發(fā)布時間：2018-09-04 20:58

【摘要】：生物材料的應(yīng)用范圍及發(fā)展水平已經(jīng)成為當今社會衡量一個國家現(xiàn)代化醫(yī)療水平的重要標志。隨著我們步入21世紀，尋找和人體組織天然結(jié)構(gòu)和性能相類似的生物材料，即可完全整合的、使受損組織可完全再生的生物材料成為當今社會生物醫(yī)用材料研究的熱點。聚乳酸材料因具有良好的生物相容性和生物可降解性，被美國FDA批準可以作為手術(shù)縫合線、人造血管、藥物載體和組織工程支架等材料，，說明聚乳酸在體內(nèi)是可以安全使用的。但是，聚乳酸缺乏人體細胞特異性識別的信號位點，不具有生物活性，限制了其在生物醫(yī)學工程領(lǐng)域的使用。因此，對聚乳酸進行仿生改性設(shè)計和研究，無疑會對這種全生物降解無殘留的生物材料的發(fā)展產(chǎn)生重要的科學意義和深遠的社會意義。 本研究以馬來酸酐改性聚乳酸（MPLA）為原料，按照材料整體仿生修飾的思路，以1-乙基-（3-二甲基氨基丙基）碳二亞胺鹽酸鹽（EDC）和N-羥基琥珀酰亞胺（NHS）為縮合劑，通過酰胺鍵將生物活性多肽力生長因子E結(jié)構(gòu)域24肽（即羧基端24肽，MGF-Ct24E）共價結(jié)合到MPLA的側(cè)鏈上，從而成功制備了一種新型聚乳酸基仿生骨組織工程支架材料（MGF-Ct24E-MPLA）。該材料具有適合于骨組織工程應(yīng)用的力學性能和良好的生物相容性、生物降解性。本文以這種新型的聚乳酸基仿生骨組織工程支架材料為研究對象，以促進骨損傷修復(fù)為研究目標，利用傅立葉變換紅外光譜儀（FTIR）、X射線光電子能譜儀（XPS）、差示掃描量熱儀（DSC）、氨基酸分析儀（AAA）、元素分析儀（EA）以及常規(guī)化學分析方法對其基本結(jié)構(gòu)和性能進行測試，并詳細考察了它們的基本物理性能及力學性能、體外降解性能、降解產(chǎn)物對炎癥反應(yīng)的影響和生物相容性。主要研究內(nèi)容和結(jié)論如下： 1.在不改變MPLA主鏈結(jié)構(gòu)的前提下，以EDC和NHS為縮合劑，通過酸酐基團與生物活性多肽MGF-Ct24E的氨基之間發(fā)生N-�；磻�(yīng)，成功將MGF-Ct24E引入到MPLA的酸酐分子骨架中從而制備出一種新型仿生聚乳酸基質(zhì)材料： ①FTIR、XPS、AAA和EA分析結(jié)果表明：MGF-Ct24E成功接枝到MPLA中，并且MGF-Ct24E在MGF-Ct24E-MPLA材料中的平均含量是1.05μmol/g，接枝效率是29.91%； ②DSC分析顯示，MPLA的玻璃化轉(zhuǎn)變溫度（Tg）為42.73℃，而MGF-Ct24E改性MPLA的玻璃化轉(zhuǎn)變溫度則升高到55.85℃。 2.通過材料的分子量、靜態(tài)水接觸角、吸水率以及材料拉伸和壓縮性能的測試，考察了PDLLA、MPLA和MGF-Ct24E-MPLA三種聚合物材料的基本物理性能和力學性能： ①三種聚合物的分子量大小依次為PDLLA MPLA MGF-Ct24E-MPLA。這是因為隨著馬來酸酐和MGF-Ct24E對PDLLA的相繼改性，PDLLA在反應(yīng)過程中也相繼發(fā)生了熱降解和氨解現(xiàn)象，使PDLLA的分子量不斷降低； ②三種聚合物的親水性依次為PDLLAMPLAMGF-Ct24E-MPLA，這是由于隨著馬來酸酐和MGF-Ct24E的依次引入使PDLLA上的親水性基團（如-COOH，-OH，-NH2，-CONH-等）相繼增多，從而提高了PDLLA的親水性； ③馬來酸酐的引入使PDLLA拉伸和壓縮性能增強，而MGF-Ct24E改性MPLA則降低了MPLA的拉伸和壓縮強度。 3.研究MGF-Ct24E-MPLA、PDLLA和MPLA三種聚合物材料的降解性能以及降解產(chǎn)物的炎癥反應(yīng)。聚合物降解性能的評價是通過體外降解實驗實現(xiàn)的，主要考察了降解過程中MGF-Ct24E-MPLA、PDLLA和MPLA三種聚合物材料降解體系的pH值的變化以及它們的表面形貌、失重率和吸水率的變化。通過將巨噬細胞和MGF-Ct24E-MPLA、PDLLA、MPLA三種聚合物材料的降解產(chǎn)物進行共培養(yǎng)，研究它們對巨噬細胞的形態(tài)和TNF-α、IL-1β和NO等炎癥因子分泌情況的影響： ①體外降解實驗說明，與PDLLA和MGF-Ct24E-MPLA相比，MPLA在降解過程中降解體系的pH值增長最快，酸致自催化降解的程度最大，由此而引起的失重率最大，降解速率最快。而與MPLA和PDLLA相比，堿性多肽MGF-Ct24E的引入使MGF-Ct24E-MPLA降解體系的pH呈上升趨勢，失重率減小，吸水率增大，酸致自催化降解引起的整體溶蝕降解的程度也降低。這說明MGF-Ct24E-MPLA具有良好的降解穩(wěn)定性； （2）炎癥反應(yīng)的實驗表明，MPLA的降解產(chǎn)物能夠刺激巨噬細胞最大限度的鋪展，偽足增多、增長，PDLLA的降解產(chǎn)物次之，MGF-Ct24E-MPLA降解產(chǎn)物的效果則沒有那么明顯。同時，和PDLLA相比，MPLA的降解產(chǎn)物能夠提高巨噬細胞產(chǎn)生的TNF-α、IL-1β和NO，而MGF-Ct24E-MPLA的降解產(chǎn)物則顯著降低了這三種炎癥因子的產(chǎn)生，說明多肽MGF-Ct24E的引入能夠緩解PDLLA降解過程中引發(fā)的炎癥反應(yīng)。 4.通過體外實驗，利用SD大鼠乳鼠的顱骨成骨細胞和MPLA、MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA三組聚合物材料共培養(yǎng)的評估模型，對成骨細胞在上述三組材料上的形態(tài)、黏附、增殖、分化和礦化情況進行了系統(tǒng)的評價和描述： ①與對照組MPLA材料相比，成骨細胞在MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA材料上黏附的數(shù)目更多，細胞形態(tài)更好，且成骨細胞在這兩種材料表面的黏附和鋪展沒有明顯差異（P 0.05）。 ②與對照組MPLA材料相比，在細胞和生物材料相互作用的早期，MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA兩種材料更有利于成骨細胞的增殖，但隨著細胞和生物材料相互作用時間的延長（大于3天），MGF-Ct24E/MPLA共混聚合物中的MGF-Ct24E大部分都已釋放出來，使其促進成骨細胞增殖的作用逐漸減弱，而MGF-Ct24E-MPLA材料中的MGF-Ct24E則隨著MGF-Ct24E-MPLA的降解緩慢的釋放出來，這使其在長時間內(nèi)能更好的促進成骨細胞的增殖。 ③與對照組MPLA材料相比，MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA兩種材料抑制成骨細胞早期的分化，促進后期的分化，其中MGF-Ct24E-MPLA材料對成骨細胞的分化促進效果更明顯。針對MGF-Ct24E-MPLA材料對成骨細胞分化的這種雙重作用，我們得出MGF-Ct24E-MPLA材料并不是抑制成骨細胞的早期分化，而是延遲了其分化過程。 ④與對照組MPLA材料相比，MGF-Ct24E-MPLA具有顯著的促進成骨細胞礦化的能力，而MGF-Ct24E/MPLA混合物和MPLA材料影響成骨細胞礦化的水平是相似的。
