Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
ObjectiveTo investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis.MethodsBMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model (n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR.ResultsAt 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B (P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days (P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A (P<0.05).ConclusionSilencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
ObjectiveTo investigate the effect of granulocyte colony-stimulating factor (G-CSF) mobilizing the bone marrow mesenchymal stem cells (BMSCs) homing to the spinal cord injury sites in rats, and to evaluate the feasibility of G-CSF mobilizing the BMSCs home to the injured spinal cord. MethodsTwenty-four healthy adult female Sprague Dawley rats were injected with 1 mL green fluorescence protein labeled BMSCs (GFP-BMSCs, 1×106 cells/mL) into tail vein at 12 hours before operation. They were randomly divided into sham operation group (group A), sham operation+G-CSF group (group B), spinal cord injury group (group C), and spinal cord injury+G-CSF group (group D), with 6 rats in each group. In groups C and D, spinal cord injury model was established by T10 level spinal cord hemisection. In groups A and B, only laminectomy was performed without injury to the spinal cord. Groups B and D were injected with G-CSF (10 μg/kg·d) at 1 hour after operation for 3 consecutive days, and groups A and C were injected with the same amount of saline. The Basso-Beattie-Bresnahan (BBB) score was used to estimate the neurological function of rats and the expressions of tumor necrosis factor α (TNF-α) and stromal-derived factor 1 (SDF-1) were detected by ELISA method at 1, 3, 7, 14, 21, and 28 days after operation. The spinal cord samples of rats were sacrificed at 28 days after operation for immunohistochemical staining to observe the expression of cytokines, including SDF-1, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and TNF-α, and immunofluorescence staining to observe GFP-BMSCs positive cells, double-stained fluorescent yellow GFP/neuronal nuclear antigen (NeuN) positive neurons, and GFP/glial fibrillary acidic protein (GFAP) positive neurons. The number of glial cells and apoptosis were detected by TUNEL method. ResultsThe BBB score of groups A and B had no significant change at each time point after operation. At 1 day after operation, the BBB score of groups C and D decreased to the lowest level, and then gradually increased. The BBB score of group D was significantly higher than that of group C at all time points except 1 day after operation (P<0.05). At 3, 7, 14, 21, 28 days after operation, the levels of TNF-α and SDF-1 in groups C and D were significantly higher than those in groups A and B (P<0.05), but the levels of TNF-α in group D were significantly lower than those in group C at each time point, and the levels of SDF-1 were significantly higher than those in group C (P<0.05). Immunohistochemical staining showed that the expressions of SDF-1, BDNF, VEGF, and TNF-α in groups C and D were significantly higher than those in groups A and B (P<0.05); the expressions of SDF-1, BDNF, and VEGF in group D were significantly higher than those in group C, and the expression of TNF-α was significantly lower than that in group C (P<0.05). Immunofluorescence staining showed that the number of GFP-BMSCs, GFP/NeuN, and GFP/GFAP positive cells in groups C and D were significantly higher than those in groups A and B, and in group D than in group C (P<0.05). TUNEL assay showed that the number of apoptotic cells in groups C and D was significantly lower than that in groups A and B, and in group D than in group C (P<0.05). ConclusionG-CSF can mobilize BMSCs to the spinal cord injury site and promote repair effect by down-regulating TNF-α to promote the anti-apoptosis function and up-regulating SDF-1, BDNF, VEGF to promote BMSCs migration.
ObjectiveTo investigate the regulation of human bone marrow mesenchymal stem cells (hBMSCs) osteogenic and adipogenic differentiations mediated by Wnt10b adenoviral vector in vitro. MethodsThe hBMSCs from ilial bone tissue in adults at passage 4 were infected by Wnt10b gene expression adenoviral vector (group A), Wnt10b-shRNA adenoviral vector (group B), and empty vector (group C), and non-transfected hBMSCs served as the blank control group. Then the cells were cultured separately in the circumstance of osteogenic induction, adipogenic induction, and non-induction. The alkaline phosphatase (ALP) staining, alizarin red staining, and oil red O staining were used to detect the osteogenic and adipogenic differentiations; real-time fluorescent quantitative PCR and Western blot were used to analyze the expressions of osteoblast and adipocyte genes and proteins. ResultsThe results of ALP staining were positive after osteogenic induction, group A showed strong staining, and group B showed the weakest staining. The results of alizarin red staining showed that there were a lot of patchy confluent brown mineralized nodules in group A; a few punctate brown mineralized nodules were seen in group B; and many punctuate brown mineralized nodules were found in groups C and D. The results of oil red O staining showed strong staining in groups B, C, and D after adipogenic induction, especially in group B; scattered or small clustered staining was observed in group A. The expressions of osteoblast genes and proteins were significantly higher in group A than groups B, C, and D, and in groups C and D than group B by real-time fluorescent quantitative PCR and Western blot test; however, the expressions of adipocyte genes and proteins showed a contrary tendency. ConclusionThe high level expression of Wnt10b can enhance osteogenic differentiation of hBMSCs, and the low level expression of Wnt10b can increase adipogenic differentiation of hBMSCs.
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
This study aimed to comprehensively evaluate the biological activity in different passage populations of mesenchymal stem cells (BMSCs) derived from bone marrow in ovariectomy osteoporotic rats (named OVX-rBMSCs), providing experimental basis for new osteoporotic drug development and research. OVX-rBMSCs were isolated and cultured in vitro by the whole bone marrow adherent screening method. The morphological observation, cell surface markers (CD29, CD45, CD90) detection, cell proliferation, induced differentiation experimental detection were performed to evaluate the biological activity of Passage 1, 2, 3, 4 populations (P1, P2, P3, P4) OVX-rBMSCs. The results showed that whole bone marrow adherent culture method isolated and differentially subcultured OVX-The morphology of P4 OVX-rBMSCs was identical fibroblast-like and had the characteristics of ultrastructure of stem cells. The CD29 positive cells rate, CD90 positive cells rate, cell proliferation index, and the osteogenic, adipogenic, chondrogenic differentiation capacities of P4 OVX-rBMSCs were significantly better than those of other populations (P < 0.05). OVX-rBMSCs purity and biological activity were gradually optimized with the passaged, and among them P4 cells were superior to all the other populations. Based on these results, we report that the P4 OVX-rBMSCs model developed in this study can be used to develop a new and effective medical method for osteoporotic drug screening.
