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SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.
La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.
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Animals , Male , Mice , Osteoporosis/drug therapy , Resveratrol/administration & dosage , Osteogenesis/drug effects , Cell Differentiation/drug effects , Blotting, Western , Disease Models, Animal , Sirtuin 1 , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Resveratrol/pharmacology , Mice, Inbred C57BLABSTRACT
Objective To investigate the efeects of microRNA(miR)-103a-3p regulates tumor protein 53-regulated inhibitor of apoptosis 1(TRIAP1)on osteoblast differentiation and bone mass in ovariectomized mice.Methods MC3T3-E1 cells were divided into normal group,miR-103a-3p-NC group,miR-103a-3p mimic group,miR-103a-3p mimic+TRIAP1-NC group,miR-103a-3p mimic+TRIAP1 mimic group.mRNA expression of miR-103a-3p,TRIAP1,P53 were detected by Real-time PCR;Cell proliferation and apoptosis were detected by MTT test and flow cytometry;cytoskeleton and mineralization of cells were detected by F-actin immunofluorescence staining and alizarin staining;alkaline phosphatase(ALP)activity was detected by ELISA.24 female mice were divided into sham group,osteoporosis(OP)group,miR-103a-3p antagonist-NC group,miR-103a-3p antagonist group(six in each group),extract bilateral ovaries to establish an OP model,sham group mice only isolated fat around ovarian tissue.mRNA expression of miR-103a-3p,TRIAP1,P53,ALP,osteocalcin(OCN),osteopontin(OPN)of bone tissue were detected;microCT detect bone mineral density(BMD),bone mineral content(BMC);haematoxylin eosin staining was used to observe pathological changes of bone tissue.Results After miR-103a-3p mimic was transfected into cells,the miR-103a-3p and P53 expression increased,TRIAP1 expression decreased,cell proliferation decreased,apoptosis increased,F-actin expression decreased,the number of calcium nodules decreased,and ALP enzyme activity decreased(P<0.01);however,after TRIAP1 mimic was additionally transfected into cells,the above result caused by miR-103a-3p mimics were significantly reversed(P<0.01).In OP group,the miR-103a-3p and P53 expression in bone tissue increased,the TRIAP1,ALP,OCN and OPN expression decreased,BMD and BMC were decreased,and bone tissue construct was damaged(P<0.05);in miR-103a-3p antagonist group,the miR-103a-3p and P53 expression in bone tissue decreased,TRIAP1,ALP,OCN,OPN expression increased,BMD and BMC increased,and bone tissue construct was improved(P<0.05).Conclusion MiRNA-103a-3p mediate TRIAP1/P53 to inhibit proliferation and mineralization of osteoblast,while miR-103a-3p antagonistic treatment reduce bone loss in OP mice.
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Osteoporosis （OP） is a skeletal metabolic disease characterized by bone loss and destruction of bone microstructure. Changes in estrogen levels are not the only pathogenic factors for the occurrence and development of OP. MicroRNA （miRNA） plays an important regulatory role in cells. The complementary sequences of miRNA and targeted mRNA combine to inhibit the expression of targeted mRNA through post-transcriptional regulation， forming a complex regulatory network. Research suggests that miRNA is closely related to the occurrence and development of various diseases， including inflammatory diseases， metabolic diseases， and cancer. Targeted mRNA participates in post-transcriptional gene expression regulation in OP， mainly regulating the balance among bone construction， bone resorption， and osteoblast differentiation. Therefore， miRNA-based gene therapy is a rapidly developing disease treatment strategy. Traditional Chinese medicine can improve bone metabolism by intervening in miRNA differential expression to target and regulate osteogenic/osteoclast differentiation. This article summarized the targeting effects of miRNAs in physiological and developmental processes such as bone cell proliferation， differentiation， survival， and apoptosis， reviewed and classified their mechanisms of action and targets， and sorted out the current treatment methods of traditional Chinese medicine for preventing and treating OP and drugs that exert bone protective functions through miRNAs. This review is expected to provide theoretical reference and research guidance for future research on OP treatment by regulating miRNA.
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Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK⁃8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1 (Wnt1), Wnt3a and Wnt3a β⁃expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real⁃time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein (DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase⁃3β(GSK⁃3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β⁃catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.
