Abstract

Stratification is required to ensure that only those patients likely to benefit, receive Autologous Chondrocyte Implantation (ACI); ideally by assessing a biomarker in the blood. This study aimed to assess differences in the plasma proteome of individuals who respond well or poorly to ACI.

Isobaric tag for relative and absolute quantitation (ITRAQ) mass spectrometry and label-free proteomics analyses were performed in tandem as described previously by our group (Hulme et al., 2017; 2018; 2021) using plasma collected from ACI responders (n=10) compared with non-responders (n=10) at each stage of surgery (Stage I, cartilage harvest and Stage II, cell implantation).

iTRAQ using pooled plasma detected 16 proteins that were differentially abundant at baseline in ACI responders compared with non-responders (n=10) (≥±2.0 fold; p<0.05). Responders demonstrated a mean Lysholm (patient reported functional score from 0–100) improvement of 33±13 and non-responders a mean worsening of −13±13 points. The most pronounced plasma proteome shift was seen in response to Stage I surgery in ACI non-responders, with 48 proteins being differentially abundant between the two surgical procedures. We have previously noted this marked shift in response to initial surgery in the SF of ACI non-responders, several of these proteins were associated with the Acute Phase Response. One of these proteins, clusterin, could be confirmed in patients’ plasma using an independent immunoassay using individual samples. Label-free proteomic data from individual samples identified only cartilage acidic protein-1 (known to associate with osteoarthritis progression) to be significantly more abundant at Stage I in the plasma of non-responders.

This study indicates that proteins can be identified within the plasma that have potential use in ACI patient stratification. Further work is required to validate the findings of this discovery-phase work in larger ACI cohorts.

The objectives of the study were to investigate demographic, injury and surgery/treatment-associated factors that could influence clinical outcome, following Autologous Chondrocyte Implantation (ACI) in a large, “real-world”, 20 year longitudinally collected clinical data set.

Multilevel modelling was conducted using R and 363 ACI procedures were suitable for model inclusion. All longitudinal post-operative Lysholm scores collected after ACI treatment and before a second procedure (such as knee arthroplasty but excluding minor procedures such as arthroscopy) were included. Any patients requiring a bone graft at the time of ACI were excluded. Potential predictors of ACI outcome explored were age at the time of ACI, gender, smoker status, pre-operative Lysholm score, time from surgery, defect location, number of defects, patch type, previous operations, undergoing parallel procedure(s) at the time of ACI, cell count prior to implantation and cell passage number.

The best fit model demonstrated that for every yearly increase in age at the time of surgery, Lysholm scores decreased by 0.2 at 1-year post-surgery. Additionally, for every point increase in pre-operative Lysholm score, post-operative Lysholm score at 1 year increased by 0.5. The number of cells implanted also impacted on Lysholm score at 1-year post-op with every point increase in log cell number resulting in a 5.3 lower score. In addition, those patients with a defect on the lateral femoral condyle (LFC), had on average Lysholm scores that were 6.3 points higher one year after surgery compared to medial femoral condyle (MFC) defects. Defect grade and location was shown to affect long term Lysholm scores, those with grade 3 and patella defects having on average higher scores compared to patients with grade 4 or trochlea defects.

Some of the predictors identified agree with previous reports, particularly that increased age, poorer pre-operative function and worse defect grades predicted poorer outcomes. Other findings were more novel, such as that a lower cell number implanted and that LFC defects were predicted to have higher Lysholm scores at 1 year and that patella lesions are associated with improved long-term outcomes cf. trochlea lesions.

Conventional proximal tibial osteotomy is a widely successful joint-preserving treatment for osteoarthritis; however, conventional procedures do not adequately control the posterior tibial slope (PTS). Alterations to PTS can affect knee instability, ligament tensioning, knee kinematics, muscle and joint contact forces as well as range of motion.

This study primarily aimed to provide a comprehensive investigation of the variables influencing PTS during high tibial osteotomy using a 3D surgical simulation approach. Secondly, it aimed to provide a simple means of implementing the findings in future 3D pre-operative planning and /or clinically.

The influence of two key variables: the gap opening angle and the hinge axis orientation on PTS was investigated using three independent approaches: (1) 3D computational simulation using CAD software to perform virtual osteotomy surgery and simulate the post-operative outcome. (2) Derivation of a closed-form mathematical solution using a generalised vector rotation approach (3) Clinical assessment of synthetically generated x-rays of osteoarthritis patients (n=28; REC reference: 17/HRA/0033, RD&E NHS, UK) for comparison against the theoretical/computational approaches.

The results from the computational and analytical assessments agreed precisely. For three different opening angles (6°, 9° and 12°) and 7 different hinge axis orientations (from −30° to 30°), the results obtained were identical. A simple analytical solution for the change in PTS, ΔP_s, based on the hinge axis angle, α, and the osteotomy opening angle, θ, was derived:

ΔP_s=sin^-1(sin α sin θ)

The clinical assessment demonstrated that the absolute values of PTS, and changes resulting from various osteotomies, matched the results from the two relative prediction methods.

This study has demonstrated that PTS is impacted by the hinge axis angle and the extent of the osteotomy opening angle and provided computational evidence and analytical formula for general use.

Abstract

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Abstract

Introduction

Primary cilia are singular structures containing a microtubule-based axoneme which are believed to not only be mechanosensitive but also to co-ordinate many cell functions via signalling pathways including Hedgehog and Wnt. Primary cilia have previously been described on cells of mouse intervertebral discs (IVDs), but not in bovine or human IVDs. Our aim was to examine primary cilia in these species.

The arcOGEN study identified the 9q33.1 locus as associated with hip osteoarthritis (OA) in females. TRIM32 lies within this locus and may have biological relevance to OA; it encodes a protein with E3 ubiquitin ligase activity.

Sanger sequencing of TRIM32 in the youngest 500 female patients with hip OA from the arcOGEN study identified genetic polymorphisms in the proximal promoter, and 3'untranslated region of TRIM32 that are disproportionately represented in female patients with hip OA compared to the control population.

Reduced expression of TRIM32 was identified in femoral head articular chondrocytes from patients with hip OA compared to control patients. Trim32 knockout resulted in increased aggrecanolysis in murine femoral head explants. Murine chondrocytes deficient in Trim32 exhibited increased expression of mature chondrocyte markers following anabolic cytokine stimulation, and increased expression of hypertrophic chondrocyte markers following catabolic cytokine stimulation.

Trim32 knockout mice demonstrated increased cartilage degradation and tibial subchondral bone changes after surgically-induced knee joint instability. Increased cartilage degradation and medial knee subchondral bone changes were also identified in aged Trim32 knockout mice.

These results further implicate TRIM32 in the genetic predisposition to OA, and indicate a role for TRIM32 in the joint degeneration evident in OA. These results support the further study of TRIM32 in the pathophysiology of OA and development of novel therapeutic strategies to manage OA.

The current study aims to ascertain the outcome of ACI with simultaneous transplantation of an autologous bone plug for the restoration of osteoarticular defects in the femoral condyle of the knee (‘Osplug’ technique).

Seventeen patients (mean age of 27±7 years), twelve with Osteochondritis dissecans (OD) and five with an osteochondral defect (OCD) was treated with unicortical autologous bone graft combined with ACI (‘Osplug’ technique). Functional outcome was assessed with Lysholm scores obtained for 5 years post-operatively. The repair site was evaluated with the Oswestry Arthroscopy Score (OAS), MOCART MRI score and ICRS II histology score.

The mean defect size was 4.5±2.6 SD cm² and mean depth was 11.3±5 SD mm. A significant improvement of Lysholm score from 45 (IQR 24, range 16–79) to 77 (IQR 28, range 41–100) at 1 year (p-value 0.001) and 70 (IQR 35, range 33–91) at 5 years (p-value 0.009). The mean OAS of the repair site was 6.2 (range 0–9) at a mean of 1.3 years. The mean MOCART score was 61 ± 22SD (range 20–85) at 2.6 ± 1.8SD years. Histology demonstrated generally good integration of the repair cartilage with the underlying bone. Poor lateral integration of the bone graft on MRI and low OAS were significantly associated with a poor outcome and failure.

The Osplug technique shows significant improvement of functional outcome for up to 5 years. This is the first report describing the association of bone graft integration with functional outcome after such a procedure.

Purpose

To assess outcomes of manipulating upper extremity fractures with conscious sedation compared with formal reduction and casting in theatre under general anaesthesia and image intensifier control.

Background

Structural and functional outcome of bone graft with first or second generation autologous chondrocyte implantation (ACI) in osteochondral defects has not been reported.

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice.

Our aim is to investigate the role of TRIM32 in human and murine articular tissue.

TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation.

TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10⁻⁵), and Sox9 (p=2.35×10⁻⁶). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006).

These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation.

By the end of training, every registrar is expected to demonstrate proficiency in total knee replacement (TKR). It is unclear whether functional outcomes for knee arthroplasty performed by training grade doctors under supervision of a consultant have equivalent functional outcomes to those performed by consultants.

This study investigated the functional outcomes following TKR in patients operated on by a supervised orthopaedic trainee compared to a consultant orthopaedic surgeon. Patients undergoing surgery by a consultant (n=491) or by a trainee under supervision (n=145) between 2003 and 2006 were included. There was a single implant, approach and postoperative rehabilitation regime. Patients were reviewed eighteen months, three years and five years postoperatively.

There were no significant differences in preoperative patient characteristics between the groups. There was no difference in length of stay or transfusion or tourniquet time. Both consultant (p<0.001) and trainee (p<0.001) groups showed significant improvement in AKSK and AKSF scores between preoperative and 18 month review and there was no difference in the magnitude of observed improvement between groups (AKSK p=0.853; AKSF p=0.970). There were no significant differences in either score between the groups preoperatively or at any review point postoperatively. At five years postoperative, both groups had a median OKS of 34 (p=0.921).

This is the largest reported series of outcomes following primary TKR examining functional outcome linked with grade of surgeon. It shows that a supervised trainee will achieve comparable functional outcomes at up to 5 years post operatively.

Paget's disease of bone (PDB) is the second most common metabolic bone disease. Osteoarthritis (OA) affects one-third of patients with PDB. The incidence of THR (total hip replacement) and TKR (total knee replacement) is 3.1- and 1.7-fold higher in PDB patients compared to non-affected age-matched controls. No large studies or joint registry reports exist describing the outcomes following THR or TKR in patients with PDB.

