Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages

INTRODUCTION: Certain amino acids within proteins have been reported to change from the L form to the D form over time. This process is known as racemization and is most likely to occur in long-lived low-turnover tissues such as normal cartilage. We hypothesized that diseased tissue, as found in an osteoarthritic (OA) joint, would have increased turnover reflected by a decrease in the racemized amino acid content. METHODS: Using high-performance liquid chromatography methods, we quantified the L and D forms of amino acids reported to racemize in vivo on a biological timescale: alanine, aspartate (Asp), asparagine (Asn), glutamate, glutamine, isoleucine, leucine (Leu), and serine (Ser). Furthermore, using a metabolically inactive control material (tooth dentin) and a control material with normal metabolism (normal articular cartilage), we developed an age adjustment in order to make inferences about the state of protein turnover in cartilage and meniscus. RESULTS: In the metabolically inactive control material (n = 25, ages 13 to 80 years) and the normal metabolizing control material (n = 19, ages 17 to 83 years), only Asp + Asn (Asx), Ser, and Leu showed a significant change (increase) in racemization with age (P < 0.01). The age-adjusted proportions of racemized to total amino acid (D/D+L expressed as a percentage of the control material) for Asx, Ser, and Leu when compared with the normal articular cartilage control were 97%, 74%, and 73% in OA meniscal cartilage and 97%, 70%, and 78% in OA articular cartilage. We also observed lower amino acid content in OA articular and meniscal cartilages compared with normal articular cartilage as well as a loss of total amino acids with age in the OA meniscal but not the OA articular cartilage. CONCLUSIONS: These data demonstrate comparable anabolic responses for non-lesioned OA articular cartilage and OA meniscal cartilage but an excess of catabolism over anabolism for the meniscal cartilage

Non-invasive assessment of lower limb alignment is accurate for pre-operative planning and post-operative follow up

Knee alignment is a fundamental measurement in the assessment, monitoring and surgical management of patients with OA. In spite of extensive research into the consequences of malalignment, there is a lack of data regarding the potential variation between supine and standing (functional) conditions. The purpose of this study was to explore this relationship in asymptomatic, osteoarthritic and prosthetic knees. Our hypothesis was that the change in alignment of these three groups would be different. Infrared position capture was used to assess knee alignment for 30 asymptomatic controls and 31 patients with OA, before and after TKA. Coronal and sagittal mechanical femorotibial (MFT) angles in extension (negative values varus/hyperextension) were measured supine and in bi-pedal stance and changes analysed using a paired t-test. To quantify this change in 3D, vector plots of ankle centre displacement relative to the knee centre were produced. Alignment in both planes changed significantly from supine to standing for all three groups, most frequently towards relative varus and extension. In the coronal plane, the mean±SD(°) of the supine/standing MFT angles was 0.1±2.5/−1.1±3.7 for asymptomatic (p=0.001), −2.5±5.7/−3.6±6.0 for osteoarthritic (p=0.009) and −0.7±1.4/ −2.5±2.0 for prosthetic knees (p<0.001). In the sagittal plane, the mean±SD(°) of the supine/standing MFT angles was −1.7±3.3/−5.5±4.9 for asymptomatic (p<0.001), 7.7±7.1/1.8±7.7 for osteoarthritic (p<0.001) and 6.8±5.1/1.4±7.6 for prosthetic knees (p<0.001). The vector plots showed that the trend of relative varus and extension in stance was similar in overall magnitude and direction between the groups. The similarities between each group did not support our hypothesis. The consistent kinematic pattern for different knee types suggests that soft tissue restraints rather than underlying joint deformity may be more influential in dynamic control of alignment from lying to standing. This potential change should be considered when positioning TKA components on supine limbs as post-operative functional alignment may be different

Inhibition of shedding of low-density lipoprotein receptor-related protein 1 reverses cartilage matrix degradation in osteoarthritis

OBJECTIVE: The aggrecanase ADAMTS-5 and the collagenase matrix metalloproteinase 13 (MMP-13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP-1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP-1. The aim of this study was to identify the LRP-1 sheddase(s) in human cartilage and to test whether inhibition of LRP-1 shedding prevents cartilage degradation in OA. METHODS: Cell-associated LRP-1 and soluble LRP-1 (sLRP-1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP-1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively. RESULTS: Shedding of LRP-1 was increased in OA cartilage compared with normal tissue. Shed sLRP-1 bound to ADAMTS-5 and MMP-13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane-bound metalloproteinases, ADAM-17 and MMP-14, were identified as the LRP-1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage. CONCLUSION: Shedding of LRP-1 is a key link to OA progression. Local inhibition of LRP-1 sheddase activities of ADAM-17 and MMP-14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach

Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage

Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88(fl/fl) transgenic mice results in up-regulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88(fl/fl) transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage

Measurements of three-dimensional glenoid erosion when planning the prosthetic replacement of osteoarthritic shoulders.

