Effects of advanced platelet-rich fibrin/chitosan thermosensitive hydrogel on full-thickness skin defect wound healing in diabetic rats
Objective To prepare advanced platelet-rich fibrin(A-PRF)/chitosan thermosensitive hydrogel(hereinafter referred to as composite hydrogel)and explore the effects of composite hydrogel on full-thickness skin defect wound healing in diabetic rats.Methods This study was an experimental study.The composite hydrogel with porous mesh structure and thermosensitive characteristics was successfully prepared,containing A-PRF with mass concentrations of 10,15,20,50,and 100 g/L.Diabetic model was successfully established in male Sprague-Dawley rats aged 6-8 weeks by intraperitoneal injection of streptozotocin,and 4 full-thickness skin defect wounds were established on the back of each rat(finally the model was successfully established in 36 rats).Three wounds of each rat were divided into blank group(no drug intervention),positive control group(dropping recombinant human granulocyte-macrophage stimulating factor gel),and chitosan hydrogel group(dropping chitosan hydrogel solution).Thirty rats were collected,and the remaining one wound of each rat(totally 30 wounds)was divided into 10,15,20,50,and 100 g/L composite hydrogel groups,with 6 wounds in each group,which were dropped with composite hydrogel solution containing 10,15,20,50,and 100 g/L A-PRF,respectively.Taking the remaining six rats,the remaining one wound from each rat was dropped with composite hydrogel solution containing 100 g/L A-PRF.On 14 d after injury,6 rats with one wound dropped with composite hydrogel containing 100 g/L A-PRF were selected for hematoxylin-eosin(HE)staining to observe the inflammation,hemorrhage,or necrosis of the heart,liver,spleen,lung,and kidney.On 10 d after injury,6 rats with one wound dropped with composite hydrogel containing 15 g/L A-PRF were selected to observe the blood perfusion of wounds in the four groups(with sample size of 6).On 7 and 14 d after injury,the wound healing rates in the eight groups were calculated.On 14 d after injury,the wound tissue in the eight groups was taken for HE and Masson staining to observe the formation of new epithelium and collagen formation,respectively;the positive expressions of CD31 and vascular endothelial growth factor A(VEGFA)were detected by immunohistochemistry,and the percentages of positive areas were calculated;the protein expressions of CD31 and VEGFA were detected by Western blotting;the mRNA expressions of CD31 and VEGFA were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction method(with all sample sizes of 4).Results On 14 d after injury,no obvious inflammation,hemorrhage,or necrosis was observed in the heart,liver,spleen,lung,and kidney in the 6 rats.On 10 d after injury,the blood perfusion volume of wound in 15 g/L composite hydrogel group was significantly more than that in blank group,positive control group,and chitosan hydrogel group,respectively(with P values all＜0.05).On 7 and 14 d after injury,the wound healing rates of blank group were(26.0±8.9)％and(75.0±1.8)％,which were significantly lower than those of positive control group,chitosan hydrogel group,and 10,15,20,50,and 100 g/L composite hydrogel groups,respectively((45.8±3.2)％,(49.8±3.7)％,(51.2±2.9)％,(68.5±2.4)％,(68.8±1.5)％,(72.7±2.1)％,(75.0±3.7)％and(79.1±1.9)％,(77.2±1.7)％,(82.3±1.3)％,(89.6±1.9)％,(89.8±1.3)％,(87.3±1.1)％,(87.9±1.3)％),P＜0.05;the wound healing rates of positive control group,chitosan hydrogel group,and 10 g/L composite hydrogel group were significantly lower than those of 15,20,50,and 100 g/L composite hydrogel groups(P＜0.05).On 14 d after injury,the wound epithelialization degrees of 15,20,50,and 100 g/L composite hydrogel groups were higher than those of the other 4 groups,the new microvascular situation was better,and the collagen was more abundant and arranged more neatly.On 14 d after injury,the percentages of CD31 and VEGFA positive areas in wounds in positive control group and the percentage of VEGFA positive area in wounds in chitosan hydrogel group were significantly higher than those in blank group(P＜0.05),the percentage of VEGFA positive area in wounds in 10 g/L composite hydrogel group was significantly higher than that in blank group,chitosan hydrogel group,and positive control group(with P values all＜0.05),and the percentages of CD31 and VEGFA positive areas in wounds in 15,20,50,and 100 g/L composite hydrogel groups were significantly higher than those in blank group,positive control group,chitosan hydrogel group,and 10 g/L composite hydrogel group(P＜0.05).On 14 d after injury,the protein and mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group,positive control group,and 10 g/L composite hydrogel group were significantly higher than those in blank group(P＜0.05);the protein expression of VEGFA in wound tissue in 10 g/L composite hydrogel group was significantly higher than that in positive control group(P＜0.05),and the mRNA expressions of CD31 and VEGFA in wound tissue in 10 g/L composite hydrogel group were significantly higher than those in positive control group and chitosan hydrogel group(P＜0.05);the protein and mRNA expressions of CD31 and VEGFA in wound tissue in 15,20,50,and 100 g/L composite hydrogel groups were significantly higher than those in blank group,positive control group,chitosan hydrogel group,and 10 g/L composite hydrogel group(P＜0.05);the mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group were significantly lower than those in positive control group(P＜0.05).Conclusions The composite hydrogel has high biological safety,can improve wound blood perfusion,effectively promote the formation of blood vessels and collagen in wound tissue,thus promoting the wound healing of full-thickness skin defects in diabetic rats.15 g/L is the optimal mass concentration of A-PRF in composite hydrogel.
NETL
NSTL
万方数据
