaded bone cement samples The original Neocement® includes: (i) a liquid phase (LP,38%wt) formed of water, citric acid (Labchem) and glucose (AppliChem Pan­ reac); (ii) a solid phase (62%wt) formed of a mixture of β-tricalcium phosphate (β-TCP, β-Ca3(PO4)2, d(50)