Objective Construction of viable tissue engineered bone is one of the most important research fields in the cl inical appl ication of bone tissue engineering, to investigate the function of nerve factors in bone tissue engineering by celldetection in vitro and construction of neurotization tissue engineered bone in vivo. Methods Fifty-four healthy New Zealandwhite rabbits, male or female, weighing 2-3 kg, were involved in this study. Bone marrow mesenchymal stem cells (BMSCs) from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates, using the LG-DMEM complete medium as control. The prol iferation and osteogenic differentiation of the cells were observed and tested by the MTT assay, alkal ine phosphatase (ALP) stain, and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in β tricalcium phosphate (β-TCP) biomaterial scaffold and cultured for 72 hours, then the β-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups (n=18): sensory nerve bundle (group A) or motor nerve bundle (group B) were transplanted into the side groove of β-TCP scaffold, group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th, 8th, and 12th weeks after operation.
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To explore the effect of age and gene therapyon the differentiation of marrow mesenchymal stem cells （MSCs） of the rats. Methods MSCs from the young (1-month-old), adult (9-month-old), and the aged(24monthold) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase(ALP), collagen Ⅰ(Col Ⅰ), bone sialoprotein(BSP) and osteopontin(OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. Results There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col-Ⅰ, OPN and BSP amongthe 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice amongthe 3 groups. Conclusion MSCs obtained from the aged ratscan restore their osteogenic activity following human BMP-2 gene transduction, therefore provides an alternative to treating the aged bone disease.
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.
Objective To monitor the stem cell migration into the bone defect following an injection of the labeled mesenchymal stem cells (MSCs) by the enha nced green fluorescent protein (EGFP)technology and to provide insights into an application of MSCs for the fracture healing. Methods Isolated MSCs from the rabbit femur marrow were culture-expanded and were labeled by the transfection with the recombinant retrovirus containing the EGFP gene. Then, some labeled MSCs were cultured under the osteogenic differentiation condition and the phenotype was examined. After the fracture of their bilateral ulna, 18 rabbits were divide d into two groups. The labeled MSCs were injected into the aural vein at 1×107 cells／kg in the experimental group and the unmarked MSCs were injected in the control group 24 hours before surgery, and 1 and 24 hours after surgery, res pectively. Necropsies were performed 2 days after surgery in the two groups. The sections from the left defects were observed under the fluorescence microscope and the others were analyzed by the bright-field microscopy after the HE staining. Results The EGFP did not affect the MSCs viability. After the labeled cells were incubated in the osteogenic medium alkaline phosphatase, the calcium nodule s were observed. All the rabbits survived. The tissue of haematoma was observed in the bone defects and the fluorescent cells were found in the experimental gr oup, but no fluorescent cells existed in the control group. Conclusion The EG FP labeled MSCs can undergo osteogenic differentiation in vitro and can mig rate into bone defects after their being injected into the peripheral vein.
Objective To review the research appl ication and advance of synovium-derived mesenchymal stem cells (SMSCs) in tissue engineering. Methods The recent related l iterature was reviewed, concerning isolation method, characteristics of SMSCs, and its appl ication in tissue engineering. Results SMSCs are multi potent cell population with characteristics of easy isolation and high prol iferation, which have been appl icated in the cartilage, tendon, l igament, and bone tissue engineering. Conclusion SMSCs is a new member of mesenchymal stem cells family. It appears to be promising seedcells for tissue engineering, but further research is needed.
ObjectiveTo construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs.MethodsBMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy.ResultsBMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system (P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well.ConclusioniPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
Objective To investigate the effect of notochordal cells (NCs) conditioned medium (NCCM) on theprol iferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods NCs and BMSCs wereisolated from the thoracolumbar intervertebral disc and the femurs of 4-week-old Japanese white rabbits, respectively. NCswere cultured with DMEM/F12 medium containing 15% FBS for 5 days to prepare NCCM. The experiment consisted of2 groups: BMSCs were cultured with NCCM in experimental group and with DMEM/F12 medium containing 15% FBSin control group. The prol iferation of BMSCs was assessed by cell counting kit-8 at 1, 3, 5, 7, 9, and 14 days after culture;the expression of proteoglycan and collagen type II were determined by immunofluorescence and real-time fluorescentquantitative PCR at 7 and 14 days after culture. Results NCs and BMSCs were successfully isloated. At 5, 7, 9, and 14days, the number of BMSCs in the experimental group was significantly more than those in the control group (P lt; 0.05).At 7 and 14 days, there was no expression or less expression of proteoglycan and collagen type II in the control group;however, there was a lot of expression of proteoglycan and collagen type II in the experimental group, and the expressionswere higher at 14 days than at 7 days. At 7 and 14 days after culture, the mRNA expressions of proteoglycan and collagentype II were significantly higher in the experimental group than in the control group (P lt; 0.05), and at 14 days than at 7days in the experimental group (P lt; 0.05). Conclusion NCCM can promote the prol iferation and the differentiation ofBMSCs into chondroyte-like cells, which provides the basis for NCs and BMSCs as seed cells in the treatment of degenerativedisc disease.
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.
Objective To investigate the dose-dependent relationship of bone marrow mesenchymal stem cells(MSCs) transplantation in improving ischemic myocardial dysfunction? in a rat ischemic heart model. Methods Myocardial infarction was induced in 32 inbred F344 rats by acute ligation of the left anterior descending(LAD) coronary artery. One week after ligation, the ratswere randomized? into four equal groups, with eight rats in each group. Equal volume Iscove’s modified Dulbecco’s medium was injected in the control group, 1×103(group 1), 1×105(group 2), and 1×107(group 3) 5-bromodeoxyuridine (BrdU) labeled bone marrow MSCs were injected into the infarcted myocardium. Cardiac function was evaluated by ultrasound before the ligation of the LAD, before the transplantation and the 4th week after transplantation. The expressions of BrdU,Connexin43,Myosin heavy chain β(MHC), and smooth muscle actin α(α-SMA) were detected by immunofluorescence and immunohistochemistry at the 4th week after transplantation. The amount of functional vessels stained by α-SMA was counted simultaneously. Results At the 4th week? after transplantation, the ejection fraction(EF) in goup 2 was more significantly improved than that in group1(0.54±0.20 vs. 0.34±0.16, P＝0.004) and EF in group 3 was more significantly improved than that in group 2(0.71±0.24 vs. 0.54±0.20,P＝0.018), whereas no significant difference between group 1 and control group was detected (0.34±0.16 vs. 0.36±0.15,Pgt;0.05). The BrdU labeled MSCs could be found in host myocardium. The number of cells in group 2 by double staining both for BrdU and for MHC observed in ischemic myocardium were significantly more than that in group 1? (323.20±91.62 n/HP vs. 51.75±27.58 n/HP,P＝0.049) and the same was true between group 3 and group 2(409.75±106.65 n/HP vs. 323.20±91.62 n/HP,Plt;0.001), whereas the result of control group was negative.The majority of transplanted cells were found positive staining both for MHC and for Connexin43 in all groups. There were lots of positive staining of α-SMA whose form were partly irregular in ischemic myocardium indicating that there was neovascularization in group1 and control group. More neovascularization in group2 was found than that in group 1 (28.38±12.79 n/HP vs. 22.75±9.07 n/HP, P＝0015) and more neovascularization in group 3 was found? than that in group 2 (35.63±13.27 n/HP vs. 28.38±12.79 n/HP, P＝0.002) . Conclusion Transplanted into infarcted myocardium, bone marrow MSCs may have significant and dose-dependent potential for cardiomyogenesis with functional recovery from myocardial ischemia.