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Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_27 | Pages 19 - 19
1 Jul 2013
Rashid M Butt U Birch A Crank S Temperley D Freemont A Trail I
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Our aim was to accurately determine whether muscle atrophy and fatty infiltration are reversible following cuff repair. Patients with a repairable cuff-tear were recruited and assessed clinically and radiologically (Magnetic Resonance Imaging). At surgery, supraspinatus was biopsied. Post-operatively, patients underwent clinical evaluation at standardised intervals, with further MRI and an ultrasound guided biopsy of supraspinatus at 12 months.

MRI was used to characterize cuff-tears and determine the degree of muscle atrophy and fatty infiltration. Biopsy samples were fixed on-site and transported for processing. Morphometric assessments of myofibres were made and mean cross-sectional areas calculated using validated techniques. The pathologist was blinded to sample details. Statistical analysis was performed to assess differences in mean myofibre area following cuff repair and correlated with radiological findings.

Eight patients were available for completed histological and radiological analysis. Six (two re-tears) demonstrated sizeable and highly statistically significant improvements in mean myofibre cross-sectional area (P=0.000–0.0253). Of the two not showing any increase in myofibre area, neither result was statistically significant (P=0.06, 0.2); one was a re-tear and one was a repair of a partial-thickness tear. Radiologically, the muscle and fatty changes had not demonstrably changed.

Our finding that myofibre cross-sectional area increases following cuff repair suggests muscle atrophy is a potentially reversible process. Even with re-tears, improvements were seen. MRI features of fatty infiltration and muscle atrophy were not seen to improve however. It is likely that radiological assessment is not sensitive enough to demonstrate the reversibility of muscle atrophy seen on histological analysis at one year.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_27 | Pages 6 - 6
1 Jul 2013
Green C Daniels J Freemont A Paul A
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Background

Although soft tissue sarcoma (STS) is a rare malignancy, myxofibrosarcoma is a common form diagnosed. Myxofibrosarcoma is complicated by a high local recurrence rate (18–54%) and significant morbidity following treatment, hence management can be challenging.

Patients and Methods

Patients treated between 2003–2012 were identified via a database within the histopathology department and case notes were retrospectively assessed. All histology samples were reviewed by a senior histopathologist to ensure a correct diagnosis.


Orthopaedic Proceedings
Vol. 92-B, Issue SUPP_I | Pages 67 - 68
1 Mar 2010
Chandrashekar S Arumilli B Conway A Hinduja K Paul A Freemont A
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Introduction: Anterior knee pain is a very common presenting symptom. Fat pad syndrome is an uncommon and a difficult condition to manage. The diagnosis is usually reached after a period of physiotherapy and investigation to rule out the more common aetiologies of anterior knee pain.

Patients & Methods: All patients who underwent excision of the infrapatellar fat pad following a diagnosis of Fat pad syndrome are included. Each patient was evaluated to exclude patellofemoral problems and intraarticular pathologies as the cause of anterior knee pain. Each patient underwent MR imaging and all the excised specimens were sent for histological analysis.

Results: The MR imaging provided with the provisional diagnosis in all patients. All the specimens were examined by a single senior histopathologist to correlate with the provisional diagnosis. The histology confirmed Hoffa’s syndrome in 5 patients and in the remaining 15 patients a spectrum of rare diagnoses as suspected by Magnetic Resonance imaging.

The more notable conditions were two synovial sarcomas, three haemangiomas and a Giant cell tumour of the tendon sheath. All patients were treated successfully with complete excision. No recurrences were recorded at the end of 3 year follow-up and all patients were symptom free.

