Advances in current clinical modalities, including magnetic resonance imaging and computed tomography, allow for earlier diagnoses of cartilage damage that could mitigate progression to osteoarthritis. However, current imaging modalities do not detect submicrometer damage. Developments in in vivo or arthroscopic techniques, including optical coherence tomography, ultrasonography, bioelectricity including streaming potential measurement, noninvasive electroarthrography, and multiphoton microscopy can detect damage at an earlier time point, but they are limited by a lack of penetration and the ability to assess an entire joint. This article reviews current advancements in clinical and developing modalities that can aid in the early diagnosis of cartilage injury and facilitate studies of interventional therapeutics.

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long-(96 h) or short-(2 and 30 min) term exposure to interleukin-1a (IL-1a), IL-6, or IL-8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase-13 (MMP-13) gene expression after prolonged cytokine exposure. Effects of short-term treatment with IL-a, IL-6, or IL-8 on endogenous GTP-bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP-13 expression. Short exposure to IL-1a, IL-6, or IL-8 decreased endogenous GTP-Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL-mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis.

Objective The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury. Methods Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and were injured by loading with 30 MPa within 1 second. Fractional dissipated energy, cell density, cell death, and gene expression were quantified. Results PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88–101%). Cell density was most dense in superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly higher cell death (13.5%, 95% CI 9.1–17.9%) than non-injured samples (6.8%, 95% CI 2.5–11.1%, p=0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp=21, 95% CI −80–122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64–213). Conclusion Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA. Clinical Relevance Understanding differences in the response to injury and potential susceptibility of OA can lead to the development of preventative or treatment strategies.

Objective Cartilage injury can lead to post-traumatic osteoarthritis (PTOA). Immediate post-trauma cellular and structural changes are not widely understood. Furthermore, current cellular-resolution cartilage imaging techniques require sectioning of cartilage and/or use of dyes not suitable for patient imaging. In this study, we used multiphoton microscopy (MPM) data with FDA-approved sodium fluorescein to identify and evaluate the pattern of chondrocyte death after traumatic injury. Method Mature equine distal metacarpal or metatarsal osteochondral blocks were injured by 30 MPa compressive loading delivered over 1 sec. Injured and control sites were imaged unfixed and in situ 1 hour post-injury with sodium fluorescein using rasterized z-scanning. MPM data was quantified in MATLAB, reconstructed in 3-D, and projected in 2-D to determine the damage pattern. Results MPM images (600 per sample) were reconstructed and analyzed for cell death. The overall distribution of cell death appeared to cluster into circular (n=7) or elliptical (n=4) patterns (p=0.006). Dead cells were also prevalent near cracks in the matrix, with only 26.3% (SE=5.0%, p<0.0001) of chondrocytes near cracks being viable. Conclusion This study demonstrates the first application of MPM for evaluating cellular-scale cartilage injury in situ in live tissue, with clinical potential for detecting early cartilage damage. With this technique, we were able to uniquely observe two death patterns resulting from the same compressive loading, which may be related to local variability in matrix structure. These results also demonstrate proof-of-concept MPM diagnostic use in detecting subtle and early cartilage damage not detectable in any other way.

Small GTPases regulate the cytoskeleton and numerous other cellular functions. In this study, the role of Rho GTPase was examined in articular chondrocytes. Chondrocytes grown in monolayer were treated with interleukin-1a (IL-1a), insulin-like growth factor-I (IGF-I), C3 Transferase, Y27632, or transfected with Rho wild type or two constitutively active mutants of Rho (Q63L and G14V). Quantitative PCR was used to determine changes in matrix metalloproteinase-13 (MMP-13), collagen types IIB (COL2A1) and type I (COL1A1), aggrecan (AGG), and SOX-9 gene expression. Affinity assays were performed to measure endogenous GTP-bound Rho, and confocal microscopy was used to assess changes in organization of the actin cytoskeleton. IL-1a and RhoG14V increased cytoplasmic actin stress fiber formation, which was blocked by C3 Transferase, and Y27632. IL-1a treatment also increased Rho activity. Conversely, IGF-I lead to formation of a cortical rim of actin and decreased Rho activity. Inhibition of Rho signaling with C3 Transferase significantly decreased Rho activity and returned IL-1a-induced Rho activity to a level not different from control. C3 Transferase treatment also increased mRNA expression of AGG, COL2A1, and SOX-9, and decreased expression of MMP-13. Expression of RhoQ63L or RhoG14V resulted in increased MMP-13 expression; however, inhibition of Rho with Y27632 was unable to inhibit IL-1a-induced MMP-13 expression. Together, these results indicate a role for increased Rho activity in mediation of chondrocyte catabolic signaling pathways. [3][4][5][6][7][8][9] Changes in cell morphology and organization of the cytoskeleton have also been associated with OA. 10,11 Defining basic intracellular signaling pathways that regulate chondrocyte phenotypic expression and morphology might further elucidate the pathogenesis of OA and reveal novel therapeutic targets.One signaling pathway that has been implicated in regulation of articular chondrocyte morphology and phenotype is the Rho subfamily of small GTPases. [4][5][6][7][8][9] The Rho subfamily of small GTPases are 21-22-kDa proteins involved in many cellular processes such as regulation of actin cytoskeleton and morphology, cell cycle progression, and integrin assembly (see Schwartz 12 for review). The Rho subfamily includes Cdc42, Rac, and Rho, which cycle between active GTP-bound and inactive GDP-bound states. Activation of Rho GTPases results in reorganization of the cytoskeleton, and only when they are in their active form can they affect downstream targets. 13 The three variants of Rho, RhoA, RhoB, and RhoC, share over 85% homology and have nearly indistinguishable actions and interactions, so the more general term Rho will be used herein. 14 The roles of small GTPases in articular chondrocytes have been partially elucidated. 8,9,15 Treatment with insulin-like growth factor-I (IGF-I) reduces actin stress fibers, diminishes activation of Cdc42 and Rac, and is associated with preservation of the normal chondrocyte phenotype. 8 In chondrocytes, assembly of an...