Cartilage Biology Research Articles

An "All-in-One" Strategy to Reconstruct Temporomandibular Joint Osteoarthritic Microenvironment Using γ-Fe2O3@TA@ALN Nanoparticles.

Current clinical strategies for the treatment of temporomandibular joint osteoarthritis (TMJOA) primarily target cartilage biology, overlooking the synergetic effect of various cells and inorganic components in shaping the arthritic microenvironment, thereby impeding the effectiveness of existing therapeutic options for TMJOA. Here, γ-Fe2O3@TA@ALN magnetic nanoparticles (γ-Fe2O3@TA@ALN MNPs) composed of γ-Fe2O3, tannic acid (TA), and alendronate sodium (ALN) are engineered to reconstruct the osteoarthritic microenvironment and mitigate TMJOA progression. γ-Fe2O3@TA@ALN MNPs can promote chondrocytes' proliferation, facilitate chondrogenesis and anisotropic organization, enhance lubrication and reduce cartilage wear, and encourage cell movement. Magnetic-responsive γ-Fe2O3@TA@ALN MNPs also exhibit pH sensitivity, which undergoes decomposition within acidic environment to release ALN on demand. Under a 0.2 T static magnetic field, γ-Fe2O3@TA@ALN MNPs accelerate the synthesis of cartilage-specific proteins, and suppress catabolic-related genes expression and reactive oxygen species generation, affording additional protection to TMJ cartilage. In TMJOA mouse models, articular injection of γ-Fe2O3@TA@ALN MNPs effectively alleviates cartilage degeneration and subchondral bone loss in short and long terms, offering promising avenues for the development of therapeutic interventions for TMJOA.

Enhanced chondrogenic potential in GelMA-based 3D cartilage model via Wnt3a surface immobilization

Cartilage tissue engineering aims to develop functional substitutes for treating cartilage defects and osteoarthritis. Traditional two-dimensional (2D) cell culture systems lack the complexity of native cartilage, leading to the development of 3D regenerative cartilage models. In this study, we developed a 3D model using Gelatin Methacryloyl (GelMA)-based hydrogels seeded with Y201 cells, a bone marrow mesenchymal stem cell line. The model investigated chondrogenic differentiation potential in response to Wnt3a stimulation within the GelMA scaffold and validated using known chondrogenic agonists. Y201 cells demonstrated suitability for the model, with increased proteoglycan content and upregulated chondrogenic marker expression under chondrogenic conditions. Wnt3a enhanced cell proliferation, indicating activation of the Wnt/β-catenin pathway, which plays a role in cartilage development. GelMA hydrogels provided an optimal scaffold, supporting cell viability and proliferation. The 3D model exhibited consistent responses to chondrogenic agonists, with TGF-β3 enhancing cartilage-specific extracellular matrix (ECM) production and chondrogenic differentiation. The combination of Wnt3a and TGF-β3 showed synergistic effects, promoting chondrogenic differentiation and ECM production. This study presents a 3D regenerative cartilage model with potential for investigating cartilage biology, disease mechanisms, and drug screening. The model provides insights into complex cartilage regeneration mechanisms and offers a platform for developing therapeutic approaches for cartilage repair and osteoarthritis treatment.

A Narrative Review of the Roles of Chondromodulin-I (Cnmd) in Adult Cartilage Tissue.

Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.

Exercise-induced modulation of myokine irisin on muscle-bone unit in the rat model of post-traumatic osteoarthritis

