Bio-Medical Materials and Engineering 24 (2014) S63–S73 DOI 10.3233/BME-140975 IOS Press

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Chitosan/hydroxyapatite hybrid scaffold for bone tissue engineering V. Brun a,b , C. Guillaume a,c , S. Mechiche Alami a,c , J. Josse a,d , J. Jing a,c , F. Draux a,c , S. Bouthors a,c , D. Laurent-Maquin a,c,e , S.C. Gangloff a,d , H. Kerdjoudj a,c,∗ and F. Velard a,c,f,∗,∗∗ a

EA 4691 «Biomatériaux et Inflammation en Site Osseux», Pôle Santé, SFR CAP-Santé (FED 4231), Université de Reims Champagne-Ardenne, Reims, France b Département de Chirurgie Orthopédique et Traumatologie, CHU de Reims, Reims, France c UFR d’Odontologie, URCA, Reims, France d UFR de Pharmacie, URCA, Reims, France e Département de Chirurgie Dentaire, CHU de Reims, Reims, France f Plateforme d’Imagerie Cellulaire et Tissulaire, URCA, Reims, France Abstract. BACKGROUND: To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of such techniques remain the availability of the autologous material and the second surgical site, inducing pain and morbidity. OBJECTIVE: Our aim was to investigate the biocompatibility in vitro of three dimensions hybrid biodegradable scaffolds combining osteoconductive properties of hydroxyapatite and anti-inflammatory properties of chitosan. METHODS: Hybrid scaffolds were characterized by microscopic observations, equilibrium swelling ratio and overtime weight loss measurements. In vitro studies were performed using primary human bone cells cultured for 7, 14 and 21 days. Cell viability, proliferation, morphology and differentiation through alkaline phosphatase (ALP) activity measurement were assessed. RESULTS: Characterization of our scaffolds demonstrated porous, hydrophilic and biodegradable characteristics. In vitro studies showed that these scaffolds have induced slight decrease in cell death and proliferation comparing to the culture plastic substrate control condition, as well as increased short term osteoinductive properties. CONCLUSIONS: In this study, we have provided evidence that our hybrid hydroxyapatite/chitosan scaffolds could be suitable for bone filling. Keywords: Hydroxyapatite, three dimensions hybrid scaffold, human primary bone cells

1. Introduction Long bone trauma can induce critical loss of bone tissue inducing an abnormal bone consolidation. The consolidation of critical bone defects, in particular in shaft, remains a major clinical challenge. Several techniques are used for the bone regeneration with a common objective: site and function restoration. In critical bone defects, such restoration is not possible using solely autologous grafts, very commonly *

Authors have contributed equally to the work. Address for correspondence: Dr. Frédéric Velard, EA 4691 «Biomatériaux et Inflammation en Site Osseux», Pôle Santé, SFR Cap-Santé (FED 4231), Université de Reims Champagne-Ardenne, 51 Rue Cognacq Jay, 51095 Reims Cedex, France. Tel.: +33 3 26 91 80 10; Fax: +33 3 26 91 86 43; E-mail: [email protected]. **

0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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used nowadays for tissue regeneration. Indeed, the bioavailability of autologous graft material does not allow the filling of longitudinal defects of more than five centimeters [1]. To overcome the limited availability of autologous graft, bone regeneration can be supported using bone substitutes. Hydroxyapatite (HAp), mostly used in orthopaedic surgery, is a calcium-phosphate material known to be the major constituent of bone mineral matrix, exhibiting osteoconductive properties [2]. Nevertheless, a limitation of its use remains its tendency to fragment and trigger inflammatory reactions, i.e. through the recruitment and the activation of inflammatory cells such as polynuclear neutrophils and monocytes [3,4]. In order to develop more efficient biomaterials for bone tissue regeneration, the use of chitosan polymer could be of interest mainly due to its biocompatibility, biodegradability and great bioavailability. In addition chitosan has anti-inflammatory capabilities, thus rendering it a suitable candidate for synthesis of hybrid scaffolds containing HAp particles [5,6]. The aim of this study is to synthesize novel, biodegradable porous scaffolds for tissue regeneration by combining the osteoconductive properties of HAp and anti-inflammatory effect of the chitosan and to assess their biocompatibility in vitro using primary human bone cells.

