Methods and Results

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.

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.

Methods and Results

We present an in vitro study looking at the effect of our Bgel hydrogel on mesenchymal stem cells (MSCs) and their bone forming capacity within lumbar cages: devices used to space the bones apart in the fusion operation, as a mechanism to improve fixation and intra cage bone formation. MSCs were isolated from human hip joint, expanded, seeded within Bgel, cast into well inserts or lumbar cages and cultured for 4 weeks. Using 3D X-ray imaging micro computed tomography (μCT) scans we show that the MSC in the presence Bgel begin to mineralise within the lumbar cages. Histology is currently ongoing and will be presented at the meeting.

Methods and Results

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.

Methods and Results

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.

Methods and Results

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.

Methods and Results

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.

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.

Methods & Results

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.