Background and aimPost-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA). Exercise may produce and release the myokine irisin through muscle fiber contraction. However, the effect of exercise-promoted irisin production on the internal interactions of the muscle–bone unit in PTOA studies remains unclear.MethodsEighteen 8-week-old Sprague–Dawley (SD) rats were randomly divided into three groups: Sham/sedentary (Sham/Sed), PTOA/sedentary (PTOA/Sed), and PTOA/treadmill-walking (PTOA/TW). The PTOA model was established by transection of anterior cruciate ligament (ACLT) and destabilization of medial meniscus (DMM). After 4 weeks of modeling, the PTOA/TW group underwent treadmill exercise (15 m/min, 30 min/d, 5 d/ week, 8 weeks), and the other two groups were free to move in the cage. Evaluation and correlation analysis of muscle, cartilage, subchondral bone and serological indexes were performed after euthanasia.ResultsEight weeks of treadmill exercise effectively alleviated the trauma-induced OA phenotype, thereby maintaining cartilage and subchondral bone integrity in PTOA, and reducing quadriceps atrophy and myofibril degradation. Exercise reversed the down-regulated expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and fibronectin type III structural domain protein 5 (FNDC5) in muscle tissue of PTOA rats, and increased the blood irisin level, and the irisin level was positively correlated with the expression of PGC-1α and FNDC5. In addition, correlation analysis showed that irisin metabolism level was strongly negatively correlated with Osteoarthritis Research Society International (OARSI) and subchondral bone loss, indicating that irisin may be involved in cartilage biology and PTOA-related changes in cartilage and subchondral bone. Moreover, the metabolic level of irisin was strongly negatively correlated with muscle fiber cross-sectional area (CSA), Atrogin-1 and muscle ring-finger protein-1(MuRF-1) expression, suggesting that irisin may alleviate muscle atrophy through autocrine action.ConclusionTreadmill exercise can alleviate the atrophy and degeneration of muscle fibers in PTOA rats, reduce the degradation of muscle fibrin, promote the expression of serum irisin, and alleviate the degeneration of articular cartilage and subchondral bone loss in PTOA rats. These results indicate that treadmill exercise can affect the process of PTOA by promoting the expression of myokine irisin in rat muscle–bone unit.

MicroRNA-138: an emerging regulator of skeletal development, homeostasis, and disease.

Noncoding microRNAs are powerful epigenetic regulators of cellular processes by their ability to target and suppress expression of numerous protein-coding mRNAs. This multitargeting function is a unique and complex feature of microRNAs. It is now well-described that microRNAs play important roles in regulating the development and homeostasis of many cell/tissue types, including those that make up the skeletal system. In this review, we focus on microRNA-138 (miR-138) and its effects on regulating bone and cartilage cell differentiation and function. In addition to its reported role as a tumor suppressor, miR-138 appears to function as an inhibitor of osteoblast differentiation. This review provides additional information on studies that have attempted to alter miR-138 expression in vivo as a means to dampen ectopic calcification or alter bone mass. However, a review of the published literature on miR-138 in cartilage reveals a number of contradictory and inconclusive findings with respect to regulating chondrogenesis and chondrocyte catabolism. This highlights the need for more research in understanding the role of miR-138 in cartilage biology and disease. Interestingly, a number of studies in other systems have reported miR-138-mediated effects in dampening inflammation and pain responses. Future studies will reveal if a multifunctional role of miR-138 involving suppression of ectopic bone, inflammation, and pain will be beneficial in skeletal conditions such as osteoarthritis and heterotopic ossification.

Efficient Generation of Chondrocytes From Bone Marrow-Derived Mesenchymal Stem Cells in a 3D Culture System: Protocol for a Practical Model for Assessing Anti-Inflammatory Therapies.

Chondrocytes are the primary cells responsible for maintaining cartilage integrity and function. Their role in cartilage homeostasis and response to inflammation is crucial for understanding the progression and potential therapeutic interventions for various cartilage-related disorders. Developing an accessible and cost-effective model to generate viable chondrocytes and to assess their response to different bioactive compounds can significantly advance our knowledge of cartilage biology and contribute to the discovery of novel therapeutic approaches. We developed a novel, streamlined protocol for generating chondrocytes from bone marrow-derived mesenchymal stem cells (BMSCs) in a 3D culture system that offers significant implications for the study of cartilage biology and the discovery of potential therapeutic interventions for cartilage-related and associated disorders. We developed a streamlined protocol for generating chondrocytes from BMSCs in a 3D culture system using an "in-tube" culture approach. This simple pellet-based 3D culture system allows for cell aggregation and spheroid formation, facilitating cell-cell and cell-extracellular matrix interactions that better mimic the in vivo cellular environment compared with 2D monolayer cultures. A proinflammatory chondrocyte model was created by treating the chondrocytes with lipopolysaccharide and was subsequently used to evaluate the anti-inflammatory effects of vitamin D, curcumin, and resveratrol. The established protocol successfully generated a large quantity of viable chondrocytes, characterized by alcian blue and toluidine blue staining, and demonstrated versatility in assessing the anti-inflammatory effects of various bioactive compounds. The chondrocytes exhibited reduced inflammation, as evidenced by the decreased tumor necrosis factor-α levels, in response to vitamin D, curcumin, and resveratrol treatment. Our novel protocol offers an accessible and cost-effective approach for generating chondrocytes from BMSCs and for evaluating potential therapeutic leads in the context of inflammatory chondrocyte-related diseases. Although our approach has several advantages, further investigation is required to address its limitations, such as the potential differences between chondrocytes generated using our protocol and those derived from other established methods, and to refine the model for broader applicability and clinical translation.