Conclusion: The work up of a patient with suspected infrapatellar fat pad syndrome must include MR imaging and the exact underlying pathology should be confirmed with histological analysis of the excised fat pad as the rare causes include soft tissue malignancy.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 14 - 14
1 Mar 2008
Giannikas K Karski M Khan A Buckley J Wilkes R Hutchinson C Freemont A
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While the early period of distraction osteogenesis has been extensively investigated, there are very few data describing the long-term morphology of the regenerate. We performed magnetic resonance scans in ten adults (men age 35+− 11 yr), seven of whom had bone transport for an iatrogenic osseous defect while further three had tibial lengthening for limb length discrepancy. Follow-up ranged between 14 and 43 months (mean : 28 + − 10 months) following the removal of the external fixator. The perimeter, cross- sectional area, volume and the mean signal intensity was calculated from the obtained T1 weighted axial images. Values were compared with the contralateral tibia that acted as control. All cases that had bone transport increased the volume of the tibia from 15.3% up to 50.8%. The regenerated segment was noted to have expanded significantly (p< 0.0001) in all cases. Mean signal intensity in the regenerate decreased in seven cases significantly (p< 0.0001) suggesting increase content of unhydrated tissue such as bone and collagen. The cross-sectional surface of the transported segment was increased in all cases (p< 0.008). Finally in cases that underwent bone transport, the docking site was noted to be obstructed by unhydrated tissue. Contrary to previous claims, the post-distraction osteogenesis tibia is far from normal, consisting of areas with potentially different biomechanical properties. Recognition of these changes is essential not only for appropriate pre-operative counselling but also for considering treatment modalities in case of a fracture.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_I | Pages 38 - 38
1 Mar 2005
Richardson S Le Maitre C Russell A Greenway E Li Y Freemont A Hoyland J
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Introduction: Intervertebral disc (IVD) degeneration involves loss of disc matrix leading to instability and pain. Autologous cells are the ideal choice for bioengineering a new IVD, but removal of cells from the IVD is problematic. Our aim was to direct mesenchymal stromal cells (MSCs) down a chondrocytic lineage to mimic disc chondrocyte phenotype.

Methods: MSCs were either maintained in monolayer, pelleted into micromass aggregates or transferred to alginate beads. Pellet cultures were used in immunohis-tochemistry for type II collagen and aggrecan and in situ hybridisation for SOX-9 mRNA. Monolayer and alginate cells were cultured in the presence or absence of chondrogenic medium for 4 and 11 days. Monolayer cultured MSCs were also transfected with a SOX-9 adenovirus and cultured in the presence or absence of TGF-_1. Realtime quantitative PCR was used to analyse expression of chondrocyte markers.

Results: IHC showed increased expression of type II collagen and aggrecan in pellet cultures, while ISH showed that SOX-9 was not expressed by monolayer MSCs, but increased after pelleting. Realtime PCR using alginate-cultured MSCs showed down regulation of type I collagen mRNA expression and up-regulation of SOX-9 that was increased by chondrocgenic medium. SOX-9 transduced monolayer MSCs showed increased type II collagen, aggrecan, SOX-6 and SOX-9 mRNA over controls, while type I collagen levels showed no significant change. Stimulation of transfected MSCs with TGF-_1 showed similar increases in chondrocyte genes.

Discussion & conclusions: Adult human MSCs were induced to differentiate along a chondrocytic phenotype, which was mediated by culture conditions. Alginate and pellet culture produce a cell that has more chondrogenic characteristics than monolayer cells. SOX-9 transduced monolayer MSCs appeared to produce a more chondrocytic phenotype which was modulated by TGF-_1. Results suggest SOX-9 transfected monolayer MSCs may be used as a source of chondrocytes for repair of degenerate IVD.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_I | Pages 79 - 79
1 Mar 2005
Talwalkar SC Kambhampati SBS Whitehouse R Stevenson AIL Freemont A
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We report a rare case of an intracortical chondroma in the region of the medial femoral condyle of the femur extending into the femoral sulcus and the patellofemoral joint.A sixteen year old Asian boy presented with repeated episodes of right sided anterior knee pain and giving way over a three year period. The patient had been treated previously for multiple bony swellings at another hospital and a diagnosis of multiple enchondromatosis had been made. Examination revealed that the patellofemoral compression test was positive with fullness over the medial eminence of the femur in the region of the trochlear groove.Pre-operative X-rays and MRI scan showed the presence of an intracortical lesion over the medial femoral condyle extending into the femoral sulcus. The lesion demonstrated intermediate signal intensity on T1 and high intensity on T2 weighted images with variable low signal intensity foci due to the presence of a calcified matrix. The patient underwent arthroscopic examination. An intra-articular lesion (2cmx 3cm.) was identified and excised through a mini-arthrotomy. The lesion was entirely intra-articular arising from the medial femoral condyle proximal to the femoral sulcus, extending partially into the supra-patellar pouch. Histopathological examination confirmed the presence of a low grade cartilaginous neoplasm best diagnosed as an atypical chondroma. At a two year follow up appointment the patient was found to be asymptomatic with no evidence of radiological recurrence.

