Abstract

Abstract

Abstract

COVID-19 confers a three-fold increased mortality risk among hip fracture patients. The aims were to investigate whether vaccination was associated with: i) lower mortality risk, and ii) lower likelihood of contracting COVID-19 within 30 days of fracture.

This nationwide cohort study included all patients aged >50 years with a hip fracture between 01/03/20-31/12/21. Data from the Scottish Hip Fracture Audit were collected and included: demographics, injury and management variables, discharge destination, and 30-day mortality status. These variables were linked to population-level records of COVID-19 vaccination and testing.

There were 13,345 patients with a median age of 82.0 years (IQR 74.0–88.0), and 9329/13345 (69.9%) were female. Of 3022/13345 (22.6%) patients diagnosed with COVID-19, 606/13345 (4.5%) were COVID-positive within 30 days of fracture. Multivariable logistic regression demonstrated that vaccinated patients were less likely to be COVID-positive (odds ratio (OR) 0.41, 95% confidence interval (CI) 0.34–0.48, p<0.001) than unvaccinated patients. 30-day mortality rate was higher for COVID-positive than COVID-negative patients (15.8% vs 7.9%, p < 0.001). Controlling for confounders (age, sex, comorbidity, deprivation, pre-fracture residence), unvaccinated patients with COVID-19 had a greater mortality risk than COVID-negative patients (OR 2.77, CI 2.12–3.62, p < 0.001), but vaccinated COVID19-positive patients were not at increased risk (OR 0.93, CI 0.53–1.60, p = 0.783).

Vaccination was associated with lower COVID-19 infection risk. Vaccinated COVID-positive patients had a similar mortality risk to COVID-negative patients, suggesting a reduced severity of infection. This study demonstrates the efficacy of vaccination in this vulnerable patient group, and presents essential data for future outbreaks.

Early changes within articular cartilage during human idiopathic osteoarthritis are poorly understood. However alterations to chondrocyte morphology occur with the development of fine cytoplasmic processes and cell clusters, potentially playing a role in cartilage degeneration. The aggrecanase ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs-4) has been implicated as an important factor in cartilage degradation, so we investigated the relationship between chondrocyte morphology and levels of ADAMTS-4 in both non-degenerate and mildly osteoarthritic human cartilage.

Human femoral heads were obtained following consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants of normal (grade 0; G0) and mildly osteoarthritic (grade 1; G1) cartilage were labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate). Explants were cryosectioned (30μm sections), and labelled for ADAMTS-4 by fluorescence immunohistochemistry. Sections were imaged with confocal microscopy, allowing the semi-quantitative analysis of ADAMTS-4 and 3D visualisation of in situ cell morphology.

With cartilage degeneration from G0 to G1, there was a decrease in the proportion of chondrocytes with normal rounded morphology (P<0.001) but an increase in the proportion of cells with processes (P<0.01) and those in clusters (P<0.001;[4(1653)]; femoral heads:cells). Although average levels of ADAMTS-4 for all cells was the same between G0 and G1 (P>0.05), a change was evident in the distribution curves for cell-specific ADAMTS-4 labelling. Cell-by-cell analysis showed that ADAMTS-4 levels were higher in chondrocytes with cytoplasmic processes compared to normal cells (P=0.044) however cells in clusters had lower levels than normal cells (P=0.003;[3(436)]). Preliminary data suggested that ADAMTS-4 levels increased with larger chondrocyte clusters.

These results suggest complex heterogeneous changes to levels of cell-associated ADAMTS-4 with early cartilage degeneration – increasing in cells with processes and initially decreasing in clusters. Increased levels of ADAMTS-4 are likely to produce focal areas of matrix weakness potentially leading to early cartilage degeneration.

The development of cytoplasmic processes from in situ chondrocytes is a characteristic feature of early osteoarthritis in human cartilage. The processes involve cytoskeletal elements and are distinct from the short primary cilia described in human chondrocytes. Vimentin is an intermediate filament playing an essential structural and signal-transduction role. We determined cellular levels and distribution of vimentin in chondrocytes of different morphologies in non-degenerate and mildly osteoarthritic cartilage.

Femoral heads were obtained after consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants were graded as non-degenerate (grade 0;G0) or mildly osteoarthritic (grade 1;G1) and labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate) for cell shape. Explants were cryosectioned and labelled for vimentin by fluorescence immunohistochemistry. In situ chondrocyte morphology was identified by confocal microscopy as either normal (rounded/elliptical) or abnormal (with one or more cytoplasmic process of ≥2µm) and vimentin levels and distribution determined semi-quantitatively and related to chondrocyte morphology.

