本文選題：SrCl2 + PRF��； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：種植義齒被譽(yù)為人類的“第三副牙齒”，它具備很多傳統(tǒng)義齒無法替代的優(yōu)點(diǎn)。但某些病例往往由于局部或大范圍的骨缺損造成了骨量不足進(jìn)而導(dǎo)致種植體無法獲得良好的初期穩(wěn)定性，限制了種植義齒修復(fù)的臨床應(yīng)用。骨移植是目前解決這一問題的主要方法，但是自體骨移植要開辟第二術(shù)區(qū)，異體骨移植由于免疫原性等原因發(fā)展受限。因此，人工骨材料成為目前研究的熱點(diǎn)。人工合成的支架材料為新生骨組織提供生長(zhǎng)空間，在骨組織工程中占有重要的地位。但是高分子支架材料的生物活性較低，力學(xué)性能較差，容易引發(fā)無菌性炎癥。因此，如何增加其表面的生物學(xué)活性以及促進(jìn)骨細(xì)胞迅速的長(zhǎng)入，是目前的主要難題之一。 鍶是人體內(nèi)的一種微量元素，1808年由Humphry Davy(英國(guó),倫敦)首次分離出。研究表明鍶對(duì)骨改建有雙重影響——促進(jìn)骨形成和抑制骨吸收。目前，鍶的主要應(yīng)用是臨床系統(tǒng)用藥治療骨質(zhì)疏松癥。但是，這種全身用藥缺少靶向性，因此，鍶的局部應(yīng)用開始為學(xué)者們所重視。 富血小板纖維蛋白也叫Choukroun’s PRF，是由法國(guó)科學(xué)家Choukroun于2001年提出。由于其制備過程不需添加任何添加劑，所以在一定程度上講，PRF為一種自體組織移植物。PRF中的細(xì)胞因子在組織改建過程中扮演了重要角色。目前，PRF廣泛應(yīng)用于軟組織缺損、骨組織缺損及軟骨組織缺損的修復(fù)中。然而其無法長(zhǎng)期保存，制作后必須即刻應(yīng)用、機(jī)械強(qiáng)度較差以及生長(zhǎng)因子釋放時(shí)間相對(duì)較短等特點(diǎn)限制了其在骨組織工程中的應(yīng)用。因此，如何解決這些問題成為PRF未來的研究熱點(diǎn)。 近些年，水凝膠作為緩釋系統(tǒng)被廣泛應(yīng)用，其特性類似細(xì)胞外基質(zhì)。PLGA-PEG-PLGA三嵌段溫敏水凝膠由Zentner等于2001年提出。這種凝膠在一定溫度范圍內(nèi)由液態(tài)交聯(lián)成為凝膠狀態(tài)，其中包括生理溫度（37℃）。然而，與PRF相同，該凝膠的機(jī)械性能較差影響了其在骨組織工程中的應(yīng)用。 可否有效的提取出PRF內(nèi)的生長(zhǎng)因子、可否將鍶與PRF內(nèi)生長(zhǎng)因子通過水凝膠包裹進(jìn)而載入人工骨支架材料中、復(fù)合骨支架材料是否可以緩釋這兩種生物因子以及該載藥復(fù)合材料是否具有促進(jìn)骨形成的作用，目前國(guó)內(nèi)外尚無研究。 本研究從體外實(shí)驗(yàn)的角度探討了載藥水凝膠與支架材料復(fù)合而形成新型骨支架材料的可行性及生物安全性；PRF內(nèi)生長(zhǎng)因子的提取方法；支架/凝膠系統(tǒng)對(duì)生物因子的緩釋作用；裝載鍶與PRF的nHA/PLGA/Gel復(fù)合材料對(duì)成骨樣細(xì)胞MG63的黏附、增殖及分化的影響；Real time PCR探討復(fù)合材料對(duì)成骨相關(guān)基因的影響；Western Blot探討其對(duì)CollagenⅠ、Runx2、OPN蛋白表達(dá)的影響。為該復(fù)合材料應(yīng)用于臨床增加骨量提供理論依據(jù)。 1.不同濃度SrCl2對(duì)MG63增殖及分化的影響。結(jié)果顯示：對(duì)于MG63細(xì)胞500g/ml濃度的SrCl2促增殖能力及促成骨細(xì)胞分化能力最強(qiáng)。