Although there have been several reports of periosteal chondromas developing around the knee the majority deal with soft tissue chondromas in para-articular locations or intra-cortical tumours in extra-articular regions. Our tumour is unique due to its intra-articular and intracortical location. A detailed review of the literature of this rare tumour is presented with a pictorial presentation of the case including arthroscopic radiological and histopathological findings.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_I | Pages 100 - 100
1 Jan 2004
Brownlow H Freemont A Even T Copeland S Levy O
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The coraco-acromial ligament (CAL) is partially resected during a subacromial decompression. Clinical studies have reported the regeneration of a structure which appears to be a new CAL. Histological studies of regenerated CAL have demonstrated an abundance of relatively acellular collagen fibrils orientated in the line of a ligament and mechanical testing of the regenerated tissue has properties similar to those of normal CAL. However it is still not clear whether this structure represents scar tissue or truly reformed ligament. Defining the major collagen constituent of this regenerated tissue would allow the distinction between ligament and scar tissue. Therefore the aim of this study was to examine the level of expression of types I and III collagen in regenerated coraco-acromial ligaments (CAL) in humans.

Samples of regenerated CAL were obtained during open surgery for repair of small rotator cuff tears at an average of 24 months (range 14 to 52) after arthroscopic subacromial decompression from 4 men and 3 women with an average age of 58 years (range 44 to 68). A standard protocol radio-active in-situ immunolocalisation technique was used to quantify the ratios of mRNA collagen I to collagen III in the samples.

The results demonstrated that the average ratio of collagen I to collagen III was 6.5. This ratio is similar to the value for normal hip capsule (5–6:1) and human posterior cruciate ligament (8:1).

We conclude that the reformed CALs are ligamentous structures, not scar tissue, and therefore represent truly regenerated ligaments.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Le Maitre C Williamson B Ross R Freemont A Hoyland J
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Objective and Background: This study investigated the effects of IL-1 on human intervertebral disc cells (IVD). IL-1 has been implicated in the degradation of IVD, in particular the up-regulation of Matrix Metalloproteinases (MMPs) and the down regulation of proteoglycan synthesis. However very little is known of the effects of IL-1 on human IVD cells. Here, we have investigated the effects of both IL-1 α and IL-1 β on nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from human degenerate IVD.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into NP and AF tissue, cells were cultured within an alginate bead system for 5 weeks before treatment with IL-1 α and IL-1 β for 48 hours. Following treatment, RNA was extracted and Real time RT-PCR was performed to investigate gene expression of IL-1 gene family, matrix proteins and degrading enzymes MMPs and ADAMTS.

Results: Interleukin 1 α showed a more potent response than IL-1 β and in addition NP cells were more sensitive than AF cells. In summary, IL-1 showed a positive feedback loop causing an up-regulation of α and β genes. IL-1 Ra was also up-regulated but to a lesser extent than IL-1 α and IL-1 β. A negative feedback loop was seen with inhibition of the IL-1 receptor gene upon treatment with IL-1. MMPs and ADAMTS showed up-regulation upon treatment with IL-1. In addition IL-1 down regulated the matrix protein’s collagen type II and Aggrecan.

Conclusions: This study demonstrates that IL-1 causes up-regulation in discal cells of the major degrading enzymes involved in discal degeneration, and a down regulation of the major matrix components within the IVD. Suggesting that IL-1 plays a major in process of discal degeneration.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Le Maitre CL Baird P Williamson B Ross R Freemont A Hoyland J
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Objective and Background: Interleukin 1 has been implicated in the progression of degenerative disc disease, however little data is available on the expression and production of IL-1 within degenerate discal cells. A few studies, have investigated herniated disc tissue but the results from these studies have been inconsistent. This study investigated the gene expression of IL-1 α, β, Ra and the receptor type I in discs removed at surgery from 7 prolapsed, 3 Scoliosis and 15 Degenerative discs (DD). In addition immunohistochemistry (IHC) was used to localise IL-1 α and IL-1 β within normal, and degenerate discs.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into nucleus pulposus (NP) and annulus fibrosus (AF) tissue, cell isolation using collagenase treatment was carried out, and RNA extraction on the cells performed immediately. Real time RT-PCR was then used to investigate gene expression of IL-1 gene family. IHC for IL-1 α and IL-1 β was also performed on paraffin embedded normal and degenerate disc samples.

