Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (22): 3573-3578.doi: 10.3969/j.issn.2095-4344.2294
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Fang Xu1, Dong Junfeng1, 2
Received:
2019-11-30
Revised:
2019-12-06
Accepted:
2020-01-10
Online:
2020-08-08
Published:
2020-04-26
Contact:
Dong Junfeng, MD, Associate chief physician, Master’s supervisor, Department of Spinal Surgery, the First Clinical Medical College of Three Gorges University (Yichang Central People’s Hospital), Yichang 443003, Hubei Province, China; Institute for Spinal Medicine and Trauma, Three Gorges University, Yichang 443003, Hubei Province, China
About author:
Fang Xu, Master candidate, Department of Spinal Surgery, the First Clinical Medical College of Three Gorges University (Yichang Central People’s Hospital), Yichang 443003, Hubei Province, China
Supported by:
CLC Number:
Fang Xu, Dong Junfeng. Surface modification of bone tissue engineering implants[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(22): 3573-3578.
2.1 无机相表面修饰 2.1.1 磷酸钙涂层 天然骨无机相约占骨质量的50%,是人体骨骼主要的机械支撑,其主要成分磷酸钙已被广泛应用于骨组织工程[1]。合成磷酸钙材料不仅与无机磷灰石具有相似的物理化学性质,而且在实际研究和临床应用中也表现出卓越的生物活性、生物相容性和骨传导 性[2-3],但是由于其力学性能较差,不能单独应用于骨组织工程。将磷酸钙涂层结合到金属植入物(如钛、镁、钴铬合金等)表面显著提高了其临床多样性。目前骨植入物材料的涂层技术包括有等离子喷涂、磁控溅射、溶胶-凝胶、脉冲激光沉积、仿生沉淀和逐层静电多层膜组装等,但是等离子喷涂由于其沉积率高和成本低一直受到广泛关注[4-5]。磷酸钙涂层包括磷酸八钙、无定形磷酸钙、二水合磷酸氢钙、磷酸三钙和羟基磷灰石,而最常用的表面修饰剂是羟基磷灰石、无定形磷酸钙和磷酸三钙[6-7]。在接触体液后,涂层中的离子会从种植体表面释放出来,刺激碳酸钙(类似于天然骨成分)的成核,可使新骨形成并与涂层种植体结合[8]。有报道称,适当比例的非晶态相(如无定形磷酸钙和磷酸三钙)可以加速初始骨生长,因为这些非晶态成分快速溶解所产生的钙基磷酸盐沉淀会吸引破骨细胞和成骨细胞,来重塑种植体和天然骨之间的局部环境,在植入物上诱导骨形成[9]。然而,较高比例的无定形磷酸钙和磷酸三钙会削弱骨固定,导致种植体松动。