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OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Subject(s)
Female , Mice , Animals , Core Binding Factor Alpha 1 Subunit/pharmacology , PPAR gamma/metabolism , Steroid 12-alpha-Hydroxylase/metabolism , Mice, Inbred C57BL , Cell Differentiation , Osteogenesis , Mesenchymal Stem Cells , Bile Acids and Salts/pharmacology , Bone Marrow Cells , Cells, Cultured , Azo CompoundsABSTRACT
Objective To investigate the targeted differentiation ability of mouse bone marrow derived mesenchymal stem cells(BM-MSCs)and adipose-derived mesenchymal stem cells(AD-MSCs).Methods BM-MSCs and AD-MSCs were isolated and cultured from bone marrow of femur and white adipose tissue of groin of C57BL/6J mice respectively,and the two types of cells were induced by osteogenic,chondrogenic and adipogenic differentiation medium respectively.Alizarin red,alcian blue and oil red O staining were used to detect the differentiated degree of osteogenic,chondrogenic and lipogenic differentiation.Real-time fluorescence quantitative PCR(qPCR)was used to identify MSCs and detected expression levels of directed differentiation-related genes Runx2,Sp7(osteoblast),Sox9,Col2a1(chondroblast),Pparg and Cebpa(lipogenesis)to determine the directed differentiation ability of cells.Based on gene expression profiles of mouse and human BM-MSCs and AD-MSCs in GEO database GSE43804 and GSE122778,the differentially expressed genes and their enrichment signal pathways were analyzed.Results The cell morphology of BM-MSCs and AD-MSCs obtained by isolation and culture was different,and spindle-shaped morphology was more obvious in AD-MSCs.Both cells expressed CD29,CD44 and CD90,but did not express CD34 and CD45.AD-MSCs showed higher osteogenic and lipogenic differentiation than those of BM-MSCs after directed induction,while chondrogenic differentiation was lower in AD-MSCs than that of BM-MSCs(P＜0.05).After directional induction,expression levels of Runx2,Pparg and Cebpa mRNA were higher in AD-MSCs than those in BM-MSCs,and Sox9 mRNA expression levels were lower than those in BM-MSCs(P＜0.05).Highly expressed genes of AD-MSCs in mice and human were enriched in PPAR and WNT signaling pathways.Highly expressed genes of BM-MSCs were enriched in cartilage and bone developmental signaling pathways.Conclusion The osteogenic and adipogenic differentiation ability of mouse AD-MSCs is stronger than those of BM-MSCs,while the chondrogenic differentiation ability AD-MSCs is weaker than that of BM-MSCs.The activation status of PPAR,WNT,cartilages and skeletal system development signaling pathways plays an important regulatory role in determining the different directional differentiation potential of AD-MSCs and BM-MSCs.
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BACKGROUND:Piezo1,a mechanosensitive protein,is tightly connected to osteogenic differentiation,and it has been demonstrated that TAZ has a role in regulating osteogenic differentiation.It is unclear whether TAZ participates in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells by Piezo1,so it is crucial to investigate its unique mechanism to prevent osteonecrosis of the femoral head. OBJECTIVE:To elucidate what function Piezo1 plays in osteogenic differentiation and TAZ expression in human bone marrow mesenchymal stem cells. METHODS:The siRNA targeting Piezo1 was constructed and transfected into 293T cells.The silencing efficiency was detected by RT-qPCR.The selected Piezo1-Home-2337 was packaged according to the silencing efficiency,and its optimal multiplicity of infection value was assayed by immunofluorescence staining.The packaged Piezo1 silencing recombinant lentivirus was transfected into human bone marrow mesenchymal stem cells,and its silencing effect was detected by RT-qPCR and western blot assay.Alizarin red staining,alkaline phosphatase activity analysis,immunofluorescence staining,RT-qPCR and western blot assay were utilized to analyze the effect of silencing Piezo1 on the osteogenic differentiation of human bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION:(1)The mRNA and protein levels of Piezo1 in human bone marrow mesenchymal stem cells transfected by si-Piezo1 were decreased significantly,with a statistically significant difference compared with normal and negative control groups.(2)The alkaline phosphatase activity in the si-Piezo1 group was much lower and the calcium deposition in the si-Piezo1 group was significantly reduced compared with the negative control group.(3)The mRNA levels of osteogenesis-related genes including Runt-related transcription factor 2(Runx2),osteopontin(OPN),distal-less homeobox 5(DLX5),osteocalcin,β-catenin and Tafazzin(TAZ)in the si-Piezo1 group were significantly decreased compared with the negative control group.Afterward,the expression levels of TAZ and β-catenin protein in the si-Piezo1 group were down-regulated significantly compared with the negative control group,whereas the expression levels of p-TAZ and p-β-catenin protein in the si-Piezo1 group had the opposite condition.(4)The results of immunofluorescence staining showed that the expression of TAZ and β-catenin in human bone marrow mesenchymal stem cells in the si-Piezo1 group was less compared with the negative control group.(5)These findings indicate that Piezo1 can promote the osteogenic differentiation of human bone marrow mesenchymal stem cells.The osteogenic ability of human bone marrow mesenchymal stem cells is significantly reduced after silencing Piezo1,and the expression of TAZ is also reduced.
