Current clinical treatment for spinal instability requires invasive spinal fusion with cages and screw instrumentation. We previously reported a novel injectable hydrogel (Bgel), which supports the delivery and differentiation of mesenchymal stem cells (MSCs) to bone forming cells and supports bone formation Bovine and Human Nucleus pulpous tissue explants were injected with Bgel with and without MSCs. Tissue samples were cultured under hypoxia (5%) in standard culture media for 4 weeks. Cell viability, histological assessment of matrix deposition, calcium formation, and cell phenotype analysis using immunohistochemistry for NP matrix and bone markers.Background
Methodology
Injectable hydrogels via minimally invasive surgery offer benefits to the healthcare system, reduced risk of infection, scar formation and the cost of treatment. Development of new treatments with the use of novel biomaterials requires significant pre-clinical testing and must comply with regulations before they can reach the bedside. In the European economic area (EEA) one of the first hurdles of this process is attaining the CE marking which protects the health, safety and environmental aspects of a product. Implanted materials fall under the class III medical device EU745 regulation standards. To attain the CE marking for a product parties must provide evidence of the materials safety with an investigational medicinal product dossier (IMPD). We have been working to develop a new thermoresponsive injectable biomaterial hydrogel (NPgel) for the treatment of intervertebral disc (IVD) disease. A large part of the IMPD requires information on how the hydrogel physical properties change over time in bodily conditions. We have been studying 6 batches of NPgel over 18 months, tracking the materials wet/ dry weight, structure and composition. To date we have found that NPgel in liquids more similar to the body (with protein and salts) appear to be stable and safe, whilst those in distilled water swell and disintegrate over time. Subtle long-term changes to the material composition were found and we are currently investigating its ramifications.Introduction
Methods and Results
Low back pain is the leading cause of musculoskeletal disease and the biggest cause of morbidity worldwide. Approximately 40% of these are cases are caused by disease of the intervertebral discs (IVDs): the shock absorbing, flexible material located between the bones (vertebrae) along the length of the spine. In severe cases, the spine becomes unstable and it becomes necessary to immobilise or fix the joint in position using a lumbar cage spacer between in the IVD and metal pins with supporting plates in the vertebrae. This is a complex, expensive, major surgery and it is associated with complications, such as spinal fusion failure and inappropriate implant position. These complications have a dramatic impact on the quality of life of the affected patients and the burden to society and the healthcare system is exacerbated. We present an Introduction
Methods and Results
We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as collagen II and aggrecan and NP phenotypic markers FOXF1 and PAX1, whereas Bgel induced expression of collagen I and osteopontin, indicative of osteogenic differentiation.Purpose of study and background
Methods and Results
IVD degeneration is a major cause of Low back pain. We have previously reported an injectable hydrogel (NPgel), which induces differentiation of human MSCs to disc cells and integrates with NP tissue following injection MSCs were cultured in NP gel under 5% O2 in either: standard culture (DMEM, pH7.4); healthy disc (DMEM, pH7.1); degenerate disc (low glucose DMEM, pH6) or degenerate disc plus IL-1β. Following 4 weeks histological staining and immunohistochemical analysis investigated viability, ECM synthesis and matrix degrading enzyme expression. Here we have shown that viability and NP cell differentiation of MSCs incorporated within NPgel was mostly unaffected by treatment with conditions such as low glucose, low pH and the presence of cytokines, all regarded as key contributors to disc degeneration. In addition, the NPgel was shown to prevent MSCs from displaying a catabolic phenotype with low expression of degradative enzymes, highlighting the potential of NPgel to differentiate hMSCs and protect them from the degenerate disc microenvironment.Purpose of study and background
Methods and Results
The intervertebral disc (IVD) is a highly hydrated and hyperosmotic tissue, water and salt content fluctuate daily due to mechanical loading. Resident IVD cells must adapt to this ever-changing osmotic environment, to maintain normal behaviour. However, during IVD degeneration the disc becomes permanently dehydrated and cells can no longer perform their correct function. Here, we investigated how human nucleus pulposus (NP) cells respond to altered osmolality with regards to cell size and the rate of water permeability, along with the potential involvement of aquaporins (AQPs) and transient receptor potential vanilloid (TRPV) membrane channels. Water permeability of NP cells exposed to altered osmolality (225–525mOsm/kg) in the presence or absence of AQP and TRPV channel inhibitors was investigated with the cell-permeable calcein-AM fluorescent dye, and cell size determined using microscopy and flow cytometry.Introduction
Methods
Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). We have developed an injectable hydrogel (NPgel), which following injection into bovine IVD explants, integrates with IVD tissue and promotes disc cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here, we investigated the injection of NPgel+MSCs into IVD explants under degenerate culture conditions. The NPgel integrated with bovine and human degenerate Nucleus Pulposus (NP) tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Significantly increased cellular immunopositivty for aggrecan was observed within native NP cells surrounding the site where NPgel+MSCs were injected (P≤0.05). In NP explants a significant decrease in catabolic factors were observed where NPgel+MSCs was injected in comparison to controls.Background
Methods and Results
The IVD is a highly hydrated, hyperosmolar tissue that allows the correct biomechanical function of the spine. When degenerated, water and ions are lost from the disc, especially within the central nucleus pulposus (NP), producing a hypoosmotic environment in which the resident cells can no longer function correctly, exacerbating the degenerative cascade. One potential way that IVD cells may adapt to their environment is through the expression and regulation of aquaporin (AQP) channels that control the movement of water in and out of cells. During human IVD degeneration AQP1 and 5 expression is decreased, highlighting AQPs may be of importance for the correct function of NP cells. The regulation of AQPs in NP cells by healthy and degenerate conditions, and the potential underlying molecular mechanisms, were investigated in both human and rat IVD cells. The gene and protein expression of AQP1 and AQP5 was upregulated by hyperosmotic conditions (425mOsm/kg H2O) in rat and human NP cells. Lentiviral knockdown of tonicity enhancer binding protein (TonEBP), a transcription factor responsible for maintaining the function of NP cells, resulted in the loss of AQP1 and 5 gene expression under hyperosmotic conditions. The maintenance of the IVD environment and adaptation of cells is vital for the function of the IVD. The regulation of AQPs by physiological conditions and TonEBP suggests a role for these water channels related to the adaptation of disc cells to their environment, which is dysregulated during degeneration.
During development the central disc contains large, vacuolated notochordal (NC) cells which in humans are replaced by mature nucleus pulposus (NP) cells during aging, but are maintained in certain breeds of dogs. During degeneration the disc becomes less hydrated which affects its normal function. Aquaporins (AQP) are a family of 13 transmembrane channel proteins that allow passage of water and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the intervertebral disc (IVD). Here, expression of AQPs in human and canine IVDs to determine expression in NC v/s NP cells and whether expression changes during degeneration. Gene expression of all 13 AQPs, were investigated in 102 human NP samples using RT-qPCR. AQPs which were expressed at gene level were further investigated by Immunohistochemistry in human and canine IVD samples.Introduction
Methods
Intervertebral disc (IVD) degeneration is a major cause of Low back pain (LBP). We have reported an injectable hydrogel (NPgel), which following injection into bovine NP explants, integrates with NP tissue and promotes NP cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here we investigated the injection of NPgel+MSCs into bovine NP explants under degenerate culture conditions to mimic the hMSCs were incorporated within liquid NPgel and injected into bovine NP explants alongside controls. Explants were cultured for 6 weeks under hypoxia (5%) with ± calcium 5.0mM CaCl2 or IL-1β individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by caspase 3 immunohistochemistry. Histological and immunohistochemical analysis was performed to investigate altered matrix synthesis and matrix degrading enzyme expression.Background
Methods
Within the intervertebral disc (IVD), nucleus pulposus (NP) cells reside within a unique microenvironment. Factors such as hypoxia, osmolality, pH and the presence of cytokines all dictate the function of NP cells and as such the cells must adapt to their environment to survive. Previously we have identified the expression of aquaporins (AQP) within human IVD tissue. AQPs allow the movement of water across the cell membrane and are important in cellular homeostasis. Here we investigated how AQP gene expression was regulated by the microenvironment of the IVD. Human NP cells were cultured in alginate beads prior to cytokine, osmolality, pH and hypoxia treatments and subsequent RT-qPCR to assess regulation of AQP gene expression.Introduction
Methods
The intervertebral disc (IVD) is a highly hydrated tissue which is reduced during degeneration leading to loss of function. Aquaporins (AQP) are a family of 13 (AQP0-12) transmembrane channel proteins that selectively allow the passage of water and other small molecules in and out of cells and are responsible for maintaining water homeostasis. AQP1, 2, 3 and 5 have been identified in the IVD. Here gene and protein expression of all 13 AQPs was investigated in a large cohort of human IVDs to investigate expression during IVD degeneration. Gene expression of all 13 AQPs was investigated in non-degenerate and degenerate tissue from 102 human NP samples using RT-qPCR. AQPs which were expressed at gene level were further investigated in 30 IVD samples by Immunohistochemistry.Introduction