Results: Expression of the IL-1 family genes was present at low levels within prolapsed disc samples. In contrast levels within scoliosis patents were the highest of the 3 disease states, however in both prolapsed discs and those from scoliosis patients a balance of IL-1 α/β to IL-1 Ra existed. Within samples from DD this balance was lost, with levels of IL-1 α and IL-1 β greatly exceeding levels of IL-1 Ra. In addition levels of IL-1 α and β showed an increase with age and were highest in those samples from the AF than the NP. IHC demonstrated both IL-1 α and IL-1 β protein within the NP and AF cells of the degenerate discs.

Conclusion: This study has demonstrated the mRNA expression of all members of the IL-1 family within IVD and in addition the chondrocytes within the disc produced IL-1 α and IL-1 β protein. The imbalance of IL-1 α/β to IL-1 Ra within those samples from degenerate discs but not prolapsed or scoliotic discs suggests a role for IL-1 within discal degeneration.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 240 - 240
1 Mar 2003
Watkins A Hoyland J Le Maitre C Baird P Ross R Williamson B Freemont A
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Aims: The intervertebral disc (IVD) consists of three structurally distinct areas; a nucleus pulposus (NP), annulus fibrosus (AF) and two cartilage endplates that together form a functional unit that allow flexibility of the spinal column and load transfer from adjoining vertebrae. The NP and AF contain cells that are phenotypically similar to chondrocytes found in articular cartilage. They also produce the 2 major matrix components aggrecan and collagen-type I and II. One feature of IVD degeneration is breakdown of the cartilage matrix. Using soluble growth factors could stimulate new matrix formation and help regenerate degraded discal cartilage. The aim of this study was to demonstrate the presence of four growth factor receptors within the IVD.

Methods and Results: Using immunohistochemsitry, we targeted expression of four growth factor receptors, (BMPRII, FGFR3, IGFR-1 and TGFβII), in biopsies taken from normal and degenerate IVD. Receptor expression was scored across regions of the disc using a peer-reviewed system that assessed the proportion of cells expressing a particular antigen and the average level of expression for those cells. For FGFR3, IGFR-1 and TGFβII, cells of the outer part of NP and inner AF expressed significantly higher receptor levels. The expression BMPRII deviated from that pattern and was present at higher levels in the inner and outer NP than in the AF. Although there were significant differences between FGFR3 expression in normal and degenerate biopsies, that was not the case for the other receptors. Growth factor receptor expression was also detectable on the ingrowing neurons and blood vessels that characterise part of the disease aetiology.

Conclusion: In conclusion, all of the receptors were found in the IVD, predominantly within the NP, suggesting that, addition of the ligands for these receptors may elicit a physiological response from disc chondrocytes.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_III | Pages 239 - 240
1 Mar 2003
Le Maitre C Williamson B Ross R Freemont A Hoyland J
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Objectives and Background: This study investigated a simple, novel, in vitro culture system which enables the in situ investigation of human intervertebral disc (IVD) cell function in healthy and diseased IVD in explant culture. Studies investigating the function of cells in IVD tissue are scarce. Whilst there is a paucity of realistic animal models of human IVD disease and in vivo study of human tissue remains impracticable, the only possible approaches remain in-situ molecular biology applied to tissue sections of biopsied tissue, which suffers from lacking a dynamic dimension. Or in vitro studies, of which cell culture lacks physiological relevance and explant cultures are subject to loss of tissue integrity and altered cellular behaviour. We have investigated a system that preserves the structure of the tissue and cellular phenotype within an explant culture system.

Methods: Human IVD tissue was obtained from disc replacement surgery and separated into nucleus pulposus (NP) and annulus fibrosus (AF) tissue, which was then cultured in either a Perspex ring or unconstrained in tissue culture medium for up to 3 weeks. The effectiveness of this system to maintain tissue integrity and cell function was tested using microscopy and either tinctoral histochemistry or immunohistochemistry.

Results: Unconstrained in medium, IVD tissue expanded and structural integrity was disturbed. The number of cells expressing type I collagen increased and aggrecan decreased by comparison with directly harvested tissue. In contrast the tissue in the Perspex rings maintained its structure and at the end of 3 weeks the cellular parameters were the same as in the newly harvested tissue.

