Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) as it is largely dispensable and readily accessible through minimally invasive procedures such as lipoaspiration. Until recently MSCs could only be isolated in a process involving ex-vivo culture. Pericytes (CD45−, CD146+, and CD34−) and adventitial cells (CD45−, CD146−, CD34+) represent two populations of MSCs (collectively termed perivascular stem cells or PSCs) that can be prospectively purified using fluorescence activated cell sorting (FACS). We performed FACS on lipoaspirate samples from n=129 donors to determine the frequency and yield of PSCs and to establish patient and processing factors that influence yield.

The mean number of stromal vascular fraction (SVF) cells from 100ml of lipoaspirate was 37.8×106. Within the SVF, mean cell viability was 82%, with 31.6% of cells being heamatopoietic (CD45+). Adventitial cells and pericytes represented 31.6% and 7.9% of SVF cells respectively. As such, 200ml of lipoaspirate would theoretically yield 24.5 million MSCs –a sufficient number to enable point-of-care delivery for use in several orthopaedic applications. The yield and prevalence of PSCs were minimally affected by donor age, sex and BMI. Storing lipoaspirate samples for up to 72 hours prior to processing had no significant deleterious effects on MSC yield or viability.

Our study confirms that pure populations of MSC-precursors (PSCs) can be prospectively isolated from adipose tissue, in sufficient quantities to negate the necessity for culture expansion while widening possible applications to include trauma, where a time delay between extraction and implantation excludes their use.

Objective:

To assess efficacy of pulsed ultrasound for accelerating regenerate consolidation.

The aim of this study was to identify risk factors for failure of exchange nailing in tibial diaphyseal fracture non-unions. The cohort comprised 99 tibial diaphyseal fracture non-unions treated by exchange nailing. The mean age of the patients at exchange nail surgery was 36 years. The median time from primary fixation to exchange nailing was 6.4 months. The main outcome measures were union, number of secondary fixation procedures required to achieve union and time to union. Univariate analysis and multiple regression were used to identify risk factors for failure to achieve union.

Multiple causes for non-union were found in 31.3% cases, with infection present in 32.3%. Further exchange procedures were required in 35.4%, 7.1% required the use of other fixation modalities. Union was ultimately achieved in 97.8%. The median time to union was 8.7 months. Univariate analysis revealed that cigarette smoking, an atrophic pattern of non-union and infection were predictive for failure of exchange nailing (p<0.05). Multi-regression analysis found that only infection was statistically significantly predictive (p<0.05) of exchange nail failure.

Exchange nailing is an effective treatment for tibial diaphyseal non-unions even in the presence of infection. Smoking, atrophic pattern of non-union and infection are associated with an increased risk of further fixation surgery.

The purpose of this study was to identify the prevalence of common mental disorders in patients undergoing complex limb reconstruction.

Patients undergoing limb reconstruction are vulnerable to mental health problem as they must adapt to significant and prolonged physical disability. Treatment emphasis has been on restoration and rehabilitation of physical health with little or no attention given to spectrum of psychological consequences. IMPARTS (Integrating Mental and Physical healthcare: Research, Training and Services) is a King's Health Partners initiative aiming to develop informatics to improve detection and management of common mental disorders in medical settings. IMPARTS screening in the King's College Hospital limb reconstruction clinic commenced in April 2012.

Outpatients attending between April 2012 and November 2013 were screened prior to their appointment. Patients were screened for symptoms of depression, anxiety, post-traumatic stress disorder (PTSD), alcohol dependence and drug use.

In total, 298 individual patients were screened. The prevalence of depression was found to be 21.8%, with 6.4% experiencing suicidal thoughts. Probable anxiety disorder was identified in 20.7% of patients. Symptoms of PTSD were reported by 29.2%, with 9.0% reporting severe symptoms. Probable alcohol dependence was identified in 2.7% of patients, and 3.0% screened positive for drug misuse.

The consequences of undergoing limb reconstruction stretch beyond the physical problem to mental well-being, rendering patients vulnerable to mental health problems and substance misuse. Early detection and management of such problems may have a significant effect on physical treatment outcome and rehabilitation to productive social life. There is urgent need to integrate mental health care as part of early management of severely injured patients.

Bone cutting produces heat which macroscopically leads to charring and the formation of bone dust. As part of a project to design a novel bone cutting device, we studied the extent of histological thermal damage from bone cutting with different cutting blades.

Three blades were used: a bone hacksaw made in the nineteenth century which was used for amputation, a sagittal saw blade made by Ortho Solutions, and a sagittal saw blade made by Stryker. Sheep femurs were harvested from recently euthanised animals and cuts were made with these three devices, producing ring-shaped bone specimens. Specimens were immediately stored in formaldehyde, decalcified, and stained with hematoxylin and eosin. The edge of the specimens was then photographed microscopically, and the images examined with the computer programme Axiovision (Carl Zeiss AG, Oberkochen, Germany). Visual examination allowed identification of live and dead osteocytes, and also to measure their depth from the surface.

