力生长因子24肽修饰的聚乳酸仿生骨基质材料的研究
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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PDLLA 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。
The application range of biomaterials and its development level has been one of theimportant sign to measure a nation's modernization medical level now. As we moveforward in the new millennium, looking for biomaterials similaring to natural humantissue structure and performance, which are designed to be completely integrated andcause the full reproduction of damaged tissue, become a research hotspot of biomedicalmaterials. PDLLA have been approved by the Food and Drug Administration of UnitedStates of America (FDA) as the safety of the biodegradable biomedical materials in thebody. However, some shortcomings of PDLLA have been limited their applications inthe field of biomedical engineering, such as lack of human cell specific recognitionsignal sites and absence of bioactive. Therefore their biomimetic modification designand research has important scientific significance and profound social significance inbiomaterials and biomedical engineering fields in future.
In this study, A novel biomimetic poly (D, L-lactic acid)(PDLLA) modification wasdesigned and synthesized based on the24amino acid peptide analog corresponding tothe unique C-terminal E-domain in mechano-growth factor (MGF-Ct24E) grafted intothe maleic anhydride modified PDLLA (MPLA). MGF-Ct24E was grafted into the sidechain of MPLA via a stable covalent amide bond by using1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide(NHS) as the condensing agent to produce biomimetic MPLA materials (MGF-Ct24E-MPLA). The characterization of the biomimetic biomaterials was by means ofthe Fourier transform infrared spectrometry (FTIR), X-ray photoelectron spectroscopy(XPS), Differential scanning calorimeter (DSC), Amino acid analyzer (AAA),Elementary analysis (EA) and classical chemical analysis to estimate the structures andthe properties. Thereafter, the basic physical properties and mechanical performance, invitro biodegradation, the influence of degradation products on the inflammatoryresponse and biocompatibility of the synthetic biomimetic biomaterials wereinvestigated. The main works and conclusions are as follows:
1. On the condition of maintaining the main chain structure, MGF-Ct24E wasgrafted into the side chain of MPLA via a stable covalent amide bond by using EDC andNHS as the condensing agent to produce novel biomimetic MPLA materials.
①FTIR, XPS, DSC AAA and EA analysis revealed that, MGF-Ct24E was successfully modified into MPLA and the average contents of MGF-Ct24E inMGF-Ct24E-MPLA materials was1.05μmol/g and the coupling efficiency was29.91%.
②DSC results showed that the glass transition temperature of MPLAwas42.73oC，but the glass transition temperature of MGF-Ct24E-MPLA was raised to55.85oC.
2. The basic physical properties and mechanical properties of the PDLLA, MPLAand MGF-Ct24E-MPLA materials were characterized by the molecular weight, staticwater contact Angle, water absorption rate, mechanical tensile tests and compressiontest.
①The molecular weight of the three kinds of polymers molecular weight was in thesequence of PDLLA> MPLA> MGF-Ct24E-MPLA. This is because that as themodification of PDLLA with maleic anhydride and MGF-Ct24E, the thermaldegradation and aminolysis phenomenon was occurred in succession in reaction process.Therefore the molecular weight of modified PDLLA continuously reduced.
②The hydrophilicity of three kinds of polymers materials was in the sequence ofPDLLA ③The maleic anhydridet grafted into PDLLAmade MPLAand the tensile andcompression performance of MPLA increased as to PDLLA. Futhermore, the MPLAmodified with MGF-Ct24E then reduced the tensile and compression strength ofMGF-Ct24E-MPLA.
3. In vitro biodegradation and the influence of degradation products on theinflammatory response of PDLLA, MPLA and MGF-Ct24E-MPLA were investigated.The evaluation indicators of in vitro biodegradation behavior were weight loss ratio,surface topography, water absorption ratio and pH value changes. Moreover, in vitro theinflammatory response of the degradation products of the three materials were evaluatedby monitoring the expression of cytokines such as the, tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β) and the release of NO by macrophages in contact withdegradation products.
①Results of hydrolytic degradation of the polymers during12weeks indicated thatthe in vitro degradation stability of MGF-Ct24E-MPLA was better than PDLLA and MPLA. This was because the alkaline polypeptides MGF-Ct24E could eliminate orweaken the acid induced auto-catalysis during the degradation of MGF-Ct24E-MPLA.
②The inflammatory response experiments indicated that macrophages culturedwith degradation products of MPLA showed increased secretion levels of TNF-α, IL-1βand NO, whereas macrophages cultured with degradation products ofMGF-Ct24E-MPLA revealed decreased secretion levels of TNF-α, IL-1β and NO,related to macrophages cultured with degradation products of PDLLA as control. Themodification of PDLLA with polypeptides MGF-Ct24E could alleviate inflammatoryresponse during the degradation of polymers.
