| BackgroundDiabetic wound including diabetic foot is a common complication of diabetic patients,which has become a worldwide problem due to its difficult healing,high disability rate and poor treatment effect.The development mechanism of diabetic wound and how to promote its healing are hot scientific issues in the field of wound repair.Peripheral nerve degeneration,peripheral small blood vessel occlusion,vascular endothelial cell proliferation restriction,wound angiogenesis obstruction,ischemia and hypoxia injury aggravation,and neurotrophic factor deficiency caused by long-term hyperglycemia have all become key problems restricting the healing of diabetic wound.Therefore,by adding various growth factors to the diabetic wound,transplanting various cells,extracellular vesicles or constructing various new wound dressings and other biological methods,researchers hope to improve the angiogenesis microenvironment on the diabetic wound to promote angiogenesis,but the effect is not satisfactory.Current studies have shown that antibiotic bone cement can affect multiple stages of fracture healing by generating highly vascularized membranous tissues,namely Masquelet induction membranes,during the treatment of patients with large bone defects and osteomyelitis,thus promoting local angiogenesis and accelerating healing.Aiming at the difficult problem of how to effectively and sustainably promote the angiogenesis and accelerate the healing of diabetic wound,the research group tried to adopt the sequential treatment strategy of debridement+antibiotic bone cement covering+skin graft sealing,and performed laser Doppler blood flow detection on the wound blood flow before debridement and after the removal of bone cement,and found that the wound blood flow was significantly improved.It is not clear whether antibiotic bone cement covering in Diabetic foot ulcer(DFU)wounds produces similar"vascularized tissue"that will improve blood flow and speed healing.By means of histomorphology,cell function and molecular biology,this project aims to systematically study the effect of antibiotic bone cement on the angiogenesis of diabetic wounds from the perspectives of prospective clinical trials,model animals and cytological studies,and conduct preliminary mechanism exploration,so as to provide a new therapeutic strategy and theoretical basis for the clinical treatment of diabetic wounds.AimThis study is based on prospective clinical trials,a diabetic rat model with full-thickness back skin defect,and HUVEC cells cultured with high glucose.1.The effect of antibiotic bone cement on improving blood flow and promoting healing in DFU wound treatment;2.The role of antibiotic bone cement in promoting angiogenesis in the full-thickness skin defect wound model of back in diabetic rats and the specific components of this role;3.The effect of antibiotic bone cement on improving the inhibition of high glucose on HUVEC cells and the preliminary mechanism of promoting DFU angiogenesis.Through this study,the promoting effect of antibiotic bone cement on DFU wound angiogenesis was verified,the specific components of its role was studied and its mechanism was preliminarily explored,providing a new treatment strategy and theoretical basis for the clinical treatment of diabetic wounds.Methods1.Use prospective randomized controlled studies.From August 2020 to January 2023,32patients with diabetic foot ulcer who met the inclusion criteria were admitted to the First Affiliated Hospital of Air Force Military Medical University.According to block randomization,the patients were divided into 2 groups with 16 patients in each group.Vascular ultrasound examination ensured the patency of at least one subknee main artery in each patient.Continuous negative pressure closed drainage was performed for 3-5 days after thorough debridement.Thereafter,the wound of antibiotic bone cement group was treated with gentamicin-laden bone cement,and the wound of control group was treated with silver sulfamazine cream dressing change.The wound was covered with a thick blade of the same lateral thigh or scalp of appropriate size.Before debridement and 3 weeks after dressing change,the blood flow intensity of wound tissue and normal skin tissue was measured by laser Doppler flow analyzer,and the percentage of relative blood flow intensity of wound and the change rate of blood flow intensity were calculated.After 3weeks of dressing change treatment,the tissue of the wound margin was taken,and immunohistochemical staining was performed to observe the number of CD31-positive neovascularization in the wound margin tissue,and the blood vessel morphology surrounded by CD31 andαsmooth muscle actin(α-SMA)double positive cells in the wound margin tissue was observed.The hyperplasia of the lesion tissues(represented by Ki67 positive cell ratio)was evaluated,and the Vascular endothelial growth factor receptor-2 was detected by Western blot(WB).VEGFR2 protein expression,Notch signaling pathway Notch1,DLL4 protein expression.Skin survival was observed 3-5 days after skin grafting,and the time of wound healing was calculated.2.Use experimental research methods.24 SPF male SD rats aged 8 weeks were selected to make diabetic model,and the full-layer skin defect wounds were made on the back of the rats.According to random number table method,the rats were divided into phosphate buffer(PBS)group,antibiotic group(gentamicin),simple bone cement group and antibiotic bone cement group(gentamicin-loaded