Distraction osteogenesis promotes temporomandibular joint self-remodeling in the treatment of mandibular deviation caused by condylar ankylosis.

Craniofacial deformity and malocclusion are primary concerns following temporomandibular joint ankylosis (TMJa) in growing patients, and they pose even greater challenges in adult patients. The treatment objectives always involve restoring proper jawbone structure, achieving stable occlusion, and attaining satisfactory joint mobility. This report presents a 4-year follow-up of an adult patient with TMJa-induced mandibular deviation, who underwent a combined treatment approach involving distraction osteogenesis (DO) and orthodontic-orthognathic surgery. Orthodontic treatment resulted in favorable occlusion and improved facial esthetics. A new condyle with a reconstructed glenoid fossa in a forward position was established after mandibular DO and the damaged TMJ experienced self-remodeling owing to functional improvement. Thus, this case demonstrates the efficacy of DO in promoting adaptive TMJ self-remodeling with long-term stability when treating mandibular deviation caused by condylar ankylosis in adult patients.

LncRNA00638 promotes the osteogenic differentiation of periodontal mesenchymal stem cells from periodontitis patients under static mechanical strain.

BACKGROUND: The osteogenic differentiation capacity of periodontal mesenchymal stem cells (PDLSCs) can be influenced by different levels of static mechanical strain (SMS) in an inflammatory microenvironment. Long non-coding RNAs (lncRNAs) are involved in various physiological processes. However, the mechanisms by which lncRNAs regulate the osteogenic differentiation of PDLSCs remain unclear. METHODS: We investigated the responses of PDLSCs obtained from periodontitis patients and healthy people to 8% and 12%SMS. Gene microarray and bioinformatics analyses were implemented and identified lncRNA00638 as a target gene for the osteogenesis of PDLSCs from periodontitis patients under SMS. Competing endogenous RNA (ceRNA) network analysis was applied and predicted interactions among lncRNA00638, miRNA-424-5p, and fibroblast growth factor receptor 1 (FGFR1). Gene expression levels were regulated by lentiviral vectors. Cell Counting Kit-8 assays, alkaline phosphatase assays, and Alizarin Red S staining were used to examine the osteogenic potential. RT-qPCR and Western blot were performed to detect the expression levels of related genes and proteins. RESULTS: We found that 8% and 12% SMS exerted distinct effects on HPDLSCs and PPDLSCs, with 12% SMS having the most significant effect. By microarray analysis, we detected differentially expressed lncRNAs/mRNAs between 12% SMS strained and static PPDLSCs, among which lncRNA00638 was detected as a positive target gene to promote the osteogenic differentiation of PPDLSCs under SMS loading. Mechanistically, lncRNA00638 may act as a ceRNA for miR-424-5p to compete with FGFR1. In this process, lncRNA00638 and miR-424-5p suppress each other and form a network to regulate FGFR1. CONCLUSIONS: Our findings demonstrate that the lncRNA00638/miRNA-424-5p/FGFR1 regulatory network is actively involved in the regulation of PDLSC osteogenic differentiation from periodontitis patients under SMS loading, which may provide evidence for optimizing orthodontic treatments in patients with periodontitis.

CXCL8, MMP12, and MMP13 are common biomarkers of periodontitis and oral squamous cell carcinoma.