The objectives of this study were to investigate the outcomes following THR and TKR in patients with PDB using national joint registry data. 144 THR and 43 TKR were identified using the Scottish Arthroplasty Project from 1996–2013.

For THR, the most common early post-operative surgical complications were haematoma formation (1.4%), and surgical site infection (1.4%). The absolute incidence during follow-up of dislocation was 2.8%, and revision hip arthroplasty was performed in 2.8% of cases. Implant survival of the primary prosthesis was 96.3% (CI: 92.8 – 99.8) at 10-years, and patient survival was 50.0% (39.6 – 60.4) at 10-years.

For TKR, the most common early post-operative surgical complication was surgical site infection (2.3%). The absolute incidence during follow-up of revision knee arthroplasty was 4.7%. On survival analysis, implant survival of the primary prosthesis was 94.5% (CI: 87.1 – 100) at 10-years, and patient survival was 38.3% (16.7 – 59.9) at 10-years.

This is the largest reported series of outcomes following primary THR and TKR in patients with PDB. PDB patients are not at increased risk of surgical complications following primary THR or TKR compared to non-PDB patients.

The natural history of primary anterior glenohumeral dislocation in adolescent patients remains unclear and no consensus exists for management of these patients. The study objectives were to report the natural history following primary anterior glenohumeral joint dislocation in adolescent patients and to identify risk factors for repeat dislocation.

We reviewed prospectively-collected clinical and radiological data of 133 adolescent patients (mean age 16.3 years (range 13–18); 115 male patients (86.5%)) diagnosed with primary anterior glenohumeral joint dislocation and managed nonoperatively from 1996 to 2008 at our institution (mean follow-up 95.2 months (range 1–215)).

During follow-up, 102 (absolute incidence of 76.7%) patients experienced repeat dislocation. Median time interval between primary and repeat dislocation was 10 months (CI: 7.4 – 12.6). On survival analysis, 59% (CI: 51.2 – 66.8%) of patients remained stable one year following initial injury, 38% (CI: 30.2 – 45.8%) after two years, 21% (CI: 13.2- 28.8%) after five years, and 7% (CI: 1.1–12.9%) after 10 years. Neither age nor gender significantly predicted repeat dislocation during follow-up.

In conclusion, adolescent patients with primary anterior glenohumeral joint dislocations have a high rate of repeat dislocation, which usually occurs within two years of initial injury, and these patients should be considered early for operative stabilisation.

Introduction:

Antimicrobial resistance is an important patient safety issue. Antibiotic Stewardship is one of the key strategies in tackling this problem. We present our data over a two year period from October 2011 to December 2013.

Introduction:

The treatment of acute rupture of the tendo-achilles remains controversial. There is good evidence to suggest that outcomes are the same for both operative and non-operative treatment when a functional rehabilitation program is utilised. However, debate continues as to whether the radiological gap-size between the proximal and distal remnants of the tendon has an influence on the suitability for non-operative management.

Summary

Randomised controlled study evaluating new bone formation in vivo in fracture non-unions by bone marrow derived stromal cells (BMSC). These cells do not show statistically significant new bone formation. Age of the patient during fracture, diabetes and doubling time had been observed to be correlated with fracture healing.

Aim:

To determine radiographic variables that predict the need for distal extension of the fusion beyond Cobb-to-Cobb levels in treating thoracolumbar/lumbar (TL/L) scoliosis (Lenke 5) in adolescent patients.

Lowest instrumented vertebra (LIV) selection is critical to preventing complications following posterior spinal arthrodesis (PSA) for thoracolumbar/lumbar adolescent idiopathic scoliosis (TL/L AIS), but evidence guiding LIV selection is limited.

This study aimed to investigate the efficacy of PSA using novel unilateral convex segmental pedicle screw instrumentation (UCS) in correcting TL/L AIS, to identify radiographic parameters correlating with distal extension of PSA, and to develop a predictive equation for distal fusion extension using these parameters.

We reviewed data (demographic, clinical, radiographic, and SRS-22 questionnaires) preoperatively to 2-years' follow-up for TL/L AIS patients treated by PSA using UCS between 2006 to 2011. 53 patients were included and divided into 2 groups: Group-1 (n=36) patients had PSA between Cobb-to-Cobb levels; Group-2 (n=17) patients required distal fusion extension.

A mean curve correction of 80% was achieved. Mean postoperative LIV angle, TL/L apical vertebra translation (AVT), and trunk shift were lower than previous studies. Six preoperative radiographic parameters significantly differed between groups and correlated with distal fusion extension: thoracic curve size, thoracolumbar curve size, LIVA, AVT, lumbar flexibility index, and Cobb angle on lumbar convex bending. Regression analysis optimised an equation (incorporating the first five parameters) which is 81% accurate in predicting Cobb-to-Cobb fusion or distal extension. SRS-22 scores were similar between groups.

We conclude that TL/L AIS is effectively treated by PSA using UCS, six radiographic parameters correlate with distal fusion extension, and a predictive equation incorporating these parameters reliably informs LIV selection and the need for fusion extension beyond the caudal Cobb level.

The aim of this study was to characterise injury patterns and examine whether survival had improved over the last decade of conflict in Iraq and Afghanistan. A logistical regression model was applied to all UK casualty data from the Joint Theatre Trauma Registry.

There were 2785 casualties over the 10-years. 72% of casualties from hostile action were injured by blast weapons. The extremities were the post commonly injured body region, being involved in 43% of all injuries sustained. The New Injury Severity Score that was observed to be associated with a 50% chance of survival rose every year from 38 in 2003 to 62 in 2012. The odds ratio of surviving with a Trauma and Injury Severity Score (TRISS) of 50% rose by 1.349 (95% CI = 1.265–1.442) per year. The actual TRISS value associated with a 50% chance of survival dropped every year from 35.3% in 2003 to 0.9 in 2010 and was un-calculable in 2011–12.

This study confirms that the last decade of conflict has been characterised by blast wounds and injuries involving the extremities. A consistent improvement in survival over the 10 years has been demonstrated, to the point that traditional metrics for measuring improvement in trauma care have been exhausted.

Aim

To evaluate the efficacy of bone marrow derived stromal cells (BMSC) for the treatment of non-unions in fractures.

Background

Intervertebral disc cells exist in a challenging physiological environment. Disc degeneration occurs early in life implying that disc cells may no longer be able to maintain a functional tissue. We hypothesise that disc cells have a stress response different from most other cells because of the disc environment. We have compared the stress response of freshly isolated and cultured bovine nucleus pulposus (NP) cells with bovine dermal fibroblasts, representative of cells from a vascularised tissue.

The potential of cells derived from human umbilical cord(UC) for orthopaedic cell engineering is evaluated by dissecting the UC into four distinct anatomical structures – cord lining (CL), Wharton's Jelly (WJ), umbilical cord artery (UCA) and umbilical cord vein (UCV). Cells from individual anatomical layers were grown by explant culture technique for 21 days. Tri-lineage differentiation and growth kinetics of cells from each layer were compared. Flowcytometry was done according to ISCT criteria to ascertain their surface antigen expressions. Cells from all four layers differentiated into bone, cartilage and fat. Osteogenic and chondrogenic differentiation was variable for each type of cells. All cells expressed surface antigens characteristic of mesenchymal stem cells (MSC). These cells can form a potential cell source in cell engineering to produce bone and cartilage although individual cell type needs to be characterised from each anatomical layer of UC and identify the best cell type for cell engineering.

Stem cells are a key component of regenerative medicine strategies. Particular areas of musculoskeletal application include cartilage and bone regeneration in arthritis and trauma. There are several types of stem cell and this article will focus on the adult derived cells. The review includes current issues and future developments.

Plantar fasciitis is thought to be a self limiting condition best treated by conservative measures, but despite this many patients have a prolonged duration of symptoms and for some surgery may be indicated. Partial plantar fascial release is reported to have a short term success rate of up 80%, but anecdotally this was not thought to represent local experience.

An audit of long term patient reported outcomes following surgery was performed. A total of 26 patients (29 feet) were identified retrospectively and case notes were reviewed for each patient. Patients were contacted by letter and invited to complete two validated patient reported outcome score questionnaires (foot and ankle visual analogue scale (VAS) and MOXFQ).

The average age of the patients was 42.4(range 28–61) for males and 46.2 (range 33–60) for female patients, with a female:male ratio of 2.7:1. Preoperative treatments included orthotics (29), steroid injections (23), physiotherapy (21) and cast immobilisation (11). The average duration of treatment prior to surgical intervention was 3.1 years (range 1–5). All patients were reviewed post operatively and discharged from follow up at an average of 31 weeks, at which time 38% remained symptomatic.

We conclude that the results from open partial plantar fascial release are poor and it is a technique of dubious clinical value.

Background

Proteoglycans (PGs) have long been known to be important to the functioning of the intervertebral disc. The most common PG is aggrecan, but there are also small leucine-rich proteoglycans (SLRPs) which constitute only a small percentage of the total PGs. However, they have many important functions, including organising the collagen, protecting it from degradation and attracting growth factors to the disc. We have examined how the core proteins of these molecules vary in intervertebral discs from patients with different pathologies.

This article reviews the current knowledge of the intervertebral disc (IVD) and its association with low back pain (LBP). The normal IVD is a largely avascular and aneural structure with a high water content, its nutrients mainly diffusing through the end plates. IVD degeneration occurs when its cells die or become dysfunctional, notably in an acidic environment. In the process of degeneration, the IVD becomes dehydrated and vascularised, and there is an ingrowth of nerves. Although not universally the case, the altered physiology of the IVD is believed to precede or be associated with many clinical symptoms or conditions including low back and/or lower limb pain, paraesthesia, spinal stenosis and disc herniation.

New treatment options have been developed in recent years. These include biological therapies and novel surgical techniques (such as total disc replacement), although many of these are still in their experimental phase. Central to developing further methods of treatment is the need for effective ways in which to assess patients and measure their outcomes. However, significant difficulties remain and it is therefore an appropriate time to be further investigating the scientific basis of and treatment of LBP.

Autologous chondrocyte implantation (ACI) has been used for many years for the treatment of symptomatic defects in articular joints, predominantly the knee. Traditionally, cells were implanted behind a periosteal membrane, but in more recent times Chondrogide, a membrane consisting of porcine collagens I and III, has been used. There have been trials comparing the clinical outcome of these two groups of patients; in this study we compare the histological outcome using the two different patch types.

