ABSTRACT
Regenerative endodontic therapy is a tissue engineering based approach of treatment for endodontic disease. Its purpose is to achieve the regeneration of the pulp-dentin complex, thus to promote root development of the immature permanent tooth with necrotic pulp. Like other treatments based on tissue engineering techniques, the success of regenerative pulp therapy depends on such three elements as seed cells, scaffold materials and growth factors. Since its inception 20 years ago, there have been various terminologies in the literature, with similarities and differences in connotation. The present article summarizes and analyzes the term evolution, biological basis, clinical considerations and future scientific research directions of regenerative endodontics, in order to find out the unsolved scientific problems and to promote the development and standardization of this technique in clinical practice.
Subject(s)
Humans , Dental Pulp , Dental Pulp Necrosis , Regeneration , Regenerative Endodontics , Root Canal Therapy , Tissue EngineeringABSTRACT
OBJECTIVE@#To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP.@*METHODS@#(1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 °C as positive control group, and co-cultured with SCAP at 4 °C as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluorescence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluorescence.@*RESULTS@#(1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 °C for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 °C for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%, and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively.@*CONCLUSION@#SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.
Subject(s)
Dental Papilla , Endocytosis , Epithelial Cells , Exosomes , Stem CellsABSTRACT
OBJECTIVE@#To evaluate the change of cell surface CXC chemokine receptor 4 (CXCR4) expression of stem cells from apical papilla (SCAP) after the inhibition of endocytotic pathway, thus to provide experimental basis for the mechanism of SCAP migration.@*METHODS@#The immunofluorescence analysis was conducted to examine the co-expression of CXCR4 and endocytotic compartments, including early endosomes, recycling endosomes and lysosomes in SCAP. Several Rab proteins were applied as markers of organelles in the endocytotic pathway, including Rab5 for early endosomes, Rab11A for recycling endosomes, and Lamp1 for lysosomes. The co-localization of CXCR4 with these endodontic compartments was further observed by proximity ligation assay (PLA). SCAP was treated with two kinds of endocytotic inhibitors, Blebbistatin and Dynasore, at a concentration of 80 μmol/L, respectively. The conditioning time was 1 hour. Flow cytometry was carried out to evaluate the proportion of SCAP that expressed CXCR4 on cell surface. The data were analysed by analysis of variance (ANOVA).@*RESULTS@#The red staining of CXCR4 on immunofluorescence confocal microscopy predominantly overlapped with the green staining of Rab5 and Rab11A, and partly overlapped with Lamp1. It indicated that most CXCR4 molecules were located in early endosomes and recycling endosomes, and some were located in lysosomes. The PLA results revealed that the co-localizaiton of CXCR4 with endocytotic compartments could be observed in early endosomes, recycling endosomes and lysosomes. According to the results of flow cytometry, the proportion of SCAP that expressed CXCR4 on cell surface was as low as 0.13%±0.10%. After the inhibition of endocytosis by pretreating the cells with the following two inhibitors, Blebbistatin and Dynasore, the percentage of SCAP that positively expressed CXCR4 on cell surface was significantly increased to 13.34%±1.31% in Blebbistatin group and 4.03%±0.92% in Dynasore group (F=16.721, P<0.001). Moreover, the number of SCAP that expressed CXCR4 on cell surface in Blebbistatin group was significantly higher than that in Dynasore group (P<0.001).@*CONCLUSION@#The inhibition of endocytotic pathway could increase the number of SCAP that expressed CXCR4 on cell surface, and provide potency for the migration of SCAP.
Subject(s)
Endocytosis , Endosomes , Lysosomes , Receptors, CXCR4 , Stem CellsABSTRACT
<p><b>OBJECTIVE</b>To explore measures to prevent motor endplate degeneration and muscular atrophy after motor nerve injury.</p><p><b>METHODS</b>Thirty Sprague-Dawley rats were randomized into 3 equal groups. In two of the groups, the right common peroneal nerves of the rats were transected and immediately sutured with implantation of collagen gel carrier of acidic fibroblast growth factor (aFGF) or the empty carrier into the denervated tibialis anterior muscles. In the control group, the transected nerves were sutured without implantation. Six weeks after the operation, morphological and electrophysiological examinations were performed.</p><p><b>RESULTS</b>In the control rats and those with empty collagen gel carrier implantation, obvious motor endplate degeneration and muscular atrophy occurred, which were not obvious in rats receiving aFGF carrier implantation. The decrement of repetitive nerve stimulation was significantly greater in the former two groups than in the latter.</p><p><b>CONCLUSION</b>Implantation of collagen gel carrier of aFGF may prevent motor endplate degeneration and facilitate functional recovery of the neuromuscular junction after motor nerve injury.</p>