【作者】 赵亮；

【导师】 李奇；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 硕士

【摘要】 研究目的:通过对纤维蛋白胶/β-磷酸三钙/磷酸二氢钙复合人工骨材料进行动物体内实验,探讨纤维蛋白胶/β-磷酸三钙/磷酸二氢钙复合人工骨材料修复骨缺损的能力及作为人工骨修复替代材料的可行性,指导临床用于骨缺损的修复。研究方法:[1]动物骨缺损模型的建立1动物骨缺损模型的建立选用健康成熟的新西兰大白兔22只,在双侧股骨外侧髁部建立骨缺损模型,用环钻分别钻取直径4mm、孔深8mm的柱状圆洞,采用随机自身对照的实验研究,随机分为两组:将纤维蛋白胶/β-磷酸三钙/磷酸二氢钙复合人工骨材料随机植入一侧兔下肢的骨缺损处,作为实验组;另一侧兔下肢骨缺损以β-磷酸三钙/磷酸二氢钙骨水泥植入,作为对照组。2植入物的准备与植入实验组将β-磷酸三钙/磷酸二氢钙骨水泥与柠檬酸溶液(500mmol/L)按固液比1g∶0.3ml混合,再用双腔注射器同时加入相同体积的纤维蛋白胶溶液和凝血酶溶液。磷酸钙骨水泥与纤维蛋白胶按凝固后体积比2∶1的比例均匀混合组成复合型磷酸钙骨水泥,用专用注射器注入骨缺损孔洞内并使复合型骨水泥外溢,同时加压固定10分钟直至硬化,对照组同样将β-磷酸三钙/磷酸二氢钙骨水泥与柠檬酸按固液比1g∶0.3ml混合,直接用专用注射器注入骨缺损孔洞内并使骨水泥外溢,同时加压固定10分钟直至硬化。[2]人工骨-骨界面力学性能研究1力学标本的制备取材时间分别在术后2、4、8和12周(n=4)。实验动物以空气栓塞方法处死。将股骨髁标本锯下后用双层生理盐水纱布包裹,置入聚乙烯袋内,在-20℃冰箱内保存备用。力学测试前24小时,将置于-20℃冰箱中保存的股骨髁标本取出,室温下自然复温解冻,再将标本用牙托粉固定准备进行力学测试。2力学标本的生物力学测试将准备好的力学标本置于测试机的底托上并固定。应用推出实验的原理,将直径4.0mm的顶冲头垂直对准植入体并与植入体长轴成一条直线,以0.5mm/min的加载速度逐渐加压,设定行程位移为15mm,记录即时加载曲线,其峰值代表界面最大剪切力;同时测量皮质骨与植入体接触的厚度(h)和植入体的直径(d)。用以下公式计算推出实验的剪切强度:最大剪切强度(MPa)=最大剪切力(N)/骨与植入体接触面积(mm~2);骨与植入体的接触面积:S=π×d×h。测试时将每组样本均按上述方法进行测试,得出剪切强度通过SPSS13.0软件包进行统计学分析。[3]人工骨-骨界面的超微结构特征1动物骨缺损标本的肉眼观察于术后2、4、8和12周分别处死新西兰兔,取双侧股骨髁标本,清除植入物表面纤维软组织,肉眼观察骨水泥表面的骨长入情况。2组织病理学观察将双侧股骨髁骨缺损标本修整后,将其置入固定脱钙液中浸泡24小时,待脱钙完全后,自来水连续冲洗3小时,直至残留硝酸冲净为止,然后将标本依次脱水、透明、石蜡包埋后,切片机沿平行于股骨髁纵轴的方向切片(片厚5μm),烘干后脱蜡,常规苏木素-伊红染色法染色,树胶封固。在OlympusBH2型光学显微镜下观察骨与磷酸钙骨水泥及骨与复合型磷酸钙骨水泥的界面结合情况及两种骨水泥的降解情况,材料周围组织内有无成骨细胞、破骨细胞及炎性细胞浸润等。3扫描电镜观察将固定脱钙后的股骨髁标本沿骨水泥植入方向剖开,修整成合适大小(2×5×5mm~3)后,磷酸缓冲液逐级浸泡,0.9%生理盐水反复冲洗,超声波振荡器内洗涤5-10分钟,30%-100%乙醇逐级脱水,临界点干燥,真空喷金,视野选定组织材料交界处,日立S-3000N型扫描电镜下观察材料与骨的界面结合情况、材料的降解及新骨生成情况等。研究结果:1实验动物数量分析:实验所选用新西兰大白兔22只(16只用于生物力学测试,6只用于结构特征检测),无一只死亡,全部进入实验结果分析。2大体观察:大部分动物术后活动进食情况基本正常,精神状态无明显变化,未见瘫痪、惊厥、呼吸抑制等不良情况。手术区均有不同程度肿胀,1周后消失,创口均无感染。材料植入部位未观察到明显的动物机体排斥反应,未见材料裸露,软组织覆盖良好,伤口愈合良好。2周时植入物外观体积和形态无明显变化,骨水泥与宿主骨之间无明显间隙,两组材料植入物均已与宿主骨纤维连接,骨水泥表面被纤维组织包裹,植入区均未见明显骨质结合,实验组植入骨水泥表面颜色由白色变成浅黄色,对照组骨水泥表面颜色仍为白色;4-8周时骨水泥被致密纤维组织包裹,表面有新生骨突起,外形不规则,表面粗糙,颜色变成红色,两组骨水泥外观区别不明显;12周时骨水泥植入体与周围骨质均匀结合,缺损区内骨外形光滑、塑型好,类似自体骨。复合型磷酸钙骨水泥植入区质地较硬,而单纯磷酸钙骨水泥植入区质地较软。3生物力学测定术后2、4、8、12周各组的推出试验所得检测结果采用SPSS13.0版软件包进行析因设计方差分析,从统计学分析结果可以得出,不同的水泥组间有显著差异(F=106.256,P=0.000),实验组复合型骨水泥最大剪切强度((?)=3.312)强于对照组骨水泥((?)=2.672);同组水泥中不同时间点的最大剪切强度有显著性差异(F=1290.166,P=0.000);结合图的多重比较结果可以看出,随着时间的增加,不同的水泥组间有显著差异(F=198.274,P=0.000)。实验组复合型骨水泥最大剪切强度2周(0.511±0.080MPa)和4周(0.938±0.067MPa)时低于对照组骨水泥。但从植入后4周到8周逐渐增强,特别是8周(5.367±0.182MPa)到12周(6.432±0.119MPa)期间,抗剪强度显著高于对照组(P＜0.05)。4组织病理学观察实验组:术后2周骨水泥疏松多孔,材料周边已少许降解,胶原纤维在材料内较多见。骨缺损区内结缔组织增生,较多成纤维细胞和间充质细胞向骨缺损区生长,可见破骨细胞活动;4周时形成新生幼稚骨小梁,胶原纤维已部分吸收,在骨-骨水泥交界处形成明显的降解带,微血管增生;8周时骨水泥降解吸收达高峰,骨水泥被大量条带状的新生骨小梁分割并包绕成块状。骨小梁较多、粗大,排列不规则,骨生长旺盛,新骨组织处于改建区,新生骨量明显增加;12周时,骨水泥降解基本完成,残余的少量骨水泥呈空泡样,缺损处基本上是骨小梁,骨小梁明显增粗成片状,新生骨基本完成了向板层骨改建。对照组:2周时可见骨缺损处未被吸收的磷酸钙骨水泥,骨生长不旺盛;4周时材料周围骨髓组织大致正常,见脂肪空泡及骨髓细胞,有纤维细胞聚集,材料与周围骨及骨髓组织接触较紧密,见少量软骨增生未见新生骨形成;8周时骨水泥部分降解,骨一骨水泥交界部位结合不紧密,可见纤维样组织条带,有软骨增生及新骨形成;12周时材料中有新生骨小梁形成及部分骨化,磷酸钙骨水泥仍有残留。5扫描电镜观察实验组:8周时,新生骨组织自宿主骨边缘向复合型磷酸钙骨水泥植块内长入,植块内充满了骨组织。宿主骨与移植物间界面可见两侧骨质致密度不同,界面两侧骨小梁、骨纤维排列不同,宿主侧排列整齐,移植侧排列不规整,新生骨板层在植块内间隙穿行,将磷酸钙骨水泥颗粒分隔成窝群并包绕;植块中的纤维蛋白胶已降解,呈现大小不一的孔隙;骨板层内可见新生的哈佛氏系统。12周时,新生骨组织改建为胶原纤维束密实、钙化程度高的板层骨组织,宿主骨与移植物间界面消失,两侧骨纤维、骨基质形态排列大致相同。对照组:12周时宿主骨与骨水泥骨界面模糊,界面两侧骨小梁、骨纤维排列不同,骨样组织及编织骨围绕磷酸钙骨水泥颗粒,骨样组织将磷酸钙骨水泥颗粒埋于其中,未见板层骨生成。结论:1.磷酸钙复合纤维蛋白胶人工骨复合材料组比磷酸钙骨水泥组能更有效地修复骨缺损;生物力学测定证明不同的水泥组间有显著差异,随着时间的延长,实验组复合型骨水泥抗剪切强度显著强于对照组单纯骨水泥抗剪强度。2.组织学电镜观察显示磷酸钙复合纤维蛋白胶人工骨复合材料组成骨性能优于磷酸钙骨水泥组;实验证实,磷酸钙复合纤维蛋白胶人工骨复合材料具有良好的可塑形性、可降解性、骨诱导性和生物相容性。3.该可注射性新型复合纤维蛋白胶的磷酸钙人工骨材料具有良好的骨传导能力、力学特性及生物相容性,有望成为骨组织工程中修复骨缺损的理想材料。更多还原