In a study of 100 patients having received ACI treatment of cartilage defects in the knee, 41 received Chondrogide (ACI-C) and 59 received periosteum (ACI-P). All of these patients had a post-operative biopsy taken at a mean of 16.9±9.2 months and 20.8±23.2 months for ACI-C and ACI-P respectively for histology using the ICRS II scoring system. Lysholm scores, a measure of knee function, were obtained pre- and post-operatively at the time of biopsy and statistical differences tested for via a Mann-Whitney U-test.

The mean age of the two groups at treatment was 37±8 and 35±10 years, the size of defect treated was 6.1±5.4 and 4.4±2.7 cm2 and the biopsy follow-up time was 50.6±22.2 and 81.2±34.8 months for ACI-C and ACI-P patients respectively. Both groups exhibited a significant improvement in Lysholm score from pre-operative to the time of biopsy (14.3±25.7; n=100), although there was no significant difference in improvement in Lysholm score between the two patch types. There was no significant difference between the histology score of the two groups, nor was the score found to correlate with the Lysholm score at that time. The individual components of the ICRS II score did not differ significantly with patch type (even for the surface architecture) apart from cellular morphology which was 6.5±3 and 8.2±1.6 for ACI-C and ACI-P respectively.

The histological quality of repair tissue formed with ACI-C differed little from that seen with ACI-P, despite the former group being biopsied ∼4 months sooner after treatment and being used to treat defects which were 39% larger. Hence Chondrogide appears just as suitable as periosteum for use as a patch in the procedure of ACI.

Background

Autologous Chondrocyte Implantation (ACI) is a procedure which is gaining acceptance for the treatment of cartilage defects in the knee with good results and a long term durable outcome. Its use in other joints has been limited, mainly to the ankle. We aimed to assess the outcome of ACI in the treatment of chondral and osteochondral defects in the hip.

Hyaline cartilage defects are a significant clinical problem for which a plethora of cartilage repair techniques are used. One such technique is cartilage replacement therapy using autologous chondrocyte or mesenchymal stem cell (MSC) implantation (ACI). Mesenchymal stem cells are increasingly being used for these types of repair technique because they are relatively easy to obtain and can be expanded to generate millions of cells. However, implanted MSCs can terminally differentiate and produce osteogenic tissue which is highly undesirable, also, MSCs generally only produce fibrocartilage which does not make biomechanically resilient repair tissue, an attribute that is crucial in high weight-bearing areas. Tissue-specific adult stem cells would be ideal candidates to fill the void, and as we have shown previously in animal model systems [Dowthwaite et al, 2004, J Cell Sci 117;889], they can be expanded to generate hundreds of millions of cells, produce hyaline cartilage and they have a restricted differential potential. Articular chondroprogenitors do not readily terminally differentiate down the osteogenic lineage.

At present, research focused on isolating tissue-specific stem cells from articular cartilage has met with modest success. Our results demonstrate that using differential adhesion it is possible to easily isolate articular cartilage progenitor populations from human hyaline cartilage and that these cells can be subsequently expanded in vitro to a high population doubling whilst maintaining a normal karyotype. Articular cartilage progenitors maintain telomerase activity and telomere length that are a characteristic of progenitor/stem cells and differentiate to produce hyaline cartilage.

In conclusion, we propose the identification and characterisation of a novel articular cartilage progenitor population, resident in human cartilage, which will greatly benefit future cell-based cartilage repair therapies.

Hyaline cartilage defects are a significant clinical problem for which a plethora of cartilage repair techniques are used. One such technique is cartilage replacement therapy using autologous chondrocyte or mesenchymal stem cell (MSC) implantation (ACI). Mesenchymal stem cells are increasingly being used for these types of repair technique because they are relatively easy to obtain and can be expanded to generate millions of cells. However, implanted MSCs can terminally differentiate and produce osteogenic tissue which is highly undesirable, also, MSCs generally only produce fibrocartilage which does not make biomechanically resilient repair tissue, an attribute that is crucial in high weight-bearing areas. Tissue-specific adult stem cells would be ideal candidates to fill the void, and as we have shown previously in animal model systems [Dowthwaite et al, 2004, J Cell Sci 117;889], they can be expanded to generate hundreds of millions of cells, produce hyaline cartilage and they have a restricted differential potential. Articular chondroprogenitors do not readily terminally differentiate down the osteogenic lineage.

At present, research focused on isolating tissue-specific stem cells from articular cartilage has met with modest success. Our results demonstrate that using differential adhesion it is possible to easily isolate articular cartilage progenitor populations from human hyaline cartilage and that these cells can be subsequently expanded in vitro to a high population doubling whilst maintaining a normal karyotype. Articular cartilage progenitors maintain telomerase activity and telomere length that are a characteristic of progenitor/stem cells and differentiate to produce hyaline cartilage.

In conclusion, we propose the identification and characterisation of a novel articular cartilage progenitor population, resident in human cartilage, which will greatly benefit future cell-based cartilage repair therapies.

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development.

Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA.

Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA.

Background

The mechanical disadvantage and detrimental effect to articular cartilage following meniscectomy has been well documented in the literature. Meniscal repair in the avascular (white on white zone) is controversial and would be deemed inappropriate by many.

Hypothesis

Avascular meniscal tears can be repaired with good clinical outcomes.

A new surgical hybrid technique involving the combination of autologous bone plug(s) and autologous chondrocyte implantation (AOsP-ACI) was used and evaluated as a treatment option in 15 patients for repair of large osteochondral defects in knee (N=12) and hip joints (N=3). Autologous Osplugs were used to contour the articular surface and the autologous chondrocytes were injected underneath a biological membrane covering the plug. The average size of the osteochondral defects treated was 4.5cm². The average depth of the bone defect was 26mm. The patients had a significant improvement in their clinical symptoms at 12 months with significant increase in the Lysholm Score and Harris Hip Score (p = 0.031). The repaired tissue was evaluated using Magnetic Resonance Imaging, Computerised Tomography, arthroscopy, histology and immunohistochemistry (for expression of type I and II collagen). Magnetic Resonance Imaging, Computerised Tomography and histology at 12 months revealed that the bone plug became well integrated with the host bone and repair cartilage. Arthroscopic examination at 12 months revealed good lateral integration of the AOsP-ACI with the surrounding cartilage. Immunohistochemistry revealed mixed fibro-hyaline cartilage. We conclude that the hybrid AOsP-ACI technique provides a promising surgical approach for the treatment of patients with large osteochondral defects. This study highlights the use of this procedure in two different weightbearing joints and demonstrates good early results which are encouraging. The long term results need to be evaluated.

Background: The cells of the intervertebral disc must synthesise and maintain their surrounding matrix for it to function normally, providing all its physiological and mechanical properties. However, disc cells survive in an environment that most cells would not tolerate, ie with a low pH and relatively little oxygen. Cells which experience such potentially damaging conditions, including excessive heat, elicit a stress response and synthesise a range of proteins, called heat shock proteins (Hsps); these facilitate repair and survival or removal of damaged cells.

Methods and Results: We have studied Hsp production by disc cells, both in vitro and in vivo. We measured Hsps produced by bovine skin and disc cells grown in monolayer and heated up to 45°C and also immunostained human surgical discs for stress proteins, Hsp27 and Hsp72.

Disc cells responded differently to dermal fibroblasts; when freshly isolated they had a reduced or attenuated stress response and produced much less Hsp 70 than freshly isolated skin cells. After culturing in monolayer (by passage 2) all cells produced more Hsps. Human surgical discs produced varying amounts of Hsp, with most being produced by cells in herniated discs, particularly those within clusters of cells.

Conclusion: Our results suggest that intervertebral disc cells in vivo normally have a reduced stress response. Hsp production is considered to protect against damage, suggesting that the reduced response may contribute to disc degeneration and back pain. The prosurvival stress response of disc cells could provide a novel therapeutic target in patients with degenerative disc disease.

Conflict of Interest: None

Source of Funding: Wolfson Charitable Trust

Introduction: Increased cell senescence has been reported in the human intervertebral disc (IVD) and was associated with degenerative pathology, particularly herniation. Increased IVD innervation and blood vessel ingrowth is associated with disc degeneration and the development of back pain. This preliminary study examines whether there is a relationship between the prevalence of senescent IVD cells and the extent to which the tissue is innervated and/or vascularised.

Methods: Specimens of herniated IVD (n=16 patients: aged 36–71) were stained for senescence associated β-galactosidase activity (SA β-gal), then snap frozen and cryosectioned prior to immunolocalisation procedures to detect nerves (NF200) or blood vessels (CD34). Stained sections were counterstained with DAPI to reveal cell nuclei. The proportion of SA β-gal +ve cells was scored and the extent of neural and blood vessel ingrowth semi-quantitated.

Results: The proportion of SA β gal +ve IVD cells ranged from 6% – 91% (median=16%) and was significantly correlated with age. The degree of neural or blood vessel ingrowth ranged from tissue which contained numerous (i.e. ≥10) positive cells/cell processes to tissue which was completely aneural or avascular. However, there was no clear relationship between the presence of SA β-gal +ve IVD cells and IVD innervation or vascularisation.

Conclusions: Cell senescence has been associated with up-regulated expression of catabolic enzymes, e.g. MMPs and increased synthesis of trophic cytokines, e.g. VEGF. Such cellular activity might by thought to contribute to the pathological ingrowth of nerves or blood vessels into the IVD. The data presented here, however, does not support such a hypothesis.

Conflicts of Interest: None

Source of Funding: Institute of Orthopaedics, RJAH Orthopaedic Hospital

Introduction: Intervertebral disc (IVD) cell transplantation is used to treat back pain. However, IVD cell activity may also contribute to pathology, e.g. IVD cells can undergo senescence or promote nerve growth, which in the IVD is associated with discogenic back pain. Serum deprivation of bovine IVD cells results in cell senescence. We have examined the influence of oxygen supply combined with serum deprivation on human IVD cells.

Methods: Cells from herniated IVD (n=3 patients) were subjected to serum deprivation and then cultured under hypoxic (1%) or atmospheric (21%) conditions for 10 days. IVD cell growth, viability and cell senescence (via Senescence Associated β-galactosidase activity; SA β-gal) were examined. The growth and migration of HMEC-1 (endothelial) and SH-SY5Y (neuronal) cells treated with conditioned medium from the IVD cell cultures (1% versus 21% oxygen) were subsequently monitored.