ObjectiveTo investigate whether transplantation of islet cells combined with bone marrow mesenchymal stem cell (BMSCs) of the pancreatic subcapsular promoting revascularization of pancreatic islets in rats, so as to reduce the loss of islet cells after transplantation and improve the success rate of islet cell transplantation. MethodsThe model of diabetic rat was established. The BMSCs and islet cells were cultured and identified, then the simple islet cells, simple BMSCs, and combination of islet cells and BMSCs were injected into the pancreatic subcapsular of the islet cell group, BMSCs group, and combination group, respectively. In addition, the same amount of normal saline was injected into the same site as the control group. There were 10 rats in each group. The changes of blood glucose and serum insulin in different time point were detected in each group. The mRNA expressions of angiogenesis factors such as hypoxia inducible factor-1α (HIF-1α), stromal cell derived factor 1α (SDF1α), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor 2 (FGF2) were determined by real-time quantitative PCR. Results① The blood glucose levels of the islet cell group and combination group were lower than those of the control group and the BMSCs group within 15 d after surgery (P<0.05) and decreased to the normal level, which of the combination group could still maintain the normal level until on day 29 (P<0.05), but which of the islet cell group began to increase on day 15 after surgery and was similar to that in the BMSCs group (P>0.05). ② Compared with the control group and the BMSCs group, the insulin levels were higher in the islet cell group and combination group on day 1, 3, 7, 15, and 29 after surgery (P<0.05), especially in the combination group. ③ The expression levels of HIF-1α, SDF1α, VEGF, and FGF2 mRNAs in the combination group were higher than those the other three groups, and the differences were statistically significant (P<0.05). ConclusionsIslet cell transplantation of pancreatic subcapsular could decrease blood glucose level in diabetic rats. Hypoglycemic effect of single islet cell transplantation gradually weakens on day 15 d after surgery. After BMSCs combined with islet cells transplantation, the glycemic effect of rat is stable for a longer time. Expressions of angiogenesis factors of BMSCs combined with islet cells transplantation rat are high, which combined with pathological sections suggests that BMSCs could promote vascular recanalization of islet transplantation.
ObjectiveTo investigate the effectiveness of autologous injectable platelet rich fibrin (i-PRF) combined with bone marrow mesenchymal stem cells (BMSCs) for sciatic nerve injury in rats.MethodsBMSCs were isolated and cultured from tibial bone marrow of Sprague Dawley (SD) neonatal rats aged 10-15 days and passaged to the 4th generation. i-PRF was prepared from posterior orbital venous blood of adult SD rats by improved low-speed centrifugation. Twenty-four adult SD rats were selected and randomly divided into 4 groups with 6 rats in each group after the sciatic nerve Ⅲ degree injury model was established by modified crush injury method. Groups A, B, C, and D were injected with BMSCs suspension+autologous i-PRF, autologous i-PRF, BMSCs suspension, and normal saline, respectively. The Basso-Beattie-Bresnahan (BBB) score was used to evaluate the recovery of neurological function of the affected limb of rats every week from 1 to 8 weeks after operation. At 2 months after operation, the rats were sacrificed and the histological changes of sciatic nerve were observed by HE staining. The microstructural changes of nerve fibers, myelin sheath, and nucleus were observed by transmission electron microscope. The expressions of N-cadherin, Nestin, and glial fibrillary acidic protein (GFAP) were detected by Western blot.ResultsNo immune rejection or death occurred in the rats after operation. There was no significant difference in BBB scores between groups at 1 week after operation (P>0.05); at 2-8 weeks after operation, BBB scores in group A were significantly higher than those in groups B, C, and D, and in groups B, C than in group D (P<0.05), there was no significant difference between groups B and C (P>0.05). HE staining showed that the nerve fibers in group A arranged in order, without defect or demyelination; the nerve fibers in group B were not clear and slightly swollen; some of the nerve fibers in group C were disordered and demyelinated; the nerve fibers in group D were not continuous, obviously demyelinated, and some of the nerve adventitia damaged. Transmission electron microscope showed that the structure of nerve fibers in group A was clear, myelin sheath was complete, and nucleus was dense; group B was slightly less than group A; group C had fuzzy structure, demyelination, and hollowing out; group D had disorder structure, demyelination, and hollowing out, and the middle part of nerve adventitia continuity. Western blot detection results showed that there was no significant difference in the relative expression of Nestin between groups (P>0.05). The relative expression of N-cadherin was significantly lower in groups B, C, and D than in group A, in groups C and D than in group B, and in group D than in group C (P<0.05). The relative expression of GFAP was significantly lower in groups B, C, and D than in group A, in group D than in groups B and C (P<0.05); there was no significant difference between groups B and C (P>0.05).ConclusionAutologous i-PRF combined with BMSCs can effectively treat sciatic nerve tissue injury in rats.
ObjectiveTo evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. ResultsThe isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). ConclusionThe prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.
Objective To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. Methods BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). Results CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic (P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group (P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group (P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group (P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant (P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP (P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group (P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group (P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group (P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased (P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group (P<0.05) except Tb.Th. Conclusion Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.