Co-culture of osteochondral explants and synovial membrane as in vitro model for osteoarthritis

The purpose of the current study was to establish an in vitro model for osteoarthritis (OA) by co-culture of osteochondral and synovial membrane explants. Osteochondral explants were cultured alone (control-1) or in co-culture with synovial membrane explants (control-2) in standard culture medium or with interleukin-1β (IL1β) and tumor necrosis factor (TNFα) added to the culture medium (OA-model-1 = osteochondral explant; OA-model-2 = osteochondroal-synovial explant). In addition, in OA-model groups a 2-mm partial-thickness defect was created in the centre of the cartilage explant. Changes in the expression of extracellular matrix (ECM) genes (collagen type-1 (Col1), Col2, Col10 and aggrecan) as well as presence and quantity of inflammatory marker genes (IL6, matrix metalloproteinase-1 (MMP1), MMP3, MMP13, a disintegrin and metalloproteinase with-thrombospondin-motif-5 (ADAMTS5) were analysed by immunohistochemistry, qPCR and ELISA. To monitor the activity of classically-activated pro-inflammatory (M1) versus alternatively-activated anti-inflammatory/repair (M2) synovial macrophages, the nitric oxide/urea ratio in the supernatant of osteochondral-synovial explant co-cultures was determined. In both OA-model groups immunohistochemistry and qPCR showed a significantly increased expression of MMPs and IL6 compared to their respective control group. ELISA results confirmed a statistically significant increase in MMP1and MMP3 production over the culturing period. In the osteochondral-synovial explant co-culture OA-model the nitric oxide/urea ratio was increased compared to the control group, indicating a shift toward M1 synovial macrophages. In summary, chemical damage (TNFα, IL1β) in combination with a partial-thickness cartilage defect elicits an inflammatory response similar to naturally occurring OA in osteochondral explants with and without osteochondral-synovial explant co-cultures and OA-model-2 showing a closer approximation of OA due to the additional shift of synovial macrophages toward the pro-inflammatory M1 phenotype.本研究旨在通过骨软骨与滑膜外植体共培养，构建骨关节炎（osteoarthritis, OA）体外模型。实验设置如下：骨软骨外植体单独培养（对照组1），或与滑膜外植体共培养（对照组2），培养介质分别为标准培养基，或添加白细胞介素-1β（interleukin-1β, IL1β）与肿瘤坏死因子-α（tumor necrosis factor, TNFα）的培养基；本研究的OA模型分为两组，OA模型1（OA-model-1）为单独培养的骨软骨外植体，OA模型2（OA-model-2）为骨软骨-滑膜共培养外植体，且在两组OA模型的软骨外植体中央制作2mm部分厚度软骨缺损。本研究通过免疫组织化学、实时荧光定量PCR（qPCR）与酶联免疫吸附测定（ELISA），分析细胞外基质（extracellular matrix, ECM）基因（Ⅰ型胶原（collagen type-1, Col1）、Ⅱ型胶原（Col2）、Ⅹ型胶原（Col10）与聚集蛋白聚糖（aggrecan））的表达变化，以及炎症标记基因（IL6、基质金属蛋白酶-1（matrix metalloproteinase-1, MMP1）、MMP3、MMP13、含血小板反应蛋白基序的解聚素样金属蛋白酶5（ADAMTS5））的存在水平与表达量；为监测经典激活的促炎型（M1）与交替激活的抗炎/修复型（M2）滑膜巨噬细胞的活性，本研究还测定了骨软骨-滑膜共培养外植体上清液中的一氧化氮/尿素比值。实验结果显示，免疫组织化学与qPCR检测表明，两组OA模型的MMP家族与IL6的表达量均显著高于各自对应的对照组；ELISA结果证实，培养周期内MMP1与MMP3的分泌量存在统计学意义的升高；在骨软骨-滑膜共培养的OA模型2组中，一氧化氮/尿素比值较对照组升高，提示滑膜巨噬细胞向促炎M1表型转化。综上，联合化学损伤（TNFα、IL1β）与部分厚度软骨缺损，可在单独或联合滑膜外植体共培养的骨软骨外植体中诱发类似自然发生的骨关节炎的炎症反应；其中OA模型2组因滑膜巨噬细胞进一步向促炎M1表型转化，其骨关节炎特征更贴近天然病变。