The management of gingival fenestration: A series of three cases.

OBJECTIVES: The aim of this article is to introduce three treatments for patients with gingival fenestration as a result of chronic apical periodontitis. Gingival fenestration is a relatively uncommon soft tissue lesion in which the root apex is exposed in the oral environment after the destruction of the overlying buccal bone plate and mucosa. At present, no clear etiology or treatment guidelines exist for gingival fenestration. This article reports three successfully treated cases of gingival fenestration associated with chronic periapical infection. This report can help contribute to treatment guidelines for gingival fenestration. METHODS: All cases were treated with apicoectomy in conjunction with a connective tissue graft (CTG). According to the different conditions of the patients, we used some slightly different treatment methods during the operation. In case 1, we treated gingival fenestration in the mandibular left first premolar by endodontic therapy with root-end resection and retrograde filling and regenerative surgical therapy using a xenograft and CTG. In case 2, we treated gingival fenestration in the maxillary left lateral incisor by endodontic therapy with root-end resection and retrograde filling in vitro and regenerative surgical therapy using advanced platelet-rich fibrin (A-PRF) and CTG. In case 3, we treated gingival fenestration in the mandibular left second premolar by endodontic therapy with root-end resection and retrograde filling and regenerative surgical therapy using CTG. RESULTS: Endodontic treatment was combined with periodontal surgery to achieve predictable treatment results. After 13 to 25 months of follow-up, all cases showed that the gingival fenestration had healed well, and the patients had no discomfort. CONCLUSIONS: These three cases show the possibility of using apical excision combined with a CTG and/or bone graft/PRF in the treatment of gingival fenestration. Reporting these three cases may help advance the field of treatments for gingival fenestration.

The Effect of an Extract of Sappanwood, Protosappanin A and Protosappanin B on Osteogenesis in Periodontitis.

BACKGROUND: Sappanwood is widely used in the prevention and treatment in diseases due to its ability to seal blood vessels, dissipate stasis, and relieve pain. Important monomer components of sappanwood, Protosappanin A (PA) and Protosappanin B (PB) have anti-tumour and antimicrobial medicinal properties. This study investigated the anti-inflammatory and osteogenic differentiation effects of a crude extract of Sappanwood (ESP), PA and PB against periodontitis in periodontal ligament stem cells (PDLSCs). METHODS: Oil Red O staining was used to assess the ability of adipocytes to differentiate. Alizarin Red staining was used to assess the capacity to differentiate into osteoblasts. Third-passage PDLSCs were grown in either basic medium alone or basic media with varying doses of ESP (0.0625 mg/mL, 0.03125 mg/mL and 0.125 mg/mL), PA and PB (2.5 µM, 5 µM, 10 µM). The CCK-8 assay was used to measure cell proliferation. Real Time PCR (RT-qPCR) and Enzyme-Linked Immunosorbnent Assay (ELISA) assay were used to measure gene expression. The capacity to differentiate into osteoblasts was evaluated using Alizarin Red staining, and Alkaline Phosphatase (ALP) staining and activity. RESULTS: The development of lipid droplets and mineralized nodules was examined using Oil Red O staining and Alizarin Red staining. Flow cytometry revealed that PDLSCs were CD29 (98.23%) and CD44 (98.81%) positive, but CD34 (0.16%) and CD45 (0.09%) negative. CCK-8 assay showed that ESP at three concentrations (0.03125 mg/mL, 0.0625 mg/mL and 0.125 mg/mL) and 2.5 µM, 5 µM and 10 µM PA and PB had no cytotoxicity at 5 and 7 days (p < 0.05). qRT-PCR and ELISA assay indicated that ESP, PA and PB downregulated the inflammatory cytokines IL-8, IL-6, IL-1ß, IL-10 and IL-4 and elevated the mRNA expression of osteogenesis cytokines RUNX2 , OSX and OCN in PDLSCs (p < 0.05). Alizarin red staining, and ALP staining and activity showed that ESP, PA and PB increased mineralized nodules and the ALP content of in PDLSCs (p < 0.05). CONCLUSIONS: ESP, PA and PB can reduce the inflammatory response and amplify the osteogenic differentiation of PDLSCs. Therefore, ESP, PA and PB may have potential pharmacological effects in controlling the progression of periodontitis and promoting periodontal tissue regeneration.

