摘要
实验目的
创伤性骨髓炎目前是临床最具挑战性的课题之一。随生产工具和交通工具的进步,高能量创伤逐步增加,伴有软组织损伤、缺损的开放性骨折不断骤升,其创面被微生物污染,首先形成创面局部感染,最终导致慢性骨髓炎,骨折不愈合。开放性骨折早期正确的创面处理对预防骨髓炎的发生至关重要。臭氧有极好的抗感染和促进创面愈合作用,但同时臭氧也有极强的氧化性,导致毒性反应发生,严重限制了其临床应用,探索研究臭氧在创面处理中合理的应用方法,扬长避短,有望达到预防骨髓炎目的。
实验方法
第一部分：臭氧水局部应用,对清洁创面的作用。
以臭氧水为冲洗液,对新鲜清洁的创面进行间断负压冲洗引流,对照组设置为生理盐水的间断负压冲洗引流,两者对比观察臭氧水对创面愈合的促进作用。
第二部分：不同浓度的臭氧水对局部组织的安全性。
以不同浓度的臭氧水采用负压冲洗引流的方式作用于创面,观察创面内肌肉和神经的组织学变化和功能学变化,以了解臭氧水对创面局部组织的损伤作用。
第三部分：臭氧水局部应用,对感染创面细菌的清除。
首先建立大鼠下肢开放性粉碎性骨折伴感染的动物模型,对感染的创面进行臭氧水负压冲洗引流,最后检测创面内组织的微生物学变化,以了解臭氧水对感染创面的抗感染作用。
结果
第一部分：
1.创面干燥,局部无渗出,无肿胀。不发红,创面明显缩小。
2.对实验组和对照组的创面生长的面积之间采用t检验进行两组样本均数比较,P=0.0047<0.05,具有统计学意义。可以认为进行臭氧水负压冲洗引流,臭氧本身具有促进创面上皮化加快,促进创面愈合的作用。
3.创面周围皮下血管增生明显,创面周围组织内无明显的炎性细胞浸润。
第二部分：
应用浓度的臭氧水采用负压冲洗引流的方法冲洗创面,在冲洗前后,和生理盐水对照方面均未见神经电生理改变。
随时间的延长,创面表面被臭氧氧化后,肌肉颜色逐渐变得深暗,但创面清洁,无明显渗出。
30分钟内肌电图正常,无改变,60分钟时发生肌电图动作电位波幅降低,潜伏期延长。
电镜结果
30分钟内,肌肉电镜检查显示：已经出现少部分的肌节损害,具体表现在肌原纤维断裂,“Z”线消失,为细胞基质所代替。
60分钟时,可见大量的肌原纤维断裂,肌节排列紊乱,“Z”线消失更为普遍,胞质中大量的线粒体空泡化,线粒体嵴小时,并出现多量的髓样小体,巨型线粒体出现,部分线粒体长轴和肌节等长。
有髓神经纤维髓鞘变形,少量的鞘膜损伤离断,轴突内出现髓样小体,线粒体空泡化,微管,微丝排列紊乱,雪旺氏细胞胞体及胞核变化不明显。髓鞘细,厚度薄的鞘膜损伤较为轻。无髓神经纤维变化较轻。
有髓神经纤维髓鞘变形更为普遍,更加明显,出现多量的鞘膜损伤离断,并出现层间解离,鞘膜重组,膜泡小体。轴突内髓样小体,线粒体空泡化,微管,微丝排列紊乱更加明显,雪旺氏细胞胞体及胞核变化不明显。髓鞘细,厚度薄的鞘膜损伤较为轻。无髓神经纤维变化较轻。
第三部分：
1.造模创面周围红肿,有脓性分泌物,患肢体肿胀明显。
2.造模创面表面金黄色葡萄球菌定性检测阳性率达到100%。
3.软组织的金黄色葡萄球菌达到3.26±1.99×105 CFU／g,达到软组织感染标准。
4.骨组织中的金黄色葡萄球菌达到5.05±3.83×105 CFU／g,达到骨感染标准。
5.经臭氧负压冲洗引流,创面清洁,无脓性分泌物,肉芽生长好。
6.经臭氧负压冲洗引流,创面表面金黄色葡萄球菌定性检测未见菌落。
7.经臭氧负压冲洗引流,创面局部组织中未检测到金黄色葡萄球。
结论
第一部分：
2～4mg／L的臭氧水局部负压冲洗引流创面,臭氧本身具有减轻创面局部的炎症反应,促进创面周围血管增生,加快创面上皮化的速度,促进创面愈合作用。
第二部分：。
高浓度的臭氧,随时间的延长,对周围组织的强氧化毒性逐渐增加,在浓度低于26mg／L-33 mg／L,时间60分钟以内,不会出现功能性神经的损伤,治疗浓度的臭氧水,局部负压冲洗引流治疗,由于其浓度和作用时间远远低于产生毒性的界限,因此可以认为其临床应用是安全的。
第三部分：
臭氧水负压冲洗引流感染创面,能有效的清除创面内的金黄色葡萄球菌,在创面的处理中起到抗感染的作用。
臭氧水结合负压引流装置,对创面进行负压冲洗引流,能够实现创面早期覆盖,清除创面的微生物感染,促进创面的愈合,从而达到预防骨髓炎发生的目的。
Objective
Posttraumatic osteomyelitis is one of the most challenging subjects for orthopaedic surgeons. With the developing of industry and traffic, high-energy trauma is increasing. More and more severe open fracture is happening, which characterized by variable degree of soft tissue and skeletal injury. contamination and infection are common. Prevention of wound infection is critical for avoiding posttraumatic osteomyelitis. And appreciated wound management is important to prevention of wound infection. ozoned water can effectively treating wound infection and accelerate wound healing. but while the same time o3 also has a strong oxidative power. unreasonable use of o3 will be toxic. the study of avoiding toxic and inducing a beneficial response of o3 will be meaningful.
