Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (34): 6193-6199.doi: 10.3969/j.issn.2095-4344.2013.34.020
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Li Zhi-ge1, Wang Yi2, Qi Yuan-yuan1, Che Xiao-qiang1, Liu Bin1
Online:
2013-08-20
Published:
2013-08-20
Contact:
Liu Bin, Professor, Doctoral supervisor, Stomatology School of Lanzhou University, Lanzhou 730000, Gansu Province, China
liubkq@lzu.edu.cn
About author:
Li Zhi-ge, Associate professor, Associate chief physician, Stomatology School of Lanzhou University, Lanzhou 730000, Gansu Province, China
lizhg@lzu.edu.cn
CLC Number:
Li Zhi-ge, Wang Yi, Qi Yuan-yuan, Che Xiao-qiang, Liu Bin. Polyvinyl alcohol and its composite materials for tissue engineering scaffolds[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(34): 6193-6199.
2.1 骨组织工程支架材料 制备能引导组织再生的复合材料是骨组织工程的重要内容之一,组织工程化骨作为骨修复材料的替代物,可避免生物源性修复材料的缺陷。天然骨是由无机矿物与生物大分子规则排列而成的复合体,骨基质中无机盐的主要化学成分是羟基磷灰石。羟基磷灰石具有与人体硬组织(如骨、牙等) 相类似的无机成分和良好的生物相容性,是一种重要的生物陶瓷材料。但羟基磷灰石纳米颗粒由于具有相当大的表面积,易发生强烈团聚,从而极大地影响了其纳米性能的发挥。羟基磷灰石粉体纳米化有望提高骨模仿自然骨的组成。聚乙烯醇因其化学组成、力学性能和可降解性等因素,越来越多的研究将其用作骨组织替代材料。虽然聚乙烯醇性能优异,但在承载一定质量的骨缺损修复中仍不能单独使用,常与无机材料如羟基磷灰石,磷酸三钙,生物活性玻璃,碳纳米管等复合来获得更大的强度和韧性。 Ikada[7]通过湿法合成制备了直径10-20 nm、长30-40 nm 的短棒状纳米羟基磷灰石,用聚乙烯醇作为表面分散剂对羟基磷灰石纳米颗粒进行了表面改性实验,实验表明经聚乙烯醇包覆后的羟基磷灰石纳米颗粒具有较好的分散性,这为以后进一步制备纳米羟基磷灰石增强聚乙烯醇水凝胶组织工程支架材料提供了可能性。李玲琍等[8]采用溶液共混法制备了含有丝素的聚乙烯醇共混膜,并采用交互浸渍法成功地在聚乙烯醇/丝素膜上生长出羟基磷灰石,使复合膜引发羟基磷灰石生长的能力增强,所得到的羟基磷灰石化学组成和结构与骨骼中的羟基磷灰石极为相似,因此认为聚乙烯醇/丝素复合材料具有较好的力学性能和生物活性,在骨组织再生材料方面具有一定的应用潜力。Xiao等[9]采用相分离法制备羟基磷灰石-聚乙烯醇/聚己内酯-壳聚糖复合多孔支架,结合了天然聚合物、合成聚合物和羟基磷灰石的优点,聚乙烯醇与羟基磷灰石结合使材料具备更好的机械强度。