载银纳米抗菌复合骨填充材料治疗兔胫骨慢性骨髓炎的实验研究
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摘要
慢性骨髓炎的治疗基本原则之一就是在充分的抗菌素治疗基础上彻底清除病灶。而病灶清除,死骨摘除后常出现不同范围的骨缺损,如骨缺损大,影响了骨干的机械强度和稳定性,则常需要植骨以促进骨愈合。尽管采用了彻底的清创方法,但缺损部位仍有一定数量的细菌残留,当有外来植入物时,这些细菌易大量繁殖,常导致治疗失败。因此,良好的骨填充材料必须既能填充骨缺损又能杀灭残留致病菌。自1980年Klemm应用抗生素骨水泥防治骨组织感染成功后,抗生素的局部应用越来越引起人们的重视。局部药物缓释系统可以直接作用于病变部位,不需血液将药物抗生素携带至病变区域,可在局部获得持续的较高药物浓度,同时保持较低的血药浓度,避免全身系统用药的毒副作用。抗生素骨水泥虽然在治疗慢性骨髓炎临床应用中取得了极大的成功,但其仍有许多不足:(1)由于骨水泥结合时有一个放热过程,温度较高,只可应用于耐高温的有限剂量的抗生素;(2)由于受抗菌谱的影响,加之近年来耐药菌株的增多使其临床应用受到了不同程度的限制;(3)部分抗生素的种类及浓度影响局部骨的生长,局部高的抗生素浓度对成骨样细胞的增殖有影响。(4)感染控制需再次手术取出珠链;(5)骨水泥无成骨作用,不能对骨缺损进行修复。纳米羟基磷灰石/聚酰胺66(n-HA/PA66)复合生物活性人工骨是由我们与四川大学联合开发出的一种新型纳米仿生复合骨修复替代材料,前期研究显示该材料的生物相容性好,机械性能佳,是一种良好的骨替代材料。我们在此基础上成功开发出二氧化钛载银纳米羟基磷灰石/聚酰胺66抗菌复合骨填充材料,本课题研究其体外抗菌特性,细胞毒性,体外Ag+释放特性及体外对MG63成骨样细胞增殖的影响并探讨其作为骨填充材料填充骨髓炎术后骨缺损的可行性及该材料在兔体内的毒副作用,以期为该材料治疗临床慢性骨髓炎术后骨缺损提供实验依据。
目的:
对二氧化钛载银纳米羟基磷灰石/聚酰胺66抗菌复合骨填充材料体外抗菌特性,细胞毒性,体外Ag+释放特性及对MG63成骨样细胞增殖的影响进行研究,建立兔胫骨近端慢性骨髓炎动物模型,并在兔胫骨近端慢性骨髓炎动物模型的基础上,研究其作为骨填充材料治疗骨髓炎术后骨缺损的治疗效果,并研究该材料在兔体内的毒副作用,期望找到一种既具有良好的局部抗感染能力又具有成骨能力的骨填充材料,为慢性骨髓炎的治疗提供一种新型的局部抗菌剂缓释系统,并为其进一步临床应用提供实验依据。
方法:本实验由四部分组成
1、载银纳米抗菌复合骨填充材料体外抗菌及释放特性测定
采用抑菌环试验、菌落数试验及扫描电镜检测载银纳米抗菌复合骨填充材料对金黄色葡萄球菌和大肠杆菌的抗菌性能及抗粘附效果;采用原子吸收光谱法检测载银纳米抗菌复合骨填充材料在模拟体液中释放银离子特性;通过MTT法,急性溶血实验检测该材料的生物相容性。
2、载银纳米抗菌复合骨填充材料对成骨样细胞生长的影响
以纳米羟基磷灰石/聚酰胺66为对照,采用相差显微镜、扫描电镜、MTT、碱性磷酸酶ALP检测、钙含量测定等方法从MG63成骨样细胞形态、增殖及生理功能等方面检测载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长的影响。
3、兔胫骨慢性骨髓炎动物模型的建立及评价
采用胫骨近段髓腔注射5%鱼肝油酸钠和不同浓度的金黄色葡萄球菌菌液的方法诱导骨髓炎,并探讨诱导的骨髓炎模型与菌液浓度的关系,为慢性骨髓炎的实验研究提供一种合适的动物模型。
4、载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究及兔体内毒副作用研究
在慢性骨髓炎造模的基础上,常规病灶清除,分别植入载银纳米抗菌复合骨填充材料,载万古霉素骨水泥填充材料,nHA/PA66复合骨填充材料及空白对照组,通过大体标本观察、放射学、细菌学和组织学方法,观察材料局部抗菌效果;通过放射学观察,组织学检查和钙黄绿素荧光标记观察材料成骨能力;并通过检测肝脏、肾脏功能及各脏器中银离子浓度,了解材料在兔体内的毒副作用。
结果:
1、载银纳米抗菌复合骨填充材料体外抗菌及释放特性测定
载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠杆菌有明显抗菌作用,尤其对金黄色葡萄球菌,且抗菌能力随银离子含量的增加而加强;该材料在模拟体液中有良好的缓释效果,对L929细胞无毒性,对红细胞不产生毒性或机械损伤,具有良好的生物相容性。
2、载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长的影响
MG63成骨样细胞与材料共培养的倒置相差显微镜可见MG63成骨样细胞在材料周围生长情况良好,且随着培养时间的延长,在材料周围的细胞数量明显增多;扫描电镜显示MG63成骨样细胞在复合材料上生长5天后可见细胞在复合材料上生长状况良好;MTT法结果显示各组细胞的数目都随着培养时间的增加而增加,第七天时抗菌材料组的MTT值与对照组间没有显著性差异(P>0.05);MG63成骨样细胞在不同材料上培养和空白对照组的ALP活性随着培养时间的增加而增加,各材料组和对照组在不同的培养时间点的ALP活性无统计学上差异(P>0.05);细胞钙离子含量测定显示各组MG63成骨样细胞钙离子含量均随时间延长呈增长趋势。
3、兔胫骨慢性骨髓炎动物模型的建立及评价
G0(注入0.1ml生理盐水)、G1(注入0.1ml 3×107 CFU/ml细菌)、G2(注入0.1ml 3×108 CFU/ml细菌)、G3(注入0.1ml 3×109 CFU/ml细菌)、G4(注入0.3ml 3×109 CFU/ml细菌)、G5(注入0.3ml 6×109 CFU/ml细菌)各组造模后4周,影像学显示均出现不同程度的软组织肿胀,骨密度降低,骨小梁纤细,数目减少,骨小梁间隙加宽,骨皮质变薄,骨溶解,死骨形成;组织学切片显示均出现不同程度的中性嗜酸性粒细胞浸润,骨髓腔内髓细胞不同程度坏死,间质出血;采用兔金黄色葡萄球菌骨髓炎模型严重程度分级大体标本观察和放射学检测标准对所诱导模型进行严重程度分级:G1组属0级,G2组属1～2级,G3组属2～3级,G4组属3～4级,G5组属4级。
4、载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究及兔体内毒副作用研究
