纳米银—小肠黏膜下层修复腹壁缺损的实验研究
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摘要
背景和目的
腹壁疝,战创伤、肿瘤等病变造成的腹壁缺损以及救治腹腔间隙综合征等危重病时,均需使用腹壁修复重建材料,临床上对此存在着巨大的需求。理想的腹壁修复重建材料应理化性质稳定、安全无毒无致癌性、耐张力优于腹壁组织、耐机械疲劳、组织相容性好、排异反应小、利于组织再生、来源及使用方便；此外最新的观点更强调材料具有抗感染性、柔软和服帖性以及能获得最小的手术痛苦以及相对最小的补片植入量等。但目前国内外公认尚未找到一种完全理想修复材料。我国人工材料疝修复术普及率远远低于西方发达国家,原因一方面是基层医院医疗技术更新速度慢,另一方面是手术中使用的修补材料在我国还没有产业化,全部依赖进口,价格昂贵。仅就我国的人口基数和美国的人口基数以及疝气的发病率来比较,不难看出腹壁修复重建材料在我国的巨大的潜在经济意义和应用价值前景。
生物材料是当下国外疝和腹壁外科研究的最新进展,包括动物源性材料如猪小肠粘膜下层(small intestinal submucosa, SIS)、牛心包等；人尸源性材料如人工脱细胞真皮(acellular dermal matrix, ADM)等。总体评价,生物材料除了拥有与合成材料一样的低疝复发率,在生物相容性、抗粘连性等方面也较人工合成材料更为理想,术后疝复发、感染,肠梗阻、瘘管形成等不良反应的发生率明显较低。
值得重视的是,生物材料的抗微生物活性优于合成材料。研究发现,醋酸消化制备的SIS细胞外基质浸出液具有抑制革兰阴性大肠杆菌和革兰阳性金黄色葡萄球菌的作用。SIS在有金黄色葡萄球菌定植时亦可实现良好的组织再塑,植入SIS伤口的感染性低于合成材料。但同时国外将生物材料试用于腹壁缺损修复或重建的初期临床经验也表明,修复污染或潜在感染的腹壁缺损失败率>50%,并且依赖于伤口感染的类型和再血管化速度。因此,提高腹壁修复材料的抗感染性仍是疝和腹壁外科的巨大挑战。
在抗菌剂方面,银作为抗菌剂,具有高效、安全无毒、抗菌谱广、无耐药性等优点,能杀灭细菌、真菌和霉菌甚至病毒。银杀菌机制主要与银离子有关,银离子可与细菌内巯基酶结合从而使后者失活,阻断微生物的能量代谢及细胞壁合成,起到杀灭细菌、病毒及真核微生物的作用。也有研究表明纳米银颗粒可以和病原体的DNA碱基结合,形成交叉链接,置换嘌呤和嘧啶中相邻氮之间的氢键,使DNA变性,不能复制,致细菌失活。此外,银还有促进伤口愈合的功能。研究发现,伤口局部涂抹银乳膏可抑制变应性接触性皮炎鼠模型炎症因子表达、诱导炎性细胞调亡。动物毒性实验和临床应用均证实银杀菌剂属实际无毒级。近年来,利用纳米技术将金属银加工成纳米级的银微粒后,其表面积极大,显示明显的表面效应、小尺寸效应和宏观隧道效应,抗菌活性大大增强,效力更持久,安全性更高。纳米银作为抗菌材料已应用于医用导管、内固定器械、创伤敷料等方面。我们从中得到启发,利用生物材料的三维网状超微结构、主要成分为胶原蛋白(90%为Ⅰ型和Ⅲ型胶原纤维)易于粒子粘附,植入粒径15nm左右的纳米银粒子形成具有抗菌性的生物材料。植入体内后随着生物材料的降解银微粒逐步释放,从而持久发挥抗细菌定植和抗感染、利于宿主细胞浸润生长和血管再生的作用。
综合上述,构建理想的新型抗菌生物一高分子复合腹壁修复材料的条件已经成熟,如有望构建成功,将为临床腹壁缺损及疝的治疗提供新的选择。经文献检索,目前国内外尚未见复合生物材料和合成材料构建腹壁补片的报道。
材料和方法
第一部分：抗菌生物—高分子复合腹壁修复材料的构建
1.按Abraham方法制备猪小肠粘膜下层(SIS)。
2.通过自组装技术将纳米银粒子植入SIS构建抗菌生物材料纳米银SIS。
3.采用扫描电镜观察材料超微结构,琼脂弥散法测试复合材料的体外抗菌能力。
第二部分：动物实验及免疫研究
1.采用复合材料、生物材料、合成材料分别修复大鼠全层腹壁缺损(3×3cm),每组12只F344成年大鼠(体重约200g),并用10^4cfu/ml的金黄色葡萄球菌污染修复区。修复后1d、3d、5d、7d取动物腹壁分泌物作细菌培养,1d、3d、5d、7d、15d、30d取动物血采用火焰原子吸收光谱法检测血液中银含量。修复后2月处死动物,腹壁注水试验检测修复区抗压情况,使用HE和甲苯胺蓝染色、扫描电镜观察修复区边缘及中央的组织结构、血管化程度,取动物脑、肝、肾组织,采用火焰原子吸收光谱检测组织中银含量。
2.SD大鼠30只,体重200～250g,手术造成3cm×2cm全层腹壁缺损,随机分为三组(n=10),分别采用相同面积的纳米银猪小肠黏膜下层(NS-SIS),单纯猪小肠黏膜下层(SIS)和Proceed(?)补片进行修补。术后4周和8周取出腹壁修复材料,进行CD3(淋巴细胞),CD68(巨噬细胞)及CD31(血管内皮)免疫组化分析。
第三部分：材料的生物安全性试验
1.进行细胞毒性试验,将复合材料放入96孔培养板,细胞培养板分3组(复合材料、阳性对照、阴性对照),每组12孔,每孔加入细胞培养液2mL,置入37℃培养箱内,24h后取出备用。将已培养48 h生长旺盛的L929细胞计算出细胞密度(细胞数/mL)：配制成4×104/mL的细胞悬液,取出96孔细胞培养板,分别加入细胞悬液0.5 mL,置37℃培养。于培养后的第1,2,4,7 d,每组各取3孔进行细胞形态观察和细胞计数。
2.进行皮肤刺激试验,于成年家兔皮内注射复合材料浸出液及对照液(生理盐水)各0.2 mL,于注射后15 min,1 h,2 d,3 d观察实验区皮肤反应。