【Abstract】 BackgroundsObjectiveUtilizing the animal experiments of fibrin glue(FG)compounded withβ-tricalcium phosphate(β-TCP)/monocalcium phosphate(MCPM) artificial bone material, to explore the feasibility of FG compoundβ-TCP/MCPM artificial bone material as bone substitute material in repairing bone defects, as well as a possible guidance for clinical applications.Methods[1] Animal model of bone defect1 Preparation of the animal model: Twenty-two healthy adult New Zealand white rabbits were selected. The models of bone defects(4 mm in diameter and 8 mm in depth) were established at the bilateral femoral condyles by drilling. The composite of FG/β-TCP/ MCPM was randomly implanted in the side of the bone defect in rabbit lower limb(as experimental group), the other side of the rabbit lower limb defects inβ-TCP/ MCPM bone cement implantation(as control group).2 Implantation: Theβ—TCP/MCPM/FG samples were prepared by mixingβ—TCP/MCPM powder with the citric acid (or sodium citrate) at powder-to-liquid mass ratios (P/L) of 1g/0.3ml, Dual-chamber syringes reused at the same time add the same volume of fibrin glue solution and thrombin solution. According to the ratio of 2:1 volume ratio, calcium phosphate bone cement with fibrin glue uniformly mixed after solidification. Then one bone defect filled with composite CPC by the special injector until the cement overflowed the pore , and pressed it for 10 minutes till the cement concreted; the operating process of the control group was the same way.[2] Study of biomechanical properties of bone interface1 Preparation of biomechanical specimens: Every four rabbits were sacrificed by air embolism at postoperative 2, 4, 8, 12 weeks. After the distal femurs were sawed down, condyle specimens wrapped with double layer of physiological saline gauze were placed into polyethylene bags and stored at -20℃refrigerator. 24hours before mechanical test, the distal femoral specimens were defrosted under room temperature, and then immobilized by Tray Powder2 Biomechanical testing: Implants were evaluated mechanically (push-out test), The mechanical specimens were fixed on the test machine, then gradually compressed at the loading speed of 0.5mm / min by a 4.0mm- diameter top punch, which was vertically aligned toward the implants and parallel with the long axis of inplants. We set compression displacement in 15mm and took a record of instant loading curve. The peak of curve corresponded to the maximum shear force at interface. Then measured the thickness(h) of contact between implants and the cortical bone, and its diameter(d). And calculated with the following equation of the experimental shear strength: Maximum shear strength (MPa) = maximum shear force (N) / bone and implant contact area (mm~2), Contact area(S) =π×d×h. Each sample data were respectively recorded for statistical analysis.