Investigating the mechanical, physiochemical and osteogenic properties in gelatin-chitosan-bioactive nanoceramic composite scaffolds for bone tissue regeneration: In vitro and in vivo.

The aim of this work was to compare the efficacy of gelatin-chitosan based bone scaffolds after incorporation of three different bioactive nanoparticles such as hydroxyapatite (HAp), β‑tricalcium phosphate (β-TCP) and 58s bio active glass by evaluating its physicochemical, mechanical and osteogenic properties. Gelatin-chitosan based scaffolds made of gelatin-chitosan (GC) and GC composites containing 30 wt% HAp, β-TCP and 58s bioactive glass nanoparticles were fabricated using freeze drying technique. The porosity and compressive strength of all the prepared scaffolds were evaluated. The average pore size of all the prepared composite scaffolds was in the range between 90 and 125 μm. Most frequent pore size in GCT 30 scaffold was the highest of 120 μm whereas that for GCH 30 was the lowest of 96 μm as suggested by Hg porosimetry analysis. GCH30 scaffolds showed the highest average compressive strength of 3.45 MPa as opposed to 2.24 MPa exhibited by GCB 30, with high degree of interconnected porosity appropriate for cellular colonization. To study the effect of different bioceramic phases on MSCs differentiation, scaffolds were cell cultured for up to 14 days in osteogenic medium. GCB30 scaffold showed higher capacity to proliferate MSCs cultured onto it as compared to other composite scaffolds. Degree of differentiation of MSCs into osteoblast was higher in GCB30 scaffolds than in the GCH30 and GCT30 composite scaffold as evident from higher amount of RUNX2 and osteocalcin expression in the former up to 14 days of cell culture. Inclusion of 58s bioactive glass particles showed positive effects on cell differentiation. In coherence with the in vitro appearance, histomorphometric analysis and fluorochrome study in a rabbit tibia model showed a significantly greater amount of new bone formation in GCB30 compared to other composite scaffolds. The results demonstrated that the prepared GCB30 scaffold could be a better candidate as bone substitute material for its higher bioactivity in bone tissue regeneration.