Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.</p><p><b>METHODS</b>GFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.</p><p><b>RESULTS</b>The expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.</p><p><b>CONCLUSIONS</b>The hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.</p>

Effects of BMP-2 gene therapy on vascularization in repairing bone defects / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the effects of BMP-2 gene therapy on vascularization in repairing bone defects.</p><p><b>METHODS</b>The isolated rabbit mesenchymal stem cells (rBMSC), after being transfected by adenovirus carrying BMP-2 gene (Ad-BMP-2) and seeded on xenogeneic bone scaffolds, were used to repair 1.5 cm-long radius bone defects. Five methods were in use in the experiments: Ad-BMP-2 infected rBMSC plus antigen-free bovine cancellous bone (BCB, Group A), rBMSC-BCB plus reconstructed hBMP-2 (Group B1), Ad-LacZ infected rBMSC-BCB (Group C), rBMSC-BCB (Group D) and only BCB scaffolds (Group E). After 4, 8, and 12 weeks of the operations, capillary vessel ink infusion, vascular endothelial growth factor ( VEGF) immunohistochemical staining and histological examination were conducted.</p><p><b>RESULTS</b>After 4 weeks of the operations, usually in Group A one newly formed artery was found in every pore between the trabeculae of the BCB. The density of these intraosseous vessels was high in the periphery and decreasing towards the center of the grafts; by transmission electron microscopy, osteoblasts were always next to vascular endothelial cells and gradually developed into osteocytes with the increase of capillary vessel; VEGF expression were apparently enhanced in mesenchymocytes.</p><p><b>CONCLUSIONS</b>BMP-2 gene therapy, by up-regulating VEGF expression, indirectly induces vascularization of grafts and is of great value to the treatment of bone in union and bone defects.</p>

Research on chondrogenic differentiation and immunologic response of allogeneic mesenchymal stem cells implanted into joint cavity / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the cartilage formation ability of allogeneic mesenchymal stem cells implanted into sheep joint cavity without the use of immunosuppressive therapy.</p><p><b>METHODS</b>Allogeneic mesenchymal stem cells (MSCs) loaded onto porous beta-tricalcium phosphate ceramic (beta-TCP) were implanted into normal sheep joint cavity. A complete mismatch between donor stem cells and recipient sheep was confirmed by mixed lymphocyte reaction assays prior to implantation. Eight weeks after implantation, the implants were taken out for histological and immunohistochemical analysis. The histological results were compared with data derived from joint cavity implantation of autologous MSCs-ceramic composites and cell-free ceramics. The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells.</p><p><b>RESULTS</b>For implantation with allogeneic MSCs, no sign of adverse immune response was detected. Histologically, few inflammation cells infiltration occurred and no antibodies against allogeneic cells were detected. Neocartilage formation in implants loaded with either allogeneic or autologous mesenchymal stem cells was revealed by histochemical and immunohistochemical analysis. In implants without stem cells, no cartilage formation was detected.</p><p><b>CONCLUSIONS</b>Allogeneic mesenchymal stem cells are capable of forming cartilage under the effect of joint cavity environment. Without the use of immunosuppressive therapy, allogeneic MSCs do not provoke an adverse immune response in vivo.</p>

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects.</p><p><b>METHODS</b>GFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine.</p><p><b>RESULTS</b>Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy.</p><p><b>CONCLUSIONS</b>hIGF-I eukaryotic expression vector has been successfully labeled with GFP.</p>

The expression of N-cadherin, fibronectin during chondrogenic differentiation of MSC induced by TGF-beta(1) / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of N-cadherin and fibronectin during chondrogenesis.</p><p><b>METHODS</b>Immunohistochemical method and autibody induced changes of aggregation of cells were used to assay the expressions of N-cadherin and fibronectin during cell differentiation.</p><p><b>RESULTS</b>The N-cadherin was present in the area of the cell nodular area in the 24 hours group after adding chondrogenic revulsant, then there was a down-regulating trend. Fibronectin was expressed in 48 and 72 hours groups after adding chondrogenic revulsant, and showed to be negative afterward. The antibody against fibronectin or N-cadherin could inhibit the formation of cellular nodule markedly.</p><p><b>CONCLUSIONS</b>Cell adhesion factors play an important role during cell differentiation. TGF-beta(1) stimulates chondrogenesis via transition from an initial N-cadherin-contributing stage to a succedent fibronectin-contributing stage during the process of chondrogenesis in MSCs. Further study is needed to evaluate whether or not it can promote chondrogenesis by transfecting cDNA of CAMs to MSCs.</p>

