The Mechanisim Of Osteogenic Differentiation For Mesenchymalstems Cells Induced By Bone Morphogenetic Protein9and Wnt3a

ObjectiveBMP9and Wnt/β-catenin signaling pathway both are able to induceosteogenic differentiation of mesenchymal stem cells (MSCs), but the roleof Wnt/β-catenin signaling pathway in BMP9-induced osteogenicdifferentiation is not well understand. Thus, our experiment was undertakenin effort to investigate the interaction between BMP and Wnt/β-cateninpathway in inducing osteogenic differentiation of MSCs.Methods1. C3H10T1/2cells was infected by recombinant adenovirusexpressing BMP9, Wnt3a, BMP9+Wnt3a, GFP.2. ALP, the early osteogenic marker, was detected by quantitative andstaining assay.3. Later osteogenic maker mineral calcium deposition was determinedby Alizarin Red S staining.4. The expression of OPN, OC, Runx2was analyzed by Real time PCR and Western blotting.5. Animal assay was carried out to further confirm the role of Wnt3ain ectopic bone formation induced by BMP9in vivo.Results1. The ALP activity was measured at day7after treated by GFP,BMP9, Wnt3a, BMP9+Wnt3a. We found that BMP9+Wnt3a significantlyincreased ALP activity in C3H10T1/2cells, compared to Wnt3a, BMP9group(P＜0.01).2. Matrix mineralization was detected at day14. We found thatWnt3a+BMP9significant increased mineralization of C3H10T1/2cellcompared to Wnt3a, BMP9group.3. The expression of OC, OPN was detected by Western blotting andQ-PCR at day9. We found the expression of the OC and OPN were morein group BMP9+Wnt3a than in BMP9group and Wnt3a group.4. The expression of Runx2was detected by Western blotting andQ-PCR at day2. We found the expression of the Runx2was more in groupBMP9+Wnt3a than in BMP9group and Wnt3a group.5. In vivo, subcutaneous osteogenetic mass size of Wnt3a+BMP9group was bigger than the Wnt3a group and BMP9group, HE staining andMasson trichrome staining showed that neo-osteogenetic cells increasedand calcification degree increased in BMP9+Wnt3a group. ConclusionsThe results indicated that Wnt3a enhanced BMP9-induced osteogenicdifferentiation of C3H10T1/2cell, and Runx2may be a linkage betweenthe two signal pathways.