Development of chitosan based composite matrices for Bone tissue engineering

Abstract

The thesis work deals with the development of chitosan (CS) based composite scaffold matrices with improved material and osteogenic differentiation property for bone tissue engineering. Pure CS and CS/ β-TCP composite scaffolds reinforced with micro and nano sized β-TCP with different CS:β-TCP ratios were successfully prepared by freeze gelation method and characterized for morphological, structural, mechanical, swelling, wettability and degradation. Pure CS and composite scaffolds possess interconnected open pore microstructure with desired pore size and porosity. The compressive strength of CS scaffold was remarkably increased by the incorporation of micro and nano sized β-TCP. However, the highest compressive strength of 2.67±0.21 MPa was achieved with CS/nano β-TCP composite scaffold at optimal CS:β-TCP ratio of 60:40. The scaffold also exhibited favourable biodegradation and improved bioactivity. The biocompatibility of the scaffold is confirmed by in-vitro cell culture study using human mesenchymal stem cells (hMSCs) seeded on the scaffold. Conjugation with fibrin is shown to be beneficial for improving cellular affinity of the scaffold which is evident by the enhanced cell attachment (FE-SEM), metabolic activity (MTT assay), proliferation (DNA quantification) and osteogenic differentiation (ALP), bio-mineralization, total calcium content and expression of osteogenic specific genes (semi quantitative RT-PCR) of seeded hMSCs. CS/nano β-TCP scaffold was cross-linked with genipin (GN) and sodium tri poly phosphate (TPP) with the aim of controlling rapid degradation rate of CS scaffold and improving mechanical strength. Thus the developed GN cross-linked CS/nano β-TCP composite scaffold has shown favourable degradation (8%) and higher compressive strength (2.78±0.11) than the scaffold cross-linked with TPP. CS/nano β-TCP/GN scaffold was further coated with fibrin thereby a significant improvement of cellular responses such as cell attachment, proliferation, metabolic activity and osteogenic differentiation was achieved. The enhanced osteogenic differentiation ability of the fibrin coated scaffold was further evident by Semi Quantitative RT-PCR study that has revealed up regulation in the expression of osteogenic specific genes like collagen 1 (COL1), osteocalcein (OC), bone sialo protein (BSP), osteonectin (ON), β-actin and ALP. In-vivo biocompatibility of the CS/nano β-TCP/GN/F composite scaffold was confirmed by animal testing using mice model. All together, the study has demonstrated that the developed CS composite scaffolds in particular CS/nano β-TCP/GN/F can be used as potential artificial ECM (extra cellular matrix) for various non-load bearing bone tissue engineering applications.