含锶及PRF的复合材料的制备、表征及体外生物活性评价

发布时间：2018-06-14 00:03

本文选题：SrCl2 + PRF　；参考：《吉林大学》2015年博士论文

【摘要】：种植义齿被誉为人类的“第三副牙齿”，它具备很多传统义齿无法替代的优点。但某些病例往往由于局部或大范围的骨缺损造成了骨量不足进而导致种植体无法获得良好的初期稳定性，限制了种植义齿修复的临床应用。骨移植是目前解决这一问题的主要方法，但是自体骨移植要开辟第二术区，异体骨移植由于免疫原性等原因发展受限。因此，人工骨材料成为目前研究的热点。人工合成的支架材料为新生骨组织提供生长空间，在骨组织工程中占有重要的地位。但是高分子支架材料的生物活性较低，力学性能较差，容易引发无菌性炎症。因此，如何增加其表面的生物学活性以及促进骨细胞迅速的长入，是目前的主要难题之一。锶是人体内的一种微量元素，1808年由Humphry Davy(英国,伦敦)首次分离出。研究表明锶对骨改建有双重影响——促进骨形成和抑制骨吸收。目前，锶的主要应用是临床系统用药治疗骨质疏松症。但是，这种全身用药缺少靶向性，因此，锶的局部应用开始为学者们所重视。富血小板纤维蛋白也叫Choukroun’s PRF，是由法国科学家Choukroun于2001年提出。由于其制备过程不需添加任何添加剂，所以在一定程度上讲，PRF为一种自体组织移植物。PRF中的细胞因子在组织改建过程中扮演了重要角色。目前，PRF广泛应用于软组织缺损、骨组织缺损及软骨组织缺损的修复中。然而其无法长期保存，制作后必须即刻应用、机械强度较差以及生长因子释放时间相对较短等特点限制了其在骨组织工程中的应用。因此，如何解决这些问题成为PRF未来的研究热点。近些年，水凝胶作为缓释系统被广泛应用，其特性类似细胞外基质。PLGA-PEG-PLGA三嵌段温敏水凝胶由Zentner等于2001年提出。这种凝胶在一定温度范围内由液态交联成为凝胶状态，其中包括生理温度（37℃）。然而，与PRF相同，该凝胶的机械性能较差影响了其在骨组织工程中的应用。可否有效的提取出PRF内的生长因子、可否将锶与PRF内生长因子通过水凝胶包裹进而载入人工骨支架材料中、复合骨支架材料是否可以缓释这两种生物因子以及该载药复合材料是否具有促进骨形成的作用，目前国内外尚无研究。本研究从体外实验的角度探讨了载药水凝胶与支架材料复合而形成新型骨支架材料的可行性及生物安全性；PRF内生长因子的提取方法；支架/凝胶系统对生物因子的缓释作用；装载锶与PRF的nHA/PLGA/Gel复合材料对成骨样细胞MG63的黏附、增殖及分化的影响；Real time PCR探讨复合材料对成骨相关基因的影响；Western Blot探讨其对CollagenⅠ、Runx2、OPN蛋白表达的影响。为该复合材料应用于临床增加骨量提供理论依据。 1.不同浓度SrCl2对MG63增殖及分化的影响。结果显示：对于MG63细胞500g/ml浓度的SrCl2促增殖能力及促成骨细胞分化能力最强。因此，在后续的工作中我们选择500g/ml作为SrCl2的最优工作浓度。 2.通过扫描电镜（SEM）及酶联免疫吸附试验（ELISA）探讨应用冻干法制备PRF的可行性，寻找提取PRF生长因子的方法。结果显示：冻干PRF较新鲜PRF疏松且有较大的孔洞，且纤维条索结构较细。冻干PRF粉末具有强烈的突释生长因子的特性，为生长因子的提取提供便利。 3.探讨载药水凝胶载入支架材料的可行性，探讨复合支架材料的表征、机械性能及其体内外降解的能力。结果显示：成功制备PLGA-PEG-PLGA温敏水凝胶,选取凝胶化温度为34℃；利用粒子沥滤法制备nHA/PLGA支架材料具有典型的多聚物支架孔状结构，孔径约为150-270m，且孔隙之间相互穿通连接；扫描电镜显示水凝胶成功注入并分布于支架材料内部的孔隙中，同时支架的内部孔隙没有被水凝胶堵塞，复合支架材料仍然拥有内部穿通的孔隙结构，复合支架材料的内部孔隙直径约为70-160m，符合成骨材料的孔隙要求；电感耦合等离子质谱仪（Inductively coupled plasma mass spectrometry，ICP-MS）及ELISA结果显示载药复合材料第1周释放较快，Sr的释放达到32.2%，之后逐渐达到线性释放模式，到12周的累计释放量达到70.3%。载药复合材料在第1周PRF衍生生长因子的平均释放量达到34.03%，之后逐渐达到线性释放模式，到12周的累计释放量达到76.5%；降解实验显示：nHA/PLGA/Gel支架材料在前两周代谢稍快，以后达到线性代谢模式，在12周末失重率达到13.4%， nHA/PLGA空白支架材料到12周末的失重率为8.2%；PH检测结果显示12周末nHA/PLGA/Gel支架材料组的PBS的PH值为6.93±0.1，而nHA/PLGA空白支架材料为6.99±0.1；压缩试验测试结果显示载入水凝胶后材料的抗压强度得到了显著的提升，nHA/PLGA支架材料的抗压强度为(2.31±0.18)MPa，nHA/PLGA/Gel支架材料的抗压强度为(2.90±0.16）MPa。 4.检测复合材料的生物安全性以及对MG63黏附、增殖及分化的影响。结果显示：本研究中所用的支架材料是安全的，无急性溶血活性。