Introduction: With the high incidence of pulpitis and obstacles of current endodontic treatments, tissue engineering has attracted increasing interest in dental tissue regeneration. Although great effort has been made on using synthetic and natural biomaterials in dentin regeneration, most materials fail to mimic the complex properties of collagen in dentin matrix, especially in inducing the biomimetic mineralization, a key factor for normal cell activity. Our study aimed to produce an elastin-like statherin peptide-containing recombinamer (st-ELR) scaffold with nanofibrous structure to promote dentin regeneration. Based on the ability of ELR fibers to undergo thermal-sensitive phase transformation, we used the biomimetic polymer induced liquid precursor (PILP) process to intra-fibrously mineralize them. We further investigated the role of mineralized ELR scaffolds in the proliferation and differentiation of human dental pulp stem cells (hDPSCs). Materials and Methods: St-ELR, and ELR without statherin-derived peptide (Ref-ELR) were electrospun to form nano-fibrous scaffolds and then subjected to supersaturated solution of Ca and PCM supplemented with poly-aspartic acid as polymeric precursor to induce intra-fibrous mineralization. Mineralization was characterized by SEM, TEM, EDS, XRD and nano-indentation after 1,3, and 7days. hDPSCs were cultured on dentin, mineralized and un-mineralized ELR surfaces. Cell proliferation was evaluated using immunofluorescence microscopy. ALP activity and expression of DMP1, DSP were analysed using ELISA assay, RT-PCR and western blot. Results and Discussion: SEM analysis revealed both St-ELR and Ref-ELR were highly infra- and extra-fibrous mineralized. A marked formation of needle-like apatitic mineral was aligned homogeneously along ELR fibers(Fig. 1C), which is similar to those seen in natural dentin. Mineral embedded within fibers were observed under SEM when looking at the cross-section surfaces (Fig.10), indicating the successful intra-fibrous mineralization. TEM images revealed that St-ELR exhibited parallel orientation of hydroxyapatite crystals to the longitudinal axis of the fibrils while Ref-ELR showed random orientated growth of minerals (Fig.2). EDS and XRD confirmed that we obtained hydroxyapatite nano-crystals. The high degree of extra- and intra-fibrous mineralization resulted in significant increase of elastic modulus and hardness to 0.99GPa and 0.058 GPa in dry state after 7 days. Cell proliferation, ALP activity, expression of DMP1 and DSP of hDPSCs were significantly enhanced on mineralized groups with respect to non-mineralized controls. This data suggests fibrous ELR scaffolds with mineralization promote stem cell activities. Conclusion: Electrospun St-ELR recombinamers along with Ref-ELR can template dentin matrix to obtain intra-fibrous mineralization using PILP process and thus, further promote dental pulp stem cell proliferation and differentiation. The combination of physical and biological properties demonstrates ELR is a potential novel scaffold for dental tissue regeneration.
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