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Facile fabrication of gradient bioactive coating with hierarchically porous structures and superior cell response
Materials Letters 133 (2014) 105–108
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Facile fabrication of gradient bioactive coating with hierarchically porous structures and superior cell response Di Huang a,b,n, Shaohui Xi a, Yi Zuo b, Yan Wei a, Meiqing Guo a, Hefeng Wang a, Weiyi Chen a,c, Yubao Li b,n a
College of Mechanics, Shanxi Key Laboratory of Material Strength & Structural Impact, Taiyuan University of Technology, Taiyuan 030024, PR China Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China c Institute of Applied Mechanics & Biomedical Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 April 2014 Accepted 27 June 2014 Available online 5 July 2014
To better construct the interface between implant and natural bone, novel gradient nano-hydroxyapatite/polyamide (nHA/PA) composite coating with hierarchically porous structures was prepared using phase-inversion technique and particle-leaching processing. Scanning electron microscopy observation shows that the size of macropores on the surface ranges from 300 to 500 μm. There are abundant micron/submicron-scale pores distributed on the walls of the macropores. Energy-dispersive X-ray spectroscopy analysis indicates that nHA component increases gradually at the interface from the porous transition region to nHA-rich coating layer and then keeps stable towards the coating surface. The osteoblast-like cells (MG63) were cultured on the samples. The results confirm that the samples with nHA-rich coatings and hierarchical structures are favorable to the attachment and proliferation of MG63 cells, and they are potentially attractive candidates for bone repair applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Microstructure Hierarchical Gradient Cytocompatibility
1. Introduction Due to the apparent advantages such as good processabilities, excellent mechanical properties and regulative features, polymers have been used increasingly in the bone repairmen [1–3]. However, polymers lack bioactivity and cannot provide Ca and P elements for the bone reconstruction. Thus the application of neat polymer in the bone repairment is limited [4,5]. To ensure their long-term clinical success, polymers are often coated with osteoconductive or/and osteointegrating biomaterials such as nanohydroxyapatite (nHA) [4,5]. In a previous study, our group has developed a novel technique to fabricate nHA/polymer composite coating on the same polymer substrate by phase-inversion method [6]. The results conformed that high bonding strength and better cytocompatibility have been obtained after nHA modification. Whereas, subsequent research demonstrated that the phaseinversion process was not efficient for creating interconnected macropores. A relatively dense layer was formed on the top surface of the coating and the pore size was less than 50 μm throughout the coating. However, it has been reported that the interconnected macropores with a diameter of 100 μm or greater is critical for bone ingrowth and n
Corresponding authors. Tel.: þ 86 351 6014477; fax: þ86 351 6011816. E-mail addresses: [email protected] (D. Huang), [email protected] (Y. Li).
http://dx.doi.org/10.1016/j.matlet.2014.06.168 0167-577X/& 2014 Elsevier B.V. All rights reserved.
cellular infiltration [7–9]. Moreover, macropores in this scale also promotes maximized bone ingrowth [10–12]. Apart from the macropores, micro/submicron-scale pores within 0.5–10 μm was also conformed to play a major role in bone reconstruction by remarkably increasing surface area for protein adsorption, cellular attachment [13], facilitating the osteoinductivity and bone regeneration in macropores [14], leading to multi-scale osteointegration [15]. However, untill now, little work has been done to investigate the osteogenetic regeneration of nHA-containing coating with simultaneous micro/submicron-scale pores on polymer substrate. In the present study, gradient nHA/polyamide (PA) composite coatings with hierarchical structures were designed using phaseinversion technique and particle-leaching method. Micro/submicron-scale pores across the entire coating were produced by phase-inversion processing. Simultaneously, macropores were generated by particle-leaching method, which only occurred on the coating surface. The coating microstructures, composition variation, and preliminary cytocompatibility were also evaluated.
2. Materials and methods Preparation of the bioactive coating: Polyamide66 (PA) was purchased from Asahi Chemical Industry, Japan. The nHA/PA composite slurry was fabricated using the co-precipitation method
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Fig. 1. SEM images of the surface of nHA/PA coated sample (a); higher magnification (b); XRD patterns of PA and nHA-rich coating sample (c).