3.进行皮肤致敏试验,于豚鼠背部脱毛区涂0.1mL复合材料浸出液和对照液(生理盐水)各0.1mL,连续观察3d,记录试验区皮肤组织反应。
4.进行致热原试验：于成年家兔耳缘静脉注射复合材料浸出液及对照液(生理盐水)各0.1 mL,于注射后1h,6h,12h,24h,48h,72h分别测量家兔体温。
5.数据采用SPSS 11.0统计软件包进行统计学处理,实验数据以均数±标准差表示,P值<0.05为有统计学意义。
结果
一、对纳米银-小肠黏膜下层修复材料(NS-SIS)的体外抗菌效果进行了初步研究,结果表明纳米银-小肠黏膜下层修复材料具有良好的体外抗菌效果。
二、纳米银-小肠黏膜下层修复材料能够用于修复大鼠污染腹壁缺损,且伤口感染率低于单纯生物腹壁修复材料(SIS)和合成材料(Proceed(?)补片)。
三、对纳米银复合生物腹壁修复材料的修复大鼠腹壁缺损后对修复区局部炎症反应和微血管密度的影响进行了初步研究,结果表明纳米银-小肠黏膜下层修复材料与单纯生物腹壁修复材料对修复区局部炎症反应及微血管密度的作用相同,两者较合成材料局部炎症反应轻,微血管密度较合成材料低。
四、对纳米银复合生物腹壁修复材料的生物安全性进行了初步评价,结果表明纳米银-小肠黏膜下层修复材料(NS-SIS)的生物安全性良好。
试验结论
纳米银-小肠黏膜下层修复材料(NS-SIS)有较好的抗菌效果,且伤口感染率和局部炎症反应低于单纯生物腹壁修复材料(SIS)和合成材料(Proceed(?)补片),并具有良好的生物安全性。
创新点
1.国内外首次将生物材料与高分子材料复合构建修复腹壁补片,集中了现有材料几乎全部的优点,为寻求理想腹壁修复材料的研究提供了新的思路；预期实验结果有望改变临床现状。
2.国内外首次将纳米技术、无机杀菌剂应用于疝和腹壁外科,改变现有修复材料抗感染性差的研究出现了新的转机。
Background and objective
Of abdominal wall hernia,war trauma, tumors and other diseases caused by abdominal wall defect as well as the treatment of abdominal compartment syndrome, critical illness, the required use of abdominal wall reconstruction materials, which there is a huge clinical demand. Abdominal wall repair and reconstruction of the ideal physical and chemical nature of the material should be stability, security, non-toxic non-carcinogenic, anti-tension of the abdominal wall superior to the organization, anti-mechanical fatigue, organizational compatibility is good, rejection is small, beneficial to tissue regeneration, source, and easy to use; In addition, the views of the latest material with more emphasis on anti-infective, soft kimono, as well as access to the minimum quote of surgical pain, as well as implantation of a relatively minimal amount of patches. However, recognized at home and abroad have yet to find a completely satisfactory repair material. My hernia repair with artificial material penetration is far lower than western countries, because on the one hand is the primary hospital medical technology update was slow, and the other hand, surgical repair materials used in the industrialization of China has not yet all rely on imports, the price expensive. Far as China's population base and the United States population base, as well as to compare the incidence of hernias, abdominal wall repair and reconstruction material is easy to see the huge potential in China's economic prospect of the meaning and application value.
