| Background:In recent years, oral soft tissue defect has benn repaired by autologous free flap, muscle flap, local mucosal flap and so on, in order to ensure the graft survival in oral special environment. But these methods also have disadvantages. Can the graft survive after transplantation? The donors become deformed because of a large amount of soft tissue defect. The flap retains the features of hair and sweat glands and so on after the flap survival. Secondary trauma increases the pain and treatment process of patients.With the development of tissue engineering, collagen membrane is used widely because of its good biocompatibility and biodegradability. There have been several studies on the adhesion, proliferation and differentiation of cells in different membrane in vitro, and the result has show collagen membrane has been the best. Collagen membrane has been widely used for oral soft tussue defect, and the effectiveness has been different.In tissue engineering and wound healing, angiogenesis is crucial. Angiogenesis can deliver oxygen and nutrients, which will be advantage of wound healing. Basic fibroblast growth factor is one of the vascular growth factor. Due to its rapid spread in body fluid, composition by protease and short half-life, it is difficult to maintain its therapeutic concentration. It can not continue to stimulate target cells to accelerate its biological effect. In order to improve its biological effect, researchers attempted to increase the dosage of bFGF. However, high doses of bFGF could lead to a series of complications, such as thrombocytopenia, renal toxicity and even the activation of certain malignant cells and so on.The binding between growth factors and biomaterial have two kinds. One is targeted binding between growth factors and biomaterials. Another is simple-absorption between growth factors and biomaterials. Targeted binding between growth factors and biomaterials make growth factors restricted to the biomaterials and reduce its dosage, which achieve better repair effect.Jianwu Dai has developed a collagen-based targeting release system. A collagen-binding domain (CBD), which contained seven amino acid peptides, was added to the N-terminal of native bFGF to allow it bind to collagen specially. The bFGF that binded with CBD was called CBD-bFGF. Meanwhile the natural bFGF which had not CBD was called NAT-bFGF. They showed that CBD-bFGF and NAT-bFGF had the same biological activity according to MTT testing. CBD-bFGF had special ability binding with collagen compared to NAT-bFGF according to ELISA testing.Objective:Prepare collagen membrane with a stable and slow-release system by loading collagen-targeting basic fibroblast growth factor. Evaluate the effect of collagen membrane loaded with collagen-targeting basic fibroblast growth factor for soft tissue wound repair in hard palate of rats. Provide theoretical basis for the effective use of bFGF to repair soft tissue defect.Methods: 96 SPF Wistar rats were 6 weeks, inbred, male and 160-200g weigh. All rats were divided into 4 groups randomly, and every group had 24 rats. A 3 mm diameter circular soft tissue defect was made in the center of hard palate of rats. The defect was covered with:A:collagen-targeting bFGF/collagen membrane, B:free bFGF/collagen membrane, C:collagen membrane, D:control group, respectively. Every 6 rats were randomly sacrificed at the 1week,2 weeks,4 weeks and 8 weeks in every group after the operation. The wound healing, histology and immunohistochemical observation were recorded. All dates were analysed with SPSS 13.0 software.Results:1. The animals recovered well after surgery and had no inflammation. No animals died. All animals achieved I period healing. The rejection reaction did not appearred because of collagen membrane.2. At 3 days, collagen membrane was light yellow, and was not degraded. At 1 week parts of the suture was off after postoperative. No abnormal leakage and no purulent was observed during wound healing. At 1 week all wound was not completely healed. The margin of the wound had a little swelling and no significant exudate. Collagen membrane was degraded partly and was light yellow. At 2 weeks all wound were healed completely and there was no difference in the naked eye view. At 4 and 8 weeks all wound were healed. The wound was close to normal undamaged tissue in collagen -targeting bFGF group.3. We could see the epithelium, fibroblast, angiogenesis and collagen membrane and so on in HE stained. At 1 week, the epithelium of all groups was imcompletely formed. Collagen membrane was incompletely degraded in collagen-targeting bFGF/collagen membrane group, in free bFGF/collagen membrane group and collagen membrane