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BMP2 Stimulation of Adipose, Bone Marrow, and Muscle-Derived Stromal Cells Fails to Augment Calvarial Repair
Sameer Shakir, BS, Dan Wang, DMD, Melissa A. Shaw, BS, Darren M. Smith, MD, Sanjay Naran, MD, James R. Gilbert, PhD, Zoe M. MacIsaac, MD, Joseph E. Losee, MD, Gregory M. Cooper, PhD.
University of Pittsburgh, Pittsburgh, PA, USA.
PURPOSE: Tissue engineering is becoming a viable adjunct - or even alternative - to autologous bone grafting in craniofacial reconstruction. Current methods focus on implantation of bioresorbable scaffolds seeded with proteins and/or osteogenic progenitor cells, although it remains unclear whether specific stromal cell types are better suited for use in craniofacial reconstruction. This study aims to determine the healing capacity of adipose (ADSC), bone marrow (BMDSC), and muscle-derived (MDSC) stromal cell populations in a calvarial defect model when this environment is optimized using bone morphogenetic protein 2 (BMP2). We hypothesize that BMP2 will augment stromal cell engraftment and differentiation within calvarial defects.
MATERIALS AND METHODS: Bone marrow, muscle, and adipose tissues were harvested from 10-week old wildtype mice (n=8). Cells were seeded overnight onto 5mm acellular dermal matrix (ADM) discs (1 x 105 cells/disc) and were osteoinduced with 150ng (30ng/mm2) BMP2. Unseeded ADM discs treated with either BMP2 or vehicle served as controls. Discs were subsequently placed into 5mm circular calvarial defects in 10-week old wildtype mice. Mice were euthanized 4 weeks postoperatively. Regenerate tissue was analyzed by 3D microCT and histology to assess percent healing and tissue morphology.
RESULTS: Differences in percent healing (mean ± SE) were observed between vehicle control (31.5%±8.8), BMP2 control (71.9%±7.0), ADSC + BMP2 (31.4%±1.8), MDCS + BMP2 (21.9%±4.9), and BMDSC + BMP2 (38.5%±20.2) groups. One-way ANOVA revealed a statistically significant main group effect (F=3.988, p<0.02). Percent healing was significantly decreased in osteoinduced stromal cell constructs and in vehicle control when compared to unseeded, BMP2 therapy. Pentachrome staining revealed histological evidence of endochondral ossification in all treatment groups. BMP2 treated defects regenerated vascularized, thick woven bone with large marrow spaces. Osteoinduced stromal cell-treated defects regenerated less bone that was also less thick when compared to BMP2-regenerated bone. However, regenerated bone was consistently limited to the region of interest in these treatment groups.
CONCLUSIONS: Low-dose BMP2 potently stimulates local osteoprogenitors to heal osseous deficiencies within the calvaria. We observed significant modulation of BMP2-induced osteogenesis with the addition of stromal cells; unlike BMP2 therapy alone, osteoinduced stromal cell therapies do not improve defect healing beyond that of vehicle control in this model. These observations collectively call into question the role of progenitor cells in tissue engineering strategies for calvarial repair, and suggest that engrafted cells may be susceptible to environmental influences that determine their ability to contribute to cranial regeneration. Based upon these findings, we suggest that the inherently heterogeneous population of cells within the stroma of adipose, bone marrow, and muscle tissues may restrict BMP2-induced calvarial defect healing.
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