Results: Hypoxia significantly decreased IVD cell proliferation, but was also found to reduce cell senescence. Hence, the proportions of SA β-gal positive IVD cells in 1% and 21% oxygen at day 10 were 18±6% and 56±10%, respectively. There was no marked difference in cell viability (> 95%). Conditioned medium from IVD cells cultured under hypoxia stimulated endothelial and neural cell growth (determined via the MTS assay) and endothelial cell migration and neurite outgrowth to an extent that was significantly greater than conditioned medium from IVD cells cultured at 21% oxygen.

Conclusions: The trophic activity of human IVD cells is responsive to oxygen supply. However, hypoxia may influence the capacity of IVD cells to reduce back pain for better or worse.

Conflicts of Interest: None

Source of Funding: Institute of Orthopaedics, RJAH Orthopaedic Hospital.

We aimed to assess the long term results of patients who underwent Autologous Chondrocyte Implantation (ACI) for osteochondral lesions of the talus. Between 1998 and 2006, 28 patients underwent ACI for osteochondral lesions of the talus. All these patients were prospectively reviewed and assessed for long term results. Outcomes were assessed using satisfaction scores, Mazur ankle score and the AOFAS score, and Lysholm knee score for donor site morbidity.

The 28 patients who underwent the procedure included 18 males and 10 females. Follow up ranged from 1–9 years. In all patients, there was an improvement in the Mazur and AOFAS ankle scores and the Lysholm scores showed minimal donor site morbidity. Improvement in ankle score was independent of age and gender. The better the pre-op score the less the difference in post-op ankle scores. Patients were unlikely to benefit with pre-op ankle scores over 75.

The mid to long term results of ACIs in the treatment of localised, contained cartilage defects of the talus are encouraging and prove that it is a satisfactory treatment modality for symptomatic osteochondral lesions of the talus. Complications are limited. However, in view of limited number of patients, a multi-centre randomised controlled study is required for further assessment.

The details of 320 consecutive patients undergoing knee microfracture, with a minimum follow up of 6 months, were taken from the Sports Injury Database at the Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry. All had same phsyiotherapy regime post operatively. Two rounds of postal questionnaires were administered to assess patient satisfaction along with Lysholm, Tegner, VAS for pain and a modified IKDC scores. 196 patients responded (61.25%).

The mean age of our patients was 40.64 years and the mean follow up 37.02 months (range 6–78 months). There were 35 smokers and 161 non-smokers. 64 patients had surgery in the medial compartment, 35 in lateral, 50 in patella-femoral and 47 belonged to the combined category. 93 patients had other surgeries (partial meniscectomies, ACL reconstruction etc) along with microfracture(47.45%).

Seventy two percent of patients were satisfied with their outcome and 18.95% weren’t. 51.43% of smokers were satisfied with their outcome and 76.88% of non smokers (p=0.021). Patients more than 50 years of age were less satisfied (p=0.023) than younger patients. Having concomitant knee surgery, including ACL reconstruction, made no difference to patient satisfaction or functional scores.

The location of the lesion in the knee did not affect patient satisfaction. However, all five post op score levels were statistically different among them. The Lysholm post op scores were significantly better in lateral and PFJ compartments than medial. Lateral and combined groups were significantly better than medial for Tegner post op scores. Lateral and PFJ groups were significantly better than medial for VAS and modified IKDC scores.

Smoking and age significantly affect patient satisfaction after knee microfracture. Having concomitant knee surgeries doesn’t make a difference to either satisfaction or functional outcome. Our results suggest that the medial compartment doesn’t do as well in functional scores as previously thought.

Radiofrequency thermal shrinkage of anterior cruciate ligament (ACL) laxity or partial injury is a relatively recent treatment. Studies have shown varied results with this technique but have had small study numbers and mixtures of both primary and reconstructed ACLs. We present our series of 109 patients.

Between 1999 and 2008 our department performed radiofrequency thermal tightening on 109 patients with partial native ACL injury or ACL laxity. Fifty three patients completed both pre and post-operative evaluations at a mean follow-up of 20.5 months. Evaluation consisted of visual analogue pain scores, Tegner activity and Lysholm scoring.

From the 110 patients that underwent thermal shrinkage for ACL instability 21 (19%) went on to require full ACL reconstruction. The decision to convert to full ACL reconstruction was made at a mean of 13 months (sd=12) following thermal shrinkage surgery. Comparing those who required ACL reconstruction with those who did not, we found those requiring reconstruction to be significantly younger. Mean = 25yrs vs. 31.5yrs. (p≤ 0.002)

Fifty three patients completed both pre and post-operative evaluations at a mean follow-up of 20.5 months. Following treatment there was a significant improvement in mean Lysholm scores from 64.4 to 79.5 (p< 8.42x10-7) and pain scores 3.7 to 2.0 (p< 3.06x10-6); however there was a reduction in patients’ activity levels as assessed by Tegner score, from 6.65 to 6.0 (p< 0.019).

Comparing those who required ACL reconstruction with those who did not, we found those requiring reconstruction to be have higher pre-operative level of activity (mean Tegner score = 7.3 vs. 6.5. (p< 0.047)).

Radiofrequency thermal shrinkage of anterior cruciate ligament significantly improves knee function but may not be appropriate for younger patients or patients with high activity levels.

Background: Degeneration of the intervertebral disc is associated with back pain. Cell transplantation to enhance disc regeneration is an attractive concept and clinical trials using autologous disc cells have begun. However, the capacity of the disc, which is poorly supplied by blood vessels, to support viable cells is currently unclear. In this study, we have assessed cell seeding densities and nutrition required to optimise nucleus pulposus (NP) cell survival and proliferation.

Methods: NP cells were cultured in alginate beads at cell seeding densities 1.25×105 – 1.0×106 cells/ml, either in 10% or 20% serum (vol/vol) ± glucose for 8 days. Cell proliferation was measured by immunopositivity for a proliferation marker, the Ki67 antigen. Cell viability was assessed by DAPI staining.

Results: NP cells grown in 10% serum with glucose proliferated and formed cell clusters at low cell seeding densities; however, this proliferative response was significantly decreased at the higher cell seeding densities. Increasing serum from 10% to 20% markedly increased the size of cell clusters that formed. Interestingly, cells grown in 20% serum but without glucose produced the largest cell clusters, some containing > 40 cells. However, DAPI staining revealed that many cells forming these clusters were dieing via apoptosis.

Conclusion: The manipulation of cells in culture, prior to transplantation into degenerate discs, may be key to optimising cell-mediated tissue regeneration. This study has shown that the number of cells transplanted and the level of nutrition available in the degenerate disc microenvironment may directly influence cell proliferation and survival potential and therefore their regenerative capability.

We present a prospective review of the two-year functional outcome of 37 Avon patellofemoral joint replacements carried out in 29 patients with a mean age of 66 years (30 to 82) between October 2002 and March 2007. No patients were lost to follow-up. This is the first independent assessment of this prosthesis using both subjective and objective analysis of outcome. At two years the median Oxford knee score was 39 (interquartile range 32 to 44), the median American Knee Society objective score was 95 (interquartile range 90 to 100), the median American Knee Society functional score was 85 (interquartile range 60 to 100), and the median Melbourne Knee score was 28 (interquartile range 21 to 30). Two patients underwent further surgery. Only one patient reported an unsatisfactory outcome.

We conclude that the promising early results observed by the designing centre are reproducible and provide further support for the role of patellofemoral joint replacement.

Introduction: Monoclonal antibodies (mAbs) recognizing linear sulphation motifs in keratan sulphate (KS) were first developed in the early 1980’s. Over the years, ELISAs using 5-D-4 or other related anti-KS mAbs have been used in many studies monitoring increased cartilage aggrecan degradation with the onset of degenerative joint diseases. However, whilst these studies have in general been useful for monitoring some aspects of disease progression (usually in parallel with other biomarker assays), many longitudinal studies have shown efficacy in only the transient (early, mid or late) stages of the degenerative joint disease process. During the onset of degenerative joint disease, the pathological tissue attempts to repair/regenerate the cartilage, the chondrocytes thus synthesizing cartilage aggrecan with KS substitution [and chondroitin sulphate (CS) isomer composition] that is more like that found in developing or immature cartilage. This immature cartilage aggrecan contains much less KS substitution with shorter chain size and less linear sulphation motifs. Thus, during the different stages of degenerative joint disease progression one would expect to find variable changes in different linear sulphation epitopes present in the serum or synovial fluids. The aim of this study was to investigate the use of several monoclonal antibodies that recognise different sulphation epitopes [high sulphation (5-D-4), low sulphation (1-B-4) and KS-stubs (BKS-1)] to see if patterns of their expression could be used to distinguish different stages of degenerative joint disease. We have also developed ELISAs using mAbs recognising the KS-proteoglycans, keratocan (Ker 1) and lumican (Lum 1) for their quantification as potential biomarkers of osteoarthritis.

Methods: Competitive ELISAs were developed using monoclonal antibodies (mAbs) 5-D-4, 1B4, BKS-1, Ker-1 and Lum-1. Bovine corneal KS-proteoglycans pre-treated with keratanase were used as both the coating antigen and “standard” antigen on the same ELISA plate. Blood, synovial fluid and cartilage samples (surgical waste) obtained from patients undergoing arthroplasty with different Kellgren & Lawrence grades were analysed.

Results and Discussion: 5-D-4 and BKS-1 showed similar inhibition curves and relative 50% inhibition points. However, the curve obtained with 1B4 indicated lower relative expression of 1B4 epitope. Analysis of serum and synovial fluid sample with 5-D-4 mAb showed the presence of the epitope in both samples, but there was significantly less KS in serum than in the synovial fluid. Our results show that competitive ELISA for quantification of several different KS sulphation or “stub” epitopes and two KS-proteoglycans can all be quantified and compared using the same experimental conditions. These studies are ongoing as part of an Arthritis Research Campaign (UK) funded study. In addition the data indicates that keratocan and lumican are also increased in their expression with the progression of disease. Future studies will be performed in an attempt to quantify increased keratocan and lumican expression as potential biomarkers of degenerative joint disease.

Introduction: Elastin is a structural protein forming a highly organised network in the annulus and nucleus of the intervertebral disc (IVD). It appears important in maintaining annulus structure as it is densely located in the interlamellar space and forms cross-bridges between lamellae. Here we have investigated elastin fibre organisation in degenerate discs and compared it to that seen in normal human and bovine discs.

Methods: Human lumbar IVD were obtained from consented patients undergoing surgery either for disc degeneration, tumour or trauma. The disc segments were collected from operating theatre and graded. A radial profile of the specimen was dissected and snap-frozen. Sections of 20μm in thickness were cut with a cryostat microtome and mounted on slides. To visualize elastin fibres, sections were digested with hyaluronidase after fixation with 10% of formalin. Elastin fibres were immunostained and fibre organisation mapped.

