Arnavaz Hakimiyan

Effects induced by BMPS in cultures of human articular chondrocytes: Comparative studies

We compared anabolic and anti-catabolic activities of selected bone morphogenetic proteins (BMP-2, -4, -6, and -7) and cartilage-derived morphogenetic proteins (CDMP-1 and -2) in human normal adult articular chondrocytes. Ankle chondrocytes were cultured in alginate beads in the presence of 10% serum and treated with either growth factors only (each at 100 ng/ml) or the combination of interleukin-1 (IL-1β) (0.1 ng/ml) and BMPs. Chondrocyte metabolism was assessed by proteoglycan (PG) synthesis, content, DNA content, and cell survival. The results showed that BMP-2, -4, and -7 were more potent in stimulating PGs than other growth factors tested. The highest levels of PG synthesis were detected at day 9 in the presence of BMP-7. With regard to anti-catabolic properties, the effect depended upon treatment scheme (simultaneous or sequential). Under simultaneous cultures, BMP-2, -4, and -6 failed to counteract IL-1β induced inhibition of PG synthesis, while the CDMPs restored this parameter to serum control levels. Only BMP-7 showed consistent and pronounced anti-catabolic activity in either culture treatment scheme. None of the factors induced cell death or chondrocyte proliferation. In conclusion, the growth factors tested showed different levels of effects on human chondrocytes in culture, but only BMP-7 displayed both strong anabolic and anti-catabolic properties.

Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage.

OBJECTIVES To investigate the effect of anti-apoptotic agents on cartilage degradation after a single impact to ankle cartilage. DESIGN Ten human normal tali were impacted with the impulse of 1 Ns generating peak forces in the range of 600 N using a 4 mm diameter indenter. Eight millimeter cartilage plugs containing the 4 mm diameter impacted core and a 4 mm adjacent ring were removed and cultured with or without P188 surfactant (8 mg/ml), caspase-3 (10 uM), or caspase-9 (2 uM) inhibitors for 48 h. Results were assessed in the superficial and middle-deep layers immediately after injury at day 0 and at 2, 7 and 14 days after injury by live/dead cell and Tunel assays and by histology with Safranin O/fast green staining. RESULTS A single impact to human articular cartilage ex vivo resulted in cell death, cartilage degeneration, and radial progression of apoptosis to the areas immediately adjacent to the impact. The P188 was more effective in preventing cell death than the inhibitors of caspases. It reduced cell death by more than 2-fold (P<0.05) in the core and by about 30% in the ring in comparison with the impacted untreated control at all time points. P188 also prevented radial expansion of apoptosis in the ring region especially in the first 7 days post-impaction (7.5% Tunel-positive cells vs 46% in the untreated control; P<0.01). Inhibitors of caspase-3 or -9 were effective in reducing cell death in the impacted core only at early time points, but were ineffective in doing so in the ring. Mankin score was significantly improved in the P188 and caspase-3 treated groups. CONCLUSIONS Early intervention with the P188 and caspase-3 inhibitor may have therapeutic potential in the treatment of cartilage defects immediately after injury.

Effect of interleukin-1β on osteogenic protein 1–induced signaling in adult human articular chondrocytes

OBJECTIVE Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1beta (IL-1beta)-induced cartilage degeneration to investigate the effect of IL-1beta on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes. METHODS Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1beta on BMP receptors was studied by reverse transcription-polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1beta (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1beta and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1beta activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38). RESULTS IL-1beta reduced the number of activin receptor-like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1-mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1beta offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38. CONCLUSION These findings describe new mechanisms of the crosstalk between OP-1 and IL-1beta in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes.

Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action.

Objective: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. Design: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. Results: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. Conclusion: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.

A novel human antimicrobial factor targets Pseudomonas aeruginosa through its type III secretion system

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen. Despite its metabolic and virulence versatility, it has not been shown to infect articular joints, which are areas that are rarely infected with bacteria in general. We hypothesized that articular joints possess antimicrobial activity that limits bacterial survival in these environments. We report that cartilages secrete a novel antimicrobial factor, henceforth referred to as the cartilage-associated antimicrobial factor (CA-AMF), with potent antimicrobial activity. Importantly, CA-AMF exhibited significantly more antimicrobial activity against P. aeruginosa strains with a functional type III secretion system (T3SS). We propose that CA-AMF represents a new class of human antimicrobial factors in innate immunity, one which has evolved to selectively target pathogenic bacteria among the beneficial and commensal microflora. The T3SS is the first example, to the best of our knowledge, of a pathogen-specific molecular target in this antimicrobial defence system.