A minimal of 7 images was obtained per blade. The hacksaw specimens had the highest percentage of live osteocytes (n=214, 59.8%), and with the shortest average depth where live osteocytes were located (169μm, SD 78.15). In comparison, the percentage of live osteocytes for the Ortho Solutions (n=156, 17.4%) and Stryker (n=168, 29.5%) blades were much lower. The difference in average depths where live osteocytes were located was statistically significant between the three groups (p < 0.001). The average depths of dead osteocytes were shallowest for the Stryker (115μm, SD 67.56) and hacksaw (118.28 μm, SD 75.16) groups with no statistical difference between them.

In conclusion the hacksaw appeared to produce the least thermal damage histologically during cutting. The results reflect a relationship between certain features in cutting blade designs and the extent of thermal damage. Future experiments to directly measure heat produced during cutting are planned.

Bone cutting produces heat which macroscopically leads to charring and the formation of bone dust. As part of a project to design a novel bone-cutting device, we studied the extent of histological thermal damage from different cutting blades. Three blades were used: a nineteenth century bone hacksaw, and modern sagittal saw blades manufactured by Ortho Solutions and Stryker. Sheep femurs were harvested from recently euthanised animals and cuts were made with these blades. Specimens were immediately stored in formaldehyde, decalcified, and stained with hematoxylin and eosin. The edge of the specimens was then photographed microscopically, and the images examined with Axiovision software (Carl Zeiss AG, Oberkochen, Germany). Visual examination allowed identification of live and dead osteocytes, and also to measure their depth from the surface. A minimal of 7 images was obtained per blade. The hacksaw specimens had the highest percentage of live osteocytes (n=214, 59.8%), and the shortest average depth where live osteocytes were located (169 μm, SD 78.15). In comparison, the percentage of live osteocytes for the Ortho Solutions (n=156, 17.4%) and Stryker (n=168, 29.5%) blades were much lower. The difference in average depths where live osteocytes were located was statistically significant between the three groups (p<0.001). In conclusion the hacksaw appeared to produce the least thermal damage histologically during cutting. The results reflect a relationship between certain features in cutting blade designs and the extent of thermal damage. Future experiments to monitor heat produced during cutting are planned.

Aim

To investigate the effects of strain rate and mineral level on the stress at failure, stiffness and toughness of whole bones.

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 type, duration and intensity of exercise required to induce mechanical hypoalgesia is poorly defined. We are interested in identifying the exercise parameters required to induce raised pressure pain thresholds. This pilot study investigates the effect of indoor rowing on pressure pain threshold (PPT) in high performance rowers. Our ultimate aim is to investigate the potential of utilising exercise in the treatment of chronic pain and specifically in relation to the management of knee osteoarthritis. 20 high performance rowers (13M:7F; Mean Age 20.8 years; SD 1.74) were recruited from the University of Nottingham and Nottingham Boat Club high performance rowing teams under a research protocol approved by the University of Nottingham Ethics Committee. PPT measurements were made in triplicate using an algometer (SOMEDIC, Sweden) at the medial knee joint line, anterior tibia and sternum, pre- and post-exercise. Anthropomorphic and rowing ergometer power output data were also recorded. There was significant increase in PPT values at all sites following exercise (Medial joint line: 127.6Nm-2, 26%, p=0.001; Tibia: 110.8Nm-2, 24.7%, p<0.001; Sternum: 48.9Nm-2, 11.7%, p=0.005 – Wilcoxon Signed Rank) statistical power was 97.1%, 100% and 88.1%, respectively. PPT was greater at baseline at the medial joint line compared to other sites, reaching highly significant relative to the sternum (p<0.001). We determined that ten minutes of high intensity indoor rowing induced hypoalgesia in high performance rowers. Further research is required to investigate the detailed interplay between exercise and hypoalgesia, including its duration post exercise, to identify suitability for use in pain management strategies.

Introduction

Bending tests are commonly used to evaluate the mechanical behaviour of small animal bones. To test whole bones, it is normal that soft tissue should be removed before testing. However, cleaning the specimens might disturb the callus, interfering with the mechanical properties. This study compares mechanical properties of rat tibia between specimen with and without muscle cleaning

INTRODUCTION

This study investigates the relationship between direct measurement of outcome and patient report of that outcome via the OKS. The stability of this relationship over time following surgery is also assessed.

Aim

Although non-union is a devastating and costly consequence of trauma for the child, family and society it is felt to be a rare complication in children. Currently there is no data available in the literature regarding its incidence either per fracture or per head of population. Should we be taking paediatric fracture non-union more seriously regarding research, resource allocation and informed consent? Our aim was to determine the incidence of non-union per child and per fracture.

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.

