Cartilage organoids for cartilage development and cartilage-associated disease modeling

Cartilage organoids have emerged as powerful modelling technology for recapitulation of joint embryonic events, and cartilage regeneration, as well as pathophysiology of cartilage-associated diseases. Recent breakthroughs have uncovered “mini-joint” models comprising of multicellular components and extracellular matrices of joint cartilage for development of novel disease-modifying strategies for personalized therapeutics of cartilage-associated diseases. Here, we hypothesized that LGR5-expressing embryonic joint chondroprogenitor cells are ideal stem cells for the generation of cartilage organoids as “mini-joints” ex vivo “in a dish” for embryonic joint development, cartilage repair, and cartilage-associated disease modelling as essential research models of drug screening for further personalized regenerative therapy. The pilot research data suggested that LGR5-GFP-expressing embryonic joint progenitor cells are promising for generation of cartilage organoids through gel embedding method, which may exert various preclinical and clinical applications for realization of personalized regenerative therapy in the future.


Introduction
Cartilage remains among the most difficult tissues to regenerate, and integration of an implant with the surrounding tissue is also a major challenge in cartilage regeneration (Huey et al., 2012;Trengove et al., 2022). Regeneration of calcified cartilage regions is also a critical issue for stable and functional integration to subchondral bone besides cartilage-cartilage integration in the field of regenerative medicine and tissue engineering.

Advancements of cartilage organoids
The development of cartilage organoid technology as useful modelling tools and robust research platforms enables the definition and disease modelling of cartilage-tissue structures ex vivo to facilitate drug screening through identification of key signaling pathways, and recapitulation of developmental events during joint embryogenesis and cartilage regeneration, dynamics of stem cell chondrogenic differentiation, and aging-induced degenerative joint diseases "in a dish" (Clevers, 2016;Lacko and Chen, 2019;O'Connor et al., 2021;Rothbauer et al., 2021;Sun et al., 2021). In the early 1990s, C. Schröter-Kermani and his colleagues successfully established an ex vivo model of a prolonged, but almost identical of chondrogenesis events in vivo prior to endochondral mineralization, providing a useful tool for investigations on cartilage differentiation, maturation, and degeneration (Schroter-Kermani et al., 1991). Further research by Irie, Yutaka, et al. developed sheet-shaped organoids (organoid-sheet) of cartilage-like tissues, in which cells formed multicellular aggregates (organoids), through an effective cartilageformation method (Irie et al., 2008). Cell clusters called spheroids exert promising therapeutic potential for cartilage tissue engineering research as building blocks Kronemberger et al., 2020). Intriguingly, recent breakthroughs have uncovered "mini-joint" models comprising of multicellular components and extracellular matrices of joint cartilage for potential realization of novel disease-modifying strategies for personalized therapeutics of cartilage-associated diseases (Delplace et al., 2021;Abraham et al., 2022). A recent striking study has developed a novel differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cells via a neural crest cell intermediate (Foltz et al., 2021).
Cartilage organoids are specific three-dimensional and functional cartilage-like tissues through self-assembled reconstruction of chondrocytes or chondroprogenitor cells (Irie et al., 2008;Schon et al., 2017;Gryadunova et al., 2021), which is of essential clinical significance for tremendous translational applications to repair various cartilaginous structures throughout the body, as well as organoid biobanking, disease modeling, drug toxicity testing, personalized regenerative therapy, host-microbe interaction studies, and omics analysis (including transcriptomics, proteomics, epigenomics, and metabolomics) (Dutta et al., 2017). Cartilage organoids have been successfully generated both from induced pluripotent stem cells or mesenchymal stem cells . Cartilage organoid formation and their assembly into neo-hyaline-  (Crispim and Ito, 2021). And the development of cartilaginous organoids has been applied to diverse implications in preclinical research during recent years (Table 1). Hyaline cartilages, fibrocartilages and elastic cartilages play multiple roles throughout human body including bearing loads in articular joints and intervertebral discs, providing joint lubrication, forming the external ears and nose, supporting the trachea, and forming the long bones during development and growth. Challenges associated with cartilage diseases include poor understanding of the etiology and pathogenesis and diagnostics due to the aneural and avascular nature of adult cartilages, and very limited chondroprogenitor cells within adult joint cartilage. (Krishnan and Grodzinsky, 2018;Bielajew et al., 2020;Liao et al., 2021;Lin et al., 2022). Age is a main risk factor for the development of rheumatoid arthritis, which is associated with accelerated immune aging and dysfunction of aging stem cells (Weyand and Goronzy, 2004;Goronzy et al., 2013;Weyand et al., 2014). Generally, joint cartilage usually degenerates spontaneously in elderly mammalians ( Figure 1). As mitochondrial dysfunctions and age-associated systemic chronic inflammation (also termed as "inflamm-aging") have been demonstrated linked to the development of diverse aging-associated degenerative diseases (Franceschi and Campisi, 2014;Laforge et al., 2016;Sanada et al., 2018;Josephson et al., 2019;Shin et al., 2022). To further decipher dynamic alterations of cellular and mitochondrial behaviors and structures (Tran-Khanh et al., 2005;Labbe et al., 2014;Guilak et al., 2018;Akatsu et al., 2019), and key signaling pathways involved in the interplay between mitochondrial remodeling and "inflamm-aging" may further advance the understanding of the pathophysiology of aging-associated cartilage degeneration.
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Involvements of GPCRS in joint embryogenesis and cartilage pathophysiology
Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. The crystal structure of LGR5 has been discovered (Peng et al., 2013).
Notably, increasing evidence has demonstrated critical involvements of GPCRs during development and tissue homeostasis and regeneration in various tissue and organ systems (Luo et al., 2009;Cui et al., 2014;Feng et al., 2019;Montgomery et al., 2019;d'Aldebert et al., 2020;Lee et al., 2021;Khedgikar et al., 2022). Crucial involvements of GPCRs, such as LGR5, in both embryonic joint development (Feng et al., 2019), and postnatal joint development in juvenile mammals (Zhou et al., 2018), as well as progression of arthritis development (Li R. et al, 2022), have been identified, suggesting targeted modulation of GPCRs on cartilage as potential novel therapeutics for arthritis management.