2. Materials and methods 2.1. Preparation of chitosan and hydroxyapatite scaffolds Chitosan of medium molecular weight (Sigma Aldrich) was dissolved (2% w/v) in 0.1 N acetic acid solution (Sigma Aldrich). Beta-glycerophosphate (β-GP, 4% w/v) (Sigma Aldrich) was added into chitosan solution, stirred to dissolve then placed at 37◦ C for 30 min to obtain a hydrogel. Atop the formed hydrogel, one milligram of hydroxyapatite (Medipure® , Medicoat) was deposited into a thin layer then covered by 500 µl of chitosan/β-GP solution and gelled at 37◦ C for 20 min. Thermogelled hybrid hydrogel was freeze-dried to generate porosity. Porous hybrid chitosan/hydroxyapatite (Chitosan + HAp) and chitosan (Chitosan) scaffolds were used thereafter. 2.2. Optical microscopy The surface of the freeze-dried scaffolds (before and after rehydration) was analyzed in 3D by digital optical microscopy with a VHX-2000 microscope at a 400× magnification (Keyence). 2.3. Water absorption To determine hydrophilicity of the scaffolds, the equilibrium swelling ratio (ESR) was calculated using dry (Wd ) and wet (Ww ) weights according to the following equation: Wa % =

Ww − Wd × 100. Wd

2.4. Scaffold degradation The weight loss of the scaffolds was evaluated after 7, 14 and 21 days of immersion in cell culture medium (DMEM + Glutamax, Life Technologies) for Chitosan and Chitosan + HAp. The samples were

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incubated at 37◦ C for the indicated period (t) and the weight loss (Wl ) was calculated after freeze-drying using the following equation with t = 0 as the reference (W0 ): Wl % =

Wt − W0 × 100. W0

2.5. Cells Human primary bone cells used in this study were obtained from patients bone explants as follows [7]. Bone explants were obtained from the femoral heads of six patients in the orthopaedic and traumatology department of the University Hospital Center (CHU) of Reims, France. Samples were collected after written informed consent was given by the donors following the guidelines approved by CHU institutional review board. The explants were cut into small pieces, washed in phosphate-buffered saline (PBS) four times for five minutes, digested in a solution of 0.5% trypsin, 5.3 mM EDTA (Life Technologies) and then in type II collagenase (1.4 mg/ml, Sigma Aldrich). The fragments obtained were placed in 25 cm2 culture flasks containing DMEM supplemented with 20% fetal calf serum (FCS) and 0.5% penicillin (10,000 IU/ml)/streptomycin sulfate (10,000 µg/ml) (Life Technologies) solution and incubated at 37◦ C in a 5% CO2 humidified atmosphere. In all experiments cells in the third passage were used. Bone cells were seeded at a density of 20,000 cells per well in 24 well culture plates (BD Falcon) which contained Thermanox® coverslips (control, Thermo Scientific), or one milligram of HAp, Chitosan or Chitosan + HAp scaffolds for 7, 14 and 21 days without medium renewal. 2.6. Cell death Cell death was determined by measuring lactate dehydrogenase (LDH) activity in cell culture supernatant according to the manufacturer’s protocol (kit Biovision # K311-400). The absorbance was read at 555 nm. LDH activity was correlated with the cell number using a standard curve of cell lysates. 2.7. Cell proliferation Cell proliferation was assessed by DNA content assay (Epicentre). Total DNA was isolated from the cultures according to the protocol provided in the MasterPureTM DNA Purification Kit (Epicentre). The concentration and purity of the DNA obtained were analyzed by spectrophotometry (Nanodrop® , Thermo Scientific) at 260 nm (A260 = one is equivalent to CADN = 50 ng/µl). 2.8. Scanning electron microscopy (SEM) SEM was performed to examine the morphology of bone cells. Cells were rinsed twice with PBS and then fixed with 2.5% glutaraldehyde (Sigma Aldrich) for one hour at room temperature. After gradual dehydration in ethanol (50, 70, 90 and 100 twice), the samples were immersed in hexamethyldizilazane (Sigma Aldrich) for five minutes, air dried at room temperature and finally sputtered with a thin gold– palladium film under a JEOL ion sputter JFC 1100. Cells were visualized using a LaB6 electron microscope (JEOL JSM-5400 LV, JEOL).

