【摘要】：背景及意義：由創(chuàng)傷、腫瘤或代謝疾病等各種原因造成的骨缺損發(fā)生率很高,骨缺損的重建一直都是骨科醫(yī)生面臨的一個極具挑戰(zhàn)的臨床難題。目前應(yīng)用于臨床的骨移植修復(fù)材料主要包括自體骨、同種異體骨、異種骨、人工骨等。自體骨作為植骨的“金標準”無可否認,但往往取骨量有限,因此很難對大范圍骨缺損進行修復(fù)。同種異體骨是目前常用的骨移植材料,主要用于修復(fù)、填充骨缺損,具有固定和支撐作用,但其來源有限,不能完全滿足臨床植骨需求。異種骨材料不受來源限制,經(jīng)適當處理即可作為骨移植替代材料修復(fù)骨缺損,其移植可很好地解決臨床上自體骨與同種異體骨來源不足的問題,但異種骨面臨的主要問題是如何降低其免疫原性及植骨排斥反應(yīng),以達到良好的植骨融合效果。我國是一個擁有眾多人口、幅員遼闊的發(fā)展中國家,地質(zhì)災(zāi)害、生產(chǎn)事故、交通意外等時有發(fā)生,由此造成人員傷亡導(dǎo)致的骨缺損病例大量存在,因此國內(nèi)市場對骨缺損材料需求量較大。異種骨來源豐富,可以滿足這一需求,但其移植后會發(fā)生免疫排斥反應(yīng),為解決其免疫排斥,相繼有許多不同方法處理的異種骨出現(xiàn),目前市場上已出現(xiàn)多種能應(yīng)用于臨床的異種骨,如Kiel骨、Bio-OSS、RBX重組合異種骨,其治療效果顯著,安全性也得到認可。 目的：本實驗分體內(nèi)實驗及體外實驗兩部分,體外實驗采用材料浸提液在體外與細胞共培養(yǎng)的方法檢測超臨界C02、環(huán)氧固定等技術(shù)制備并復(fù)合骨形態(tài)發(fā)生蛋白-2(bone morphogenetic protein BMP-2)的生物型異種豬松質(zhì)骨對成骨細胞增殖活性、遷移能力及細胞周期的影響,評價與成骨細胞的體外生物相容性。體內(nèi)實驗評價生物型異種骨應(yīng)用于山羊頸椎的椎間融合效果,進一步為生物型異種骨移植材料應(yīng)用于臨床提供安全性和有效性的依據(jù)。 材料：生物型異種松質(zhì)骨：由廣東冠昊生物科技股份有限公司采用已獲得美國專利授權(quán)(US6106555A,US6231614B1)的技術(shù)制備；SD大鼠成骨細胞株由廣州軍區(qū)總醫(yī)院骨科實驗室液氮凍存；成年本地山羊18只,體重25-30kg,由廣東冠昊生物科技有限公司動物實驗中心提供。 第一章異種骨與成骨細胞體外生物相容性研究 方法：將SD大鼠來源的成骨細胞分別在普通培養(yǎng)基和含異種骨浸提液的培養(yǎng)基中培養(yǎng),于1、3、5、7天用四甲基偶氮唑鹽(methyl thiazolyltetrazolium, MTT)法測定吸光度值,以評價細胞的增殖情況。采用Transwell試驗,在倒置相差顯微鏡下觀察細胞的遷移情況,計算穿膜成骨細胞數(shù)量,檢測成骨細胞的遷移能力。應(yīng)用流式細胞儀檢測細胞周期變,以評價異種骨浸提液對成骨細胞細胞周期的影響。 實驗數(shù)據(jù)采用SPSS20.0統(tǒng)計軟件進行分析,以均數(shù)±標準差(x±s)表示,組間比較采用兩獨立樣本t檢驗,P0.05為有統(tǒng)計學(xué)意義。 結(jié)果：1成骨細胞增殖活性培養(yǎng)1、3、5、7d后在顯微鏡下觀察可見實驗組與對照組成骨細胞形態(tài)均良好。