[Abstract]:The application scope and development level of biomaterials have become an important symbol to measure the modern medical level of a country in today's society. As we enter the 21st century, we are looking for biomaterials similar to the natural structure and properties of human tissues, which can be fully integrated, so that damaged tissue can be completely regenerated biomaterials become today's society. Polylactic acid (PLA) has been approved by FDA to be used as surgical suture, artificial blood vessels, drug carriers and tissue engineering scaffolds because of its good biocompatibility and biodegradability, indicating that PLA can be used safely in vivo. However, PLA lacks the specificity of human cells. Sexually identifiable signal sites have no biological activity, which limits their use in biomedical engineering. Therefore, biomimetic modification design and research of PLA will undoubtedly have important scientific and far-reaching social significance for the development of this biodegradable biomaterial.
In this study, poly (lactic acid) (MPLA) modified with maleic anhydride (MAH) was used as raw material, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used as condensation agents to bind 24 peptides (MGF-Ct24E) of biologically active polypeptide force growth factor E domain by amide bonding. A novel polylactic acid-based biomimetic bone tissue engineering scaffold material (MGF-Ct24E-MPLA) was successfully prepared by covalently bonding to the side chain of MPLA. The material has good mechanical properties, good biocompatibility and biodegradability, which is suitable for bone tissue engineering. The basic structure and properties of the materials were tested by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), amino acid analyzer (AAA), elemental analyzer (EA) and conventional chemical analysis methods, and their properties were investigated in detail. Basic physical and mechanical properties, in vitro degradation, effects of degradation products on inflammatory response and biocompatibility. The main research contents and conclusions are as follows:
1. Without changing the main chain structure of MPLA, a novel biomimetic polylactic acid matrix material was prepared by N-acylation reaction between anhydride group and amino group of bioactive polypeptide MGF-Ct24E in the presence of EDC and NHS as condensation agent.
The results of FTIR, XPS, AAA and EA analysis showed that MGF-Ct24E was successfully grafted into MPLA, and the average content of MGF-Ct24E in MGF-Ct24E-MPLA was 1.05 micromol/g, and the grafting efficiency was 29.91%.
(2) DSC analysis showed that the glass transition temperature (Tg) of MPLA was 42.73 C, while that of MGF-Ct24E modified MPLA was 55.85 C.
2. The basic physical and mechanical properties of PDLLA, MPLA and MGF-Ct24E-MPLA were investigated by measuring the molecular weight, static water contact angle, water absorption and tensile and compressive properties of the materials.
(1) The molecular weight of the three polymers is PDLLA MPLA MGF-Ct24E-MPLA in turn. This is because with the successive modification of PDLLA by maleic anhydride and MF-Ct24E, the molecular weight of PDLLA decreases with the thermal degradation and ammonolysis of PDLLA.
(2) The hydrophilicity of the three polymers was PDLLAMPLAMGF-Ct24E-MPLA in turn, which was due to the increase of hydrophilic groups (such as-COOH, -OH, -NH2, -CONH-etc.) on PDLLA with the introduction of maleic anhydride and MFG-Ct24E.
(3) The introduction of maleic anhydride enhanced the tensile and compressive properties of PDLLA, while MGF-Ct24E modified MPLA decreased the tensile and compressive strength of MPLA.
