Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
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.
ObjectiveTo review the role of stem cell niches in maintaining cardiac stem cells homeostasis, and to foresee its prospects. MethodsThe literature on cardiac stem cells niches was extensively reviewed. The roles of stem cell niches components, extracellular matrix, and secretory factors in maintaining cardiac stem cell homeostasis were analysed and reviewed. ResultsLots of experiments reveal that stem cell niches are able to delay the aging of cardiac stem cells, protect from external damage, keep stem properties, and improve the purity and quantity. However, the mechanism is not fully understood. ConclusionThe stem cell niches have a very bright application prospect in homeostasis, purification, and amplification for the cardiac stem cells, and it needs further study.
Retinal degeneration mainly include age-related macular degeneration, retinitispigmentosa and Stargardt’s disease. Although its expression is slightly different, its pathogenesis is photoreceptor cells and/or retinal pigment epithelial (RPE) cel1 damage or degeneration. Because of the 1ack of self-repairing and renewal of retinal photoreceptor cells and RPE cells, cell replacement therapy is one of the most effective methods for treating such diseases.The stem cells currently used for the treatment of retinal degeneration include embryonicstem cells (ESC) and various adult stem cells, such as retinal stem cells (RSC), induced pluripotent stem cells (iPSC). and mesenchyma1 stem cells (MSC). Understanding the currentbasic and clinical application progress of ESC, iPSC, RSC, MSC can provide a new idea for the treatment of retinal degeneration.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To study the effect of core-binding factor α1(Cbfa1)on the mesenchymal stem cells(MSCs) osteoblastic differentiation.Methods The MSCs were isolated from Japan white rabbits and cultured in vitro. The 3rd generation MSCs were infected with Cbfa1 recombinant adenovirus. The expression of Cbfa1 was detected by immunofluorescence after being infected for 3 days and the proliferation was estimated by MTT method from the 1st day to the 7th day. Then the MSCs were divided into four groups: the commonly cultured group, the simply induced group, the control adenovirus treatment group, and the Cbfa1 adenovirus treatment group. The expressions of mRNA for a various of osteoblast gene markers such as alkaline phosphatase, osteocalcin, osteopontin and type I collagen were analyzed based on reverse transcriptase polymerase chain reaction (RT-PCR). The change of adipose and myoblastic differentiation gene marker PPARγ2 and MyoD expression were detected by RT-PCR respectively.Results Positive staining of Cbfa1 was found in the MSCs infected with Cbfa1 adenovirus, and there was no significant difference in cell proliferation among the experimental groups（Pgt;0.05）. The RT-PCR indicated that all the osteoblast gene markers except type I collagen were up-regulated in the Cbfa1 adenovirus treatment group. In contrast, the expressions of PPARγ2 and MyoD were restrained. Conclusion Cbfa1 can directly promote the differentiation of MSCs into osteoblasts.
Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
ObjectiveTo summarize the research progress of mesenchymal stem cells in acute pancreatitis animal models, and to explore the potential of mesenchymal stem cells in the treatment of acute pancreatitis.MethodWecollected domestic and foreign studies on mesenchymal stem cells in acute pancreatitis animal models and made a review.ResultsIn the animal model of acute pancreatitis, the infusion of mesenchymal stem cells could reduce the expression of inflammatory factors, regulate the expression of immune cells, inhibit pancreatic cell apoptosis and autophagy, resist oxidative stress, and promote angiogenesis. Mesenchymal stem cells had a relieving effect on acute pancreatitis.ConclusionThe infusion of mesenchymal stem cells can relieve acute pancreatitis in animal models, and has great potential in the clinical application of acute pancreatitis.