目的 建立猪关节软骨外植体模型，为药物递送修复骨关节炎关节软骨病损的作用提供一种简单经济的研究策略。 方法 体外无菌解剖分离猪膝关节软骨，使用活检组织穿孔器取直径为40 mm的小圆柱体。用无血清培养基培养外植体，荧光标记的Rhodamine B - Kartogenin（KGN）进行小分子药物渗透测试。药物渗透试验12 h、24 h、48 h，每个时间点设置3个重复组，获取外植体进行冰冻切片，利用激光共聚焦显微镜观察外植体的结构及其体外培养系统中小分子药物在软骨层的渗透情况，通过Image J分析荧光强度。 结果 激光共聚焦显微镜观察，软骨外植体无血清培养模型在体外12 h、24 h、48 h均可维持正常的形态结构，小分子药物Rhodamine B-KGN呈红色荧光，48 h组荧光信号明显增强（P<0.01），随着时间的推移，药物不断渗入软骨外植体，进入外植体的药物逐渐增多。 结论 本研究建立一种猪软骨外植体模型，检测了小分子化合物KGN在软骨外植体中的渗透分布情况，为探讨药物递送对骨关节炎软骨病损的修复作用提供一个简单经济的评价模型。
Abstract
Objective To establish a pig articular cartilage explant model and explore its role in drug delivery evaluation to repair osteoarthritis articular cartilage lesion, thus providing a simple and economical strategy for ex vivo study. Methods The pig knee joint cartilage was aseptically dissected ex vivo. The cylindric cartilage in diameter of 40 mm was taken using a biopsy tissue puncher. Then, the explants were cultured in a serum-free medium. The penetration study of small molecule compounds was performed using the Rhodamine B labeled- Kartogenin(KGN). After penetration for 12 h, 24 h, and 48 h, each time point was set up for three repeat groups , the explants were obtained for frozen section. The structure of the explants and the penetration process of small molecule drugs in the cartilage of explants were observed by laser confocal microscope, and the fluorescence intensity was analyzed by Image J. Results The cartilage explants can maintain the normal morphology and structure in the serum-free culture for 12 h, 24 h, and 48 h ex vivo. The small molecule drug Rhodamine B-KGN emitted red fluorescence under laser confocal microscope. The fluorescence signal in the 48 h group significantly enhanced (P<0.01). The amount and depth of drugs entrance into the explants were gradually increasing. Conclusions An evaluation model is successfully established to trace the penetration and distribution of the small molecule compound KGN in the cartilage explant. This study can provide a simple and economical model to evaluate the effect of drug delivery on osteoarthritis cartilage repair.
关键词
软骨外植体； /
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小分子药物递送； /
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药物渗透
Key words
Cartilage explant; /
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Small molecule drug delivery; /
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Drug penetration
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参考文献
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基金
国家自然科学基金（81972116；81972085；81772394）；广东省自然科学基金-重点项目（2018B0303110003）；广东省国际科技合作项目（2021A0505030011）；深圳市科创委科技计划项目（JCYJ20170817172023838；SGDX20201103095800003；GJHZ20200731095606019）；中国博士后科学基金（2020M682907）；广东省高水平医院建设专项资金
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