Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness.

During angiogenesis, endothelial cells (ECs) use both soluble and insoluble cues to expand the existing vascular network to meet the changing trophic needs of the tissue. Fundamental to this expansion are physical interactions between ECs and extracellular matrix (ECM) that influence sprout migration, lumen formation and stabilization. These physical interactions suggest that ECM mechanical properties may influence sprouting ECs and, therefore, angiogenic responses. In a three-dimensional angiogenic model in which a monolayer of ECs is induced to invade an underlying collagen matrix, angiogenic responses were measured as a function of collagen matrix stiffness by inducing collagen crosslinking with microbial transglutaminase (mTG). By biaxial mechanical testing, stiffer collagen matrices were measured with both mTG treatment and incubation time. Using two-photon excited fluorescence (TPF) and second harmonic generation (SHG), it was shown that collagen TPF intensity increased with mTG treatment, and the TPF/SHG ratio correlated with biaxially tested mechanical stiffness. SHG and OCM were further used to show that other ECM physical properties such as porosity and pore size did not change with mTG treatment, thus verifying that matrix stiffness was tuned independently of matrix density. The results showed that stiffer matrices promote more angiogenic sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened matrices, but greater remodeling was revealed in stiffer gels using SHG and OCM. The results of this study show that angiogenic responses are influenced by stiffness and suggest that ECM properties may be useful in regenerative medicine applications to engineer angiogenesis.

Normal basilar artery structure and biaxial mechanical behaviour.

Much is known about cerebral vasospasm, a devastating sequela to ruptured intracranial aneurysms, yet underlying mechanisms remain unclear and clinical treatments have proven unsatisfactory. We have hypothesised that biochemical stimuli associated with the formation of extravascular blood clots dominate early maladaptive responses, leading to marked structural and functional changes in affected cerebral arteries. Before a precise picture of vasospasm can be obtained, however, we must understand better the structure and mechanical behaviour of normal cerebral arteries. Basilar arteries from rabbits were tested mechanically under biaxial loading conditions with and without active tone, segments were imaged using intravital nonlinear optical microscopy to quantify transmural orientations of fibrillar collagen, and passive mechanical data were fit with a four-fiber family stress-stretch relation. This constitutive model predicted well the overall mechanical behaviour and mean collagen fiber distributions, and thereby has promise to contribute to analyses of the biochemomechanics of cerebral vasospasm and similar cerebral pathologies. It is now time, therefore, to focus on mechanisms of vasospasm via mathematical models that incorporate growth and remodelling in terms of changes in the cross-linking and distributions of adventitial and medial collagen, primary contributors to the structural integrity of the arterial wall.

A theoretically-motivated biaxial tissue culture system with intravital microscopy.

Many cell types produce, remodel, and degrade extracellular matrix in response to diverse stimuli, including mechanical loads. Much is known about the molecular biology and biochemistry of the deposition and degradation of collagen, the primary structural constituent of the extracellular matrix in many tissues, yet there has been little modeling of the associated mechanobiology. For example, we do not have quantitative descriptions, or rules, for the kinetics of collagen turnover as a function of altered mechanical loading and we do not know what governs the orientation and pre-stretch at which new fibers are incorporated within extant tissue. In this paper, we use a constrained mixture theory for growth and remodeling of planar soft tissues to motivate a new experimental approach for investigating competing hypotheses on, for example, how new collagen is aligned by synthetic cells. In particular, because stress and strain fields can be homogeneous in a central region of a biaxially tested tissue, and because biaxial testing admits diverse protocols wherein equal stresses can be imposed in the presence of unequal strains or stresses can be maintained in the absence of strain, we report simulations that illustrate the potential utility of biaxial culture studies. Finally, we describe the associated design of a computer-controlled system that allows intravital microscopic quantification of collagen density, orientation, and cross-linking at various stages during the adaptation of a native tissue or the development of a tissue engineered equivalent, each subjected to well controlled biaxial loads.