生物型异种骨体内外实验的相关研究
发布时间：2018-07-15 18:47
【摘要】：背景及意义：由创伤、肿瘤或代谢疾病等各种原因造成的骨缺损发生率很高,骨缺损的重建一直都是骨科医生面临的一个极具挑战的临床难题。目前应用于临床的骨移植修复材料主要包括自体骨、同种异体骨、异种骨、人工骨等。自体骨作为植骨的“金标准”无可否认,但往往取骨量有限,因此很难对大范围骨缺损进行修复。同种异体骨是目前常用的骨移植材料,主要用于修复、填充骨缺损,具有固定和支撑作用,但其来源有限,不能完全满足临床植骨需求。异种骨材料不受来源限制,经适当处理即可作为骨移植替代材料修复骨缺损,其移植可很好地解决临床上自体骨与同种异体骨来源不足的问题,但异种骨面临的主要问题是如何降低其免疫原性及植骨排斥反应,以达到良好的植骨融合效果。我国是一个拥有众多人口、幅员辽阔的发展中国家,地质灾害、生产事故、交通意外等时有发生,由此造成人员伤亡导致的骨缺损病例大量存在,因此国内市场对骨缺损材料需求量较大。异种骨来源丰富,可以满足这一需求,但其移植后会发生免疫排斥反应,为解决其免疫排斥,相继有许多不同方法处理的异种骨出现,目前市场上已出现多种能应用于临床的异种骨,如Kiel骨、Bio-OSS、RBX重组合异种骨,其治疗效果显著,安全性也得到认可。 目的：本实验分体内实验及体外实验两部分,体外实验采用材料浸提液在体外与细胞共培养的方法检测超临界C02、环氧固定等技术制备并复合骨形态发生蛋白-2(bone morphogenetic protein BMP-2)的生物型异种猪松质骨对成骨细胞增殖活性、迁移能力及细胞周期的影响,评价与成骨细胞的体外生物相容性。体内实验评价生物型异种骨应用于山羊颈椎的椎间融合效果,进一步为生物型异种骨移植材料应用于临床提供安全性和有效性的依据。 材料：生物型异种松质骨：由广东冠昊生物科技股份有限公司采用已获得美国专利授权(US6106555A,US6231614B1)的技术制备；SD大鼠成骨细胞株由广州军区总医院骨科实验室液氮冻存；成年本地山羊18只,体重25-30kg,由广东冠昊生物科技有限公司动物实验中心提供。 第一章异种骨与成骨细胞体外生物相容性研究 方法：将SD大鼠来源的成骨细胞分别在普通培养基和含异种骨浸提液的培养基中培养,于1、3、5、7天用四甲基偶氮唑盐(methyl thiazolyltetrazolium, MTT)法测定吸光度值,以评价细胞的增殖情况。采用Transwell试验,在倒置相差显微镜下观察细胞的迁移情况,计算穿膜成骨细胞数量,检测成骨细胞的迁移能力。应用流式细胞仪检测细胞周期变,以评价异种骨浸提液对成骨细胞细胞周期的影响。 实验数据采用SPSS20.0统计软件进行分析,以均数±标准差(x±s)表示,组间比较采用两独立样本t检验,P0.05为有统计学意义。 结果：1成骨细胞增殖活性培养1、3、5、7d后在显微镜下观察可见实验组与对照组成骨细胞形态均良好。MTT法测定OD值可间接反应细胞增殖数量,实验组与对照组相比,随着培养时间延长,OD值均升高,在1、3、5、7d时间点,数据统计学分析显示两组间吸光度值差别均无统计学意义(P0.05),表明异种骨浸提液对成骨细胞增殖活性无不良影响,与成骨细胞相容性良好,无明显细胞毒性。 2成骨细胞的迁移能力Transwell试验中通过在高倍镜下(×200)随机取5个视野计数,计数每个视野的平均细胞数量评价成骨细胞在普通培养基与含异种骨浸提液培养基的迁移能力。本实验中实验组迁移的细胞数目为：13.40±2.51个／视野,对照组迁移的细胞数目为：8.00±1.87个／视野,数据统计学分析显示两组差别有统计学意义(P0.05),实验组细胞迁移数量多于对照组,表明异种骨复合BMP-2后可促进成骨细胞迁移。 3流式细胞术检测成骨细胞周期实验组与对照组成骨细胞培养24小时后流式细胞术检测,Millipore软件分析实验结果示：实验组G0/G1、S、G2/M分别所占细胞周期百分比为：69.92±1.31%、3.83±0.31%、16.56±1.20%；对照组G0/G1、S、G2/M分别所占细胞周期百分比为：71.61±1.69%、4.06±0.28%、17.24±0.94%,数据统计学分析显示两组间差别均无统计学意义(P0.05)可见两组细胞各期分布相似。表明异种骨浸提液对成骨细胞细胞周期无不良影响。 结论：采用超临界CO2、环氧固定等技术制备并复合骨形态发生蛋白-2的生物型异种猪松质骨对成骨细胞增殖能力及细胞周期无不良影响,能促进成骨细胞迁移,具有良好的生物相容性。 第二章生物型异种骨应用于山羊颈椎融合的实验研究 方法：18只成年本地山羊,体重25-30kg,随机分成A组：自体髂骨组,B组：PEEK融合器加自体骨组,C组：PEEK融合器加生物型异种骨组,每组6只。三组均行C3-C4椎间盘切除术并植入以上内植物。