Background. 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 in vivo. Here, we investigated whether this system could be utilised to induce bone formation within intervertebral disc tissue as a potential injectable spinal fusion approach. Methodology. 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. Results. Following injection of B-gel into tissue explants following culture for 4 weeks, cells were visualized within the regions of the B-gel. Demonstrating that native cells were able to migrate into regions of B-gel. Increased collagen deposition was seen in tissue explants injected with Bgel, with increased collagen type I and X but decreased collagen type II staining in explants injected with Bgel. Tissue explants, in the absence of Bgel, showed limited calcium deposition, which was increased in B-gel injected explants. Furthermore, disc cells increased expression of bone markers (alkaline phosphatase & osteocalcin), but decreased NP matrix (Aggrecan and Collagen type II) following Bgel injection. Conclusion. This system could have potential to support spinal fusion via direct injection into the disc. Conflict of interest: C Le Maitre & C Sammon are inventors on the hydrogel discussed. Funding: This work was funded by GrowMed Tech Proof of Concept funding

Introduction. 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. Methods. 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. Results. Physiological conditions observed within the native IVD regulated AQP gene expression in human NP cells. Hyperosmotic treatment up-regulated the expression of AQP1 and 5 during hypoxic conditions, whereas AQP4 expression was down-regulated. During hypoxia and physiological pH treatments AQP5 expression was increased. Pro-inflammatory cytokines, increased during IVD degeneration, also altered AQP gene expression. Interleukin-1β (IL-1β) decreased expression of AQP1 and 3 yet up-regulated AQP9, interleukin-6 (IL-6) increased expression of AQP1, 3, and 9 and tumour necrosis factor α (TNFα) upregulated the gene expression of both AQP2 and 9. Conclusion. The microenvironment in which NP cells reside in vivo directly contributes to their correct function and survival. AQP gene expression was differentially regulated under healthy compared to degenerate conditions; this potentially highlights that during IVD degeneration NP cells differentially express AQPs. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Introduction. We have developed a new synthetic hydrogel that can be injected directly into the intervertebral disc (IVD) without major surgery. Designed to improve fixation of joint prosthesis, support bone healing or improve spinal fusion, the liquid may support the differentiation of native IVD cells towards osteoblast-like cells cultured within the hydrogel. Here we investigate the potential of this gel system (Bgel) to induce bone formation within intervertebral disc tissue. Methods. IVD tissue obtained from patients undergoing discectomy, or cadaveric samples, were cultured within a novel explant device. The hydrogel was injected, with and without mesenchymal stem cells (MSCs), and cultured under hypoxia, to mimic the degenerate IVD environment, for 4 weeks. Explants were embedded to wax and native cellular migration into the hydrogel was investigated, together with cellular phenotype and matrix deposition. Results. Increased collagen deposition was seen in tissue explants injected with Bgel, with evidence of elevated native cell migration towards the hydrogel. Increased collagen staining was seen in explants injected with Bgel together with MSCs. Alizarin red staining was utilised to investigate calcium deposition. Tissue explants, in the absence of Bgel, showed limited calcium deposition. This was increased in hydrogel-treated samples, with large clumping regions in the tissue that was injected with Bgel and MSCs. Conclusion. The injection of our synthetic hydrogel into disc tissue explants increased the amount of collagen and calcium deposition. This was further enhanced by the incorporation of MSCs, suggesting the promotion of bone formation. Current work is investigating phenotypic markers for bone formation within these tissues. CS and CLM have a patent on the hydrogel system described in this abstract. Funded by EPSRC and Grow MedTech

Background. 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 in vivo environment of the degenerate IVD. Methods. 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 CaCl. 2. 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. Results. CFSe positive hMSCs were identified in all NPgel injected explants and cell viability was maintained. The NPgel integrated with NP tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Increased cellular immunopositivty for aggrecan and collagen type II as well as decreased cellular immunopositivity for degrading enzyme expression was observed within NP tissue removed from the injection site. Conclusion. MSCs incorporated within NPgel could be used to regenerate the NP and restore the healthy NP phenotype of degenerate NP cells as a treatment strategy for LBP. We are currently investigating the survival and differentiation capacity of hMSCs delivered via the NPgel into degenerate human NP explants and thus ascertain the future clinical success of this therapy. Conflicts of Interest: None. Funding: BMRC, MERI Sheffield Hallam University

Background. Intervertebral disc degeneration is implicated as a major cause of chronic lower back pain. Current therapies for lower back pain are aimed purely at relieving the symptoms rather than targeting the underlying aberrant cell biology. As such focus has shifted to development of cell based alternatives. Notochordal cells are progenitors to the adult nucleus pulposus that display therapeutic potential. However, notochordal cell phenotype and suitable culture conditions for research or therapeutic application are poorly described. This study aims to develop a suitable culture system to allow comprehensive study of the notochordal phenotype. Methods & Results. Porcine notochordal cells were isolated from 6 week post natal discs using dissection and enzymatic digestion and cultured in vitro under different conditions: (1)DMEM vs αMEM (2)laminin-521, fibronectin, gelatin and untreated tissue culture plastic (3)2% 02 vs normoxia (4)αMEM (300 mOsm/L) vs αMEM (400 mOsm/L). Notochordal cells were cultured in alginate beads as a control. Adherence, cell viability, morphology and expression of known notochordal markers (CD24, KRT8, KRT18, KRT19 and T) were assessed throughout the culture period. Use of αMEM media and laminin-521 coated surfaces displayed the greatest cell adherence, viability and retention of notochordal cell morphology and gene expression, which was further enhanced through culture in hypoxia and hyperosmolar media mimicking the intervertebral disc niche. Conclusions. Assessment of the therapeutic potential of notochordal cells is potentially valuable to development of a cell based therapy for chronic lower back pain. Our model has provided a system in which notochordal cells can be studied extensively. Conflicts of Interest: None. Funding obtained from the Henry Smith Charity, London