Biological material is immediate, and abdominal wall hernia surgery abroad, the latest progress of research, including animal-derived materials such as porcine small intestinal submucosa (small intestinal submucosa, SIS), bovine pericardium, were dead-derived materials such as artificial acellular dermal matrix (acellular dermal matrix, ADM) and so on. Overall assessment [7], biological materials and synthetic materials, like the addition to the low hernia recurrence rate, biocompatibility, anti-adhesive, it is also more desirable than synthetic materials, postoperative hernia recurrence, infection, bowel obstruction, fistula formation and other adverse reactions were significantly lower.
Worthy of note is that the anti-microbial activity of biological material is superior to synthetic materials. The study found [8], acetic acid digest prepared SIS extracellular matrix leaching solution can inhibit the Gram-negative Escherichia coli and gram-positive Staphylococcus aureus role. SIS in a Staphylococcus aureus colonization can also be achieved when the well-organized remodeling, implantation of SIS wound infection than synthetic materials [9]. At the same time foreign biological material tested in the repair or reconstruction of abdominal wall defects in the initial clinical experience also shows that repair contaminated or potentially infected abdominal wall defect failure rate 50%, and depends on the type of wound infection rate of revascularization. Therefore, raising the abdominal wall repair material is still resistance to infection and abdominal wall hernia surgery enormous challenge.
In the antibacterial agent, the silver as an antimicrobial agent, a highly efficient, safe non-toxic, broad spectrum anti-bacterial, non-resistance, etc., can kill bacteria, fungi and fungi and even viruses. Silver sterilization mechanism is mainly related with the silver ions, silver ions with bacterial sulfhydryl enzyme inactivation combined so that the latter, blocking the energy metabolism and microbial cell wall synthesis, play to kill bacteria, viruses and eukaryotic microorganisms role. There are also studies have shown that nano-silver particles and pathogens of the DNA bases can be combined to form cross-links, replacement of purine and pyrimidine hydrogen bonds between the adjacent nitrogen, so that DNA denaturation can not be copied, caused by bacteria inactivation. In addition, Silver also promote wound healing functions. The study found that topical wound silver cream inhibit mouse model of allergic contact dermatitis expression of inflammatory cytokines induce apoptosis of inflammatory cells. Animal toxicity experiments and clinical application of fungicides were found to belong to the actual non-toxic silver-level. In recent years, the use of metallic silver nano-technology will be processed into nano-scale particles of silver, its surface area dramatically, indicating significant surface effect, small size effect and the macro-tunnel effect, antibacterial activity greatly enhanced the effectiveness of more durable and more secure. Nano-silver as an antibacterial material has been applied to medical catheters, internal fixation devices, wound dressings and so on. We drew inspiration from the use of biological materials, three-dimensional ultrastructure, the main component of collagen (90% for theⅠ-and typeⅢcollagen fibers) easy-to-particle adhesion, implant diameter of about 15nm with the formation of nano-silver particles antibacterial activity of biological materials. Implantation in vivo degradation of biological material with the gradual release of silver particles, and thus sustain the anti-bacterial colonization and infection, which will help the growth of the host cell infiltration and blood vessel regeneration.
The above, build an ideal new antibacterial bio-polymer composite abdominal wall repair material conditions are ripe, as is expected to build successful, will be the clinical treatment of abdominal wall hernia defect and to provide new options. The literature search, at home and abroad have not yet see the complex biological materials and synthetic materials to build the abdominal wall patches were reported.
Materials and Methods
PartⅠ:anti-bacterial bio-polymer composite materials to build the abdominal wall repair
1. Prepared according to Abraham pig small intestinal submucosa (SIS).
2. Through self-assembly technology to nano-silver particles embedded SIS biological materials to build anti-bacterial silver nano-SIS.
3. Materials using scanning electron microscopy the ultrastructure of agar diffusion method testing composite materials in vitro antimicrobial capacity of.