于术前、术后即刻及术后4、8、12、24周时分别拍摄颈椎正、侧位X光线片,并在侧位X线片上测量C3-C4的平均椎间高度(discspace height, DSH),术后12、24周行CT检查,并每组处死3只山羊,取C3-C4节段标本通过大体观察及组织学方法评价脊柱愈合情况。 实验所得数据采用SPSS20.0统计软件进行分析,以均数±标准差(x±s)表示,椎间高度及CT影像学评分整体采用单因素方差分析(One-way ANOVA),组间两两比较采用Bonferroni检验,P0.05为有统计学意义。 结果：1一般情况1只山羊术后当天因麻醉相关并发症死亡,随后补上1只替代。术后山羊正常进食及放养,术后切口轻度肿胀,于第5天肿胀基本消失。术口愈合良好,无感染、渗液及排斥反应等。 2标本大体观察术后12周时A组上下椎体间大部分融合,融合节段可见大量纤维性骨痂；B组及C组融合器与上下椎体融合较为紧密,结合处也可见大量纤维性骨痂。术后24周时A组两椎体完全融合,B组及C组融合器与上下椎体结合紧密,未见明显间隙。 3影像学分析 3.1X线检查融合情况术后即刻X线片显示3组山羊融合节段区可见较明显间隙,无骨小梁通过。术后4周时,A组可见融合节段区密度有所增高,B、C组融合节段区略模糊,密度较A组稍低,周围可见软组织影。术后12周时,A组融合节段区密度较前增高,与椎体骨界面模糊,可见骨小梁通过,B组融合节段区密度较A组低,骨小梁较少,C组融合节段密度与A组相比无明显差别,也可见较多骨小梁通过。术后24周时,A组融合节段区密度较前明显增高,与正常骨分界消失,B组融合节段密度较A组稍低,也可见大量骨小梁通过,与正常骨分界基本消失,C组融合节段与正常骨分界消失,密度与A组相比无明显差别。术前及术后即刻三组动物所测量DSH无组间差异。术后4、8、12及24周B组及C组平均DSH值均大于A组(P0.05),差异有显著性意义,而B组、C组间差异无统计学意义(P0.05)。 3.2CT扫描及评分结果术后12周时A组部分CT层面可见融合器与椎体界面间隙稍模糊,部分新生骨形成,少见骨性连接；B、C组部分CT层面可见新生骨形成,融合器与椎体界面间隙模糊程度较A组轻。术后24周时,A组绝大部分CT层面均可见新生骨形成,融合器与椎体之间已形成骨性连接；B、C组同样可观察到大部分CT层面新生骨形成,融合器与椎体之间有大量骨小梁,形成骨性连接。术后12周时,A组融合度评分较B、C组高(P0.05),B组与C组间数据统计差异无显著性意义(P0.05)；术后24周时,三组间数据统计差异无显著性意义(P0.05)。 4组织学检查术后12周时,A组可见自体髂骨块被纤维骨痂包裹,大量新骨形成骨小梁结构,并可见毛细血管形成；B组PEEK融合器周围可见纤维骨痂包裹,融合器界面可见大量新骨生长入,较多毛细血管形成；C组PEEK融合器周围也可以大量纤维骨痂包裹,大量新骨长入,异种松质骨部分降解,较多毛细血管形成。术后24周时,A组的自体髂骨块与椎体间的新生骨小梁已改建为成熟的骨小梁,纤维骨痂被成熟骨性骨痂代替,植骨块与椎体间已完全骨性融合；B组PEEK融合器周围纤维骨痂被骨性骨痂代替,可见成熟骨小梁,融合器与椎体形成骨性融合；C组在PEEK融合器边缘可见大量成熟的骨小梁组织,异种松质骨已完全降解被正常骨替代,大量骨性骨痂,融合器已椎体已完全形成骨性融合。 结论：在颈椎椎间融合中,椎间融合器加生物型异种骨植骨融合比自体三面皮质髂骨植骨融合可更好地维持颈椎生理曲度,降低椎间隙塌陷发生率,可获得与自体三面皮质髂骨植骨融合及椎间融合器加自体骨植骨融合同样的融合效果,是一种理想的椎间融合材料,在临床上有广阔的应用前景。
[Abstract]:Background and significance: the incidence of bone defects caused by various causes such as trauma, tumor or metabolic disease is very high, and the reconstruction of bone defect has always been a very challenging clinical problem in the Department of orthopedics. The clinical bone graft repair materials include autogenous bone, allograft, xenogeneic bone, artificial bone and so on. As an undeniable "gold standard" for bone graft, it is often difficult to repair large bone defects. Allograft