Objective To investigate the feasibility of imaging of bone marrow mesenchymal stem cells (BMMSCs) labeled with superparamagnetic iron oxide(SPIO) transplanted into coronary artery in vivo using magnetic resonance imaging (MRI), and the redistribution of the cells into other organs. Methods BMMSCs were isolated, cultured from bone marrow of Chinese mini swine, and double labeled with SPIO and CMDiI(Cell TrackerTM C-7001). The labeled cells were injected into left anterior descending coronary artery through a catheter. The injected cells were detected by using MRI at 1 week,3weeks after transplantation. And different organs were harvested and evaluated the redistribution of transplanted cells through pathology. Results The SPIO labeled BMMSCs injected into coronary artery could be detected through MRI and confirmed by pathology and maintained more than 3 weeks. The SPIO labeled cells could be clearly imaged as signal void lesions in the related artery. The pathology showed that the injected cells could be distributed into the area of related artery, and the cells injected into coronary artery could be found in the lung, spleen, kidney, but scarcely in the liver, the structures of these organs remained normal. Conclusion The SPIO labeled BMMSCs injected into coronary artery can be detected by using MRI, the transplanted cells can be redistributed into the non-targeted organs.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.
OBJECTIVE To investigate the ectopic osteogenesis of bone marrow stromal cells (MSC) induced by bone morphogenetic protein(BMP) in vitro and in vivo, providing the experimental evidence for making an artificial bone with its own capacity of bone formation. METHODS MSC were separated and cultured from bone marrow of Wistar rats, MSC were co-cultured with BMP in vitro (cultured in plate and diffuse chamber). Artificial coral hydroxyapatites (CHA) with MSC and BMP were implanted into dorsal muscles of Wistar rats, their bone formation were observed by morphological examination, histochemistry and immunohistochemistry. RESULTS Only cartilaginous matrix were produced by MSC in vitro (cultured in plate and diffuse chamber), and both cartilaginous and bone matrix production within the combined grafts were seen. The bone formation of experimental groups (CHA + BMP + MSC) was ber than that of control A(CHA + MSC) and control B(CHA). CONCLUSION It may be possible to produce an artificial bone with its own capacity of bone formation by combined graft (CHA + BMP + MSC). There may be multiple factors as well as BMP inducing bone formation both in the whole body and the location of the implantation. Further research on these factors will have the significance for making the ideal artificial bone.
OBJECTIVE: To investigate the feasibility of coralline hydroxyapatite (CHA) as scaffolds in bone tissue engineering. METHODS: The bone marrow stromal cells from 4-month New Zealand rabbits were harvested and cultured in vitro. After multiplied, dexamethasone was used to promote the osteoblastic phenotype of the cells. The cells were harvested and then seeded into CHA. By means of tissue engineering technique, osteoblastic cells/CHA complex were formed. The complex were implanted subcutaneously in nude mice. The CHA alone was implanted as control. Bone regeneration was assessed 6, 8 weeks after implantation by histological and roentgenographic analysis. RESULTS: After six weeks of implantation, x-ray film showed high-density signal, osteoid tissue formed under histological examination. Large amount of new bone were formed and connected to trabecularism 8 weeks after implantation in the experimental group. While in the control group, there were no new bone formation, but amount of fiber tissue grew into the pore of CHA 8 weeks after implantation. CONCLUSION: CHA may be used as a good scaffold material for bone tissue engineering.
Objective To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithel ial reconstruction. Methods The related l iterature about BMDSCs heterogeneity and its role in intestinal epithel ial repair was reviewed and analyzed. Results The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithel ium included direct implantation into intestinal epithel ium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction. Conclusion BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.
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 The combined appl ication of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods. Methods After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was ampl ified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of l iposome. Positive PT67 cells were picked out with G418, and prol iferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and prol iferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and prol iferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts. Results The isolated cells were fibroblast-l ike morphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The prol iferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, prol iferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation. Conclusion The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.