PartⅡ:Animal experiments and Immunity
1. Use of composite materials, biological materials, synthetic materials, respectively, full-thickness abdominal wall defect repair in rats (3×3cm), each group of 12 adult F344 rats (weighing about 200g), and use 10 ^ 4cfu/ml of Staphylococcus aureus contamination Repair area. After the repair 1d,3d,5d,7d take animal secretions for bacterial culture of abdominal wall, 1d,3d,5d,7d,15d,30d to take animal blood by flame atomic absorption spectrometry detection of silver content in the blood. Animals were killed in February after the repair, abdominal wall injection test for detection Restoration Area compression, use HE and toluidine blue staining, scanning electron microscopy Restoration Area and the central edge of the organizational structure, the degree of blood vessel, take animals, brain, liver, kidney, using flame atomic absorption spectroscopy detection of tissue silver content.
2. SD 30 rats, weighing 200～250g, surgery caused by 3cm×2cm full-thickness abdominal wall defect, were randomly divided into three groups (n=10), respectively, the same area of nano-silver porcine small intestine submucosa (NS-SIS) Simply porcine small intestine submucosa (SIS), and Proceed(?)patch to patch.4 weeks and 8 weeks after removal of abdominal wall repair material, for CD3 (lymphocytes), CD68 (macrophages) and CD31 (vascular endothelium) Immunohistochemical analysis.
PartⅢ:Materials, bio-safety tests
1. Cytotoxicity test, the composite material into 96-well culture plate, cell culture plate divided into 3 groups (composite materials, positive control, negative control), each 12 holes, each hole by adding cell culture medium 2mL, placed at 37℃box,24 h after the stand-out. Will have strong growth of 48 h cultured L929 cells, calculate the cell density (cells/mL):preparation of 4×104/mL the cell suspension, remove the 96-hole cell culture plates, were added to cell suspension 0.5 mL, at 37℃for. After culture the first 1,2,4,7 d, each depicting three holes, cell morphology and cell count.
2. The skin stimulation test, in adult rabbit skin injection of composite materials at home leaching solution and the control solution (saline) of 0.2 mL, was injected 15 min,1 h,2 d,3 d experimental zone observed skin reactions.
3. The skin sensitization test in guinea pigs back hair removal zone Tu 0.1mL composite materials leaching solution and the control solution (saline) Each 0.1 mL, a continuous observation of 3d, recording test area of skin tissue reaction.
4. For pyrogen test:in the adult rabbit ear vein injection of composite materials leaching solution and the control solution (saline) of 0.1 mL, after injection, 1h,6h,12h,24h,48h,72h body temperature were measured in rabbits.
5. The data using SPSS 11.0 statistical package for statistical analysis, experimental data indicated that the mean±standard deviation, P value of 0.05 was considered statistically significant.
Results
1. For bio-nano-silver abdominal wall repair materials (NS-SIS) in vitro antibacterial effect of the preliminary study results show that the nano-silver biological abdominal wall repair material has good in vitro antibacterial effects.
2. Nano-silver biological materials can be used to repair abdominal wall to repair contaminated abdominal wall defects in rats, and the wound infection rate of less than the purely biological abdominal wall repair material (SIS) and synthetic materials (Proceed(?)patch).
Three pairs of nano-silver compound bio-repair materials, repair of abdominal wall abdominal wall defects in rats after the Restoration Area local inflammatory response and the impact of microvessel density, a preliminary study results show that nano-silver material and purely biological repair of abdominal wall abdominal wall repair of biological materials on the Restoration Area the local inflammatory response and the role of microvessel density in the same synthetic material between the local inflammatory response than the light, microvessel density, lower than the synthetic materials.
Fourth, nano-silver compound bio-bio-safety of the abdominal wall repair material to conduct a preliminary evaluation results show that the nano-silver bio-abdominal repair materials (NS-SIS) of the bio-security was good.
Conclusion
Nano Silver Bio-abdominal repair materials (NS-SIS) has good anti-bacterial effect, and the wound infection and local inflammatory response than a simple biological abdominal wall repair material (SIS) and synthetic materials (Proceed(?)patch), and has a good bio-security.
Innovation
1. At home and abroad for the first time of biological materials and polymer composite materials to build abdominal wall repair patch, brings together almost all the advantages of existing materials, in order to find the ideal study of abdominal wall repair material provides a new idea; the expected clinical results are expected to change the status quo.
2. At home and abroad for the first time nanotechnology, inorganic fungicides used in hernia and abdominal wall surgery, and changes to existing repair materials with poor anti-infective research there is a new turn for the better.
