No abstract

No abstract

To build anisotropic, mechanically functioning tissue, it is essential to understand how cells orient in response to mechanical stimuli. Therefore, a computational model was developed which predicts cell orientation, based on the actin stress fiber distribution inside the cell. In the model, the stress fiber distribution evolves dynamically according to the following: (1) Stress fibers contain polymerized actin. The total amount of depolymerized plus polymerized actin is constant. (2) Stress fibers apply tension to their environment. This active tension is maximal when strain rate and absolute strain are zero and reduces with increasing shortening rate and absolute strain. (3) A high active fiber stress in a direction leads to a large amount of fibers in this direction. (4) The cell is attached to a substrate; all fiber stresses are homogenized into a total cell stress, which is in equilibrium with substrate stress. This model predicts that on a substrate of anisotropic stiffness, fibers align in the stiffest direction. Under cyclic strain when the cellular environment is so stiff that no compaction occurs (1 MPa), the model predicts strain avoidance, which is more pronounced with increasing strain frequency or amplitude. Under cyclic strain when the cellular environment is so soft that cells can compact it (10 kPa), the model predicts a preference for the cyclically strained compared to the compacting direction. These model predictions all agree with experimental evidence. For the first time, a computational model predicts cell orientation in response to this range of mechanical stimuli using a single set of parameters.

Use of biomaterial scaffolds for tendon repair often results in tissue formation characteristic of scar tissue, rather than the highly aligned type-1 collagen matrix of healthy tendons. We hypothesized that non-optimal biomaterial surfaces may play a role in these outcomes, specifically randomly oriented biomaterial surfaces that unintentionally mimic structure of pathological tendon. We observed that disorganized scaffold surfaces do adversely affect early cell attachment and gene expression. We further identified that disorganized fiber surfaces can prime tendon cells toward pro-inflammatory signaling. These findings represent provocative evidence unstructured fiber surfaces may underlie inflammatory responses that drive aberrant collagen matrix turnover. This work could be highly relevant for the design of cell instructive biomaterial therapies that yield positive clinical outcomes.

A feedback mechanism between different tissues in a growing bone is thought to determine the bone's morphogenesis. Cartilage growth strains the surrounding tissues, eliciting alterations of its matrix, which in turn, creates anisotropic stresses, guiding directionality of cartilage growth. The purpose of this study was to evaluate this hypothesis by determining whether collagen fiber directions in the perichondrium and periosteum align with the preferential directions of long bone growth. Tibiotarsi from chicken embryos across developmental stages were scanned using optical projection tomography (OPT) to assess preferential directions of growth at characteristic sites in perichondrium and periosteum. Quantified morphometric data were compared with two-photon laser-scanning microscopy images of the three-dimensional collagen network in these fibrous tissues. The diaphyseal periosteum contained longitudinally oriented collagen fibers that aligned with the preferential growth direction. Longitudinal growth at both metaphyses was twice the circumferential growth. This concurred with well-developed circumferential fibers, which covered and were partly interwoven with a dominant network of longitudinally oriented fibers in the outer layer of the perichondrium/periosteum at the metaphysis. Toward both articulations, the collagen network of the epiphyseal surface was randomly oriented, and growth was approximately biaxial. These findings support the hypothesis that the anisotropic architecture of the collagen network, detected in periosteum and perichondrium, concurs with the assessed growth directions. ß

In the cardiac microenvironment, cardiomyocytes (CMs) are embedded in an aligned and structured extracellular matrix (ECM) to maintain the coordinated contractile function of the heart. The cardiac fibroblast (cFB) is the main cell type responsible for producing and remodeling this matrix. In cardiac diseases, however, adverse remodeling and CM death may lead to deterioration of the aligned myocardial structure. Here, we present an in vitro cardiac model system with uniaxial and biaxial constraints to induce (an)isotropy in 3D microtissues, thereby mimicking 'healthy' aligned and 'diseased' disorganized cardiac matrices. A mixture of neonatal mouse CMs and cFBs was resuspended in a collagen-matrigel hydrogel and seeded to form microtissues to recapitulate the in vivo cellular composition. Matrix disarray led to a stellate cell shape and a disorganized sarcomere organization, while CMs in aligned matrices were more elongated and had aligned sarcomeres. Although matrix disarray has no detrimental effect on the force generated by the CMs, it did have a negative effect on the homogeneity of contraction force distribution. Furthermore, proliferation of the cFBs affected microtissue contraction as indicated by the negative correlation between the percentage of cFBs in the microtissues and their beating frequency. These results suggest that in regeneration of the diseased heart, reorganization of the disorganized matrix will contribute to recover the coordinated contraction but restoring the ratio in cellular composition (CMs and cFBs) is also a prerequisite to completely regain tissue function.

Tendinopathy is a widespread and unresolved clinical challenge, in which associated pain and hampered mobility present a major cause for work-related disability. Tendinopathy associates with a change from a healthy tissue with aligned extracellular matrix (ECM) and highly polarized cells that are connected head-to-tail, towards a diseased tissue with a disorganized ECM and randomly distributed cells, scar-like features that are commonly attributed to poor innate regenerative capacity of the tissue. A fundamental clinical dilemma with this scarring process is whether treatment strategies should focus on healing the affected (disorganized) tissue or strengthen the remaining healthy (anisotropic) tissue. The question was thus asked whether the intrinsic remodeling capacity of tendon-derived cells depends on the organization of the 3D extracellular matrix (isotropic vs anisotropic). Progress in this field is hampered by the lack of suitable in vitro tissue platforms. We aimed at filling this critical gap by creating and exploiting a next generation tissue platform that mimics aspects of the tendon scarring process; cellular response to a gradient in tissue organization from isotropic (scarred/non-aligned) to highly anisotropic (unscarred/aligned) was studied, as was a transient change from isotropic towards highly anisotropic. Strikingly, cells residing in an 'unscarred' anisotropic tissue indicated superior remodeling capacity (increased gene expression levels of collagen, matrix metalloproteinases MMPs, tissue inhibitors of MMPs), when compared to their 'scarred' isotropic counterparts. A numerical model then supported the hypothesis that cellular remodeling capacity may correlate to cellular alignment strength. This in turn may have improved cellular communication, and could thus relate to the more pronounced connexin43 gap junctions observed in anisotropic tissues. In conclusion, increased tissue anisotropy was observed to enhance the cellular potential for functional remodeling of the matrix. This may explain the poor regenerative capacity of tenocytes in chronic tendinopathy, where the pathological process has resulted in ECM disorganization. Additionally, it lends support to treatment strategies that focus on strengthening the remaining healthy tissue, rather than regenerating scarred tissue.

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well-characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load-deprived environment or in a custom-designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum-free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load-deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative-like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low-magnitude mechanical loading. Thus a minimal explant culture model featuring serum-free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1383-1390, 2018.