【作者】 潘月海；

【导师】 路来金；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 人工关节置换术已成为治疗髋关节、膝关节和手足部各关节等关节严重病变的主要手段，被誉为20世纪骨科发展史中重要的里程碑之一。但用于生产人工关节的材料尚不尽人意。一方面要求其要具有优异的生物相容性、不产生任何的毒副作用；同时应具有寿命长、不磨损、不产生松动等特性。聚酰亚胺材料是我国自行研发的一类高分子聚合材料，具有优良的物理化学稳定性、易加工性、表面粘附性、自润滑性、生物力学性能，是体内植入材料的理想选择。但目前国内尚无聚酰亚胺树脂作为体内植入材料的相关报道，国外仅限于口腔内假体的研究，并且国内外的聚酰亚胺的分子结构亦有不同。本文对由长春高琦聚酰亚胺材料有限公司生产的碳纤维增强的聚酰亚胺纤维为材料，对其体外的细胞毒性、体内植入毒性、遗传毒性等进行生物相容性的评价，为其临床应用奠定基础。目的：本研究旨在探索长春高琦聚酰亚胺材料有限公司所生产的聚酰亚胺树脂作为体内植入材料的生物学特性；并利用CF作为填充相，改善该材料的生物力学性能，为该材料在人体应用提供理论依据，争取为临床人工关节和骨折内固定材料的应用奠定基础。方法：1.材料制备：热固性PMR型聚酰亚胺的合成过程称为原位单体聚合反应物，比热塑性聚酰亚胺更容易加工成高质量的复合结构。碳纤维增强聚酰亚胺复合材料通常采用PMR型聚酰亚胺浸渍碳纤维来制成，树脂的聚合通过交联封端基团和环化脱水产生的二酰亚胺聚合物链。本研究的聚酰亚胺由长春高琦聚酰亚胺材料有限公司提供：在室温下将预聚物粉末50克放在直径为10厘米的金属模具内，放在预热到220度的压榨机上加热，当温度达到280℃，压力3-4Mpa。释放热量1分钟，以使低分子量的分子逸出。然后重新加压，加热温度到达320℃。在这个温度下固话2小时，然后在相应压力下冷却到200度，释放压力，在室温下取出复合材料。2.将108只大鼠随机分成6组，正常组、阳性对照组、假手术组、PI材料低剂量组、PI材料中剂量组和PI材料高剂量组，每组18只。采用背部植入法，用钝器解剖法在一皮肤切口部位制备皮下囊，底部和皮肤切口应为10mm以上，每个囊内植入一个植入物，植入物之间不能互相接触。分别于手术后1、4、12周取血、取尿检测血常规、血生化、尿常规和尿生化。脱臼处死大鼠，取下材料植入区的皮肤、肝脏、肾脏、胰腺和脾脏进行理切片分析。3.取健康、成年的白色家兔6只，在试验前4h-24h将动物背部脊柱两侧被毛除去（约10cm×15cm区域），作为试验和观察部位。将0.5mL的PI浸提液直接滴到2.5cm×2.5cm大小的吸收性纱布块上，浸提液的用量以能浸透纱布块为宜。用绷带固定敷贴片至少4h。接触期结束后取下敷贴片，用持久性墨水对接触部位进行标记，并用适当的方法除去残留试验材料。在自然光线或全光谱灯光下观察皮肤反应。记录每一接触部位在每一规定时间内皮肤红斑和水肿反应情况。4.用无水乙醇超声清洗试样20min，然后用蒸馏水反复清洗，常规消毒、干燥。根据GB/T16886.12标准中规定，材料表面积与浸提介质6cm2·ml-1的比例加入含10%FBS的RPMI-1640培养液，39℃度轻摇72h，制备浸提液。取出Vero细胞，用含10%FBS的RPMI-1640培养基培养，制备成细胞悬浮液。参照国家标准GB/T16886.5-1997及相关参考文献，采用MTT法检测PI材料对Vero细胞毒性作用，观察24h，48h和72h PI材料和浸提液对vero细胞存活率的影响。进行HE染色，检测接触PI材料对细胞形态的影响5.取50只昆明小鼠随机分为5组，分别为PI浸提液低剂量组、PI浸提液中剂量组、PI浸提液高剂量组、阳性对照组以及阴性对照组。PI浸提液低剂量组给予材料浸提液5ml/kg，连续灌胃给药30天；PI浸提液中剂量组给予材料浸提液10ml/kg，连续灌胃给药30天；PI浸提液高剂量组给予材料浸提液20ml/kg，连续灌胃给药30天，阴性对照组给予生理盐水，连续灌胃给药30天；阳性对照组给予环磷酰胺80mg/kg，于处死动物前30小时和6小时各经腹腔注射给药一次。进行微核实验和染色体畸变分析实验。结果：1.制备出抗压强度、抗弯强度和抗拉强度优于纯聚酰亚胺的碳纤维增强的聚酰亚胺复合材料。其中聚酰亚胺由长春高琦聚酰亚胺材料有限公司提供：在室温下将预聚物粉末50克放在直径为10厘米的金属模具内，放在预热到220度的压榨机上加热，当温度达到280℃，压力3-4Mpa。释放热量1分钟，以使低分子量的分子逸出。然后重新加压，加热温度到达320℃。在这个温度下固话2小时，然后在相应压力下冷却到200度，释放压力，在室温下脱模。机械加工成各种试样。2.于术后第1、4和12周取大鼠全血检测血常规，各组大鼠淋巴细胞绝对值、单核细胞绝对值、血红蛋白、红细胞比容和平均血小板体积不全相等，差异具有统计学意义。淋巴细胞绝对值、单核细胞绝对值阳性对照组水平显著高于其它组（P<0.05），血红蛋白和平均血小板体积阳性对照组的水平显著低于其它组（P<0.05），红细胞比容和血小板分布宽度的水平在各处理间差异无统计学意义（P<0.05）。于术后第1、4和12周取大鼠血清检测血生化，各组大鼠间天门冬氨酸氨基转移酶、丙氨酸氨基转移酶、总蛋白、球蛋白、白/球比例和尿素氮的水平不全相等，差异具有统计学意义（P<0.05）。阳性对照组天门冬氨酸氨基转移酶、丙氨酸氨基转移酶的水平高于正常组、假手术组、低剂量组、中剂量组和高剂量组（P<0.05）。总蛋白水平在阳性对照组中最高，显著高于其它处理组（P<0.05）。球蛋白水平和白/球比例阳性对照组高于其它处理组，差异均具有统计学意义（P<0.05）。阳性对照组尿素氮水平高于其它组（P<0.05）。于术后第1、4和12周取大鼠尿液检测尿常规，阳性对照组大鼠尿白细胞个数和尿病理管型数量显著高于其它组。于术后第1、4和12周取大鼠尿液检测尿胆原，各组大鼠尿胆原阳性率不等，差异具有统计学意义（P<0.001）。其中阳性对照组尿胆原阳性率最高，显著高于其它组，其它组间阳性率差异无统计学意义，。