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. 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 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. Methods and Results. 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. Conclusions. In agreement with our previous findings NPgel was sufficient alone to induce NP cell differentiation of MSCs following injection into NP tissue explants. Here, we have shown that viability is maintained even in degenerate conditions. Injection of NPgel with MSCs increased aggrecan expression and reduced MMP3 and IL-1R1 expression by native NP cells. The NPgel with incorporated MSCs has the potential to regenerate the NP and provide mechanical support, whilst reducing the catabolic phenotype of degenerate NP cells, as a treatment strategy for IVD degeneration. No conflicts of interest. Sources of funding: Funded by ARUK and MRC

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. Degeneration of the intervertebral disc (IVD) is a major cause of Low back pain. We have recently reported a novel, injectable liquid L-pNIPAM-co-DMAc hydrogel (NPgel), which promote differentiation of MSCs to nucleus pulposus (NP) cells without the need for additional growth factors. Here, we investigated the behaviour of hMSCs incorporated within the hydrogel injected into NP tissue. Methods. hMSCs were injected either alone or within NPgel, into bovine NP tissue explants and maintained at 5% O. 2. for up to 6wks. Media alone and acellular NPgel were also injected into NP explants to serve as controls. Cell viability was assessed by Caspase 3 immunohistochemistry and the phenotype of injected hMSC was assessed by histology and immunohistochemistry. Mechanical properties were also assessed via dynamic mechanical analysis (DMA). Results. No significant difference in the elastic modulus was observed between NPgel injected NP tissue and media injected controls. CFSe positive hMSCs were identified in all injected tissue samples and cell viability was maintained. Where hMSCs were delivered via NPgel, the hydrogel integrated with native NP tissue and cells producing NP matrix components: aggrecan; collagen type II and chondroitin sulphate. Conclusion. hMSC incorporated within L-pNIPAM-co-DMAc hydrogel and injected into NP explants, integrate with native NP tissue and promote differentiation towards the NP phenotype; thus potentially could be used to regenerate the NP as a treatment strategy for LBP. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University

Background. For bone grafting procedures, the use of autologous bone is considered the gold standard, as it is has a better healing capacity compared to other alternatives as allograft and synthetic bone substitutes. However, as there are several drawbacks related to autografting (infection, nerve- or vascular damage, chronic pain problems, abdominal herniation), there has been a targeted effort to improve the healing capacities of synthetic bone substitutes. Aim. To evaluate the performance of a carbonated osteoionductive hydroxyapatite (CHA) scaffold of clinical relevant size (Ø=15mm, H=50mm) in a sheep model of multi level posterolateral intertransverse lumbar spine fusion after activation with autologous bone marrow nuclear cells (BMNC) in a flow perfusion bioreactor. Method. Two groups were included in the study, autograft (n=6) and CHA scaffold (n=6) CHA. A paired design was used between and within the groups as lumbar posterolateral arthrodesis was performed in sheep on two levels (L2-L3, L5-L6) +/− BMNC, respectively. Before implantation, the CHA scaffold was cultured in a flow perfusion bioreactor system with BMNC for 21 days, and the autograft group was supplemented with isolated BMNC during the procedure. Micro tomography was used to evaluate fusion rate and the microarchitectural properties of the explants after an observation period of four months. Results. In the autograft group, the healing rate was 83.3% irrespective of the presence BMNC, and in the CHA group, 66.7% fused in the presence of BMNC, and 33.3% without. The microarchitectural data suggested the autograft group to be superior to the CHA scaffold regarding mechanical properties, however porosity decreased significantly (p=0.001) in the CHA scaffold group suggesting deposition of mineralized bone matrix. Conclusion. Based on the fusion rate and micro architectural properties, we consider the CHA scaffold fully capable of new bone formation, and that the presence of BMNC has a positive effect on the fusion rate in a challenging model of bone healing

Aims

During revision procedures for aseptic reasons, there remains a suspicion that failure may have been the result of an undetected subclinical infection. However, there is little evidence available in the literature about unexpected positive results in presumed aseptic revision spine surgery. The aims of our study were to estimate the prevalence of unexpected positive culture using sonication and to evaluate clinical characteristics of these patients.