Comparison of Kindlin-2 deficiency-stimulated osteoarthritis-like lesions induced by Prg4CreERT2 versus AggrecanCreERT2 transgene in mice

BackgroundGenetically modified mice are the most useful tools for investigating the gene functions in articular cartilage biology and the pathogenesis of osteoarthritis. The AggrecanCreERT2 mice are one of the most reported mouse lines used for this purpose. The Prg4 (proteoglycan 4) gene encodes the lubricin protein and is expressed selectively in chondrocytes located at the superficial layer of the articular cartilage. While the Prg4GFPCreERT2 knock-in inducible-Cre transgenic mice were generated a while ago, so far, few studies have used this mouse line to perform gene functional studies in cartilage biology. MethodsWe have recently reported that deleting the Fermt2 gene, which encodes the key focal adhesion protein Kindlin-2, in articular chondrocytes by using the AggrecanCreERT2 transgenic mice, results in spontaneous osteoarthritis (OA) lesions, which highly mimics the human OA pathologies. In this study, we have compared the Kindlin-2 deficiency-caused OA phenotypes induced by Prg4GFPCreERT2 with those caused by AggrecanCreERT2 using imaging and histological analyses. ResultsWe find that Kindlin-2 protein is deleted in about 75% of the superficial articular chondrocytes in the tamoxifen (TAM)-treated Prg4GFPCreERt2/+; Fermt2fl/fl mice compared to controls. At 6 months after TAM injections, the OARSI scores of AggrecanCreERT2/+; Fermt2fl/fl and Prg4GFPCreERt2/+; Fermt2fl/fl mice were 5 and 3, respectively. The knee joints histological osteophyte and synovitis scores were also significantly decreased in Prg4GFPCreERT2/+; Fermt2fl/fl mice compared to those in AggrecanCreERT2/+; Fermt2fl/fl mice. Furthermore, magnitudes of upregulation of the extracellular matrix-degrading enzymes Mmp13 and hypertrophic chondrocyte markers Col10a1 and Runx2 were decreased in Prg4GFPCreERT2/+; Fermt2fl/fl versus AggrecanCreERT2/+; Fermt2fl/fl mice. We finally examined the susceptibility of Prg4GFPCreERT2/+; Fermt2fl/fl mouse model to surgically induce OA lesions. The pathological features of OA in the TAM-DMM model exhibited significant enhancement in cartilage erosion, proteoglycan loss, osteophyte, and synovitis and an increase in OARSI score in articular cartilage compared with those in corn-oil DMM mice. ConclusionKindlin-2 loss causes milder OA-like lesions in Prg4GFPCreERT2/+;Fermt2fl/fl than in AggrecanCreERT2/+; Fermt2fl/fl mice. In contrast, Kindlin-2 loss similarly accelerates the destabilization of the medial meniscus-induced OA lesions in both mice.Translational Potential of this Article: Our study demonstrates that Prg4GFPCreERT2 is a useful tool for gene functional study in OA research. This study provides useful information for investigators to choose appropriate Cre mouse lines for their research in cartilage biology.

Lineage-specific differences and regulatory networks governing human chondrocyte development.

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology.

Bone and Cartilage Biology

Recent technical and conceptual advances in molecular and cellular biology have dramatically advanced bone and cartilage biology [...].

Techniques for Visualization and Quantification of Primary Cilia in Chondrocytes.

Primary cilia regulate and coordinate a variety of cell signaling pathways important in chondrocyte physiology and cartilage development, health, and disease. Despite this, the chondrocyte primary cilium and its associated role in cartilage biology remains poorly understood. Key to elucidating primary cilia structure and function in chondrocytes is the ability to visualize this unique structure. Here we describe materials and methods for immunofluorescence labeling, microscopy, and measurement of chondrocyte primary cilia.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Biochemical characterization of a disease-causing human osteoprotegerin variant

Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

The Critical Role of Hypoxia in the Re-Differentiation of Human Articular Chondrocytes.

The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.