4.1载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究
抗菌效果:术后12周对各组Norden改良骨髓炎分值进行统计学分析显示:A组(载银纳米抗菌复合骨填充材料)、B组(载万古霉素骨水泥填充材料)分别与C组(nHA/PA66复合骨填充材料)、D组(空白组)比较,差异有非常显著性(P<0.01),A、B组比较差异无统计学意义(P>0.05),C、D组比较差异无统计学意义(P>0.05);术后12周,大体标本可见A、B组保持较正常外观形状,未发现明显的骨质破坏及髓腔脓液,其中A组材料与骨接触处紧密连接,其余各组标本外形粗大,骨质破坏明显,骨缺损边缘有不规则增生及硬化,其中C组材料与骨接触处连接不紧密,其间可见脓性分泌物;术后4周和8周时,每克标本细菌计数转换成对数后进行统计学分析结果显示:A、B组细菌计数明显小于C、D组,差异有非常显著性(P<0.01),A、B组比较,B组细菌计数小于A组,差异有显著性(P<0.05),C、D组比较,D组细菌计数小于C组,差异有显著性(P<0.05);组织学观察显示术后12周,A、B组炎症反应明显较C、D组轻,B组又较A组炎症反应轻。
成骨效果:术后12周X线显示A组植入的填充材料与周围骨皮质界线模糊,与周围骨组织基本愈合,B、C组材料与周围骨组织未见明显愈合征象;钙黄绿素荧光标记显示术后12周,A组材料与骨界面可见大量绿色荧光,B、C组材料边缘及D组荧光标记明显较A组少;组织学观察显示术后12周, A组胶原数量多,可见编织骨形成,新生骨小梁及新生血管,可见多数成骨细胞,成骨活动明显;B组无编织骨小梁,未见明显胶原存在;C、D组均未见到骨形成,无编织骨小梁,可见部分胶原存在。
4.2载银纳米抗菌复合骨填充材料动物体内毒副作用研究
血清生化检验结果显示,天门冬酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)及尿素氮(BUN)、肌酐(Cr)水平在各时相点实验组与对照组两两比较均无显著性差异(P>0.05),实验组内各时相点两两比较均无显著性差异(P>0.05);血液中Ag+浓度检测结果显示各时间点实验组中血液中Ag+浓度均低于10ppb;组织学观察显示实验组肝肾均未见异常变化;术后12周时,实验组肝脏、肾脏中Ag+浓度分别为(224.46±83.04) bbp和(88.57±75.72) bbp。
结论:
1、载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠杆菌有明显抗菌作用,尤其对金黄色葡萄球菌,且抗菌能力随银离子含量的增加而加强;在模拟体液中有良好的缓释效果,对L929细胞无毒性,对红细胞不产生毒性或机械损伤,具有良好的生物相容性。
2、载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长没有抑制作用,对成骨样细胞的碱性磷酸酶活性及钙离子含量均无影响,对MG63细胞具有良好的生物相容性。
3、载银纳米抗菌复合骨填充材料是具有一定的抗感染能力和成骨能力的骨填充材料,其抗感染能力较载万古霉素骨水泥稍弱,但成骨能力较载万古霉素骨水泥强,短期内在兔体内无明显毒副作用;在彻底病灶清除的前提下,能一期填充慢性骨髓炎术后骨缺损,有望成为治疗慢性骨髓炎的较为理想的骨填充材料。
One of the basic principles of the treatment of chronic osteomyelitis is adequate antibiotic treatment on the basis of completely clear the lesions.but the bone defect exist after debridement and removal of sequestrum which often need bone graft to promote bone healing. Good bone fill material must not only fill bone defects but also to kill residual bacteria. Antibiotic bone cement was more and more attentioned since Klemm successful prevention of bone tissue infections by antibiotic bone cement in 1980. Local drug delivery system can be directly on the lesion site, and didn't carry the drug to the lesions by blood and can be sustained in a high local drug concentration. Although antibiotic bone cement in the treatment of chronic osteomyelitis achieved great clinical success, but there is still much to be desired. (1)Antibiotics have to be water soluble and resistant to heat, because temperature sometimes exceeds 100°C during polymerization of the bone cement;(2)Antibiotic bone cement was limited in clinical application because of resistant strains in recent years;(3)The types and concentrations of some antibiotics affect the growth of local bone;(4)The beads must take out reoperation after control infection;(5)The bone cement no effect of osteogenesis, and can not repair the bone defect.N-HA/PA66 composite materials was a new type of bone