腹壁疝,战创伤、肿瘤等病变造成的腹壁缺损以及救治腹腔间隙综合征等危重病时,均需使用腹壁修复重建材料,临床上对此存在着巨大的需求。理想的腹壁修复重建材料应理化性质稳定、安全无毒无致癌性、耐张力优于腹壁组织、耐机械疲劳、组织相容性好、排异反应小、利于组织再生、来源及使用方便；此外最新的观点更强调材料具有抗感染性、柔软和服帖性以及能获得最小的手术痛苦以及相对最小的补片植入量等。但目前国内外公认尚未找到一种完全理想修复材料。我国人工材料疝修复术普及率远远低于西方发达国家,原因一方面是基层医院医疗技术更新速度慢,另一方面是手术中使用的修补材料在我国还没有产业化,全部依赖进口,价格昂贵。仅就我国的人口基数和美国的人口基数以及疝气的发病率来比较,不难看出腹壁修复重建材料在我国的巨大的潜在经济意义和应用价值前景。
生物材料是当下国外疝和腹壁外科研究的最新进展,包括动物源性材料如猪小肠粘膜下层(small intestinal submucosa, SIS)、牛心包等；人尸源性材料如人工脱细胞真皮(acellular dermal matrix, ADM)等。总体评价,生物材料除了拥有与合成材料一样的低疝复发率,在生物相容性、抗粘连性等方面也较人工合成材料更为理想,术后疝复发、感染,肠梗阻、瘘管形成等不良反应的发生率明显较低。
值得重视的是,生物材料的抗微生物活性优于合成材料。研究发现,醋酸消化制备的SIS细胞外基质浸出液具有抑制革兰阴性大肠杆菌和革兰阳性金黄色葡萄球菌的作用。SIS在有金黄色葡萄球菌定植时亦可实现良好的组织再塑,植入SIS伤口的感染性低于合成材料。但同时国外将生物材料试用于腹壁缺损修复或重建的初期临床经验也表明,修复污染或潜在感染的腹壁缺损失败率>50%,并且依赖于伤口感染的类型和再血管化速度。因此,提高腹壁修复材料的抗感染性仍是疝和腹壁外科的巨大挑战。
在抗菌剂方面,银作为抗菌剂,具有高效、安全无毒、抗菌谱广、无耐药性等优点,能杀灭细菌、真菌和霉菌甚至病毒。银杀菌机制主要与银离子有关,银离子可与细菌内巯基酶结合从而使后者失活,阻断微生物的能量代谢及细胞壁合成,起到杀灭细菌、病毒及真核微生物的作用。也有研究表明纳米银颗粒可以和病原体的DNA碱基结合,形成交叉链接,置换嘌呤和嘧啶中相邻氮之间的氢键,使DNA变性,不能复制,致细菌失活。此外,银还有促进伤口愈合的功能。研究发现,伤口局部涂抹银乳膏可抑制变应性接触性皮炎鼠模型炎症因子表达、诱导炎性细胞调亡。动物毒性实验和临床应用均证实银杀菌剂属实际无毒级。近年来,利用纳米技术将金属银加工成纳米级的银微粒后,其表面积极大,显示明显的表面效应、小尺寸效应和宏观隧道效应,抗菌活性大大增强,效力更持久,安全性更高。纳米银作为抗菌材料已应用于医用导管、内固定器械、创伤敷料等方面。我们从中得到启发,利用生物材料的三维网状超微结构、主要成分为胶原蛋白(90%为Ⅰ型和Ⅲ型胶原纤维)易于粒子粘附,植入粒径15nm左右的纳米银粒子形成具有抗菌性的生物材料。植入体内后随着生物材料的降解银微粒逐步释放,从而持久发挥抗细菌定植和抗感染、利于宿主细胞浸润生长和血管再生的作用。
综合上述,构建理想的新型抗菌生物一高分子复合腹壁修复材料的条件已经成熟,如有望构建成功,将为临床腹壁缺损及疝的治疗提供新的选择。经文献检索,目前国内外尚未见复合生物材料和合成材料构建腹壁补片的报道。
材料和方法
第一部分：抗菌生物—高分子复合腹壁修复材料的构建
1.按Abraham方法制备猪小肠粘膜下层(SIS)。
2.通过自组装技术将纳米银粒子植入SIS构建抗菌生物材料纳米银SIS。
3.采用扫描电镜观察材料超微结构,琼脂弥散法测试复合材料的体外抗菌能力。
第二部分：动物实验及免疫研究
1.采用复合材料、生物材料、合成材料分别修复大鼠全层腹壁缺损(3×3cm),每组12只F344成年大鼠(体重约200g),并用10^4cfu/ml的金黄色葡萄球菌污染修复区。修复后1d、3d、5d、7d取动物腹壁分泌物作细菌培养,1d、3d、5d、7d、15d、30d取动物血采用火焰原子吸收光谱法检测血液中银含量。修复后2月处死动物,腹壁注水试验检测修复区抗压情况,使用HE和甲苯胺蓝染色、扫描电镜观察修复区边缘及中央的组织结构、血管化程度,取动物脑、肝、肾组织,采用火焰原子吸收光谱检测组织中银含量。
2.SD大鼠30只,体重200～250g,手术造成3cm×2cm全层腹壁缺损,随机分为三组(n=10),分别采用相同面积的纳米银猪小肠黏膜下层(NS-SIS),单纯猪小肠黏膜下层(SIS)和Proceed(?)补片进行修补。术后4周和8周取出腹壁修复材料,进行CD3(淋巴细胞),CD68(巨噬细胞)及CD31(血管内皮)免疫组化分析。
第三部分：材料的生物安全性试验
1.进行细胞毒性试验,将复合材料放入96孔培养板,细胞培养板分3组(复合材料、阳性对照、阴性对照),每组12孔,每孔加入细胞培养液2mL,置入37℃培养箱内,24h后取出备用。将已培养48 h生长旺盛的L929细胞计算出细胞密度(细胞数/mL)：配制成4×104/mL的细胞悬液,取出96孔细胞培养板,分别加入细胞悬液0.5 mL,置37℃培养。于培养后的第1,2,4,7 d,每组各取3孔进行细胞形态观察和细胞计数。
2.进行皮肤刺激试验,于成年家兔皮内注射复合材料浸出液及对照液(生理盐水)各0.2 mL,于注射后15 min,1 h,2 d,3 d观察实验区皮肤反应。
