骨修复因子功能化聚乳酸的制备及其生物相容性研究

发布时间：2018-05-14 21:54

本文选题：骨修复 + 功能聚乳酸前体　；参考：《重庆大学》2012年博士论文

【摘要】：目前，因自然灾害、交通事故、工伤、运动创伤、骨肿瘤切除引起的骨损伤，先天性骨疾病、代谢性骨质疏松(OP)、以及各类骨修复后再骨折造成的骨损伤成为威胁人们健康最大的疾病，受到世界范围的广泛关注。据美国统计,全球每年对此的医疗花费约170亿美元，我国每年也有3000万人次进入临床治疗。这已构成强大的全球性经济社会需求。因此制备具有临床应用意义的骨修复替代材料至关重要。本研究从现有骨修复植入体进入人体后面临的问题出发，以FDA获准的临床骨修复材料聚乳酸（Polylactic acid, PLA）为基本原料，以促黏附因子RGDS/胶原、抗应力遮挡因子MGF-Ct24E、抗血小板凝聚/抗炎症因子甘油磷脂胆碱为模型活性分子，建立起了共价引入RGDS/胶原、MGF-Ct24E和甘油磷脂胆碱的技术平台，并以成骨细胞为模型细胞，体外考察了这些因子的浓度对成骨细胞的影响规律，寻求最适浓度，旨在为设计和制备具有全面生物活性功能的骨修复植入体提供理论和技术基础，同时也为制备兼具促黏附活性、抗应力遮挡活性和抗血小板凝聚/抗炎症活性的新型多功能骨修复材料奠定基础。本文的主要研究内容和结论如下： 1.功能聚乳酸前体的制备与表征 ①侧链接枝改性的功能聚乳酸前体Ⅰ、Ⅱ的制备 1）功能聚乳酸前体Ⅰ即马来酸酐改性聚乳酸（MPLA）的制备：以分子量分布在1.3以内，数均分子量为10万左右的聚乳酸为反应原料，通过自由基反应在聚乳酸主链CH上引入马来酸酐，采用FTIR、13C NMR进行定性评价，采用罗丹明比色法对MPLA中的MAH进行定量分析。结果表明，在聚乳酸的侧链上成功引入了马来酸酐。在分子量为10万左右的聚乳酸侧链上可实现至少三个马来酸酐浓度梯度：1.53%、2.45%、3.04% 2）功能聚乳酸前体Ⅱ即二胺改性聚乳酸（DPLA）的制备：以上述三个浓度的马来酸酐改性聚乳酸为反应基材，利用脂肪族二胺与酸酐基团可以发生酰化反应的特点，在聚乳酸的侧链上引入了具有活性反应末端的脂肪族二胺，从而为后续引入各类活性因子提供反应有效活性位点。采用FTIR、13C NMR进行定性评价。结果表明，在聚乳酸的侧链上成功引入了丁二胺。茚三酮显色法测试可知，所得二胺改性聚乳酸中二胺的含量分别为：1.37%、2.20%、2.59%，从而制备了后续反应所需的功能聚乳酸前体Ⅱ。 ②主链共聚改性的功能聚乳酸前体Ⅲ的制备为了进一步提高聚乳酸上的活性反应位点，从而引入更高含量的活性因子，本研究还采用主链共聚的方法引入了反应活性基团，制备了功能聚乳酸前体Ⅲ。 1）衣糠酸酐改性聚乳酸（PITLA）的制备：采用D,L-丙交酯为原料，Sn(Oct)2为引发剂，通过熔融聚合的方法先合成低分子量的聚乳酸。为提高后续反应的速率，聚乳酸的分子量控制在5000。然后将其与甲基丙烯酸酐（MAA）进行反应，制得PDLLA-MAA。最后，在过氧化二苯甲酰（BPO）引发下，PDLLA-MAA与衣康酸酐发生自由基反应，制得衣糠酸酐改性的聚乳酸，缩写为PITLA。 2）功能聚乳酸前体Ⅲ（DPITLA）的制备：以PITLA为原料，与脂肪族二胺进行反应，制备二胺改性的功能聚乳酸前体Ⅲ（DPITLA）。FTIR、1HNMR的分析结果表明DPITLA已成功合成，茚三酮显色法测试可知合成的DPITLA中的HMD的接枝率为4.23%、5.79%。 2.梯度浓度功能聚乳酸的制备与表征 ①黏附性多肽的引入——促黏附功能聚乳酸的制备以DPLA作为功能聚乳酸前体，，以二环己基碳二亚胺（DCC）为偶联剂，通过粘附性四肽RGDS和胶原蛋白上的氨基与功能聚乳酸前体DPLA上的羧基发生酰胺化反应，从而将RGDS和胶原蛋白引入到DPLA侧链中，制备出具有促细胞黏附活性的功能聚乳酸。通过控制反应混合物中粘附性多肽RGDS或胶原的浓度可以调节聚合物链上多肽的浓度。由实验结果可知：RGDS浓度低时，其转化率为40%~60%，而RGDS浓度高时，其转化率只有10%~30%；在DPLA上引入胶原时存在着同样的规律，但是总体而言，胶原的转化率明显低于RGDS的转化率。 ②M GF-Ct24E肽的引入——抗应力遮挡功能聚乳酸的制备 MGF-Ct24E是一种含有24个氨基酸的多肽，采用类似于引入RGDS的方法将MGF-Ct24E引入到DPLA中，制得MGF-PLA。采用FTIR和1H NMR对MGF-PLA的结构进行了定性表征，结果表明，MGF已成功引入到DPLA中。采用氨基酸分析法进一步定量检测MGF-PLA中MGF-Ct24E的含量。从分析结果可见，相对于RGDS含量的分析，MGF的分析难度提高，与理论值存在偏差。选择结构较为稳定的氨基酸作为基准进行分析时，得到MGF-Ct24E在MGF-PLA中的含量为0.31umol/g、0.55umol/g、0.83umol/g。 ③甘油磷脂胆碱的引入——抗血小板凝聚/抗炎症活性功能聚乳酸的制备以MPLA为前体，通过甘油磷脂胆碱（GPC）中的羟基与MPLA中的酸酐直接进行反应，从而将甘油磷脂胆碱引入到MPLA侧链中，制得GPC-PLA。FTIR、13C NMR的表征结果表明甘油磷脂胆碱已经成功接枝到聚乳酸分子上，由XPS的定量结果可知聚合物中各原子个数百分比。 3.功能聚乳酸的结构及性能研究 ①化学结构：通过侧链接枝的方法引入不同的活性因子，为聚乳酸表面提供了不同化学官能团，尤其是通过侧链接枝的方法在聚乳酸上引入了不同的支化结构，从而为后续细胞相容性实验提供不同基材。 ②拓扑结构：由于不同的活性因子具有不同的分子结构及分子量，在溶剂中溶解时具有不同的团状结构，从而获得具有不同拓扑结构的聚乳酸表面。 ③亲疏水性：亲疏水性的结果表明，引入不同的活性因子后，随着活性因子分子量的增大，功能聚乳酸的亲水性提高；随着引入活性因子浓度的提高，功能聚乳酸的亲水性提高。 4.功能聚乳酸细胞相容性研究本研究以成骨细胞为模型细胞，从细胞形态、粘附力和铺展、细胞增殖能力、功能特性及血液相容性几个方面考察了各类活性因子对功能化聚乳酸生物相容性的影响。结果表明： ①低浓度黏附因子就可以提高细胞的生物相容性。 ②高浓度黏附因子与低浓度时相比，对细胞的黏附性能影响不明显，但是细胞的增殖活力明显提高。 ③MGF-Ct24E通过侧链接枝的方式引入到聚乳酸中，随着MGF-Ct24E含量的提高，材料表面的细胞生长能力增强， MGF-Ct24E引入后成骨细胞具有显著分化、矿化功能，但相对于黏附肽因子，其分化和矿化延后。MGF-Ct24E引入后在细胞上可以获得与应力加载一样的效果，有望为制备抗应力遮挡骨修复材料提供基础。 ④磷脂胆碱通过侧链接枝的方法引入聚乳酸中，降低了纤连蛋白在材料表面的吸附；降低了血小板在聚乳酸上的黏附；磷脂胆碱模拟细胞膜的结构，较好地保持了吸附蛋白的特异构象，进而促进细胞的黏附和生长。这为骨修复用聚乳酸的应用提供了一个契机。
