Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Computational identification and functional validation of regulatory motifs in cartilage-expressed genes.

Chondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Several regulatory factors have been identified that play a role in chondrogenesis, including the positive transacting factors of the SOX family such as SOX9, SOX5, and SOX6, as well as negative transacting factors such as C/EBP and delta EF1. However, a complete understanding of the intricate regulatory network that governs the tissue-specific expression of cartilage genes is not yet available. We have taken a computational approach to identify cis-regulatory, transcription factor (TF) binding motifs in a set of cartilage characteristic genes to better define the transcriptional regulatory networks that regulate chondrogenesis. Our computational methods have identified several TFs, whose binding profiles are available in the TRANSFAC database, as important to chondrogenesis. In addition, a cartilage-specific SOX-binding profile was constructed and used to identify both known, and novel, functional paired SOX-binding motifs in chondrocyte genes. Using DNA pattern-recognition algorithms, we have also identified cis-regulatory elements for unknown TFs. We have validated our computational predictions through mutational analyses in cell transfection experiments. One novel regulatory motif, N1, found at high frequency in the COL2A1 promoter, was found to bind to chondrocyte nuclear proteins. Mutational analyses suggest that this motif binds a repressive factor that regulates basal levels of the COL2A1 promoter.

Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-associated adaptations to biomechanical and anatomical roles

OBJECTIVE Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focussing on structural issues which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. METHODS Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20 to 40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy and unbiased stereological methods. Variations associated with joint (knee versus ankle) and biomechanical role (convex versus concave articular surfaces) were identified by 2-way analysis of variance and post-hoc analysis. RESULTS Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex versus concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2×10(6) chondrons/cm(3). CONCLUSION Variation of cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help identify conditions where OA is likely to occur.

Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Mucosal Erosion of the Cricoid Cartilage After the Use of an i-Gel Supraglottic Airway Device in a Patient with Diffuse Idiopathic Skeletal Hyperostosis

After standard hip arthroplasty, an 82-year-old patient with previously undiagnosed diffuse idiopathic skeletal hyperostosis of the cervical spine experienced life-threatening side effects after use of a supraglottic airway device (i-gel). Extensive mucosal erosion and denudation of the cricoid cartilage caused postoperative supraglottic swelling and prolonged respiratory failure requiring tracheostomy. In this case report, we highlight the importance of evaluating risk factors for failure of supraglottic airway devices.

Mapping tibiofemoral gonarthrosis: an MRI analysis of non-traumatic knee cartilage defects

OBJECTIVE Arthroscopy is "the gold standard" for the diagnosis of knee cartilage lesions. However, it is invasive and expensive, and displays all the potential complications of an open surgical procedure. Ultra-high-field MRI now offers good opportunities for the indirect assessment of the integrity and structural changes of joint cartilage of the knee. The goal of the present study is to determine the site of early cartilaginous lesions in adults with non-traumatic knee pain. METHODS 3-T MRI examinations of 200 asymptomatic knees with standard and three-dimensional double-echo steady-state (3D-DESS) cartilage-specific sequences were prospectively studied for early degenerative lesions of the tibiofemoral joint. Lesions were classified and mapped using the modified Outerbridge and modified International Cartilage Repair Society classifications. RESULTS A total of 1437 lesions were detected: 56.1% grade I, 33.5% grade II, 7.2% grade III and 3.3% grade IV. Cartographically, grade I lesions were most common in the anteromedial tibial areas; grade II lesions in the anteromedial L5 femoral areas; and grade III in the centromedial M2 femoral areas. CONCLUSION 3-T MRI with standard and 3D-DESS cartilage-specific sequences demonstrated that areas predisposed to early osteoarthritis are the central, lateral and ventromedial tibial plateau, as well as the central and medial femoral condyle. ADVANCES IN KNOWLEDGE In contrast with previous studies reporting early cartilaginous lesions in the medial tibial compartment and/or in the medial femoral condyle, this study demonstrates that, regardless of grade, lesions preferentially occur at the L5 and M4 tibial and L5 and L2 femoral areas of the knee joint.

Accuracy of cartilage-specific 3-Tesla 3D-DESS magnetic resonance imaging in the diagnosis of chondral lesions: comparison with knee arthroscopy

BACKGROUND Arthroscopy is considered as "the gold standard" for the diagnosis of traumatic intraarticular knee lesions. However, recent developments in magnetic resonance imaging (MRI) now offer good opportunities for the indirect assessment of the integrity and structural changes of the knee articular cartilage. The study was to investigate whether cartilage-specific sequences on a 3-Tesla MRI provide accurate assessment for the detection of cartilage defects. METHODS A 3-Tesla (3-T) MRI combined with three-dimensional double-echo steady-state (3D-DESS) cartilage specific sequences was performed on 210 patients with knee pain prior to knee arthroscopy. Sensitivity, specificity, and positive and negative predictive values of magnetic resonance imaging were calculated and correlated to the arthroscopic findings of cartilaginous lesions. Lesions were classified using the modified Outerbridge classification. RESULTS For the 210 patients (1260 cartilage surfaces: patella, trochlea, medial femoral condyle, medial tibia, lateral femoral condyle, lateral tibia) evaluated, the sensitivities, specificities, positive predictive values, and negative predictive values of 3-T MRI were 83.3, 99.8, 84.4, and 99.8 %, respectively, for the detection of grade IV lesions; 74.1, 99.6, 85.2, and 99.3 %, respectively, for grade III lesions; 67.9, 99.2, 76.6, and 98.2 %, respectively, for grade II lesions; and 8.8, 99.5, 80, and 92 %, respectively, for grade I lesions. CONCLUSIONS For grade III and IV lesions, 3-T MRI combined with 3D-DESS cartilage-specific sequences represents an accurate diagnostic tool. For grade II lesions, the technique demonstrates moderate sensitivity, while for grade I lesions, the sensitivity is limited to provide reliable diagnosis compared to knee arthroscopy.