3.进行皮肤致敏试验,于豚鼠背部脱毛区涂0.1mL复合材料浸出液和对照液(生理盐水)各0.1mL,连续观察3d,记录试验区皮肤组织反应。
4.进行致热原试验：于成年家兔耳缘静脉注射复合材料浸出液及对照液(生理盐水)各0.1 mL,于注射后1h,6h,12h,24h,48h,72h分别测量家兔体温。
5.数据采用SPSS 11.0统计软件包进行统计学处理,实验数据以均数±标准差表示,P值<0.05为有统计学意义。
结果
一、对纳米银-小肠黏膜下层修复材料(NS-SIS)的体外抗菌效果进行了初步研究,结果表明纳米银-小肠黏膜下层修复材料具有良好的体外抗菌效果。
二、纳米银-小肠黏膜下层修复材料能够用于修复大鼠污染腹壁缺损,且伤口感染率低于单纯生物腹壁修复材料(SIS)和合成材料(Proceed(?)补片)。
三、对纳米银复合生物腹壁修复材料的修复大鼠腹壁缺损后对修复区局部炎症反应和微血管密度的影响进行了初步研究,结果表明纳米银-小肠黏膜下层修复材料与单纯生物腹壁修复材料对修复区局部炎症反应及微血管密度的作用相同,两者较合成材料局部炎症反应轻,微血管密度较合成材料低。
四、对纳米银复合生物腹壁修复材料的生物安全性进行了初步评价,结果表明纳米银-小肠黏膜下层修复材料(NS-SIS)的生物安全性良好。
试验结论
纳米银-小肠黏膜下层修复材料(NS-SIS)有较好的抗菌效果,且伤口感染率和局部炎症反应低于单纯生物腹壁修复材料(SIS)和合成材料(Proceed(?)补片),并具有良好的生物安全性。
创新点
1.国内外首次将生物材料与高分子材料复合构建修复腹壁补片,集中了现有材料几乎全部的优点,为寻求理想腹壁修复材料的研究提供了新的思路；预期实验结果有望改变临床现状。
2.国内外首次将纳米技术、无机杀菌剂应用于疝和腹壁外科,改变现有修复材料抗感染性差的研究出现了新的转机。
Background and objective
Of abdominal wall hernia,war trauma, tumors and other diseases caused by abdominal wall defect as well as the treatment of abdominal compartment syndrome, critical illness, the required use of abdominal wall reconstruction materials, which there is a huge clinical demand. Abdominal wall repair and reconstruction of the ideal physical and chemical nature of the material should be stability, security, non-toxic non-carcinogenic, anti-tension of the abdominal wall superior to the organization, anti-mechanical fatigue, organizational compatibility is good, rejection is small, beneficial to tissue regeneration, source, and easy to use; In addition, the views of the latest material with more emphasis on anti-infective, soft kimono, as well as access to the minimum quote of surgical pain, as well as implantation of a relatively minimal amount of patches. However, recognized at home and abroad have yet to find a completely satisfactory repair material. My hernia repair with artificial material penetration is far lower than western countries, because on the one hand is the primary hospital medical technology update was slow, and the other hand, surgical repair materials used in the industrialization of China has not yet all rely on imports, the price expensive. Far as China's population base and the United States population base, as well as to compare the incidence of hernias, abdominal wall repair and reconstruction material is easy to see the huge potential in China's economic prospect of the meaning and application value.