Fig. 2. The cross-section microstructure of the substrate/transition region/coating layer and EDS element line-scanning: SEM image and element line scanning of the crosssection of sample (a); and the interface between the transition region and coating layer (b). The inset is the EDS spectrum reflected the spectrum 1.
in ethanol [6]. Briefly, PA granules were completely dissolved into ethanol at 80 1C for 2 h. The PA to ethanol ratio (w/v) was fixed at 1:10. The nHA crystals slurry, prepared by wet synthesis [16], was dispersed by anhydrous ethanol and then gradually added into PA ethanol solution with vigorously stirring at 80 1C for another 2 h. The weight ratio of nHA crystals to PA was 1:1. When a homogeneous system was obtained, the mixture was kept in sealing container at room temperature. PA laminae of 1 mm thickness were prepared by injection molding machine (KTC-200, Kinki, China). The injection temperature ranged from 240 to 270 1C under 30 MPa pressure. The PA laminae were cut into rectangle shapes of 10 20 mm2, and then vertically immersed in nHA/PA composite slurry with gentle stirring at 37 1C for 6 h. Subsequently, the samples were removed from the composite slurry and placed horizontally. NaCl particles (300–500 μm) were added onto the coating surface and shaken gently. NaCl particles were uniformly adhered onto the samples’ surface duo to the high viscosity of the composite slurry. Then the redundant NaCl particles were discarded and the final density of NaCl particles was about 0.5 g/cm2. Afterwards, the samples were dried in air for 24 h. Finally, the obtained samples were soaked in distilled water for 24 h to leach out salt particles and then airdried at room temperature. Characterization: The samples were analyzed by X-ray diffraction (XRD, DX2500, China). Scan was performed with 2θ value from 101 to 601 at a rate of 0.051s 1. The hierarchical structures of samples were observed using scanning electron microscopy (SEM, JSM-6500LV, Japan). Energy-dispersive X-ray spectroscopy (EDS, Oxford, UK) on the SEM was used to analyze the elements.
Cytocompatibility: Human osteosarcoma cell lines (MG63) were employed to evaluate the cytocompatibility of PA substrate and nHA/PA coated samples. The samples were cut into discs of φ10 1 mm and sterilized by ethylene oxide gas. The disinfected specimens were put in 24-well plates. Afterwards, suspension of MG63 cells with number of 2 104 per milliliter was added to 24-well plates, and cultured for 1, 3, 5 and 7 day(s), respectively. MTT assay was selected and the OD values at 570 nm were measured. MG63 cells cultured on the specimens for 4 days were observed with SEM. Three samples were tested for each group. Statistical analysis: The data were collected in Origin 8.0 software and the results were expressed as means and standard deviations. Two experimental groups were evaluated by Student’s t-test. A value of p o0.05 was considered statistically significant.
3. Results and discussion The nHA/PA composite coated samples exhibit a porous morphology as shown in Fig. 1. The macropore size is about 300– 500 μm. Micropores about 10 μm and submicron pores about 500 nm (as arrows pointed in Fig. 1b) present on the interior surface of the macropores. Fig. 1c shows the XRD patterns of the sample with and without coating. The main characteristic peaks of both HA and PA are present in the sample with coating. Previous results of the samples fabricated by phase-inversion technique demonstrated that the process was not efficient for creating large interconnected pores and the pore size was less than 50 μm [6]. At present study, apart from the micron/submicron-scale pores generated from phase separation processing, macropores and high
D. Huang et al. / Materials Letters 133 (2014) 105–108
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Fig. 3. The morphology of MG63 cells cultured for 4 days on nHA/PA-coated samples (a); higher magnification of MG63 cells on the surface (b) and inside the macropores (c); MTT assay for proliferation of cells cultured on nHA/PA-coated samples for 1, 3, 5 and 7 day(s), compared with the pure PA samples under the same culture condition (d). Significantly different at np o 0.05 and nnp o 0.01.
