Objective
To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs).
Methods
The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg (control group), 75 mm Hg (low negative pressure group), and 150 mm Hg (high negative pressure group) (1 mm Hg=0.133 kPa), and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase (ALP) activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I (COL-I) and osteocalcin (OC) in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot.
Results
The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group (P<0.05), but there was no significant difference between the low negative pressure group and the control group (P>0.05); at 5-7 days, the cell number showed significant difference between 3 groups (P<0.05). The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction (P>0.05); the ALP activity showed significant difference (P<0.05) between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction (P<0.05). The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group (P<0.05), and in the high negative pressure group than the low negative pressure group (P<0.05).
Conclusion
With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.
Objective To summarize the research progress of bioactive scaffolds in the repair and regeneration of osteoporotic bone defects. Methods Recent literature on bioactive scaffolds for the repair of osteoporotic bone defects was reviewed to summarize various types of bioactive scaffolds and their associated repair methods. Results The application of bioactive scaffolds provides a new idea for the repair and regeneration of osteoporotic bone defects. For example, calcium phosphate ceramics scaffolds, hydrogel scaffolds, three-dimensional (3D)-printed biological scaffolds, metal scaffolds, as well as polymer material scaffolds and bone organoids, have all demonstrated good bone repair-promoting effects. However, in the pathological bone microenvironment of osteoporosis, the function of single-material scaffolds to promote bone regeneration is insufficient. Therefore, the design of bioactive scaffolds must consider multiple factors, including material biocompatibility, mechanical properties, bioactivity, bone conductivity, and osteogenic induction. Furthermore, physical and chemical surface modifications, along with advanced biotechnological approaches, can help to improve the osteogenic microenvironment and promote the differentiation of bone cells. ConclusionWith advancements in technology, the synergistic application of 3D bioprinting, bone organoids technologies, and advanced biotechnologies holds promise for providing more efficient bioactive scaffolds for the repair and regeneration of osteoporotic bone defects.
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.
ObjectiveTo study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity.MethodsIsoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR).ResultsCCK-8 assay showed that the absorbance (A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D (P>0.05), but there were significant differences between other groups (P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points (P<0.05).Conclusion3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.
ObjectiveTo investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot.ResultsCompared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased (P<0.05); the above indexes in group D were significantly decreased (P<0.05); the above indexes between groups C, E and group A were not significantly different (P>0.05).ConclusionmiR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.
This study investigated the early mechanical adaptability and osteogenic differentiation of mouse bone marrow mesenchymal stem cells (M-BMSCs) under micro-vibration stimulation (MVS). M-BMSCs were stimulated by MVS in vitro, cell proliferation, alkaline phosphatase (ALP) activity assay, and cytoskeleton were measured, and cell apoptosis was observed by flow cytometry. Early osteoblast-associated genes, runt-related transcription factor 2 (Runx2), Collagen Ⅰ (Col-Ⅰ) and ALP, were observed by RT-PCR and the activation of extracellular regulated protein kinases 1/2 (ERK1/2) was determined by Western blotting. The results showed that MVS had no significant effect on the proliferation of M-BMSCs. The early apoptosis was induced by mechanical stimulation (for one day), but the apoptosis was decreased after cyclic stimulation for 3 days. At the same time, MVS significantly accelerated the expression of F-actin protein in cytoskeleton, the synthesis of ALP and the ERK1/2 pathway, also up-regulated the expressions of Runx2, Col-Ⅰ and ALP genes. This study indicates that MVS could regulate cellular activity, alter early adaptive structure and finally promote the early osteogenic differentiation of M-BMSCs.
Objective To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice. Methods Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. Results On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group (P<0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were significantly lower than that at 0 day (P<0.05); then, the relative expressions at 2 and 3 days significantly increased, showing no significant difference when compared with that at 0 day (P>0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day (P<0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group (P<0.05) and higher in mimics group than in NC mimics group (P<0.05). Conclusion Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.
ObjectiveTo explore the effect and mechanism of miR-21 down-regulated which was induced by H2O2 on osteogenic differentiation of MC3T3-E1 cells.MethodsMC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H2O2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H2O2 was obtained. To analyze the effect of H2O2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H2O2 group (group B), osteogenic induction group (group C), and H2O2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H2O2 group (group A1), H2O2+osteogenic induction group (group B1), H2O2+osteogenic induction+miR-21 inhibitor group (group C1), and H2O2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H2O2 group (group A2), H2O2+osteogenic induction group (group B2), H2O2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H2O2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining.ResultsThe results of MTS and RT-PCR showed that the appropriate concentration of H2O2 was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks (P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D (P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 (P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks (P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks (P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week (P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2.ConclusionH2O2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.