Biological material is immediate, and abdominal wall hernia surgery abroad, the latest progress of research, including animal-derived materials such as porcine small intestinal submucosa (small intestinal submucosa, SIS), bovine pericardium, were dead-derived materials such as artificial acellular dermal matrix (acellular dermal matrix, ADM) and so on. Overall assessment [7], biological materials and synthetic materials, like the addition to the low hernia recurrence rate, biocompatibility, anti-adhesive, it is also more desirable than synthetic materials, postoperative hernia recurrence, infection, bowel obstruction, fistula formation and other adverse reactions were significantly lower.
Worthy of note is that the anti-microbial activity of biological material is superior to synthetic materials. The study found [8], acetic acid digest prepared SIS extracellular matrix leaching solution can inhibit the Gram-negative Escherichia coli and gram-positive Staphylococcus aureus role. SIS in a Staphylococcus aureus colonization can also be achieved when the well-organized remodeling, implantation of SIS wound infection than synthetic materials [9]. At the same time foreign biological material tested in the repair or reconstruction of abdominal wall defects in the initial clinical experience also shows that repair contaminated or potentially infected abdominal wall defect failure rate 50%, and depends on the type of wound infection rate of revascularization. Therefore, raising the abdominal wall repair material is still resistance to infection and abdominal wall hernia surgery enormous challenge.
In the antibacterial agent, the silver as an antimicrobial agent, a highly efficient, safe non-toxic, broad spectrum anti-bacterial, non-resistance, etc., can kill bacteria, fungi and fungi and even viruses. Silver sterilization mechanism is mainly related with the silver ions, silver ions with bacterial sulfhydryl enzyme inactivation combined so that the latter, blocking the energy metabolism and microbial cell wall synthesis, play to kill bacteria, viruses and eukaryotic microorganisms role. There are also studies have shown that nano-silver particles and pathogens of the DNA bases can be combined to form cross-links, replacement of purine and pyrimidine hydrogen bonds between the adjacent nitrogen, so that DNA denaturation can not be copied, caused by bacteria inactivation. In addition, Silver also promote wound healing functions. The study found that topical wound silver cream inhibit mouse model of allergic contact dermatitis expression of inflammatory cytokines induce apoptosis of inflammatory cells. Animal toxicity experiments and clinical application of fungicides were found to belong to the actual non-toxic silver-level. In recent years, the use of metallic silver nano-technology will be processed into nano-scale particles of silver, its surface area dramatically, indicating significant surface effect, small size effect and the macro-tunnel effect, antibacterial activity greatly enhanced the effectiveness of more durable and more secure. Nano-silver as an antibacterial material has been applied to medical catheters, internal fixation devices, wound dressings and so on. We drew inspiration from the use of biological materials, three-dimensional ultrastructure, the main component of collagen (90% for theⅠ-and typeⅢcollagen fibers) easy-to-particle adhesion, implant diameter of about 15nm with the formation of nano-silver particles antibacterial activity of biological materials. Implantation in vivo degradation of biological material with the gradual release of silver particles, and thus sustain the anti-bacterial colonization and infection, which will help the growth of the host cell infiltration and blood vessel regeneration.
The above, build an ideal new antibacterial bio-polymer composite abdominal wall repair material conditions are ripe, as is expected to build successful, will be the clinical treatment of abdominal wall hernia defect and to provide new options. The literature search, at home and abroad have not yet see the complex biological materials and synthetic materials to build the abdominal wall patches were reported.
Materials and Methods
PartⅠ:anti-bacterial bio-polymer composite materials to build the abdominal wall repair
1. Prepared according to Abraham pig small intestinal submucosa (SIS).
2. Through self-assembly technology to nano-silver particles embedded SIS biological materials to build anti-bacterial silver nano-SIS.
3. Materials using scanning electron microscopy the ultrastructure of agar diffusion method testing composite materials in vitro antimicrobial capacity of.
