学科：口腔临床医学,出版时间：2010,导师：艾红军指导,学位授予单位：中国医科大学,论文作者：孙伟著,副题名：,学科专业：,关键词：,馆藏号：,
中文
目的 随着生物材料的不断更新及医学新技术的飞跃发展，镁作为具有特殊理化性能的新材料吸引了很多学者的注意力。镁是人类新陈代谢所需的物质之一，也是骨组织的重要组成部分。镁在人体内生理活动中起重要作用，但血清镁过高会引起肌肉麻痹，低血压，呼吸性窘迫。镁可以在短时间内降解，但在一定生理条件下镁腐蚀速度过快，引起局部氢气生成和pH值升高，同时材料结构也会破坏，导致严重的不良组织反应和机械性能的过早丧失。应用恰当的表面处理方法既可以调控镁及其合金的抗腐蚀性，常用的表面处理方法主要有采用等离子法，但是这些涂层本身不可降解，影响了镁合金可降解性的发挥。因此找到一种合适的表面处理方法对镁合金材料的开发具有重要意义。 前期研究结果显示AZ31B镁合金植入早期阶段对动物是安全的，具有明显的诱导动物机体新骨生成的作用，但是镁合金快速降解产生的氢气和局部pH值过度升高影响了周围组织的正常愈合。为了改进其组织相容性和生物学活性，采用不同的化学方法将钙磷及氟作用于实验材料表面。本实验将经过低温化学表面氟涂层和钙磷涂层处理和未处理的AZ31B镁合金螺钉植入兔股骨和下颌骨，以钛合金为对照，对不同涂层处理的镁合金在体内的降解过程和特点进行观察和检测；研究不同镁合金表面处理后其对周围局部组织变化和成骨作用的影响；检测合金植入后BMP-2和I型胶原的基因和蛋白表达情况，研究不同涂层处理的AZ31B镁合金的生物活性等三部分实验过程，为临床选择合适的镁合金表面处理技术提供理论基础，也为医用可降解镁合金材料的临床商业化推广提供实验支持。 方法 1、实验动物 健康成年雄性日本大耳白兔60只，随机分成A组无涂层处理的AZ31B镁合金螺钉组；F组氟表面处理的AZ31B镁合金螺钉组；T组钙磷表面处理的AZ31B镁合金螺钉组；B组钛(Ti)合金螺钉对照组，每组15只。每组观察时间分别为1周(1w)、2周(2w)、1个月(1m)、2个月(2m)、3个月(3m)。 2、手术及术后处理 将四组螺钉种植入兔股骨区和下颌骨区，消毒，缝合，进行分笼饲养。术后1周、2周、1个月、2个月、3个月处死实验动物，每次处死12只，进行相应部位的取材和处理。 3、扫描电子显微镜(SEM)观察 术后1周、2周、1个月、2个月、3个月时，取出兔子股骨区螺钉，观察降解情况。 4、能谱分析检测(EDS) 术后1周、2周、1个月、2个月、3个月应用能谱分析检测股骨区合金/骨组织界面元素构成。 5、大体标本观察 充分暴露手术部位，对1周、2周、1个月、2个月、3个月时的股骨区标本进行观察并用数码相机进行图像采集。 6、硬组织切片观察 将4%多聚甲醛固定的股骨标本，脱水，透明，包埋，打磨、抛光，切割，研磨，采用亚甲基蓝-碱性品红法进行染色观察，并采集图像。 7、HE染色观察 下颌骨区标本进行脱钙和HE染色 8、免疫组织化学染色观察 1个月、2个月、3个月时下颌骨区标本采用S-P法免疫组织化学染色，进行BMP-2，I型胶原蛋白表达的检测。 9、RT-PCR检测 提取下颌骨区螺钉接触组织总RNA，进行逆转录、PCR扩增，观察BMP-2， I型胶原mRNA的表达情况。 结果 1、扫描电镜观察(SEM) 扫描电镜结果显示，A组早期降解速度较快，降解表面粗糙，这表明镁合金在植入初期即开始降解。1个月和2个月可见层状降解层。F组和T组1个月时可见部分合金表面变形，覆盖一层降解产物，3个月时，合金-组织界面可见明显降解层，形态不规则。 2、能谱分析检测(EDS) A组镁合金与骨组织界面主要成分有C，O，Na，Mg，P，Ca，Zn等元素。F组检测到的元素为C，O，Mg，Al，P，Ca，2个月、3个月时P和Ca含量逐渐增加， 3个月时Mg含量增加。T组检测到C，O，Na，Mg，P，Ca等元素，2个月时P的含量下降，Mg元素的含量增加，3个月时C，O，Mg的含量增加。随着时间的增加，A组镁元素含量没有明显变化，随着涂层的降解F组和T组镁元素的含量逐渐增加。 3、大体标本观察 A组植入1个月时可见螺钉周围出现新生骨组织，出血且伴有缺损，并有局部炎症反应表现。3个月时，螺钉帽大部分已经被周围新生骨组织包裹。F组无明显炎症反应，骨组织逐渐增加，3个月时黑色涂层区全部消失，螺钉部分表面被新生骨组织包裹。T组局部无明显炎症反应，3个月时钙磷涂层基本消失，骨组织生成增加。B组各时间点间螺钉周围新生骨组织增生程度相近，均表现为轻度增生。 4、硬组织切片观察 A组可见1个月时与螺钉相邻的骨组织增厚，新骨生成显著提高。3个月时大量新生骨组织已完全包绕螺钉帽，而且螺钉表面降解明显，植入体/骨组织界面不连续。F组可见1个月时与螺钉相邻的骨组织轻度增生，3个月时新骨生成大量增加，可见骨皮质明显增厚，但植入体/骨组织界面仍连续。T组可见1个月时与螺钉相邻的骨组织增厚，中度增生，3个月时螺钉表面的降解程度与F组相近，植入体/骨组织界面基本保持连续。B组可见螺钉周围新骨组织轻度增生。3个月时螺钉外形清晰，植入体/骨组织界面连续一致。 5、HE染色观察 A组，在1周时可见纤维结缔组织中炎性细胞浸润。2周以后时可见局部出血，纤维结缔组织中炎性细胞浸润。F组，1周时镜下可见少量出血，局部有炎性细胞浸润。2个月时无出血、无炎症反应。3个月时镜下局部出血，无炎症反应表现。T组，1周和2周时局部镜下可见少量红细胞，无炎症反应。1个月时局部有少量，无炎症反应，。3个月时镜下无炎症反应表现，局部可见少量出血。B组早期局部有炎症反应，少量炎性细胞浸润。1个月时无明显炎症反应。 A组在1周时可见骨小梁和毛细血管。2周时可见大量成纤维细胞，核深染的破骨细胞。3个月时可见成型骨板。F组1周、2周时，可见纤维结缔组织中散在新生骨组织。3个月时骨组织明显增加。T组1周、2周时可见成纤维细胞条带， 2个月时可见部分相对成熟的骨组织，3个月时骨组织增加明显。B组2周时，局部镜下可见大量纤维结缔组织。3个月可见骨组织生成增加不明显。 6、免疫组化染色观察 A组BMP-2前期表达较高，主要表达在细胞浆。2个月、3个月时时可见被染成棕色的成骨细胞数增多，可见一些破骨细胞。T组和F组前期表达相对较低，逐渐增加，3个月时蛋白表达增加。B组BMP-2表达量增加不显著 A组的I型胶组织1个月时在成骨细胞胞浆内可见棕色的染色颗粒，随着时间增加颜色增强，2个月时可见分泌到细胞外基质的I型胶原蛋白被染成棕色，3个月时可见胞浆被染成深棕色。T组和F组前期表达相对较低，逐渐增加，3个月时组织增加显著。B组I型胶原的表达整体水平较低，随着时间的增加I型胶原蛋白表达增多。 7、RT-PCR检测 各组的BMP-2 mRNA相对表达量均表现为逐渐增加的趋势，1个月时A组表达量明显高于涂层组，3个月时表达最多。1个月时，F组和T组BMP-2 mRNA表达较低，3个月时增加明显。 A组随着时间的增加I型胶原mRNA变化不如涂层组明显，1个月时I型胶原mRNA表达量稍低于涂层组，3个月时表达水平明显低于其它两组。F组2个月时相对表达量有所增加，而3个月时其表达水平明显增加。T组I型胶原mRNA水平随时间增加而增加，在1个月和2个月时稍高于F组，3个月时表达水平低于F组，高于A组。 结论 1、未处理AZ31B镁合金植入物在体内早期就开始降解，速度较快。而钙磷涂层和氟涂层可以有效控制早期AZ31B镁合金的腐蚀速率，减少镁离子的释放。 2、未处理的镁合金在降解过程中能够刺激成骨作用，但同时局部有溶骨现象发生，这会影响植入体的早期骨结合，不利于植入体稳定和固位。 3、钙磷涂层和氟涂层都能够控制镁合金的降解，减少因过多镁离子释放所引起的不良组织反应，表现出良好的生物相容性和生物活性。 4、植入早期，钙磷涂层对镁合金的降解调控作用及其表现的生物活性要优于氟涂层。 5、未处理组的材料在体内降解产生碱性环境，能够有利于周围组织内BMP-2表达，进而引起局部的新骨生成和I型胶原表达增加。但过量的BMP-2分泌会导致溶骨现象的发生。 6、磷涂层和氟涂层在控制镁离子释放的同时，在基因和蛋白水平上都能够促进BMP-2和I型胶原的表达，刺激局部新骨组织的生成，有利于早期成骨过程，表现出良好的生物活性。 关键词 镁合金；氟；磷酸钙；表面处理；生物降解；生物相容性；成骨细胞；BMP-2； I型胶原
