Cartilage regeneration is a priority in medicine for the treatment of osteoarthritis and isolated cartilage defects. Several molecules with potential for cartilage regeneration are under investigation. Unfortunately, in vitro chondrogenesis assays do not always predict the stability of the newly formed cartilage in vivo. Therefore, there is a need for a stringent, quantifiable assay to assess in vivo the capacity of molecules to promote the stable formation of cartilage that is resistant to calcification and endochondral bone formation. We developed an ectopic cartilage formation assay (ECFA) that enables one to assess the capacity of bioactive molecules to support cartilage formation in vivo using cartilage organoids. The ECFA predicted good clinical outcomes when used as a quality control for efficacy of chondrocyte preparations before implantation in patients with cartilage defects. In this assay, articular chondrocytes from human donors or animals are injected either intramuscularly or subcutaneously in nude mice. As early as 2 weeks later, cartilage organoids can be retrieved. The size of the implants and their degree of differentiation can be assessed by histomorphometry, immunostainings of molecular markers and real-time PCR. Mineralization can be assessed by micro-computed tomography or by staining. The effects of molecules on cartilage formation can be tested following the systemic administration of the molecule in mice previously injected with chondrocytes, or after co-injection of chondrocytes with cell lines overexpressing and secreting the protein of interest. Here we describe the ECFA procedure, including steps for harvesting human and bovine articular cartilage, isolating primary chondrocytes, preparing overexpression cell lines, injecting the cells intramuscularly and retrieving the implants. This assay can be performed by technicians and researchers with appropriate animal training within 3 weeks.

软骨再生是治疗骨关节炎和孤立性软骨缺损的医学重点。几种具有软骨再生潜力的分子正在研究中。不幸的是，体外软骨形成试验并不总是能预测体内新形成的软骨的稳定性。因此，需要一种严格的、可量化的测定来评估分子在体内促进稳定形成抗钙化和软骨内骨形成的软骨的能力。我们开发了一种异位软骨形成测定法 (ECFA)，使人们能够使用软骨类器官评估生物活性分子在体内支持软骨形成的能力。ECFA 在软骨缺损患者植入前用作软骨细胞制剂疗效的质量控制时，预测了良好的临床结果。在该测定中，来自人类供体或动物的关节软骨细胞被肌肉内或皮下注射到裸鼠中。早在 2 周后，就可以取出软骨类器官。植入物的大小及其分化程度可以通过组织形态学、分子标记的免疫染色和实时 PCR 来评估。矿化可以通过微型计算机断层扫描或染色来评估。分子对软骨形成的影响可以在以前注射过软骨细胞的小鼠全身给药后进行测试，或在将软骨细胞与过表达和分泌目的蛋白的细胞系共同注射后。在这里，我们描述了 ECFA 程序，包括收获人和牛关节软骨、分离原代软骨细胞、制备过表达细胞系、肌内注射细胞和取回植入物的步骤。该测定可由技术人员和研究人员在 3 周内进行适当的动物训练。