Chondrosarcoma with Target-Like Chondrocytes: Update on Molecular Profiling and Specific Morphological Features.

This is the first histological and molecular analysis of two chondrosarcomas with target-like chondrocytes that were compared with a group of conventional chondrosarcomas and enchondromas. The unique histological feature of target-like chondrocytes is the presence of unusual hypertrophic eosinophilic APAS-positive perichondrocytic rings (baskets). In the sections stained with Safranin O/Fast green, the outer part of the ring was blue and the material in the lacunar space stained orange, similarly to intercellular regions. Immunohistochemical examination showed strong positivity for vimentin, factor XIIIa, cyclin D1, osteonectin, B-cell lymphoma 2 apoptosis regulator (Bcl-2), p53 and p16. The S-100 protein was positive in 25 % of neoplastic cells. Antibodies against GFAP, D2-40 (podoplanin), CD99, CKAE1.3 and CD10 exhibited weak focal positivity. Pericellular rings/baskets contained type VI collagen in their peripheral part, in contrast to the type II collagen in intercellular interterritorial spaces. Ultrastructural examination revealed that pericellular rings contained an intralacunar component composed of microfibrils with abundant admixture of aggregates of dense amorphous non-fibrillar material. The outer extralacunar zone was made up of a layer of condensed thin collagen fibrils with admixture of non-fibrillar dense material. NGS sequencing identified a fusion transcript involving fibronectin 1 (FN1) and fibroblast growth factor receptor 2 (FGFR2) at the RNA level. At the DNA level, no significant variant was revealed except for the presumably germline variant in the SPTA1 gene.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

An injectable hydrogel having proteoglycan-like hierarchical structure supports chondrocytes delivery and chondrogenesis.

The ECM of cartilage is composed of proteoglycans (PG) that contain glycosaminoglycan (GAG), aggrecan, hyaluronic acid (HA) and other molecular components which play an important role in regulating chondrocyte functions via cell-matrix interactions, integrin-mediated signalling etc. Implantation of chondrocytes encapsulated in scaffolds that mimic the micro-architecture of proteoglycan, is expected to enhance cartilage repair. With an aim to create a hydrogel having macromolecular structure that resembles the cartilage-specific ECM, we constructed a hierarchal structure that mimic the PG. The bottle brush structure of the aggrecan was obtained using chondroitin sulphate and carboxymethyl cellulose which served as GAG and core protein mimic respectively. A proteoglycan-like structure was obtained by cross-linking it with modified chitosan that served as a HA substitute. The physico-chemical characteristics of the above cross-linked injectable hydrogel supported long term human articular chondrocyte subsistence and excellent post-injection viability. The chondrocytes encapsulated in the PMH expressed significant levels of articular cartilage specific markers like collagen II, aggrecan, GAGs etc., indicating the ability of the hydrogel to support chondrocyte differentiation. The biocompatibility and biodegradability of the hydrogels was confirmed using suitable in vivo studies. The results revealed that the PG-mimetic hydrogel could serve as a promising scaffold for chondrocyte implantation.

Local anisotropy in mineralized fibrocartilage and subchondral bone beneath the tendon-bone interface.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Bioorthogonal hydroxyethyl cellulose-based scaffold crosslinked via click chemistry for cartilage tissue engineering applications.

In this study, azide and alkyne moieties were introduced to the structure of citric acid-modified hydroxyethyl cellulose (HEC) and then through a bioorthogonal click chemistry method: Strain-promoted azide-alkyne cycloaddition, a novel crosslinked HEC scaffold (click sample) was obtained. Chemical modifications and successful crosslinking of the samples were assessed with FTIR and 1H NMR spectroscopy. Lyophilized samples exhibited a porous interconnected microarchitecture with desirable features for commensurate cartilage tissue engineering applications. As the stability of scaffolds improved upon crosslinking, considerable water uptake and swelling degree of ~650% could still be measured for the click sample. Offering Young's modulus of ~10 MPa and tensile strength of ~0.43 MPa, the mechanical characteristics of click sample were comparable with those of normal cartilage tissue. Various in vitro biological assays, including MTT analysis, cellular attachment, histological staining with safranin O, and real-time PCR decisively approved significant biocompatibility, chondrogenic ability, and bioorthogonal features of click sample.

The hypertrophic chondrocyte: To be or not to be.