The treatment of chronic osteomyelitis requires both appropriate surgical and antibiotic management. Prolonged intravenous antibiotic therapy followed by oral therapy is widely utilised. Despite this, the long-term recurrence rate is approximately 25%. The aim of this cohort study was to examine the effectiveness of marginal surgical resection in combination with local application of antibiotics (Collatamp G - gentamicin in a collagen fleece). Post-operatively this was followed by a short course of intravenous antibiotics, then oral antibiotics, to 6 weeks in total. A cohort of 50 patients from a 10-year period, 2000 to 2010, with chronic osteomyelitis was identified. Most were male (n= 35, 70%) and the average age is 40.9 years (SD 15.9). The mean follow-up duration was 3.2 years (SD 1.8). The average length of admission was 9.8 days (SD 11.4). 6 patients (12%) suffered recurrence of infection requiring further treatment. We used the Cierny and Mader classification to stratify the patients further. There were 24 (48%) ‘A’ hosts and 26 (52%) ‘B’ hosts. ‘A’ hosts had a shorter duration of admission (7.1 days) than ‘B’ hosts (12.3 days). There was no significant difference between recurrence rates of ‘A’ and ‘B’ hosts. The available pre-operative C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) levels did not predict disease recurrence. Overall, the disease-free probability for this cohort was 0.80. A similar cohort treated with prolonged systemic and oral antibiotics reported by Simpson and colleagues (JBJS Br 2001) had a disease-free probability at 0.68. Local administration of gentamicin in a collagen fleece leads to improved disease-free probability when compared with prolonged systemic antibiotic treatment. We believe this is a useful component in the management of chronic osteomyelitis.

Aims

The aim of this study was to compare biomechanical properties of pre-contoured plate fixation using different screw fixation modes in a mid-shaft clavicle fracture model.

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.

There is an established link between bone quality and fracture risk. It has been suggested that reduced bone quality will also reduce the toughening mechanisms displayed during loading at a high strain rate. We hypothesised that partially decalcified bone will not demonstrate an increase in force required to cause failure when comparing low and high strain rate loading.

Mechanical properties were defined by the maximum force at failure. Bone quality was defined by the mineral content. This was altered by subjecting the bones to ultrasonically assisted decalcification in 10M EDTA to achieve an average 18% mineral reduction (A 70 yr old woman has approx 18% of her peak bone mass). 20 pairs of sheep femurs were harvested and split into four equal groups: normal bone quality, fast strain rate (NF); normal bone quality, slow strain rate (NS); low bone quality, fast strain rate (LF) and low bone quality, slow strain rate (LS). All mechanical testing was carried out by means of 3-point bending. Load representing the slow strain rate was applied by a mechanical testing machine (Zwick) at a rate resulting in a deflection of 1mm/s. The dynamic loading was applied by a custom designed pneumatic ram at a mean rate of deflection between the specimens of 2983 mm/s (±SD 1155), this equates to strain rates experienced in a road traffic accident.

The following results for force at failure were found (mean ± SD). NF: Force 5503N (± 1012); NS: Force 3969N (± 572); LF: Force 3485N (± 772); LS: Force 3165N (± 605). Groups were compared using a Mann-Whitney U test. Significant results were found between the following groups: Normal bone quality, strain rate compared (NF-NS) p<0.002; Fast strain rate, bone quality compared (NF-LF) p=0.008; Slow strain rate, bone quality compared (NS-LS) p=0.02. No statistical significance was found when comparing low bone quality, strain rate compared (LF-LS) p=0.47.

These results show that normal healthy bone has an ability to withstand higher strain rates which protects it against fracture. This ability to withstand high strain rates is lost in decalcified bone making it more susceptible to fracture. The results of this study indicate the importance of strain rate reduction as well as energy absorption in the design of hip protectors and in environmental modifications.

End-stage osteoarthritis is characterised by pain and reduced physical function, for which total knee arthroplasty (TKA) is recognised to be a highly effective treatment. Most implants are multi radius in design, though modern kinematic theory suggests a single flexion/extension axis is located in the femur. A recently launched TKA implant (Triathlon, Stryker US), is based on this theory, adopting a single radius of curvature femoral component. It is hypothesised that this design allows better function, and specifically, that it results in enhanced efficiency of the quadriceps group through a longer patello-femoral moment arm.

Change in power output was compared between single and multi radius implants as part of a larger ongoing randomised controlled trial to benchmark the new implant. Power output was assessed using a Leg Extensor Power Rig, well validated for use with this population, pre-operatively and at 6, 26 and 52 weeks post-operatively in 101 Triathlon and 82 Kinemax implants. All patients were diagnosed with osteoarthritis, and drawn from a single centre. Output was reported as maximal wattage (W) generated in a single leg extension, and expressed as a proportion of the contralateral limb power output to act as an internal control.

The results are shown in the table below. Two-way repeated measures ANOVA demonstrated a significant effect of TKA on the quadriceps power output, F = 249.09, p = <0.001 and also a significant interaction of the implant group on the output F = 11.33, p = 0.001. Independent samples t-tests of between group differences at the four assessment periods highlighted greater improvement in the single radius TKA group at all post-operative assessments (p <0.03), see table.

The theoretical enhanced quadriceps efficiency conferred by single radius design was found in this study. Power output was significantly greater at all post-operative assessments in the single radius compared to the multi radius group. This difference was particularly relevant at early 6 week and 1 year assessment. Lower limb power output is known to link positively to functional ability. The results support the hypothesis that TKAs with a single radius design have enhanced recovery and better function.

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.