庆大霉素脂质体抗菌骨治疗兔慢性感染性骨缺损的实验研究
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摘要
细菌生长主要以浮游和贴附两种形式进行。细菌贴附于实体表面后一方面进行分裂增殖,另一方面分泌胞外多糖基质,将细菌包裹于基质内,形成细菌生物膜。由于胞外基质的物理和电荷屏障以及内部特殊的生态环境,细菌生物膜所致感染较浮游细菌更难防治,是包括慢性骨髓炎在内的多种慢性感染性疾病的主要病因之一。一旦细菌生物膜在骨缺损周围骨组织、死骨、内固定或人工关节表面滋生,将导致反复发作的慢性骨髓炎,给患者带来极大的痛苦。PMMA、Osteoset T在局部缓释抗生素,提高了骨髓炎的治愈率,但PMMA需二次手术取出,同时研究表明单纯清创与清创后植入Osteoset T在骨髓炎的治愈率方面无明显差异。此外,高浓度庆大霉素可抑制人成骨细胞和内皮细胞的增殖,从而影响骨折愈合。慢性感染性骨缺损实际上是一种合并骨缺损的慢性骨髓炎,其主要病因仍是细菌生物膜的形成。
脂质体是磷脂分散在水中定向排列而形成的囊泡,可包封抗生素,按表面电荷不同分为阳离子、阴离子和中性脂质体。研究表明,阳离子脂质体青霉素对金黄色葡萄球菌(Staphylococcus aureus,S.aureus)生物膜的抑制作用显著强于青霉素,其机制与S.aureus生物膜带负电荷有关;另一方面,脂质体可使包封药物在骨髓浓聚,因此静脉注射脂质体抗生素可增强骨感染灶局部抗生素浓度,进而消除感染。同时,脂质体可降低高浓度抗生素对成骨细胞、内皮细胞的毒性作用。然而人体网状内皮系统对血液中的脂质体具有很强的清除作用,大大降低了脂质体抗生素的半衰期,削弱其抗感染能力。由于抗生素缓释系统可在感染灶释放高浓度抗生素,因此,将脂质体抗生素负载于载体在理论上可解决这一问题。但脂质体抗生素负载于载体后再释放的抗细菌生物膜作用是否受到影响尚不明确。本研究首先制备阳离子脂质体庆大霉素,将其负载于同种异体骨和硫酸钙,分别进行释放实验,并探讨释放的阳离子脂质体庆大霉素对S.aureus ATCC 29213生物膜生长的抑制作用,然后构建兔慢性感染性骨缺损模型,了解S.aureus ATCC 29213注射剂量对该模型的影响,并行阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用。此外,研究还探讨了脂质体抗生素静脉注射对兔慢性感染性骨缺损的治疗作用。
本课题包括四部分:1、S.aureus注射剂量对兔慢性感染性骨缺损形成的影响。2、脂质体庆大霉素静脉注射对兔慢性感染性骨缺损的治疗作用。3、阳离子脂质体抗菌骨的制备以及体外对S.aureus生物膜生长的抑制作用。4、阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用。
第一部分S.aureus注射剂量对兔慢性感染性骨缺损形成的影响
实验方法
1构建兔慢性感染性骨缺损模型,制备骨缺损,微量注射器先后注入S.aureusATCC 29213,注射总量分别为6×10~1-6×10~6CFU。
2术后3w处死所有实验兔。计算体重变化,评估肉眼观察评分及兔放射学改变。将兔左下肢胫骨取出进行细菌学检查,测量并计算平均每克标本中的细菌数。
主要实验结果
注射剂量达6×10~5CFU即可使所有实验兔在肉眼观察评分、放射学检查及细菌学检查呈现出明显感染征象。
第二部分脂质体庆大霉素静脉注射治疗兔慢性感染性骨缺损的实验研究
实验方法
1不同脂质体庆大霉素的制备
三种原材料(阳、阴离子脂质体)或两种(中性脂质体)按照一定比例混合,旋转蒸干制成脂质膜,加入庆大霉素溶液,振荡制成脂质悬液,超声处理后离心去除上清中尚未包被的庆大霉素,沉淀以ddH_2O重悬制成庆大霉素脂质体。
2兔慢性感染性骨缺损模型的制备
兔麻醉后消毒铺巾,切开皮肤,暴露胫骨近端骨面,制造骨缺损,骨蜡封闭,先后注入精氨酸和S.aureus ATCC 29213,缝合皮肤。造模术后行大体观察,X线检查,X线检查提示慢性骨髓炎后开始治疗实验。
3脂质体庆大霉素静脉注射治疗兔慢性感染性骨缺损
造模成功后分组进行不同脂质体庆大霉素静脉注射治疗,治疗3w后处死实验动物。处死前抽血、X检查,处死后取左下肢近端胫骨骨髓行细菌学检查。
主要实验结果:
对照组血细菌培养和骨细菌培养均为阳性;阳离子脂质体庆大霉素治疗组血培养均为阴性,骨培养4只阴性;阴离子脂质体庆大霉素组和单独庆大霉素治疗组血培养呈阴性,但骨培养均为阳性;中性脂质体庆大霉素治疗组血培养阴性,骨培养2只阴性;
第三部分阳离子脂质体抗菌骨的制备及体外对葡萄球菌生物膜的抑制作用
实验方法
1阳离子脂质体抗菌骨的制备
阳离子脂质体庆大霉素的制备与第二部分相同。无菌取兔胫骨及股骨干骺端,经脱脂、脱蛋白、脱钙后制得兔同种异体骨。将上述同种异体骨块及硫酸钙浸泡至阳离子脂质体庆大霉素溶液中,蒸干,~(60)Co辐照消毒。
2阳离子脂质体抗菌骨的体外释放实验
将阳离子脂质体庆大霉素抗菌骨置于微孔板,加入ddH_2O,室温振荡。于振荡后不同时间更换ddH_2O,并将所更换出的液体以Triton X-100进行处理,测定庆大霉素浓度。
3阳离子脂质体抗菌骨体外对葡萄球菌生物膜的抑制作用
培养S.aureus ATCC 29213生物膜。分别取释放实验中8个不同时相点释放的阳离子脂质体庆大霉素溶液,并以上述8个时相点的释放浓度配制新鲜阳离子脂质体庆大霉素、庆大霉素,将各组抗生素分别加入S.aureus ATCC29213生物膜,1h后弃去上清,加入培养基,并于此后每隔30min检测每孔溶液在630nm的吸光度。计算细菌生长速率,并得出最大细菌生长速率及所需时间。
主要实验结果:
阳离子脂质体庆大霉素抗菌骨在释放前期具有爆发释放效应,且在释放前期的特殊时间段所释放的庆大霉素总百分比与(时间)~(1/2)成线性关系(R~2＞0.9)。由阳离子脂质体庆大霉素抗菌骨中释放的阳离子脂质体庆大霉素对S.aureus ATCC 29213生物膜生长的抑制作用与新配制的同浓度阳离子脂质体庆大霉素无显著差异(P＞0.05),二者在低浓度时对S.aureus ATCC 29213生物膜生长的抑制作用显著强于单独使用庆大霉素(P＜0.01)。此外,庆大霉素对S.aureus ATCC 29213的MIC为230.4 mg/L。
