Abstract
Introduction
Given the predominant functional role which aggrecan has in the intervertebral disc, particularly within the nucleus pulposus, it is necessary to evaluate the quality of aggrecan produced by cells within tissue engineered disc constructs. The aim here was to characterise the nanostructure of aggrecan synthesised by nucleus pulposus cells treated with growth differentiation factor [GDF]-6) seeded in hydrogels in comparison to aggrecan isolated from healthy disc.
Methods
Aggrecan was isolated from bovine nucleus pulposus (NP) tissue (n=3 [<18 months old]) and primary bovine NP cells cultured with (+GDF6) or without GDF6 (−GDF6) for 28 days (n=2) in type I collagen hydrogels. Isolated aggrecan monomers were visualised by atomic force microscopy and categorised as either intact (globular domains visible at both the N and C termini) or non-intact. Core protein contour length (LCP) was calculated for intact molecules. The proportion of non-intact/fragmented to intact aggrecan and the molecular area of all monomers was determined.
Results
Very few aggrecan molecules were intact (1.3% in NP compared to 4.3% +GDF6 and 0% -GDF6). There was no significant difference in the mean LCP between NP (389 ± 37 nm) compared to +GDF6 (379.2 ± 26 nm) or the molecular area between NP (3560 ± 2179 nm2) and –GDF6 (3586 ± 2071 nm2). However, the molecular area in both cases was significantly lower than +GDF6 (4774 ± 3715 nm2) p≤0.0001.
Discussion & conclusions
Aggrecan structure can be altered by culture conditions. GDF6 treatment promoted the synthesis of more intact monomers, with greater over all molecular area.
Conflicts of interest: None
Funding: Impact Research Scholarship and the Presidents Doctoral Scholarship, provided by the University of Manchester.
