Composite diaphragm inflation: A method for probing the rheological functions of cell-cell anchoring junctions and cytoskeletal networks within a living normal human epidermal keratinocyte sheet.

摘要

Normal human epidermal keratinocytes (NHEKs), like most all nucleated human cells, possess a filamentous cytoskeleton, composed of actin microfilaments (MFs), microtubules (MTs), and intermediate filaments (IFs). Distinct from connective tissues cells like the dermal fibroblast, however, keratinocytes in vivo are organized as a multicellular epithelium. In the absence of extracellular matrix for mechanical support, keratinocytes express specialized cell-cell anchoring junctions that interconnect MFs and IFs within adjacent cells in the formation of a mesoscopic network cytoarchitecture. Despite advances in our understanding of the proteins that regulate cytoskeletal filament and anchoring junction assembly, the biophysical mechanisms by which these structures provide the epidermis an innate mechanical resilience are, at present, not fully understood.; In this thesis, we validate a new method for the exploration of keratinocyte rheology, referred to as the technique of composite diaphragm inflation (CDI). Sheets of living NHEKs were reconstituted in vitro on tensed but highly compliant, freestanding polydimethylsiloxane (PDMS) elastomer membranes, 5.0 mm in diameter and 10.0 mum thick. NHEK-PDMS composite diaphragm (CD) specimens were then subjected to a series of quasi-static axisymmetric inflation tests to examine the stress response of the epithelial sheet at physiologically severe deformations (∼50% nominal biaxial strains). During these experiments, living NHEK sheets exhibited several unique rheological behaviors, including viscoelasticity, plasticity, and the process of biological adaptation and recovery or a restitutio ad integrum. In addition to a rigorous accounting of the experimental instrumentation and protocol distinct to CDI, a finite elasticity model is proposed for analyzing the mechanics of the associated inflation test. Numerical solution procedures are formulated to predict the quasi-static load-deformation response of a prestretched clamped circular isotropic incompressible hyperelastic membrane inflated into a horizontally semi-infinite incompressible liquid reservoir of finite vertical depth. Assuming a Mooney-Rivlin (MR) constitutive model, we quantitatively demonstrate a new non-traditional regression analysis for estimating values of the MR material parameters and residual membrane tension that best describe a set of experimental inflation response data. Combining improved culture techniques with the more advanced tools of the molecular cell biologist, CDI experiments can potentially transform morphological observations of NHEK cytoarchitecture into well-posed boundary value problems amenable to mechanical experimentation and hypothesis testing.