is a common bone graft material, which is used to repair and fill bone defects with fixed and supporting functions, but its source is limited and can not fully meet the needs of clinical bone graft. Xenogenic bone materials It can be used as a substitute for bone graft to repair bone defect without the restriction of source, and the transplantation can solve the problem of the shortage of autogenous bone and allograft bone, but the main problem is how to reduce its immunogenicity and bone graft rejection, so as to achieve good bone graft fusion effect. A large population, a vast developing country, geological disasters, production accidents, traffic accidents and so on occur, resulting in a large number of cases of bone defects caused by casualties, so the domestic market has a large demand for bone defect materials. The rich and rich bone sources can meet this demand, but it will take place after transplantation. In order to solve the immune rejection, in order to solve the immune rejection, many different kinds of bone have been treated in different ways. At present, many kinds of xenogenic bone, such as Kiel bone, Bio-OSS and RBX, have appeared in the market. The therapeutic effect is remarkable and the safety is also recognized.
Objective: this experiment was divided into two parts: in vitro experiment and in vitro experiment. In vitro experiment, the proliferation activity of xenogeneic pig cancellous bone with bone morphogenetic protein -2 (bone morphogenetic protein BMP-2) was prepared by using material leaching solution in vitro and co culture method to detect the proliferation of osteoblast with bone morphogenetic protein -2 (bone protein BMP-2). The effects of migration and cell cycle on the biocompatibility of osteoblasts in vitro were evaluated. In vivo experiments were conducted to evaluate the effect of biogenic xenogeneic bone on the intervertebral fusion of the cervical vertebrae in goats, and to provide the basis for the application of biotype xenograft materials to clinical safety and effectiveness.