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2.9. Alkaline phosphatase activity assay Bone cells differentiation was evaluated by determining alkaline phosphatase (ALP) activity. Cells were lysed in buffer containing 0.06% Triton X-100, 1.2% Tris and 5.8% NaCl (w/v in distilled water, pH 10.2. All from Sigma Aldrich, pH 10.2). Then, 50 µl of sample were mixed with 50 µl of 0.0742% p-nitrophenylphosphate (w/v, Fisher Bioreagents) in AminoMethylPropanol (AMP, Acros Organics) buffer and incubated for two hours at 37◦ C. The absorbance was read at 400 nm with correction at 700 nm. The ALP activity was normalized to the amount of DNA and reported as ALP activity/µg of total DNA content. 2.10. Statistical analysis Statistical analyses were performed using an exact non-parametric and stratified permutation test (StatXact 7.0, Cytel Inc., Cambridge, MA, USA). Nonparametric statistics were used because of the few numbers of donors (n = 6), and the stratification allowed taking into account the impact of interdonor variability. Differences were considered significant at p < 0.05. 3. Results 3.1. Physico-chemical characterization of the matrices Cellular behaviour on a matrix depends in big part on the physical and morphological characteristics of the matrix. Therefore, we first aimed to determine the physical and morphological characteristics of the scaffolds, such as their surface morphology and porosity (Fig. 1). At the freeze-dried state, each scaffold contained pores that varied in size from tens to hundreds of micrometers in diameter, and tens of micrometers of depth to the maximum, without inter-connection (analysis was performed on the side of cracked scaffolds, data not shown), and some rugosity. Once rehydrated, the scaffolds did not present any porosity and formed a hydrogel-like structure in which also rugosity has decreased. HAp particles, which were only flush at the dried state, became highly visible at the surface of the rehydrated scaffold. Next, we determined the rehydration potential of the scaffolds following immersion in cell culture medium (DMEM). Chitosan scaffold exhibited a statistically increased ESR (mean = 170 ± 14%) compared to Chitosan + HAp scaffolds (mean = 104 ± 7%; p < 0.05; n = 8) (Fig. 2(A)). Finally, overtime degradation rate was assessed on both scaffolds. A passive degradation of scaffolds was observed at each time point and for each condition (following a single immersion). In fact, Chitosan scaffold exhibited a mean 55±2%, 58±5% and 63±2% loss of weight after 7, 14 and 21 days compared to first day synthesis, respectively. Addition of HAp to the scaffold resulted in a significant decrease in these values to 40 ± 3%, 40 ± 4% and 28 ± 6%, respectively (p < 0.05 for all three values) (Fig. 2(B)). 3.2. Evaluation of scaffolds’ cytotoxicity In order to evaluate the toxicity of the scaffolds, we proceeded to the measurement of LDH release by human primary bone cells in the culture supernatants. Apoptotic cells harboring membrane damage release LDH, and LDH accumulation in cell supernatants following a stimulus signifies the cytotoxicity of the latter. Following seven days of culture on both scaffolds, human primary bone cells demonstrated a modest increase in cell death (Fig. 3) (mean 1.53 fold) as compared to the control culture (Thermanox® )

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Fig. 1. Representative photographs of Chitosan (A)–(B) and Chitosan + HAp (C)–(D) scaffolds in freeze-dried (A), (C) or rehydrated state (B), (D) by digital optical microscopy (Keyence VHX-2000, magnification ×400). (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140975.)