MTT法測定OD值可間接反應(yīng)細胞增殖數(shù)量,實驗組與對照組相比,隨著培養(yǎng)時間延長,OD值均升高,在1、3、5、7d時間點,數(shù)據(jù)統(tǒng)計學(xué)分析顯示兩組間吸光度值差別均無統(tǒng)計學(xué)意義(P0.05),表明異種骨浸提液對成骨細胞增殖活性無不良影響,與成骨細胞相容性良好,無明顯細胞毒性。 2成骨細胞的遷移能力Transwell試驗中通過在高倍鏡下(×200)隨機取5個視野計數(shù),計數(shù)每個視野的平均細胞數(shù)量評價成骨細胞在普通培養(yǎng)基與含異種骨浸提液培養(yǎng)基的遷移能力。本實驗中實驗組遷移的細胞數(shù)目為：13.40±2.51個／視野,對照組遷移的細胞數(shù)目為：8.00±1.87個／視野,數(shù)據(jù)統(tǒng)計學(xué)分析顯示兩組差別有統(tǒng)計學(xué)意義(P0.05),實驗組細胞遷移數(shù)量多于對照組,表明異種骨復(fù)合BMP-2后可促進成骨細胞遷移。 3流式細胞術(shù)檢測成骨細胞周期實驗組與對照組成骨細胞培養(yǎng)24小時后流式細胞術(shù)檢測,Millipore軟件分析實驗結(jié)果示：實驗組G0/G1、S、G2/M分別所占細胞周期百分比為：69.92±1.31%、3.83±0.31%、16.56±1.20%；對照組G0/G1、S、G2/M分別所占細胞周期百分比為：71.61±1.69%、4.06±0.28%、17.24±0.94%,數(shù)據(jù)統(tǒng)計學(xué)分析顯示兩組間差別均無統(tǒng)計學(xué)意義(P0.05)可見兩組細胞各期分布相似。表明異種骨浸提液對成骨細胞細胞周期無不良影響。 結(jié)論：采用超臨界CO2、環(huán)氧固定等技術(shù)制備并復(fù)合骨形態(tài)發(fā)生蛋白-2的生物型異種豬松質(zhì)骨對成骨細胞增殖能力及細胞周期無不良影響,能促進成骨細胞遷移,具有良好的生物相容性。 第二章生物型異種骨應(yīng)用于山羊頸椎融合的實驗研究 方法：18只成年本地山羊,體重25-30kg,隨機分成A組：自體髂骨組,B組：PEEK融合器加自體骨組,C組：PEEK融合器加生物型異種骨組,每組6只。三組均行C3-C4椎間盤切除術(shù)并植入以上內(nèi)植物。于術(shù)前、術(shù)后即刻及術(shù)后4、8、12、24周時分別拍攝頸椎正、側(cè)位X光線片,并在側(cè)位X線片上測量C3-C4的平均椎間高度(discspace height, DSH),術(shù)后12、24周行CT檢查,并每組處死3只山羊,取C3-C4節(jié)段標本通過大體觀察及組織學(xué)方法評價脊柱愈合情況。 實驗所得數(shù)據(jù)采用SPSS20.0統(tǒng)計軟件進行分析,以均數(shù)±標準差(x±s)表示,椎間高度及CT影像學(xué)評分整體采用單因素方差分析(One-way ANOVA),組間兩兩比較采用Bonferroni檢驗,P0.05為有統(tǒng)計學(xué)意義。 結(jié)果：1一般情況1只山羊術(shù)后當天因麻醉相關(guān)并發(fā)癥死亡,隨后補上1只替代。術(shù)后山羊正常進食及放養(yǎng),術(shù)后切口輕度腫脹,于第5天腫脹基本消失。術(shù)口愈合良好,無感染、滲液及排斥反應(yīng)等。 2標本大體觀察術(shù)后12周時A組上下椎體間大部分融合,融合節(jié)段可見大量纖維性骨痂；B組及C組融合器與上下椎體融合較為緊密,結(jié)合處也可見大量纖維性骨痂。