Materials: biotype xenogeneic cancellous bone: the technical preparation of US6106555A (US6106555A, US6231614B1) was adopted by the Guangdong crown Hao biological Polytron Technologies Inc; the osteoblast of the rat was frozen in the laboratory of the Department of orthopedics, General Hospita of Guangzhou Military Region; 18 adult local goats, weight 25-30kg, and Guangdong Guan Hao biological technology The animal experiment center of the limited company is provided.
Chapter 1 biocompatibility of xenogeneic bone and osteoblasts in vitro
Methods: the osteoblasts from SD rats were cultured in ordinary medium and culture medium containing dissimilar bone extract, and the absorbance value was measured by four methyl azazoles (methyl thiazolyltetrazolium, MTT) on 1,3,5,7 days to evaluate the cell proliferation. The Transwell test was used to observe the cells under the inverted phase contrast microscope. The number of transmembrane osteoblasts was calculated and the migration ability of osteoblasts was detected. Cell cycle changes were detected by flow cytometry in order to evaluate the effect of xenoextraction on the cell cycle of osteoblasts.
The experimental data were analyzed with SPSS20.0 statistical software, and the average number of standard deviation (x + s) was expressed. The two independent sample t test was used in the group. The P0.05 was statistically significant.
Results: 1 osteocyte proliferation activity was cultured for 1,3,5,7d. The morphology of the bone cells in the experimental group and the control was observed under the microscope, and the.MTT method was good for determining the number of cell proliferation. Compared with the control group, the OD value increased with the time of culture, and the data statistical analysis showed two at the time point of 1,3,5,7d. The difference of absorbance between groups was not statistically significant (P0.05), indicating that the xenoextraction had no adverse effects on the proliferation of osteoblasts, and had good compatibility with osteoblasts, and no obvious cytotoxicity was found.
The migration ability of 2 osteoblasts in the Transwell test was measured by a random number of 5 visual fields at high magnification (x 200), counting the average number of cells in each field to evaluate the migration ability of osteoblasts in common medium and xenoextraction liquid medium. In this experiment, the number of migrating cells in the experimental group was 13.40 + 2.51 / 200. The number of cells migrated in the group was 8 + 1.87 / visual field. Statistical analysis showed that there were significant differences between the two groups (P0.05). The number of cell migration in the experimental group was more than that of the control group, indicating that the migration of osteoblasts could be promoted after the xenogeneic bone combined with BMP-2.
3 flow cytometry was used to test the osteoblast cycle test group and the control group for the flow cytometry after 24 hours of bone culture. The results of Millipore software analysis showed that the percentage of cell cycle of G0/G1, S and G2/M in the experimental group was 69.92 + 1.31%, 3.83 + 0.31% and 16.56 + 1.20%, and the control group was G0/G1, S, and G2/M, respectively. The ratio was 71.61 + 1.69%, 4.06 + 0.28%, and 17.24 + 0.94%. Statistical analysis showed that there was no statistical difference between the two groups (P0.05), which showed that the distribution of cells in the two groups was similar, indicating that the xenoextraction was not harmful to the cell cycle of osteoblasts.
Conclusion: the biotype pig cancellous bone with bone morphogenetic protein -2, using the technique of supercritical CO2 and epoxy immobilization, has no adverse effect on the proliferation and cell cycle of osteoblasts, and can promote the migration of osteoblasts and have good biocompatibility.
The second chapter biological xenogeneic bone should be used in goat cervical vertebrae fusion.
Methods: 18 adult local goats, weight 25-30kg, were randomly divided into group A: autogenous iliac bone group, group B: PEEK fusion apparatus and autogenous bone group, C group: PEEK fusion apparatus and biogenic xenogeneic bone group, each group were treated with C3-C4 discectomy and implanted in the above plant. The cervical vertebrae were taken immediately after operation, immediately after operation and at 4,8,12,24 weeks after operation. In the side position X ray, the mean intervertebral height of C3-C4 (discspace height, DSH) was measured on the lateral X-ray film. The CT examination was performed at 12,24 weeks after the operation. 3 goats were killed in each group. The specimens of C3-C4 segment were taken to evaluate the spinal union by gross observation and histological method.