ObjectiveTo explore the effects of concentrated growth factor (CGF) combined with mineralized collagen (MC) materials on the adhesion, proliferation, and differentiation of bone marrow mesenchymal stem cells (BMSCs) and their osteogenic effects in vivo, and to provide a theoretical basis for the combined application of CGF and MC materials in bone defect regeneration and repair.MethodsCGF was prepared from venous blood of healthy volunteers, and then CGF extracts (CGFe) were prepared. In vitro experiment: human BMSCs (hBMSCs) were divided into 4 groups. Groups A, B, and C were cultured with α-MEM medium [containing 10% fetal bovine serum (FBS) and 1% double antibody] containing 2%, 5%, and 10%CGFe, respectively; group D was cultured with α-MEM medium (containing 10%FBS and 1% double antibody) without CGFe. Scanning electron microscopy was used to observe the effect of CGFe on cell adhesion. Cell counting kit 8 (CCK-8) was used to detect the effect of CGFe on cell proliferation. After osteogenic induction, alkaline phosphatase (ALP) activity was detected and Western blot was performed to detect osteopontin (OPN) expression. In vivo experiment: Eighteen New Zealand big-eared rabbits were used to prepare circular bone defect models on the left and right mandibles, and implant CGF gel (prepared from autologous venous blood)+MC material (volume ratio 1∶1, experimental group) and simple MC material (control group), respectively. At 4, 8, and 12 weeks after operation, 6 rabbits were sacrificed respectively to obtain materials, and Micro-CT scanning was performed to observe the formation of new bone and material degradation in vivo.ResultsIn vitro experiments: Scanning electron microscopy showed that the cells of groups A, B, and C spread better on MC materials than group D, with more pseudopodia. CCK-8 method showed that different concentrations of CGFe could promote cell proliferation, and the absorbance (A) value of cells cultured for 2, 3, 5, and 7 days was in the order of group C>group B>group A>group D, the differences were significant (P<0.05). ALP activity test showed that its activity was proportional to the osteogenic induction time and CGFe concentration (P<0.05). Western blot analysis of osteogenic induction culture for 14 days showed that the relative expression of OPN protein in groups A, B, and C was significantly higher than that in group D, and the higher the CGFe concentration, the higher the relative expression of OPN protein (P<0.05). In vivo experiment: Micro-CT observation showed that the new bone formation and material degradation of the experimental group were better than those of the control group at 4, 8, and 12 weeks after operation. Quantitative detection showed that the volume of new bone volume, new bone volume fraction, trabeculae number, and trabecular thickness of the experimental group were significantly higher than those of the control group at each time point, the residual material volume, residual material volume fraction, and trabecular separation were significantly lower than those of the control group, all showing significant differences (P<0.05).ConclusionCGF can effectively promote the adhesion, proliferation, and osteogenic differentiation of BMSCs on MC materials, and 10%CGFe has the most significant effect. The combined application of CGF and MC material can significantly promote bone formation in vivo.
ObjectiveTo prepare a bone tissue engineering scaffold for repairing the skull defect of Sprague Dawley (SD) rats by combining exogenous transforming growth factor β1 (TGF-β1) with gelatin methacryloyl (GelMA) hydrogel.MethodsFirstly, GelMA hydrogel composite scaffolds containing exogenous TGF-β1 at concentrations of 0, 150, 300, 600, 900, and 1 200 ng/mL (set to groups A, B, C, D, E, and F, respectively) were prepared. Cell counting kit 8 (CCK-8) method was used to detect the effect of composite scaffold on the proliferation of bone marrow mesenchymal stem cells (BMSCs) in SD rats. ALP staining, alizarin red staining, osteocalcin (OCN) immunofluorescence staining, and Western blot were used to explore the effect of scaffolds on osteogenic differentiation of BMSCs, and the optimal concentration of TGF-β1/GelMA scaffold was selected. Thirty-six 8-week-old SD rats were taken to prepare a 5 mm diameter skull bone defect model and randomly divided into 3 groups, namely the control group, the GelMA group, and the GelMA+TGF-β1 group (using the optimal concentration of TGF-β1/GelMA scaffold). The rats were sacrificed at 4 and 8 weeks after operation, and micro-CT, HE staining, and OCN immunohistochemistry staining were performed to observe the repair effect of skull defects.ResultsThe CCK-8 method showed that the TGF-β1/GelMA scaffolds in each group had a promoting effect on the proliferation of BMSCs. Group D had the strongest effect, and the cell activity was significantly higher than that of the other groups (P<0.05). The results of ALP staining, alizarin red staining, OCN immunofluorescence staining, and Western blot showed that the percentage of ALP positive area, the percentage of alizarin red positive area, and the relative expressions of ALP and OCN proteins in group D were significantly higher than those of the other groups (P<0.05), the osteogenesis effect in group D was the strongest. Therefore, in vitro experiments screened out the optimal concentration of TGF-β1/GelMA scaffold to be 600 ng/mL. Micro-CT, HE staining, and OCN immunohistochemistry staining of rat skull defect repair experiments showed that the new bone tissue and bone volume/tissue volume ratio in the TGF-β1+GelMA group were significantly higher than those in the GelMA group and control group at 4 and 8 weeks after operation (P<0.05).ConclusionThe TGF-β1/GelMA scaffold with a concentration of 600 ng/mL can significantly promote the osteogenic differentiation of BMSCs, can significantly promote bone regeneration at the skull defect, and can be used as a bioactive material for bone tissue regeneration.
