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 explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
OBJECTIVE: To isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI). METHODS: BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation. RESULTS: Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days. CONCLUSION: Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
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 study the adhesion characteristic in vitrobetween porous biphasic calcium phosphate(BCP) nanocomposite and bone marrow mesenchymal stem cells (MSCs) that have been induced and proliferated. Methods MSCs obtained from SD ratbone marrow were in vitro induced and proliferated. After their osteoblastic phenotype were demonstrated, MSCs were seeded onto prepared porous BCP nanocomposite(experiment group)and common porous hydroxyapatite (control group). Their adhesion situation was analyzed by scanning electron microscope. The initial optimal cell seeding density was investigated between new pattern porous BCP nanocomposite and MSCs by MTT automated colormetric microassay method. Results The differentiation of MSCs to osteoblastic phenotype were demonstrated by the positive staining of mineralized node, alkaline phosphatase (ALP) and collagen typeⅠ, the most appropriate seeding density between them was 2×106／ml. The maximal number which MSCs could adhere to porous BCP nanocomposite was 1.28×107/cm3. Conclusion MSCs can differentiate to osteoblastic phenotype.The MSCs were well adhered to porous BCP nanocomposite.
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 investigate effects of the autologous bone mesenchymal stem cells (MSCs) enriched by the small intestinal submucosa (SIS) film implantation on the myocardial structure, cardiac function, and compensator y circulation after myocardial infarction in the goats. Methods Sixteen black goats were selected and divided randomly into the control group (n=8)and the experimental group (n=8). The chronic myocardial infarction models were made by the ligation of the far end of the left anterior desc ending coronary artery. At the same time, MSCs were aspired from the thigh bone of the goats in the experimental group. MSCs were isolated by the centrifu gation through a percoll step gradient and purified by the plating culture and depletion of the non-adherent cells. Primary MSCs were cultured in the DMEM me dium supplemented with the fetal bovine serum in vitro. After that, the cultures were labeled by 5- BrdU. The active cells were transplanted into the SIS film. Six weeks after the ligation, the MSCs-SIS film was implanted by its being sutured onto the infarction area; whereas, the control group underwent a shamoperation. In both groups, echocardiographic measurements were performed before infarction, 6 weeks after infarction and 6 weeks after the MSC-collagen mplantion, respectively, to assess the myocardial structure and ca rdiac function. The left coronary artery angiography was performed with the digi tal subtraction angiography. Results In an assessment of the left ventricular function, at 6 weeks after operation, t he stroke volume and the ejection fraction of the control group and the experim ental group were 42.81±4.91, 37.06±4.75 ml and 59.20%±5.41%, 44.56%±4.23%, respectively (Plt;0.05). The enddisatolic volume and the endsystolic volume of the control group and the experimental group were 72.55±8.13, 83.31±8.61 ml and 29.75±5.98, 46.25±6.68 ml, respectively (Plt;0.05). The maximal velocity of peak E of contral group and experimental group were 54.8 5±6.35 cm/s and 43.14±4.81cm/s (Plt;0.01); and the maximal velocity of peak A o f control group and experimental grouop were 52.33±6.65 cm/s and 56.91±6.34 cm/s (Pgt;0.05). Echocowdiogr aphy sho wing a distinctly dilatation of left ventricle with the ventricular dyskinesia i n contral group, but without the ventricular dyskinesia in experimental group. T he selective-coronary evngiography revealed that the obvious compensatory circu l ation established between the anterior descending branch and the left circumflex branch in the experimental group. Conclusion Implantation of the autologus MSCs enriched by the SIS film can prevent dilatation of the left ventricular chamber and can improve the contractile ability of the myocardium, cardiac function, and collateral perfusion.
Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
Objective To investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs）and to evaluate the osteoinductivity and dosedependence of the BMP-2 derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 μg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP）and the amount of calciumdeposition were meassured at 5，10，15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type Ⅰ, osteopontin (OPN), and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（Plt;0.05）. However,there was no significant difference between Group A and Group B （Pgt;0.05）. Theresult of FQPCR showed that after MSCs were cultured in the different doses of theconditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ, OPN andOCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: Agt;Bgt;Cgt;D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B thanin Groups C and D（Plt;0.05）. However, there was no significant difference between Group A and Group B （Pgt;0.05）.ConclusionThe BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dosedependent pattern, and the best inducing dosage is 200 μg/ml.
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.