Method
Part 1:The role of 03 when the fresh wound is irrigated by ozoned water.
There were 40 Wister mice, divided into 2 groups, ozoned water were treated group and saline were control group, every group had 20 mice. the fresh wound was cut on the animal back .the wound was covered with vacuum-assisted closure (VAC) device, and ozoned water as an instilling water irrigated wound through the VAC device.
The concentration of the ozoned water was 2～4mg/L.
After the irrigation, there were 3 facets were detected:
1、The common assignment around the wound.
2、The surface area of the wound.
3、Histological examination of the wound.
Statistical analysis has performed with t-test for Equality of Means,p values < 0.05 were considered significant. Part 2 the toxic of the local used ozoned water.
step 1: The safety of treated concentration ozoned water on wound.
The nerve was irrigated by treated concentration ozoned water, neural physiological examination of the sciatic nerve was detected,. Statistical analysis has performed with repeated measured data ANOVA.
Step 2:the safety of superier concentration ozoned water on wound.
There were 60 Wister mice ,divided into 4 groups (0、5、30、60min) according to the irrigation time of the ozoned water.
The sciatic nerve and Biceps femoris were discovered in the wound.
Ozoned water irrigated wound through the VAC device, but the concentration was 26.7mg/L-33.4mg/L.
After the irrigation, there were also 3 facets were detected:
1、The common assignment around the wound.
2、Neural physiological examination of the sciatic nerve.
3、Transmission electron microscope examination of the sciatic nerve and Biceps femoris
Part 3 The role of 03 when the infection wound is irrigated by ozoned water.
step 1:Animal model of infection wound of open fracture.
Middle shaft of tibia was revealed, and smashed into fragile .the wound was inoculated with S. aureus, after 24 hours ,the soft tissue and the bone were taken into biological examination.
step 2:the role of o3 when the infection wound is irrigated by ozoned water.
The infection wound was covered with VAC device, and ozoned water was used to irrigate the wound, the saline water irrigation was considered as control group.
5 days later, the irrigated wound was detected
Result
Part 1
1、The fresh wound was clearance, the wound area was contracted.
2、Compared with the control group ,statistical analyze tested the difference of wound area within two groups was significance (p<0.05).
3、Histological examination: compared with the control group, the treated group, Inflammation cells was reduced around the wound, and the capillary propagation was more manifested.
part 2
Step 1:
It was no difference between before and after treated ozoned water irrigation,so was the comparing ozoned water with saline water.
Step 2:As the irrigation time prolonged:
1、The color of the wound get darker, and the excludative fluid was increasing.
2、The latency had a tendency of longer and maximum amplitude had a tendency of shorten within 30 mins the difference amongst the groups Was not significant, but 60 mins group the difference amongst the groups Was significant (p<0.05).
3、The injury of sciatic nerve and the Biceps femoris had a tendency of severer .the 60 mins group the injury of the muscle and nerve was more manifest.
Part 3
Step 1:
The average S. aureus counts was 3.26±1.99×105 CFU/g and 5.05±3.83×105 CFU/g respectively in soft tissue and bone.
step 2:
After 5 days ozoned water irrigation, the S. aureus was not detected both in soft tissue and bone tissue.
conclusion
part 1:2～4mg/L ozoned water had the ability of prompting the wound heal accelerating the epithelium velocity.
part 2:the injury of the nerve and muscle was severer as the time of ozoned water irrigation prolonged, the concentration above 26.7mg/L-33.4mg/L and the time longer than 60 mins the injury will be manifest, otherwise, it was safety.
part 3:2～4mg/L ozoned water irrigated wound through VAC device,the effect of local anti-infection was satisfactory.
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