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BACKGROUND:Irisin,a myokine isolated from the transmembrane protein FNDC5 by muscle cells during exercise,has the function of inducing the browning of white adipose tissue,but its effect on lipotoxicity-induced osteogenic differentiation and the mechanism is unclear. OBJECTIVE:To investigate the effect of irisin on the osteogenic ability of palmitic acid-induced bone marrow mesenchymal stem cells and the mechanism of action. METHODS:CCK-8 assay was used to detect the effect of different concentrations of palmitic acid on the proliferation of mouse bone marrow mesenchymal stem cells and the effect of irisin on the proliferation of mouse bone marrow mesenchymal stem cells in the presence of palmitic acid.After pretreatment with irisin and palmitic acid for 24 hours,osteogenic differentiation of mouse bone marrow mesenchymal stem cells was induced by alkaline phosphatase staining as well as qRT-PCR was performed to detect the expression of osteogenesis-related genes on day 7 of osteogenic induction culture.The expression of proteins related to the AMPK/BMP2/SMAD signaling pathway was detected by western blot assay.Alizarin red staining was conducted on day 21 to detect osteogenic differences. RESULTS AND CONCLUSION:(1)The CCK-8 assay results suggested that the amplification of bone marrow mesenchymal stem cells was inversely proportional to the concentration of palmitic acid,but at 0.02 mmol/L concentration,palmitic acid had no significant effect on the amplification of bone marrow mesenchymal stem cells,and irisin did not affect the proliferation of bone marrow mesenchymal stem cells when its mass concentration was in the range of 0.1-20 μg/L.(2)Alkaline phosphatase staining and alizarin red staining showed that palmitic acid inhibited the osteogenic differentiation ability of bone marrow mesenchymal stem cells.Irisin improved palmitic acid-induced osteogenic inhibition of bone marrow mesenchymal stem cells.qRT-PCR results showed that palmitic acid could cause the downregulation of osteogenic-related genes,and irisin could inhibit this trend.(3)Western blot assay results showed that compared with the palmitic acid intervention group,irisin treatment enhanced AMPK/BMP2/SMAD signal transduction in bone marrow mesenchymal stem cells.It is found that irisin can improve the osteogenic differentiation ability of bone marrow mesenchymal stem cells pretreated with palmitic acid,and proposed that the specific mechanism might be mediated by AMPK/BMP/SMAD signaling pathway.
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BACKGROUND:Studies have shown that long non-coding RNA small nucleolar RNA host gene 4(lncRNA SNHG4)is involved in the progress of many inflammatory diseases,but the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells during the treatment of periodontitis is still unclear. OBJECTIVE:To investigate the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells by regulating miR-152-3p. METHODS:Human periodontal ligament stem cells were isolated from periodontal membranes of premolars extracted for orthodontic purposes.After human periodontal ligament stem cells were induced to differentiate into osteoblasts for 0,7,and 14 days,the expression levels of RUNX family transcription factor 2,osteocalcin mRNA,lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells were detected by qRT-PCR.The third-generation human periodontal ligament stem cells were divided into the NC group,pcDNA group,pcDNA-SNHG4 group,inhibitor NC group,miR-152-3p inhibitor group,pcDNA-SNHG4+mimic NC group,and pcDNA-SNHG4+miR-152-3p mimic group.The expression of lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells was detected by qRT-PCR.The proliferation of human periodontal ligament stem cells was detected by CCK-8 assay.Alkaline phosphatase activity was detected by colorimetry.The formation of mineralized nodules was detected by alizarin red staining.Western blot assay was used to detect the expression of RUNX family transcription factor 2,osteocalcin and alkaline phosphatase proteins.A double luciferase reporter gene experiment was applied to verify the relationship between lncRNA SNHG4 and miR-152-3p. RESULTS AND CONCLUSION:(1)The expression of RUNX family transcription factor 2,osteocalcin mRNA and lncRNA SNHG4 in human periodontal ligament stem cells after 7 and 14 days of osteogenic induction was higher than that after 0 days of osteogenic induction,while the expression of miR-152-3p was lower(P<0.05).(2)Overexpression of lncRNA SNHG4 or inhibition of miR-152-3p was able to enhance the proliferation of human periodontal ligament stem cells,the alkaline phosphatase activity,mineralized nodule formation,the expression of RUNX family transcription factor 2,osteocalcin,and alkaline phosphatase proteins(P<0.05).miR-152-3p mimic attenuated the promoting effect of overexpression of lncRNA SNHG4 on osteogenic differentiation of human periodontal ligament stem cells.LncRNA SNHG4 had a targeting relationship with miR-152-3p.(3)These findings indicate that overexpression of lncRNA SNHG4 may promote the osteogenic differentiation of human periodontal ligament stem cells by inhibiting miR-152-3p.
