Superficial Zone Of Cartilage Research Articles

Elevated glucose promotes MMP13 and ADAMTS5 production by osteoarthritic chondrocytes under oxygenated but not hypoxic conditions.

Type 2 diabetes is linked with increased incidence and severity of osteoarthritis. The purpose of this study was to determine the effect of extracellular glucose within the normal blood glucose and hyperglycemic range on catabolic enzyme production by chondrocytes isolated from osteoarthritic (OA) and macroscopically normal (MN) human cartilage under oxygenated (18.9% oxygen) and hypoxic (1% oxygen) conditions. OA and MN chondrocytes were maintained in 4, 6, 8, or 10 mM glucose for 24 h. Glucose consumption, GLUT1 glucose transporter levels, MMP13 and ADAMTS5 production, and levels of RUNX2, a transcriptional regulator of MMP13, ADAMTS5, and GLUT1, were assessed by enzyme-linked assays, RT-qPCR and/or western blot. Under oxygenated conditions, glucose consumption and GLUT1 protein levels were higher in OA but not MN chondrocytes in 10 mM glucose compared to 4 mM. Both RNA and protein levels of MMP13 and ADAMTS5 were also higher in OA but not MN chondrocytes in 10 mM compared to 4 mM glucose under oxygenated conditions. Expression of RUNX2 was overall lower in MN than OA chondrocytes and there was no consistent effect of extracellular glucose concentration on RUNX2 levels in MN chondrocytes. However, protein (but not RNA) levels of RUNX2 were elevated in OA chondrocytes maintained in 10 mM versus 4 mM glucose under oxygenated conditions. In contrast, neither RUNX2 levels or MMP13 or ADAMTS5 expression were increased in OA chondrocytes maintained in 10 mM compared to 4 mM glucose in hypoxia. Elevated extracellular glucose leads to increased glucose consumption and increased RUNX2 protein levels, promoting production of MMP13 and ADAMTS5 by OA chondrocytes in oxygenated but not hypoxic conditions. These findings suggest that hyperglycaemia may exacerbate chondrocyte-mediated cartilage catabolism in the oxygenated superficial zone of cartilage in vivo in patients with undertreated type 2 diabetes, contributing to increased OA severity.

KARTOGENIN-ENCAPSULATED COAXIAL PGS/PCL-ALIGNED NANOFIBRES FOR ARTICULAR CARTILAGE REGENERATION

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1].In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2].The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies.Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020).

Effect of Moderate Exercise on the Superficial Zone of Articular Cartilage in Age-Related Osteoarthritis.

This study aimed to evaluate the effect of exercise on the superficial zone of the osteoarticular cartilage during osteoarthritis progression. Three-month-old, nine-month-old, and eighteen-month-old Sprague Dawley rats were randomly divided into two groups, moderate exercise and no exercise, for 10 weeks. Histological staining, immunostaining, and nanoindentation measurements were conducted to detect changes in the superficial zone. X-ray and micro-CT were quantitated to detect alterations in the microarchitecture of the tibial subchondral bone. Cells were extracted from the superficial zone of the cartilage under fluid-flow shear stress conditions to further verify changes in vitro. The number of cells and proteoglycan content in the superficial zone increased more in the exercise group than in the control group. Exercise can change the content and distribution of collagen types I and III in the superficial layer. In addition, TGFβ/pSmad2/3 and Prg4 expression levels increased under the intervention of exercise on the superficial zone. Exercise can improve the Young's modulus of the cartilage and reduce the abnormal subchondral bone remodeling which occurs after superficial zone changes. Moderate exercise delays the degeneration of the articular cartilage by its effect on the superficial zone, and the TGFβ/pSmad2/3 signaling pathways and Prg4 play an important role.

Development of a computational-experimental framework for enhanced mechanical characterization and cross-species comparison of the articular cartilage superficial zone

To provide a better understanding of the contribution of specific constituents (i.e. proteoglycan, collagen, fluid) to the mechanical behavior of the superficial zone of articular cartilage, a complex biological tissue with several time-dependent properties, a finite element model was developed. Optimization was then used to fit the model to microindentation experiments. We used this model to compare superficial zone material properties of mature human vs. immature bovine articular cartilage. Non-linearity and stiffness of the fiber-reinforced component of the model differed between human and bovine tissue. This may be due to the more complex collagen architecture in mature tissue and is of interest to investigate in future work.