The experimental data were analyzed with SPSS20.0 statistical software, and the average number of standard deviation (x + s) was expressed. The intervertebral height and CT imaging score were analyzed by single factor analysis of variance (One-way ANOVA), and 22 of the groups were compared with Bonferroni test. P0.05 was statistically significant.
Results: 1 in general, 1 goats were killed on the day of anesthesia related complications, and then 1 were replaced. After the operation, the goats were normally fed and fed, the incision was slightly swollen after the operation, and the swelling disappeared basically in fifth days. The healing of the operation, no infection, infiltration and rejection.
2 the total fusion of the upper and lower vertebrae of group A was observed at 12 weeks after the operation, and a large amount of fibrous callus was seen in the fusion segment, and the fusion device in group B and C was closely integrated with the upper and lower vertebrae, and a large number of fibrous callus were found at the junction. The two vertebral body was completely fused at 24 weeks after the operation, and the fusion apparatus of group B and C group was closely associated with the upper and lower vertebrae. Clear gap.
3 imaging analysis
3.1 X - ray examination showed that the fusion segmental area of the 3 groups of goats showed obvious clearances and no trabeculae passed. At 4 weeks after the operation, the fusion segment area density increased in group A, B, C group was slightly blurred in the fusion segment area, and the density was slightly lower than that of the A group. The density of the fusion segment area in the A group was increased at 12 weeks after the operation. The bone trabecular density of the B group was lower and the bone trabecula was less than that of the A group. The density of the fusion segment of the C group was not significantly different from that of the A group, and there were more bone trabeculae in the group C. The density of the fusion segmental area in the A group was significantly higher than that of the normal bone, and the density of the fusion segment of the B group was less than that of the A group. A large number of bone trabeculae passed, and a large number of bone trabeculae disappeared and the normal bone demarcation disappeared. The fusion segment of group C disappeared and the normal bone demarcation disappeared. The density was not significantly different from that of the A group. The difference between the three groups of animals before and after the operation was measured by three groups. The average DSH value of group 4,8,12 and 24 weeks after operation was greater than that of A group (P0.05), and the difference was significant, but B was significant, B. There was no significant difference between group C (P0.05).
At 12 weeks after the 3.2CT scan and score, part CT of group A showed that the interspace of the fusion device and the vertebral body was slightly blurred, some new bone formed and the bone connection was rare; B, the formation of new bone was visible on the part of CT, and the blurred degree of the interface gap between the fusion apparatus and the vertebral body was lighter than that of the A group. At the 24 week after the operation, most of the CT layers in A group were all visible new bone. Formation, bone connection between the fusion cage and the vertebral body, B, group C can also observe the formation of most of the CT layers, and there are a large number of bone trabeculae between the fusion apparatus and the vertebral body to form the bone connection. At 12 weeks after the operation, the fusion degree score of group A is higher than that of B and C group (P0.05), and there is no significant difference between the B and C group (P0.05), and 24 weeks after the operation. There was no significant difference in data statistics between the three groups (P0.05).
4 after 12 weeks of histological examination, the A group showed that the autogenous iliac bone block was wrapped by the fibrous callus, a large number of new bone formed the trabecular structure, and the capillaries were formed. The fibrous callus was wrapped around the PEEK fusion device in group B, a large number of new bone growth was seen in the fusion interface, more capillary vessels were formed, and the PEEK fusion apparatus in group C could also be found in large quantities. 24 weeks after the operation, 24 weeks after the operation, the autogenous iliac bone block and the new bone trabecula between the vertebral body and the vertebral body had been rebuilt into a mature trabecula, the fibrous callus was replaced by the mature osseous callus, and the bone graft was completely fused with the vertebral body, and the B group was surrounded by the PEEK fusion apparatus. The fibrous callus is replaced by bone callus, the mature trabecula is visible, the fusion apparatus and the vertebral body form bone fusion; in the C group, a large number of mature trabecular tissue are visible on the edge of the PEEK fusion device. The xenogeneic cancellous bone has completely degraded by the normal bone substitute, a large number of bone callus, and the fusion body already formed the bone fusion completely.