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BACKGROUND:The repair of maxillofacial bone tissue defects is a hot and difficult point in current research and the selection of seed cells is the key.Jaw bone marrow mesenchymal stem cells are adult mesenchymal stem cells that exist in the jaw bone.They have advantages in the application of maxillofacial tissue regeneration. OBJECTIVE:To summarize the biological characteristics,osteogenic differentiation advantages of jaw bone marrow mesenchymal stem cells,and the effects of drugs,in vivo environment,and microRNAs on the osteogenic differentiation of jaw bone marrow mesenchymal stem cells. METHODS:Computers were used to perform literature retrieval in PubMed and CNKI.Chinese and English search terms were"oral,bone tissue engineering,stem cells".405 articles were retrieved and downloaded.The articles were screened according to the inclusion and exclusion criteria and 70 articles were finally included for literature review. RESULTS AND CONCLUSION:Jaw bone marrow mesenchymal stem cells were excellent seed cells for oral bone tissue engineering,and had good proliferation and osteogenic differentiation potential.Drugs,in vivo environment and microRNAs could regulate the osteogenic differentiation of jaw bone marrow mesenchymal stem cells.However,the research on jaw bone marrow mesenchymal stem cells was still in the initial stage,so more research with strong demonstration is needed to confirm that jaw bone marrow mesenchymal stem cells have more advantages in the application of maxillofacial bone tissue regeneration.
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BACKGROUND:Sema3A is a power secretory osteoprotective factor.However,studies about Sema3A-modified dental pulp stem cells(Sema3A-DPSCs)are rare. OBJECTIVE:To explore the osteogenic differentiation ability of Sema3A-DPSCs and their regulatory effect on the osteogenic differentiation of the pre-osteoblast cell line MC3T3-E1. METHODS:First,Sema3A-DPSCs were constructed using a lentivirus infection system carrying the Sema3A gene.Control lentivirus-treated DPSCs(Vector-DPSCs)were used as controls.Sema3A-DPSCs or Vector-DPSCs were co-cultured with proosteoblast line MC3T3-E1 at the ratio of 1∶1 and 1∶3 for 24 hours.Finally,the Sema3A-DPSCs,Vector-DPSCs and their co-cultured cells with MC3T3-E1 were cultured for osteogenic induction and differentiation.Osteogenic gene expression was detected by alkaline phosphatase staining,alizarin red staining and real-time quantitative RT-PCR to evaluate osteogenic differentiation ability. RESULTS AND CONCLUSION:(1)Sema3A mRNA and protein expression levels in Sema3A-DPSCs were significantly up-regulated.The level of secreted Sema3A in cell supernatant was up-regulated.(2)Compared with the Vector-DPSCs,mRNA expressions of osteogenic genes alkaline phosphatase,Runt-related transcription factor 2,osteocalcin and Sp7 transcription factors in Sema3A-DPSCs were up-regulated;the activity of alkaline phosphatase was enhanced,and the formation of mineralized nodules increased.(3)There were no obvious differences in proliferation between Sema3A-DPSCs and Vector-DPSCs.(4)Compared with MC3T3-E1/Vector-DPSCs co-culture system,the expression of MC3T3-E1 osteogenic genes was up-regulated,and the total alkaline phosphatase activity was enhanced and more mineralized nodules were formed in the MC3T3-E1/Sema3A-DPSCs co-culture system.(5)The results suggest that overexpression of Sema3A can enhance the osteogenic differentiation of DPSCs.Overexpression of Sema3A in DPSCs can promote osteogenic differentiation of MC3T3-E1 in the DPSCs/MC3T3-E1 co-culture system.
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BACKGROUND:In response to the limitations of traditional repair methods for bone defects,stem cells are widely used in the research of regenerative medicine.Chemical factors are the current research hotspots,but recent studies confirm that the application of physical factors to regulate stem cell differentiation at home and abroad has been intensifying,and physical factors combined with biological scaffolds in bone tissue engineering provide a new idea and method for solving the difficult problem of bone defect repair,with good development prospects. OBJECTIVE:To summarize the molecular mechanisms of physical factors such as electromagnetic fields and ultrasound on osteogenic differentiation of stem cells as well as the regulation of signaling pathways and the feasibility of their application in bone tissue engineering. METHODS:A computerized search of the CNKI and PubMed for the last 20 years was conducted.In the title and abstract,we used"stem cell,bone defect,osteogenic differentiation,electromagnetic fields,ultrasound,shock wave,low-level laser therapy,mechanical force,bone tissue engineering"in Chinese and"stem cell,osteoporosis,osteogenic differentiation,electromagnetic fields,ultrasound,bone tissue engineering"in English as search terms.A total of 94 relevant articles were included for review. RESULTS AND CONCLUSION:(1)As a non-invasive,non-contact adjuvant therapy,physical factors have a significant impact on bone tissue engineering,and have a positive effect on regulating osteogenic differentiation of stem cells,promoting cell proliferation and viability in bone engineering scaffolds.(2)In addition to activating signaling pathways and osteogenic gene transcription,physical factors can also improve vascularization,increase the volume,area and thickness of bone formed in the stent,promote osseointegration,and improve the success rate of bone scaffolds in regenerating healthy bone tissue.(3)However,the use of physical factors for bone tissue engineering uses different experimental conditions,such as scaffold type,cell type,and intervention conditions,and cannot be directly compared to determine the best parameter settings.There is also a lack of consistency in the effectiveness of these different interventions in promoting fracture healing in clinical use.Therefore,it is necessary to further determine the optimal parameters of physical factors for bone tissue engineering in the future.(4)In general,as an ideal adjuvant therapy,physical factors have great potential in combining with various biomaterials and applying them in bone tissue engineering.
