Myogenic differentiation is an important stage within the multi-step process of skeletal muscle development. We previously found that excess retinoic acid (RA) could induce derangement of myofilaments in the embryonic tongue by inhibiting differentiation through Wnt5a/CaMKIIδ (calcium/calmodulin-dependent protein kinase II delta) pathway. Furthermore, our recently studies indicated that excess RA can directly induce abnormal expression of series of miRNAs in embryonic tongue, including miR-27b-3p. miR-27b-3p has been reported as a regulator involved in kinds of tumors development. In addition, miR-27 was proved to negatively regulate adipogenesis. It has been indicated that miR-27b could down-regulate the expression of Pax3 and Pax7 and thus inhibits myoblast proliferation. Here, we found that the expression of miR-27b-3p increased at early stage of myogenic differentiation (differentiation day 2, D2) in RA-treated C2C12 cells, but CaMKIIδ expression was reduced. Furthermore, bioinformatics analysis predicted that miR-27b-3p targets the 3’UTR of CaMKIIδ. The direct interaction between of miR-27b-3p and CaMKIIδ was confirmed by luciferase reporter assays. More importantly, rescue experiments indicated that CaMKIIδ mediated miR-27b-3p to regulate the early differentiation defects induced by excess RA, and which was achieved mainly via Myogenin. In conclusion, excess RA disturbed the early differentiation of C2C12 cells by stimulating miR-27b-3p expression which targeted CaMKIIδ, and then controlled the expression of Myogenin, those above implying new mechanism in myogenic differentiation and miR-27b-3p may act as a new biomarker for muscle disease.
Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital deformity worldwide with multifaceted etiology. The interactions of genes and environmental factors may be related to NSCL/P susceptibility. In the present study, we aimed to identify the relationship between ABCA4 (rs481931, rs560426) and MAFB (rs17820943) polymorphisms and NSCL/P in a northeastern Chinese population. A total of 248 patients, including 98 nuclear families, and 280 healthy controls were recruited for this study. The three SNPs were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Differences in the allele and genotype frequency between cases and controls were evaluated by chi-square test with OR values and 95% confidence intervals. The family-based association test 5.5 (FBAT 5.5) software package for genetic analysis of pedigrees with complex diseases was used to evaluate the patients and their parents' core families. In MAFB, statistical evidence of an association between rs17820943 (p=0.022) and NSCL/P was observed in case-control analyses. FBATs showed over-transmission of the C allele at the rs481931 polymorphism (p=0.039). However, no statistically significant difference was observed at rs560426. In addition, the C (rs481931)-G (rs560426) haplotype (P=0.004) was associated with the occurrence of NSCL/P. This study shows that rs481931 in ABCA4 and rs17820943 in MAFB are involved in the occurrence of NSCL/P in a northeastern Chinese population.
The regeneration of large bone defects is limited by reduced angiogenesis and cell migration from the remaining bone and tissue. Although scaffolds are required as autogenous bone grafts or artificial substrates for efficient bone tissue engineering, cellular implants can also facilitate bone regeneration, and bone marrow is frequently applied to treat such defects. Here, we attempted to reconstruct a three-dimensional hematopoietic environment on a newly developed porous polyimide membrane using a modified Dexter culture method and found that the membrane could maintain CD34-, CD45-, and TER-119-positive cells. We further applied membrane-enclosed, hematopoietic-lineage cells to a rat calvarial bone defect model to evaluate the effects of a multicellular environment on bone regeneration. Our results suggested that the cultured hematopoietic environment on the porous membrane facilitated new bone formation. The bone volume and bone mineral content values of the coculture group at 8 weeks post-surgery were significantly higher than those in samples where only bone marrow stromal cells were used. Thus, coculture with multiple cell types accelerated bone formation, and culturing diverse cells on a membrane may facilitate cell transplantation in bone tissue engineering.