第四部分阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用
实验方法
1阳离子脂质体抗菌骨及兔慢性感染性骨缺损模型的制备与第三部分相同。
2实验兔分组,进行静脉注射、手术清创植入抗菌骨治疗。2w后行X线检查,抽静脉血行血液培养,处死后取局部骨髓组织行细菌学检查。
主要实验结果:
对照组没有抗生素治疗,均出现明显感染,血液及骨髓培养均呈S.aureus阳性。单独手术植入同种异体骨或硫酸钙组评分较A组更高,但差异不显著(P＞0.05)。单独使用庆大霉素治疗2w可使兔血液培养阴性,且感染评分低于对照组,二者相差显著(P＜0.05),且全部骨髓培养均阳性。使用阳离子脂质体庆大霉素静脉注射治疗可有效清除血液感染,评分低于A、B、H、I组,且与上述4组相差显著(P＜0.05),同时评分高于F、G组,相差显著(P＜0.05),治疗的6只兔中有2只骨髓培养阴性。采用庆大霉素同种异体骨和庆大霉素硫酸钙治疗效果相似,低于F、G组(P＜0.05),治疗的6只兔中有3只骨髓培养阴性。两种阳离子脂质体抗菌骨疗效相似,可完全清除血液、骨髓感染,感染评分低于上述各组,且相差显著(P＜0.05)。
主要结论
1、耐药S.aureus可诱导明显的胫骨感染性骨缺损形成,6×10~5 CFU/5ul的剂量不仅可使慢性骨髓炎模型制备成功率达100%,而且能够出现较明显的放射学和细菌学改变。
2、脂质体庆大霉素可有效治疗慢性感染性骨缺损,其中以阳离子脂质体庆大霉素效果最为明显,中性次之,阴离子脂质体庆大霉素与单独使用庆大霉素相同,但任何一种脂质体庆大霉素均不能完全清除感染。
3、低温真空负压吸引法制备阳离子脂质体庆大霉素抗菌骨并不影响所释放的阳离子脂质体庆大霉素对S.aureus生物膜的抑制作用,且在低浓度对S.aureus生物膜抑制显著强于单独使用庆大霉素。
4、手术清创后植入阳离子脂质体庆大霉素抗菌骨对兔胫骨S.aureus慢性感染性骨缺损的治疗效果优于庆大霉素抗菌骨以及静脉输注阳离子庆大霉素、庆大霉素。
Both bacterial biofilms and floating bacteria are two forms of bacterial growth. The bacterial biofilms can formed as surface-bound organisms begin to actively replicate (and die) and extracellular components generated by attached bacteria interact with organic and inorganic molecules in the immediate environment to create the glycocalyx. Biofilm organisms are far more resistant to antimicrobial agents than organisms in suspension. In some extreme cases, the concentrations of antibiotics required to achieve bactericidal activity against adherent organisms can be three to four orders of magnitude higher than for planktonic bacteria, depending on the species-drug combination, which is the main reason of the recrudescence of chronic osteomyelitis. Once bacteria have irreversibly attached to a surface of bone, dead bone, internal fixation or artificial joint, the process of biofilm maturation begins, and the infection of bone is recurrent attacks, which bring more pain to the patients. Polymethylmethacrylate(PMMA) and Osteoset T can delayed release antibiotics in the infected bone, and improve the healing rate. However, PMMA need to be taken out after operation. At the same time, the cure rate of implantation of Osteoset T after debridement is similar to debridement alone. In addition, it is indicated that high concentration of gentamicin can inhibit the proliferation of osteoblast and endothelial cell, which can inhibit the union of fracture.