Mansur等[10]用溶胶凝胶法和发泡方法成功制备三维多孔聚乙烯醇/生物活性玻璃和聚乙烯醇/壳聚糖/生物活性玻璃两种有机无机混合支架,通过改变聚乙烯醇加入量和水解度改变凝胶时间,可改变泡沫孔隙率、孔径分布等。随聚乙烯醇水解度增高,支架的机械性能提高,适合松质骨的修复。但是细胞生存能力不因聚乙烯醇/生物活性玻璃的比例或加入壳聚糖的量而存在明显差异,两种水凝胶都表现出良好的机械性能,形态学特点和保持细胞活力等。牟元华等[11]采用乳化发泡-冷冻固化-去除表面活性剂法制取了聚乙烯醇和纳米聚乙烯醇两种多孔材料,所得样品的孔径均匀,孔隙率高孔之间相互贯通,说明此方法是一种制取多孔材料的新方法。聚乙烯醇的浓度也对孔径和孔隙率有影响,低浓度有利于形成大孔和高的孔隙率,高浓度有利于形成小孔和低的孔隙率。因羟基磷灰石能吸附表面活性剂,对形成孔有一定阻碍作用,其浓度的增减对孔径和孔隙率也有一定的影响。目前,纳米材料和纳米技术在生物医学工程领域的地位日益突出。用于创伤修复的纳米纤维,以及用于组织工程的纳米结构材料等更是引起了人们的重视。静电纺丝纳米纤维能够从纳米尺度上模仿天然胞外基质,可用作骨细胞和间叶干细胞的生长基底。Kim等[12]利用静电纺丝法制备了聚乙烯醇/羟基磷灰石复合纳米纤维,纤维的形貌和晶体结构显示棒状的羟基磷灰石粒子嵌入纤维内部并表现出纳米多孔形貌,并且羟基磷灰石取向于平行纤维的方向排列,类似于骨组织的自然矿化。总之,用作骨组织工程支架的材料必须满足力学与生物学方面的基本性能要求,聚乙烯醇作为制备基体是与其良好的性能密切相关的,在与高强度的材料复合时能发挥出各自的优势[13-16] 。 2.2 软骨组织工程支架材料 软骨组织工程支架材料要求具有特定的合适的生物降解性、可控的孔径大小、足够的孔隙率等。高分子水凝胶是线性高分子链通过交联形成三维网状结构,再经过大量溶剂溶胀形成的一种胶态物质。由于聚乙烯醇水凝胶除了具备一般水凝胶的性能外,特别具有低毒性、机械性能良好(高弹性模量和高机械强度)、吸水量高和生物相容性好等优点,因而备受研究者青睐。聚乙烯醇水凝胶具有与关节软骨相似的结构和力学性能,弹性模量与摩擦系数很低,有助于实现液膜润滑,减少磨损及松动。现在采用高聚合度(5 000-8 000)聚乙烯醇- DMSO-H2O 混合体系可以制造出耐磨损性优异的水凝胶材料,可望用于人工软骨,置换病变或损伤的软骨。聚乙烯醇水凝胶复合材料也被广泛用于软骨组织工程支架材料[17-20] 。近年来研究者们对聚乙烯醇水凝胶进行适当改性,制得高强度、高含水量的弹性材料,促进了它在生物医学方面的应用。常见与羟基磷灰石、壳聚糖、有机小分子和生物活性分子复合制备复合水凝胶支架。卢华定等[21]将聚乙烯醇/纳米羟基磷灰石复合水凝胶植入兔膝关节缺损处,12 周后植入物与周围软骨交界面有大量的软骨细胞增殖,并且结合紧密,有骨样组织长入植入物,未发现软骨退变。Bodugoz-Senturk等[22]制备聚乙烯醇-丙烯酰胺水凝胶,丙烯酰胺的应用提高了孔隙率,平衡了水凝胶的水分含量,降低了摩擦因数,抗拉强度、抗蠕变性都得到了提高。Bonakdar等[23]制备不同配比聚乙烯醇/聚亚胺酯可降解水凝胶,其压缩模量在关节软骨范围内,人软骨细胞在支架上生长好。Bichara等[24]在多孔聚乙烯醇-藻酸盐凝胶支架上培养人鼻中隔细胞修复鼻中隔软骨,材料在组织结构上类似自然组织,在与组织培养中承受剪切力10 d后,发现DNA、黏多糖和羟基脯氨酸成分都有所增加,且能保持机械完整性。Oka 等[25]报道聚乙烯醇水凝胶软骨材料植入人体8-52 周后基本无炎症现象和退化行为出现。开发了一种商业名称为SalubriaTM (Salum edica, Atlanta, GA,USA)的有机聚合物水凝胶材料,这种材料由聚乙烯醇分子构架和9 g/L NaCl溶液经过冷冻/解冻过程合成,具有与自然关节组织极为相似的含水量,并具有良好机械整体性、生物相容性及易成型为固定解剖学形状等优点。