Intra-operative fluoroscopy in thumb metacarpophalangeal joint arthrodesis has been recommended as a means of achieving optimal alignment more consistently. This is not our current practice. A patient attending dissatisfied with an arthrodesis in excessive flexion performed outwith our unit highlighted the potential for problems, and we therefore elected to review our own outcomes. An evaluation of the alignment achieved in thumb metacarpophalangeal joint arthrodeses, to determine if current outcomes satisfactory or if fluoroscopic assistance should be considered. Radiological review of alignment of thumb metacarpophalangeal joint arthodeses carried out by two Consultant Surgeons with specialist interests in upper limb surgery in a District General hospital. Cases were predominantly identified retrospectively from sequential review of operating lists. The radiological images were, or had been taken, as part of routine follow-up and were not standardised. The alignment was also assessed independently by a junior doctor with no involvement in the patient's surgical treatment and no knowledge of the intended alignment. Recommended positions for arthrodesis have covered a range from 0 to 30 degrees, so for the purposes of analysis that range was considered acceptable. 14 cases had an average fusion position of 18 degrees flexion (range 6 to 30 degrees). 6 underwent concurrent ipsilaterel trapeziectomy. The series achieved satisfactory alignment radiologically without the routine use of intra-operative fluoroscopy

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

The internal fixation of osteochondral fragments in fractures normally utilizes intra-articular screws inserted through a pilot hole drilled into cartilage/bone. This trauma causes cartilage injury leading to chondrocyte death. We have quantified the cell death following cartilage drilling and identified irrigation conditions that can protect chondrocytes. Articular cartilage of bovine metacarpophalangeal joints of 3yr-old cows was irrigated in the presence/absence of saline of various compositions. Holes were then made using a standard 1.5mm drill (Ortho Solutions Ltd.) at 18,000 rpm through the articular cartilage into bone. Osteochondral explants were then harvested and cultured in Dulbecco's Modified Eagle's Medium containing chloromethylfluorescein-di-acetate and propidium iodide (10uM each), to label living chondrocytes green and dead cells red, respectively. Axial images were taken by confocal microscopy and the width of the zone of cell death (ZCD) around the hole determined. With no irrigation, new drills caused a ZCD of 171±25um, which was increased when drills used 50+ times were tested (279±31um;p=0.03). With saline irrigation, the ZCD was reduced for old drills (150±6um;p=0.016) but not for new drills (124±8um) suggesting the heating effect of the old drills caused additional chondrocyte death. However for new drills, the ZCD was further reduced significantly to 82±7um when the osmolarity of the saline irrigation solution was raised to 480mOsm using sucrose. Data are mean±s.e.m., from at least 5 separate experiments each with a minimum of 3 replicates. The results demonstrate a chondroprotective effect of raising the osmolarity of saline used during drilling of cartilage which could be clinically beneficial

Few studies have investigated the direct effect of bacteria and their products on articular cartilage chondrocytes ex vivo. An ex vivo model that allows the analysis of chondrocytes in situ would therefore be an important and exciting area of future research. It was hypothesised that a bovine cartilage explant model of septic arthritis would be an ideal model for providing fundamental information on the basic cellular mechanisms of cartilage destruction and chondrocyte death induced by bacterial infection uncomplicated by the immune response. A fresh metacarpophalangeal joint from an abattoir slaughtered 3-year-old cow was skinned, rinsed in water and opened under sterile conditions. The cartilage explants were harvested using surgical scalpels and placed into a total of three tissue culture bottles (2 explants per bottle) containing 10ml Dulbecco's Modified Eagle Medium (DMEM). 50ml of a knee aspirate from a patient with septic arthritis, containing Group B streptococci (GBS), was added to bottle 1, 50ml of a negative knee aspirate was added to bottle 2 and 50ml DMEM to bottle 3. The explants were incubated at 37°C for 24 hours. They were then stained with the fluorescent probes Chloromethylfluorescein Di-acetate (CMFDA) and Propidium Iodide and analysed using a Confocal Scanning Laser Microscope. Cell counts to assess percentage cell death were performed using Velocity 4 software. There was strikingly more cell death observed at 24 hours in the cartilage explant exposed to bacteria in comparison to the non-infected controls. The percentage chondrocyte death was 43% in the presence of GBS, 0.8% in the presence of the negative aspirate and 0.2% in the presence of the DMEM control. Although this is a very preliminary pilot study, it demonstrates an extremely rapid effect on the cartilage. Future bovine explant studies of septic arthritis will therefore be feasible and achievable

Intra-articular screw fixation is indicated for internal fixation of large osteochondral fragments secondary to trauma or osteochondritis dissecans. During surgery, orthopaedic drills are used to prepare a hole through which the screw can pass. Previous work has shown that mechanical injury to articular cartilage results in a zone of cell death adjacent to the traumatised articular cartilage (1). Here, we characterise and quantify the margin of in situ chondrocyte death surrounding drill holes and screws (standard cortical and headless compression designs) placed in mature bovine articular cartilage to model the orthopaedic procedure. Drill holes (1mm) were made through the articular cartilage and bone of intact bovine metacarpophalangeal joints obtained from 3-yr old cows within 12hrs of slaughter. Osteochondral explants (∼1cm square and 2-3mm thick) encompassing the drilled holes in articular cartilage and subchondral bone were harvested using a chisel. Explants were then incubated in Dulbecco's modified Eagle's medium for 45mins with CMFDA (5-chloromethylfluorescein diacetate) and PI (propidium iodide; both at 10micromolar) to identify/quantify living and dead in situ chondrocytes respectively in a consecutive series of axial optical sections using confocal scanning laser microscopy (CLSM). The drill holes through cartilage appeared to have clearly defined edges with no macroscopic evidence of cartilage splitting. However visualisation of fluorescently-labelled in situ chondrocytes by CLSM demonstrated clear cell death around the periphery of the drilled hole which was 166±19 micrometers in width. This increased with a larger diameter (1.5mm) drill to 450±151 micrometers (all data are means±s.e.m.; n=3). Preliminary experiments indicated that the margin of chondrocyte death around a 1.5mm hole was dramatically increased further by the insertion of screws into pre-drilled holes. These results suggest that the mechanical trauma associated with cartilage drilling and the insertion of intra-articular screws occurs with marked death of in situ chondrocytes extending into normal cartilage beyond the area occupied by the screw. As chondrocytes are not replaced in mature cartilage, their loss around the hole/screw will mean that the extracellular matrix is not maintained, inevitably leading to cartilage failure

Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine cartilage explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and cartilage explants harvested. Explants were placed into flasks containing Dulbecco's Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability (<1% cell death) over 40hrs incubation with the negative aspirate. However, for the aspirate from a patient positive for S. aureus, there was a rapid increase in cell death between 18 and 24hrs (0.2 +/− 0.3% to 23 +/− 5% cell death respectively) and almost complete cell death at 40hrs (80 +/− 12%; data are means +/− s.d; n=4). These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine cartilage explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, cartilage integrity

Saline (0.9%) is typically used to rinse joints during osteo-articular surgery. It is not unusual for cartilage to then be exposed to the air of the operating theatre for 1-2hrs, which can lead to chondrocyte death. We have compared the survival of in situ chondrocytes within bovine cartilage which has been rinsed in various solutions or simply drained of synovial fluid (SF) and then allowed to dry, to identify approaches that could reduce chondrocyte death arising from cartilage drying. Metacarpophalangeal joints from 3yr-old cows were opened under aseptic conditions. The joints were then (a) rinsed with saline (Baxter's Healthcare, Newbury), (b) rinsed with saline+glucose (20mM; both 300mOsm) or (c) drained of SF, and allowed to dry at room temperature. Full depth cartilage explants were taken after 2hrs, placed into Dulbecco's modified Eagle's medium and incubated with CMFDA (5-chloromethyl-fluorescein diacetate; 10microM) and propidium iodide (10microM) for the identification/quantification of living and dead cells respectively by confocal scanning laser microscopy and image analysis. After 2hrs, the appearance and properties of the cartilage of the drying joints were clearly different. Saline-rinsed cartilage was dark purple and appeared dull with the cartilage difficult to sample. However when the rinsing solution was saline+glucose, or when joints were drained of SF, the cartilage was almost identical to the freshly-opened joint with a pearly-blue, shiny appearance, and cartilage sampling was easy. Chondrocyte death was markedly increased in saline rinsed/dried joints after 2hrs (21±9% cell death). In contrast, there was no significant (P>0.05) death in saline+glucose rinsed/dried (2±1%) or SF-drained joints (3±2%;means±s.e.m.;n=5). The loss of cartilage wet weight over 2hrs (time=0 taken as 100%) was almost identical between cartilage rinsed in saline (73.6±1.6%), saline + glucose (78.6±1.1%) or SF (75.0±0.2%; data means±s.d.;n=2). These results suggest that it was not the loss of water per se during cartilage drying that was the key determinant of chondrocyte viability. As chondrocytes are normally anaerobic, the rise in cartilage pO2 which occurs during exposure to air could have a deleterious effect on cell viability however the presence of glucose or SF protects through an anti-oxidant effect

The aim of this study was to determine whether subchondral bone influences in situ chondrocyte survival. Bovine explants were cultured in serum-free media over seven days with subchondral bone excised from articular cartilage (group A), subchondral bone left attached to articular cartilage (group B), and subchondral bone excised but co-cultured with articular cartilage (group C). Using confocal laser scanning microscopy, fluorescent probes and biochemical assays, in situ chondrocyte viability and relevant biophysical parameters (cartilage thickness, cell density, culture medium composition) were quantified over time (2.5 hours vs seven days). There was a significant increase in chondrocyte death over seven days, primarily within the superficial zone, for group A, but not for groups B or C (p < 0.05). There was no significant difference in cartilage thickness or cell density between groups A, B and C (p > 0.05). Increases in the protein content of the culture media for groups B and C, but not for group A, suggested that the release of soluble factors from subchondral bone may have influenced chondrocyte survival. In conclusion, subchondral bone significantly influenced chondrocyte survival in articular cartilage during explant culture.

The extrapolation of bone-cartilage interactions in vitro to the clinical situation must be made with caution, but the findings from these experiments suggest that future investigation into in vivo mechanisms of articular cartilage survival and degradation must consider the interactions of cartilage with subchondral bone.

The aim of this randomised, controlled in vivo study in an ovine model was to investigate the effect of cylic pneumatic pressure on fracture healing. We performed a transverse osteotomy of the right radius in 37 sheep. They were randomised to a control group or a treatment group where they received cyclic loading of the osteotomy by the application of a pressure cuff around the muscles of the proximal forelimb. Sheep from both groups were killed at four or six weeks. Radiography, ultrasonography, biomechanical testing and histomorphometry were used to assess the differences between the groups. The area of periosteal callus, peak torsional strength, fracture stiffness, energy absorbed over the first 10° of torsion and histomorphometric analysis all showed that the osteotomies treated with the cyclic pneumatic pressure at four weeks were not significantly different from the control osteotomies at six weeks.

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.

Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.

There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor.