Speaker
Description
Osteoarthritis is a progressive inflammatory disease characterized by articular cartilage (AC) degeneration, affecting millions globally. Recapitulating AC’s arcade-like collagen structure is key to engineering functional grafts. AC progenitor cells (ACPs) have a unique ability to maintain a stable hyaline phenotype. ACP derived microtissues can potentially be used as biological building blocks for the biofabrication of scaled-up grafts as they support cell-cell and cell-extracellular matrix (ECM) interactions. Here we first explored a temporal BMP-9 stimulation to generate hyaline cartilage microtissues using ACPs and evaluated their fusion and (re)modelling. We then used decellularized AC ECM, known to be chondro-inductive [1], as a scaffold to support ACP microtissues as they self-organise forming a functional osteochondral graft.
ACP microtissues (1,000 or 2,000 cells/microtissue) were produced using a high throughput microwell mould [2]. After 2 days of maturation, 2,000, 4,000 or 6,000 microtissues were fused in a 6mm cylindrical mould and cultured for 6 weeks (Fig. 1A). We then evaluated the effect of temporal stimulation with BMP-9, with or without TGF-β3 and dexamethasone, for 2 days during microtissue formation, before fusing 2,000 microtissues and culturing for 4 weeks in chondrogenic media without BMP-9 (Fig. 1B). Finally, 4,000 microtissues (1,000 cells/microtissue) were fused for 4 hours in a 5mm cylindrical agarose mould before adding the ECM scaffold. On day 3, the scaffold was flipped and cultured for 4 weeks (Fig. 1C). Chondrogenesis was assessed through histology, immunohistochemistry, and biochemical assays.
ACP microtissues rapidly fused and supported robust chondrogenesis in all conditions as evident by the positive staining for sulphated glycosaminoglycan (sGAG) and collagen (Fig 1A). In the 6000 microtissues group, some necrosis was evident in the centre of the construct. Temporal stimulation of ACP microtissues with BMP-9 induced volumetric expansion during fusion, with an increase in sGAG and collagen synthesis while maintaining a hyaline-like phenotype with an intense collagen type II stain (Fig 1B). Microtissues that were allowed to fuse on the surface of the AC ECM scaffolds formed a white smooth and homogeneous layer of tissue that stained positive for sGAG and collagen. Polarized light microscopy revealed an arcade-like collagen organization with horizontal fibres on the surface and vertical fibres in the middle/deep zone (Fig. 1C).
ACP microtissues exhibited robust fusion and chondrogenesis, with radial confinement in the 6mm cylindrical mould promoting biomimetic collagen alignment, consistent with previous findings [3]. The ACP microtissues formed a smooth, uniform layer of cartilage on the surface of the AC ECM derived scaffold. Temporal BMP-9 stimulation enhanced ECM production, highlighting its potential for improving graft functionality. Future studies will integrate BMP-9 stimulated ACPs with ECM scaffolds, assess mechanical properties, and evaluate graft performance in a goat model of joint injury.
References
[1] 10.1016/j.mtbio.2022.100343
[2] 10.3389/fbioe.2021.661989
[3] 10.1002/adhm.202300174
Acknowledgments
This research was supported by Emirates NBD, Sharjah Electricity, Water & Gas Authority (SEWA), and the Technology Innovation Institute (TII), who served as the golden sponsors of the 5th Forum for Women in Research (QUWA): Together Innovating to Shape the Future at the University of Sharjah and ERC grant 4D-Boundaries #101019344.