Cytokines regulate stemness of mesenchymal stem cells via miR-628-5p during periodontal regeneration.

BACKGROUND: Cytokines play key roles in stimulating periodontal regeneration; however, their exact mechanisms of action remain unclear. Mesenchymal stem cells (MSCs) are multipotent cells that have self-renewal abilities and can differentiate into periodontal tissues such as bone, cementum, and periodontal ligaments following transplantation, like periodontal progenitor cells. Here, we used MSCs to identify the regulatory genes induced by periodontal regenerative cytokines. METHODS: Human MSCs (hMSCs) were cultured under conditions of periodontal regenerative cytokine stimulation or silencing of undifferentiated hMSC transcription factors. To characterize the changes associated with periodontal regenerative cytokine-regulated microRNAs (miRNAs), miRNA, and mRNA expression was evaluated using miRNA arrays and quantitative real-time polymerase chain reaction, respectively. One of the identified miRNAs, miR-628-5p, was then overexpressed or suppressed in hMSCs during osteogenesis; the effect of these changes on osteogenesis was investigated. RESULTS: Cytokine-stimulated MSCs showed characteristic miRNA profiles and mRNA levels of undifferentiated hMSC transcription factors ETV1, SOX11, and GATA6. Next, we silenced these transcription factors in MSCs and examined the miRNA profiles. The levels of miR-628-5p were decreased upon all cytokine treatments and were increased upon silencing of ETV1, SOX11, and GATA6. Overexpression of miR-628-5p suppressed osteogenesis; however, its inhibition enhanced OPN, ALP, OC, BMP2, and RUNX2 mRNA levels, and bone matrix mineralization, but not OSX mRNA or ALP activity. CONCLUSIONS: miR-628-5p negatively regulates MSC stemness during periodontal regeneration. Periodontal regenerative cytokines act as miR-628-5p suppressors to support periodontal regeneration. Thus, selection of effective cytokines for different MSCs, based on miRNA profiling, is important for advancing regenerative therapies.

Identification of regulatory mRNA and microRNA for differentiation into cementoblasts and periodontal ligament cells.

OBJECTIVE: Periodontitis causes periodontal tissue destruction and results in physiological tooth dysfunction. Therefore, periodontal regeneration is ideal therapy for periodontitis. Mesenchymal stem cells (MSCs) are useful for periodontal regenerative therapy as they can differentiate into periodontal cells; however, the underlying regulatory mechanism is unclear. In this study, we attempted to identify regulatory genes involved in periodontal cell differentiation and clarify the differentiation mechanism for effective periodontal regenerative therapy. BACKGROUND: The cementum and periodontal ligament play important roles in physiological tooth function. Therefore, cementum and periodontal ligament regeneration are critical for periodontal regenerative therapy. Mesenchymal stem cell transplantation can be a common periodontal regenerative therapy because these cells have multipotency and self-renewal ability, which induces new cementum or periodontal ligament formation. Moreover, MSCs can differentiate into cementoblasts. Cementoblast- or periodontal ligament cell-specific proteins have been reported; however, it is unclear how these proteins are regulated. MicroRNA (miRNA) can also act as a key regulator of MSC function. Therefore, in this study, we identified regulatory genes involved in cementoblast or periodontal cell differentiation and commitment. METHODS: Human MSCs (hMSCs), cementoblasts (HCEM), and periodontal ligament cells (HPL cells) were cultured, and mRNA or miRNA expression was evaluated. Additionally, cementoblast-specific genes were overexpressed or suppressed in hMSCs and their expression levels were investigated. RESULTS: HCEM and HPL cells expressed characteristic genes, of which we focused on ets variant 1 (ETV1), miR-628-5p, and miR-383 because ETV1 is a differentiation-related transcription factor, miR-628-5p was the second-highest expressed gene in HCEM and lowest expressed gene in HPL cells, and miR-383 was the highest expressed gene in HCEM. miR-628-5p and miR-383 overexpression in hMSCs regulated ETV1 mRNA expression, and miR-383 overexpression downregulated miR-628-5p expression. Moreover, miR-383 suppression decreased miR-383 expression and enhanced ETV1 mRNA expression, but miR-383 suppression also decreased miR-628-5p. Furthermore, silencing of ETV1 expression in hMSCs regulated miR-628-5p and miR-383 expression. Concerning periodontal cell commitment, miR-628-5p, miR-383, and ETV1 regulated the expression of HCEM- or HPL cell-related genes by adjusting the expression of these miRNAs. CONCLUSION: HCEM and HPL cells show characteristic mRNA and miRNA profiles. In particular, these cells have specific miR-383, miR-628-5p, and ETV1 expression patterns, and these genes interact with each other. Therefore, miR-383, miR-628-5p, and ETV1 are key genes involved in cementogenesis or HPL cell differentiation.

Combined endodontic and periodontal management of a class 3 invasive cervical resorption in a mandibular first molar.

Dental radiography and cone-beam computed tomography revealed the left mandibular first molar in a 68-year-old female patient with Heithersay Class 3 invasive cervical resorption (ICR). The inhibition of ICR progression and environmental improvement in and around the affected tooth through combined endodontic and periodontal treatments led to a favorable clinical outcome.

Detecting gene mutations in Japanese Alzheimer's patients by semiconductor sequencing.

Alzheimer's disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD.