The Effect of Microsphere Growth and Static Magnetic Field on Osteogenic Differentiation of Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) have great potential in regenerative medicine due to their noninvasive extraction, low immunogenicity, and multipotency [1]. Previous research revealed that under the influence of chemical stimuli, DPSCs can differentiate into functional osteoblasts that secrete extracellular matrix (ECM) [1, 2]. The osteogenic potential of DPSCs is of great importance due to its therapeutic applications in bone mineralization and reconstruction [3]. Through careful modifications of the cell microenvironment such as three-dimensional culture methods [4] and magneto-mechanical stimulations [5], the osteogenic differentiation of DPSCs can be enhanced significantly. This study aims to investigate the effect of microsphere formation and static magnetic fields on osteogenic differentiation.<br/><br/>After trypsinization after passage four of DPSCs, cells were placed in U-bottom non-adhesive suspension wells or flat-bottom wells covered with 50, 100, and 150 uL 2% agarose to induce microsphere formation. Different volumes of agarose produce different degrees of curvature, thereby influencing the compactness of the microspheres. Through EVOS imaging, we observed that agarose wells were better at forming dense aggregates compared to suspension wells. At different timepoints after microsphere growth (5 and 7 days in wells), the cells were plated onto 6-well tissue culture plates. After 7 days of plating, ascorbic acid and β-glycerophosphate were added into the medium to induce osteogenic differentiation. After 28 days of plating, PCR was performed to quantify mRNA of osteocalcin (OCN) and dentin sialophosphoprotein (DSPP). All results were normalized with the housekeeping gene GAPDH related to Day 0. The results confirmed the observations from EVOS that agarose wells induced greater osteogenic potential. DPSCs in 100 uL agarose wells plated 5 days after growth expressed the greatest level of OCN. Unexpectedly, suspension wells seem to induce greater odontoblastic potential than agarose wells. The expression of DSPP was highest in suspension wells plated 5 days and 7 days after growth.<br/><br/>The synergistic effect of magnetic fields and magnetic scaffolds has been proposed to enhance osteogenesis through activating signaling pathways [5]. For the magneto-mechanical stimuli, cells were plated onto different scaffolds and incubated in the presence or absence of magnetic fields. The three types of compression-molded scaffolds were pure polylactic acid (PLA), a biodegradable polyester frequently used in tissue engineering [6], 95% PLA and 5% RDP clay, and 90% PLA and 10% magnetic iron oxide microparticles. Using atomic force microscopy (AFM) to characterize surface topology, we found that all three scaffolds were of nanoscale roughness, with the scaffolds containing iron oxide being the roughest. After 28 days of incubation, EVOS was used to image cell morphology and biomineralization. Cells were stained with Alexa Fluor 488, DAPI, and Xylenol Orange to visualize actin, DNA, and calcium deposition respectively. We observed calcium deposition between the DPSCs. PCR results of the osteogenic-related genes confirmed the synergistic effect as the expression level of DSPP and OCN in cells grown in presence of magnets on iron oxide scaffolds were significantly higher than cells grown on pure PLA with magnets present and cells grown on iron oxide scaffolds without magnets. SEM will be used to determine the conformation of extracellular matrix proteins deposited on the different scaffolds and their ability to template the mineralized deposits. Raman spectroscopy will be used to determine the nature of the mineralized tissue.