【作者】 夏学颖；

【导师】 姜平；

【作者基本信息】 南方医科大学 ， 外科学， 2010， 硕士

【摘要】 研究背景临床上因各种原因所造成的软组织缺损的修复是整形修复外科面对的主要问题之一。软组织的缺损畸形不仅严重影响患者功能和外观,而且影响其心理健康和生活质量。各种充填材料的应用是修复畸形缺损的有效手段之一,但目前临床常用的材料都不同程度地存在一些问题而不能完全满足临床治疗的要求。近年来,组织工程与再生医学发展迅速,特别是再生医学理论为理想的软组织材料研究指明了前景。再生医学是研究组织器官缺损生理性修复以及功能重建的新兴学科,其重要的技术方法即是采用人工生物组织移植并通过激活局部干细胞、改变局部微环境来诱导组织再生和替代,从而达到生理性修复。异种基质材料因其结构与人体组织相似,与细胞亲和性强,能为细胞分化、增殖、生长及功能发挥提供近似体内组织发生发育的细胞外基质(ECM)支架条件,加之其来源丰富,故逐渐被应用到再生医学领域。采用各种脱细胞技术处理的异种基质材料,如膀胱、输尿管、骨组织、心瓣膜、硬脑膜以及脱细胞真皮等,部分已进入临床试验阶段,但在整形外科领域,除针对皮肤组织缺损的脱细胞真皮材料基质(AlloDerm)外,目前还鲜有其它异种软组织材料的应用研究报道。异种材料在制作过程中需进行交联处理,以解决生物来源材料的病菌传播问题,并增强其力学强度,增进其生物稳定性并最终解决生物来源材料的储存问题。同时,通过交联处理保持了生物材料的天然结构,使有利于材料与其植入的生物体融合。戊二醛为目前应用最广泛的交联剂,虽然能起到组织固定及去抗原双重功能,但也存在去除抗原不彻底,特别是固定的组织降解物存在醛类,在组织处理过程中引入了残留毒性,这不利于细胞增殖、分化、迁徙以及功能建立。另外,还会引起宿主一定的体液及细胞免疫排斥反应,而且,用戊二醛进行化学交联后材料容易变得僵硬,柔韧性下降,在医用时常引起严重的钙化。“再生型医用植入器械国家工程实验室”(广东冠昊生物科技股份有限公司承担)采用—种高反应活性的环氧化物作为交联剂(专利号：US6106555、US623161481)解决了这一难题,研究证实此种环氧化物可与蛋白质分子形成稳定的交联键,大大提高材料的稳定性,且无残留毒性,使被处理的异种材料不仅稳定性好,不引起免疫排异反应,还能诱导机体组织向其中生长,并和机体组织融为一体。采用此环氧化物交联处理研制的异种脑膜材料已获得SFDA认证,并在临床广泛应用。以猪的肌腱为原料,采用同一种环氧化物作为交联剂,应用组织固定、去抗原等系列专利技术,“再生型医用植入器械国家工程实验室”研制出了新型异种肌腱基质材料(xenogenic tendon matrix materials, XTMM)。本研究拟通过光镜、扫描电镜观察XTMM的超微结构,参照医用植入材料的国家标准对XTMM的拉伸强度、扯断伸长率、蛋白含量、及重金属和环氧化物残留量进行研究,评价材料的理化性能,进一步通过急性全身毒性试验、致敏试验、皮内刺激试验、细胞毒性试验和皮下埋植试验研究其安全性、生物相容性和生物学转归,探讨XTMM的生物学性能和应用机制,为异种基质材料的研究和应用奠定基础。研究方法一、XTMM的制备取新鲜猪肌腱,消毒后首先作预处理,脱细胞处理后环氧交联固定,加上采用自主研发的系列专利技术,包括多方位除抗原技术,蛋白质分子修饰技术等,作进一步处理后,裁剪、成形、包装,钴60照射灭菌,备用。二、XTMM的形态学观察制备的XTMM分别行大体观察、常规HE染色后光镜下观察材料的超微结构以及扫描电镜观察材料的微观结构。扫描电镜观察：取材料的切片,2.5%(V／V)戊二醛4℃固定24 h,经30%-100%梯度乙醇脱水、醋酸异戊酯浸泡30 min、等电点干燥。干燥后的样品表面喷铂金,扫描电镜观察XTMM的微观结构。三、XTMM的理化性能检测使用微控电子式拉力试验机测量该材料的拉伸强度和拉断伸长率等；采用凯氏定氮法粗略测量材料的总蛋白含量；测量材料的重金属含量和环氧化物残留量；通过测量材料在PBS溶液中的PH值,绘制时间-pH图像,研究材料的化学稳定性。四、XTMM的生物相容性研究参照医疗器械生物学评价标准和要求,采用标准的毒理学方法进行急性毒性试验、致敏试验、皮内刺激实验、细胞毒性试验和皮下埋植试验,对制备的XTMM进行系统的生物相容性及生物安全性评价。五、XTMM的长期转归的实验研究选用健康成年杂种犬24只,雌雄不限,体重10—13公斤,按实验后取材时间不同随机分为4组,每组6只,将材料埋置于狗的鼻骨骨膜下,以目前临床最常用的隆鼻材料硅胶作为对照,3、6、9、12月定期取材,处死动物后取材,H&E染色,光镜下研究XTMM的生物学转归。六、统计方法采用SPSS 13.0统计软件进行分析,数据以均数±标准差表示,材料的理化性能检测采用单样本t检验,皮下埋植试验采用两相关样本非参数检验,P值<0.05为差异有统计学意义。结果一、XTMM的形态学观察材料成均匀的乳白色或浅黄色,表面隐约可见条纹状天然结构,质地均一,材料柔韧性尚可,易塑形,易缝合,可按照所需的大小剪裁。HE染色光镜下见材料为红染纤维束样物,可见缝隙,未见细胞碎屑及蓝染的核物质,脱细胞完全。扫描电镜观察结果见走向一致的发状纤维样物紧密纵行平行排列,平直,无明显弯曲,分叉不明显,表面可见棉絮样物附着,未见细胞成份。的理化性能检测XTMM二、材料的拉伸强度为11-16 MPa,拉断伸长率为52-67%,均高于标准要求。粗测XTMM总蛋白含量为94%,证明材料组成较为单一。重金属含量结果小于1ug/ml,符合不得高于1 ug/ml的标准要求；环氧化物的残留量检测结果为5ug/ml,完全达到应小于10 ug/ml的标准规定。材料化学稳定性检测24h pH值连续监测显示,XTMM在PBS溶液中的pH值稳定,pH值为7.25-7.32。三、XTMM的生物相容性研究急性毒性实验：各组小鼠活动正常,72 h小鼠无死亡,各组动物未见中毒症状或不良反应。致敏试验显示材料组和阴性对照组的皮肤反应指数均为0,无致敏性。皮内刺激实验观察显示,材料浸提液和生理盐水处无明显红斑、水肿和皮肤坏死,极轻微刺激。材料的细胞毒性结果示该材料的相对增殖度较高,细胞毒性分级在0-1级。皮下埋植试验显示各组动物术后生存良好,植入部位均未见组织坏死、积液及化脓感染。组织学观察显示材料在植入14天时引起轻到中度炎症反应,但随时间延长炎症反应逐渐消失。术后不同时间点植入材料组与对照组组织学评分比较差异无显著性意义(P>0.05)。四、XTMM的长期转归的动物实验研究XTMM按预期的设计显示了植入后的转归,材料内部可见自体新生纤维结缔组织呈“蟹足样”长入,形成由原植入物和新生自体组织交织在一起的“过渡态结构”。呈现新生组织逐渐增加,最终取代残留植入材料的趋势。经历1年的缓慢降解,植入的材料残留部分仍然保持了比较完整的微观结构,并与周围新生的纤维组织交织在一起。结论本研究制备的XTMM脱细胞完全,未见细胞成分与碎片,理化性能稳定,同时具有良好的生物相容性及安全性,因此,该材料有望成为一种良好的软组织缺损修复材料。在XTMM植入后可见材料降解的同时伴有自体组织呈“蟹足状”长入材料内部,形成由新生组织和XTMM组成的“过渡态结构”,随着时间的推移而不断地改变着组合比例,这种“爬行替代”的模式能使XTMM成为宿主组织的一部分,从而实现了植入组织的同化,完成了组织再生过程。更多还原

【Abstract】 Background:Treatment of soft tissue defects caused by trauma, tumour surgery or pressure sores is a challenge to the plastic and reconstructive surgeon. Soft tissue defects may not only cause aesthetic and functional problems if not properly repaired, but also affect their mental health and quality of life. Current treatment modalities for soft tissue defects due to various pathologies and trauma include autologous grafting and commercially available fillers. However, these treatment methods present a number of challenges and limitations, such as donor site morbidity and volume loss over time. As such, improved therapeutic modalities need