Developmentally inspired model of endochondral ossification

et al. (2023) Developmentally inspired model of endochondral ossification. International Journal of Experimental Pathology, 104 (2), A12-A12. (doi:10.1111/iep.12471).

Abstract

Introduction: In postnatal development, chondro-osseous transitions such as endochondral ossification (EO) are regulated by rapid matrix turnover, mineralisation and chondro-osseous transdifferentiation, all disrupted in osteoarthritis (OA). We are exploring how force governs these transitions of cartilage to bone; previous studies from our group indicate cartilage matrix and chondrocyte phenotypic plasticity in the growth plate, and stability in articular cartilage, is mechanoregulated. Here, we describe exploitation of a human ‘developmental biology-inspired platform’ alongside in vivo studies, to study cartilage mineralisation.

Materials and Methods: In vivo studies reveal chondro-osseous transitions are inhibited by forces. We synthesise GelMA using click chemistry to generate hydrogels with compressive moduli of 3–5 kPa. Buoyancy-driven gradients of BMP-2 within hydrogels seeded with hMSCs were cultured for 28 days. qPCR, histology and immunohistochemistry assessed, cartilage, hypertrophic and bone markers. Uniaxial cyclic compression (0.5 Hz, 10% strain) was applied using Electroforce5500. Cells from Confetti-UBCre mice are being used for lineage tracing studies.

Results: MSC-laden GelMA constructs showed differential gene expression across the gradient, indicating tri-phasic osteochondral tissue formation within 28 days. Runx2/Sox9 immunostaining confirmed osteochondral differentiation, increased expression of SPP1, SP7, Runx2, Col1 indicated osteogenesis at one end, while distinct expression of Col10 and Runx2 in the central region marked cellular hypertrophy. The effects of cyclic loading on cell signalling, hyaline cartilage formation and thickness, calcification and phenotypic plasticity/stability are being assessed and compared with in vivo findings.

Discussion: These data validate the formation of a humanised osteochondral gradient recapitulating developmental processes, in vitro. It is hypothesised that the generated osteochondral tissues will reflect the results of phenotypic changes in cells and ECM regulation, under physiological and pathological loads. The model will be integrated with snRNA sequencing and lineage tracing, to study trans-differentiation. This approach provides an experimentally tractable mechanobiology model and clinically conformant osteochondral tissue development model enabling fundamental biology and disease modelling across scales.

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Contributors

Author: Angus Wann

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