Differential expression and methylation patterns of NFATC1, NADSYN1 and JAK3 gene in equine chondrocytes expanded in monolayer culture

Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcript abundance and performed DNA bisulfite sequencing of upstream located elements. It seems that anabolic-like effects of chondrogenic differentiation were observed in form of NFATC1 and NADSYN1 upregulation in chondrocytes at the earlier stages of passaging whereas JAK3 upregulation at the 11th passage was the sign of chondrocytes dedifferentiation. Summarizing the inversely correlated DNA methylation and expression patterns in NFATC1 and JAK3 locus might be relevant for cellular dedifferentiation during chondrocyte expansion in monolayer. Obtained results are supportive for further studies on the role of encoded proteins in regenerative biology of articular cartilage using in vitro expanded chondrocytes.

Extracellular biomolecular free radical formation during injury

ObjectiveDetermine if oxidative damage increases in articular cartilage as a result of injury and matrix failure and whether modulation of the local redox environment influences this damage.Osteoarthritis is an age associated disease with no current disease modifying approaches available. Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical to early degeneration, but these mechanisms have not been described in intact tissue. To assess free radical production as a result of traumatic injury, we measured biomolecular free radical generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2 J/cm2 impact to swine articular cartilage explants. This technique allows visualization of free radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant increases with impact injury, particularly at sites of cartilage cracking. Increases remain in the absence of live chondrocytes but are diminished; thus, they appear to be a cell-dependent and -independent feature of injury. We then modulated the extracellular environment with a pulse of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology. Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study directly confirms the production of biomolecular free radicals from articular trauma, providing a rigorous characterization of their formation by injury.

Efficient TGF-β1 Delivery to Articular Chondrocytes In Vitro Using Agro-Based Liposomes.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-β1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-β1 to rat chondrocytes. Results showed that TGF-β1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-β1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Rare SLC13A1 variants associate with intervertebral disc disorder highlighting role of sulfate in disc pathology

Back pain is a common and debilitating disorder with largely unknown underlying biology. Here we report a genome-wide association study of back pain using diagnoses assigned in clinical practice; dorsalgia (119,100 cases, 909,847 controls) and intervertebral disc disorder (IDD) (58,854 cases, 922,958 controls). We identify 41 variants at 33 loci. The most significant association (ORIDD = 0.92, P = 1.6 × 10−39; ORdorsalgia = 0.92, P = 7.2 × 10−15) is with a 3’UTR variant (rs1871452-T) in CHST3, encoding a sulfotransferase enzyme expressed in intervertebral discs. The largest effects on IDD are conferred by rare (MAF = 0.07 − 0.32%) loss-of-function (LoF) variants in SLC13A1, encoding a sodium-sulfate co-transporter (LoF burden OR = 1.44, P = 3.1 × 10−11); variants that also associate with reduced serum sulfate. Genes implicated by this study are involved in cartilage and bone biology, as well as neurological and inflammatory processes.

Maturation- and degeneration-dependent articular cartilage metabolism via optical redox ratio imaging.

From the two metabolic processes in healthy cartilage, glycolysis has been associated with proliferation and oxidative phosphorylation (oxphos) with matrix synthesis. Recently, metabolic dysregulation was significantly correlated with cartilage degradation and osteoarthritis progression. While these findings suggest maturation predisposes cartilage to metabolic instability with consequences for tissue maintenance, these links have not been shown. Therefore, this study sought to address three hypotheses (a) chondrocytes exhibit differential metabolic activity between immaturity (0-4 months), adolescence (5-18 months), and maturity (>18 months); (b) perturbation of metabolic activity has consequences on expression of genes pertinent to cartilage tissue maintenance; and (c) severity of cartilage damage is positively correlated with glycolysis and oxphos activity as well as optical redox ratio in postadolescent cartilage. Porcine femoral cartilage samples from pigs (3 days to 6 years) underwent optical redox ratio imaging, which measures autofluorescence of NAD(P)H and FAD. Gene expression analysis and histological scoring was conducted for comparison against imaging metrics. NAD(P)H and FAD autofluorescence both demonstrated increasing intensity with age, while optical redox ratio was lowest in adolescent samples compared to immature or mature samples. Inhibition of glycolysis suppressed expression of Col2, Col1, ADAMTS4, and ADAMTS5, while oxphos inhibition had no effect. FAD fluorescence and optical redox ratio were positively correlated with histological degeneration. This study demonstrates maturation- and degeneration-dependent metabolic activity in cartilage and explores the consequences of this differential activity on gene expression. This study aids our basic understanding of cartilage biology and highlights opportunity for potential diagnostic applications.