to be developed. In recent years, tissue engineering and regenerative medicine have developed rapidly, especially in the theory of regenerative medicine. Regenerative medicine is the process of creating living, functional tissues to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects. This field holds the promise of regenerating damaged tissues and organs in the body by stimulating previously irreparable tissues to heal themselves. Xenogenic materials has been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. It was gradually applied to the field of regenerative medicine, because it can provide the ECM environments which regulate the migration, proliferation, and differentiation of progenitor cells in and around a scaffold during tissue repair. It has been previously reported that acellular xenogeneic extracellular matrix facilitate the constructive, tissue-specific replacement of diverse tissue structure such as urinary bladder, urethra, bone, dura mater, bioprosthetic heart valve (BPHVs), and acellular dermal materials (ADM) in experimental animals and in human patients. However, there were few reports about xenogenic materials used in the field of plastic surgery, except for the skin defects with ADM.Many attempts have been made to produce long-lasting, biocompatible implants. To overcome the mechanical and biological limitations of synthetic implants, various researchers have begun to focus on the development of a naturally derived biomaterial. In order for materials to be transplanted to a patient from a donor, especially an animal donor, the tissue must be modified to increase resistance to degradation and to decrease immunogenicity, while maintaining natural mechanical properties. Glutaraldehyde (GA) is the first and most prevalent fixative of Fixation with glutaraldehyde was once thought to largely mitigate the immune response to connective tissue xenografts by irreversibly cross-linking graft matrix proteins. It is now clear that both humoral and cell-mediated immune responses to glutaraldehyde-fixed xenografts occur. Moreover, this treatment causes altered mechanical properties, increased calcification, and cytotoxicity. In this study a novel polyepoxy compounds was used as an alternative fixative (Patent number: US6106555, US6231614B1). In some previous reports, xenogenic dura fixed with this polyepoxy compounds represents an effort to overcome some of the drawbacks that are typically encountered with GA. The xenogenic dura was approved by SFDA and widely used in clinical practices. In this study, xenogenic tendon matrix materials were prepared by means of treating porcine tendon with epoxy cross-linking fixation, diversified antigen minimization process, mechanic enhancement modification and surface activating process. The purpose of this study was to evaluate the application potential of the recently developed xenogenic tendon matrix materials as a novel soft-tissue implant by investigating its properties in vivo and in vitro.Methods1. Preparation of xenogenic tendon matrix materialsTaking fresh pig tendon→bio-burden→eliminating impurities and making pretreatments→epoxy cross-linking fixation→protein molecules modification raising the mechanical strength of the material→diversified eliminations of antigen→surface activity modification making the material capable for adhesion to enriched growing factors→packaging→terminal sterilization usingγ-ray→finished.2. Morphologic Observation of xenogenic tendon matrix materialsGross examination; HE stained and observed under the optical microscope; Sample preparation for SEM (scanning electron microscope). Parallel and cross sections were taken, fixed 24 hours at 4℃with 2.5%(v/v) glutaraldehyde, via gradient dehydration with 30%-100% alcohol, soaked