全层软骨缺损大鼠模型的开发与评价
1The First Affiliated Hospital,Zhejiang Chinese Medical University, 2Fuyang Orthopaedics and Traumatology Affiliated Hospital,Zhejiang Chinese Medical University, 3College of Pharmaceutical Science,Zhejiang Chinese Medical University, 4Affiliated Zhejiang Hospital,Zhejiang University School of Medicine, 5Xianju County Hospital of Chinese Medicine
Summary
该协议通过在大鼠的股骨滑车沟中钻孔并测量随后的疼痛行为和组织病理学变化来建立全层软骨缺陷（FTCD）模型。
Abstract
创伤引起的膝关节软骨缺损是临床上常见的运动关节损伤，这些缺损会导致关节疼痛、运动障碍，最终导致膝关节骨关节炎（kOA）。然而，对软骨缺陷甚至kOA的有效治疗很少。动物模型对于开发治疗药物很重要，但现有的软骨缺陷模型并不令人满意。本工作通过在大鼠股骨滑车槽钻孔建立了全层软骨缺损（FTCD）模型，并将后续的疼痛行为和组织病理学变化作为读数实验。术后机械戒断阈值降低，损伤部位软骨细胞丢失，基质金属蛋白酶MMP13表达升高，II.型胶原表达降低，与人软骨缺损中观察到的病理变化一致。这种方法操作简单易行，可以在损伤后立即进行粗略观察。此外，该模型可以成功模拟临床软骨缺损，从而为研究软骨缺损的病理过程和开发相应的治疗药物提供平台。
Introduction
关节软骨是由软骨细胞和细胞外基质组成的高度分化和致密的组织1。关节软骨的表层是透明软骨的一种形式，具有表面光滑，摩擦力低，强度和弹性好，机械应力耐受性优异2。细胞外基质由胶原蛋白蛋白聚糖和水组成，II型胶原蛋白是胶原蛋白的主要结构成分，约占胶原蛋白总量的90%3。由于软骨组织中不存在血管或神经，因此在受伤后缺乏自我修复的能力4。因此，创伤引起的软骨缺损一直是临床上顽固的关节疾病;此外，这种关节疾病往往会袭击年轻人，全球发病率正在上升5,6。膝关节是软骨缺损最常见的部位，这里的缺损伴有关节疼痛、关节功能障碍和关节软骨变性，最终导致膝关节骨关节炎（kOA）7。膝关节软骨缺损给患者带来经济和生理负担，严重影响患者的生活质量8.这种疾病构成了重大而紧迫的临床挑战，没有迫在眉睫的解决方案。目前，手术是治疗软骨缺损的主要手段，但其长期结果仍不令人满意9。
临床软骨缺陷最终导致kOA，因此kOA动物模型常用于软骨缺陷的病理研究和药物开发。动物模型的建立对于理解软骨缺损修复的病理生理过程具有重要意义，可用于观察软骨再生和纤维软骨与透明软骨之间的改变10。然而，常用的kOA动物模型，如前交叉韧带横断（ACLT）、内侧半月板不稳定（DMM）、卵巢切除术（OVX）和Hulth的手术模型，通常需要长期建模，并且只允许病理和疼痛评估，这限制了药物开发的效率11。除了手术模型外，化学模型，如单碘乙酸酯（MIA）和木瓜蛋白酶注射液，也会导致软骨缺陷，但缺陷的程度无法很好地控制，条件与临床现实相去甚远11。碰撞是模拟大型动物软骨缺陷的另一种方法，但这种方法取决于特定仪器的使用，很少应用12。
综上所述，现有的kOA模型对于研究软骨缺损的发病机制或开发新药并不理想，需要针对软骨缺损建立特异性和标准化的模型。本研究通过在大鼠股骨滑车槽中钻孔建立了全层软骨缺损（FTCD）模型。进行大体观察、疼痛行为测试和组织病理学分析以进行模型评估。与其他kOA动物模型不同，该模型对大鼠的一般状况几乎没有影响。这种建模方法是可访问的，可以很好地管理，并支持了解从软骨缺陷到kOA的进展以及有效疗法的开发。该模型还可用于测试通过治愈骨关节炎前关节缺陷来预防kOA的疗法。
Protocol
动物实验经浙江中医药大学医学标准伦理委员会批准，符合中国关于实验动物使用和护理的立法。在本研究中，使用体重150-180g的6周龄雄性Sprague-Dawley（SD）大鼠。这些动物是从商业来源获得的（见 材料表）。 1. 大鼠全层软骨缺损模型的建立 适应新环境1周后，将大鼠随机平均分为两组（n = 8只大鼠/组）。假手术组的大鼠将进行假手术，而模?…
Representative Results
本工作通过在股骨滑车槽上钻孔并检测后续疼痛行为和组织病理学变化，建立了FTCD大鼠模型。如图1所示，建模后3天，与假手术组相比，模型组大鼠MWT明显降低，提示FTCD引起的痛觉过敏。建模后17 d，模型组大鼠机械戒断阈值保持在较低水平，表明疼痛致敏性至少可持续17 d。组织病理学染色结果显示，假手术组关节软骨结构清晰，软骨表面完整，软骨细胞分布均匀，II.型胶?…
Discussion
本研究描述了一种通过在大鼠股骨滑车槽中钻孔来模拟临床软骨缺陷的动物模型（补充图1）。软骨损伤后，外周伤害感受器的兴奋性或反应性增强，这可能导致疼痛阈值降低和对刺激的反应性增强18。在临床前研究中，不同种类动物软骨缺陷的建模总是引起疼痛19。临床研究还表明，软骨损伤患者的疼痛视觉模拟量表（VAS）评分明显低?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了浙江省自然科学基金（批准号LQ20H270009）、中国自然科学基金（批准号82074464和82104890）、浙江省中医药科学基金（批准号2020ZA039、2020ZA096和2022ZB137）和浙江省卫生健康委员会医疗卫生科技项目（批准号2016KYA196）的支持。
Materials
| 3, 3 '-diaminobenzidine | Hangzhou Zhengbo Biotechnology Co., Ltd. | ZLI-9019 | The dye for IHC staining |
| Anti-Collagen III antibody | Novus | NB600-594 | Primary antibody for IHC |