interconnectivity have been produced by the addition of NaCl particle as a porogen. The hierarchical porous microstructure is implemented via integrating phase-inversion technique and salt leaching method. Fig. 2 shows SEM images and composition distribution of the cross-section of nHA/PA composite coated samples. The crosssection microstructure in Fig. 2a shows the dense substrate, transition region ( 200 μm), and nHA-rich coating layer ( 500 μm). The depth of the macropores on the top surface ranges from 250 μm to 350 μm, which is suitable for growth of bony tissues. The interconnective micron/submicron-scale pores across the porous layer are helpful for nutrient transportation [7]. EDS analysis indicates that nHA composition increases at the interface from the porous transition region to nHA-rich coating layer and then keeps stable towards the coating surface. EDS spectrum (the inset in Fig. 2b) shows the existence of C, Ca and P in the area nearby the surface. It means that nHA is the main inorganic composition of the coating, which will accelerate new bone formation. The magnified interface (Fig. 2b) shows that nHA content is gradually increasing from neat PA transition region to rough nHA embedded coating layer. Fig. 3a–c shows the SEM photographs of MG63 cells cultured on composite coated samples for 4 days. From the photographs, after 4 days culture, large amount of cells proliferate (Fig. 2a). The cells with predominant fusiform shape have attached on the surface of the coating (Fig. 2b). Moreover, cells migrate into the macropores and stretch their pseudopodia to connect with each other (Fig. 2c). After 1, 3, 5 and 7 day(s) of culturing, the cell proliferation in each group was assessed using MTT test. As represented in Fig. 2d, after 5 and 7 days, the proliferation of cells cultured on composite coated samples shows significant difference from the pure PA group (day 5, p o0.05; day 7, p o0.01). The results demonstrate that the nHA/PA composite coated samples with hierarchical structures have superior cytocompatibility.
4. Conclusions Gradient nHA/PA composite coating with hierarchically porous structures was successfully fabricated via integrating phaseinversion technique and salt leaching method. The macropores on the surface ranges from 300 to 500 μm and micron/submicronscale pores present throughout the cross-section of the coating. The nHA content increases gradually at the interface from the transition region to nHA-rich coating and then keeps stable towards the coating surface. The nHA-rich coating with hierarchical porous structure have superior cell response, which have great potential applications in orthopedic fields.
Acknowledgements This work has been supported by the Natural Science Foundation of China (no. 31370971, 11032008 and 51203028), the support of the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (2013111), Natural Science Foundation of Shanxi Province (no. 2013021014-2, 2013021003-1 and 2013021013-5), Program for the Outstanding Innovative Teams of Higher Learning Institutions of Shanxi is also acknowledged with gratitude. References [1] Estrin Y, Koh YH. Mater Lett 2014;116:20–2. [2] Huang D, Zuo Y, Zou Q, Zhang L, Li JD, Cheng L, et al. J Biomater Sci, Polym Ed 2011;22:931–44. [3] Zhang JC, Lu HY, Lv GY, Mo AC, Yan YG, Huang C. Int J Oral Maxillofac Surg 2010;39(5):469–77. [4] Kim MS, Khang G, Lee HB. Prog Polym Sci 2008;33:138–64. [5] Song L, Gan L, Xiao YF, Wu Y, Wu F, Gu ZW. Mater Lett 2011;65:974–7.
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[6] Huang D, Zuo Y, Li J, Zou Q, Zhang L, Gong M, et al. J R Soc Interface 2012;9:1450–7. [7] Woodard JR, Hilldore AJ, Lan SK, Park CJ, Morgan AW, Eurell JAC, et al. Biomaterials 2007;28:45–54. [8] Huang D, Zuo Y, Zou Q, Wan YY, Gao SB, Wang XY, et al. J Biomed Mater Res Part B 2012;100B:51–7. [9] Xu Q, Lu HY, Zhang JC, Lu GY, Deng ZN, Mo AC. Int J Nanomed 2010;5:331–5. [10] Zhang J, Zhou H, Yang K, Yuan Y, Liu C. Biomaterials 2013;34:9381–92. [11] Baino F, Vitale-Brovarone C. Mater Lett 2014;118:27–30. [12] Guo J, Meng ZS, Chen G, Xie D, Chen YL, Wang H, et al. Tissue Eng Part A 2012;18(11–12):1239–52.
