Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength.

Hydroxyapatite-chitosan/gelatin (HA:Chi:Gel) nanocomposite scaffold has potential to serve as a template matrix to regenerate extra cellular matrix of human bone. Scaffolds with varying composition of hydroxyapatite, chitosan, and gelatin were prepared using lyophilization technique where glutaraldehyde (GTA) acted as a cross-linking agent for biopolymers. First, phase pure hydroxyapatite-chitosan nanocrystals were in situ synthesized by coprecipitation method using a solution of 2% acetic acid dissolved chitosan and aqueous solution of calcium nitrate tetrahydrate [Ca(NO3)2,4H2O] and diammonium hydrogen phosphate [(NH4)2H PO4]. Keeping solid loading constant at 30 wt% and changing the composition of the original slurry of gelatin, HA-chitosan allowed control of the pore size, its distribution, and mechanical properties of the scaffolds. Microstructural investigation by scanning electron microscopy revealed the formation of a well interconnected porous scaffold with a pore size in the range of 35-150 μm. The HA granules were uniformly dispersed in the gelatin-chitosan network. An optimal composition in terms of pore size and mechanical properties was obtained from the scaffold with an HA:Chi:Gel ratio of 21:49:30. The composite scaffold having 70% porosity with pore size distribution of 35-150 μm exhibited a compressive strength of 3.3-3.5 MPa, which is within the range of that exhibited by cancellous bone. The bioactivity of the scaffold was evaluated after conducting mesenchymal stem cell (MSC) - materials interaction and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay using MSCs. The scaffold found to be conducive to MSC's adhesion as evident from lamellipodia, filopodia extensions from cell cytoskeleton, proliferation, and differentiation up to 14 days of cell culture.