ABSTRACT
Objective: To assess the effect of application of Biodentine (BD), Photobiomodulation (PBM) using 810 nm diode laser and both on the proliferation and odontogenic differentiation of human dental pulp stem cells (HDPSCs). Material and Methods: HDPSCs were collected, isolated, and characterized and then divided into six groups: groups 1, control; groups 2, biodentine (BD); group 3, irradiation at 1 J/cm 2 of 810-nm diode laser; group 4, irradiation at 1 J/cm 2 and culture with BD; group 5, irradiation at 2 J/cm 2, and group 6, irradiation at 2 J/cm 2 and culture with BD. Viability assay was measured through MTT assay and Alkaline phosphatase (ALP) enzyme activity and mRNA levels of RUNX2, collagen 1 (Col-1) and BMP2 were also assessed. Results: Photobiomodulation at 1 and 2 J/cm 2 combined with biodentine significantly promoted HDPSCs proliferation (in MTT assay results) and odontogenic differentiation (through the gene expression of RUNX2, Col-1 and BMP2 levels (p < 0.05). Conclusion: Photobiomodulation at 2 J/cm 2 combined with biodentine enhanced proliferation and odontogenic differentiation of cultured HDPSCs and thus could further be beneficial for dentin regeneration (AU)
Objetivo: Avaliar o efeito da aplicação de Biodentina (BD), Fotobiomodulação (PBM) usando diodo de laser de 810 nm e ambos na proliferação e diferenciação odontogênica de células tronco cultivadas da polpa dental (HDPSCs). Material e Métodos: HDPSCs foram coletadas, isoladas, caracterizadas e então divididas em seis grupos: grupo 1, controle; grupo 2, biodentina (BD); grupo 3, irradiação com diodo de laser a 1 J/cm2 de 810- nm; grupo 4, irradiação a 1 J/cm 2 e cultivo com BD; grupo 5, irradiação a 2 J/cm2, e grupo 6, irradiação a 2 J/cm2 e cultivo com BD. A viabilidade foi mensurada através do teste MTT e a atividade da enzima Fosfatase alcalina (ALP), e níveis de RNAm de RUNX2, de colágeno 1 (Col-1) e de BMP2 foram também mensurados. Resultados: Fotobiomodulação a 1 e 2 J/cm 2 combinada com biodentina promoveu significativa proliferação de HDPSCs (nos resultados do teste MTT) e diferenciação odontogênica (através da expressão genética dos níveis de RUNX2, Col-1 e BMP2 (p < 0.05)). Conclusão: Fotobiomodulação a 2 J/cm2 combinada com biodentina aumentou a proliferação e diferenciação odontogênica de HDPSCs cultivadas e dessa forma poderia ser benéfica para a regeneração dentinária. (AU)
Subject(s)
Stem Cells , Collagen Type I , Core Binding Factor Alpha 1 SubunitABSTRACT
OBJECTIVE@#To detect the genetic variant of a child with cleidocranial dysplasia (CCD) and to find out the causation of the illness.@*METHODS@#Gene variant was identified by the second generation targeted sequencing and Sanger sequencing.@*RESULTS@#The gene sequencing revealed that the RUNX2 gene had c.196C>T(p.Glu66*) nonsense variant, which was predicted to be a pathogenic variant according to the ACMG guidelines(PVS1+PS2).@*CONCLUSION@#The variant of c.196C > T in the RUNX2 gene may be the cause of the child with CCD, and the novel variant enriches the RUNX2 gene variant spectrum.
Subject(s)
Child , Humans , Asian People/genetics , China , Cleidocranial Dysplasia/genetics , Core Binding Factor Alpha 1 Subunit/genetics , MutationABSTRACT
OBJECTIVE@#To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.@*METHODS@#Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining.@*RESULTS@#The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).@*CONCLUSION@#Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.