Depleting transforming growth factor beta receptor 2 signalling in the cartilage of itga1-null mice attenuates spontaneous knee osteoarthritis

ObjectivesIntegrin α1β1 protects against osteoarthritis (OA) when it is upregulated in the superficial zone of cartilage in the early stages of disease. However, the mechanism behind this protection is unknown. Integrin α1β1 moderates transforming growth factor β receptor II (TGFBR2) signalling, a critical regulator of chondrocyte anabolic activity. To this end, mice lacking integrin α1β1 have increased baseline activation of TGFBR2 signalling and overall fibrosis. The purpose of this study was to evaluate the interplay between integrin α1β1 and TGFBR2 in the development of spontaneous OA. We hypothesized that dampening TGFBR2 signalling in the cartilage of itga1-null mice would attenuate OA. MethodsBehavioural and histological manifestations of spontaneous knee OA were measured at 4, 8, 12 and 16 months in mice with and without a ubiquitous itga1 deletion and with and without a tamoxifen-induced cartilage specific TGFBR2 depletion. ResultsKnee cartilage degeneration, collateral ligament ossification and pain responses increased with age. Itga1-null mice with intact TGFBR2 signalling developed earlier and more severe OA compared to controls. In agreement with our hypothesis, depleting TGFBR2 signalling in the cartilage of itga1-null mice attenuated OA progression. ConclusionIntact TGFBR2 signalling drives early and worse knee OA in itga1-null mice. This result supports the hypothesis that the increased expression of integrin α1β1 by superficial zone chondrocytes early in OA development dampens TGFBR2 signalling and thus protects against degeneration.

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

Differential Effects of Hypoxia versus Hyperoxia or Physoxia on Phenotype and Energy Metabolism in Human Chondrocytes from Osteoarthritic Compared to Macroscopically Normal Cartilage.

Chondrocyte phenotype and energy metabolism are altered in osteoarthritis (OA). However, most studies characterising the change in human chondrocyte behaviour in OA have been conducted in supraphysiological oxygen concentrations. The purpose of this study was to compare phenotype and energy metabolism in chondrocytes from macroscopically normal (MN) and OA cartilage maintained in 18.9% (standard tissue culture), 6% (equivalent to superficial zone of cartilage in vivo) or 1% oxygen (equivalent to deep zone of cartilage in vivo). MMP13 production was higher in chondrocytes from OA compared to MN cartilage in hyperoxia and physoxia but not hypoxia. Hypoxia promoted SOX9, COL2A1 and ACAN protein expression in chondrocytes from MN but not OA cartilage. OA chondrocytes used higher levels of glycolysis regardless of oxygen availability. These results show that differences in phenotype and energy metabolism between chondrocytes from OA and MN cartilage differ depending on oxygen availability. OA chondrocytes show elevated synthesis of cartilage-catabolising enzymes and chondrocytes from MN cartilage show reduced cartilage anabolism in oxygenated conditions. This is relevant as a recent study has shown that oxygen levels are elevated in OA cartilage in vivo. Our findings may indicate that this elevated cartilage oxygenation may promote cartilage loss in OA.

HMGB1 can activate cartilage progenitor cells in response to cartilage injury through the CXCL12/CXCR4 pathway.

Recent studies have suggested that cartilage progenitor cells (CPCs) could be activated and differentiated into chondrocytes to produce matrix and to restore the integrity of damaged cartilage after injury. However, the mechanism involved in CPC activation upon damage is still unclear. This study aims to investigate the role of high mobility group box chromosomal protein 1 (HMGB1) in both activation and migration of CPCs during cartilage injury. Explants harvested from mature bovine stifle joints were used for impact injury. The proliferation and migration of CPCs were examined via confocal imaging. Gene and protein expression of Hmbg1, Cxcl12, and Cxcr4 was also examined by quantitative polymerase chain reaction (qPCR), ELISA, and western blot. Each experiment was repeated 3 times. ANOVA and Student's t-test were performed for statistical analysis. HMGB1 released from dead and damaged chondrocytes after an impact injury could activate CPCs in the superficial zone of cartilage and promote their migration and proliferation to injury sites. However, the block of HMGB1 activation with its specific binding inhibitor glycyrrhizin inhibits the proliferation and migration of CPCs. Further investigations demonstrate that HMGB1 promotes CPCs migration through the pathway of C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4. Quantitative analysis of HMGB1 in cell culture medium also indicates that CPCs may have a self-activation property after the HMGB1 released from dead cells has been exhausted. HMGB1 is a pivotal factor that could enhance the migration and proliferation of CPCs through the CXCL12/CXCR4 pathway after cartilage injury, which could provide useful information for cartilage repair and osteoarthritis treatment.