Ozone is currently being considered as a potential oral antiseptic agent because it is highly antimicrobial and does not induce microbial resistance. In this study, we demonstrated that an optimal dosage of ozone gel enhanced the proliferation, type 1 collagen production, and alkaline phosphatase (ALP) secretion of Saos-2 cells in vitro. Proliferation of Saos-2 cells was assessed by MTT and DNA synthesis assays. Type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay (ELISA) and ALP assays. The cells were treated with/without 0.05, 0.5, 5 ppm ozone gel for 24 h. Ozone gel (0.5 ppm) significantly induced the proliferation of Saos-2 cells. At this concentration, ozone gel enhanced type 1 collagen production and ALP secretion. The results indicated that ozone gel controls the cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.
Oxidative stress in keratinocytes induces cytoprotective events, such as autophagy and cellular senescence. The present study investigated whether an induction of autophagy and cellular senescence can be observed in oxidative-stressed keratinocytes to allow those cells to maintain a cytoprotecitve state. We examined that the effect of various inhibitors on the induction of both autophagy and senescence in H2O2-treated HaCaT cells via Western blotting and immunocytochemical assays. H2O2-treated cells exhibited increased expression of the senescent markers, p21 and Decades (Dec1), in addition to increased and decreased numbers of senescence-associated β-galactosidase (SA-β-gal) – and Ki-67–positive cells, respectively. These senescent cells also displayed upregulation of the autophagy marker, LC3-II. Attenuation of LC3-II expression using 3-methyladenin inhibited H2O2- autophagy and cellular senescence. Our Western blotting results revealed that H2O2-induced autophagy was regulated independently by the negative feedback pathway of a mammalian target of rapamycin. By contrast, H2O2-induced autophagy and cellular senescence depended on the activation of the p38 mitogen-activated protein kinase α (MAPKα) pathway mediated by the intracellular reactive oxygen species (ROS) production. Furthermore, a suppression of autophagy by 3-methyladenine promoted an induction of apoptosis in H2O2-treated cells, suggesting that autophagy, in association with the cellular senescence, may induce the cytoprotection under the oxidative stress. Our findings suggest that the acceleration of both events may allow stressed cells to maintain the cytoprotective effects and may be regulated, in part, by p38 MAPK activation through the intracellular production of ROS.
Ozone is currently being considered as a possible oral antiseptic agent because it is strongly antimicrobial and does not induce microbial resistance. Here, we examined the effects of ozone exposure on the production of collagen type-1 and inflammatory cytokines in primary human gingival fibroblasts (HGFs) in vitro using enzyme-linked immunosorbent assays. In this study, we demonstrated that ozone ointment increased type 1 collagen production and hindered pro-inflammatory cytokine secretion from primary HGFs in vitro. HGFs were isolated from a 65-year-old patient who had undergone surgery due to chronic periodontitis. The cells were exposed to media with or without 0.05, 0.5 and 5 ppm ozone ointment for 24 hours 2 min. No cytotoxic effect of the ozone ointment was observed up to the concentration of 0.5 ppm, cell viability was attenuated at the dose of 5 ppm. When ozone ointment was used at the non-cytotoxic concentration of 0.5 ppm, it significantly enhanced type 1 collagen production by HGFs within for 24 hours. Secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 by HGFs treated with lipopolysaccharide (LPS) decreased when ozone ointment was present in the medium. These results suggest that the therapeutic effect of ozone ointment against periodontal disease is partially due to modulation of the function of HGFs.
Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine known to cause bone resorption, swelling and edema during tissue organization. Conversely, TNF-α has also been shown to participate in tissue regeneration during the wound healing process. We have previously investigated the effects of TNF-α on human dental pulp cell differentiation. Dental pulp cells are composed of different cell types including primary odontoblasts and fibroblasts. We determined that the ratio of stem cells within the pulp cell population was increased following short-term stimulation with TNF-α. The aim of this study therefore was to investigate the effect of short-term stimulation with TNF-α on osteoblast-like MC3T3-E1 cell growth and differentiation. MC3T3-E1 cells were cultured in standard growth medium and on reaching sub-confluence were exposed to recombinant TNF-α (10 and 100 ng/ml) for 2 days prior to assessing their cell proliferation and differentiation properties in comparison to non-stimulated MC3T3-E1 cells (control). Although no significant differences in cell proliferation were observed between the TNF-α-stimulated and control groups, cell differentiation was delayed in the TNF-α-stimulated groups. In summary, short-term stimulation of cultured MC3T3-E1 cells with TNF-α had only minimal effect on their growth and differentiation.