此外,高结晶度和低溶解度的纯羟基磷灰石涂层具有较差的初始固定,因此不是骨组织修复的好选择,即使它具有显著的长期骨整合[10]。因此,准确控制好非晶体相和羟基磷灰石之间的比例是磷酸钙涂料应用于临床需要优化的问题。此外,由于磷酸钙涂层的热膨胀系数与种植体基体表面的热膨胀系数有一定差异,在长期使用中也应考虑由于涂层热膨胀系数而产生的残余应力。残余应力应力在一定程度上增加了涂层开裂、分层和变形的风险,可能会破坏涂层的整体生理和力学性能。已有很多关于全髋关节置换的研究证明,与未涂层假体相比,涂层假体周围的骨整合得到了改善。 2.1.2 其他矿物离子涂层 由于天然骨中含有多种无机离子,因此将这些离子元素引入到涂层中对骨植入物的表面修饰有重要作用。此前已有研究表明,离子掺杂可以影响涂层的化学组成、结晶度、形貌和溶解速率,并改变其力学性能和生物相容性。与各种生物蛋白活性相比,无机离子用于骨修复具有成本低、安全性好等优点。作为一种金属植入物,钛具有良好的力学性能、耐腐蚀性和生物相容性,在过去几十年里一直是人们研究的对象。WU等[11]发现,在钛植入物表面涂覆锶基陶瓷可以下调导致破骨细胞分化的白细胞介素6型细胞因子,从而促进骨愈合。OFFERMANNS等[12]在兔模型中研究了牙种植体不同骨整合阶段锶功能化钛种植体对骨-种植体接触和骨形成的影响,组织形态数据显示锶功能化的钛表面可加速骨整合,这可能会对牙科植入装置改造提供新的方向。锌是许多酶的组成部分,锌离子涂层钛植入物也被证明能促进成骨细胞和成纤维细胞的生长,提高细胞活力,并具有额外的抗菌功能[13-14]。掺硅羟基磷灰石涂层已被证明能通过增加细胞膜上的肌动蛋白应力纤维促进细胞骨架的成熟,进而显著增强了细胞的附着性[15]。用硅基化合物进一步修饰植入物,发现其对小鼠前成骨细胞MC3T3-E1增殖和磷灰石形成有积极影响[16-17]。镁离子被发现有助于成骨分化、成骨细胞黏附与细胞外基质蛋白表达[18-19]。另外,镁离子可以通过为磷灰石的形成提供成核位点,来促进有机基质中的生物矿化[20]。有学者观察到将氟离子并入羟基磷灰石结构可增强基底涂层的黏附强度和相关骨细胞(例如MG63和MC2T3-E1细胞)的活性,金属植入物上的氟表面修饰可显著抑制细菌活性[21]。锂离子可影响涂层的沉积和生物相容性,加速骨细胞的附着及早期增殖[22]。此外,其他主要元素如钠、钾和氯也在骨矿化、吸收和代谢中起着不可或缺的作用。 尽管将这些离子渗入到骨植入物涂层中已显示出良好的效果,但这些离子在长期植入后可能会产生不良影响。例如,快速退化镁离子进入周围组织可能导致氢气积聚和体液碱化,延缓骨骼愈合,导致组织坏死,甚至因氢气泡阻塞血管而死亡[23]。低浓度锌离子能促进成骨细胞生长,但高浓度锌涂层的长期研究证明其会加快骨髓腔区域的骨吸收[24]。最近一项研究表明,成骨细胞、破骨细胞和上皮样细胞的活性与钛离子剂量存在依赖关系,钛离子浓度在1-9 ppm时,对这些细胞影响不大,浓度达到9 ppm时,促进了他们的生长,但当浓度达到20 ppm时会对这些细胞存在明显毒性作用[25]。为了获得最佳的生理反应,这些无机离子在体内环境中的剂量、持续时间和释放动力学有待进一步研究。 2.1.3 种植体表面地形的物理修饰 骨组织工程植入物的表面地形(如表面粗糙度和晶体大小)是宿主组织和生物材料之间直接相互作用的主要界面,因而备受关 注[26]。近年来,纳米技术已逐渐应用于骨植入物涂层中,其通过产生更薄的涂层来调节炎症,减少细菌黏附,获得更好的骨诱导性,这是传统的涂层种植体所不具备 的[27-29]。通过将表面几何结构改为微/纳米结构可以增加成骨细胞的增殖率,使蛋白质(结构蛋白和非胶原蛋白)从周围体液中吸收,并最终决定成骨细胞的黏附、运动、增殖和分化[30]。