When all cells in G0 and G1 cartilage were compared, there was no difference between average levels of vimentin per cell (P=0.144)[6(261)];femoral heads:cells). When cells were separated on the basis of morphology, there was no difference between vimentin levels in cells with one or more cytoplasmic process compared to those of normal morphology (P>0.05;[6(261)]). However vimentin levels were much greater at the base of cytoplasmic processes compared to distant areas of the same cells (P=0.021)[5(29)]).

Although overall levels of chondrocyte vimentin do not change in these early stages of osteoarthritis, the formation and structure of these substantial chondrocyte cytoplasmic processes involves changes to its distribution. These morphological changes are similar to those occurring during chondrocyte de-differentiation to fibroblasts reported in osteoarthritis which results in the formation of mechanically-inferior fibro-cartilage. Alterations to chondrocyte vimentin distribution either directly or indirectly may play a role in cartilage degeneration.

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.

The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, Imaris^TM and ImageJ software.

There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.

These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.

Articular cartilage has poor repair potential and the tissue formed is mechanically incompetent. Mesenchymal stromal cells (MSCs) show chondrogenic properties and the ability to re-grow cartilage, however a viable human model for testing cartilage regeneration and repair is lacking. Here, we describe an ex vivo pre-clinical femoral head model for studying human cartilage repair using MSCs.

Human femoral heads (FHs) were obtained following femoral neck fracture with ethical permission/patient consent and full-depth cartilage wells made using a 3mm biopsy punch. Pancreas-derived mesenchymal stromal cells (P-MSC) were prepared in culture media at ~5000 cells/20µl and added to each well and leakage prevented with fibrin sealant. After 24hrs, the sealant was removed and medium replaced with StemPro^TM chondrogenesis differentiation medium. The FHs were incubated (37^oC;5% CO₂) for 3wks, followed by a further 3wks in standard medium with 10% human serum with regular medium changes throughout. Compared to wells with medium only, A-MSCs produced a thin film across the wells which was excised en-block, fixed with 4% paraformaldehyde and frozen for cryo-sectioning.

The cell/tissue films varied in thickness ranging over 20-440µm (82±21µm; mean±SEM; N=3 FHs). The thickness of MSC films abutting the cartilage wells was variable but generally greater (15-1880µm) than across the wells, suggesting an attachment to native articular cartilage. Staining of the films using safranin O (for glycosaminoglycans; quantified using ImageJ) was variable (3±8%; mean±SEM; N=3) but in one experiment reached 20% of the adjacent cartilage. A preliminary assessment of the repair tissue gave an O'Driscoll score of 10/24 (24 is best).

These preliminary results suggest the ex vivo femoral head model has promise for studying the capacity of MSCs to repair cartilage directly in human tissue, although optimising MSCs to produce hyaline-like tissue is essential.

Supported by the CSO (TCS/17/32).

Hip fracture patients are vulnerable to delirium. This study examined the associations between delirium and outcomes including mortality, length of stay, post-discharge care requirements, and readmission.

This cohort study collected validated healthcare data for all hip fracture patients aged ≥50 years that presented to a high-volume centre between March 2020-November 2021. Variables included: demographics, delirium status, COVID-19 status, treatment factors, and outcome measures. Wilcoxon rank sum or Chi-squared tests were used for baseline differences, Cox proportional hazard regression for mortality, logistic regression for post-discharge care requirements and readmission, and linear regression for length of stay. Analyses were adjusted for age, sex, deprivation, pre-fracture residence type and COVID-19.

There were 1822 patients (mean age 81 years; 72% female) of which 496/1822 (27.2%) had delirium (4AT score ≥4). Of 371/1822 (20.4%) patients that died within 180 days of admission, 177/371 (47.7%) had delirium during the acute stay. Delirium was associated with an increased 30- and 180-day mortality risk (adjusted HR 1.74 (95%CI 1.15-2.64; p=0.009 and 1.74 (1.36-2.22; p<0.001), respectively), ten day longer total inpatient stay [adj. B.coef 9.80 (standard error 2.26); p<0.001] and three-fold greater odds of higher care requirements on discharge [Odds Ratio 3.07 (95% Confidence Interval 2.27-4.15; p<0.001)].

More than a quarter of patients had delirium during the hip fracture stay, and this was independently associated with increased mortality, longer length of stay, and higher post-discharge care requirements. These findings are relevant for prognostication and service planning, and emphasise the importance of effective delirium screening and evidence-based interventions in this vulnerable population.