Liposomes are the vesicles which are formed from the arrangement of phospholipid during the dispersion in the water. Liposomes can encapsule antibiotics and are devided to cation, anion and neutral liposomes. It is indicated that the inhibited effect of the penicillin encapsulated in the cation liposomes to the Staphylococcus aureus(S. aureus) biofilms is much better than the penicillin alone, and the mechanism is related to the negative charge of the surface of the S. aureus biofilms. On the other hand, liposomes can make the drug gathered in the bone marrow, which make it possible that infection of bone is eliminated by intravenous injection of antibiotics impregnated in the liposomes. At the same time, liposomes can reduce the toxic effect of high concentration antibiotics to the osteoblast and the endothelial cell significantly. However, the reticuloendothelial system (RES) of human body can eliminate the liposomes in the blood, which induces the half life period of the liposomes and weakens the anti-infection ability of the liposomal antibiotics. Due to the delayed release of high concentration antibiotics of antibiotics delayed released system in the area of bone infection, the weaken of RES to the liposomal antibiotics can be solved by impregnating the liposomal antibiotics to the carrier of antibiotics delayed released system. However, it is unknow whether the anti-biofilm ability of the liposomal antibiotics released from the antibiotics delayed released system is affected. The cation liposomal gentamicin was prepared in our reasearch firstly. Then the cation liposomal gentamicin was impregnated in allogeneic cortical bone of rabbit or calcium sulfate, and the release experiment was carried out. The anti-biofilm effect of cation liposomal gentamicin released from the substitution of bone was explored. During the preparation of the mode of chronic bone infection with bone defect, the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic osteomy elitis with bone defect. In addition, the treatment of injection of three kinds of liposomal gentamicin to the chronic bone infection with bone defect of rabbit was also done.
The study includes four parts. Part one: the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic bone infection with bone defect of rabbit. Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit. Part three: the preparation of cation liposomal getamicin antimicrobial bone and the effect of anti-biofilm in vitro. Part four: the treatment of cation liposomal antimicrobial bone to the chronic tibial infection with bone defect of rabbit.
Main experiments show below:
Part one: the affection of the injected dose of S. aureus to the formation of chronic bone infection with bone defect of rabbit.