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering.</p><p><b>METHODS</b>DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope (SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated.</p><p><b>RESULTS</b>DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs.</p><p><b>CONCLUSION</b>dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.</p>

Experimental study on the prevention of epidural scar adhesion with polycaprolactone/polylactic acid membrane / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the ability of a polycaprolactone/polylactic acid (PCL/PLA) membrane to inhibit epidural scar adhesion after laminectomy, and observe the responsive changes of the pain media in the spinal cord.</p><p><b>METHODS</b>L(1), L(3) laminectomies were performed on 96 Wistar rats. The rats were divided into 3 groups: None-implant Control Group (NC), Autologous free fat graft group (AFFG) and PCL/PLA membrane group (PCL/PLAm). The rats were killed at 1, 3, 6, and 12 weeks postoperatively. Epidural scar formation and adhesion were observed grossly and histologically. Reverse transcription polymerase chain reaction (RT-PCR) were used to analyses the expression of Transforming growth factor beta (TGF-beta) in the epidural scar. Immunohistochemistry stain and RT-PCR were performed to evaluate the expression of the substance P and the c-fos gene in the relevant spinal cord, and the results were analyzed statistically.</p><p><b>RESULTS</b>Gross evaluation and histological evaluation showed that in the NC lamina defect site had much scar tissue and had wide and tight adhesions to the dura; in the AFFG, with the fat degrading gradually, the adhesions were increased; whereas in the PCL/PLAm group, there were slightly adhesions to the dura. RT-PCR showed that the expression of the TGF-beta was much less in the PCL/PLAm group than in the NC group. The insertion of the PCL/PLA membrane and the fat patch reduced the expression of the substance P and the c-fos gene in the spinal cord.</p><p><b>CONCLUSION</b>The insertion of the PCL/PLA membrane reduces scar formation and separates fibrosis tissue from the dura, the results indicate that PCL/PLA membrane is an effective way of reducing peridural scar formation and preventing the failed back surgery syndrome.</p>

Repair of segmental bone defect by using gene modified tissue engineering bone combined with vascular bundle implantation / 中华创伤杂志

Objective To evaluate the effects of bone morphogenetic protein 2(BMP-2)gene modified tissue engineering bone combined with vascular bundle implantation in repairing segmental bone defect.Methods The isolated rabbit hone marrow stromal cells(MSCs),after being transfected by adenovirus carrying BMP-2 gene(Ad-BMP-2),were seeded on bovine cancellous bone scaffolds(BCB) to construct gone modified tissue engineering hone.The rabbit models with radial defects(2.0 cm long) were made and repaired with four methods including gene modified tissue engineering bone with vascular bundle implantation(Group A),gene modified tissue engineering bone(Group B),nongene modified tissue engineering bone with vascular bundle implantation(Group C),and only BCB scaffolds(Group D).After 4,8,and 12 weeks of operation,X-ray,histological examination,biomechanics analysis and capillary vessel ink infusion were conducted to observe angiopoiesis and osteogenesis.Results Group A gained better effect in the volume and activity of new bones than other groups,with vascular bundle sending out new branches into the transplanted bones and productive regeneration of capillary vessel.The defect in Group A was repaired satisfactorily.Group B showed better effect in speed and quality of bone formation than Group C under induction of BMP-2 gent.Mainly fibrous tissues but not new bones were observed in Group D.Conclusion BMP-2 gene therapy with vascular bundle implantation has very strong osteoinduction ability and quite good vascularization effect and is of great value to the treatment of bone nonunion and bone defects.