复合材料对成骨细胞的早期黏附、增殖以及分化均有一定的促进作用。 5.探讨载药复合材料对MG63相关功能基因及蛋白的影响。PCR检测成骨相关基因表达结果：MG63细胞内CollagenⅠ基因的表达在第1天，四组没有显著性差异；第3天，实验组一明显高于其他组，其中实验组二高于空白支架组；第5天，实验组一显著的高于其他各组；OPN基因表达量，第1天，实验组一、实验组二和空白支架组均显著高于空白组，其中实验组一表达量最高；第3天，实验组一与实验组二明显高于其他组，其中实验组一明显高于实验组二；第5天，实验组一与实验组二的表达量高于其他组；Runx2基因表达量，第1天，实验组一高于其他组，其他三组未见统计学差异；第3天，实验组一与实验组二明显高于其他组，，其中实验组一明显高于实验组二；第5天，实验组一与实验组二高于其他组；SP7基因表达量，第1天，四组间无统计学差异；第3天，实验组一与实验组二和空白支架组的表达明显高于空白组，其中实验组一表达量最高；第5天，实验组一与实验组二高于其他组。 Western Blot检测Runx2的蛋白表达结果，第1天，实验组一与空白组显著高于实验组二和空白支架组；第3天，空白支架组和空白对照组显著高于实验组一与实验组二；第5天，实验组一和实验组二明显高于空白支架组和空白组，其中实验组一高于实验组二；OPN的蛋白表达，第1天，实验组一与实验组二显著高于空白支架组和空白组，其中实验组一高于实验组二；第3天，实验组一，实验组二和空白支架组明显高于空白组，其中实验组一明显高于实验组二和空白支架组；第5天，实验组一和空白组明显高于实验组二和空白支架组，其中实验组一高于空白组；CollagenⅠ的蛋白表达结果，第1天，空白支架组显著高于其他三组；第3天，实验组一与空白支架组明显高于实验组二和空白组，其中实验组一明显高于空白支架组，实验组二明显高于空白组；第5天，实验组一明显高于其他三组，其中实验组二和空白支架组高于空白组。综上所述，500g/ml的浓度的SrCl2对成骨细胞有着显著的促增殖和分化作用；冻干后研磨可有效的提取PRF中的生长因子；载药水凝胶可以成功的注入并分布于支架内表面行使缓释功能且不影响成骨材料的内部孔隙结构，为今后凝胶/支架这种新型支架材料的应用打下实验基础；本实验制备的nHA/PLGA/Gel复合材料生物安全性可靠，缓释作用、降解过程及机械性能符合成骨材料的要求；载药复合材料对成骨细胞具有促早期黏附、增殖及分化的作用；载药复合材料在早期可以促进成骨相关基因及蛋白的表达。本研究为缓释锶及PRF生长因子的凝胶/支架复合材料应用于口腔种植领域提供了理论基础。
[Abstract]:The implant denture is known as the "third tooth" of human. It has many advantages that can not be replaced by a lot of traditional dentures. However, some cases often cause insufficient bone mass due to local or large bone defects, which can lead to the implants' inability to obtain good initial stability and limit the clinical application of implant prosthesis. Bone transplantation is now the present. The main method to solve this problem is to open up the second area of autogenous bone graft, and the allograft bone graft is limited by the reason of immunogenicity. Therefore, artificial bone material has become a hot spot of research. Artificial scaffold material provides a growing space for new bone tissue, and occupies an important position in bone tissue engineering. The biological activity of molecular scaffold materials is low, the mechanical properties are poor, and it is easy to cause aseptic inflammation. Therefore, how to increase the biological activity of its surface and promote the rapid growth of bone cells is one of the main problems at present.
