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.
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 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 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.
Objective To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Methods Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. ResultsThere were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that \begin{document}$\hat y $\end{document}=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. Conclusion The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.
ObjectiveTo review the related studies on the application of nanomaterials in the treatment of osteomyelitis, and to provide new ideas for the research and clinical treatment of osteomyelitis.MethodsThe literature about the treatment of osteomyelitis with nanomaterials at home and abroad in recent years was reviewed and analyzed.ResultsAt present, surgical treatment and antibiotic application are the main treatment options for osteomyelitis. But there are many defects such as antibiotic resistance, residual bone defect, and low effective concentration of local drugs. The application of nanomaterials can make up for the above defects. In recent years, nanomaterials play an important role in the treatment of osteomyelitis by filling bone defects, establishing local drug delivery system, and self-antibacterial properties.ConclusionIt will provide a new idea and an important research direction for the treatment of osteomyelitis to fully study the related characteristics of nanomaterials and select beneficial materials to make drug delivery system or substitute drugs.
Objective To investigate the effects of titanium modified by ultrasonic acid etching/anodic oxidation (UAT) loaded with endothelial progenitor cells-exosome (EPCs-exo) on proliferation and osteogenic and angiogenic differentiations of adipose-derived stem cells (ADSCs). Methods The adipose tissue and bone marrow of 10 Sprague Dawley rats were harvested. Then the ADSCs and EPCs were isolated and cultured by collagenase digestion method and density gradient centrifugation method, respectively, and identified by flow cytometry. Exo was extracted from the 3rd to 5th generation EPCs using extraction kit, and CD9 and CD81 were detected by Western blot for identification. The three-dimensional printed titanium was modified by ultrasonic acid etching and anodic oxidation to prepare the UAT. The surface characteristics of UAT before and after modification was observed by scanning electron microscopy; UAT was placed in EPCs-exo solutions of different concentrations (100, 200 ng/mL), and the in vitro absorption and release capacity of EPCs-exo was detected by BCA method. Then, UAT was placed in DMEM medium containing different concentrations of EPCs-exo (0, 100, 200 ng/mL), and co-cultured with the 3rd generation ADSCs to construct UAT-ADSCs-exo. Cell morphology by laser confocal microscopy, live/dead cell staining, and cell proliferation were observed to evaluate biocompatibility; alkaline phosphatase (ALP) staining and alizarin red staining, RT-PCR detection of osteogenesis-related genes [osteocalcin (OCN), RUNT-related transcription factor 2 (Runx2), ALP, collagen type 1 (COL-1)] and angiogenesis-related gene [vascular endothelial growth factor (VEGF)], immunofluorescence staining for osteogenesis (OCN)- and angiogenesis (VEGF)-related protein expression were detected to evaluate the effect on the osteogenic and angiogenic differentiation ability of ADSCs. Results Scanning electron microscopy showed that micro-nano multilevel composite structures were formed on the surface of UAT. About 77% EPCs-exo was absorbed by UAT within 48 hours, while EPCs-exo absorbed on the surface of UAT showed continuous and stable release within 8 days. The absorption and release amount of 200 ng/mL group were significantly higher than those of 100 ng/mL group (P<0.05). Biocompatibility test showed that the cells in all concentration groups grew well after culture, and the 200 ng/mL group was better than the other groups, with fully spread cells and abundant pseudopodia, and the cell count and cell activity were significantly higher than those in the other groups (P<0.05). Compared with the other groups, 200 ng/mL group showed enhanced ALP activity and mineralization ability, increased expressions of osteogenic and angiogenic genes (OCN, Runx2, COL-1, ALP, and VEGF), as well as increased expressions of OCN and VEGF proteins, with significant differences (P<0.05). Conclusion EPCs-exo can effectively promote the adhesion, proliferation, and osteogenic and angiogenic differentiation of ADSCs on UAT surface, the effect is the most significant when the concentration is 200 ng/mL.
Objective To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells. MethodsThe 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein. ResultsThe CCK-8 assay showed that the absorbance (A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups (P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture (P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A (P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A (P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups (P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited (P<0.05). There was no significant difference between groups B and C and between groups D and E (P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups (P<0.05). Conclusion MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.
Objective To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased (P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day (P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points (P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity (P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased (P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups (P>0.05). Conclusion Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.