| Refractory wounds occured when the wounds happen with severe diseases brought negative effects on healing, which makes wound heal slowly and even cause serious consequences. Typical refractory are diabetic ulcers, radiation ulcers, bed sores and so on. Patients suffer painfully from these wounds and have to endure tough course of clinical treatment. In 2004, autologous platelet-rich gel was applied on treatment for refractory diabetic foot ulcer successfully. Since then, the local application of autologous platelet concentrate has become a research hotspot of treat refractory injure. However, there are some obstacles difficult to get over in the application process of autologous platelet-rich concentrates (such as autologous platelet extraction is relatively complicated, the platelets are difficult to be stored and quantified, and release of the growth factor from platelets is also usually impacted by the clinical conditions). Therefore, some negative results occurred in the randomized clinical trials and consequently the application of platelet has been limited. However, it is indisputable that the topical use by high concentrations of platelets can provide a large number of growth factors to promote wound healing. In this study, we used lyophilized platelets to treat refractory wounds, in order to improve the availability and operability of platelet concentrates.Establishment of the animal model for diabetes: in the experimental studies on refractory trauma, the wound models bulit oversea are generally based on hereditary diabetes rats and mice. This kind of transgenic animal model for diabetes mellitus is optimal to use. But the model animals are too expensive and the conditions for raising and breeding are usually unaffordable to most of the labs. So the hereditary models are less applicated in domestic studies, meanwhile, the drug-induced diabetes animals are widely used to establish the refractory wound model. The method is relatively simple and can be used commonly. In this study, we did intraperitoneal injection of STZ (65mg/kg.b.w) to the rats to build the model of diabetes. After 72 hours, the blood glucose was significantly increased with the values all higher than 16.7mmol/L. The values of urine sugar are 4+. Polydipsia and polyuria signs occured obviously. These symptoms lasted at least 9 weeks. At the 8th week, some rats grew with cataracts symptoms. The weights of the diabetic rats significantly decreased compared to the normal rats; diabetic rat epidermis was getting thinner skin, capillaries were getting less and collagen was degenerated. 5 weeks after injection, the weights were no longer growing; diabetic kidney weight/body weight ratio was significantly increased. Through the HE dyeing picture, it was observed that synthesis of extracellular matrix was increased, basement membranes were getting thick and mesangial matrixs were increased in the kidneys. In summary, the symptoms occurred in the model were similar to the ones happen in advanced diabetes of human, which indicated refractory trauma could be developed in the model.Establishment animal model for refractory trauma: The experimental animals were divided into 5 groups: control rats, rats of 3 weeks, 5 weeks, 7 weeks and 9 weeks after blood glucose increased. All rats were operated in intact cortical defect surgery to establish the wounds with 2cm diameter. Then the the re-epithelialization rate and healing time were compared between different groups. In the healing process, the re-epithelialization rate had no significant difference (p>0.05) between control group and 3 weeks group. The healing time for control group and 3 weeks group were 15.7±1.4 days and 17.0±2.6 days respectively without significant difference (p>0.05), but the re-epithelialization rate in 5 weeks and 7 weeks group were lower than the rates in control group (p<0.05); healing time in 5 weeks group and 7 weeks group were 24.8±4.2 days and 25.2±3.7 days respectively, 10 days longer than control group; the healing rate and healing time in 5 weeks group and 7 weeks group had no significant difference (p>0.05) compared to control. Many animals in 9 week group died during the experiment because of high blood glucose during long time. The lesions were getting worse with long time high blood glucose. When high blood glucose last short time, refractory wounds were difficult to obtain. But if time of high blood glucose last too long, the lesions were getting worse and leaded high mortality of the animals. So we decided to establish the whole cortical defect model at 5-7 weeks after the emergence of diabetes, and use the model to study the platelets role in the refractory trauma repair.Content analysis for EGF and VEGF of lyophilized platelets: platelets can release the major six kinds of growth factors (TGF, PDGF, EGF, VEGF, IGF and FGF) to promote wound healing. In the pre-experiment, we analyzed the release ability of fresh platelets and lyophilized platelets by comparing the contents of PDGF and TGF. In this study, we further compared the contents of EGF and VEGF. The results show that PPP reconstituted lyophilized platelets after activation by thrombin, the release contents of EGF and VEGF were 312.9±67.3 pg/mL and 512.2±43.8 pg/mL, had no significant difference compared with fresh platelets (298.8±65.9 pg/mL and 506.6±38.9 pg/mL) (p>0.05); the release content of EGF and VEGF of lyophilized platelets reconstituted with saline were 295.7±45.2 pg/mL and 489.3±62.3 pg/mL, had no significant difference compared with lyophilized platelets reconstituted with PPP (p>0.05). Based on the results, it was further confirmed that the lyophilized platelets and fresh platelets have the same abilities to release growth factors; the lyophilized platelets reconstituted with PPP and the saline have the same capacity for release of the growth factors. These results provide the theoretical basis of the different lyophilized platelets preparation for clinic application.Study on the role of lyophilized platelets in repair process for cortical defect of diabetic rats: In the experiment, diabetic rats were divided into 4 groups: not treatment group (NT), rhEGF treatment group (GFT), lyophilized platelet saline treatment group (LYPT1), lyophilized platelet gel treatment group (LYPT2). In LYPT1 group, lyophilized platelets were reconstituted by saline and sprayed to the wound directly right after thrombin activation. LYPT2 was lyophilized platelets reconstituted by the PPP. Thrombin and calcium were added to prepare PRG. The results show that: in NT group, wound healing time was 25.4±3.8 days; in GFT group, wound healing time was 20.6±2.7 days; in LYPT1 group, wound healing time was 16.2±1.3 days; in LYPT2 group, wound healing time was 17.8±2.3 days; Compared with the NT group, wound healing time of GFT, LYPT1 and LYPT2 group were significantly shorter (p<0.05). This indicates that growth factor has therapeutic effect on the difficult healing model group. In LYPT1 and LYPT2 groups, the wounds were healed faster than the wounds in GFT group (p<0.05). The results suggested that the therapy effect of the natural platelet growth factor was superior to a single equivalent rhEGF. There was no significant difference for the healing time in group LYPT1 and LYPT2 (p>0.05), which shows lyophilized platelets reconstituted with saline and platelets gel have the same therapeutic effect.In summary, the SD rats injected by STZ (65mg/kg.b.w) can be used as a stable diabetic animal model, maintaining high blood sugar and urine sugar at least more than 9 weeks. We chose the SD rats of which blood sugar and urine sugar have maintain persistently high level for 5 weeks to establish refractory experimental animal models. Application of lyophilized platelets for treatment can significantly promote wound healing; lyophilized platelets reconstituted with saline and PPP have the same therapy effects. The above results demonstrated that the large number of growth factors in platelets is the key for promoting wound refractory healing, which provide theoretical basis for the clinic treatment of refractory injury (such as: diabetic foot, etc.). |
PDF Full Text Request