Multiligament knee injuries (MLKI) are associated with significant morbidity and healthcare requirements. The primary aim of this study is to report the patient reported outcomes measures (PROMs) after reconstructive surgery.

Patients undergoing surgery for MLKI between 2014 and 2018 in the single large-volume trauma centre were included. Electronic patient records were reviewed for demographic data, details of surgery and complications. PROMs collected were EQ-5D-5L, Lysholm Knee Score (LKS), UCLA Activity and Sport and patient satisfaction.

Thirty-five patients were included. Mean age was 31 years (range 16-66), and 71% were male. The most common mechanism of injury was sports-related (71%). Obesity was present in eight (23%) patients. No vascular injuries were recorded and four patients sustained nerve injuries. PROMs were available for 18 patients (51%) with a median follow up of 4.5 years. Median EQ-5D-5L was 0.78 (IQR 0.14). Median LKS was 84.5 (IQR 21) and there was no correlation with time to surgery (p=0.43). Grade of MLKI did not impact LKS (p=0.09). Fifteen patients (83%) saw a reduction in their activity level. All patients were satisfied with their surgical treatment. Recurrent instability was noted in four patients (11%). Three patients (8%) required further surgery (one revision reconstruction, one meniscectomy, one conversion to a hinged knee replacement.

This study demonstrates two groups of patients who sustain MLKI: the sporting population and obese patients. Health related quality of life, functional outcomes and satisfaction are high after surgery. Time to surgery did not impact on functional outcomes.

This international multicentre retrospective cohort study aimed to assess: 1) prevalence of COVID-19 in hip fracture patients, 2) effect on mortality, and 3) clinical factors associated mortality among COVID-19-positive patients.

A collaboration among 112 centres in 14 nations collected data on all patients with a hip fracture between 1st March-31st May 2020. Patient, injury and surgical factors were recorded, and outcome measures included admission duration, COVID-19 and 30-day mortality status.

There were 7090 patients and 651 (9.2%) were COVID-19-positive. COVID-19 was independently associated with male sex (p=0.001), residential care (p<0.001), inpatient fall (p=0.003), cancer (p=0.009), ASA grade 4–5 (p=0.008; p<0.001), and longer admission (p<0.001). Patients with COVID-19 had a significantly lower chance of 30-day survival versus those without (72.7% versus 92.6%, p<0.001), and COVID-19 was independently associated with increased 30-day mortality risk (p<0.001). Increasing age (p=0.028), male sex (p<0.001), renal (p=0.017) and pulmonary disease (p=0·039) were independently associated with higher 30-day mortality risk in patients with COVID-19 when adjusting for confounders.

The prevalence of COVID-19 in hip fracture patients was 9% and was independently associated with a three-fold increased 30-day mortality risk. Clinical factors associated with mortality among COVID-19-positive hip fracture patients were identified for the first time. This is the largest study, and the only global cohort, reporting on the effect of COVID-19 in hip fracture patients. The findings provide a benchmark against which to determine vaccine efficacy in this vulnerable population and are especially important in the context of incomplete vaccination programmes and the emergence of vaccine-resistant strains.

Abstract

Abstract

Abstract

Background

National hip fracture programmes are becoming widespread, but this practice is nascent and varied. The Scottish Hip Fracture Audit (SHFA) was an early adopter of this strategy and is credited with substantial systemic improvements in quality and outcomes.

Hip fracture care is complex multi-disciplinary. We hypothesise that quality of care is affected by variance in resources between ‘in-hours’ (Monday-Friday, 0800–1700) and ‘out-of-hours’ services.

This prospective multicentre national cohort study assessed quality of care by evaluating adherence to the evidence-based Scottish Standards of Care for Hip Fracture Patients. Data was collected by the Scottish Hip Fracture Audit for 15174 patients admitted to any of 22 Scottish hospitals from January 2014-April 2018.

11197/15174 (73.8%) patients were admitted out-of-hours. They were significantly less likely to meet the following Standards: ED Big-6-Bundle (OR 0.85, p= 0.002); Time in ED <4 hours (OR 0.76, p< 0.001); avoidance of repeated fasting (OR 0.80, p< 0.001), and avoidance of prolonged fluid fasting (OR 0.83, p< 0.001). Out-of-hours admissions were more likely to receive: geriatric assessment <3 days (OR 1.16, p< 0.001); OT input <3 days (OR 1.10, p= 0.013), and PT input <2 days (OR 1.44, p< 0.001). There were no significant differences for: Time to Theatre <36 hours; Inpatient Care Bundle <24 hours, and Post-op Day 1 Mobilisation.