Subject(s)
Animals , Humans , Male , Rabbits , Calcium Carbonate/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Exosomes/metabolism , Osteogenesis/genetics , RNA, Messenger/metabolismABSTRACT
OBJECTIVE@#To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene.@*METHODS@#The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription.@*RESULTS@#We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively.@*CONCLUSION@#pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Subject(s)
Humans , Core Binding Factor Alpha 1 Subunit/genetics , Lipopolysaccharides/pharmacology , Luciferases/genetics , Metformin/pharmacology , NFATC Transcription Factors/genetics , Promoter Regions, Genetic , T-Lymphocytes , Transcription, Genetic/drug effects , TransfectionABSTRACT
OBJECTIVE@#To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC).@*METHODS@#Mouse models of VC were established in ApoE-deficient (ApoE-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with Nε-carboxymethyl-lysine for 16 weeks. ApoE-/- mice (control group), ApoE-/- diabetic mice (VC group), ApoE-/- diabetic mice with BI-1 overexpression (VC + BI-1TG group), and ApoE-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1TG+OPA1-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining.@*RESULTS@#ApoE-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007).@*CONCLUSION@#BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Subject(s)
Animals , Mice , Apolipoproteins E/metabolism , Calcium/metabolism , Caspase 3/metabolism , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Diabetes Mellitus, Experimental/pathology , GTP Phosphohydrolases/metabolism , Membrane Proteins/metabolism , Mice, Knockout , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Optic Atrophy, Autosomal Dominant/pathology , Osteogenesis , Vascular Calcification/pathology , bcl-2-Associated X Protein/metabolismABSTRACT
SUMMARY: Dental caries corresponds to an ecological and non-contagious, dynamic and chronic disease of multifactorial origin; currently there is evidence of how genetic factors could be included as predisposing agents to suffer it, however this evidence is diverse and incipient. a cross-sectional study was p erformed to investigate the possible associations of DSPP (rs36094464), RUNX2 (rs566712) and KLK4 (rs198968) polymorphisms in early childhood caries. Saliva samples of children (2-11years old) were collected and genotyped for DSPP (rs36094464), RUNX2 (rs566712) and KLK4 (rs198968) polymorphisms. Through the ceft index their caries history was determined and the gene variants were students through molecular biology techniques. polymorphisms of the DSSP (rs36094464) and RUNX2 (rs566712) are associated and contribute to the susceptibility of dental caries disease in early childhood, as they are related to their history of caries. KLK4 (rs198968) polymorphisms are not associated. In conclusions, the studied polymorphisms on DSSP and RUNX2 genes are associated with changes in the tooth microarchitecture, favoring the appearance of microlesions that would contribute to dental caries disease susceptibility in early childhood. Also, no association was found for the studied polymorphism of the KLK4 gene with dental caries disease susceptibility.
RESUMEN: La caries dental corresponde a una enfermedad crónica, no contagiosa, dinámica y de origen multifactorial. Actualmente existe evidencia de cómo los factores genéticos podrían incluirse como agentes predisponentes, sin embargo, esta evidencia es diversa e incipiente. Se realizó un estudio transversal para investigar las posibles asociaciones entre los polimorfismos DSPP (rs36094464), RUNX2 (rs566712) y KLK4 (rs198968) y la caries en la infancia. Se colectaron muestras de saliva de niños (de 2 a 11 años de edad) y se genotipificaron para los polimorfismos DSPP (rs36094464), RUNX2 (rs566712) y KLK4 (rs198968). Mediante el índice ceft se determinó su historial de caries y se estudiaron las variantes genéticas mediante técnicas de biología molecular. Los datos obtenidos indican que los polimorfismos del DSSP (rs36094464) y RUNX2 (rs566712) están asociados y contribuyen a la susceptibilidad de la enfermedad de caries dental en la infancia, ya que están - además - relacionados con el historial de caries. En conclusión, los polimorfismos estudiados en los genes DSSP y RUNX2 se asocian a la aparición de microlesiones que contribuirían a la susceptibilidad a la enfermedad de caries dental en la infancia. Creemos que este estudio es importante para la odontopediatría porque destaca el papel de DSSP (rs36094464) y RUNX2 (rs566712) y la susceptibilidad a la caries dental durante la infancia, además resalta la utilidad de la evaluación genética para la predicción y prevención de la caries dental y porque aporta evidencia que indica que los factores genéticos están implicados en la etiología de la caries.
Subject(s)
Humans , Male , Female , Child, Preschool , Child , Dental Caries/genetics , Dental Caries/epidemiology , Phosphoproteins/genetics , Polymorphism, Genetic , Saliva/chemistry , Sialoglycoproteins/genetics , Kallikreins/genetics , Cross-Sectional Studies , Dental Caries Susceptibility/genetics , Dentin , Core Binding Factor Alpha 1 Subunit/genetics , Genotype , Molecular BiologyABSTRACT
OBJECTIVE@#To explore the genetic basis for a Chinese patient featuring cleidocranial dysplasia(CCD).@*METHODS@#Genomic DNA was extracted from peripheral blood samples of the patient and his parents. Whole exome sequencing (WES) was carried out for the patient, and suspected variant was verified by Sanger sequencing.@*RESULTS@#WES has identified a missense c.460G>T (p.Val154Phe) (GRCh37/hg19) variant of the RUNX2 gene. The variant was located in the Runt domain, a highly conserved region (PM1); it was not present in either the Genome Aggregation Database or the 1000 Genomes Project (PM2), and was predicted to have a deleterious effect on the gene product by multiple in silico prediction tools (PP3); the clinical phenotype of the patient was highly consistent with that of cleidocranial dysplasia (PP4). Furthermore, the variant was unreported in medical literature and was absent in both parents (PS2). Based on the American College of Medical Genetics and Genomics guidelines, the c.460 G>T variant of RUNX2 gene was predicted to be pathogenic (PS2+PM1+PM2+PP3+PP4).@*CONCLUSION@#The c.460G>T (p.Val154Phe) variant of the RUNX2 gene probably underlay the clinical phenotype in the patient. Above finding has enabled accurate diagnosis and expanded the spectrum of RUNX2 variants.
Subject(s)
Humans , China , Cleidocranial Dysplasia/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Mutation , Exome SequencingABSTRACT
OBJECTIVE@#To explore effect of tobramycin (TOB) on healing of femoral fractures in rats.@*METHODS@#Totally 32 male sprague-dawley (SD) rats were selected and randomly divided into sham group (group A), fracture group (group B), fracture with TOB group (group C) and fracture + TOB + IWR-1 group (group D), 8 rats in each group. Close femoral fracture model in rats were established in group B, C and D, group A was sham operation without otherwise process. Group D was intraperitoneal injected 100 μl (8 μM) of Wnt pathway inhibitor IWR-1-endo (IWR-1) before molding at 1 day. At 1 day after molding, 100 μl (100 μM) of TOB was intraperitoneally injected into group C and D at once a day for 7 days. At 7 weeks after modling, fracture healing of group B, C and D were observed by X-ray, Western blotting was appilied to detect alkaline phosphatase(ALP) and Runt related transcription factor 2 (RUNX2) and β-catenin of Wnt passway.@*RESULTS@#X-ray results showed fracture line disappeared, callus formation and fracture healing well in group C compared with begning of molding; while a little fracture line, callus formation and fracture malunion in group B and d could be seen. Western blotting results showed ALP, RUNX2 and expression of β-catenin in group B, C and D were higher than that of group A (@*CONCLUSION@#Tobramycin could promote osteoblast differentiation and fracture healing by stimulating Wnt / β-catenin signaling pathway, up regulating expression of ALP and RUNX2.
Subject(s)
Animals , Male , Rats , Alkaline Phosphatase , Cell Differentiation , Core Binding Factor Alpha 1 Subunit/genetics , Femoral Fractures , Fracture Healing , Osteogenesis , Tobramycin , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
ABSTRACT Introduction: Vascular calcification is a common complication of chronic kidney disease. Osteoblast differentiation factor (Cbfa1) is present in histologic sections of arteries from patients with end-stage renal disease. Vascular smooth muscle cells (VSMC) can dedifferentiate to osteoblast-like cells, possibly by up-regulation of Cbfa1. There is evidence that the production of nitric oxide (NO) may have an important role in the regulation of osteoblast metabolism. The aim of this study is to evaluate whether increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC. Methods: VSMC were obtained from renal artery of Wistar male rats, treated for 72 hours with lipopolysaccharide (LPS), ß-glycerophosphate (BGF), a donor of phosphate and aminoguanidine (AG), an inhibitor of iNOS, in the following groups: CTL (control), LPS, BGF, LPS + BGF, and LPS + AG. NO synthesis was determined by chemiluminescence. Cbfa1 and iNOS mRNA expressions were analyzed by RT-PCR, Cbfa1 protein expression by immunohistochemistry and cellular viability by acridine orange. Results: Cbfa1 and iNOS mRNA expressions were higher in LPS and LPS+ BGF vs CTL (p < 0.05), and they were lower in LPS+AG vs LPS (p < 0.05). The Cbfa1 in the groups LPS and LPS+BGF also resulted in a higher value compared to CTL (p < 0.05), and in LPS+AG it was lower compared to LPS (p < 0.05). NO was higher in LPS and LPS+BGF compared to CTL group (p < 0.05) and lower in LPS + AG compared to LPS group (p < 0.05). Cellular viability showed no statistical difference among groups. Conclusion: This study showed that increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.
