脱碳软骨衍生基质支架的制备
Summary
脱形软骨衍生支架可用作指导软骨修复的支架, 也可用作骨软骨组织再生的手段。本文详细介绍了脱髓化过程, 并对在体外环境中使用这些支架提出了建议。
Abstract
骨软骨缺损缺乏足够的内在修复能力来再生功能健全的骨骼和软骨组织。在这方面, 软骨研究的重点是再生支架的开发。本文介绍了支架的发展, 这是完全来自天然软骨细胞外基质, 来自一个马供体。支架的潜在应用包括生产用于软骨修复的同种异体移植, 作为骨软骨组织工程的支架, 以及提供研究组织形成的体外模型。通过去细胞化组织, 供体细胞被切除, 但许多天然的生物活性线索被认为是被保留的。与合成支架相比, 使用这种天然支架的主要优点是, 不需要聚合物的进一步功能化来推动骨软骨组织再生。软骨衍生基质支架可用于体内和体外组织的骨和软骨组织再生。
Introduction
创伤事件引起的膝关节关节软骨缺损会导致不适, 最重要的是会对年轻和活跃人群的生活产生很大的影响 1,2,3。此外, 年轻时的软骨损伤可能会导致以后更迅速地开始骨关节炎 4.目前, 对全身性膝关节骨关节炎的唯一抢救疗法是关节置换术。由于软骨是一种下细胞、听觉和血管组织, 其再生能力受到严重限制。因此, 我们寻求再生医学的方法来帮助和刺激原生组织的再生能力。为此, 支架被设计并用作细胞载体或诱导材料, 通过人体的原生细胞促进组织的分化和再生5。
脱光支架在再生医学中得到了广泛的研究。例如, 它在帮助皮肤7、腹部结构8和肌腱9的再生方面取得了一些成功。使用脱细胞支架的优点是它们的自然来源和保留生物活性线索的能力, 这些线索既能吸引和诱导细胞分化, 也能诱导细胞分化为组织修复所需的适当谱系 6,10.此外, 由于细胞外基质 (ecm) 是一种天然的生物材料, 去细胞化通过去除细胞或遗传含量来防止潜在的免疫反应, 从而克服了生物相容性和生物降解性方面的问题。
软骨衍生基质 (cdm) 支架在体外实验中显示出巨大的软骨生质潜力, 当使用间充质基质细胞 11播种时。此外, 这些支架还显示了在体内环境中通过软骨内骨化在异位形成骨组织的潜力.由于 cdm 支架引导骨和软骨组织的形成, 这些支架除了软骨修复外, 还可能具有骨软骨缺损修复的潜力。
本文介绍了杨等人 (2010年)13所采用的一种协议, 用于生产从马窒息软骨中脱病毒的 cdm 支架。这些支架富含 ii 型胶原蛋白, 没有细胞, 脱髓化后不含任何糖胺多糖 (gag)。在 (骨) 软骨缺损修复的体外和体内实验都可以使用这些支架进行。
Protocol
在这个协议中, 马窒息软骨是从死于骨关节炎以外的其他原因的马身上获得的。根据机构道德条例, 组织是经业主许可获得的。 请注意:该协议描述了脱细胞马软骨支架的制作, 可用于体外组织培养平台或体内植入再生医学策略等应用。酶处理步骤必须按所述的时间顺序进行。 1. 从供体 (尸体) 关节中收集关节软骨 在收获步…
Representative Results
cdm 支架的去序列化必须始终使用组织学标记以及 dna 定量来测量 dna 残留量来确认。去细胞化不足可能会导致不想要的免疫反应, 影响在体内的结果15,16,17。对于这种特定的去细胞化方法, dna 低于检测范围, 从 13.6±2.3 ng/mg dna/干重 (n = 3) 开始。使用此协议的完全去电化将导致一个支架的生产, 该支架富含 i…
Discussion
关节软骨的 ecm 是非常密集的, 对不同的酶处理有相当的弹性。本文描述的多步骤去电磁化协议解决了这种电阻问题, 并成功地生成了去电磁化矩阵。为了实现这一目标, 这一过程持续了几天。针对不同类型的组织18提出了许多脱髓化过程,本文介绍了一种适用于软骨去细胞化的方案。然而, 在这个协议中, 有必要遵循酶处理与洗涤剂步骤, 以消除所有细胞。dna 的数量在涉及胰蛋白酶?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者希望感谢 w. boot 在制作脚手架方面的帮助。benders 由大学医学中心的亚历山大·苏尔曼基金会提供支持。r. levato 和 j. malda 得到荷兰关节炎基金会的支持 (分别为 co-14-001 和 llp-12 赠款协议)。
Materials
| Cadaveric joint | This can be obtained as rest material from the local butcher or veterinary center. | ||
| Sterile phosphate-buffered saline (PBS) | |||
| Penicillin-Streptomycin | Gibco | 15140 | |
| Amphotericin B | Thermo Fischer Scientific | 15290026 | |
| Liquid nitrogen | |||
| Trypsin-EDTA (0.25%), phenol red | Thermo Fischer Scientific | 25200072 | |
| Tris-HCl pH 7.5 | |||
| Deoxyribonuclease I from bovine pancreas | Sigma-Aldrich | DN25 | |
| Ribonuclease A from bovine pancreas | Sigma-Aldrich | R6513 | |
| Triton X-100 (octoxynol-1) | Sigma-Aldrich | X100 | |
| Papain | Sigma-Aldrich | P3125 | |
| Trisodium citrate dihydrate | Sigma-Aldrich | S4641 | |
| Alginate | Sigma-Aldrich | 180947 | |
| Formalin | |||
| CaCl2 | |||
| Ethanol | |||
| Xylene | |||
| Paraffin | |||
| Ethylene oxide sterilization | Synergy Health, Venlo, the Netherlands | ||
| Multipotent Stromal cells/chondrocytes from equine donors | MSCs and chondrocytes can be isolated from donor joints that are rest material, coming from the local butcher or veterinary center. | ||
| MEM alpha | Thermo Fischer Scientific | 22561 | |
| L-ascorbic acid 2-phosphate | Sigma-Aldrich | A8960 | |
| DMEM | Thermo Fischer Scientific | 41965 | |
| Heat inactivated bovine serum albumin | Sigma-Aldrich | 10735086001 | |
| Fibroblast growth factor-2 (FGF-2) | R & D Systems | 233-FB | |
| DNA quantification kit (Quant-iT PicoGreen dsDNA Reagent) | Thermo Fischer Scientific | P7581 | |
| 1,9-Dimethyl-Methylene Blue zinc chloride double salt | Sigma-Aldrich | 341088 | |
| Freeze-dryer | SALMENKIPP | ALPHA 1-2 LD plus | |
| Analytical mill | IKA | A 11 basic | |
| mortar/pestle | Haldenwanger 55/0A | ||
| Roller plate | CAT | RM5 | |
| Centrifuge (for 50 mL tubes) | Eppendorf | 5810R | |
| Capsule (cylindric mold) | TAAB | 8 mm flat | |
| Superlight S UV | Lumatec | 2001AV | |
| Incubator | |||
| Microtome | |||
| Sieve (mesh size 0.71 mm) | VWR | 34111229 | |
| Scalpel | |||
| Scalpel holder | |||
| Small laddle |
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Citer Cet Article
Benders, K. E., Terpstra, M. L., Levato, R., Malda, J. Fabrication of Decellularized Cartilage-derived Matrix Scaffolds. J. Vis. Exp. (143), e58656, doi:10.3791/58656 (2019).