Hypertrophic chondrocytes are the master regulators of endochondral ossification; however, their ultimate cell fates cells remain largely elusive due to their transient nature. Historically, hypertrophic chondrocytes have been considered as the terminal state of growth plate chondrocytes, which are destined to meet their inevitable demise at the primary spongiosa. Chondrocyte hypertrophy is accompanied by increased organelle synthesis and rapid intracellular water uptake, which serve as the major drivers of longitudinal bone growth. This process is delicately regulated by major signaling pathways and their target genes, including growth hormone (GH), insulin growth factor-1 (IGF-1), indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic proteins (BMPs), sex determining region Y-box 9 (Sox9), runt-related transcription factors (Runx) and fibroblast growth factor receptors (FGFRs). Hypertrophic chondrocytes orchestrate endochondral ossification by regulating osteogenic-angiogenic and osteogenic-osteoclastic coupling through the production of vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kappa-B ligand (RANKL) and matrix metallopeptidases-9/13 (MMP-9/13). Hypertrophic chondrocytes also indirectly regulate resorption of the cartilaginous extracellular matrix, by controlling formation of a special subtype of osteoclasts termed "chondroclasts". Notably, hypertrophic chondrocytes may possess innate potential for plasticity, reentering the cell cycle and differentiating into osteoblasts and other types of mesenchymal cells in the marrow space. We may be able to harness this unique plasticity for therapeutic purposes, for a variety of skeletal abnormalities and injuries. In this review, we discuss the morphological and molecular properties of hypertrophic chondrocytes, which carry out important functions during skeletal growth and regeneration.

Moderate mechanical stress suppresses the IL-1ß-induced chondrocyte apoptosis by regulating mitochondrial dynamics.

Mitochondrial dysfunction contributes to osteoarthritis (OA) onset and progress. Mitochondrial dynamics, coupled with mitophagy, is critical for the maintenance of mitochondrial fitness, involving many cellular processes, such as proliferation and apoptosis. Excessive mechanical stress induces chondrocyte apoptosis; however, the effects of mechanical stress on mitochondrial dynamics remain elusive. In this study, we performed fluorescence staining, flow cytometry, transmission electron microscope, Western blot analysis, and RNA-sequencing to assess the effects of different strength of mechanical stimulation on mitochondrial functions of chondrocyte treated with interleukin-1ß (IL-1ß). We found that moderate mechanical stress reduced the IL-1ß-induced apoptosis by maintaining mitochondrial function and scavenging the reactive oxygen species, while excessive mechanical stress induced strong mitochondrial dysfunction and apoptosis. Moreover, RNAsequencing revealed that mitophagy and mitochondrial dynamics were involved in the regulation of mechanical stress on chondrocyte biology. In addition to the elevated mitophagy, moderate mechanical stress also promoted mitochondrial dynamics by enhancing the expression of MFN1/2 and OPA1 and the translocation of dynamin-related protein 1 from the cytoplasm to the mitochondria. However, an uncoupling of mitochondrial dynamics, characterized by strongly elevated fission, resulted in the unfavorable apoptosis of excessive mechanical stress-stimulated chondrocytes. This study revealed the effects of mechanical stress upon mitochondrial dynamics in chondrocyte.

Ultrastructural analysis of different skeletal cell types in mucopolysaccharidosis dogs at the onset of postnatal growth.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Cell Lineage Tracing: Colocalization of Cell Lineage Markers with a Fluorescent Reporter.

Cell lineage tracing, an old technique which originated in the nineteenth century, regains popularity and relevance due to introduction of a more sensitive tomato fluorescent protein under the control of a ubiquitous promoter (Rosa 26 gene). In addition, various tissue specific CreERT2 mouse lines are widely available, making cell lineage tracing studies more specific and powerful. In this protocol, we provide a practical guide for researchers to map progeny of specific cells such as chondrocytes during development using a fluorescent reporter (tomato, red) and multiple chondrocyte Cre lines. Further, we provide valuable examples in which these tracing lines, combined with a bone reporter mouse line (2.3 Col 1a1-GFP) or costained with different immunofluorescent proteins, revealed how a chondrocyte transdifferentiates into a bone cell in vivo.

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptg knockout (Gnptgko ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgko chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptab knock-in (Gnptabki ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.

Structure and mechanical properties of high-weight-bearing and low-weight-bearing areas of hip cartilage at the micro- and nano-levels.

