【作者】 李毅；

【导师】 李少萍；

【作者基本信息】 第一军医大学 ， 颌面外科， 2004， 硕士

【摘要】 研究背景 目前临床广泛应用的金属骨折内固定材料，由于存在应力遮挡效应、腐蚀刺激周围组织、易感染等弊端，不利于骨折愈合，一般需二次手术取出；而可吸收性骨折内固定材料，主要以聚乳酸为代表，具有无应力遮挡效应、避免再次手术取出等优点，被认为是理想的骨折内固定材料。然而，随着研究和应用的进一步深入，聚乳酸作为骨折内固定材料的缺陷也进一步暴露：首先，聚乳酸降解时生成的乳酸等酸性降解产物，可使局部组织内的pH值下降，诱发迟发性异物炎性反应；其次，聚乳酸的体内降解周期过长，影响骨折愈合后的骨组织改建，也容易导致感染等，限制了聚乳酸的应用。 甲壳素是自然界中少见的带正电荷的高分子聚合物，与软骨基质糖氨多糖（GAG）结构相似。该材料具有良好的生物相容性，无毒，降解过程中产生的低分子寡聚糖在体内不积累，几乎没有免疫原性，对人体无不良反应，目前已应用于伤口敷料、骨缺损充填及药物缓释系统等生物医学领域，其医学应用研究十分活跃。但甲壳素在水及有机溶剂中不溶解，壳聚糖只溶于稀酸中，所以其应用范围受到限制。在甲壳素分子结构中含有很多活泼的氨基（-NH₂）和羟基（-OH），易于进行化学修饰和改性，如果引入多功能基团，还可拓宽其应用领域。 根据其生物学特性，我们研制了新型的骨折内固定材料——聚乳酸-甲壳素接骨板（PL刀CHI）及螺钉。甲壳素降解时释放的氨基葡萄糖等碱性小分子物质，可中和聚乳酸降解时释放出的乳酸等酸性产物，抑制迟发性异物炎性反应的发生;甲壳素中轻基（一OH）、氨基（一HZ）等大量活性基团，可与聚乳酸分子中的活性基团轻基（一OH）、梭基（一COOH）通过氢键、酷键、氨键、离子键等多种形式结合，增强聚乳酸一甲壳素接骨板的机械强度，满足骨折内固定的需要;另外，聚乳酸的降解过程，是无需酶参与的简单水解过程，而甲壳素具有良好的亲水性，可加速水分子扩散渗透进入聚乳酸一甲壳素接骨板内的速度，促进了聚乳酸降解，缩短其体内降解吸收周期。二者相互促进、相互补充，因此，聚乳酸一甲壳素接骨板具有良好的性能，有望成为一种具有良好发展前景的可吸收骨折内固定材料。研究目的 通过体外细胞培养技术，采用不同的细胞毒性试验方法，检测聚乳酸一甲壳素接骨板接触细胞后细胞发生的特异性生物学变化，并预测最终体内应用时的组织细胞反应，在短期内较经济、简便地检测出聚乳酸一甲壳素接骨板的细胞毒性，为动物实验的进行与否提供先决条件，并为今后聚乳酸一甲壳素接骨板的临床应用提供理论依据和实验基础。研究方法1.分别用聚乳酸一甲壳素接骨板浸提液（测试样品，实验组）、聚乳酸接 骨板浸提液（参照样品组）和即Mn 640培养基（空白对照组）培 养L929细胞，通过细胞增殖度法（RGR法）、细胞生长抑制法（MTT 法）、流式细胞仪法（F CM法），观察其生长、增殖及形态学变化， 对聚乳酸一甲壳素接骨板的细胞毒性进行评价;2.将L929细胞直接接种于聚乳酸一甲壳素接骨板表面，进行直接接触 法实验，通过光镜、扫描电镜及透射电镜观察细胞的生长、增殖及 形态学变化，进一步对聚乳酸一甲壳素接骨板的细胞毒性进行评价。3.用SD大鼠成骨细胞进行MTT法和直接接触法实验，观察细胞的生 一2一 长、增殖及形态学变化，评价聚乳酸一甲壳素接骨板的骨细胞相容性。4.统计方法:采用SPsslo.0 forwindows统计软件，将所得实验数据进 行统计学处理。结果1.细胞增殖度法:用聚乳酸一甲壳素接骨板浸提液（实验组）培养的L929细胞在第2、4、7d的细胞相对增殖率均在100%以上，细胞毒性评分均为O级;各观察期中，实验组细胞呈短梭形，细胞突充分伸展，形态良好，并可见大量分裂细胞，胞核未见异形性，而且随着时间延长，细胞数量增多，与空白对照组相比，P>O.05，无显著性差异，说明聚乳酸一甲壳素接骨板浸提液对L929细胞的增殖有促进作用，无明显的细胞毒性。2.MTT法:随培养时间延长，聚乳酸一甲壳素接骨板的50%、100%浓度浸提液组（实验组）和RPMI 1 640培养液组（空白对照组）的OD值均逐渐增加，50%、100%浓度浸提液组在第2、4、7d的细胞相对增殖率均在90%以上，聚乳酸一甲壳素接骨板的细胞毒性为0级或1级，提示聚乳酸一甲壳素接骨板浸提液对L929细胞增殖无明显抑制作用，说明聚乳酸一甲壳素接骨板无明显的细胞毒性。3.流式细胞仪法:聚乳酸一甲壳素接骨板组（实验组）和聚乳酸接骨板组（参照样品组）细胞的GOG;期（DNA合成前期）的DNA百分含量低于空白对照组;在S期（DNA合成期），实验组（49.6%）高于参照样品组（38.07%）和空白对照组（22.57%），提示聚乳酸一甲壳素接骨板能促使更多的L929细胞进入S期，使DNA合成加速，导致细胞增殖，无明显的细胞毒性。4.直接接触法: 光镜下观察，聚乳酸一甲壳素接骨板组（实验组）可见大量 L929细胞附着于聚乳酸一甲壳素接骨板表面生长，形态良好。细胞与聚乳酸一甲壳素接骨板紧密贴合，并随着时间延长，大量增殖。与聚乳酸接骨板组(参照样品更多还原

