同种异体富血小板血浆对兔骨缺损修复作用的实验研究

发布时间：2018-01-24 06:11

本文关键词： 兔骨缺损植骨同种异体富血小板血浆修复　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本实验旨在观察不同时间同种异体富血小板血浆联合同种异体骨植入对骨缺损修复的影像学和组织学变化,以探讨同种异体富血小板血浆对骨缺损植骨修复的疗效,从而为临床工作提供实验理论依据及指导意见。方法:1实验分组:选取健康新西兰大白兔36只,于兔的双下肢股骨内侧髁建立骨缺损的模型后按分笼饲养编号随机分为A、B两组,A组(实验组,n=18),同种异体骨复合同种异体富血小板血浆植入;B组(对照组,n=18),单纯植入同种异体骨,另取6只单纯建立骨缺损模型,缺损不做处理作为模型组。2同种异体PRP的制备:心脏取血50ml,分别移入10ml离心管进行二次离心,第一次以200g离心力,离心10min,第二次同样以200g离心力离心,时间同上,制备PRP约1ml,将PRP与同种异体骨按2:1比例混合激活后,制成复合型PRP凝胶。3血小板计数:心脏取血后,使用血细胞分析仪,取少量血液进行血小板计数,记录全血中血小板数,参考Landesberg法通过两次离心法制备PRP,同样对PRP进行血小板计数,验证二次离心法制备出的PRP是否满足实验要求。4观察及评价指标:第4、8、12周时,采用过量麻醉的方法处死各组实验动物,首先设定特定的曝光条件摄股骨髁侧位X光片(距离1m,条件220KV,30mA.曝光时间0.15s),观察骨缺损植骨愈合情况。参照Yang氏放射学评分标准进行评分,并通过X线平片测量记录其骨缺损区上、中、下三点的阻射密度值,取平均值进行统计分析。而后截取标本对其进行大体标本观察,观察植入材料的表面变化和骨组织缺损区域植入骨的生长修复情况。制作病理组织切片及免疫组化染色,光学显微镜下观察兔骨缺损再生与修复情况;并根据Lane氏组织学评分标准对每组动物的骨修复情况进行评分;应用显微图像分析系统(HMIAS-2000)和专业图像分析软件(Image-Pro Plus 6.0)计算其免疫组化切片的染色阳性细胞平均光密度(Mean Optical Density,MOD)值,进行统计学分析。结果:1一般情况及大体观察:术后所有动物切口愈合良好,从缺损边界和骨痂形成情况,术后4周和8周时,A组比B组缺损区组织修复骨缺损较平整,修复较好,术后12周,A组和B组骨组织缺损区域均得到完全修复,边界已无法分辨,部分可见异位骨化;2全血及PRP血小板计数情况:PRP中血小板数量为全血中的(4.30±0.28)倍,符合Landesberg法通过两次离心制备PRP的要求,满足了目前实验需求;3影像学评分及观察结果:术后4、8、12周各时间点A组与B组两组放射学评分比较差异具有统计学意义(P0.05),A组评分显著高于B组;而A组与B组两组术后各期X线阻射密度比较,A组阻射密度值优于B组,差异具有统计学意义(P0.05);4组织学评分及镜下观察情况:术后4、8、12周各时期A组和B组Lane组织学评分比较,实验组评分均高于对照组,且比较差异具有统计学意义(P0.05),光学显微镜下观察结果显示术后各时间点实验组成骨的面积均高于对照组,两组比较差异具有统计学意义(P0.05);而免疫组化观察结果显示第4和8周时A组PDGF、TGF-β免疫组化切片平均光密度(MOD)明显高于B组,比较差异具有统计学意义(P0.05),说明第4和8周时PDGF、TGF-β蛋白的表达量高于对照组;术后第12周时,PDGF、TGF-β蛋白的表达量均较第4和8周时下降,且实验组与对照组比较差异没有统计学意义(P0.05)。结论:1同种异体富血小板血浆与异体骨组织复合后,能够促进骨组织生成,加快骨缺损修复。2血小板源性生长因子(PDGF)和转化生长因子(TGF-β)于8周后显著下降,是早期诱导骨组织再生的重要生长因子。3术后12周时模型组的骨缺损处仍未完全修复,证明实验模型成立。
[Abstract]:Objective: To study the change of this experiment is to observe the different time of allogeneic platelet rich plasma combined with bone allograft implantation for repairing bone defect imaging and tissue, to investigate the effect of allogeneic platelet rich plasma clinical effects of bone grafting on the repair of bone defect, so as to provide the experimental basis and theoretical guidance for clinical work. Methods: 1 experimental groups: 36 New Zealand rabbits were selected in rabbit limb medial femoral condyle to establish bone defect model by feeding numbers were randomly divided into A, B two groups, A group (experimental group, n=18), allogeneic bone allograft combined with platelet rich plasma implantation group (control group, B; n=18), simple implantation of allograft bone, another 6 only to establish bone defect model, defect not treated as model group.2 allogenic PRP preparation: heart blood 50ml, were transferred to 10ml centrifuge tube two centrifugal, first to 200g centrifugal Centrifugal force, 10min, second times the same with 200g centrifugal force, the time above, the preparation of PRP about 1ml, PRP and allograft bone mixed according to the proportion of 2:1 after activation, made of composite PRP gel.3 platelet count: blood from the heart, blood cell analyzer, take a small amount of blood in the platelet count, recording whole blood platelet count, Landesberg method two PRP were prepared by centrifugal method, also the platelet count of PRP, verify whether the prepared two PRP centrifugal method can meet the requirement of.4 observation and evaluation index: 4,8,12 weeks, using the method of excessive anesthesia sacrificed animal, first set the exposure condition specific perturbation the lateral femoral condyle X ray (1m 220KV, 30mA. distance, exposure time 0.15s), to observe the bone defect bone