[Abstract]:At present, natural disasters, traffic accidents, injuries, sports injuries, bone injury caused by bone tumor resection, congenital bone disease, metabolic osteoporosis (OP), and bone damage caused by fracture of bone after various kinds of bone repair have become the greatest health diseases that threaten people, and are widely concerned around the world. According to us statistics, this is a year in the world. The cost of medical treatment is about 17 billion dollars, and 30 million people enter clinical treatment each year in our country. This has formed a strong global economic and social demand. Therefore, the preparation of bone repair substitute materials with clinical significance is very important. This study starts with the problems faced by the existing bone repair and reimplantation into the body, and the clinical bone approved by FDA. Polylactic acid (PLA) is used as the basic raw material to promote adhesion factor RGDS/ collagen, anti stress shielding factor MGF-Ct24E, anti platelet aggregation / anti inflammatory factor glycerin choline as model active molecule, and establish a technical platform to covalently introduce RGDS/ collagen, MGF-Ct24E and glycerin phosphatidylcholine, and take osteoblast as the osteoblast. In the model cells, the effects of these factors on osteoblasts were investigated in vitro, and the optimum concentration was sought. The aim was to provide a theoretical and technical basis for the design and preparation of bone repair and replants with full biological activity, as well as for the preparation of adhesion activity, anti stress shielding activity and anti platelet aggregation / anti-inflammatory activity. It lays the foundation for the new multifunctional bone repair material.
The main contents and conclusions of this paper are as follows:
Preparation and characterization of 1. functional poly (lactic acid) precursor
Preparation of functional poly lactic acid precursor I, II modified by side link
