Chondrocyte Response to Fresh Autologous Conditioned Serum Versus Freeze-Dried Allogenic Conditioned Serum.

OBJECTIVE: To investigate the cytokine release profile and histological response of human cartilage after exposure to autologous conditioned serum (ACS) and freeze-dried allogenic conditioned serum (FD-CS). DESIGN: Cartilage explants were collected from 6 patients undergoing total knee arthroplasty. ACS and FD-CS were created from patient serum samples. Cartilage samples were divided into 6 groups: (1) untreated control, (2) ACS, (3) FD-CS, (4) untreated interleukin (IL)-1ß (5 ng/ml), (5) IL-1ß + ACS, and (6) IL-1ß + FD-CS. After 12 days, cartilage samples were analyzed with glycosaminoglycan (GAG) concentration normalized to wet weight while comparing cytokine concentrations, and histological scoring. RESULTS: There was a significant decrease in pathology scoring for ACS (P = 0.0368) and FD-CS (P = 0.0368) in the IL-1ß injury groups compared with the untreated IL-1ß insult group. ACS and FD-CS significantly mitigate the IL-1ß induced increase in basic fibroblast growth factor (bFGF) (P = 0.0009 and P = 0.0002, respectively). FD-CS showed a significant decrease in IL-1ß concentration in the presence of IL-1ß insult compared with the untreated IL-1ß group (P < 0.0001). ACS-treated samples had significantly higher concentration of tumor necrosis factor (TNF)-α independent of IL-1ß when compared with samples not treated with biologics (P = 0.0053). CONCLUSIONS: Explanted osteoarthritic cartilage responds favorably and equivalently to treatment with ACS and FD-CS from a histological perspective. Both ACS and FD-CS were able to mitigate the IL-1ß-induced increases in bFGF and FD-CS lowered IL-1ß concentration while increasing interleukin-1 receptor antagonist (IL-1Ra) concentration. Although the cytokine profile of cartilage tissue explants treated with FD-CS appears to be different than that of ACS, this difference does not seem to affect biologic activity of FD-CS.

Biologic Characteristics of Shoulder Articular Cartilage in Comparison to Knee and Ankle Articular Cartilage From Individual Donors.

OBJECTIVE: To describe histological and metabolic characteristics of glenohumeral joint (GHJ) articular cartilage and compare to knee and ankle joints. DESIGN: Macroscopically healthy human humeral head, glenoid, knee, and ankle articular cartilage were obtained from tissue donors (N = 16, 9 males, 7 females; age 45-78 years), within 24 hours of death. Gross morphology of each joint was assessed using Collins grading. Cartilage explants were removed from the entire surface of each joint, cultured for 48 hours with or without interleukin-1ß and processed for histology with Safranin O, proteoglycan (PG) synthesis/content, and polymerase chain reaction for collagen II, aggrecan, and SOX9. Results were compared between uncultured and cultured controls and across all 3 joints. RESULTS: Structural differences were seen on histology between GHJ cartilage and knee and ankle cartilage of the same Collins grade, specifically, depletion of Safranin O staining in the extracellular matrix. Treatment of glenoid and humerus specimens with IL-1ß demonstrated a trend toward decreased PG synthesis in each explant but this decrease did not reach significance. There was no significant difference in PG synthesis between humerus, glenoid, knee, and ankle samples at baseline, day-0 control, 48-hour control, and 48 hours after treatment with 0.1 ng or 10 ng of IL-1ß. There were no significant increases in collagen II, SOX9, and aggrecan expression in glenoid and humeral head cartilage samples treated with IL-1ß compared to baseline controls. CONCLUSIONS: GHJ articular cartilage did not significantly differ from ankle or knee cartilage with regard to PG synthesis and gene expression. However, it did differ in its histological appearance in normal state.

TGF-ß type 2 receptor-mediated modulation of the IL-36 family can be therapeutically targeted in osteoarthritis.

Mechanisms that govern the shift from joint homeostasis to osteoarthritis (OA) remain unknown. Here, we identify a pathway used for joint development and homeostasis, and its role in OA. Using a combination of transgenic, pharmacological, and surgical conditions in mouse and human tissues, we found that TGF-ß signaling promotes joint homeostasis through regulation of the IL-36 family. We identified IL-36 receptor antagonist (IL-36 in mice and IL-36RN in humans) as a potential disease-modifying OA drug. Specifically, OA development was associated with IL-36α up-regulation and IL-36Ra down-regulation in mice with tissue-specific postnatally induced ablation of Tgfbr2, mice treated with a TGF-ß signaling inhibitor, mice with posttraumatic OA, and aging mice with naturally occurring OA. In human cartilage, OA severity was associated with decreased TGFBR2 and IL-36RN, whereas IL-36α increased. Functionally, intra-articular treatment with IL-36Ra attenuated OA development in mice, and IL-36RN reduced MMP13 in human OA chondrocytes. These findings highlight the relevance of TGFBR2-IL-36 interplay in joint homeostasis and IL-36RN as a potential therapeutic agent for OA.