英文
The Effects on Degradation and Osteogenesis of AZ31B Magnesium Alloy with Different Coatings In Vivo Objective Magnesium and magnesium alloy as biomaterials attracted more and more attention from research recently. Magnesium is a light metal, with great breaking strength, compressive yield strength and elastic modulus of cortical bone tissue, which close to the features of natural Cortical. Magnesium is a required substance human metabolism, and is also an important component of bone tissue. Magnesium plays an important role in physical activity in vivo, but high magnesium in serum can cause muscle paralysis, hypotension, respiratory distress. Magnesium can be degraded in a short time. But under certain physiological conditions, corrosion rate of magnesium is too fast and causes the local hydrogen production, pH value increase, material structure destroy and serious tissue reaction happens and mechanical premature losses. Application of appropriate surface treatment methods could control the corrosion resistance of magnesium and magnesium alloys. Common surface treatment methods include plasma method, but the non-degradable coating itself affect the degradation of magnesium alloy can play. So finding a suitable surface treatment of magnesium alloy material is very important. Early research results showed that the early stages of AZ31B magnesium alloy implants were safe in animals, with significant new bone formation function, but the increased local pH value and hydrogen which caused by the rapid degradation of magnesium alloy influenced the healing of normal surrounding tissues. In order to improve its tissue compatibility and biological activity, fluorine coating and calcium phosphate coatings were applied AZ31B magnesium alloy by low temperature chemical surface treatment technology. In this research, the calcium phosphate coating and fluoride coatings and untreated AZ31B magnesium alloy screw were implanted into femur and mandible of rabbits, to observe the degradation characteristics and the degradation process of different coatings of magnesium alloy in vivo, and titanium alloy as the control; to observe and study the changes and the effects of surrounding local bone tissues after implanting different surface treatment of magnesium alloys into bones; to test mRNA and protein expression of BMP-2 and Collagen Type I after implantation of different coatings of AZ31B magnesium alloys. The three parts of the research aimed to study the biological activity of different coatings of magnesium alloy in vivo, to provide a theoretical foundation for the clinical usage of coating method of magnesium alloy and promote clinical application and commercialization of biodegradable magnesium alloy materials. Methods 1. Experimental animals 60 healthy adult male Japanese white rabbits were randomly divided into A group:non-processed AZ31B magnesium alloy screw group; F group: fluorine (F) coating magnesium alloy AZ31B screw group; T group: calcium phosphate (Ca-P) coating magnesium alloy AZ31B screw group; B group: titanium (Ti) alloy screw as the control group, n = 15. Each observation time was 1 week (1w), 2 week (2w), 1 month (1m), 2 month (2m), 3 month (3m). 