Hydrothermal fabrication of hydroxyapatite/chitosan/carbon porous scaffolds for bone tissue engineering Teng Long,1* Yu-Tai Liu,2,3* Sha Tang,2 Jin-Liang Sun,3 Ya-Ping Guo,2 Zhen-An Zhu1 1
Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China 2 The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China 3 The Education Ministry Engineering Research Center of Materials Composition and Advanced Dispersion Technology, Shanghai University, Shanghai 200072, China Received 31 October 2013; revised 28 January 2014; accepted 13 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33151 Abstract: Porous carbon fiber felts (PCFFs) have great applications in orthopedic surgery because of the strong mechanical strength, low density, high stability, and porous structure, but they are biologically inert. To improve their biological properties, we developed, for the first time, the hydroxyapatite (HA)/ chitosan/carbon porous scaffolds (HCCPs). HA/chitosan nanohybrid coatings have been fabricated on PCFFs according to the following stages: (i) deposition of chitosan/calcium phosphate precursors on PCFFs; and (ii) hydrothermal transformation of the calcium phosphate precursors in chitosan matrix into HA nanocrystals. The scanning electron microscopy images indicate that PCFFs are uniformly covered with elongated HA nanoplates and chitosan, and the macropores in PCFFs still remain. Interestingly, the calcium-deficient HA crystals exist as plate-like shapes with thickness of 10–18 nm,
width of 30–40 nm, and length of 80–120 nm, which are similar to the biological apatite. The HA in HCCPs is similar to the mineral of natural bone in chemical composition, crystallinity, and morphology. As compared with PCFFs, HCCPs exhibit higher in vitro bioactivity and biocompatibility because of the presence of the HA/chitosan nanohybrid coatings. HCCPs not only promote the formation of bone-like apatite in simulated body fluid, but also improve the adhesion, spreading, and proliferation of human bone marrow stromal cells. Hence, HCCPs have great potentials as scaffold materials for bone tissue C 2014 Wiley Periodicals, Inc. J engineering and implantation. V Biomed Mater Res Part B: Appl Biomater 00B:000–000, 2014.
Key Words: porous carbon fiber felts, hydroxyapatite, hydrothermal method, bioactivity, biocompatibility
How to cite this article: Long T, Liu Y-T, Tang S, Sun J-L, Guo Y-P, Zhu Z-A. 2014. Hydrothermal fabrication of hydroxyapatite/ chitosan/carbon porous scaffolds for bone tissue engineering. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Bone tissue engineering is widely recognized as a substitute for autografts and allogeneic bone grafts, which combines the technology of material engineering and biological factors to regenerate damaged bone tissues.1–3 An ideal bone scaffold not only needs to have chemical composition similar to natural bone, but also have suitable porous structure and mechanical properties, which are especially important in the reconstruction of critical-sized bone defects in weightbearing locations. Natural bone is in fact a fiber-reinforced hybrid material, which consists of around 65 wt % mineral phase, 25 wt % organic fiber, and 10 wt % water.4,5 The main mineral phase of natural bone is hydroxyapatite (HA) crystals with thickness of 3210 nm and width and length
of 302200 nm.4,6 The corresponding synthetic HA has been widely used as bone filling material to repair injured bone tissues caused by trauma, disease, or genetic disorders, because it exhibits outstanding bioactivity, biocompatibility, and osteoconductivity.7–9 Unfortunately, pure HA scaffold is poor in mechanical properties, which limits its clinical application to the restoration of small non-weight-bearing locations.10–12 To improve the mechanical properties, a plausible strategy is to mimic the collagen fiber/HA hybrid structure of natural bone. Recently, fiber/HA composites have been fabricated by using poly(L-lactic acid) fibers,13 silk fibers,14 cellulose fibers,15 or carbon fibers16 as templates to induce the nucleation and growth of HA nanocrystals on their surfaces.
*Both authors contributed equally to this work. Correspondence to: Z.-A. Zhu (e-mail: [email protected]) or Y.-P. Guo (e-mail: [email protected]) Contract grant sponsor: Key Disciplines of Shanghai Municipal Education Commission; contract grant number: J50206 Contract grant sponsor: Natural Science Foundation of China; contract grant numbers: 51002095, 51372152, and 30973038 Contract grant sponsor: Science and Technology Commission of Shanghai Municipality; contract grant number: 12JC1405600 Contract grant sponsor: Program of Shanghai Normal University; contract grant numbers: DZL124 and DCL201303 Contract grant sponsor: Innovation Foundation of Shanghai Education Committee; contract grant number: 14ZZ124
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Among these reinforcing fibers, carbon fibers are attracting increasing attention because of good mechanical properties,16 light density,17 and cytocompatibility.18 Carbon fibers simulate dimensions of collagen fibers (0.128 lm in diameter) in natural bone.18 Tanaka et al.19 found that the Young’s moduli of the bulk polyacrylonitrile-based carbon fibers ranged from 200 to 500 GPa, and their axial shear modulus was about 20 GPa. The fractured force and fractured stress of high-tenacity carbon fibers may approach 21.10 cN and 4.72 GPa, respectively.20 Besides the good mechanical properties, carbon fibers do not cause rejection and have no toxic effects on cells.21 However, the biological inertness of carbon fibers hinders the extensive application in clinic, whereas HA has been confirmed to have excellent bioactivity. Therefore, HA/carbon fiber composite is a very promising biomaterial for bone tissue engineering, which combines the advantages of HA and carbon fibers. Many efforts have been put into the fabrication of HA/carbon fiber composite by plasma spraying technique,22 in situ polymerization with a later solution co-mixing approach,23 or electrochemical deposition.24 However, these synthetic scaffolds are different from natural bone in the chemical composition, microstructure, and biological property. Herein, we developed, for the first time, the HA/chitosan/carbon porous scaffolds (HCCPs) according to the following stages: (i) deposition of chitosan/calcium phosphate precursors in porous carbon fiber felts (PCFFs); and (ii) hydrothermal transformation of the calcium phosphate precursors in chitosan matrix into HA nanocrystals. The main aims of this study were to fabricate HCCPs, and to investigate the in vitro bioactivity by simulated body fluid (SBF) tests and the biocompatibility using human bone marrow stromal cells (hBMSCs) as the cell model. MATERIALS AND METHODS
Materials Biomedical-grade chitosan (viscosity-average molecular weight 3.4 3 105 g/mol) was supplied by the Qingdao Haihui Bioengineering Co., with 91% degree of deacetylation. PCFFs were supplied by Education Ministry Engineering Research Center of Materials Composition and Advanced Dispersion Technology, Shanghai University. The other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Preparation of HCCPs 1.0 g of chitosan powder was dissolved in 100 mL of acetic acid solution (2 wt%) under vigorous agitation to obtain a homogeneous solution. 2.3615 g of Ca(NO3)24H2O and 0.9361 g of NaH2PO42H2O were added into the chitosan/ acetic acid solution at room temperature. The PCFFs (12 mm 3 12 mm 3 0.9 mm) were immersed into the above mixed solution for 5 min. After drying at 50 C for 48 h, the obtained samples were immersed in NaOH solution (5 wt %). The mixtures were sealed in autoclaves, and hydrothermal reaction was allowed to take place at 160 C for 24 h. Finally, the products (HCCPs) were washed with deionized water and then dried at 60 C for 48 h.