Evaluation of articular cartilage wear against pyrolytic carbon in the context of spherical interposition shoulder arthroplasty

Metal shoulder hemiarthroplasty is known to cause failure due to glenoid cartilage erosion. Pyrolytic carbon (PyC), a non-metallic biomaterial, may be an attractive alternative due to its good general tribological characteristics and biocompatibility. We investigated the in vitro biotribological properties of PyC articulated against living cartilage for use as spherical shoulder interposition implant. The evaluation includes a comparison with the typically used cobalt‑chromium‑molybdenum alloy (CoCrMo) and zirconia toughened alumina (ZTA), a more recent biomaterial for hemiarthroplasty. The rate of cartilage degradation was assessed by means of cartilage wear biomarkers (glycosaminoglycans and hydroxyproline) in lubricant and histological changes of the cartilage superficial zone. In addition, the percentage of cell viability and metabolic activity of chondrocytes was assessed. The data revealed that PyC has similar tribological performance in terms of cell viability, cell metabolic activity, and tissue degradation compared to ZTA. Although not significant, CoCrMo affected chondrocyte metabolism and induced a thinner superficial layer with structural defects. There was a combined adsorption of phospholipids and biomarkers on the biomaterials; sGAGs adsorbed between 23 and 43% of the total concentration, and the adsorption rate of phospholipids increased significantly with friction. Biomaterial analysis is required to obtain the full picture of cartilage biomarker release and lubrication mechanisms. Our results suggest that PyC has good tribological performance and compares well with established biomaterials for hemiarthroplasty. It supports the use of PyC in spherical shoulder interposition implant as a viable alternative to conventional metal hemiarthroplasty. Further evaluation of the clinical performance of PyC is required.

Toward defining the role of the synovium in mitigating normal articular cartilage wear and tear

Cartilage repair has been studied extensively in the context of injury and disease, but the joint’s management of regular sub-injurious damage to cartilage, or ‘wear and tear,’ which occurs due to normal activity, is poorly understood. We hypothesize that this cartilage maintenance is mediated in part by cells derived from the synovium that migrate to the worn articular surface. Here, we demonstrate in vitro that the early steps required for such a process can occur. First, we show that under physiologic mechanical loads, chondrocyte death occurs in the cartilage superficial zone along with changes to the cartilage surface topography. Second, we show that synoviocytes are released from the synovial lining under physiologic loads and attach to worn cartilage. Third, we show that synoviocytes parachuted onto a simulated or native cartilage surface will modify their behavior. Specifically, we show that synoviocyte interactions with chondrocytes lead to changes in synoviocyte mechanosensitivity, and we demonstrate that cartilage-attached synoviocytes can express COL2A1, a hallmark of the chondrogenic phenotype. Our findings suggest that synoviocyte-mediated repair of cartilage ‘wear and tear’ as a component of joint homeostasis is feasible and is deserving of future study.

Establishment of an Ex Vivo Inflammatory Osteoarthritis Model With Human Osteochondral Explants.

