骨修复因子功能化聚乳酸的制备及其生物相容性研究
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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。
Bone fractures or defects resulted from various reasons such as natural disasters,traffic accidents, occupational injury, sport, bone tumor incision, congenital bonediseases or metabolic osteoporosis have become one of the most popular diseases. It isestimated that the world-wide medical cost for bone fractures or defects has reachedabout$17billion per year, and the clinical treatment of bone fractures or defects inChina is about30million person-time per year. Therefore, it is highly important andurgent to design and prepare a bone repair/replacement biomaterial with clinicaleffectiveness. In this study, a series of polylactic acid (PLA) based bioactive materialswere design and prepared by using adhesive molecules RGDS/collagen, stressshielding-prevention factor MGF-Ct24E and anti-coagulation factorglycerophosphorylcholine (GPC) as model bioactive factors and PLA as an initialpolymer. The platforms for covalent incorporation of these bioactive factors have beenestablished, and the effects of the concentration of various bioactive factors in thepolymers on osteoblasts were evaluated and an optimal concentration was proposed,aiming to provide a theoretical and technological support for design and preparation ofclinically effective bone implants integrated with multiple bioactive functions and toprepare a specific biomaterial which combines the merits to promote specific celladhesion and prevent stress shielding and blood coagulation. The main works andconclusions are listed as follows.
1. Preparation of the reactive PLAs
①Preparation of type I and Ⅱreactive PLAvia grafting method
1) Preparation of type I reactive PLA from maleic anhydride-grafted PLA (MPLA):Maleic anhydride was grafted onto poly(D,L-lactic acid)(PDLLA)(100kDa,polydispersity index (PDI)<=1.3) by using free radical reactions. FTIR and13C NMRwere used to qualitatively characterize MPLA. The results indicated that MAH has beensuccessful grafted onto PDLLA. When the Mw of PDLLA is100kDa, three MPLApolymers with a MAH content of1.53%,2.45%and3.04%were prepared.
2) Preparation of type Ⅱ reactive PLA from butanediamine-grafted MPLA: TheMPLAs with MAH content of1.53%,2.45%and3.04%were used to react withbutanediamine (BDA) through the reaction of anhydride with-NH2, giving the desiredtype Ⅱ reactive PLA (DPLA). FTIR and13C NMR were used to qualitatively characterize DPLA.Ninhydrin test results showed that the content of BDA in DPLAswere1.37%,2.20%and2.59%, respectively.
②Preparation of type Ⅲ reactive PLAvia copolymerization method
In order to increase the reactive sites and thus introduce more bioactive factors, atype Ⅲ reactive PLA was designed and prepared through the copolymerization ofPDLLA oligomer with methacrylic anhydride (MAA) and itaconic anhydride (ITA).
Firstly, PDLLA oligomer (=5000) was synthesized, and then reacted with MAA toproduce PDLLA-MAA. Secondly, PDLLA-MAA further reacted with ITA via meltingfree radical copolymerization using benzoyl peroxide (BPO) as an initiator, resulting inITA-modified PLA (PITLA).
Finally, PITLA reacted with aliphatic diamine to prepare the type Ⅲ reactive PLA--DPITLA. FTIR,1HNMR showed that DPITLA was prepared successfully. Ninhydrintest results showed that the content of HMD in DPITLA were4.23%,5.79%.
2. Preparation of a series of functionalized PLAs
①Incorporation of adhesive molecules RGDS/collagen
Cell adhesive molecules RGDS or collagen was incorporated into DPLA via thereaction between–NH2in RGDS or collagen and–COOH in DPLA withdicyclohexylcarbodiimide (DCC) as a condensation agent, producing the desiredbioactive PLA with enhanced cell adhesive activity (RGDS-PLA or COL-PLA). Thecontent of RGDS or collagen in the polymers could be controlled by regulating theirconcentrations in the raw materials. The results of amino acid analysis (AAA)demonstrated that the conversion of RGDS for low RGDS concentration had a higherconversion40%-60%while for high RGDS concentration the conversion was10%-30%.The conversion of collagen followed the similar trend although it was obviously lowerthan that of RGDS.
②Incorporation of MGF-Ct24E
MGF-Ct24E is a kind of peptide consisting of24amino acids. It was incorporatedinto DPLA by using the similar technology for RGDS peptide. The obtained polymerMGF-PLA was qualitatively characterized by means of FTIR and1H NMR. The resultsindicated that MGF-Ct24E has been successfully incorporated into DPLA. The AAAresults indicated that the content of MGF-Ct24E in MGF-PLA is0.31umol/g、0.55umol/g and0.83umol/g.
③Incorporation of glycerophosphorylcholine
Glycerophosphorylcholine (GPC) was incorporated into MPLA through the direct reaction of–OH in GPC with the anhydride groups in MPLA. The results of FTIR and1H NMR showed that GPC has been successfully introduced into DPLA. XPS detectionshowed that the atom percentage of GPC-PLA.
3. Structures and performance study on functionalized PLAs
①Chemical structures: Various active factors, especially various branchedstructures were introduced by the means of grafting in side chains to provide differentchemical functional groups on the surface of polylactic acid, which provided diverseparent metals for subsequent cell compatible experiments.
②Topological structures: Various polylactic acid surfaces of distinct topologicalstructure were obtained based on: first, different active factors possess differentmolecular structures and weights, second different active factors form differentrotational structure when dissolve in different solvents.
③Hydrophilicity and hydrophobicity: The results of hydrophilicity andhydrophobicity show that the hydrophilicity of functional polylactic acid increased afterthe introduction of different active factors, and the more molecular weight with higherhydrophilicity; with increased active factor concentration, the hydrophilicity offunctionalized polylactic acid improved.
4. Cytocompatibility evaluation of functionalized PLAs
The cell morphology, adhesion and spreading, proliferation, differentiation andmineralization of osteoblasts on various bioactive polymers and the blood compatibilitywere were mainly investigated. The results revealed that:
①Low concentration of bioactive factor might improve the cytocompatibility.
②Compared to low concentration of bioactive factor, high concentration ofbioactive factors was good for proliferation but not good for cell adhesion.
③With the increasing content of MGF-Ct24E in MGF-PLA, the cell proliferationwas enhanced. Incorporation of MGF-Ct24E significantly improved the differentiationand mineralization of osteoblasts, however, they were delayed compared to RGDS. Thecells show same performance after the introduction of MGF-Ct24E or stress loading,which provides basis for the preparation of anti-stress shieding bone repair materials.
④Incorporation of glycerophosphorylcholine to MPLA decreased the adsorptionof fibronectin and blood platelet on GPC-PLA. Glycerophosphorylcholine has similarchemical structure to cell membrane so that it is good for the natural conformation ofproteins, and thus promote the adhesion and growth of osteoblasts. This study providesopportunity for the application of polylactic acid for bone wound repair.
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。
Bone fractures or defects resulted from various reasons such as natural disasters,traffic accidents, occupational injury, sport, bone tumor incision, congenital bonediseases or metabolic osteoporosis have become one of the most popular diseases. It isestimated that the world-wide medical cost for bone fractures or defects has reachedabout$17billion per year, and the clinical treatment of bone fractures or defects inChina is about30million person-time per year. Therefore, it is highly important andurgent to design and prepare a bone repair/replacement biomaterial with clinicaleffectiveness. In this study, a series of polylactic acid (PLA) based bioactive materialswere design and prepared by using adhesive molecules RGDS/collagen, stressshielding-prevention factor MGF-Ct24E and anti-coagulation factorglycerophosphorylcholine (GPC) as model bioactive factors and PLA as an initialpolymer. The platforms for covalent incorporation of these bioactive factors have beenestablished, and the effects of the concentration of various bioactive factors in thepolymers on osteoblasts were evaluated and an optimal concentration was proposed,aiming to provide a theoretical and technological support for design and preparation ofclinically effective bone implants integrated with multiple bioactive functions and toprepare a specific biomaterial which combines the merits to promote specific celladhesion and prevent stress shielding and blood coagulation. The main works andconclusions are listed as follows.
1. Preparation of the reactive PLAs
①Preparation of type I and Ⅱreactive PLAvia grafting method
1) Preparation of type I reactive PLA from maleic anhydride-grafted PLA (MPLA):Maleic anhydride was grafted onto poly(D,L-lactic acid)(PDLLA)(100kDa,polydispersity index (PDI)<=1.3) by using free radical reactions. FTIR and13C NMRwere used to qualitatively characterize MPLA. The results indicated that MAH has beensuccessful grafted onto PDLLA. When the Mw of PDLLA is100kDa, three MPLApolymers with a MAH content of1.53%,2.45%and3.04%were prepared.
