**16. Conclusions**

*N*-terminal interactions of aggrecan with HA forms macro-aggregate structures that are physically entrapped within the type II collagen networks of cartilaginous tissues. These along with the C-terminal G3 mediated interactions of aggrecan EGF-like, Complement-like and C-type lectin domains with Type VI collagen lattices forms an extended tissue-wide cooperative network. This network provides a bio-sensory platform that not only dissipates loading in cartilaginous tissues but also facilitates an appropriate metabolic response by chondrocytes within the tissue to the loads they perceive to assemble an optimal functional and protective matrix, thus preventing cellular damage through point loading. Matrilin-3, SLRPs, COMP and fibulin also stabilise G3 interactions in this network. Collectively, these N- and C-terminal interactions tether aggrecan at both ends in tissues where its dynamic water imbibing properties with HA convey weight-bearing properties to tissues. In cartilaginous tissues, incorporation of aggrecan into an extended mechano-transductive sensory network allows the resident chondrocytes to perceive and respond to biomechanical changes to maintain biosynthetic responses which ensure tissue homeostasis and optimal tissue properties. The interactive properties of the G1 domain of aggrecan with HA undergoes a maturational phase where initially it does not interact with HA for 24 h which allows the newly secreted aggrecan to diffuse away from the cell into the interstitial matrix, as confirmed through pulse-chase radiosulphate labelling experiments. Immunohistochemistry also demonstrates a high density of aggrecan in the pericellular matrix around chondrocytes where network formation transmits regulatory cues to the chondrocyte to effect tissue homeostasis and aid in the stabilisation of cartilage. The variable functional attributes of aggrecan in particular tissue contexts is due to the diverse structure of its attached glycan side chains, allowing it to act as a space-filling molecule with an ability to entrap water in weight-bearing tissues such as articular cartilage and IVD but also as an interactive molecule with morphogens, growth factors and cells in growth plate cartilage and embryonic tissues. This functional diversity arises through substitution and post-translational modifications of aggrecan's attached GAG chains, for example post-translational modification of CS can occur such as disulphation on some non-reducing termini or variation in the disaccharide composition along the CS chain. These disulphated terminal groups regulate collagen fibrillogenesis in growth plate cartilage and are interactive with morphogenetic proteins such as IHH and SHH. These morphogenetic proteins direct maturational changes in the growth plates by regulating spatial and temporal chondrocyte differentiation eventually leading to growth plate closure and mineralisation at the cartilage–bone interface as part of the endochondral ossification process to extend the axial skeleton. The molecular composition of aggrecan thus varies in specific cellular and developmental contexts. Aggrecan in early embryonic development of the neural tube and notochord does not contain KS chains and some of its CS chains are replaced by the HNK-1 trisaccharide motif. This reduces aggrecans charge density and its solvation volume but conveys homophilic and heterophilic HNK-1 mediated properties and interactions with NC cells and ECM glycoproteins which direct NC cell

migration, development of the neural tube and notochord and migration of precursor cells involved in the development of the neural network, heart and brain stem. Aggrecan also forms specialised ECM structures such as PNNs in the CNS/PNS, which are neuroprotective and important for synaptic plasticity and cognitive learning. The form of aggrecan in these structures contains KS, CS and the HNK-1 glycan motif; however, the density of attached CS and KS side chains in brain aggrecan is less than in cartilage aggrecan. While aggrecan has essential cell directive properties in embryonic heart formation, it also has important supportive roles to play in mature heart tissues. The heart has a complex structure, aggrecan is an important functional component of a number of its tissues providing mechanical strength and resilience for the demanding continuous cycles of compression and relaxation which occur throughout an animals life-time. Aggrecan also stabilises the attachment points of valve leaflets facilitating the co-ordinated flow of blood between the ventricles and strengthens the heart tendon chordae tendineae which attach the papillary muscles of the internal heart wall to the atrioventricular valve. These are important internal stabilising structural components of the heart. This review shows that aggrecan has a diverse range of functional attributes and is of major importance not only in embryonic skeletal development but also in mature tissues where it maintains homeostasis and functionality. Due recognition of aggrecans attached GAG chains is important and explains its diverse tissue context driven properties. A greater understanding of the glyco-code and its cell directive properties may one day provide important insights as to how specific tissue repair and regenerative strategies may be directed more effectively in repair biology.

**Author Contributions:** Conceptualisation, J.M. and A.J.H.; Methodology, A.J.H.; Validation, J.M. and A.J.H.; Formal Analysis, A.J.H.; Writing—Original Draft Preparation, J.M.; Writing—Review and Editing, J.M. and A.J.H; and Project Administration, J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Melrose Personal Fund, Sydney, Australia.

**Conflicts of Interest:** The authors declare no conflict of interest and have no financial disclosures to make.