Osteoarthritis (OA) is the most common degenerative joint disease without clear pathophysiological mechanism and effective drugs for treatment. Although various animal models exist, the translation of the outcome into clinics remains difficult due to species differences. In this study, an ex vivo inflammatory OA model was induced using different concentrations of interleukin one beta (IL-1β) and tumor necrosis factor α (TNF-α) on explants from the human femoral head. In the inflammatory OA groups, the gene expression levels of cartilage catabolism (matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3)), and inflammation (interleukin 6 (IL-6), interleukin 8 (IL-8)) markers were significantly upregulated, while the anabolic genes (collagen 2 (COL2), aggrecan (ACAN), and proteoglycan 4 (PRG4)) were downregulated compared to the control group. The release of cytokines (IL-6, IL-8) and nitric oxide (NO) in the conditioned medium was also upregulated in inflammatory OA groups. The Safranin O/Fast Green staining showed loss of proteoglycan in the superficial zone cartilage after cytokine treatment. The results indicated that an ex vivo inflammation and degeneration model was successfully established using osteochondral explants from the human femoral head. This model can be used to elucidate the in-depth mechanism of inflammatory OA and to screen new drugs for OA treatment.

Matrix metalloproteinase-13 is fully activated by neutrophil elastase and inactivates its serpin inhibitor, alpha-1 antitrypsin: Implications for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage exvivo, in an MMP-dependent manner. Invitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

HMGB2 promotes chondrocyte proliferation under negative pressure through the phosphorylation of AKT

Cells in articular cartilage are zonal arranged. Cells in superficial zone cartilage are generally small and proliferative. Appropriate negative pressure stimulation is beneficial to cell survival and tissue repair. Whether negative pressure has promotive impact on the proliferation activity of the superficial zone chondrocytes is of interest. In this study, we isolated superficial chondrocytes from the mandibular condylar cartilage of rats. After negative pressure treatment, the cells were collected for RNA-sequencing, quantitative real-time PCR and western blotting assays, aiming to detect the proliferative responses of chondrocytes to negative pressure and explore the potential molecular mechanisms. Data from RNA-sequencing analysis indicated that the superficial chondrocytes responded to the 4 h −10 kPa treatment by a significant increase in proliferation. In addition, the expression of high-mobility group box 2 (HMGB2) and the phosphorylation of AKT were obviously promoted. Knockdown of HMGB2 decreased AKT phosphorylation and diminished the negative pressure-induced proliferation of chondrocytes, as shown by decreased expression of Ki67 and cyclin-dependent kinase 6 (CDK6). In contrast, overexpression of HMGB2 enhanced AKT phosphorylation and further promoted proliferative activity. Moreover, LY294002, an AKT inhibitor, suppressed the proliferative activity of chondrocytes under negative pressure, while SC79, an activator of AKT phosphorylation, enhanced the proliferation of chondrocytes. Our data demonstrated that HMGB2 exhibits a promotion impact on chondrocyte proliferation under negative pressure via the phosphorylation of AKT. These results provide a new perspective for superficial zone chondrocytes proliferation under negative pressure, which should be benefit for cartilage regeneration.

Microindentation of cartilage before and after articular loading in a bioreactor: assessment of length-scale dependency using two analysis methods.

Microindentation is a technique with high sensitivity and spatial resolution, allowing for measurements at small-scale indentation depths. Various methods of indentation analysis to determine output properties exist. Here, the Oliver-Pharr Method and Hertzian Method were compared for stiffness analyses of articular cartilage at varying length-scales before and after bioreactor loading. Using three different conospherical tips with varying radii (20, 100, 793.75 μm), a bioreactor-indenter workflow was performed on cartilage explants to assess changes in stiffness due to articular loading. For all data, both the Oliver-Pharr Method and Hertzian Method were applied for indentation analysis. The reduced moduli calculated by the Hertzian Method were found to be similar to those of the Oliver-Pharr Method when the 20 μm tip size was used. The reduced moduli calculated using the Hertzian Method were found to be consistent across the varying length-scales, whereas for the Oliver-Pharr Method, adhesion/suction led to the largest tip exhibiting an increased average reduced modulus compared to the two smaller tips. Loading induced stiffening of articular cartilage was observed consistently, regardless of tip size or indentation analysis applied. Overall, geometric linearity is preserved across all tip sizes for the Hertzian Method and may be assumed for the two smaller tip sizes using the Oliver-Pharr Method. These findings further validate the previously described stiffening response of the superficial zone of cartilage after articular loading and demonstrate that the finding is length-scale independent.

