Abstract
Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.
Highlights
The field of regenerative medicine and tissue engineering (TE) has emerged as a necessity for tissue substitutes in the case of major trauma
We developed a new scaffold composed of fish gelatin/chitosan crosslinked with genipin (GCsGp) and reinforced with various graphene oxide (GO):biopolymer mass ratios [15,42]
The fish gelatin/chitosan crosslinked with genipin and reinforced with different GO biopolymer scaffolds have been previously analyzed by our group for their good physico-chemical properties and biocompatibility [15,42]
Summary
The field of regenerative medicine and tissue engineering (TE) has emerged as a necessity for tissue substitutes in the case of major trauma. The development of novel biomaterials to efficiently support tissue repair and regeneration is a serious matter in this research area. The goal of BTE relies on generating the expected support for the repair of bone defects based on biocompatible scaffolds with unique properties that enhance cell adhesion and formation of new bone extracellular matrix (bECM) and tissue. High-performing artificial bone substitutes raise critical issues since engaging in the fabrication of autografts with equal performance to the “gold standard” is still technologically out of reach. In BTE, calcium (Ca2+) mineral-enriched scaffolds possess the ability to generate osteogenic signaling in osteoprogenitor populations [1]. Immunogenicity and unpredictable resorption rates can occur with low prospects of restraint [3]
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