Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide1. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain2. In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach3. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction. Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments). The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group. Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance4. Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition.
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Low back pain affects 80% of the population with half of cases attributed to intervertebral disc (IVD) degeneration. However, the majority of treatments focus on pain management, with none targeting the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of the pro-inflammatory cytokine IL-1, which orchestrates the catabolic degeneration of the IVD. Our objective here is to determine the ability of PCRX-201 to infect human nucleus pulposus (NP) cells and tissue to increase the production of IL-1Ra and assess downstream effects on catabolic protein production. Degenerate human NP cells and tissue explants were infected with PCRX-201 at 0 or 3000 multiplicities of infection (MOI) and subsequently cultured for 5 days in monolayer (n=7), 21 days in alginate beads (n=6) and 14 days in tissue explants (n=5). Cell culture supernatant was collected throughout culture duration and downstream targets associated with pain and degeneration were assessed using ELISA. IL-1Ra production was increased in NP cells and tissue infected with PCRX-201. The production of downstream catabolic proteins such as IL-1β, IL-6, MMP3, ADAMTS4 and VEGF was decreased in both 3D-cultured NP cells and tissue explants. Here, we have demonstrated that a novel gene therapy, PCRX-201, is able to infect and increase the production of IL-1Ra in degenerate NP cells and tissue in vitro. The increase of IL-1Ra also resulted in a decrease in the production of a number of pro-inflammatory and catabolic proteins, suggesting PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. At present, no treatments for IVD degeneration target the underlying pathology. The ability of FX201 to elicit anti-catabolic responses is promising and warrants further investigation in vitro and in vivo, to determine the efficacy of this exciting, novel gene therapy.
Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints. Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation. A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of
The effect of high-fat diet and testosterone replacement therapy upon bone remodelling was investigated in orchiectomised male APOE-/- mice. Mice were split in to three groups: sham surgery + placebo treatment (control, n=9), orchiectomy plus placebo treatment (n=8) and orchiectomy plus testosterone treatment (n=10). Treatments were administered via intramuscular injection once a fortnight for 17 weeks before sacrifice at 25 weeks of age. Tibiae were scanned ex-vivo using µCT followed by post-analysis histology and immunohistochemistry. Previously presented µCT data demonstrated orchiectomised, placebo treated mice exhibited significantly reduced trabecular bone volume, number, thickness and BMD compared to control mice despite no significant differences in body weight. Trabecular parameters were rescued back to control levels in orchiectomised mice treated with testosterone. No significant differences were observed in the cortical bone. Assessment of TRAP stained FFPE sections revealed no significant differences in osteoclast or osteoblast number along the endocortical surface. IHC assessment of osteoprotegerin (OPG) expression in osteoblasts is to be quantified alongside markers of osteoclastogenesis including RANK and RANKL. Results support morphological analysis of cortical bone where no change in cortical bone volume or density between groups is in line with no significant change in osteoblast or osteoclast number and percentage across all three groups. Future work will include further IHC assessment of bone remodelling and adiposity, as well as utilisation of mechanical testing to establish the effects of observed morphological differences in bone upon mechanical properties. Additionally, the effects of hormone treatments upon murine-derived bone cells will be investigated to provide mechanistic insights.
An increasing elderly population means joint replacement surgery numbers are projected to increase, with associated complications such as periprosthetic joint infections (PJI) also rising. PJI are particularly challenging due to antimicrobial resistant biofilm development on implant surfaces and surrounding tissues, with treatment typically involving invasive surgeries and systemic antibiotic delivery. Consequently, functionalisation of implant surfaces to prevent biofilm formation is a major research focus. This study characterises clinically relevant antimicrobials including gentamicin, clindamycin, daptomycin, vancomycin and caspofungin within a silica-based, biodegradable sol-gel coating for prosthetic devices. Antimicrobial activity of the coatings against clinically relevant microorganisms was assessed via disc diffusion assays, broth microdilution culture methods and the MBEC assay used to determine anti-biofilm activity. Human and bovine cells were cultured in presence of antimicrobial sol-gel to determine cytotoxicity using Alamar blue and antibiotic release was measured by LC-MS. Biodegradability in physiological conditions was assayed by FT-IR, ICP-MS and measuring mass change. Effect of degradation products on osteogenesis were studied by culturing mesenchymal stem cells in the presence of media in which sol-gel samples had been immersed. Antimicrobial-loaded coatings showed strong activity against a wide range of clinically relevant bacterial and fungal pathogens with no loss of activity from antibiotic alone. The sol-gel coating demonstrated controlled release of antimicrobials and initial sol-gel coatings showed no loss of viability on MSCs with gentamicin containing coatings. Current work is underway investigating cytotoxicity of sol-gel compositions against MG-63 cells and primary osteoblasts. This research forms part of an extended study into a promising antimicrobial delivery strategy to prevent PJI. The implant coating has potential to advance PJI infection prevention, reducing future burden upon healthcare costs and patient wellbeing.