OBJECTIVE: To analysis the relationship between periodontitis (PD) and oral squamous cell carcinoma (OSCC) by bioinformatic analysis. MATERIALS AND METHODS: We analyzed the gene expression profiles of PD (GSE16134) from the Gene Expression Omnibus (GEO) database and OSCC samples from TCGA-HNSC (head and neck squamous cell carcinoma) and identified common differentially expressed genes (DEGs) in PD and OSCC. Then, functional annotation and signaling pathway enrichment, protein interaction network construction, and hub gene identification were performed. Subsequently, the function and signaling pathway enrichment of hub genes, miRNA interaction, and transcription factor interaction analyses were carried out. We analyzed GSE10334 and GSE30784 as validation datasets, and performed qRT-PCR experiments simultaneously for validation, and obtained 4 hub genes. Finally, immune infiltration analysis and clinical correlation analysis of 4 hub genes and related miRNAs were performed. RESULTS: We identified 31 DEGs (16 up-regulated and 15 down-regulated). Four hub genes were obtained by qRT-PCR and validation dataset analysis, including IL-1ß, CXCL8, MMP12, and MMP13. The expression levels of them were all significantly upregulated in both diseases. The functions of these genes focus on three areas: neutrophil chemotaxis, migration, and CXCR chemokine receptor binding. Key pathways include IL-17 signaling pathway, chemokine signaling pathway, and cytokine-cytokine receptor interactions pathway. Immune infiltration analysis showed that the expressions of 4 hub genes were closely related to a variety of immune cells. ROC curve analysis indicated that AUCs of 4 hub genes are all greater than 0.7, among which MMP12 and MMP13 were greater than 0.9. Kaplan-Meier survival analysis indicated that worse OS was strongly correlated with CXCL8 and MMP13 high-expression groups. MMP12 low-expression group was strongly associated with worse OS. The results of multivariate Cox regression analysis showed that age, N stage, CXCL8, MMP12, and MMP13 were independent prognostic factors for OS. We also identified 3 miRNAs, including hsa-miR-19b-3p, hsa-miR-181b-2-3p, and hsa-miR-495-3p, that were closely related to 4 hub genes. Hsa-miR-495-3p is closely related to the diagnosis and prognosis of OSCC. CONCLUSIONS: We identified 4 hub genes between PD and OSCC, including IL-1ß, CXCL8, MMP12, and MMP13. These genes may mediate the co-morbid process of PD and OSCC through inflammation-related pathways such as the IL-17 signaling pathway. It is worth noting that CXCL8, MMP12, and MMP13 have great significance in the diagnosis and prognosis of OSCC.

The role of the Smad2/3/4 signaling pathway in osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells.

BACKGROUND: ClC-3 chloride channels promote osteogenic differentiation. Transforming growth factor-ß1 (TGF-ß1) and its receptors are closely related to ClC-3 chloride channels, and canonical TGF-ß1 signaling is largely mediated by Smad proteins. The current study aimed to explore the role of the Smad2/3/4 signaling pathway in the mechanism by which ClC-3 chloride channels regulate osteogenic differentiation in osteoblasts. METHODS: First, real-time PCR and western blotting were used to detect the expression of Smad and mitogen-activated protein kinase (MAPK) proteins in response to ClC-3 chloride channels. Second, immunocytochemistry, coimmunoprecipitation (Co-IP) and immunofluorescence analyses were conducted to assess formation of the Smad2/3/4 complex and its translocation to the nucleus. Finally, markers of osteogenic differentiation were determined by real-time PCR, western blotting, ALP assays and Alizarin Red S staining. RESULTS: ClC-3 chloride channels knockdown led to increased expression of Smad2/3 but no significant change in p38 or Erk1/2. Furthermore, ClC-3 chloride channels knockdown resulted in increases in the formation of the Smad2/3/4 complex and its translocation to the nucleus. In contrast, the inhibition of TGF-ß1 receptors decreased the expression of Smad2, Smad3, p38, and Erk1/2 and the formation of the Smad2/3/4 complex. Finally, the expression of osteogenesis-related markers were decreased upon ClC-3 and Smad2/3/4 knockdown, but the degree to which these parameters were altered was decreased upon the knockdown of ClC-3 and Smad2/3/4 together compared to independent knockdown of ClC-3 or Smad2/3/4. CONCLUSIONS: The Smad2/3 proteins respond to changes in ClC-3 chloride channels. The Smad2/3/4 signaling pathway inhibits osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells.

Transplantation of bone marrow mesenchymal stem cells and fibrin glue into extraction socket in maxilla promoted bone regeneration in osteoporosis rat.

