發(fā)布時間：2018-06-28 19:41

  本文選題：磷酸化殼聚糖 + 1型膠原　； 參考：《浙江大學》2017年碩士論文

【摘要】：背景骨、軟組織的損傷和缺失不僅影響患者的咀嚼功能和面部美觀,更成為影響人類生命健康的重大威脅。隨著人類經(jīng)濟不斷發(fā)展,口腔頜面部嚴重的創(chuàng)傷,腫瘤,炎癥等疾病日益增多。不僅如此,在牙科修復領域中,由于酸蝕深度與樹脂單體滲透深度的不匹配,粘接界面中已脫礦的膠原纖維裸露而樹脂未能滲透包裹富水區(qū)域形成。缺乏樹脂及礦物保護的裸露膠原機械性能差,且極易被基質(zhì)金屬蛋白酶(MatrixMetalProteinases,MMPs)降解,進一步破壞粘接界面影響樹脂-牙本質(zhì)粘接的耐久性。所以,對骨及牙本質(zhì)等生物硬組織的修復再生成為當前口腔領域研究的重要課題。仿生礦化是一個應用納米技術理論來模仿生物礦化過程的概念驗證策略。無定形相廣泛存在于生物礦化過程中,并作為形成無機相的前驅體參與體內(nèi)硬組織的形成。通過與礦化相關的蛋白調(diào)控無定形相的尺寸以及相轉變,以更好的滲透到膠原纖維內(nèi)部空間形成高度復雜的有機-無機復合生物材料,這種礦化策略是一種很有前景的硬組織修復方法,之前的研究工作中,我們在體外模擬與生物礦化相關的非膠原蛋白(non-collagenous proteins,NCP)的功能特點,使用聚丙烯酸(poly acry lic acid,PAA)穩(wěn)定的亞穩(wěn)態(tài)無定形相(amorphous calcium phosphate,ACP)轉化為羥基磷灰石(hydroxyaptite,HAP)并促進脫礦牙本質(zhì)的仿生再礦化,值得一提的是,再礦化牙本質(zhì)的結構和顯微機械性能與天然牙本質(zhì)幾乎相似。但是,該方法存在耗時過久的問題。因此,探索更完善耗時更短的的礦化體系是亟須解決的問題。殼聚糖,完全或部分脫乙酰形式的幾丁質(zhì),在自然界中作為幾種活生物體的必需支持結構,其獨特的大分子結構具有無毒,抗菌和鎮(zhèn)痛的優(yōu)點。此外,它有良好的粘膜粘附性,顯示止血性能,廣泛用于止血輔料中,并且是酶促可生物降解的。通過化學修飾合成新的生物功能性殼聚糖衍生物已被廣泛研究,通過化學修飾既可保持殼聚糖的原始物理化學和生物化學性質(zhì),同時新引入的官能團賦予其新的性質(zhì)。這種天然聚合物復合材料廣泛傾向于合成復合生物材料。在近年來興起的骨組織工程中,殼聚糖及其衍生材料廣泛用于支架材料緩釋遞送生長因子,促進新骨形成。目的:基于磷酸化殼聚糖良好的生物相容性和螯合鈣離子等特性,本實驗擬探究磷酸化殼聚糖對1型膠原對生物礦化進程的影響。采用透射電鏡(Transmission Electron Microscope,TEM)、傅里葉紅外光譜(Fourier Transform Infrared,FTIR)表征ACP顆粒的粒徑和相變過程以及1型膠原的礦化情況。探索磷酸化殼聚糖促進1型膠原礦化進程的機制及對體系中磷酸鈣礦物相變動力學影響。方法:1.1型膠原溶液的配制:將小牛表皮凍干粉溶于0.1mol/L乙酸溶液(pH = 3.0)配制0.2g/L的膠原溶液,4度儲存24h備用。2.1型膠原自組裝:0.2g/L的膠原溶液置入含碳透射鎳網(wǎng)浸泡,置于密閉含1%氨水的容器,中和4h后移去繼續(xù)培養(yǎng)21h。為穩(wěn)定膠原結構,用0.5%戊二醛交聯(lián)1h,制備透射觀察的樣本60個。3.磷酸化殼聚糖制備:將1g殼聚糖粉末,5g尿素和10mL磷酸加入到40mL二甲基甲酰胺(DMF)中,并將混合物在150℃下在油浴中連續(xù)攪拌1小時。冷卻至室溫后,過濾溶液,沉淀用蒸餾水和無水乙醇充分洗滌沉淀,然后真空干燥將干燥的Pchi溶解在蒸餾水中得到將50mg/ml的溶液備用。4.再礦化液的配制與膠原礦化:配制氯化鈣溶液的中含有計算量的tris,以維持反應液的pH約為7.4-7.6。在鈣液中添加一定量的PAA溶液,配制一定濃度的磷酸氫二鈉溶液。含一定濃度的氯化鈣溶液和磷酸氫二鈉溶液各25 ml滴定混合后獲得礦化液,置于無菌燒杯中,并加入0.2 wt%NaN3溶液以抑制細菌增殖。最終實驗組礦化液分別為CaC121OmM,NaHPO4 6mM,PAA350ug/ml。最后在實驗組中加入配制好的Pchi溶液,其終濃度為0.2mg/ml。將組裝好的含膠原的鎳網(wǎng)(n = 60)隨機分為兩組置于實驗組和空白組礦化液中反應按照特定時間點反應后取出。結果:1.1型膠原礦化進程及微觀形貌。TEM結果示實驗組樣本處理36h后開始礦化,72h后礦化完成,密度更高,膠原增粗,與對照組相比明顯加速礦化進程。2.紅外光譜結果。實驗組中Pchi能與膠原反應修飾膠原表面形成功能基團。3.相變過程。FT-IR結果示36h時,實驗組與對照組的ACP均發(fā)生了相變,實驗組中礦化液中的磷酸鈣在較短時間內(nèi)發(fā)生相變�？瞻捉M礦化液的磷酸鈣48小時開始相變。4.礦物形貌。TEM電鏡結果示實驗組中的無機物顆粒聚集粘附,而對照組中均一分布。結論:磷酸化殼聚糖(PhosphorylatedChitosan,Pchi)能夠有效的促進1型膠原的仿生礦化。紅外光譜證實了 Pchi能修飾膠原表面,與膠原發(fā)生反應。透射電鏡觀察Pchi作用下1型膠原的礦化進程加快,膠原表面的無機物含量增多,ACP的相變加快,短時間內(nèi)形成的等級排列的膠原,與天然礦化膠原一致。根據(jù)生物礦化相關蛋白的結構特征,本實驗在體外模擬了 NCP調(diào)控成礦的作用。通過Pchi對膠原的修飾作用和良好的鈣離子螯合作用,結合其良好的抗菌性和良好的生物相容性,在短時間內(nèi)完成脫礦膠原的再礦化,為提高牙本質(zhì)樹脂粘接復合體的耐久性及骨組織工程的構建提供了一個有意義的仿生策略。
[Abstract]:The damage and loss of the background bone and soft tissue not only affect the chewing function and facial beauty of the patients, but also become a major threat to the human life and health. With the continuous development of the human economy, the severe trauma, tumor, and inflammation of the oral and maxillofacial regions are increasing. In the field of dental repair, the depth of acid erosion and the resin are in the field of dental repair. The mismatching of the penetrant depth of the monomer, the demineralized collagen fibers exposed in the bonding interface and the resin failed to permeate the water rich region. The lack of mechanical properties of the exposed collagen, which is not protected by the resin and minerals, is very easy to be reduced by the matrix metalloproteinase (MatrixMetalProteinases, MMPs), which further destroys the influence of the bonding interface on the resin dentin. Therefore, the repair and regeneration of biological hard tissues such as bone and dentin have become an important topic in the field of oral field. Biomimetic