RESUMO Introdução: A calcificação vascular é uma complicação comum da doença renal crônica. O fator de diferenciação osteoblástica (Cbfa1) está presente em cortes histológicos das artérias de pacientes com doença renal em estágio terminal. As células do músculo liso vascular (CMLV) podem desdiferenciar para células do tipo osteoblastos, possivelmente pela regulação positiva da Cbfa1. Há evidências de que a produção de óxido nítrico (NO) pode ter um papel importante na regulação do metabolismo dos osteoblastos. O objetivo deste estudo é avaliar se o aumento da expressão de NO/iNOS causa um aumento na expressão de cbfa1 nas CMLV. Métodos: As CMLV foram obtidas da artéria renal de ratos machos Wistar, tratados por 72 horas com lipopolissacarídeo (LPS), ß-glicerofosfato (BGF), um doador de fosfato e aminoguanidina (AG), um inibidor da iNOS, nos seguintes grupos: CTL (controle), LPS, BGF, LPS + BGF e LPS + AG. A síntese de NO foi determinada por quimioluminescência. As expressões de mRNA de Cbfa1 e iNOS foram analisadas por RT-PCR, a expressão da proteína Cbfa1 por imunohistoquímica e viabilidade celular por laranja de acridina. Resultados: As expressões de mRNA de Cbfa1 e iNOS foram maiores em LPS e LPS + BGF v.s. CTL (p < 0,05) e menores em LPS + AG v.s. LPS (p <0,05). O Cbfa1 nos grupos LPS e LPS + BGF também resultou em um valor maior em comparação ao CTL (p < 0,05), e no LPS + AG foi menor em comparação ao LPS (p < 0,05). NO foi maior no LPS e LPS + BGF em comparação ao grupo CTL (p < 0,05) e menor no LPS + AG em comparação ao grupo LPS (p < 0,05). A viabilidade celular não mostrou diferença estatística entre os grupos. Conclusão: Este estudo mostrou que o aumento da expressão de NO/iNOS causa um aumento na expressão de cbfa1 nas CMLV.
Subject(s)
Humans , Animals , Male , Rats , Muscle, Smooth, Vascular , Nitric Oxide , Renal Artery , Lipopolysaccharides , Rats, Wistar , Core Binding Factor Alpha 1 SubunitABSTRACT
PURPOSE: Osteitis refers to the development of new bone formation and remodeling of bone in chronic rhinosinusitis (CRS) patients; it is typically associated with eosinophilia, nasal polyps (NPs), and recalcitrant CRS. However, the roles of ossification in CRS with or without NPs remain unclear due to the lack of appropriate animal models. Thus, it is necessary to have a suitable animal model for greater advances in the understanding of CRS pathogenesis.METHODS: BALB/c mice were administered ovalbumin (OVA) and staphylococcal enterotoxin B (SEB). The numbers of osteoclasts and osteoblasts and bony changes were assessed. Micro computed tomography (micro-CT) scans were conducted to measure bone thickness. Immunofluorescence, immunohistochemistry, and quantitative polymerase chain reaction were performed to evaluate runt-related transcription factor 2 (RUNX2), osteonectin, interleukin (IL)-13, and RUNX2 downstream gene expression. Gene set enrichment analysis was performed in mucosal tissues from control and CRS patients. The effect of resveratrol was evaluated in terms of osteogenesis in a murine eosinophilic CRS NP model.RESULTS: The histopathologic changes showed markedly thickened bones with significant increase in osteoblast numbers in OVA/SEB-treated mice compared to the phosphate-buffered saline-treated mice. The structural changes in bone on micro-CT were consistent with the histopathological features. The expression of RUNX2 and IL-13 was increased by the administration of OVA/SEB and showed a positive correlation. RUNX2 expression mainly co-localized with osteoblasts. Bioinformatic analysis using human CRS transcriptome revealed that IL-13-induced bony changes via RUNX2. Treatment with resveratrol, a candidate drug against osteitis, diminished the expression of IL-13 and RUNX2, and the number of osteoblasts in OVA/SEB-treated mice.CONCLUSIONS: In the present study, we found the histopathological and radiographic evidence of osteogenesis using a previously established murine eosinophilic CRS NP model. This animal model could provide new insights into the pathophysiology of neo-osteogenesis and provide a basis for developing new therapeutics.