The three-dimensional (3D) correction of glenoid erosion is critical to the long-term success of total shoulder replacement (TSR). In order to characterise the 3D morphology of eroded glenoid surfaces, we looked for a set of morphological parameters useful for TSR planning. We defined a scapular coordinates system based on non-eroded bony landmarks. The maximum glenoid version was measured and specified in 3D by its orientation angle. Medialisation was considered relative to the spino-glenoid notch. We analysed regular CT scans of 19 normal (N) and 86 osteoarthritic (OA) scapulae. When the maximum version of OA shoulders was higher than 10°, the orientation was not only posterior, but extended in postero-superior (35%), postero-inferior (6%) and anterior sectors (4%). The medialisation of the glenoid was higher in OA than normal shoulders. The orientation angle of maximum version appeared as a critical parameter to specify the glenoid shape in 3D. It will be very useful in planning the best position for the glenoid in TSR

Transcriptome‐Wide Analysis of Messenger RNA Decay in Normal and Osteoarthritic Human Articular Chondrocytes

Objective Messenger RNA (mRNA) decay rates control not only gene expression levels, but also responsiveness to altered transcriptional input. We undertook this study to examine transcriptome‐wide posttranscriptional regulation in both normal and osteoarthritic (OA) human articular chondrocytes. Methods Human articular chondrocytes were isolated from normal or OA tissue. Equine articular chondrocytes were isolated from young or old horses at a commercial abattoir. RNA decay was measured across the transcriptome in human cells by microarray analysis following an actinomycin D chase. Messenger RNA levels in samples were confirmed using quantitative reverse transcription–polymerase chain reaction. Results Examination of total mRNA expression levels demonstrated significant differences in the expression of transcripts between normal and OA chondrocytes. Interestingly, almost no difference was observed in total mRNA expression between chondrocytes from intact OA cartilage and those from fibrillated OA cartilage. Decay analysis revealed a set of rapidly turned over transcripts associated with transcriptional control and programmed cell death that were common to all chondrocytes and contained binding sites for abundant cartilage microRNAs. Many transcripts exhibited altered mRNA half‐lives in human OA chondrocytes compared to normal cells. Specific transcripts whose decay rates were altered were generally less stable in these pathologic cells. Examination of selected genes in chondrocytes from young and old healthy horses did not identify any change in mRNA turnover. Conclusion This is the first investigation into the “posttranscriptome” of the chondrocyte. It identifies a set of short‐lived chondrocyte mRNAs likely to be highly responsive to altered transcriptional input as well as mRNAs whose decay rates are affected in OA chondrocytes

Morphometric Alteration of Femoral Condyles Due to Knee Osteoarthritis

Aim of this study was to estimate how knee osteoarthritis (OA) affects the shape of femoral condyles by comparing the radiuses of condylar curves between healthy and OA knees. Seventeen female and five male patients with established diagnosis of knee OA were included in the study. Radiuses of medial and lateral condylar curves were calculated from the side view knee X-ray by original mathematical equation and compared to referent values of healthy knees, after adjusting to body height. The average radiuses of condylar curves were between 52.6 ± 6.2 and 17.6 ± 3.5 mm medially, and between 43.3 ± 8.4 and 15.4 ± 3.7 mm laterally, for 0° and 90° femoral flexion contact points, respectively. The OA knees had longer curve radiuses medially and laterally at 0°, 10°, and 20° femoral flexion contact points in comparison to the healthy sample (P<0.001; t-test). Our results suggest that the shape of the femoral condyles in OA knees is changed. It should be aware not only in researching of OA etiology, but also in designing of knee endoprostheses, in a manner to achieve better individual sizing

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

INTRODUCTION: Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA. METHODS: MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA). RESULTS: In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125. CONCLUSIONS: Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA

MUSCULAR CO-ACTIVATION IN SUBJECTS AFFECTED BY FEMOROACETABULAR IMPINGEMENT

Femoroacetabular Impingement (FAI) is a hip deformity that causes hip and groin pain. Previous research showed that FAI patients have altered hip kinetics and kinematics and pelvic kinematics. Whether or not this is due to different muscular strategies is still unclear. The purpose of this study was to investigate the muscular co-activation in FAI patients. Electromyographic signals were recorded from 16 hip muscles. The coactivation index was calculated for FAI symptomatic, asymptomatic and control groups. Even if not statistically significant, the co-activation measurements showed a trend similar to the findings for osteoarthritic (OA) patients. Additional investigations are warranted to confirm this analogy that could further relate FAI and OA development, and to confirm the hypothesis that FAI anomalies are also due to altered muscular strategies