CAIRNS等[31]研究成骨样细胞MG63对不同表面形貌的反应,发现与未改性的表面相比,具有规则纳米结构薄膜的磷酸钙促进了碱性磷酸酶和骨钙素的表达。AJAMI等[32]研究了种植体表面地形对骨重建的影响,发现纳米表面修饰的钛植入物优于微米尺寸的表面修饰,同时两种表面修饰都显示在健康或高血糖条件下骨传导得到改善。ZHOU等[33]采用微弧氧化对钛种植体的拓扑结构和表面化学进行了改性,通过微孔表面形成分层纳米点来增强了骨再生和植骨接触。此外,有学者通过控制微弧氧化的退火温度来调节羟基磷灰石纳米离子在钛种植体上表面形貌,并证明控制退火温度在650 ℃时的钛/羟基磷灰石植入体不仅支持成骨细胞和内皮细胞的增殖和分化,而且抑制巨噬细胞的炎症反应,并具有良好的骨免疫调节功能、促进成骨和血管生成功能[34]。可以清楚地看到,多种骨植入物的表面形貌修饰在生物医学假体的骨整合中发挥着巨大作用[35]。 表面粗糙度是影响种植体固定和成骨的另一个关键因素,适当的粗糙度可促进成骨细胞的增殖和分化,促进新生血管的形成,增强碱性磷酸酶活性,上调骨相关基因表达,抑制破骨细胞活性,减少细菌黏附[36-37]。与普通机械加工的植入物相比,粗糙的表面不仅可增加骨植入物的接触面积和植骨量,还能改善其稳定性和机械性能[38]。此外,表面的粗糙度通过改变局部机械环境在一定程度上影响着植入物向周围骨细胞的应力传递[39]。虽然许多文献报道了表面粗糙度和机械应力对宿主细胞反应的影响,但很少有文献报道表面粗糙度和机械应力与宿主细胞之间的相互反应。尽管微纳米制造技术的快速发展赋予了精确控制骨植入物表面形貌的能力,但仍需要更深入的研究,来全面比较不同表面形貌在细胞反应中的作用[40]。建立一个具有多种标准化地形表面修改的系统数据库,可以用来破译骨细胞与表面地形之间复杂的相互作用,从而实现骨植入物的最佳修饰材料表面。 2.2 骨有机相表面修饰 2.2.1 含有细胞天然蛋白的涂层 由于骨组织工程植入物的宿主反应受骨细胞和细胞外基质之间相互作用的调节,骨细胞与不同细胞外基质蛋白(胶原、明胶、纤维连接蛋白、卵黄蛋白和纤维蛋白原)的相互调控作用,被许多研究者认为是一种很有前途的骨修复方法[41-42]。如HE等[43]利用胶原渗透羟基磷灰石涂层构建纤维网络,并报道了这种新涂层增强了骨髓间充质干细胞的黏附、增殖和分化;体内实验表明,在胶原改性羟基磷灰石涂层中加入骨形态发生蛋白可使骨早期固定,骨生长率高达29%,这种复合涂层具有良好的骨传导和骨诱导性能。通过用Ⅰ型胶原和硫酸软骨素(骨组织细胞外基质中普遍存在的一种糖胺聚糖)涂覆钛植入物,观察到骨与骨组织工程植入物的结合加快,显著降低了无菌性松动率[44]。碱性磷酸酶是一种金属酶,它能催化无机磷酸盐的成核,而无机磷酸盐是硬组织矿化所必需的。通过电喷涂在金属基底上制备了活性碱性磷酸酶涂层,并将其加载到无机钙基生物陶瓷涂层上,结果表明磷酸盐成核速度加快,成骨细胞的活力增强[45]。植入式材料的表面特性对于蛋白质相关涂层和宿主组织的细胞反应至关重要。蛋白质在材料表面的黏附能力很大程度上受表面张力、极性、表面电荷、润湿性和形貌的影响[46-47]。蛋白质通常倾向于黏附在非极性、高表面张力和带电的底物上[48]。此外,还应考虑外部参数如涂层温度、pH值、离子强度和溶液的化学成分对其吸附的影响[49-50]。由于蛋白质的多样性和周围环境的复杂性,使得利用机蛋白涂层进行植入物的表面修饰难以达到统一的标准。 2.2.2 细胞结合肽涂层 目前,在植入物表面涂敷活性因子涂层以赋予植入物生物特性是研究的热点,但以正确的构象嫁接和吸收这些蛋白是不容易实现的。考虑到与细胞外基质生物分子相关的这些缺点,已经逐步建立了具有特定生物活性序列的人工细胞结合多肽。目前研究最广泛的底物涂层多肽序列是精氨酸-甘氨酸-天冬氨酸,它存在于大多数细胞外基质蛋白中,包括纤维连接蛋白、维生素c和层粘连蛋白[51]。