Pulpitis in primary teeth, a condition caused by presence of bacteria, is highly prevalent worldwide. The use of biocompatibility materials with anti-inflammatory, anti-bacterial, and regenerative properties is critical for prognosis of this endodontic disease. This study aimed to identify expression of human β defensin 4 (HBD4) in stem cells derived from human exfoliated deciduous teeth (SHED) and characterize the effects of HBD4 on SHED. Quantitative polymerase chain reaction (qPCR) was used to detect HBD4 expression in SHED and the effect of HBD4 on inflammatory factors in lipopolysaccharide (LPS)-stimulated SHED. Affinity measurement was made by the Fortebio Octet System to explore the potential interaction between LPS and HBD4. Western blot analysis was used to explore the effect of HBD4 on mitogen-activated protein kinase (MAPK) pathway. Colony-forming unit methods and scanning electron microscopy were applied to study antimicrobial effect of HBD4 on Fusobacterium nucleatum and Porphyromonas gingivalis. Alkaline phosphatase staining, alizarin red staining, qPCR and western blot were taken to detect effects of HBD4 on osteoblast/odontoblast differentiation of SHED. RT2 Profiler PCR Array was used to explore the potential signaling pathways involved in the osteogenic/odontogenic differentiation. HBD4 was highly expressed in SHED stimulated by TNF-α and IL-1α. HBD4 could bind to LPS directly and down-regulate IL-1α, IL-1β, IL-6, TNF-α in LPS-stimulated SHED, thus the activation of MAPK pathway decreased. HBD4 was sensitive to P. gingivalis and enhanced osteoblast/odontoblast differentiation potential of SHED by modulating Notch pathway. HBD4 was highly expressed in SHED stimulated by proinflammatory cytokines, and possessed anti-inflammatory, anti-bacterial activity. HBD4 promoted osteogenic/odontogenic differentiation of SHED. HBD4 may thus represent a suitable agent for vital pulp therapy in future clinic application.

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Objective To evaluate the therapeutic potential of stem cells from human exfoliated deciduous teeth (SHED) for diabetic peripheral neuropathy. Methods The biological characteristics of SHED were identified by flow cytometric study and evaluation of differentiation potential. Using high-fat feeding, diabetes was induced in GK rats, and SHED were transplanted into the caudal veins of these rats. Immunohistochemical analysis was used to compare the capillary to muscle fiber ratio and intra-epidermal nerve fiber density between SHED- and saline-treated diabetic rats. Further, the expressions of angiogenesis-related and neurotrophic factors were quantified by real-time PCR and western blot. Results SHED had a capacity of multiple differentiation and shared typical characteristics of mesenchymal stem cells. SHED transplantation relieved diabetic neuropathic pain, enabled functional recovery of the peripheral nerves, and increased the capillary to muscle fiber ratio and intra-epidermal nerve fiber density compared to the saline group and normal controls. Real-time PCR results showed that the expressions of CD31, vWF, bFGF, NGF, and NT-3 in the skeletal muscles were higher in the SHED group than in the saline groups. Western blot results indicated that the levels of the CD31 and NGF proteins were higher in the SHED transplantation group than the saline group. Conclusion SHED transplantation ameliorated diabetic peripheral neuropathy in diabetic GK rats. Thus, systemic application of SHED could be a novel strategy for the treatment of diabetic peripheral neuropathy.