Transient stiffening of cartilage during joint articulation: A microindentation study

As a mechanoactive tissue, articular cartilage undergoes compression and shear on a daily basis. With the advent of high resolution and sensitive mechanical testing methods, such as micro- and nanoindentation, it has become possible to assess changes in small-scale mechanical properties due to compression and shear of the tissue. However, investigations on the changes of these properties before and after joint articulation have been limited. To simulate articular loading of cartilage in the context of human gait, a previously developed bioreactor system was used. Immediately after bioreactor testing, the stiffness was measured using microindentation. Specifically, we investigated whether the mechanical response of the tissue was transient or permanent, dependent on counterface material, and an effect limited to the superficial zone of cartilage. We found that cartilage surface stiffness increases immediately after articular loading and returns to baseline values within 3 hr. Cartilage-on-cartilage stiffening was found to be higher compared to both alumina- and cobalt chromium-on-cartilage stiffening, which were not significantly different from each other. This stiffening response was found to be unique to the superficial zone, as articular loading on cartilage with the superficial zone removed showed no changes in stiffness. The findings of this study suggest that the cartilage superficial zone may adapt its stiffness as a response to articular loading. As the superficial zone is often compromised during the course of osteoarthritic disease, this finding is of clinical relevance, suggesting that the load-bearing function deteriorates over time.

A Biomimetic Nano‐Engineered Platform for Functional Tissue Engineering of Cartilage Superficial Zone

Articular cartilage has limited regeneration capacity because of its acellular and avascular nature. Although tissue engineering has been shown to be life-saving, reforming cartilage zones required by the appropriate tissue functions are challenging. Herein, the need is addressed by designing and producing a nano-engineered structure mimicking the superficial zone (SZ) of articular cartilage. The substrate is based on silk with good mechanical properties in conjunction with nano-topographical and biochemical cues. Nanopillar arrays are produced on the silk surface to regulate the stem cell morphology rendering them with a flattened ellipsoidal shape that is similar to that of chondrocytes in the SZ of natural cartilage. The cell interactions are enhanced by nitrogen ion implantation and the biomolecule, kartogenin (KGN), is loaded to promote chondrogenesis of the stem cells and furthermore, a thermosensitive chitosan hydrogel is formed on the nanopatterned silk to produce rheological properties similar to those of a synovial fluid. Based on the in vitro results and mechanical properties, it is a desirable implantable smart structure mimicking the cartilage SZ with the ability of continuous drug release for cartilage regeneration.

Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Cartilage surface integrity influences contrast enhanced ct imaging of osteoarthritis

Purpose: To assess the utility of contrast-enhanced uCT (CE-uCT) as an imaging biomarker for preclinical assessment of osteoarthritis in a sheep model using IA injection of iodinated contrast medium and short exposure times that can be obtained in vivo. Methods: An in-vitro pilot study was conducted to assess differences in the apparent density between damaged (fibrillated/incised or shaved/superficial zone removed) and intact bovine osteochondral blocks after exposure to an anionic contrast medium (Conray 60) for 10 minutes (n=22). The sidewalls of blocks were wrapped in Parafilm™ to limit contrast contact to the cartilage surface only. Fibrillated (n=6), shaved (n=7), and intact (n=4) blocks were immersed in the Conray 60 and intact control (N=5) blocks were immersed in lactated ringer’s solution (LRS) for 10 minutes prior to uCT imaging at 45 micron resolution. Uptake of contrast was measured by calculation of the average apparent density in Hounsfield Units (HU). Twenty-four sheep were enrolled in a follow-up, ex-vivo, study. Osteoarthritis was induced by arthroscopic meniscal destabilization in n=20 sheep and n=4 remained as naive controls. The x-ray attenuation of cartilage was measured in animals with early (3 month) and late stage (12 month) OA after a 10-minute exposure to anionic contrast media (Conray 60). Concentration (ug/mg) of sulfated-glycosaminoglycan (sGAG) and histological OARSI scores for proteoglycan (PG) staining were also collected and recorded from the medial tibial plateau (MTP) of the operated leg. ANOVA was used to analyze variance in sGAG and HU between sites on the MTP or between groups. Correlation coefficients between sGAG and HU were analyzed using Pearson’s correlation coefficients and simple linear regression analysis. Spearmann-rank coefficients were used to examine the association between histological PG score and either HU or sGAG. We hypothesized apparent density (HU) would vary with respect to concentration of local sGAG, and that HU would be higher in regions where sGAG is depleted. Results: In the in-vitro CE-uCT study we observed a significant increase in apparent density of intact cartilage samples exposed to Conray 60 for 10 minutes compared to LRS controls (p=0.03). HU was also significantly higher in fibrillated (p=0.030) and shaved (p=0.0007) blocks compared to non-contrasted samples. In ex-vivo CE-uCT imaging of the MTP location accounted the variance in HU for both 3 month (p=0.09) and 12 month (p=0.0074) groups but not controls (p=0.32). Early 3 month OA sheep had elevated levels of sGAG that were significantly higher than the late phase 12 month group group (Fig 1, p =0.0032). A significant, negative correlation between sGAG concentration and HU of the same location was observed (r= -0.71, p = 0.047) in the early stage (3 month) sheep. Linear regression analysis revealed that every one unit (ug CSC/mg cartilage) increase in sGAG resulted in a reduction in HU by 8.62 units (R2 = 0.43, p< 0.05). Conclusions: Ten minutes of exposure to contrast media was sufficient to clearly outline cartilage borders due to the increase in apparent density and measured HU. Differences in cartilage HU of CE-uCT images can be used to estimate differences in local sGAG concentrations in animals with thin cartilage during early and late OA but variance in local sGAG concentration cannot exclusively explain the resulting variance in HU, suggesting that other factors may play a role such as an the integrity of the superficial cartilage zone. Elevated sGAG cartilage concentrations at three months returned to near normal levels at 12 months suggesting the hypertrophic phase of OA may be longer than expected. During this time matrix depletion as measured by CE-CT may not be a reliable imaging biomarker.