Salvage of contaminated osteochondral allografts: the effects of chlorhexidine on human articular chondrocyte viability.

Background: Because chondrocyte viability is imperative for successful osteochondral allograft transplantation, sterilization techniques must provide antimicrobial effects with minimal cartilage toxicity. Chlorhexidine gluconate (CHG) is an effective disinfectant; however, its use with human articular cartilage requires further investigation. Purpose: To determine the maximal chlorhexidine concentration that does not affect chondrocyte viability in allografts and to determine whether this concentration effectively sterilizes contaminated osteoarticular grafts. Study Design: Controlled laboratory study. Methods: Osteochondral plugs were subjected to pulse lavage with 1-L solutions of 0.002%, 0.01%, 0.05%, and 0.25% CHG and cultured for 0, 1, 2, and 7 days in media of 10% fetal bovine serum and antibiotics. Chondrocyte viability was determined via LIVE/DEAD Viability Assay. Plugs were contaminated with Staphylococcus aureus and randomized to 4 treatment groups. One group was not contaminated; the 3 others were contaminated and received no treatment, saline pulse lavage, or saline pulse lavage with 0.002% CHG. Serial dilutions were plated and colony-forming units assessed. Results: The control group and the 0.002% CHG group showed similar cell viability, ranging from 67% ± 4% to 81% ± 22% (mean ± SD) at all time points. In the 0.01% CHG group, cell viability was reduced in comparison with control by 2-fold at day 2 and remained until day 7 (P < .01). The 0.05% and 0.25% CHG groups showed a 2-fold reduction in cell viability at day 1 (P < .01). At day 7, cell viability was reduced to 15% ± 18% (4-fold decrease) for the 0.05% CHG group and 10% ± 19% (6-fold decrease) for the 0.25% CHG group (P < .01). Contaminated grafts treated with 0.002% CHG demonstrated no colony-forming units. Conclusion: Pulse lavage with 0.002% CHG does not cause significant cell death within 7 days after exposure, while CHG at concentrations >0.002% significantly decreases chondrocyte viability within 1 to 2 days after exposure and should therefore not be used for disinfection of osteochondral allograft. Pulse lavage does not affect chondrocyte viability but cannot be used in isolation to sterilize contaminated fragments. Overall, 0.002% CHG was shown to effectively decontaminate osteoarticular fragments. Clinical Relevance: This study offers a scientific protocol for sterilizing osteochondral fragments that does not adversely affect cartilage viability.

The Effect of Surgical Insertion and Proinflammatory Cytokines on Osteochondral Allograft Survival and Metabolism

Objective To investigate the responses of refrigerated osteochondral allograft cartilage (OCA) and fresh cartilage (FC), including cell survival and metabolism, to surgical impaction and proinflammatory cytokines. Design Osteochondral plugs (8 mm diameter) were harvested from prolonged-refrigerated (14-28 days) and fresh (≤24 hours postmortem) human femoral hemicondyles and subjected to a 0.2 N s pneumatic impaction impulse. Cartilage explants were removed from subchondral bone and randomized to 1 of 6 treatment groups: (1) Unimpacted control (UIC), (2) Impacted control (IC), (3) Impacted + interleukin (IL)-1β (0.1 ng/mL), (4) Impacted + IL-1β (0.1 ng/mL) + IL-6, (5) Impacted + IL-1β (10 ng/mL), and (6) Impacted + IL-1β (10 ng/mL) + IL-6. Samples were measured for cell viability, histology, and proteoglycan (PG) content at days 0, 2, 7, and 14 of culture. Results In UIC, cell viability was indistinguishable between OCA and FC and remained constant. Impaction alone decreased cell viability by 30% (P < 0.01) in the OCA superficial layer and by 26% (P < 0.01) in the entire tissue, but did not affect viability in FC. Cytokine addition did not further influence cell viability. Impaction alone did not affect PG synthesis. Addition of cytokines to impacted tissue decreased PG synthesis by ~3-fold in both tissue types in comparison with corresponding impacted controls (P < 0.01). Throughout 2-week culture, PG release remained stable in all FC groups, but peaked at day 14 in OCA cartilage subjected to cytokines. Conclusions Mechanical impaction, mimicking surgical insertion, has a more profound effect on cell viability in OCA than in FC. Addition of proinflammatory cytokines further decreases OCA tissue metabolism and integrity.