Background Treatments based on stem cell-derived small extracellular vesicles (sEVs) have been explored as an alternative to stem cell transplantation-based therapies in periodontal regeneration. Dental follicle stem cells (DFSCs) have shown great potential for regenerative medicine applications. However, it is unclear whether sEVs derived from DFSCs (DFSCs-sEVs) could be used in periodontal regeneration. This study investigates whether DFSCs-sEVs could regenerate damaged periodontal tissue and the potential underlying mechanism. Methods DFSCs-sEVs were isolated and identified, and periodontal ligament stem cells (PDLSCs) were cocultured with the isolated sEVs. The effect of DFSCs-sEVs on the biological behaviour of PDLSCs was examined using EdU assay, CCK-8 assay, cell cycle analysis, wound healing, alizarin red staining, qRT-PCR, and western blot analysis. RNA sequencing and functional enrichment analysis were used to detect the signal pathway involved in the effect of DFSCs-sEVs on PDLSCs. PDLSCs were pretreated with ERK1/2 or p38 MAPK inhibitors to investigate the possible involvement of the ERK1/2 and p38 MAPK pathways. Additionally, DFSCs-sEVs were combined with collagen sponges and transplanted into the periodontal defects in SD rats, and then, pathological changes in periodontal tissue were examined using haematoxylin and eosin (HE) staining and micro-CT. Results PDLSCs could internalize DFSCs-sEVs, thereby enhancing the proliferation assessed using EdU assay, CCK-8 assay and cell cycle analysis. DFSCs-sEVs significantly enhanced the migration of PDLSCs. DFSCs-sEVs promoted osteogenic differentiation of PDLSCs, showing deep Alizarin red staining, upregulated osteogenic genes (RUNX2, BSP, COL1), and upregulated protein expression (RUNX2, BSP, COL1, ALP). We found that p38 MAPK signalling was activated via phosphorylation. Inhibition of this signalling pathway with a specific inhibitor (SB202190) partially weakened the enhanced proliferation. After DFSCs-sEVs transplantation, new periodontal ligament-like structures and bone formation were observed in the damaged periodontal area in rats. Labelled DFSCs-sEVs were observed in the newly formed periodontal ligament and soft tissue of the defect area. Conclusions Our study demonstrated that DFSCs-sEVs promoted periodontal tissue regeneration by promoting the proliferation, migration, and osteogenic differentiation of PDLSCs. The effect of DFSCs-sEVs in promoting PDLSCs proliferation may be partially attributed to the activation of p38 MAPK signalling pathway. DFSCs-sEVs provide us with a novel strategy for periodontal regeneration in the future.

Retinal degeneration is characterized by the progressive loss of photoreceptors, and stem cell therapy has become a promising strategy. Many studies have reported that mesenchymal stem cell transplantation can sustain retinal structure and prolong retinal functions based on two mechanisms. One is cell replacement, and the other is the paracrine action of stem cells. Cells from human exfoliated deciduous teeth (SHEDs) show characteristics typical of mesenchymal stem cells. They are derived from the neural crest and are a potential cellular source for neural regeneration in stem cell therapy. In this study, we explored the potential of SHEDs to be induced towards the retinal photoreceptor phenotype and to be sustainable in an animal model of retinal degeneration. A factor-cocktail protocol was used to induce SHEDs towards retinal photoreceptors for 24 days, and the characteristics of the induced cells were identified in terms of morphological changes, biomarker expression and subcellular distribution, and calcium influx. SHEDs were labeled with firefly luciferase for in vivo tracking by bioluminescent imaging and then transplanted into the subretinal space of mice. Our results showed that SHEDs successfully transdifferentiated into photoreceptor-like cells, which displayed neuron-like morphology, and expressed specific genes and proteins associated with retinal precursors, photoreceptor precursors, and mature photoreceptors. In addition, calcium influx was significantly greater in the retinal-induced than in noninduced SHEDs. In vivo tracking confirmed at least 2 weeks of good survival by bioluminescent imaging and 3 months of sustainability of SHEDs by histological analysis. We conclude that SHEDs have the potential to transdifferentiate into retinal photoreceptor-like cells in vitro and maintain good viability in vivo after transplantation into mice with a normal immune system. This demonstrates preliminary success in generating photoreceptor-like cells from SHEDs and applying SHEDs in treating retinal degeneration.