Evaluation of the donor cell contribution in rhBMP-2 mediated bone formation with chitosan thermogels using fluorescent protein reporter mice.

Abstract

Our current understanding regarding the contribution of donor cells in growth factor and cell based tissue regeneration strategies is limited. The present study attempts to utilize fluorescent protein reporter mice [Col3.6Topaz (enhanced yellow fluorescent protein, EYFP) as host and Col3.6Cyan (enhanced cyan fluorescent protein, ECFP) as donor] to determine donor cell contribution in bone regeneration using a bilateral calvarial defect model. Thermogelling chitosan hydrogels (Chi-AHP) were used as bone marrow stromal cell (BMSC) delivery vehicle in the presence and absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). Co-delivery of rhBMP-2 and donor BMSCs led to a significant increase in bone formation indicating the potential of using the combination approach for improved regeneration. On a cellular level, presence of rhBMP-2 resulted in an increased host cell derived osteoblast infiltration at 8 weeks. However, the new mineralized tissue presented two distinct morphological features based on its cellular origin. Regenerated tissue associated with ECFP positive donor cells showed a woven bone-like structure with diffuse alizarin complexone (AC) label and minimal tartrate resistant acid phosphatase (TRAP) activity, indicating the presence of immature early osteoblast cells depositing mineralized tissue without progressing to a mature lamellar bone. Host derived bone showed sharp mineralization line (AC), strong alkaline phosphatase (ALP) and TRAP activity, indicating the presence of actively mineralizing and remodeling, mature lamellar bone matrix. The study demonstrated the remarkable potential of transgenic reporters to improve our understanding of donor cell contribution during bone formation.