Application of chitosan hydrogels in cartilage repair

doi:10.3969/j.issn.2095-4344.1620

Abstract

Abstract:

BACKGROUND: Chitosan has become a research hotspot of cartilage repair materials because of a wide range of biological sources, simple preparation processes and excellent biocompatibility.

OBJECTIVE: To review the characteristics of chitosan hydrogels and their application in cartilage repair.

METHODS: PubMed and CNKI databases were searched by computer with the keywords of “chitosan; chitosan hydrogel; cartilage repair; tissue engineering” in English and Chinese, respectively. The time range was from January 2000 to March 2018. After initial screening, the retained articles were further analyzed, concluded and summarized.

RESULTS AND CONCLUSION: Chitosan has good biocompatibility, low toxicity, low immunogenicity, and can load multiple growth factors and seed cells with different physical or chemical improvements. Encouraging progress in tissue engineering cartilage repair has been achieved. With the development of technology and the understanding of cartilage and subchondral bone structure, the application range of chitosan biomimetic hydrogel has been broadened. However, the molecular mechanism and degradation process of chitosan in vivo still have a lot of controversies, and further research and exploration are needed.

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: Chitosan;, Hydrogels, Cartilage, Articular, Tissue Engineering

CLC Number:

R459.9

R318.08

Yang Lingjian, Li Yanlin, Jia Di, He Yinghong, Xiang Yaoyu. Application of chitosan hydrogels in cartilage repair[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(10): 1611-1616.

