The effect of MICRORNA-675 with silk / polycaprolactone and MTA scaffolding on odontoblastic differentiation and biomineralization of human dental pulp stem cells (in-vitro study)

Abstract

Abstract: Background: Regenerative treatments to restore pulp tissue or maintain pulp life in immature teeth have been the focus of recent dental studies. The aim of this study is to produce a biocompatible polycaprolactone/silk nanofiber scaffold to transfer miRNA-675 and create a suitable substrate for the proliferation of odontoblast cells or the differentiation of dental pulp stem cells to form dentin and ultimately preserve the vital dental pulp tissue. Materials and methods: Silk fibrin was prepared. The silk/polycaprolactone nanofibrous scaffolds were converted with different electrospinning ratios and water angle and tensile strength were evaluated. Also, the structure of the scaffolds was examined by SEM, FTIR and XRD. miRNA-675 was loaded with TPP in chitosan and the structure of nanoparticles was evaluated by DLS. Chitosan nanoparticles containing miRNA-675 were loaded on the surface of nanofibrous scaffolds. The biocompatibility of the obtained compound on human dental pulp stem cells on the surface of the scaffold produced was checked by MTT test. Also, the morphology of the cells cultured on the scaffolds was examined by SEM. Finally, the level of mineralization was evaluated by the expression level of dentin sialophosphoprotein gene through western blot test. Results: Silk/polycaprolactone nanofiber scaffold was prepared with different ratios. According to SEM images, the average diameter size of nanofibers is equal to: 97.2, 104.86, 118.02, 144.92, and 102.1 nm in the order of increasing the amount of silk. Also, by increasing the amount of silk up to 40% in the scaffolds' structure, the amount of water contact angle decreased to 59°. The evaluation of the scaffold with dopamine coating also showed a uniform and healthy nanofiber structure. The results of deposition of chitosan nanoparticles on nanofibers showed that at pH=5, nanoparticles with the smallest diameter are uniformly loaded on the scaffold nanofibers. Chitosan nanoparticles and chitosan nanoparticles containing miRNA-675 had diameters of 343 and 194.6 nm and zeta potential of 26 and 21 mV, respectively. The evaluation of the biocompatibility of different nanoparticles (50 to 300 nM) showed that all environments are compatible and the highest percentage of living cells is related to 200 nM. Biocompatibility evaluation of the produced scaffolds showed that during 24 and 48 hours, all scaffolds supported cell proliferation and the biocompatibility was related to the scaffold containing chitosan-miR-675 nanoparticles. Conclusion: In this study, miR-675 was encapsulated in chitosan nanoparticles and loaded in silk/polycaprolactone scaffold after evaluation. The silk/polycaprolactone scaffold containing chitosan-miR-675 nanoparticles is a suitable substrate for the attachment and proliferation of DPSCs cells with the aim of expanding and differentiating into odontoblasts and finally mineralization.