Extracellular matrix (ECM) is a complex mixture of molecules that exist at the interface between cells in biological tissues. Many ECM proteins consist of conserved functional domains and are heavily decorated with glycosaminoglycans [1, 2]. These domains act as the binding sites for other ECM proteins, cellular receptors and soluble growth factors . Over the past decades, knowledge on the functional roles of ECMs have been generated from in vitro experiments, in which cells are co-cultured with purified ECM components in two- or three-dimensions. For example, Schedin et al.  used ECMs isolated from rat mammary glands to generate 3D culture scaffolds. Mammary epithelial cells seeded in ECMs isolated from nulliparous rats developed epithelial ducts in vitro, whereas the same type of cells died in ECMs isolated from mid-involuting mammary glands. More recently, ECMs have been found to induce the direct transdifferentiation of bone vascular cells into lymphatic endothelium . Mouse embryonic stem cells (ESCs) seeded onto ECMs isolated from the osteogenic cell line MC3T3E1 differentiate into osteoblasts more efficiently than on ECMs from a non-osteogenic cell line . Similarly, specific combinations of ECM proteins have been shown to direct the differentiation of primary human neural precursor cells into either neurons or glial cells . These remarkable observations suggest that ECMs contain biological cues that can instruct cell fate determination, and different combinations of ECM components may harbor specific signals for particular cell types and for effecting different phenotypic consequences. Furthermore, the density of ECMs is known to influence cell spreading and migration speed , possibly due to the effect of ECM concentration on the clustering of integrins on cell membranes .
While these in vitro models are powerful in delineating the functions and dosage effect of specific ECM components, much less is known about the biological roles of the spatial arrangement and orientation of individual ECM molecules. Many organs contain ECM components arranged in highly distinctive spatial organizations . Among them, tendons are one of the most structurally organized tissues and the best illustration of the level of structural organization of ECMs in vivo . Tendons are mainly composed of Collagen I, with small amounts of Collagens III and V . The collagen fibres in tendons are bundled as highly organized parallel arrays , which serve to transmit force generated from muscle to bone . The building blocks of collagen fibers are triple helix collagen monomers assembled from three collagen polypeptides. Each monomer is composed of two α1 and one α2 collagen chains. The triple-helical monomer is approximately 300 nm in length and 1.5 nm in diameter. Recent studies have indicated that the interaction sites for collagen binding proteins are highly ordered on a three-dimensional structure of collagen fibrils. These ligand-binding sites are assembled on collagen monomers, and depending on how the collagen aggregates, these sites are exposed or hidden . Hence, cell interaction and ligand interaction domains are arranged in a specific pattern on collagen fibres.
A number of techniques have been recently developed to control the spatial arrangement of collagen proteins in artificial scaffolds. Collagen fibres can be physically aligned by electrospinning , microfluidics [16, 17], molecular crowding , electrochemical fabrication , magnetic fields , strain pulling  and extrusion methods . However, the resulting fibrils, albeit aligned to various extents, are still very disorganized when compared to natural collagen fibrils, and lack the architectural complexity observed in naturally occurring ECMs. Nevertheless, the structural alignment of collagen fibres generated by these techniques can affect cell morphology. For example, human fibroblasts seeded on collagen conduits are significantly aligned along the collagen fibril axis  and a similar alignment has been observed for bovine aortic endothelial cells cultured on aligned collagen scaffolds produced by microfluidics . Conversely, mesenchymal stem cells (MSC) and rotator cuff fibroblasts cultured on anisotropic substrates secreted aligned collagen fibres [23, 24]. These studies suggest the mutual relationship between the physical packing of ECMs and cell morphology, but systematic and quantitative understanding of how the spatial organization of ECMs affects cellular functions is still lacking.
Our group has recently developed a novel approach for studying the functional roles of the tissue microenvironment , in which histological sections from intact mammalian tissues are directly used as cell culture substrates. Cells seeded on these sections are exposed to native ECM scaffolds, and adopted morphological and biochemical changes as a result. We have envisaged a mammalian tendon as a relatively acellular block of collagen with a strikingly ordered organization, and can be viewed as a convenient, naturally occurring collagen scaffold for cell culturing. In this study, we have explored this property and created cell culture substrates from bovine Achilles tendon sectioned at different angles. These surfaces contain the same biochemical composition, but different in the arrangement of collagen fibrils. Human MSC were seeded onto these sections, hence exposed to collagen fibrils with different arrangements. This experimental design provides a simple but novel way for the systematic analysis of the influence of ECM arrangements on cell behaviour.