Si-doping bone composite based on protein template-mediated assembly for enhancing bone regeneration

doi:10.1007/s11706-017-0375-y

Front. Mater. Sci.

2017, Vol. 11

Issue (2) : 106-119 https://doi.org/10.1007/s11706-017-0375-y

RESEARCH ARTICLE

Si-doping bone composite based on protein template-mediated assembly for enhancing bone regeneration

Qin YANG^1,², Yingying DU^1,², Yifan WANG^1,², Zhiying WANG^1,², Jun MA^1,², Jianglin WANG^1,²(

), Shengmin ZHANG^1,²(

)

¹. Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074, China
². Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

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Abstract

Bio-inspired hybrid materials that contain organic and inorganic networks interpenetration at the molecular level have been a particular focus of interest on designing novel nanoscale composites. Here we firstly synthesized a series of hybrid bone composites, silicon-hydroxyapatites/silk fibroin/collagen, based on a specific molecular assembled strategy. Results of material characterization confirmed that silicate had been successfully doped into nano-hydroxyapatite lattice. In vitro evaluation at the cellular level clearly showed that these Si-doped composites were capable of promoting the adhesion and proliferation of rat mesenchymal stem cells (rMSCs), extremely enhancing osteoblastic differentiation of rMSCs compared with silicon-free composite. More interestingly, we found there was a critical point of silicon content in the composition on regulating multiple cell behaviors. In vivo animal evaluation further demonstrated that Si-doped composites enabled to significantly improve the repair of cranial bone defect. Consequently, our current work not only suggests fabricating a potential bone repair materials by integrating element-doping and molecular assembled strategy in one system, but also paves a new way for constructing multi-functional composite materials in the future.

Keywords silicate-doped molecular assembly biomimetic bone bone regeneration osteoblastic differentiation

Corresponding Author(s): Jianglin WANG,Shengmin ZHANG

Online First Date: 13 April 2017 Issue Date: 26 May 2017

Cite this article:

Qin YANG,Yingying DU,Yifan WANG, et al. Si-doping bone composite based on protein template-mediated assembly for enhancing bone regeneration[J]. Front. Mater. Sci., 2017, 11(2): 106-119.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-017-0375-y
https://academic.hep.com.cn/foms/EN/Y2017/V11/I2/106

Fig.1 General idea of this work. Silicon-doped HA was induced by collagen/SF bi-template to generate a specific composite of SF-collagen/silicon-HA. The interactions between the powder samples and rMSCs were systematically investigatedin vitro, and the derivative 3D porous scaffolds were evaluated by the rat cranial defect model in vivo.

Fig.2 Characterization of materials. (a) XRD analysis showed both Si-free and Si-doped HA groups exhibited the main diffraction peaks of HA in comparison with HA standard card (JCPDS-PDF 09-0432).(b) FTIR analysis indicated that each group of template-induced materials displayed the typical phosphate group and main functional groups of amides I and II due to the presence of organic protein templates.(c) TGA analysis confirmed that the template induced materials were organic-inorganic composites consisting of 20%–30% organics and 70%–80% inorganics.(d) Silicomolybdenum blue spectrophotometry confirmed that the actual value of silicon content was quite close to the theory value.(e)(f) XPS analysis showed the typical peaks of Ca and P in Si-0 group, and the typical peaks of Si in Si-doped groups. The mole ratio ofn(Ca)/n(P) or n(Ca)/[n(P) +n(Si)] of the sample was close to the theoretical value of HA (n(Ca)/n(P) = 1.67).

Fig.3 TEM images and SAED from HR-TEM analysis. The results showed that all those HA crystals presented a needle-like nano-crystal with 100 nm in length and 20 nm in width. There was on obvious difference no crystallite morphology between Si-doped and Si-free composites that mean the doping of silicon will not change the crystallite structure of HA. SAED images also confirmed that there was no preferential growth of HA crystals induced by bi-template of silk fibroin and collagen.

Fig.4 Cell proliferation and adhesion. (a) Cell proliferation of rMSCs cultured on the representative samples in growth medium for 1, 3, 5 and 7 d. It showed that both Si-doped and Si-free group exhibited excellent biocompatibility, and presented no obvious toxicity, and were able to well support cell proliferation over the time.(b) Confocal microscope images of rMSCs cultured on the samples for 12 h. (c) SEM images of rMSCs cultured on the samples for 12 h.

Fig.5 Osteoblastic differentiation of stem cell. (a) ALP activities and (c) ALP staining of each group indicated the early differentiation of rMSCs. (b) Real-time PCR showed each sample significantly enhanced osteoblastic differentiation of MSCs based on the analysis of specific osteogenic genes including osteocalcin (OCN), osteonectin (ONN), and collagen I (COL I) on day 21. The Si-0.8 group showed the best osteogenic capacity among all samples.(d) Immunofluorescence staining showed that all corresponding proteins of osteogenic genes exhibited positive staining, and the results were highly consistent with mRNA level in (b).(e) The calcium nodule staining were generated from each group. OCN, ONN, and COL I were stained by Rhodamine-labeled antibody (red), cell nuclei were stained by DAPI (blue), and F-actin were stained by FITC-labeled phalloidin (green).

Fig.6 Micro-CT analysis: (a) micro-CT scan images of the rat cranial bone defect in four groups at each time-point of 8 weeks; (b) the percentages of newly formed bone in the bone-defect cavities were significantly higher in PLGA/Si-0.8 group than others (p<0.05); (c) the BMD were significantly higher in PLGA/Si-0.8 scaffolds than the other groups (p<0.05).

Fig.7 Analysis of histological staining. Eight (8) weeks after implantation, both Hematoxylin-Eosin (H&E) and Masson Trichrome (M&T) staining showed that PLGA/Si-0.8 group was able to enhance newly formed bone compared with other three groups. It was consistent with micro-CT analysis. For H&E staining, the cytoplasm and nuclei show red and blue, respectively. For M&T staining, the cytoplasm and collagen matrixes were stained by red and blue, respectively.

Fig. S1&chsp;?The porous scaffolds were prepared by ice-templating process, and the morphology and microstructure of the scaffolds were examined using SEM. PLGA, PLGA/Si-0 and PLGA/Si-0.8 scaffolds with high?permeability?of?pore space were obtained and Si-0 and Si-0.8 particles were distributed within the pore walls of the scaffolds and no large aggregates appeared in pores.

Fig. S2?&chsp;Rat MSCs were used to evaluate the biological function of PLGA/Si-0 and PLGA/Si-0.8 scaffold and its potential application on bone repair. We found that the scaffolds were able to well support cell adhesion, and the resident cells exhibited obviously outspread pseudopod attaching to the adjacent pore.

Fig. S3&chsp;?In vivo evaluation of the scaffolds. (a) Hematoxylin-Eosin and (b) Masson Trichrome staining after hypodermic?implantation for 3 weeks.

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