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. It has been proven that mesenchymal stem cells (MSCs) have therapeutic effects on kidney disease. Stem cells from human exfoliated deciduous teeth (SHED) are MSCs that are derived from dental pulps in exfoliated deciduous teeth from young patients and therefore have a high proliferation rate and an easy access. Hence, we aimed to explore the effect of SHED on DN in Goto-Kakizaki (GK) rats. SHED were administered via the tail vein. Blood glucose, serum triglycerides and cholesterol, body weight, and urinary albumin were measured before and after administration. At 8 weeks after administration, real-time PCR, immunohistochemistry (IHC), and electron microscopy were employed to examine pathological changes in glomerular and tubulointerstitial tissue. Kidney weight and serum IL-1, IL-10, TNF-α, TGF-β, and HGF levels were measured. SHED engraftment in the kidneys was detected by transfecting green fluorescence protein (GFP). Type II epithelial-mesenchymal transition (EMT) in the tubule-interstitial and arteriolar regions has been reported to be an important pathological characteristic of DN. This study is the first to apply a transwell system for coculture to explore the effects of MSCs on the EMT of human proximal tubular epithelial (HK-2) cells. The effects of SHED on advanced glycation end product- (AGE-) activated EMT in HK-2 cells were explored by real-time PCR and western blot. At 8 weeks after administration, renal injury, including hyperglycemia, hyperlipidemia, increased urinary albumin excretion, ECM accumulation, and a fractional mesangial area, was dramatically attenuated. The serum levels of IL-1, TNF-α, and TGF-β were significantly downregulated, whereas the serum levels of IL-10 and HGF were upregulated by SHED. GFP expression confirmed the engraftment of SHED in diabetic kidneys. In addition, cocultured SHED inhibited AGE-induced EMT in HK-2 cells. In conclusion, SHED offer a novel potential effective therapeutic approach for ameliorating DN.

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Background By 2030, diabetes mellitus (DM) will be the 7th leading cause of death worldwide. Type 2 DM (T2DM) is the most common type of DM and is characterized by insulin resistance and defective β-cell secretory function. Stem cells from human exfoliated deciduous teeth (SHED) are favorable seed cells for mesenchymal stem cells (MSCs)-based therapy due to their higher proliferation rates and easier access to retrieval. Currently, no study has revealed the therapeutic efficiency of MSCs for T2DM in Goto-Kakizaki (GK) rats. Hence, we aimed to explore the effect of SHED on T2DM in GK rats. Materials and methods We investigated the effects of SHED on the progression of T2DM in GK rats. SHED and bone marrow mesenchymal stem cells (BMSCs) were injected via the tail vein. Body weight, fasting blood glucose and non-fasting blood glucose were measured before and after administration. At 8 weeks after injection, intraperitoneal insulin tolerance tests (IPITTs) and insulin release tests (IRTs) were performed. Additionally, hematoxylin–eosin (HE) staining, periodic acid-Schiff (PAS) staining and double-label immunofluorescence staining were used to explore the pathological changes in pancreatic islets and the liver. Immunohistochemistry (IHC) was employed to detect SHED engraftment in the liver. Additionally, real-time PCR and western blotting were used to explore glycogen synthesis, glycolysis and gluconeogenesis in the liver. Results At 8 weeks after SHED injection, T2DM was dramatically attenuated, including hyperglycemia, IPGTT and IRT. Additionally, histological analysis showed that SHED injection improved pancreatic islet and liver damage. Real-time PCR analysis showed that SHED significantly reversed the diabetic-induced increase of G-6-Pase, Pck1 and PK; and significantly reversed the diabetic-induced decrease of GSK3β, GLUT2, and PFKL. In addition, western blotting demonstrated that SHED significantly reversed the diabetic-induced increase of G-6-Pase and reversed the diabetic-induced decrease of GLUT2, GSK3β and PFKM. Conclusion Stem cells from human exfoliated deciduous teeth offers a potentially effective therapeutic modality for ameliorating T2DM, including hyperglycemia, insulin resistance, pancreatic islets and liver damage, and decreased glycogen synthesis, inhibited glycolysis and increased gluconeogenesis in the liver.