The impact of early intra-articular administration of interleukin-1 receptor antagonist on lubricin metabolism and cartilage degeneration in an anterior cruciate ligament transection model.

OBJECTIVE: Study the impact of intra-articular interleukin-1 receptor antagonist (IL-1 ra) treatment on lubricin biosynthesis following anterior cruciate ligament transection (ACLT) in the rat and evaluate the effect of combined IL-1 ra and recombinant human lubricin (rhPRG4) treatments on chondrocyte apoptosis. METHODS: ACLT was performed in male Lewis rats. Treatments included IL-1 ra or vehicle (n = 36 in each group). IL-1 ra intra-articular dosing was performed on days 1, 3, 5 and 7 following ACLT using Anakinra (150 mg/ml; 40 µl). At 3 and 5 weeks, animals were sacrificed and RNA was isolated. Histological analyses included Safranin O and H&E. Lubricin synovial fluid (SF) lavage concentrations were determined at 5 weeks. ACLT animals were treated with a single injection of vehicle, IL-1 ra (75 mg/ml; 40 µl), rhPRG4 (200 µg/ml; 40 µl), or IL-1 ra + rhPRG4 (75 mg/ml + 200 µg/ml; 40 µl) (n = 6 in each group) on day 7 following ACLT and cartilage was probed for cleaved caspase-3 at 5 weeks. RESULTS: IL-1 ra treatment improved lubricin expression (P < 0.001) and lubricin SF lavage concentrations in the IL-1 ra group was higher (P = 0.005) than the vehicle. IL-1 ra treatment reduced cartilage and synovial scores (P < 0.001) compared to vehicle. IL-1 ra and rhPRG4 acted synergistically to reduce caspase-3 positive chondrocytes (P < 0.001) compared to individual treatments. CONCLUSION: IL-1 ra treatment preserved lubricin following ACLT and a combined treatment of IL-1 ra + rhPRG4 may act synergistically to reduce cartilage catabolism.

Loss of extracellular matrix from articular cartilage is mediated by the synovium and ligament after anterior cruciate ligament injury.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) occurs after anterior cruciate ligament (ACL) injury. PTOA may be initiated by early expression of proteolytic enzymes capable of causing degradation of the articular cartilage at time of injury. This study investigated the production of three of these key proteases in multiple joint tissues after ACL injury and subsequent markers of cartilage turnover. METHODS: ACL transection was performed in adolescent minipigs. Collagenase (MMP-1 and MMP-13) and aggrecanase (ADAMTS-4) gene expression changes were quantified in the articular cartilage, synovium, injured ligament, and the provisional scaffold at days 1, 5, 9, and 14 post-injury. Markers of collagen degradation (C2C), synthesis (CPII) and aggrecan synthesis (CS 846) were quantified in the serum and synovial fluid. Histologic assessment of the cartilage integrity (OARSI scoring) was also performed. RESULTS: MMP-1 gene expression was upregulated in the articular cartilage, synovium and ligament after ACL injury. MMP-13 expression was suppressed in the articular cartilage, but upregulated 100-fold in the synovium and ligament. ADAMTS-4 was upregulated in the synovium and ligament but not in the articular cartilage. The concentration of collagen degradation fragments (C2C) in the synovial joint fluid nearly doubled in the first five days after injury. CONCLUSION: We conclude that upregulation of genes coding for proteins capable of degrading cartilage ECM is seen within the first few days after ACL injury, and this response is seen not only in chondrocytes, but also in cells in the synovium, ligament and provisional scaffold.

Pharmaceutical nanocarrier association with chondrocytes and cartilage explants: influence of surface modification and extracellular matrix depletion.

OBJECTIVE: To evaluate cartilage diffusion and isolated chondrocyte association of micelles and liposomes and to determine the effect of cell-penetrating peptide (CPP) surface functionalization and extracellular matrix depletion on chondrocyte association and cartilage diffusion, respectively. METHODS: Rhodamine-labeled micelles and liposomes were incubated with bovine chondrocytes and cell-associated fluorescence was quantified using flow cytometry. Rhodamine-labeled CPP-modified micelles and liposomes were incubated with chondrocytes and cell-associated fluorescence was compared to unmodified nanocarriers. Rhodamine-labeled micelles and liposomes were incubated with bovine cartilage explants for 1, 2 and 4 h and cartilage-associated fluorescence was compared across groups. Cartilage explants were treated with interleukin-1 alpha (IL-1α) or with 0.25% trypsin. Rhodamine-labeled micelles and liposomes were incubated with control, IL-1 and trypsin-treated explants and cartilage-associated fluorescence was compared across groups. RESULTS: Chondrocyte-associated fluorescence following treatment with micelles was significantly higher (P < 0.001) than fluorescence in the cells treated with liposomes while there was no difference between cell-associated fluorescence in the liposomes-treated and untreated controls. CPP-modified nanocarriers exhibited a significant increase in chondrocyte association compared to unmodified nanocarriers (P < 0.001). Micelles exhibited a time and concentration-dependent diffusion in cartilage explants while liposomes showed no diffusion. Following IL-1 and trypsin treatments, micelle diffusion in articular cartilage was significantly higher (P < 0.001) than their diffusion in untreated explants. CONCLUSION: Micelles exhibit superior association with isolated chondrocytes compared to liposomes. Surface modification with a CPP enhances chondrocyte association of both nanocarriers. 15 nm diameter micelles are better than 138 nm diameter liposomes in penetrating articular cartilage and extracellular matrix depletion enhances micelle penetration.

