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 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 investigate the results of human amniotic membrane(HAM) which are loaded with marrow mesenchymal stem cells(MSCs) and epidermis cells in treating fullthickness skin defect combined with radiation injury. Methods Eight minipigs were used in this study. Three round fullthickness wounds（Ф3.67cm）, which combined with radiation injury, were created on the dorsum of each side close to the vertebral column in each animal. Among 48 wounds, 24 left side wounds were treated with HAM loaded with MSCs and epidermis cells as experimental group (group A), 16 right side wounds with simple HAM (HAM group, group B) and 8 right side wounds with oil gauze as control (group C). The granulation tissue, reepithelization and wound area were observed after 1,2 and 3 weeks. Immunohistochemistry was performed using vWF as a marker for blood vessels.Image analysis was employed to test new area of wound at different interval time and healing rate of wound.Results The healing time of group A was 6 to 7 days faster than that of group C and 5 to 6 days faster than that of group B. After 15-17 days of graft, there were significant differences in new area of wound and healing rate between group A and groups B,C(Plt;001). New epidermis fully covered whole wound surface in group A, and their granulation tissue, which contained a lot of vWF, fibroblasts, capillaries and collagen, grew well. Many inflammatory cells still were seen in groups B and C, and their contents of vWF, fibroblasts, capillaries and collagen in granulation tissue were smaller than that in group A.Conclusion The graft of HAM loaded with MSCs and epidermis cells played an effective role in promoting healing of wound combined radiation injury with high quality.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
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 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 study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
Objective To evaluate the effect of nano-hydroxyapatit e collagen (nHAC) bone and marrow mesenchymal stem cells (MSCs) on the treatment of rabbit osteonecrosis of the femoral head (ONFH) defect. Methods From June to October 2004, animal models of ONFH defect were established i n 45 New Zealand rabbits. They were divided into 3 groups randomly:In group A, as the control group, defect was not filled with any implants; In group B with nHAC; In group C with nHAC+MSC. Imaging and histological observation were made 4, 8, 12 weeks after operation. Results group C had a better o steogenesis ability than group B and group A. group B had a better osteogenesis ability than group A. Obvious new bones and osteogenesis were observed in group C 4 weeks after operation. The defect areas in group C were almost repaired 12 weeks after operation. Conclusion nHAC has a better effect of o steoconduction and it is a superior material for repairing bone defect of ONFH a nd of great value in treating ONFH when compounded with MSCs.
Objective To optimize the in vitro culture system of C57/BL6 marrow mesenchymal stem cells (MSCs) and to investigate the effect of alcohol and acetaldehyde on MSCs. Methods The MSCs were isolated from the femur marrow of C57/BL6 mice and were cultured in the optimized system, so that highlypurified MSCs were harvested and identified by immunohistochemistry. Then, MSCs were cultured in the medium containing alcohol or its metabolic product acetaldehyde, with the following concentration groups: alcohol 5.7，17.0，50.0，100.0 and 150.0 mmol/L; acetaldehyde 4.5, 0.9, 0.18, 0.036, 0.007 2, 0.001 44 , 0.000 28 mmol/L. MSCs were cultured with α-MEM as the control group. After 3 days, their proliferation activity was measured by the MTT method. Results MSCs within 6 passages had a good stability and a high proliferation activity. They were identified to express CD90 but no CD34. The MTT assay showed that alcohol at the concentration greater than 100.0 mmol/L and acetaldehyde at the concentration greater than 4.5 mmol/L could inhibit proliferation of MSCs(P＜0.05) . But the proliferation activity might rise with an increase in the acetaldehyde concentration smaller than 0.18 mmol/L(P＜0.05) . Conclusion Theoptimized culture system can effectively isolate and culture MSCs. Both alcoholand acetaldehyde can inhibit proliferation of MSCs but toxicity of acetaldehydeis more serious.
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.