graft healing. According to Yang's radiological score standard for evaluation, and by measuring the bone defect records of X Xianping In the area, resistance, density three point value, average value of statistical analysis. Then the interception were gross observation on the repair of growth observed surface changes of implant materials and bone defect area of bone. Making histological and immunohistochemical staining, observe the regeneration of bone defect in rabbits with the restoration of optical microscope; and score according to the standard for evaluation of Lane's bone repair in each animal; the application of image analysis system (HMIAS-2000) and the professional image analysis software (Image-Pro Plus 6) positive cells to calculate the average optical density of immunohistochemical slices (Mean Optical Density, MOD) the value for statistical analysis. Results: 1 the general situation and general observation: after all animal wound healing well from the defect boundary and callus formation, after 4 and 8 weeks, A group than in B group of defects Tissue repair bone defect repair is better than the flat, 12 weeks after operation, A group and B group of bone defects were completely repaired, the boundary has been unable to distinguish, partially visible heterotopic ossification; 2 PRP and blood platelet count: the number of platelets in the blood of PRP (4.30 + 0.28) times, according to Landesberg by the method of two centrifugal PRP preparation requirements, meet the experimental requirements; score and observation results of 3 imaging: statistically significant 4,8,12 weeks each time point in A group and B group two radiology group after the score difference (P0.05), A group was significantly higher than B group; compared their X-ray the density of A group and B group after operation in two groups, A group resistance, density value is better than that of B group, the difference was statistically significant (P0.05); 4 histological grading and microscopic observation: 4,8,12 weeks after surgery in each period of Lane group A group and B group was compared with the experimental group were higher than those in the control group and more, The difference was statistically significant (P0.05), optical microscope observation showed that the experimental group at each time point after operation of bone area were higher than the control group, with significant differences between the two groups (P0.05); and immunohistochemistry results showed that fourth and 8 weeks in group A, PDGF, TGF- P immunohistochemistry average light density (MOD) was significantly higher than that of B group, the difference was statistically significant (P0.05), fourth and 8 weeks of PDGF, the expression of TGF- protein was higher than that in the control group; twelfth weeks after the operation, PDGF, the expression of TGF- beta protein were fourth and 8 weeks of the fall, and the comparison between the experimental group and the the control group was not statistically significant difference (P0.05). Conclusion: 1 allogeneic platelet rich plasma and bone allograft composite tissue, can promote bone formation and accelerate bone repair.2 platelet-derived growth factor (PDGF) and transforming growth factor (TGF-) in 8 weeks decreased significantly early The bone defect of the model group was still not completely repaired at 12 weeks after the important growth factor.3, which proved that the experimental model was established.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687.3

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