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In vivo potency assay for the screening of bioactive molecules on cartilage formation

Abstract

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.

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Fig. 1: The ECFA distinguishes between articular and epiphyseal cartilage formation.
Fig. 2: Flow chart describing the ECFA.
Fig. 3: Isolation of bovine chondrocytes.
Fig. 4: Isolation of human articular chondrocytes.
Fig. 5: In vitro validation of secretion and bioactivity of the overexpressed gene in cell lines following growth inhibition with mitomycin C.
Fig. 6: Intramuscular injection and retrieval site.
Fig. 7: Histological expected outcome.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

This work was supported by grants from FOREUM, grant/award number 1016807; Versus Arthritis, grant/award number 21515 and 22628; Rosetrees Trust, grant/award numbers A589 and A2575; the MRC grant/award numbers MR/L022893/1 and MR/K013076/1; the St Bartholomew’s Hospital Medical College Trust (James Paget Studentship Award) and Versus Arthritis Centre for Osteoarthritis Pathogenesis grant number 20205. We also thank P. Achan and M. Ramachandran (Barts and the London National Health Service Trust, London, England, UK) for the supply of human knee samples, and C. Mole (School of Engineering and Materials Science, QMUL) for helping with the provision of the hooves.

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A-S.T., S.C., G.N. and S.E.E. designed and performed the co-injection experiments, analyzed the data and wrote the manuscript; B.L.T. performed experiments and contributed to writing the manuscript; Y.S. performed experiments; F.P.L. designed the ECFA and contributed to writing the manuscript; F.D.A. designed and developed the ECFA, supervised the co-injection experiments and contributed to writing the manuscript. F.D.A. and S.E.E. share senior authorship.

Corresponding authors

Correspondence to Francesco Dell’Accio or Suzanne E. Eldridge.

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Competing interests

F.P.L and F.D.A. are co-inventors on patent WO2008061804 (A2): ‘In vivo assay and molecular markers for testing the phenotypic stability of cell populations, and selecting cell populations for autologous transplantation’. The other authors declare no competing interests.

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Thorup, AS., Caxaria, S., Thomas, B.L. et al. In vivo potency assay for the screening of bioactive molecules on cartilage formation. Lab Anim 51, 103–120 (2022). https://doi.org/10.1038/s41684-022-00943-y

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