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.
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
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
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
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
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
Mechanical overloading initiates intervertebral disc degeneration, presumably because cells break down the extracellular matrix (ECM). We used Fourier Transform Infrared Spectroscopy (FTIR) imaging to identify, visualize and quantify the ECM and aimed to identify spectroscopic markers for early disc degeneration. In seven goats, one disc was injected with chondroitinase ABC (mild degeneration) and after three months compared to control.
Purpose of study and background
Methods and Results
Musculoskeletal diseases are the biggest cause of morbidity worldwide, with low back pain (LBP) being the leading cause. Forty percent of LBP cases are caused by disease of shock absorbers in the spine known as intervertebral discs (IVDs). The IVDs enable the spine to twist and bend, whilst absorbing load during normal daily activities. The durability of this tissue is sustained by the cells of the spine and so during disease or mechanical damage these cells can behave abnormally further damaging the disc and stimulating local nerves causing extreme pain. Degradation of the intervertebral disc (IVD) currently has no preventative treatment; an injectable hydrogel biomaterial could reinforce disc mechanical properties and promote tissue regeneration. We present an injectable range of hydrogel biomaterials made from water, clay and polymer that set at 37°C. The materials were made at 80°C polymerised in water and stored at 70°C to remain liquid. The physical properties of the materials were assessed using various methods, including mechanical assessment using temperature-controlled rheometry to monitor the liquid-hydrogel transition.Introduction
Methods and Results
Low back pain affects 80% of the population at some point in their lives with 40% of cases attributed to intervertebral disc (IVD) degeneration. A number of potential regenerative approaches are under investigation worldwide, however their translation to clinic is currently hampered by an appropriate model for testing prior to clinical trials. Therefore, a more representative large animal model for IVD degeneration is needed to mimic human degeneration. Here we investigate a caprine IVD degeneration model in a loaded disc culture system which can mimic the native loading environment of the disc. Goat discs were excised and cultured in a bioreactor under diurnal, simulated-physiological loading (SPL) conditions, following 3 days pre load, IVDs were degenerated enzymatically for 2hrs and subsequently loaded for 10 days under physiological loading. A PBS injected group was used as controls. Disc deformation was continuously monitored and changes in disc height recovery quantified using stretched-exponential fitting. Histological staining was performed on caprine discs to assess extracellular matrix production and immunohistochemistry performed to determine expression of catabolic protein expression. The injection of collagenase and cABC induced mechanical behavior akin to that seen in human degeneration. A decrease in collagens and glycosaminoglycans (GAGs) was seen in enzyme injected discs, which was accompanied by increased cellular expression for degradative enzymes and catabolic cytokines.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
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
Injectable hydrogels via minimally invasive surgery reduce the risk of infection, scar formation and the cost of treatment. Degradation of the intervertebral disc (IVD) currently has no preventative treatment. An injectable hydrogel material could restore disc height, reinforce local mechanical properties, and promote tissue regeneration. We present a hydrogel material Laponite® associated poly(N-isopropylacrylamide)-co-poly(dimethylacrylamide) (NPGel). Understanding how the components of this hydrogel system influence material properties, is crucial for tailoring treatment strategies for the IVD and other tissues. The effect of hydrogel wt./wt., clay and co-monomer percentages were assessed using a box-Behnken design. Rheometry, SEM, FTIR and swelling was used to measure changes in material properties in simulated physiological conditions. Rheometry revealed gelation temperature of hydrogel materials could be modified with dimethyl-acrylamide co-monomer; however, final maximum mechanical properties remained unaffected. Increasing the weight % and clay % increased resultant mechanical properties from ∼500–2500 G' (Pa), increased viscosity, but retained the ability to flow through a 26G needle at 39°C.Introduction
Methods & Results
Biomechanical overloading initiates intervertebral disc degeneration. We hypothesized that this is due to mechanosensitivity of the cells, which break down the extracellular matrix. Previously, we found that overloading in a loaded disc culture system causes upregulation of remodeling- and inflammatory gene expressions. Fourier Transform Infrared Spectroscopy is a novel technique to identify, visualize and quantify ECM. In this research, we first identified novel spectroscopic markers for disc degeneration, and then applied these markers to investigate the first steps into disc degeneration by overloading. In dataset 1, 18 discs of 9 goats were injected with chondroitinase ABC (degenerated) or not (control), and obducted 3 months after injection. This was used to find new spectroscopic markers for degeneration. In dataset 2, 42 goat discs were loaded with a physiological loading regime (50–150N) or overloading (50–400N) in a loaded disc culture system. In 18 of these discs, the cell activity was diminished in advance by freeze-thaw cycles and culturing on saline alone (non-vital group)). 24 additional discs were cultured in culture medium immediately post-mortem (vital group). Thereby, we are able to control whether the effect of the overloading is due to cell activity. The discs were fixed in formaldehyde, and 4 μm mid-sagittal were mounted to steel reflectance slides. Infrared spectroscopic mosaic images (23 × 57 images) were collected in transflectance mode at a spectral region of 1025–1150 cm−1. Data was pre-processed by second derivative transformation and MCR-MALS with two factors. The two factors were transferable between datasets, confirming the reliability. The first factor represents proteoglycans, as confirmed by Saffrin-O staining. In dataset 1, the degenerated group had less proteoglycan factor overall, especially in the nucleus (p<0.05). The second factor was found to have a lower entropy (p<0.01), showing a disorganization in the matrix. In dataset 2, no significant reduction in proteoglycan was found due to overloading in any group. However, the entropy was lower in the overloaded vital group (p<0.05), but not in the overloaded non-vital group (p>0.5). Therefore, we conclude that infrared spectroscopy is a promising tool to investigate early disc degeneration. Overloading can cause changes in the extracellular matrix, but only due to cell activity. Entropy is an early marker for early disc degeneration, implying that cutting of the extracellular matrix by cell activity is the first step into intervertebral disc degeneration.
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
We have reported an injectable L-pNIPAM-co-DMAc hydrogel with hydroxyaptite nanoparticles (HAPna) which promotes mesenchymal stem cell (MSC) differentiation to bone cells without the need for growth factors. This hydrogel could potentially be used as an osteogenic and osteoconductive bone filler of spinal cages to improve vertebral body fusion. Here we investigated the biocompatibility and efficacy of the hydrogel Rat sub-cut analysis was performed to investigate safety Background
Methods
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 hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5%) with ± calcium (2.5mM and 5.0mM CaCl2), 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.Introduction
Methods
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. hMSCs were injected either alone or within NPgel, into bovine NP tissue explants and maintained at 5% O2 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).Background
Methods