Conclusions: This is the first reported system to preserve cell function of discal explants for long periods in tissue culture. This system will be a useful tool for a wide range of investigations of IVD biology that have not hitherto been possible.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_II | Pages 141 - 141
1 Jul 2002
Freemont A Hoyland J Byers R Bartley C Baird P Jeziorska M Knight M Ross R O’Brien J Sutcliffe J LeMaitre C Goswami A
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Purpose and Background: We have previously reported our investigations of nerve ingrowth into intervertebral discs (IVD) from patients with mechanical low back pain. We have shown that in discs that are painful on discography (pain level discs) nerves actively grow into the deep annulus fibrosus and nucleus pulposus. Nerve ingrowth accompanies blood vessel ingrowth and advances into the nucleus pulposus from the end plate. The morphology and neurochemistry of these nerves indicate them to be nociceptive.

The growth of non-myelinated pain fibres in other settings is regulated by the cytokine Nerve Growth Factor (NGF). In this study, we have investigated the production and distribution of NGF, or more particularly its active isoform – NGF-β, and its receptors, in diseased intervertebral discs in order to establish whether this cytokine might be responsible for the observed nerve ingrowth in this situation.

Methods: Tissue sections of 21 pain level, 15 non-pain level diseased and 12 normal intervertebral discs, taken at the time of spinal surgery, and from cadavers, were probed by radioactive in situ hybridisation (ISH) for expression of NGF-β, and by immunohistochemistry (IHC) for its high and low affinity receptors (trk-A and p75 respectively). In addition, either serial sections were stained with cell specific markers (CD31 – endothelial cell, PGP9.5 – neurones, GAP43 – actively growing nerves) or sections were doubled stained (two antibodies or both ISH and IHC).

Results: We have demonstrated that NGF-β is synthesised by the endothelial cells of blood vessels growing into the IVD from the end plate. The high affinity receptor is expressed by those small nerve fibres that accompany the vessels and in their offshoots in pain level discs that are growing from perivascular nerves into the disc. In addition to their expressing the nerve specific molecule PGP9.5, the trk-A positive cells also express the nerve growth associated protein GAP43.

Conclusion: The data indicate that nerve ingrowth into IVD is regulated by NGF-β. We have localised this production to the endothelial cells of ingrowing blood vessels. NGF-β is a potential therapeutic target for the management of back pain.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_II | Pages 143 - 143
1 Jul 2002
Goswami A Knight M Freemont A
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Introduction: Recent cadaveric studies have identified neovascularisation and neoneuralisation as probable mechanisms in the causation of discogenic pain. Calcium pyrophosphate deposits have been observed in discs in several studies. Their significance in the causation of discogenic pain is unclear. Direct correlation between the pain site and histological features can be verified by aware state endoscopic visualisation.

Aim and Objectives: The study aims to examine and correlate the presence of neovascularisation, crystalline pyrophosphate deposits in the disc, and discogenic pain by spinal probing and discography under endoscopic visualisation.

Material and Methods: Tissue removed from intervertebral discs of 224 patients during surgery was examined directly, and polarised microscopy was used to identify the presence of calcium pyrophosphate and neovascularisation. Their presence was correlated to diagnostic provocative findings of spinal probing and discography and intradiscal distortion during aware state endoscopy.

Results: Calcium Pyrophosphate: Twenty out of 224 patients (9%) demonstrated calcium pyrophosphate in the discs. Fourteen had pain reproduced on probing or discography. Thirteen out of 20 patients (65%) had either an annular collection or leak at the index level. 6 had an extradiscal cause of pain. One hundred percent of the patients with annular collections or leaks had pain on spinal probing or discography. Sixteen patients with pyrophosphate deposits did not have neovascularisation.

Neovascularisation: Thirty seven out of 224 patients (16.5%) showed neovascularisation in the disc. Four discs had crystalline pyrophosphate deposits. Thirty three out of 37 (90%) had pain on probing and/or discography. Out of four patients who had no pain on probing or discography, two had demonstrated tears during previous discographic procedures which were treated with laser annealing. These patients had disc bulges and compressive radiculopathy.