bone cement),with 3 rats in each group,and were treated accordingly.On day 0,3,7,10 and 14 after injury,the wound healing was observed and the healing rate was calculated.The blood intensity of the wound base was measured by Doppler flow meter,and the relative percentage of blood intensity of the wound base in each group was calculated and compared on day 14 after injury.At 10 days after injury,the number of Ki67 positive cells in the wound was detected by immunofluorescence method,and the number of new vessels with CD31 positive staining and tubular structure in the wound was detected by immunohistochemistry method.HE staining was used to detect the defect length of skin tissue on 14 days after injury,immunofluorescence double staining was used to detect the content and distribution of type I collagen and type III collagen in the wound,and WB was used to detect the expression of VEGFR2 protein in the wound and Notch1 and DLL4 protein in Notch signaling pathway.The number of samples in each group was 3.Data rows were repeatedly measured by ANOVA,one-way ANOVA and Tukey test.3.Use experimental research methods.HUVEC cells of the 3rd～5th generation at logarithmic growth stage were selected for experiment.First,HUVEC cells were inoculated on self-made simple bone cement and antibiotic bone cement slices,and the adherent growth of HUVEC cells on the surface of bone cement was observed by scanning electron microscopy.Four batches of cells were collected,and each batch of cells were divided into normal control group,high glucose culture group(glucose content16.7mmol/L)and high glucose+antibiotic bone cement culture group(high glucose+antibiotic bone cement impregnation culture).The first batch of cells were cultured for24h after the completion of treatment,and the cell proliferation level was detected by Edu inclusion method.After the second batch of cells were treated,the scratch test was carried out.The migration of cells was observed under inverted phase contrast microscope at 0h(immediately),24h and 48h after the scratch.After the third batch of cells were treated,the experiment of tubule formation was carried out.Cells were cultured for 6 h after gel and inoculation,and the tubule formation was observed under an inverted phase contrast microscope,and then the number of tubule formation was calculated.The expression of VEGFR2,Notch1 and DLL4 proteins in the fourth batch of cells was detected by WB.The number of samples in each group was 3,and the mean value of the results was taken.Each experiment was repeated three times.Data were analyzed by one-way analysis of variance and Tukey test.Results1.A total of 32 patients were enrolled,including 16 in each group,including 10 males and6 females in the antibiotic bone cement group.The average age of diabetic foot ulcer was(63.6±8.5)years old,the average time of ulcer was(55.6±6.4)d,and the average area of ulcer was(6-68)cm~2,(41.2±18.8)cm~2.WagnerⅢgrade 6 cases,Ⅳgrade 10 cases;In the silver sulfadiazine group,there were 9 males and 7 females,aged(61.6±8.5)years,the mean time of diabetic foot ulcer was(56.9±5.8)d,the mean area of ulcer was(5～62)cm~2,the mean was(37.6±17.1)cm~2,8 cases of WagnerⅢgrade and 8 cases ofⅣgrade.There were no significant differences in age,gender,course of disease,ulcer area and Wagner grade between the two groups before surgery(P>0.05).The percentage of wound relative blood flow intensity before debridement was similar between the two groups(P<0.01);After 3 weeks of dressing change in antibiotic bone cement group,the percentage of relative blood flow intensity in wound tissue and the change rate of blood flow intensity were(43.40±2.87)%and(122.5±13.47)%,respectively.The values of(29.72±2.91)%and(47.46±14.29)%of silver sulfadiazine group were significantly higher than those of silver sulfadiazine group(P<0.01).After 3 weeks of dressing change,HE staining showed that the blood vessels in the wound margin tissue of the antibiotic bone cement group were more than those in the silver sulfadiazine group,and the blood vessels were arranged in a more orderly manner.The number of CD31-positive new vessels in the wound margin of antibiotic bone cement group was(83.7±4.3)/field,significantly higher than that in silver sulfadiazine group(36.9±3.4)/field,(P<0.01),and the vascular wall continuity of CD31 andα-SMA positive cells was better than that of silver sulfadiazine group.The proportion of Ki67 positive cells,VEGFR2 positive cells and VEGFR2 protein expression in the wound margin of antibiotic bone cement group were(3.54±0.28)%,(1.57±0.18)%and 0.841±0.046,respectively.Those of silver sulfadiazine group were significantly higher than(1.89±0.14)%,(0.11±0.02)%and 0.321±0.038(P<0.01);The expression levels of Notch1 protein and DLL4 protein in the wound tissue of antibiotic bone cement group were 0.227±0.030 and 0.080±0.017,respectively,significantly lower than those of silver sulfadiazine group(0.743±0.028 and 0.521±0.034)(P<0.01).At 3-5days after skin grafting,all patients in the antibiotic bone cement group and 10 patients in the silver sulfadiazine group had good skin survival.6 patients in silver sulfadiazine group showed spot-like/flaky skin necrosis and were cured after corresponding treatment.The wound healing time of antibiotic bone cement group was(42.6±2.3)d,which was significantly shorter than that of silver sulfadiazine group(51.1±4.2)d(P<0.01).2.3 days after injury,the wounds of rats in the 4 groups were all redness and