Methods:
1 S. aureus(ATCC 29213) with total number of colony from 6×10~1 to 6×10~6 was prepared. The bacteria were injected in the tibia after an area of bone defect was made.
2 The inoculated lesions were assessed for changes in macroscopic observation, radiological and bacteriological parameters at 3 weeks after infection.
Main results:
Obvious infection sign can be seen in the E and F groups due to the macroscopic observation, radiological and bacteriological parameters. There is significant difference between both E, F groups and other groups. However there is no difference between E and F group.
Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit.
Methods:
1 The preparation of different liposomal gentamicin
Three liposomal compositions were mixed in a round-bottomed flask and dried to a thin lipid film with a rotary evaporator. The lipids were then hydrated by agitation in aqueous solution of gentamicin. Then the lipid suspensions were sonicated for 40 s in an ultrasonic bath. Unencapsulated gentamicin was removed by centrifugation. The final pellets were resuspended in distilled water.
2 The preparation of chronic tibial infection with bone defect of rabbit.
After anesthetized, the tibial skin of rabbit was cut off, and the tibia was exposed. The bone defect was made and sealed with bone wax. The ATCC 29213 was injected in the bone defect. Treatment was done till the infection formed through the macroscopic and radiological observation.
3 The treatment of liposomal gentamicin to the chronic tibial infection with bone defect.
The treatment with injection of liposomal gentamicin began after the chronic tibial infection with bone defect of rabbit was made successfully. The rabbit was executed at 3m after the treatment and the macroscopic observation, radiological and bacteriological examinations were done.
Main results:
The bacterial culture of blood was posive in A group, and all negative in other groups. The bacterial culture of tibia was posive in A, C, E groups, 4 negative in B group, and 2 negative in D group.
Part three: Preparation of liposomal gentamicin-impregnated allogeneic cortical bone and the effect of antin-biofilm in vitro.
Methods:
1 Preparation of the cation liposomal antimicrobial bone
The preparation of the cation liposomal gentamicin is the same with part two. The metaphysic of tibia and femur was obtained, defated in mixature of chloroform and methanol, deproteinized in H_2O_2, and decalcificated in HC1. The production was observed by scanning electron microscope. The allogeneic cortical bone and calcium sulfate were impregnated in the cation liposomal gentamicin, evaporation to dryness, and sterilized by irradiation of ~(60)Co.
2 Release experiment of cation liposomal antimicrobial bone in vitro
Specimens of liposomal gentamicin-impregnated allogeneic cortical bone and CS were immersed in dd H_2O on a shaking device. The water was replaced at different time points. The replaced water at different time points was added Triton X-100. After rupturing the liposomes, gentamicin concentrations were measured using an AxSym System.
3 Release experiment and regrowth assay
Liposomal gentamicin released at eight time points during the release experiment was chosen to detect the effectiveness of anti-biofilm which was assessed from the rate of regrowth of bacteria from the biofilms after exposure to the cation liposomal gentamicin, relative to regrowth after exposure to an equivalent concentration of free drug or liposomal gentamicin prepared freshly. After exposure of the biofilms to the test samples the plate wells were washed with PBS and growth medium was added and the absorbance of the growth medium was measure at 30 min intervals at 630nm over a period of 24h. The plate reader was programmed to read the optical density vs. time profile and determine the time to reach maximum growth rate(Dod/dt)max. These times were compared with those from growth of biofilms exposed to only nutrient broth(positive controls).
Main results:
The combination of liposomal gentamicin and allogeneic cortical bone showed initial burst-release of active liposomal gentamicin and had continuous-release.
Liposomal gentamicin released from allogeneic cortical bone had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration.
Part four: The treatment of osteomyelitis with bone defect by liposomal gentamicin-impregnated allogeneic cortical bone or calcium sulfate
Methods:
1 The preparation of the cation liposomal antimicrobial bone and the chronic tibial infection with bone defect was the same with part three.
2 After the preparation of the chronic tibial infection, rabbits were treated with intravenous injection of liposomal gentamicin or debridement with implantion of the cation liposomal antimicrobial bone. At 2w after treatment, radiological and bacteriological examinations were done.
Main results:
Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with cation liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was lower slightly than the result treated with implantation of gentamicin-impregnated calcium sulfate(66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin anti microbial bone treated for 14 days.
Conclusions:
1. Development of significant histological and radiological signs of infected bone defect required an inoculum of at least 6×10~5 CFU/5 ul.
