The fibrous cellular microenvironment, and how cells make sense of a tangled web

The physiology and fate of living cells have long been known to be guided by their niche-specific microenvironments. Certain lineage-specific traits arise in mesenchymal stem cells from the elastic stiffness of the substratum on which they are cultured (1). This observation helped launch mechanobiology as a modern field, and has motivated a decade of research on how cells sense stiffness. A number of factors have been identified experimentally for transduction of mechanics to cells, including the local surface topology of the substratum to the cell through structural features, such as porosity (2). Mathematical models have determined that cells interact with the materials around them through dynamically cycling focal adhesions; this has led to an understanding of a molecular clutch that transmits forces and stabilizes larger focal adhesions on stiffer substrata (3, 4). These models have recently been extended to account for a broad range of detailed biophysical and kinetic phenomena within the cell, including intracellular transport and the spatial disposition of the cell, and have also accounted for nonlinearity of the extracellular material (5). The picture seemed fairly complete until focal adhesions were studied on a different type of substratum, a nonwoven mesh of nanofibers (6). Here, the same cells that consistently formed larger focal adhesions on stiffer substrata seemingly violated their rules. Does this apparent contradiction mean that cells follow different rules on fibrous substrata than they do on continuous substrata? Cao et al. (7) reveal in PNAS that there may indeed be a single set of rules that can explain both sets of results. The discovery that focal adhesions follow a single rulebook on fibrous and continuous substrata is of pivotal importance as the study of cellular mechanobiology progresses toward the goal of controlling how cells connect to and feel the microenvironment around them.

This transition in thinking was made necessary not only by the need to understand the cell’s microenvironment as it interfaces with extracellular matrix, but also by the need to understand how tissues interact with one another. Even “solid” tissues in the body present cells with a meshwork of discrete, charged, and cross-linked fibers rather than with a smooth, homogenous continuum, and in many cases interface with more fibrous tissues (for example, the insertion of tendon into bone). The fields of musculoskeletal mechanics and tissue engineering are founded on representing living tissues as mechanical continua. Using this approach, the continuum responses of the native tissue must be known under all possible loadings, and that understanding then defines the design criteria for tissue replacements. Seminal models in the field of biomechanics, such as the Y. C. Fung “quasilinear viscoelastic model” (8), draw upon continuum representations of tissues that account, under certain cases (9, 10), for the ways that fibers and their solvent interact, but hide the fibrous nature of the tissue. Flory-type treatment of tissues such as polymers with prescribed orientation distributions, as established for musculoskeletal tissues by Lanir (11), Sacks and colleagues (12), and Zahalak and colleagues (13), are a mainstay of modeling of fibrous collagenous tissues and their attachments to bone (14, 15). Furthermore, biphasic mixture models underlie nearly all modern successes in cartilage tissue engineering (16, 17). In the case of hydrogels, Xin–Lu acoustomechanical wave theory is a novel example of how molecular-level interactions can be deduced from acoustic conduction (18, 19). By accounting faithfully for the physics of both the material constituents and their loading, molecular-level interactions can be elucidated through readily available modalities, such as ultrasound. However, as we probe deeper into the hierarchical structures of tissue systems, many tissues no longer appear continuous, but rather present discrete fibrous structures. For example, fibrous networks appear to drive the mechanics of the attachment of tendon to bone (20, 21) and the pericellular environment (22).

Continuum models are well established for estimating the stresses and strains that cells feel (13); however, these models have failed to predict experimental results (7). Specifically, when cells were cultured on fibrous mats of nanofibers, their focal adhesions under certain circumstances did the opposite of what the models predicted they would do. Therefore, either our models for the cell responses were wrong, or the “well-established” continuum models were wrong. Cao et al. (7) reveal that cells still operate with the same molecular clutches they always have, but that it is the fibrous nature of the substratum that determines what a cell feels. How do molecular clutches know the difference between a fibrous substratum and a continuous substratum of the same stiffness? The key is that fibrous mats and Maxwell–Wiechert viscoelastic continua differ in their loading responses (23–25): both scenarios typically present concave-down force-displacement relationships in response to a first loading (Fig. 1), but only the fibers within the fibrous mats can be easily modified locally by the cells in a way that changes their local stiffness. This can be achieved, for example, by breaking cross-links between fibers, particularly in the vicinity of protrusions where focal adhesions form, or by physical rearrangement of fibers. As a result, the overall stiffness of the fibrous mat might not change substantially over the course of remodeling by a cell, but the local stiffness around the cell can change substantially. Specifically, the plasticity of fibrous substrata enables creation of stiff bands that can dominate the mechanics of the cell microenvironment in the vicinity of focal adhesions. Therefore, although a cell cannot change a viscoelastic solid substantially, it can modify a fibrous extracellular matrix so that its local stiffness far exceeds its homogenized global stiffness.

Fig. 1.

Cells dictate and respond to changes in local stiffness within fibrous matrices. Although solid and fibrous substrata can have identical mechanical responses to an initial ramped displacement loading, the plasticity of fibrous substrata enables creation of stiff bands in subsequent loadings. This local alignment of fibers and the associated local stiffening can dominate the mechanics of the cell microenvironment in the vicinity of focal adhesions.

Notably, composite material engineers have been applying these principles for decades without knowing that cells have been applying them for billions of years. In engineering materials, although macroscale phenomena, such as elastic and plastic deformation, are often well modeled by homogenized material representations, local phenomena, such as fracture and corrosion,

With predictive mathematical models for how cells respond to their local microenvironments, the tools are now available for transforming basic cellular discovery into breakthroughs in tissue engineering and wound healing.

are governed by local phenomena, and structural factors commonly enter the equation for phenomena occurring over length scales comparable to the sizes of a material’s constituents (26, 27). Engineers have harnessed this to great effect with tough ceramic composites that combine the strength of thin fibers with the toughness afforded by the stochastic failure of fiber bundles. Models for this behavior have enabled the prediction of strength, ductility, and fracture, and thus the design of the lightweight material systems in the engines and fuselages of modern aircraft (28).

This new understanding of how cells respond to bulk versus fibrous materials has critical implications for the development of tissue-engineered materials, and should be incorporated into the new generation of fibrous-based biomaterials (29). The picture is still not complete, and additional factors will certainly emerge over time as our understanding of the detailed feedback between cellular remodeling and extracellular synthesis, degradation, and remodeling is refined. However, we now have a solid, predictive foundation for future analysis, and an understanding of how cells feel their microenvironment. Engineers have long exploited the difference between a bundle of fibers and a bulk solid, and that approach can now be taken thoughtfully in the context of biologic cellular materials. With predictive mathematical models for how cells respond to their local microenvironments, the tools are now available for transforming basic cellular discovery into breakthroughs in tissue engineering and wound healing.