The impact of forced joint exercise on lubricin biosynthesis from articular cartilage following ACL transection and intra-articular lubricin's effect in exercised joints following ACL transection.

OBJECTIVE: To evaluate the impact of forced joint exercise following acute knee injury on lubricin metabolism and its relationship to cartilage degeneration and to assess chondroprotection of a single-dose purified human lubricin injection in exercised injured joints. METHODS: Anterior cruciate ligament transection (ACLT) was performed in rats with six experimental groups; 3-week post-ACLT, 3-week post-ACLT + exercise, 5-week post-ACLT, 5-week post-ACLT + exercise, and 5-week post-ACLT + exercise treated with intra-articular phosphate buffered saline (PBS) or lubricin. Joint exercise was achieved using a rotating cylinder at a speed of 6 rpm for 30 min daily, 5 days a week starting 1 week following surgery. Cartilage lubricin expression in injured joints was determined. Histological analyses included Safranin O/Fast Green, activated caspase-3, and lubricin mRNA in-situ hybridization. Assessment of cartilage damage was performed by osteoarthritis research society international (OARSI) modified Mankin scoring and urinary CTXII (uCTXII) levels. RESULTS: At 3 weeks, lubricin expression in exercised ACLT joints was significantly (P < 0.001) lower compared to ACLT joints. The OARSI scores were significantly (P < 0.001) higher in the ACLT + exercise animals compared to ACLT animals at 5 weeks. Compared to 3-week ACLT, 3-week ACLT + exercise cartilage showed increased caspase-3 staining. Compared to ACLT + exercise and PBS-treated ACLT + exercise, lubricin intra-articular treatment resulted in a significant increase (P < 0.001) in cartilage lubricin gene expression and a reduction (P < 0.05) in uCTXII levels. CONCLUSION: Joint exercise resulted in decreased lubricin cartilage expression, increased cartilage degeneration and reduced superficial zone chondrocyte viability in the ACLT joint. Intra-articular lubricin administration ameliorated cartilage damage due to exercise and preserved superficial zone chondrocytes' viability.

The impact of anterior cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor necrosis factor alpha on chondroprotection in an animal model.

OBJECTIVE: To examine the effects of anterior cruciate ligament transection (ACLT) in a rat model on lubricin metabolism and its relationship to markers of inflammation and cartilage damage, and to determine whether blocking the metabolic effects of tumor necrosis factor alpha (TNFalpha) by etanercept increases the chondroprotection provided by lubricin. METHODS: Unilateral ACLT was performed in Lewis rats. Levels of lubricin, TNFalpha, interleukin-1beta (IL-1beta), and sulfated glycosaminoglycans (sGAG) in synovial fluid (SF) lavage specimens and synovial tissue lubricin gene expression were evaluated at 1 week and 4 weeks following ACLT. Histologic evaluation of articular cartilage included staining with lubricin-specific monoclonal antibody 9G3 and Safranin O. The percentage of lubricin staining on the surface of articular cartilage in weight-bearing areas was estimated by digital imaging. Blocking of TNFalpha was performed using etanercept, which was administered subcutaneously at a dose of 0.5 mg/kg around the ACL-transected joints, using different dosing strategies. The ACL-transected and contralateral joints of these rats were harvested 4 weeks following surgery. RESULTS: Four weeks following ACLT, SF lubricin concentrations and the percentage of cartilage surface lubricin staining were significantly lower in the injured joints compared with the contralateral joints. A significant decrease in synovial tissue lubricin gene expression was associated with elevated TNFalpha and IL-1beta concentrations in SF lavage samples. With all of the etanercept treatment strategies, blocking of TNFalpha significantly increased the amount of lubricin bound to cartilage, coupled with a significant decrease in sGAG release. However, changes in the concentrations of lubricin in SF were variable. CONCLUSION: Blocking TNFalpha resulted in a chondroprotective effect, exemplified by increased lubricin deposition on articular cartilage and a decrease in sGAG release from articular cartilage in an animal model of posttraumatic arthritis.

Decreased lubricin concentrations and markers of joint inflammation in the synovial fluid of patients with anterior cruciate ligament injury.

