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.
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.
ObjectiveTo explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.
ObjectiveTo summarize the mechanism of long non-coding RNA (lncRNA) in signal pathways related to osteogenic differentiation. Methods Relevant domestic and foreign researches in recent years were consulted. The characteristics and biological functions of lncRNA were introduced, and the specific mechanism of lncRNA regulating related signal pathways in osteogenic differentiation was elaborated. Results The exertion and maintenance of normal function of bone requires the closed coordination of transcription networks and signal pathways. However, most of these signal pathways or networks are dysregulated under pathological conditions that affect bone homeostasis. lncRNA can regulate the differentiation of various bone cells by activating or inhibiting signal pathways to achieve the balance of bone homeostasis, thereby reversing the pathological state of bones and achieving the purpose of treating bone metabolic diseases. Conclusion At present, the research on the mechanism of lncRNA regulating various osteogenic differentiation pathways is still in the early stage. Its in-depth regulator mechanism, especially the cross-talk of complex signal pathways needs to be further studied. And how to apply these molecular targets to clinical treatment is also a big challenge.
ObjectiveTo investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs).MethodsThe fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group.ResultsMEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs (P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs (P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I (P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days (P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo.ConclusionThe combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.
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.
Chit-oligosaccharide (COS) is a low-molecular, water-soluble mass with higher biological activity, which can be absorbed by human body easily and interact with cells directly. Based on the excellent biological properties of collagen (Col) and COS, a series of Col and COS composite hydrogel (Col/COSn) was constructed in this study. The effect of composite hydrogel on cells proliferation, differentiation and related osteogenic gene expression was evaluated on pre-osteoblast MC3T3-E1s. The experimental results showed that all the Col/COS composite gels could promote the growth of MC3T3-E1s, proliferation and bone related gene expression compared to that of pure Col gels. And there was significant difference among the composite hydrogel groups with different degrees of polymerization of COS. The effect of the composite gel which contained chitotetraose (COS4) or chitohexaose (COS6) on the cells proliferation was better than that of other groups, while on cells differentiation and related osteogenic gene expression the composite gel contained chitopentaose (COS5) was the best in all the groups.
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 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 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.