BACKGROUND: Articular cartilage has a high-weight-bearing area and a low-weight-bearing area, the macroscopic elastic moduli of the two regions are different. Chondrocytes are affected by the applied force at the microscopic level. Currently, the modulus of the two areas at the micro and nano levels is unknown, and studies on the relationship between macro-, micro- and nano-scale elastic moduli are limited. Such information may be important for further understanding of cartilage mechanics. Moreover, the surface morphology, proteoglycan content, and micro and nano structure of the two areas, which influences the mechanical properties of cartilage should be discussed. METHODS: Safranin-O/Fast Green staining was used to evaluate the surface morphology and semi-quantify proteoglycan content of porcine femoral head cartilage between the two weight-bearing areas. The unconfined compression test was used to determine the macro elastic modulus. Atomic force microscope was used to measure the micro and nano compressive elastic modulus as well as the nano structure. Scanning electron microscope was employed to evaluate the micro structure. RESULTS: No significant differences in the fibrillation index were observed between two areas (P = 0.5512). The Safranin-O index of the high-weight-bearing area was significantly higher than that of the low-weight-bearing area (P = 0.0387). The compressive elastic modulus of the high-weight-bearing area at the macro and micro level was significantly higher than that of the low-weight-bearing area (P = 0.0411 for macro-scale, and P = 0.0001 for micro-scale), while no statistically significant differences were observed in the elastic modulus of collagen fibrils at the nano level (P = 0.8544). The density of the collagen fibers was significantly lower in the high-weight-bearing area (P = 0.0177). No significant differences were observed in the structure and diameter of the collagen fibers between the two areas (P = 0.7361). CONCLUSIONS: A higher proteoglycan content correlated with a higher compressive elastic modulus of the high-weight-bearing area at the micro level than that of the low-weight-bearing area, which was consistent with the trend observed from the macroscopic compressive elastic modulus. The weight-bearing level was not associated with the elastic modulus of individual collagen fibers and the diameter at the nano level. The micro structure of cartilage may influence the macro- and micro-scale elastic modulus.

The endoplasmic reticulum stress induced by tunicamycin affects the viability and autophagy activity of chondrocytes.

Osteoarthritis (OA) is attributed to a reduction in chondrocytes within joint cartilage, and research has shown that endoplasmic reticulum (ER) stress and autophagy play important roles in the survival of chondrocytes. However, the relationship between ER stress and autophagy in chondrocytes remains unclear. In this study, we investigated the changes in apoptotic and autophagic activity in chondrocytes under ER stress. Following treatment with tunicamycin, the rate of apoptosis among chondrocytes increased. Western blot analysis showed the levels of unfolded protein response (UPR) related proteins increased, followed by elevated expression of light chain 3B-II (LC3B-II) and Beclin-1. An ultrastructural investigation showed that a large number of pre-autophagosomal structures or autophagosomes formed under tunicamycin treatment. However, the autophagy activity was significantly inhibited in chondrocytes after suppression of GRP78 by siRNA. The apoptosis ratio of chondrocytes pre-treated with 3-methyladenine was much higher than that of normal chondrocytes after exposure to tunicamycin. Our study revealed that the tunicamycin-induced persistent UPR expression led to apoptosis of chondrocytes and activation of autophagy incorporation with GRP78. Blocking autophagy accelerated the apoptosis induced by ER stress, which confirmed the protective function of autophagy in the homeostasis of chondrocytes. These findings advance our understanding of chondrocyte apoptosis and provide potential molecular targets for preventing apoptotic death of chondrocytes.

Imperatorin suppresses IL-1ß-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes.

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1ß (IL-1ß) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 µM) followed by IL-1ß (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1ß induced expression of iNOS and NO production. Imperatorin blocked the IL-1ß-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Therapeutic Potential of Dental Pulp Stem Cells and Leukocyte- and Platelet-Rich Fibrin for Osteoarthritis.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.

Phenome-based approach identifies RIC1-linked Mendelian syndrome through zebrafish models, biobank associations and clinical studies.

Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.

Loss of autophagy in chondrocytes causes severe growth retardation.

Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of Atg7, a gene essential for autophagy. Mice lacking Atg7 after the transition exhibited phenotypes severer than mutant mice in which Atg7 was removed before the transition. Atg7-deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates.Abbreviations: DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1.

MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint.

A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early stage both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes. At late stage, MTORC1-p-EIF2AK3-mediated ERS apoptosis were predominant, while p-ERN1 and autophagic flux were inhibited. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis, and reduced TMJ cartilage loss induced by UAC. In contrast, MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1, an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA.Abbreviations : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A1; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4',6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.

Three-dimensional architecture of the acetabular labrum in the human hip joint.

The acetabular labrum is frequently damaged with advancing age. As collagen fibers are the main sources of strength, knowledge of their ultrastructure is important to determine the cause of age-induced changes. We aimed to investigate the ultrastructure of collagen fibers constituting the acetabular labrum using scanning electron microscopy (SEM). Acetabular labrum samples obtained during total hip arthroplasty were studied. The samples were specially prepared to observe the steric construction of collagen fibrils constituting the acetabular labrum under light microscopy followed by SEM. The acetabular labrum was mostly composed of cartilage tissue, consisting of chondrocytes and collagen type II, with a layer of collagen type I. In adults, chondrocytes with a rich cytoplasm were surrounded by a dense network of fine type II collagen fibrils, and small bundles of type I collagen fibrils were interposed in the cartilage layer. In elderly individuals, the chondrocytes atrophied and both type I and II collagen fibrils were sparse. We suggest that cartilage has three to five layers, consisting of type I and type II collagen fibrils with a solid cartilage substrate. In elderly individuals, the density of chondrocytes decreases and the cellular shape and architecture of collagen fibrils also changes.