From cells to organs: progress and potential in cartilaginous organoids research

Cartilage, a specialized form of connective tissue, is characterized by its low cell density and high matrix composition. The extracellular matrix (ECM) of cartilage consists of a complex arrangement of collagen fibres and proteoglycan molecules, providing the tissue with a unique ability to withstand compression. Chondrocytes, the primary cell population in cartilage, play a crucial role in synthesizing and maintaining the matrix in response to various genetic and environmental cues, including growth factors and physiological loading [20]. However, under pathological or injurious conditions, chondrocytes may transition to a degradative and inflammatory phenotype [21].

As a vital component of the musculoskeletal system, cartilage possesses distinctive features such as the absence of blood vessels and nerves. It serves as a structural support system and enables smooth joint movement by cushioning bones and reducing friction between them [22]. Articular cartilage, specifically, is anatomically and functionally divided into four distinct zones, characterized by distinct morphological, chemical, and collagen density properties [23] (Fig. 2). Despite its critical role, cartilage has limited self-repair capacity, rendering it vulnerable to various pathological changes and diseases such as osteoarthritis (OA), chondromalacia patellae, and chondral injuries [24].

OA, a degenerative joint disease, is characterized by the progressive loss of articular cartilage. With a growing prevalence affecting more than 30 million Americans, the incidence of OA is escalating due to the aging population and increasing obesity rates [25]. While the exact mechanisms underlying OA remain incompletely understood, it is considered a complex interplay of mechanical, biochemical, and cellular factors [26, 27].

Preclinical models play a crucial role in the advancement of treatments for cartilage diseases by enabling the investigation of underlying mechanisms, testing the effectiveness and safety of new therapies, and predicting their clinical potential (Fig. 3).

During various developmental processes, the gradients of morphogens and physical cues play a critical role in determining the polarity and diversity of structures that form in vivo. Similarly, self-organizing organoid systems have the ability to establish complex cellular patterns through successive modifications of the local microenvironment, driving organoid morphogenesis in vitro.

Currently, the utilization of cartilaginous organoids remains somewhat restricted. However, as research progresses, it is foreseeable that the utilization of cartilaginous organoids will flourish in the upcoming decade (Fig. 5).

One of the paramount applications of cartilaginous organoids resides in the realm of disease modelling. These organoids can be derived from patients afflicted with joint ailments such as OA and rheumatoid arthritis, thereby providing researchers with a more physiologically relevant system to investigate the disease. In a recent study, Cullier et al. [108] constructed an equine organoid model for OA by inducing it with IL-1β. The researchers discovered that a combination of BQ-123-CHI and R-954-HA (BR5) yielded the most notable reduction in inflammatory and catabolic markers. This finding underscores the potential of organoids as a potent tool for studying the pathophysiology of joint diseases and developing innovative therapies to combat them.

Another potential application of cartilaginous organoids lies in drug screening. In comparison to conventional animal models, organoids provide a more precise and cost-effective means of assessing the efficacy and toxicity of new drug candidates. This capability not only accelerates the drug development process but also diminishes the necessity for animal testing.

Within the realm of tissue engineering, cartilaginous organoids can be harnessed to pioneer innovative approaches for repairing damaged cartilage tissue. Presently, the existing methods rely on synthetic scaffolds that are seeded with or without chondrocytes or stem cells [109‐112]. However, these approaches frequently yield tissue that lacks the functional and mechanical properties of native cartilage. In contrast, cartilaginous organoids present a more intricate and dynamic system for advancing tissue engineering endeavors. Recent studies have underscored the potential of cartilaginous organoids in the realm of tissue repair. For instance, Kengo Abe et al. [113] demonstrated that allogeneic iPSC-derived cartilaginous organoids exhibit remarkable survival and integration capacities, as well as the ability to remodel articular cartilage, when tested in a primate model with chondral defects in the knee joints. Furthermore, Hall et al. [50] ingeniously incorporated genetically distinct populations of cartilaginous tissue intermediates into a single implant, creating an osteochondral tissue unit. By combining human iPSC-derived cartilage microtissues with callus organoids derived from human PDCs, they successfully formed a dual structure of cartilage and bone after implantation. These studies demonstrate the immense potential of cartilaginous organoids as a promising alternative for the repair of damaged cartilage tissue.

