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.

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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