Evaluating the effect of preheating on resin composites in pit-and-fissure caries treatments with a digital intraoral scanner.

OBJECTIVE: To evaluate the effect of preheating on the microleakage and surface hardness of resin composites in the treatment of pit-and-fissure caries with various widths, as measured by an intraoral scanner. METHODS: A total of 153 L-shaped cavities with different widths (1 mm, 1.6 mm and 2 mm) were prepared on the buccal or palatal/lingual surfaces of human molars. The cavities were measured in three dimensions by a TRIOS scanner and then filled with various resins (room temperature Z350 flowable resin and room temperature and 60 â„ƒ Z350 universal resin). Microleakage and gap formation at 2 sites were evaluated by stereomicroscopy and scanning electron microscope. Resin samples were prepared, and the top surface Vickers hardness (VHNtop) of all samples was measured at 1 day and 30 days postirradiation. RESULTS: No difference were observed in the 3D scans for the cavities sizes among groups with the same width. For the 1 mm-wide cavity, the lowest microleakage was obtained with the flowable group; for the 1.6 mm-wide cavity, the nonpreheating universal group showed the highest microleakage at site 1, and the preheating group exhibited lower microleakage than that of the nonpreheating universal group at site 2; and for the 2 mm-wide cavity, the preheating group presented lower microleakage at site 2. The gap formations were consistent with the microleakage degrees. The preheating group exhibited the highest VHNtop at 1 day and 30 days postirradiation. SIGNIFICANCE: A digital intraoral scanner could be used to scan the cavities in three dimensions. Preheating technology could reduce the microleakage of Z350 universal resin and enhance its surface hardness.

Effect of dermatopontin on osteogenic differentiation of periodontal ligament stem cells.

Human periodontal ligament stem cells (hPDLSCs) are promising seed cells for oral bone tissue engineering. Dermatopontin (DPT) is a small-molecule protein recognized as a non-collagenous component of the extracellular matrix and is associated with a variety of biological processes. In this study, we first determined that DPT was elevated during the osteogenic differentiation of hPDLSCs. HPDLSCs interfering with DPT expression were established by lentiviral infection. It was found that the proliferation and osteogenic differentiation ability of hPDLSCs were inhibited after interfering DPT with lentivirus. Exogenous recombinant DPT treatment could not alter the proliferation of hPDLSCs. Coincidentally, exogenous DPT can only enhance the osteogenic differentiation of hPDLSCs in the control lentivirus group, but had no significant effect on the DPT interference group. This study expands the understanding of DPT function and implicates DPT as an important target for enhancing osteogenic differentiation of hPDLSCs.

Small extracellular vesicles from dental follicle stem cells provide biochemical cues for periodontal tissue regeneration.

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.

TGF-ß2 and TGF-ß1 differentially regulate the odontogenic and osteogenic differentiation of mesenchymal stem cells.