3. The degradation performance and the inflammatory reaction of the degradation products of MGF-Ct24E-MPLA, PDLLA and MPLA were studied. The evaluation of the degradation performance of the polymers was carried out by degradation experiments in vitro. The pH values of the degradation systems of MGF-Ct24E-MPLA, PDLLA and MPLA and their surface were investigated. By co-culturing macrophages with degradation products of MGF-Ct24E-MPLA, PDLLA and MPLA, their effects on the morphology of macrophages and the secretion of inflammatory factors such as TNF-a, IL-1beta and NO were studied.
(1) Compared with PDLLA and MGF-Ct24E-MPLA, the pH value of the degradation system of MPLA increased fastest and the degree of acid-induced autocatalytic degradation was the greatest, resulting in the greatest weight loss and the fastest degradation rate. Compared with MPLA and PDLA, the introduction of basic polypeptide MGF-Ct24E resulted in the pH of the degradation system of MGF-Ct24E-MPLA. The results showed that MGF-Ct24E-MPLA had good degradation stability.
(2) Inflammation experiments showed that the degradation products of MPLA could stimulate the maximum spread of macrophages, increase pseudopodia, PDLLA degradation products followed by MGF-Ct24E-MPLA degradation products were not so obvious. At the same time, compared with PDLA, the degradation products of MPLA could increase the production of TNF-a, IL-1beta and NO by macrophages, while MGF-Ct24E-MPLA degradation products could increase the production of TNF-a, IL-1beta and NO by macrophages. The degradation products of -Ct24E-MPLA significantly reduced the production of these three inflammatory factors, suggesting that the introduction of polypeptide MGF-Ct24E can alleviate the inflammation induced by PDLLA degradation.
4. The morphology, adhesion, proliferation, differentiation and mineralization of osteoblasts on the above three groups of materials were systematically evaluated and described in vitro by using the co-culture evaluation model of SD rat skull osteoblasts and MPLA, MGF-Ct24E/MPLA mixture and MGF-Ct24E-MPLA.
(1) Compared with the control group, osteoblasts adhered to the MGF-Ct24E/MPLA mixture and the MGF-Ct24E-MPLA material more and had better morphology, and there was no significant difference in the adhesion and spread of osteoblasts on the surface of the two materials (P 0.05).
(2) Compared with control MPLA, MGF-Ct24E/MPLA mixture and MGF-Ct24E-MPLA were more conducive to the proliferation of osteoblasts in the early stage of cell-biomaterial interaction, but most of MGF-Ct24E in MGF-Ct24E/MPLA blends were released with the prolongation of interaction time (more than 3 days). The effect of MGF-Ct24E in MGF-Ct24E-MPLA material was slowly released with the degradation of MGF-Ct24E-MPLA, which made MGF-Ct24E-MPLA promote the proliferation of osteoblasts in a long time.
(3) Compared with the control group, the mixture of MGF-Ct24E/MPLA and MGF-Ct24E-MPLA can inhibit the early differentiation of osteoblasts and promote the late differentiation of osteoblasts. The MGF-Ct24E-MPLA material can promote the differentiation of osteoblasts more obviously. 24E-MPLA material did not inhibit the early differentiation of osteoblasts, but delayed the differentiation process.
(4) Compared with the control group, MGF-Ct24E-MPLA had significant ability to promote osteoblast mineralization, while the effect of MGF-Ct24E/MPLA mixture and MPLA material on osteoblast mineralization was similar.
【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.08

【引證文獻】

相關(guān)碩士學位論文 前1條

1 王品品;新型骨修復(fù)用反應(yīng)性聚氨酯的研究[D];重慶大學;2013年

本文編號：2223311