Novel bone substitute composed of chitosan and strontium-doped α-calcium sulfate hemihydrate: Fabrication, characterisation and evaluation of biocompatibility

Materials Science and Engineering C 66 (2016) 84–91

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Novel bone substitute composed of chitosan and strontium-doped α-calcium sulfate hemihydrate: Fabrication, characterisation and evaluation of biocompatibility Yirong Chen a,1, Yilin Zhou a,1, Shenyu Yang b, Jiao Jiao Li c, Xue Li a, Yunfei Ma a, Yilong Hou a, Nan Jiang a, Changpeng Xu a, Sheng Zhang a, Rong Zeng b, Mei Tu b,⁎, Bin Yu a,⁎ a b c

Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People's Republic of China Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, People's Republic of China Biomaterials and Tissue Engineering Research Unit, School of AMME, University of Sydney, Sydney, NSW 2006, Australia

a r t i c l e

i n f o

Article history: Received 29 January 2016 Received in revised form 11 April 2016 Accepted 18 April 2016 Available online 20 April 2016 Keywords: Chitosan Calcium sulfate Strontium Microcapsules Bone substitute Biocompatibility

a b s t r a c t Calcium sulfate is in routine clinical use as a bone substitute, offering the benefits of biodegradability, biocompatibility and a long history of use in bone repair. The osteoconductive properties of calcium sulfate may be further improved by doping with strontium ions. Nevertheless, the high degradation rate of calcium sulfate may impede bone healing as substantial material degradation may occur before the healing process is complete. The purpose of this study is to develop a novel composite bone substitute composed of chitosan and strontium-doped αcalcium sulfate hemihydrate in the form of microcapsules, which can promote osteogenesis while matching the natural rate of bone healing. The developed microcapsules exhibited controlled degradation that facilitated the sustained release of strontium ions. In vitro testing showed that the microcapsules had minimal cytotoxicity and ability to inhibit bacterial growth. In vivo testing in a mouse model showed the absence of genetic toxicity and low inflammatory potential of the microcapsules. The novel microcapsules developed in this study demonstrated suitable degradation characteristics for bone repair as well as favourable in vitro and in vivo behaviour, and hold promise for use as an alternative bone substitute in orthopaedic surgery. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The increasing prevalence of bone defects caused by trauma, osteomyelitis, tumour and other types of bone diseases has created an urgent need for synthetic bone substitutes which are biodegradable and have properties similar to natural bone. Many inorganic materials have been investigated for this purpose due to their chemical similarity to the mineral phase of bone, which promotes the formation of a direct bond with host bone and enhanced osteogenesis [1]. Of these materials, α-calcium sulfate hemihydrate (commonly referred to in the medical literature as calcium sulfate) stands out as a candidate material due to its good biocompatibility, biodegradability, osteoconductive properties and a long history of use in bone repair [2–5]. Upon degradation, αcalcium sulfate hemihydrate forms a slightly acidic environment and results in a high local calcium concentration which promotes osteogenesis [6]. α-Calcium sulfate hemihydrate can be doped with strontium to further improve its ability to promote bone repair. The concentration of ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Tu), [email protected] (B. Yu). 1 Equally contributing to the manuscript.

http://dx.doi.org/10.1016/j.msec.2016.04.070 0928-4931/© 2016 Elsevier B.V. All rights reserved.

strontium ions is maintained at a high level during new bone formation, after which the concentration declines and is held at a constant level in the extracellular fluid of bone cells to facilitate the normal function of bone [7]. The mechanism of action of strontium ions in promoting bone formation is to increase the expression of osteogenic genes and alkaline phosphatase activity in bone marrow-derived mesenchymal stem cells (BMSCs), as well as to inhibit their secretion of receptor activator of nuclear factor-κB ligand (RANKL) which is essential for osteoclast differentiation [8,9]. By releasing strontium ions upon degradation, strontium-doped α-calcium sulfate hemihydrate allows the strontium ion concentration to be maintained at high levels within the defect site, which is advantageous for bone repair as shown in our previous report [10]. The current problem which remains to be solved before strontiumdoped α-calcium sulfate hemihydrate can be considered for clinical application is its fast degradation rate. α-Calcium sulfate hemihydrate in current clinical use undergoes complete degradation in 6 to 9 weeks, which is much shorter than the period of time required for satisfactory bone healing to occur (12 weeks) [11]. Following degradation of the implanted material, an empty space remains within the defect site which becomes occupied by fibrous tissue and results in incomplete healing of the defect. The same problem applies to strontium-doped α-