2) Preparation of type Ⅱ reactive PLA from butanediamine-grafted MPLA: TheMPLAs with MAH content of1.53%,2.45%and3.04%were used to react withbutanediamine (BDA) through the reaction of anhydride with-NH2, giving the desiredtype Ⅱ reactive PLA (DPLA). FTIR and13C NMR were used to qualitatively characterize DPLA.Ninhydrin test results showed that the content of BDA in DPLAswere1.37%,2.20%and2.59%, respectively.
②Preparation of type Ⅲ reactive PLAvia copolymerization method
In order to increase the reactive sites and thus introduce more bioactive factors, atype Ⅲ reactive PLA was designed and prepared through the copolymerization ofPDLLA oligomer with methacrylic anhydride (MAA) and itaconic anhydride (ITA).
Firstly, PDLLA oligomer (=5000) was synthesized, and then reacted with MAA toproduce PDLLA-MAA. Secondly, PDLLA-MAA further reacted with ITA via meltingfree radical copolymerization using benzoyl peroxide (BPO) as an initiator, resulting inITA-modified PLA (PITLA).
Finally, PITLA reacted with aliphatic diamine to prepare the type Ⅲ reactive PLA--DPITLA. FTIR,1HNMR showed that DPITLA was prepared successfully. Ninhydrintest results showed that the content of HMD in DPITLA were4.23%,5.79%.
2. Preparation of a series of functionalized PLAs
①Incorporation of adhesive molecules RGDS/collagen
Cell adhesive molecules RGDS or collagen was incorporated into DPLA via thereaction between–NH2in RGDS or collagen and–COOH in DPLA withdicyclohexylcarbodiimide (DCC) as a condensation agent, producing the desiredbioactive PLA with enhanced cell adhesive activity (RGDS-PLA or COL-PLA). Thecontent of RGDS or collagen in the polymers could be controlled by regulating theirconcentrations in the raw materials. The results of amino acid analysis (AAA)demonstrated that the conversion of RGDS for low RGDS concentration had a higherconversion40%-60%while for high RGDS concentration the conversion was10%-30%.The conversion of collagen followed the similar trend although it was obviously lowerthan that of RGDS.
②Incorporation of MGF-Ct24E
MGF-Ct24E is a kind of peptide consisting of24amino acids. It was incorporatedinto DPLA by using the similar technology for RGDS peptide. The obtained polymerMGF-PLA was qualitatively characterized by means of FTIR and1H NMR. The resultsindicated that MGF-Ct24E has been successfully incorporated into DPLA. The AAAresults indicated that the content of MGF-Ct24E in MGF-PLA is0.31umol/g、0.55umol/g and0.83umol/g.
③Incorporation of glycerophosphorylcholine
Glycerophosphorylcholine (GPC) was incorporated into MPLA through the direct reaction of–OH in GPC with the anhydride groups in MPLA. The results of FTIR and1H NMR showed that GPC has been successfully introduced into DPLA. XPS detectionshowed that the atom percentage of GPC-PLA.
3. Structures and performance study on functionalized PLAs
①Chemical structures: Various active factors, especially various branchedstructures were introduced by the means of grafting in side chains to provide differentchemical functional groups on the surface of polylactic acid, which provided diverseparent metals for subsequent cell compatible experiments.
②Topological structures: Various polylactic acid surfaces of distinct topologicalstructure were obtained based on: first, different active factors possess differentmolecular structures and weights, second different active factors form differentrotational structure when dissolve in different solvents.
③Hydrophilicity and hydrophobicity: The results of hydrophilicity andhydrophobicity show that the hydrophilicity of functional polylactic acid increased afterthe introduction of different active factors, and the more molecular weight with higherhydrophilicity; with increased active factor concentration, the hydrophilicity offunctionalized polylactic acid improved.
4. Cytocompatibility evaluation of functionalized PLAs
The cell morphology, adhesion and spreading, proliferation, differentiation andmineralization of osteoblasts on various bioactive polymers and the blood compatibilitywere were mainly investigated. The results revealed that:
①Low concentration of bioactive factor might improve the cytocompatibility.
②Compared to low concentration of bioactive factor, high concentration ofbioactive factors was good for proliferation but not good for cell adhesion.
③With the increasing content of MGF-Ct24E in MGF-PLA, the cell proliferationwas enhanced. Incorporation of MGF-Ct24E significantly improved the differentiationand mineralization of osteoblasts, however, they were delayed compared to RGDS. Thecells show same performance after the introduction of MGF-Ct24E or stress loading,which provides basis for the preparation of anti-stress shieding bone repair materials.
④Incorporation of glycerophosphorylcholine to MPLA decreased the adsorptionof fibronectin and blood platelet on GPC-PLA. Glycerophosphorylcholine has similarchemical structure to cell membrane so that it is good for the natural conformation ofproteins, and thus promote the adhesion and growth of osteoblasts. This study providesopportunity for the application of polylactic acid for bone wound repair.
引文
[1] Richards J B, Rivadeneira F, Inouye M, et al. Bone mineral density, osteoporousis andosteoporotic fractures: a genome-wide association study [J]. Lancet.2008,371:1505-1512.
[2]胡蕴玉.“骨与关节十年”与中国[M].国外医学内分泌学分册.2003.
[3]谢恩.新型仿生BMP-BG-COL-HYA-PS复合支架的实验研究[D].重庆:第四军医大学.2008.
[4]崔少千. BMP-2基因修饰的骨髓基质干细胞复合纳米骨促进兔腰椎融合的实验研究[D].沈阳:中国医科大学.2007.
[5]唐尤超. bBmp-2基因修饰促进骨质疏松症骨愈合的实验研究[D].成都:四川大学.2005.
[6] Augat P, Simon U, Leiedert A, Claes L. Mechanics and mechano-biology of fracture healingin normal and osteoporotic bone [J]. Osteoporos Int.2005,16: S36-S43.
[7]黄霞,陈家昌,申长雨等.骨组织工程支架材料研究进展[J].化工新型材料,2010,38(9):65-68.
[8] L. Calandrelli,G. De Rosa,M. E. Errico, et al.. Novel graft PLLA－based copolymers:Potential of their application to particle technology[J]. Journal of Biomedical MaterialsResearch Part A，2002，62(2):244-253.
[9] Freed L E, Vunjak-Novakovic G, Biron RJ. Biodegradable polymer scaffolds for tissueengineering [J]. Nat Biotechnol.1994,12(7):689-693.
[10]郑磊,王前.骨组织工程基质材料的现状及展望[J].生物医学工程学杂志,2001,18(3):470-474.
[11] Kim B S, Mooney D J. Development of biocompatible synthetic extracellular matrices fortissue engineering [J]. Trends Biotechnol.1998,16(5):224-230.
[12] Niu X F, Wang Y L, Luo Y F, et al. Arg-Gly-Asp (RGD) modified biomimetic polymericmaterials [J]. J. Mater Sci Technol.2005,21(4):571-576.
[13] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated celladhesion and beyond [J]. Biomaterials,2003,24:4385-4415.
[14] Langer R, Tirrell D A. Designing materials for biology and medicine [J]. Nature,2004,428:487-492.
[15] Drotleff S, Lungwitz U, Breunig M, et al. Biomimetic polymers in pharmaceutical andbiomedical sciences [J]. Eur. J. Pharm and Biopharm.2004,58:385-407.
[16] Sanchez C, Arribart H, Guille MMG. Biomimetism and bioinspiration as tools for the designof innovative materials and systems [J]. Nat Mater.2005,4:277-283.
[17] Healy K E, Rezania A, Stile R A. Designing biomaterials to direct biological responses [J].Ann Ny Acad Sci,1999,875:24-35.
[18] Langer R, Tirrell D A. Designing materials for biology and medicine [J]. Nature428,487-492
[19] Holmes T C. Novel peptide-based biomaterial scaffolds for tissue engineering [J]. Trends inBiotechnology,2002,20:16-21.
[20] Pan J, Zhao M, Liu Y, et al. Development of a new poly(ethylene glycol)-graft-poly(D,L-lactic acid) as potential drug carriers [J]. J. Biomed Mater Res. A,2008,89:160-167.
[21] Petrak K. Essential properties of drug-targeting delivery systems [J]. Drug Discovery Today10,1667-1673.
[22] Niu X F, Wang Y L, Luo Y F, Xin J, Li YG. Arg-Gly-Asp (RGD) modified biomimeticpolymeric materials [J]. Journal of Materials Science&Technology,2005,21:571-576.
[23] Anselme K, Linezb P, Bigerelle M, et al. The relative influence of the topography andchemistry of TiAl6V4surfaces on osteoblastic cell behaviour [J]. Biomaterials,2000,21(15):1567-1577.