AIMS: Bone defect repair in osteoporosis remains a tremendous challenge for clinicians due to increased bone metabolism resulted from estrogen deficiency. This study aims to investigate the effect of bone marrow mesenchymal stem cells (BMSCs) combined with fibrin glue (FG) in the extraction socket healing process of osteoporosis rats, as well as estimate the role of estrogen receptors (ERs) played in BMSCs differentiation in vitro and in the alveolar bone reconstruction process in vivo. MAIN METHODS: Forty rats were randomly divided into four groups, under general anesthesia, three groups underwent bilateral ovariectomy(OVX) and one group with the sham operation. Three months later, the osteogenic ability of BMSCs, isolated from healthy and osteoporosis rats, respectively, was tested. The ERα and ERß mRNA expression in BMSCs was also evaluated by RT-PCR analysis. In vivo experiment, Micro-CT detection, histological and immunofluorescent analysis, tissue PCR was conducted up to 2, 4 and 6 weeks after transplantation of BMSCs/FG to assess the newly formed bone in the extraction socket. KEY FINDINGS: The BMSCs from osteoporosis rats displayed weaker osteogenic potential and lower ERs expression compared with the BMSCs from healthy rats. Newly formed bone tissue filled the socket defect in BMSCs/FG treated VOX rats after six weeks, which was comparable to the sham group, while reduced ERs expression was found in the regenerated bone of the OVX group. SIGNIFICANCE: The BMSCs seeded within FG might provide an alternative therapeutic method for repairing the extraction socket defect in osteoporosis condition.

LncRNA FER1L4 Promotes Oral Squamous Cell Carcinoma Progression via Targeting miR-133a-5p/Prx1 Axis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common cancer especially young people in the world. The long non-coding RNA Fer-1-like protein 4 (FER1L4) has been reported to be closely associated with the progression of various human cancers. However, the role of FER1L4 in OSCC remains unclear. METHODS: The expression level of FER1L4 in OSCC tissues and cancer cell lines was detected by using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay and EdU staining assay. Cell invasion and migration were evaluated by Transwell assay. Cell apoptosis was detected by flow cytometry. Luciferase reporter assay was performed to determine the targeting relationship between FER1L4, miR-133a-5p and Prx1. The protein expression of Prx1 was detected by Western blot. In addition, a xenograft tumor model in vivo was constructed to confirm the function of FER1L4. RESULTS: FERIL4 was significantly upregulated in OSCC tissues and cancer cell lines. Moreover, high level of FER1L4 predicted a poor prognosis of OSCC patients. Silencing of FER1L4 not only significantly inhibited cell growth, invasion, migration and induced apoptosis in SCC-9 and HN4 cells in vitro, but also effectively suppressed the tumorigenesis of OSCC cells in vivo. Knockdown of FER1L4 significantly enhanced the expression of miR-133a-5p by sponging it, and then downregulated Prx1 expression. CONCLUSION: Our study elucidated a new mechanism of lncRNA FER1L4 that promoting OSCC progression by directly targeting miR-133a-5p/Prx1 axis and provided novel therapeutic targets for OSCC.

Local SDF-1α application enhances the therapeutic efficacy of BMSCs transplantation in osteoporotic bone healing.

Bone defect healing is markedly impaired in osteoporotic patient due to poor bone regeneration ability. Stromal cell derived factor-1α (SDF-1α) plays a pivotal role in the repair of various injured tissues including bone. Here, we definite that SDF-1α hydrogels potentiates in vivo osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in osteoporosis. The characteristics of rat primary BMSCs including superficial markers by flow cytometry and multi-lineage differentiation by induction were determined. At different time intervals, the release media from the SDF-1α-releasing hydrogels were collected to identificate SDF-1α exhibited a sustained release profile and maintained its bioactivity after release from the hydrogels to stimulate chemotaxis of BMSCs in a time dependent manner. Bilateral alveolar defects were operated in ovariectomized (OVX) rats and repaired with systemic BMSCs transplantation with or without the hydrogels. Local administration of SDF-1α significantly enhanced BMSCs recruitment and promoted more bone regeneration as well as the expression of OCN and Runx2 compared with the effect of BMSCs transplantation alone. Moreover, after BMSCs transplantation with SDF-1α delivery, macrophage polarization was promoted toward the M2 phenotype, that is identified as an important symbol in tissue regeneration process. Taken together, local SDF-1α application enhances the efficacy of BMSCs transplantation therapy in osteoporotic bone healing, suggesting clinical potential of SDF-1α to serve as a therapeutic drug target for osteoporosis treatment.

Periostin Mediates Oestrogen-Induced Osteogenic Differentiation of Bone Marrow Stromal Cells in Ovariectomised Rats.

