软骨
软骨发生
体内
软骨内骨化
软骨细胞
骨关节炎
再生(生物学)
化学
体外
细胞生物学
病理
生物医学工程
解剖
医学
生物
生物技术
生物化学
替代医学
作者
Anne-Sophie Thorup,Sara Caxaria,B.L. Thomas,Yasir Suleman,Giovanna Nalesso,Frank P. Luyten,Francesco Dell'Accio,S. Eldridge
出处
期刊:Lab Animal
[Springer Nature]
日期:2022-03-31
卷期号:51 (4): 103-120
被引量:2
标识
DOI:10.1038/s41684-022-00943-y
摘要
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. This protocol describes an in vivo cartilage formation assay. Human or bovine articular chondrocytes injected in nude mice form cartilage organoids that can be used for the screening of molecules that promote cartilage formation.
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