OBJECTIVE: To explore the effects of transforming growth factor-ß2 (TGF-ß2) and TGF-ß1 on the odontogenic and osteogenic differentiation of mesenchymal stem cells (MSCs). DESIGN: We used lentiviral transduction to knock down TGF-ß1 or TGF-ß2 in stem cells from dental apical papilla (SCAPs), and to generate bone marrow mesenchymal stem cells (BMSCs) with overexpression of TGF-ß1 or TGF-ß2. We investigated the odontogenic and osteogenic differentiation abilities of these transductants in vitro and in vivo. RESULTS: In vitro, TGF-ß2 knockdown in SCAPs reduced the expression of odontoblast-related markers DSPP and DMP-1, and increased the expression of osteoblast-related markers OCN and RUNX-2. Conversely, TGF-ß1 knockdown had the opposite effects. TGF-ß2 overexpression promoted expression of odontoblast-related markers in BMSCs at early differentiation, but inhibited the expression of odontoblast-related markers at later stages. TGF-ß2 overexpression attenuated expression of osteogenic-related markers in BMSCs, while TGF-ß1 overexpression enhanced odontoblast-related and osteoblast-related markers. SCAP or BMSC transductants were transplanted underneath kidneys in vivo. Masson staining showed that knockdown of TGF-ß1, but not TGF-ß2 promoted the expression of type I collagen in SCAPs. Immunohistochemical staining showed that TGF-ß2 knockdown inhibited DSPP expression in SCAPs, but TGF-ß1 knockdown had no obvious effect on DSPP expression. In vivo, TGF-ß1 overexpression and TGF-ß2 overexpression had no effect on the expression of type I collagen and DSPP in BMSCs. CONCLUSIONS: TGF-ß2 promotes odontogenic differentiation of SCAPs and attenuates osteogenic differentiation of SCAPs and BMSCs. TGF-ß1 promotes osteogenic differentiation of BMSCs and plays a complex role in regulating odontogenic differentiation of MSCs.

Key Markers and Epigenetic Modifications of Dental-Derived Mesenchymal Stromal Cells.

As a novel research hotspot in tissue regeneration, dental-derived mesenchymal stromal cells (MSCs) are famous for their accessibility, multipotent differentiation ability, and high proliferation. However, cellular heterogeneity is a major obstacle to the clinical application of dental-derived MSCs. Here, we reviewed the heterogeneity of dental-derived MSCs firstly and then discussed the key markers and epigenetic modifications related to the proliferation, differentiation, immunomodulation, and aging of dental-derived MSCs. These messages help to control the composition and function of dental-derived MSCs and thus accelerate the translation of cell therapy into clinical practice.

Stem Cells from Human Exfoliated Deciduous Teeth Ameliorate Diabetic Nephropathy In Vivo and In Vitro by Inhibiting Advanced Glycation End Product-Activated Epithelial-Mesenchymal Transition.

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-ß, 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-ß 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.

Activation and Biological Properties of Human ß Defensin 4 in Stem Cells Derived From Human Exfoliated Deciduous Teeth.

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 ß 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-1ß, 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.

Therapeutic effects of stem cells from human exfoliated deciduous teeth on diabetic peripheral neuropathy.

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.

Stem cells from human exfoliated deciduous teeth ameliorate type II diabetic mellitus in Goto-Kakizaki rats.

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 ß-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 GSK3ß, 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, GSK3ß 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.

Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth into Retinal Photoreceptor-Like Cells and Their Sustainability In Vivo.

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.

[Skeletal Class â ¢ patients treated with Fränkel function regulator type â ¢ in the early and late mixed dentition].

OBJECTIVE: To investigate the outcome of skeletal Class Ⅲ patients treated with Fränkel function regulator type Ⅲ (FR Ⅲ)in the early mixed and late mixed dentition. METHODS: The samples consisted of 45 mild and moderate skeletal Class Ⅲ patients(26 males, 19 females; meanage, [7.9±1.3] years) treated with FR Ⅲ. According to Hellman's dental developmental stages, these samples were divided into early-treated group(n=24) and late-treated group(n=21). Lateral cephalograms were taken at the beginning and the end of treatment. Twenty-one measurements on hard and soft tissue were included. RESULTS: After treatment, SNA, ANB, NA-Apo, Wits, U1-SN, U1-NA, Overjet, UL-EP were significantly increased (1.0±1.9)°, (1.2±1.6)°, (2.6±4.2)°, (1.8±2.7) mm, (4.2±7.6)°, (2.6±7.5)°, (3.6±2.3) mm and (0.8±2.2) mm(P<0.05). OP-SN and IMPA were significantly decreased (1.5±3.7)°and (1.4±4.2)°(P<0.05). There were significant differences in SNA, ANB, UL-EP, IMPA, L1-NB between early-treated group and late-treated group(P<0.05). CONCLUSIONS: FR Ⅲ was suitable for the treatment of mild and moderate skeletal Class Ⅲ patients. The result was better in the early-treated patients than in late-treated ones.