Osteoporosis is a metabolic disease that results in the progressive loss of bone mass, which, in postmenopausal women, is related to oestrogen deficiency. Periostin (POSTN) plays a key role in the early stages of bone formation. However, whether POSTN participates in oestradiol regulation of osteogenic differentiation of bone marrow stromal cells (BMSCs) from ovariectomised (OVX) rats remains unclear. In vivo, using microcomputed tomography (micro-CT), immunohistochemistry, and dynamic analysis of femurs, we found that 17ß-E2 promotes bone formation and POSTN expression at the endosteal surface. In vitro, 17ß-E2 upregulated POSTN expression in OVX-BMSCs. POSTN overexpression activated the Wnt/ß-catenin signalling pathway and enhanced osteogenic differentiation of OVX-BMSCs. Furthermore, knockdown of Postn blocks the involvement of 17ß-E2 in the osteogenic differentiation of OVX-BMSCs. Collectively, our study indicated the role of POSTN in the osteogenesis and stemness of OVX-BMSCs and proves that 17ß-E2 reduces osteoporosis and promotes osteogenesis through the POSTN-Wnt/ß-catenin pathway. POSTN could, therefore, be a novel target gene for anti-osteoporosis therapies.

LncRNA-TWIST1 Promoted Osteogenic Differentiation Both in PPDLSCs and in HPDLSCs by Inhibiting TWIST1 Expression.

HPDLSCs derived from periodontal ligament tissues contribute to tooth development and tissue regeneration. Exploring the effects of long noncoding RNAs (lncRNAs) in the process of osteogenic differentiation of periodontal ligament stem cells would provide novel therapeutic strategies for tissue regeneration. The expression levels of lncRNA, which significantly changed during osteogenic differentiation, were observed by real-time quantitative PCR (q-PCR). Then, we screened for osteogenic-related lncRNA, which was initially named lncRNA-TWIST1. Moreover, we detected the mRNA expression levels of TWIST1 and osteogenesis-related genes after upregulating and downregulating lncRNA-TWIST1 in PPDLSCs (periodontal mesenchymal stem cells from periodontitis patients) and HPDLSCs (periodontal mesenchymal stem cells from healthy microenvironment), respectively. The osteogenic degree was verified by detecting ALP activity and alizarin red staining. LncRNA-TWIST1 decreased the mRNA levels of TWIST1 and promoted osteogenic differentiation in PPDLSCs, which was confirmed by the increase in osteogenesis-related gene levels (Runx2, ALP, and OCN), the increase in ALP activity, and the formation of more osteogenic nodules. In contrast, downregulating lncRNA-TWIST1 decreased the expression of osteogenesis-related genes, ALP activity, and osteogenic nodules both in PPDLSCs and in HPDLSCs. LncRNA-TWIST1 promoted osteogenic differentiation both in PPDLSCs and in HPDLSCs by inhibiting the TWIST1 expression. LncRNA-TWIST1 may be a novel therapeutic strategy to regenerate dental tissues.

COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair. Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT. Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/ß-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a. Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/ß-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.

miR­494­3p regulates lipopolysaccharide­induced inflammatory responses in RAW264.7 cells by targeting PTEN.

MicroRNAs (miRNAs/miRs) serve important roles in regulating inflammatory responses at the post­transcriptional level. In the present study, the limma package was used to analyze the GSE43300 array dataset downloaded from the Gene Expression Omnibus database. It was identified that several miRNAs, including miR­494­3p, were upregulated in lipopolysaccharide (LPS)­treated RAW264.7 macrophages compared to control cells. Transfection experiments indicated that overexpressing miR­494­3p inhibited production of LPS­induced proinflammatory cytokines, including interleukin­1ß and tumor necrosis factor­α. Conversely, knockdown of miR­494­3p enhanced cytokine expression. Bioinformatics prediction and luciferase assay both revealed that miR­494­3p could directly target phosphatase and tensin homolog (PTEN) and upregulate protein kinase B activity. In addition, miR­494­3p mimics suppressed p65 translocation to the nucleus. Similar effects were observed following PTEN silencing. In conclusion, the results of the present study revealed that miR­494­3p may act as an important immune regulator in LPS­stimulated macrophages, and be an effective therapeutic target for treating infections in the future.

