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Exploration of changes in spatial chondrocyte organisation in human osteoarthritic cartilage by means of 3D imaging.

ABSTRACT: Using two-dimensional top-down view microscopy, researchers have recently described chondrocytes as being spatially arranged in distinct patterns such as strings, double strings, and small and large clusters. Because of the seeming association of these changes with tissue degeneration, they have been proposed as an image-based biomarker for early osteoarthritis (OA) staging. The aim of our study was to investigate the spatial arrangement of chondrocytes in human articular cartilage in a 3D fashion and to evaluate the 3D changes of these patterns in the context of local tissue destruction. Decalcified femoral condyle resections from the load-bearing area were analysed in 3D for their spatial chondrocyte organisation by means of fluorescence microscopy and synchrotron-radiation micro-computed tomography (SR-µCT). In intact cartilage chondrocyte strings can be found in the superficial, transitional and deep zones. The proposed pattern changes accompanying tissue destruction could be located not just along the surface but also through all layers of cartilage. Each spatial pattern was characterised by a different cellular density (the only exception being between single and double strings with p = 0.062), with cellular density significantly increasing alongside the increase in local tissue degeneration as defined by the chondrocyte patterns. We can thus corroborate that the proposed cellular spatial changes are a three-dimensional function of local tissue degeneration, underlining their relevance as an image-based biomarker for the early diagnosis and description of OA.Clinical trial registration number: Project number of the ethics committee of the University of Tübingen:171/2014BO2.

SUBMITTER: Danalache M

PROVIDER: S-EPMC8105369 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveDegeneration of articular cartilage is central to the pathology of osteoarthritis (OA). However, the molecular mechanisms leading to these irreversible changes are still poorly understood. This study was undertaken to investigate how changes in the chondrocyte translational apparatus may contribute to the development and progression of knee OA.MethodsArticular cartilage from the knees of normal healthy subjects and patients with OA was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and nonlesional areas of the human OA cartilage were used to estimate the relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament of rats to investigate changes in the translational apparatus associated with OA. The role of interleukin-1β (IL-1β) signaling was assessed in vitro using rat articular chondrocytes. In human or rodent knee cartilage, messenger RNA expression was analyzed by quantitative polymerase chain reaction, and protein levels were determined by immunohistochemistry and Western blotting.ResultsSeveral novel traits of OA chondrocytes were identified, including up-regulation of the serine/threonine kinases Akt-2 and Akt-3 at the posttranscriptional level and an increased rate of total protein synthesis, likely attributable to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a known repressor of cap-dependent translation. Inactivation of 4E-BP1 was dependent on the activity of mechanistic target of rapamycin and was crucial for the up-regulation of protein synthesis in general and expression of matrix metalloproteinase 13 and ADAMTS-5 in particular. In addition, treatment of articular chondrocytes with IL-1β led to inactivation of 4E-BP1 and up-regulation of protein synthesis.ConclusionPrecise control of protein synthesis is vital for cartilage homeostasis, and its dysregulation contributes to the molecular pathology of OA. The results of this study therefore identify a novel set of potential therapeutic targets to ameliorate the effects of knee OA.

Project description:Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

Project description:Yougui pills (YGPs) have been used for centuries in the treatment of Chinese patients with Kidney-Yang Deficiency Syndrome. Despite the fact that the efficiency of YGPs on treating osteoarthritis has been verified in clinic, the underlying mechanisms are not totally understood. The present study observes the therapeutic role of YGPs and mechanisms underlying its chondroprotective action in osteoarthritic cartilage. To evaluate the chondroprotective effects of YGPs, we examined the impact of orally administered YGPs in a model of destabilization of the medial meniscus (DMM). Male C57BL/6J mice were provided a daily treatment of YGPs and a DMM surgery was performed on the right knee. At 12 weeks post-surgery, the joints were harvested for tissue analyses, including histomorphometry, OARSI scoring, micro-CT and immunohistochemistry for COL-2, MMP-13 and pSMAD-2. We also performed the relative experiments mentioned above in mice with Tgfbr2 conditional knockout (TGF-?RIICol2ER mice) in articular cartilage. To evaluate the safety of YGPs, hematology was determined in each group. Amelioration of cartilage degradation was observed in the YGPs group, with increases in cartilage area and thickness, proteoglycan matrix, and decreases in OARSI score at 12 weeks post surgery. In addition, reduced BV/TV and Tb. Th, and elevated Tb. Sp were observed in DMM-induced mice followed by YGPs treatment. Moreover, the preservation of cartilage correlated with reduced MMP-13, and elevated COL-2 and pSMAD-2 protein expressional levels were also revealed in DMM-induced mice treated with YGPs. Similarly, TGF-?RIICol2ER mice exhibited significant OA-like phenotype. However, no significant difference in cartilage structure was observed in TGF-?RIICol2ER mice after YGPs treatment. Interestingly, no obvious adverse effects were observed in mice from each group based on the hematologic analyses. These findings suggested that YGPs could inhibit cartilage degradation through enhancing TGF-?/Smad signaling activation, and be considered a good option for the treatment of osteoarthritis.

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Exploration of changes in spatial chondrocyte organisation in human osteoarthritic cartilage by means of 3D imaging.

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