Introduction

Non-union is an unfortunate outcome of the fracture healing process for some patients; with an estimated annual incidence of 17.4- 18.9 per 100,00. The management of these patients depicts a significant clinical challenge for surgeons and financial burden to health services. External ultrasound stimulation devices (Exogen^TM) have been highlighted as a novel non invasive therapy to achieve union in cases of delayed and non-union. The aim of the current study was to assess the rate of union in patients using Exogen^TM therapy for delayed fracture union in a district general hospital.

Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p<0.001) compared to the alpha-toxin knockout strain, which was negligible (4.1+/−1.7%; means+/−SEM; N=4 independent experiments). In this in vitro bovine cartilage explant model, whereby the effects of defined toxins were determined in isolation of a complex host immune response, in situ chondrocyte viability was dramatically and exclusively reduced by alpha-toxin. This work forms the basis for developing a rational treatment to reduce the extent of cartilage destruction during an episode of septic arthritis. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh.

We have demonstrated that toxins produced by Staphylococcus aureus, a common infective agent in septic arthritis (SA), cause rapid in situ chondrocyte death. Here, we have compared the sensitivity of chondrocytes within the superficial and deep zones (SZ, DZ) of cartilage to the same toxins. Culture medium containing the toxins produced by S. aureus strain 8325-4, which include alpha-, beta-, and gamma-toxin, was prepared. Cartilage explants free of subchondral bone were taken from the metacarpophalangeal joints of 3-year-old cows, and incubated (37°C) with the toxins. Explants were stained after 6hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Full-thickness coronal sections were imaged by confocal microscopy and the percentage cell death within the SZ (100μm from articular surface) and DZ (100μm from subchondral bone interface) determined. Both zones were incubated with the same toxin culture medium for the same time period. At 0hrs, chondrocytes within all zones were >98% viable. However, after incubation with toxin-containing culture medium for 6hrs, 71.9+/−11.2% of the SZ cells were dead compared to only 47.4+/−6.7% in the DZ (p=0.03;data are means+/−SEM;N=4). These results suggest that SZ chondrocytes are considerably more sensitive to S. aureus toxins than those within deeper zones. As SZ chondrocytes are close to the synovial fluid harbouring bacterial toxins, these data emphasise the need to remove bacteria and their products aggressively as part of the treatment of SA. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh.

Staphylococcus aureus is a highly virulent pathogen and is implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections have suggested that alpha (Hla), beta (Hlb) and gamma (Hlg) toxins are key virulence factors. In particular, the ‘pore-forming’ alpha toxin is believed to be most potent. In this study, we have assessed the influence of alpha toxin on in situ chondrocyte viability.

Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and placed into flasks containing Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with the following isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-) or DU1090 (Hla-Hlb+Hlg+).

The explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death obtained using Volocity 4 software.

The alpha toxin-producing S. aureus caused rapid cell death, with 24.8+/−3.7% at 18hrs and 44.6+/−7.2% at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%; p<0.001) compared to the alpha toxin knockout strain (4.1+/−1.7%; means +/− SEM; n=4).

In situ chondrocyte viability was significantly compromised by alpha toxin, with beta and gamma toxins having minimal effect. Further work will clarify the exact mechanism through which this important toxin induces chondrocyte death. Thereafter, it is hoped that targeted treatments can be developed to reduce the extent of cartilage destruction during, and after, an episode of septic arthritis.