Conclusion: in cervical interbody fusion, the fusion of interbody fusion and biogenic xenograft fusion can better maintain the physiological curvature of the cervical vertebra and reduce the incidence of intervertebral collapse, and can obtain the same fusion effect as the fusion of autogenous three facial cortical iliac bone graft and the fusion of intervertebral fusion and autogenous bone graft. Fruit is an ideal intervertebral fusion material and has broad application prospects in clinic.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R683
本文编号：2125041
[Abstract]:Background and significance: the incidence of bone defects caused by various causes such as trauma, tumor or metabolic disease is very high, and the reconstruction of bone defect has always been a very challenging clinical problem in the Department of orthopedics. The clinical bone graft repair materials include autogenous bone, allograft, xenogeneic bone, artificial bone and so on. As an undeniable "gold standard" for bone graft, it is often difficult to repair large bone defects. Allograft is a common bone graft material, which is used to repair and fill bone defects with fixed and supporting functions, but its source is limited and can not fully meet the needs of clinical bone graft. Xenogenic bone materials It can be used as a substitute for bone graft to repair bone defect without the restriction of source, and the transplantation can solve the problem of the shortage of autogenous bone and allograft bone, but the main problem is how to reduce its immunogenicity and bone graft rejection, so as to achieve good bone graft fusion effect. A large population, a vast developing country, geological disasters, production accidents, traffic accidents and so on occur, resulting in a large number of cases of bone defects caused by casualties, so the domestic market has a large demand for bone defect materials. The rich and rich bone sources can meet this demand, but it will take place after transplantation. In order to solve the immune rejection, in order to solve the immune rejection, many different kinds of bone have been treated in different ways. At present, many kinds of xenogenic bone, such as Kiel bone, Bio-OSS and RBX, have appeared in the market. The therapeutic effect is remarkable and the safety is also recognized.
Objective: this experiment was divided into two parts: in vitro experiment and in vitro experiment. In vitro experiment, the proliferation activity of xenogeneic pig cancellous bone with bone morphogenetic protein -2 (bone morphogenetic protein BMP-2) was prepared by using material leaching solution in vitro and co culture method to detect the proliferation of osteoblast with bone morphogenetic protein -2 (bone protein BMP-2). The effects of migration and cell cycle on the biocompatibility of osteoblasts in vitro were evaluated. In vivo experiments were conducted to evaluate the effect of biogenic xenogeneic bone on the intervertebral fusion of the cervical vertebrae in goats, and to provide the basis for the application of biotype xenograft materials to clinical safety and effectiveness.
Materials: biotype xenogeneic cancellous bone: the technical preparation of US6106555A (US6106555A, US6231614B1) was adopted by the Guangdong crown Hao biological Polytron Technologies Inc; the osteoblast of the rat was frozen in the laboratory of the Department of orthopedics, General Hospita of Guangzhou Military Region; 18 adult local goats, weight 25-30kg, and Guangdong Guan Hao biological technology The animal experiment center of the limited company is provided.
Chapter 1 biocompatibility of xenogeneic bone and osteoblasts in vitro
Methods: the osteoblasts from SD rats were cultured in ordinary medium and culture medium containing dissimilar bone extract, and the absorbance value was measured by four methyl azazoles (methyl thiazolyltetrazolium, MTT) on 1,3,5,7 days to evaluate the cell proliferation. The Transwell test was used to observe the cells under the inverted phase contrast microscope. The number of transmembrane osteoblasts was calculated and the migration ability of osteoblasts was detected. Cell cycle changes were detected by flow cytometry in order to evaluate the effect of xenoextraction on the cell cycle of osteoblasts.