This study aimed to characterise the microarchitecture of bone in different species of animal leading to the development of a physiologically relevant 3D printed cellular model of trabecular (Tb) and cortical bone (CB). Using high resolution micro-computed tomography (μ-CT) bone samples from multiple species were scanned and analysed before creating Porcine and murine bone samples were scanned using μ-CT, with a resolution of 4.60 μM for murine and 11 μM for porcine and reconstructed to determine the architectural properties of both Tb and CB independently. A region of interest, 1 mm in height, will be used to generate an A 1 mm section of each bone was analysed, to determine the differences in the microarchitecture with the intent of setting a benchmark for the developmental 3D μ-CT scanning and analysis permits tessellation of the 3D output which will lead to the generation of an
A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats. This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration.
Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source.
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition. Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel with maintenance of morphology and low caspase 3 staining. NPgel and Albugel maintained NC cell markers (brachyury and cytokeratin 8/18/19) and extracellular matrix (collagen type II and aggrecan). Whilst Brachyury and Cytokeratin were decreased in dNCM/NPgel biomaterials, Aggrecan and Collagen type II was seen in acellular and NC containing dNCM/NPgel materials. NC containing constructs excreted more GAGs over the four weeks than the acellular controls. NC cells maintain their phenotype and characteristic features in vitro when encapsulated into biomaterials. NC cells and biomaterial construct could potentially become a therapy to treat and regenerate the IVD.
An improved understanding of intervertebral disc (IVD) structure and function is required for treatment development. Loading induces micro-fractures at the interface between the nucleus pulposus (NP) and the annulus fibrosus (AF), which is hypothesized to induce a cascade of cellular changes leading to degeneration. However, there is limited understanding of the structural relationship between the NP and AF at this interface and particularly response to load. Here, X-ray scattering is utilised to provide hierarchical morphometric information of collagen structure across the IVD, especially the interface region under load. IVDs were imaged using the I22 SAXS/WAXS beamline at Diamond Light Source. Peaks associated with the D-banded structure of collagen fibrils were fitted to quantify their azimuthal distribution, as well the magnitude and direction of internal strains under static and applied strain (0–20%).Background
Methodology
Low back pain resulting from Intervertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect, however, their behaviour in the harsh degenerate environment is unknown. Thus, we aimed to investigate and compare their physiological behaviour in Porcine NC cells were encapsulated in 3D alginate beads to maintain their phenotype then cultured in media to mimic the healthy and degenerate disc environment, together with control NC media for 1 week. Following which viability using PI and Calcein AM, RNA extraction and RT-PCR for NC cell markers, anabolic and catabolic genes analysed. Proteomic analysis was also performed using Digiwest technology.Backgrounds and aim
Methodology
Involving research users in setting priorities for research is essential to ensure research outcomes are patient-centred and to maximise research value and impact. The Musculoskeletal (MSK) Disorders Research Advisory Group Versus Arthritis led a research priority setting exercise across MSK disorders. The Child Health and Nutrition Research Initiative (CHRNI) method of setting research priorities with a range of stakeholders were utilised. The MSKD RAG identified, through consensus, four research Domains: Mechanisms of Disease; Diagnosis and Impact; Living Well with MSK disorders and Successful Translation. Following ethical approval, the research priority exercise involved four stages and two surveys, to: 1) gather research uncertainties; 2) consolidate these; 3) score uncertainties using agreed criteria of importance and impact on a score of 1–10; and 4) analyse scoring, for prioritisation.Background
Methods
This study aims to investigate whether bacteria are present in intervertebral discs (IVDs) and their influence. Causality between chronic infection of the IVD and its degenerative process gained great interest recently. Granville Smith Human IVD tissue was fixed in paraffin and Immunohistochemical stained for Gram-positive bacteria. NP cells in monolayer have been stimulated with LPS (0.1–50 µg/ml) and Peptidoglycan (0.1–50 µg/ml) for 24, 48 and 72 hrs to investigate their influence. The concentration of proinflammatory and catabolic cytokines in the media is being measured using ELISA. RNA extracted and RT-qPCR utilised for factors associated with disc degeneration matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors.Objectives
Methods
Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated. Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition.Objectives
Methodology
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
We have previously reported an injectable hydrogel (NPgel), which could deliver patients own stem cells, via small bore needles, decreasing damage to the annulus fibrosus. NPgel drives differentiation to NP cells and can inhibit the degenerate niche. However, clinical success of NPgel is dependent on the capacity to inject NPgel into naturally degenerate human discs, restore mechanical function to the IVD, prevent extrusion during loading and induce regeneration. This study assessed injectability of NPgel into human IVD, influence on mechanical properties, regeneration ability in an Cadaveric human discs were used to calculate disc height and to determine Youngs Modulus during simulated walking pre and post injection of NPgel, extrusion testing performed. Whole human IVDs were injected with NPgel +/− human BMPCs and maintained in culture under physiological loading regime for 4 weeks. Pre and post culture MRI imaging and in line biomechanical characteristics determined. Histology and immunochemistry performed for anabolic and catabolic factors.Background
Methodology
The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses. Coatings containing gentamicin at a concentration of 1.25% weight/volume (wt/vol), similar to that found in commercially available antibiotic-loaded bone cement, were prepared and tested in the laboratory for: kinetics of antibiotic release; activity against planktonic and biofilm bacterial cultures; biocompatibility with cultured mammalian cells; and physical bonding to the material (n = 3 in all tests). The sol-gel coatings and controls were then tested in vivo in a small animal healing model (four materials tested; n = 6 per material), and applied to the surface of commercially pure HA-coated titanium rods.Aims
Methods
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
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. 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.Introduction
Methods
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
