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目的:应用可吸收复合材料替代传统的金属材料作骨折内固定装置是当前骨科研究的热点之一。聚乳酸(PLA)和聚乙醇酸(PGA)及其共聚物等可吸收高分子材料越来越受到人们的关注。然而,PLA和PGA虽具有良好的生物相容性和可吸收性,但由于其不具备生物活性、降解速度较快、力学强度和弹性模量不如正常皮质骨和降解时的酸性产物导致炎症反应的缺点,限制了它在骨科领域的应用。PLA和PGA的共聚物聚乳酸乙醇酸(PLGA)具有较高的力学强度,但仍不具备生物活性。羟基磷灰石(HA)在生物学特性方面,具有良好的生物相容性和骨传导性,与骨组织能形成直接的骨性结合,但HA材料缺乏足够的强度和疲劳承受能力,不能作为结构材料使用。因此,我们以PLGA和聚L-乳酸(PLLA)为基质,以HA作为生物加强相,制成PLGA/PLLA/HA复合材料,并通过体内外实验和力学实验评价材料的生物相容性和力学性能。方法:(1)试验材料的制备:将PLGA、PLLA、HA粉末[PLLA:(PLGA+HA)的摩尔比均为3:1]成比例混合,研磨均匀、混匀后液压成形。按HA比例5%、10%、15%分为试验组A、B、C。(2)细胞毒性试验:将MG-63细胞接种到96孔板内,培养24h后将受试材料的浸提液及阴阳性对照加入96孔板共同培养,分别于1、3、5、7d各取出一块96孔板,MTT比色法检测吸光度值,计算细胞相对增殖率并进行细胞毒性分级。(3)粘附试验:将MG-63细胞接种到含粘附标本的6孔板,培养3天后取出标本,PBS液漂洗,2%冷戊二醛和1%锇酸固定,梯度酒精脱水,临界点干燥,喷金后行电镜扫描,观察细胞形态。(4)碱性磷酸酶(ALP)活性检测:将MG-63细胞接种到6孔板,培养24h后将受试材料的浸提液及阴性对照加入6孔板共同培养,5d后将细胞消化下来制成细胞匀浆液。以碱性磷酸酶试剂盒和Braford蛋白含量检测试剂盒步骤测定细胞匀浆液中碱性磷酸酶活性。(5)动物体内埋置试验:取新西兰纯种兔12只,在背部肌肉植X3组实验材料。分别于术后1、4、12w取材,石蜡切片,HE染色,光学显微镜下观察组织反应情况进行评级。结果:(1)细胞毒性试验:PLLA/PLGA/HA复合材料细胞毒性为0级或1级,表明PLLA/PLGA/HA复合材料无或仅具有极轻微的细胞毒性。(2)粘附试验:扫描电镜显示MG-63细胞在PLLA/PLGA/HA复合材料表面生长良好。(3)碱性磷酸酶(ALP)活性检测:阴性对照组中的碱性磷酸酶表达很低,与试验组A、B、C有显著性差异,结果表明PLLA/PLGA/HA复合材料具有良好生物相容性,且有利于成骨细胞生长分化及成骨活性表达。(4)动物体内埋置试验:通过国家标准GB/T 16175-1996对其进行组织反应分级,PLLA/PLGA/HA复合材料符合标准,说明材料有良好的组织相容性。结论:PLLA/PLGA/HA复合材料具有良好的生物相容性,且有利于成骨细胞生长分化及成骨活性表达,已达到生物安全性初级检测及临床应用前试验的要求,为下一步的临床应用提供了可靠的实验室依据,作为传统金属材料内固定的替代有着广阔前景。
 
      【英文摘要】 Objective:Absorbable synthetic material applying to substitute material for fracture fixation is one of central issues in Orthopedics. Polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers has attracted more and more attentions. Among absorbable fixation materials, PLA and PGA tend to be used more frequently than the others because their favorable biocompatibility and absorbability. However, several complications have been reported, including biological inactivity, fast degradation rate, lower mechanical strength and elastic modulus than natural cortical bone, aseptic inflammation caused by degradable product. Poly lactic-co-glycolic acid (PLGA), the copolymer of PLA and PGA, raised higher mechanical strength, but still biological inactivity. Hydroxylapatite (HA), with favorable biocompatibility and bone conduction, can form direct combination with bone structure.So, we choose the PLLA and PLGA as the matrix of the absorbable materials, and the Hydroxylapatite as the bioactive composition, fabricate PLGA/PLLA/HA Synthetic Material. Investigations in vitro and vivo biocompatibility assays and mechanics experiments of PLGA/PLLA/HA Synthetic Material were carried out. Methods:(1) Preparation of PLGA/PLLA/HA Synthetic Material: Mix the PLGA, PLLA and HA powder as certain proportion [PLLA: (PLGA+HA) =3:1, mol ratio], mill uniformly, blend, and shape as rods by hydraulic forming. The test materials were grouped by proportion of HA (5%,10%, 15%) as Group A, Group B, Group C.(2)Cytotoxicity test: Mg-63 cell were incubated on 96 pore plate, cultured with the leaching liquor of 3 group of test material after 24h, tested optical density value in MTT assay in 1d, 3d, 5d, 7d. With the results, we calculated the cell proliferation levels (RGR) and assessed the cytotoxicity grade.(3) Adherence test: Mg-63 cell were incubated on 6 pore plate with adherence materials and cultured for 3d. The test materials, washed with PBS liquid, fixed by 2% cold glutaric dialdehyde and 1% osmic acid, dehydrated with gradient of alcohol, dried in critical point, sprayed metal, were observed with Scanning Electron Microscopy (SEM).(4)Alkaline Phosphatase (ALP) activity test: Mg-63 cell were incubated on 6 pore plate, cultured with the leaching liquor of 3 group of test material after 24h. Make cell homogenate liquid by digesting cell after 5d. Alkaline Phosphatase (ALP) activity of cell homogenate liquid was measured by alkaline Phosphatase test kit and Braford protein content test kit. (5)Implant test: Test material was implanted in back muscle bladder in 12 New Zealand rabbits. These rabbits were executed after anesthesia at 1w, 4w, 12w post-operation to conduct paraffin section and hematoxylin-eosin (HE) staining in the surrounding tissues of materials and observe the tissue reaction degree.Results:(1) Cytotoxicity test: The toxic levels of test materials are ranged from 0 to 1 degree, which indicates PLGA/PLLA/HA Synthetic Material has no or only minimal cytotoxity.(2)Adherence test: Observed with Scanning Electron Microscopy (SEM), the morphology of the cells close to test materials was normal.(3)Alkaline Phosphatase (ALP) activity test: Alkaline Phosphatase (ALP) activity is very lowly expressed in negative control group, has significant difference with test groups. It indicates PLGA/PLLA/HA Synthetic Material is favorable biocompatible, and can stimulate the growth and osteogenic activity of osteoplast.(4)Implant test: Ranged the tissue reaction degree with national standard GB/T 16175-1996, PLGA/PLLA/HA Synthetic Material meet the criterion, has favorable biocompatibility.Conclusions:PLGA/PLLA/HA Synthetic Material has favorable biocompatibility, and can stimulate the growth and osteogenic activity of osteoplast. It has reached the requirements of the bio-safety initial testing and the clinical application pre-test, provides a reliable laboratory basis for the next phase of clinical application. It has broad prospects as a substitute material for conventional metallic materials internal fixation.
 
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