其多功能性可归因于其与24种已知人类整合蛋白的广泛结合能力[51]。在各种体内研究中,精氨酸-甘氨酸-天冬氨酸涂层种植体不仅能促进粘连,还能增加骨细胞的增殖和存活,改善骨整 合[52]。由于精氨酸-甘氨酸-天冬氨酸序列只能模拟细胞外基质蛋白中众多生物活性位点中的一个,因此在骨缺损修复中也合成和评估了其他多肽。例如,GFOGER作为一种类似胶原的三重螺旋六肽,通过与α2β1受体结合诱导成骨细胞分化,增强成骨细胞的活性,而精氨 酸-甘氨酸-天冬氨酸肽对此受体的骨诱导活性可忽略不 计[53-54]。用DLTIDDSYWYRI基序(来源于人层粘连蛋白2α2链)涂层的钛植入物通过加速胶原合成和碱性磷酸酶表达,改善了骨形成[55]。由于细胞外基质蛋白中不同活性位点之间存在多样的位置和空间关系,仅保证植入物表面覆盖人工多肽有时不足以充分发挥再生潜能。因此,人们的研究兴趣已经转移到同时结合生物蛋白质和多肽的表面涂层上,在这里称这种表面改性方法为“混合涂层”。与传统的单肽或蛋白质涂层相比,这种新的涂层技术在骨愈合方面表现出更好的性能[56]。含有多个肽序列的蛋白质或材料表面特殊排列的多肽,也提供了更多改善植入物骨整合途径。然而,如何以精确控制的方式稳定它们的构象和片段长度仍是待以解决的问题[57]。 2.2.3 生长因子涂层 缺损修复和再生是一个复杂的生物学工程,需要再现体内联级信号,使多个过程(即软骨生成、成骨和血管生成)以有序的空间和时间进行。为了改善骨形成,大量生长因子(骨形态发生蛋白、胰岛素样生长因子Ⅰ/Ⅱ、转化生长因子β、甲状旁腺激素、成纤维细胞生长因子、血小板衍生生长因子和Wnt)被克隆和重组表达[58],这些趋化因子和有丝分裂因子不仅能促进成骨细胞的增殖和分化,而且能促进成胶原基质和磷灰石的合 成[59-60]。在这些生长因子中,骨形态发生蛋白2、骨形态发生蛋白7、甲状旁腺激素、甲状旁腺激素相关蛋白和血小板衍生生长因子已被美国食品药品管理局和欧洲药品管理局批准用于临床应用[58]。通过使用交替沉积技术得到聚电解质多层涂层,MACDONALD等[61]发现在最初的2 h内骨形态发生蛋白2的释放低于1%,在最初的2周内骨形态发生蛋白2的释放低于10 μg。与商用胶原基质中近60%的释放量相比,这种交替沉积技术膜可以很好地控制宿主对骨科设备的反应。MIN等[62]通过自组装、水解降解的交替沉积涂层依次输送骨形态发生蛋白2和庆大霉素,成功地对诱导性骨髓炎大鼠进行了骨组织修复和抗菌治疗的双重治疗。在LA等[63]将氧化石墨烯通过逐层组装包覆到钛基体上,并且再将最外层涂层负载骨形态发生蛋白2,氧化石墨烯层作为蛋白质的有效载体导致骨形态发生蛋白2可控释放,从而加速骨形成。为了改善传统涂层的成骨诱导,各种生长因子(如甲状旁腺激素、骨形态发生蛋白2和成骨蛋白1)也被嵌入到多孔结构中以制造双功能植入物[64-65]。然而,当这些生物分子被用于骨再生时,额外的并发症和安全性问题应该被关注。CARRAGEE等[66]发现在脊柱融合术中应用重组人骨形态发生蛋白治疗的患者中,近40%可能会出现多种不良反应,包括神经根炎、骨溶解、意外异位骨形成,甚至死亡。骨形态发生蛋白2的使用会使发生癌症的潜在风险增高,相对风险达到1.98[95%CI(0.86,4.54)][67]。这可能与骨形态发生蛋白2过量的给药量有关,导致局部浓度急剧增加,显著超过骨组织的生理水平[68]。近年来,将生长因子与其他非胶原蛋白和生物活性聚合物结合在一起进行表面修饰的方法越来越受欢迎[69-70]。然而,如何高效地结合生物分子,以可控的方式调节其释放并保持其各自的生物学功能,对于这些新型骨科涂层的开发具有重要意义。 "
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