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Fig. 1. Schematic illustrating the synthesis of microcapsules (CS-C-CSH(Sr)) composed of chitosan (CS) and strontium-doped α-calcium sulfate hemihydrate (CSH(Sr)).

calcium sulfate hemihydrate, with the additional complication that fast degradation will result in the rapid release of strontium ions at a rate that is mismatched to the rate of new bone formation. The purpose of this study is to develop a novel composite bone substitute composed of strontium-doped α-calcium sulfate hemihydrate as the base material, with controlled degradation that facilitates the release of strontium ions with a linear release profile to match the rate of new bone formation. To achieve this goal, chitosan was chosen as the material used to encapsulate strontium-doped α-calcium sulfate hemihydrate in order to control its degradation rate. Chitosan is a linear polysaccharide derived from the natural biopolymer chitin, which is found in crustacean exoskeletons [12]. The benefits of using chitosan as a biomaterial, particularly for orthopaedic applications, include biocompatibility and minimal inflammatory potential, intrinsic antibacterial properties, biodegradability, and the ability to support bone formation on its own or when incorporated into various composite systems [13]. Chitosan has also been frequently investigated as a vehicle for drug delivery in the form of micro- and nanoparticles, which can provide controlled and versatile release profiles depending on the method of preparation [14]. A further advantage of using chitosan to form the composite microcapsules in this study is that it has a natural positive charge, which can be anticipated to interact with the negative charges present in strontium-doped α-calcium sulfate hemihydrate to result in a stable complex with controlled release characteristics (Fig. 1). The present study explores, for the first time, the synthesis of novel microcapsules composed of chitosan and strontium-doped α-calcium sulfate hemihydrate, and reports their composition, morphology and degradation characteristics, as well as the evaluation of their in vitro and in vivo biocompatibility. The main novelties of the microcapsules are: 1) preservation of the good bioactivity and biodegradation properties of strontium-doped α-calcium sulfate hemihydrate, which are advantageous in promoting bone healing, 2) simultaneous introduction of the properties of chitosan as a sustained release vehicle with antibacterial effects, and 3) ability to treat osteomyelitis as well as the bone defect when applied, as these two conditions frequently occur together in the clinical situation, due to the combined properties of the two materials.

Fig. 2. XRD patterns of CS-C-CSH(Sr) and CSH(Sr).

another 8 h for the reaction to reach completion. The reaction product was filtered, washed with deionised water and ethanol, and dried at 65 °C. The resulting Sr-CaSO4·2H2O powder was ground and sieved through 200 meshes. To prepare Sr-α-CaSO4·0·5H2O powder, 300 mL of 15% NaCl solution was heated to 104 °C while stirring, and the solution was adjusted to pH = 5 using diluted HCl. 45 g of Sr-CaSO4·2H2O powder was added into the solution while stirring at room temperature, and the mixture was stirred for another 4 h for the reaction to reach completion. The reaction product was filtered while hot, washed several times with boiling deionised water and then ethanol, and dried at 100 °C. The resulting Srα-CaSO4·0·5H2O powder, hereon referred to as CSH(Sr), was used for subsequent experiments. 2.2. Preparation of microcapsules composed of chitosan and strontiumdoped α-calcium sulfate hemihydrate (CS-C-CSH(Sr)) To prepare microcapsules composed of chitosan and strontiumdoped α-calcium sulfate hemihydrate, chitosan solution adjusted to pH = 5 was heated to 100–102 °C while stirring, and strontiumdoped α-calcium sulfate hemihydrate powder was slowly added into the solution (molar ratio: CSH(Sr)/H2O = 0.15:1). The mixture was stirred for 1 h for the reaction to reach completion. The reaction product was filtered while hot, washed several times with boiling deionised water and then ethanol, and dried at 80 °C for 8 h. The resulting microcapsules, hereon referred to as CS-C-CSH(Sr), was used for subsequent experiments. 2.3. Physical properties of the samples