in isoamyl acetate 30 minutes, and dried at isoelectric point. The dried surface of the sample was sprayed with platinum. Then, the morphology and structure was observed under SEM.3. Mechanical and chemical testing on of xenogenic tendon matrix materialsTensile properties were determined by using a testing machine (Micro-controlled electron tension-testing device). The protein content of xenogenic tendon matrix materials was tested by Kjeldahl method. Heavy metal content of materials and epoxide residue were examined. The chemical stability of materials was tested by measuring the PH value of the material in PBS solution.4. Biocompatibility of xenogenic tendon matrix materialsAccording to the standard for the biological evaluation of the medical devices, the acute toxicity test, sensitization test, skin stimulating test, cellular toxicity test, and subcutaneous embedded test were carried out to evaluate the biocompatibility of the prepared implants.5. Evaluation of xenogenic tendon matrix materialsTo observe evolution of the xenogenic tendon matrix materials in different implanting periods by means of a canine model.24 mongrel dogs were divided into 6 groups (4 in each group). The animals in each group were sacrificed at 3,6,9,12 months after implantation, respectively.6. StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by t-test. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS 13.0. The accepted level of significance was P<0.05.Results1. Morphologic Observation of xenogenic tendon matrix materialsHistology observation suggested that the xenogenic implant material consisted primarily of collagen without cell fragments. Scanning electron micrograph demonstrated that collagenous fibers were arranged uniformly.2. Mechanical and chemical testing on of xenogenic tendon matrix materialsThe tensile strength was at 11～16MPa.The breaking elongation rate was at 52～67%. The contents of protein was 94%.The residual of polyepoxy compound was lower than the standard line. The pH value in PBS kept stable around 7.24-7.32.3. Biocompatibility of xenogenic tendon matrix materialsNo animal died and no toxicity symptom or adverse effects were shown within 72 hours. No obvious sensitivity were observed. Skin stimulating reactions were not found in the experimental groups and negative control group by intradermal stimulation test. The toxicity of materials leaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All dogs survived well during the embedded test. There was no tissue necrosis, effusion or inflammation at all implantation sites. The xenogenic implant materials promoted slight to moderate inflammation process at 14 days, however, at 30 days, there was a regression of inflammation. After 60 days, it was observed the presence of well-organized connective tissue, and few inflammatory cells. Score evaluation of inflammation response at different time after operation of two groups showed no statistically significant difference (P> 0.05).4. Histological evaluation of xenogenic tendon matrix materials in animalsThe xenogenic tendon matrix materials indicated that regenerated collagen fibers had gradually encroached the implant and became a "composite"biomaterial with both original implanting tissue and host living tissue. After a year of remodeling the remaining materials, although reverse substituted half way through, was still kept its microscopic structure. The process of evaluation can be described as "creeping substitution", which is similar to the process of bone remodeling. ConclusionsThe xenogenic tendon matrix materials consisted primarily of collagen without cell fragments, and it has acceptable biomechanical properties and superior biocompatibility. It may be served as a promising implant material.更多还原