Actuality and challenge of biomaterials in annulus fibrosus repair

doi:10.3969/j.issn.2095-4344.0075

Abstract

Abstract:

BACKGROUND: Recently, tissue-engineered biomaterials for annulus fibrosus repair in the treatment of intervertebral disc degeneration have aroused wide attentions.

OBJECTIVE: To investigate the research progress in biomaterials for the repair of annulus fibrosus in intervertebral disc tissue engineering.

METHODS: PubMed database (1991-2017) was retrieved by the first author with the key words of “intervertebral disc, annulus fibrosus, material, scaffold” to search relevant articles about the use of biomaterials in annulus fibrosus repair.

RESULTS AND CONCLUSION: Biomaterials play important roles in annulus fibrosus repair, which mainly function to recover the physical structure and mechanical function of the annulus fibrosus by promoting extracellular matrix secretion and tissue regeneration. Current materials mainly used for the repair of annulus fibrosus include natural materials, polymer materials, and biomaterials. Natural materials have good biocompatibility, biodegradability and no cytotoxicity, but their mechanical strength is poor. Polymer materials which overcome the lack of mechanical strength have repeatability, controllability, no immunogenicity, and are easy to be processed, but they have poor biocompatibility and cell affinity, as compared with the natural materials. Therefore, the selection of composite materials by integrating the advantages of different materials becomes the main trend in the annulus fibrosus repair.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Biocompatible Materials, Intervertebral Disk, Tissue Engineering

CLC Number:

R318

Zhang Lei, Zhou Song, Cheng Bao-chang. Actuality and challenge of biomaterials in annulus fibrosus repair[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(6): 958-963.