Quality of hip fracture care is affected by time of admission. ED care is poorer out-of-hours, which may reflect limited resources, and out-of-hours admissions are more likely to be excessive fasted excessively. Weekday in-hours admissions are less likely to receive geriatric and allied health professional input in the days following admission, which may reflect the reduced weekend services. Examination of out-of-hours service organisation is required for the pursuit of consistent, equitable care for hip fracture patients.

Introduction

Subacromial corticosteroid injection is widely used in the treatment of Subacromial Impingement Syndrome (SIS). There is increasing interest in using ultrasound (US) to improve the accurate placement of injections. This study investigated whether the accuracy of placement of US-guided subacromial corticosteroid injections influences patients' outcome of pain and function.

Chondrocytes are essential to the maintenance of articular cartilage and it is thought that chondrocyte death occurs early in septic arthritis. Understanding the causes of chondrocyte death will allow the development of chondroprotective strategies to improve long-term outcomes following septic arthritis.

We utilised a murine model of septic arthritis using intra-articular injection of 10µL of a 10⁷ concentration of S. aureus suspended in PBS. Seventy-five adult male C57/Bl6 mice were randomised to receive injection of either S. aurues 8325-4 (a wild-type of S. aurues capable of alpha toxin production), DU1090 (an isogenic mutant of 8325-4 that is identical to 8325-4 other than being incapable of producing alpha toxin) or a PBS control. Establishment of septic arthritis was confirmed through gait changes (5 mice/group), limb swelling and histological changes (10 mice/group). 10 animals from each group were sacrificed at 48 hours and the injected knee joints were dissected before being stained with CFMDA (labelling live chondrocytes green) and PI (labelling dead chondrocytes red). The samples were imaged using a confocal laser scanning microscope and the percentage of chondrocyte death was calculated.

Mice injected with S. aureus 8325-4 or DU1090 developed septic arthritis with evidence of weight loss, limb swelling and gait changes whereas these were absent in the control group. There was a significantly higher level of chondrocyte death in the group infected with 8325-4 (2.7% chondrocyte viability) when compared to DU1090 (73.9% chondrocyte viability) and PBS injected mice (95% chondrocyte viability). One-Way ANOVA revealed that the difference between each group was statistically different (p < 0.05).

Alpha toxin is the major damaging toxin in S. aurues septic arthritis. Any adverse effect of the immune system is negligible in comparison. Development of treatments counteracting the effect of alpha toxin is required.

Articular cartilage has very poor repair potential, however it has an extraordinary capacity to withstand physiological mechanical loads in an intact joint. The nature and extent of chondrocyte death in articular cartilage following many forms of injury (trephine, scalpel, osteotome, sutures and drilling) has been characterised, but the ability to bear mechanical injury from iatrogenic surgical interventions is still unknown.

A standard arthroscopic probe was moved at varying physiological pressures along the articular cartilage of joint before staining with fluorescent dyes to allow live/dead cell imaging using laser confocal scanning microscopy and imaging software, Image J. Bovine metatarsal phalangeal joints and fresh human cadaveric femoral condyles were used.

The probe caused statistically significant chondrocyte death in bovine cartilage (p=0.02). Mild pressure 5% cell death, moderate (standard arthroscopic technique pressure) 22% and severe pressure 38%. A similar result was seen in human tissue with 24% cell death at moderate pressure compared to a control (p=0.0699).

The widely assumed benign arthroscopic probe produces significant cell death in articular cartilage when used at standard operating pressures.

Purpose of the study and background

The CONNECT trial evaluated a theory-based intervention to increase low back pain patients' adherence to treatment recommendations through physiotherapists' communication behaviour. Bridging the gap between evidence and evidence-based practice, we aimed to develop an implementation intervention to support physiotherapists in translating the CONNECT communication training into practice.

Purpose of the study and background

Physical activity and exercise therapy are among the guideline recommendations for the rehabilitation of people with chronic low back pain (CLBP ≥ 3 months); however, patient adherence is often poor. CONNECT is a theory-based communication skills training programme designed to enhance physiotherapists' support of their CLBP patients' psychological needs in order to increase adherence to their home-based rehabilitation programme.

This study investigated confocal laser scanning microscopy (CLSM) as a novel method of imaging of chondrocytes on a collagen membrane used for articular cartilage repair. Cell viability and the effects of surgery on the cells were assessed.