substitute materials which developed by Sichuan University and Us which has good biocompatibility, good mechanical properties, and is a good bone substitute materials. We developed the TiO_2-Ag-nHA/PA66 composite bone filling materials on the based of it.In this paper, the antibacterial activity ,cytotoxicity ,silver ion release characteristics and the effect on MG63 osteobalst-like cells growth in vitro of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied, and the effect of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied on the base of rabbit models of chronic osteomyelitis.and hope to find an materials to treat osteomyelitis and infective bone defect, and provide experimental evidence for clinical applications.
Objective
To study the antibacterial activity ,cytotoxicity ,silver ion release characteristics and the effect on MG63 osteobalst-like cells growth in vitro of TiO_2-Ag-nHA/PA66 composite bone filling materials. Rabbit models of chronic osteomyelitis at proximal tibia were established.The effect of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied on the base of rabbit models of chronic osteomyelitis.Hope to find an materials to treat osteomyelitis and infective bone defect, and provide experimental evidence for clinical applications.
Method : The experiment was divided into four parts.
1. Experimental research in vitro on antibacterial property and release characteristics of TiO_2-Ag-nHA/PA66 composite bone filling materials
The inhibition ring test and plate-counting method was used to evaluate anti-bacterial performance against Staphylococcus aureus and Escherichia coli, the effect of anti adhesion of Staphylococcus aureus and Escherichia coli were observe by scanning electron microscope(SEM), The cytotoxicity was detected via MTT and the biocompatibil ity of composite bone filling materials was evaluated by acute haemolysis test.
2. Effeets of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro
Nano hydroxyapatite/polyamide 66 composite bone filling materials as the control, Phase contrast microscopy, MTT, Alkaline phosphatase and calcium determination were used to detect the effects of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro from MG63 osteoblast-like cell morphology, proliferation and physiological function.
3. The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in rabbits
Five percent sodium morrhuate and serial dilutions of the bacteria of Staphylococcus aureus suspended in saline were inoculated into the proximal metaphysis of the tibia. the relationship between the inoculation dose of the bacteria of Staphylococcus aureus and the severity of experimental Staphylococcus aureus osteomyelitis in rabbits was studied to provide a suitable animal model of chronic osteomyelitis.
4. Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials and side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
In the basis of chronic osteomyelitis model of rabbit, Conventional debridement, TiO_2-Ag-nHA/PA66 composite bone filling materials, Vancomycin-loaded bone cement and nano-hydroxyapatite/polyamide 66 composite bone filling materials were implanted, the antibacterial effect of material in local was observed by general radiographic observation, bacteriological and histological methods; The osteogenesis of of material in local was observed by general radiographic observation, histological and Fluorescent calcein methods.The toxic side effects of materials in rabbits was studied by detect the liver function, kidney function and the concentration of silver ions in organs.
Results
1.Experimental research in vitro on antibacterial property and release characteristics of TiO_2-Ag-nHA/PA66 composite bone filling materials
TiO_2-Ag-nHA/PA66 composite bone filling materials has excellent antibacterial property against Staphylococcus aureus and Escherichia coli, especially for Staphylococcus aureus , and the antibacterial property increased with the silver ion content increases of TiO_2-Ag-nHA/PA66 composite bone filling materials, and has a good release effect in a simulated body fluid. There was no obvious cytotoxicity to L929 cell and erythrocyte destruction was no found which indicate that TiO_2-Ag-nHA/PA66 composite bone filling materials has good biocompatibility.
2. Effeets of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro
The number of MG63 osteobalst-like cells around the material increased with culture time and can be seen by inverted phase contrast microscope;MTT assay showed that the number of cells in each group are increased with time, and there no significant difference between the TiO_2-Ag-nHA/PA66 composite bone filling materials group and control group on the seventh day (P>0.05);The alkaline phosphatase activity of MG63 osteobalst-like cells which culture on different materials and the control group increased with the increase of culture time,and there was no significant difference in ALP activity of MG63 osteobalst-like cells which cultured with different material groups and control group at different culture time points(P>0.05). The Ca2+ concentration of MG63 osteobalst-like cells which cultured with different material groups and control group all increased with the increase of culture time.
3. The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in rabbits
Four weeks after modeling of G0 group(0.1ml Normal Sodium was inoculated)、G1 group (0.1ml 3×107 CFU/ml Staphylococcus aureus ATCC25923 suspension was inoculated)、G2 group (0.1ml 3×108 CFU/ml)、G3 group (0.1ml 3×109 CFU/ml)、G4 group (0.3ml 3×109 CFU/ml)、G5 group (0.3ml 6×109 CFU/ml), radiography show that there were different degrees of soft tissue swelling, bone mineral density reduced, bone trabecular rare and bone trabecular space widening,cortical thinning, osteolysis, sequestra formation in experimental groups ;histomorphology indicated that there were varying degree of neutrophil infiltration, Necrosis of Marrow Cells and interstitial hemorrhage; Gross Pathologic and Radiographic Criteria was used to Grading the Severity of Experimental Staphylococcus aureus osteomyelitis in rabbits.The grading of experimental groups G1、G2、G3、G4、G5 were respectively stage 0,stage 1 to 2 osteomyelitis, stage 2 to 3 osteomyelitis, stage 3 to 4 osteomyelitis,stage 4.
4. Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials and side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
4.1 Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials
Antibacterial effect:Statistical analysis indicate that there was a significant difference between group A(TiO_2-Ag-nHA/PA66),group B(Vancomycin-loaded bone cement) and group C(nHA/PA66),group D(control group) ,no significant difference between group A and group B,no significant difference between group C and group D of Norden osteomyelitis scores after 12 weeks. Bacterial count per gram indicate that group A and group B significantly less than group C and group D, group B less than group A, group D less than group C,and there was a significant difference(P<0.05).
Osteogenic effect:Radiography indicate that materials and cortical bone around the materials was healing in group A, but there were not healing in group B and C after 12 weeks;Fluorescent calcein indicate that the interface of materials and bone shows a lot of green fluorescent at group A, group B,group C and groupD significantly less than group A;Histomorphology indicate that there were abundant collagen, Newly formed trabecular bone can be seen,and significantly osteogenesis at group A,there were no collagen and newly formed trabecular bone can be seen at group B,there were no newly formed trabecular bone can be seen,and only little collagen at group C and D.
4.2 Side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
Serum biochemical tests indicate that there were no significant difference between experimental group and control group of AST、ALT、BUN、Cr at each time point(P>0.05),and there were no significant differences among all subgroups of the experimental group at each time point(P>0.05).Ag + concentration in the blood were lower than10ppb of experimental group at each time point.Histomorphology indicate that there were no abnormal changes in liver and kidney of experimental group. Ag + concentration in the liver and kidney of experimental group were (224.46±83.04)bbp and (88.57±75.72)bbp ,respectively.
Conclusion
1. The bone filling materials of TiO_2-Ag- nHA/PA66 has antibacterial property against staphylococcus aureus and Escherichia coli, and has a good release effect in a simulated body fluid. There was no obvious cytotoxicity to L929 cell and erythrocyte destruction was no found which indicate that TiO_2-Ag-nHA/PA66 composite bone filling materials has good biocompatibility.
2. TiO_2-Ag-nHA/PA66 composite bone filling materials did not inhibit growth of MG63 osteoblast-like cells, no effect on ALP activity and calcium content of MG63 osteoblast-like cells, and have a good biocompatibility on MG63 osteoblast-like cells.
3. TiO_2-Ag-nHA/PA66 composite bone filling materials have a certain resistance to infection and osteoblast function.and be suitable for use with osteomyelitis surgery in the premise of thorough debridement, and without adverse effects in rabbits, It's a good material which has the function of bone plerosis and anti-infection.
目的:
对二氧化钛载银纳米羟基磷灰石/聚酰胺66抗菌复合骨填充材料体外抗菌特性,细胞毒性,体外Ag+释放特性及对MG63成骨样细胞增殖的影响进行研究,建立兔胫骨近端慢性骨髓炎动物模型,并在兔胫骨近端慢性骨髓炎动物模型的基础上,研究其作为骨填充材料治疗骨髓炎术后骨缺损的治疗效果,并研究该材料在兔体内的毒副作用,期望找到一种既具有良好的局部抗感染能力又具有成骨能力的骨填充材料,为慢性骨髓炎的治疗提供一种新型的局部抗菌剂缓释系统,并为其进一步临床应用提供实验依据。
方法:本实验由四部分组成
1、载银纳米抗菌复合骨填充材料体外抗菌及释放特性测定
采用抑菌环试验、菌落数试验及扫描电镜检测载银纳米抗菌复合骨填充材料对金黄色葡萄球菌和大肠杆菌的抗菌性能及抗粘附效果;采用原子吸收光谱法检测载银纳米抗菌复合骨填充材料在模拟体液中释放银离子特性;通过MTT法,急性溶血实验检测该材料的生物相容性。
2、载银纳米抗菌复合骨填充材料对成骨样细胞生长的影响
以纳米羟基磷灰石/聚酰胺66为对照,采用相差显微镜、扫描电镜、MTT、碱性磷酸酶ALP检测、钙含量测定等方法从MG63成骨样细胞形态、增殖及生理功能等方面检测载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长的影响。
3、兔胫骨慢性骨髓炎动物模型的建立及评价
采用胫骨近段髓腔注射5%鱼肝油酸钠和不同浓度的金黄色葡萄球菌菌液的方法诱导骨髓炎,并探讨诱导的骨髓炎模型与菌液浓度的关系,为慢性骨髓炎的实验研究提供一种合适的动物模型。
4、载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究及兔体内毒副作用研究
在慢性骨髓炎造模的基础上,常规病灶清除,分别植入载银纳米抗菌复合骨填充材料,载万古霉素骨水泥填充材料,nHA/PA66复合骨填充材料及空白对照组,通过大体标本观察、放射学、细菌学和组织学方法,观察材料局部抗菌效果;通过放射学观察,组织学检查和钙黄绿素荧光标记观察材料成骨能力;并通过检测肝脏、肾脏功能及各脏器中银离子浓度,了解材料在兔体内的毒副作用。
结果:
1、载银纳米抗菌复合骨填充材料体外抗菌及释放特性测定
载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠杆菌有明显抗菌作用,尤其对金黄色葡萄球菌,且抗菌能力随银离子含量的增加而加强;该材料在模拟体液中有良好的缓释效果,对L929细胞无毒性,对红细胞不产生毒性或机械损伤,具有良好的生物相容性。
2、载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长的影响
MG63成骨样细胞与材料共培养的倒置相差显微镜可见MG63成骨样细胞在材料周围生长情况良好,且随着培养时间的延长,在材料周围的细胞数量明显增多;扫描电镜显示MG63成骨样细胞在复合材料上生长5天后可见细胞在复合材料上生长状况良好;MTT法结果显示各组细胞的数目都随着培养时间的增加而增加,第七天时抗菌材料组的MTT值与对照组间没有显著性差异(P>0.05);MG63成骨样细胞在不同材料上培养和空白对照组的ALP活性随着培养时间的增加而增加,各材料组和对照组在不同的培养时间点的ALP活性无统计学上差异(P>0.05);细胞钙离子含量测定显示各组MG63成骨样细胞钙离子含量均随时间延长呈增长趋势。
3、兔胫骨慢性骨髓炎动物模型的建立及评价
G0(注入0.1ml生理盐水)、G1(注入0.1ml 3×107 CFU/ml细菌)、G2(注入0.1ml 3×108 CFU/ml细菌)、G3(注入0.1ml 3×109 CFU/ml细菌)、G4(注入0.3ml 3×109 CFU/ml细菌)、G5(注入0.3ml 6×109 CFU/ml细菌)各组造模后4周,影像学显示均出现不同程度的软组织肿胀,骨密度降低,骨小梁纤细,数目减少,骨小梁间隙加宽,骨皮质变薄,骨溶解,死骨形成;组织学切片显示均出现不同程度的中性嗜酸性粒细胞浸润,骨髓腔内髓细胞不同程度坏死,间质出血;采用兔金黄色葡萄球菌骨髓炎模型严重程度分级大体标本观察和放射学检测标准对所诱导模型进行严重程度分级:G1组属0级,G2组属1～2级,G3组属2～3级,G4组属3～4级,G5组属4级。
4、载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究及兔体内毒副作用研究
4.1载银纳米抗菌复合骨填充材料治疗兔胫骨近段慢性骨髓炎的实验研究
抗菌效果:术后12周对各组Norden改良骨髓炎分值进行统计学分析显示:A组(载银纳米抗菌复合骨填充材料)、B组(载万古霉素骨水泥填充材料)分别与C组(nHA/PA66复合骨填充材料)、D组(空白组)比较,差异有非常显著性(P<0.01),A、B组比较差异无统计学意义(P>0.05),C、D组比较差异无统计学意义(P>0.05);术后12周,大体标本可见A、B组保持较正常外观形状,未发现明显的骨质破坏及髓腔脓液,其中A组材料与骨接触处紧密连接,其余各组标本外形粗大,骨质破坏明显,骨缺损边缘有不规则增生及硬化,其中C组材料与骨接触处连接不紧密,其间可见脓性分泌物;术后4周和8周时,每克标本细菌计数转换成对数后进行统计学分析结果显示:A、B组细菌计数明显小于C、D组,差异有非常显著性(P<0.01),A、B组比较,B组细菌计数小于A组,差异有显著性(P<0.05),C、D组比较,D组细菌计数小于C组,差异有显著性(P<0.05);组织学观察显示术后12周,A、B组炎症反应明显较C、D组轻,B组又较A组炎症反应轻。