Introduction. Current strategies to treat back pain address the symptoms but not the underlying cause. Here we are investigating a novel hydrogel material (NPgel) which can promote MSC differentiation to Nucleus pulposus cells. Current in vitro studies have only explored conditions that mimic the native disc microenvironment. Here, we aim to determine the stem cells regenerative capacity under conditions that mimic the degenerate environment seen during disc degeneration. Methods. hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl. 2. ), IL-1β and TNFα either individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by Alamar blue assay. Histological and immunohistochemical analysis investigated altered matrix and matrix degrading enzyme expression. Results. Viability of hMSCs was maintained under all culture conditions. Matrix deposition of glycosaminoglycans were observed under all conditions, MMP13 expression was upregulated by calcium but not by pro-inflammatory cytokines IL-1β and TNFα. Conclusions. We are developing an in vitro modelling system which can be used to test novel therapies within a degenerate microenvironment. Interestingly, our preliminary findings suggest calcium is a major contributor to regulating MMP13 in this model system. Investigating the degenerate niche will identify targets for inhibition to provide the correct niche to promote regeneration of the IVD. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University, for joint funding the Daphne Jackson Trust fellowship

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Aims

In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD.

This review provides a concise outline of the advances made in the care of patients and to the quality of life after a traumatic spinal cord injury (SCI) over the last century. Despite these improvements reversal of the neurological injury is not yet possible. Instead, current treatment is limited to providing symptomatic relief, avoiding secondary insults and preventing additional sequelae. However, with an ever-advancing technology and deeper understanding of the damaged spinal cord, this appears increasingly conceivable. A brief synopsis of the most prominent challenges facing both clinicians and research scientists in developing functional treatments for a progressively complex injury are presented. Moreover, the multiple mechanisms by which damage propagates many months after the original injury requires a multifaceted approach to ameliorate the human spinal cord. We discuss potential methods to protect the spinal cord from damage, and to manipulate the inherent inhibition of the spinal cord to regeneration and repair. Although acute and chronic SCI share common final pathways resulting in cell death and neurological deficits, the underlying putative mechanisms of chronic SCI and the treatments are not covered in this review.

We assessed the frequency and causes of neurological deterioration in 59 patients with spinal cord injury on whom reports were prepared for clinical negligence litigation. In those who deteriorated neurologically we assessed the causes of the change in neurology and whether that neurological deterioration was potentially preventable. In all 27 patients (46%) changed neurologically, 20 patients (74% of those who deteriorated) had no primary neurological deficit. Of those who deteriorated, 13 (48%) became Frankel A. Neurological deterioration occurred in 23 of 38 patients (61%) with unstable fractures and/or dislocations; all 23 patients probably deteriorated either because of failures to immobilise the spine or because of inappropriate removal of spinal immobilisation. Of the 27 patients who altered neurologically, neurological deterioration was, probably, avoidable in 25 (excess movement in 23 patients with unstable injuries, failure to evacuate an epidural haematoma in one patient and over-distraction following manipulation of the cervical spine in one patient). If existing guidelines and standards for the management of actual or potential spinal cord injury had been followed, neurological deterioration would have been prevented in 25 of the 27 patients (93%) who experienced a deterioration in their neurological status.

Cite this article: Bone Joint J 2015;97-B:527–31.

Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc.

Cite this article: Bone Joint Res 2013;2:169–78.

Despite the increasing prevalence of sleep apnoea, little information is available regarding its impact on the peri-operative outcome of patients undergoing posterior lumbar fusion. Using a national database, patients who underwent lumbar fusion between 2006 and 2010 were identified, sub-grouped by diagnosis of sleep apnoea and compared. The impact of sleep apnoea on various outcome measures was assessed by regression analysis. The records of 84 655 patients undergoing posterior lumbar fusion were identified and 7.28% (n = 6163) also had a diagnostic code for sleep apnoea. Compared with patients without sleep apnoea, these patients were older, more frequently female, had a higher comorbidity burden and higher rates of peri-operative complications, post-operative mechanical ventilation, blood product transfusion and intensive care. Patients with sleep apnoea also had longer and more costly periods of hospitalisation.

In the regression analysis, sleep apnoea emerged as an independent risk factor for the development of peri-operative complications (odds ratio (OR) 1.50, confidence interval (CI) 1.38;1.62), blood product transfusions (OR 1.12, CI 1.03;1.23), mechanical ventilation (OR 6.97, CI 5.90;8.23), critical care services (OR 1.86, CI 1.71;2.03), prolonged hospitalisation and increased cost (OR 1.28, CI 1.19;1.37; OR 1.10, CI 1.03;1.18).

Patients with sleep apnoea who undergo posterior lumbar fusion pose significant challenges to clinicians.

Cite this article: Bone Joint J 2014;96-B:242–8.