(p < 0.05 for chitosan). However, no significant change in cell death was observed when cells were cultured in presence of HAp particles only. After 14 and 21 days of culture, no significant difference was detected in LDH release by bone cells in contact with HAp particles, Chitosan or Chitosan + HAp scaffolds. Of note in all tested conditions, we had noticed an accumulation of LDH overtime (data not shown). In order to know if the developed scaffolds could act as a proliferative support for bone cells, the cellular proliferation was studied through the DNA quantification in the samples (Fig. 4). We demonstrated that Thermanox® and HAp particles support cell proliferation (mean 1227 and 1762 ng of DNA, respectively) and the amounts of DNA were statistically higher in these conditions compared to both scaffolds at seven days (p < 0.05; mean 562 and 734 ng of DNA for Chitosan and Chitosan + HAp, re-

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Fig. 2. Measurement of the rehydration capabilities (ESR) of the scaffolds (n = 8) (A) and weight loss after 7, 14 and 21 days of incubation (n = 4 independent experiments in duplicate for each time point) (B). Red bar shows median value, lower and upper limits of each box show 1st and 9th decile, respectively. $ p < 0.05 compared to the Chitosan scaffold. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140975.)

Fig. 3. Cell death evaluation by measurement of the LDH concentration in cell supernatants (n = 6). Red bar shows median value, lower and upper limits of each box show first and ninth decile, respectively. ∗ p < 0.05 compared to control. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140975.)

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Fig. 4. Evaluation of the cellular proliferation by quantification of total DNA (n = 6). Red bar show median value, lower and upper limits of each box shows 1st and 9th decile, respectively. ∗ p < 0.05 compared to control. # p < 0.05 compared to HAp particles. $ p < 0.05 compared to the Chitosan scaffold. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140975.)

spectively). Interestingly, Chitosan + HAp exhibited a significant increase in cell number in comparison with Chitosan (p < 0.05) after seven days. After 14 days, a decrease in cell number was observed with both of the scaffolds (mean 789 and 641 ng of DNA for Chitosan and Chitosan + HAp, respectively) as compared to Thermanox® and HAp particles conditions (p < 0.05; mean 1559 and 1620 ng of DNA, respectively). After 21 days only Chitosan scaffold exhibited reduced DNA amounts (541 ng of total DNA) compared to control and HAp particles (p < 0.05; mean 1391 and 1418 ng of DNA for control and HAp particles, respectively). No statistical difference was observed between the amounts of DNA in Chitosan and Chitosan + HAp (883 ng of DNA). Of note, highest cell density was reached after seven days of culture when bone cells were in contact with HAp particles, after 14 days on Thermanox® or on Chitosan scaffold and after 21 days on Chitosan + HAp scaffold. 3.3. Cell morphology study After scaffolds biocompatibility assessment, SEM was used to assess the morphology and adhesion of bone cells on the scaffolds (Fig. 5). As early as seven days in culture, human primary bone cells in basal culture condition were flattened and well spread for the major part whereas some cells remained in fibroblast-like shape in control conditions. Importantly, it has been observed that bone cell climbed onto the HAp particles. On the Chitosan scaffold, cells kept a round morphology and were only seen at the bottom of the pores. Cell behaviour on Chitosan + HAp scaffold exhibited distinct responses to the substrate as they were round when no HAp particles were accessible and they were well adhered on such particles. After 14 days, on Thermanox® and in contact with HAp particles, cells reached the confluence whereas no progression could be noticed on the scaffolds. After 21 days, the only variation

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Fig. 5. Representative SEM micrographs showing the morphology of bone cells on Thermanox® (control), in presence of HAp particles or on Chitosan or Chitosan + HAp scaffolds (white scale bar = 50 µm).