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Characterization The crystalline phases of samples were examined with X-ray powder diffraction (XRD; D/max-II B, Japan). The microstructure of samples was investigated by using transmission electron microscopy (TEM; CM200/FEG, Philips) and scanning electron microscopy (SEM; MX2600, CamScan). Fourier transform infrared (FTIR) spectra (Vector22, Bruker) were collected by using the KBr pellet technique. In vitro bioactivity of HCCPs SBF with ion concentrations approximately equal to those of human blood plasma is widely used for assessment of in vitro bioactivity of biomaterials.25 HCCPs were soaked in 25 mL of SBF and kept at 37 C. The SBF was prepared by dissolving the reagent-grade chemicals NaCl, NaHCO3, KCl, K2HPO43H2O, MgCl26H2O, CaCl2, Na2SO4, and (CH2OH)3CNH2 in deionized water and buffering it at pH 7.40 with hydrochloric acid. At given time point, specimens were removed from SBF, washed with deionized water, and dried at room temperature. Biocompatibility of HCCPs hBMSCs were isolated and expanded using a modification of standard methods as described previously.26 The study was approved by the Ethic Committee of the Ninth People’s Hospital of Shanghai Jiao Tong University. Cells were grown in complete alpha minimum essential medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Tauranga, New Zealand) and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL; Hyclone, Logan, UT) in a 37 C humidified atmosphere with 5% CO2. Cells at a passage from P2 to P4 were used for the following experiments. hBMSCs were seeded on the samples at a density of 1 3 104 cells/sample. The number of viable cells was measured with cell counting kit-8 (Dojindo, Kumamoto, Japan). At each time point, 100 lL of water-soluble tetrazolium-8 (CCK-8) solution was added to each well, and plates were incubated for 2 h at 37 C. 200 lL solution of each well was added into a new 96-well plate, and absorbance was measured at 450 nm by a microplate spectrophotometer (BioRad Laboratories, Hercules, CA). The cytoskeleton of hBMSCs on the samples was observed by double fluorescence staining. Briefly, after cultured at a density of 1 3 104 cells/sample for 24 h, samples were gently washed with PBS and maintained in 4% paraformaldehyde for 15 min, followed by immersing in 0.1% Triton X-100 solution for 15 min. Tetramethylrhodamine B isothiocyanate–phalloidin was used to stain the actin filaments of cells shown as red fluorescent light, and 40 ,6-diamidino-2-phenylindole was used to stain the nucleus of cells shown as blue fluorescent light. The cytoskeleton of hBMSCs was observed under laser scanning confocal microscope (Leica TCS SP2, Heidelberg, Germany). Statistical analysis Results were presented as means 6 SD. Statistical analysis was conducted by analysis of variance. All experiments were performed in triplicate. (*) denotes significant
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image reveals that HA crystals exhibit plate-like structure, which is in good agreement with the results of SEM observation [Figure 2(c)]. The elongated HA nanoplates are single crystals with the [01I¯] zone axis, as confirmed by the corresponding electron diffraction pattern [Figure 3(d)]. The lattice fringes with d spacing of 0.35 nm on the highresolution TEM image are corresponding to the interplanar spacing of (002) planes for hexagonal HA [Figure 3(c)]. The results of electron diffraction pattern and high-resolution TEM images demonstrate that the c-axis of HA is parallel to the long axis of the nanocrystals. The same crystal orientation is also observed in bone mineralization. Namely, HA in HCCPs is described as parallel nanocrystals with the c-axis aligned with the long axis.6 FIGURE 1. Photograph of PCFFs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
difference with p < 0.05, and (**) denotes significant difference with p < 0.01.
RESULTS AND DISCUSSION
Morphology of HCCPs Recently, three-dimensional fibrous artificial matrices with high porosity and high spatial interconnectivity have been widely applied for bone tissue engineering.27,28 PCFFs not only possess the advantages of carbon fibers such as excellent mechanical properties and light density, but also provide the three-dimensional porous structure. In the present work, PCFFs, which are composed of carbon fibers, serve as reinforcing materials (Figure 1). The cross-sections of carbon fibers in the felts have a smooth surface and uniform diameter of about 8 lm [Figure 2(a)]. To improve the biological properties, a layer of HA/chitosan nanohybrid composites was precipitated on PCFFs by hydrothermal method. HCCPs are composed of a core of carbon fibers and a shell of HA/ chitosan layers [Figure 2(b)]. Under the high-resolution SEM image, lots of HA nanocrystals are dispersed within the gellike chitosan [Figure 2(c)]. HA crystals exist as plate-like shapes with thickness of 10218 nm, width of 30240 nm, and length of 802120 nm, as confirmed by the TEM images [Figure 3(a,b)]. Interestingly, the morphology and particle size of HA crystals in HCCPs are similar to that of the biological apatite. In the natural calcified tissue, HA exhibits platelike structure with thickness of 3210 nm and width and length of 302120 nm.6 As shown in Figure 2(d), the corresponding energy dispersive X-ray spectroscopy (EDS) spectrum reveals that the chemical elements of HCCPs mainly include Ca, P, O, C, N, and Na. Ca, P, and O are derived from HA. Na may be due to the remained Na2HPO4 or the partial substitution of Ca21 ions in HA crystal lattice by Na1 ions. C is derived from chitosan and carbon fibers, and N is due to chitosan. The average Ca/P molar ratio of HA is about 1.36, which is lower than that of stoichiometric HA (1.67),29 indicating that HA in HCCPs is calcium deficient. HA crystals in HCCPs are characterized further by TEM imaging, as shown in Figure 3. The low-resolution TEM
Phase and structure of HCCPs The phases and structures of pure chitosan and samples scraped from HCCPs have been characterized by XRD patterns and FTIR spectra, as shown in Figures 4 and 5. Chitosan, a random copolymer of N-acetyl-D-glucosamine and Dglucosamine, is the partially de-acetylated derivative of chitin. The chitosan exhibits a broad peak at around 20 because it is a semicrystalline material [Figure 4(a)]. The functional groups in chitosan are characterized further by the FTIR spectrum [Figure 5(a)]. The band at 1589 cm21 is assigned to the NAH bending vibration overlapping the amide II vibration. CAN stretching vibrations occur at around 1030 cm21 and overlap the vibrations from the carbohydrate ring. The broad band at around 3417 cm21 is corresponding to stretching vibration of NAH and OH groups. ACH2 bending vibration occurs at about 1420 cm21.30–32 As we know, HA and chitosan possess good bioactivity, biocompatibility, and biodegradability; so, they are precipitated on the surface of PCFFs to improve the biological properties of PCFFs. Under the hydrothermal conditions, the chitosan/calcium phosphate precursors on PCFFs