"INTRODUCTION: Back pain is one of the leading causes of disability, with an estimated 540 million sufferers worldwide. Degeneration of the intervertebral discs (IVDs) is implicated in 40% of cases; however, treatment remains limited and regenerative therapies are becoming essential. 3D bioprinting provides a powerful tool to create biomimetic tissue analogues with properties closely matched to complex tissues. This study therefore attempts to use shear-thinning alginate-collagen hydrogels to replicate the complex mechanical, biochemical, and structural cues present within the IVD by 3D bioprinting a range of models for the developing, healthy, and degenerating IVD. It is hypothesised that by varying bioprinted hydrogel stiffness, incorporating important extracellular matrix components such as laminin, and by varying the IVD models’ regionally specific macro- and micro-architecture, healthier disc cell phenotypes can be encouraged and the results used to inform regenerative strategies.
METHODOLOGY: Immortalised human cells from the IVD’s central gelatinous nucleus pulposus (NP) and surrounding fibrous annulus fibrosus (AF) regions were suspended in blended alginate-collagen hydrogels using a protocol previously developed within the lab group1. Cell-laden hydrogels were then bioprinted using Suspended Layer Additive Manufacturing (SLAM), a novel technique appropriate for bioprinting low-stiffness (<~100kPa) materials. IVD models were designed using BioCAD and BioCAM, based on patient data. The mechanical properties of the bioprinted constructs were then characterised using rheology and compressive testing. Live/Dead staining was used to optimise bioprinting settings. Cells were tagged with cell-permanent tracker dyes and monitored for a period of 28 days using stereo fluorescence microscopy and confocal laser scanning microscopy. Cell phenotype was assessed using qPCR, for a panel of genes previously defined by the research group2, whilst tissue development was examined using immunostaining, for a range of key healthy and degenerate IVD markers.
RESULTS: A range of physiologically relevant stiffnesses and regionally specific stiffness gradients have been achieved within the bioprinted IVD models. All models supported high cell viability. Cell tracker studies indicated that the NP, AF and NP-AF interface regions were maintained over a culture period of at least 28 days. Immunostaining revealed substantial production of HA and ACAN, whilst qPCR demonstrated phenotypic responses to changes in stiffness and gel composition. The introduction of laminin into the hydrogels was shown to encourage NP cell clustering.
CONCLUSION: Using SLAM, soft hydrogels have been patterned to replicate an IVD with regionally specific NP-like, AF-like and interface-like regions, containing both human NP and AF cells. Softer and stiffer variants of the IVD can now be created to deliver ‘healthy’ and ‘degenerate’ IVD models based on patient data. The discovery that laminin encourages clustering is a particularly interesting result that must be investigated further when applied to a model of the foetal IVD, since laminin is especially present at this early stage. The introduction of growth factors (TGFβ, GDF-5/6) is also of particular interest, since bioprinted gel composition has been shown to influence NP cell phenotype using both qPCR and immunostaining. The platforms developed will ultimately be applied to optimise protocols for IVD regeneration using primary NP cells and mesenchymal stem cells."