2. Materials and methods All reagents were obtained from Sigma-Aldrich, USA unless otherwise specified. 2.1. Preparation of strontium-doped α-calcium sulfate hemihydrate (CSH (Sr)) powder Strontium-doped α-calcium sulfate hemihydrate (Sr-αCaSO4·0·5H2O) was synthesised from Sr-CaSO4·2H2O powder. SrCaSO4·2H2O and Sr-α-CaSO4·0·5H2O powders were prepared according to our previously published procedures [10]. Briefly, to prepare SrCaSO4·2H2O powder, Ca(OH)2 and Sr(OH)2 (molar ratio: Ca(OH)2/Sr (OH)2 = 9:1) were dispersed in a mixture of ethanol and water (volume ratio: ethanol/water = 4:3). At room temperature and under rapid mechanical stirring at 800–1000 rpm, 2 mol H2SO4 solution was added dropwise into the mixture and the mixture was stirred for

Samples of CS-C-CSH(Sr) and CSH(Sr) were examined by X-ray diffraction (XRD; MaxRC, Rigaku, Japan) with a voltage of 36 kV and current of 20 mA, at a scanning rate of 4° min−1, step width of 0.02° and 2θ range from 7° to 65°. Morphology and microstructure of the samples were examined by scanning electron microscopy (SEM; Ultra 55, Carl Zeiss, Germany) after sputter coating with gold. 2.4. Degradation and ion release test Samples of CS-C-CSH(Sr), CSH(Sr) and α-calcium sulfate hemihydrate (CSH) were soaked in deionised water for 12 weeks. At the end of each week, the concentration of strontium ions in solution was determined using inductive coupled plasma atomic emission spectroscopy (ICP-AES; Optima 2000DV, PerkinElmer, USA) to obtain the release profile of strontium ions for each of the materials over 12 weeks. The release rate of strontium ions is the most important outcome resulting

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from degradation of the materials and gives an indication of their respective degradation rates in vitro. 2.5. Antibacterial test Escherichia coli were used as they represented one of the most common infecting organisms in clinical cases of osteomyelitis. E. coli CICC 10662 were cultured in Luria–Bertani (LB) broth with pH 7.2 at 37 °C for 24 h (first generation). An adequate volume of this suspension was then cultured in fresh LB broth at 37 °C for 6 h (second generation). The concentration of this suspension was determined using an ultraviolet-visible spectrophotometer (Shimadzu, Japan), and diluted to appropriate concentration in phosphate buffered saline (PBS, pH 7.2) for use in the antibacterial test. Samples of chitosan, CS-C-CSH (Sr) and CSH(Sr) were pressed into uniform disks of 1 cm diameter by 1 mm height and placed into culture dishes. E. coli were seeded onto the surface of the samples in 600 μL of suspension containing 1 × 105 cells, and incubated at 37 °C for 2 h. The negative control group consisted of E. coli seeded on a culture dish without any materials. After incubating for 2 h, 10 min of ultrasonic treatment was applied to each sample to remove the attached E. coli on the sample surface. The suspension containing the detached cells was diluted 10-fold, and 50 μL of this suspension was transferred to an agar plate and incubated at 37 °C for 24 h. At the end of the incubation period, the antibacterial rate of samples was calculated as a percentage based on the coverage area of the agar plate by bacterial colonies and normalised to the negative control, according to the following equation: Antibacterial rate ð%Þ ¼ 1−

Asample =Aplate  100: Acontrol =Aplate

Here, Asample and Acontrol represent the coverage area of bacterial colonies on the agar plate from the sample (chitosan, CS-C-CSH(Sr) or CSH (Sr)) and negative control, respectively, while Aplate is the total area of the agar plate.

