Abstract
Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells.ObjectivesThis study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Material and MethodsHuman ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. ConclusionsWe concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.
Highlights
Mesenchymal stem cells (MSCs) have been receiving considerable attention in bone regeneration, both for tissue engineering or cellular therapy
This study evaluated the effect of a recombinant Bone morphogenetic proteins (BMPs)-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate
We concluded that human Adipose-derived stem cells (ASCs) expressed endogenous BMP-4 and BMP-7
Summary
Mesenchymal stem cells (MSCs) have been receiving considerable attention in bone regeneration, both for tissue engineering or cellular therapy. These cells are capable of self-renewal or differentiation into multi-lineage cells, including osteoblasts and condroblasts, under the appropriate conditions. BMP-2 has been used for treatment of bone defects in orthopedic, spine, and maxillofacial surgeries1,5 It was observed during the fracture repair that an increase of BMP-4 and BMP-7 occurs in response to the presence of BMP-227, and it was suggested that BMP-4 and BMP-7 might be able to substitute each other during bone healing, as has been shown in other tissues where these BMPs are co-expressed. Et al. (2009) observed that bone marrow-derived stem cells promoted osteogenesis by endogenous BMP-2, BMP-4, and BMP-6
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