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.