2.6. Evaluation of in vitro biocompatibility 2.6.1. Preparation of material extracts Samples of CS-C-CSH(Sr) were immersed in medium composed of Dulbecco's Modified Eagle Medium (DMEM; GE Healthcare, USA) + 10% fetal bovine serum (FBS; GE Healthcare) at a ratio of 0.2 g/mL for 24 h at 37 °C, and the supernatant was collected to prepare the material extracts. Each specimen was sterilised by 60Co irradiation before use. 2.6.2. Cytotoxicity test (MTT assay) L929 mouse fibroblast cells were cultured in DMEM containing 10% FBS, 200 U mL−1 penicillin and 200 μg/mL streptomycin. Cell suspension at a concentration of 1 × 105 mL−1 was seeded in 100 μL aliquots into 96-well microtitre plates. The cells were incubated for 24 h (37 °C, 5% CO2) until most of the cells have attached. The original culture medium was then discarded and each well was washed twice with PBS (pH 7.4, concentration 10 mM). 6 groups of samples were added into the wells at 100 μL per well with each group occupying 18 wells: 4 experimental groups of 100%, 75%, 50% and 25% material extract of CS-CCSH(Sr), positive control group of 0.64% phenol solution, and negative control group of fresh culture medium. MTT assay was performed in 6 wells of each group after culturing for 24, 48 and 72 h. At each time point, the original culture medium was discarded and 20 μL of colorimetric reagent MTT (5 mg/mL) was added to each well. After incubating for 4 h at 37 °C, the MTT solution was discarded and 150 μL dimethyl sulfoxide (DMSO) was added to each well, after which the plate was shaken for 10 min. The absorbance of each well was measured at 490 nm using a plate reader (Biotek, USA) and measurements were repeated 3 times. The relative growth rate of cells (RGR) was calculated as RGR = A/A0 × 100%, where A is the absorbance of the experimental group and A0 is the absorbance of the negative control group. Cytotoxicity assessment criteria: Class 0 for RGR ≥ 100%, Class I for RGR = 75–99%, Class II for RGR = 50–74%, Class III for RGR = 25–49%, Class IV for RGR = 1–25% and Class V for RGR = 0%.

Fig. 3. SEM images of (A) CSH(Sr) and (B) CS-C-CSH(Sr) at the same magnification, and (C) CS-C-CSH(Sr) at lower magnification showing the size distribution of the microcapsules.

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2.7.1.2. Smear observation. The slides were viewed using a light microscope (BX53F, Olympus, Japan) at low magnification to select areas showing evenly distributed and well-stained cells. The cells were then viewed and counted using the oil immersion lens, with polychromatic erythrocytes (PCE) appearing grayish blue and eosinophilic normochromatic erythrocytes (NCE) appearing orange. 2.7.1.3. Toxicity assessment index. The frequency of micronucleated polychromatic erythrocytes (MPCE) was used as an indicator of genetic toxicity, which was calculated for each animal as the number of MPCE present among 1000 PCE observed. Genetic toxicity was also evaluated by calculating the PCE/NCE ratio from 200 erythrocytes in each animal, where the PCE/NCE ratio for the experimental group should not be lower than 20% of the value for the control groups.

Fig. 4. Release of strontium ions into solution over 12 weeks for CS-C-CSH(Sr), CSH(Sr) and CSH.

2.7. Evaluation of in vivo biocompatibility All experimental protocols were approved by the Institutional Animal Ethics Committee. All animals were provided by the Southern Medical University Experimental Animal Center. 2.7.1. Genetic toxicity test Genetic toxicity of the microcapsules was tested using 30 Kunming mice (20–25 g). Three groups were tested: material extracts of CS-CCSH(Sr) at a concentration of 0.2 g/mL (experimental group), 0.9% saline (negative control group), and cyclophosphamide (positive control group), with each group being tested in 5 male animals and 5 female animals. The samples were administered in two doses by intraperitoneal injection. All animals received injections of the first dose almost simultaneously and the second dose was administered after 24 h. The same dosage was used for the two doses, which was 20 mL/kg body weight for the experimental and negative control groups and 40 mL/kg body weight for the positive control group. Injection volume was accurate to 0.01 mL. The animals were sacrificed 18 h after administration of the second dose. 2.7.1.1. Preparation of bone marrow smears. The bilateral femurs of each animal were excised and attached muscles removed. The femoral head and femoral condyles were removed from each femur to expose both ends of the marrow cavity. The bone marrow was removed by suction using a syringe containing 1 mL FBS and made into a cell suspension inside a centrifuge tube by mixing with a dropper pipette. The cell suspension was centrifuged at 1000 rpm for 10 min, after which the supernatant was removed. One drop of the remaining suspension was smeared onto a microscope slide and dried over an alcohol lamp. The slide was fixed in methanol solution for 10 min and then stained with 10% Giemsa dye solution for 10 min. The slide was washed several times with PBS, rinsed with distilled water and dried.

