Imaging via widefield surface plasmon resonance microscope for studying bone cell interactions with micropatterned ECM proteins

Abstract

The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5-, 10-, 25-, 50- and 100-μm-wide repeat grating. MG63 bone cells were seeded at 50,000 cells per 25 cm(2) and cell alignment was determined from micrographs taken at time-points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin-patterned cover slips especially to the 50- and 100-μm-wide patterns, although in this case cells did not position themselves in the middle of each fibronectin-coated region, but instead aligned to the small features associated with the edges of the fibronectin-coated regions. Patterned and un-patterned cells also had quite different morphologies. The un-patterned cells had a more rounded morphology and lengths of 25 to 35 μm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50-70 μm. The widefield surface plasmon resonance imaging indicated that cells on un-patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin-patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin-coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.