ATP6V1H regulates the growth and differentiation of bone marrow stromal cells.

ATP6V1H encodes subunit H of vacuolar ATPase (V-ATPase) and may regulate osteoclastic function. The deficiency of ATP6V1H caused bone loss in human, mouse and zebrafish. In this report, we identified the mechanisms by which ATP6V1H regulates proliferation and differentiation of bone marrow stromal cells (BMSCs). We found that ATP6V1H was expressed in BMSCs, and Atp6v1h+/- BMSCs exhibited the lower proliferation rate, cell cycle arrest and reduced osteogenic differentiation capacity, as well as the increased adipogenic potentials. Histologic analysis confirmed less bone formation and more fatty degeneration in Atp6v1h+/- mice in the different age groups. Q-PCR analysis revealed that loss of ATP6V1H function downregulated the mRNA level of TGF-ß1 receptor, and its binding molecule, subunit ß of adaptor protein complex 2 (AP-2), suggesting ATP6V1H regulates the proliferation and differentiation of BMSCs by interacting with TGF-ß receptor I and AP-2 complex.

In vitro cell behaviors of bone mesenchymal stem cells derived from normal and postmenopausal osteoporotic rats.

Postmenopausal osteoporosis (PMO) increases bone fragility and the risk of fractures, and impairs the healing procedure of bone defects in aged women. The stromal cell­derived factor-1α (SDF-1α)/CXC chemokine receptor type 4 (CXCR4) axis helps to maintain the biological and physiological functions of bone marrow mesenchymal stem cells (BMSCs) and increase the homing efficiency of BMSCs. The present study aimed to provide insights into the possible association between migration and osteogenic ability and the SDF-1α/CXCR4 axis in BMSCs derived from a rat model of PMO. In order to do this, the general and SDF-1α/CXCR4-associated biological characteristics as well as associated molecular mechanisms in BMSCs isolated from a PMO rat model (OVX-BMSCs) and normal rats (Sham­BMSCs) were investigated and compared. In comparison with Sham-BMSCs, OVX-BMSCs exhibited an impaired osteo-genic ability, but a stronger adipogenic activity as well as a higher proliferative ability. In addition, OVX-BMSCs presented a lower chemotactic activity towards SDF-1α, lower expression levels of CXCR4 and reduced levels of phosphorylated AKT (p-AKT). Therefore, the lower expression levels of CXCR4 and p-AKT may be responsible for the impaired osteogenic ability and lower chemotactic activity towards SDF-1α of OVX-BMSCs.

MiR-106a* inhibits oral squamous cell carcinoma progression by directly targeting MeCP2 and suppressing the Wnt/ß-Catenin signaling pathway.

MicroRNAs (miRNAs) play critical roles in the tumorigenesis and progression of oral squamous cell carcinoma (OSCC). MiR-106a* functions as a tumor suppressor miRNA in several cancers; however, its role in OSCC has not been elucidated. We investigated the role of miR-106a* in human OSCC and explored its relevant mechanisms. The expression of miR-106a* was significantly downregulated in OSCC tissues and cell lines. The overexpression of miR-106a* inhibited OSCC cell proliferation and the cell cycle G1-S transition, and induced apoptosis. In contrast, inhibition of miR-106a* promoted cell proliferation and G1-S transition and suppressed apoptosis. The expression of miR-106a* inversely correlated with methyl-CpG binding protein 2 (MeCP2) expression in OSCC tissues. Using a luciferase reporter assay, MeCP2 was determined to be a direct target of miR-106a*. Overexpression of miR-106a* decreased MeCP2 expression at both the mRNA and protein levels, while inhibition of miR-106a* increased MeCP2 expression. Importantly, overexpression of MeCP2 eliminated the effects of miR-106a* overexpression in OSCC cells and silencing of MeCP2 recapitulated the cellular and molecular effects observed with miR-106a* overexpression. MeCP2 may promote OSCC cell proliferation by activating the Wnt/ß-Catenin signaling pathway. Taken together, our study demonstrated that miR-106a* inhibited OSCC cell proliferation by suppression of the Wnt/ß-Catenin signaling pathway and induced apoptosis through regulation of Caspase 3/9 expression via targeting MeCP2. These findings suggest that miR-106a* acted as a tumor suppressor in the progression of OSCC and may be a potential new target for OSCC diagnosis and therapy.

