| Summary: | <p> There are multiple causes of tooth loss can be caused by multiple reasons, including caries, periodontal disease, trauma and genetic disease. The dental pulp has important functions to sustain teeth, by providing nutrient and oxygen supply, innervation and immune response. Our <i>objective</i> was to determine whether incorporating extracellular matrix derived from decellularized pig tooth buds (tECM) into silk scaffolds would enhance dental pulp derived dental mesenchymal stem cell (DMSC) differentiation. Nine different types of silk scaffolds were fabricated: 1) 6% silk; 2) 6% silk+tECM; 3) 6% silk+collagen; 4) 3% silk, 4g salt; 5) 3% silk, 4g salt + tECM; 6) 3% silk, 4g salt + collagen; 7) 3% silk, 2g salt; 8) 3% silk, 2g salt + tECM; and 9) 3% silk, 2g salt + collagen. Unseeded silk scaffolds were used as controls. Three replicates were used for each experimental condition and controls. Porcine DMSCs (pDMSCs) at passage 2 were seeded into silk scaffolds (3×10<sup>5</sup> cells/scaffold, ~3×10<sup>3</sup>cells/mm<sup>3</sup>) and cultured <i> in vitro</i> in osteogenic media for 24 hours, or for 2 and 4 weeks. Histological analyses of paraffin embedded and sectioned constructs revealed pDMSC attachment and proliferation after two and four-week <i>in vitro </i> culture. Immunofluorescent (IF) analysis showed robust dentin sialoprotein (DSP) expression in all pDMSC seeded silk scaffolds. DSP expression was enhanced in scaffolds containing tECM. Statistical analysis demonstrated significant differences in DSP expression in scaffolds with versus without tECM (P≤0.01). Silk scaffolds supported the attachment, proliferation and differentiation of seeded pDMSCs. Furthermore, our research is the first study to demonstrate that tECM can enhance the pDMSC differentiation, suggesting that tECM incorporated scaffolds show promise for dental pulp and/or whole tooth regeneration. Studies of in vivo implanted constructs are being performed to further investigate the effect of tECM on DMSC differentiation and mineralized tissue formation. </p><p>
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