Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate.

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2CreERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

The role of hypertrophic chondrocytes in regulation of the cartilage-to-bone transition in fracture healing.

Endochondral bone formation is an important pathway in fracture healing, involving the formation of a cartilaginous soft callus and the process of cartilage-to-bone transition. Failure or delay in the cartilage-to-bone transition causes an impaired bony union such as nonunion or delayed union. During the healing process, multiple types of cells including chondrocytes, osteoprogenitors, osteoblasts, and endothelial cells coexist in the callus, and inevitably crosstalk with each other. Hypertrophic chondrocytes located between soft cartilaginous callus and bony hard callus mediate the crosstalk regulating cell-matrix degradation, vascularization, osteoclast recruitment, and osteoblast differentiation in autocrine and paracrine manners. Furthermore, hypertrophic chondrocytes can become osteoprogenitors and osteoblasts, and directly contribute to woven bone formation. In this review, we focus on the roles of hypertrophic chondrocytes in fracture healing and dissect the intermingled crosstalk in fracture callus during the cartilage-to-bone transition.

Activating EGFR Signaling Attenuates Osteoarthritis Development Following Loading Injury in Mice.

Posttraumatic osteoarthritis (PTOA) results in joint pain, loss of joint function, and impaired quality of daily life in patients with limited treatment options. We previously demonstrated that epidermal growth factor receptor (EGFR) signaling is essential for maintaining chondroprogenitors during articular cartilage development and homeostasis. Here, we used a nonsurgical, loading-induced PTOA mouse model to investigate the protective action of EGFR signaling. A single bout of cyclic tibial loading at a peak force of 6 N injured cartilage at the posterior aspect of lateral femoral condyle. Similar loading at a peak force of 9 N ruptured the anterior cruciate ligament, causing additional cartilage damage at the medial compartment and ectopic cartilage formation in meniscus and synovium. Constitutively overexpression of an EGFR ligand, heparin binding EGF-like growth factor (HBEGF), in chondrocytes significantly reduced cartilage injury length, synovitis, and pain after 6 N loading and mitigated medial side cartilage damage and ectopic cartilage formation after 9 N loading. Mechanistically, overactivation of EGFR signaling protected chondrocytes from loading-induced apoptosis and loss of proliferative ability and lubricant synthesis. Overexpressing HBEGF in adult cartilage starting right before 6 N loading had similar beneficial effects. In contrast, inactivating EGFR in adult cartilage led to accelerated PTOA progression with elevated cartilage Mankin score and synovitis score and increased ectopic cartilage formation. As a therapeutic approach, we constructed a nanoparticle conjugated with the EGFR ligand TGFα. Intra-articular injections of this nanoconstruct once every 3 weeks for 12 weeks partially mitigated PTOA symptoms in cartilage and synovium after 6 N loading. Our findings demonstrate the anabolic actions of EGFR signaling in maintaining articular cartilage during PTOA development and shed light on developing a novel nanomedicine for PTOA. © 2022 American Society for Bone and Mineral Research (ASBMR).

EGFR Signaling Is Required for Maintaining Adult Cartilage Homeostasis and Attenuating Osteoarthritis Progression.

The uppermost superficial zone of articular cartilage is the first line of defense against the initiation of osteoarthritis (OA). We previously used Col2-Cre to demonstrate that epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, plays an essential role in maintaining superficial chondrocytes during articular cartilage development. Here, we showed that EGFR activity in the articular cartilage decreased as mice age. In mouse and human OA samples, EGFR activity was initially reduced at the superficial layer and then resurged in cell clusters within the middle and deep zone in late OA. To investigate the role of EGFR signaling in postnatal and adult cartilage, we constructed an inducible mouse model with cartilage-specific EGFR inactivation (Aggrecan-CreER EgfrWa5/flox , Egfr iCKO). EdU incorporation revealed that postnatal Egfr iCKO mice contained fewer slow-cycling cells than controls. EGFR deficiency induced at 3 months of age reduced cartilage thickness and diminished superficial chondrocytes, in parallel to alterations in lubricin production, cell proliferation, and survival. Furthermore, male Egfr iCKO mice developed much more severe OA phenotypes, including cartilage erosion, subchondral bone plate thickening, cartilage degeneration at the lateral site, and mechanical allodynia, after receiving destabilization of the medial meniscus (DMM) surgery. Similar OA phenotypes were also observed in female iCKO mice. Moreover, tamoxifen injections of iCKO mice at 1 month post-surgery accelerated OA development 2 months later. In summary, our data demonstrated that chondrogenic EGFR signaling maintains postnatal slow-cycling cells and plays a critical role in adult cartilage homeostasis and OA progression. © 2022 American Society for Bone and Mineral Research (ASBMR).

Automatic Detection of Medial and Lateral Compartments from Histological Sections of Mouse Knee Joints Using the Single-Shot Multibox Detector Algorithm.