PartⅡ:Animal experiments and Immunity
1. Use of composite materials, biological materials, synthetic materials, respectively, full-thickness abdominal wall defect repair in rats (3×3cm), each group of 12 adult F344 rats (weighing about 200g), and use 10 ^ 4cfu/ml of Staphylococcus aureus contamination Repair area. After the repair 1d,3d,5d,7d take animal secretions for bacterial culture of abdominal wall, 1d,3d,5d,7d,15d,30d to take animal blood by flame atomic absorption spectrometry detection of silver content in the blood. Animals were killed in February after the repair, abdominal wall injection test for detection Restoration Area compression, use HE and toluidine blue staining, scanning electron microscopy Restoration Area and the central edge of the organizational structure, the degree of blood vessel, take animals, brain, liver, kidney, using flame atomic absorption spectroscopy detection of tissue silver content.
2. SD 30 rats, weighing 200～250g, surgery caused by 3cm×2cm full-thickness abdominal wall defect, were randomly divided into three groups (n=10), respectively, the same area of nano-silver porcine small intestine submucosa (NS-SIS) Simply porcine small intestine submucosa (SIS), and Proceed(?)patch to patch.4 weeks and 8 weeks after removal of abdominal wall repair material, for CD3 (lymphocytes), CD68 (macrophages) and CD31 (vascular endothelium) Immunohistochemical analysis.
PartⅢ:Materials, bio-safety tests
1. Cytotoxicity test, the composite material into 96-well culture plate, cell culture plate divided into 3 groups (composite materials, positive control, negative control), each 12 holes, each hole by adding cell culture medium 2mL, placed at 37℃box,24 h after the stand-out. Will have strong growth of 48 h cultured L929 cells, calculate the cell density (cells/mL):preparation of 4×104/mL the cell suspension, remove the 96-hole cell culture plates, were added to cell suspension 0.5 mL, at 37℃for. After culture the first 1,2,4,7 d, each depicting three holes, cell morphology and cell count.
2. The skin stimulation test, in adult rabbit skin injection of composite materials at home leaching solution and the control solution (saline) of 0.2 mL, was injected 15 min,1 h,2 d,3 d experimental zone observed skin reactions.
3. The skin sensitization test in guinea pigs back hair removal zone Tu 0.1mL composite materials leaching solution and the control solution (saline) Each 0.1 mL, a continuous observation of 3d, recording test area of skin tissue reaction.
4. For pyrogen test:in the adult rabbit ear vein injection of composite materials leaching solution and the control solution (saline) of 0.1 mL, after injection, 1h,6h,12h,24h,48h,72h body temperature were measured in rabbits.
5. The data using SPSS 11.0 statistical package for statistical analysis, experimental data indicated that the mean±standard deviation, P value of 0.05 was considered statistically significant.
Results
1. For bio-nano-silver abdominal wall repair materials (NS-SIS) in vitro antibacterial effect of the preliminary study results show that the nano-silver biological abdominal wall repair material has good in vitro antibacterial effects.
2. Nano-silver biological materials can be used to repair abdominal wall to repair contaminated abdominal wall defects in rats, and the wound infection rate of less than the purely biological abdominal wall repair material (SIS) and synthetic materials (Proceed(?)patch).
Three pairs of nano-silver compound bio-repair materials, repair of abdominal wall abdominal wall defects in rats after the Restoration Area local inflammatory response and the impact of microvessel density, a preliminary study results show that nano-silver material and purely biological repair of abdominal wall abdominal wall repair of biological materials on the Restoration Area the local inflammatory response and the role of microvessel density in the same synthetic material between the local inflammatory response than the light, microvessel density, lower than the synthetic materials.
Fourth, nano-silver compound bio-bio-safety of the abdominal wall repair material to conduct a preliminary evaluation results show that the nano-silver bio-abdominal repair materials (NS-SIS) of the bio-security was good.
Conclusion
Nano Silver Bio-abdominal repair materials (NS-SIS) has good anti-bacterial effect, and the wound infection and local inflammatory response than a simple biological abdominal wall repair material (SIS) and synthetic materials (Proceed(?)patch), and has a good bio-security.
Innovation
1. At home and abroad for the first time of biological materials and polymer composite materials to build abdominal wall repair patch, brings together almost all the advantages of existing materials, in order to find the ideal study of abdominal wall repair material provides a new idea; the expected clinical results are expected to change the status quo.
2. At home and abroad for the first time nanotechnology, inorganic fungicides used in hernia and abdominal wall surgery, and changes to existing repair materials with poor anti-infective research there is a new turn for the better.
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