95 ANALYSIS OF SYNOVIAL FLUID BIOMARKERS IN PATIENTS WITH RHEUMATOID ARTHRITIS, OSTEOARTHRITIS, AND NORMAL DONORS

Purpose: To compare synovial fluid (SF) biomarker levels in samples from patients with rheumatoid (RA), osteoarthritis (OA), and normal organ donors for prognostic/diagnostic purposes. Methods: Synovial fluid (SF) was collected from the knees of 45 (6 males and 39 females) OA, 22 (14 females and 8 males) RA patients and 20 (15 females and 5 males) normal organ donors. Eight biomarkers analyzed by ELISA methods were evaluated: interleukin (IL)-1, Il-6, Il-8, Il-11, leukemia inflammatory factor (LIF), cartilage oligomeric protein (COMP), osteocalcin, and osteogenic protein-1 (OP-1). Multivariate analysis assessed the effects of gender and disease activity: WOMAC scores for OA samples, and SF WBC, ESR, CRP for RA samples. Multivariant KruskalWallis and Mann-Whitney Tests were used; p< 0.05 was considered significant. Results: The mean (±SD) age was: 53±9 years for OA, 54±11 for RA, and 52±7 for donors. No gender differences were identified between markers. In RA SF, the levels of 4 out of 5 tested cytokines/chemokines were higher than in OA and normal SF. The most significant differences were found for Il-6 and Il-8, where Il-6 concentration was 2.5-fold higher than in OA, 2587.59±6393.69 vs. 1045.68±5451.13 og/ml, respectively (p< 0.005), and 8-fold higher than in normal, 2587.59±6393.69 vs. 339.06±906.28, respectively (P< 0.002). The levels of Il-8 were 8-fold higher than in OA, 6490.04±16550.74 vs 849.48 og/ml, respectively (P< 0.0005) and normal, 6490.04±16550.74 vs 788.69±837.705 og/ml, respectively (P< 0.028). The differences for Il-11 were not as substantial, especially between RA and OA samples, while it was a 5-fold difference between OA and normal SF, 654.11±1411.844 og/ml, respectively (P< 0.003). The value of Il-1 was significantly higher in normal than OA, 9.81±2.72 vs 8.56±7.06 og/ml, respectively (P< 0.004); in the RA, it was a tendency for a higher concentration of Il-1 than in OA and normal, but it did not reach significance. Surprisingly, concentration of LIF was higher in normal samples than in RA (P< 0.044) and OA (P< 0.001). The highest levels of OP-1 were found in normal SF (541.92±28.61 ng/ml), which were almost 5-fold higher than in OA (112.40±124.64) (P< 0.001) and more than 2-fold higher than in RA SF (202.25±149.13 ng/ml, P< 0.001). No differences were detected in the levels of COMP or osteocalcin between the experimental groups. Although the differences between active and inactive states of OA or RA were insignificant, for cytokines and OP-1 each state was statistically different from normal. SF from both OA and RA had higher biomarker levels than normals, regardless of disease state, though there was a trend of higher chemokine levels in less active RA, and higher Il-1, Il-6, and Il-8 levels in active OA. Conclusions: Our findings suggest that the levels of pathophysiologically important biomarkers in SF of patients with OA and RA differ according to the mechanisms that drive each disease. Thus, Il-1, Il-11, LIF and OP-1 appear to be significant for OA; while Il-6, Il-8, and OP-1 may have significance for RA. As previously observed in cartilage, a strong negative correlation between the levels of OP-1 and Il-6 family of chemokines was seen. Larger studies are necessary to develop a biomarker algorithm that would be of diagnostic/prognostic use.