成骨效果:术后12周X线显示A组植入的填充材料与周围骨皮质界线模糊,与周围骨组织基本愈合,B、C组材料与周围骨组织未见明显愈合征象;钙黄绿素荧光标记显示术后12周,A组材料与骨界面可见大量绿色荧光,B、C组材料边缘及D组荧光标记明显较A组少;组织学观察显示术后12周, A组胶原数量多,可见编织骨形成,新生骨小梁及新生血管,可见多数成骨细胞,成骨活动明显;B组无编织骨小梁,未见明显胶原存在;C、D组均未见到骨形成,无编织骨小梁,可见部分胶原存在。
4.2载银纳米抗菌复合骨填充材料动物体内毒副作用研究
血清生化检验结果显示,天门冬酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)及尿素氮(BUN)、肌酐(Cr)水平在各时相点实验组与对照组两两比较均无显著性差异(P>0.05),实验组内各时相点两两比较均无显著性差异(P>0.05);血液中Ag+浓度检测结果显示各时间点实验组中血液中Ag+浓度均低于10ppb;组织学观察显示实验组肝肾均未见异常变化;术后12周时,实验组肝脏、肾脏中Ag+浓度分别为(224.46±83.04) bbp和(88.57±75.72) bbp。
结论:
1、载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠杆菌有明显抗菌作用,尤其对金黄色葡萄球菌,且抗菌能力随银离子含量的增加而加强;在模拟体液中有良好的缓释效果,对L929细胞无毒性,对红细胞不产生毒性或机械损伤,具有良好的生物相容性。
2、载银纳米抗菌复合骨填充材料对MG63成骨样细胞生长没有抑制作用,对成骨样细胞的碱性磷酸酶活性及钙离子含量均无影响,对MG63细胞具有良好的生物相容性。
3、载银纳米抗菌复合骨填充材料是具有一定的抗感染能力和成骨能力的骨填充材料,其抗感染能力较载万古霉素骨水泥稍弱,但成骨能力较载万古霉素骨水泥强,短期内在兔体内无明显毒副作用;在彻底病灶清除的前提下,能一期填充慢性骨髓炎术后骨缺损,有望成为治疗慢性骨髓炎的较为理想的骨填充材料。
One of the basic principles of the treatment of chronic osteomyelitis is adequate antibiotic treatment on the basis of completely clear the lesions.but the bone defect exist after debridement and removal of sequestrum which often need bone graft to promote bone healing. Good bone fill material must not only fill bone defects but also to kill residual bacteria. Antibiotic bone cement was more and more attentioned since Klemm successful prevention of bone tissue infections by antibiotic bone cement in 1980. Local drug delivery system can be directly on the lesion site, and didn't carry the drug to the lesions by blood and can be sustained in a high local drug concentration. Although antibiotic bone cement in the treatment of chronic osteomyelitis achieved great clinical success, but there is still much to be desired. (1)Antibiotics have to be water soluble and resistant to heat, because temperature sometimes exceeds 100°C during polymerization of the bone cement;(2)Antibiotic bone cement was limited in clinical application because of resistant strains in recent years;(3)The types and concentrations of some antibiotics affect the growth of local bone;(4)The beads must take out reoperation after control infection;(5)The bone cement no effect of osteogenesis, and can not repair the bone defect.N-HA/PA66 composite materials was a new type of bone substitute materials which developed by Sichuan University and Us which has good biocompatibility, good mechanical properties, and is a good bone substitute materials. We developed the TiO_2-Ag-nHA/PA66 composite bone filling materials on the based of it.In this paper, the antibacterial activity ,cytotoxicity ,silver ion release characteristics and the effect on MG63 osteobalst-like cells growth in vitro of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied, and the effect of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied on the base of rabbit models of chronic osteomyelitis.and hope to find an materials to treat osteomyelitis and infective bone defect, and provide experimental evidence for clinical applications.
Objective
