Abstract
Limited self-regenerating capacity of human skeleton makes the reconstruction of critical size bone defect a significant challenge for clinical practice. Aimed for regenerating bone tissues, this study was designed to investigate osteogenic differentiation, along with bone repair capacity of 3D chitosan (CHT) scaffolds enriched with graphene oxide (GO) in critical-sized mouse calvarial defect. Histopathological/histomorphometry and scanning electron microscopy(SEM) analysis of the implants revealed larger amount of new bone in the CHT/GO-filled defects compared with CHT alone (p < 0.001). When combined with GO, CHT scaffolds synergistically promoted the increase of alkaline phosphatase activity both in vitro and in vivo experiments. This enhanced osteogenesis was corroborated with increased expression of bone morphogenetic protein (BMP) and Runx-2 up to week 4 post-implantation, which showed that GO facilitates the differentiation of osteoprogenitor cells. Meanwhile, osteogenesis was promoted by GO at the late stage as well, as indicated by the up-regulation of osteopontin and osteocalcin at week 8 and overexpressed at week 18, for both markers. Our data suggest that CHT/GO biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.
Highlights
Ceramic materials were extensively used as bone tissue engineering substrates, owing to their high strength, tissue compatibility and good osteoconductivity, while biodegradation, difficulty of shaping and fragile mechanical properties are important limits to be considered ideal material for clinical applications[10]
alkaline phosphatase (ALP) activity was significantly increased in cells cultured in CHT/graphene oxide (GO) 3 wt.% (p < 0.05), as compared to the control CHT
The effect of CHT/ GO 0.5 wt.% material on cell osteogenic differentiation was not as constant and remarkable as of chitosan/graphene oxide (CHT/GO) 3 wt.%- after 14 days, ALP activity registered an important increase (p < 0.01) as compared to the control (CHT), whereas after 28 days of differentiation this difference in ALP activity related to the control was not statistically visible anymore, suggesting that the addition of a low percentage of GO to material composition could have an impact on cell osteogenic differentiation, but unstable on a long term
Summary
Ceramic materials were extensively used as bone tissue engineering substrates, owing to their high strength, tissue compatibility and good osteoconductivity, while biodegradation, difficulty of shaping and fragile mechanical properties are important limits to be considered ideal material for clinical applications[10]. Contrariwise, polymeric-based biomaterials were noticed for their biodegradation properties combined with excellent tissue-compatibility[11,12], poor mechanical features or irregular release of osteoinductive factors for many polymer materials were noticed[6]. Graphene oxide (GO) has attracted much interest for uses in bone tissue engineering[15], because of its unique physico-chemical and mechanical properties, as well as good biocompatibility[16,17]. The addition of GO to the tridimesional composite scaffold of the CHT/ GO increased proliferation profile of MC-2T3 murine preosteoblasts after 7 days of direct contact with materials and the extracts released by these composites in the surrounding environment exhibit no significant cytotoxic effects after 24 h of seeds[24]. The potential advantages of using GO-based CHT scaffolds directly as factors inducing cellular differentiation, as well as bone tissue regeneration are not been explored
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