【Abstract】 BACKGROUND:Internal fixation devices have long been used to reduce and stabilize bone fractures, including metal wires, metal plates, et al. The advantage of metal plates in osteosynthesis includes accurate three-dimensional stabilization of fracture segments, direct bone healing, and early mobilization. Nevertheless, permanent metal plating systems have several shortcomings. Palpability, infection, migration, and extrusion are the most commonly reported complications. The metal plates have also been reported to hinder local structural growth and interfere with diagnostic and therapeutic radiation. Other reported drawbacks of metal plates include stress shielding, cortical osteopenia, thermal sensitivity, and potential for more severe damage with repeated injury. Moreover, many trauma surgeons, including those of the AO-ASIF school, recommend that all metallic implants used for the fixation of fractures be removed in due course. The development of biodegradable internal bone fixation was fueled by these disadvantages. Over the last three decades, there has been considerable development and subsequent application of degradable biomaterials. One of the most commonly tested biodegradable biomaterials is polylactic acid (PLA), which has excellent biocompatibilityand can disintegrate completely after a sufficient time of osseofixation in the body. However, with the development of the study and application, the shortcomings of PLA have become clear. First, the lactic acid piled up locally may cause down-regulation of pH, which triggers the aseptic inflammation. Second, PLA biodegrades slowly, which also might hinder local structural growth. What’s more, the compatibility of PLA needs more development. In some cases, foreign body reaction is severe.Chitin, rich in nature, resembles GAG (glycosaminoglycan) in structure and turns into Chitosan after deacetylation. Chitosan takes the positive charge. And it contains many (-NEs and (-OH)s, which is easy for chemical modification and characteristic deformation. The chitosan also has good biological compatibility and its degradation products (N-acetylglucose and aminoglucose) will not store up in vivo or do harm to human body.In this study, PLA-Chitin Compound was made into Plates and Screws as internal bone fixation devices. On the one hand, PLA has lots of (-OH)s and (-COOH)s; while chitin has lots of (-NH2)s and (-OH)s. These activity groups can combine together, which will strengthen the mechanical power of the compound and satisfied the need of bone fracture fixation. On the other hand, as PLA-Chitin plates degrade in the body, PLA discharge lactic acid which could be neutralized by the basic molecules that Chitin discharges. This process avoids lactic acid piled up locally, and might eliminate aseptic inflammation. What’s more, the excellent hydrophilic affinity of Chitin enhances water to enter PLA-Chitin Compound, which will accelerate the degradation of PLA and shorten the degradation period of PLA-Chitin Compound. On the whole, we assume that PLA-Chitin Compound is one of the most prospective implants in osteosynthesis.OBJECTIVES:1. To evaluate the cytotoxicity of the Biodegradable Polylactic Acid-ChitinPlates in different assays using the cell line L-929 in vitro, for all medical devices are required to be evaluated before their clinical application. 2. To investigate the osteocompatibility of the Biodegradable Polylactic Acid-Chitin Plates using osteoblast in vitro.Methods:1. The cytotoxicity of the Polylactic Acid-Chitin Plates was evaluated in extract assay (the RGR assay and the MTT assay), direct contact assay, and FCM assay using the cell line L-929 in vitro.2. The cytotoxicity and osteocompatibility of the Polylactic Acid-Chitin Plates was evaluated in the MTT assay and the direct contact assay using osteoblast in vitro. Cell growth, proliferation, and attachment were studied when osteoblasts, together with Polylactic Acid-Chitin Plates, were cultured.3. Statistical method: All data were analyzed with SPSS 10.0 statist更多还原