[24] Fittkau M H, Zillaa P, Bezuidenhout D, et al. The selective modulation of endothelial cellmobility on RGD peptide containing surfaces by YIGSR peptides [J]. Biomaterials,2005,26(2),167-174.
[25] Ohga Y, Katagiri F, Takeyama K, et al. Design and activity of multifunctional fibrils usingreceptor-specific small peptides [J]. Biomaterials,2009,30(35):6731-6739.
[26] Shin H, Jo S, Mikos A G. Biomimetic materials for tissue engineering [J]. Biomaterials24,4353-4364.
[27] Niu X F, Luo Y, Li Y, Fu C, Chen J, Wang Y. Design of bioinspired polymeric materials basedon poly(D,L-lactic acid) modifications towards improving its cytocompatibility [J]. J. BiomedMater Res A,2008,84(4):908-916.
[28] Luo Y F, Wang Y L, Niu X F,Shang J F. Evaluation of the cytocompatibility ofbutanediamine-and RGDS-grafted poly(DL-lactic acid)[J]. Eur. Polym J44:1390-1402.
[29] Pierschbacher M D, Ruoslahti E. Cell Attachment Activity of Fibronectin Can Be Duplicatedby Small Synthetic Fragments of the Molecule [J]. Nature,1984,309,30-33.
[30] Massia S P, Hubbell J A. An RGD spacing of440nm is sufficient for intergrin ανβ3-mediatedfibroblast spreading and140nm for focal contact and stress fiber formation [J]. Journal of CellBiology,1991,114:1089-1100.
[31] Hozumi K, Dai O, Yuji Y, et al. Cell surface receptor-specific scaffold requirements foradhesion to laminin-derived peptide-chitosan membranes [J]. Biomaterials,2010,31(12):3237-3243.
[32] Toshihiro K, Yoshio O, Masayuki N. Preparation and mechanical properties of polylactic acidcomposites containing hydroxyapatite fibers [J]. Biomaterials,2001,22(1):19-23
[33] Quirk R A, Davies W C, Tendler S J B. Poly(L-lysine)-GRGDS as a biomimetic surfacemodifier for poly(lactic acid)[J]. Biomaterials,2001,22:865-872)
[34] Keilhoff G, Stang F, Wolf G, et al. Biocompatibility of type I/III collagen mat rixforperipheral nerve reconst ruction [J]. Biomaterials,2003,24:2779-2787.
[35] Li J, Tao R, Wu W, et al.3D PLGA scaffolds improve differentiation and function of bonemarrow mesenchymal stem cell-derived hepatocytes [J]. Stem Cells and Development.2010,19(9):1427-36.
[36]王晶,周庆颂,袁悦等.生物降解聚合物PLGA-PEG-PLGA的合成及表征[J].沈阳药科大学学报,2005(5):348-351.
[37] Donga Y, Feng S S. In vitro and in vivo evaluation of methoxy polyethyleneglycol–polylactide (MPEG–PLA) nanoparticles for small-molecule drug chemotherapy [J]Biomaterials.2007,28:4154-4160.
[38] Miura H, Onishi H, Sasatsu M, et al. Antitumor characteristics of methoxypolyethyleneglycol-poly(DL-lactic acid) nanoparticles containing camptothecin [J]. Journal of ControlledRelease,2004,97(1):101-113.
[39] Ruan G, Feng S S. Preparation and characterization of poly(lactic acid)–poly(ethyleneglycol)–poly(lactic acid)(PLA–PEG–PLA) microspheres for controlled release of paclitaxel[J]. Bromaterial,2003,24(27):5037-5044.
[40]葛建华,王迎军,郑裕东等. PLA-PEG-PLA嵌段共聚物的合成及研究[J].材料科学与工程学报.2003,21(6):817-820
[41]崔国振,贺继东. PLLA-PEG-PLLA三嵌段共聚物的合成及性能[J].青岛科技大学学报.2010,31(4):395-399.
[42] Achim G, Susam J P. Modulation of marrow stromal cell function using poly(D,L-lactic Acid)-block-poly(Ethylene Glycol)-monomethyl ether surfaces [J]. Journal of Biomedical MaterialsResearch.1999,46:390-398.
[43] Luo Y F, Wang Y L, Niu X F, et al. Synthesis and Characterization of a Novel Biomaterial:Maleic Anhydride-modified Poly(dl-lactic acid)[J]. Chinese Chemical Letters.2004,15(5):521-524.
[44]牛旭锋,王远亮,罗彦凤等.聚(D，L-乳酸)基仿生聚合物材料的合成与表征[J].功能材料.2006,11(37):1790-1794.
[45] Kang G, Lee S J, Lee J H, et al. Interaction of fibroblast cells on poly(lactide-co-glycolide)surface with wettability chemogradient [J]. Biomedical Materials and Engineering.1999;9:179-187.
[46] Kang G, Jeon J H, Lee J W, et al. Cell and platlet adhesions on plasma glow discharge-treatedpoly(lactide-co-glycolide)[J]. Biomedical Materials and Engineering.1997,7:357-368.
[47] Kim B S, Mooney D J. Development of biocompatible synthetic extracellular matrices fortissue engineering [J]. Trends Biotechnol.1998,16(5):224-230.
[48] Holmes TC. Novel peptide-based biomaterial scaffolds for tissue engineering. Trends [J].Biotechnol.2002,20(1):16-21.
[49] Sanchez C, Arribart H, Guille MMG. Biomimetism and bioinspiration as tools for the designof innovative materials and systems [J]. Nat Mater.2005,4:277-283.
[50] Lee K Y, Mooney D J. Cell-interactive polymers for tissue engineering [J]. Fibers Polym.2001,2(2):51-57.
[51] Lebaron R G, Athanasiou K A. Extracellular matrix cell adhesion peptides: functionalapplications in orthopedic materials [J]. Tissue Eng.2000,6(2):85-103.
[52] Cook A D, Hrkach J S, Gao N N, et al. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial [J]. J BiomedMater Res.1997,35:513-523.
[53]牛旭锋.聚(D,L-乳酸)基仿生细胞外基质的骨组织工程基质材料研究[D].重庆:重庆大学.2006.
[54] Yang S, Alnaqeeb M, Simpson H,et al. Cloning and characterization of an IGF-1isoformexpressed in skeletal muscle subjected to stretch [J].J Muscle Res Cell Motil,1996,17(4):487-495.
[55] Goldspink G. Mechanical signals, IGF-I gene splicing, and muscle adaptation [J]. Physiology(Bethesda).2005,20:232-238.
[56] Xian C Y, Wang Y L, Zhang B B, et al. Alternative splicing and expression of the insulin-likegrowth factor gene (IGF-1)in osteoblasts under mechanical stretch [J].Chin Sci Bull.2006,51(22):2731-2736.
[57] Tang L L, Xian C Y, Wang Y L. The MGF expression of osteoblasts in response to mechanicaloverload [J]. Arch Oral Biol.2006,51:1080-1085.
[58] Goldspink G, Scutt A, Loughna PT, et al. Gene expression in skeletal muscle in response tostretch and force generation [J].Am J Physiol,1992,262(3Pt2):R356-363.
[59] Hill M, Wernig A, Goldspink G. Muscle satellite (stem) cell activation during local tissueinjury and repair [J].J Anat.2003,203(1):89-99.
[60] Deng M, Zhang B B, Wang K, et.al. Mechano growth factor E peptide promotes osteoblastsproliferation and bone-defect healing in rabbits [J]. International Orthopaedics.2011,35:1099-1106.
[61] Ishihara K, Ueda T, Nakabayashi N. Preparation of phospholipid polymers and theirproperties as hydrogel sheet [J]. Polym J.1990,22:355-360.
[62] Jasmine D P, Yasuhiko I, Kazuhiko I, James M. Phospholipid polymer surfaces reducebacteria and leukocyte adhesion under dynamic flow conditions [J]. J Biomed Mater Res2005,73A:359-366.
[63]罗娟,王立坚,陈元维等.含生物功能基团磷脂胆碱的聚乳酸亲水性及细胞亲合性研究[J].生物医学工程学杂志.2008.25(6):1344-1348.
[64] Yasuhiko I, Yoko T, Tomoyuki K, Nobuo N. Reduced adhesion of blood cells tobiodegradable polymers by introducing phosphorylcholine moieties [J]. Journal of biomedicalmaterials research.2003,65A:164-169.
[65] Yasuhiko I, Shin-ichi S, Kazuhiko I, et al. Reduction of surface-induced inflammatoryreaction on PLGA/MPC polymer blend [J]. Biomaterials.2002,23:3897-3903.
