力生长因子24肽修饰的聚乳酸仿生骨基质材料的研究
发布时间：2018-09-04 20:58
【摘要】：生物材料的应用范围及发展水平已经成为当今社会衡量一个国家现代化医疗水平的重要标志。随着我们步入21世纪，寻找和人体组织天然结构和性能相类似的生物材料，即可完全整合的、使受损组织可完全再生的生物材料成为当今社会生物医用材料研究的热点。聚乳酸材料因具有良好的生物相容性和生物可降解性，被美国FDA批准可以作为手术缝合线、人造血管、药物载体和组织工程支架等材料，，说明聚乳酸在体内是可以安全使用的。但是，聚乳酸缺乏人体细胞特异性识别的信号位点，不具有生物活性，限制了其在生物医学工程领域的使用。因此，对聚乳酸进行仿生改性设计和研究，无疑会对这种全生物降解无残留的生物材料的发展产生重要的科学意义和深远的社会意义。 本研究以马来酸酐改性聚乳酸（MPLA）为原料，按照材料整体仿生修饰的思路，以1-乙基-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为缩合剂，通过酰胺键将生物活性多肽力生长因子E结构域24肽（即羧基端24肽，MGF-Ct24E）共价结合到MPLA的侧链上，从而成功制备了一种新型聚乳酸基仿生骨组织工程支架材料（MGF-Ct24E-MPLA）。该材料具有适合于骨组织工程应用的力学性能和良好的生物相容性、生物降解性。本文以这种新型的聚乳酸基仿生骨组织工程支架材料为研究对象，以促进骨损伤修复为研究目标，利用傅立叶变换红外光谱仪（FTIR）、X射线光电子能谱仪（XPS）、差示扫描量热仪（DSC）、氨基酸分析仪（AAA）、元素分析仪（EA）以及常规化学分析方法对其基本结构和性能进行测试，并详细考察了它们的基本物理性能及力学性能、体外降解性能、降解产物对炎症反应的影响和生物相容性。主要研究内容和结论如下： 1.在不改变MPLA主链结构的前提下，以EDC和NHS为缩合剂，通过酸酐基团与生物活性多肽MGF-Ct24E的氨基之间发生N-酰化反应，成功将MGF-Ct24E引入到MPLA的酸酐分子骨架中从而制备出一种新型仿生聚乳酸基质材料： ①FTIR、XPS、AAA和EA分析结果表明：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分析显示，MPLA的玻璃化转变温度（Tg）为42.73℃，而MGF-Ct24E改性MPLA的玻璃化转变温度则升高到55.85℃。 2.通过材料的分子量、静态水接触角、吸水率以及材料拉伸和压缩性能的测试，考察了PDLLA、MPLA和MGF-Ct24E-MPLA三种聚合物材料的基本物理性能和力学性能： ①三种聚合物的分子量大小依次为PDLLA MPLA MGF-Ct24E-MPLA。这是因为随着马来酸酐和MGF-Ct24E对PDLLA的相继改性，PDLLA在反应过程中也相继发生了热降解和氨解现象，使PDLLA的分子量不断降低； ②三种聚合物的亲水性依次为PDLLAMPLAMGF-Ct24E-MPLA，这是由于随着马来酸酐和MGF-Ct24E的依次引入使PDLLA上的亲水性基团（如-COOH，-OH，-NH2，-CONH-等）相继增多，从而提高了PDLLA的亲水性； ③马来酸酐的引入使PDLLA拉伸和压缩性能增强，而MGF-Ct24E改性MPLA则降低了MPLA的拉伸和压缩强度。 3.研究MGF-Ct24E-MPLA、PDLLA和MPLA三种聚合物材料的降解性能以及降解产物的炎症反应。聚合物降解性能的评价是通过体外降解实验实现的，主要考察了降解过程中MGF-Ct24E-MPLA、PDLLA和MPLA三种聚合物材料降解体系的pH值的变化以及它们的表面形貌、失重率和吸水率的变化。通过将巨噬细胞和MGF-Ct24E-MPLA、PDLLA、MPLA三种聚合物材料的降解产物进行共培养，研究它们对巨噬细胞的形态和TNF-α、IL-1β和NO等炎症因子分泌情况的影响： ①体外降解实验说明，与PDLLA和MGF-Ct24E-MPLA相比，MPLA在降解过程中降解体系的pH值增长最快，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。而与MPLA和PDLLA相比，碱性多肽MGF-Ct24E的引入使MGF-Ct24E-MPLA降解体系的pH呈上升趋势，失重率减小，吸水率增大，酸致自催化降解引起的整体溶蚀降解的程度也降低。这说明MGF-Ct24E-MPLA具有良好的降解稳定性； （2）炎症反应的实验表明，MPLA的降解产物能够刺激巨噬细胞最大限度的铺展，伪足增多、增长，PDLLA的降解产物次之，MGF-Ct24E-MPLA降解产物的效果则没有那么明显。同时，和PDLLA相比，MPLA的降解产物能够提高巨噬细胞产生的TNF-α、IL-1β和NO，而MGF-Ct24E-MPLA的降解产物则显著降低了这三种炎症因子的产生，说明多肽MGF-Ct24E的引入能够缓解PDLLA降解过程中引发的炎症反应。 4.通过体外实验，利用SD大鼠乳鼠的颅骨成骨细胞和MPLA、MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA三组聚合物材料共培养的评估模型，对成骨细胞在上述三组材料上的形态、黏附、增殖、分化和矿化情况进行了系统的评价和描述： ①与对照组MPLA材料相比，成骨细胞在MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA材料上黏附的数目更多，细胞形态更好，且成骨细胞在这两种材料表面的黏附和铺展没有明显差异（P 0.05）。 ②与对照组MPLA材料相比，在细胞和生物材料相互作用的早期，MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA两种材料更有利于成骨细胞的增殖，但随着细胞和生物材料相互作用时间的延长（大于3天），MGF-Ct24E/MPLA共混聚合物中的MGF-Ct24E大部分都已释放出来，使其促进成骨细胞增殖的作用逐渐减弱，而MGF-Ct24E-MPLA材料中的MGF-Ct24E则随着MGF-Ct24E-MPLA的降解缓慢的释放出来，这使其在长时间内能更好的促进成骨细胞的增殖。 ③与对照组MPLA材料相比，MGF-Ct24E/MPLA混合物和MGF-Ct24E-MPLA两种材料抑制成骨细胞早期的分化，促进后期的分化，其中MGF-Ct24E-MPLA材料对成骨细胞的分化促进效果更明显。针对MGF-Ct24E-MPLA材料对成骨细胞分化的这种双重作用，我们得出MGF-Ct24E-MPLA材料并不是抑制成骨细胞的早期分化，而是延迟了其分化过程。 ④与对照组MPLA材料相比，MGF-Ct24E-MPLA具有显著的促进成骨细胞矿化的能力，而MGF-Ct24E/MPLA混合物和MPLA材料影响成骨细胞矿化的水平是相似的。
[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.
【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
本文编号：2223311
[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.
【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
【引证文献】
相关硕士学位论文 前1条
1 王品品;新型骨修复用反应性聚氨酯的研究[D];重庆大学;2013年
本文编号：2223311
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