An ECHO of Cartilage: In Silico Prediction of Combinatorial Treatments to Switch Between Transient and Permanent Cartilage Phenotypes With Ex Vivo Validation.

A fundamental question in cartilage biology is: what determines the switch between permanent cartilage found in the articular joints and transient hypertrophic cartilage that functions as a template for bone? This switch is observed both in a subset of OA patients that develop osteophytes, as well as in cell-based tissue engineering strategies for joint repair. A thorough understanding of the mechanisms regulating cell fate provides opportunities for treatment of cartilage disease and tissue engineering strategies. The objective of this study was to understand the mechanisms that regulate the switch between permanent and transient cartilage using a computational model of chondrocytes, ECHO. To investigate large signaling networks that regulate cell fate decisions, we developed the software tool ANIMO, Analysis of Networks with interactive Modeling. In ANIMO, we generated an activity network integrating 7 signal transduction pathways resulting in a network containing over 50 proteins with 200 interactions. We called this model ECHO, for executable chondrocyte. Previously, we showed that ECHO could be used to characterize mechanisms of cell fate decisions. ECHO was first developed based on a Boolean model of growth plate. Here, we show how the growth plate Boolean model was translated to ANIMO and how we adapted the topology and parameters to generate an articular cartilage model. In ANIMO, many combinations of overactivation/knockout were tested that result in a switch between permanent cartilage (SOX9+) and transient, hypertrophic cartilage (RUNX2+). We used model checking to prioritize combination treatments for wet-lab validation. Three combinatorial treatments were chosen and tested on metatarsals from 1-day old rat pups that were treated for 6 days. We found that a combination of IGF1 with inhibition of ERK1/2 had a positive effect on cartilage formation and growth, whereas activation of DLX5 combined with inhibition of PKA had a negative effect on cartilage formation and growth and resulted in increased cartilage hypertrophy. We show that our model describes cartilage formation, and that model checking can aid in choosing and prioritizing combinatorial treatments that interfere with normal cartilage development. Here we show that combinatorial treatments induce changes in the zonal distribution of cartilage, indication possible switches in cell fate. This indicates that simulations in ECHO aid in describing pathologies in which switches between cell fates are observed, such as OA.

MicroRNA Regulation of Bone Marrow Mesenchymal Stem Cell Chondrogenesis: Toward Articular Cartilage.

The production of a clinically useful engineered cartilage is an outstanding and unmet clinical need. High-throughput RNA sequencing provides a means of characterizing the molecular phenotype of populations of cells and can be leveraged to better understand differences among source cells, derivative engineered tissues, and target phenotypes. In this study, small RNA sequencing is utilized to comprehensively characterize the microRNA transcriptomes (miRNomes) of native human neonatal articular cartilage and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) differentiating into cartilage organoids, contrasting the microRNA regulation of engineered cartilage with that of a promising target phenotype. Five dominant microRNAs are upregulated during cartilage organoid differentiation and disproportionately regulate transcription factors: miR-148a-3p, miR-140-3p, miR-27b-3p, miR-140-5p, and miR-181a-5p. Two microRNAs that dominate the miRNomes of hBM-MSCs, miR-21-5p and miR-143-3p, persist throughout the differentiation process and may limit the ability of these cells to differentiate into an engineered cartilage resembling target native articular cartilage. By using predictive bioinformatics tools and antagomir inhibition, these persistent microRNAs are shown to destabilize the mRNA of genes with known or potential roles in cartilage biology including FGF18, TGFBR2, TET1, STOX2, ARAP2, N4BP2L1, LHX9, NFIA, and RPS6KA5. These results shed light on the extent to which only a few microRNAs contribute to the complex regulatory environment of hBM-MSCs for engineered tissues. Impact statement MicroRNAs are emerging as important controlling elements in the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). By using a robust bioinformatic approach and further validation in vitro, here we provide a comprehensive characterization of the microRNA transcriptomes (miRNomes) of a commonly studied and clinically promising source of multipotent cells (hBM-MSCs), a gold standard model of in vitro chondrogenesis (hBM-MSC-derived cartilage organoids), and an attractive in vivo target phenotype for clinically useful engineered cartilage (neonatal articular cartilage). These analyses highlighted a specific set of microRNAs involved in the chondrogenic program that could be manipulated to acquire a more robust articular cartilage-like phenotype. This characterization provides researchers in the cartilage tissue engineering field a useful atlas with which to contextualize microRNA involvement in complex differentiation pathways.