Gene Expression of Semaphorin 7A in Human Dental Follicle Cells

The human dental follicle is an ectomesenchymally derived connective tissue surrounding the tooth germ, containing stem cells of the periodontium has been reported (Morsczeck et al., 2005). Human dental follicle cells (hDFCs) have the capacity to mineralize under in vitro culture using osteogenic induction medium (OIM). Moreover, the transplanted hDFCs with OIM were more capable of mineralization when compared to GM cultured hDFCs under in vivo. However, there is little known that the mechanism or key factor of osteogenic differentiation in hDFCs. The aim of this study was to investigate the key genes in hDFCs during osteogenic differentiation and mineralization using microarray analysis.

   hDFCs, which isolated from dental follicle tissue by impacted third moller teeth, were were digested in 0.1 U/ml collagenase type I and 1 U/ml dispase. Experiments using hDFCs were performed according to the guidelines established by the ethics committee of Nihon University School of Dentistry at Matsudo (Recognition number: EC10-036). To investigate the capacity for mineralization in vivo, hDFCs from the 5th to 8th passage were grown in a three-dimensional (3D) culture and cells were transplanted onto calvaria of F344/NJcl-rnu/rnu male rat. Hematoxylin and eosin (H-E) staining and immunohistochemistry were performed, and newly formed bone was evaluated at micro-computed tomography (micro-CT) in day 28. In vitro studies, hDFCs were cultured with OIM and Alkaline phosphatase activity was measured. For mineralization, Alizarin red and von kossa staining were performed. To investigate the gene expression during osteogenic differentiation was performed by microarray, RT-PCR or real time-PCR curtured with OIM at Day0 and Day17 compared to Growth medium (GM) cultured.

   In vivo study, micro-CT stigmatically showed that transplanted hDFCs promoted better bone quality and bone volume compared to control on day 28. In vitro study, the ALP activity in hDFCs cultured with OIM was significantly higher than in those cultured in GM on day 13 and 16. We found several Semaphorin family genes were expressed by microarray analysis. Among these semaphorin families, semaphorin7A was up regulated cultured in OIM compared to culture in GM. Moreover, real time-PCR showed that sema-7A and their receptors were also up regulated with OIM cultured compared to GM cultured.

The Semaphorin family is a large family of phylogenetically, secreted, and membrane-bound proteins that are divided into eight classes based on sequence similarities and distinct structural features. Among these Semaphorin family, It is well known that Sema-7A plays a role in axon guidance through the MAPK (mitogen-activated protein kinase) pathway (Pasterkamp RJ et al., 2003). Resent study reported that Sema-7A play prominent roles in mesenchymal stem cells to induce osteogenic differentiation and promote osteoblast migration. Our finding suggest that sema- 7a was upregulated in OIM cultured compared to GM cultured suggest the sema-7A is a one of gene that related in osteogenic differentiation of hDFCs. Finally human dental follicles might be a potentially useful source for regenerative therapy.

Morsczeck C, Götz W, Schierholz J, Zeilhofer F, Kühn U, Möhl C, Sippel C, Hoffmann KH. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 2005; 24(2):155-65

Pasterkamp RJ, Peschon JJ, Spriggs MK, Kolodkin AL. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature. 2003; 424(6947):398-405