swelling around the wound,and the wounds did not decrease significantly;7 days after the injury,the wounds of the 4 groups were gradually reduced and healed.On day 10 after injury,the wound in the antibiotic bone cement group was significantly reduced compared with the other three groups.14 days after injury,the wound in the antibiotic bone cement group was basically healed,while the residual wound in the other three groups was found.14days after injury,the wound healing rate of antibiotic bone cement group was significantly higher than that of control group,antibiotic group and bone cement group(P<0.01).On day 3 after injury,the blood supply of the trauma group was poor in the 4 groups.On the7th day after injury,the invasive blood flow in the antibiotic bone cement group began to increase,while the other 3 groups only had traumatic hyperemia.10 days after injury,there was still no obvious blood flow outside the invasive group in the PBS group and the antibiotic group,and the other two groups had microangiogenesis.On 14 days after injury,the blood flow signal in the antibiotic bone cement group was stronger than that in the other three groups,and the distribution was uniform.14 days after the injury,The percentage of relative blood flow intensity in antibiotic bone cement group(72.93±2.40)%was significantly higher than that in PBS group(33.53±1.72)%,antibiotic group(37.73±0.47)%and bone cement group(53.23±2.14)%(P<0.01);The proportion of Ki67 positive cells in the antibiotic bone cement group(7.60±0.26)%was significantly higher than that in PBS group(2.20±0.36)%,antibiotic group(2.20±0.36)%and bone cement group(2.47±0.21)%(P<0.01);In the antibiotic bone cement group,there were34.7±1.5 new blood vessels per field under 100 times visual field.Significantly more than PBS group(13.7±1.5)/field,antibiotic group(15.3±2.1)/field and bone cement group(18.7±2.1)/field(P<0.01).14 days after the injury,The tissue defect length(0.25±0.01)cm in antibiotic bone cement group was significantly shorter than that in PBS group(0.83±0.01)cm,antibiotic group(0.64±0.01)cm and bone cement group(0.45±0.01)cm(P<0.01);On day 14 after injury,the contents of typeⅠcollagen and typeⅢcollagen in antibiotic bone cement group were significantly higher than those in PBS group,antibiotic group and bone cement group(P<0.01).On day 14 after injury,VEGFR2 protein expression in the wound tissue of antibiotic bone cement group was significantly higher than that of PBS group,antibiotic group and bone cement group(P<0.01);The expressions of Notch1 protein and DLL4 protein in the wound tissue of antibiotic bone cement group were significantly lower than those in PBS group,antibiotic group and bone cement group(P<0.01).3.Scanning electron microscopy showed that the surface of antibiotic bone cement was uneven,and HUVEC cells could not grow on the surface.Compared with the normal control group,the proportion of Edu positive HUVEC cells in the high glucose culture group was significantly decreased after 24 h culture(P<0.01),24h Edu positive cells in high glucose culture+antibiotic bone cement group were higher than those in high glucose culture group(P<0.01).24h and 48h after scratch,compared with the normal control group,the open wound width of(213±2)um and(22±3)um increased significantly in the high glucose culture group(398±4)um and(163±4)um)(P<0.01),the open width of wound in the high sugar+antibiotic bone cement treatment group was lower(349±4)um and(100±6)um in the high sugar culture group(P<0.01).After 6h of tube culture,compared with the normal control group(25.3±2.1)cells/field,the number of cell tubes in the high glucose culture group(2.0±1.0)cells/field was significantly reduced(P<0.01),the number of cell tubules in the high glucose+antibiotic bone cement treatment group was(10.3±1.5)higher than that in the high glucose culture group(P<0.01).WB assay showed that the expression of VEGFR2 protein was significantly decreased in the high glucose culture group compared with the normal control group(P<0.01),the VEGFR2protein expression in the high sugar+antibiotic bone cement group was higher than that in the sugar culture group(P<0.01);Notch1 and DLL4 protein expressions of Notch signaling pathway were significantly increased in the high-glucose culture group compared with the normal control group(P<0.01),the expressions of Notch1 and DLL4protein in the high glucose+antibiotic bone cement treatment group were decreased in the high glucose culture group(P<0.01).Conclusions1.Compared with silver sulfadiazine cream,antibiotic bone cement in the treatment of diabetic foot ulcer has the characteristics of shorter healing time and better local blood flow reconstruction,which may be closely related to antibiotic bone cement promoting effective local blood vessel formation on the wound by enhancing the expression of VEGFR2.2.Antibiotic bone cement can increase neovascularization in DFU tissue,thus significantly promoting full-layer skin defect wound healing in diabetic rats.The mechanism may be related to antibiotic bone cement improving VEGFR2 protein expression in wound tissue by down-regulating Notch signaling pathway.3.Antibiotic bone cement may enhance VEGFR2 protein expression,promote HUVEC cell proliferation,migration and tubulogenesis by inhibiting the over-activated Notch signaling pathway in a high-glucose environment,and ultimately promote angiogenesis in a high-glucose environment. |
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