178 Differential Behavior of Porcine Mesenchymal Stem Cells from Bone Marrow and Adipose During Osteogenic Differentiation on a Glycosaminoglycan Hydrogel Scaffold for Bone and Cartilage Tissue Engineering

Abstract

The pig is an ideal species for use in tissue engineering studies targeted towards repair of bone and cartilage defects. Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. In order to determine the suitability of these scaffolds for use in porcine model systems for bone and cartilage tissue engineering, we have begun to investigate the behaviour of porcine mesenchymal stem cells on this material. The purpose of this study was to determine whether mesenchymal stem cells from adipose (ASC) and bone marrow (BMSC) form bone on a CG scaffold material. Primary BMSC and ASC from 6-month-old Yorkshire pigs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum. The ASC and BMSC were then trypsinized at passage 4 or 5 and used to seed ~4-mm-diameter CG scaffolds with 2 million cells/scaffold. Scaffolds were seeded by suspending the cells in medium that had been equilibrated for 30 min, and then placing the CG scaffold into the medium. This method of seeding was determined to be most effective in previous experiments. Scaffolds were then cultured for 7 days in DMEM followed by 21 days in osteogenic media. At the conclusion of the incubation period, the diameter of the scaffolds was measured, and they were fixed with 4% paraformaldehyde and cryosectioned. Then, 10-µm sections were stained with Alizarin Red to assay for mineralization, a hallmark of osteogenic differentiation. Both ASC- and BMSC-loaded scaffolds showed Alizarin Red staining throughout the section after incubation, demonstrating that both undergo osteogenesis on the scaffold material (n = 4). During osteogenic differentiation, scaffolds seeded with both ASC and BMSC showed a decrease in diameter. Unseeded scaffolds showed no decrease in size when in media. The BMSC scaffolds demonstrated a more extensive decrease in size than ASC. The average diameter of ASC loaded scaffolds after differentiation was 2.49 ± 0.39 mm, and that of BMSC-loaded scaffolds was 1.47 ± 0 0.18 mm (n = 3, P < 0.05, Student’s t-test). This suggests a differential ability of ASC and BMSC to break down and metabolize the scaffold matrix, and may indicate that one cell type may be preferable to the other for repairing osteogenic defects using these scaffolds. Current experiments underway will analyse expression of matrix-degrading enzymes to determine the source of the difference between cell types in scaffold shrinkage during differentiation. We will also quantify mineralization in ASC- v. BMSC-loaded scaffolds and assay gene expression of osteogenic markers to determine if there is a difference in osteogenic potential between sources of mesenchymal stem cells on these scaffolds.