VEGF Modulates BMP-induced Osteogenesis By Human Adipose Derived Stem Cells

Background and Purpose:Traumatic bone defect and fracture nonunion are crucial problems in orthopedics. In order to effectively promoting healing of nonunion and bone defect, the stem cells and synthetic graft material combined with growth factors is the best treating strategy, and are the focuses of orthopedic research.Adipose derived stem cells (ADSCs) have multi-potent differentiation abilities which can differentiate into fat, bone, cartilage, nerve or muscle etc. while stimulated by different external growth factors. Therefore, applying ADSCs in promoting bone repairing has been became one of important research topic in orthopedics. Current research mainly focuses on some specific bone induction growth factors such as vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) etc. BMPs, one kind of cytokines which belong to transforming growth factor-β(TGF-β) superfamily, is an important factor for embryonic development and the inductions of adult bone and cartilage differentiation. VEGF is known as the most important regulator on angiogenesis which promotes vascular endothelial cell proliferation, induce angiogenesis and promote bone healing. Recently, several studies demonstrated that combined treatments of VEGF and BMPs showed synergistic effects on bone regeneration and repair in vitro and in vivo studies. Enhanced osteogenesis by VEGF/BMPs is vary depending on the ratio of VEGF and BMPs. However, whether different ratios of VEGF/BMPs have effects on osteogenesis by ADSCs is rarely investigated. In this study, the effects of various ratios of VEGF/BMPs on osteogenesis of human ADSCs (hADSCs) were examined. The specific aims of this study are shown as following:1. To verify whether hADSCs have adipogenic and osteogenic abilities.2. To test the effects of different exogenous recombinant human bone morphogenetic proteins (rhBMPs) on osteogenic differentiation of hADSCs in vitro.3. To verify the optimal ratio of rhBMPs and rhVEGF for promoting hADSC osteogenesis to establish the foundation for future in vivo studies and clinical translation for treatment of large bone defects and non-unions.Material and Methods:1. Induction of the adipogenic and osteogenic differentiation of hADSCs, the differentiation status was determined using Sudan IV and ARS staining at 21 days.2. Three different exogenous rhBMPs were used to induce osteogenic differentiation on hADSCs in vitro. Five groups for each time point were used for this study design:1) BM (DMEM containing 10% FBS with 50μg/ml ascorbic acid and 10-7M dexamethasone),2) OM (DMEM containing 10% FBS with 50μg/ml ascorbic acid,10 mMβ-glycerophosphate, and 10-7 M dexamethasone),3) BMP-2 group; 4) BMP-6 group; 5) BMP-9 group. ARS staining was used to test the Mineralization at 14,21 and 28 days. RT-PCR was used to detect the expression of ALP, OCN, Coll, BSP, Runx2, OSX, Dlx5 and Msx2 at 14,21 and 28 days.3. Optimizing rhBMP and rhVEGF ratio to induce osteogenic differentiation of hADSCs. Twelve groups for each time point were used for this study design:1) BM group, 2) OM group, 3) 1ng BMP-6 group, 4) 10ng BMP-6 group, 5) 0.1ng VEGF group, 6) 1ng VEGF group, 7) 10ng VEGF group, 8) 1ng BMP-6+0.1ng VEGF group, 9) 1ng BMP-6+1ng VEGF group, 10) 1ng BMP-6+10ng VEGF group, 11) lOng BMP-6+1ng VEGF group, 12) 10ng BMP-6+10ng VEGF group. ARS staining was used to test the Mineralization at 14,21 and 28 days. RT-PCR was used to detect the expression of ALP, OCN, ColI, BSP, Runx2, OSX, Dlx5 and Msx2 at 14, 21 and 28 days.Results:1. Adipogenic induction at 21 days:Sudan IV staining showed significant red dyed lipid droplets in BM group and 10-4M dexamethasone group.2. Osteogenic induction at 21 days:ARS staining showed significant red dyed calcium nodules in OM group.3. Results of different exogenous rhBMPs to induce osteogenic differentiation of hADSCs in vitro:ARS staining showed that the OD increased significantly in BMP-9 group compared with OM group at 14 days (P<0.01). RT-PCR results showed that at 14 days, the expression of OCN (P<0.05), BSP and Runx2 (P<0.01) increased significantly in BMP-9 group, the expression of OCN increased significantly (P<0.01) in BMP-6 group; at 21 days, the expression of OCN (P<0.01), ColI and Runx2 (P<0.05) increase significantly in BMP-6 and BMP-9 groups; at 28 days, the expression of Runx2 (P<0.05), ALP and BSP (P<0.01) increased significantly in BMP-9 group, the expression of ALP (P<0.01) and BSP (P<0.05) increased significantly in BMP-2 group.4. The results of rhVEGF regulating the osteogenic differentiation on hADSCs induced by rhBMP-6:ARS staining showed that at 14 days, the OD increased significantly in 1ng BMP-6 group and 10ng VEGF group (P<0.05), 10ng BMP-6+1ng VEGF group (P<0.01); at 28 days, the OD increased significantly in 0.1ng VEGF group, 1ng VEGF group and lOng VEGF group (P<0.01), 1ng BMP-6+0.1 ngVEGF group and 1ng BMP-6+10ng VEGF group (P<0.05); at 14 days, the OD increased significantly in lOng BMP-6+1ng VEGF group compared with 10ng BMP-6 group or 1ng VEGF group (P<0.01); at 28 days, the OD increased significantly in 1ng VEGF group compared with 1ng BMP-6+1ng VEGF group and 10ng BMP-6+1ng VEGF group (P<0.01); the OD increased significantly in 10ngVEGF group compared with lOng BMP-6+10ng VEGF group (P<0.01).RT-PCR results showed that at 14 days, the expression of ALP in 1ng VEGF group (p<0.01), the expression of OCN in 1ng VEGF group, 1ng BMP-6+1ng VEGF group and 1ng BMP-6+10ng VEGF group (P<0.05), and 10ngVEGF group (P<0.01), OSX and Msx2 in lOngVEGF group (P<0.05) increased significantly compared with OM group; at 21 days, the expression of Coll increased significantly (P<0.01) in all groups except the BM and OM groups; the expression of Coll increased significantly (P<0.05) in 1ng BMP-6+1ng VEGF group compared with 1ng VEGF group, the expression of ColI(P<0.01) and OSX(P<0.05) increased significantly in 1ng BMP-6+10ng VEGF group compared with 1ng BMP-6 group and 10ng VEGF group.Conclusions:1. hADSCs cultivated and induced in vitro have the ability of adipogenic and osteogenic differentiation.2. Osteogenic media and rhBMPs have the effect of osteogenic differentiation on hADSCs.3. hADSCs respond to all studied rhBMPs based on the results of gene expression. rhBMP-9 and rhBMP-6 have the best ability of osteogenic differentiation of hADSCs.4. At molecular level and cell signal pathway, combination of rhVEGF and rhBMP-6 can significantly promote the osteogenic differentiation of hADSCs compared with rhVEGF alone or rhBMP-6 alone. Furthermore, the ability of osteogenic differentiation of rhBMPs/rhVEGF ratio 1:10 is better than 1:1 or 10:1.