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BACKGROUND:Adjustable piezoelectric effect can promote tissue regeneration and repair.Piezoelectric materials are widely used in weight-bearing tissue engineering. OBJECTIVE:To prepare a piezoelectric film material that can promote bone regeneration,and to explore its structural characterization,electrical output performance,biocompatibility,and effect of electrical output on osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. METHODS:Using poly-3-hydroxybutyrateco/4-hydroxybutyrate(P34HB)as raw material,barium calcium stannate titanate powder(Ba0.94Ca0.06Sn0.08Ti0.92O3,BCST)was added according to mass ratios of 0%,5%,10%,15%,and 20%.Dichloromethane was added to solve P34HB,and the thickness of 150-200 μm BCST/P34HB piezoelectric film was prepared by vacuum drying method.After polarization in the oil bath,the surface morphology,crystal phase composition,piezoelectric coefficient and open circuit voltage were tested.The effect of BCST/P34HB electrical output at 110 Hz and 0.25 N force on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells was tested. RESULTS AND CONCLUSION:(1)Scanning electron microscopy,X-ray diffraction,water contact angle,piezoelectric coefficient and electrical output performance tests showed that when the mass ratio of BCST increased to 20%,the BCST/P34HB piezoelectric film had good piezoelectric properties(d33=5.9 pC/N)and electrical output performance(180 mV),which was closer to the suitable range of 500 mV for electrical stimulation.(2)Live and dead staining showed that on the first day of co-culture,15%group and 20%group showed less red fluorescence.On the 5th day of culture,the number of green fluorescence in each group was significantly higher than that on the first day,and the red fluorescence was not observed in the 10%,15%and 20%groups,and only a small amount of red fluorescence was observed in the 0%and 5%groups.(3)On the 1st,3rd and 5th days of co-culture with rabbit bone marrow mesenchymal stem cells,Almar blue staining exhibited that the number of cells in each group showed an increasing trend with the increase of time.On the 5th day of culture,the number of cells in the 20%group was significantly more than that in the 0%group(P<0.05).(4)On day 10 of osteogenic induction,alkaline phosphatase staining results showed that the positive rate of the 20%group was significantly higher than that of the 0%group(P=0.000 1).On day 21,alizarin red staining and quantitative analysis of calcium nodules showed a similar trend to alkaline phosphatase staining.Compared with the 0%group,the 15%group and 20%group showed significant differences(P<0.01,P<0.000 1).(5)The results showed that 20%BCST/P34HB films had good piezoelectric properties,electrical output properties,biocompatibility and the ability of promoting osteogenic differentiation of bone marrow mesenchymal stem cells.
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BACKGROUND:microRNA-26b(miR-26b)plays an important regulatory role in a variety of stem cell functions,but its effects on the biological properties of stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells are unknown. OBJECTIVE:To investigate the effects of miR-26b on the proliferation,migration and osteogenic differentiation of stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells. METHODS:Stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells were cultured and identified.miR-26 mimics(experimental group)and miRNAs mimics control(control group)were used to transfect above mentioned two kinds of cells and construct overexpressed models for subsequent experiments.CCK-8 assay was applied to detect the proliferation ability of overexpressed miR-26b cells.Transwell and scratch assay were employed to analyze the migration ability of overexpressed miR-26b cells.RT-qPCR was utilized to examine the expression of osteogenic markers after osteogenic induction of overexpressed miR-26b cells. RESULTS AND CONCLUSION:(1)Transfection of miR-26b mimics increased miR-26b expression in the two kinds of cells and promoted the proliferation of stem cells from human exfoliated deciduous teeth,with no significant effect on the amplification of human umbilical cord mesenchymal stem cells.(2)Compared with the control group,the migration ability was enhanced after two types of cells overexpressing miR-26b.(3)miR-26b expression decreased during osteogenic differentiation of the two kinds of cells.(4)Compared with the control group,the levels of osteogenesis-related genes osteocalcin,osteopontin,alkaline phosphatase,and human type I collagen mRNA were downregulated after overexpression of miR-26b in the two kinds of cells.The results showed that overexpression of miR-26b promoted the proliferation and migration of stem cells from human exfoliated deciduous teeth and inhibited their osteogenic differentiation;it promoted the migration of human umbilical cord mesenchymal stem cells and inhibited their osteogenic differentiation,but had no significant effects on their proliferation.
