A prospective randomized controlled study of antibiotic bone cement in the treatment of diabetic foot ulcer
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摘要:
目的 探讨用抗生素骨水泥治疗糖尿病足溃疡（DFU）的临床效果及其相关机制。 方法 采用前瞻性随机对照研究方法。2020年8月—2022年8月，空军军医大学第一附属医院收治24例符合入选标准的DFU患者。按区组随机化法，将患者分为2组（每组12例），抗生素骨水泥组患者中男7例、女5例，年龄（64±8）岁，溃疡面积（41±21）cm 2；磺胺嘧啶银组患者中男8例、女4例，年龄（62±8）岁，溃疡面积（38±19）cm 2。在确保每例患者膝下主要动脉至少1条通畅的情况下，彻底清创后行持续负压封闭引流3~5 d，此后对抗生素骨水泥组创面采用载庆大霉素骨水泥、磺胺嘧啶银组创面采用磺胺嘧啶银乳膏行换药治疗，换药3周后，均取患侧大腿外侧刃厚皮覆盖创面。清创前与换药3周后均用激光多普勒血流仪检测创面组织及足部正常皮肤组织血流强度，计算创面相对血流强度百分比及血流强度改变率。换药3周后取创缘组织，行免疫组织化学染色观测CD31阳性新生血管数与血管形貌，行免疫荧光染色观察CD31与α平滑肌肌动蛋白（α-SMA）双阳性细胞所围成的血管形态，行免疫荧光染色评估细胞增殖活性（以Ki67阳性细胞比表示），采用蛋白质印迹法检测血管内皮生长因子受体2（VEGFR2）蛋白表达。观察植皮后3~5 d皮片存活情况，记录创面愈合时间。对数据行独立样本 t检验及Fisher确切概率法检验。 结果 2组患者清创前创面相对血流强度百分比相近（ P>0.05）；抗生素骨水泥组患者换药3周后创面相对血流强度百分比及血流强度改变率分别为（44.7±2.0）%、（129±12）%，均显著高于磺胺嘧啶银组的（28.3±1.2）%、（41±8）%（ t值分别为24.15、20.97， P<0.05）。换药3周后，与磺胺嘧啶银组比较，抗生素骨水泥组患者创缘组织中CD31阳性新生血管数显著增多（ t=33.81， P<0.05）且管径更大、排列更有序，CD31与α-SMA双阳性细胞所围成的血管管壁连续性更好，Ki67阳性细胞比、VEGFR2蛋白表达均显著增高（ t值分别为40.97、47.38， P<0.05）。植皮后3~5 d，抗生素骨水泥组所有患者及磺胺嘧啶银组8例患者皮片均存活良好；磺胺嘧啶银组4例患者皮片出现点状/片状坏死，经相应处理后治愈。抗生素骨水泥组患者创面愈合时间为（47.1±2.9）d，明显短于磺胺嘧啶银组的（58.8±2.3）d（ t=10.86， P<0.05）。 结论 相较于磺胺嘧啶银，临床应用抗生素骨水泥治疗DFU具有加快创面愈合、更好重建局部血流的特点，这可能与抗生素骨水泥通过增强VEGFR2的表达促进了创面局部血管有效新生密切相关。 Abstract:Objective To investigate the clinical effects and related mechanism of antibiotic bone cement in treating diabetic foot ulcer (DFU). Methods A prospective randomized controlled study was conducted. From August 2020 to August 2022, 24 patients with DFU who met the inclusion criteria were admitted to the First Affiliated Hospital of Air Force Medical University. According to the block randomization, the patients were divided into 2 groups, with 12 patients in each group. In antibiotic bone cement group, there were 7 male and 5 female patients, aged (64±8) years, with the ulcer area of (41±21) cm 2. In silver sulfadiazine group, there were 8 male and 4 female patients, aged (62±8) years, with the ulcer area of (38±19) cm 2. Under the condition of ensuring the patency of at least one main inferior genicular artery in each patient, the continuous vacuum sealing drainage was performed for 3-5 days after thorough debridement. Thereafter, the wounds in antibiotic bone cement group were treated with gentamicin-laden bone cement, and the wounds in silver sulfadiazine group were treated with silver sulfadiazine cream for dressing change. After 3 weeks of dressing change, the wound was covered with split-thickness skin graft from the lateral thigh on the affected side. Before debridement and after 3 weeks of dressing change, the blood flow intensities of wound tissue and normal skin tissue in foot were measured using laser Doppler flowmeter, and then, the percentage of relative blood flow intensity of wound and the change rate of blood flow intensity were calculated. After 3 weeks of dressing change, the wound margin tissue was taken, the number of CD31-positive neovascular and the vascular morphology were observed and detected by immunohistochemical staining, the morphology of blood vessels surrounded by CD31 and α-smooth muscle actin (α-SMA) double-positive cells was observed by immunofluorescence staining, the cell proliferation activity was evaluated by immunofluorescence staining (denoted as the ratio of Ki67 positive cells), and the protein expression of vascular endothelial growth factor receptor 2 (VEGFR2) was detected by Western blotting. The skin graft survival was observed 3-5 days after skin grafting, and the wound healing time was recorded. Data