Figures/Tables 1

壳聚糖复合水凝胶主要有智能体系壳聚糖水凝胶、种子细胞-壳聚糖体系水凝胶、生长因子-壳聚糖体系水凝胶、基因-壳聚糖体系水凝胶、基质-壳聚糖体系水凝胶、3D打印仿生壳聚糖体系水凝胶，各类壳聚糖复合水凝胶体系的优点及不足，见表1。 2.1 智能体系壳聚糖水凝胶智能型壳聚糖水凝胶是一类对外界物理/化学环境变化敏感、随着环境变化而自身理化性质发生改变的智能组织工程材料[9]，大致可分为：pH敏感型、温度敏感型、电场敏感型、光敏感型、磁场敏感型和多重敏感型。智能型壳聚糖水凝胶具有以下优点：①可模拟细胞外基质环境，给组织修复提供支架和细胞生长微环境[2]；②以半溶液性质载药，药物在局部温度的影响下形成水凝胶，靶向治疗且避免药物过快流失；③与不同化合物通过物理/化学方法结合，构成三维网状交联结构，形成新兴组织工程修复支架等[10]。温敏型、磁场敏感型水凝胶在关节软骨修复中被广泛研究，其中温敏型水凝胶尤为突出，其余智能型水凝胶在软骨修复中尚未报道。 2.1.1 温敏型壳聚糖水凝胶 Chenite等[11]最早发现单独壳聚糖溶液不具备温敏性，而在β-甘油磷酸钠干预下，壳聚糖/β-甘油磷酸钠溶液在一定温度时发生凝胶化。其原理可能为壳聚糖/β-甘油磷酸钠溶液在达到临界温度时，壳聚糖分子上氨基发生去质子化，分子间静电作用降低，同时分离的质子与β-甘油磷酸钠羟基相结合，疏水基团凝聚形成分子内氢键，发生凝胶化[12]。由于壳聚糖/β-甘油磷酸钠温敏性凝胶依靠分子内氢键及分子间作用力相连接，故存在其稳定性欠佳、容易快速降解及药物半衰期短等缺点[13]。Song等[14]将壳聚糖/β-甘油磷酸酯/明胶混合构建水凝胶，当其处于生理体温37 ℃时发生凝胶化，扫描电镜发现水凝胶结构稳定；体外联合培养脂肪干细胞并诱导成软骨分化，干细胞在5 d内表现高生存力形态；体内实验显示，诱导分化细胞在凝胶支架材料维持下可增殖更多同种细胞，并且细胞外基质产生一定血管化。虽然此类温敏性凝胶结构稳定，但机械性能较差，抗阻抗旋转等方面有所欠缺，未来研究可进一步改善。 Deng等[15]利用NaHCO3作为偶联剂，发现随着NaHCO3浓度的增加，水凝胶凝胶化时间缩短，机械强度增强，但高渗性溶液会引起细胞溶解。若将150 mL 50%β-甘油磷酸钠溶于0.4 mol/L NaHCO3与壳聚糖溶液中制备温敏性凝胶，可缩短凝胶化时间，并且热稳定性和机械强度符合人体膝关节内负荷，同时可将细胞毒性降至最低，扫描电镜观察水凝胶呈现不规则粗糙多孔形态，有利于细胞的附着和生长，为进一步研究提供了借鉴。随着研究的不断推进，壳聚糖温敏水凝胶有望制备成为可注射型试剂，在体外物理诱导条件下将药物局部定点传递至病变部位，在减轻全身药物毒性反应的同时也达到了精准医疗，取得更加有效的治疗效果。 2.1.2 磁场敏感型凝胶 Su等[16]在磁场作用下使氧化铁磁性纳米颗粒标记的软骨细胞均匀分布于聚乳酸-羟基乙酸共聚物与壳聚糖-Ⅱ型胶原双层支架，细胞支架染色证实支架材料逐渐转变为软骨组织，软骨外基质填充支架空隙中，RT-qPCR也证实了特异基因的表达。该水凝胶运用磁场达到种子细胞的移动性，使负载的细胞均匀覆盖损伤部位，仿生双层结构上层的细胞在适宜环境下短时间内促进软骨细胞增殖，为分泌的细胞外基质提供黏附点，同时为再生血管化提供条件，下层聚乳酸-羟基乙酸共聚物层则成为软骨细胞迁移的过渡区，为整个支架结构的移植提供机械稳定性。但目前磁铁矿纳米粒子的体内相容性尚不明确，还需进一步研究。 2.2 种子细胞-壳聚糖体系水凝胶种子细胞作为组织工程重要组成部分之一，同种软骨细胞获取简单，且同种自体具有低免疫原性风险。Chen等[17-18]将兔自体软骨细胞封装于乙二醇壳聚糖水凝胶支架内，用于修复兔膝关节软骨缺损，发现体外维持软骨细胞形态并使细胞增殖超过21 d，植入体内后，水凝胶为携带的软骨细胞分泌细胞外基质提供附着，缺损部位Ⅱ型胶原蛋白呈高表达，软骨缺损明显改善。但从自体非负重区可取软骨有限，体外扩增自体软骨移植在短期内需进行2次手术治疗，对患者的经济及身心压力产生了较大考验，极大限制了该手术的临床应用[19]。Man等[20]运用去矿化骨基质-壳聚糖水凝胶支架搭载同种异体软骨细胞体内修复兔软骨损伤，术后24周，支架组未见明显炎症反应，支架具备良好组织相容性，组织学评分明显优于对照组，MRI显示支架组软骨缺损被成功填充，去矿化骨基质给予水凝胶稳定的支架结构，壳聚糖为搭载种子细胞提供了良好的黏附环境，有效修复了软骨缺损。但同种异体细胞可能存在免疫排斥反应和疾病传播风险[21]。无论自体或异体软骨细胞负载，软骨细胞在体外扩增培养细胞过程中存在去分化现象，可能失去原有细胞的形态及功能[8]。自体和异体软骨细胞移植受到来源及运用的限制，间充质干细胞、脂肪源性干细胞逐渐成为再生医学中使用最广泛的干细胞[22-23]，因为它们具有丰富的细胞来源和低免疫原性，畸胎瘤风险极小，在三维环境下接受特定物理刺激或化学诱导具有增殖、分化成软骨细胞的能力[24-26]。Lu等[27]将间充质干细胞搭载于壳聚糖膜并诱导其成软骨分化，在染色鉴定下证实中等分子质量(190-310 kDa)的壳聚糖薄膜更适于诱导间充质干细胞向软骨分化，分泌软骨形成蛋白及软骨内标记呈现高表达。