[3] Ultrastructural characteristics of bone interface1 Visual observation of the specimens: Before sacrificing rabbits each time at postoperative 2, 4, 8, 12 weeks, removed the soft tissue from the surface of specimens, and then observed bone growth from the surface of implants.2 Histopathology: The specimens were decalcified, dehydrated, hyalinized, and paraffin-embedded in turn, then cut the sample into 5μm slices. After conventional HE staining, observed bone and bone cement interfaces and the degradation of bone cement, as well as the surrounding inflammatory cell infiltrations, osteoblasts and osteoclasts, with optical microscope.3 Scanning electron microscope observation: Split the decalcified specimens along the direction of bone cement implanted, then trimmed into appropriate sizes (2×5×5mm~3). After handling with a series of steps, samples were placed under electron microscope to observe the interface between material and bone, material degradation, and new bone formation.Results1 A quantitative analysis of experimental animals: All the 22 rabbits survived till result analysis2 Appearance observation: After surgery no infection was observed. No obvious rejection was observed at the site of implanting site. 2 weeks, the size and appearance of implant were not significantly changed. There was no significant gap between the host bone and implant. Two group materials implants had fiber connection with the host bone, and implants were wrapped by fibrous tissue. Implant bone areas were not found obvious bone combined. The materials of experimental group became light yellow, while white was still in control group. 4-8 weeks, the implants were coated dense fibrous tissue and there was new bone protruding which was irregular and roughness in implant surface. Implants block turns red, the different appearance of the two group was not obvious. 12 weeks, the implants combine evenly with the surrounding bone. Appearance of bone defects was similar to autogenous bone.3 Statistical analyses: The results of the push-out test showed that the difference of biomechanics had statistical significance between experimental and control groups(F=198.274, P=0.000). After 2 weeks the shear strength of the CPC/FG was 0.511+/-0.080MPa (average+/-sd), which increased to 5.367+/-0.182MPa at 8 weeks and finally resulted in 6.432+/-0.119MPa at 12 weeks. The shear stress of experimental group was lower than that of the control group in 2 weeks and 4 weeks. However, from 4 weeks to 8 weeks, the shear stress of experimental group became gradually higher than that of the control group, especially during the 8 to 12 weeks. There were significant differences in every weeks in the experimental group.4 Histological analysis under light microscope: Experimental group: Histological evaluation revealed that there were some more collagen fibers in the materials and bone cement was porous at 2 weeks. Meanwhile more fibroblasts and mesenchymal cells could be seen in the bone defect area. At 4 weeks of engraftment, we observed a region of absorption at the junction of bone - bone cement. Collagen fibers had been partially absorbed, newborn trabecular bone and newly formed vessels could be seen. In all sections, what remained of the graft appeared actively to be in the process of being resorbed by osteoclasts. 