observed was on the chitosan scaffold where some cells exhibited a bubbling surface which could be a sign of cellular suffering. 3.4. Study of alkaline phosphatise activity To emphasize the scaffolds ability to enhance bone cells differentiation, ALP activity was assessed in cells (Fig. 6). After seven days of culture, bone cells in contact with HAp particles had a decreased ALP activity compared to Thermanox® control condition (p < 0.05; mean 0.3 IU/ml/µg versus 0.47 IU/ml/µg, respectively) while cells cultured on the Chitosan and Chitosan + HAp scaffolds exhibited an increased ALP activity compared to the control and HAp particles conditions (p < 0.05 for both; mean 1.83 IU/ml/µg and 0.76 IU/ml/µg, respectively). After 14 days, no significant differences were seen in ALP activity between the different conditions. After 21 days, only Chitosan + HApcultured bone cells exhibited a reduced ALP activity as compared to HAp particles (p < 0.05; mean 1.21 IU/ml/µg versus 2.77 IU/ml/µg, respectively). Of note bone cells on Thermanox® and HAp demonstrated a time-dependent increase in ALP activity whereas cells on scaffolds maintained the same ALP activity over time (data not shown). 4. Discussion One of the major challenges of long bone defect regeneration is to provide to the patients scaffolds for bone filling which are at the same time biocompatible, porous, osteoconductive and osteoinductive hence supporting bone regeneration. Mixing a β-glycerophosphate salt to an acidic chitosan aqueous solution

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Fig. 6. Measurement of the ALP activity (n = 6). Red bar shows median value, lower and upper limits of each box show 1st and 9th decile, respectively. ∗ p < 0.05 compared to control. # p < 0.05 compared to HAp particles. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140975.)

induces chitosan gelation at 37◦ C [8]. This thermogelling process, which occurs by a combination of charge neutralization along with ionic and hydrogen bonding and hydrophobic interactions [9], has been used to develop injectable systems to locally deliver therapeutic agents [10]. In this study, we focused on development and characterization of freeze-dried chitosan/hydroxyapatite scaffolds. On one hand, the scaffolds developed here match with the physical criteria needed to guide bone tissue regeneration as they present pores of tens to hundreds of micrometers in diameter at a freeze-dried state, independently of the presence of HAp particles. On the other hand, rehydration induces a loss of porosity at the scaffolds’ surface to form a hydrogel-like structure whereas pores are preserved deeper in the scaffolds (biphoton confocal microscopy, data not shown). We hypothesized that bonds created during thermogelling process could be disturbed preferentially at the surface and preserved inside of the scaffold. As a consequence HAp particles seemed preserved and chitosan collapsed. It has already been demonstrated that porosity and hydration of hybrid scaffolds containing chitosan and hydroxyapatite may vary depending on the concentration of calcium phosphate [11,12]. This sustains our measure of ESR and weight loss, which highlight the fact that HAp particles induced a decrease in both of these parameters. Therefore, we propose the use of our reliable and cost effective thermogelling approach to obtain a wide panel of structural properties for scaffolds intended for bone regeneration. To address this application, we cultured human primary bone cells in contact with Chitosan and Chitosan + HAp scaffolds. We demonstrated that cellular proliferation peak was delayed on both of the scaffolds and that there was a slight increase in cell death only at early time point on Chitosan scaffolds. These results were consistent with SEM observations, which have demonstrated impaired cell distribution on Chitosan scaffolds (either at the surface or inside the scaffolds as demonstrated by SEM analysis, data not shown). HAp particles even when included in the polymer, acted as a support for

