Structural Characterisation of Aggrecan in Cartilaginous Tissues and Tissue Engineered Constructs

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Abstract

Collagen II and the proteoglycan aggrecan are key extracellular matrix (ECM) proteins in cartilaginous tissues such as the intervertebral disc (IVD). Given the functional role that these structural and functional proteins have in the IVD, ECM in tissue engineered intervertebral disc (TE IVD) constructs needs to recapitulate native tissue. As such, there is a need to understand the structure and mechanical function of these molecules in native tissue to inform TE strategies. The aims here were to characterise aggrecan and collagen II using atomic force microscopy (AFM), size-exclusion chromatography multi angle light scattering (SEC-MALS), histology, quantitative PCR, nanomechanical and computational modelling in: (i) skeletally immature and mature bovine articular cartilage (AC) and nucleus pulposus (NP), (ii) TE IVD constructs cultured in hypoxia or treated with transforming growth factor beta [TGFβ3] or growth differentiation factor [GDF6]), and (iii) porcine AC and NP tissue. No variation in collagen II structure was observed although the proportion of organised fibrillar collagen varied between tissues. Both intact (containing all three globular domains) and non-intact (fragmented) aggrecan monomers were isolated from both AC and IVD and TE IVD constructs. Mature intact native NP aggrecan was ~60 nm shorter (core protein length) compared to AC. In skeletally mature bovine NP and AC tissue, most aggrecan monomers were fragmented (99% and 95%, respectively) with fragments smaller and more structurally heterogeneous in NP. Similar fragmentation was observed in skeletally immature bovine AC (99.5%), indicating fragmentation occurs developmentally at an early age. Fragmentation was not a result of enhanced gelatinase activity. Aggrecan monomers isolated from notochordal cell rich porcine NP were also highly fragmented, similar to bovine NP. Application of a computational packing model suggested fragmentation may affect porosity and nutrient transfer. The reduced modulus was greater in AC than NP (497 kPa and 76.7 kPa, respectively) with the difference likely due to the organisation and abundance of ECM molecules, rather than individual structure. Growth factors (GDF6 and TGFβ3), and not oxygen tension treated TE IVD constructs were structurally (with >95% fragmented monomers), histologically and mechanically (GDF6: 60.2 kPa; TGFβ3; 69.9 kPa) similar to native NP tissue (76.7 kPa) and there was evidence of gelatinase activity. To conclude, these results show that the ultrastructure of intact aggrecan was tissue and cell dependent, and could be modified by manipulation of cell culture conditions, specifically GDF6 which may play a role in aggrecan glycosylation.

Keyword(s)

Aggrecan; Atomic force microscopy; Protein characterisation; mechanics; tissue engineering

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