术后第1、4和12周取大鼠尿液检测尿亚硝酸盐，各组大鼠尿亚硝酸盐阳性率不全相等，差异具有统计学意义（P<0.001）。其中阳性对照组大鼠尿亚硝酸盐阳性率最高，显著高于其它组，其它组间阳性率差异无统计学意义。术后第1、4和12周取大鼠尿液检测尿蛋白，各组大鼠尿蛋白阳性分布情况不全相等，差异具有统计学意义（P<0.001）。阳性对照组的大鼠尿蛋白水平和阳性率最高，显著高于其它组，其它组间阳性率差异无统计学意义。术后第1、4和12周取大鼠粪便进行隐血试验，各组大鼠隐血分布情况不全相等，差异具有统计学意义（P<0.001）。其中阳性对照组大鼠隐血阳性率最高，其它组间阳性率差异无统计学意义。术后第1周、第4周和第12周对大鼠肝、脾、肾、胰腺和手术局部皮肤组组进行病理学观察，正常组、假手术组、PI低剂量组、PI中剂量组和PI高剂量组皮肤组织无明显变化，肝脏、脾脏、肾脏和胰腺组织无明显病理变化。第1周阳性对照组大鼠皮肤手术局部有炎性浸润细胞，第4周发现炎性肉芽，第12周可见植入材料出现纤维包裹。肝脏切片见棕色颗粒，肝细胞出现坏死变性。脾脏、肾脏和胰腺内偶见棕色颗粒。3. PI浸提液组家兔背部皮肤未见红斑和水肿反应，阳性药物组动物皮肤见有轻微红斑。4.分别观察PI材料浸提液孵育Vero细胞24h、48h和72h后细胞的存活率变化，随着PI浸提液浓度的增加，细胞存活率无明显改变，而PVC对照组的细胞存活率明显下降，说明在孵育PI材料与PVC材料相比，对Vero细胞存活率的几乎没有影响PI材料浸提液组和材料直接接触组孵育Vero细胞24、48、72小时后细胞形态未见异常，细胞形态完整，胞核浆界限清楚，与正常组细胞无显著性差异。5.阳性对照组（环磷酰胺）有较高的小鼠骨髓微核发生率，阴性对照组与PI浸提液各剂量组小鼠骨髓微核发生率都很低。其中PI浸提液低剂量组、PI浸提液中剂量组和PI浸提液高剂量组的细胞微核率分别为3‰、4‰和3‰，与阴性对照组（3‰）相比，差异没有统计学意义。只有阳性对照组（环磷酰胺）发生了染色体畸变，染色体畸变率为7%；PI浸提液低剂量组、PI浸提液中剂量组、PI浸提液高剂量组以及阴性对照组染色体畸变率均为0。结论：通过体内植入试验、皮肤致敏试验、细胞毒性试验和遗传毒性评价等生物毒理试验，证实碳纤维增强的聚酰亚胺材料生物安全性良好，对生物体无毒副作用，可以作为人工关节的生物材料，应用于临床。本文的创新之处在于材料方面，研究我国最近生产的具有自主知识产权的聚酰亚胺树脂的生物学特性；工艺方面，利用聚酰亚胺树脂的表面粘附性，将碳纤维作为填充材料，进一步改善聚酰亚胺的生物力学性能；应用方面，将材料在人工仿真关节制造中应用。更多还原

【Abstract】 Artificial joints displacement has become one of the major strategies that are used intreatment of diseases in joints. Joints displacement has been given the reputation of a famousmilestone in the development history of bone science. Materials used for the artificial jointsneed to be of excellent biocompatibility, of no potential to producing side effects, of longduration, of sustained wearability, and of no subject to loosing. Polyimide material possessesthe characteristics of physical and chemical stability, flexibility, surface adherence,lubrication, biomechanical support and is an ideal transplanting material. So far, there is noreport on the PI as a transplanting material in domestic and limited research in abroad. Thepresent study will investigate the biocompatibility of the polyimide material enhanced bycarbon fiber designed and produced by Applied Chemistry institute of Changchun and textileindustry design institute of Jilin Province through in vitro cytotoxicity evaluation, in vivotransplanting experiment, genetic toxicity assessment to provide experimental evidence forthe application of polyimide in clinical practice.Objectives: To explore the biocompatibility of the polyimide carbon fiber reinforcedmaterials and evaluate the potential of utilizing PI in artificial displacement in clinicalpractice.Materials and Methods1.108Wistar rats were divided into six groups, control