| Anti-Collagen II antibody | Abcam (UK) | 34712 | Primary antibody for IHC |
| Anti-Collagen I antibody | Novus | NB600-408 | Primary antibody for IHC |
| Bouin solution | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
| Celestite blue | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
| Corncob paddings | Xiaohe Technology Co., Ltd | Bedding for animal | |
| Eosin | Sigma-Aldrich | 861006 | The dye for HE staining |
| Fast Green FCF | Sigma-Aldrich | F7252 | The dye for SO staining |
| Goat anti-mouse antibody | ZSGQ-BIO (Beijing, China) | PV-9002 | Secondary antibody for IHC |
| Goat anti-rabbit antibody | ZSGQ-BIO (Beijing, China) | PV-9001 | Secondary antibody for IHC |
| Hematoxylin | Sigma-Aldrich | H3163 | The dye for HE staining |
| Masson | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
| Microdrill | Rwd Life Science Co., Ltd | 78001 | Equipment for surgery |
| MMP13 | Cell Signaling Technology, Inc. (Danvers, MA, USA) | 69926 | Primary antibody for IHC |
| Modular tissue embedding center | Thermo Fisher Scientific (USA) | EC 350 | Produce paraffin blocks |
| Neutral resin | Hangzhou Zhengbo Biotechnology Co., Ltd. | ZLI-9555 | Seal for IHC |
| Nonabsorbable suture | Hangzhou Huawei Medical Supplies Co.,Ltd. | 4-0 | Equipment for surgery |
| Pentobarbital sodium | Hangzhou Zhengbo Biotechnology Co., Ltd. | WBBTN5G | Anesthetized animal |
| phosphomolybdic acid | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
| Ponceau fuchsin | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
| Rotary and Sliding Microtomes | Thermo Fisher Scientific (USA) | HM325 | Precise paraffin sections |
| Safranin-O | Sigma-Aldrich | S2255 | The dye for SO staining |
| Scalpel blade | Shanghai Lianhui Medical Supplies Co., Ltd. | 11 | Equipment for surgery |
| Sodium citrate solution (20x) | Hangzhou Haoke Biotechnology Co., Ltd. | HK1222 | Antigen retrieval for IHC |
| Sprague Dawley (SD) rats | Shanghai Slake Experimental Animal Co., Ltd. | SD | Experimental animal |
| Tissue-Tek VIP 5 Jr | Sakura (Japan) | Vacuum Infiltration Processor | |
| Toluidine Blue | Sigma-Aldrich | 89640 | The dye for TB staining |
| Von Frey filament | UGO Basile (Italy) | 37450-275 | Equipment for MWT assay |
| Wire mesh platform | Shanghai Yuyan Instruments Co.,Ltd. | Equipment for MWT assay |
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Cite This Article
Zhang, H., Bao, R., Xu, J., Ge, Y., Chen, Z., Fan, M., Yu, G., Zhou, L., Guo, L., Shan, L., Bao, H. Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects. J. Vis. Exp. (195), e64475, doi:10.3791/64475 (2023).