Cell images were acquired under four conditions: 1, Pre-operative 2, After handling 3, Heavily grasped with forceps 4, Cut around the edge. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 1.12–1.68 ± 0.22 × 10⁶ cells/cm² in specimens without significant trauma (n=25 images), this decreased to 0.253 × 10⁶ cells/cm² in the specimens that had been grasped with forceps (p <0.001) (5 images). Cell viability on delivery grade membrane was 86.8±2.1%. The viability dropped to 76.3 ± 1.6% after handling and 35.1 ± 1.7% after crushing with forceps. Where the membrane was cut with scissors, there was a band of cell death where the viability dropped to 17.3 ± 2.0% compared to 73.4 ± 1.9% in the adjacent area (p <0.001). Higher magnification revealed cells did not have the rounded appearance of chondrocytes.

CLSM can quantify and image the fine morphology of cells on a MACI membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery.

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.

The cartilage diseases such as osteoarthritis and chondral injuries are considered irreversible and the result of recent treatments remains not optimal. One of the reasons is due to the poor understanding of chondrocyte behaviours. To understand more about cartilage, we designed a series of novel experiments. First, a total joint of bovine metatarsophalanges was isolated as our novel model. We chose it because the configuration and the healing potential were similar to human, and many variables of large animal studies could be controlled in laboratory. The model not only provided a good ex vivo platform for cartilage researches but also connected in vitro cellular studies and in vivo animal studies. To mimic joint movement a special driving machine was designed. To characterise the novel model viabilities of chondrocytes and contents of sulphated glycosaminoglycan (GAGs) in extracellular matrixes were measured every seven days. The preliminary results revealed the viabilities of chondrocytes remained above 80% alive in the middle zone after four-weeks culture. The GAGs contents decreased after this culturing period. The experiments still carry on going to compare the static and dynamic models which joint movement could be a determinative factor to the viability of chondrocytes. Cellular treatment is the recent mainstream for cartilage diseases. If advanced knowledge in chondrocyte behaviours could be obtained from this model, development of optimal treatment will be possible in the future.

In macroscopically-normal and early degenerate human articular cartilage, chondrocytes often exhibit increased volume and abnormal morphology with cytoplasmic processes. With further degeneration, chondrocyte clusters are a characteristic feature. These changes can influence matrix metabolism leading to matrix loss and predisposition to osteoarthritis (OA). Here, we report that articular chondrocytes cultured in a weak 3D agarose gel develop some of the morphological changes observed in degenerate cartilage. Cells were isolated from bovine metacarpal-phalangeal joints using collagenase. Gels were prepared with agarose (2% or 0.2% (v/v)) and cultured for 7 days (Dulbecco's modified Eagle's medium;37superscriptC;pH7.4;fetal calf serum (FCS; 1–10%)). Cells were fluorescently-labelled and volume/morphology examined by confocal microscopy. After one week of culture, chondrocytes in 2% gels (10% FCS) were mostly spheroidal; only 18.5±1% cells exhibited fine processes and 42.5±0.1% formed small clusters. However, in weak (0.2%) gels 66.9±1.3% (P=0.011) of chondrocytes had processes with lengths 7–63μm and 80.8±0.2% (P<.005) formed large clusters. In the weak gel after seven days, increasing FCS concentration markedly elevated the %age of chondrocytes in clusters from 31.1±0.2% in 1% FCS, to 87.3±0.27% in 10% FCS (P⊖.05). (Data from a minimum of three separate experiments at each condition with at least three replicates). These results suggest some similarities between the morphological changes to chondrocytes with OA development and those observed in weak agarose gels. The increased prevalence of abnormal chondrocytes with raised FCS concentration suggests that action of e.g. growth factors on chondrocytes is a more potent controller of cell shape than the strength of agarose.

Objective

There is mounting evidence to suggest a vascular insult is responsible for Perthes' disease, and it is suggested that this may have long-term implications for the vascular health of affected individuals. This study sought to use ultrasound measures to investigate vascular structure and function in children affected by Perthes' disease.

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.

Objective

The relationship between the index (2D) to ring finger (4D) is one of the most commonly studied anthropometric measures, which is believed to offer insight into early growth and the foetal environment. This study aimed to determine the relationship between the 2D:4D ratio and the risk of Perthes' disease in children.