are converted to the elongated HA nanoplates within the chitosan matrix. Interestingly, chitosan remains on the surfaces of HCCPs [Figure 4(b)], as confirmed by the SEM images [Figure 2(c)]. Besides chitosan, the characteristic peaks due to HA crystals are detected in the XRD patterns [Figure 4(b)]. The sharp narrow diffraction peaks suggest that HA in HCCPs exhibits great crystallinity, although part of the Ca21 and PO432 ions in the HA crystal lattices are replaced by the impurities such as Na1, CO322, and HPO422 ions [Figures 2(d) and 5(b)]. The FTIR spectrum in Figure 5(b) indicates the characterized functional groups of both HA and chitosan. The intense absorption peak at 1030 cm21 is ascribed to the stretching vibration (v3) of the phosphate (PO432) groups, and the absorption peaks at 563 and 604 cm21 are ascribed to the bending vibration (v4) of the phosphate (PO432) groups.33 The characteristic band of B-type CO322 substitution is observed at 1420 cm21 (v3).34 The absorption band due to HPO422 at around 1130 cm21 indicates that the HA is calcium-deficient HA.35 The band at 3455 cm21 is corresponding to adsorbed water in HCCPs.36,37 The hydroxyl absorption bands at 636 and 3703 cm21 are
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FIGURE 2. SEM images of samples: (a) PCFFs; (b,c) HCCPs; (d) Reflects the corresponding EDS spectrum in (c).
characteristic of a typical HA FTIR spectrum.38 The weak bands at around 3571 cm21 are ascribed to OH group with weak interactions to the environment.36,37 Based on the results of FTIR spectra and EDS spectrum, HA crystals in HCCPs may be expressed as Ca102x2y/22z/2Naz(HPO4)x (PO4)62x2y(CO3)y(OH)22x. In addition, the characteristic bands at 1037, 1420, and 3417 cm21 due to chitosan are overlapped by those of HA crystals [Figure 5(b)]. Formation mechanism of HCCPs The fabrication of a bone scaffold, which is similar to natural bone in chemical composition, morphology, and macropore, remains a big challenge. Fortunately, researches on the chemical composition, microstructure, and biomineralization mechanism of natural bone provide some important clues to design and fabricate the ideal bone scaffold. Natural bone is the complex hierarchical composites, consisting of a mineral phase (HA) and a protein phase (mostly type 1 collagen).4–6 Recently, Olszta et al.4 proposed that HA crystals in mineralized collagenous tissues such as bone and dentin do not initially nucleate within hole zones, but rather an amor-
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phous calcium phosphate serves as precursor to create a highly loaded mineral/organic composite with HA nanocrystals existing within the collagen fiber matrix. In this study, HCCPs have been fabricated based on the above biomineralization mechanism. PCFFs served as the substrate of bone scaffolds, providing good mechanical property and porous structure. The chitosan/calcium phosphate precursors were then deposited on PCFFs. Under the hydrothermal conditions, HA nucleated within the chitosan matrix and grew into the plate-like structure. During the in situ crystallization of HA, the Na1, HPO422, and CO322 ions may enter into the crystal lattice, and replace part of the Ca21 and PO432 ions in HA to form the calcium-deficient HA [Figures 2(d) and 5(b)]. Moreover, chitosan plays an important role in the in situ formation of HA on PCFFs. First, chitosan holds back the diffusion of the calcium phosphate precursors into the NaOH solution because it does not dissolve in an alkaline solution; second, the amino groups and hydroxyl groups in chitosan may attract the calcium ions and phosphate ions via chelation and hydrogen bonding, and thus improve the nucleation and growth of HA within the chitosan matrix.
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FIGURE 3. (a,b) TEM images of HA in HCCPs; (c) High-resolution TEM image; (d) Electron diffraction pattern acquired in the region marked with a square in (b).
FIGURE 4. XRD patterns of the samples: (a) chitosan; (b) samples scraped from HCCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 5. FTIR spectra of the samples: (a) chitosan; (b) samples scraped from HCCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 6. SEM images of samples after soaking in SBF for 48 h: (a,b) PCFFs; (c,d) HCCPs.
Bioactivity of HCCPs A bioactive material is a material that may have apatite form on its surface after it is immersed in a serum-like solution and bonds to living bone through this apatite layer.39,40 Therefore, the formation of apatite on the surface of a material dipped in SBF is a proof of its bioactivity and can be used to anticipate its bone bonding ability in vivo, not only qualitatively but also quantitatively. In this study, the bioactivity of PCFFs and HCCPs has been investigated by examining apatite formation on the surfaces after soaking them in SBF for 48 h. As shown in Figure 6(a,b), no bone-like apatite precipitates were observed on the surface of PCFFs, suggesting that PCFFs possess poor in vitro bioactivity. On the contrary, lots of plate-like apatite particles deposited on the surface of HCCPs [Figure 6(c,d)]. EDS analyses revealed that the formed apatite was also calcium-deficient, with an average Ca/P molar ratio of 1.48. In view of the above, a conclusion could be made that HCCPs have great bioactivity, which may be attributed to the following reasons. First, the elongated HA nanoplates in HCCPs are calcium-deficient, and therefore possess greater bioactivity compared with stoichiometric HA.41,42 The dissolution of HA increases the super-
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saturation degree in SBF, and thus accelerates the nucleation and growth of bone-like apatite nanocrystals.36 Second, chitosan contains functional groups like the amino groups and hydroxyl groups [Figure 5(a)], which may create a favorable environment to induce the formation of bonelike apatite. Biocompatibility of HCCPs The biocompatibility of a scaffold for bone tissue engineering refers to the ability to perform as a substrate that will support the appropriate cellular activity, including the facilitation of cell spreading, proliferation, and differentiation, in order to optimize bone regeneration and bone remodeling, without eliciting any undesirable effects in those cells. Good biocompatibility is an important preferable attribute for bone scaffold material. Recently, Im et al.27 reported the biomimetic synthesis of three-dimensional nanocrystalline HA and single-walled carbon nanotube chitosan nanocomposite. The nanocrystalline HA and carbon nanotube in the nanocomposite not only enhance the mechanical properties of the scaffold, but also improve cytocompatibility for osteoblast adhesion and proliferation. In the present work, PCFFs
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FIGURE 7. CCK-8 assay results of hBMSCs cultured on PCFFs and HCCPs at different days. The data are presented as the means 6 SD; n 5 3. (*) denotes significant difference with p < 0.05, and (**) denotes significant difference with p < 0.01.
