Mcroencapsulated VEGF Genetically Modified Cells Transplantation Improves Angiogenesis Of Acellular Dermis

Skin defect wound repairing is always topic studied deeply in surgical field, and ideal wound healing should contain dermal structure. So far it has demonstrated that xenogeneic acellular dermis is satisfactory substitute of dermis, and there have been some reports about transplantation of xenogenic ADM and autologous split thickness skin graft on wound at home and aboard. Broad source and lower cost of porcine ADM make its application become promising in clinic. But ADM without vessel structure develops angiogenesis slowly, which could lead to insufficient nutrition of epidermic cells, regional infection and failure of transplantation. Therefore it is crucial for successful transplantation that early angiogenesis of ADM is accelerated.In experimental studies of plastic surgery, many angiogenic factors have been applied in study of skin graft to promote survival rate. Of which VEGF is a most effective and highly specific factor, and it is well known that its angiogenic effect is obvious. However, short half-life of VEGF protein limits clinical application. To make VEGF exert more bioactive effect, now there has been no perfect releasing and delivering system to lengthen effective time in tissue. VEGF gene therapy may overcome drawback of protein therapy, therefore we try to undertake gene therapy. We may adopt two pathways, namely in vivo and in vitro, but common problem encountered in reality include delivering gene hard to persistent express in vivo, some gene hard to efficaciously transfer, existing safety tissue, cells in vitro proliferating difficultly, short resource of autlogous cell and low survival rate after cell implantation. In 1993, Sun and Chang in Canada firstly raised strategy of allogenic cell gene therapy, namely making desired gene delivering into cell line, and making these cells express gene product, then making them implant into body. Thus we may greatly enlarge resource of cell, but immune rejection reactions limit their application. Microencapsulated cell transplantation technique provides a novel approach for resolving the problem. Microencapsulated allogenic or xenogenic cells transplantation may avoid immune rejection reaction, and simultaneously don't influence their secreting function.In the early 20th century 80, microcapsule technique combined with tissue and cell transplantation, characteristic of which is its selective permeable membrane, and shelter cells from the recipient's immune system, but allows free exchange of small molecular nutrient, bioactive agent and metabolites. Since then, microencapsulating technique has been widely used in experimental and clinical study on neuroendocrine disease, and it has achieved gratifying results. To the 1990s,with the development of gene recombination technology, people try to use microcapsule as immune isolation and their means of delivery, and to regulate physical function and treat associated disease by metabolic product of genetically modified allogenic or xenogenic cells.Mircroencapsulation of recombinant cell lines is viewed as a new delivery system for gene therapy. This approach would allow nonautologous genetically modified cells to be implanted into any host to deliver the desired gene product without triggering graft rejection. The advantages of nonautologous method of gene delivery are: it does not require modification of the host's genome, thus providing additional measures of safety and cost saving; it provide ample material for quality assessment before implantation, a safety feature not available to most other forms of in vivo delivery. Therefore in theory by microencapsulation technique combining with genetically engineering and tissue transplantation technique, cells in microcapsule may secret VEGF through transferring gene, and promote angiogenesis of implanted tissue. However, so far there have been no reports about whether microencapsulated VEGF-secreting cells play a key role in accelerating angiogenesis for implanted ADM. So we designed this present experiment, and undertake following studies.Part one: Objective: To construct the recombinant adenovirus vector containing human vascular endothelial growth factor-165(hVEGF₁₆₅), so as to lay a foundation for study on genetically modified cells. Methods: pAxCAwt.VEGF₁₆₅ and DNA-TPC cotransfected into 293 cells by lipofection method. Being propagated in HEK293 cells and purified by cesium chloride gradient centrifugation, recombinant replication-deficient adenovirus named Ad.VEGF₁₆₅ was obtained. Then the titer of virus was detected by TCID₅₀ method. The Ad. VEGF₁₆₅ was identified by PCR restriction enzyme digestion and DNA sequencing methods. Results: An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-deficient adenovirus vectors coding for VEGF₁₆₅ DNA were generated in high titer. 