Subject(s)
Animals , Humans , Mice , Computational Biology , Core Binding Factor Alpha 1 Subunit , Enterotoxins , Eosinophilia , Eosinophils , Fluorescent Antibody Technique , Gene Expression , Immunohistochemistry , Interleukin-13 , Interleukins , Models, Animal , Mucous Membrane , Nasal Polyps , Nose , Osteitis , Osteoblasts , Osteoclasts , Osteogenesis , Osteonectin , Ovalbumin , Polymerase Chain Reaction , Sinusitis , Transcription Factors , TranscriptomeABSTRACT
Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.
Subject(s)
Humans , Osteogenesis/drug effects , Stanozolol/pharmacology , Gene Expression/drug effects , Anabolic Agents/pharmacology , Osteoblasts/drug effects , Time Factors , Calcification, Physiologic/drug effects , Linear Models , Osteonectin/analysis , Osteonectin/drug effects , Reproducibility of Results , Analysis of Variance , Receptors, Calcitriol/analysis , Receptors, Calcitriol/drug effects , Cell Line, Tumor/drug effects , Core Binding Factor Alpha 1 Subunit/analysis , Core Binding Factor Alpha 1 Subunit/drug effects , Osteopontin/analysis , Osteopontin/drug effects , Real-Time Polymerase Chain ReactionABSTRACT
Cleidocranial dysplasia is a rare autosomal dominant hereditary disease characterized by abnormal skeletal and dental development. In this work, a case of cleidocranial dysplasia is reported, and a new frameshift mutation is confirmed by gene detection.
Subject(s)
Humans , Cleidocranial Dysplasia , Core Binding Factor Alpha 1 Subunit , Diagnostic Tests, Routine , MutationABSTRACT
OBJECTIVE@#To analyze variants of RUNX2 gene in two pedigrees affected with cleidocranial dysplasia and provide prenatal diagnosis for them.@*METHODS@#For the two probands, the coding sequences of the RUNX2 gene were analyzed with PCR and bidirectional Sanger sequencing. To verify the results, peripheral blood samples were collected from their parents and 100 healthy controls. For family 1, umbilical cord blood was also collected for prenatal genetic diagnosis.@*RESULTS@#In family 1, the proband and the fetus both carried a heterozygous c.578G>C (p.Arg193Pro) mutation. For family 2, the proband was found to carry a heterozygous c.909C>A (p.Tyr303X) mutation. The same mutations were not found among their parents and 100 healthy controls. Neither mutation was reported previously.@*CONCLUSION@#Variants of the RUNX2 gene probably underlie the cleidocranial dysplasia in both pedigrees. The results enabled prenatal diagnosis for the affected family.
Subject(s)
Female , Humans , Pregnancy , Cleidocranial Dysplasia , Diagnosis , Genetics , Core Binding Factor Alpha 1 Subunit , Genetics , Exons , Mutation , Pedigree , Prenatal DiagnosisABSTRACT
OBJECTIVE@#The purpose of this study is to investigate the potential effects of sclerostin (SOST) on the biological funtions and related mechanisms of cementoblasts under mechanical stress.@*METHODS@#OCCM-30 cells were treated with varying doses of SOST (0, 25, 50, and 100 ng·mL⁻¹) and were loaded with uniaxial compressive stress (2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase (ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), bone sialoproteins (BSP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA.@*RESULTS@#The expression of p-smad 1/5/8 was significantly downregulated with increasing SOST. β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under mechanical compressive stress with increasing SOST concentrations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased.@*CONCLUSIONS@#Under mechanical comprehensive stress, SOST can adjust the bone morphogenetic protein (BMP) /smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors (Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors (RANKL/OPG) through BMP signal pathways.