serve as reinforcing materials because they have many merits such as strong mechanical strength, light density, high stability, and porous structure.43 However, the biological inertness of PCFFs hinders their extensive application in clinical field. To improve the biological properties, HA/chitosan composites were deposited on PCFFs. hBMSCs were
seeded on HCCPs to evaluate the effects of HA/chitosan coating on the biocompatibility, with PCFFs as the control. First, the cell proliferation and viability were investigated by the CCK-8 assay. As shown in Figure 7, a similar trend of cell number increase in a time-dependent manner was observed for the two groups. It is noteworthy that more viable cells were observed on HCCPs than on PCFFs at each time point, which suggests that HA/chitosan coating may promote the adhesion and proliferation of hBMSCs. Then, an actin cytoskeleton and focal adhesion staining kit were used to examine the morphology of hBMSCs after culture on HCCPs for 24 h. The orientation of actin filaments was mapped with tetramethylrhodamine B isothiocyanate–phalloidin, and nuclei were labeled with 40 ,6-diamidino-2-phenylindole. The typical images of hBMSCs on HCCPs and PCFFs are shown in Figure 8. Compared with PCFFs, there are more viable cells present on HCCPs, and cells display better cell-spreading cytoskeleton morphology. Taken together, HCCPs have higher biocompatibility compared with PCFFs, which may be attributed to the following reasons. First, HA in HCCPs is highly biocompatible because of its similar chemical composition and crystallinity to the bone mineral, whereas PCFFs are biologically inert. Second, chitosan is well recognized as a suitable functional material because it has many good properties such as biocompatibility and biodegradability. The introduction of chitosan in
FIGURE 8. Typical images of hBMSCs cultured on different samples: (a–c) PCFFs; (d–f) HCCPs. The actin filaments stained as red fluorescent light, and the nucleus stained as blue fluorescent light. The images were observed under a laser scanning confocal microscopy.
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HCCPs may also promote the adhesion and proliferation of hBMSCs.
16.
CONCLUSIONS
An ideal bone scaffold needs to have good bioactivity, biocompatibility, suitable porous structure, and mechanical properties. In this work, HCCPs have been fabricated by the hydrothermal method according to the biomineralization mechanism of natural bone. The obtained HA nanocrystals are calcium-deficient and similar in size to the biological apatite. Because the HA/chitosan nanohybrid coatings are similar to natural bone in the chemical composition and microstructure, HCCPs exhibit higher in vitro bioactivity and biocompatibility compared with PCFFs. HCCPs not only promote the formation of bone-like apatite in SBF, but also improve the adhesion, spreading, and proliferation of hBMSCs, suggesting that they have great potentials as scaffold materials for bone tissue engineering and implantation. REFERENCES 1. Bose S, Roy M, Bandyopadhyay A. Recent advances in bone tissue engineering scaffolds. Trends Biotechnol 2012;30(10): 5462554. 2. Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari F. Nanostructured scaffolds for bone tissue engineering. J Biomed Mater Res A 2013;101(8):242422435. 3. Holzwarth JM, Ma PX. Biomimetic nanofibrous scaffolds for bone tissue engineering. Biomaterials 2011;32(36):962229629. 4. Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y-Y, Kaufman MJ, Douglas EP, Gower LB. Bone structure and formation: A new perspective. Mater Sci Eng R 2007;58(325):772116. 5. Nudelman F, Pieterse K, George A, Bomans PH, Friedrich H, Brylka LJ, Hilbers PA, de With G, Sommerdijk NA. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 2010;9(12):100421009. 6. Hassenkam T, Fantner GE, Cutroni JA, Weaver JC, Morse DE, Hansma PK. High-resolution AFM imaging of intact and fractured trabecular bone. Bone 2004;35(1):4210. 7. Lima PA, Resende CX, Soares GD, Anselme K, Almeida LE. Preparation, characterization and biological test of 3D-scaffolds based on chitosan, fibroin and hydroxyapatite for bone tissue engineering. Mater Sci Eng C Mater Biol Appl 2013;33(6):338923395. 8. Liu H, Peng H, Wu Y, Zhang C, Cai Y, Xu G, Li Q, Chen X, Ji J, Zhang Y, Ouyang HW. The promotion of bone regeneration by nanofibrous hydroxyapatite/chitosan scaffolds by effects on integrin-BMP/Smad signaling pathway in BMSCs. Biomaterials 2013; 34(18):440424417. 9. Luo Y, Wu C, Lode A, Gelinsky M. Hierarchical mesoporous bioactive glass/alginate composite scaffolds fabricated by threedimensional plotting for bone tissue engineering. Biofabrication 2013;5(1):015005. 10. De Groot K. Bioceramics of Calcium Phosphate. Boca Raton, FL: CRC Press; 1983. 11. LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev 2008;108(11):474224753. 12. Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop Relat Res 1981;157:2592278. 13. Yanagida H, Okada M, Masuda M, Narama I, Nakano S, Kitao S, Takakuda K, Furuzono T. Preparation and in vitro/in vivo evaluations of dimpled poly(L-lactic acid) fibers mixed/coated with hydroxyapatite nanocrystals. J Artif Organs 2011;14(4):3312341. 14. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006;27(16): 311523124. 15. Stanishevsky A, Chowdhury S, Chinoda P, Thomas V. Hydroxyapatite nanoparticle loaded collagen fiber composites: Microarchi-
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36. Badri A, Binet C, Lavalley J-C. An FTIR study of surface ceria hydroxy groups during a redox process with H2. J Chem Soc Faraday Trans 1996;92(23):466924673. 37. Panda RN, Hsieh MF, Chung RJ, Chin TS. FTIR, XRD, SEM and solid state NMR investigations of carbonate-containing hydroxyapatite nano-particles synthesized by hydroxide-gel technique. J Phys Chem Solids 2003;64(2):1932199. 38. Ye F, Guo H, Zhang H, He X. Polymeric micelle-templated synthesis of hydroxyapatite hollow nanoparticles for a drug delivery system. Acta Biomater 2010;6(6):221222218. 39. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006;27:290722915.