To study the antibacterial activity ,cytotoxicity ,silver ion release characteristics and the effect on MG63 osteobalst-like cells growth in vitro of TiO_2-Ag-nHA/PA66 composite bone filling materials. Rabbit models of chronic osteomyelitis at proximal tibia were established.The effect of TiO_2-Ag-nHA/PA66 composite bone filling materials was studied on the base of rabbit models of chronic osteomyelitis.Hope to find an materials to treat osteomyelitis and infective bone defect, and provide experimental evidence for clinical applications.
Method : The experiment was divided into four parts.
1. Experimental research in vitro on antibacterial property and release characteristics of TiO_2-Ag-nHA/PA66 composite bone filling materials
The inhibition ring test and plate-counting method was used to evaluate anti-bacterial performance against Staphylococcus aureus and Escherichia coli, the effect of anti adhesion of Staphylococcus aureus and Escherichia coli were observe by scanning electron microscope(SEM), The cytotoxicity was detected via MTT and the biocompatibil ity of composite bone filling materials was evaluated by acute haemolysis test.
2. Effeets of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro
Nano hydroxyapatite/polyamide 66 composite bone filling materials as the control, Phase contrast microscopy, MTT, Alkaline phosphatase and calcium determination were used to detect the effects of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro from MG63 osteoblast-like cell morphology, proliferation and physiological function.
3. The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in rabbits
Five percent sodium morrhuate and serial dilutions of the bacteria of Staphylococcus aureus suspended in saline were inoculated into the proximal metaphysis of the tibia. the relationship between the inoculation dose of the bacteria of Staphylococcus aureus and the severity of experimental Staphylococcus aureus osteomyelitis in rabbits was studied to provide a suitable animal model of chronic osteomyelitis.
4. Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials and side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
In the basis of chronic osteomyelitis model of rabbit, Conventional debridement, TiO_2-Ag-nHA/PA66 composite bone filling materials, Vancomycin-loaded bone cement and nano-hydroxyapatite/polyamide 66 composite bone filling materials were implanted, the antibacterial effect of material in local was observed by general radiographic observation, bacteriological and histological methods; The osteogenesis of of material in local was observed by general radiographic observation, histological and Fluorescent calcein methods.The toxic side effects of materials in rabbits was studied by detect the liver function, kidney function and the concentration of silver ions in organs.
Results
1.Experimental research in vitro on antibacterial property and release characteristics of TiO_2-Ag-nHA/PA66 composite bone filling materials
TiO_2-Ag-nHA/PA66 composite bone filling materials has excellent antibacterial property against Staphylococcus aureus and Escherichia coli, especially for Staphylococcus aureus , and the antibacterial property increased with the silver ion content increases of TiO_2-Ag-nHA/PA66 composite bone filling materials, and has a good release effect in a simulated body fluid. There was no obvious cytotoxicity to L929 cell and erythrocyte destruction was no found which indicate that TiO_2-Ag-nHA/PA66 composite bone filling materials has good biocompatibility.