Results: In degenerate disc, the elastin fibre network appeared sparse and disorganised in comparison to that seen in non-degenerate human or in bovine discs in which elastin fibres are well organised. In addition, in degenerate discs the elastin fibres appear fragmented. Fragmentation of the elastin network within lamellae of the annulus in particular increased with both degeneration grade and with age.

Discussion: The loss of elastic network integrity observed in degenerate discs could contribute to loss of annulus integrity and affect disc mechanical properties adversely. Furthermore, our initial results have suggested fragmented elastin degradation products could upregulate MMP expression by disc cells thus stimulating a degenerative cascade.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different soft and hard musculosk-eletal tissues. In the intervertebral disc (IvD) the major SLRPs involved in regulation of types I & II collagen fibril size are believed to be decorin, fibromodulin and lumican. Research into IvD degeneration and backpain is hampered by a lack of specific biomarkers to detect and monitor the disease process. We have discovered that two keratan sulphate (KS) substituted members of the SLRP family, Keratocan and Lumican (that are major KS-pro-teoglycans found in cornea) were unusually expressed in extracts from degenerative disc tissues.

Methods: Non-degenerate disc tissue (n=10) was obtained from 2 scoliosis patients and degenerate disc tissue from 11 patients undergoing surgery. The degenerate discs were graded using criteria described by Pfir-rman et al (Spine26: 1873; 2001). Tissue samples were extracted with 4M guanidine HCl and after dialysis subjected to SDS-PAGE and Western blot analyses using monoclonal antibodies that recognise epitopes on kera-tocan and lumican.

Results & Discussion: Keratocan was not found in the non-degenerate disc tissue but was present in all degenerate IvD tissues tested. Lumican showed and increased expression in extracts of degenative IvD tissues. Our working hypothesis is that the increased expression of these two SLRPs in degenerative disc tissue results from a reparative depostion of a type I collagen fibrillar ‘scar’. This unusual expression suggests their potential as biomarkers for detecting the onset of degenrative disc disease.

Background: Many studies have examined magnetic resonance images (MRI) with a view to the anatomy and signaling properties of the intervertebral disc and adjacent tissues in asymptomatic populations. In this study we have examined MRIs of a discrete population of patients undergoing surgery for symptomatic disc herniations.

Methods: Sixty patients (aged 23–66 years, mean 41.5±8.4) had sagittal T1 and T2- weighted turbo spin echo imaging of the lumbar spine prior to surgery. One disc was herniated at L2-3, 3 at L3-4, 22 at L4-5 and 31 at L5-S1; 3 patients had herniations at both L4-5 and L5-S1. The images were scored for disc narrowing and signal, degree of anterior and posterior bulging and herniation, and assessed for Modic I and II endplate changes and fatty degeneration within the vertebrae. These were carried out for each of 6 discs (T12-S1) for all patients (ie 360 discs and 720 endplates).

Results: There were trends of increasing disc narrowing, disc bulging and fatty degeneration with increasing age in these patients. 83% of patients had disc bulging, 53% had endplate irregularities and 44% had fatty degeneration. There was a significant correlation between patient weight and fatty degeneration. 7.5% of vertebrae (in 22% of patients) demonstrated Modic I changes whilst Modic II changes were seen in 14% of vertebrae (40% of patients). This is considerably higher than the incidence reported in asymptomatic individuals where Modic I changes were seen in 0.7% of vertebrae (3% of individuals) and Modic II changes in 1.9% of vertebrae (10% of individuals).

Conclusion: There is a higher incidence of Modic I and II changes in disc herniation patients than in asymptomatic individuals.

Cells of the intervertebral disc exist in an unusual environment compared to those of other tissues. Within the disc there are low levels of nutrients available, low oxygen levels and it is an acidic environment due to high lactate levels. Apoptosis (programmed or controlled cell death) has been reported in intervertebral discs, as well as necrosis (uncontrolled cell death). This study has focused on examining the sensitivity of nucleus pulpo-sus (NP) cells to several stimuli, in comparison to two other cells types.

Ultra violet (UV) irradiation, serum starvation (with no foetal calf serum) and treatment with 2mM hydrogen peroxide were used to induce apoptosis in cultured bovine NP cells, HeLa (cancer cell line) and 293T cells (human embryo kidney derived) cells. Apoptosis was identified by nuclear morphology following staining with fluorescent Hoechst 33342 dye and propidium iodide; the incidence was measured at 24, 48 and 72 hours. Untreated controls were used for each treatment and at each time point.

The incidence of apoptosis increased with time for all treatments. After 72 hours, UV treatment produced the highest levels of apoptosis with levels of apoptosis occurring in the order of HeLa (94%) > NP cells (29%) > 293T cells (15%). Treatment with hydrogen peroxide and serum starvation induced apoptosis at lower levels in all three cell types (maximum of 30%). Serum starvation induced apoptosis in only 10% of NP cells at 72 hours, compared to 20% in HeLa cells. None of the controls contained apoptotic cells.

NP cells are stimulated to apoptose in response to UV irradiation, hydrogen peroxide and serum starvation. However, levels of apoptosis are much lower after UV treatment in comparison to HeLa cells (3 times lower), suggesting that they may have a protective mechanism to this apoptotic stimulus, compared to HeLa cells. The low levels of apoptosis observed in NP cells with serum starvation may be due to the low nutrient environment that they exist in normally.

Introduction: Before proceeding to long-term studies, we studied early clinical results of combined Autologous Chondrocyte Implantation (ACI) and Allogenic Meniscus Transplantation (AMT). Meniscus deficient knees develop early osteo-arthritis (OA) of the knee joint. Autologous Cartilage Implantation (ACI) is contraindicated in case of meniscus deficient knees. And on contrary the Allogenic Meniscus Transplantation (AMT) is contraindicated in cartilage defects in the knee joint. But a combination of the two procedures for bone on bone OA might be a solution for this problem. This was the main purpose of our study.

Methods: We studied a consecutive series of eight patients (7 males and 1 female), with an average age= 43 years (29–58), presenting with painful secondary arthritis, due to premature loss of meniscus and chondral defect/s. Median size of the femoral defects was 8.16 cm2 and of the tibial side 2.69 cm2 All patients were treated with a combination of Autologous Chondrocyte implantation (ACI) and Allogenic Meniscus Transplantation (AMT). Chondral defects were covered with periosteum/ Chondroguide membrane, secured in place with in-vitro cultured autologous chondrocytes injected underneath the path. Meniscus placed as load-bearing washer on the surface of ACI of tibia. ACI rehabilitation protocol followed post-operatively. Assessment at the end of one year was done with self-assessed Lysholm score, histology and the MRI scan.

Results: Mean pre-operaive Lysholm score was 49 (17–75). This increased to a mean of 66 (26–87) at 1 year, an average increase of 16.4 points. Average one-year satisfaction score was 3 and they were back to all active life style. Five out of eight patients showed significant functional improvement at last post-operative follow-up (2 to 6 years; mean of 3.2 years). Complications were aseptic synovitis in 3 cases. Three failures were noted showig persistant pain and swelling in one, rupture of meniscus in second and third patient had a knee replacement. Arthroscopy at 1 year showed a stable meniscus with all healed peripheral margins in all except in one case with some thinning with no evidence of rejection. Histology of meniscus showed a fibrocartilage well populated with viable cells and the peripheral zone was well vascularised and integrated with capsule. Biopsy of ACI site was predominantly of fibrocartilage with good basal integration with subchondral bone. On MRI scan, allogenic meniscus was well integrated with capsule along the line of repair, showing foci of variable signal intensities within the meniscus. There was no evidence of meniscal subluxation in all but one case showing mild extrusion. ACI graft site showed a varied appearance, with 3 grafts showing focal grade 3to 4 changes.

Conclusion: Seven out of eight patients improved post-operatively at one year, in terms of pain relief and increased activity. It’s possible to combine these two techniques together. Short-term outcomes are satisfactory. We could not find any deleterious effects of combining these two techniques together. So we conclude that, this might act as a one step towards a biological knee replacement.

Background: Articular cartilage injuries are very common. Small defects don’t heal on their own and large defects can’t regenerate new cartilage. This would largely be due to the fact that chondrocytes are embedded in a firm and tough matrix and hence can’t migrate to the defect site to regenerate a new cartilage tissue. So ultimate fate is patient getting early osteoarthritis. Cartilage defects in the knee may be symptomatic and cause pain, swelling and catching. There are several different surgical procedures available to treat cartilage injuries, but no method has been judged superior. The ultimate aim of the treatment is restoration of normal knee function by regeneration of hyaline cartilage in the defect, and to achieve a complete integration to the surrounding cartilage and underlying bone. Arthroscopic debridement and lavage may give symptomatic relief for a limited time. Autologous Chondrocytes Implantation (ACI) was first described in 1994. Encouraging primary results were reported, and further research was promoted. Long-term results are encouraging. ACI is being done in Robert Jones & Agnes Hunt orthopaedic Hospital, Oswestry since last 8 years.

Methods: We studied a cohort of first 118 patients who underwent ACI for knee joint in this institute, focussing on their mid-term results. Patients having chondral defects were offered ACI. They all were explained the procedure and informed written consent was obtained. Patients filled in a self-assessed Lysholm forms before the operation. They also underwent pre-operative MRI scan of knee joint. ACI procedure consisted of three stages— Stage I —Arthroscopic harvest biopsy of cartilage and chondrocytes culture in lab. Stage II—Arthrotomy of the knee. The defect edges were freshened, covered by periosteum or chondroguide, which was sutured to the cartilage with 6-0 vicryl. Chondrocytes were injected underneath this patch. Post-op CPM and Physiotherapy. Stage III—1-year arthroscopic surgery. Assessment was done with Lysholm score, MRI scan, histological and arthroscopic analysis. Patients were followed up clinically thereafter with yearly Lysholm scores.

Results: 118 patients with an average age of 35 years (15–59) underwent ACI for knee in last 8 years. 93 patients had single defect, 24 had multiple (> 1) chondral defects, with mean area 4.81 cm2. MRI showed a good integration of defect with surrounding cartilage with varied signal intensities. About 55–56% patients underwent some or other form of trimming, which improved immediate results. However only 50 % of these were symptomatic. Defects on MFC did well as compared to other sites, followed by on trochlea. Defects on patella showed poor results, though the number is less for comparison. Total 79 specimens of 1-year histology showed good healing with formation of fibrocartilage (40), mixed (20) and hyaline (8), fibrous tissue (6), bone in 1 case and inconclusive in 2 cases. Mean pre-op Lysholm score was 50.16. Average score at one year was found to be 69.52.

