There is a large biomanufacturing and clinical need for cost-effective and simple techniques to expand mesenchymal stem cells whilst retaining their multipotency. Endosteum-derived particles were prepared, characterised and examined as a biomaterial to facilitate the in vitro expansion of human mesenchymal stem cells. Bovine endosteum-derived particles are composed of chondroitin sulphate glycosaminoglycans with 4- and 6-sulphation and N-sulphated heparan sulphate glycosaminoglycans. The particles were positive for perlecan, laminin and fibronectin by immunohistochemistry and alpha-mannose, alpha-glucose, terminal N-acetyl-alpha-D-glucosamine, N-acetyl-alpha-galactosamine and alpha-fucose, using lectin binding. Human mesenchymal stem cells showed greater than 96% attachment to the particles after one day in spinner culture. After 7 days, the stem cells on decalcified particles were viable and had a 5-fold higher growth than the stem cells grown on Cytodex-2 beads. Significantly more stem cells were recovered from decalcified particles compared with mineralised particles (P < 0.05). Differentiation to chondrogenic, osteogenic and adipogenic lineages was maintained after culturing stem cells on the demineralised particles. We conclude that bovine endosteum-derived particles can be extracted from bone marrow to retain sulphated proteoglycans and glycosylated proteins. These particles are a suitable biomaterial for supporting the growth and retaining the multipotency of human mesenchymal stem cells.