Autophagy In Tooth Development And Pulp Regeneration

Part One:Autophagy Appears in developing Mouse Tooth GermsObjectives:Odontogenesis consists of a series of consecutive tooth morphogenesis stages, in which apoptosis is involved to eliminate the unnecessary cells. Autophagy, a self-degradation process, is indicated to participate in embryogenesis and tissue morphogenesis associated with apoptosis. This study hypothesized that autophagy may be involved and associate with apoptosis in odontogenesis.Methods:To test the appearance of autophagy in odontogenesis, quantitative real-time PCR, Western blot were firstly performed. Immunohistochemistry staining of LC3and Beclinl were used to detect the autophagic cells. Then, double and triple immunofluorescence (LC3/TUNEL; LC3/Beclinl/Cl-Caspase-3), and further transmission electron microscopy (TEM), were employed to test the possible colocalization of autophagy and apoptosis.Results:The transcripts of autophagy related genes (Atg5, Atg7and Atg12), the expression of Atg5-Atgl2conjugate, lipidation of LC3, and Beclin1(full length and cleaved fragments) were detected in the developing tooth germs. Meanwhile, LC3was immunolocalized in the molars at E13.5-P15.5, especially E14.5cervical loop and the PEK facing the mesenchyme. The expression pattern of Beclinl does not exactly match that of LC3, especially the nucleus localization of Beclinl was detected in part of the tooth germs. Moreover, the partial colocalization of LC3and TUNEL signal was revealed in developing tooth germs. Nevertheless, LC3was also found in non-apoptotic cells. TEM images showed the presence of autophagic vacuoles and its partial colocalization with apoptotic nuclei. In addition, partial colocalization of Beclinl, LC3, or cleaved Caspase-3was detected in the E14.5, especially the Beclin1+LC3+Cl-Caspase-3+cells in the PEK.Conclusions:Our findings demonstrated the appearance of autophagy and its partial colocalization with apoptosis in odontogenesis, and indicated that Beclinl might be responsible for the crosstalk between autophagy and apoptosis. Part Two:Role of Autophagy in SDF-1?-mediated DSPCs migration and Ectopic Pulp RegenerationObjectives:Critical morphologic requirements for pulp regeneration are dental pulp-like tissues replete with vascularization, neuron formation, and dentin deposition. Stromal cell-derived factor-la (SDF-la) belongs to the CXC chemokine subfamily, which plays a key role in tissue vascularisation, neurogenesis, and osteogenesis. Iohara et al. reported complete pulp regeneration by transplantation of SDF-la and DPSCs in dog teeth after pulpectomy. However, the mechanism involved in SDF-1?-mediated pulp regeneration has yet to be studied. The present study aimed to investigate the possible involvement of autophagy in SDF-la-mediated DPSCs migration and pulp regeneration.Methods:DPSCs were isolated using the enzymatic digestion method and characterized by colony forming assay, immunofluorescence, flow cytometric analysis, and multiple lineage differentiation assay. The effect of SDF-1? on DPSCs vialibity and migration was examined by CCK8and cell migration assay. And immunofluorescence staining was employed to detect the cytoskeletal arrangement and focal contacts of DPSCs challenged with SDF-1?. Then, immunofluorescence, Western blot, inhibitory assay, and TEM were used to verify the involvement of autophagy in SDF-la-mediated DPSCs migration. Porous silk fibroin scaffolds were fabricated using freeze-drying technique (with or without SDF-la incorporation), and characterized by scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. Then, the ectopic pulp regeneration model was established through the subcutaneous transplantation of DPSCs-seeded silk fibroin scaffold-tooth fragment complex (TSS, insertion the scaffold into the pre-treated tooth fragment). The histologic characterization of the regenerated tissues was analyzed by HE, Masson's trichrome, picrosirius staining, Western blot, and immunohistochemical staining. Furthermore, immunostaining, Western blot, and double immunofluorescence analysis were used to clarify the possible involvement of autophagy in pulp regeneration.Results:DPSCs, which could form single cell derived colonies, express mesenchymal stem cell markers, and possessed multi-directional differentiation capabilities, were successfully isolated. In vitro studies showed that SDF-1? had no effect on DPSCs viability, while enhanced DPSCs migration, accompanied by focal adhesion formation and stress fiber assembly. Autophagy related proteins and autophagic vacuoles were upregulated in SDF-la-treated DPSCs. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1?-stimulated DPSCs migration. Then the porous silk fibroin scaffold was fabricated with the pore size about200±46?m, the incorporation of SDF-1? had no effect on the structure and molecular conformation of the scaffold. DPSCs seeded in the TSS survived, exhibited cytoplasmic elongation and proliferated in the scaffold for at least four weeks in culture. Interestingly, after ectopic transplantation of TSS with DPSCs into nude mice, there generated pulp-like tissues with vascularity and well-organized fibrous matrix formation, as well as newly dentin deposition along the dentinal wall in SDF-1?-loaded samples. Moreover, autophagy related proteins (Atg5, LC3) were expressed and autophagic vacuoles were present in the regenerated tissues. In addition, partial colocalization of LC3and CD31, as well as the presence of autophagic vacuoles in the endothelial cells, might indicate the possible involvement of autophagy in the neovascularization during the ectopic pulp-dentin complex regeneration. It is noteworthy that the regenerated pulp-dentin complex was composed of both donor and host derived cells. SDF-la could also effectively regenerate vascularized connective tissue through recruiting host-derived cells in non-DPSCs loading TSS, accompanied by the expression of autophagy related proteins.Conclusions:SDF-1?-loaded silk fibroin scaffold could successfully be used for ectopic pulp regeneration. Autophagy was involved in the process of pulp regeneration, possibly through the positive regulation of SDF-la-mediated DPSCs migration and autologous cell homing. Our findings provided novel insights into the mechanism of pulp regeneration. Part Three:"Stem Cell Homing"-based SDF-la-mediated In Situ Pulp Regeneration in Mature Dog Teeth Accompanied by AutophayObjectives:Compared with cell transplantation, cell homing may offer a more clinically translational approach. SDF-1?, as a chemotaxic factor, has been shown the use for ectopic pulp-like tissue regeneration without DPSCs transplantation from previous results. This study was aimed to further explore its possible use for in situ pulp regeneration through "cell homing" stragety.Methods:Apical periodontitis was induced in8lower closed-apex premolars of2dogs. After biomechanical preparation, enlargement to a#80K file, and disinfection with a triantibiotic paste for28days, the roots were randomly assigned to2treatment groups:blood clot (BC), BC+SDF-la-loaded silk fibroin scaffold. The teeth left untreated were used as negative controls. After a3-month follow-up period, the animals were sacrificed. HE, Masson's trichrome and picrosirius staining were used to analyze the histological characterization of the tissue regenerated in the canal. Immunostaining was further used to detect the expression of autophagy related proteins LC3and Atg5.Results:Histologic analysis showed the presence of newly formed vital tissues in both groups. In the BC group, there generated cement-like and bone-like tissues, and little connective tissue in the canal. In the BC+SDF-la-loaded silk fibroin scaffold group, there formed pulp-like tissues in the canal. Moreover, autophagy related proteins were demonstrated to be present in the regenerated tissues.Conclusion:SDF-la-loaded silk fibroin scaffold could lead to in situ pulp regeneration through "cell homing" strategy, accompanied by the accurance of autophagy. This study might provide a basis for clinical transformation of dental pulp regeneration.