were statistically analyzed with independent sample t test and Fisher's exact probability test. Results The percentages of relative blood flow intensity of wounds of patients before debridement were similar between the two groups ( P>0.05). After 3 weeks of dressing change, the percentage of relative blood flow intensity of wounds and the change rate of blood flow intensity of patients in antibiotic bone cement group were (44.7±2.0)% and (129±12)%, respectively, which were significantly higher than (28.3±1.2)% and (41±8)% in silver sulfadiazine group (with t values of 24.15 and 20.97, respectively, P<0.05). After 3 weeks of dressing change, compared with those in silver sulfadiazine group, the number of CD31-positive neovascular in the wound margin tissue of patients in antibiotic bone cement group was significantly increased ( t=33.81, P<0.05) with larger diameter and more regular arrangement, the vascular wall continuity surrounded by CD31 and α-SMA double-positive cells was better, and the ratio of Ki67 positive cells and protein expression of VEGFR2 were significantly increased (with t values of 40.97 and 47.38, respectively, P<0.05). On post skin grafting day 3-5, all the patients in antibiotic bone cement group and 8 patients in silver sulfadiazine group had good skin graft survival, while 4 patients in silver sulfadiazine group showed spotted/patchy skin graft necrosis, which were cured after corresponding treatment. The wound healing time of patients in antibiotic bone cement group was (47.1±2.9) d, which was significantly shorter than (58.8±2.3) d in silver sulfadiazine group ( t=10.86, P<0.05). Conclusions Compared with silver sulfadiazine, clinical application of antibiotic bone cement for treating DFU has the characteristics of accelerating wound healing and better reconstruction of local blood flow, which may be closely related to the fact that antibiotic bone cement promoted the local angiogenesis effectively in the wound through enhancing the expression of VEGFR2. -
参考文献
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1 2组糖尿病足溃疡患者换药3周后创缘组织中CD31阳性（棕色）新生血管情况 辣根过氧化物酶-二氨基联苯胺-苏木精×100。1A、1B.分别为磺胺嘧啶银组、抗生素骨水泥组，图1B中新生血管数多于图1A且血管管径更大、排列更有序
注：骨水泥所载抗生素为庆大霉素
2 2组糖尿病足溃疡患者换药3周后创缘组织中CD31与α-SMA双阳性细胞围成血管形态 花青素3-Alexa Fluor 488-4',6-二脒基-2-苯基吲哚×200。2A、2B、2C、2D.分别为磺胺嘧啶银组细胞核染色（蓝色）、α-SMA染色（绿色）、CD31染色（红色）、细胞核与α-SMA和CD31染色重叠图片，双染阳性的细胞数量较少，分布分散；2E、2F、2G、2H.分别为抗生素骨水泥组细胞核染色、α-SMA染色、CD31染色、细胞核与α-SMA和CD31染色重叠图片，图2H中双染阳性的细胞围成血管管壁连续性较图2D好
注：骨水泥所载抗生素为庆大霉素，α-SMA为α平滑肌肌动蛋白
3 2组糖尿病足溃疡患者换药3周后创缘组织中Ki67阳性细胞（红色）情况 花青素3-4′,6-二脒基-2-苯基吲哚×100。3A、3B.分别为磺胺嘧啶银组、抗生素骨水泥组，图3B中Ki67阳性细胞数多于图3A
注：骨水泥所载抗生素为庆大霉素
4 蛋白质印迹法检测的2组糖尿病足溃疡患者换药3周后创缘组织中VEGFR2蛋白表达
注：VEGFR2为血管内皮生长因子受体2，GAPDH为3-磷酸甘油醛脱氢酶；条带上方的1指示磺胺嘧啶银组，2指示抗生素骨水泥组；骨水泥所载抗生素为庆大霉素
5 清创+负压封闭引流+抗生素骨水泥覆盖+刃厚皮移植治疗抗生素骨水泥组患者糖尿病足溃疡的效果。5A.入院时第2~5趾坏疽，足背部溃疡且周围炎症严重；5B.行球囊扩张术开通右胫前动脉后的CT血管造影最大密度投影图（左图）及反最大密度投影图（右图），显示血管通畅；5C.清创+负压封闭引流治疗5 d后，创面炎症得以改善，第2~4跖骨远端外露；5D.抗生素骨水泥覆盖创面后即刻；5E.抗生素骨水泥覆盖3周后，去除抗生素骨水泥后的肉芽组织创面，可见外露跖骨已完全被肉芽组织覆盖；5F.大张刃厚皮移植到创面后即刻；5G.植皮后3 d打开敷料，见皮片存活良好；5H.术后半年，患足创面无破溃，局部皮肤色素沉着
注：骨水泥所载抗生素为庆大霉素
表1 2组糖尿病足溃疡患者临床资料比较
组别 例数 性别（例） 年龄（岁， 病程（d， 溃疡面积（cm 2， Wagner分级（例） 男 女 Ⅲ级 Ⅵ级 抗生素骨水泥组 12 7 5 64±8 55±6 41±21 4 8 磺胺嘧啶银组 12 8 4 62±8 58±6 38±19 6 6 t值 — 0.72 0.90 0.51 — P值 0.673 0.477 0.380 0.618 0.680 注：“—”表示无此统计量值；骨水泥所载抗生素为庆大霉素 
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表2 2组糖尿病足溃疡患者创面血流强度变化情况比较（%，
组别 例数 相对血流强度百分比 血流强度改变率 清创前 换药3周后 抗生素骨水泥组 12 19.5±0.9 44.7±2.0 129±12 磺胺嘧啶银组 12 20.1±1.1 28.3±1.2 41±8 t值 1.47 24.15 20.97 P值 0.155 <0.001 <0.001 注：骨水泥所载抗生素为庆大霉素
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