Nanostructure of Aggrecans in Natural and Engineered Intervertebral Discs

UoM administered thesis: Master of Philosophy

  • Authors:
  • Jude Aldaco

Abstract

Degeneration of the intervertebral disc (IVD) is considered to be a major cause of low back pain. The IVD is composed of the nucleus pulposus (NP), surrounded by concentric layers of annulus fibrosus (AF). The NP is composed predominantly of fibrillar type II collagen and the hydrophilic proteoglycan aggrecan which resist tensile and compressive loads respectively. The degenerate IVD is characterised by the loss of aggrecan in the NP, which in turn is linked to a reduced load bearing capacity and hence low back pain. Given the important role that aggrecan plays in the IVD and that previous work has demonstrated that aggrecan ultrastructure varies species and tissue type, this project aimed to determine if aggrecan synthesized by cultured cells was comparable to aggrecan derived from tissue.The first aim of this project was to adapt existing methodologies developed for cartilage to enable the visualization and characterisation of extracted aggrecan from IVD by atomic force microscopy (AFM). Following initial extraction, the presence of isolated aggrecan was confirmed with immunoblotting and the suitability of APTES-coated mica surfaces for aggrecan immobilisation and visualisation was determined by contact angle measurements (to confirm the hydrophilicity of the substrate) and AFM roughness analysis. Subsequent AFM imaging demonstrated that bovine tissue derived aggrecan was characterised by a core protein (CP) adorned with individual glycosaminoglycan (GAG) side chains. Although aggrecan CP length for IVD derived molecules was biomodally distributed (215 ± 2 nm and 371 ± 6 nm) and the CP of the shorter population was comparable to that reported for cartilage derived aggrecan (220 ± 142 nm), IVD extracted molecules were predominantly in the shorter form. The above optimised methodologies were subsequently applied to aggrecan isolated from alginate constructs seeded with human NP cells and cultured in standard or chondrogenic media with TGF-β3 for 21 days. Newly synthesised aggrecan was isolated by GuHCl solubilisation and ultracentrifugation under dissociative conditions (D1) with size exclusion chromatography. Aggrecan molecules isolated from human NP cells cultured in standard and chondrogenic media (n= 300/culture condition) conditions were imaged by PeakForce AFM. AFM height imaging and data analysis revealed a significant difference (p

Details

Original languageEnglish
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Award date1 Aug 2014