We describe the results for the management of chronic osteomyelitis in long bones using a new single stage procedure, the Cardiff technique. Fourteen consecutive patients were treated prospectively. The patients had a mean age of 52; all patients had a mean stay in hospital of 5 days with outpatient dressings. All patients had tibial fractures, 2 were infected non unions. The origin of infection was traumatic in 13 cases and haematogenous in 1 case. This one stage procedure or The Cardiff technique, uses techniques described by both Papineau's 3 stage method and Lautenbach's procedure. It involves removal of all avascular and infected tissue via marginal debridement, opening of the medullary cavity proximally and distally, compartmental debridement and copious saline irrigation. The wound is then packed with Jelonet and kaltostat and fresh dressings applied weekly. The wound is left to heal via secondary intention. There is no routine use of antibiotics. Antibiotics were only used when required, i.e. Clinical evidence of cellulitis or infection. 11 patients healed with no further surgery, no skin grafting, no bone grafting and no flap coverage. Mean time to healing was 5 months; both non unions united giving results similar to other accepted techniques. There were 3 residual infections, 2 due to inadequate debridement which required a single further compartmental debridement (Lautenbach method), 1 had an avascular bone edge requiring further local debridement. We feel this procedure confers the following advantages; minimal surgical insult, early discharge, out patient management, good outcomes and most importantly, high patient satisfaction. It also serves as a reminder that simple wounds do well with simple solutions.

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.

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.

In some centres, serial bedside aspirations, in association with intravenous antibiotics, are still an accepted treatment for septic arthritis (Mathews, Postgraduate Medical Journal, 2008). However, there is a risk that bacterial products remain in the joint, even when the bacteria have been destroyed. We have conducted a study to ascertain whether bacterial products alone have an effect on in situ chondrocyte viability.

A hip aspirate (25μl), containing Staphylococcus aureus, from a patient with septic arthritis was added to 5ml culture medium and incubated (37°C) for 48hrs. The solution was then centrifuged (3400g for 10mins) and the supernatant removed.

Cartilage explants were harvested from a bovine metacarpophalangeal joint, placed into the bacterial supernatant and incubated at 37°C. Explants were removed at hourly intervals over a 6-hour period and stained with the fluorescent probes chloromethylfluorescein di-acetate (10μM) and propidium iodide (10μM) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal microscopy, the percentage cell death at each time point was obtained using Volocity 4 software.

Chondrocyte death increased markedly with time: 0.04% at 2hrs, 28% at 4hrs and 39% at 6hrs.

This study shows that bacterial products rapidly penetrate the cartilage matrix and have a damaging effect on in situ chondrocyte viability. Further work will clarify the contributions made by the various toxic components in the culture supernatant, but these data support the need to remove the bacteria and their products aggressively as part of the treatment of septic arthritis.

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.

Articular cartilage is attached to subchondral bone but little is known regarding bone-cartilage interactions important for chondrocyte survival. In this study, bovine articular cartilage has been evaluated in vitro to determine if the presence of subchondral bone influences chondrocyte survival. We hypothesised that

Excision of subchondral bone from articular cartilage would increase in situ chondrocyte death in explant culture and,

Articular cartilage explants (n=132) harvested from the metacarpophalangeal joints of three-year old cows (N=12) were placed into three groups:

subchondral bone excised from articular cartilage (Group A)

Explants were cultured in serum-free media over 7 days with or without media changes to assess the effect of potential soluble mediators. Using confocal laser scanning microscopy to image in situ chondrocytes, fluorescent probes to determine cell viability and biochemical assays to detect alterations in the culture media, differences in the chondrocyte responses (cell density, spatial distribution, percentage cell death) and culture medium composition between Groups A, B and C were quantified over time (2.5 hours versus 7 days).

There was no significant change in cell density for Groups A, B and C over 7 days (t-test, p> 0.05). With excision of subchondral bone from articular cartilage (Group A), there was a marked increase in chondrocyte death over 7 days primarily within the superficial zone involving an extensive area of the articular surface (p< 0.05). There was no significant increase in chondrocyte death over the same time period for Groups B and C (p> 0.05). Corresponding 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 the superficial zone.

Subchondral bone interacts with articular cartilage in vitro and promotes chondrocyte survival in the superficial zone. These data support the concept of a functional bone-cartilage system in vivo.

Chondrocytes are responsible for the mechanical resilience of cartilage by controlling the synthesis/degradation of the extracellular matrix. In osteoarthritis (OA), increased activity of cytokines/degradative enzymes (e.g. IL-1beta, MMP-13) play a key role leading to matrix breakdown/cartilage loss. Studying early events in OA might identify targets for limiting the deleterious changes to cartilage stability. Human chondrocyte shape in situ is normally elipsoidal/spheroidal however abnormal forms within otherwise macroscopically normal cartilage are present. Changes to cell shape can alter ECM metabolism and thus these abnormal forms might be an early event in OA. We have investigated whether levels of IL-1beta and MMP-13 are altered in human chondrocytes of abnormal morphology.