Age-Independent Cartilage Generation for Synovium-Based Autologous Chondrocyte Implantation.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

T1ρ MRI detects cartilage damage in asymptomatic individuals with a cam deformity

Hips with a cam deformity are at risk for early cartilage degeneration, mainly in the anterolateral region of the joint. T1ρ MRI is a described technique for assessment of proteoglycan content in hyaline cartilage and subsequently early cartilage damage. In this study, 1.5 Tesla T1ρ MRI was performed on 20 asymptomatic hips with a cam deformity and compared to 16 healthy control hips. Cam deformity was defined as an alpha angle at 1:30 o'clock position over 60° and/or at 3:00 o'clock position over 50.5°. Hip cartilage was segmented and divided into four regions of interest (ROIs): anterolateral, anteromedial, posterolateral and posteromedial quadrants. Mean T1ρ value of the entire weight bearing cartilage in hips with a cam deformity (34.0 ± 4.6 ms) was significantly higher compared to control hips (31.3 ± 3.2 ms, p = 0.050). This difference reached significance in the anterolateral (p = 0.042) and posteromedial quadrants (p = 0.041). No significant correlation between the alpha angle and T1ρ values was detected. The results indicate cartilage damage occurs in hips with a cam deformity before symptoms occur. A significant difference in T1ρ values was found in the anterolateral quadrant, the area of direct engagement of the deformity, and in the posteromedial quadrant. To conclude, T1ρ MRI can detect early chondral damage in asymptomatic hips with a cam deformity. This article is protected by copyright. All rights reserved.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Studies of pseudoachondroplasia chondrocytes and knockdown of the cartilage oligomeric matrix protein

Cartilage oligomeric matrix protein (COMP) is a large, homopentameric, extracellular matrix glycoprotein. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EMD1). These dwarfing conditions are caused by retention of misfolded mutant COMP with type IX collagen and matrilin-3 (MATN3) in the rough endoplasmic reticulum (rER) of the chondrocyte. These proteins form a matrix in the rER that continues to expand until it fills the entire cell, eventually causing cell death. Interestingly, loss of COMP in COMP null mice does not affect normal bone development or growth, suggesting that elimination of COMP (wildtype and mutant) expression may prevent PSACH. The hypothesis of these studies was that a hammerhead ribozyme could eliminate or knockdown COMP mRNA expression in PSACH chondrocytes . To test this hypothesis, a human chondrocyte model system that recapitulates the PSACH chondrocyte phenotype was developed by over-expressing mutant (mt-) COMP in normal chondrocytes using a recombinant adenovirus. Chondrocytes over-expressing mt-COMP developed giant rER cisternae containing COMP, type IX collagen and MATN3. Deconvolution microscopy and computer modeling showed that these proteins formed an ordered matrix surrounding a type II pro-collagen core. Additionally, the results show that a hammerhead ribozyme, ribozyme 56 (Ribo56) reduced over-expressed mt-COMP in COS cells and endogenous COMP in normal chondrocytes and mt-COMP in three PSACH chondrocytes cell line (with different mutations) by 40-70%. Altogether, these studies show that the PSACH cellular phenotype can be created in vitro and that the mt-COMP protein burden can be reduced by the presence of a COMP-specific ribozyme. Future studies will focus on designing ribozymes or short interfering RNA (siRNA) technologies that will result in better knockdown of COMP expression as well as the temporal constraints imposed by the PSACH phenotype. ^

Approximate Analytical Model for the Squeeze-Film Lubrication of the Human Ankle Joint with Synovial Fluid Filtrated by Articular Cartilage

The aim of this article is to propose an analytical approximate squeeze-film lubrication model of the human ankle joint for a quick assessment of the synovial pressure field and the load carrying due to the squeeze motion. The model starts from the theory of boosted lubrication for the human articular joints lubrication (Walker et al., Rheum Dis 27:512–520, 1968; Maroudas, Lubrication and wear in joints. Sector, London, 1969) and takes into account the fluid transport across the articular cartilage using Darcy’s equation to depict the synovial fluid motion through a porous cartilage matrix. The human ankle joint is assumed to be cylindrical enabling motion in the sagittal plane only. The proposed model is based on a modified Reynolds equation; its integration allows to obtain a quick assessment on the synovial pressure field showing a good agreement with those obtained numerically (Hlavacek, J Biomech 33:1415–1422, 2000). The analytical integration allows the closed form description of the synovial fluid film force and the calculation of the unsteady gap thickness.