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cell adhesion. It has been proposed that porosity and stiffness of the biomaterials are prerequisites for bone cell proliferation inside such constructs [13]. Therefore, our data suggest that reduction of porosity observed immediately after rehydration, in combination with quite high degradation rate (up to 65%) of the scaffolds, could bear the responsibility for defective cellular distribution. Furthermore, the high biodegradability of our scaffolds could be due to the viscosity of the chitosan polymer we used herein [200,000 cps, 10 times greater than the ones frequently found in the literature (20,000 cps)] displaying potentially higher biological properties [14,15]. Moreover, the viscosity of the chitosan is linked to both molecular weight and degree of acetylation of the polymer, both known to greatly impact tissue regeneration using chitosan [16]. Concerning osteoinductive properties of the scaffolds, we have highlighted that bone cells’ ALP activity was increased when cells are cultured on both of the scaffolds at early time point even when compared with HAp particles, known to favour osteogenic phenotype [7]. This could be linked to the progressive release of the β-glycerophosphate during degradation of the scaffolds, which is widely used to stimulate bone cell-mediated mineralization processes. 5. Conclusion Based on in vitro analyses, this work has demonstrated that hybrids and porous scaffolds containing chitosan and HAp were a suitable matrix for bone tissue engineering. Moreover, the original and simple thermogelling then freeze-drying approach ensure an easy transfer to the orthobiological industries. Nevertheless, in vivo studies are needed in order to assess the suitability of our scaffolds for use in human bone grafts. Acknowledgements Author would like to thank the CHU de Reims for providing biological samples and especially K. Madi, MD and C. Mensa, MD, and URCA for funding V. Brun. We also warmly thank D. Al Alam, PhD (Developmental Biology and Regenerative Medicine Program, Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA, USA) for having carefully proof read this manuscript. References [1] A.J. Weiland, T.W. Phillips and M.A. Randolph, Bone graft: a radiological, histological and biomechanical model comparing auto grafts, free vascularized bone allografts and grafts, Plast. Reconstr. Surg. 74 (1984), 368. [2] J. Sales de Gauzy, F. Accadbled, A. Aziz, G. Knorr and P. Darodes, Résection-reconstruction dans les tumeurs osseuses maligne chez l’enfant, EMC Techniques Chirurgicales Orthopédie–Traumatologie 44 (2009), 100. [3] H. Benhayoune, E. Jallot, P. Laquerriere, G. Balossier, P. Bonhomme and P. Frayssinet, Integration of dense HA rods into cortical bone, Biomaterials 21 (2000), 235. [4] F. Velard, J. Braux, J. Amédée and P. Laquerrière, Inflammatory cell response to calcium phosphate biomaterial particles: An overview, Acta Biomater. 9 (2013), 4956. [5] A. Di Martino, M. Sittinger and V. Risbud Makarand, Chitosan: A versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005), 5983. [6] S. Seung-yun, P. Ho-Nam, K. Kyoung-Hwa and C. Chong-Pyoung, Biological evaluation of chitosan nanofiber membrane for guided bone regeneration, J. Periodontol. 76 (2005), 1778. [7] J. Braux, F. Velard, C. Guillaume, S. Bouthors, E. Jallot, J.M. Nedelec et al., A new insight into the dissociating effect of strontium on bone resorption and formation, Acta Biomater. 7 (2011), 2593.

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[8] A. Chenite, C. Chaput, D. Wang, C. Combes, M.D. Buschmann, C.D. Hoemann et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ, Biomaterials 21 (2000), 2155. [9] J. Cho, M.C. Heuzey, A. Bégin and P.J. Carreau, Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature, Biomacromolecules 6 (2005), 3267. [10] C. Gong, T. Qi, X. Wei, Y. Qu, Q. Wu, F. Luo and Z. Qian, Thermosensitive polymeric hydrogels as drug delivery systems, Curr. Med. Chem. 20 (2013), 79. [11] Y. Zhang and M. Zhang, Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering, J. Biomed. Mater. Res. 55 (2000), 304. [12] H. Qiaoling, L. Baoqiang, W. Mang and S. Jiacong, Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture, Biomaterials 25 (2004), 779. [13] A. Di Martino, M. Sittinger and M. Risbud, Chitosan: A versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005), 5983. [14] Z. Yong and Z. Miqin, Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants, J. Biomed. Mater. Res. 61 (2002), 301. [15] A. Abarrategi, Y. Morales, V. Ramos, A. Civantos, L. Dura, F. Marco et al., Chitosan scaffolds for osteochondral tissue regeneration, J. Biomed. Mater. Res. A 95 (2010), 1132. [16] G. Zigang, S. Baguenardb, L. Lima, A. Weec and E. Khorb, Hydroxyapatite–chitin materials as potential tissue engineered bone substitutes, Biomaterials 25 (2004), 1049.

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hydroxyapatite hybrid scaffold for bone tissue engineering.

To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of su...
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