Furthermore, cartilaginous organoids present a valuable platform for investigating the long-term behavior of cells and tissues in vitro, which is essential for the development of effective and safe clinical therapies. In pursuit of this objective, Thorup et al. [114] devised an ectopic cartilage formation assay utilizing organoids derived from articular chondrocytes of human donors, which were injected into nude mice to evaluate the potential of bioactive molecules in promoting in vivo cartilage formation. Such test systems based on organoids offer a potent tool for screening molecules with regenerative potential for cartilage.

Moreover, cartilaginous organoids hold significant promise in the advancement of individualized treatments for cartilage-related ailments. By generating organoids from a patient's own cells, therapies can be tailored to the specific biology of each patient, thereby enhancing the likelihood of successful outcomes while mitigating the risk of adverse events. Notably, recent breakthroughs have led to the development of "mini-joint" models that incorporate multicellular components and extracellular matrices of joint cartilage. These models present a novel approach for devising strategies to modify diseases and crafting personalized therapeutics for conditions associated with cartilage [115, 116].

Organoids have emerged as potent instruments in fundamental research and have made significant contributions to advancements in the biomedical domain. Nonetheless, the utilization of cartilaginous organoids in translational studies remains restricted due to the intricate and demanding process involved in their translation into practical applications, and achieving maturation and functionality in cartilaginous organoids, remains a significant challenge. This challenge stems from the limited understanding of the molecular mechanisms governing organ development.

Recent studies have demonstrated the remarkable capability of single-cell sequencing (scRNA-seq) in elucidating the cellular heterogeneity and lineage specification of chondrocytes derived from hiPSCs [107], identifying rare cell populations within osteoarthritic cartilage [117], and unveiling the molecular signatures of ferroptotic chondrocyte clusters in human OA cartilage [118]. These investigations underscore the emerging role of single-cell "omics" approaches in chondrocyte research, offering potential insights into innovative therapeutic strategies for OA and other cartilage-related disorders.

The combination of organoid technology and scRNA-seq holds immense potential in addressing the limitations associated with each approach. Significant advancements in stem cell biology have facilitated the precise regulation of differentiation pathways within organoids [119, 120]. Through the integration of cutting-edge technologies for organoid culture and scRNA-seq, researchers have acquired a powerful tool for exploring organ development and diseases [121]. Although the application of these techniques to cartilaginous organoids is still in its early stages, the future appears promising for unlocking their complete potential.

Among the various gene-editing techniques available, CRISPR/Cas9 stands out due to its precise targeting and shearing capabilities, cost-effectiveness, and user-friendliness, making it indispensable in biological research [125, 126].

CRISPR/Cas9 screens provide an unbiased approach to establish the causal relationship between genotype and phenotype. By enabling genome-wide knockout of gene expression and subsequent analysis of resulting phenotypic changes, these screens have proven valuable [127, 128]. Additionally, the CRISPR system has been utilized for gene knockout and genetic mutation repair to facilitate in vitro disease modelling, offering significant time and labor savings compared to constructing animal models [129].

In recent years, CRISPR/Cas9 has emerged as a robust tool for investigating cartilage diseases. More recently, Chaudhry et al. [130] developed an efficient CRISPR/Cas9-mediated strategy for gene editing in primary human chondrocytes, enabling the investigation of miR-140-dependent mechanosensitive gene regulation. However, conventional 2D culture conditions still limit the assessment of intricate cellular functions and physiological characteristics of cartilage.

The integration of gene editing techniques with organoids has provided researchers with a powerful tool to investigate intrinsic developmental mechanisms through loss-of-function and gain-of-function studies conducted in vitro. Recently, CRISPR/Cas9 technology has been successfully applied to iPSC-based organoids, including neuronal, brain, intestinal, and colonic organoids. These advancements have significantly contributed to our improved understanding of human organogenesis, normal physiology, and disease pathology [131‐134].

In a pioneering study by Ruiz et al. [135], CRISPR/Cas9 technique was employed on cartilaginous organoids to conduct functional characterization of the effects of OPG-XL in joint tissues. The researchers utilized hiPSCs derived from individuals within the affected CCAL1 family and employed CRISPR/Cas9 to repair hiPSCs, creating isogenic controls. These isogenic control cells were then utilized to establish in vitro organoid models of cartilage and bone, providing valuable insights into the effects of OPG-XL.

The combination of CRISPR/Cas9 and organoid technologies exhibits significant potential for advancing the study and treatment of genetic cartilaginous diseases, including Achondroplasia (ACH). ACH results from a mutation in the FGFR3 gene located on chromosome 4p16.31 [136]. Moreover, integrating CRISPR/Cas9 and organoid technologies can bridge the gap between potential therapeutic molecular mechanisms and their translation into treatments for human patients with OA and other cartilaginous diseases. Organoids, especially those derived from hiPSCs, serve as highly representative models of human diseases. By applying CRISPR-based mutagenesis to hiPSC-derived cartilaginous organoids, personalized therapy or precision medicine for cartilaginous diseases can be facilitated [137, 138].