Figures/Tables 1

2.1 纤维环的结构特点纤维环主要由内纤维环和外纤维环两部分构成，内层主要由类软骨细胞及其分泌的Ⅱ型胶原组成，承受椎间盘的压力；外层主要由类成纤维细胞及其分泌的Ⅰ型胶原和纤维蛋白组成，维持纤维环的张力[13-14]。纤维环在椎间盘内受到机械负荷的影响，其结构特点有利于纤维环承受均匀分布的压力，避免椎间盘纤维环因局部压力过大而导致破损[15]。纤维在椎间盘内排列呈同心圆状，胶原纤维在椎体间呈斜行排列，各层纤维环之间的角度约为60°，这些独特的构造使纤维环形成特殊的交叉网状结构，使其具有强大的抗拉伸能力[14-16]，能防止髓核向外突出，对维持椎间盘的稳定性起重要作用。然而，内层纤维环胶原蛋白表达的减少，将降低椎间盘对外界机械负荷的承受能力；而纤维环中蛋白聚糖的损失将影响纤维环细胞的功能，进一步引起椎间盘纤维变性，降低椎间盘的高度，加快椎间盘的退变[17]。纤维环是维持椎间盘正常生理功能的重要结构，椎间盘的退变过程也伴随着纤维环结构和组分的改变，纤维环退变也同样加快了椎间盘的退变进程。因此，构建具有生物学功能的材料以修复损伤的纤维环，对于椎间盘退变的治疗有重要意义[18-19]。此外，由于退变及椎间盘内压力等微环境引起的纤维环组织损伤(如纤维层分离、纤维环断裂等)是引起整个椎间盘退变的重要原因[8]。近年来，组织工程学的不断发展给学者们带来希望，生物材料在组织器官修复再生方面具有突出的优点，通过修复损伤的纤维环以修复退变椎间盘受到了广泛关注。 2.2 生物材料修复纤维环生物材料在纤维环组织工程中扮演着重要作用，主要通过促进细胞外基质的产生和组织再生，以恢复纤维环的力学功能和结构的完整性[20]，因此，作为纤维环修复的材料应具备一些基本要求[15，19-22]：①低免疫原性：材料在人体内不会产生明显的免疫排斥反应；②生物相容性：将生物材料植入椎间盘后，对外源性细胞及体内组织无毒害作用；③适当的孔隙：便于种子细胞在其内部生长并分泌细胞外基质；④生物可降解性：在体内可降解，并且降解速率与组织的再生速率相匹配；⑤良好的力学性能：在椎间盘的压力下，能保持材料的稳定性。目前，用于纤维环修复的生物材料种类繁多，根据材料本身的来源可分为：一是天然材料，包括丝素蛋白、透明质酸、胶原、藻酸盐等；二是合成高分子材料，包括聚氨酯、聚三亚甲基碳酸酯、聚己内酯、聚乙二醇、聚乳酸-乙醇酸共聚物等[19]；三是生物组织材料，如来源于椎间盘纤维环的脱细胞基质材料、小肠黏膜脱细胞基质材料等(表1)。 2.2.1 天然材料由于天然材料具有良好的生物相容性、对细胞无明显毒性等优点，在纤维环组织工程中被广泛利用，目前已报道的天然材料包括丝素蛋白、纤维蛋白、藻酸盐、胶原、透明质酸等。丝素蛋白作为一种天然材料，具有力学强度高、生物降解速率可控、生物相容性好和结构稳定等特点，其不仅能独立用于组织工程修复纤维环，也可与其他生物材料联用构建生物材料，因此在纤维环组织工程中应用前景广阔。Park等[23]根据纤维环的板层状特点，将丝素蛋白制作成与椎间盘纤维环结构相似的支架材料，将纤维环细胞接种于材料中培养2周，结果显示纤维环细胞能在板层状支架材料中良好生长，并且能较好地分泌细胞外基质。Bhattacharjee等[24]采用蚕丝构建与纤维环结构相似的薄层状纤维支架，通过与硫酸软骨素交联，增强生物材料的力学性能，在修复纤维环时，使其能在椎间盘压力微环境下保持材料的稳定性，此外，将其与软骨细胞共培养后，能显著分泌与纤维环组织相似的细胞基质。这些研究显示，丝素蛋白可能是一种理想的纤维环组织工程支架生物材料。由于纤维蛋白生物材料良好的生物相容性、有利于细胞合成细胞外基质及蛋白聚糖等特点，被认为是椎间盘组织工程中理想的生物材料[25-26]。Colombini等[26]将纤维蛋白与胶原合成可注射性的纤维蛋白水凝胶，将纤维环细胞种植于凝胶后植入裸鼠皮下，结果显示与植入前相比，纤维环细胞能在材料内长期存在并合成大量的细胞外基质及与天然纤维软骨组织相似的Ⅰ型胶原。 Guillaume等[27]制作了一种形状记忆藻酸盐-胶原复合材料，该材料具有较好的弹性性能，在外界压力下，能快速恢复其原有形状，此外，纤维环细胞在材料中具有良好的生物活性，并且能很好地黏附于材料中并迁移。体内实验显示，间充质干细胞在材料中也能分泌大量细胞外基质，修复缺损的纤维环。Fuller等[28]制作了一种透明质酸寡糖，体外研究显示该材料对椎间盘细胞无毒性作用，并且能下调MMP13、ADAMTS1、ADAMTS4等的表达，这种改变有望清除缺损部位的瘢痕组织，有利于细胞外基质的形成，羊纤维环损伤的体内实验也证实其能促进椎间盘纤维环的修复。 Grunert等[29]制作了一种含有核黄素的高密度胶原凝胶材料，该材料具有较好的刚性和渗透性能，而核黄素能显著提高材料的这种特性，发现纤维原细胞在水凝胶内逐渐形成纤维帽，以修复缺损的纤维环；动物体内研究同样显示，该材料能更好地维持髓核的结构，增强椎间盘内蛋白聚糖的水合作用，保证盘内的渗透性，抑制椎间盘退变的进展。Borde等[30]同样制备了一种可注射型的高密度胶原凝胶材料，注入鼠纤维环缺损处取得了理想的修复效果。由于京尼平交联剂能显著增强材料的力学性能，被广泛用于椎间盘组织工程中[31]。Schek等[32]将京尼平交联的纤维蛋白凝胶材料用于修复纤维环，结果显示在超负荷应力下，细胞也能在其表面较好地生长，但是，纤维环细胞在京尼平交联纤维蛋白凝胶材料中生长缓慢，这提示该材料可能更适用于修复较小纤维环缺口，或者用于修复大纤维环缺口的附加手段。Long等[33]采用纤维蛋白-京尼平水凝胶作为修复牛纤维环缺损模型的黏合剂，获得了较理想的结果，此外，其还能恢复椎间盘的生物力学强度，降低椎间盘突出风险。 