Conclusion: Results of ACI are encouraging. Patients continued to improve slowly over a period of time, achieving maximum function between one and 2 years post-surgery. Our study showed that there after their scores remained static.

Introduction: The search continues for ideal markers and methods of monitoring cartilage degeneration. Various cartilage components, whole or fragmented, have been measured in synovial fluids. A common problem in quantitating these markers is often the unknown dilution of synovial fluid which can occur in obtaining the samples. In this study we have used urea (ratio in synovial fluid:serum) as a method to correct for the dilution of synovial fluid, and hence to quantify enzyme levels in patients with a spectrum of cartilage degradation, in addition to identifying aggrecan degradation products, many of them for the first time in such samples.

Methods: Forty synovial fluid samples were obtained from 4 groups of individuals (10 in each):

normal,

Levels of matrix metalloproteinases (MMPs) 2 and 3 and the inhibitor, TIMP 1, were measured in the fluids via ELISA assays. Urea levels were measured in blood and synovial fluids and enzymes and their inhibitors were normalized according to the ratio of serum:SF urea, to account for the dilution factor of the SF (Kraus et al 2001). Western blotting was used to identify the presence of aggrecan components (chondroitin-4-sulphate: 2B6 antibody; C-6-S: 3B3 and C-0-S: 1B5; keratan sulphate: BKS-1; the G1 domain: 7D1; interglobular domain: 6B4) and also enzyme degradation products of MMPs (BC14) and aggrecanases (BC3; BC-13).

Results: MMPs 2 and 3 and TIMP 1 were all significantly increased in the synovial fluids from OA patients compared to normals (P< 0.01, 0.001 and 0.01 respectively) and MMP3 was greater in the grade IV chondral and osteochondral defect groups than the normals (P< 0.01). Western blotting demonstrated fragmented aggrecan components with a range of molecular weights. Aggrecanase activity was seen in the OA and grade IV chondral damage groups but not in the osteochondral or normal groups, whereas MMP activity was seen in all 3 groups showing cartilage damage but not in the normals.

Conclusion: Dilution of the synovial fluid, either due to inflammation or joint lavage, is often a problem in quantitating metabolites and markers in joint cavities. This pilot study of a limited number of samples from well characterized patient groups indicates that using urea concentrations in synovial fluid relative to serum provides a mechanism to overcome this. It confirms elevated enzyme activity, both aggrecanase and MMPs, in the joints of patients with degenerate cartilage, compared to normals.

Introduction: Autologous chondrocyte implantation is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogenous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.

Methods: Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into poly vinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM+10% FBS within 15ml conical tubes rotated at 37°C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H& E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X, and to the glycosaminoglycans, chondroitin-4- and -6-sulphate and keratan sulphate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein, and small leucine-rich PGs.

Results: H& E staining of 10μm-thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining revealed the presence of GAGs, increasing with time. Ki-67 revealed approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X and the glycosaminoglycans, chondroitin-4-, -6 and keratan sulphate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.

Conclusions: Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.

Background: Unfortunately ACL injuries are not uncommon in the young: the majority however occurring after skeletal maturity.

Aim: To perform an internal audit of the demand, methods and results of ACL reconstruction in young patients at a tertiary referral centre.

Methods: Patients were identified through electronic patient records, and all operation notes and follow up records were scrutinised.

Results: 84 cases under 20 years of age (range 14–19) were reviewed from 2000–2004 with a minimum follow-up of 6 months. Over 10% had undergone previous surgery or had documented articular injury. 42 cases required further meniscal surgery at the time of reconstruction: 12% repairs (20/168 menisci), 18% partial menisectomy (30/168). The median time to reconstruction from injury was 9 months (range 1–72). No case was delayed for growth plate maturation. Reconstruction methods were partly surgeon dependent, following adult themes. Occasionally tibial fixation was away from the growth plate with low profile screws and washers. We are only aware of 1 failure during this short follow-up.

Conclusion: We believe that the use of techniques similar to those used on adults is appropriate for adolescents. However the high comorbidity is of some concern, demonstrating that this age range is as challenging as their older counterparts.

Introduction: Proteoglycans are found both in the annulus fibrosus and nucleus pulposus of the intervertebral disc and contribute to the hydration of the tissue (aggrecan) and the regulation of matrix assembly (small proteoglycans) [1]. Whilst loss of proteoglycan is the main chemical change in disc degeneration seen in back pain patients, little is known of the events leading to and controlling this loss. In this study the metabolism of the most common proteoglycan, aggrecan, and others including decorin, biglycan, lumican, fibromodulin and versican, together with collagen types I and II were studied in diseased and normal discs.

Methods: Ten discs from patients aged 11–57 years (mean:39±15) with scoliosis (n=1), spondylolisthesis (n=1) and low back pain (n=8), were graded for macroscopic degeneration (Grades 1–4). Three ‘normal’ cadaveric discs from 3 individuals aged 25–27 years (mean 26±1) were also investigated. Disc was either snap-frozen (for RNA isolation) or the proteoglycans extracted with 4M GuHCl. Total RNA was isolated and RT-PCR performed using various oligonucleotide primers. GuHCl-extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and small leucine-rich proteoglycans.

Results: Intervertebral discs contain a very heterogenous population of proteoglycans demonstrating extensive enzymic degradation, particularly with increasing age and macroscopic degeneration such as is seen in back pain patients. Younger, less degenerate discs contained more biglycan than the older, more degenerate discs. However, the mRNA gene expression analyses demonstrated little cellular activity and potential synthetic response, there was very little expression of particularly in comparison to osteoarthritic cartilage cells which show considerable synthetic capability for all the major matrix components.

Discussion: Our analyses indicate that several biochemical, catabolic and biosynthetic changes occur in disc matrix molecules which are likely to contribute to loss of disc function with ageing and degeneration. The loss of biosynthetic capability of cells is very important in considering the potential of newer therapeutic modalities such as cellular repair and genetic engineering for the treatment of degenerative disc disease.

Introduction: Massive irreparable degenerative rotator cuff tears are amongst the most difficult conditions for treatment in shoulder surgery. These involve usually elderly patients, which present with severely painful and restricted active shoulder movement. These patients have low demand from their shoulders, mainly for pain relief and performing their simple activities of daily living. Major surgery for major tendon transfer will not be advisable in these cases in view of the morbidity involved and the questionable outcome. We suggest a simple non-surgical rehabilitation treatment consisting on anterior deltoid strengthening exercises in the supine position for re-education of the anterior deltoid to compensate for the absent rotator cuff.

Methods: 17 patients with degenerative (non traumatic) Massive irreparable rotator cuff tears were recruited. They were all greater than 70 years of age and of mixed gender. Patients were English speaking, had full mental faculties and gave informed consent. They suffered no other shoulder pathology and were not participants in any other upper limb rehabilitation. All patients complained on severe shoulder pain and severely limited active range of motion with inability to actively elevate the arm to the horizontal. They all had full passive range of motion.

The diagnosis of a Massive irreparable rotator cuff tear was confirmed by diagnostic ultrasound scan. The shoulder function was evaluated using the Constant Score. Patients’ active shoulder ranges of motion were recorded and video-recorded as well. Each participant was taught the initial 6-week of self Deltoid muscle exercise, executed in supine, at least three times a day. They were instructed that when they felt better control on their active shoulder movements to gradually recline up the head of the bed and continue with the same simple exercise. They were reviewed at 6 weeks re-assessed and re-taught the same exercise, with a 2kg weight in their hand. At the 12th week they were reassessed using the constant score, and their active range of motion was video recorded again.

Results: 90% of the participants expressed a significant improvement in their upper limb function already after 6 weeks of treatment. All components of the Constant score (beside the strength) have improved. 90% reported less pain and found general activities of daily living easier to execute and a diminished level of muscle fatigue. 10% of the patients were able to establish a recording of > 1.26kg on the myometer in 90 degrees of abduction. 10% failed to report any benefit.

Discussion and Conclusion: Anterior deltoid strengthening exercises in the supine position for re-education of the anterior deltoid seem to have a significant beneficial effect for restoration of shoulder function and pain relief in the majority of patients with Massive irreparable degenerative rotator cuff tears. Using this simple non-invasive rehabilitation technique helps to re-educate the anterior deltoid to compensate for the absent rotator cuff and restore shoulder function.

Problem Chronic disabling pain in the sacrococcygeal region is regarded by clinicians with great dismay because of unpredictability of the treatment outcome. The subject is under- represented in the literature.

Method Thirty eight patients with intractable coccydynia had imaging investigations for the spine other than X-rays. Six of these patients were also investigated by means of sacrococcygeal and intercoccygeal discography. The excised specimen with intact sacrococcygeral joint was sent for histological examination in 22 patients. Patients’ assessment of the benefit of coccygectomy was conducted by telephonic interview.

Results After a mean post surgical follow up of 6.75 years (range 2–16 yrs), results were available for 31 out of 38 patients.

16 patients benefited greatly from the surgery and 6 benefited to some extent, giving an overall good result of 71%. 7 patients had no or little relief from surgery (29%).

Moderate to severe degenerate changes in SC and IC joints on histology were found in 59% of patients. 91.6 % of these patients did well with surgery. Only 60 % of those with mild changes did well.

Discography was possible in five out of six attempted cases. Two were positive and both did well from surgery. Three patients had negative discographies and two of them had a poor result and one had only some relief.

Conclusions Degenerate changes in sacrococcygeal discs give rise to pain. Surgical results are better in those with a severe degree of degenerative change. It is possible to identify these with discography, though a larger study needs to be carried out.

Background: Growth and development of the intervertebral disc and its adjacent vertebrae is regulated via relative levels of cell proliferation, cell death and hypertrophy, and through extracellular matrix synthesis or degradation [1]. The synthesis of matrix molecules in the growing spine of embryonic rats has been reported in some detail [2,3]. In addition, increased levels of apoptotic disc cell death have been described in normal ageing, disc degeneration and in a murine model of disc spondylosis [4,5]. However, levels of cell proliferation in the developing spine have not been formally investigated.