Subject(s)
Bone Morphogenetic Proteins , Metabolism , Core Binding Factor Alpha 1 Subunit , Dental Cementum , Osteocalcin , Smad Proteins , Metabolism , Stress, MechanicalABSTRACT
Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Subject(s)
Animals , Mice , Cell Differentiation , Physiology , Core Binding Factor Alpha 1 Subunit , Homeodomain Proteins , Metabolism , Mesenchymal Stem Cells , Metabolism , Mice, Nude , Osteoblasts , Metabolism , Osteocalcin , Osteogenesis , Physiology , Transcription Factors , Metabolism , Up-RegulationABSTRACT
Cleidocranial dysplasia (CCD) is an autosomal-dominant disease characterized by the delayed closure of cranial sutures, defects in clavicle formation, supernumerary teeth, and delayed tooth eruption. Defects in the Runt-related transcription factor 2 (RUNX2), a master regulator of bone formation, have been identified in CCD patients. The aim of this study was to identify the molecular genetic causes in a CCD family with delayed tooth eruption.The 23-year-old female proband and her mother underwent clinical and radiographic examinations, and all coding exons of the RUNX2 were sequenced. Mutational analysis revealed a single nucleotide deletion mutation (NM_001024630.4 : c.357delC) in exon 3 in the proband and her mother. The single C deletion would result in a frameshift in translation and introduce a premature stop codon [p.(Asn120Thrfs*24)]. This would result in the impaired function of RUNX2 protein, which may be the cause of delayed eruption of permanent teeth in the family.
Subject(s)
Female , Humans , Young Adult , Clavicle , Cleidocranial Dysplasia , Clinical Coding , Codon, Nonsense , Core Binding Factor Alpha 1 Subunit , Cranial Sutures , Exons , Molecular Biology , Mothers , Osteogenesis , Sequence Deletion , Tooth , Tooth Eruption , Tooth, Supernumerary , Transcription FactorsABSTRACT
Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.
Subject(s)
Humans , Male , Wound Healing/physiology , Bone Remodeling/physiology , Tooth Socket/physiology , Tooth Socket/diagnostic imaging , Reference Values , Time Factors , Tooth Extraction , Transcription Factors/analysis , Immunohistochemistry , Gene Expression , Osteocalcin/analysis , Reproducibility of Results , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Core Binding Factor Alpha 1 Subunit/analysis , Osteopontin/analysis , RANK Ligand/analysis , Osteoprotegerin/analysis , X-Ray Microtomography , Tartrate-Resistant Acid Phosphatase/analysisABSTRACT
Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed - Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.
Subject(s)
Animals , Male , Pineal Gland/surgery , Osseointegration/drug effects , Bone Density Conservation Agents/pharmacology , Bone-Implant Interface , Melatonin/pharmacology , Tibia/drug effects , Tibia/injuries , Tibia/pathology , Titanium , Immunohistochemistry , Osteocalcin/analysis , Reproducibility of Results , Treatment Outcome , Rats, Wistar , Implants, Experimental , Dental Implantation, Endosseous , Alkaline Phosphatase/analysis , Core Binding Factor Alpha 1 Subunit/analysis , Osteopontin/analysis , X-Ray Microtomography , Fluorescent DyesABSTRACT
Abstract Objective: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.
Subject(s)
Animals , Male , Skull/drug effects , Wound Healing/drug effects , Bone Regeneration/drug effects , Bone Substitutes/pharmacology , Hydroxyapatites/pharmacology , Skull , Skull/pathology , Time Factors , Wound Healing/physiology , Bone Regeneration/physiology , Immunohistochemistry , Bone Density , Osteocalcin/analysis , Treatment Outcome , Rats, Wistar , Bone Substitutes/therapeutic use , Vascular Endothelial Growth Factor A/analysis , Core Binding Factor Alpha 1 Subunit/analysis , Tartrate-Resistant Acid Phosphatase/analysis , Hydroxyapatites/therapeutic useABSTRACT
Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.