Background/Purpose: Septic arthritis (SA) caused by bacterial species, such as Staphylococcus aureus, has high morbidity and mortality¹. Currently diagnosis is often prolonged and unreliable, with no suitable near-patient biomarkers available². To generate more reliable biomarkers and to understand pathogenesis we sought to develop a novel ex vivo system to explore the effect of pathogenic and mutant Staphylococcus strains in promoting cartilage degradation.

Methods: Human cartilage explants were obtained from femoral heads being surgically removed following trauma. Explants were infected for 48h with 10⁶ cfu bacteria from two Staph. aureus SA-derived patient isolates (28g & 36v) or with a mutated bacterial strain preventing biofilm formation and adhesion, Staphylococcus epidermidis RP62A wild type strain or RP62A mutated strain. In the final 24h of bacterial infection, neutrophils purified from healthy donor blood were added to explant cultures at 3 x 10⁶ cells/well. Chondrocyte viability was assessed using CellTracker green CMFDA and propidium iodide. Images were captured using confocal microscopy (LSM880) and cells counted using Imaris software. Structural damage was measured by glycosaminoglycan (GAG) release and statistical analysis performed using GraphPad Prism software.

Results: When cartilage explants were co-cultured with bacteria +/- neutrophils, cell death was significantly increased compared to the negative control or addition of neutrophils alone, (one-way ANOVA, Holm-Sidak’s multiple comparisons test, N=3, bacteria – neutrophils p < 0.05, bacteria + neutrophils p < 0.01). Cartilage breakdown, estimated via GAG release, was induced by Staph. aureus alone, whereas it was significantly enhanced upon neutrophil addition in the final 24h of co-culture, Figure 1. Using the model, we investigated the effect of bacterial cell adhesion using a Staph. epidermidis adhesion mutant. The mutated strain did not significantly release more GAG compared to the negative control, whereas the wild type strain caused significant damage (2-way ANOVA, Holm-Sidak’s multiple comparisons test, N=3, p < 0.05). Moreover, the effect of neutrophil stimulation was attenuated when bacteria were unable to adhere to the cartilage.

Conclusion: A co-culture model of septic arthritis has been developed which allows precise examination of the contribution of the host neutrophil response and (mutant) bacterial species to cartilage damage. We used this to define the importance of bacterial adherence to cartilage for subsequent degradation. This novel model will be a valuable tool in understanding pathology and identifying biomarkers of joint infection in the future.

References

1. Gupta, M. N., Sturrock, R. D. & Field, M. A prospective 2-year study of 75 patients with adult-onset septic arthritis. Rheumatology40, 24–30 (2001).
2. Swan, A., Amer, H. & Dieppe, P. The value of synovial fluid assays in the diagnosis of joint disease: a literature survey. Ann. Rheum. Dis.61, 493–8 (2002).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-ex-vivo-model-of-septic-arthritis-identifies-role-of-neutrophils-in-joint-destruction/