Methods/Results: BALB/c mice were injected with the thymidine analogue, bromodeoxyuridine (BrdU), at weeks 1–4 postnatally and killed 1 or 24 hours later. The lumbar spines were decalcified and tissue sections immunostained for BrdU-incorporation. The intervertebral disc was fully formed at weeks 1–4, consisting of a notochordal nucleus pulposus, lamellar anulus fibrosus, and cartilaginous endplates between the disc and vertebral growth-plates. BrdU-immunopositivity was most marked in 1 week old mice, particularly in the proliferative zone of the growth-plate and the apophyseal ring. By 4 weeks, few, if any, BrdU-labelled cells were present in the disc, but some positivity remained in the apophyses. There were more paired BrdU-labelled cells at 24 hours than 1 hour post-injection in all regions, indicating likely clonal growth of these cells.

Conclusions: Cell proliferation forms an important part of the growth of the vertebrae, but also features in the early postnatal growth of the murine intervertebral disc. An understanding of how proliferation in these cell populations is regulated will help augment repair and regenerative responses in damaged adult discs or scoliosis.

Introduction: Degeneration of the intervertebral disc is characterised by loss of normal cell activity, disc matrix and loss of disc height. There is currently much interest in using cells to effect a biological repair in connective tissues, eg autologous chondrocyte implantation for cartilage repair. Intervertebral discs have a low cell density, with those cells present often being unhealthy and necrotic. Hence, identification of an alternative source of cells for autologous disc repair could be beneficial. Thus we have investigated other types of connective tissue cells to determine if they may be encouraged to undertake a disc cell phenotype.

Materials and Methods: Cells were enzymatically/mechanically extracted from bovine coccygeal discs (annulus and nucleus), skin, bone marrow, periosteum and tendon and the efficiency and proliferation rates assessed. Dermal fibroblasts and bone marrow cells were also grown in a 3D alginate system and compared to disc nucleus pulposus cells for phenotypic expression from 0–28 days. Cell phenotype was assessed via morphology, immunohistochemistry, Western blotting and RT-PCR for mRNA expression.

Results: All cell types could be extracted and proliferated in monolayer, with a flattened and fibroblast-like morphology. Proliferation was slowest for bone marrow cells (4 times slower than nucleus pulposus cells). Cells cultured in alginate became rounded with chondrocyte-like morphology. They remained viable for 4 weeks, but with little replication. Expression or production of proteoglycans, both aggrecan and the small proteoglycans (especially fibromodulin) and collagen types I, II and X was demonstrated for all cell types. There was, however, a difference in the timescale of production between some cell types.

Conclusions: Plasticity of different cell types is well known and the connective tissue cells investigated in this study are capable of responding to the environment in which they are cultured. They can synthesise matrix molecules typically produced by disc cells in vivo and hence warrant further investigation as a potential source of cells for biological repair of the intervertebral disc.

Background: Increased nerve growth into degenerated intervertebral discs is associated with discogenic low back pain [1]. Many of these growing nerves are in neo-vascularised areas of the tissue [1,2] and endothelial cells that penetrate the disc express neurotrophic factors [3]. Thus, disc neovascularisation and disc innervation may be closely linked. Whilst disc aggrecan has been found to inhibit sensory nerve growth in vitro [4], the effects of disc aggrecan on endothelial cells are unknown.

Methods/Results: Adapting in vitro assays used previously [4], with HMEC-1 and EAhy-926 cell lines as models of endothelial cell growth, we found that disc aggrecan inhibited endothelial cell migration in a dose-dependent manner. Endothelial cells traversed over collagen substrates until they encountered disc aggrecan substrates (1mg/ml human aggrecan), where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the collagen:aggrecan border (Figure 1). After reaching the aggrecan border, some endothelial cells also migrated away from the disc aggrecan. At lower concentrations of disc aggrecan (0.01mg/ml), no such inhibition of endothelial cell growth was seen.

Conclusions: Loss of aggrecan, increased innervation and neovascularisation are all marked features of disc degeneration [1,2,5]. This study provides evidence that disc aggrecan inhibits endothelial migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predisposes the tissue to vascular invasion.

Autologous chondrocyte implantation (ACI) has been used most commonly as a treatment for cartilage defects in the knee and there are few studies of its use in other joints. We describe ten patients with an osteochondral lesion of the talus who underwent ACI using cartilage taken from the knee and were prospectively reviewed with a mean follow-up of 23 months. In nine patients the satisfaction score was ‘pleased’ or ‘extremely pleased’, which was sustained at four years. The mean Mazur ankle score increased by 23 points at a mean follow-up of 23 months. The Lysholm knee score returned to the pre-operative level at one year in three patients, with the remaining seven showing a reduction of 15% at 12 months, suggesting donor-site morbidity. Nine patients underwent arthroscopic examination at one year and all were shown to have filled defects and stable cartilage. Biopsies taken from graft sites showed mostly fibrocartilage with some hyaline cartilage. The short-term results of ACI for osteochondral lesions of the talus are good despite some morbidity at the donor site.

Aims: To examine vibration levels produced by orthopaedic air tools and the prevalence of upper limb symptoms in orthopaedic surgeons. Methods: A preliminary measurement of vibration levels produced by six air powered orthopaedic saws was followed by a national survey of orthopaedic surgeons and controls. A health surveillance questionnaire of symptoms associated with Hand Arm Vibration Syndrome (HAVS) was sent to 1200 orthopaedic surgeons (test group) and 1200 gynaecological surgeons (controls). Results: Measured accelerations of the saws were 3.42 to 10.7 m/sec² using BSI standards and 90.5 to 182 m/sec⁻² using NIOSH standards. These vibration levels are compatible with those reported to cause significant upper limb symptoms

Survey responses were received from 741(61.7%) of the test group and 748 (62.3%) of the control group. A statistically significant increase in the prevalence of the neurological symptoms was seen among orthopaedic surgeons (p< 0.001). A significant increase in musculoskeletal problems (p< 0.008) and muscle pain (p< 0.004) was also found. No significant difference was seen in the prevalence of vascular symptoms. The neurological symptoms were not related to other potential medical causes. Conclusions: Orthopaedic surgeons report an excess of upper limb symptoms and these may be linked to vibration exposure at work.

Objective: To determine if (a) inflammatory mediators are present in herniated intervertebral discs and (b) if their presence correlates with inflammation of nerve roots or symptoms.

Design: Inflammation was assessed with gadolinium enhancement of MRI. Neurological compromise was measured. Disc tissue was examined for inflammatory mediators IL-1α and β, IL-6, MCP-1, TSG-6, iNOS, TNFα and thromboxane.

Patients: Sixty-five discs were removed from 64 patients undergoing surgery for disc prolapse.

Outcome measures: We developed (i) an MRI score to assess inflammation radiologically prior to surgery (n=28, mean 4.9±6.8 days), (ii) a Surgical Score to assess inflammation of the nerve roots at surgery (n=44), (iii) a Clinical Score to determine pain, disability and neurological compromise (n=17) and (iv) a Mediator Score to reflect the number and amount of inflammatory mediators present (n=20).

Results: Thirty percent of the prolapses in this study were extrusions, 19% sequestrations and 51% protrusions. Sixteen of the 28 patients with gadolinium had nerve root enhancement (86% of the extrusions, 57% of sequestrations, and 43% of protrusions), whilst 19 had enhancement of or around the disc herniation itself (71% of the extrusions, 86% of sequestrations and 57% of protrusions). The Mediator Scores were highest for the sequestrations (as was the Surgical Score) and lowest for the protrusions, but extruded discs had most IL-1α and β, IL-6, TNFα and thromboxane. Extruded discs had the highest Clinical Score and sequestrated the lowest.

Conclusions: Mediators produced in prolapsed disc appear to play an important role in inflammation of adjacent tissue and nerve roots. The type of mediator present and proximity of the prolapse to the nerve root may be the important factors in determining which pro-lapses are the most painful.

Objective: The shape of articular chondrocytes regulates their function, changes in response to mechanical load and is altered in osteoarthritis. We aimed to identify the shape of intervertebral disc cells in pathological and normal tissue.

Design: Immunohistology of human intervertebral discs using cytoskeletal markers to examine disc cell shape.

Subjects: Intervertebral discs from patients with degeneration (n=3), scoliosis (n=3), spondylolisthesis (n=3) and from non-pathological cadaveric spines (n=3).

Outcome measures: (i). Cell shape and (ii). Organisation/ content of cytoskeleton.

Results: In degenerate and normal discs, cells of the anulus fibrosus were generally elongated and bipolar, whilst those of the nucleus pulposus were rounded/oval. However, in localised areas, cells were observed with multiple cytoplasmic processes that extended into the discal matrix. In central regions of scoliotic and, most markedly, spondylolisthetic discs, such cells were more frequent. Their processes were vimentin positive (but F-actin negative) and reached up to 80μm in length. F-actin was clearly present in endothelial cells of blood vessels but absent in disc cells. In contrast, vimentin was expressed by disc cells within the discs’ inner regions, but not towards the outer anulus fibrosus.

Conclusions: The altered shape of disc cells in pathological tissue may reflect areas of abnormal loading. These changes are also likely to affect/reflect altered cell function and therefore have a role to play in the pathological process.

Background: The healthy, adult human disc is innervated but the nerves are restricted to the outer few millimetres of the annulus fibrosus. In degenerate discs with associated back pain, however, the nerves are more numerous and penetrate further in.

We have used a sheep model of intervertebral disc degeneration to monitor the presence and organisation of nerves in the disc as degeneration progresses. This model has been used to study morphological and bio-chemical changes of the disc as it degenerates, in addition to associated alterations in end-plate vascularity and vertebral bone remodelling. One aspect of this model which has not been studied to date is how the innervation of the disc may change with the onset of degeneration. This is the object of the present study.

Materials and Methods: Four-year old, skeletally mature Merino wether sheep (n=64) were divided randomly into lesion and control groups. A surgical incision was created in the anterolateral annulus in the L1–L2 and L3–L4 discs of the lesion group. The control group received the same retroperitoneal surgical approach but the annulus was not incised. Intact lumbar discs encompassed by adjacent vertebral bodies were removed at 3,6,12 and 26 months post operation. Specimens were fixed, decalcified and paraffin embedded before sectioning (7μ thick, vertical sagittal sections) and stained immunohistochemically with the neuronal marker, PGP9.5, together with standard histological stains.