Recent advancements in scRNA-seq technology, combined with CRISPR/Cas9 gene editing, have provided an unbiased approach to uncovering genotype–phenotype relationships at the single-cell level. For example, Dicks et al. [139] employed a CRISPR/Cas9-edited COL2A1-GFP knock-in reporter hiPSC line to identify a unique subpopulation of CPs with high chondrogenic potential. Subsequent analysis using scRNA-seq revealed distinct clusters within this population.

As highlighted earlier, the utilization of CRISPR/Cas9 gene editing technology in cartilaginous organoids has proven effective in elucidating human organogenesis, normal physiology, and disease pathology. Furthermore, implementing more sophisticated cartilaginous organoid culture systems can contribute to the evaluation of genome editing technology in terms of safety and efficiency.

However, CRISPR/Cas9 screening in organoids still faces certain limitations, primarily due to challenges associated with the manual handling of 3D organoids on a large scale. Improvements in sgRNA design, specifically tailored for organoids, can enhance phenotypic induction and penetrance, ultimately improving the CRISPR/Cas9 organoid screening platform to target patient-specific mutations or vulnerabilities. In due course, the emergence of microfluidic technology is expected to provide favorable conditions and timing for further advancements.

Despite the significant progress achieved in the development of physiologically relevant cartilaginous organoids, several challenges persist. These challenges include heterogeneity, higher costs, and the need for high throughput in practical applications [140]. Addressing the limitations related to reproducibility and automation is crucial for the widespread utilization of cartilaginous organoids in clinical research. The integration of cartilaginous organoids with microfluidic systems, based on microphysiological technology, offers a promising avenue to overcome these technological challenges.

Microfluidic technology presents a powerful approach to establish complex biomolecule gradients that better mimic physiological conditions compared to conventional cell culture models. By replicating perfusion, mechanical forces, and other essential parameters for tissue and organ physiology, microfluidic systems enable a more comprehensive understanding of specific cellular responses and the fluidic and mechanical aspects of the cellular microenvironment [141]. Organ-on-a-chip devices, also known as organ chips, are microfluidic cell culture systems maintained under constant fluid flow. They serve as a means to bridge the gap between in vitro models and in vivo physiology [142].

The combination of organoid culture with microfluidic technologies offers several advantages [143]. First, microfluidic devices provide enhanced control over spontaneous morphogenesis, thus reducing variability. Second, automated operation reduces labor costs and minimizes human error. Third, miniaturized culture systems result in reduced reagent usage. Finally, microfluidic systems can expedite the maturation of organoids. Given the avascular, aneural state, and fibrillar composition of cartilage, microfluidic systems are particularly well suited for cartilaginous organoid studies. They enhance mass transfer, regulate cellular interactions, and allow for the adjustment of porosity.

A recent breakthrough by Rothbauer et al. [144] successfully established a microfluidic joint-on-a-chip organoid system. This system allows for the investigation of reciprocal cross-talk between individual synovial and cartilaginous organoids at the tissue level, providing a valuable model for studying arthritic diseases.

Microfluidic hydrogel-based scaffolds, fabricated using microfluidic devices, offer promising alternatives to traditional hydrogels used in cartilage tissue engineering [145]. The incorporation of microchannels enhances mass transfer, allowing for precise control over the distribution of chemical substances. This feature facilitates the creation of 3D structures that more accurately replicate native tissue, enabling the estimation of a particular tissue's functional performance [146, 147].

In the preceding discussion, we emphasize the capacity of cartilaginous organoids to simulate both physiological and pathological states of cartilage, including molecular mechanisms and signal transduction. However, despite the prevalent focus on chondrocyte pathobiology in most microphysiological models of OA, it is important to acknowledge that this disease affects the entire joint, including the influence of intra-articular pressure (IAP) and synovial fluid. Imbalanced mechanical stresses are identified as contributing factors to the pathogenesis of OA. Thus, mechanosensory activation during the onset and progression of OA represents a crucial yet often overlooked aspect of microsystems, demanding careful examination [148].

Moreover, systemic factors like age and sex, recognized as risk factors for OA, prove challenging to investigate using current joint-on-a-chip organoids.