2. Cationic liposomes change the pharmacokinetics of gentamicin which can improve the concentration of gentamicin in the bone marrow. Cationic liposomal gentamicin was most effect to cure the chronic osteomyelitis with bone defect among the three liposomal gentamicin.
3. The anti-biofilm effciency of liposomal gentamicin was not affected by the impregnating process. Compared with free drug, liposomal gentamicin is very effective in inhibiting the regrowth of cells from S. aureus biofilms at low drug concentration.
4. The new drug delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.
脂质体是磷脂分散在水中定向排列而形成的囊泡,可包封抗生素,按表面电荷不同分为阳离子、阴离子和中性脂质体。研究表明,阳离子脂质体青霉素对金黄色葡萄球菌(Staphylococcus aureus,S.aureus)生物膜的抑制作用显著强于青霉素,其机制与S.aureus生物膜带负电荷有关;另一方面,脂质体可使包封药物在骨髓浓聚,因此静脉注射脂质体抗生素可增强骨感染灶局部抗生素浓度,进而消除感染。同时,脂质体可降低高浓度抗生素对成骨细胞、内皮细胞的毒性作用。然而人体网状内皮系统对血液中的脂质体具有很强的清除作用,大大降低了脂质体抗生素的半衰期,削弱其抗感染能力。由于抗生素缓释系统可在感染灶释放高浓度抗生素,因此,将脂质体抗生素负载于载体在理论上可解决这一问题。但脂质体抗生素负载于载体后再释放的抗细菌生物膜作用是否受到影响尚不明确。本研究首先制备阳离子脂质体庆大霉素,将其负载于同种异体骨和硫酸钙,分别进行释放实验,并探讨释放的阳离子脂质体庆大霉素对S.aureus ATCC 29213生物膜生长的抑制作用,然后构建兔慢性感染性骨缺损模型,了解S.aureus ATCC 29213注射剂量对该模型的影响,并行阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用。此外,研究还探讨了脂质体抗生素静脉注射对兔慢性感染性骨缺损的治疗作用。
本课题包括四部分:1、S.aureus注射剂量对兔慢性感染性骨缺损形成的影响。2、脂质体庆大霉素静脉注射对兔慢性感染性骨缺损的治疗作用。3、阳离子脂质体抗菌骨的制备以及体外对S.aureus生物膜生长的抑制作用。4、阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用。
第一部分S.aureus注射剂量对兔慢性感染性骨缺损形成的影响
实验方法
1构建兔慢性感染性骨缺损模型,制备骨缺损,微量注射器先后注入S.aureusATCC 29213,注射总量分别为6×10~1-6×10~6CFU。
2术后3w处死所有实验兔。计算体重变化,评估肉眼观察评分及兔放射学改变。将兔左下肢胫骨取出进行细菌学检查,测量并计算平均每克标本中的细菌数。
主要实验结果
注射剂量达6×10~5CFU即可使所有实验兔在肉眼观察评分、放射学检查及细菌学检查呈现出明显感染征象。
第二部分脂质体庆大霉素静脉注射治疗兔慢性感染性骨缺损的实验研究
实验方法
1不同脂质体庆大霉素的制备
三种原材料(阳、阴离子脂质体)或两种(中性脂质体)按照一定比例混合,旋转蒸干制成脂质膜,加入庆大霉素溶液,振荡制成脂质悬液,超声处理后离心去除上清中尚未包被的庆大霉素,沉淀以ddH_2O重悬制成庆大霉素脂质体。
2兔慢性感染性骨缺损模型的制备
兔麻醉后消毒铺巾,切开皮肤,暴露胫骨近端骨面,制造骨缺损,骨蜡封闭,先后注入精氨酸和S.aureus ATCC 29213,缝合皮肤。造模术后行大体观察,X线检查,X线检查提示慢性骨髓炎后开始治疗实验。
3脂质体庆大霉素静脉注射治疗兔慢性感染性骨缺损
造模成功后分组进行不同脂质体庆大霉素静脉注射治疗,治疗3w后处死实验动物。处死前抽血、X检查,处死后取左下肢近端胫骨骨髓行细菌学检查。
主要实验结果:
对照组血细菌培养和骨细菌培养均为阳性;阳离子脂质体庆大霉素治疗组血培养均为阴性,骨培养4只阴性;阴离子脂质体庆大霉素组和单独庆大霉素治疗组血培养呈阴性,但骨培养均为阳性;中性脂质体庆大霉素治疗组血培养阴性,骨培养2只阴性;
第三部分阳离子脂质体抗菌骨的制备及体外对葡萄球菌生物膜的抑制作用
实验方法
1阳离子脂质体抗菌骨的制备
阳离子脂质体庆大霉素的制备与第二部分相同。无菌取兔胫骨及股骨干骺端,经脱脂、脱蛋白、脱钙后制得兔同种异体骨。将上述同种异体骨块及硫酸钙浸泡至阳离子脂质体庆大霉素溶液中,蒸干,~(60)Co辐照消毒。
2阳离子脂质体抗菌骨的体外释放实验
将阳离子脂质体庆大霉素抗菌骨置于微孔板,加入ddH_2O,室温振荡。于振荡后不同时间更换ddH_2O,并将所更换出的液体以Triton X-100进行处理,测定庆大霉素浓度。
3阳离子脂质体抗菌骨体外对葡萄球菌生物膜的抑制作用
培养S.aureus ATCC 29213生物膜。分别取释放实验中8个不同时相点释放的阳离子脂质体庆大霉素溶液,并以上述8个时相点的释放浓度配制新鲜阳离子脂质体庆大霉素、庆大霉素,将各组抗生素分别加入S.aureus ATCC29213生物膜,1h后弃去上清,加入培养基,并于此后每隔30min检测每孔溶液在630nm的吸光度。计算细菌生长速率,并得出最大细菌生长速率及所需时间。
主要实验结果:
阳离子脂质体庆大霉素抗菌骨在释放前期具有爆发释放效应,且在释放前期的特殊时间段所释放的庆大霉素总百分比与(时间)~(1/2)成线性关系(R~2＞0.9)。由阳离子脂质体庆大霉素抗菌骨中释放的阳离子脂质体庆大霉素对S.aureus ATCC 29213生物膜生长的抑制作用与新配制的同浓度阳离子脂质体庆大霉素无显著差异(P＞0.05),二者在低浓度时对S.aureus ATCC 29213生物膜生长的抑制作用显著强于单独使用庆大霉素(P＜0.01)。此外,庆大霉素对S.aureus ATCC 29213的MIC为230.4 mg/L。
第四部分阳离子脂质体抗菌骨对兔慢性感染性骨缺损的治疗作用
实验方法
1阳离子脂质体抗菌骨及兔慢性感染性骨缺损模型的制备与第三部分相同。