Results: The incised region of the outer annulus underwent collagenous re-organisation, consistent with an active repair process as early as three months post-operatively. However, the inner annular lesion had a poor repair response and propagated with time, sometimes through to the nucleus. In contrast, remodelling of the outer annular lamellae occurred across the cut region. For example, in one sample at two years post injury there were up to six lamellae “bridging the gap”. Nerves were present in all samples but in the sham animals they were very few and confined to the very outer annulus or longitudinal ligament. In the operated animals, nerves were more extensive, occurring in the matrix adjacent to the fissure where there was often blood vessel ingrowth. The maximum number of nerves was seen at 12 months post-operatively, before diminishing in number at 24 months post-op. This paralleled the presence and extent of blood vessel penetration in this experimental model.

Conclusions: We have used an animal model to follow longitudinally the penetration of nerves into the ovine intervertebral disc in association with disc degeneration. Whilst we obviously cannot assess back pain in these animals, and not all nerves are nociceptive, nerves nevertheless are a pre-requisite for the perception of pain. Hence the greater numbers, size and penetration of nerves into degenerate discs demonstrated here has important implications not only for the aetiopathogenesis of degenerative disc disease but also for the treatment of its associated symptoms. Further characterisation of this innervation, i.e. whether autonomic or sensory, may provide an indication as to its nociceptive potential.

Introduction: Intervertebral disc degeneration occurs with ageing and is often associated with back pain. During such degeneration, gross morphological differences between the central nucleus pulposus (NP) and outer annulus fibrosus (AF) are lost and the disc loses hydration and height due to decreased proteoglycan content. The cartilage endplate may also become calcified and this blocks the passage of nutrients into the disc, causing cell death and further degeneration. A potential therapy of degeneration is “re-inflation” of the disc with the use of hydrogels seeded with autologous disc cells. In this study, we have assessed the ability of a variety of hydrogels to support intervertebral disc cell growth.

Method: Intervertebral disc cells were isolated enzymatically from bovine tails and cultured as a monolayer in 10% foetal calf serum in DMEM containing antibiotics and ascorbic acid. This stimulates the cells to proliferate and thereby produces increased cell numbers. The cells were then seeded onto various hydrogels including hyaluronic acid (HA), 2-hydroxyethyl methacrylate (HEMA), N’N’ dimethyl methacrylate (NNDMA) and polyacryloyl morpholine (AMO) before harvesting at set time points of 1, 3, 6 and 9 days for hyaluronic acid and 1, 7, 14, 21, and 28 days for the other hydrogels. Cell number, morphology, viability and adherence to or migration into the hydrogels were assessed. Cell proliferation was also determined by immunostaining for the Ki67 antigen.

Results: Disc cells became incorporated in the HA gel, adopted a spherical morphology and remained viable for up to nine days. However, after a few days, a large proportion of the cells began to migrate through the gel to form a monolayer on the bottom of the tissue culture well. These monolayered cells became fibroblastic and proliferated. NP cells appeared to proliferate to a greater extent than AF cells both in monolayer and in suspension. Ki67 antigen immunostaining confirmed cell proliferation. On the non-porous HEMA, NNDMA and AMO, both cell types adhered and adopted a fibroblast-like morphology. Cell adhesion was greatest to the HEMA. NNDMA and AMO had lower levels of cell adherence. Both cell types became incorporated into the porous materials and adopted a rounded morphology. Cell incorporation appeared to be greatest into porous HEMA.

Conclusion: These initial studies show that intervertebral disc cells will adhere to or migrate into a variety of hydrogels and remain viable. The morphology and proliferative capacity of cells derived from both the AF and NP were responsive to the structure of the hydrogel with which they were cultured. Thus, cells were able to become fibroblastic or chondrocytic. Further analyses will reveal whether matrix synthesis by disc cells is similarly responsive to the hydrogel format. The results of these experiments suggest that the hydrogels tested have potential as support matrices in intervertebral disc repair to provide relief from discogenic low-back pain.

Mature human intervertebral disc cells have generally been described as being either fibroblast-like or chondrocyte-like; i.e. appearing either elongated and bipolar or rounded/oval. Fibroblast-like cells are observed within the outer regions of the anulus fibrosus whilst chondrocyte-like cells are found in the more central regions of the disc. However, a few reports have noted that in some circumstances disc cells appear to extend more elaborate cytoplasmic processes into their surrounding extracellular matrix. In this study, we have examined healthy and pathological human intervertebral discs for the presence of the cytoskeletal elements, F-actin and vimentin.

Tissues examined included discs of no known pathology, discs with spondylolithesis, scoliosis specimens taken from the convex and concave sides, and degenerated discs. F-actin was not readily observed within discs cells but was a marked feature of vascular tissue within the disc and occasionally seen in infiltrating cells. Vimentin was more readily seen within cells of the inner anulus fibrosus and nucleus pulposus. In general, disc cell morphology was fibrocyte or chondrocyte-like; however, in spondylolisthetic discs, cells with numerous cytoplasmic projections were frequently observed.

The differential morphologies and cytoskeletal composition observed in disc cells may be indicative of variations in mechanical strains and/or pathologies, or indeed of cell function.

The aim of this study was to identify potential inflammatory mediators in herniated and non-herniated intervertebral disc. It has been suggested that inflammation of the nerve root is a pre-requisite for disc herniations to be symptomatic. What leads to this inflammation is a matter of conjecture; one possible cause may be inflammatory mediators released from the herniated disc tissue itself. In this study we have examined discs from individuals with and without disc herniations to determine if there is a different degree of occurrence.

Twenty two discs from 21 patients with disc herniation were examined together with four discs from patients with other disc disorders and five age-matched discs from individuals obtained at autopsy. Samples were studied for the presence of blood vessels and inflammatory cytokines: IL-1α and β, IL-6, INOS, MCP1, TNFα, TSG-6 and thromboxane.

Of the herniated discs 10 were protrusions, six extrusions and six sequestrations. There was less of all the cytokines in the non-herniated discs than found in the herniated, with very little immunostaining for iNOS or IL-1α in any samples. Staining was seen in all herniated samples for IL-1β, but in fewer for IL-six and MCP1 (86%), thromboxane (68%), TNFα (64%) and TSG-6 (59%). The presence of cytokines was strongly associated with the presence of blood vessels. Protruded discs had less TNFα and thromboxane than sequestrated or extruded discs.

Cytokines appear to play an active role in the aetiopathogenesis of disc herniations. Some may be involved in the stimulation of degradative enzymes and hence resorption of, for example, sequestrations, whereas others may be responsible for an inflammatory response in the surrounding tissues such as nerve roots.

Although an increased and deeper innervation of painful and degenerate intervertebral discs (IVDs) has been reported, the mechanisms that regulate nerve growth into the IVD are largely unknown. In other tissues, proteoglycans have been found to act as nerve guidance molecules that, generally speaking, inhibit nerve growth. As disc degeneration is characterised by a loss of proteoglycans, we assessed the effects of IVD proteoglycans on nerve growth and guidance.

Using in vitro assays of nerve growth, we found that human disc proteoglycans inhibited nerve attachment, neurite extension and induced sensory growth cone turning in a dose-dependent manner. Digestions with chondroitinase ABC or keratinase abrogated these inhibitory effects. Proteoglycans of the anulus fibrosus were more inhibitory than those from the nucleus pulposus.

Disc proteoglycans inhibit nerve growth and this inhibitory activity may dependent on proteoglycan glycosylation and/or sulfation. A loss of proteoglycans from degenerative discs may therefore predispose the discs to nerve invasion.

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage.

Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.

The articular surface of the humeral head is usually described as facing posteromedially, making an angle of between 16 degrees and 35 degrees with the transepicondylar plane. At hemiarthroplasty the articular surface also appears to be offset posteriorly with respect to the humeral shaft. Coracoid impingement may occur if this offset is not accommodated. An analysis was made of 29 cadaveric humeri using an industrial co-ordinate measuring machine. The position of the centre of the head was defined with respect to the humeral shaft and transepicondylar plane. The humeral articular surface was found to be retroverted by 21.4 degrees and its centre offset posteriorly by 4.7 mm. Previous interpretation of retroversion did not take into account the posterior displacement, and this may be of importance in improving future prosthetic design.

Articular cartilage from the femoral heads of 27 patients having an arthroplasty for subcapital fracture was studied, and its mechanical and chemical properties compared to those of a group of 33 age-matched macroscopically normal autopsy specimens. Water and proteoglycan contents were measured, as were swelling ability, compressive and tensile strength of the cartilage, and the density of the underlying bone. Cartilage from the fracture specimens had a significantly reduced proteoglycan content, as measured by fixed charge density, and increased swelling ability. These results indicate that this group differs from the "normal" population and care should be taken before they are accepted as control material for studies on osteoarthritic cartilage. Another finding was that bone density was much the same in the fracture and the normal group. This casts some doubt upon the concept that patients who sustain subcapital fractures are more osteoporotic than the average for the same age range.

We studied the mechanical and biochemical properties of articular cartilage from 22 osteoarthritic femoral heads obtained at operation and 97 femoral heads obtained at autopsy. Cartilage from the zenith and from the antero-inferior aspect of each head was tested both in tension and in compression. Water content, swelling ability and proteoglycan content were measured, the cartilage was examined histologically and the density of the underlying bone was assessed. Fifty-five of the autopsy specimens were defined as macroscopically normal because they exhibited no progressive fibrillation patterns on staining with Indian ink; but significant changes in water content, bone density and tensile strength related to age were seen in this group. In 20 pairs of femoral heads which were both macroscopically normal, we found, surprisingly, that cartilage from the left and right sides of the same patient was sometimes very different. Compared with the normal autopsy specimens the osteoarthritic specimens had a significantly increased swelling ability, a lower proteoglycan content and impaired mechanical properties, being both weaker in tension and softer in compression. Abnormal autopsy specimens had values intermediate between those of osteoarthritic and normal groups. Results from this abnormal group suggest that there is no primary loss of proteoglycan in early osteoarthritis.

Specific antisera to collagen Types I, II and III and proteoglycan were used to investigate the distributions of these molecules in normal human intervertebral discs. Immunofluorescent staining indicated the presence of small amounts of Type III collagen located pericellularly in normal adult intervertebral discs. This finding had not been demonstrated previously by other methods. Similar specimens of intervertebral discs from 17 patients with scoliosis of varying aetiologies were examined, but no evidence was obtained for primary connective tissue defects. Secondary changes, especially marked vascularisation of the inner annulus, were apparent in a number of scoliotic discs, and some of these showed enhanced staining for collagen Type I and proteoglycan, and intercellular matrix staining for Type III collagen.