RGD Surface Modification On Decellularized And Photooxidated Bovine Jugular Vein Matrix Treated By Photochemical Grafting And It's Reendothelialization

PartⅠFabrication of decellularized and photooxidated bovine jugular vein matrix & RGD surface modificationObjective:To investigate the mode of photocrosslinking RGD peptide onto the decellularized and photooxidated bovine jugular vein matrix surface and the persistence of RGD in vivo.Methods:1. Bovine jugular vein was decellularized and photooxidated, then cut into 48 pieces of 1×1cm~2 and divided into 12 groups randomly. The solutions of RGD covalent labelled with FITC and photochemical crosslinker SANPAH were prepared, their concentrations were 0.12mM, 0.6mM, 1.2mM and 6mM. The reaction between them were performed with 3 different mole ratios in 2 hours away from light, which were 1∶0, 1∶1 and 1∶10. 100μl reacted solution of them were dropped onto the prepared BJV pieces, then irradiated under 365nm ultraviolet ray for 5 minutes. The pieces were rinsed in a shaking machine for 6 hours. The fast frozen sections of the specimens were observed under the fluorescent microscope. The grafting effect was viewed by different fluorescent brightness, so the suitable reactive concentration and ratio were educed. This reactive concentration and ratio were selected for further experiment in vivo as below.2. Four RGD modified BJV patches were prepared which were treated with RGD-FITC and SANPAH of 0.6mM and 1∶1 mole ratio by the same method as above mentioned(SANPAH group). Another four patches were only coated with RGD-FITC directly(control group). Eight Sprague-Dawley rats (SD rats) were divided into this two groups equally. All the patches were transplanted into murine inferior vena cava wall and harvested at the 5th, 10th, 15th, 20th day postoperatively. Their fast frozen sections were observed and pictured under fluorescent microscope as above procedure.Results: The brighter fluorescence on the side of endangium was viewed on the SANPAH photocrosslinking group. The higher the concentrations of RGD and SANPAH were, the brighter the fluorescence was. But when the concentration of them was higher than 0.6mM, the difference of fluorescent brightness was not so clear. In the same concentration, the fluorescence was brightest if the reactive mole ratio between them was 1∶1. In vivo fluorescence still existed after 20 days postoperatively in SANPAH group even though attenuating slightly. At 5th day postoperatively, the fluorescence disapeared in RGD group. Conclusion: RGD peptide could be covalent grained onto the BJVC surface by photochemical cross-linker SANPAH. The optimal concentration of RGD and SANPAH and reactive mole ratio between them were 0.6mM and 1∶1. The persistence time of RGD treated with photochemical cross-linker was over 20 days at least in vivo. 10~5/ml density and cultured for 5 days. The cells were departed with enzyme from the surface of these pieces at 1st, 3th, 5th, their numbers were counted. The slices of BJV implanted with cells were HE stained and pictured. The difference between two groups was compared to verify whether BJV modified with RGD could promote the cell adhesion and proliferation.2. In vivo: Eight SD rats were divided into RGD modified group and control group equally. BJV patches were implanted in their IVC wall. Continuous suture technique was performed. The rats were fed with atorvastatin of 10mg/kg/d. The patches were harvested at 5th, 10th, 15th, 20th day postoperatively. Gross view was achieved. The samples were HE stained and scanned under scanning electron microscope and tested ofⅧfactor dyeing with immunohistochemistry method. The thickness of neointima were measured and compared between two groups by using Image Pro Plus software.Results:1. In vitro: (1) Cells counting: The cells number in RGD group was more than control group at 1st, 3rd, 5th day (p＜0.05). (2) At 1st day, the cells arrayed intensively and not spreaded completely in each group's surface. At 3rd day, the cells on BJV surface decreased apparently and disrupt among themselves in control group, while the RGD group gained a cell-layer well. At 5th day, this phenomenon was more obvious. 2.In vivo: (1) Gross view: The patches were harvested from IVC of the rats. Adherence with surrounding tissue was slight. The color of methylthioninium chloride disappeared and the patches turned white and were still soft. The inner side of all patches was smooth. No thrombus was found in both two groups. The suture line was seen clearly. A thickening lamella was viewed on all patches.(2)HE(hematoxylin - eosin) stain: BJV treated with photooxidation could inhibit cell infiltration. The infiltration occurred firstly in anastomotic area and was not through the whole midpiece wall after 20 days in both two groups except in a very small area. Inside the neointima there were more cells in RGD group than control group. As soon as the neointima was shucked, cells were seen on BJV surface in RGD group while few cells in control group at 5th day postoperatively by HE stain. Endothelioid cells were viewed in RGD group at 5th day and it completely covered neointima at 10th day. While this phenomenon occurred at 20th day in control group.(3)Ⅷfactor staining: Positive staining was viewed at 5th day in RGD group, stronger positive at 10th day postoperatively, and there was neocapillary in neointima at 5th day postoperatively. There were fewer cells on the neointimal surface at 15th in control group. Negative staining still existed at 20th in control group postoperatively, though lots of cells were seen on the neointimal surface at this time. (4) Scanning electron microscope: Lots of endothelioid cells along with the direction of blood flow were observed but there was still distance between the cells in RGD group at 5th day. A layer of endothelial cells were compacted and well-arrayed, the cell junction was tight at 10th day in RGD group. There was only cellulose and macromolecule substance on the neointima in control group at these times. Though it was seems that there were cells under the surface but the endothelial cell covering was not observed.(5) Thickness of neointima: The neointima was thicker in RGD group than in control group at 5th day postoperatively, but it was not significant(P＞0.5). It was thinner in RGD group than in control at 10th, 15th, 20th day postoperatively(P≤0.01). In general, the neointima turned thicker when time went on, which got to the peak at 15th day and then went down in both group. The thickness of neointima at 20th day decreased to the level of that at 5th day postoperatively in RGD group, while the thickness of neointima was still large at 20th day in control group.Conclusions:RGD surface modification could promote the cell adhesion and prolification on BJV in vivo. Fast reendothelialization was achieved on the BJV patches modified with RGD in vivo. Even still accompanying with intima hyperplasia, but the thickness of neointima was slighter than that untreated with RGD.