[13] Hing KA, Annaz B, Saeed S, Revell PA, Buckland T. J Mater Sci—Mater Med 2005;16:467–75. [14] Lan Levengood SK, Polak SJ, Poellmann MJ, Hoelzle DJ, Maki AJ, Clark SG, et al. Acta Biomater 2010;6:3283–91. [15] Lan Levengood SK, Polak SJ, Wheeler MB, Maki AJ, Clark SG, Jamison RD, et al. Biomaterials 2010;31:3552–63. [16] Li YB, de Wijn J, Klein CPAT, Van der Meer S, de Groot K. J Mater Sci—Mater Med 1994;5:252–5.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Facile fabrication of gradient bioactive coating with hierarchically porous structures and superior cell response Di Huang a,b,n, Shaohui Xi a, Yi Zuo b, Yan Wei a, Meiqing Guo a, Hefeng Wang a, Weiyi Chen a,c, Yubao Li b,n a
College of Mechanics, Shanxi Key Laboratory of Material Strength & Structural Impact, Taiyuan University of Technology, Taiyuan 030024, PR China Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China c Institute of Applied Mechanics & Biomedical Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 April 2014 Accepted 27 June 2014 Available online 5 July 2014
To better construct the interface between implant and natural bone, novel gradient nano-hydroxyapatite/polyamide (nHA/PA) composite coating with hierarchically porous structures was prepared using phase-inversion technique and particle-leaching processing. Scanning electron microscopy observation shows that the size of macropores on the surface ranges from 300 to 500 μm. There are abundant micron/submicron-scale pores distributed on the walls of the macropores. Energy-dispersive X-ray spectroscopy analysis indicates that nHA component increases gradually at the interface from the porous transition region to nHA-rich coating layer and then keeps stable towards the coating surface. The osteoblast-like cells (MG63) were cultured on the samples. The results confirm that the samples with nHA-rich coatings and hierarchical structures are favorable to the attachment and proliferation of MG63 cells, and they are potentially attractive candidates for bone repair applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Microstructure Hierarchical Gradient Cytocompatibility
1. Introduction Due to the apparent advantages such as good processabilities, excellent mechanical properties and regulative features, polymers have been used increasingly in the bone repairmen [1–3]. However, polymers lack bioactivity and cannot provide Ca and P elements for the bone reconstruction. Thus the application of neat polymer in the bone repairment is limited [4,5]. To ensure their long-term clinical success, polymers are often coated with osteoconductive or/and osteointegrating biomaterials such as nanohydroxyapatite (nHA) [4,5]. In a previous study, our group has developed a novel technique to fabricate nHA/polymer composite coating on the same polymer substrate by phase-inversion method [6]. The results conformed that high bonding strength and better cytocompatibility have been obtained after nHA modification. Whereas, subsequent research demonstrated that the phaseinversion process was not efficient for creating interconnected macropores. A relatively dense layer was formed on the top surface of the coating and the pore size was less than 50 μm throughout the coating. However, it has been reported that the interconnected macropores with a diameter of 100 μm or greater is critical for bone ingrowth and n
Corresponding authors. Tel.: þ 86 351 6014477; fax: þ86 351 6011816. E-mail addresses: [email protected] (D. Huang), [email protected] (Y. Li).
http://dx.doi.org/10.1016/j.matlet.2014.06.168 0167-577X/& 2014 Elsevier B.V. All rights reserved.
cellular infiltration [7–9]. Moreover, macropores in this scale also promotes maximized bone ingrowth [10–12]. Apart from the macropores, micro/submicron-scale pores within 0.5–10 μm was also conformed to play a major role in bone reconstruction by remarkably increasing surface area for protein adsorption, cellular attachment [13], facilitating the osteoinductivity and bone regeneration in macropores [14], leading to multi-scale osteointegration [15]. However, untill now, little work has been done to investigate the osteogenetic regeneration of nHA-containing coating with simultaneous micro/submicron-scale pores on polymer substrate. In the present study, gradient nHA/polyamide (PA) composite coatings with hierarchical structures were designed using phaseinversion technique and particle-leaching method. Micro/submicron-scale pores across the entire coating were produced by phase-inversion processing. Simultaneously, macropores were generated by particle-leaching method, which only occurred on the coating surface. The coating microstructures, composition variation, and preliminary cytocompatibility were also evaluated.
2. Materials and methods Preparation of the bioactive coating: Polyamide66 (PA) was purchased from Asahi Chemical Industry, Japan. The nHA/PA composite slurry was fabricated using the co-precipitation method
106
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Fig. 1. SEM images of the surface of nHA/PA coated sample (a); higher magnification (b); XRD patterns of PA and nHA-rich coating sample (c).