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BACKGROUND:Nano-zirconium dioxide has good application potential in the field of bone tissue repair.Studying the effect of nano-zirconium dioxide on osteogenic differentiation will help to promote the clinical application of nano-zirconium dioxide in the treatment of bone defects. OBJECTIVE:To explore the effect of nano-zirconium dioxide on the osteogenic differentiation of ectomesenchymal stem cells in the nasal mucosa. METHODS:Ectomesenchymal stem cells derived from rat nasal mucosa were isolated and cultured,and the biotoxicity of nano-zirconium dioxide to the cells was detected by CCK-8 assay.The biosafety concentration was selected according to the cytotoxicity,and the cells were randomly divided into a control group,a nano-zirconium dioxide group,and a nano-hydroxyapatite group.Osteogenic differentiation of cells was directionally induced in each group.On day 7 of induced differentiation,alkaline phosphatase staining was performed.qRT-PCR and western blot assay were used to detect the expression of early osteogenic markers(Runx2 and Osx).On day 21 of induced differentiation,alizarin red staining was conducted.qRT-PCR and western blot assay were utilized to determine the expression levels of late osteogenic markers(OPN and OCN). RESULTS AND CONCLUSION:(1)The median lethal concentration of nano-zirconium dioxide on ectomesenchymal stem cells in nasal mucosa was 0.6 mg/mL.In the experiment,the mass concentration of 200 μg/mL was selected for intervention.Zirconium dioxide had no significant effect on the proliferation of the cells.(2)Compared with the control group,the alkaline phosphatase staining of the cells in the nano-zirconium dioxide group was more obvious and the level of cell mineralization was higher,but there was no significant difference compared with the nano-hydroxyapatite.(3)Compared with the control group,the expression of bone-related genes and proteins increased significantly,but there was no significant difference compared with nano-hydroxyapatite.(4)The results show that nano-zirconium dioxide has good biological safety and can promote the osteogenic differentiation of ectomesenchymal stem cells in the nasal mucosa.This promoting effect is equivalent to that of nano-hydroxyapatite.
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BACKGROUND:Oral and maxillofacial bone tissue defects can seriously affect the physical and mental health of patients.When bone defects occur in diabetic patients,bone metabolism disorders caused by abnormal blood sugar make it more difficult to repair and treat. OBJECTIVE:To attempt to apply AOPDM1,a polypeptide with potential bioactivity to the osteogenic treatment of diabetic patients. METHODS:In normal or high-glucose environment,different concentrations of AOPDM1 were used to interfere with mouse bone marrow mesenchymal stem cells,and cell proliferation,alkaline phosphatase activity,mineralization nodules formation and osteogenic differentiation gene expression were detected.The polycaprolactone scaffold was prepared by electrospinning technology,and the scaffold was modified by polydopamine to prepare the polycaprolactone-polydopamine composite scaffold.Finally,the scaffolds were placed in AOPDM1 solution to prepare polycaprolactone-polydopamine-AOPDM1 scaffolds.The water contact angle and mechanical properties of the scaffolds were tested in the three groups.In normal or high-glucose environment,the three groups of scaffolds were co-cultured with mouse bone marrow mesenchymal stem cells,respectively,and cell adhesion,alkaline phosphatase activity and osteopontin expression were detected. RESULTS AND CONCLUSION:(1)Compared with normal environment,high-glucose environment inhibited the proliferation of bone marrow mesenchymal stem cells.In the same environment,AOPDM1 could promote the proliferation of mouse bone marrow mesenchymal stem cells.When AOPDM1 concentration was the same,alkaline phosphatase activity,mineralization ability and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase,and Runx2 of bone marrow mesenchymal stem cells were decreased in high-glucose environment compared with normal environment.Under the same environment,AOPDM1 could improve the alkaline phosphatase activity,mineralization ability,and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase and Runx2 of bone marrow mesenchymal stem cells.(2)The hydrophilicity of polycaprolactone-polydopamine scaffold and polycaprolactone-polydopamine-AOPDM1 scaffold was higher than that of polycaprolactone scaffold(P<0.001),and there was no significant difference in tensile strength and elastic modulus among the three groups(P>0.05).Compared with the other two groups of scaffolds,the cells on the polycaprolactone-polydopamine-AOPDM1 scaffold had better adhesion morphology.When the scaffolds were identical,compared with normal environment,high-glucose environment inhibited alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells.When the environment was the same,the alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells on the polycaprolactone-polydopamine-AOPDM1 scaffold were higher than those on the other two scaffolds.(3)The above results prove that polycaprolactone-polydopamine-AOPDM composite scaffold can promote the osteogenic properties of bone marrow mesenchymal stem cells in high-glucose environment.
