In situ intercellular mechanics of the bovine outer annulus fibrosus subjected to biaxial strains

In situ intercellular strains in the outer annulus fibrosus of bovine caudal discs were determined under two states of biaxial strain. Confocal microscopy was used to track and capture images of fluorescently labelled nuclei at applied Lagrangian strains in the axial direction (E(A)(S)) of 0%, 7.5% and 15% while the circumferential direction (E(C)(S)) was constrained to either 0% or -2.5%. The position of the nuclear centroids were calculated in each image and used to investigate the in situ intercellular mechanics of both lamellar and interlamellar cells. The intercellular Lagrangian strains measured in situ were non-uniform and did not correspond with the biaxial Lagrangian strains applied to the tissue. A row-oriented analysis of intercellular unit displacements within the lamellar layers found that the magnitudes of unit displacements between cells along a row (delta;(II)) were small (|delta;(IIavg)|=1.6% at E(C)(S)=0%, E(A)(S)=15%; |delta;(IIavg)|=3.0% at E(C)(S)=-2.5%, E(A)(S)=15%) with negative unit displacements occurring greater than one-third of the time. Evidence of interlamellar shear and increased intercellular Lagrangian strains among the cells within the interlamellar septa suggested that their in situ mechanical environment may be more complex. The in situ intercellular strains of annular cells were strongly dependent upon the local structure and behaviour of the extracellular matrix and did not correspond with applied tissue strains. This knowledge has immediate relevance for in vitro investigations of disc mechanobiology, and will also provide a base to investigate the mechanical implications of disc degeneration at the cellular level.