[66] Junji W, Fredrik N, Bjorn A, et al. Cytocompatible biointerface on poly(lactic acid) byenrichment with phosphorylcholine groups for cell engineering [J]. Materials Science andEngineering C.2006,2:227-231.
[67] Junji W, Kazuhiko I. Change in cell adhesion property on cytocompatible interface usingphospholipids polymer grafted with poly(D,L-lactic acid) segment for tissue engineering [J].Science and Technology of Advanced Materials.2004,4:539-544.
[68] Augat P, Margevicius K, Simon J, Wolf S, Suger G, Claes L. Local tissue properties in bonehealing: influence of size and stability of the osteotomy gap [J]. J. Orthop Res.1998,16:475-481.
[69] Pankovic A M, Tarabishy E, Yelda S. Flexible intramedullary nailing of tibial-shaft fractures[J]. Clin Orthop,1981,160:185-195.
[70] Koch P P, Gross D F, Gerber C. The results of functional (Sarmiento) bracing of humeral shaftfractures [J]. J Shoulder Elbow Surg,2002,11:143-150.
[71] Claes L E, Wilke H J, Augat P, Rubenacker S, Margevicius K J. Effect of dynamization ongap healing of diaphysealfractures under external fixation [J]. Clin Biomech.1995,10:227-234.
[72] Kusec V, Jelic M, Borovecki F, Kos J, Vukicevic S, Korzinek K. D. Istraction osteogenesis byIlizarov and unilateral external fixators in a canine model [J]. Int. Orthop.2003,27:47-52.
[73] Weiss S, Henle P, Bidlingmaier M, et al. Systemic response of the GH/IGF-1axis in timelyversus delayed fracture healing [J]. Growth Hormone&Res.2008,18:205-212.
[74]刘振东,马梦然.从坚强固顶到生物学固定-----历史的回归[J].中华创伤骨科杂志.2004,6(8):910-912.
[75] Mueller C A. Primary stability of various forms of osteosynthesis in the treatment of fracturesof the proximal tibia [J]. J. Bone Joint Surg (Br).2005,873:426.
[76] Shtansky D V, Gloushankova N A, Sheveiko A N, et al. Design, characterization and testing ofTi-based multicomponent coatings for load-bearing medical applications [J]. Biomaterials.2005,26(16):2909.
[77]余贯华.生物功能表面活性剂和生物功能微球改性聚乳酸表面的研究[D].浙江:浙江大学.2005.
[78] Anselme K. Osteoblast adhesion on biomaterials [J].Biomaterials,2000,21:667-681.
[79] Beman A E, Kozlova N I and Morozevich G E. Integrins: Structure and signaling [J].Biochemistry-Moscow,2003,68(12):1284-1299
[80] Klaus Kühn. Integrin–Ligand Interaction [M]. Chapman and Hall, New York,1997.
[81] Lee J H, Jung H W, Kang I K and Lee H B. Cell behavior on polymer surfaces with differentfunctional groups [J]. Biomaterials,1994,15:705-711.
[82] Webb K, Hiady V and Tresco P A. Relationships among cell attachment, spreading,cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces[J]. J. Biomed Mater Res,2000,49:362-368.
[83] Humphries M J, Akiyama S K, Komoriya A, Olden K, et al. Indentification of an alternativelyspliced site in human plasma fibronectin that mediates cell type specific adhesion [J]. J. CellBiol,1986,103:2637-2647.
[84] Arnold M, Cavalcanti-Adam E A, Glass R, et al. Activation of integrin function bynanopatterned adhesive interfaces [J]. Chemphyschem,2004,5:383-388.
[85] Sinhgvi R, StePhnaoPoulos G, Wnag D I C. Eeffets of substratum moprhology on cellPhysiology [J]. Bioteehnology and Bioengineering,1994,43:764-71.
[86] Hatano K, Inoue H, Kojo T, Matsunaga T, et al. Effect of surface roughness on proliferationand alkaline phosphate expression of rat calvarial cells cultured on polystyrene [J]. Bone,1999,25:439-445.
[87] Matsuzaka K, Walboomers F, Ruijter A and Jansen J A. Effect of microgrooved poly-lactic(PLA) surfaces on proliferation, cyyoskeletal organization, and mineralized matrix formationof rat bone marrow cells [J]. Clinical oral implants research,2000,11(4):325-333.
[88]颜泽萱.骨修复用形状记忆聚氨酯的细胞相容性研究[D].重庆:重庆大学.2009.
[89]毛学理等.表面改性聚合物左旋聚乳酸-多聚赖氨酸可促进骨髓基质细胞的初始黏附[J].中国组织工程研究与临床康复.2011,15(38):7100-7104.
[90] Qiu Q, Sayer M, Kawaja M, Shen X and Davies J E. Attachment, morphology, and proteinexpression of rat marrow stromal cells cultured on charged substrate surfaces [J]. J. BiomedMater Res,1998,42:117-127.
[91] Boyan B D, Hummert T W, Dean D D and Schwartz Z. Role of material surfaces in regulatingbone and cartilage cell response [J]. Biomaterials,1996,17:137-146.
[92]马祖伟.聚乳酸软骨组织工程支架制备、改性及其细胞相容性研究[D].浙江:浙江大学.2003.
[93]罗彦凤.聚(DL-乳酸)的改性及体外降解和细胞相容性研究[D].重庆:重庆大学.2003.
[94] Peppas HA,Lanser R. New challenges in biomaterials [J]. Science,1994,263(5154):1715-1720.
[95] Kang I K, Kwon B K, Lee J H, et al. Immobilization of proteins on poly(methyl methacrylate)films [J]. Biomaterials,1993,14(10):787-792.
[96] Zhu Y B, Gao C Y, Liu X Y, et al. Immobilization of biomacromolecules onto aminolyzedpoly(L-lactic acid) toward acceleration of endothelium regeneration [J]. Tissue Eng.2004,10(1-2):53.
[97]姚康德,尹玉姬.组织工程相关生物材料[M].北京:化学工业出版社.2003.
[98] Healy KE, Rezania A, Stile RA. Designing biomaterials to direct biological responses[J]. AnnNY Acad Sci.1999,875:24-35.
[99] Horhertt T A, Schway M. Correlations between mouse3T3cell spreading and serumfibronextin adsorption on glass and hydroxyethymethacrylate-ethymethacrylate copolymers[J]. J. Biomed Mater Res.1998,22:763-793.
[100] Pettit D K, Hoffman A S, Horbett T A. Correlation between corneal epithelial cell outgrowthand monoclonal antibody binding to the cell binding domain of adsorbed fibronectin [J]. J.Biomed Mater Sci.1994,28:685-691.
[101] Chinn J, Horbeet T, Ratner B, et al. Enhancement of serum fibronectin adsorption and theclonal plating efficiencies of swiss mouse3T3fibroblast and MM14mouse myoblast cells onpolymer substrates modified by radiofrequency plasma deposition [J]. J Colloid and InterfaceSci.1989,127:67-87.
[102] Drumheller P D, Hubbell J A. Polymer networks with grafted cell adhesion peptides forhighly biospecific cell adhesive substrates [J]. Analytical Chem.1994,222:380-388.
[103] Drumheller P D, Ebert D L, Hubbell J A. Multifunctional poly(ethyleneglycol)semi-interpenetrating polymer networks as highly selective adhesive substrates forbioadhesive peptide grafting [J]. Biotech and Bioeng.1994,43:772-780.
[104] Zheng J S, Nothrup R, Hornsby P J. Modification of materials formed from poly(L-lactic acid)to enable covalent binding of biopolymers:Application to high-density three-dimensional cellculture in foams with attached collagen [J]. In Vitro Cell Dev Biol-animal.1998.34.679-684.
[105] Brandly B, Schnaar R. Covalent attachment of an Arg-Gly-Asp sequence peptide toderivatizable polyacrylamide surfaces: Support of fibroblast adhesion and long-term growth[J]. Analytical Biochem.1988,172:270-278.
[106]楚万忠,李晓光,刘百川,张凯宁,吴莹光.紫外分光光度法在退变腰椎间盘髓核胶原蛋白水平测试中的应用[J].山东医药.2007,47(29):35-36.
[107] Brenda K M, Annabel T T, Tmothy S B, et al. Modification of surfaces with cell adhesionpeptides alters extracellular matrix deposition [J]. Biomaterials.1999,20:2281-2286.
[108]王镜岩,朱圣庚,徐长法.生物化学(上)(第三版)[M].北京:高等教育出版社.2002,152-153.
[109] Qiu K, Wan C X, Chen X, et al. Acrylic acid-RGD as a biomimetic surface modifier forpoly(ethylene terephthalate)[J]. Chin J Biomed Eng.2003,12(1):22-31.