Increased Substance P Immunoreactivity in Ipsilateral Knee Cartilage of Rats Exposed to Lumbar Spine Injury

The present study aimed to investigate whether experimentally induced lumbar facet-joint OA lead to degenerative changes and enhanced SP expression within the ipsilateral neurosegmentally linked tibiofemoral cartilage. Adult male Sprague-Dawley rats were assigned to left side L5-L6 facet mechanical compression injury (surgery) (n = 6), L5-L6 facet exposure with no compression (sham) (n = 5), or naïve (no surgery) (n = 4) groups. The morphology of the tibiofemoral articular cartilage was assessed using a modified Mankin scoring system. Immunohistochemistry was used to examine the density of chondrocytes stained positive for SP (cells/cm2) in the ipsilateral tibiofemoral cartilage at 28 days postintervention. Tibiofemoral cartilage in the surgery group showed consistent loss of superficial zone chondrocytes, mild roughening of the articular surface and occasional chondrocyte clusters as well as a greater density of SP mainly in the superficial cartilage zone compared with sham and naïve groups, although they also had a basic SP-expression. Our results support the hypothesis that neurogenic mechanisms may mediate the spread of SP to neurosegmentally linked heterologous joints affecting the distal cartilage homeostasis. These findings contribute additional insight into the potential role of neurogenic inflammation with implications in the pathophysiology of chronic inflammatory joint disease and OA.

A compression system for studying depth-dependent mechanical properties of articular cartilage under dynamic loading conditions

The biological activities of chondrocytes are influenced by the mechanical characteristics of their environment. The overall real-time mechanical response of cartilage has been investigated earlier. However, the instantaneous local mechano-biology of cartilage has not been investigated in detail under dynamic loading conditions. In order to address this gap in the literature, we designed a compression testing device and implemented a dual photon microscopy technique with the goal of measuring local mechanical and biological responses of articular cartilage under dynamic loading conditions. The details of the compression system and results of a pilot study are presented here. A 15% ramp compression at a rate of 0.003/s with a subsequent stress relaxation phase was applied to the cartilage explant samples. The extra cellular matrix was imaged throughout the entire thickness of the cartilage sample, and local tissue strains were measured during the compression and relaxation phase. The axial compressive strains in the middle and superficial zones of cartilage were observed to increase during the relaxation phase: this was a new finding, suggesting the importance of further investigations on the real-time local behavior of cartilage. The compression system showed promising results for investigating the dynamic, real-time mechanical response of articular cartilage, and can now be used to reveal the instantaneous mechanical and biological responses of chondrocytes in response to dynamic loading conditions.