Methods
Cytokines produced within the degenerate disc induce expression of neurotrophic factors and pain related peptides which could be important in nerve ingrowth and pain sensitisation leading to low back pain. The intervertebral disc (IVD) is considered the largest aneural and avascular structure within the human body, yet during degeneration vascularisation of the IVD is seen to be accompanied by nociceptive nerves. Low back pain is a highly debilitating condition affecting around 80% of the population, 40% of which are attributed to IVD degeneration. Discogenic pain was largely thought to be a result of irritation and compression of the nerve root, yet recent data suggests that pain may be attributed to the sensitisation of sensory nerves by the synthesis of pain related peptides, calcitonin gene related peptide (CGRP) and substance P. It is known that cytokines and chemokines produced by nucleus pulposus cells elicit various effects including the production of matrix degrading enzymes, and decreased matrix molecules. Here, we investigate the hypothesis that cytokines regulate both neurotrophic factor and pain related peptide synthesis within nucleus pulposus and nerve cells which may elicit algesic effects. Real-Time PCR was performed to investigate gene expression of the neurotrophic factors NGF, BDNF, NT3 and their receptors Trk A, B and C along with Substance P and CGRP on directly extracted RNA from human NP cells and NP cells cultured in alginate for 2 weeks prior to treatment for 48hours with IL-1, IL-6 or TNFα at 0–100ng/mL. Similarly SH-SY5Y neuroblastoma cells were differentiated in retinoic acid for 7 days prior to stimulation with IL-1, IL-6 or TNFα at 0ng/mL and 10ng/mL for 48hours. Immunohistochemistry was used to localise neurotrophic factor receptors Trk A, B and C in both degenerate discs and neuronal cells. NGF expression was present in normal and degenerate disc samples, however only degenerate discs expressed the high affinity receptor TrkA. Similarly Trk B was present in 22% of normal samples increasing to 100% expression within degenerate disc samples. All cytokines increased expression of NGF in NP cells (P≤0.05). TNFα also increased BDNF significantly, whereas no significant affects were seen in NT3 expression in NP cells. Trk B expression was significantly increased by IL-1 and TNFα treatment of NP cells. Conversely Trk C was down regulated by IL-6. Substance P was significantly increased by IL-1 and TNFα treatments whilst IL-6 and TNFα increased CGRP expression in NP cells. In SH-SY5Y cells, IL-1 significantly increased BDNF whilst IL-6 and TNFα failed to induce significant differences in neurotrophic factors. All cytokines increased Trk expression in the nerve cell line; however this failed to reach significance. Immunohistochemistry confirmed the presence of Trk receptors within the neuronal cell line. Here we have demonstrated that a number of cytokines known to be up regulated during disc degeneration and disc prolapse, induce expression of various neurotrophic factors, their receptors and pain related peptides within human NP cells, as well as SH-SY5Y cells. This data suggests that the presence and production of cytokines within the degenerate disc may be responsible for nerve ingrowth and sensitisation of nerves which may result in discogenic pain.Summary
Anabolic and catabolic signalling processes within IVDs display overlapping pathways, however some pathways were identified as selective to catabolic signalling and inhibition of one of these pathways inhibited some of the catabolic factors induced by IL-1 although NFkB inhibition also affected anabolic expression. Degeneration of intervertebral discs (IVDs) is implicated in 40% of low back pain cases. In the normal disc the balance between anabolic and catabolic processes are carefully balanced. During degeneration this balance is lost in favour of catabolic processes which lead to degradation of the IVD, infiltration of blood vessels and nerves and release of cytokines which sensitise nerves to pain. Interleukin 1 (IL-1) is known to be important in the pathogenesis of IVD degeneration, here we investigated the intracellular signalling pathways activated by IL-1 and those activated by an anabolic factor (CDMP-1) to investigate differential pathways. Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 or CDMP-1 for 30 minutes. Site-specific phosphorylation of 46 signalling molecules were identified using R&D proteome array. The activation of ERK1/2, p38, c-jun, and IkB were confirmed using cell based ELISAs, in addition pNFκB localisation in stimulated cells was determined using immunohistochemisty. Pre-treatment with inhibitors to p38, and NFkB for 30 minutes, followed by stimulation with IL-1 (10ng/mL) or CDMP-1 (10ng/mL) for 24 hours was investigated to determine effects on anabolic and catabolic factors. In addition localisation of phosphorylated c-jun, p38 and NFkB were investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways Twenty intracellular signalling pathways were activated following CDMP-1 treatment and 8 signalling pathways activated by IL-1. Of note key classical IL-1 signalling pathways p38 MAPK, ERK 1/2 and JNK were activated by IL-1, however of these ERK 1/2 particularly was also activated by CDMP-1, whilst p38 and c-jun were only activated by IL-1. IL-1 induced activation of NFkB signalling to a greater extent than CDMP-1, these results were confirmed by the ‘in cell ELISAs’. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, NFkB and p38. Inhibition of p38 signalling inhibited IL-1 induced MMP 13 expression, but had little effect on the induction of IL-8. However inhibitors of NFkB signalling pathway failed to inhibit the induction of MMP 13 but abrogated the induced IL-6 and IL-8 expression. IL-1 induced a complete aberration of aggrecan expression by NP cells in alginate culture, this effect was partly inhibited by p38 MAPK inhibitor but was completely restored by inhibiting NFkB signalling. However the aggrecan expressed in CDMP-1 treated cells was decreased by inhibiting NFkB but not p38. Here, we have shown that anabolic and catabolic signalling processes within IVDs show a number of overlapping pathways, however a number of differential pathways were identified and inhibition of p38 MAPK and NFkB pathways inhibited a number of catabolic processes investigated which were induced by IL-1. Thus inhibition of signalling pathways could be a novel mechanism of inhibiting catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc.Summary
The primary aim of the study was to test the feasibility of conducting a full RCT with economic analysis and help to inform the provision of physiotherapy in a specific sub-group of patients with sciatica 60 patients waiting for primary, unilateral, single level, lumbar micro-discectomy surgery were recruited and randomised into two groups. The intervention group received a new spinal physiotherapy regimen. Primary outcome measure was the number of patients who did not require surgery at the time of consent clinic. Secondary measures were the Visual Analogue Scale (VAS) Oswestry Disability Index (ODI) and EQ5DL, taken at recruitment, 1 week before surgery and 2 weeks and 3 months after surgery.Purpose of study and background
Methods
To investigate the views and experiences of patients with sciatica who have undergone a bespoke physiotherapy programme whilst awaiting primary lumbar microdiscectomy. This is a qualitative study, nested within a preliminary RCT. All patients were listed for primary, single-level microdiscectomy surgery. In the experimental arm of the study 29 patients had up to 6 sessions of physiotherapy over an 8 week period while on the waiting list for lumbar microdiscectomy. After surgery, they were invited to participate in an in-depth semi-structured interview. At this time patients had either decided not to have the surgery, or had undergone surgery. Interviews were audio-recorded, transcribed, and thematically analysed. Two researchers were involved in the analysis of the data to ensure the interpretation of the findings was robust, credible and trustworhy.Objectives
Methods
Spinal infections constitute a spectrum of disease comprising pyogenic, tuberculous, nonpyogenic-nontuberculous and postoperative spinal infections. The aim of this study was to review the epidemiology, diagnostic yield of first and second biopsy procedures and microbiology trends from Sheffield Spinal Infection Database along with analysing prognostic predictors in spinal infections. Sheffield Spinal Infection Database collects data prospectively from regularly held Spinal infection MDTs. We accrued 125 spinal infections between September 2008 and October 2010. The medical records, blood results, radiology and bacteriology results of all patients identified were reviewed. In patients with negative first biopsy, second biopsy is contemplated and parenteral broad spectrum antibiotic treatment initiated.Introduction
Materials & methods
Spinal infections constitute a spectrum of disease comprising pyogenic, tuberculous, nonpyogenic-nontuberculous and postoperative spinal infections. The aim of this study was to review the epidemiology, diagnostic yield of first and second biopsy procedures and microbiology trends from Sheffield Spinal Infection Database along with analysing prognostic predictors in spinal infections. Sheffield Spinal Infection Database collects data prospectively from regularly held Spinal infection MDTs. We accrued 125 spinal infections between September 2008 and October 2010. The medical records, blood results, radiology and bacteriology results of all patients identified were reviewed. In patients with negative first biopsy, second biopsy is contemplated and parenteral broad spectrum antibiotic treatment initiated.Introduction
Materials and Methods
To question the reliability of Thoracic Spine pain as a red flag and symptoms of a possible cause of Serious Spinal Pathology (SSP). The clinical notes and Magnetic Resonance Imaging (MRI) results of patients presenting to the Sheffield Spinal Service with Thoracic spine symptoms but no signs were retrospectively reviewed over the period of 2 year (September 2008-August 2010). The clinical reason for request of Thoracic MRIs were noted and the patient notes were reviewed to determine their presentation, length of time of symptoms, age and also it was noted whether any other recognized red flag symptoms were present. Exclusion criteria consisted of patients referred with known SSP or myelopathic symptoms.Purpose
Methods
