Enhancing Osteogenic Differentiation of Dental Pulp Stem Cells Via Iron Oxide-Infused PLA in a Static Magnetic Field

Dental pulp stem cells (DPSCs) have promising potential for stem cell-based regenerative dental therapy due to their high capacity for proliferation and differentiation. Polylactic acid (PLA) is a degradable biocompatible polymer often used for bone regenerative scaffolds [1]. Exposure to a static magnetic field (SMF) can enhance biomineralization [2]. Here, we present the results of a study where DPSCs were plated on PLA scaffolds with and without iron oxide particles and cultured for 28 days in an external SMF of B=0.0375T, generated by neodymium magnets. Previous studies have used iron oxide nanoparticles to enhance the magnetic field, but these particles were observed to have entered the cells, resulting in potential toxicity. We therefore compounded the particles within the PLA scaffolds, where the particles were not available for cellular uptake. Furthermore, the cells were initially cultured in 3-D microspheres, which have been postulated to enhance differentiation [3], and then collected for plating on the PLA scaffolds.<br/><br/>To form spheroid-derived DPSCs (sd-DPSCs), DPSCs of strain 13, passage 7 were cultured in round bottom 96-well plates for 7 days without dexamethasone, an osteogenic inducer, and then resuspended. Sigma Aldrich Iron-Oxide Nanopowder and Nature Works PLA were combined in a twin screw extruder Brabender to produce 10% iron-oxide-infused PLA (IO-PLA). The scaffolds were produced via molding into 1.38 and 0.63 cm discs, which were then submerged in 70% ethanol, sterilized by UV exposure, and placed into 6-well plates. To ensure cell adhesion, a thin coating of 10 µg/mL collagen was applied prior to cell plating. Three different substrates were probed: Tissue Culture Plastic, PLA, and IO-PLA. Half of these plates were placed in the static magnetic field while the other half were not, as a control.<br/><br/>The elastic moduli of mesenchymal stem cells correlate to their differentiation potential and lineage – cells in the osteoblast lineage tend to have a higher elastic modulus pre-differentiation. Although there was no difference between the elastic moduli of sd-DPSCs and 2D-DPSCs on day 1, more than a two-fold difference was observed in the elastic moduli of sd-DPSCs compared to 2D-DPSCs on day 4, suggesting that sd-DPSCs are likely to exhibit more robust osteogenic differentiation than 2D-DPSCs.<br/><br/>The substrates were imaged on day 28 with SEM and EDX. The data clearly showed the deposition of collagen fibers, distinct triple helix spacing, and templated CaP mineralization on the substrates exposed to the magnetic fields, with the samples containing iron oxide particles exhibiting greater deposition of both minerals and ordered collagen fibers. Despite collagen deposition, minimal fiber structures were observed in the absence of the magnetic field. Even though some mineral deposition was observed, it was of a particulate nature, without specific templating. RT-PCR and Raman scattering results will be reported, which will identify whether differentiation occurred along osteogenic or odontogenic lineages.<br/><br/>We gratefully acknowledge the Louis Morin Charitable Trust for their support of this work.<br/><br/>[1] Barchiki, Fabiane, et al. “Biocompatibility of ABS and PLA Polymers with Dental Pulp Stem Cells Enhance Their Potential Biomedical Applications.” <i>Polymers</i>, vol. 15, no. 24, 2023, p. 4629.<br/>[2] Xia, Yang, et al. “Novel Magnetic Calcium Phosphate-Stem Cell Construct with Magnetic Field Enhances Osteogenic Differentiation and Bone Tissue Engineering.” <i>Materials Science and Engineering</i>: C, vol. 98, Elsevier BV, May 2019, pp. 30–41.<br/>[3] Bu, Nam-Ung, et al. “In Vitro Characterization of Dental Pulp Stem Cells Cultured in Two Microsphere-Forming Culture Plates.” <i>Journal of Clinical Medicine</i>, vol. 9, no. 1, 2020, p. 242.