Wiliams[6]曾对这种材料做过粉碎、拉伸及压缩实验以评价其力学性能,发现这种水凝胶的机械强度基本满足关节应用。Bajpai等[26]合成了由聚乙烯醇和壳聚糖组成的互穿聚合物网络,实验结果显示其有很好的电敏性。将溶胀的聚乙烯醇-壳聚糖互穿聚合物网络水凝胶放在2个电极之间,当有外加电压时水凝胶会产生弯曲;随着NaCl水溶液浓度的增加和施加电压的增大,水凝胶的弯曲角度和速度都会相应增大。 2.3 皮肤组织工程支架材料 理想的皮肤替代物应该具有一些基本的特征:易加工处理、细胞容易黏附、有适当的物理或机械稳定性、无毒、无抗原性,并且还要易于血管再生、创伤小、尽可能地减少瘢痕形成。组织工程皮肤构建的关键是设计能够有效支撑皮肤组织再生修复行为的皮肤再生支架,从组分仿生角度,采用一种或多种细胞外基质如胶原、硫酸软骨素等为原料构建皮肤支架已有大量的研究报道。聚乙烯醇及复合材料在制备皮肤组织工程支架方面的研究也越来越多。Lee等[27]制备了聚乙烯醇/硫酸软骨素凝胶支架,能促进细胞吸附和生长,在支架上连接成密集的片状。硫酸软骨素促进了细胞与支架的相互作用,聚乙烯醇为赐予支架良好的机械性能促进细胞培养。由于聚乙烯醇水凝胶具有良好的生物相容性,对机体和组织细胞无任何毒性和刺激作用。Costa-Júnior等[28]制备聚乙烯醇和壳聚糖交联水凝胶,发现成纤维细胞在支架上的黏附好,无毒,可用作伤口敷料或皮肤组织工程支架。组织或器官的细胞外基质纳米微结构对调控细胞行为具有重要的作用,如纳米纤维结构的高比表面特性,可提高细胞在材料表面的黏附能力。模拟皮肤组织的细胞外基质纳米结构,制备具有纳米纤维结构的皮肤组织工程支架逐步受到重视。Duan等[29]用电纺技术制备了具有多孔纳米结构的聚乙烯醇/聚乳酸乙醇酸共聚物/壳聚糖薄膜。体外细胞培养实验证明,这种复合多孔纳米结构能更好地支持人胚胎皮肤成纤维细胞的黏附和增殖,并能迁移和渗透到薄膜内部,可望促进皮肤创面的修复,该纳米纤维薄膜具有良好的创面贴附性能,且不易导致创面积液。具有复杂结构的皮肤组织再生修复过程是多种细胞协同作用的结果。此外,各种细胞的增殖、迁移及细胞外基质分泌等行为需要通过生长因子、激素等信号分子的调控来实现。近来,将血管内皮细胞生长因子、成纤维细胞生长因子、血小板衍生因子等活性因子通过化学固定、微载体复合等方法与组织工程皮肤支架结合,构建活性皮肤支架的研究也较多,也是聚乙烯醇下一步应用的方向之一[30-31] 。王宏昕等[32]通过溶剂浇铸/粒子沥滤的方法制备pNSR16/聚乙烯醇支架材料及其浸提液。将小鼠胚胎成纤维细胞( NIH-3T3 细胞) 与浸提液培养,采用MTT法检测培养1,3,5 d 时支架材料的细胞毒性。苏木精-伊红染色示细胞能在支架表面黏附和生长,且随培养时间的延长,细胞向支架内部迁移。免疫组织化学检测到NIH-3T3 细胞分泌的碱性成纤维细胞生长因子,细胞能进行正常分化。pNSR16/聚乙烯醇支架材料具有良好的细胞相容性,有望作为一种较理想的组织工程支架材料。Duan等[33]用聚乙烯醇、壳聚糖和聚乳酸乙醇酸共聚物做成静电纺丝,然后把静电纺丝制成纳米纤维复合膜,通过戊二醛蒸汽处理,使得复合膜交联,很好地维持了膜的力学性能及湿润状态下的纤维形态。聚乙烯醇和壳聚糖的亲水性使得复合膜磷酸盐缓冲液的吸附量较纯聚乳酸乙醇酸共聚物静电纺丝膜有所提高。细胞培养表明,静电纺丝聚乳酸乙醇酸共聚物-壳聚糖/聚乙烯醇膜可以促进成纤维细胞的贴附和增殖。静电纺丝聚乳酸乙醇酸共聚物-壳聚糖/聚乙烯醇膜有望被应用在皮肤重建等皮肤组织工程方面。Pal等[34]通聚乙烯醇羟基和明胶羧基的酯化,形成了聚乙烯醇-明胶水凝胶膜,此膜有良好持水能力和适当的强度,溶血性试验显示没有溶血或接近0%的溶血率。