This study aimed to evaluate the efficacy of rabbit demineralized dentin matrix (DDM) as a recombinant human bone morphogenetic protein-2 (rhBMP-2) carrier using the subcutaneous tissues of mice and rabbit calvarial critical-sized defects. DDM of rabbit, combined with rhBMP-2 (DDM/rhBMP-2) was transplanted into the subcutaneous tissues of 6 mice and 6 rabbit calvarial critical-sized defects (DDM = 0.03 g, control; DDM/rhBMP-2 = 0.03 g of DDM, 0.2 mg/ml, 5.0 μg of rhBMP-2, experimental). Both DDM and DDM/rhBMP-2 was transplanted into the left and right subcutaneous tissues of mice symmetrically. For rabbits, 4 round critical-sized defects (8 mm diameter) were formed on the exposed skull. DDM was transplanted into the 2 defects on the left sides (n = 12) and DDM/rhBMP-2 into the right sides (n = 12). Two animals among 6 mice and 6 rabbits were sacrificed respectively at the 1, 2, and 4 experimental weeks for the histological and histomorphometrical evaluations with hematoxylin and eosin staining. Tissues from rabbits were imaged via micro-computed tomography (μCT). DDM/rhBMP-2 in mice induced new bone formation at 2 weeks and maturation with bone marrow at 4 weeks. DDM/rhBMP-2 in rabbit calvarium induced new bone formation remarkably at 4 weeks 21.77–47.99% compared to the DDM. These observations suggest that DDM could be considered a potential carrier of rhBMP-2. The rhBMP-2 loaded on DDM enhanced bone formation.
Pro-inflammatory cytokines activate the fibroblasts that reside in periodontal tissues and the polymorphonuclear leukocytes (PMNs) that are responsible for a substantial part of the destruction caused by the host response. The resident anaerobic bacteria interact with host inflammatory reactions leading to a hypoxic environment in the periodontal pocket. Expression of the transcription factor Differentiated Embryonic-Chondrocytes Expressed Gene 1 (DEC1) is regulated by hypoxia, growth factors and cytokines. To reveal the biological relevance of hypoxia in periodontal inflammation, we examined the role of DEC1 in the responses of human periodontal ligament cells to treatment with lipopolysaccharide (LPS), P. gingivalis and/or hypoxia. We found significantly increased levels of DEC1 after hypoxic or inflammatory stimulation of periodontal ligament cells. To further characterize the role of DEC1 in that process, inhibition by DEC1 siRNA was used in the experiments. The results demonstrated that treatment with the DEC1 siRNA inhibited the effects of LPS and hypoxia. Immunohistochemistry performed on an experimental animal model of periodontitis revealed an increase of PMN immunoreactivity, which validated the importance of the in vitro results. Treatment of DEC1-knockout mice with P. gingivalis decreased the production of PMN in periodontal ligament cells. These results demonstrate that DEC1 interference in the pathophysiological process of hypoxia has fundamental relevance for periodontal defenses.
Melanocytes are widely distributed in the skin and often present in oral mucosa, but they do not normally exist in bone tissue. Previous reports have shown that melanin pigmentation was seen in some odontogenic lesions of the jaw bone. In this study, the presence of melanin pigmentation and melanocytes was analyzed in ameloblastomas, odontogenic keratocysts (OKCs), and radicular cysts. The aim of this study was to compare the existence of melanin pigmentation and melanocytes in ameloblastomas, radicular cysts, and OKCs, and to clarify the different origins of these odontogenic lesions. Melanin pigmentation was detected using hematoxylin and eosin (HE) staining and Schmorl’s method staining. The presence of melanocytes was confirmed with Melan-A immunohistochemical staining. Melanin pigmentation and melanocytes were shown in OKCs, only melanocytes appeared in ameloblastomas, and neither melanin pigmentation nor melanocytes were present in radicular cysts. Comparing younger and older cases of OKC, both Melan-A and Schmorl’s reaction-positive rates were higher in the younger cases. In conclusion, these data raise the important possibility that the origin of OKC epithelium differs from that of ameloblastoma and radicular cyst based on the expression of melanin pigmentation and melanocytes. These findings also underscore the fact that the origin of OKC differs between younger and older patients. It is suggested that melanin pigmentation and melanocyte expression may assist in the classification of OKCs and may be useful in the development of new therapies in the future.
