Osteogenic Differentiation of Dental Pulp Stem Cells via Titanium Deposition in a Static Magnetic Field

Osteogenic differentiation of dental pulp stem cells (DPSCs) offers potential treatments for bone-related diseases such as osteoporosis. DPSCs are preferred over other stem cells due to their ease of preservation and relative ease to obtain. Previous literature has shown that continuous exposure to a static magnetic field (SMF) induces osteogenic differentiation in rat DPSCs [1]. Past studies also suggest titanium-treated surfaces improve the rate of differentiation of DPSCs. [2] We report the degree of osteogenic differentiation with the combined effects of titanium deposition and an SMF, and compare each treatment individually. Investigating these factors will provide crucial insight to the differentiation of DPSCs and the discovery of a stem cell-based treatment for endemic bone diseases.<br/> DPSCs of strain 13 were isolated from third molar teeth and prepared via trypsinization every 4 days for 16 days. 4x104 cells were plated on each sample with agarose, along with MEM-α, 10% fetal bovine serum, and 1% glutaMAX. After 1 day, the media was changed to ascorbic acid and β-glycerophosphate, which has been shown to induce cell biomineralization. [2] 4 groups contained 2-inch x 2-inch silicon wafers while 2 others were in tissue culture plastic (TCP). All wafers were coated with Polylactic Acid (PLA). Half the wafers were further treated with titanium via atomic layer deposition (ALD). Half of all groups were placed in an SMF parallel to the surface for the duration of their incubation period of 28 days.<br/> Images produced by atomic force microscopy for ALD and PLA groups were analyzed via NanoScope Analysis to quantify the roughness of the wafers. Roughness did not play a role in differentiation, as it was in the nanometer scale for all groups. Cells were also counted via alamarBlue staining on days 1 and 5 to ensure similar proliferation rates.<br/> RT-PCR was performed after incubation to measure the expression of the following genetic markers: osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and bone sialoprotein (BSP). RNA lysed from DPSCs on day 0 was used as a baseline to compare the relative expression levels of each gene for each treatment group after the 28 days. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the housekeeping gene. Our results show an upregulation of BSP expression for the ALD/SMF group compared to PLA/SMF and TCP/SMF groups, confirming titanium deposition’s inductive effect on osteogenic differentiation. ALD/SMF produces the greatest osteogenic differentiation with respect to BSP expression. OCN expression was also upregulated by a factor of two for ALD groups compared to PLA, and by a factor of four when compared to TCP. DSPP expression was downregulated for ALD groups in comparison to PLA and TCP, and further downregulated for ALD/SMF. Addition of other magnetic materials, such as iron oxide, may further induce osteogenic differentiation.<br/> Collagen triple helices are abundant in osteoblasts. Scanning electron microscopy will be used to further analyze collagen triple helix formation, allowing us to understand the role of an SMF on the rate of collagen triple helix formation and thus osteogenic differentiation. Raman spectroscopy will be used to determine the nature of mineralization.<br/><br/>1. Hsu, S. H., & Chang, J. C. The static magnetic field accelerates the osteogenic differentiation and mineralization of dental pulp cells. Cytotechnology 62(2). 143–155 (2010).<br/>2. Chuang, Y.C,. et al. The Role of Titania Surface Coating by Atomic Layer Deposition in Improving Osteogenic Differentiation and Hard Tissue Formation of Dental Pulp Stem Cells. Adv. Engi. Mater. 23(9). 2100097 (2021)