Organ-on-a-chip platforms confront numerous practical considerations contingent on their intended application. Specifically, Joint-on-a-chip organoids are influenced by factors including device material selection, and the capacity for functional integration.

In the long term, the discovery of novel materials promises to foster technological advancements, potentially facilitating the large-scale fabrication of cartilaginous organoids integrated with microfluidic systems.

To enhance current disease models based on cartilaginous organoids, we propose the integration of anatomical and biomechanical considerations into next-generation microfluidic systems used in this context. The structure‒function relationship of an articular joint is highly intricate and multifaceted, involving diverse cellular, biochemical, and critical biophysical factors. Therefore, future models of OA that utilize cartilaginous organoids must enhance the control and precision of fluid-mechanical cues at the microscale.

To establish organoids as a reliable evaluation platform, it is essential to define specific technical standards and ensure the generation of functional units within a predetermined size range while maintaining consistent functionality. 3D bioprinting represents a novel approach that enables the creation of highly organized constructs.

3D bioprinting is an advanced technology that provides precise control over biophysical properties, such as organoid size, cell number, and structure, enabling the creation of tissue-like structures that closely resemble natural tissues [98].

While extensive discussions on the principles, classifications, characteristics, and applications of 3D bioprinting can be found in the literature, they surpass the scope of this paper [149, 150].

The application of 3D bioprinting to cartilaginous organoids is still in its early stages. However, recent advancements in fabrication techniques have made it feasible to utilize these methodologies for cartilaginous organoids using ADSCs and chondrocytes. Although hiPSCs hold immense potential for cartilaginous organoids and regenerative medicine, there are limited reports on bioprinting human 3D constructs based on hiPSCs. This is primarily due to the unique characteristics of hiPSCs, which present challenges for bioprinting. First, hiPSCs have low survival rates as single cells in culture, and dissociation into single cells is often a necessary step in most bioprinting procedures. Second, hiPSCs exhibit high responsiveness to environmental cues due to their embryonic-like nature and ability to respond to developmental signals. Last, hiPSCs tend to form clusters or colonies because of their epithelial character, which must be taken into account when employing nozzle-based bioprinting methods [151].

Cartilage tissue exhibits cellular heterogeneity and hierarchical organization across different zones of varying depth. However, accurately replicating zone-dependent characteristics in cartilaginous organoids, including size, ECM composition, and expression of anabolic and catabolic molecules, remains a challenge [152]. Traditionally, specific culture conditions are required to expand chondrocytes derived from different regions of the cartilage or stem cells undergoing distinct in vitro cartilage differentiation processes. Jonathan A [153] introduced a novel bioprinting-assisted tissue emergence (BATE) printing technology that utilizes stem cells and organoids as self-organizing building blocks. These blocks can be spatially arranged to form interconnected and evolving cellular structures. Through 3D bioprinting, individual cells or cellular aggregates that typically develop into randomly shaped small organoids can be induced to fuse and reorganize according to the imposed geometry and constraints. BATE printing technology holds promise for generating large-scale cartilaginous organoids with biological complexity.

Furthermore, Ludovicserex et al. [154] introduced a microfluidic-based print head that enables real-time adjustment of the print unit concentration, allowing for fibroblast bioprinting at concentrations of up to 10 million cells/mL. As the cell inoculation concentration plays a crucial role in 3D organoid culture, this method can yield more reliable and repeatable results.

Nonetheless, the technology remains in its experimental phase, signifying that various technical challenges must be addressed.

The generation of realistic cartilage incorporates diverse cell types of disparate shapes, thereby presenting a difficulty in determining the optimal printing parameters. Characteristically, 3D bioprinting exhibits slow printing speed and the attributes of the bio-ink, which include viscosity and cell density, are prone to alterations over time. This can notably influence the printing quality of cartilaginous organoids. Consequently, it is crucial to implement appropriate strategies to prevent ink desiccation during the printing process.

The hurdles that exist between laboratory research discoveries and commercial production are substantial. As it stands, 3D bioprinting within a laboratory context remains nascent and large-scale production of bio-products with high efficiency is currently unattainable. Hence, optimization of experimental apparatus and methodologies is a requisite for achieving rapid, and efficient large-scale biomanufacturing.

Although 3D bioprinting of cartilaginous organoids is still in its early stages, it offers immense potential. Future efforts should focus on improving cell viability postprinting, addressing limitations such as poor cytocompatibility and degradation-associated toxicity, and enhancing printing accuracy using existing technology. The integration of 3D bioprinting and microfluidic technologies may open new avenues for the development of cartilaginous organoids.