OBJECTIVE: Although mouse osteoarthritis (OA) models are widely used, their histological analysis may be susceptible to arbitrariness and inter-examiner variability in conventional methods. Therefore, a method for the unbiased scoring of OA histology is needed. In this study, as the first step for establishing this system, we developed a computer-vision algorithm that automatically detects the medial and lateral compartments of mouse knee sections in a rigorous and unbiased manner. DESIGN: A total of 706 images of coronal sections of mouse knee joints stained by hematoxylin and eosin, safranin O, or toluidine blue were randomly divided into training and validation images at a ratio of 80:20. A model to detect both compartments automatically was built by machine learning using a single-shot multibox detector (SSD) algorithm with training images. The model was tested to determine whether it could accurately detect both compartments by analyzing the validation images and 52 images of sections stained with Picrosirius red, a method not used for the training images. RESULTS: The trained model accurately detected both medial and lateral compartments of all 140 validation images regardless of the staining method employed, severity of articular cartilage defects, and the anatomical positions and conditions of the sections. Our model also correctly detected both compartments of 50 of 52 Picrosirius red-stained images. CONCLUSIONS: By applying deep learning based on the SSD algorithm, we successfully developed a model that detects the locations of the medial and lateral compartments of tissue sections of mouse knee joints with high accuracy.

4PBA reduces growth deficiency in osteogenesis imperfecta by enhancing transition of hypertrophic chondrocytes to osteoblasts.

Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood, and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts, and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.

Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving the synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-d-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment, and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups was higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from the upregulated expression of genes involved the synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization.

Osteochondroma Pathogenesis: Mouse Models and Mechanistic Insights into Interactions with Retinoid Signaling.

Osteochondromas are cartilage-capped tumors that arise near growing physes and are the most common benign bone tumor in children. Osteochondromas can lead to skeletal deformity, pain, loss of motion, and neurovascular compression. Currently, surgery is the only available treatment for symptomatic osteochondromas. Osteochondroma mouse models have been developed to understand the pathology and the origin of osteochondromas and develop therapeutic drugs. Several cartilage regulatory pathways have been implicated in the development of osteochondromas, such as bone morphogenetic protein, hedgehog, and WNT/ß-catenin signaling. Retinoic acid receptor-γ is an important regulator of endochondral bone formation. Selective agonists for retinoic acid receptor-γ, such as palovarotene, have been investigated as drugs for inhibition of ectopic endochondral ossification, including osteochondromas. This review discusses the signaling pathways involved in osteochondroma pathogenesis and their possible interactions with the retinoid pathway.

Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated.

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans.

OBJECTIVE: The purposes of this study are to evaluate which growth plate parameters are associated with bone growth in mice and to compare the mouse results with those in humans. DESIGN: The sagittal sections of the proximal growth plate of the mouse tibia from neonate to young adult stages were subjected to histomorphometric and functional analyses. The radiographic images of tibias of human patients until puberty were analyzed to obtain the tibia length and the proximal growth plate height. It was found that a linear correlation best modeled the relationship between the growth plate variables with the tibia growth rate and length. RESULTS: In mice, total height, resting zone height, combined height of the proliferation and prehypertrophic zones, proliferation activity, and the total width of tibia growth plate showed high linear correlation with tibia bone length and bone growth rate, but the hypertrophic zone height and the growth plate area did not. In both mice and humans, the total growth plate width of tibia was found to have the strongest correlation with tibia length and growth rate. CONCLUSIONS: The results validated that growth plate total height, the height of the resting zone and cell proliferation activity are appropriate parameters to evaluate the balance between growth plate activity and bone growth in mice, consistent with previous reports. The study also provided a new growth plate parameter candidate, growth plate width for growth plate activity evaluation in both mouse and human tibia bone.

Decorin regulates cartilage pericellular matrix micromechanobiology.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2-4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Early changes in cartilage pericellular matrix micromechanobiology portend the onset of post-traumatic osteoarthritis.

The pericellular matrix (PCM) of cartilage is a structurally distinctive microdomain surrounding each chondrocyte, and is pivotal to cell homeostasis and cell-matrix interactions in healthy tissue. This study queried if the PCM is the initiation point for disease or a casualty of more widespread matrix degeneration. To address this question, we queried the mechanical properties of the PCM and chondrocyte mechanoresponsivity with the development of post-traumatic osteoarthritis (PTOA). To do so, we integrated Kawamoto's film-assisted cryo-sectioning with immunofluorescence-guided AFM nanomechanical mapping, and quantified the microscale modulus of murine cartilage PCM and further-removed extracellular matrix. Using the destabilization of the medial meniscus (DMM) murine model of PTOA, we show that decreases in PCM micromechanics are apparent as early as 3 days after injury, and that this precedes changes in the bulk ECM properties and overt indications of cartilage damage. We also show that, as a consequence of altered PCM properties, calcium mobilization by chondrocytes in response to mechanical challenge (hypo-osmotic stress) is significantly disrupted. These aberrant changes in chondrocyte micromechanobiology as a consequence of DMM could be partially blocked by early inhibition of PCM remodeling. Collectively, these results suggest that changes in PCM micromechanobiology are leading indicators of the initiation of PTOA, and that disease originates in the cartilage PCM. This insight will direct the development of early detection methods, as well as small molecule-based therapies that can stop early aberrant remodeling in this critical cartilage microdomain to slow or reverse disease progression. STATEMENT OF SIGNIFICANCE: Post-traumatic osteoarthritis (PTOA) is one prevalent musculoskeletal disease that afflicts young adults, and there are no effective strategies for early detection or intervention. This study identifies that the reduction of cartilage pericellular matrix (PCM) micromodulus is one of the earliest events in the initiation of PTOA, which, in turn, impairs the mechanosensitive activities of chondrocytes, contributing to the vicious loop of cartilage degeneration. Rescuing the integrity of PCM has the potential to restore normal chondrocyte mechanosensitive homeostasis and to prevent further degradation of cartilage. Our findings enable the development of early OA detection methods targeting changes in the PCM, and treatment strategies that can stop early aberrant remodeling in this critical microdomain to slow or reverse disease progression.