Strontium is a trace element in the human body, which was first isolated in 1808 by Humphry Davy (UK, London). Studies have shown that strontium has a dual effect on bone remodeling - promoting bone formation and inhibiting bone absorption. Currently, strontium is mainly used in clinical systems for osteoporosis. The application of the ministry began to be valued by scholars.
Platelet rich fibrin is also called Choukroun 's PRF, which was proposed by French scientist Choukroun in 2001. As the preparation process does not need to add any additives, to a certain extent, PRF plays an important role in the remodeling process of an autologous tissue graft.PRF. Currently, PRF is widely used in the process. Soft tissue defect, bone tissue defect and cartilage tissue defect repair. However, it can not be preserved for a long time. It must be used immediately after making, the poor mechanical strength and the relatively short release time of growth factor limit its application in bone tissue engineering. Therefore, how to solve these problems has become a hot research topic in the future of PRF.
In recent years, hydrogels have been widely used as a sustained-release system. Their properties are similar to the extracellular matrix.PLGA-PEG-PLGA three block thermosensitive hydrogels, which are proposed by Zentner in 2001. This gel is linked to the gel state by liquid crosslinking within a certain temperature range, including the physiological temperature (37 degrees C). However, the mechanical properties of the gel are the same as that of PRF. The poor influence on its application in bone tissue engineering.
Can the growth factor in PRF be extracted effectively, and can the strontium and PRF growth factor be encapsulated through hydrogel and then loaded into the artificial bone scaffold materials. Whether the composite scaffold materials can release these two biological factors and whether the drug composite material has the effect of promoting bone formation, there is no research at home and abroad.
In this study, the feasibility and biological safety of a new type of scaffold material formed by the combination of drug loading hydrogel and scaffold material in vitro, the extraction method of growth factor in PRF, the sustained release effect of scaffold / gel system on biological factors, and the adhesion of strontium and PRF nHA/PLGA /Gel composite to osteoblast MG63 The effect of Real time PCR on the gene of osteogenesis, and the effect of Western Blot on the expression of Collagen I, Runx2 and OPN protein were investigated by PCR, which provided a theoretical basis for the application of the composite to the increase of bone mass.
1. the effects of different concentrations of SrCl2 on the proliferation and differentiation of MG63. The results showed that the SrCl2 proliferation ability of MG63 cells and the ability to induce osteoblast differentiation were the strongest. Therefore, we chose 500g/ml as the optimal working concentration of SrCl2 in the follow-up work.
2. by scanning electron microscopy (SEM) and enzyme linked immunosorbent assay (ELISA), the feasibility of using freeze-drying to prepare PRF was explored and the method of extracting PRF growth factor was found. The results showed that the freeze-dried PRF was looser than the fresh PRF and had a larger hole and the structure of the fiber cord was fine. The freeze-dried PRF powder had a strong characteristic of the sudden release growth factor, which was a growth factor. The extraction of factors is convenient.
3. the feasibility of carrying the scaffold material loaded with hydrogel was discussed, and the characterization, mechanical properties and the ability to degrade in vivo and in vivo were discussed. The results showed that the PLGA-PEG-PLGA thermosensitive hydrogel was prepared successfully and the gelation temperature was 34. The nHA /PLGA scaffold material was prepared by the particle leaching method, which had a typical polymer scaffold hole. The pore size is about 150-270m, and the pores are connected with each other. The scanning electron microscope shows that the hydrogel is successfully injected and distributed in the pores inside the scaffold material, and the internal pores of the scaffold are not blocked by hydrogel, and the composite scaffold material still has internal perforated pore structure and the internal pore diameter of the composite scaffold material. It is about 70-160m, which meets the pore requirement of the bone forming material; the results of the inductively coupled plasma mass spectrometry (Inductively coupled plasma mass spectrometry, ICP-MS) and ELISA show that the drug loading composite is released rapidly for first weeks and the release of Sr reaches 32.2%, and then gradually reaches the linear release mode, and the cumulative release amount to 12 weeks is reached to the 70.3%. loading. The average release of PRF derived growth factor reached 34.03% after first weeks, and then gradually reached the linear release mode, and the cumulative release amount reached 76.5% to 12 weeks. The degradation experiment showed that nHA/PLGA/Gel scaffold metabolized slightly faster in the first two weeks, then reached the linear metabolic pattern, and the weight loss rate reached 13.4%, nHA/PLGA blank at the end of the 12 weekend. The weight loss rate of the frame material at the end of the 12 week was 8.2%, and the PH test results showed that the pH value of the nHA/PLGA/Gel scaffold in the 12 weekend was 6.93 + 0.1 and the nHA/PLGA blank holder was 6.99 + 0.1. The compression test results showed that the compressive strength of the material after the loading hydrogel was significantly improved and the compressive strength of the nHA/PLGA scaffold material was the compressive strength. For (2.31 + 0.18) MPa, the compressive strength of nHA/PLGA/Gel scaffolds was (2.90 + 0.16) MPa.
