ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
ObjectiveTo evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection. MethodsBMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO[green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor). IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPMI 1640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. ResultsThe IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P<0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P<0.05); there was no significant difference between experimental group 4 and the positive control group (P>0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P<0.05) except experimental group 4 (P>0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5±0.6) days in group A, (14.5±0.8) days in group B, and (9.0±0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P<0.05). ConclusionIDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.
Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCRto detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
ObjectiveTo review the research progress of induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) transfected by double-gene. MethodsThe recent literature concerning the comparative research of induced osteogenesis of BMSCs transfected by double-gene was extensively reviewed. The characteristics of BMSCs, the advantage and effect of synergistic inductive osteogenesis, the application prospect and problems of BMSCs transfected by double-gene were summarized. ResultsThe effect of induced osteogenesis concerning BMSCs transfected by double-gene is far superior to single gene transfection and the activity of osteoblast is also significantly increased. The research used in bone tissue engineering experiment also obtain good effect. ConclusionInduced osteogenesis of BMSCs transfected by double-gene is able to make up for the lack of a single gene transfection and has great development prospects in the orthopaedic field.
Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
ObjectiveTo investigate the anti-apoptotic ability of synovium-derived mesenchymal stem cells (SMSCs) by comparing the apoptosis induced by tumor necrosis factor α (TNF-α) between SMSCs and bone marrow mesenchymal stem cells (BMSCs). MethodSMSCs and BMSCs were isolated with tissue adhering and density gradient centrifugation respectively, and cells at passages 3-5 were used in further experiments. After immunophenotype identification and differentiation induction, cells were divided into 4 groups. In the experimental groups, apoptosis of SMSCs and BMSCs were induced by 20 ng/mL TNF-α and 10 μg/mL cycloheximide, and cells were cultured in normal culture medium in the control groups. Cellular morphology were observed by inverted phase contrast microscope. After apoptosis induction for 24 hours, cell viability was determined by cell counting kit 8 assay and apoptotic index was detected by flow cytometer. Moreover, the level of Cleaved Caspase-8, 3 were determined by Western blot. ResultsBoth SMSCs and BMSCs accorded with the definition criteria of MSCs according to results of immunophenotype identification and differentiation induction. After apoptosis induction, cells became shrinking and partially floated and cellular morphologies became worse than those in the control groups. After apoptosis induction for 24 hours, cell viabilities of SMSCs and BMSCs in the control groups were both 100%, and no apoptotic cells were observed. However, cell viabilities of SMSCs and BMSCs in the experimental groups were 60.13%±8.63% and 46.55%±10.54% respectively, which were both significantly lower than those in the control groups (P<0.05) , and cell viability in the SMSCs experimental group was significantly higher than that in the BMSCs experimental group (t=3.152, P=0.006) . The apoptotic index was 36.54%±8.63% in the SMSCs experimental group and was 53.77%±11.52% in the BMSCs experimental group, both were significantly higher than the control groups (1.12%±0.24% and 1.35%±0.31%) (P<0.05) . What's more, it was significantly lower in SMSCs experimental group than that in BMSCs experimental group (t=3.785, P=0.001) . Moreover, no expression of Cleaved Caspase-8, 3 was detected in the control groups. But the levels of Cleaved Caspase-8, 3 were significantly enhanced in the experimental groups and they were lower in SMSCs than in BMSCs (t=13.870, P=0.000; t=7.309, P=0.000) . ConclusionsTNF-α induced apoptosis is lower in SMSCs than in BMSCs, which means that SMSCs may have stronger anti-apoptosis ability than BMSCs.
