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 effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
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 elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
Abstract： Objective To study the effect of different numbers of bone marrow mesenchymal stem cells（MSCs） transplanted into rats with pulmonary arterial hypertension （PAH）induced by monocrotaline（MCT）and their influence on the expression of endothelin-1（ET-1）. Methods Forty healthy male Wistar rats（weight，from 180 to 250 g） were divided into four groups by random number table（n=10）：group A：Wistar rats were intraperitoneally injected with MCT 60 mg/ kg， and then injected with 1×106 MSCs via the external jugular vein；group B：Wistar rats were intraperitoneally injected with MCT 60 mg/kg，and then injected with 5×105 MSCs via the external jugular vein；MCT group：Wistar rats were intraperitoneally injected with MCT 60 mg/kg， and then injected with equal amount of PBS via the external jugular vein； control group：Wistar rats were intraperitoneally injected with equal amount of saline and then injected with equal amount of PBS via the external jugular vein. Four weeks after MSCs transplantation，right ventricular systolic pressure（RVSP） and ventricular weight ratio of right ventricle/ （left ventricle＋ventricular septum）were measured. Histomorphology of lung tissue was observed. Genetic expression of ET-1 in lungs and serum peptide of ET-1 were also measured. Results Four weeks after MSCs transplantation，both RVSP and ventricular weight ratio decreased significantly in rats of group Acompared with those of MCT group（RVSP：35.8±4.2 mm Hg vs. 47.2±10.1 mm Hg，P＜ 0.01； ventricular weight ratio：0.357±0.032 vs. 0.452±0.056，P＜0.01）， but these two parameters didn’t decrease significantly in rats of group B（P＞ 0.05）. By histopathological staining， the percentage of medial wall thickness of the pulmonary arterioles was significantly less in rats of group A than that of MCT group（19.7%±3.0% vs. 26.8%±3.6%， P＜ 0.01）. There was no statistical difference in the percentage of medial wall thickness of the pulmonary arterioles between group B and MCT group. Reverse transcriptase-polymerase chain reaction （RTase-PCR）results showed that ET-1messenger ribonucleic acid（mRNA）expression was highest in MCT group and MSCs transplantation significantly decreasedits expression in group A， while its expression was similar between group B and MCT group. The expression ofET-1 in plasma was also significantly decreased in group A than that in MCT group. Conclusion Intravenous MSCs transplantation can significantly inhibit MCT-induced PAH，and reduce both ET-1 mRNA expression in lung and ET-1 peptide level in plasma. It’s a better choice to transplant 1×106 MSCs to inhibit PAH in rats.
Objective To explore a method to isolate, culture and multiplicate the placentaderived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit，and to compare their biological characteristics. Methods PMSCs were isolated from placenta of 1fetation rabbitby Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culturemethod. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials，(1.0-1.5)×106 cells in one biomaterial， and then observed by aematoxylinstaining after 5 days,and by SEM after 3 days and 8 days. Results PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite b and declined with passages. After cultured 10 passages in vitro, its growthslowed. Both PMSCs and BMSCs expressed CD44 and CD105,but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation ofSEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew,arranged in layers andsecreted many matrices; the reticular collagen formed arround cells after 8 days. Conclusion PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seedingcells for tissue engineering.
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.