4. to detect the biological safety of the composite and the effect on the adhesion, proliferation and differentiation of MG63. The results show that the scaffold materials used in this study are safe and have no acute hemolytic activity. The composites have a certain effect on the early adhesion, proliferation and differentiation of osteoblasts.
5. study on the effect of drug loading composite on MG63 related functional genes and proteins.PCR detection of bone related gene expression: the expression of Collagen I gene in MG63 cells was first days, and there was no significant difference in the four groups. On the third day, the experimental group was significantly higher than the other groups, and the experimental group was two higher than the blank stents group; the fifth day, the experimental group showed a significant difference. The expression of OPN gene was higher than that of the other groups, first days, the experimental group two and the blank stents were significantly higher than that in the blank group, and the one in the experimental group was the highest. On the third day, the experimental group and the experimental group two were significantly higher than the other groups. The experimental group was significantly higher than the experimental group two; the fifth day, the experimental group and the experimental group two. The expression of Runx2 was higher than that of the other groups, first days, the experimental group was higher than the other groups, and the other three groups did not have statistical difference. On the third day, the experimental group and the experimental group two were significantly higher than the other groups, and the experimental group was significantly higher than the experimental group two; the fifth day, the experimental group and the experimental group two were higher than the other groups; the SP7 gene expression, first, was higher than that of the experimental group. There was no statistical difference between the four groups. On the third day, the expression of two and the blank stents in the experimental group was significantly higher than that in the blank group, and the one in the experimental group was the highest, and on the fifth day, the experimental group and the experimental group two were higher than the other groups.
Western Blot detection of Runx2 protein expression results, first days, the experimental group and the blank group was significantly higher than the experimental group two and the blank stents group, third days, the blank stents group and the blank control group were significantly higher than the experimental group and the experimental group two; fifth days, the experimental group and the experimental group two were significantly higher than the blank stents group and the blank group, among the experimental group A Higher than experimental group two; OPN protein expression, first days, experimental group 1 and experimental group two was significantly higher than the blank stents group and blank group, one of the experimental group was higher than the experimental group two; third days, experimental group one, the experimental group two and blank stents were significantly higher than the blank group, and the experimental group was significantly higher than the experimental group two and the blank stents group; fifth days, The experimental group one and the blank group were significantly higher than the experimental group two and the blank stents group, one of the experimental group was higher than the blank group; the protein expression results of Collagen I, first days, the blank stents group were significantly higher than the other three groups, and on the third day, the experimental group and the blank stents group were significantly higher than the experimental group two and the blank group, of which the experimental group was obviously higher than the blank group. The stent group, two in the experimental group, was significantly higher than that in the blank group. On the fifth day, the experimental group was significantly higher than the other three groups, and the experimental group two and the blank stent group were higher than the blank group.
To sum up, the SrCl2 concentration of 500g/ml has a significant effect on the proliferation and differentiation of osteoblasts; the lapping after freeze drying can effectively extract the growth factor in PRF; the drug loading hydrogel can be successfully injected and distributed on the inner surface of the scaffold to exercise the sustained release function without affecting the internal pore structure of the osteoblast material, for the future gel / branch. The application of this new type of scaffold material has laid the experimental foundation. The nHA/PLGA/Gel composites prepared in this experiment have reliable biological safety, sustained release effect, the degradation process and mechanical properties conform to the requirements of the bone forming materials; the drug loading composites have the effect of promoting early adhesion, colonization and differentiation on osteoblasts; the drug carrying composite material is in the early stage. This study provides a theoretical basis for the application of Sr and PRF growth factor gel / scaffold composites in the field of oral implant.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

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