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 study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
Objective To test the hypothesis that marrow stromal cells （MSCs）, when implanted into selfmyocardium in rabbits, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes and enhance cardiac function of ischemic hearts, through establish a clinically relevant model for autologous MSCs transplantation, Methods Thirteen New Zealand White rabbits were randomly divided into experimental group （n= 7） and control group （n= 6）. In experimental group, autotogous MSCs（3× 106 cells/30μl） labeled with Bromodeoxyuridine （BrdU） were respectively injected into superior, central and inferior sites in the periphery of the myocardial infarct region. Phosphate buffer saline （PBS） was injected into the scar of the control group hearts according to the same procedure used in the experimental group. Four weeks later, the transplanted labeled MSCs were detected by laser scanning confocal microscopy and the cardiac function were examined by echocardiogram and muhichannel physiologic recorder. Results After 4 weeks, transplanted MSCs were demonstrated myogenic differentiation with the expression of α-sarcomeric actin and connexin 43 located in intercalated disk. MSCs increased the number of vessels compared with controls in myocardial ischemia area. MSCs implantation resulted in markedly improved left ventricular contractility[left ventricular ejection fraction （LVEF）： 0. 51 ± 0.07 vs. 0. 43 ± 0.06 ,left ventricular lateral wall motion distance （LVLWMD） ：1. 75±0. 42mm vs. 1.09±0. 28mm, left ventricular systolic wall thickening ratio（LVAT） ：0. 19%±0.05% vs. 0. 11%±0.04%, left ventricular systolic pressure （LVSP）： 113. 1± 6.3mmHg vs. 99, 5 ± 5, lmmHg, left ventricular end diastolic pressure （LVEDP）： 11. 5±2. lmmHg vs, 14, 3 ±3. lmmHg, maximum rate of left ventricular pressure rise （＋dp/dtmax）：4 618. 3±365. 2 mmHg/s vs. 3 268. 1± 436.9 mmHg/s, maximum rate of left ventricular pressure fall （-dp/dtmax） ：3 008.8±346.7 mmHg/s vs. 2 536.9± 380.4 mmHg/s, P〈0.05]. Conclusion Transplanted autologous MSCs are able to undergo differentiation to form myocardial cells and improve the cardiac function of ischemia myocardium effectively. Autologous MSCs transplantation may have significant clinical potential in treatment myocardial ischemia.
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 integration of rat marrow stromal stem cells (MSCs) after transplantation into acellular extracellular matrix (AECM). Methods We got 16 femurs from 8 Kunming rats, the femurs were treated by Triton X100 toget AECM, MSCs were collected from femoral marrow of 20 Kunming rats about a mouth old by PBS 4ml, centrifugalized and primary cultured in bottles,then therat MSCs were transplanted into AECM at a concentration of 5×106/ml and culturedfor 7 days. The integration of the donor cells was observed using one phase contrast microscope, a light microscope and a scanning electron microscope (SEM).Results In AECM bone lacunas there were MSCs nucleuses stained blue. The nucleuses were unevenly distributed in AECM with more in the peripheral AECM than in the central AECM and with more in the layer anear culture medium than in the layer far away from culture medium.AECM possessed a good spatial scaffold structure, the marrow stromal stem cells were well integrated into AECM.Conclusion AECM can be usedas a good scaffold material for tissue engineered bone construction.
Objective To investigate the possibility of constructing eukaryoticexpression vector for human angiopoietin 1（hAng-1），transfecting it to bonemarrow mesenchymal stem cells (MSCs) so as to repair bone defect. Methods The eukaryotic expression vector pcDNA3-hAng-1 was constructed by recombinant DNA technique, transfected into MSCs by liposome DOTAP, and selected with G418. The hAng-1 expression of mRNA and protein was detected by reverse transcript-PCR and Western Blot. Results After the recombinant eukaryotic expressionvector for hAng-1 was digested with Xho-I and BamH-I, electrophoresis revealed 1.4 kb fragment for hAng-1 gene and 5.4 kb fragment for pcDNA3 vector. In the transfected MSCs, the mRNA and protein expression of hAng-1 gene were detected with reverse transcriptPCR and Western Blot. Conclusion The constructed eukaryotic expression vector hAng-1 could be expressed in the transfected MSCs, thus to provide the basis for bone repair with tissue engineering.
ObjectiveTo explore the effectiveness of vacuum sealing drainage (VSD) combined with open bone graft for tibial traumatic osteomyelitis. MethodsBetween June 2007 and December 2012, 23 cases of tibial traumatic osteomyelitis were treated, including 15 males and 8 females with an average age of 32.5 years (range, 22-48 years). The time from injury to admission was 7-18 months (mean, 8.6 months). There was local bone scarring in 15 cases, the size ranged from 8 cm×4 cm to 15 cm×8 cm. The CT multi-planar reconstruction was carried out preoperatively. Eleven cases had segmental bone sclerosis with a length of 1.5 to 3.8 cm (mean, 2.6 cm); 12 cases had partial bone sclerosis with a range of 1/3 to 2/3 of the bone diameter. On the basis of complete debridement, infection was controlled by VSD; bone defect was repaired by VSD combined with open bone graft. After there was fresh granulation tissue, the wound was repaired by free skin graft or local skin flap transfer. ResultsNail infection occurred in 2 cases, which was cured after the use of antibiotics. The wound healed at the first stage after repairing. All cases were followed up 10-18 months (mean, 13.5 months). In 11 cases of segmental bone sclerosis, the infection control time was 7-14 days (mean, 8.8 days); the bone healing time was 32-40 weeks (mean, 34.4 weeks); and the frequency of VSD was 3-6 times (mean, 4.5 times). In 12 cases of partial bone sclerosis, the infection control time was 7-12 days (mean, 8.3 days); the bone healing time was 24-31 weeks (mean, 27.3 weeks); and the frequency of VSD was 3-5 times (mean, 3.6 times). Infection recurred in 1 case, and the patient gave up the therapy. No infection recurrence was observed in the other patients. ConclusionThe VSD combined with open bone graft is an effective method for the treatment of tibial traumatic osteomyelitis.