[110]赵强,万昌秀,刘建伟等.生物活性短肽RGD在PET表面接枝方法的研究[J].生物医学工程学杂志,2003,20(3):384-387.
[111] Philippe M, Jean-Francois L, Basma Fattouma B, et al. A new pro-migratory activity onhuman myogenic precursor cells for a synthetic peptide within the E domain of the mechanogrowth factor [J]. Exp Cell Res,2007,313(3):527-537．
[112] Ates K, Yang S Y, et al. The IGF-I splice variant MGF increases progenitor cells in ALS,dystrophic, and normal muscle [J]. FEBS Letters,2007,581(14):2727-2732.
[113] Dluzniewska J, Sarnowska A, Beresewicz M, et al. A strong neuroprotective effect of theautonomous C-terminal peptide of IGF-1Ec (MGF) in brain ischemia [J]. FASEB J,2005,19(13):1896-1898．
[114] Carpenter V, Matthews K, Devlin G, et al. Mechano-growth factor reduces loss of cardiacfunction in acute myocardial infarction [J]. Heart Lung Circ,2008,17(1):33-39.
[115]张静平.空间位阻在有机化学反应中的应用[J].池州师专学报.2001,15(3):17-18.
[116]王喜云. EMPLA分子量与接枝率的测定及其细胞生物相容性研究[D].重庆:重庆大学.2007.
[117]张俐娜等.高分子物理近代研究方法[M].武汉:武汉大学出版社.2003.
[118] Podximek S. The use of GPC coupled with a multi-angle laser light scattering photometer forthe characterization of polymers on the determination of molecular weight, size, andbranching [J]. Journal of applied polymer science,1994,54:91-103
[119] Leong K W, Brott B C, Langer R. Bioerodible polyanhydrides as drug-carrier matrices. I:Characterization, biodegradation and release characteristics [J]. J. Biomed Mater Res.1985,19:941-955.
[120]李玉宝.生物医学材料[M].北京:化学工业出版社.2003.
[121]俞耀庭,张兴栋.生物医用材料[M].天津:天津大学出版社.2000.
[122]林福春.力生长因子E肽（MGF-Ct24E）与应力作用对成骨细胞基因表达影响的基因芯片分析[D].重庆:重庆大学.2011.
[123] Small J V, Stradal T, Vignal E, et al. The lamellipodium: where motility begins [J]. TrendsCell Biol,2002,12:112-118.
[124] Critchley D R, Holt M R, Barry S T, et al. Integrin-mediated cell adhesion: The cytoskeletalconnection [J]. Biochem Soc Symp,1999,65:7-13.
[125] Neff J A, Tresco P A, Caldwell K D. Surface modification for controlled studies of cell-ligandinteractions [J]. Biomaterials,1999,20:2377-2393.
[126] Mann B K, West J L. Cell adhesion peptides alter smooth muscle cell adhesion, proliferation,migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds [J]. JBiomed Mater Res.2002,60:86-93.
[127] McCabe L R，Kundu B R，Harrison R J，et al．Developmental expression and activities ofspecific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2and jun D during differentiation [J]. Endocrinology,1996,137(10):4398-4408．
[128] Owen T A, Aronow M, Shalhoub V, et al. Progressive development of the rat osteoblastphenotype in vitro：reciprocal relationships in expression of genes associated with osteoblastproliferation and differentiation during formation of the bone extracellular matrix [J]. J CellPhysiol,1990,143(3):420-430.
[129] Hallab N J, Bundy K J, O’Connor K, et al. Evaluation of metallic and polymeric biomaterialsurface energy and surface roughness characteristics for directed cell adhesion [J]. Tissue Eng.2001,7(1):55-71.
[130] Deligianni D D, Katsala N D, Koutsoukos P G, et al. Effect of surface roughness ofhydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation anddetachment strength [J]. Biomaterials,2001,22(1):87-96.
[131] Curtis A, Wilkinson C. Topographical control of cells [J]. Biomaterials,1997,18(24):1573-1583.
[132] Flemming R G, Murphy C J, Abrams G A, et al. Effects of synthetic micro-andnano-structured surfaces on cell behavior [J]. Biomaterials,1999,20(6):573-588.
[133]文学军,王小祥,薛淼.金属生物材料的微粗糙表面及其生物学效应(I)－金属生物材料的微粗糙表面[J].生物医学工程学杂志,1997,14(1):77-80.
[134]牛旭锋,王远亮,潘君.成骨细胞在生物活性材料中黏附性能研究进展[J].生物医学工程杂志,2005,22(4):848-852．
[135]邓霞.新型骨组织工程类细胞外基质复合生物材料的研究[D].成都:四川大学.2006.
[136] Atarashiya. Aluminum nitride/metal composites using ultra-fine aluminum particles and theirapplication for joining [J]. J Mater Proc Technol,1995,54(1-4):54-59.
[137] Anselme K, Bigerelle M, Noel B, et al. Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses [J]. J Biomed Mater Res,2000,49:155-166.
[138] Langer R, Lüthen F, Beck U, et al. Cell-extracellular matrix interaction and physicochemicalcharacteristics of titanium surfaces depend on the roughness of the material [J]. BiomolecularEngineering,2002,19:255-263.
[139] Horbett T A, Schway M B, Ratner B D. Hydrophilic-hydrophobic copolymers as cellsubstrates: effect on3T3cell growth rates [J]. J Coll Interf Sci.1985,104(1):28-39.
[140] Tziampazis E, Kohn J, Moghe P V. PEG-variant biomaterials as selectively adhesive proteintemplates: model surfaces for controlled cell adhesion and migration [J]. Biomaterials,2000,21(5):511-520.
[141] Christopher M Y, Robin S, David S, et al. Strong, tailored, biocompatible shape-memorypolymer networks [J]. Adv Funct Mater,2008,18:2428-2435.
[142] Wachem P B V, Hogt A H, Beugeling T, et al. Adhesion of cultured human endothelial cellsonto methacrylate polymers with varying surface wettability and charge [J]. Biomaterials,1987,8(5):323-328.
[143] Lee J H, Khang G, Lee J W, et al. Interaction of different types of cells on polymer surfaceswith wettability gradient [J]. J Coll Interf Sci.1998,205(2):323-330.
[144] Webb K, Hlady V, Tresco P A. Relative importance of surface wettability and chargedfunctional groups on NIH3T3fibroblast attachment, spreading and cytoskeletal organization[J]. J Biomed Mater Res.1998,41(3):422-430.
[145] Peluso G, Petillo O, Anderson JM, et al. The differential effects of poly (2-hydroxyethylmethacrylate) and poly (2-hydroxyethyl methacrylate)/poly (caprolactone) polymers on cellproliferation and collagen synthesis by human lung fibroblasts [J]. J Biomed Mater Res,1997,34(3):327-336.
[2]胡蕴玉.“骨与关节十年”与中国[M].国外医学内分泌学分册.2003.
[3]谢恩.新型仿生BMP-BG-COL-HYA-PS复合支架的实验研究[D].重庆:第四军医大学.2008.
[4]崔少千. BMP-2基因修饰的骨髓基质干细胞复合纳米骨促进兔腰椎融合的实验研究[D].沈阳:中国医科大学.2007.
[5]唐尤超. bBmp-2基因修饰促进骨质疏松症骨愈合的实验研究[D].成都:四川大学.2005.
[6] Augat P, Simon U, Leiedert A, Claes L. Mechanics and mechano-biology of fracture healingin normal and osteoporotic bone [J]. Osteoporos Int.2005,16: S36-S43.
[7]黄霞,陈家昌,申长雨等.骨组织工程支架材料研究进展[J].化工新型材料,2010,38(9):65-68.
[8] L. Calandrelli,G. De Rosa,M. E. Errico, et al.. Novel graft PLLA－based copolymers:Potential of their application to particle technology[J]. Journal of Biomedical MaterialsResearch Part A，2002，62(2):244-253.
[9] Freed L E, Vunjak-Novakovic G, Biron RJ. Biodegradable polymer scaffolds for tissueengineering [J]. Nat Biotechnol.1994,12(7):689-693.
[10]郑磊,王前.骨组织工程基质材料的现状及展望[J].生物医学工程学杂志,2001,18(3):470-474.
[11] Kim B S, Mooney D J. Development of biocompatible synthetic extracellular matrices fortissue engineering [J]. Trends Biotechnol.1998,16(5):224-230.
[12] Niu X F, Wang Y L, Luo Y F, et al. Arg-Gly-Asp (RGD) modified biomimetic polymericmaterials [J]. J. Mater Sci Technol.2005,21(4):571-576.