2.7.2. Intramuscular implantation test The CS-C-CSH(Sr) microcapsules were processed into cylinders of 3 mm diameter by 10 mm height for intramuscular implantation in 9 Sprague Dawley rats (180–200 g). Hair around the spine region of the animals was shaved 24 h prior to operation. During surgery under general anaesthesia (induced by intraperitoneal injection of 3% sodium pentobartital at 30 mg/kg body weight) and aseptic conditions, bilateral intramuscular pockets were created in each animal approximately 1 cm from the midline of the spine. The experimental group was implanted into the left side while the right side was a sham control. After surgery, the animals were injected with gentamicin for 3 days to prevent infection. The animals were sacrificed at 1, 4 and 12 weeks with 3 animals being sacrificed at each time point. 2.7.2.1. Histological analysis. Tissue samples were harvested from each animal at the bilateral operation sites, embedded in paraffin and cut into 6 μm thick sections using a microtome. The sections were stained with hematoxylin and eosin (H&E) and mounted to slides for observation using a light microscope. 2.8. Statistical analysis Data for all experiments were obtained from at least 3 independent samples unless otherwise specified. All data were expressed as mean ± standard deviation and analysed using SPSS13.0 statistical analysis software. ANOVA was used for comparisons between multiple groups. Differences were considered as significant for p b 0.05. 3. Results 3.1. Physical properties of the samples The XRD patterns of CS-C-CSH(Sr) and CSH(Sr) showed characteristic peaks at 2θ = 14.75, 25.71, 29.76 and 31.91 which corresponded to the (110), (310), (220) and (−114) crystal planes of α-calcium sulfate hemihydrate crystals (Fig. 2). The peak intensity of CS-C-CSH(Sr) was weaker than that of CSH(Sr). XRD analysis indicated that the crystal structure of CSH(Sr) was preserved after formation of the CS-C-CSH (Sr) microcapsules. SEM examination showed that the CSH(Sr) crystals

Fig. 5. The antibacterial properties of CS, CS-C-CSH(Sr) and CSH(Sr) were tested by culturing E. coli on the surface of the materials and comparing the number of colonies generated (right dish) to the negative control which was grown in culture medium (left dish).

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were rod-like in appearance with relatively uniform length averaging 50–70 μm (Fig. 3A). In comparison, the CS-C-CSH(Sr) microcapsules consisted of bundles of CSH(Sr) crystals encapsulated in a chitosan shell, which were spherical-shaped and averaged 120 μm in diameter (Fig. 3B), with a relatively uniform size distribution (Fig. 3C).

3.2. Degradation and ion release test The release of strontium ions from CSH(Sr) over 12 weeks followed a logarithmic relation, with rapid release of over 50% of the strontium ions into solution during the first week and progressively more limited amounts being released during the remaining 11 weeks (Fig. 4). In contrast, the release of strontium ions from CS-C-CSH(Sr) over 12 weeks followed a linear relation, with the release rate remaining relatively constant over the entire 12 week period. No strontium was found in α-calcium sulfate hemihydrate (CSH) over the 12 weeks except error.

3.4. Evaluation of in vitro biocompatibility After culturing for 72 h, cells in the material extract groups and negative control group exhibited fibroblast-like morphology with good spreading and growth, in contrast to the positive control group where the cells appeared to have diminished volume, rounded morphology and lack of spreading and multiplication (Fig. 6A). At each time point, there were no significant differences in cell proliferation between any of the material extract groups and the negative control group, while the positive control group showed significantly lower cell proliferation compared to all other groups (Fig. 6B). The overall cytotoxicity at 72 h was Class 0 for the material extract groups and negative control group, and Class III for the positive control group (Table 1). The results indicated that material extracts of CS-C-CSH(Sr) had no cytotoxic effects and did not affect the normal proliferation of L-929 fibroblast cells.