To evaluate the competencies of spinal extended scope physiotherapists (ESP) following the introduction of requesting rights for magnetic resonance imaging (MRI) one year later. From September 2009 to August 2010 each MRI scan requested by the 2 spinal ESPs within the orthopaedic clinic was recorded along with their clinical diagnosis to ascertain why the scan was requested. This was indicated on a four point scale of likelihood of pathology which had been introduced to give evidence for MRI requesting rights. This was then audited to determine the total number of scans requested along with the accuracy or justification of the request.Purpose
Methods
Medical Exposure Directive of the European Commission, 97/43/Euratom recommended setting-up local national diagnostic reference levels (DRLs) for the most common radiological examinations in order to comply with the law and to maintain safe clinical practice. There are no guidelines for spinal diagnostic and therapeutic procedures. The aims of this study were to evaluate local radiation doses & screening times for diagnostic spinal blocks, to look at PACS image intensifier films for diagnostic representation and to assess the accuracy of data in IR(ME) document. Between 1/01/2009 and 15/07/2010, all spinal blocks done under care of three spinal surgeons (LB/NC/AAC) were reviewed. Images revisited on PACS for confirmation. We reviewed 229 patients (included single & two levels nerve root blocks, facet joint and lysis blocks). Data were collected with regard to radiation dose, screening times, third-quartile values used to establish DRLs, IR(ME) documentation and PACS fluoroscopic image documentation.Introduction
Materials and Methods
To investigate, through a randomised, single blind, Quasi-experimental trial, whether immediate physiotherapy after lumbar micro-discectomy enables patients to become independently mobile more rapidly with no increase in risk of complications. Although studies have demonstrated the efficacy of rehabilitation after lumbar discectomy, nos have looked at physiotherapy commencing immediately post-operatively.Objective
Background data
Results: The Pearson product moment correlation coefficients were calculated for the group using SPSS v.13. The results show low negative correlations for the whole group with low to moderate negative correlations for the male group. There were no statistically significant correlations for the physical performance measures and ODI in the female sub-group.
Group 4 = Very high suspicion of pathology (n=41) Group 3 = Moderate suspicion of pathology (n=21) Group 2 = Some suspicion of pathology (n=10) Group 1 = Pathology unlikely but scan indicated eg thoracic pain (n=4).
Group 4: 88% Group 3: 67% Group 2: 40% Group 1: 0%
Outcome measures used were post operative mortality, Post operative improvement in Frankel score, level of pain perception, level of mobility and ability to perform activities of daily living.
Traumatic atlanto-occipital dislocation in adults is usually fatal and survival without neurological deficit is rare. The surgical management of those who do survive is difficult and controversial. Most authorities recommend posterior occipitoaxial fusion, but this compromises cervical rotation. We describe a case in which a patient with a traumatic atlanto-occipital disruption but no neurological deficit was treated by atlanto-occipital fusion using a new technique consisting of cancellous bone autografting supported by an occipital plate linked by rods to lateral mass screws in the atlas. The technique is described in detail. At one year the neck was stable, radiological fusion had been achieved, and atlantoaxial rotation preserved. The rationale behind this approach is discussed and the relevant literature reviewed. We recommend the technique for injuries of this type.
Following fixation of proximal femoral fractures in the elderly the operating surgeon may request that the patient be mobilised partially weight bearing on the injured limb. This instruction is most likely if the bone quality is very poor or the fracture pattern unstable, despite evidence that full weight bearing does not affect outcome. 98 elderly patients with proximal femoral fractures treated by either hip screw device, cannulated screws or hemiarthroplasty, who were previously independently mobile, have been followed prospectively to determine their ability to comply with partial weight bearing instructions. A specially designed capacitance foot pressure device was used to determine percentage body weight transferred through the injured limb on mobilising under physiotherapy instruction over 5 days, and factors thought to be predictive of success in partially weight bearing were measured using simple ward tests. 14 patients failed to mobilise independently prior to discharge from hospital and were excluded from further analysis. Of the remaining 84 patients only 24 (28%) successfully managed to partially weight bear 30-50% of their body weight on the injured limb. Only six of those who were unable to partially weight bear on starting to walk after surgery had learnt to do so by the fifth day. Factors indicating success or failure were mental test score, grip strength and straight leg raise on the unaffected side. This study has shown that the majority of elderly hip fracture patients are unable to partially weight bear but, if required, success can be predicted by a few simple ward tests. It is hoped that this information will lead to the more appropriate use of inpatient physiotherapy resources.