2. Surgery and postoperate treatment Four groups of screws were implanted into the femur area and mandibular area, sterilized, sutured, housed separately. Rabbits were killed after 1 week, 2 weeks, 1 month, 2 months, 3 months, each 12. 3. Scanning electron microscopy (SEM) After 1 week, 2 weeks, 1 month, 2 months, 3 months, removed the screws from rabbit femoral area and observed degradation by SEM. 4. The energy distribution spectrum probe (EDS) After 1 week, 2 weeks, 1 month, 2 months, 3 months, used the energy distribution spectrum probe to detect element composition of alloy area / femoral bone interface. 5. General observation Fully exposed the surgical site on the femoral region and mandibular area, taked photos with a digital camera after 1 week, 2 weeks, 1 month, 2 months, 3 months. 6. Observation of hard tissuesection 4% paraformaldehyde fixed femoral specimens, dehydrated, transparenting, embeding, grinding, polishing and cutting, grinding, methylene blue – basic fuchsin staining, observing and image acquisition. 7. HE staining Specimens from mandibular area were stained. 8. Immunohistochemistry Mandible bone area samples were stained by immunohistochemical staining (S-P) to detect protein expression of BMP-2, Collagen Type I. 9. RT-PCR Extracted Total RNA from close contact area of mandible bone and screws. Made reverse transcription, PCR amplification of BMP-2, Collagen Type I. Results 1. SEM Group A showed fast degradation, and the degradation surface was rough. At 1 month and 2 month the degradation layer was very obvious. After 1 month part of the alloy of F group and T group showed visible surface deformation. After 3 month, the degradation layer was obvious and the interface was irregular. 2. EDS Group A magnesium alloy and bone interface’s major components were C, O, Na, Mg, P, Ca, Zn. Group F magnesium alloy and bone interface’s major components were C, O, Mg, Al, P, Ca. P and Ca content increased after 2 months. After 3 months, Mg content increased. T group detected C, O, Na, Mg, P, Ca. 2 months down the content of P, Mg elements were increased, 3 months, C, O, Mg content increased. There was no significant change in magnesium content group A. The magnesium content of F group and T group gradually increased with the coating degradation. 3. General observation After 1 month around the group A screw appeared new bone tissue, bleeding and associated with defects, and have the performance of local inflammatory reaction. After 3 months, the screw cap has been largely wrapped with the new bone tissue. There was no significant inflammatory response in group A, and new bone tissue gradually increased. After 3 months, the black coating almost disappeared. The screw part of the surface was surrounded by new bone tissue. T group had no significant local inflammatory reaction. After 3 months, calcium phosphate coatings disappeared, osteogenesis increased, and new bone had grown to the lower part of the screw cap. The osteogenesis of group B increased mildly, shared similar conditions at different times. 