40. Bohner M, Lemaitre J. Can bioactivity be tested in vitro with SBF solution? Biomaterials 2009;30:217522179. 41. Valletregi M. Calcium phosphates as substitution of bone tissues. Prog Solid State Chem 2004;32(122):1231. 42. Spence G, Phillips S, Campion C, Brooks R, Rushton N. Bone formation in a carbonate-substituted hydroxyapatite implant is inhibited by zoledronate: The importance of bioresorption to osteoconduction. J Bone Joint Surg Br 2008;90(12): 163521640. 43. Wu M, Wang Q, Liu X, Liu H. Biomimetic synthesis and characterization of carbon nanofiber/hydroxyapatite composite scaffolds. Carbon 2013;51:3352345.
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Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China 2 The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China 3 The Education Ministry Engineering Research Center of Materials Composition and Advanced Dispersion Technology, Shanghai University, Shanghai 200072, China Received 31 October 2013; revised 28 January 2014; accepted 13 March 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33151 Abstract: Porous carbon fiber felts (PCFFs) have great applications in orthopedic surgery because of the strong mechanical strength, low density, high stability, and porous structure, but they are biologically inert. To improve their biological properties, we developed, for the first time, the hydroxyapatite (HA)/ chitosan/carbon porous scaffolds (HCCPs). HA/chitosan nanohybrid coatings have been fabricated on PCFFs according to the following stages: (i) deposition of chitosan/calcium phosphate precursors on PCFFs; and (ii) hydrothermal transformation of the calcium phosphate precursors in chitosan matrix into HA nanocrystals. The scanning electron microscopy images indicate that PCFFs are uniformly covered with elongated HA nanoplates and chitosan, and the macropores in PCFFs still remain. Interestingly, the calcium-deficient HA crystals exist as plate-like shapes with thickness of 10–18 nm,
width of 30–40 nm, and length of 80–120 nm, which are similar to the biological apatite. The HA in HCCPs is similar to the mineral of natural bone in chemical composition, crystallinity, and morphology. As compared with PCFFs, HCCPs exhibit higher in vitro bioactivity and biocompatibility because of the presence of the HA/chitosan nanohybrid coatings. HCCPs not only promote the formation of bone-like apatite in simulated body fluid, but also improve the adhesion, spreading, and proliferation of human bone marrow stromal cells. Hence, HCCPs have great potentials as scaffold materials for bone tissue C 2014 Wiley Periodicals, Inc. J engineering and implantation. V Biomed Mater Res Part B: Appl Biomater 00B:000–000, 2014.
Key Words: porous carbon fiber felts, hydroxyapatite, hydrothermal method, bioactivity, biocompatibility
How to cite this article: Long T, Liu Y-T, Tang S, Sun J-L, Guo Y-P, Zhu Z-A. 2014. Hydrothermal fabrication of hydroxyapatite/ chitosan/carbon porous scaffolds for bone tissue engineering. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Bone tissue engineering is widely recognized as a substitute for autografts and allogeneic bone grafts, which combines the technology of material engineering and biological factors to regenerate damaged bone tissues.1–3 An ideal bone scaffold not only needs to have chemical composition similar to natural bone, but also have suitable porous structure and mechanical properties, which are especially important in the reconstruction of critical-sized bone defects in weightbearing locations. Natural bone is in fact a fiber-reinforced hybrid material, which consists of around 65 wt % mineral phase, 25 wt % organic fiber, and 10 wt % water.4,5 The main mineral phase of natural bone is hydroxyapatite (HA) crystals with thickness of 3210 nm and width and length
of 302200 nm.4,6 The corresponding synthetic HA has been widely used as bone filling material to repair injured bone tissues caused by trauma, disease, or genetic disorders, because it exhibits outstanding bioactivity, biocompatibility, and osteoconductivity.7–9 Unfortunately, pure HA scaffold is poor in mechanical properties, which limits its clinical application to the restoration of small non-weight-bearing locations.10–12 To improve the mechanical properties, a plausible strategy is to mimic the collagen fiber/HA hybrid structure of natural bone. Recently, fiber/HA composites have been fabricated by using poly(L-lactic acid) fibers,13 silk fibers,14 cellulose fibers,15 or carbon fibers16 as templates to induce the nucleation and growth of HA nanocrystals on their surfaces.
*Both authors contributed equally to this work. Correspondence to: Z.-A. Zhu (e-mail: [email protected]) or Y.-P. Guo (e-mail: [email protected]) Contract grant sponsor: Key Disciplines of Shanghai Municipal Education Commission; contract grant number: J50206 Contract grant sponsor: Natural Science Foundation of China; contract grant numbers: 51002095, 51372152, and 30973038 Contract grant sponsor: Science and Technology Commission of Shanghai Municipality; contract grant number: 12JC1405600 Contract grant sponsor: Program of Shanghai Normal University; contract grant numbers: DZL124 and DCL201303 Contract grant sponsor: Innovation Foundation of Shanghai Education Committee; contract grant number: 14ZZ124
C 2014 WILEY PERIODICALS, INC. V
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Among these reinforcing fibers, carbon fibers are attracting increasing attention because of good mechanical properties,16 light density,17 and cytocompatibility.18 Carbon fibers simulate dimensions of collagen fibers (0.128 lm in diameter) in natural bone.18 Tanaka et al.19 found that the Young’s moduli of the bulk polyacrylonitrile-based carbon fibers ranged from 200 to 500 GPa, and their axial shear modulus was about 20 GPa. The fractured force and fractured stress of high-tenacity carbon fibers may approach 21.10 cN and 4.72 GPa, respectively.20 Besides the good mechanical properties, carbon fibers do not cause rejection and have no toxic effects on cells.21 However, the biological inertness of carbon fibers hinders the extensive application in clinic, whereas HA has been confirmed to have excellent bioactivity. Therefore, HA/carbon fiber composite is a very promising biomaterial for bone tissue engineering, which combines the advantages of HA and carbon fibers. Many efforts have been put into the fabrication of HA/carbon fiber composite by plasma spraying technique,22 in situ polymerization with a later solution co-mixing approach,23 or electrochemical deposition.24 However, these synthetic scaffolds are different from natural bone in the chemical composition, microstructure, and biological property. Herein, we developed, for the first time, the HA/chitosan/carbon porous scaffolds (HCCPs) according to the following stages: (i) deposition of chitosan/calcium phosphate precursors in porous carbon fiber felts (PCFFs); and (ii) hydrothermal transformation of the calcium phosphate precursors in chitosan matrix into HA nanocrystals. The main aims of this study were to fabricate HCCPs, and to investigate the in vitro bioactivity by simulated body fluid (SBF) tests and the biocompatibility using human bone marrow stromal cells (hBMSCs) as the cell model. MATERIALS AND METHODS
Materials Biomedical-grade chitosan (viscosity-average molecular weight 3.4 3 105 g/mol) was supplied by the Qingdao Haihui Bioengineering Co., with 91% degree of deacetylation. PCFFs were supplied by Education Ministry Engineering Research Center of Materials Composition and Advanced Dispersion Technology, Shanghai University. The other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Preparation of HCCPs 1.0 g of chitosan powder was dissolved in 100 mL of acetic acid solution (2 wt%) under vigorous agitation to obtain a homogeneous solution. 2.3615 g of Ca(NO3)24H2O and 0.9361 g of NaH2PO42H2O were added into the chitosan/ acetic acid solution at room temperature. The PCFFs (12 mm 3 12 mm 3 0.9 mm) were immersed into the above mixed solution for 5 min. After drying at 50 C for 48 h, the obtained samples were immersed in NaOH solution (5 wt %). The mixtures were sealed in autoclaves, and hydrothermal reaction was allowed to take place at 160 C for 24 h. Finally, the products (HCCPs) were washed with deionized water and then dried at 60 C for 48 h.