1) the preparation of functional poly (lactic acid) precursor: maleic anhydride modified polylactic acid (MPLA): maleic anhydride was introduced into the main chain of polylactic acid on CH with molecular weight distribution within 1.3 and the molecular weight of about 100 thousand of polylactic acid as reaction raw material, FTIR, 13C NMR was used for qualitative evaluation by free radical reaction, and the Luo Danming colorimetric method was used for MPLA MAH was used for quantitative analysis. The results showed that maleic anhydride was successfully introduced on the side chain of polylactic acid. At least three maleic anhydride concentration gradients were achieved on the polylactic acid side chain with a molecular weight of about 100 thousand: 1.53%, 2.45%, 3.04%.
2) the preparation of functional poly (lactic acid) precursor II is the preparation of two amine modified polylactic acid (DPLA): using the three concentrations of maleic anhydride modified polylactic acid as the substrate, the aliphatic two amine and the anhydride group can be acylated, and the aliphatic two amine with the active reaction end is introduced on the side chain of the polylactic acid. FTIR, 13C NMR were used to evaluate the active active sites of various active factors. The results showed that two amine was successfully introduced on the side chain of polylactic acid. It was found that the content of two amine in the two amine modified polylactic acid was 1.37%, 2.20%, 2.59%, and the function needed for subsequent reaction was prepared. The precursor of polylactic acid.
Preparation of functional poly (lactic acid precursor III) modified by main chain copolymerization
In order to further improve the active reaction site on polylactic acid and to introduce a higher content of active factors, the reactive active group was introduced by the main chain copolymerization method, and the functional poly (lactic acid) precursor was prepared.
1) preparation of poly (lactic acid) modified polylactic acid (PITLA): using D, L- lactide as raw material and Sn (Oct) 2 as initiator, low molecular weight polylactic acid was synthesized by melting polymerization. To improve the rate of subsequent reaction, the molecular weight of polylactic acid was controlled at 5000. and then it was reacted with methacrylic anhydride (MAA) to produce PDLLA-MAA. finally. A free radical reaction of PDLLA-MAA with itaconic anhydride was initiated by peroxide benzoyl peroxide (BPO), and the polylactic acid anhydride modified poly (lactic acid) was abbreviated as PITLA.. Two
2) preparation of functional poly (lactic acid) precursor III (DPITLA): using PITLA as raw material and reacting with aliphatic two amines to prepare functional poly (DPITLA).FTIR modified by two amines, 1HNMR analysis showed that DPITLA had been successfully synthesized. The grafting rate of HMD in DPITLA was 4.23%, 5.79%.
