Fibrin and collagen, biopolymers occurring naturally in the body, are commonly-used biomaterials as scaffolds for tissue engineering. How collagen and fibrin interact to confer macroscopic mechanical properties in collagen-fibrin composite systems remains poorly understood. In this study, we formulated collagen-fibrin co-gels at different collagen-to-fibrin ratios to observe changes in overall mechanical behavior and microstructure. A modeling framework of a two-network system was developed by modifying our micro-scale model, considering two forms of interaction between the networks: (a) two interpenetrating but non-interacting networks (“parallel”), and (b) a single network consisting of randomly alternating collagen and fibrin fibrils (“series”). Mechanical testing of our gels show that collagen-fibrin co-gels exhibit intermediate properties (UTS, strain at failure, tangent modulus) compared to those of pure collagen and fibrin. Comparison with model predictions show that the parallel and series model cases provide upper and lower bounds respectively for the experimental data, suggesting that a combination of such interactions exist between collagen and fibrin in co-gels. A transition from the series model to the parallel model occurs with increasing collagen content, with the series model best describing predominantly fibrin co-gels, and the parallel model best describing predominantly collagen co-gels.

While collagen is recognized as the predominant mechanical component of soft connective tissues, the role of the non-fibrillar matrix (NFM) is less well understood. Even model systems, such as the collagen-agarose co-gel, can exhibit complex behavior, making it difficult to identify relative contributions of specific tissue constituents. In the present study, we developed a two-component microscale model of collagen-agarose tissue analogs and used it to elucidate the interaction between collagen and NFM in uniaxial tension. Collagen fibers were represented with Voronoi networks, and the NFM was modeled as a neo-Hookean solid. Model predictions of total normal stress and Poisson’s ratio matched experimental observations well (including high Poisson’s values of ~3), and the addition of NFM led to composition-dependent decreases in volume change and increases in fiber stretch. Because the NFM was more resistant to volume change than the fiber network, extension of the composite led to pressurization of the NFM. Within a specific range of parameter values (low shear modulus and moderate Poisson’s ratio), the magnitude of the reaction force decreased relative to this pressurization component resulting in a negative (compressive) NFM stress in the loading direction, even though the composite tissue was in tension.

Collagen and fibrin are important extra-cellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen-fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via 3-D network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in the co-gels compared to the pure gels; this microstructural change was accompanied by increased failure strain and decreased tangent modulus for both collagen and fibrin following selected digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated presence of very long fibers and clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels.

Tissue-equivalents (TEs), simple model tissues with tunable properties, have been used to explore many features of biological soft tissues. Absent in most formulations however, is the residual stress that arises due to interactions among components with different unloaded levels of stress, which has an important functional role in many biological tissues. To create a pre-stressed model system, co-gels were fabricated from a combination of hyaluronic acid (HA) and reconstituted Type-I collagen (Col). When placed in solutions of varying osmolarity, HA-Col co-gels swell as the HA imbibes water, which in turn stretches (and stresses) the collagen network. In this way, co-gels with residual stress (i.e., collagen fibers in tension and HA in compression) were fabricated. When the three gel types tested here were immersed in hypotonic solutions, pure HA gels swelled the most, followed by HA-Col co-gels; no swelling was observed in pure collagen gels. The greatest swelling rates and swelling ratios occurred in the lowest salt concentration solutions. Tension on the collagen component of HA-Col co-gels was calculated from a stress balance and increased nonlinearly as swelling increased. The swelling experiment results were in good agreement with the stress predicted by a fibril network + non-fibrillar interstitial matrix computational model.

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*|−1 − |X*|−1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.