術(shù)后24周時A組兩椎體完全融合,B組及C組融合器與上下椎體結(jié)合緊密,未見明顯間隙。 3影像學(xué)分析 3.1X線檢查融合情況術(shù)后即刻X線片顯示3組山羊融合節(jié)段區(qū)可見較明顯間隙,無骨小梁通過。術(shù)后4周時,A組可見融合節(jié)段區(qū)密度有所增高,B、C組融合節(jié)段區(qū)略模糊,密度較A組稍低,周圍可見軟組織影。術(shù)后12周時,A組融合節(jié)段區(qū)密度較前增高,與椎體骨界面模糊,可見骨小梁通過,B組融合節(jié)段區(qū)密度較A組低,骨小梁較少,C組融合節(jié)段密度與A組相比無明顯差別,也可見較多骨小梁通過。術(shù)后24周時,A組融合節(jié)段區(qū)密度較前明顯增高,與正常骨分界消失,B組融合節(jié)段密度較A組稍低,也可見大量骨小梁通過,與正常骨分界基本消失,C組融合節(jié)段與正常骨分界消失,密度與A組相比無明顯差別。術(shù)前及術(shù)后即刻三組動物所測量DSH無組間差異。術(shù)后4、8、12及24周B組及C組平均DSH值均大于A組(P0.05),差異有顯著性意義,而B組、C組間差異無統(tǒng)計學(xué)意義(P0.05)。 3.2CT掃描及評分結(jié)果術(shù)后12周時A組部分CT層面可見融合器與椎體界面間隙稍模糊,部分新生骨形成,少見骨性連接；B、C組部分CT層面可見新生骨形成,融合器與椎體界面間隙模糊程度較A組輕。術(shù)后24周時,A組絕大部分CT層面均可見新生骨形成,融合器與椎體之間已形成骨性連接；B、C組同樣可觀察到大部分CT層面新生骨形成,融合器與椎體之間有大量骨小梁,形成骨性連接。術(shù)后12周時,A組融合度評分較B、C組高(P0.05),B組與C組間數(shù)據(jù)統(tǒng)計差異無顯著性意義(P0.05)；術(shù)后24周時,三組間數(shù)據(jù)統(tǒng)計差異無顯著性意義(P0.05)。 4組織學(xué)檢查術(shù)后12周時,A組可見自體髂骨塊被纖維骨痂包裹,大量新骨形成骨小梁結(jié)構(gòu),并可見毛細血管形成；B組PEEK融合器周圍可見纖維骨痂包裹,融合器界面可見大量新骨生長入,較多毛細血管形成；C組PEEK融合器周圍也可以大量纖維骨痂包裹,大量新骨長入,異種松質(zhì)骨部分降解,較多毛細血管形成。術(shù)后24周時,A組的自體髂骨塊與椎體間的新生骨小梁已改建為成熟的骨小梁,纖維骨痂被成熟骨性骨痂代替,植骨塊與椎體間已完全骨性融合；B組PEEK融合器周圍纖維骨痂被骨性骨痂代替,可見成熟骨小梁,融合器與椎體形成骨性融合；C組在PEEK融合器邊緣可見大量成熟的骨小梁組織,異種松質(zhì)骨已完全降解被正常骨替代,大量骨性骨痂,融合器已椎體已完全形成骨性融合。 結(jié)論：在頸椎椎間融合中,椎間融合器加生物型異種骨植骨融合比自體三面皮質(zhì)髂骨植骨融合可更好地維持頸椎生理曲度,降低椎間隙塌陷發(fā)生率,可獲得與自體三面皮質(zhì)髂骨植骨融合及椎間融合器加自體骨植骨融合同樣的融合效果,是一種理想的椎間融合材料,在臨床上有廣闊的應(yīng)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R683

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