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-1 beta) (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-1 beta 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.

Response of human chondrocytes prepared for autologous implantation to growth factors.

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.

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)-1ß (0.1 ng/mL), (4) Impacted + IL-1ß (0.1 ng/mL) + IL-6, (5) Impacted + IL-1ß (10 ng/mL), and (6) Impacted + IL-1ß (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.

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.

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.

The role of synovial fluid markers of catabolism and anabolism in osteoarthritis, rheumatoid arthritis and asymptomatic organ donors.

INTRODUCTION: The purpose of this study was to correlate the level of anabolic and catabolic biomarkers in synovial fluid (SF) from patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA) and asymptomatic organ donors. METHODS: SF was collected from the knees of 45 OA, 22 RA patients and 20 asymptomatic organ donors. Eight biomarkers were selected and analyzed by using an enzyme-linked immunosorbent assay: interleukin (IL)-1, IL-6, IL-8 and IL-11; leukemia-inhibitory factor (LIF); cartilage oligomeric protein (COMP); osteocalcin; and osteogenic protein 1 (OP-1). Data are expressed as medians (interquartile ranges). The effects of sex and disease activity were assessed on the basis of the Western Ontario and McMaster Universities index score for patients with OA and on the basis of white blood cell count, erythrocyte sedimentation rate and C-reactive protein level for patients with RA. RESULTS: The mean ages (± SD) of the patients were as follows: 53 ± 9 years for patients with OA, 54 ± 11 years for patients with RA and 52 ± 7 years for asymptomatic organ donors. No effect of participants' sex was identified. In the SF of patients with RA, four of five cytokines were higher than those in the SF of patients with OA and those of asymptomatic organ donors. The most significant differences were found for IL-6 and IL-8, where IL-6 concentration in SF of patients with RA was almost threefold higher than that in patients with OA and fourfold higher than that in asymptomatic donor controls: 354.7 pg/ml (1,851.6) vs. 119.4 pg/ml (193.2) vs. 86.97 pg/ml (82.0) (P < 0.05 and P < 0.05, respectively). IL-8 concentrations were higher in SF of patients with RA than that in patients with OA as well as that in asymptomatic donor controls: 583.6 pg/ml (1,086.4) vs. 429 pg/ml (87.3) vs. 451 pg/ml (170.1) (P < 0.05 and P < 0.05, respectively). No differences were found for IL-11 in the SF of patients with RA and that of patients with OA, while a 1.4-fold difference was detected in the SF of patients with OA and that of asymptomatic donor controls: 296.2 pg/ml (257.2) vs. 211.6 pg/ml (40.8) (P < 0.05). IL-1 concentrations were the highest in the SF of RA patients (9.26 pg/ml (11.1)); in the SF of asymptomatic donors, it was significantly higher than that in patients with OA (9.083 pg/ml (1.6) vs. 7.76 pg/ml (2.6); P < 0.05). Conversely, asymptomatic donor control samples had the highest LIF concentrations: 228.5 pg/ml (131.6) vs. 128.4 pg/ml (222.7) in the SF of patients with RA vs. 107.5 pg/ml (136.9) in the SF of patients with OA (P < 0.05). OP-1 concentrations were twofold higher in the SF of patients with RA than those in patients with OA and threefold higher than those in asymptomatic donor control samples (167.1 ng/ml (194.8) vs. 81.79 ng/ml (116.0) vs. 54.49 ng/ml (29.3), respectively; P < 0.05). The differences in COMP and osteocalcin were indistinguishable between the groups, as were the differences between active and inactive OA and RA. CONCLUSIONS: Activation of selected biomarkers corresponds to the mechanisms that drive each disease. IL-11, LIF and OP-1 may be viewed as a cluster of biomarkers significant for OA; while profiling of IL-1, IL-6, IL-8, LIF and OP-1 may be more significant in RA. Larger, better-defined patient cohorts are necessary to develop a biomarker algorithm for prognostic use.

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 NH2-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.

Effect of interleukin-1beta 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.

Antisense inhibition of osteogenic protein 1 disturbs human articular cartilage integrity.

OBJECTIVE: To delineate the role of endogenous osteogenic protein 1 (OP-1) in human articular cartilage homeostasis via the inhibition of OP-1 gene expression by antisense oligonucleotides. METHODS: Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high-density monolayers for 48 hours in the presence of OP-1 antisense or sense oligonucleotides. The effect of OP-1 antisense inhibition was evaluated by reverse transcription-polymerase chain reaction, (35)S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti-proOP-1, anti-mature OP-1, and anti-aggrecan antibodies. RESULTS: Antisense treatment inhibited OP-1 gene expression by a mean +/- SD of 34 +/- 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 +/- 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP-1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP-1 antisense reduced aggrecan messenger RNA expression by 42 +/- 17% (P < 0.05) and proteoglycan synthesis by 48 +/- 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti-aggrecan staining (primarily in the superficial and middle cartilage layers) with OP-1 antisense treatment. CONCLUSION: Our results suggest that OP-1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up-regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP-1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.