FIGURE 4
Establishment of cartilage organoid cell atlas through RNA sequencing-based drug discovery and single cell multi-omics analysis. Targeted organoid sequencing through a high-throughput, high-content drug discovery platform targeting RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in cartilage organoids generated from pluripotent or adult stem cells.
Frontiers in Cell and Developmental Biology frontiersin.org Interestingly, a recent breakthrough by Rothbauer, M. et al. has successfully established microfluidic joint-on-a-chip organoid system to investigate reciprocal cross-talk between individual synovial and chondral organoids on tissue-level for modelling of arthritic diseases (Rothbauer et al., 2021). And our ongoing research suggest that LGR5-GFP + embryonic joint progenitors embedded within hydrogels enable the generation of organoid-structures under appropriate culture conditions with expression of LGR5-GFP signal (Figure 2), suggesting that LGR5-expressing joint chondroprogenitor cells are potential ideal cells for cartilage-like organoids formation, disease modelling for cartilage-associated diseases, drug screening and cartilage regeneration for realization of personalized medicine.

Conclusions and future perspectives
Organoids have been firmly established as a robust platform to investigate organ development, normal and pathological processes, and drug screening in both basic preclinical science and translational research, to overcome the limitations associated with animal models (Singh et al., 2021). Optimization of superior cell source, and ex vivo culture conditions for phenotypic control of cartilage organoids after transplantation deserve further exploitation. Integrated with advanced technologies (such as 3D bioprinting, bio-assembly, and organon-chip-based models, and comprehensive in-depth organoid single-cell genomic atlas mapping through high-spatialresolution multi-omics sequencing), cartilage organoid models may provide novel molecular, spatial, and temporal insights of embryonic joint development, and (patho)-physiology of cartilageassociated diseases for boosting the development of personalized regenerative therapy for treating cartilage-associated diseases (Liu et al., 2020;Singh et al., 2021). Cartilage organoids-based research on basic preclinical study and clinical transformation of personalized regenerative therapy will put forward a new era of regeneration medicine (Figure 3). Cartilage organoids provide an ideal platform for mechanistic biology at scale for establishment of cartilage organoid cell atlas through high-throughput drug screening or tissue-on-a-chip systems with molecular and phenotypic readout, and single cell multi-omics analysis (Figure 4; Figure 5). Collaborations among bioengineers, pharmacologists, clinicians, and developmental biologists, integrated with cutting-edge technologies and multi-disciplinary platforms, may accelerate the pace of discovery and precision of future clinical translation based on preclinical models of cartilage organoids (Li and Izpisua Belmonte, 2019;Xinaris, 2019;Berishvili et al., 2021;Bhamidipati and Wei, 2022).

Data availability statement
The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding authors.

FIGURE 5
LGR5-joint progenitors-based cartilage organoids for realization of novel drug discovery (identification of novel cytokines, small molecules, and natural compounds), and personalized regenerative therapy of cartilage repair.
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Ethics statement
The animal study was reviewed and approved by LX; Guangzhou University of Chinese Medicine. Fund (19-093-GRF, 14120118, 9054014, N_CityU102/15, and 14119115), and Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR, P. R. China.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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