3.5. Evaluation of in vivo biocompatibility 3.3. Antibacterial test The antibacterial properties of chitosan (CS), CS-C-CSH(Sr) and CSH (Sr) were demonstrated by comparing the amount of E. coli resulting from culture on the surface of each of the materials compared to the negative control (culture medium) (Fig. 5). CS exhibited the best antibacterial properties as shown by an almost complete absence of E. coli colonies in the agar plate (99.2% antibacterial rate), indicating that chitosan could effectively inhibit bacterial growth. In contrast, CSH(Sr) had no effect in inhibiting bacterial growth as shown by the large amount of E. coli colonies (0% antibacterial rate), which had similar appearance to the negative control. The CS-C-CSH(Sr) exhibited good inhibition of bacterial growth as only a few isolated colonies were visible in the agar plate (92.0% antibacterial rate), indicating that the beneficial antibacterial properties of chitosan were preserved in the CS-C-CSH(Sr) microcapsules.

3.5.1. Genetic toxicity test All animals survived until the time of sacrifice after both doses of the tested groups were administered. Genetic toxicity was evaluated by determining the frequency of MPCE and PCE/NCE ratio for the tested groups (Table 2). In both male and female animals, the material extract group showed no significant differences in the frequency of MPCE when compared to the negative control group, and there was also no evidence of dose-dependent changes. The positive control group, consisting of cyclophosphamide which is known to cause chromosomal aberrations in mice, showed significantly higher frequency of MPCE compared to the other two groups. In both male and female animals, the PCE/NCE ratio of the material extract group showed no significant changes when compared to the control groups, and was also not lower than 20% of the values for the control groups. The results indicated that material extracts of CS-C-CSH(Sr) did not induce in vivo genetic toxicity in male and female mouse bone marrow.

Fig. 6. (A) Morphology of cells cultured for 72 h on the experimental (100% material extract of CS-C-CSH(Sr)), negative control (culture medium) and positive control (0.64% phenol) groups; scale bar = 100 μm. (B) Proliferation of L-929 fibroblast cells cultured on the experimental and control groups for 24, 48 and 72 h.

Y. Chen et al. / Materials Science and Engineering C 66 (2016) 84–91 Table 1 Results of cytotoxicity evaluation after 72 h of culture.

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Table 2 Results of in vivo genetic toxicity evaluation, *p b 0.05.

Groups

Absorbance at 490 nm

RGR (%)

Cytotoxicity

Animals

Groups

Frequency of MPCE

PCE/NCE ratio

Negative control 100% material extract 75% material extract 50% material extract 25% material extract Positive control

0.145 ± 0.013 0.237 ± 0.051 0.216 ± 0.031 0.200 ± 0.027 0.181 ± 0.039 0.069 ± 0.015

100 163 149 138 125 48.1

Class 0 Class 0 Class 0 Class 0 Class 0 Class III

Male

Material extract Negative control Positive control Material extract Negative control Positive control

0.22 0.20 3.14* 0.12 0.08 2.92*

1.12 1.06 0.98 1.08 1.04 0.92

3.5.2. Intramuscular implantation test All animals survived until the time of sacrifice with no adverse events. The animals exhibited normal movement and feeding behaviour after operation, with no redness, effusion or implant extrusion observed at the wound site. Capsule formation was not noted at 1 week after operation but became visible at 4 and 12 weeks. Histological examination showed that at 1 week after operation, there were lymphocytic infiltration and the presence of macrophages around the implant but no visible fibrous capsule formation (Fig. 7A). At 4 weeks after operation, some lymphocytes and multinucleated giant cells were found surrounding the implant, with evidence of fibroblast proliferation and fibrous capsule formation (Fig. 7B). At 12 weeks after operation, few lymphocytes were visible around the implant and fibrous capsule formation was complete (Fig. 7C). The sham controls showed normal tissue structures at each time point (data not shown). The results indicated that CS-CCSH(Sr) was well tolerated during intramuscular implantation in a rat model which confirmed its in vivo biocompatibility.

