Nanofibrous engineered matrix induces odontoblast differentiation through Wnt signaling pathways

Abstract

Nanofibrous engineered matrix induces odontoblast differentiation through Wnt signaling pathways Saeed Ur Rahman Molecular Genetics Major, Department of Dental Science The Graduate School of Seoul National University (Directed by Prof. Kyung Mi Woo, D.D.S., PhD)

Biomaterials have an important role in tissue engineering and regenerative medicine. In this aspect, the nanofibrous engineered matrix holds high potential for cellular differentiation and tissue regeneration that mimic the morphology of natural extracellular matrix microenvironment. Nanofiber induces the differentiation of cells not only committed to specific lineages, but also undifferentiated mesenchymal stem cells. Stem cells may require the specific extracellular signals that lead to induce in different lineages. However, the mechanisms by which nanofibrous engineered matrix promotes mesenchymal stem cells response differentially that lead to specific differentiation are mostly unknown. Some specific inductive factors might be involved in the initiation of such stimuli. However, the first aim of the present study was to investigate the responsiveness of mesenchymal stem cells on the nanofibrous matrix. Here, our study demonstrated that nanofibrous engineered matrix induces the growth factors expression profile differentially among mesenchymal stem cells at same substrate and in same culture condition. Among growth factors, Wnts and BMPs signaling molecules were highly expressed in dental pulp-derived mesenchymal stem cells on the nanofibrous matrix. Among differentiation markers, dentin sialophosphoprotein (DSPP) expression was highly increased in human dental pulp-derived stem cells (hDPSCs) on polystyrene nanofibrous matrix (PSF), bone sialoptotein (BSP) expression was increased in hADSCs, while osteocalcin (OCN) expression was increased in human bone marrow-derived stem cells (hBMSCs) on PSF during osteogenic condition. It has been reported that Wnt signaling pathway has an important role in odontoblast differentiation in-vitro and tooth development in-vivo. However, the underlying mechanism was largely unknown. Here, we found that Wnt3a induces DSPP expression directly by canonical Wnt/β-catenin signaling pathway by using the nanofibrous engineered matrix. MDPC23 pre-odontoblasts cultured on electrospun PSF exhibited intense alizarin-red staining and abundant expressions of DSPP and DMP1, indicating that PSF promoted odontoblast differentiation. Canonical Wnt signaling pathway was involved in the PSF-induced DSPP expression indicating that, PSF maintained high level of β-catenin, and the β-catenin in the cells grown on PSF was trans-localized to nucleus. Further, we investigated that PSF increased activities of an LEF/TCF reporter. Knockdown of β-catenin and treatment with rDkk1 abrogated PSF-induced DSPP expression, while the forced expression of LEF1 and treatment with LiCl enhanced DSPP expression. In addition, adding the rhWnt3a protein to the culture of dental pulp cells increased DSPP expression. Furthermore, transplantations of MDPC cells seeded on PSF to the subcutaneous tissue of nude mice confirmed the association of PSF-stimulated Wnt3a with the DSPP expression in an in vivo condition. Overall, these results demonstrate that Wnt3a induces DSPP expression through canonical Wnt signaling pathway when cells cultured on nanofibrous engineered matrix. The nanofibrous matrix that mimics the feature of natural extracellular microenvironment not only binds the cells but also exert their impact on cell survival, shape, and reorganization. Therefore, the nanofibrous matrix implicates to facilitate cellular behavior such as cellular attachment, differentiation, morphology, and activate the intracellular signaling that lead to induce these characteristics. However, the morphological alterations and actin reorganization in dental pulp cells during differentiation induced by the nanofibrous matrix was unknown. Cellular polarization is an important morphological feature of odontoblast differentiation. Here we investigated that morphological alteration of MDPC-23 cells induced by noncanonical Wnt signaling pathway that has an important role in the generation of cellular polarization. Next, we found that Wnt5a is involved in morphological alterations of cells during odontoblast differentiation. The cells height and cell processes formation on PSF was increased and was further increased with rWnt5a treatment. The cell processes formation in cells on PSF was induced through CDC42 while the stress fibers formation was induced through RhoA in cells on TCD. Knockdown of Wnt5a, Ror2 or CDC42 abrogated PSF-induced cell processes formation. The stress fiber formation was increased in cells on TCD and was further increased with rWnt5a treatment

this stress fiber formation was reduced with knockdown of RhoA. The CDC42 activation was increased in cells on PSF while RhoA activation was increased on TCD. Collectively, the results in this study provide evidence that nanofibrous engineered matrix induces odontoblast differentiation through Wnt signaling pathways. Key word: Nanofiber, odontoblast, Wnt3a, β-catenin, Wnt5a Student number: 2009-31329