2.2.2 合成高分子材料合成高分子材料具有较好的可塑性，可克服天然材料力学性能差的不足，同时还具有可控性好、无免疫原性且易于加工等优点。 Wismer等[34]采用静电纺丝技术构建有方向性和无方向性的聚氨酯静电纺丝支架，结果显示，有方向性静电纺丝支架生物材料能更好地促进细胞黏附，维持细胞表型，促进胶原、蛋白多糖等细胞外基质的沉积，此外，其良好的力学强度使其在椎间盘压力微环境下可保持材料的稳定性，有望成为修复纤维环理想的生物材料。Liu等[35]同样证实了有序静电纺丝生物材料能为纤维环来源干细胞分化为纤维环细胞提供良好的微环境，还能更多地分泌细胞外基质。 Turner等[36]通过静电纺丝技术制备了聚氨酯纳米纤维静电纺丝支架，将其与纤维环细胞共培养，结果显示纤维环细胞能在聚氨酯纳米纤维静电纺丝支架中较好地增殖及合成胶原，而外界拉力能抑制纤维环细胞的细胞学行为，这种抑制作用与材料本身的弹性模量有关。Iu等[37]将内层纤维环细胞和外层纤维环细胞分别接种于聚氨酯支架生物材料内，通过测定并对比多功能蛋白聚糖和COL1A1等基因的表达水平，发现内层纤维环细胞高表达 COL2A1、聚集蛋白聚糖、多功能蛋白聚糖基因，而COL1A1基因低表达，此外，与外层纤维环细胞相比，内层纤维环细胞能合成大量的胶原和蛋白聚糖，该研究认为聚氨酯支架生物材料可维持内层纤维环细胞的表型，该研究可能对未来纤维环组织工程中选择合适的种子细胞修复纤维环具有重要意义。 Zhu等[38]制作了一种6聚氨酯静电纺丝支架材料，将纤维环来源的干细胞种植于材料内，结果显示，纤维环干细胞能很好地贴附于生物材料上，并且分泌大量细胞外基质，随着时间的推移，细胞增殖也越来越多。此外，该研究还显示，在不同弹性模量的聚氨酯静电纺丝生物材料中接种纤维环干细胞后，细胞的基质组成、基因表达和细胞力学方面受到该生物材料的弹性模量调控，并有着向纤维环结构所特有的细胞类型分化的趋势，这为椎间盘纤维环修复提供了新的研究思路与方向。他们还发现，随着该材料刚度的增加，骨髓间充质干细胞在聚氨酯静电纺丝材料中Ⅰ型胶原的表达也增加，而Ⅱ型胶原和蛋白聚糖则呈现相反的趋势[39]。 Pirvu等[40]同样制备了一种聚三亚甲基碳酸酯支架生物材料，该材料表面给予聚氨酯包被，将其移植入破损的纤维环中，结果显示在一定的压力下，其在椎间盘内能保持材料的稳定性，阻止髓核突出，恢复并维持椎间盘的高度，体外也证实其对间充质干细胞无细胞毒性，具有较好的生物相容性，间充质干细胞在材料中同样能分泌细胞外基质，以进一步修复纤维环。Blanquer等将[41]脂肪干细胞接种于聚三亚甲基碳酸酯生物材料中，再加入转化生长因子β3，证实了脂肪干细胞能在生物材料中更好地向纤维环组织方向分化，并且分泌大量胶原蛋白，显示出用于纤维环修复的巨大潜力。 Van Uden等[42]介绍了聚己内酯用于纤维环组织工程的能力，它模拟椎间盘的生理结构及纤维环组织的各向异性，对纤维环细胞无毒害作用，具有良好的细胞相容性，同时可获得比人椎间盘更好的力学性能。Nerurkar等[43]制备了一种纳米纤维聚己内酯生物材料，能模拟纤维环结构的非线性和各向异性特点，有利于纤维环细胞和干细胞的生长、增殖并分泌细胞外基质，增强材料的力学性能。 Jeong等[44]将透明质酸和聚乙二醇制备成复合水凝胶生物材料，结果显示纤维环细胞能在材料内大量增殖，并且分泌细胞外基质和蛋白聚糖产物。由于聚乙酸合成高分子材料具有良好生物相容性和可降解等特性，引起了广大研究者的兴趣[45-47]，Xin等[48]制作了一种聚乳酸-乙醇酸共聚物支架材料，将其植入兔椎间盘纤维环缺损模型中，结果显示该材料能显著修复缺损的纤维环，恢复椎间盘的高度，展现出其修复纤维环的潜力。从上述研究可看出，合成高分子材料在纤维环修复中具有较广阔的应用前景和空间，但合成高分子材料仍有一些不足，如缺少生物活性、细胞亲和力差等。 2.2.3 生物组织材料脱细胞基质材料是通过物理和化学等方法去除组织中的细胞成分，保留以细胞外基质为主的成分，不仅维持了组织的生物学功能和力学性能，还能显著降低生物材料的免疫原性，具有良好的生物相容性，有利于各种细胞在生物材料中良好的增殖、分化，促进组织再生，可作为生物组织工程的支架材料[49]。此外，脱细胞基质还可为细胞提供接近其体内生长的微环境。 Xu等[50]分别采用Triton X-100、十二烷基硫酸钠及胰蛋白酶对猪来源纤维环进行去细胞化，制作脱细胞基质材料，结果显示这3种方式制作的脱细胞基质材料对细胞均无毒性作用，纤维环细胞在能在脱细胞材料中生长良好，展示了脱细胞基质材料良好的生物相容性。然而，经十二烷基硫酸钠和胰蛋白酶方法处理后，纤维环原有的同心圆排列结构被破坏，而采用Triton X-100方法制作的脱细胞基质材料能保留主要的细胞外基质及纤维环薄层状结构，其力学强度也明显优于十二烷基硫酸钠和胰蛋白酶处理组。具有良好生物相容性脱细胞基质材料，很有可能成为修复纤维环的理想生物材料。McGuire等[51]将猪来源心包制作成与纤维环结构相似的多层结构脱细胞基质材料，结果显示该材料具有良好的抗拉伸能力，其弹性模量与人纤维环相似，将纤维环细胞种于材料后，细胞能在材料内正常增殖和生长，并且分泌细胞外基质。Le Visage等[52]用猪来源小肠黏膜下层制备了一种脱细胞基质材料，将纤维环细胞接种于材料后，细胞生长良好，细胞外基质分泌增加，蛋白聚糖、胶原、Sox-9 基因显著表达。但脱细胞基质力学性能较差，同时还受制于生物学变异情况，如依赖宿主年龄、组织来源和处理具体方法，仍不能单独作为纤维环组织工程支架材料，限制了其在纤维环组织工程中的应用，因而需要适宜的材料加强其力学性能。 "

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