Fig. 2. The cross-section microstructure of the substrate/transition region/coating layer and EDS element line-scanning: SEM image and element line scanning of the crosssection of sample (a); and the interface between the transition region and coating layer (b). The inset is the EDS spectrum reflected the spectrum 1.
in ethanol [6]. Briefly, PA granules were completely dissolved into ethanol at 80 1C for 2 h. The PA to ethanol ratio (w/v) was fixed at 1:10. The nHA crystals slurry, prepared by wet synthesis [16], was dispersed by anhydrous ethanol and then gradually added into PA ethanol solution with vigorously stirring at 80 1C for another 2 h. The weight ratio of nHA crystals to PA was 1:1. When a homogeneous system was obtained, the mixture was kept in sealing container at room temperature. PA laminae of 1 mm thickness were prepared by injection molding machine (KTC-200, Kinki, China). The injection temperature ranged from 240 to 270 1C under 30 MPa pressure. The PA laminae were cut into rectangle shapes of 10 20 mm2, and then vertically immersed in nHA/PA composite slurry with gentle stirring at 37 1C for 6 h. Subsequently, the samples were removed from the composite slurry and placed horizontally. NaCl particles (300–500 μm) were added onto the coating surface and shaken gently. NaCl particles were uniformly adhered onto the samples’ surface duo to the high viscosity of the composite slurry. Then the redundant NaCl particles were discarded and the final density of NaCl particles was about 0.5 g/cm2. Afterwards, the samples were dried in air for 24 h. Finally, the obtained samples were soaked in distilled water for 24 h to leach out salt particles and then airdried at room temperature. Characterization: The samples were analyzed by X-ray diffraction (XRD, DX2500, China). Scan was performed with 2θ value from 101 to 601 at a rate of 0.051s 1. The hierarchical structures of samples were observed using scanning electron microscopy (SEM, JSM-6500LV, Japan). Energy-dispersive X-ray spectroscopy (EDS, Oxford, UK) on the SEM was used to analyze the elements.
Cytocompatibility: Human osteosarcoma cell lines (MG63) were employed to evaluate the cytocompatibility of PA substrate and nHA/PA coated samples. The samples were cut into discs of φ10 1 mm and sterilized by ethylene oxide gas. The disinfected specimens were put in 24-well plates. Afterwards, suspension of MG63 cells with number of 2 104 per milliliter was added to 24-well plates, and cultured for 1, 3, 5 and 7 day(s), respectively. MTT assay was selected and the OD values at 570 nm were measured. MG63 cells cultured on the specimens for 4 days were observed with SEM. Three samples were tested for each group. Statistical analysis: The data were collected in Origin 8.0 software and the results were expressed as means and standard deviations. Two experimental groups were evaluated by Student’s t-test. A value of p o0.05 was considered statistically significant.
3. Results and discussion The nHA/PA composite coated samples exhibit a porous morphology as shown in Fig. 1. The macropore size is about 300– 500 μm. Micropores about 10 μm and submicron pores about 500 nm (as arrows pointed in Fig. 1b) present on the interior surface of the macropores. Fig. 1c shows the XRD patterns of the sample with and without coating. The main characteristic peaks of both HA and PA are present in the sample with coating. Previous results of the samples fabricated by phase-inversion technique demonstrated that the process was not efficient for creating large interconnected pores and the pore size was less than 50 μm [6]. At present study, apart from the micron/submicron-scale pores generated from phase separation processing, macropores and high
D. Huang et al. / Materials Letters 133 (2014) 105–108
107
Fig. 3. The morphology of MG63 cells cultured for 4 days on nHA/PA-coated samples (a); higher magnification of MG63 cells on the surface (b) and inside the macropores (c); MTT assay for proliferation of cells cultured on nHA/PA-coated samples for 1, 3, 5 and 7 day(s), compared with the pure PA samples under the same culture condition (d). Significantly different at np o 0.05 and nnp o 0.01.