2实验兔分组,进行静脉注射、手术清创植入抗菌骨治疗。2w后行X线检查,抽静脉血行血液培养,处死后取局部骨髓组织行细菌学检查。
主要实验结果:
对照组没有抗生素治疗,均出现明显感染,血液及骨髓培养均呈S.aureus阳性。单独手术植入同种异体骨或硫酸钙组评分较A组更高,但差异不显著(P＞0.05)。单独使用庆大霉素治疗2w可使兔血液培养阴性,且感染评分低于对照组,二者相差显著(P＜0.05),且全部骨髓培养均阳性。使用阳离子脂质体庆大霉素静脉注射治疗可有效清除血液感染,评分低于A、B、H、I组,且与上述4组相差显著(P＜0.05),同时评分高于F、G组,相差显著(P＜0.05),治疗的6只兔中有2只骨髓培养阴性。采用庆大霉素同种异体骨和庆大霉素硫酸钙治疗效果相似,低于F、G组(P＜0.05),治疗的6只兔中有3只骨髓培养阴性。两种阳离子脂质体抗菌骨疗效相似,可完全清除血液、骨髓感染,感染评分低于上述各组,且相差显著(P＜0.05)。
主要结论
1、耐药S.aureus可诱导明显的胫骨感染性骨缺损形成,6×10~5 CFU/5ul的剂量不仅可使慢性骨髓炎模型制备成功率达100%,而且能够出现较明显的放射学和细菌学改变。
2、脂质体庆大霉素可有效治疗慢性感染性骨缺损,其中以阳离子脂质体庆大霉素效果最为明显,中性次之,阴离子脂质体庆大霉素与单独使用庆大霉素相同,但任何一种脂质体庆大霉素均不能完全清除感染。
3、低温真空负压吸引法制备阳离子脂质体庆大霉素抗菌骨并不影响所释放的阳离子脂质体庆大霉素对S.aureus生物膜的抑制作用,且在低浓度对S.aureus生物膜抑制显著强于单独使用庆大霉素。
4、手术清创后植入阳离子脂质体庆大霉素抗菌骨对兔胫骨S.aureus慢性感染性骨缺损的治疗效果优于庆大霉素抗菌骨以及静脉输注阳离子庆大霉素、庆大霉素。
Both bacterial biofilms and floating bacteria are two forms of bacterial growth. The bacterial biofilms can formed as surface-bound organisms begin to actively replicate (and die) and extracellular components generated by attached bacteria interact with organic and inorganic molecules in the immediate environment to create the glycocalyx. Biofilm organisms are far more resistant to antimicrobial agents than organisms in suspension. In some extreme cases, the concentrations of antibiotics required to achieve bactericidal activity against adherent organisms can be three to four orders of magnitude higher than for planktonic bacteria, depending on the species-drug combination, which is the main reason of the recrudescence of chronic osteomyelitis. Once bacteria have irreversibly attached to a surface of bone, dead bone, internal fixation or artificial joint, the process of biofilm maturation begins, and the infection of bone is recurrent attacks, which bring more pain to the patients. Polymethylmethacrylate(PMMA) and Osteoset T can delayed release antibiotics in the infected bone, and improve the healing rate. However, PMMA need to be taken out after operation. At the same time, the cure rate of implantation of Osteoset T after debridement is similar to debridement alone. In addition, it is indicated that high concentration of gentamicin can inhibit the proliferation of osteoblast and endothelial cell, which can inhibit the union of fracture.
Liposomes are the vesicles which are formed from the arrangement of phospholipid during the dispersion in the water. Liposomes can encapsule antibiotics and are devided to cation, anion and neutral liposomes. It is indicated that the inhibited effect of the penicillin encapsulated in the cation liposomes to the Staphylococcus aureus(S. aureus) biofilms is much better than the penicillin alone, and the mechanism is related to the negative charge of the surface of the S. aureus biofilms. On the other hand, liposomes can make the drug gathered in the bone marrow, which make it possible that infection of bone is eliminated by