[13] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated celladhesion and beyond [J]. Biomaterials,2003,24:4385-4415.
[14] Langer R, Tirrell D A. Designing materials for biology and medicine [J]. Nature,2004,428:487-492.
[15] Drotleff S, Lungwitz U, Breunig M, et al. Biomimetic polymers in pharmaceutical andbiomedical sciences [J]. Eur. J. Pharm and Biopharm.2004,58:385-407.
[16] Sanchez C, Arribart H, Guille MMG. Biomimetism and bioinspiration as tools for the designof innovative materials and systems [J]. Nat Mater.2005,4:277-283.
[17] Healy K E, Rezania A, Stile R A. Designing biomaterials to direct biological responses [J].Ann Ny Acad Sci,1999,875:24-35.
[18] Langer R, Tirrell D A. Designing materials for biology and medicine [J]. Nature428,487-492
[19] Holmes T C. Novel peptide-based biomaterial scaffolds for tissue engineering [J]. Trends inBiotechnology,2002,20:16-21.
[20] Pan J, Zhao M, Liu Y, et al. Development of a new poly(ethylene glycol)-graft-poly(D,L-lactic acid) as potential drug carriers [J]. J. Biomed Mater Res. A,2008,89:160-167.
[21] Petrak K. Essential properties of drug-targeting delivery systems [J]. Drug Discovery Today10,1667-1673.
[22] Niu X F, Wang Y L, Luo Y F, Xin J, Li YG. Arg-Gly-Asp (RGD) modified biomimeticpolymeric materials [J]. Journal of Materials Science&Technology,2005,21:571-576.
[23] Anselme K, Linezb P, Bigerelle M, et al. The relative influence of the topography andchemistry of TiAl6V4surfaces on osteoblastic cell behaviour [J]. Biomaterials,2000,21(15):1567-1577.
[24] Fittkau M H, Zillaa P, Bezuidenhout D, et al. The selective modulation of endothelial cellmobility on RGD peptide containing surfaces by YIGSR peptides [J]. Biomaterials,2005,26(2),167-174.
[25] Ohga Y, Katagiri F, Takeyama K, et al. Design and activity of multifunctional fibrils usingreceptor-specific small peptides [J]. Biomaterials,2009,30(35):6731-6739.
[26] Shin H, Jo S, Mikos A G. Biomimetic materials for tissue engineering [J]. Biomaterials24,4353-4364.
[27] Niu X F, Luo Y, Li Y, Fu C, Chen J, Wang Y. Design of bioinspired polymeric materials basedon poly(D,L-lactic acid) modifications towards improving its cytocompatibility [J]. J. BiomedMater Res A,2008,84(4):908-916.
[28] Luo Y F, Wang Y L, Niu X F,Shang J F. Evaluation of the cytocompatibility ofbutanediamine-and RGDS-grafted poly(DL-lactic acid)[J]. Eur. Polym J44:1390-1402.
[29] Pierschbacher M D, Ruoslahti E. Cell Attachment Activity of Fibronectin Can Be Duplicatedby Small Synthetic Fragments of the Molecule [J]. Nature,1984,309,30-33.
[30] Massia S P, Hubbell J A. An RGD spacing of440nm is sufficient for intergrin ανβ3-mediatedfibroblast spreading and140nm for focal contact and stress fiber formation [J]. Journal of CellBiology,1991,114:1089-1100.
[31] Hozumi K, Dai O, Yuji Y, et al. Cell surface receptor-specific scaffold requirements foradhesion to laminin-derived peptide-chitosan membranes [J]. Biomaterials,2010,31(12):3237-3243.
[32] Toshihiro K, Yoshio O, Masayuki N. Preparation and mechanical properties of polylactic acidcomposites containing hydroxyapatite fibers [J]. Biomaterials,2001,22(1):19-23
[33] Quirk R A, Davies W C, Tendler S J B. Poly(L-lysine)-GRGDS as a biomimetic surfacemodifier for poly(lactic acid)[J]. Biomaterials,2001,22:865-872)
[34] Keilhoff G, Stang F, Wolf G, et al. Biocompatibility of type I/III collagen mat rixforperipheral nerve reconst ruction [J]. Biomaterials,2003,24:2779-2787.
[35] Li J, Tao R, Wu W, et al.3D PLGA scaffolds improve differentiation and function of bonemarrow mesenchymal stem cell-derived hepatocytes [J]. Stem Cells and Development.2010,19(9):1427-36.
[36]王晶,周庆颂,袁悦等.生物降解聚合物PLGA-PEG-PLGA的合成及表征[J].沈阳药科大学学报,2005(5):348-351.
[37] Donga Y, Feng S S. In vitro and in vivo evaluation of methoxy polyethyleneglycol–polylactide (MPEG–PLA) nanoparticles for small-molecule drug chemotherapy [J]Biomaterials.2007,28:4154-4160.
[38] Miura H, Onishi H, Sasatsu M, et al. Antitumor characteristics of methoxypolyethyleneglycol-poly(DL-lactic acid) nanoparticles containing camptothecin [J]. Journal of ControlledRelease,2004,97(1):101-113.
[39] Ruan G, Feng S S. Preparation and characterization of poly(lactic acid)–poly(ethyleneglycol)–poly(lactic acid)(PLA–PEG–PLA) microspheres for controlled release of paclitaxel[J]. Bromaterial,2003,24(27):5037-5044.
[40]葛建华,王迎军,郑裕东等. PLA-PEG-PLA嵌段共聚物的合成及研究[J].材料科学与工程学报.2003,21(6):817-820
[41]崔国振,贺继东. PLLA-PEG-PLLA三嵌段共聚物的合成及性能[J].青岛科技大学学报.2010,31(4):395-399.
[42] Achim G, Susam J P. Modulation of marrow stromal cell function using poly(D,L-lactic Acid)-block-poly(Ethylene Glycol)-monomethyl ether surfaces [J]. Journal of Biomedical MaterialsResearch.1999,46:390-398.
[43] Luo Y F, Wang Y L, Niu X F, et al. Synthesis and Characterization of a Novel Biomaterial:Maleic Anhydride-modified Poly(dl-lactic acid)[J]. Chinese Chemical Letters.2004,15(5):521-524.
[44]牛旭锋,王远亮,罗彦凤等.聚(D，L-乳酸)基仿生聚合物材料的合成与表征[J].功能材料.2006,11(37):1790-1794.
[45] Kang G, Lee S J, Lee J H, et al. Interaction of fibroblast cells on poly(lactide-co-glycolide)surface with wettability chemogradient [J]. Biomedical Materials and Engineering.1999;9:179-187.
[46] Kang G, Jeon J H, Lee J W, et al. Cell and platlet adhesions on plasma glow discharge-treatedpoly(lactide-co-glycolide)[J]. Biomedical Materials and Engineering.1997,7:357-368.
[47] Kim B S, Mooney D J. Development of biocompatible synthetic extracellular matrices fortissue engineering [J]. Trends Biotechnol.1998,16(5):224-230.
[48] Holmes TC. Novel peptide-based biomaterial scaffolds for tissue engineering. Trends [J].Biotechnol.2002,20(1):16-21.
[49] Sanchez C, Arribart H, Guille MMG. Biomimetism and bioinspiration as tools for the designof innovative materials and systems [J]. Nat Mater.2005,4:277-283.
[50] Lee K Y, Mooney D J. Cell-interactive polymers for tissue engineering [J]. Fibers Polym.2001,2(2):51-57.
[51] Lebaron R G, Athanasiou K A. Extracellular matrix cell adhesion peptides: functionalapplications in orthopedic materials [J]. Tissue Eng.2000,6(2):85-103.
[52] Cook A D, Hrkach J S, Gao N N, et al. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial [J]. J BiomedMater Res.1997,35:513-523.
[53]牛旭锋.聚(D,L-乳酸)基仿生细胞外基质的骨组织工程基质材料研究[D].重庆:重庆大学.2006.
[54] Yang S, Alnaqeeb M, Simpson H,et al. Cloning and characterization of an IGF-1isoformexpressed in skeletal muscle subjected to stretch [J].J Muscle Res Cell Motil,1996,17(4):487-495.
[55] Goldspink G. Mechanical signals, IGF-I gene splicing, and muscle adaptation [J]. Physiology(Bethesda).2005,20:232-238.
[56] Xian C Y, Wang Y L, Zhang B B, et al. Alternative splicing and expression of the insulin-likegrowth factor gene (IGF-1)in osteoblasts under mechanical stretch [J].Chin Sci Bull.2006,51(22):2731-2736.