interconnectivity have been produced by the addition of NaCl particle as a porogen. The hierarchical porous microstructure is implemented via integrating phase-inversion technique and salt leaching method. Fig. 2 shows SEM images and composition distribution of the cross-section of nHA/PA composite coated samples. The crosssection microstructure in Fig. 2a shows the dense substrate, transition region ( 200 μm), and nHA-rich coating layer ( 500 μm). The depth of the macropores on the top surface ranges from 250 μm to 350 μm, which is suitable for growth of bony tissues. The interconnective micron/submicron-scale pores across the porous layer are helpful for nutrient transportation [7]. EDS analysis indicates that nHA composition increases at the interface from the porous transition region to nHA-rich coating layer and then keeps stable towards the coating surface. EDS spectrum (the inset in Fig. 2b) shows the existence of C, Ca and P in the area nearby the surface. It means that nHA is the main inorganic composition of the coating, which will accelerate new bone formation. The magnified interface (Fig. 2b) shows that nHA content is gradually increasing from neat PA transition region to rough nHA embedded coating layer. Fig. 3a–c shows the SEM photographs of MG63 cells cultured on composite coated samples for 4 days. From the photographs, after 4 days culture, large amount of cells proliferate (Fig. 2a). The cells with predominant fusiform shape have attached on the surface of the coating (Fig. 2b). Moreover, cells migrate into the macropores and stretch their pseudopodia to connect with each other (Fig. 2c). After 1, 3, 5 and 7 day(s) of culturing, the cell proliferation in each group was assessed using MTT test. As represented in Fig. 2d, after 5 and 7 days, the proliferation of cells cultured on composite coated samples shows significant difference from the pure PA group (day 5, p o0.05; day 7, p o0.01). The results demonstrate that the nHA/PA composite coated samples with hierarchical structures have superior cytocompatibility.
4. Conclusions Gradient nHA/PA composite coating with hierarchically porous structures was successfully fabricated via integrating phaseinversion technique and salt leaching method. The macropores on the surface ranges from 300 to 500 μm and micron/submicronscale pores present throughout the cross-section of the coating. The nHA content increases gradually at the interface from the transition region to nHA-rich coating and then keeps stable towards the coating surface. The nHA-rich coating with hierarchical porous structure have superior cell response, which have great potential applications in orthopedic fields.
Acknowledgements This work has been supported by the Natural Science Foundation of China (no. 31370971, 11032008 and 51203028), the support of the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (2013111), Natural Science Foundation of Shanxi Province (no. 2013021014-2, 2013021003-1 and 2013021013-5), Program for the Outstanding Innovative Teams of Higher Learning Institutions of Shanxi is also acknowledged with gratitude. References [1] Estrin Y, Koh YH. Mater Lett 2014;116:20–2. [2] Huang D, Zuo Y, Zou Q, Zhang L, Li JD, Cheng L, et al. J Biomater Sci, Polym Ed 2011;22:931–44. [3] Zhang JC, Lu HY, Lv GY, Mo AC, Yan YG, Huang C. Int J Oral Maxillofac Surg 2010;39(5):469–77. [4] Kim MS, Khang G, Lee HB. Prog Polym Sci 2008;33:138–64. [5] Song L, Gan L, Xiao YF, Wu Y, Wu F, Gu ZW. Mater Lett 2011;65:974–7.
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[6] Huang D, Zuo Y, Li J, Zou Q, Zhang L, Gong M, et al. J R Soc Interface 2012;9:1450–7. [7] Woodard JR, Hilldore AJ, Lan SK, Park CJ, Morgan AW, Eurell JAC, et al. Biomaterials 2007;28:45–54. [8] Huang D, Zuo Y, Zou Q, Wan YY, Gao SB, Wang XY, et al. J Biomed Mater Res Part B 2012;100B:51–7. [9] Xu Q, Lu HY, Zhang JC, Lu GY, Deng ZN, Mo AC. Int J Nanomed 2010;5:331–5. [10] Zhang J, Zhou H, Yang K, Yuan Y, Liu C. Biomaterials 2013;34:9381–92. [11] Baino F, Vitale-Brovarone C. Mater Lett 2014;118:27–30. [12] Guo J, Meng ZS, Chen G, Xie D, Chen YL, Wang H, et al. Tissue Eng Part A 2012;18(11–12):1239–52.
[13] Hing KA, Annaz B, Saeed S, Revell PA, Buckland T. J Mater Sci—Mater Med 2005;16:467–75. [14] Lan Levengood SK, Polak SJ, Poellmann MJ, Hoelzle DJ, Maki AJ, Clark SG, et al. Acta Biomater 2010;6:3283–91. [15] Lan Levengood SK, Polak SJ, Wheeler MB, Maki AJ, Clark SG, Jamison RD, et al. Biomaterials 2010;31:3552–63. [16] Li YB, de Wijn J, Klein CPAT, Van der Meer S, de Groot K. J Mater Sci—Mater Med 1994;5:252–5.