The experimental data were analyzed with SPSS20.0 statistical software, and the average number of standard deviation (x + s) was expressed. The two independent sample t test was used in the group. The P0.05 was statistically significant.
Results: 1 osteocyte proliferation activity was cultured for 1,3,5,7d. The morphology of the bone cells in the experimental group and the control was observed under the microscope, and the.MTT method was good for determining the number of cell proliferation. Compared with the control group, the OD value increased with the time of culture, and the data statistical analysis showed two at the time point of 1,3,5,7d. The difference of absorbance between groups was not statistically significant (P0.05), indicating that the xenoextraction had no adverse effects on the proliferation of osteoblasts, and had good compatibility with osteoblasts, and no obvious cytotoxicity was found.
The migration ability of 2 osteoblasts in the Transwell test was measured by a random number of 5 visual fields at high magnification (x 200), counting the average number of cells in each field to evaluate the migration ability of osteoblasts in common medium and xenoextraction liquid medium. In this experiment, the number of migrating cells in the experimental group was 13.40 + 2.51 / 200. The number of cells migrated in the group was 8 + 1.87 / visual field. Statistical analysis showed that there were significant differences between the two groups (P0.05). The number of cell migration in the experimental group was more than that of the control group, indicating that the migration of osteoblasts could be promoted after the xenogeneic bone combined with BMP-2.
3 flow cytometry was used to test the osteoblast cycle test group and the control group for the flow cytometry after 24 hours of bone culture. The results of Millipore software analysis showed that the percentage of cell cycle of G0/G1, S and G2/M in the experimental group was 69.92 + 1.31%, 3.83 + 0.31% and 16.56 + 1.20%, and the control group was G0/G1, S, and G2/M, respectively. The ratio was 71.61 + 1.69%, 4.06 + 0.28%, and 17.24 + 0.94%. Statistical analysis showed that there was no statistical difference between the two groups (P0.05), which showed that the distribution of cells in the two groups was similar, indicating that the xenoextraction was not harmful to the cell cycle of osteoblasts.
Conclusion: the biotype pig cancellous bone with bone morphogenetic protein -2, using the technique of supercritical CO2 and epoxy immobilization, has no adverse effect on the proliferation and cell cycle of osteoblasts, and can promote the migration of osteoblasts and have good biocompatibility.
The second chapter biological xenogeneic bone should be used in goat cervical vertebrae fusion.
Methods: 18 adult local goats, weight 25-30kg, were randomly divided into group A: autogenous iliac bone group, group B: PEEK fusion apparatus and autogenous bone group, C group: PEEK fusion apparatus and biogenic xenogeneic bone group, each group were treated with C3-C4 discectomy and implanted in the above plant. The cervical vertebrae were taken immediately after operation, immediately after operation and at 4,8,12,24 weeks after operation. In the side position X ray, the mean intervertebral height of C3-C4 (discspace height, DSH) was measured on the lateral X-ray film. The CT examination was performed at 12,24 weeks after the operation. 3 goats were killed in each group. The specimens of C3-C4 segment were taken to evaluate the spinal union by gross observation and histological method.
The experimental data were analyzed with SPSS20.0 statistical software, and the average number of standard deviation (x + s) was expressed. The intervertebral height and CT imaging score were analyzed by single factor analysis of variance (One-way ANOVA), and 22 of the groups were compared with Bonferroni test. P0.05 was statistically significant.
Results: 1 in general, 1 goats were killed on the day of anesthesia related complications, and then 1 were replaced. After the operation, the goats were normally fed and fed, the incision was slightly swollen after the operation, and the swelling disappeared basically in fifth days. The healing of the operation, no infection, infiltration and rejection.