4. In this study, we evaluated the cytocompatibility of MGF-Ct24E chemicallygrafted and physically blended with MPLA, relative to maleic MPLA as the control. Ratcalvarial osteoblasts were seeded on the three polymer films, and cell adhesion,spreading, proliferation, differentiation and mineralization were assessed.
①Compared with MPLA, MGF-Ct24E-MPLA and MGF-Ct24E/MPLA blendspromoted osteoblasts adhesion and spreading, and there was no significant difference incell viability between the MGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends (p <0.05).
②The proliferation ability of rat calvarial osteoblasts cultured on theMGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends films was significantly strongerthan that on MPLA. There was no significant difference in cell proliferation behavior onthe MGF-Ct24E-MPLA and MGFCt24E/MPLA films at the prophase of osteoblastsgrowth stage. Interestingly, the cell proliferation activity on the MGFCt24E-MPLAfilms was greater than that on the MGF-Ct24E/MPLA blends films at the anaphase ofosteoblasts growth stage. The phenomenon occurred because MGF-Ct24E blending intoMPLA easily decreases. Then the MGF-Ct24E alone was metabolized quickly duringcell metabolism over time. However, when MGF-Ct24E was grafted into MPLA byusing the chemical method, it was sustained and released as MGF-Ct24E-MPLAdegraded. Therefore, MGFCt24E-MPLA has a sustaining effect on promoting cellgrowth, compared to the MGFCt24E/MPLA blends over time
③MGF-Ct24E-MPLA and MGF-Ct24E/MPLA materials blends had dual influenceon the differentiation of rat calvarial osteoblasts. In the early period of cell growth, theMGFCt24E-MPLA and MGF-Ct24E/MPLA blends promoted osteoblast proliferationand temporarily restrained differentiation. During the late period of cell growth, theMGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends enhanced osteoblast differentiation. MGF-Ct24E-MPLA has a stronger effect on promoting cells differentiation compared tothe MGF-Ct24E/MPLA blends. Therefore it can be conjectured thatMGF-Ct24E-MPLA did not inhibit differentiation but delayed.
④MGF-Ct24E-MPLA significantly enhanced mineralization of the rat calvarialosteoblasts compared with the MGFCt24E/MPLA blends and MPLA, between whichthere was no significant difference.
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PDLLA
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。
The application range of biomaterials and its development level has been one of theimportant sign to measure a nation's modernization medical level now. As we moveforward in the new millennium, looking for biomaterials similaring to natural humantissue structure and performance, which are designed to be completely integrated andcause the full reproduction of damaged tissue, become a research hotspot of biomedicalmaterials. PDLLA have been approved by the Food and Drug Administration of UnitedStates of America (FDA) as the safety of the biodegradable biomedical materials in thebody. However, some shortcomings of PDLLA have been limited their applications inthe field of biomedical engineering, such as lack of human cell specific recognitionsignal sites and absence of bioactive. Therefore their biomimetic modification designand research has important scientific significance and profound social significance inbiomaterials and biomedical engineering fields in future.
In this study, A novel biomimetic poly (D, L-lactic acid)(PDLLA) modification wasdesigned and synthesized based on the24amino acid peptide analog corresponding tothe unique C-terminal E-domain in mechano-growth factor (MGF-Ct24E) grafted intothe maleic anhydride modified PDLLA (MPLA). MGF-Ct24E was grafted into the sidechain of MPLA via a stable covalent amide bond by using1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide(NHS) as the condensing agent to produce biomimetic MPLA materials (MGF-Ct24E-MPLA). The characterization of the biomimetic biomaterials was by means ofthe Fourier transform infrared spectrometry (FTIR), X-ray photoelectron spectroscopy(XPS), Differential scanning calorimeter (DSC), Amino acid analyzer (AAA),Elementary analysis (EA) and classical chemical analysis to estimate the structures andthe properties. Thereafter, the basic physical properties and mechanical performance, invitro biodegradation, the influence of degradation products on the inflammatoryresponse and biocompatibility of the synthetic biomimetic biomaterials wereinvestigated. The main works and conclusions are as follows:
1. On the condition of maintaining the main chain structure, MGF-Ct24E wasgrafted into the side chain of MPLA via a stable covalent amide bond by using EDC andNHS as the condensing agent to produce novel biomimetic MPLA materials.