intravenous injection of antibiotics impregnated in the liposomes. At the same time, liposomes can reduce the toxic effect of high concentration antibiotics to the osteoblast and the endothelial cell significantly. However, the reticuloendothelial system (RES) of human body can eliminate the liposomes in the blood, which induces the half life period of the liposomes and weakens the anti-infection ability of the liposomal antibiotics. Due to the delayed release of high concentration antibiotics of antibiotics delayed released system in the area of bone infection, the weaken of RES to the liposomal antibiotics can be solved by impregnating the liposomal antibiotics to the carrier of antibiotics delayed released system. However, it is unknow whether the anti-biofilm ability of the liposomal antibiotics released from the antibiotics delayed released system is affected. The cation liposomal gentamicin was prepared in our reasearch firstly. Then the cation liposomal gentamicin was impregnated in allogeneic cortical bone of rabbit or calcium sulfate, and the release experiment was carried out. The anti-biofilm effect of cation liposomal gentamicin released from the substitution of bone was explored. During the preparation of the mode of chronic bone infection with bone defect, the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic osteomy elitis with bone defect. In addition, the treatment of injection of three kinds of liposomal gentamicin to the chronic bone infection with bone defect of rabbit was also done.
The study includes four parts. Part one: the affection of the injected dose of S. aureus ATCC29213 to the formation of chronic bone infection with bone defect of rabbit. Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit. Part three: the preparation of cation liposomal getamicin antimicrobial bone and the effect of anti-biofilm in vitro. Part four: the treatment of cation liposomal antimicrobial bone to the chronic tibial infection with bone defect of rabbit.
Main experiments show below:
Part one: the affection of the injected dose of S. aureus to the formation of chronic bone infection with bone defect of rabbit.
Methods:
1 S. aureus(ATCC 29213) with total number of colony from 6×10~1 to 6×10~6 was prepared. The bacteria were injected in the tibia after an area of bone defect was made.
2 The inoculated lesions were assessed for changes in macroscopic observation, radiological and bacteriological parameters at 3 weeks after infection.
Main results:
Obvious infection sign can be seen in the E and F groups due to the macroscopic observation, radiological and bacteriological parameters. There is significant difference between both E, F groups and other groups. However there is no difference between E and F group.
Part two: the treatment of intravenous injection of liposomal gentamicin to the chronic bone infection with bone defect of rabbit.