mineralization is a conceptual verification strategy using nanotechnology to imitate the process of biomineralization. The amorphous phase is widely used in the process of biological mineralization and is a precursor to the formation of inorganic phases. The body participates in the formation of hard tissues in the body. Through the mineralization related proteins, the size and phase transition of the amorphous phase are regulated to better penetrate into the internal space of the collagen fibers to form highly complex organic inorganic composite biomaterials. This mineralization strategy is a promising method of hard tissue repair. We simulate the functional characteristics of non collagen (non-collagenous proteins, NCP) related to biomineralization in vitro, and use polyacrylic acid (poly acry LIC acid, PAA) stable metastable amorphous phase (amorphous calcium phosphate, ACP) to be transformed into hydroxyapatite and promote the biomimetic remineralization of the demineralized dentine. It is mentioned that the structure and micromechanical properties of remineralized dentin are almost similar to that of natural dentine. However, this method has a long time-consuming problem. Therefore, it is an urgent problem to explore a more time-consuming and shorter mineralizing system. Chitosan, complete or partially deacetyl form of chitin, is used as a few kinds of activities in nature. The necessary support structure of organisms, its unique macromolecular structure has the advantages of non-toxic, antibacterial and analgesic. In addition, it has good mucous adhesion, hemostasis, widely used in hemostasis excipients and biodegradable. The synthesis of new biofunctional chitosan derivatives by chemical modification has been widely studied. Chemical modification can not only maintain the original physicochemical and biochemical properties of chitosan, but also new properties of the newly introduced functional groups. This natural polymer composite is widely used in the synthesis of composite biomaterials. In recent years, chitosan and its derived materials are widely used in scaffold material in bone tissue engineering. Release growth factor to promote the formation of new bone. Objective: Based on the good biocompatibility of phosphorylated chitosan and chelating calcium ion, the effect of phosphorylated chitosan on the biological mineralization process of type 1 collagen was investigated. Transmission electron microscopy (Transmission Electron Microscope, TEM), Fourier infrared spectroscopy (Fourier Transfor) M Infrared, FTIR) characterized the particle size and phase transition of ACP particles as well as the mineralization of type 1 collagen. Explore the mechanism of the phosphorylated chitosan to promote the mineralization process of type 1 collagen and the effect on the kinetics of calcium phosphate mineral transformation in the system. Method: the preparation of type 1.1 collagen solution: calf surface freeze dry powder in 0.1mol/L acetic acid solution (pH = 3). The collagen solution of 0.2g/L was prepared at 4 degrees to store 24h backup.2.1 collagen self assembly: 0.2g/L collagen solution was immersed in a carbon containing transmission nickel net and was placed in a closed container containing 1% ammonia water. After neutralizing 4h, it was removed to continue to cultivate 21h. as a stable collagen structure, and 1H was crosslinked with 0.5% glutaraldehyde to prepare 60.3. phosphorylated chitosan samples for penetration observation: 1g Chitosan powder, 5g urea and 10mL phosphoric acid were added to 40mL two methyl formamide (DMF), and the mixture was stirred for 1 hours in the oil bath at 150 C. After cooling to room temperature, the filter solution was filtered, the precipitate was washed with distilled water and anhydrous ethanol, and then the dry Pchi was dissolved in distilled water to dissolve 50mg/ml in distilled water. Preparation and collagen mineralization of liquid reserve.4. remineralizing solution: the preparation of calcium chloride solution contains a calculated amount of Tris, in order to maintain the pH of the reaction liquid to add a certain amount of PAA solution in the calcium solution to a certain concentration of two sodium hydrogen phosphate solution. After a certain concentration of calcium chloride solution and two sodium hydrogen phosphate solution are mixed with 25 ml titrations, the mixture is titrated by a mixture of 25 ml. The mineralized liquid was placed in the aseptic beaker, and 0.2 wt%NaN3 solution was added to inhibit the bacterial proliferation. Finally, the experimental group was CaC121OmM, NaHPO4 6mM, and PAA350ug/ml. was added to the experimental group at the end of the experimental group. The final concentration was 0.2mg/ml. and the assembled collagen containing nickel net (n = 60) was randomly divided into two groups. The reaction of the mineralized fluid in the group and the blank group was taken out according to the specific time points. Results: the mineralization process and Micromorphology.TEM of type 1.1 collagen showed that the samples of the experimental group began to mineralized after 36h, and the mineralization was completed after 72h, and the density was higher and the collagen was thickened. Compared with the control group, the mineralization process was obviously accelerated by the.2. infrared spectrum. The Pchi energy in the experimental group was Pchi. When the collagen reaction modified the collagen surface to form the functional group.3. phase transition.FT-IR, the results showed that the ACP of the experimental group and the control group were all phase transition. The phase transition of calcium phosphate in the mineralized fluid in the experimental group was in a short time. The phase transformation of calcium phosphate in the blank group began to phase the phase transformation.4. mineral morphology.TEM electron microscope results showed in the experimental group in the experimental group. Conclusion: PhosphorylatedChitosan (Pchi) can effectively promote the biomimetic mineralization of type 1 collagen. Infrared spectra confirm that Pchi can modify collagen surface and react with collagen. Transmission electron microscope observed that the mineralization process of type 1 collagen was accelerated, collagen was accelerated, collagen was observed. The content of the surface inorganic substance increased, the phase transition of ACP accelerated, and the grade of collagen formed in a short time was in accordance with the natural mineralized collagen. According to the structural characteristics of the biomineralized related proteins, this experiment simulated the role of NCP in regulating the mineralization in vitro. The modification of collagen by Pchi and the good calcium ion chelation were combined with it. Good antibacterial and good biocompatibility and complete remineralization of demineralized collagen in a short time provide a meaningful bionic strategy for improving the durability of dentin resin adhesive complex and the construction of bone tissue engineering.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781
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本文編號：2079128