Introduction Several studies have identified multiple cell populations in the intervertebral disc (IVD) [5, 6, 11, 16, 18, 22, 28, 29]. Similarities between cells of the nucleus pulposus (NP) and articular chondrocytes (ACs), and cells of the annulus fibrosus (AF) and fibroblasts of fibrous connec- tive tissues have previously been assumed, largely on the basis of their rounded (chondrocytic) or more elongated (fibroblastic) spindle-shaped morphologies. As NP cells synthesise type II collagen and aggrecan, both of which are markers of a chondrocytic phenotype, these morpho- logical criteria may have some validity. However, while the aggrecan synthesised by NP cells is similar to that synthesised by ACs, it is not identical. Aggrecan from the NP differs not only in terms of its keratan sulphate (KS) and chondroitin sulphate (CS) substitution patterns, but also in how effectively it can form macromolecular com- plexes with hyaluronan, which are important for the vis- coelastic and hydrodynamic properties of this tissue [14, 15]. Aggrecan from the NP contains significantly more KS and is more heterogeneous than its counterpart in ar- ticular cartilage [14, 15, 17]. As shown in the present in- vestigation, NP cells can also synthesise type I in addition to type II collagen, thus it is an oversimplification to as- sume that NP cells are a chondrocytic cell type. Similarly, unlike fibroblasts AF cells synthesise type I and II colla- Abstract The objective of this study was to compare and contrast the cel- lular heterogeneity of ovine interver- tebral disc (IVD) annulus fibrosus (AF) and nucleus pulposus (NP) cells, synovial fibroblasts (SFs) and articular chondrocytes (ACs) grown in monolayer and alginate bead cul- ture. Phase contrast microscopy of the monolayers indicated that the SF and AF cells, and the AC and NP cells had similar morphologies. Im- munolocalisation of type I, II and VI collagen epitopes in the monolayers, however, demonstrated distinct quan- titative and relative differences be- tween the SF and AF, and the AC and NP cells. Immunolocalisation of bead-incorporated proteoglycans (PGs) also demonstrated quantitative and qualitative differences between the SF and AF cells. Quantitation of 35 S-bead PGs indicated that the AF cells synthesised significantly higher PG levels than the SFs, and AC cells synthesised significantly higher PG levels than the NP cells. These data were also consistent with the relative metabolic activities and cellular via- bilities displayed by each cell type in bead culture. AF cells shared mor- phological traits with SFs in mono- layer, but displayed distinctly differ- ent characteristics in bead culture. Similarly, NP cells shared similari- ties with ACs in monolayer and bead culture, but overall were less meta- bolically active. Data presented here support the proposal that the AF and NP cells of the ovine IVD should be considered as specialised fibrochon- drocytic cells. Keywords Disc cell heterogeneity · Annulus fibrosus · Nucleus pulposus · Mesenchymal cells · Fibrocartilage ORIGINAL ARTICLE Eur Spine J (2003) 12 : 57–65 DOI 10.1007/s00586-002-0434-6 James Melrose Susan Smith Peter Ghosh Assessment of the cellular heterogeneity of the ovine intervertebral disc: comparison with synovial fibroblasts and articular chondrocytes Received: 1 November 2001 Revised: 25 February 2002 Accepted: 30 April 2002 Published online: 6 July 2002 © Springer-Verlag 2002 J. Melrose (✉) · S. Smith · P. Ghosh Raymond Purves Bone and Joint Research Laboratories, The Institute of Bone and Joint Research, The University Clinic, Level 5, Building B26, The Royal North Shore Hospital of Sydney, Reserve Road, St. Leonards, NSW 2065, Australia e-mail: jmelrose@mail.usyd.edu.au, Tel.: +61-2-99267395/6535, Fax: +61-2-99266539