①FTIR, XPS, DSC AAA and EA analysis revealed that, MGF-Ct24E was successfully modified into MPLA and the average contents of MGF-Ct24E inMGF-Ct24E-MPLA materials was1.05μmol/g and the coupling efficiency was29.91%.
②DSC results showed that the glass transition temperature of MPLAwas42.73oC，but the glass transition temperature of MGF-Ct24E-MPLA was raised to55.85oC.
2. The basic physical properties and mechanical properties of the PDLLA, MPLAand MGF-Ct24E-MPLA materials were characterized by the molecular weight, staticwater contact Angle, water absorption rate, mechanical tensile tests and compressiontest.
①The molecular weight of the three kinds of polymers molecular weight was in thesequence of PDLLA> MPLA> MGF-Ct24E-MPLA. This is because that as themodification of PDLLA with maleic anhydride and MGF-Ct24E, the thermaldegradation and aminolysis phenomenon was occurred in succession in reaction process.Therefore the molecular weight of modified PDLLA continuously reduced.
②The hydrophilicity of three kinds of polymers materials was in the sequence ofPDLLA
3. In vitro biodegradation and the influence of degradation products on theinflammatory response of PDLLA, MPLA and MGF-Ct24E-MPLA were investigated.The evaluation indicators of in vitro biodegradation behavior were weight loss ratio,surface topography, water absorption ratio and pH value changes. Moreover, in vitro theinflammatory response of the degradation products of the three materials were evaluatedby monitoring the expression of cytokines such as the, tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β) and the release of NO by macrophages in contact withdegradation products.
①Results of hydrolytic degradation of the polymers during12weeks indicated thatthe in vitro degradation stability of MGF-Ct24E-MPLA was better than PDLLA and MPLA. This was because the alkaline polypeptides MGF-Ct24E could eliminate orweaken the acid induced auto-catalysis during the degradation of MGF-Ct24E-MPLA.
②The inflammatory response experiments indicated that macrophages culturedwith degradation products of MPLA showed increased secretion levels of TNF-α, IL-1βand NO, whereas macrophages cultured with degradation products ofMGF-Ct24E-MPLA revealed decreased secretion levels of TNF-α, IL-1β and NO,related to macrophages cultured with degradation products of PDLLA as control. Themodification of PDLLA with polypeptides MGF-Ct24E could alleviate inflammatoryresponse during the degradation of polymers.
4. In this study, we evaluated the cytocompatibility of MGF-Ct24E chemicallygrafted and physically blended with MPLA, relative to maleic MPLA as the control. Ratcalvarial osteoblasts were seeded on the three polymer films, and cell adhesion,spreading, proliferation, differentiation and mineralization were assessed.
①Compared with MPLA, MGF-Ct24E-MPLA and MGF-Ct24E/MPLA blendspromoted osteoblasts adhesion and spreading, and there was no significant difference incell viability between the MGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends (p <0.05).
②The proliferation ability of rat calvarial osteoblasts cultured on theMGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends films was significantly strongerthan that on MPLA. There was no significant difference in cell proliferation behavior onthe MGF-Ct24E-MPLA and MGFCt24E/MPLA films at the prophase of osteoblastsgrowth stage. Interestingly, the cell proliferation activity on the MGFCt24E-MPLAfilms was greater than that on the MGF-Ct24E/MPLA blends films at the anaphase ofosteoblasts growth stage. The phenomenon occurred because MGF-Ct24E blending intoMPLA easily decreases. Then the MGF-Ct24E alone was metabolized quickly duringcell metabolism over time. However, when MGF-Ct24E was grafted into MPLA byusing the chemical method, it was sustained and released as MGF-Ct24E-MPLAdegraded. Therefore, MGFCt24E-MPLA has a sustaining effect on promoting cellgrowth, compared to the MGFCt24E/MPLA blends over time
③MGF-Ct24E-MPLA and MGF-Ct24E/MPLA materials blends had dual influenceon the differentiation of rat calvarial osteoblasts. In the early period of cell growth, theMGFCt24E-MPLA and MGF-Ct24E/MPLA blends promoted osteoblast proliferationand temporarily restrained differentiation. During the late period of cell growth, theMGF-Ct24E-MPLA and MGF-Ct24E/MPLA blends enhanced osteoblast differentiation. MGF-Ct24E-MPLA has a stronger effect on promoting cells differentiation compared tothe MGF-Ct24E/MPLA blends. Therefore it can be conjectured thatMGF-Ct24E-MPLA did not inhibit differentiation but delayed.
④MGF-Ct24E-MPLA significantly enhanced mineralization of the rat calvarialosteoblasts compared with the MGFCt24E/MPLA blends and MPLA, between whichthere was no significant difference.
引文
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