2. Effeets of TiO_2-Ag-nHA/PA66 composite bone filling materials on MG63 osteobalst-like cells growth in vitro
The number of MG63 osteobalst-like cells around the material increased with culture time and can be seen by inverted phase contrast microscope;MTT assay showed that the number of cells in each group are increased with time, and there no significant difference between the TiO_2-Ag-nHA/PA66 composite bone filling materials group and control group on the seventh day (P>0.05);The alkaline phosphatase activity of MG63 osteobalst-like cells which culture on different materials and the control group increased with the increase of culture time,and there was no significant difference in ALP activity of MG63 osteobalst-like cells which cultured with different material groups and control group at different culture time points(P>0.05). The Ca2+ concentration of MG63 osteobalst-like cells which cultured with different material groups and control group all increased with the increase of culture time.
3. The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in rabbits
Four weeks after modeling of G0 group(0.1ml Normal Sodium was inoculated)、G1 group (0.1ml 3×107 CFU/ml Staphylococcus aureus ATCC25923 suspension was inoculated)、G2 group (0.1ml 3×108 CFU/ml)、G3 group (0.1ml 3×109 CFU/ml)、G4 group (0.3ml 3×109 CFU/ml)、G5 group (0.3ml 6×109 CFU/ml), radiography show that there were different degrees of soft tissue swelling, bone mineral density reduced, bone trabecular rare and bone trabecular space widening,cortical thinning, osteolysis, sequestra formation in experimental groups ;histomorphology indicated that there were varying degree of neutrophil infiltration, Necrosis of Marrow Cells and interstitial hemorrhage; Gross Pathologic and Radiographic Criteria was used to Grading the Severity of Experimental Staphylococcus aureus osteomyelitis in rabbits.The grading of experimental groups G1、G2、G3、G4、G5 were respectively stage 0,stage 1 to 2 osteomyelitis, stage 2 to 3 osteomyelitis, stage 3 to 4 osteomyelitis,stage 4.
4. Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials and side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
4.1 Experimental osteomyelitis treatment with TiO_2-Ag-nHA/PA66 composite bone filling materials
Antibacterial effect:Statistical analysis indicate that there was a significant difference between group A(TiO_2-Ag-nHA/PA66),group B(Vancomycin-loaded bone cement) and group C(nHA/PA66),group D(control group) ,no significant difference between group A and group B,no significant difference between group C and group D of Norden osteomyelitis scores after 12 weeks. Bacterial count per gram indicate that group A and group B significantly less than group C and group D, group B less than group A, group D less than group C,and there was a significant difference(P<0.05).
Osteogenic effect:Radiography indicate that materials and cortical bone around the materials was healing in group A, but there were not healing in group B and C after 12 weeks;Fluorescent calcein indicate that the interface of materials and bone shows a lot of green fluorescent at group A, group B,group C and groupD significantly less than group A;Histomorphology indicate that there were abundant collagen, Newly formed trabecular bone can be seen,and significantly osteogenesis at group A,there were no collagen and newly formed trabecular bone can be seen at group B,there were no newly formed trabecular bone can be seen,and only little collagen at group C and D.
4.2 Side effects of TiO_2-Ag-nHA/PA66 composite bone filling materials in rabbit
Serum biochemical tests indicate that there were no significant difference between experimental group and control group of AST、ALT、BUN、Cr at each time point(P>0.05),and there were no significant differences among all subgroups of the experimental group at each time point(P>0.05).Ag + concentration in the blood were lower than10ppb of experimental group at each time point.Histomorphology indicate that there were no abnormal changes in liver and kidney of experimental group. Ag + concentration in the liver and kidney of experimental group were (224.46±83.04)bbp and (88.57±75.72)bbp ,respectively.
Conclusion
1. The bone filling materials of TiO_2-Ag- nHA/PA66 has antibacterial property against staphylococcus aureus and Escherichia coli, and has a good release effect in a simulated body fluid. There was no obvious cytotoxicity to L929 cell and erythrocyte destruction was no found which indicate that TiO_2-Ag-nHA/PA66 composite bone filling materials has good biocompatibility.
2. TiO_2-Ag-nHA/PA66 composite bone filling materials did not inhibit growth of MG63 osteoblast-like cells, no effect on ALP activity and calcium content of MG63 osteoblast-like cells, and have a good biocompatibility on MG63 osteoblast-like cells.
3. TiO_2-Ag-nHA/PA66 composite bone filling materials have a certain resistance to infection and osteoblast function.and be suitable for use with osteomyelitis surgery in the premise of thorough debridement, and without adverse effects in rabbits, It's a good material which has the function of bone plerosis and anti-infection.
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