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BACKGROUND:Bone is a remarkable natural material possessing piezoelectric properties.By harnessing the biomimetic piezoelectric effect,tissue engineering materials can be employed to effectively address bone tissue defects and facilitate their repair. OBJECTIVE:Using a solid-phase force chemistry technique,a piezoelectric scaffold with inherent osteogenic properties was meticulously fabricated.This unique scaffold was then assessed for its impact on osteoblast adhesion,proliferation,and osteogenic differentiation. METHODS:Polyvinylidene fluoride(PVDF)powders,along with commercially available NaCl(mass ratios are 60:40,50:50,40:60,and 30:70,respectively),were subjected to solid-phase shear milling technology,resulting in a homogenous mixture.Through a melting process,a substantial material was formed,and subsequent treatment with a pure water solution effectively eliminated the NaCl.Consequently,PVDF piezoelectric foam scaffolds with varying pore sizes were successfully prepared.These materials were categorized as PVDF-40,PVDF-50,PVDF-60,and PVDF-70,denoting the respective mass percentages of NaCl during preparation.The surface morphology,crystal phase composition,thermodynamic behavior,mechanical properties,and piezoelectric properties of each group were meticulously characterized.The four kinds of piezoelectric foam scaffolds were co-cultured with the MG63 osteoblast cell line to evaluate its biocompatibility and potential to promote bone differentiation. RESULTS AND CONCLUSION:(1)The scanning electron microscopy,four groups of scaffolds had multi-level pores.As the NaCl mass fraction in the mixed powder increased,the porosity of the scaffolds increased.X-ray energy dispersion spectrum,X-ray diffraction,Fourier transform infrared spectroscopy,and thermogravimetric analysis collectively revealed the scaffold predominantly comprised the α phase,which inherently lacked piezoelectric properties.However,the application of solid-phase force chemistry successfully stimulated the formation of the β phase,thereby enhancing the scaffold's piezoelectric properties.Notably,the PVDF-60 group exhibited the highest proportion of the β phase among all the tested groups.The results of cyclic compression testing and piezoelectric performance assessment demonstrated that the PVDF-60 group exhibited superior compressive strength and piezoelectric performance compared to the other groups.(2)The findings from scanning electron microscopy and laser confocal microscopy exhibited that MG63 cells adhered well to the surface of the four groups of scaffolds,with good morphology,extended more pseudopods,and secreted a large amount of extracellular matrix.CCK-8 assay revealed that the proliferative absorbance of PVDF-60 cells cultured for 4 days was higher than that of the other three groups(P<0.000 1).Alkaline phosphatase staining and alizarin red staining showed that the expression of alkaline phosphatase and the number of calcified nodules in the PVDF-60 group were higher than those in the other three groups(P<0.01,P<0.000 1).(3)The piezoelectric PVDF foam-based scaffolds demonstrated favorable cytocompatibility.Notably,the PVDF-60 group showed superior mechanical properties,piezoelectric performance,and bone-inducing capabilities.
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BACKGROUND:The repair of large-scale bone defects is still facing serious challenges.It is of great significance to develop personalized,low-cost,and osteogenic-inducing tissue engineering scaffolds for bone repair. OBJECTIVE:To explore the process of 3D printing bone tissue engineering scaffold containing pearl composite material by low-temperature condensation deposition method,and further test the physicochemical properties and in vitro biological functions of the composite scaffold. METHODS:Pearl powder was prepared by grinding and sieving.The pearl powder of different qualities was added into the poly-L-lactic acid ink,so that the mass ratio of pearl powder to poly-L-lactic acid was 0,0.1,0.2,0.3,and 0.5,respectively.The 3D-printed poly-L-lactic acid/pearl powder scaffolds were prepared using the low-temperature condensation deposition method.The microstructure,compressive properties,water contact angle,cytocompatibility,and in vitro bone differentiation ability of the printed poly-L-lactic acid/pearl powder composite scaffolds were detected. RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the five groups of scaffolds all had micropores with a diameter of 2 μm or even smaller,irregular shapes and interconnectivity.(2)All the five groups had good compressive properties.The compressive strength of the pearl powder 0.5 group was higher than that of the other four groups(P<0.05).The water contact angle of the pearl powder 0.2 group and the pearl powder 0.5 group was smaller than that of the pearl powder 0 group(P<0.01,P<0.001).(3)Bone marrow mesenchymal stem cells were co-cultured with five groups of scaffolds for 1,3,and 5 days,respectively.The cell proliferation in pearl powder 0.1,0.2,0.3,and 0.5 groups cultured for 3 and 5 days was faster than that in pearl powder 0 group(P<0.05).After 1 day of culture,live-dead staining exhibited that the number of cells on the scaffold was small,but all of them were living cells.(4)Bone marrow mesenchymal stem cells were inoculated on the scaffold surface of the pearl powder 0 group and pearl powder 0.1 group respectively for osteogenic differentiation.The alkaline phosphatase activity induced for 4 and 6 days in the pearl powder 0.1 group was higher than that in the pearl powder 0 group(P<0.05).(5)The results showed that the poly-L-lactic acid/pearl powder composite scaffold had good compressive strength,hydrophilicity,cytocompatibility,and osteogenic properties.