4. Observation of hard tissue section After a month, the bone tissue around the group A screw became thicker, new bone increased significantly. After 3 months, the screw cap was completely surrounded by the new bone tissues, and screw surface degraded obvious while the implant / bone interface is not continuous. After a month, the bone tissue around the group F screw increased mildly, while the ossification significant increased, at the same time the cortical bone was thicker and the implant / bone interface is still continuous. After 3 month the degradation of group T screw was similar to group F and implant / bone interface remained continuous. The bone tissue around the group B screw increased mildly. After 3 months, the screw shape was clear, and the implant / bone interface was continuous. 5. HE staining Group A: There were inflammatory cells among fibrous connective tissue after 1 week. After 2 weeks, there were inflammatory cells and localized hemorrhage. Group F: At the 1st week there was localized hemorrhage, and no inflammatory cells at the 2nd month. After 3 months, there were localized bleeding and no inflammatory response. Group T, there were a few red blood cells and no inflammatory response by microscopy at the 1st and 2nd week. At the 3rd month, there were a little bleeding and no inflammatory response. Group B: After 2 months the small blood vessels to dilated and congested, with many inflammatory cells infiltration. After 3 months, there was partial visible hemorrhage, and infiltration of inflammatory cells. Group A: At the 1st week there were trabecular bone and capillaries. After 2 weeks, there were a large number of fibroblasts, osteoclasts which nuclear deep-stained. After 3 months there were bone plates. Group F: Between 1 week and 2 weeks, new bone tissue could be seen scattered in the fibrous connective tissue. After 3 months the bone tissue increased. Group T: At 1st week, 2nd weeks there were visible bands of fibroblasts. After 2 months relatively mature bone tissue could be observed, and bone tissue increased significantly after 3 months. Group B: At 2nd week, a large number of fibrous connective tissue could be seen under the microscope. After 3 months no significant new bone tissues increase was showed. 6. Immunohistochemistry Group A: BMP-2 strongly early expressed in the cytoplasm. At 2nd month and 3rd month, the number of brown osteoblasts increased, and some osteoclasts were seen. At early stage, expression of group T and group F is relatively low, and gradually increased. After 3 months, the protein expression of BMP-2 significant increased. BMP-2 protein of group B was not significant increased. Group A: Collagen type I was brown in the cytoplasm of osteoblasts, and the color became darker. At the 2nd month collagen type I