The aim of this experimental study was to examine the effect of Caffeic acid phenethyl ester (CAPE) on steroid-induced osteonecrosis of femoral head (ONFH) in rats. Thirty-one male Wistar albino rats were divided into 4 groups: control group (7 rats), methylprednisolone treatment group (MPS, 8 rats), CAPE treatment group (8 rats) and MPS+CAPE administered group (8 rats). The rats of group MPS and CAPE+MPS, On days 2, 3 and 4 were treated with 20 mg/kg/day methylprednisolone (MPS; Pfizer Pharmaceutical, Puurs, Belgium) intramuscularly. 10 μmol/kg/day CAPE was intraperitoneally injected to the rats of group CAPE from 13 weeks of age for 4 weeks. The control group was fed and housed under identical conditions without any treatment. All rats were sacrificed at 17 weeks of age by taking blood from the heart. Both proximal femoral parts were taken for histopathological and immunohistochemical analysis . Total oxidant status, total antioxidant status, and oxidative stress index (OSI), lipid parameters, coagulation parameters were assessed in blood specimens. Much lesser amount of osteonecrosis lesions were observed in the MPS+CAPE group compared to MPS group. In immunhisochemical analysis, oxidative stress was found significantly decreased in CAPE+MPS group compared to MPS group. OSI levels were significantly decreased in CAPE+MPS group compared to MPS group (p<0.001). In CAPE+MPS group lipid and coagulation parameters were found positively affected compared to MPS group. In conclusion, CAPE has strong protective effect against the steroid induced femoral head osteonecrosis in rats.
Identifying the differentially expressed miRNAs in cleft palate, in order to study the molecular mechanism in the development and progress of cleft palate. C57BL/6J mice were used to establish the RA induced cleft palate mouse model. Nine pairs of tissues were obtained on embryonic day 15.5 (E15.5). Total RNA was extracted and miRNAs microarray chip was used to screen the miRNAs. Real-time quantitative PCR (RT-qPCR) was used to verify the miRNAs microarray chip results. Cleft palate related genes targeted by the miRNAs were predicted by TargetScan and miRTarBase, and functional annotation clustering of Gene Ontology (GO) term and KEGG signaling pathways in DAVID were used to analysis these target genes. 1265 miRNAs were identified in cleft palate, and among them 31 were differentially expressed (p < 0.01, fold change > 1.5), including 17 up-regulated and 14 down-regulated miRNAs in cleft palate. 7 up-regulated miRNAs (mmu-miR-181a-5p, mmu-miR-410-3p, mmu-miR-3960, mmu-miR-1224-5p, mmu-miR-3970, mmu-let-7e-5p and mmu-miR-1907) and 3 down-regulated miRNAs (mmu-miR-140-3p, mmu-miR-351-5p and mmu-miR-503-5p) were validated by RT-qPCR, and mmu-miR-181a-5p and mmu-miR-410-3p were in concordance with those of miRNAs microarray chip detection. 484 target genes were predicted and proven by TargetScan and miRTarBase. GO term showed that RA-induced cleft palate was associated with enrichments in miRNAs involved in embryo development, osteoblast differentiation and so on. KEGG signaling pathways analysis indicated that the differentially expressed miRNAs were involved in MAPK, TGFβ and WNT signaling pathways. 10 differentially expressed miRNAs in cleft palate have been identified. These miRNAs and their target genes may become new therapeutic targets for cleft palate.
