Method: Gelatin-based Gelfoam® sponges were cut into 10x5x7 mm3 pieces, seeded with 2x106 pig bone marrow stromal cells (pBMSCs), and incubated with serum-free regular medium (TCM) or osteogenic medium (ODM). Scaffolds were collected at day 1, 3, 5 to analyze cell integration/growth by DNA assay and histological analyses. Image series from H&E sections were captured randomly and cells were counted in a blinded fashion. For in vivo testing, bone marrow was aspirated from a 3-month-old pig, from which 100-million pBMSCs were expanded. Then bilateral mandibular osteotomies were performed and Gelfoam scaffolds (60x20x7mm3) with or without autologous BMSCs (5days in TCM) were transplanted into either osteotomy. After a 2-day latency, 10 mm-distraction (1mm/day) and 4-week-consolidation, distraction-site regenerates were analyzed by Cone-beam CT (250mm voxel-size) for remaining gap width and by micro-CT (27mm voxel-size) for stereology parameters. Bone volume fraction (BV/TV) of 6-12 3x3x3mm3 regions in the central and peripheral (near host bone) regenerates was quantified.
Result: Live cell content (DNA assay) in the Gelfoam scaffold peaked at 3 and 5 days of incubation in ODM and TCM media, respectively, with ODM being higher (119% vs. 80% of initial cell-loading number). Histology confirmed that cell density reached peaks (ANOVA, p<0.05) at 3 and 5 days for ODM (surface/interior: 2766/1633cells/mm2) and TCM (surface/interior: 2266/1126cells/mm2), respectively. In-vivo testing demonstrated that the cell-scaffold side had substantially smaller remaining gap (1.3mm vs. 3.6mm) and higher BV/TV (central/peripheral zones: 0.46/0.63) than the scaffold-only side (central/peripheral zones: 0/0.30).
Conclusion: This study established optimal incubation times for integrating/growing pBMSC on Gelfoam scaffolds in serum-free media before in-vivo transplantation, which may strongly enhance mandibular distraction osteogenesis.
Keywords: Bioengineering, Bone repair, Cell culture, Distraction osteogenesis and Regeneration