597bp and 146bp were obtained by NcoI restriction enzyme .the result was consistent with that of Gene Tool software calculating, virus titers was 2.2×10¹² pfu / ml. Conclusions: pAxCAwt.VEGF₁₆₅ and DNA-TPC can be used to construct replication-deficient recombinant Ad vectors with high titer and purity. It is proved to be efficacy and reliable.Part two: Objective: To investigate the expression of adenovirus transfected hVEGF165 in NIH3T3 cells in vitro and the effect of transfection on NIH3T3 cells proliferation. Methods: NIH3T3 cells were passaged and expanded, then infected by Ad. VEGF and Ad. GFP. The infection efficiency of adenovirus vector to NIH3T3 cells was tested by Ad.GFP infection procedure. Ad.VEGF expression in NIH3T3 cells was detected by immunohistochemical staining and RT-PCR, and its secretion in culture medium were measured with ELISA method. Proliferation of cells was determined by MTT. Results: NIH3T3 cells could be effectively transfected by adenovirus containing hVEGF165 gene in vitro, the transfection efficiency has the dose-effect relationship with multiplicities of infection (MOI). When MOI was 100, the infection efficiency was more than 95% and stable. The expression of VEGF was traced both in cell lysate and in culture medium. A maximum production of VEGF was observed at 5～9 days after infection (1052 pg/mL at the 7th day), and VEGF was found even in day 13. The result of MTT demonstrated there was no significant difference between infected cells and uninfected cells (P>0.05). Conclusion: Gene transfer technology mediated by adenoviral vector can transfect VEGF gene into NIH3T3 cells with high efficiency. NIH3T3 cells transfected by hVEGF165 gene could efficiently express VEGF.Part three: Objective: To prepare microencapsulated VEGF-expressing NIH-3T3 cells , and study the effect of microencapsulated process on cellular metabolic functions and proliferation. Methods: Microencapsulated VEGF-expressing NIH-3T3 cell was made by Alginate-BaCl2 process. Morphological appearances of the microcapsule were observed under inverted phase microscope. The concentrations of VEGF in culture supernatant were measured by ELISA; The proliferation of microencapsulated VEGF-expressing NIH-3T3 cell was detected by MTT, and viability was tested by PI and flow cytometry. Unencapsulated VEGF-expressing NIH3T3 cells were used as control. Results: The morphological appearances of microcapsules were round and uniform, and the microencapsulated VEGF-expressing NIH-3T3 cells in vitro survived well. There was no statistical significance in concentration of VEGF, MTT value and viability of cells between two groups in vitro culture(P>0.05). Conclusion: The physiologically metabolic functions of NIH3T3 cells within Alginate-BaCl2 microencapsule have not been impacted by microcapsule membrane. In vitro culture, there is no significant difference in biocharacteristic between microencapsulated VEGF-expressing NIH3T3 cells and unencapsulated cells. Then it lays experimental foundation for exploring further transplantation of microencapsulated VEGF-expressing NIH3T3 cells in vivo.Part four: Objective: To investigate the angiogenic effect of microencapsulated VEGF-expressing NIH3T3 cells transplantation on xenogenic acellular dermis on acute wound. Methods: Each guinea pig dorsal was divided into four symmetrical areas, cutting off the skin to fascia, causing the size of 2cm x 2cm skin defect region, then composite skin (porcine acellular dermis + autologous split thickness skin) were use to cover wound. According to different injected agent under ADM, experiment were further divided into four groups: Microencapsulated VEGF-NIH3T3 cells group (A group), unencapsuled VEGF-NIH3T3 group (B group) ,empty microcapsules grafted group (C group ) , PBS blank control (D group ). The morphological appearances of microcapsules and cells, the early angiogenesis of acellular ADM at one week and survival of composite skin at two week were observed, the expression of VEGF and CD34 were detected by IHC on the 3th, 7th and the 14th day. The microvessel density (MVD) was calculated. Results: The morphological appearances of microcapsules were smooth and round one week after implantation, of which the cells surviving well, and immersion of lymphocytes were not obvious surrounding microcapsule, but there were obvious lymphocytes surrounding unencapsuled cells, the extent of angiogenesis at one week in A group was obvious.the survived rate of composite skin at end of two weeks in A group(91±7%) was higher than that in other three groups(79±5%,76±2%,77±4%,respectively in B,C,D group) (P<0.01), but there was no statistical difference among B,C,D groups(P>0.05).Compared with other three groups, the expression of hVEGF and CD34 were marked in A group, and MVD at 7,14 day in A group was higher than other three groups (P<0.01). Conclusion: Transplantation of microencapsulated VEGF-expressing NIH3T3 cells could augment angiogenesis of ADM in wound healing early stage, and elevate survive rate of composite skin.To sum up, the results of experiment demonstrate that preparation of microencapsulated genetically modified cells is feasible, which may accelerate early angiogenesis of implanted ADM, and improve quality of wound healing. With study deeply in this regard, a serial of achievement will be to bring about expectantly, providing a novel strategy of augmenting angiogenesis for tissue transplantation and refractory wound.