The effect of CaTiO3 compounded with different amounts of CaCO3 on osteoblastic KUSA/A1 cells was evaluated. CaTiO3-CaCO3 composites were obtained by alkoxide method, a simple, low-cost and reproducible technique used for large-scale production of material. The content of CaCO3 in our samples was controlled by varying the sintering time of the overall process. Composite morphology was assessed by scanning electron microscopy (SEM) showing particles with sizes ranging from100 to 500 nm. The presence of CaCO3 was revealed by XRD and thermogravimetric analyses, which suggested that samples treated at 650℃ for 30 min contained higher amounts of CaCO3 than samples treated for 2 and 10 h. Additionally, in vitro studies demonstrated that CaTiO3–CaCO3 composites sintered for 30 min induced augmented cell proliferation and mineralization in comparison to composites sintered for longer periods of time. Hence, our findings clearly suggest that the amount of CaCO3 within CaTiO3-CaCO3 composites exerts a critical effect on osteoblastic cells response. Enhanced bone regeneration could be achieved by increasing the content of CaCO3 within the composites, thus establishing CaTiO3-CaCO3 as a promising material for bone augmentation procedures in dental field.
Human dental pulp cells significantly contribute to the generation of patient-specific human induced pluripotent stem cells (hiPSCs) because dental pulp is easily accessible and contains high-quality mesenchymal stem cells. This study aimed to generate hiPSCs from deciduous dental pulp cells, using three factors, OCT3/4, SOX2, and KLF4, and to evaluate the feasibility of hiPSCs as substitutes for odontogenic cells. HiPSCs were mixed with heterogeneous cells of porcine third molar tooth germs extracted from the mandibles of six-month-old pigs. The mixed cells were seeded in a disc-shaped poly (D, L-lactic-co-glycolic acid) scaffold. The cell–scaffold complexes were then wrapped around the omentum of immunocompromised rats as recipients to promote vascularization and maturation of the implants; the implants were harvested at 16 weeks after transplantation. The paraffin-embedded sections of the implants were used for histological observation and for immunohistochemical and immunofluorescence analyses. Histologically, several small pieces of odontogenic tissue were observed in the implants. The enamel organ-like structures were observed and the tall and columnar-shaped cells facing the enamel stained positive for anti-human nuclei and amelogenin antibodies. Dentin–pulp complexes with dentinal tubules were observed and the columnar-shaped cells facing the dentin stained positive for anti-human nuclei and dentin sialophosphoprotein antibodies. Dental root-like structures accompanying the Hertwig’s epithelial root sheath (HERS)-like bilayer were observed and cells constituting the HERS-like bilayer stained positive for anti-human nuclei and cytokeratin 14 antibodies and negative for anti-vimentin antibody. The cementum adjacent to the dentin was recognized, and staining for bone sialoprotein (BSP) was observed to be intense at the cementum–dentin border. Cementoblasts and cementocytes stained positive for anti-human nuclei and BSP antibodies. These results suggest that hiPSCs have the potential to differentiate into ameloblasts, odontoblasts, and cementocytes, and are capable of generating odontogenic tissues.
Healthy bone has many physiological microcracks, which may be involved in the release of growth factors and act to accelerate bone remodeling process. In this study, the cortical bone fragments (5 x 5 x 1 mm3) from rat skull were ultrasonically demineralized with acidic electrolyzed water (AEW: pH 2.7) in ultrasonic bath (120 W, 38 kHz, 20 min) designated as ultrasonic bone. Fresh bone and ultrasonic bone were grafted into subcutaneous tissue of 4 week-old male rats, and explanted at 2 and 4 weeks. Fresh bone had physiological micro-cracks, while ultrasonic bone showed clear enlargement and union of cracks on SEM. Ultrasonic bone revealed bone induction locally at 2 weeks, while fresh bone didn’t induce bone and cartilage until 4 weeks. The new combination technique of ultrasonic irradiation and AEW will contribute to improve the surface area and the 3D-structure of the dense bone and should promote bone induction in the initial stage.