Effect Of SATB2on Osteoblast Differentiation Of Mandible Bone Marrow Stromal Cells

[Background] Bone marrow stromal cells (BMSCs) are multipotential stem cellsthat give rise to a variety of different cell types. However, many studies haveindicated that BMSCs from the mandible and tibia have phenotypic and functionaldifferences. Compared with tibia BMSCs(T-BMSCs), mandible BMSCs(M-BMSCs)have a stronger osteogenic potential. Recent studies demonstrated that specialAT-rich binding proteins2(SATB2) plays an important role in osteoblastdifferentiation. To investigate the mechanisms regulating osteoblast differentiation intwo different types of BMSCs, we compared the temporal and spatial mRNA andprotein expression patterns of Satb2in M-BMSCs and T-BMSCs undergoingosteoblast differentiation. Moreover, we investigated the contribution of SATB2tothe stronger osteogenic potential of M-BMSCs, which will make some informationavailable for us to application of SATB2in craniofacial bone healing andregeneration in future.[Methods]1. BMSCs isolated from the mandible and tibia using Friedenstein’s methods. They were allowed to attach for3days, at which point nonadherent cellswere removed by changing the culture medium. Subsequently the media was changedevery3days. The primary cells were used to perform colony forming efficiency assayand third passage cells were used to MTT assay. In addition, we investigated theexpression of keratins8, keratins18, E-cadherin, desmin, GFAP, vimentin and N-cadherin in the two types of BMSCs using fluorescent immunostaining.2. The thirdpassage cells were cultured in osteogenic medium for2weeks. ALP activity wasdetermined colorimetrically using p-nitrophenyl phosphate as a substrate aftercultured in osteogenic medium3,5,7and9days. The third passage cells cultured intraditional medium and osteogenic medium for1week were performed ALPcytologic staining respectively. Cultured in osteogenic medium for7,10and14days,cells were fixed with70%ethanol and stained with2%Alizarin Red S to assesscalcium accumulation. The temporal and spatial mRNA and protein expressionpatterns of Satb2, Hoxa2, Runx2, Ocn and Opn were examed using real-time PCR,western blotting and fluorescent immunostaining in M-BMSCs and T-BMSCsundergoing osteoblast differentiation.[Results]1. Spindle-shaped adherent cells did not show the morphologicaldifferences between mandible groups and tibia groups under a phase-contrastmicroscope. Immunophenotypic properties of BMSCs between two groups alsodemonstrated some similarities. Both M-BMSCs and T-BMSCs showed the negativeexpression of epithelium markers for keratins K8, K18and E-cadherin, and thepositive expression of desmin, GFAP, vimentin, and N-cadherin. However, morecolony forming units were observed in M-BMSCs than T-BMSCs after10daysculture. The proliferation rates were much higher in M-BMSCs compared toT-BMSCs.2. Higher levels of alkaline phosphatase, greater calcium accumulation,earlier and more expression of Runx2, Ocn and Opn were observed in osteogenic-induced M-BMSCs compared to T-BMSCs.3. Satb2was expressedearlier in M-BMSCs, and Hoxa2, a downstream target of Satb2, was not expressed inuninduced M-BMSCs or during osteoblast differentiation. In contrast, Hoxa2wasreactivated in T-BMSCs during osteoblast differentiation.[Conclusion] This study further confirmed the effect of embryonic origins on theosteogenic potential of BMSCs. In addition, we have found that SATB2plays animportant role in regulating these differences in BMSCs osteoblast differentiation.The earlier activation of Satb2expression in M-BMSCs compared to T-BMSCs mayexplain the stronger osteogenic potential of M-BMSCs.