group.There was lots of fibroblasts, a lot of new blood vessels with larger diameter in collagen-targeting bFGF/collagen membrane group. At 2 weeks, the epithelium of all groups was completely formed. The most of collagen membrane was degraded in collagen-targeting bFGF/collagen membrane group, in free bFGF/collagen membrane group and collagen membrane group. At 2 weeks there was new formed epithelium and thick epithelial spikes in collagen-targeting bFGF/collagen membrane group. There was new formed epithelium and thin epithelial spikes in free bFGF/collagen membrane group and collagen membrane group. There was new formed epithelium and no epithelial spikes in control group. At 4 and 8 weeks there was new formed epithelium and thick epithelial spikes.4. Statistical analysis:(1) the length of epithelial gap:At 1 week, there was statistically significant each other through one-way ANOVA (F=18.904,P<0.001). The length of epithelial gap in collagen-targeting bFGF/collagen membrane group was the shortest. The length of epithelial gap in control group was the longest. The length of epithelial gap in free bFGF/collagen membrane group and collagen membrane group was the medium.(2) the degree of re-epithelialization:At 1 week, there was statistically significant each other through one-way ANOVA (F=18.904,P<0.001). The degree of re-epithelialization in collagen-targeting bFGF/collagen membrane group was the hightest. The degree of re-epithelialization in control group was the lowest. The degree of re-epithelialization in free bFGF/collagen membrane group and collagen membrane group was the medium.(3) the number of new blood vessel:There was statistically significant of the number of new blood vessel in different time through factorial classification (F=161.167, P<0.001). There was statistically significant of the number of new Wood vessel between experimental and control groups through factorial classification (F=114.871, P<0.001). There was statistically significant between time and group (F=17.791, P<0.001). So we should compare the number of new blood vessel between treatment groups in different time through one-way ANOVA.At 1,2 and 4 weeks, there was statistically significant in the number of new blood vessel among four groups (P<0.001). The number of new blood vessel of collagen-targeting bFGF/collagen membrane group was the most. The number of new blood vessel of control group was the least. The number of new blood vessel of free bFGF/collagen membrane group and collagen membrane group was the medium. At 8 weeks, there was no statistically significant in the number of new blood vessel among four groups (P=0.782). From 1 week to 8 weeks, the number of new blood vessel increased to the peak at 2 weeks and then decreased.(4) the mean optical density of myofibroblast:There was statistically significant of the mean optical density of myofibroblast in different time through factorial classification (F=934.804, P< 0.001). There was statistically significant of the mean optical density of myofibroblast between experimental and control groups through factorial classification (F=86.549, P< 0.001). There was statistically significant between time and group (F= 11.768, P< 0.001). So we should compare the mean optical density of myofibroblast between treatment groups in different time through one-way ANOVA.At 1,2 and 4 weeks, there was statistically significant in the mean optical density of myofibroblast among four groups (P<0.001). The mean optical density of myofibroblast of collagen-targeting bFGF/collagen membrane group was the lowest. The mean optical density of myofibroblast of collagen membrane group and control group was the highest. The mean optical density of myofibroblast of free bFGF/collagen membrane group was the medium. At 8 weeks, there was no statistically significant in the mean optical density of myofibroblast among four groups (P=0.392). From 1 week to 8 weeks, the mean optical density of myofibroblast increased to the peak at 2 weeks and then decreased.Conclusion:1. Collagen membrane loaded with collagen-targeting basic fibroblast growth factor could slowly release bFGF, could have a better role in angiogenesis and could be good to soft tissue wound repair.2. Collagen-targeting bFGF/collagen membrane could increase the process of wound healing in hard palate of rats and promote the re-epithelialization of oral soft tissue defect.3. Collagen-targeting bFGF/collagen membrane could reduce wound contraction and improve the quality of wound healing in the wound healing process, because the mean optical density of myofibroblast was the lowest in the collagen-targeting bFGF/collagen membrane. |
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