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.