[57] Tang L L, Xian C Y, Wang Y L. The MGF expression of osteoblasts in response to mechanicaloverload [J]. Arch Oral Biol.2006,51:1080-1085.
[58] Goldspink G, Scutt A, Loughna PT, et al. Gene expression in skeletal muscle in response tostretch and force generation [J].Am J Physiol,1992,262(3Pt2):R356-363.
[59] Hill M, Wernig A, Goldspink G. Muscle satellite (stem) cell activation during local tissueinjury and repair [J].J Anat.2003,203(1):89-99.
[60] Deng M, Zhang B B, Wang K, et.al. Mechano growth factor E peptide promotes osteoblastsproliferation and bone-defect healing in rabbits [J]. International Orthopaedics.2011,35:1099-1106.
[61] Ishihara K, Ueda T, Nakabayashi N. Preparation of phospholipid polymers and theirproperties as hydrogel sheet [J]. Polym J.1990,22:355-360.
[62] Jasmine D P, Yasuhiko I, Kazuhiko I, James M. Phospholipid polymer surfaces reducebacteria and leukocyte adhesion under dynamic flow conditions [J]. J Biomed Mater Res2005,73A:359-366.
[63]罗娟,王立坚,陈元维等.含生物功能基团磷脂胆碱的聚乳酸亲水性及细胞亲合性研究[J].生物医学工程学杂志.2008.25(6):1344-1348.
[64] Yasuhiko I, Yoko T, Tomoyuki K, Nobuo N. Reduced adhesion of blood cells tobiodegradable polymers by introducing phosphorylcholine moieties [J]. Journal of biomedicalmaterials research.2003,65A:164-169.
[65] Yasuhiko I, Shin-ichi S, Kazuhiko I, et al. Reduction of surface-induced inflammatoryreaction on PLGA/MPC polymer blend [J]. Biomaterials.2002,23:3897-3903.
[66] Junji W, Fredrik N, Bjorn A, et al. Cytocompatible biointerface on poly(lactic acid) byenrichment with phosphorylcholine groups for cell engineering [J]. Materials Science andEngineering C.2006,2:227-231.
[67] Junji W, Kazuhiko I. Change in cell adhesion property on cytocompatible interface usingphospholipids polymer grafted with poly(D,L-lactic acid) segment for tissue engineering [J].Science and Technology of Advanced Materials.2004,4:539-544.
[68] Augat P, Margevicius K, Simon J, Wolf S, Suger G, Claes L. Local tissue properties in bonehealing: influence of size and stability of the osteotomy gap [J]. J. Orthop Res.1998,16:475-481.
[69] Pankovic A M, Tarabishy E, Yelda S. Flexible intramedullary nailing of tibial-shaft fractures[J]. Clin Orthop,1981,160:185-195.
[70] Koch P P, Gross D F, Gerber C. The results of functional (Sarmiento) bracing of humeral shaftfractures [J]. J Shoulder Elbow Surg,2002,11:143-150.
[71] Claes L E, Wilke H J, Augat P, Rubenacker S, Margevicius K J. Effect of dynamization ongap healing of diaphysealfractures under external fixation [J]. Clin Biomech.1995,10:227-234.
[72] Kusec V, Jelic M, Borovecki F, Kos J, Vukicevic S, Korzinek K. D. Istraction osteogenesis byIlizarov and unilateral external fixators in a canine model [J]. Int. Orthop.2003,27:47-52.
[73] Weiss S, Henle P, Bidlingmaier M, et al. Systemic response of the GH/IGF-1axis in timelyversus delayed fracture healing [J]. Growth Hormone&Res.2008,18:205-212.
[74]刘振东,马梦然.从坚强固顶到生物学固定-----历史的回归[J].中华创伤骨科杂志.2004,6(8):910-912.
[75] Mueller C A. Primary stability of various forms of osteosynthesis in the treatment of fracturesof the proximal tibia [J]. J. Bone Joint Surg (Br).2005,873:426.
[76] Shtansky D V, Gloushankova N A, Sheveiko A N, et al. Design, characterization and testing ofTi-based multicomponent coatings for load-bearing medical applications [J]. Biomaterials.2005,26(16):2909.
[77]余贯华.生物功能表面活性剂和生物功能微球改性聚乳酸表面的研究[D].浙江:浙江大学.2005.
[78] Anselme K. Osteoblast adhesion on biomaterials [J].Biomaterials,2000,21:667-681.
[79] Beman A E, Kozlova N I and Morozevich G E. Integrins: Structure and signaling [J].Biochemistry-Moscow,2003,68(12):1284-1299
[80] Klaus Kühn. Integrin–Ligand Interaction [M]. Chapman and Hall, New York,1997.
[81] Lee J H, Jung H W, Kang I K and Lee H B. Cell behavior on polymer surfaces with differentfunctional groups [J]. Biomaterials,1994,15:705-711.
[82] Webb K, Hiady V and Tresco P A. Relationships among cell attachment, spreading,cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces[J]. J. Biomed Mater Res,2000,49:362-368.
[83] Humphries M J, Akiyama S K, Komoriya A, Olden K, et al. Indentification of an alternativelyspliced site in human plasma fibronectin that mediates cell type specific adhesion [J]. J. CellBiol,1986,103:2637-2647.
[84] Arnold M, Cavalcanti-Adam E A, Glass R, et al. Activation of integrin function bynanopatterned adhesive interfaces [J]. Chemphyschem,2004,5:383-388.
[85] Sinhgvi R, StePhnaoPoulos G, Wnag D I C. Eeffets of substratum moprhology on cellPhysiology [J]. Bioteehnology and Bioengineering,1994,43:764-71.
[86] Hatano K, Inoue H, Kojo T, Matsunaga T, et al. Effect of surface roughness on proliferationand alkaline phosphate expression of rat calvarial cells cultured on polystyrene [J]. Bone,1999,25:439-445.
[87] Matsuzaka K, Walboomers F, Ruijter A and Jansen J A. Effect of microgrooved poly-lactic(PLA) surfaces on proliferation, cyyoskeletal organization, and mineralized matrix formationof rat bone marrow cells [J]. Clinical oral implants research,2000,11(4):325-333.
[88]颜泽萱.骨修复用形状记忆聚氨酯的细胞相容性研究[D].重庆:重庆大学.2009.
[89]毛学理等.表面改性聚合物左旋聚乳酸-多聚赖氨酸可促进骨髓基质细胞的初始黏附[J].中国组织工程研究与临床康复.2011,15(38):7100-7104.
[90] Qiu Q, Sayer M, Kawaja M, Shen X and Davies J E. Attachment, morphology, and proteinexpression of rat marrow stromal cells cultured on charged substrate surfaces [J]. J. BiomedMater Res,1998,42:117-127.
[91] Boyan B D, Hummert T W, Dean D D and Schwartz Z. Role of material surfaces in regulatingbone and cartilage cell response [J]. Biomaterials,1996,17:137-146.
[92]马祖伟.聚乳酸软骨组织工程支架制备、改性及其细胞相容性研究[D].浙江:浙江大学.2003.
[93]罗彦凤.聚(DL-乳酸)的改性及体外降解和细胞相容性研究[D].重庆:重庆大学.2003.
[94] Peppas HA,Lanser R. New challenges in biomaterials [J]. Science,1994,263(5154):1715-1720.
[95] Kang I K, Kwon B K, Lee J H, et al. Immobilization of proteins on poly(methyl methacrylate)films [J]. Biomaterials,1993,14(10):787-792.
[96] Zhu Y B, Gao C Y, Liu X Y, et al. Immobilization of biomacromolecules onto aminolyzedpoly(L-lactic acid) toward acceleration of endothelium regeneration [J]. Tissue Eng.2004,10(1-2):53.
[97]姚康德,尹玉姬.组织工程相关生物材料[M].北京:化学工业出版社.2003.
[98] Healy KE, Rezania A, Stile RA. Designing biomaterials to direct biological responses[J]. AnnNY Acad Sci.1999,875:24-35.
[99] Horhertt T A, Schway M. Correlations between mouse3T3cell spreading and serumfibronextin adsorption on glass and hydroxyethymethacrylate-ethymethacrylate copolymers[J]. J. Biomed Mater Res.1998,22:763-793.
[100] Pettit D K, Hoffman A S, Horbett T A. Correlation between corneal epithelial cell outgrowthand monoclonal antibody binding to the cell binding domain of adsorbed fibronectin [J]. J.Biomed Mater Sci.1994,28:685-691.
[101] Chinn J, Horbeet T, Ratner B, et al. Enhancement of serum fibronectin adsorption and theclonal plating efficiencies of swiss mouse3T3fibroblast and MM14mouse myoblast cells onpolymer substrates modified by radiofrequency plasma deposition [J]. J Colloid and InterfaceSci.1989,127:67-87.
