Growth Factor Dose Tuning for Bone Progenitor Cell Proliferation and Differentiation on Resorbable Poly(propylene fumarate) Scaffolds.

One approach to the development of an artificial graft material could rely on uniform coverage of a resorbable biomaterial with bone extracellular matrix (ECM). To achieve this on the surface of poly(propylene fumarate) (PPF) scaffolds, we selected a growth factor regime of basic fibroblast growth factor (FGF-2) (5 ng/mL), platelet-derived growth factor (PDGF-BB) (40 ng/mL), and epidermal growth factor (EGF) (20 ng/mL) to stimulate proliferation of bone marrow-derived human mesenchymal stem cells (BM-hMSCs). Bone morphogenetic protein (BMP) 4 (50 ng/mL), 6 (50 ng/mL), and 7 (27 ng/mL) in the presence of the following osteogenic substances: dexamethasone (10(-7) M), ß-glycerophosphate (10 mM), and ascorbic acid (50 µg/mL) were chosen to induce differentiation of BM-hMSCs into ECM-secreting osteoblasts. These growth factors were also studied at 10× concentration to determine dose effect. Proliferation was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, scanning electron microscopy (SEM), and toluidine blue staining, whereas differentiation was analyzed through alizarin red S staining and assay, alkaline phosphatase (ALP) staining and assay, and SEM. The proliferation study suggests that a combination of EGF, PDGF-BB, and FGF-2 growth factors at optimal concentration over a period of 1 week exhibits significantly (p = 0.001) higher number of cells (116,024 ± 5165) than these cytokines without EGF (91,706 ± 11,965). Increasing the dosage does not show any significant effect. The BM-hMSC differentiation study results show that ALP enzyme production and mineral deposition increase from day 14 to day 21 in all groups containing BMPs and osteogenic medium. However, mineralization is significantly higher in the BMP-7 group. Furthermore, the feasibility of translating the results from two dimensional thin films to three dimensional-printed PPF scaffolds was determined through uniform initial seeding and spreading of BM-hMSCs. Therefore, we have determined the optimum dose of growth factors for proliferation and differentiation of BM-hMSCs on the surface of PPF scaffolds, which can be used to produce ECM-coated implants for the treatment of bone defects.