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BACKGROUND:The implant osseointegration rate of patients with diabetes is low,and the failure rate is high,which seriously affects the quality of life.It is urgent to improve the implant osseointegration of patients with diabetes by effective means to elevate the success rate.Exploring the effect of berberine on the osteogenic differentiation of bone marrow mesenchymal stem cells under a high-glucose environment and its specific mechanism will provide effective theoretical support for solving the above problems. OBJECTIVE:To explore the effect of natural extract berberine on the osteogenic differentiation of rat bone marrow mesenchymal stem cells under the high-glucose microenvironment. METHODS:Bone marrow mesenchymal stem cells of SD rats were cultured by the whole bone marrow adherence method.CCK-8 assay was used to detect the effects of different concentrations of berberine on the proliferation of bone marrow mesenchymal stem cells under the high-glucose environment and to screen out the optimal berberine concentration.The expressions of Runx2 and Osx were detected by alkaline phosphatase activity,alicarin red staining and PCR to determine the effect of berberine on osteogenic differentiation of bone marrow mesymal stem cells under the high-glucose environment.To further explore the underlying mechanism,we introduced the AMPK-specific inhibitor Dorsomorphin and used a DCFH-DA reactive oxygen species fluorescent probe to examine reactive oxygen species levels.The p-AMPK expression was also determined by western blot assay. RESULTS AND CONCLUSION:(1)10 μmol/L was the optimal concentration of berberine to promote bone marrow mesenchymal stem cell proliferation.(2)Alberberine promoted alkaline phosphatase viability of bone marrow mesenchymal stem cells and mineralized nodule formation in a high-glucose microenvironment.(3)Alberberine promoted the expression of Runx2 and OSx in a high-glucose microenvironment.(4)Alberensine effectively inhibited the reactive oxygen species level of bone marrow mesenchymal stem cells in a high-glucose environment.(5)The effects of berberine on promoting bone marrow mesenchymal stem cell osteogenesis and inhibition of reactive oxygen species were reversed by the AMPK inhibitor.(6)Berberine activated AMPK and promoted p-AMPK expression.(7)The above results indicate that berberine(10 μmol/L)promotes the osteogenic differentiation of bone marrow mesenchymal stem cells in a high-glucose environment by activating AMPK and reducing intracellular reactive oxygen species levels.
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BACKGROUND:Diabetic osteoporosis is gaining public attention.However,few studies have reported the effect of a high-glucose environment on the osteogenic differentiation of human umbilical cord mesenchymal stem cells and the corresponding therapeutic strategies. OBJECTIVE:To investigate whether vitamin D3 can restore the osteogenic differentiation potential of human umbilical cord mesenchymal stem cells in a high-glucose environment. METHODS:The viability of human umbilical cord mesenchymal stem cells was detected by CCK-8 assay to screen the appropriate vitamin D3 intervention concentration.Under the high-glucose environment,RT-qPCR,western blot assay,immunofluorescence,JC-1 mitochondrial membrane potential,alizarin red staining,and β-galactosidase staining were used to evaluate the osteogenic differentiation potential,intracellular reactive oxygen species accumulation,mitochondrial membrane potential alteration,and cell senescence of human umbilical cord mesenchymal stem cells after vitamin D3 intervention.The underlying mechanism was also discussed. RESULTS AND CONCLUSION:(1)Vitamin D3 significantly promoted the proliferation of human umbilical cord mesenchymal stem cells in the range of 0.1 μmol/L to 1 mmol/L.(2)High-glucose environment down-regulated the mRNA and protein level expressions of osteogenic-related genes α1-I collagen,alkaline phosphatase,Runt-associated transcription factor 2,and osteocalcin in human umbilical cord mesenchymal stem cells,which induced oxidative stress and cellular senescence.(3)Vitamin D3 at an intervention concentration of 10 μmol/L significantly restored the osteogenic phenotype of human umbilical cord mesenchymal stem cells under high-glucose conditions and attenuated intracellular oxidative stress and cellular senescence by activating the Nrf2/HO-1 signaling pathway.(4)These findings suggested that the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells was reduced in the high-glucose environment,and vitamin D3 could partially improve their osteogenic differentiation ability and reduce cell damage.