Evaluation of BMMSCs-EPCs sheets for repairing alveolar bone defects in ovariectomized rats.

The aim of this paper is to investigate the effect that bone marrow mesenchymal stem cells (BMMSCs) - endothelial progenitor cells (EPCs), BMMSCs and EPCs sheets have on repairing maxillary alveolar defects in ovariectomized (OVX) rats. In this study, after identification using multi-lineage differentiation and flow cytometry, BMMSCs and EPCs were isolated from female rats. The BMMSCs-EPCs, BMMSCs and EPCs sheets were detected by hematoxylin-eosin (H&E) staining, alkaline phosphatase (ALP) staining and qRT-PCR. Defects were created in maxillary alveoli and repaired with BMMSCs-EPCs, BMMSCs and EPCs sheets in OVX rats. The repair effects were determined by histological staining and micro-CT analysis at 2, 4 and 8 weeks after implantation. We aim to clarify whether BMMSCs-EPCs sheets are more effective in repairing alveolar bone defects than are BMMSCs and EPCs sheets in OVX rats. The results show that the osteogenic potential and the effect of bone repair are greater in the BMMSCs-EPCs sheet group and that this group has a higher ability to repair alveolar bone defects in OVX rats. These results suggest that BMMSCs-EPCs sheets have potential in clinical applications for treating humans with osteoporosis.

Regulation of OPG and RANKL expressed by human dental follicle cells in osteoclastogenesis.

We investigate whether the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in human dental follicle cells (HDFCs) regulated by colony stimulating factor 1 (CSF-1), parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein-2 (BMP-2) contributes to osteoclastogenesis. Adolescent human impacted third mandibular molars were used to separate HDFCs. These cells were incubated with PTHrP (10 ng/ml), CSF-1 (25 ng/ml), or BMP-2 (100 ng/ml) for 0.5, 1, 3, 6 and 12 h. The expression of OPG and RANKL was investigated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Two co-culture systems and tartrate-resistant acid phosphatase (TRAP) staining were used to examine osteoclast formation. Scanning electron microscopy was utilized for the resorption pit assay. RANKL and OPG were expressed innately in HDFCs. Exogenous PTHrP, CSF-1 and BMP-2 chronologically regulated the expression of RANKL and OPG in HDFCs. PTHrP and CSF-1 had similar regulative patterns leading to the up-regulated expression of RANKL and the down-regulated expression of OPG and opposite for BMP-2. The number of TRAP-positive peripheral blood mononuclear cells (PBMCs) slightly increased in contacted co-culture of HDFCs and PBMCs, whereas secreted OPG from HDFCs inhibited osteoclastogenesis in the transwell co-culture system. Contacted co-culture of HDFCs and PBMCs exhibited small and shallow resorption pits, whereas in the transwell co-culture system, secreted OPG from HDFCs reduced the resorption pits, reflecting the difference in osteoclast production. Collectively, we found a dual action of HDFCs in osteoclastogenesis; moreover, PTHrP, CSF-1 and BMP-2 might influence osteoclastogenesis by regulating the expression of RANKL and OPG in HDFCs.

Dental follicle cells rescue the regenerative capacity of periodontal ligament stem cells in an inflammatory microenvironment.