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Characterization The crystalline phases of samples were examined with X-ray powder diffraction (XRD; D/max-II B, Japan). The microstructure of samples was investigated by using transmission electron microscopy (TEM; CM200/FEG, Philips) and scanning electron microscopy (SEM; MX2600, CamScan). Fourier transform infrared (FTIR) spectra (Vector22, Bruker) were collected by using the KBr pellet technique. In vitro bioactivity of HCCPs SBF with ion concentrations approximately equal to those of human blood plasma is widely used for assessment of in vitro bioactivity of biomaterials.25 HCCPs were soaked in 25 mL of SBF and kept at 37 C. The SBF was prepared by dissolving the reagent-grade chemicals NaCl, NaHCO3, KCl, K2HPO43H2O, MgCl26H2O, CaCl2, Na2SO4, and (CH2OH)3CNH2 in deionized water and buffering it at pH 7.40 with hydrochloric acid. At given time point, specimens were removed from SBF, washed with deionized water, and dried at room temperature. Biocompatibility of HCCPs hBMSCs were isolated and expanded using a modification of standard methods as described previously.26 The study was approved by the Ethic Committee of the Ninth People’s Hospital of Shanghai Jiao Tong University. Cells were grown in complete alpha minimum essential medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Tauranga, New Zealand) and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL; Hyclone, Logan, UT) in a 37 C humidified atmosphere with 5% CO2. Cells at a passage from P2 to P4 were used for the following experiments. hBMSCs were seeded on the samples at a density of 1 3 104 cells/sample. The number of viable cells was measured with cell counting kit-8 (Dojindo, Kumamoto, Japan). At each time point, 100 lL of water-soluble tetrazolium-8 (CCK-8) solution was added to each well, and plates were incubated for 2 h at 37 C. 200 lL solution of each well was added into a new 96-well plate, and absorbance was measured at 450 nm by a microplate spectrophotometer (BioRad Laboratories, Hercules, CA). The cytoskeleton of hBMSCs on the samples was observed by double fluorescence staining. Briefly, after cultured at a density of 1 3 104 cells/sample for 24 h, samples were gently washed with PBS and maintained in 4% paraformaldehyde for 15 min, followed by immersing in 0.1% Triton X-100 solution for 15 min. Tetramethylrhodamine B isothiocyanate–phalloidin was used to stain the actin filaments of cells shown as red fluorescent light, and 40 ,6-diamidino-2-phenylindole was used to stain the nucleus of cells shown as blue fluorescent light. The cytoskeleton of hBMSCs was observed under laser scanning confocal microscope (Leica TCS SP2, Heidelberg, Germany). Statistical analysis Results were presented as means 6 SD. Statistical analysis was conducted by analysis of variance. All experiments were performed in triplicate. (*) denotes significant
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image reveals that HA crystals exhibit plate-like structure, which is in good agreement with the results of SEM observation [Figure 2(c)]. The elongated HA nanoplates are single crystals with the [01I¯] zone axis, as confirmed by the corresponding electron diffraction pattern [Figure 3(d)]. The lattice fringes with d spacing of 0.35 nm on the highresolution TEM image are corresponding to the interplanar spacing of (002) planes for hexagonal HA [Figure 3(c)]. The results of electron diffraction pattern and high-resolution TEM images demonstrate that the c-axis of HA is parallel to the long axis of the nanocrystals. The same crystal orientation is also observed in bone mineralization. Namely, HA in HCCPs is described as parallel nanocrystals with the c-axis aligned with the long axis.6 FIGURE 1. Photograph of PCFFs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
difference with p < 0.05, and (**) denotes significant difference with p < 0.01.
