Differences in pathways contributing to thyroid hormone effects on postnatal cartilage calcification versus secondary ossification center development

The proximal and distal femur epiphysis of mice are both weight bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH) in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in Thyroid stimulating hormone receptor (Tshr -/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo, and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Bsp, Ocn, Dmp1, Opn, and Alp). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.

Chondrocyte Adipogenic Differentiation in Softening Osteoarthritic Cartilage

A chondrocyte-to-osteoblast lineage continuum exists in the growth plate. Adipogenic differentiation of chondrocytes in vivo should be investigated. Here, unilateral anterior crossbite (UAC), which can induce osteoarthritic lesions in the temporomandibular joint (TMJ), was applied to 6-wk-old C57BL/6 mice. Matrix loss in TMJ cartilage was obvious, as demonstrated by safranin O staining, and the condylar cartilage elastic modulus values, detected by using atomic force microscopy (AFM), were reduced, indicating cartilage softening that might be linked with loss of the highly charged proteoglycan. By crossing the Rosa26/tdTomato (TdT) mice with Sox9;CreERT2 mice or with Col10;CreERT2 mice, we obtained the Sox9-TdT and Col10-TdT strains, respectively, in which the Sox9- or Col10-expressing cells, accordingly, were labeled by TdT. A few TdT-labeled cells in both strains expressed AdipoQ or DMP-1. The Sox9-TdT+AdipoQ+ cells were primarily located in the deep zone cartilage and then in the whole cartilage. Col10-TdT+AdipoQ+ cells, Sox9-TdT+DMP-1+ cells, and Col10-TdT+DMP-1+ cells were located in the deep zone region. UAC promoted AdipoQ and DMP-1 expression in cartilage. The percentages of Sox9-TdT+AdipoQ+ and Col10-TdT+AdipoQ+ cells to Sox9-TdT+ and Col10-TdT+ cells, respectively, were increased (both P < 0.05), implying that more chondrocytes were undergoing adipogenic differentiation in the UAC group, the cartilage of which was softened. The percentages of Sox9-TdT+DMP-1+ and Col10-TdT+DMP-1+ cells to Sox9-TdT+ cells and Col10-TdT+ cells, respectively, were increased (both P < 0.05), consistent with our report that UAC enhanced deep zone cartilage calcification, causing stiffening of the deep zone cartilage. Our present data demonstrated that TMJ chondrocyte descendants can become adipogenic in vivo in addition to becoming osteogenic. This potential was promoted in osteoarthritic cartilage, in which deep zone cartilage calcification-associated cartilage stiffening and proteoglycan loss-associated cartilage softening were both stimulated.

Association of Quantified Costal Cartilage Calcification and Long-Term Cumulative Blood Glucose Exposure: The Multi-Ethnic Study of Atherosclerosis

AimsAnecdotal reports have suggested increased soft tissue calcification in individuals with long-term exposures to high blood glucose. The association of costal cartilage calcification (CCC), a reliably quantifiable marker obtainable from non-contrast cardiac computed tomography (CT) with cumulative fasting blood glucose (FBG) exposure, is unknown. In this study, we aimed to determine the association between quantified CCC and cumulative glucose exposure using non-contrast coronary artery calcium (CAC) scoring computed tomography (CT) images in the Multi-Ethnic Study of Atherosclerosis (MESA).MethodsThe volume of bilateral CCC was quantified in high-density pixels (threshold of Hounsfield Unit&gt;180) using the CAC scoring CT images acquired in the 5th MESA exam. Prior long-term cumulative exposure to FBG was calculated by area under the FBG-time curve over ten years before the time of the CT exam.ResultsA total of 2,305 participants (mean age: 69, female/male: 1.3) were included in this study. The median CCC volume was lower in females than males (1158 mm3 [IQR: 1751] vs. 3054 mm3 [3851], p&lt;0.001). In cross-sectional analysis, quantified CCC was associated with FBG (9% increase per SD) and HbA1c (7% increase per SD) at the CT exam only in female participants after adjustment for age, race, BMI, and glomerular filtration rate. Only in female participants, quantified CCC was also associated with prior cumulative FBG (3% increase per decile change). In the subgroup of females with zero CAC scores, the adjusted CCC was still associated with FBG (13% increase per SD) at the time of CT exam and with prior cumulative FBG exposure (4% increase per decile change) before the CT exam.ConclusionsThe CCC, a reliably quantified marker in non-contrast cardiac CT, is associated with 10-year cumulative FBG exposure only in female participants, even those with zero CAC.

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage

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.

Abstract 13595: Association of Costal Cartilage Calcification With Cardiovascular Events and Risk Factors in Multi-Ethnic Study of Atherosclerosis (MESA); The MESArthritis Study

Introduction: Costal cartilage calcification (CCC) is an evident yet usually neglected finding in calcium scoring CT scans. Although CAC score is a well-known predictive marker for CVD, little is known about the potential association between CCC and CVD risk factors (RFs) and events. Hypothesis: CCC is associated with CVD RFs and is predictive of CVD events independent from CAC score. Methods: Cardiac CAC score images from Johns Hopkins Hospital field center acquired within the fifth exam of the MESA cohort (2010-2012) were re-analyzed as part of the MESArthritis ancillary study. The CCC was measured bilaterally for the first pair of cartilages at the superior end of image FOVs using calcium scoring package (VScore, Vitrea 7.11, Vital Images) with a 180 HU cut-off. Outlier values were excluded using the IQR-based outlier fence estimates. Association of CCC with CVD RFs, and CVD and mortality time-to-event was investigated by multivariable linear regression and Cox proportional hazard models, respectively. Results: After exclusion of 3 outliers, 329 participants (53% female) with a mean age of 70.1 (±8.75) were included. In cross-sectional analysis, in addition to age (β=8.86, p:.003) and gender (376.0, p<.001), CCC was associated with diabetes (141.42, p:.019) and glucose level (2.85, p:.006). CCC was correlated with Framingham global CVD risk score (FRS) (coefficient:0.39, p<.001), but not with CAC when adjusted for age and gender. In time-to-event analysis, adjusted CCC was positively associated with CHD (HR:1.17, p:.039) and CVD (1.14, p:.012) risk. Compared with CAC score (C-index: 0.76) and FRS (0.74) individually, models with CCC addition to FRS (0.85) and both CAC and FRS (0.84) had higher C-index for CHD prediction. Conclusions: CCC is associated with diabetes and can be used to predict CHD and CVD events and augment the predictive power of the highly validated CAC and FRS for CHD events. However, this scoring should be validated in other larger cohorts.