An intense stimulus can cause death of odontoblasts and initiate odontoblastic differentiation of stem/progenitor cell populations of dental pulp cells (DPCs), which is followed by reparative dentin formation. However, the mechanism of odontoblastic differentiation during reparative dentin formation remains unclear. This study was to determine the role of β-catenin, a key player in tooth development, in reparative dentin formation, especially in odontoblastic differentiation. We found that β-catenin was expressed in odontoblast-like cells and DPCs beneath the perforation site during reparative dentin formation after direct pulp capping. The expression of β-catenin was also significantly upregulated during odontoblastic differentiation of in vitro cultured DPCs. The expression pattern of runt-related transcription factor 2 (Runx2) was similar to that of β-catenin. Immunofluorescence staining indicated that Runx2 was also expressed in β-catenin–positive odontoblast-like cells and DPCs during reparative dentin formation. Knockdown of β-catenin disrupted odontoblastic differentiation, which was accompanied by a reduction in β-catenin binding to the Runx2 promoter and diminished expression of Runx2. In contrast, lithium chloride (LiCl) induced accumulation of β-catenin produced the opposite effect to that caused by β-catenin knockdown. In conclusion, it was reported in this study for the first time that β-catenin can enhance the odontoblastic differentiation of DPCs through activation of Runx2, which might be the mechanism involved in odontoblastic differentiation during reparative dentin formation.

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Brain tissues that are severely damaged by traumatic brain injury (TBI) is hardly regenerated, which leads to a cavity or a repair with glial scarring. Stem-cell therapy is one viable option to treat TBI-caused brain tissue damage, whose use is, whereas, limited by the low survival rate and differentiation efficiency of stem cells. To approach this problem, we developed an injectable hydrogel using imidazole groups-modified gelatin methacrylate (GelMA-imid). In addition, polydopamine (PDA) nanoparticles were used as carrier for stromal-cell derived factor-1 (SDF-1α). GelMA-imid hydrogel loaded with PDA@SDF-1α nanoparticles and human amniotic mesenchymal stromal cells (hAMSCs) were injected into the damaged area in an in-vivo cryogenic injury model in rats. The hydrogel had low module and its average pore size was 204.61 ± 41.41 nm, which were suitable for the migration, proliferation and differentiation of stem cells. In-vitro cell scratch and differentiation assays showed that the imidazole groups and SDF-1α could promote the migration of hAMSCs to injury site and their differentiation into nerve cells. The highest amount of nissl body was detected in the group of GelMA-imid/SDF-1α/hAMSCs hydrogel in the in-vivo model. Additionally, histological analysis showed that GelMA-imid/SDF-1α/hAMSCs hydrogel could facilitate the regeneration of regenerate endogenous nerve cells. In summary, the GelMA-imid/SDF-1α/hAMSCs hydrogel promoted homing and differentiation of hAMSCs into nerve cells, and showed great application potential for the physiological recovery of TBI.

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Following a limited number of cell divisions, mesenchymal stem cells (MSCs) undergo senescence, and these senescent cells maintain metabolic modification and remain viable for long periods. Autophagy, an intracellular bulk degradation process, provides a survival effect for cells under stress. In this study, the effect of autophagy on senescent MSCs was analyzed. Following serial passaging, rat MSCs underwent replicative senescence, characterized by positive staining for senescence-associated β-galactosidase (SA-β-gal), and increased expression levels of p16 and p21. During MSC senescence, the levels of autophagic activity were increased, a greater number of autophagic vacuoles were observed in senescent MSCs by transmission electron microscopy, acridine orange staining was elevated and the expression levels of autophagy‑related proteins (microtubule‑associated protein 1A/1B‑light chain 3-II, Atg7 and Atg12) were increased. The role of autophagy in MSC senescence was further investigated through pharmacological inhibition of autophagy with bafilomycin A1 and 3-methyladenine. Inhibition of autophagy by pharmacological means reduced the rate of positive staining for SA-β-gal and the expression levels of senescence‑related proteins. In conclusion, these findings suggest that autophagy is activated during senescence and the autophagic activity may be a requirement for maintaining the senescent state of MSCs.

To explore the mechanisms underlying spontaneous transformation of mesenchymal stem cells (MSCs), changes in senescence-associated molecules, particularly the epigenetic modification of the p16(INK4a) gene, including histone H3 lysine 27/9 methylation (H3K27/9me) and DNA methylation, were investigated in cultured adult rat bone marrow MSCs at different stages during the transformation process. It was shown that the MSCs underwent replicative senescence after 24 to 25 population doublings, characterized by positive staining for senescence-associated β-galactosidase, increased expression of p16(INK4a) and p21, and downregulated phosphorylation of Rb. The upregulation of p16(INK4a) was associated with decreased expression of enhancer of the zeste homolog 2 (Ezh2), and reduced levels of H3K27me and DNA methylation in the p16(INK4a) gene. At week 4 of senescence, reproliferating cells emerged among the senescent MSCs. These senescence-escaped MSCs lost their senescence-related markers (including p16(INK4a)) and became highly proliferative. In addition to H3K27me, another H3 modification pattern, H3K9me, appeared in the p16(INK4a) gene, accompanied by an enhanced DNA methylation. With continued culture, the senescence-escaped MSCs did not show any sign of growth arrest and gained the capacity for anchorage-independent growth. These immortalized (transformed) MSCs showed further enhanced DNA methylation of the p16(INK4a) gene by increased H3K9me. Ezh2 knockdown with shRNA eliminated H3K27me-mediated DNA methylation of the p16(INK4a) gene in presenescent MSCs, but had no effect on H3K9me-enhanced DNA hypermethylation in the cells after senescence escape. These findings identify an Ezh2- and H3K27me-independent, but H3K9me-enhanced, DNA hypermethylation of the p16(INK4a) gene, which might be an epigenetic signature for MSC spontaneous transformation.

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