A bioinspired three-dimensional graphene/chitosan hybrid scaffold for bone tissue engineering

Abstract

An important aspect of bone tissue engineering (BTE) is bone regeneration, an interdisciplinary process involving the use of cells, biomaterials, and therapeutic agents. Biomaterials, being an essential part of this process, still face challenges in mimicking the three-dimensional (3D) architecture of natural bone and inducing the functions of cells and tissues during bone tissue formation. Electrical stimulation (ES), as one of the signals governing the functions of cells and tissues, has been proved to induce the bone formation of damaged tissues in vivo with outstanding efficiency. However, the research on its potential to improve the performance of bone regeneration with tissue-engineered materials in vitro has been challenging due to the lack of conductive 3D scaffolds. It has been found that graphene with great conductivity could be hybridized to conductive 3D scaffolds composited with biocompatible chitosan (CHI). Thus, it could be hypothesized that assembling graphene and CHI into a 3D construct mimicking the environment of bone tissues and the conductive property for the functioning of ES could generate a novel conductive hybrid scaffold for bone tissue applications.

Inspired by bone’s internal structure and its ability to be electrically stimulated, a 3D graphene/CHI scaffold combined with ES for inducing osteoblast functions in vitro was investigated in this thesis. Figure 1 shows the overall schematics of this thesis. To achieve this overall objective, a 3D conductive graphene/CHI scaffold was synthesized after first acquiring a full understanding of its material properties. As current viable approaches of preparing graphene for biomedical applications are limited due to the toxic residuals or undesirable structures of the products, an environmentally friendly way to obtain graphene/CHI scaffold was developed in this study via the in situ electrochemical reduction of the graphene oxide (GO)/CHI scaffold to a reduced GO (RGO)/CHI scaffold. In addition, the effects of various RGO contents in the scaffold on THE morphology, mechanical strength, and conductivity of the scaffolds were explored in order to find the optimal content for later studies. Subsequently, to understand the performance of scaffolds in biological environments, the degradation against enzymes, bioactivity for inducing apatite formation, and capability of supporting human fetal osteoblast (hFOB) proliferation were measured. The scaffolds presented a tunable degradation rate by adjusting RGO contents, which enabled the scaffolds to provide space for new tissue formation in bone regeneration with controllable rates according to cell types. Attributed to the wrinkled layer surface of the RGO/CHI scaffold, it also presented higher bioactivity compared to CHI scaffolds for bone-like apatite formation. Similar behaviors of hFOB proliferation and viability were observed on the RGO/CHI scaffolds compared with the CHI scaffolds. This suggested the need of ES for further improvement.

The effects of scaffolds with ES on inducing hFOB functions were subsequently characterized. As there was no protocol in the literature, the parameters of ES were firstly optimized in terms of current density, frequency, and seeding density by comparing their proliferative effects on hFOBs. These optimal parameters were subsequently used to investigate the effects of RGO/CHI scaffolds with ES on hFOB functions, including cell alignment, proliferation, alkaline phosphatase (ALP) activity, and mineralization deposition. According to the results, electrical signals combined with RGO/CHI scaffolds not only affect the early stage of hFOB differentiation by inducing its proliferation and matrix organization but also accelerate the progress of its mineralization stages by improving mineral deposition. Taken together, 3D conductive graphene/CHI scaffolds combined with ES could act as a promising platform by inducing the functions of osteoblasts in vitro for the development of BTE.