RESULTS AND DISCUSSION
Morphology of HCCPs Recently, three-dimensional fibrous artificial matrices with high porosity and high spatial interconnectivity have been widely applied for bone tissue engineering.27,28 PCFFs not only possess the advantages of carbon fibers such as excellent mechanical properties and light density, but also provide the three-dimensional porous structure. In the present work, PCFFs, which are composed of carbon fibers, serve as reinforcing materials (Figure 1). The cross-sections of carbon fibers in the felts have a smooth surface and uniform diameter of about 8 lm [Figure 2(a)]. To improve the biological properties, a layer of HA/chitosan nanohybrid composites was precipitated on PCFFs by hydrothermal method. HCCPs are composed of a core of carbon fibers and a shell of HA/ chitosan layers [Figure 2(b)]. Under the high-resolution SEM image, lots of HA nanocrystals are dispersed within the gellike chitosan [Figure 2(c)]. HA crystals exist as plate-like shapes with thickness of 10218 nm, width of 30240 nm, and length of 802120 nm, as confirmed by the TEM images [Figure 3(a,b)]. Interestingly, the morphology and particle size of HA crystals in HCCPs are similar to that of the biological apatite. In the natural calcified tissue, HA exhibits platelike structure with thickness of 3210 nm and width and length of 302120 nm.6 As shown in Figure 2(d), the corresponding energy dispersive X-ray spectroscopy (EDS) spectrum reveals that the chemical elements of HCCPs mainly include Ca, P, O, C, N, and Na. Ca, P, and O are derived from HA. Na may be due to the remained Na2HPO4 or the partial substitution of Ca21 ions in HA crystal lattice by Na1 ions. C is derived from chitosan and carbon fibers, and N is due to chitosan. The average Ca/P molar ratio of HA is about 1.36, which is lower than that of stoichiometric HA (1.67),29 indicating that HA in HCCPs is calcium deficient. HA crystals in HCCPs are characterized further by TEM imaging, as shown in Figure 3. The low-resolution TEM
Phase and structure of HCCPs The phases and structures of pure chitosan and samples scraped from HCCPs have been characterized by XRD patterns and FTIR spectra, as shown in Figures 4 and 5. Chitosan, a random copolymer of N-acetyl-D-glucosamine and Dglucosamine, is the partially de-acetylated derivative of chitin. The chitosan exhibits a broad peak at around 20 because it is a semicrystalline material [Figure 4(a)]. The functional groups in chitosan are characterized further by the FTIR spectrum [Figure 5(a)]. The band at 1589 cm21 is assigned to the NAH bending vibration overlapping the amide II vibration. CAN stretching vibrations occur at around 1030 cm21 and overlap the vibrations from the carbohydrate ring. The broad band at around 3417 cm21 is corresponding to stretching vibration of NAH and OH groups. ACH2 bending vibration occurs at about 1420 cm21.30–32 As we know, HA and chitosan possess good bioactivity, biocompatibility, and biodegradability; so, they are precipitated on the surface of PCFFs to improve the biological properties of PCFFs. Under the hydrothermal conditions, the chitosan/calcium phosphate precursors on PCFFs are converted to the elongated HA nanoplates within the chitosan matrix. Interestingly, chitosan remains on the surfaces of HCCPs [Figure 4(b)], as confirmed by the SEM images [Figure 2(c)]. Besides chitosan, the characteristic peaks due to HA crystals are detected in the XRD patterns [Figure 4(b)]. The sharp narrow diffraction peaks suggest that HA in HCCPs exhibits great crystallinity, although part of the Ca21 and PO432 ions in the HA crystal lattices are replaced by the impurities such as Na1, CO322, and HPO422 ions [Figures 2(d) and 5(b)]. The FTIR spectrum in Figure 5(b) indicates the characterized functional groups of both HA and chitosan. The intense absorption peak at 1030 cm21 is ascribed to the stretching vibration (v3) of the phosphate (PO432) groups, and the absorption peaks at 563 and 604 cm21 are ascribed to the bending vibration (v4) of the phosphate (PO432) groups.33 The characteristic band of B-type CO322 substitution is observed at 1420 cm21 (v3).34 The absorption band due to HPO422 at around 1130 cm21 indicates that the HA is calcium-deficient HA.35 The band at 3455 cm21 is corresponding to adsorbed water in HCCPs.36,37 The hydroxyl absorption bands at 636 and 3703 cm21 are
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FIGURE 2. SEM images of samples: (a) PCFFs; (b,c) HCCPs; (d) Reflects the corresponding EDS spectrum in (c).
characteristic of a typical HA FTIR spectrum.38 The weak bands at around 3571 cm21 are ascribed to OH group with weak interactions to the environment.36,37 Based on the results of FTIR spectra and EDS spectrum, HA crystals in HCCPs may be expressed as Ca102x2y/22z/2Naz(HPO4)x (PO4)62x2y(CO3)y(OH)22x. In addition, the characteristic bands at 1037, 1420, and 3417 cm21 due to chitosan are overlapped by those of HA crystals [Figure 5(b)]. Formation mechanism of HCCPs The fabrication of a bone scaffold, which is similar to natural bone in chemical composition, morphology, and macropore, remains a big challenge. Fortunately, researches on the chemical composition, microstructure, and biomineralization mechanism of natural bone provide some important clues to design and fabricate the ideal bone scaffold. Natural bone is the complex hierarchical composites, consisting of a mineral phase (HA) and a protein phase (mostly type 1 collagen).4–6 Recently, Olszta et al.4 proposed that HA crystals in mineralized collagenous tissues such as bone and dentin do not initially nucleate within hole zones, but rather an amor-
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phous calcium phosphate serves as precursor to create a highly loaded mineral/organic composite with HA nanocrystals existing within the collagen fiber matrix. In this study, HCCPs have been fabricated based on the above biomineralization mechanism. PCFFs served as the substrate of bone scaffolds, providing good mechanical property and porous structure. The chitosan/calcium phosphate precursors were then deposited on PCFFs. Under the hydrothermal conditions, HA nucleated within the chitosan matrix and grew into the plate-like structure. During the in situ crystallization of HA, the Na1, HPO422, and CO322 ions may enter into the crystal lattice, and replace part of the Ca21 and PO432 ions in HA to form the calcium-deficient HA [Figures 2(d) and 5(b)]. Moreover, chitosan plays an important role in the in situ formation of HA on PCFFs. First, chitosan holds back the diffusion of the calcium phosphate precursors into the NaOH solution because it does not dissolve in an alkaline solution; second, the amino groups and hydroxyl groups in chitosan may attract the calcium ions and phosphate ions via chelation and hydrogen bonding, and thus improve the nucleation and growth of HA within the chitosan matrix.
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FIGURE 3. (a,b) TEM images of HA in HCCPs; (c) High-resolution TEM image; (d) Electron diffraction pattern acquired in the region marked with a square in (b).
FIGURE 4. XRD patterns of the samples: (a) chitosan; (b) samples scraped from HCCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 5. FTIR spectra of the samples: (a) chitosan; (b) samples scraped from HCCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 6. SEM images of samples after soaking in SBF for 48 h: (a,b) PCFFs; (c,d) HCCPs.