Wnt signaling in chondroprogenitors during long bone development and growth.

Wnt signaling together with other signaling pathways governs cartilage development and the growth plate function during long bone formation and growth. ß-catenin-dependent Wnt signaling is a specific lineage determinant of skeletal mesenchymal cells toward chondrogenic or osteogenic direction. Once cartilage forms and the growth plate organize, Wnt signaling continues to regulate proliferation and differentiation of the growth plate chondrocytes. Although chondrocytes in the growth plate have a high capacity to proliferate, new cells must be supplied to the growth plate from chondroprogenitor population. Advances in in vivo cell tracking techniques have demonstrated the importance of Wnt signaling in driving tissue renewal. The Wnt-responsive cells, genetically marked by the Wnt-reporter system, are found as stem cells in various tissues. Similarly, Wnt-responsive cells are found in the periphery of the growth plate and expanded to constitute entire column structure, indicating that Wnt signaling participates in the regulation of chondroprogenitors in the growth plate. This review will discuss advancements in research of progenitors in the growth plate, specifically focusing on Wnt/ß-catenin signaling.

Understanding the Action of RARγ Agonists on Human Osteochondroma Explants.

Osteochondromas are cartilage-capped growths located proximate to the physis that can cause skeletal deformities, pain, limited motion, and neurovascular impingement. Previous studies have demonstrated retinoic acid receptor gamma (RARγ) agonists to inhibit ectopic endochondral ossification, therefore we hypothesize that RARγ agonists can target on established osteochondromas. The purpose of this study was to examine the action of RARγ agonist in human osteochondromas. Osteochondroma specimens were obtained during surgery, subjected to explant culture and were treated with RARγ agonists or vehicles. Gene expression analysis confirmed the up-regulation of RARγ target genes in the explants treated with NRX 204647 and Palovarotene and revealed strong inhibition of cartilage matrix and increased extracellular matrix proteases gene expression. In addition, immunohistochemical staining for the neoepitope of protease-cleaved aggrecan indicated that RARγ agonist treatment stimulated cartilage matrix degradation. Interestingly, cell survival studies demonstrated that RARγ agonist treatment stimulated cell death. Moreover, RNA sequencing analysis indicates changes in multiple molecular pathways due to RARγ agonists treatment, showing similarly to human growth plate chondrocytes. Together, these findings suggest that RARγ agonist may exert anti-tumor function on osteochondromas by inhibiting matrix synthesis, promoting cartilage matrix degradation and stimulating cell death.

Influence of Genetic Background and Sex on Gene Expression in the Mouse (Mus musculus) Tail in a Model of Intervertebral Disc Injury.

To facilitate rational experimental design and fulfill the NIH requirement of including sex as a biologic variable, we examined the influences of genetic background and sex on responses to intervertebral disc (IVD) injury in the mouse tail. The goal of this study was to compare gene expression and histologic changes in response to a tail IVD injury (needle puncture) in male and female mice on the DBA and C57BL/6 (B6) backgrounds. We hypothesized that extracellular matrix gene expression in response to IVD injury differs between mice of different genetic backgrounds and sex. Consistent changes were detected in gene expression and histologic features after IVD injury in mice on both genetic backgrounds and sexes. In particular, expression of col1a1 and adam8 was higher in the injured IVD of DBA mice than B6 mice. Conversely, col2a1 expression was higher in B6 mice than DBA mice. Sex-associated differences were significant only in B6 mice, in which col2a1 expression was greater in male mice than in female. Histologic differences in response to injury were not apparent between DBA and B6 mice or between males and females. In conclusion, mouse tail IVD showed sex- and strain-related changes in gene expression and histology after needle puncture. The magnitude of change in gene expression differed with regard to genetic background and, to a lesser degree, sex.

Mediation of Cartilage Matrix Degeneration and Fibrillation by Decorin in Post-traumatic Osteoarthritis.

OBJECTIVE: To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS: Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (Dcni KO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1ß (IL-1ß) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1ß and release of sGAGs were quantified. RESULTS: In both Dcn-/- and Dcni KO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1ß, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION: In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus.

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1+/- mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS-2/8 Chondrosarcoma Cells.

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020.

Decorin Regulates the Aggrecan Network Integrity and Biomechanical Functions of Cartilage Extracellular Matrix.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.