group, positive control group,pseudo-operation group, PI low dosage group, PI medium dosage group and PI high dosagegroup, each group having18rats. PI was transplanted into the back of PI group. Inpseudo-operation group, rats were cut in the back, but no PI was transplanted. PVC was putinto the back of positive group rats. On the day of one week, four weeks and12weeks afterPI was transplanted into the rats,6rats were randomly selected from each group and wereanesthetized and killed, blood and urine were collected for the test; skin around the PI material, kidney, liver, pancreas and spleen were removed and pathological test wasconducted.2. Fur in the back of6white rabbits were removed4or24h prior to experiment, thesurface area was about10cm×15cm square.0.5ml of extract from PI material was directlyput on the back skin which was covered by bandage for at least4h. Ink was used to mark thearea with PI. Under natural light or full spectrum light, the skin in contact with PI extract wasobserved and skin erythema and swelling were recorded.3.PI material was washed with ethanol by ultrasonic cleaning for20min，then rinsed bydistilled water several times, dried in dryer and sterilized. Based on the regulations ofGB/T16886.12, PI material and extract liquid should be at the ratio of6cm2·ml-1. The extractliquid was RPMI-1640supplemented with10%FBS. The extract process was performed at39℃for72h in the constant temperature shaker. Vero cells were used for the detection ofcytotoxicity of PI. MTT assay was conducted to determine the cell survival rate. After verocells were co-cultured with PI for24h，48h and72h, the cell survival rate was determined.4.50kunming mice were randomly divided into5groups, PI extract low dosage group(5ml/kg), medium dosage group (10ml/kg), high dosage group (20ml/kg), positive controlgroup and control group. Cyclophosphamide was administered to positive group rats at thedosage of80mg/kg30h and6h prior to the end of the experiment respectivelyintraperitoneally. PI extract was administered to mice through gastric injection forconsecutive30days. Saline was administered to control group mice through gastric injectionfor consecutive30days. Micronuclear and chromosome aberration was detected in thesemice.Results1. Blood test results showed that the number of lymphocytes and monocytes of rats frompositive group were significantly higher than those from control, pseudo-operation and PIgroups on one week, four weeks and12weeks after transplanting (P<0.05). Bloodhemoglobin content and mean blood platelet of rats from positive