secreted into the extracellular matrix were seen. At 3rd months, the cytoplasm was stained dark brown. At early stage, protein expression of group T and group F is relatively low, and gradually increased. At 3rd month, the protein expression of Collagen type I increased significantly. Collagen type I of group B was low and increased as time went on. 7. T-PCR BMP-2 mRNA expression showed a relatively gradual increase among 3 groups. At 1st month, the expression of A group was significantly higher than coated group, the expression of BMP-2 increased a lot at 3rd month. At 1st month, BMP-2 mRNA expression of group F and T was lower, while increased significantly after 3 months. The increase of collagen type I mRNA of group A was not much significantly than the coating groups. At 1st month, collagen type I mRNA expression of group A was slightly lower than coated groups, and the expression of 3rd month was significantly lower than the other two groups. At 2nd month, expression level of Fluorine coated group relatively increased, while the expression of type I collagen mRNA were significantly increased at 3rd month. The collagen type I mRNA of group T increased with time, and was slightly higher than that of group F at 1st month and 2 months coated group compared with the expression of high levels of fluoride, while is lower than that of group F and higher than group A. Conclusions 1. Naked AZ31B magnesium alloy implants degraded early and quickly in vivo. Applying calcium and phosphorus, fluorine coating of AZ31B magnesium alloy could effectively inhibit the early degradation of early AZ31B magnesium alloy and controlled the release. 2. Untreated AZ31B magnesium alloy could stimulate ossification in the degradation process of magnesium, but locally osteolytic phenomena would affect the implants combination with bone which was not unfavorable to implant stability and solid position. 3. Calcium phosphate coating and fluorine coating could control the degradation of magnesium alloy, reduced the excessive release of magnesium ions and adverse tissue reaction, also showed good biocompatibility and biological activity. 4. Calcium phosphate coating magnesium alloys performed better regulation of degradation and biological activity than fluoride coating magnesium alloys. 5. The degradation of AZ31B magnesium alloy released magnesium ions and caused alkaline surrounding environment. It could promote the increased expression of collagen type I and BMP -2. The latter stimulated new bone formation, but excessive concentration of magnesium ions and high pH value would lead to excessive secretion of BMP-2 and resulted in osteolytic phenomena. 6. Calcium phosphate coating and fluoride coating alloy controlled the release of magnesium, and also promoted the mRNA and protein levels of BMP-2 and collagen type I and stimulated locally osteogenesis. Calcium phosphate coating and fluoride coating treatments were beneficial to the early osteogenesis, showed good biological activity. Key words Magnesium alloy; Fluride; Calcium phosphate; surface treatment; Biodegradable; Biocompatibility; Osteoblast; BMP-2; Type I collagen