表明膜有很好的血液相容性,可用在伤口辅料及植入性药物递送系统等生物医学领域;另外凝胶聚乙烯醇-明胶良好的成膜性使其有望在人工皮肤上得到应用。 2.4 血管组织工程支架材料 血管支架材料在结构上分为双层,内层为与血液相容性好的生物活性材料,该类材料要求不仅具有生物活性,同时还要具有抗凝血和抗溶血作用,这类材料一般为经过表面修饰的降解材料。将肝素以离子键或共价键的形式有机地结合在聚乙烯醇-海藻酸钙网状高分子材料中,可使材料具有良好抗凝血性能和生物相容性,基本能够达到预期的要求[34-35] 。并且该复合材料在结合肝素后,仍能保留原有材料的强度和高弹性,满足力学相容性的要求。体内外实验均表明,血小板在聚乙烯醇表面黏附性差,接触时间短促,并且在如此短促的时间仍足以激活血小板并使之在血液循环中很快消失,而不致在聚乙烯表面发生凝血现象,这可能与聚乙烯醇表面具有低粗糙度和高的活化能等特点有关。外层材料必须为保证内层材料细胞生长提供一定的支撑强度、抗拉强度和韧性。总体上,血管组织工程支架材料要求具有生物相容性,适于细胞贴附和基质沉积,同时还应具有传递弹性的作用,以适应血管在体内的功能。小口径血管的替换问题是血管组织工程亟待解决的关键,首先要求支架材料具有优良的机械性能,聚乙烯醇等人工合成材料的优点是它们的强度、降解速度、微结构和渗透性均可在生产过程中进行控制;而天然材料的优点则在于包含有生物信息(如特殊的氨基酸序列),可促进细胞吸附或使细胞保留分化功能。现在研究人员试图综合两类材料的最佳优点来设计新一代复合材料。聚乙烯醇水凝胶能满足脉管系统的机械性能要求[36-37] 。Wan等[38]已优化聚乙烯醇水凝胶的拉伸性能,可匹配猪主动脉根部。Millon等[39]也证明了猪主动脉的机械性能与聚乙烯醇管状结构相似,但是聚乙烯醇水凝胶因其高度的亲水性不利于细胞的黏附,为克服此不足,常将聚乙烯醇与明胶、右旋糖苷、胶原蛋白、壳聚糖、透明质酸等复合。Liu等[40]用冷冻干燥法制备聚乙烯醇与壳聚糖,聚乙烯醇与明胶,聚乙烯醇与淀粉的复合水凝胶,评价其用于人造血管的性能,牛血管内皮细胞黏附性能提高,可形成完整的内皮细胞层。魏梅红等[41]基因重组蛛丝蛋白溶解于98%甲酸,采用冷冻干燥粒子滤沥法制备重组蛛丝蛋白-聚乙烯醇复合多孔支架;采用乙醇作变性剂制得的多孔支架力学性能较好,支架的断裂应力、断裂比强度均提高5倍以上,断裂伸长率可达12.2%。以粒径<500 pm的NaCl为致孔剂制得的多孔支架力学性能更好,高分子材料聚乙烯醇能明显改善重组蛛丝蛋白多孔支架的性能,有望在组织工程领域得以应用。利用相分离、电纺丝、结构蛋白多肽的自组装等技术可构建纳米血管。目前应用较多,发展较迅速的是电纺丝技术。支架纳米技术在血管组织工程中的应用主要是能够建立一个适于细胞定向分化和组织增殖的细胞外微环境。在这个环境中,纳米支架能够可控地使血管细胞的表型得以表达,同时使细胞表达自身的生物学功能[42-43] 。 2.5 其他组织工程支架材料 聚乙烯醇还常被用作角膜、主动脉瓣膜、腺体、神经等组织工程支架,在修复缺损方面具广泛应用前景。Mottaghitalab等[44]制备了纳米结构的壳聚糖/聚乙烯醇支架,并增强了神经细胞株的增殖,具有较好的应用前景。Chen等[45]研究大鼠腮腺腺泡细胞在表面涂有聚乙烯醇的支架上的生长聚集,体内试验尤其聚集更明显,形成腺泡细胞球。Teramura等[46]将聚乙烯醇为表面的复合膜制备成胰岛微囊,微囊化后胰岛的数量没有增加,胰岛表面的聚乙烯醇不会损害胰岛素的释放。Bader 等[47]制备甲基丙烯酸缩水甘油酯改性的聚乙烯醇水凝胶可用作脊髓核替代材料,可模仿天然髓核的黏弹性。"
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