Progress towards endometrial tissue engineering for modelling endometrial diseases and infertility is frustrated by the inability to mimic the fibronectin (FN) extracellular matrix required by human endometrial stromal cells (EnSCs). Here we show that this is because of the requirement to present integrin alpha 5 beta 1 (the FN receptor) ligands in specifically oriented, polyvalent displays; by engineering controlled self-assembly of the 9th-10th type III FN domain pair (FIII9-10, the minimal integrin alpha 5 beta 1 ligand) immobilised in a specific orientation to cell culture surfaces. The fraction of adherent EnSCs seen to spread increased significantly for the multimeric ligand surfaces in the order: tetramer > trimer > dimer > monomer. The extent of EnSC spread morphology also increased in the same order, with the tetrameric ligand supporting a morphology most similar to that supported by FN. Our data suggest that only higher-order multimers of FIII9-10 will fully promote cell spreading mediated through integrin alpha 5 beta 1 binding. (C) 2011 Elsevier Inc. All rights reserved.