Paul A. DiMilla
Studies of the molecular mechanisms of cell detachment from self-assembled monolayers using advanced microscopies
Although molecular biology provides the means to identify the molecular components involved in adhesive interactions between cells and natural biological or artificial "engineered" substrata, the actual molecular mechanisms responsible for the detachment of cells under flow or during migration are unclear. For example, under what conditions does detachment occur by reversible dissociation of receptors from ligands, extraction of receptors from the cell membrane, or removal (or rearrangement) of ligands from the substratum? The answer to this question has profound implications for interpreting the role of receptor-mediated adhesive events in cell spreading and motility, for optimizing therapies involving adhesive processes, and for designing biomaterial supports for tissue engineering. To address this problem we applying quantitative optical microscope imaging to observe in situ how cells attach to and detach from model biomaterial surfaces, including self-assembled organic monolayers (SAMs) supported on transparent films of gold, under hydrodynamic shear and how cells migrate on these same surfaces in the absence of applied fluid shear.
One main focus of our studies has been to identify quantitatively the strength of adhesion and dynamics of detachment of 3T3 fibroblasts from SAMs. The radial-flow chamber we have developed in our laboratory, which allows in situ imaging of adherent cells exposed to a continuous spatially-dependent range of well-defined fluid shear stresses, is ideal for this study because it allows measurement of both shear- and temporally-dependent probabilities of detachment. We have demonstrated that shear-dependent patterns of detachment can be characterized with two parameters that are independent of applied volumetric flow rate: twc and stw, reflecting the mean strength of adhesion and the heterogeneity in the strength of adhesion, respectively. We also have demonstrated that initial detachment during the first 0.3 seconds of flow is a first-order homogeneous process but that subsequent detachment is described better as a first-order heterogeneous process; the rates of detachment, including heterogeneity in rate, increase with increasing shear stress.
We currently are applying these approaches to examine the effects of systematic variations in the mean and randomness in substratum chemistry on both cell adhesive and motile properties. Recent results from our laboratory, in collaboration with Steven Garoff (Physics, CMU), using SAMs degraded photolytically with UV light demonstrate that the mean strength of adhesion of fibroblasts to SAMs increases dramatically with modest increases in the hysteresis of wetting of water on these surfaces. These observations are consistent with the hypothesis that increasing the concentration of "defects" on a surface "pins" adherent cells -- suggesting that the cellular mechanism of detachment depends on surface order -- and have profound implications for how biomaterials are designed and fabricated. We will test this hypothesis and the role of adsorbed fibronectin on adhesion by simultaneously imaging patterns of cell detachment using phase-contrast optics and fluorescently identifying whether individual cells labeled with the fluorescent lipid analogue DiI-C18(3) detach by membrane rupture. Densities of fibronectin adsorbed on SAMs will be measured using in situ reflectometry in collaboration with Robert Tilton (ChE, CMU); this technique allows adaptation for simultaneous use with the radial-flow chamber to detect the removal of ligand upon detachment. Other current studies focus on verifying functional consequences of changes in adhesion on the rate and dynamics of migration of cells on identical SAMs. We also have been investigating the relative roles of cell/substratum and cell/cell adhesion in cell motility. We recently have demonstrated in our laboratory the application of the radial-flow chamber to measure the adhesive properties of semi-confluent and confluent sheets of cells. We have observed that the rate of peeling of a monolayer of murine stromal cells depends on both the confluency of the sheet and the fluid shear stress. In collaboration with Horace DeLisser and Steven Albelda (University of Pennsylvania), we are applying this approach to identify the role of cell-cell interactions and signaling mediated by cell-cell PECAM receptors on the motility of isolated cells and wounded sheets of cells on collagen- and fibronectin-coated surfaces. Our system allows in vitro comparison of the adhesive and motile behavior (in either the absence or presence of soluble anti-PECAM antibodies) among human mesothelioma cells transfected with cDNA encoding complete and incomplete constructs of PECAM. These studies are designed to provide rationales explaining changes in angiogenesis observed in vivo upon alteration in PECAM expression.
In 1998 Dr. DiMilla joined Organogenesis, Inc. to direct the development of an extracorporeal liver-assist device based on ex vivo culture of hepatocytes for treatment of liver failure. More recently, he led the development of biodegradable pharmacologically-active polymers for controlled-release therapies and as coatings for implantable medical devices as the Director of Research at Polymerix Corporation. He was a recipient of the inaugural Whitaker Young Investigator Award from the Biomedical Engineering Society.