ObjectiveTo evaluate the effect of tissue engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) as supporting scaffolds of tissue engineering periosteum. MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured from 1-month-old New Zealand Rabbit and osteogenetically induced into osteoblasts. Porcine small intestinal submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then the adhesion of cells to scaffolds was observed by scanning electron microscope (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineered periosteum/DPB complex was constructed by tissue engineered periosteum and DPB. Tissue engineered periosteum was "coat-like" package the DPB, and bundled with absorbable sutures. Forty-eight New Zealand white rabbits (4-month-old) were randomly divided into 4 groups (groups A, B, C, and D, n=12). The bone defect model of 3.5 cm in length in the left radius was created. Defect was repaired with tissue engineered periosteum in group A, with DPB in group B, with tissue engineered periosteum/DPB in group C; defect was untreated in group D. At 4, 8, and 12 weeks after operation, 4 rabbits in each group were observed by X-ray. At 8 weeks after operation, 4 rabbits of each group were randomly sacrificed for histological examination. ResultsSEM observation showed that abundant seeding cells adhered to tissue engineered periosteum. At 4, 8, and 12 weeks after operation, X-ray films showed the newly formed bone was much more in groups A and C than groups B and D. The X-ray film score were significantly higher in groups A and C than in groups B and D, in group A than in group C, and in group B than in group D (P<0.05). Histological staining indicated that there was a lot of newly formed bone in the defect space in group A, with abundant newly formed vessels and medullary cavity. While in group B, the defect space filled with the DPB, the degradation of DPB was not obvious. In group C, there was a lot of newly formed bone in the defect space, island-like DPB and obvious DPB degradation were seen in newly formed bone. In group D, the defect space only replaced by some connective tissue. ConclusionTissue engineered periosteum constructed by SIS and BMSCs has the feasibility to repair the large diaphysis defect in rabbit. DPB isn't an ideal support scaffold of tissue engineering periosteum, the supporting scaffolds of tissue engineered periosteum need further exploration.
Objective To investigate the effects of allogenic transplantation of acellular muscle bioscaffolds (AMBS) seeded with bone marrow mesenchymal stem cells (BMSCs) on the repair of acute hemi-transection spinal cord injury (SCI) in rats. Methods AMBS were prepared by reformed chemical approach and sterilized by compound cold sterilization; BMSCs were harvested by density gradient centrifugation and cultured with adherent method. The 3rd generation BMSCs labeled by Hoechst 33342 were injected into AMBS to construct the BMSCs-AMBS composite scaffolds; the biocompatibility was observed under scanning electron microscope (SEM) and fluorescence microscope in vitro at 14 days. Forty-eight adult female Sprague Dawley rats were used to build SCI model by hemi-transecting at T9-11 level, then randomly divided into 4 groups (n=12). Defects were repaired with BMSCs-AMBS composite scaffolds, BMSCs, and AMBS in groups A, B, and C, respectively; group D was blank control by injecting PBS. At 1, 2, 3, and 4 weeks after surgery, the functional recovery of the hind limbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. At 4 weeks after surgery, HE staining and immunofluorescent assay were adopted. Results Masson staining and HE staining showed that AMBS was mainly of the collagen fibers in parallel arrange, without muscle fibers. After 14 days of BMSCs and AMBS co-culture, a large number of survival BMSCs labeled by Hoechst 33342 were seen under fluorescence microscope; SEM showed that BMSCs grew and attached to the inner surfaces of AMBS. At 2-4 weeks, the BBB score in group A was significantly higher than that in groups B, C, and D (P lt; 0.05), and it was significantly lower in group D than in the other 3 groups (P lt; 0.05); at 4 weeks, the BBB score in group B was significantly higher than that in group C (t=10.352, P=0.000). HE staining revealed that the area of spinal cord cavity after SCI was markedly smaller in group A than in the other 3 groups; immunofluorescent assay showed that more neurofilament 200 positive fibers and Nestin positive cells were detected in group A than in groups B, C, and D, but glial fibrillary acidic protein (GFAP) positive cells significantly decreased. The integral absorbance (IA) values of GFAP were 733.01 ± 202.04, 926.42 ± 59.46, 1 069.37 ± 33.42, and 1 469.46 ± 160.53 in groups A, B, C, and D, respectively; the IA value of group A was significantly lower than that of groups B, C, and D (P lt; 0.05), and it was significantly higher in group D than in groups A, B, and C (P lt; 0.05). Conclusion With relatively regular internal structures and good biocompatibility, AMBS can inhibit glial scar and enhance the survival, migration, and differentiation of BMSCs, so AMBS is the ideal nature vector for cell transplantation. Co-transplantation of AMBS and BMSCs has synergistic effect in treating SCI, it can promote rat motor function recovery.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.