2 the total fusion of the upper and lower vertebrae of group A was observed at 12 weeks after the operation, and a large amount of fibrous callus was seen in the fusion segment, and the fusion device in group B and C was closely integrated with the upper and lower vertebrae, and a large number of fibrous callus were found at the junction. The two vertebral body was completely fused at 24 weeks after the operation, and the fusion apparatus of group B and C group was closely associated with the upper and lower vertebrae. Clear gap.
3 imaging analysis
3.1 X - ray examination showed that the fusion segmental area of the 3 groups of goats showed obvious clearances and no trabeculae passed. At 4 weeks after the operation, the fusion segment area density increased in group A, B, C group was slightly blurred in the fusion segment area, and the density was slightly lower than that of the A group. The density of the fusion segment area in the A group was increased at 12 weeks after the operation. The bone trabecular density of the B group was lower and the bone trabecula was less than that of the A group. The density of the fusion segment of the C group was not significantly different from that of the A group, and there were more bone trabeculae in the group C. The density of the fusion segmental area in the A group was significantly higher than that of the normal bone, and the density of the fusion segment of the B group was less than that of the A group. A large number of bone trabeculae passed, and a large number of bone trabeculae disappeared and the normal bone demarcation disappeared. The fusion segment of group C disappeared and the normal bone demarcation disappeared. The density was not significantly different from that of the A group. The difference between the three groups of animals before and after the operation was measured by three groups. The average DSH value of group 4,8,12 and 24 weeks after operation was greater than that of A group (P0.05), and the difference was significant, but B was significant, B. There was no significant difference between group C (P0.05).
At 12 weeks after the 3.2CT scan and score, part CT of group A showed that the interspace of the fusion device and the vertebral body was slightly blurred, some new bone formed and the bone connection was rare; B, the formation of new bone was visible on the part of CT, and the blurred degree of the interface gap between the fusion apparatus and the vertebral body was lighter than that of the A group. At the 24 week after the operation, most of the CT layers in A group were all visible new bone. Formation, bone connection between the fusion cage and the vertebral body, B, group C can also observe the formation of most of the CT layers, and there are a large number of bone trabeculae between the fusion apparatus and the vertebral body to form the bone connection. At 12 weeks after the operation, the fusion degree score of group A is higher than that of B and C group (P0.05), and there is no significant difference between the B and C group (P0.05), and 24 weeks after the operation. There was no significant difference in data statistics between the three groups (P0.05).
4 after 12 weeks of histological examination, the A group showed that the autogenous iliac bone block was wrapped by the fibrous callus, a large number of new bone formed the trabecular structure, and the capillaries were formed. The fibrous callus was wrapped around the PEEK fusion device in group B, a large number of new bone growth was seen in the fusion interface, more capillary vessels were formed, and the PEEK fusion apparatus in group C could also be found in large quantities. 24 weeks after the operation, 24 weeks after the operation, the autogenous iliac bone block and the new bone trabecula between the vertebral body and the vertebral body had been rebuilt into a mature trabecula, the fibrous callus was replaced by the mature osseous callus, and the bone graft was completely fused with the vertebral body, and the B group was surrounded by the PEEK fusion apparatus. The fibrous callus is replaced by bone callus, the mature trabecula is visible, the fusion apparatus and the vertebral body form bone fusion; in the C group, a large number of mature trabecular tissue are visible on the edge of the PEEK fusion device. The xenogeneic cancellous bone has completely degraded by the normal bone substitute, a large number of bone callus, and the fusion body already formed the bone fusion completely.
Conclusion: in cervical interbody fusion, the fusion of interbody fusion and biogenic xenograft fusion can better maintain the physiological curvature of the cervical vertebra and reduce the incidence of intervertebral collapse, and can obtain the same fusion effect as the fusion of autogenous three facial cortical iliac bone graft and the fusion of intervertebral fusion and autogenous bone graft. Fruit is an ideal intervertebral fusion material and has broad application prospects in clinic.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R683
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