Preparation and characterization of poly (lactic acid) with 2. gradient concentration
Introduction of adhesion peptides -- Preparation of poly (lactic acid) for adhesion function
DPLA is used as functional poly (lactic acid) precursor and dicyclohexyl carbon two imide (DCC) as coupling agent. Amidating reaction of the amino group on the carboxyl group DPLA on the functional polylactic acid precursor by the amino group on the adhesive four peptide RGDS and collagen is introduced, and RGDS and collagen are introduced into the DPLA side chain, and the functional polylactic acid with cell adhesion activity is prepared. The concentration of polypeptides on the polymer chain can be regulated by controlling the concentration of RGDS or collagen in the reaction mixture. It is known from the experimental results that when the concentration of RGDS is low, the conversion rate is 40%~60%, while the conversion rate is only 10%~30% when the concentration of RGDS is high; there is the same rule when the collagen is introduced to DPLA, but collagenous as a whole, collagen is in general. The conversion rate is obviously lower than the conversion rate of RGDS.
Introduction of M GF-Ct24E peptide -- Preparation of poly (lactic acid) with stress shielding function
MGF-Ct24E is a polypeptide containing 24 amino acids. MGF-Ct24E is introduced into DPLA by a method similar to the introduction of RGDS. The structure of MGF-PLA is characterized by FTIR and 1H NMR. The results show that MGF has been successfully introduced into DPLA. The amino acid segregation method is used to further determine the content of MGF-PLA in the MGF-PLA. From the analysis results, compared with the analysis of RGDS content, the analysis of MGF is more difficult to improve, and there is a deviation from the theoretical value. The content of MGF-Ct24E in MGF-PLA is 0.31umol/g, 0.55umol/g, 0.83umol/g. when selecting the more stable amino acid as the benchmark.
Introduction of glyceryl phosphatidylcholine -- Preparation of functional poly (lactic acid) against platelet aggregation / anti-inflammatory activity
Using MPLA as the precursor, the glycerin phosphatidylcholine was directly reacted with the acid anhydride in MPLA, and glycerin phosphatide choline was introduced into the MPLA side chain, and GPC-PLA.FTIR was prepared. The characterization of 13C NMR showed that the glycerin phosphatide choline had been successfully grafted onto the polylactic molecules and the atoms in the polymer were identified by the quantitative results of XPS. The percentage of the number.
Study on the structure and properties of 3. functional polylactic acid
(1) chemical structure: the introduction of different active factors through the side chain grafting method provides different chemical functional groups for the polylactic acid surface, especially by introducing different branching structures on polylactic acid by side chain grafting, thus providing different substrate for the subsequent cell compatibility test.
(2) topological structure: because different active factors have different molecular structures and molecular weights, they have different mass structure in solvent dissolving, so that the surface of polylactic acid with different topological structures is obtained.
Hydrophobicity: the hydrophobicity results showed that the hydrophilicity of functional polylactic acid was improved with the increase of the molecular weight of active factors, and the hydrophilicity of functional polylactic acid increased with the increase of the concentration of active factors.
Study on the compatibility of 4. functional polylactic acid cells
In this study, the effects of various active factors on the biocompatibility of functional polylactic acid were investigated from the aspects of cell morphology, adhesion and spreading, cell proliferation, functional properties and blood compatibility.
(1) low concentration of adhesion factors can enhance cell biocompatibility.
(2) compared with low concentration, high concentration of adhesion factor had little effect on cell adhesion, but cell proliferation activity increased significantly.
(3) MGF-Ct24E was introduced into polylactic acid by side chain grafting. With the increase of MGF-Ct24E content, the cell growth ability of the surface was enhanced. After MGF-Ct24E was introduced, the osteoblasts had significant differentiation and mineralization, but relative to the adhesion peptide factor, the differentiation and mineralization after the introduction of.MGF-Ct24E could be obtained and stress on the cell. Loading the same effect is expected to provide a basis for preparing stress shielding bone repair materials.
(4) phosphatidylcholine is introduced into polylactic acid by side chain grafting, which reduces the adsorption of fibronectin on the surface of the material, reduces the adhesion of platelets on the polylactic acid, and the structure of the phosphatidylcholine mimic cell membrane, which keeps the specific conformation of the adsorbed protein, and thus promotes cell adhesion and growth. This is a polyemulsion for bone repair. The application of acid provides an opportunity.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

【相似文献】