此外，Zhu等[28]模拟关节运动剪切力，在壳聚糖/明胶复合水凝胶支架中种植人脂肪源性干细胞，运用旋转壁容器生物反应器干预，动态与静态培养构建工程软骨仿生材料，结果证实在诱导培养基动态培养下，脂肪源性干细胞呈现软骨细胞外基质广泛表达，有效促进了脂肪源性干细胞的增殖及成软骨分化。随着体外研究成果的进一步推进，有研究在兔软骨缺损体内植入负载骨髓间充质干细胞的聚乙烯醇/壳聚糖复合水凝胶，验证得到复合支架材料体外细胞相容性良好，体内对兔软骨缺损有明显的修复效果，但可能由于体内观察时间较短和植入材料过少的缘故，软骨缺损未完全修复。林涛等[29]运用复合兔脂肪源性干细胞及肋软骨细胞的壳聚糖水凝胶修复兔膝关节软骨缺损，发现双细胞支架组在12周时达到完全修复且缺损边界模糊，再生组织富含Ⅱ型胶原蛋白，达到透明软骨修复的效果。但干细胞存在多向分化能力，存在诱发体内肿瘤发病的潜在风险，如何定向诱导干细胞成软骨及成骨分化还需进一步的深入研究。 2.3 生长因子-壳聚糖体系水凝胶干细胞是软骨再生的起点，但必须将干细胞诱导分化形成软骨组织，可最终达到形成透明软骨。大量实验证明，转化生长因子β、胰岛素样生长因子、骨形态发生蛋白和地塞米松等是最广泛用于刺激干细胞软骨形成、成骨分化的活性因子与激素[30-33]。其中生长转化因子β是一种具有成熟二聚体，的25 kDa多肽[34]。大量体内外实验证明，活化的转化生长因子β信号通路是胚胎发生软骨形成的驱动因素，不同来源干细胞在与活化转化生长因子β共培养下可形成软骨组织[31-35]。Merlin Rajesh Lal等[36]将壳聚糖的细胞黏附性和琼脂糖优良的机械稳定性相结合，采用冷冻干燥合成了壳聚糖-琼脂糖凝胶，封装人脐带华通胶间充质干细胞后，在双生长因子骨形态发生蛋白2、转化生长因子β3诱导下成功向软骨分化，壳聚糖-琼脂糖凝胶的低免疫原性可更好地支持同种异体人脐带华通胶间充质干细胞分化，双因子支架对人脐带华通胶间充质干细胞的诱导分化较单纯支架更为显著，软骨标志物COL2A1、ACAN、SOX6和SOX9均呈高表达。但无论骨形态发生蛋白2或转化生长因子β3，体外制备复杂、昂贵且体外保存稳定性较差，使其临床运用受到一定限制。富血小板血浆来源于自体血小板浓缩物，分离与提取快捷方便，而血小板在凝血酶或CaCl2作用下活化形成α纯富血小板血浆，后者含有多种生长因子(转化生长因子β、胰岛素样生长因子或血管内皮生长因子)[37-39]。Sancho-Tello等[40]在稳定多孔壳聚糖上结合富血小板血浆，结果显示与空载支架对比，富血小板血浆负载壳聚糖支架在多重生长因子及稳定纤维网络影响下可促使软骨细胞Ⅱ型胶原蛋白高表达，且电镜扫描观察到富血小板血浆诱导分化软骨细胞与正常软骨细胞形态结构相一致，为软骨修复提供了新的思考方向。 2.4 基因-壳聚糖体系水凝胶生长因子对加速、诱导细胞增殖有不可或缺的作用，研究者逐渐将目光转移到转染生长因子，提高生长因子生物效力，从基因分子机制层面来增强软骨修复效率的研究中。赵荣兰等[41-42]研究发现，壳聚糖/胰岛素样生长因子基因复合物在体外能促进间充质干细胞的增殖速度并向软骨分化。随后的兔体内实验中，运用荧光定量PCR对修复部位基因进行定量检测，发现壳聚糖/胰岛素样生长因子基因干预组Ⅱ型胶原、聚集蛋白聚糖基因均呈上调趋势，组织修复效果明显高于对照组。此外，该课题组还构建了磷酸化短肽偶联壳聚糖基因载体，负载胰岛素样生长因子1和白细胞介素1受体拮抗剂双基因，用于修复兔软骨损伤，验证得到双基因在软骨修复过程中存在协同作用，聚集蛋白聚糖无论基因或蛋白层面均呈现高表达，组织学染色中发现软骨基质的分解代谢被抑制，合成代谢被增强[43-44]。 2.5 基质-壳聚糖体系水凝胶种子细胞、生长因子的负载存在一定潜在风险，广大学者开始探索运用软骨细胞外基质类似物制作脱细胞支架，从而规避疾病传播、诱发潜在畸胎瘤等临床风险，同时为原有组织增殖、黏附提供骨架材料[45]。Sivandzade等[46]在电喷雾技术下将人软骨组织脱细胞细胞外基质包封于壳聚糖水凝胶中，制备缓释微球，1%细胞外基质：2%壳聚糖的缓释微球在孔径、密度、孔隙率和机械性质方面表现出独特优势，此外在体外动态与静态共培养原代人软骨细胞对比中发现，动态培养下1%细胞外基质缓释微球具备更佳的细胞迁移、增殖等能力，能达到最大的细胞附着，为后期软骨修复支架材料提供参考。 2.6 3D打印仿生壳聚糖体系水凝胶随着对软骨组织结构了解的不断深入，3D打印技术突飞猛进，模拟软骨结构修复支架材料的理念进入学者们的视野[7，47]。Reed等[48]应用3D打印技术模拟软骨及软骨下骨结构，将天然关节软骨细胞外基质糖胺聚糖类似多糖与壳聚糖-藻酸盐定向冷冻制备为多微孔无细胞支架，该支架实现分层模拟关节软骨分层体系，有助于摄取内源性骨髓、再生细胞迁移分布和新生软骨血管化。Wang等[49]利用壳聚糖改性多糖-羟丁基壳聚糖，通过控制其表面羟丁基取代度调控多糖-羟丁基壳聚糖溶液临界温度溶胶-凝胶状态的转变，于10%NaCl溶液中，支架在73.2 kPa- 40 MPa下表现出优异的可调弹性和弹力回缩性，具有良好的生物降解性和细胞相容性。该水凝胶在3D打印技术支持下可制得具有强机械性能和精细结构的25层支架材料，有望应用于软骨组织的修复。"

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