Tibial plateau cartilage was obtained from patients undergoing knee arthroplasty and only areas graded 0 or 0–1 studied. The shape of fluorescently-labelled in situ chondrocytes was classified by confocal scanning laser microscopy with cartilage depth, and cells characterised as normal (no cytoplasmic processes) or abnormal (one/more cytoplasmic process). Within grade 0 cartilage about 40% of the cells demonstrated abnormal morphology with a reduced proportion in deep zones. Fluorescence immunohistochemistry of antibodies for IL-1beta or MMP-13 was studied in the same cells and quantified. There was an increase in IL-1beta fluorescence with abnormal chondrocytes within the superficial (p=0.033; 21 joints > 190 cells) and deep zones (p=0.001; 8 joints > 100 cells). There were no differences between MMP-13 labelling of normal compared to abnormal chondrocytes within either the superficial or deep zones.

Our results suggest that in relatively non-degenerate cartilage, a proportion of the chondrocyte population demonstrated abnormal morphology and that these cells have elevated levels of IL-1beta but not MMP-13. However, we do not know if chondrocyte shape alters cytokine levels, or vice versa. Additionally, the role of cartilage age is unclear, as although the cartilage samples were relatively normal they were obtained from aged individuals. Nevertheless these results show changes to chondrocyte morphology and increased levels of IL-1beta, and thus presumably matrix catabolism - in relatively normal human articular cartilage, raising the possibility that this is an early event in cartilage degeneration.

Supported by the Wellcome Trust (075753).

0.9% Saline and Hartmann’s are commonly used joint irrigating solutions during articular surgery. The objective of the study was to determine whether the osmolarity of these solutions affects chondrocyte death in mechanically injured articular cartilage.

The osmolarity of 0.9% Saline (285 mOsm) and Hartmann’s (255 mOsm) solutions was varied from 100–600 mOsm by the addition of distilled water or sucrose. Osteochondral explants (rectangular blocks, n=72) harvested from the metacarpophalangeal joints of six different three-year old cows were exposed to prepared solutions of different osmolarity for 2 minutes to allow in situ chondrocytes (cells embedded within their native extracellular matrix) to respond to the altered osmotic environment. Explants were then mechanically injured through the full thickness of articular cartilage with a fresh scalpel and incubated in the same solution for 2.5 hours. Using confocal laser scanning microscopy (CLSM) and fluorescent probes to determine cell viability, percentage cell death (PCD, 100 × number of dead cells/number of dead and live cells) was quantified within the full thickness of mechanically injured articular cartilage as a function of solution osmolarity.

Cell death was localised to the superficial zone (first 100 microns from the articular surface) of injured cartilage for explants exposed to the control 0.9% Saline (285 mOsm) and Hartmann’s (255 mOsm) solutions, with relative sparing of the middle and deep zones (analysis of variance (ANOVA), p< 0.05). Compared to the control explants exposed to 0.9% Saline, PCD in the superficial zone was greatest for the low osmolarity (100 mOsm) saline solution and least for the high osmolarity (600 mOsm) saline solution (ANOVA, p=0.04). PCD in the superficial zone significantly decreased for explants exposed to 600 mOsm solutions of 0.9% Saline and Hartmann’s, compared to their respective control solutions (p< 0.05 for paired comparisons). There was no significant difference in the PCD between 600 mOsm solutions of 0.9% Saline and Hartmann’s (p=0.5).

Increasing the osmolarity of 0.9% Saline and Hartmann’s solutions is chondroprotective in a surgically relevant model of mechanical cartilage injury. These experiments have important clinical relevance for the design of irrigation solutions during arthroscopic and open articular surgery.

