Macromechanics and polycaprolactone fiber organization drive macrophage polarization and regulate inflammatory activation of tendon in vitro and in vivo.

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.

The relationship between metastatic potential and in vitro mechanical properties of osteosarcoma cells.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.

Substrate fiber alignment mediates tendon cell response to inflammatory signaling.

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. STATEMENT OF SIGNIFICANCE: 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.

Macrophage Polarization by Titanium Dioxide (TiO2) Particles: Size Matters.

Wear particles of total joint replacements may lead to an inflammatory response driven by cells of the monocyte/macrophage lineage. Today, there is a general agreement that the continuous release of wear particles by the implant has a critical impact on periprosthetic osteolysis, which can eventually lead to aseptic loosening of the implant. The focus of this study lay on the determination of the polarization of macrophages (M0) toward the pro-inflammatory M1 phenotype or the anti-inflammatory M2-like phenotype upon exposure to differently sized TiO2 particles. The analysis was done with an in vitro model using THP-1 monocytes. It offers a direct characterization of the polarization profile of the macrophages exposed to nano- (<100 nm, measured hydrodynamic diameter: 518.5 nm) and micro- (<5 µm, measured hydrodynamic diameter: 2213 nm) sized TiO2 particles in different concentrations (4 × 104 -4 × 106 particles/mL). The polarization profile was analyzed by the quantitative assessment of relative gene expression levels as well as by the determination of specific proteins by enzyme linked immunosorbent assay (ELISA). Analysis by qRT-PCR revealed significantly elevated levels of pro-inflammatory markers such as TNF-α and CD197 at the highest concentration of stimulation by the microsized particles. This was confirmed on the protein level in the cytokine expression profile of TNF-α. Furthermore, no significant differences were found for the markers CCL22 and CD206, which are specific for the M2-like phenotype. In contrast, stimulation by nanoparticles did not induce macrophage polarization toward M1 or M2-like phenotype in any applied concentration. We conclude that the size of the particle is a determinant factor in driving the biological response of macrophages and an increased understanding of this relationship may potentially guide the design of new biomaterials.