Conclusion: The presence of pyrophosphate in the disc without a tear or leak does not directly render them tender to provocation. The presence of pyrophosphate is not correlated to neovascularisation. Annular tears or leaks are not directly correlated to the presence of pyrophosphates. There is a high correlation between pain provocation and neovascularisation.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_II | Pages 142 - 142
1 Jul 2002
Le Maitre C Rajpura A Staley W Byers R Knight M Ross R Freemont A Hoyland J
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Background: Low back pain (LBP) is a major cause of disability. However, current treatments are often empirical and few are directed at the underlying disorder, altered discal cell metabolism, which precipitates the problem. The use of gene therapy to manipulate discal metabolism to treat LBP is an interesting possibility. The Intervertebral disc (IVD) is a therapeutic target in LBP, and one approach to gene therapy would be to isolate IVD chondrocytes (IVDC) and transfer genes ex vivo into these cells. Subsequent reinjection of these genetically altered cells into the lumbar IVD, would permit the expression of the transgene in vivo, generating the therapeutic protein within the IVD.

Methods: To test the viability of this approach, we isolated human IVDC from patients undergoing surgery, grew them ex vivo and transfected them with the marker gene LacZ, using an adenovirus vector and the CMV promoter. Expression of the gene was then measured using X-gal staining for the gene product _-galactosidase. Post infection, some cells were treated with forskolin for 24 hours to assess whether expression of the transgene could be manipulated.

Results: IVDC infected with adenovirus/CMV-LacZ showed maximal LacZ expression 2 days post infection, with almost 50% of cells displaying X-gal positivity. Cells maintained a low level of expression for the remaining 12 days of the study. Control cultures showed no LacZ expression. Cells treated with forskolin after infection with adenovirus/CMV-LacZ exhibited 4 times the level of _-galactosidase activity seen in unstimulated cultures.

Conclusion: This study shows that human IVDC can be transfected with a foreign gene using the adenovirus vector. The gene transduction of a therapeutic gene into IVDC could provide a long lasting effect. In addition, the use of inducible promoters could allow for the autoregulation of gene expression.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_II | Pages 141 - 141
1 Jul 2002
Freemont A Hoyland J Rajpura A Byers R Bartley C Jeziorska M Knight M Ross R O’Brien J Sutcliffe J LeMaitre C Goswami A
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Purpose and Background: There is increasing evidence that events within the diseased intervertebral disc (IVD) are mediated by locally synthesised cytokines. A prominent histological, imaging and surgical feature of IVD disease is degradation of the cartilaginous discal matrix. Whilst the mechanism by which this is mediated is unknown, in other situations where connective tissues are degraded degradation is the result of production of matrix-degrading enzymes by local connective tissue cells stimulated by cytokines, particularly the beta isoform of interleukin-1 (IL-1β). Included amongst these disorders is osteoarthritis (OA) of diarthrodial joints. OA has many similarities to the discal “degeneration” seen in mechanical back pain syndromes. In the current study, we have used a combination of in-situ techniques to establish if IL-1β is responsible for stimulating matrix degradation in the IVD.

Methods: Using a combination of radioactive in-situ hybridisation (ISH) and competitive in situ zymography (ISZ) we have studied expression of IL-1β and IL-1R – its type 1 receptor (ISH) and matrix degradation (ISZ) in five diseased lumbar IVD taken at spinal fusion surgery and 10 cadaveric IVD (five normal and five diseased). The nucleus pulposus (NP) was separated from the annulus fibrosus and diced into 0.5cm cubes. Half the cubes (typically three) were fixed in formalin and processed into paraffin wax for ISH, and half were used for ISZ. For ISH, 5 μm sections of paraffin-embedded tissue were reacted with cDNA probes radiolabelled with 35S to 580 and 530 base segments of the IL-1β and IL-1R molecules. Hybridisation was disclosed using autoradiography. For ISZ, 50 μm vibratome sections were placed into wells on microscope slides precoated with gelatin. Sections were incubated for 10 days, half in culture medium and half in medium supplemented with human recombinant IL-1 receptor antagonist (IL-1Ra – an inhibitor of IL-1). Sections were photographed at daily intervals to detect evidence of gel degradation.

Results: Chondrocytes within patient and cadaveric diseased but not normal discs expressed mRNA for both IL-1β and IL-1R. By ISZ, the same cells degraded gelatin. Degradation was inhibited by recombinant IL-1Ra.

Conclusion: This study shows that chondrocytes of diseased discs express IL-1 and its receptor. The same cells produced matrix-degrading enzymes by a mechanism that can be inhibited by the IL-1 inhibitor IL-1Ra. IL-1 is a potential therapeutic target for the management of IV disc disease.