Appropriate macrophage response to an implanted biomaterial is crucial for successful tissue healing outcomes. In this work we investigated how intrinsic topological cues from electrospun biomaterials and extrinsic mechanical loads cooperate to guide macrophage activation and macrophage-tendon fibroblast cross-talk. We performed a series of in vitro and in vivo experiments using aligned or randomly oriented polycaprolactone nanofiber substrates in both mechanically loaded and unloaded conditions. Across all experiments a disorganized biomaterial fiber topography was alone sufficient to promote a pro-inflammatory signature in macrophages, tendon fibroblasts, and tendon tissue. Extrinsic mechanical loading was found to strongly regulate the character of this signature by reducing pro-inflammatory markers both in vitro and in vivo. We observed that macrophages generally displayed a stronger response to biophysical cues than tendon fibroblasts, with dominant effects of cross-talk between these cell types observed in mechanical co-culture models. Collectively our data suggest that macrophages play a potentially important role as mechanosensory cells in tendon repair, and provide insight into how biological response might be therapeutically modulated by rational biomaterial designs that address the biomechanical niche of recruited cells.

Healthy tendon tissue features a highly aligned extracellular matrix that becomes disorganized with disease. Recent evidence suggests that inflammation coexists with early degenerative changes in tendon, and that crosstalk between immune-cells and tendon fibroblasts (TFs) can contribute to poor tissue healing. We hypothesized that a disorganized tissue architecture may predispose tendon cells to degenerative extracellular matrix remodeling pathways, particularly within a pro-inflammatory niche. This hypothesis was tested by analyzing human TFs cultured on electrospun polycaprolactone (PCL) mats with either highly aligned or randomly oriented fiber structures. We confirmed that fibroblast morphology, phenotype, and markers of matrix turnover could be significantly affected by matrix topography. More strikingly, the TF response to paracrine signals from polarized macrophages or by stimulation with pro-inflammatory cytokines featured significant downregulation of signaling related to extracellular synthesis, with significant concomitant upregulation of gene and protein expression of matrix degrading enzymes. Critically, this tendency towards degenerative re-regulation was exacerbated on randomly oriented PCL substrates. These novel findings indicate that highly aligned tendon cell scaffolds not only promote tendon matrix synthesis, but also play a previously unappreciated role in mitigating adverse resident fibroblast response within an inflammatory milieu. 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.

Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. Here we show that human FHOD1 is lacking actin nucleation and elongation capacity, but acts as actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin and confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

Significance: Cell instructive biomaterial cues are a major topic of interest in both basic and applied research. In this work, we clarify how surface energy of soft biomaterials can dramatically affect mesenchymal stem cell receptor recruitment and downstream signaling related to cell fate. We elucidate how surface protein self-assembly and the resulting surface topology can act to steer mechanotransduction and related biological response of attached cells. These findings fill a critical gap in our basic understanding of cell–biomaterial interaction and highlight soft biomaterial surface energy as a dominant design factor that should not be neglected.

The accurate determination of cellular forces using Traction Force Microscopy at increasingly small focal attachments to the extracellular environment presents an important yet substantial technical challenge. In these measurements, uncertainty regarding accuracy is prominent since experimental calibration frameworks at this size scale are fraught with errors – denying a gold standard against which accuracy of TFM methods can be judged. Therefore, we have developed a simulation platform for generating synthetic traction images that can be used as a benchmark to quantify the influence of critical experimental parameters and the associated errors. Using this approach, we show that TFM accuracy can be improved >35% compared to the standard approach by placing fluorescent beads as densely and closely as possible to the site of applied traction. Moreover, we use the platform to test tracking algorithms based on optical flow that measure deformation directly at the beads and show that these can dramatically outperform classical particle image velocimetry algorithms in terms of noise sensitivity and error. We then report how optimized experimental and numerical strategy can improve traction map accuracy, and further provide the best available benchmark to date for defining practical limits to TFM accuracy as a function of focal adhesion size.