OBJECTIVE: To study the effect of anterior cruciate ligament (ACL) injury on lubricin concentrations in synovial fluid (SF) and its correlation with time postinjury, inflammatory cytokines, lubricin-degrading enzymes, and SF proteoglycan content. METHODS: SF samples were obtained from both knees of 30 patients with unilateral ACL insufficiency, 32-364 days postinjury. Lubricin, inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], and IL-6), and catabolic enzymes (procathepsin B and neutrophil elastase) were measured in SF from injured and contralateral (uninjured) joints, by enzyme-linked immunosorbent assay. Sulfated glycosaminoglycan (sGAG) levels in the SF were measured by Alcian blue binding assay. RESULTS: SF lubricin concentrations were significantly (P < 0.001) reduced at an early stage following ACL injury when compared with those in the contralateral joint. Within 12 months, the lubricin concentration in the injured knee (slope = 0.006, SE = 0.00010, P < 0.001) approached that in the contralateral knee, which did not change with time (slope = -0.0002, SE = 0.00050, P = 0.71). TNFalpha levels showed a significant negative relationship with log2 lubricin levels. IL-1beta, TNFalpha, IL-6, procathepsin B, and neutrophil elastase concentrations in SF from injured knees were greater in samples from recently injured knees compared with those that were chronically injured. There were no detectable cytokines or enzymes in the SF of contralateral joints. Concentrations of sGAG were significantly (P = 0.0002) higher in the SF from injured knees compared with the contralateral joints. CONCLUSION: The decrease in SF lubricin concentrations following ACL injury may place the joint at an increased risk of wear-induced damage as a consequence of lack of boundary lubrication, potentially leading to secondary osteoarthritis. The decrease in SF lubricin was associated with an increase in levels of inflammatory cytokines.

Review: Collagen markers in early arthritic diseases.

In arthritic diseases e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), the stability of the collagen type II (CII) fibers, a major component of articular cartilage, is compromised with extensive proteolytic breakdown leading to cartilage erosion and joint deterioration. A clinical need for molecular markers that give instantaneous measure of rate of joint deterioration has developed, as other measurements e.g. arthroscopy, and joint space narrowing are insensitive to small changes in disease status over short periods of time. Owing to its exclusive presence in cartilaginous tissues, markers of CII synthesis and degradation have been extensively studied. Assays that measure these markers in biological fluids e.g. synovial fluid (SF), serum, and urine have been developed and applied to detect early disease onset, monitor disease progression, and response to anti-arthritic drugs. CII synthesis markers include the procollagen type II C-propeptide (PIICP) and the procollagen type IIA N-propeptide (PIIANP). CII degradation markers include CII C-telopeptide (CII-X), CII neoepitope (TIINE), helix II, C2C, CNBr 9.7, Coll 2-1, and Coll 2-1 NO(2). Most of these markers differentiate between early stages of OA, RA and reference controls. The best correlations with structural changes occur when measurements are made in SF while serum measurement frequently did not correlate with structural changes. Although the selection of an optimal marker or a set of markers is still problematic, few markers are of considerable utility in early detection and monitoring of arthritic diseases. The current challenge is to improve the discriminatory power of these markers so they can be used to guide therapeutic decisions.

Association of articular cartilage degradation and loss of boundary-lubricating ability of synovial fluid following injury and inflammatory arthritis.

OBJECTIVE: To study the relationship between the boundary-lubricating ability of synovial fluid (SF) and articular cartilage damage in a rabbit knee injury model, to correlate collagen markers of such damage with SF boundary-lubricating ability and elastase activity, and to examine the lubricating ability of SF, together with collagen markers of articular cartilage damage, under the inflammatory conditions of knee joint synovitis (KJS) and rheumatoid arthritis (RA). METHODS: SF was aspirated weekly from the affected knee joints of 10 adult rabbits following transection of the anterior and posterior cruciate ligaments. The boundary-lubricating ability of SF was determined in vitro using a previously described friction apparatus. Lubricin concentrations and type II collagen (CII) peptides were quantified by sandwich enzyme-linked immunosorbent assays (ELISAs). Levels of the C-terminal neoepitope 9A4 (derived from collagenase degradation of CI, CII, and CIII) and of epitope 5-D-4 of keratan sulfate (a marker of proteoglycan depletion) were quantified by inhibition ELISAs. Elastase activity was measured spectrophotometrically. The sensitivity of purified human lubricin to digestion by neutrophil elastase (NE) was examined by Western blotting. RESULTS: The lubricating ability of SF from injured rabbit knees was significantly decreased at weeks 2 and 3 compared with week 1 after injury. Lubricin concentrations were significantly higher at week 1 than at weeks 2 and 3. CII peptide concentrations increased significantly at weeks 2 and 3 compared with week 1, while 9A4 neoepitope concentrations increased significantly at week 3 compared with weeks 1 and 2. There were no significant differences in epitope 5-D-4 concentrations among the 3 weeks. Elastase activity in SF increased significantly at weeks 2 and 3 compared with week 1. Elastase activity correlated significantly with diminishing lubrication at weeks 1, 2, and 3. SF from patients with KJS or RA exhibited deficient lubrication and elevated levels of CII peptides compared with SF from normal controls. NE was shown to completely degrade purified human lubricin in vitro. CONCLUSION: Loss of boundary-lubricating ability of SF after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from the injury, particularly in the early phases. This association also exists in patients with acute knee injuries or progressive chronic inflammatory arthritis.

Detection of collagen type II and proteoglycans in the synovial fluids of patients diagnosed with non-infectious knee joint synovitis indicates early damage to the articular cartilage matrix.

OBJECTIVE: We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls. METHODS: CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate. RESULTS: The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS (P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower (P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher (P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS. CONCLUSION: The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.