[102] Drumheller P D, Hubbell J A. Polymer networks with grafted cell adhesion peptides forhighly biospecific cell adhesive substrates [J]. Analytical Chem.1994,222:380-388.
[103] Drumheller P D, Ebert D L, Hubbell J A. Multifunctional poly(ethyleneglycol)semi-interpenetrating polymer networks as highly selective adhesive substrates forbioadhesive peptide grafting [J]. Biotech and Bioeng.1994,43:772-780.
[104] Zheng J S, Nothrup R, Hornsby P J. Modification of materials formed from poly(L-lactic acid)to enable covalent binding of biopolymers:Application to high-density three-dimensional cellculture in foams with attached collagen [J]. In Vitro Cell Dev Biol-animal.1998.34.679-684.
[105] Brandly B, Schnaar R. Covalent attachment of an Arg-Gly-Asp sequence peptide toderivatizable polyacrylamide surfaces: Support of fibroblast adhesion and long-term growth[J]. Analytical Biochem.1988,172:270-278.
[106]楚万忠,李晓光,刘百川,张凯宁,吴莹光.紫外分光光度法在退变腰椎间盘髓核胶原蛋白水平测试中的应用[J].山东医药.2007,47(29):35-36.
[107] Brenda K M, Annabel T T, Tmothy S B, et al. Modification of surfaces with cell adhesionpeptides alters extracellular matrix deposition [J]. Biomaterials.1999,20:2281-2286.
[108]王镜岩,朱圣庚,徐长法.生物化学(上)(第三版)[M].北京:高等教育出版社.2002,152-153.
[109] Qiu K, Wan C X, Chen X, et al. Acrylic acid-RGD as a biomimetic surface modifier forpoly(ethylene terephthalate)[J]. Chin J Biomed Eng.2003,12(1):22-31.
[110]赵强,万昌秀,刘建伟等.生物活性短肽RGD在PET表面接枝方法的研究[J].生物医学工程学杂志,2003,20(3):384-387.
[111] Philippe M, Jean-Francois L, Basma Fattouma B, et al. A new pro-migratory activity onhuman myogenic precursor cells for a synthetic peptide within the E domain of the mechanogrowth factor [J]. Exp Cell Res,2007,313(3):527-537．
[112] Ates K, Yang S Y, et al. The IGF-I splice variant MGF increases progenitor cells in ALS,dystrophic, and normal muscle [J]. FEBS Letters,2007,581(14):2727-2732.
[113] Dluzniewska J, Sarnowska A, Beresewicz M, et al. A strong neuroprotective effect of theautonomous C-terminal peptide of IGF-1Ec (MGF) in brain ischemia [J]. FASEB J,2005,19(13):1896-1898．
[114] Carpenter V, Matthews K, Devlin G, et al. Mechano-growth factor reduces loss of cardiacfunction in acute myocardial infarction [J]. Heart Lung Circ,2008,17(1):33-39.
[115]张静平.空间位阻在有机化学反应中的应用[J].池州师专学报.2001,15(3):17-18.
[116]王喜云. EMPLA分子量与接枝率的测定及其细胞生物相容性研究[D].重庆:重庆大学.2007.
[117]张俐娜等.高分子物理近代研究方法[M].武汉:武汉大学出版社.2003.
[118] Podximek S. The use of GPC coupled with a multi-angle laser light scattering photometer forthe characterization of polymers on the determination of molecular weight, size, andbranching [J]. Journal of applied polymer science,1994,54:91-103
[119] Leong K W, Brott B C, Langer R. Bioerodible polyanhydrides as drug-carrier matrices. I:Characterization, biodegradation and release characteristics [J]. J. Biomed Mater Res.1985,19:941-955.
[120]李玉宝.生物医学材料[M].北京:化学工业出版社.2003.
[121]俞耀庭,张兴栋.生物医用材料[M].天津:天津大学出版社.2000.
[122]林福春.力生长因子E肽（MGF-Ct24E）与应力作用对成骨细胞基因表达影响的基因芯片分析[D].重庆:重庆大学.2011.
[123] Small J V, Stradal T, Vignal E, et al. The lamellipodium: where motility begins [J]. TrendsCell Biol,2002,12:112-118.
[124] Critchley D R, Holt M R, Barry S T, et al. Integrin-mediated cell adhesion: The cytoskeletalconnection [J]. Biochem Soc Symp,1999,65:7-13.
[125] Neff J A, Tresco P A, Caldwell K D. Surface modification for controlled studies of cell-ligandinteractions [J]. Biomaterials,1999,20:2377-2393.
[126] Mann B K, West J L. Cell adhesion peptides alter smooth muscle cell adhesion, proliferation,migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds [J]. JBiomed Mater Res.2002,60:86-93.
[127] McCabe L R，Kundu B R，Harrison R J，et al．Developmental expression and activities ofspecific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2and jun D during differentiation [J]. Endocrinology,1996,137(10):4398-4408．
[128] Owen T A, Aronow M, Shalhoub V, et al. Progressive development of the rat osteoblastphenotype in vitro：reciprocal relationships in expression of genes associated with osteoblastproliferation and differentiation during formation of the bone extracellular matrix [J]. J CellPhysiol,1990,143(3):420-430.
[129] Hallab N J, Bundy K J, O’Connor K, et al. Evaluation of metallic and polymeric biomaterialsurface energy and surface roughness characteristics for directed cell adhesion [J]. Tissue Eng.2001,7(1):55-71.
[130] Deligianni D D, Katsala N D, Koutsoukos P G, et al. Effect of surface roughness ofhydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation anddetachment strength [J]. Biomaterials,2001,22(1):87-96.
[131] Curtis A, Wilkinson C. Topographical control of cells [J]. Biomaterials,1997,18(24):1573-1583.
[132] Flemming R G, Murphy C J, Abrams G A, et al. Effects of synthetic micro-andnano-structured surfaces on cell behavior [J]. Biomaterials,1999,20(6):573-588.
[133]文学军,王小祥,薛淼.金属生物材料的微粗糙表面及其生物学效应(I)－金属生物材料的微粗糙表面[J].生物医学工程学杂志,1997,14(1):77-80.
[134]牛旭锋,王远亮,潘君.成骨细胞在生物活性材料中黏附性能研究进展[J].生物医学工程杂志,2005,22(4):848-852．
[135]邓霞.新型骨组织工程类细胞外基质复合生物材料的研究[D].成都:四川大学.2006.
[136] Atarashiya. Aluminum nitride/metal composites using ultra-fine aluminum particles and theirapplication for joining [J]. J Mater Proc Technol,1995,54(1-4):54-59.
[137] Anselme K, Bigerelle M, Noel B, et al. Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses [J]. J Biomed Mater Res,2000,49:155-166.
[138] Langer R, Lüthen F, Beck U, et al. Cell-extracellular matrix interaction and physicochemicalcharacteristics of titanium surfaces depend on the roughness of the material [J]. BiomolecularEngineering,2002,19:255-263.
[139] Horbett T A, Schway M B, Ratner B D. Hydrophilic-hydrophobic copolymers as cellsubstrates: effect on3T3cell growth rates [J]. J Coll Interf Sci.1985,104(1):28-39.
[140] Tziampazis E, Kohn J, Moghe P V. PEG-variant biomaterials as selectively adhesive proteintemplates: model surfaces for controlled cell adhesion and migration [J]. Biomaterials,2000,21(5):511-520.
[141] Christopher M Y, Robin S, David S, et al. Strong, tailored, biocompatible shape-memorypolymer networks [J]. Adv Funct Mater,2008,18:2428-2435.
[142] Wachem P B V, Hogt A H, Beugeling T, et al. Adhesion of cultured human endothelial cellsonto methacrylate polymers with varying surface wettability and charge [J]. Biomaterials,1987,8(5):323-328.
[143] Lee J H, Khang G, Lee J W, et al. Interaction of different types of cells on polymer surfaceswith wettability gradient [J]. J Coll Interf Sci.1998,205(2):323-330.
[144] Webb K, Hlady V, Tresco P A. Relative importance of surface wettability and chargedfunctional groups on NIH3T3fibroblast attachment, spreading and cytoskeletal organization[J]. J Biomed Mater Res.1998,41(3):422-430.
[145] Peluso G, Petillo O, Anderson JM, et al. The differential effects of poly (2-hydroxyethylmethacrylate) and poly (2-hydroxyethyl methacrylate)/poly (caprolactone) polymers on cellproliferation and collagen synthesis by human lung fibroblasts [J]. J Biomed Mater Res,1997,34(3):327-336.