因此，在后續(xù)的工作中我們選擇500g/ml作為SrCl2的最優(yōu)工作濃度。 2.通過掃描電鏡（SEM）及酶聯(lián)免疫吸附試驗(yàn)（ELISA）探討應(yīng)用凍干法制備PRF的可行性，尋找提取PRF生長(zhǎng)因子的方法。結(jié)果顯示：凍干PRF較新鮮PRF疏松且有較大的孔洞，且纖維條索結(jié)構(gòu)較細(xì)。凍干PRF粉末具有強(qiáng)烈的突釋生長(zhǎng)因子的特性，為生長(zhǎng)因子的提取提供便利。 3.探討載藥水凝膠載入支架材料的可行性，探討復(fù)合支架材料的表征、機(jī)械性能及其體內(nèi)外降解的能力。結(jié)果顯示：成功制備PLGA-PEG-PLGA溫敏水凝膠,選取凝膠化溫度為34℃；利用粒子瀝濾法制備nHA/PLGA支架材料具有典型的多聚物支架孔狀結(jié)構(gòu)，孔徑約為150-270m，且孔隙之間相互穿通連接；掃描電鏡顯示水凝膠成功注入并分布于支架材料內(nèi)部的孔隙中，同時(shí)支架的內(nèi)部孔隙沒有被水凝膠堵塞，復(fù)合支架材料仍然擁有內(nèi)部穿通的孔隙結(jié)構(gòu)，復(fù)合支架材料的內(nèi)部孔隙直徑約為70-160m，符合成骨材料的孔隙要求；電感耦合等離子質(zhì)譜儀（Inductively coupled plasma mass spectrometry，ICP-MS）及ELISA結(jié)果顯示載藥復(fù)合材料第1周釋放較快，Sr的釋放達(dá)到32.2%，之后逐漸達(dá)到線性釋放模式，到12周的累計(jì)釋放量達(dá)到70.3%。載藥復(fù)合材料在第1周PRF衍生生長(zhǎng)因子的平均釋放量達(dá)到34.03%，之后逐漸達(dá)到線性釋放模式，到12周的累計(jì)釋放量達(dá)到76.5%；降解實(shí)驗(yàn)顯示：nHA/PLGA/Gel支架材料在前兩周代謝稍快，以后達(dá)到線性代謝模式，在12周末失重率達(dá)到13.4%， nHA/PLGA空白支架材料到12周末的失重率為8.2%；PH檢測(cè)結(jié)果顯示12周末nHA/PLGA/Gel支架材料組的PBS的PH值為6.93±0.1，而nHA/PLGA空白支架材料為6.99±0.1；壓縮試驗(yàn)測(cè)試結(jié)果顯示載入水凝膠后材料的抗壓強(qiáng)度得到了顯著的提升，nHA/PLGA支架材料的抗壓強(qiáng)度為(2.31±0.18)MPa，nHA/PLGA/Gel支架材料的抗壓強(qiáng)度為(2.90±0.16）MPa。 4.檢測(cè)復(fù)合材料的生物安全性以及對(duì)MG63黏附、增殖及分化的影響。結(jié)果顯示：本研究中所用的支架材料是安全的，無急性溶血活性。復(fù)合材料對(duì)成骨細(xì)胞的早期黏附、增殖以及分化均有一定的促進(jìn)作用。 5.探討載藥復(fù)合材料對(duì)MG63相關(guān)功能基因及蛋白的影響。PCR檢測(cè)成骨相關(guān)基因表達(dá)結(jié)果：MG63細(xì)胞內(nèi)CollagenⅠ基因的表達(dá)在第1天，四組沒有顯著性差異；第3天，實(shí)驗(yàn)組一明顯高于其他組，其中實(shí)驗(yàn)組二高于空白支架組；第5天，實(shí)驗(yàn)組一顯著的高于其他各組；OPN基因表達(dá)量，第1天，實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和空白支架組均顯著高于空白組，其中實(shí)驗(yàn)組一表達(dá)量最高；第3天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二明顯高于其他組，其中實(shí)驗(yàn)組一明顯高于實(shí)驗(yàn)組二；第5天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二的表達(dá)量高于其他組；Runx2基因表達(dá)量，第1天，實(shí)驗(yàn)組一高于其他組，其他三組未見統(tǒng)計(jì)學(xué)差異；第3天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二明顯高于其他組，，其中實(shí)驗(yàn)組一明顯高于實(shí)驗(yàn)組二；第5天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二高于其他組；SP7基因表達(dá)量，第1天，四組間無統(tǒng)計(jì)學(xué)差異；第3天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二和空白支架組的表達(dá)明顯高于空白組，其中實(shí)驗(yàn)組一表達(dá)量最高；第5天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二高于其他組。 Western Blot檢測(cè)Runx2的蛋白表達(dá)結(jié)果，第1天，實(shí)驗(yàn)組一與空白組顯著高于實(shí)驗(yàn)組二和空白支架組；第3天，空白支架組和空白對(duì)照組顯著高于實(shí)驗(yàn)組一與實(shí)驗(yàn)組二；第5天，實(shí)驗(yàn)組一和實(shí)驗(yàn)組二明顯高于空白支架組和空白組，其中實(shí)驗(yàn)組一高于實(shí)驗(yàn)組二；OPN的蛋白表達(dá)，第1天，實(shí)驗(yàn)組一與實(shí)驗(yàn)組二顯著高于空白支架組和空白組，其中實(shí)驗(yàn)組一高于實(shí)驗(yàn)組二；第3天，實(shí)驗(yàn)組一，實(shí)驗(yàn)組二和空白支架組明顯高于空白組，其中實(shí)驗(yàn)組一明顯高于實(shí)驗(yàn)組二和空白支架組；第5天，實(shí)驗(yàn)組一和空白組明顯高于實(shí)驗(yàn)組二和空白支架組，其中實(shí)驗(yàn)組一高于空白組；CollagenⅠ的蛋白表達(dá)結(jié)果，第1天，空白支架組顯著高于其他三組；第3天，實(shí)驗(yàn)組一與空白支架組明顯高于實(shí)驗(yàn)組二和空白組，其中實(shí)驗(yàn)組一明顯高于空白支架組，實(shí)驗(yàn)組二明顯高于空白組；第5天，實(shí)驗(yàn)組一明顯高于其他三組，其中實(shí)驗(yàn)組二和空白支架組高于空白組。 綜上所述，500g/ml的濃度的SrCl2對(duì)成骨細(xì)胞有著顯著的促增殖和分化作用；凍干后研磨可有效的提取PRF中的生長(zhǎng)因子；載藥水凝膠可以成功的注入并分布于支架內(nèi)表面行使緩釋功能且不影響成骨材料的內(nèi)部孔隙結(jié)構(gòu)，為今后凝膠/支架這種新型支架材料的應(yīng)用打下實(shí)驗(yàn)基礎(chǔ)；本實(shí)驗(yàn)制備的nHA/PLGA/Gel復(fù)合材料生物安全性可靠，緩釋作用、降解過程及機(jī)械性能符合成骨材料的要求；載藥復(fù)合材料對(duì)成骨細(xì)胞具有促早期黏附、增殖及分化的作用；載藥復(fù)合材料在早期可以促進(jìn)成骨相關(guān)基因及蛋白的表達(dá)。本研究為緩釋鍶及PRF生長(zhǎng)因子的凝膠/支架復(fù)合材料應(yīng)用于口腔種植領(lǐng)域提供了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08

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