Bioactivity of HCCPs A bioactive material is a material that may have apatite form on its surface after it is immersed in a serum-like solution and bonds to living bone through this apatite layer.39,40 Therefore, the formation of apatite on the surface of a material dipped in SBF is a proof of its bioactivity and can be used to anticipate its bone bonding ability in vivo, not only qualitatively but also quantitatively. In this study, the bioactivity of PCFFs and HCCPs has been investigated by examining apatite formation on the surfaces after soaking them in SBF for 48 h. As shown in Figure 6(a,b), no bone-like apatite precipitates were observed on the surface of PCFFs, suggesting that PCFFs possess poor in vitro bioactivity. On the contrary, lots of plate-like apatite particles deposited on the surface of HCCPs [Figure 6(c,d)]. EDS analyses revealed that the formed apatite was also calcium-deficient, with an average Ca/P molar ratio of 1.48. In view of the above, a conclusion could be made that HCCPs have great bioactivity, which may be attributed to the following reasons. First, the elongated HA nanoplates in HCCPs are calcium-deficient, and therefore possess greater bioactivity compared with stoichiometric HA.41,42 The dissolution of HA increases the super-
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saturation degree in SBF, and thus accelerates the nucleation and growth of bone-like apatite nanocrystals.36 Second, chitosan contains functional groups like the amino groups and hydroxyl groups [Figure 5(a)], which may create a favorable environment to induce the formation of bonelike apatite. Biocompatibility of HCCPs The biocompatibility of a scaffold for bone tissue engineering refers to the ability to perform as a substrate that will support the appropriate cellular activity, including the facilitation of cell spreading, proliferation, and differentiation, in order to optimize bone regeneration and bone remodeling, without eliciting any undesirable effects in those cells. Good biocompatibility is an important preferable attribute for bone scaffold material. Recently, Im et al.27 reported the biomimetic synthesis of three-dimensional nanocrystalline HA and single-walled carbon nanotube chitosan nanocomposite. The nanocrystalline HA and carbon nanotube in the nanocomposite not only enhance the mechanical properties of the scaffold, but also improve cytocompatibility for osteoblast adhesion and proliferation. In the present work, PCFFs
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FIGURE 7. CCK-8 assay results of hBMSCs cultured on PCFFs and HCCPs at different days. The data are presented as the means 6 SD; n 5 3. (*) denotes significant difference with p < 0.05, and (**) denotes significant difference with p < 0.01.
serve as reinforcing materials because they have many merits such as strong mechanical strength, light density, high stability, and porous structure.43 However, the biological inertness of PCFFs hinders their extensive application in clinical field. To improve the biological properties, HA/chitosan composites were deposited on PCFFs. hBMSCs were
seeded on HCCPs to evaluate the effects of HA/chitosan coating on the biocompatibility, with PCFFs as the control. First, the cell proliferation and viability were investigated by the CCK-8 assay. As shown in Figure 7, a similar trend of cell number increase in a time-dependent manner was observed for the two groups. It is noteworthy that more viable cells were observed on HCCPs than on PCFFs at each time point, which suggests that HA/chitosan coating may promote the adhesion and proliferation of hBMSCs. Then, an actin cytoskeleton and focal adhesion staining kit were used to examine the morphology of hBMSCs after culture on HCCPs for 24 h. The orientation of actin filaments was mapped with tetramethylrhodamine B isothiocyanate–phalloidin, and nuclei were labeled with 40 ,6-diamidino-2-phenylindole. The typical images of hBMSCs on HCCPs and PCFFs are shown in Figure 8. Compared with PCFFs, there are more viable cells present on HCCPs, and cells display better cell-spreading cytoskeleton morphology. Taken together, HCCPs have higher biocompatibility compared with PCFFs, which may be attributed to the following reasons. First, HA in HCCPs is highly biocompatible because of its similar chemical composition and crystallinity to the bone mineral, whereas PCFFs are biologically inert. Second, chitosan is well recognized as a suitable functional material because it has many good properties such as biocompatibility and biodegradability. The introduction of chitosan in
FIGURE 8. Typical images of hBMSCs cultured on different samples: (a–c) PCFFs; (d–f) HCCPs. The actin filaments stained as red fluorescent light, and the nucleus stained as blue fluorescent light. The images were observed under a laser scanning confocal microscopy.
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HCCPs may also promote the adhesion and proliferation of hBMSCs.
16.
CONCLUSIONS
An ideal bone scaffold needs to have good bioactivity, biocompatibility, suitable porous structure, and mechanical properties. In this work, HCCPs have been fabricated by the hydrothermal method according to the biomineralization mechanism of natural bone. The obtained HA nanocrystals are calcium-deficient and similar in size to the biological apatite. Because the HA/chitosan nanohybrid coatings are similar to natural bone in the chemical composition and microstructure, HCCPs exhibit higher in vitro bioactivity and biocompatibility compared with PCFFs. HCCPs not only promote the formation of bone-like apatite in SBF, but also improve the adhesion, spreading, and proliferation of hBMSCs, suggesting that they have great potentials as scaffold materials for bone tissue engineering and implantation. REFERENCES 1. Bose S, Roy M, Bandyopadhyay A. Recent advances in bone tissue engineering scaffolds. Trends Biotechnol 2012;30(10): 5462554. 2. Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari F. Nanostructured scaffolds for bone tissue engineering. J Biomed Mater Res A 2013;101(8):242422435. 3. Holzwarth JM, Ma PX. Biomimetic nanofibrous scaffolds for bone tissue engineering. Biomaterials 2011;32(36):962229629. 4. Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y-Y, Kaufman MJ, Douglas EP, Gower LB. Bone structure and formation: A new perspective. Mater Sci Eng R 2007;58(325):772116. 5. Nudelman F, Pieterse K, George A, Bomans PH, Friedrich H, Brylka LJ, Hilbers PA, de With G, Sommerdijk NA. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 2010;9(12):100421009. 6. Hassenkam T, Fantner GE, Cutroni JA, Weaver JC, Morse DE, Hansma PK. High-resolution AFM imaging of intact and fractured trabecular bone. Bone 2004;35(1):4210. 7. Lima PA, Resende CX, Soares GD, Anselme K, Almeida LE. Preparation, characterization and biological test of 3D-scaffolds based on chitosan, fibroin and hydroxyapatite for bone tissue engineering. Mater Sci Eng C Mater Biol Appl 2013;33(6):338923395. 8. Liu H, Peng H, Wu Y, Zhang C, Cai Y, Xu G, Li Q, Chen X, Ji J, Zhang Y, Ouyang HW. The promotion of bone regeneration by nanofibrous hydroxyapatite/chitosan scaffolds by effects on integrin-BMP/Smad signaling pathway in BMSCs. Biomaterials 2013; 34(18):440424417. 9. Luo Y, Wu C, Lode A, Gelinsky M. Hierarchical mesoporous bioactive glass/alginate composite scaffolds fabricated by threedimensional plotting for bone tissue engineering. Biofabrication 2013;5(1):015005. 10. De Groot K. Bioceramics of Calcium Phosphate. Boca Raton, FL: CRC Press; 1983. 11. LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev 2008;108(11):474224753. 12. Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop Relat Res 1981;157:2592278. 13. Yanagida H, Okada M, Masuda M, Narama I, Nakano S, Kitao S, Takakuda K, Furuzono T. Preparation and in vitro/in vivo evaluations of dimpled poly(L-lactic acid) fibers mixed/coated with hydroxyapatite nanocrystals. J Artif Organs 2011;14(4):3312341. 14. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006;27(16): 311523124. 15. Stanishevsky A, Chowdhury S, Chinoda P, Thomas V. Hydroxyapatite nanoparticle loaded collagen fiber composites: Microarchi-
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