4. Discussion α-Calcium sulfate hemihydrate has excellent features as a bone substitute material as it is readily available, can undergo complete resorption without eliciting a significant inflammatory response, biocompatible and osteoconductive [6]. A primary mechanism by which calcium sulfate can accelerate bone formation is by supplying a high concentration of extracellular calcium ions upon degradation, which is known to have the effects of providing positive stimulation to osteoblasts [15,16] and inhibiting osteoclast formation [17]. However, a major concern regarding the clinical application of this material is its rapid degradation rate [18], as disappearance of the material before bone healing is complete may often result in the reappearance of smaller defects that prevent bone union at the defect site. Doping α-calcium sulfate hemihydrate with a small amount of strontium results in a new compound that can accelerate bone repair [10], by introducing a high local concentration of strontium ions at the defect site which act to promote bone formation and inhibit bone resorption [19]. Nevertheless, in order for strontiumdoped α-calcium sulfate hemihydrate to be translated for clinical applications, its rapid degradation rate must be adjusted to match the rate of new bone formation and also to facilitate the sustained, rather than rapid, release of strontium ions after implantation. A novel composite bone substitute was developed in this study by using chitosan to encapsulate strontium-doped α-calcium sulfate hemihydrate to form microcapsules with controlled degradation. Chitosan not only offers the unique combination of antibacterial properties, biocompatibility and biodegradability, but can also be easily fabricated into vehicles to control the release rate of drugs or improve the bioavailability of degradable materials [13,14]. Chitosan also has specific properties which are beneficial for applications in bone regeneration, as it is known to participate in wound healing and has the ability to induce or accelerate bone formation and osseointegration [20–22]. Through physical and chemical fabrication methods, CS-C-CSH(Sr) microcapsules were created with the intention of combining the beneficial properties of chitosan and strontium-doped α-calcium sulfate hemihydrate, in order to produce a composite bone substitute that better meets the clinical needs.

Female

XRD analysis showed that the incorporation of strontium ions into CSH(Sr) and its combination with chitosan to form CS-C-CSH(Sr) microcapsules did not alter the crystallisation behaviour of these materials when compared to simple α-calcium sulfate hemihydrate [23]. The lower peak intensity of CS-C-CSH(Sr) compared to CSH(Sr) was likely due to the masking effect of the chitosan shell in the CS-C-CSH(Sr) microcapsules which was important for their controlled degradation. SEM examination confirmed that the rod-like appearance of CSH(Sr), which was characteristic of α-calcium sulfate hemihydrate crystals [24], was converted into spherical microcapsules of CS-C-CSH(Sr) after the addition of chitosan. The relatively uniform size distribution of the CS-C-CSH(Sr) microcapsules was important for the controlled release of strontium ions. The preparation method described in this study could be used to reliably produce CS-C-CSH(Sr) microcapsules with defined characteristics. The CS-C-CSH(Sr) microcapsules exhibited favourable degradation characteristics over 12 weeks compared to CSH(Sr), with a linear release profile of strontium ions over the entire test period and mitigation of the burst release behaviour observed for CSH(Sr). Chitosan encapsulation was therefore an effective method of controlling the degradation rate of CSH(Sr). The resulting CS-C-CSH(Sr) microcapsules could be expected to consistently promote bone formation at the defect site through the sustained release of strontium ions, as well as degrade at a moderate rate corresponding to the rate of bone healing such that the material would be completely replaced by new bone at the end of the healing period. The benefits of using chitosan as the encapsulating material was demonstrated by an antibacterial test, where the presence of the chitosan shell resulted in the ability of CS-C-CSH(Sr) microcapsules to inhibit bacterial growth on the surface. As infection is a common complication of orthopaedic procedures and often occurs in conjunction with bone defects, the surface antibacterial properties of CS-C-CSH(Sr) microcapsules are advantageous for their clinical application. The interactions between a biomaterial and the human body result in complex physical, chemical and biological reactions. Biocompatibility is defined by the types of reactions elicited by a biomaterial and the body's tolerance to these reactions. Biocompatibility is particularly important for a biomaterial intended for orthopaedic applications, where the material should support normal cellular activity including molecular signalling systems at the defect site without any local or systemic toxic effects to the host tissue [25]. In this study, the in vitro and in vivo biocompatibility of the developed CS-C-CSH(Sr) microcapsules were evaluated by tests for cytotoxicity, genetic toxicity and intramuscular implantation. The CS-C-CSH(Sr) microcapsules showed absence of in vitro cytotoxicity and supported the viability of L-929 fibroblast cells, producing higher cell numbers than the negative control (cells grown in culture medium) at 72 h which was suggestive of their bioactivity. In vivo evaluation of genetic toxicity in a mouse model confirmed that the CS-C-CSH(Sr) microcapsules had minimal potential of causing mutations or chromosomal aberrations in bone marrow cells. In vivo evaluation of local toxicity was performed by intramuscular implantation of CS-C-CSH(Sr) microcapsules in a rat model. The infiltration of inflammatory cells observed at 1 week after operation was possibly due to the implant eliciting a foreign body reaction alongside the acute injury caused by surgical trauma. The inflammatory response had begun to subside by 4 weeks after operation and had ceased to progress at 12 weeks. At the latest time point, the implant was surrounded by a