AIMS: Periodontal ligament stem cells (PDLSCs) are one of the best candidates for periodontal regeneration. Their function could be impaired in periodontitis microenvironment. Dental follicle cells (DFCs), serving as precursor cells and mesenchymal stem cells, have intimate connection with PDLSCs. However, it is still unknown whether DFCs could provide a favorable microenvironment to improve the proliferation and differentiation capacity of PDLSCs from healthy subjects (HPDLSCs) and patients diagnosed with periodontitis (PPDLSCs). METHODS: HPDLSCs, PPDLSCs and DFCs were harvested and identified using microscopic and flow cytometric analysis. Then, the coculture systems of DFCs/HPDLSCs and DFCs/PPDLSCs were established with 0.4 µm transwell, in which all the detection indexs were obtained from HPDLSCs and PPDLSCs. The expression of stemness-associated genes was detected by real-time PCR, and the proliferation ability was assessed using colony formation and cell cycle assays. The osteogenic differentiation capacity was evaluated by real-time PCR, western blot, ALP activity, Alizarin Red S staining and calcium level analysis, while the adipogenic differentiation capacity was determined by real-time PCR and Oil Red O staining. The cell sheet formation in vitro was observed by HE staining and SEM, and the implantation effect in vivo was evaluated using HE staining and Masson's trichrome staining. RESULTS: PPDLSCs had a greater proliferation capability but lower osteogenic and adipogenic potential than HPDLSCs. DFCs enhanced the proliferation and osteogenic/adipogenic differentiation of HPDLSCs and PPDLSCs to different degrees. Moreover, coculture with DFCs increased cell layers and extracellular matrix of HPDLSCs/PPDLSCs cell sheets in vitro and improved periodontal regeneration by HPDLSCs/PPDLSCs in vivo. CONCLUSIONS: Our data suggest that the function of PPDLSCs could be damaged in the periodontitis microenvironment. DFCs appear to enhance the self-renewal and multi-differentiation capacity of both HPDLSCs and PPDLSCs, which indicates that DFCs could provide a beneficial microenvironment for periodontal regeneration using PDLSCs.

Restoration of miR-34a in p53 deficient cells unexpectedly promotes the cell survival by increasing NFκB activity.

Upregulation of miR-34a by p53 is recently believed to be a key mediator in the pro-apoptotic effects of this tumor suppressor. We sought to determine whether restoration of miR-34a levels in p53 deficient cells could rescue the response to DNA damage. Compared with the p53 wildtype U2OS cells, miR-34a expression was much lower in p53 deficient Saos2 cells upon cisplatin treatment. Unexpectedly, delivery of miR-34a in Saos2 cells does not increase the cell sensitivity to apoptosis. This effect was mediated by direct downregulation of SirT1 expression by miR-34a, which in turn increased the NFκB activity. Inhibition of NFκB activity in Saos2 cells by Aspirin sensitized the miR-34a overexpressing cells to cell death. Thus, in tumors with p53 deficiency, miR-34a restoration alone confers drug resistance through Sirt1-NFκB pathway and combination of miR-34a and NFκB inhibitor could be considered as a promising therapeutic strategy.

Inhibition of NFkappaB by activated c-Jun NH2 terminal kinase 1 acts as a switch for C2C12 cell death under excessive stretch.

Mechanical stimuli play an important role in adaptive response of skeletal muscles, while inappropriate stretches result in apoptosis. In this study, we investigated the contribution of cyclic stretches to apoptosis of myoblast C2C12 cells. A 24-h cyclic stretch at an extension level of over 15%/10 cycles/min produced abundant reactive oxygen species, leading to significant apoptosis. Under such stretches, JNK1 phosphorylation increased while NFkappaB activity decreased. Inhibition of JNK by a specific chemical inhibitor or RNAi increased cell survival, which was blocked by additional NFkappaB inhibition. Furthermore, inhibition of JNK rescued the transcriptional activity of NFkappaB and upregulated the expression of Bcl2 through NFkappaB nuclear localization, suggesting the existence of inhibition of NFkappaB by activated JNK pathway. Taken together, our study suggests that excessive ROS induced by undue stretch favor the activation of JNK1, which in turn inhibit the activity of NFkappaB, resulting in significant apoptosis.

Plasma exchange therapy holds promise in the management of advanced cancer.

Cancer is now emerging as the second most common cause of death worldwide. Currently the most widely used treatments for solid cancer are surgery, chemotherapy, radiotherapy and hormone therapy, all of which are of limited effects in improvement of the survival. It is well-established that circulating factors are involved in the development of cancer. And plasma exchange is a well-established therapeutic procedure commonly used in many autoimmune diseases through the elimination of pathologic mediators, including auto-antibodies, complement components, and cytokines. Thus, we hypothesize that plasma exchange therapy alone or in combination with the routine therapeutics should be considered as an alternative for the present treatments. Future clinical studies are needed to validate the hypothesis.