Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen GAG scaffold

Abstract

Anchorage dependent cells respond to mechanical and physical properties of biomaterials. One such cue is a material?s mechanical stiffness. We compared the osteogenic potential of collagen glycosaminoglycan (CG) scaffolds with varying stiffness up to 6 weeks in culture. The mechanical stiffness of CG scaffolds were varied by crosslinking via physical (dehydrothermal [DHT]) and chemical, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide [EDAC] and glutaraldehyde [GLUT]) methods. Results showed that all CG substrates allowed for cellular attachment, infiltration and osteogenic differentiation. CG scaffolds treated with EDAC and GLUT, were mechanically stiffer, retained their original scaffold structure and resisted cellular contraction. Consequently they facilitated a 2-fold greater cell number probably due to pore architecture being maintained allowing for improved diffusion of nutrients. On the other hand, the less stiff substrates crosslinked with DHT allowed for increased cell-mediated scaffold contraction; contracting by 70% following 6 weeks (p<0.01) of culture. This reduction in scaffold area resulted in cells reaching the centre of the scaffold quicker up to 4 weeks; however, at 6 weeks all scaffolds showed similar levels of cellular infiltration with higher cell numbers found on the stiffer EDAC and GLUT? treated scaffolds. Analysis of osteogenesis showed, that scaffolds crosslinked with DHT expressed higher levels of the late stage bone formation markers osteopontin and osteocalcin (p<0.01) and increased levels of mineralisation. In conclusion, the more compliant CG scaffolds allowed for cell-mediated contraction and supported a greater level osteogenic maturation of MC3T3 cells while the stiffer, non contractible scaffolds resulted in lower levels of cell maturation but higher cell numbers on the scaffold. Therefore, we find scaffold stiffness has different effects on differentiation and cell number whereby the increased cell-mediated contraction facilitated by the less stiff scaffolds positively modulates osteoblast differentiation while reducing cell numbers.