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Fig. 7. H&E staining images of tissue samples harvested around intramuscular implants of CS-C-CSH(Sr) in a rat model at (A) 1 week, (B) 4 weeks, and (C) 12 weeks after operation; scale bar = 50 μm.

fibrous capsule and only a small amount of inflammatory cells, indicating that the CS-C-CSH(Sr) microcapsules produced interactions with the living tissue that were well-tolerated and resulted in the reorganisation of tissue around the implant to reach a stable state. The in vitro and in vivo evaluations performed in this study confirmed the feasibility of developing CS-C-CSH(Sr) microcapsules for clinical application, and active investigations are in progress to assess their performance in larger in vivo models and through implantations at orthotopic sites. The combination of chitosan and α-calcium sulfate hemihydrate to form composite bone substitutes has been explored in other studies, including chitosan-coated α-calcium sulfate hemihydrate pellets [26], coated pellets loaded with antibiotics [27], and coated pellets loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) [28–30]. The composite pellets composed of chitosan coating and calcium sulfate were found to be more effective at facilitating new bone formation compared to monolithic pellets of chitosan or calcium sulfate [26,29] in rabbit models. Composite pellets containing antibiotic showed significant antibacterial effects in a rabbit model of osteomyelitis, and the independent therapeutic benefits of chitosan itself irrespective of the presence of antibiotic was also noted [27]. In a rabbit radial segmental defect model, the composite pellets showed slower resorption compared to monolithic calcium sulfate pellets that coincided closely with the growth rate of new bone, and the outcome of osteogenesis was further enhanced by the inclusion of rhBMP-2 [28–30]. Although the bone repair performance of the CS-C-CSH(Sr) microcapsules developed in this study remains to be confirmed at orthotopic sites in a clinically relevant large animal model, similar outcomes of improved biodegradation and enhanced osteogenesis can be anticipated based on the results of other studies. Furthermore, the CSC-CSH(Sr) microcapsules have potential to achieve even better outcomes for two main reasons: 1) they contain strontium-doped αcalcium sulfate hemihydrate instead of α-calcium sulfate hemihydrate, which can promote osteogenesis to a greater extent due to the release of strontium ions, and 2) their size distribution is in the micron-scale, which can facilitate more uniform degradation and ion release, provide much greater surface area for cell interaction, and allow better packing into the defect compared to macroscopic composite pellets with size distribution in the millimetre-scale. Similar drug loading strategies as those described in other studies can also be employed to further enhance the antibacterial properties and osteogenic ability of the CS-CCSH(Sr) microcapsules, in order to increase their suitability for applications in the clinical treatment of bone defects and/or osteomyelitis. The capacity to use the CS-C-CSH(Sr) microcapsules as delivery vehicles for the sustained release of antibiotics or osteogenic growth factors in addition to strontium ions will be investigated in future studies. 5. Conclusions Microcapsules of chitosan and strontium-containing α-calcium sulfate hemihydrate were developed in this study as a novel composite

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