Methods:
1 The preparation of different liposomal gentamicin
Three liposomal compositions were mixed in a round-bottomed flask and dried to a thin lipid film with a rotary evaporator. The lipids were then hydrated by agitation in aqueous solution of gentamicin. Then the lipid suspensions were sonicated for 40 s in an ultrasonic bath. Unencapsulated gentamicin was removed by centrifugation. The final pellets were resuspended in distilled water.
2 The preparation of chronic tibial infection with bone defect of rabbit.
After anesthetized, the tibial skin of rabbit was cut off, and the tibia was exposed. The bone defect was made and sealed with bone wax. The ATCC 29213 was injected in the bone defect. Treatment was done till the infection formed through the macroscopic and radiological observation.
3 The treatment of liposomal gentamicin to the chronic tibial infection with bone defect.
The treatment with injection of liposomal gentamicin began after the chronic tibial infection with bone defect of rabbit was made successfully. The rabbit was executed at 3m after the treatment and the macroscopic observation, radiological and bacteriological examinations were done.
Main results:
The bacterial culture of blood was posive in A group, and all negative in other groups. The bacterial culture of tibia was posive in A, C, E groups, 4 negative in B group, and 2 negative in D group.
Part three: Preparation of liposomal gentamicin-impregnated allogeneic cortical bone and the effect of antin-biofilm in vitro.
Methods:
1 Preparation of the cation liposomal antimicrobial bone
The preparation of the cation liposomal gentamicin is the same with part two. The metaphysic of tibia and femur was obtained, defated in mixature of chloroform and methanol, deproteinized in H_2O_2, and decalcificated in HC1. The production was observed by scanning electron microscope. The allogeneic cortical bone and calcium sulfate were impregnated in the cation liposomal gentamicin, evaporation to dryness, and sterilized by irradiation of ~(60)Co.
2 Release experiment of cation liposomal antimicrobial bone in vitro
Specimens of liposomal gentamicin-impregnated allogeneic cortical bone and CS were immersed in dd H_2O on a shaking device. The water was replaced at different time points. The replaced water at different time points was added Triton X-100. After rupturing the liposomes, gentamicin concentrations were measured using an AxSym System.
3 Release experiment and regrowth assay
Liposomal gentamicin released at eight time points during the release experiment was chosen to detect the effectiveness of anti-biofilm which was assessed from the rate of regrowth of bacteria from the biofilms after exposure to the cation liposomal gentamicin, relative to regrowth after exposure to an equivalent concentration of free drug or liposomal gentamicin prepared freshly. After exposure of the biofilms to the test samples the plate wells were washed with PBS and growth medium was added and the absorbance of the growth medium was measure at 30 min intervals at 630nm over a period of 24h. The plate reader was programmed to read the optical density vs. time profile and determine the time to reach maximum growth rate(Dod/dt)max. These times were compared with those from growth of biofilms exposed to only nutrient broth(positive controls).
Main results:
The combination of liposomal gentamicin and allogeneic cortical bone showed initial burst-release of active liposomal gentamicin and had continuous-release.
Liposomal gentamicin released from allogeneic cortical bone had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration.
Part four: The treatment of osteomyelitis with bone defect by liposomal gentamicin-impregnated allogeneic cortical bone or calcium sulfate
Methods:
1 The preparation of the cation liposomal antimicrobial bone and the chronic tibial infection with bone defect was the same with part three.
2 After the preparation of the chronic tibial infection, rabbits were treated with intravenous injection of liposomal gentamicin or debridement with implantion of the cation liposomal antimicrobial bone. At 2w after treatment, radiological and bacteriological examinations were done.
Main results:
Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with cation liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was lower slightly than the result treated with implantation of gentamicin-impregnated calcium sulfate(66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin anti microbial bone treated for 14 days.
Conclusions:
1. Development of significant histological and radiological signs of infected bone defect required an inoculum of at least 6×10~5 CFU/5 ul.
2. Cationic liposomes change the pharmacokinetics of gentamicin which can improve the concentration of gentamicin in the bone marrow. Cationic liposomal gentamicin was most effect to cure the chronic osteomyelitis with bone defect among the three liposomal gentamicin.
3. The anti-biofilm effciency of liposomal gentamicin was not affected by the impregnating process. Compared with free drug, liposomal gentamicin is very effective in inhibiting the regrowth of cells from S. aureus biofilms at low drug concentration.
4. The new drug delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.
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