Chitosan-based membranes regulate human mesenchymal stem cell response in guided bone regeneration

Designing an implantable construct requires the simulation of microenvironmental cues that drive cellular response in vivo. Guided bone regeneration (GBR) is a technique that utilizes a physical barrier membrane to facilitate healing of a bony defect. The major challenge in the design of GBR membrane materials is combining both mechanical stability and biological properties. Chitosan is a semi-synthetic cationic polysaccharide that is biocompatible, biodegradable and antimicrobial, giving chitosan-based barrier membranes the potential to overcome this challenge. Human mesenchymal stem cells are multipotent cells that have been extensively investigated for therapeutic application for numerous diseases and defects, and their proliferative capacity and osteogenic differentiation potential makes them a suitable model cell population to represent the regenerative osteoprogenitor in GBR. It is well established that the cascade of cellular events leading to hMSC osteogenic differentiation is dictated by microenvironmental cues, and the physical, chemical and biological properties of tissue engineered substrates must be tailored to elicit the desired cellular response. Cells attach and respond to an implant material substrate through adsorbed proteins and diffusing cytokines. In this dissertation, the physical, chemical and biological properties of chitosan-based membranes were evaluated. Hydroxyapatite-chitosan-gelatin membranes were fabricated and the mechanical properties and bioadsorption kinetics were analyzed. hMSC proliferation and osteogenic differentiation on the HCG membranes was investigated. HCG was found to have appropriate mechanical and biological properties for application in GBR. Plasmid DNA encoding for the reporter protein GFP and functional growth factor BMP-2 was incorporated into HCG membranes and the plasmid retention kinetics, transgene expression kinetics and subsequent osteogenic differentiation were examined. Sustained DNA retention and cell transfection throughout 28 days of culture was achieved in the HCG GAMs and pre-condensation of the plasmid DNA prior to membrane fabrication resulted in greater retention and transfection efficacy of plasmid as culture time increased. Investigation of the effects of chitosan degree of deacetylation on the physical and biological properties of chitosan-based membranes revealed distinct trends in biological and physical properties. Increasing the DDA% of chitosan within the chitosan-gelatin and hydroxyapatite-chitosan-gelatin membranes resulted in an increase in the elastic modulus and ultimate stress. The adsorption of the proteins fibronectin, vitronectin and laminin also increased with increasing DDA%. Additionally, hMSCs cultured on membranes with higher DDA% chitosan have faster proliferation and more desirable morphology. Overall, chitosan-based membranes have the potential for optimization of cellular response through the design of the physical, chemical and biological properties of the composite formulation.