group were significantlylower than those from other groups (P<0.05); There was no significant difference of specificred blood cell volume and blood platelet range (P﹥0.05). The levels of aspartate aminotransferase and Alanine aminotransferase of rats frompositive group were significantly higher than those from control, pseudo-operation group, PIlow dosage group, medium dosage group an d high dosage group (P<0.05). Total proteinlevels of rats from positive group were the highest among the rats of all groups (P<0.05).Globulin levels and ratio of albumin to globulin of rats from positive group weresignificantly higher than those from others groups (P<0.05). Urea nitrogen levels of ratsfrom positive group were significantly higher than those from other groups (P<0.05).On one week, four weeks and12weeks after transplanting, the number of white bloodcells and pathological tube in urine of rats from positive group were significantly higherthan those from other groups.Pathological detection showed that there was no pathological changes in the skin, liver,kidney, pancreas and spleen of rats from control, pseudo-operation, PI low dosage group,medium dosage group and high dosage group on one week, four weeks and12weeks aftertransplanting. For the positive control group, on the first week, inflammatory cell infiltrationwas seen in transplanted skin; on four week, inflammatory granulation was seen in thetransplanted skin; on12week, fiber wrap was in the transplanted skin, brown particles wereseen in the liver and liver cells manifested necrotic degeneration; brown particles wereoccasionally seen in the cells of kidney, spleen and pancreas.2.Mild erythema was seen on the skin of rabbits in contact with PVC extract while noerythema and swelling was seen on the skin of rabbits in contact with PI material.3. There was no significant difference in the survival rate of vero cells between cellsco-cultured with PI extract and control cells. However, the survival rate of vero cells in PVCextract was significantly decreased.4. Micronuclear rate of mice from positive group was significantly higher than that fromPI groups. There was no significant difference of micronuclear rate between control groupmice and PI groups.Chromosome aberration was seen in mice from positive group while no chromosomeaberration was found in mice from control and PI extract groups. ConclusionPI was of excellent biocompatibility based on the results from in vivo transplantingexperiment evaluation, skin sensitization test, cytotoxicity test and genetic toxicityassessment.更多还原