8 weeks after engraftment, we observed extensive recruitment of activated osteoblastic cells. Most of the CPC was degradation, many trabeculae and plastic osteoids were detected . Trabecular bone was disorder and irregular. 12 weeks after engraftment, bone cement basically completed degradation, defects are basically full of trabecular bone, and new lamellar bones grew together. New bone gradually converted into a lamellar bone structure. Control group: Substantial CPC residued in the bone defect at 2 weeks. 4-8 weeks after engraftment bone cement begun degradation, especially at the junction of the bone - bone cement site, there is cartilage proliferation and new bone formation in this period. New trabecular bone formation and ossification in part could observed, calcium phosphate cement was still residual at 12 weeks.5 Evaluation of scanning electron microscope: Experimental group: SEM examination showed that the implant completely embedded in new bone at 8 weeks. There was different density in host bone and graft bone. The degradation rate of the material was relatively high but compatible with the ingrowth of bone trabeculae within the resorbing material, newly-formed trabeculae were observed around and on the surface of the implant, the trabeculae grew on to the material surface from the edges of the implantation site. Lamellar new bone pass through the gap of implant, and implant particles are separated in groups. The FG was dramatically degradated after 8 weeks with a few FG microspheres, and new bone replaced the degradated FG. The Harvard’s System was seen in woven bone structures. At 12 weeks lamellar bone tissue was more mature. Control group: The implants were absorbed slowly compared with experimental group, the trabeculae formation was still observed and implant was still degrading. So SEM examination showed that bone formation in the experimental group was significantly higher than in the control group after the 12 week of implantation.Conclusion:1 It is revealed in the experiment thatβ—TCP/MCPM/FG is more effective thanβ—TCP/MCPM in repairing bone defect. Biomechanical test showed that there is statistical significance between experimental group and control group (P<0.05). With the elongation of healing time, the effect of anti-shearing force inβ—TCP/MCPM/FG group was shown to be stronger thanβ-TCP/MCPM group.2 Histology microscopic studies indicated that the ossification capacity of experimental group is superior to that of control group. The material ofβ—TCP/MCPM/FG rendered favorable properties of biocompatibility, osteoinductivity, degradability, as well as plasticity.3 In this study, this novel injectable material ofβ—TCP/MCPM/FG performed satisfactory osteoacusis ablity, mechanical property and biocompatibility , which is a promising material in repairing bone defect in osseous tissue engineering.更多还原