Autologous osteochondral cylinder transfer is a treatment option for small articular defects, especially those arising from trauma or osteochondritis dissecans. There are concerns about graft integration and the nature of tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of quality of repair. The aim was to evaluate cell viability at the graft edge from ex vivo human femoral condyles, after harvest using conventional technique. With ethical approval and patient consent, fresh human tissue was obtained at total knee arthroplasty. Osteochondral plugs were harvested using the commercially available Acufex 4.5mm diameter mosaicplasty osteotome from regions of the lateral femoral condyle (anterior cut) that were macroscopically non-degenerate and microscopically non-fibrillated. Plugs were assessed for chondrocyte viability at the graft edge using confocal laser scanning microscopy (CLSM), fluorescent indicators and image analysis. The central portions of the plugs remained healthy, with > 99% cell viability (n=5). However, there was substantial marginal cell death, of thickness 382 ± 68.2 microm in the superficial zone (SZ). Demi-plugs were created by splitting the mosaicplasty explants with a fresh No. 11 scalpel blade. The margin of SZ cell death was 390.3 ± 18.8 microm at the curved edge of the Acufex, significantly (Mann-Whitney; P= 0.0286; n =4) greater than that at the scalpel cut (34.8 ± 3.2 microm). Findings were similar when the cartilage was breached but the bone left intact. In time-course experiments, the SZ marginal zone of cell death after Acufex harvest showed no increase over the time period 15 minutes to 2 hours. Mathematical modelling of the mosaicplasty surface shows that cell death of this magnitude results in a disturbing 33% of the superficial graft area being non-viable. In conclusion, mosaicplasty, though capable of transposing viable hyaline cartilage, is associated with an extensive margin of cell death that is likely to compromise lateral integration. There would appear to be considerable scope for improvement of osteochondral transplant techniques which may improve graft-recipient healing and clinical outcomes.

Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (1,3). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (1). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy.

Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (2). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones.

Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm³ and 5227±1974μm³ respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively. Chondrocytes within the proliferative zone then recovered in volume by ~60% over the following 20mins (p=0.04), whereas no volume recovery was detected in hypertrophic cells (p=0.94).

Conclusions: For the increase in growth plate chondrocyte volume to produce hypertrophy it is essential that the membrane transporters which normally prevent cell swelling are suppressed, otherwise the increase in volume will be compromised. These results suggest that chondrocyte hypertrophy is associated with reduced activity of the swelling-stimulated osmolyte transporter whereas the pathway is active in proliferating chondrocytes. Changes in the activity of this pathway are likely to be an important component in the control of chondrocyte hypertrophy. It is clear that the contributions of other membrane transporters in mediating chondrocyte swelling must be identified in order to understand the overall hypertrophic process.

The hip joints are commonly affected in Juvenile Idiopathic Arthritis (JIA) in childhood. Common features are pain, subluxation, femoral anteversion, coxa valga, significant fixed flexion deformity and a true arthritis, with loss of articular cartilage principally from the femoral head but also the acetabulum.

In children with JIA, it is accepted that a medial soft tissue release of the hips, dividing adductor longus, adductor brevis and the ilio-psoas, is a useful tool in the management of significant hip joint involvement. The principal indication for surgery is the relief of pain, but other benefits are correction of fixed flexion deformity, restoration of articular cartilage, increased abduction of the hips and, in those children who are unable to walk, frequently a transition to the potential to walk. The procedure is nearly always performed bilaterally.

Our study aimed to document the restoration of articular cartilage at the hips following soft tissue release. It has been noted in the literature that there is regrowth of articular cartilage in the hip but there has been no true documentation of this and x-ray studies are unreliable as the elimination of fixed flexion deformity can prejudice accurate analysis of femoral head geometry on 2 –dimensional views.

We therefore carried out MRI scanning of the hips, immediately prior to the soft tissue release and 12–18 months post-operatively. In 10 consecutive patients analysed, scans demonstrated true articular cartilage regrowth in 8 cases.

We thus conclude that soft tissue release of the hips in JIA is a useful management tool, and may to some extent reverse the severe articular cartilage loss seen in these children. The next stage of our study is to analyse the articular cartilage at the time of subsequent hip arthroplasty to determine whether true hyaline cartilage is reformed or whether the reconstitute represents fibrocartilage.

Perthes' disease is common in certain urban areas within Britain. It is one manifestation of a generalised growth disorder and nutritional causes are suspected. Orthopaedic surgeons throughout the Yorkshire region recorded all new patients with Perthes' disease over two years. There were large geographical differences in incidence which could not be explained by urban-rural or social class differences. No cases were recorded in a large area within the eastern part of the region, which is in high-grade farming land and has had a relatively low infant mortality throughout this century.

There is a high incidence of Perthes' disease among the children of unskilled manual workers in underprivileged urban areas in Britain. The skeletal measurements of 38 Liverpool children with Perthes' disease were compared with those of their siblings and of normal children from the inner and outer city. Children in families where Perthes' disease occurs have retarded growth of the trunk, with reduced sitting height and bi-acromial diameter. Among those who develop the disease there is also retarded limb growth, most evident as unusually small feet.