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Objective: To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes., Methods: Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs., Results: Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2., Conclusions: CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Recent developments on high resolution micro computed tomography (μCT) allow imaging of soft tissues in small animal joints. Nevertheless, μCT images cannot distinguish soft tissues from synovial fluid due to their similar mass density, limiting the 3D assessment of soft tissues volume and thickness. This study aimed to evaluate a lead chromate contrast agent for μCΤ arthrography of rat knee joints ex vivo., Design: Intact tibiofemoral rat joints were injected with the contrast agent at different concentrations and imaged using a μCT at 2.7 μm isotropic voxel size. Cartilage thickness was measured using an automated procedure, validated against histological measurements, and analyzed as a function of μCT image resolution. Changes in hard and soft tissues were also analyzed in tibiofemoral joints 4 weeks after surgical destabilization of the medial meniscus (DMM)., Results: The contrast agent diffused well throughout the whole knee cavity without penetrating the tissues, therefore providing high contrast at the boundaries between soft tissues and synovial fluid space. Thickness analysis of cartilage demonstrated a high similarity between histology and μ-arthrography approaches (R(2) = 0.90). Four weeks after surgical DMM, the development of osteophytes (Oph) and cartilage ulcerations was recognizable with μCT, as well as a slight increase in trabecular bone porosity, and decrease in trabecular thickness., Conclusions: A lead chromate-based contrast agent allowed discriminating the synovial fluid from soft tissues of intact knee joints, and thus made possible both qualitative and quantitative assessment of hard and soft tissues in both intact and DMM tibiofemoral joints using high resolution μCT., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)