Ultrasoft microgels displaying emergent platelet-like behaviours.

Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.

Vascularization of Human Acellular Dermal Matrices: A Comparative Study in a Nonhuman Primate Model.

Four human acellular dermal matrices (hADMs) were characterized in a nonhuman primate abdominal wall repair model by evaluating host immune response, vascularization, and incorporation into host tissues. AlloDerm™ (electron beam-sterilized hADM [e-hADM]), AlloMax™ (gamma beam-sterilized hADM, freeze-dried [g-hADM-FD]), DermaMatrix™ (hADM, freeze-dried [hADM-FD]), and FlexHD™ (ethanol-treated hADM [EtOH-hADM]) were each implanted in an abdominal wall-bridging defect in nonhuman primates (n = 3 animals/time point, n = 36 animals). Immunohistochemical and histological assessments were conducted on biopsies from each hADM at 1-, 3-, and 6-months postimplantation to assess vascularization (hematoxylin and eosin [H&E], CD31, alpha smooth muscle actin [αSMA], collagen IV), inflammatory/immune response (H&E, CD3, CD20, CD68), and collagen turnover (H&E, matrix metalloproteinase-9 [MMP-9]). MMP-9 immunolabeling was similar among different hADMs at 1 month; however, hADM-FD and EtOH-hADM showed higher total mean MMP-9-immunopositive areas at approximately 16% compared with <1% for e-hADM and g-hADM at 6 months postimplantation. Cells that stained positively for CD68, CD3, and CD20 were generally higher for hADM-FD and EtOH-hADM compared with other hADMs. The mean CD31-immunopositive area, CD31 vessel density, CD31 vessel diameter, and collagen IV-immunopositive area increased over time. Among all the hADM types, e-hADM had the highest mean (±standard deviation [SD]) CD31-immunopositive area at 1.54% ± 1.01%, vessel density at 7.86 × 10-5 ± 3.96 × 10-5 vessels/µm2, and collagen IV-immunopositive area at 2.55% ± 0.73% 1-month postimplantation. The pattern of αSMA immunolabeling varied among the hADMs. Histology showed that overall inflammation was mild at 1 month. Overall fibroblast repopulation and collagen remodeling increased over time from 1 to 6 months postimplantation. Fibroblast infiltration was minimal to mild at 1 month, with e-hADM showing the highest mean (±SD) score at 2.00 ± 0.00 compared with other hADMs. Only hADM-FD was not completely replaced by neotissue formation at 6 months postimplantation. All hADMs promoted vascularization, cell infiltration, and incorporation into host tissue, which were associated with acute inflammation and immune responses, within a 6-month period. A trend toward relatively enhanced early vascularization in e-hADM compared with other hADMs was observed. Immunogenic responses among the hADMs in the present study showed a slight distinction toward more quiescent terminally sterilized hADMs (e-hADM, g-hADM-FD) versus aseptically processed hADMs (EtOH-hADM, hADM-FD).

Design matters: A comparison of natural versus synthetic skin substitutes across benchtop and porcine wound healing metrics: An experimental study.

Background and Aims: Skin substitutes, essential tools for helping close full thickness wounds with minimal scarring, are available in both collagen-based and synthetic polyurethane constructions. Here we explore fundamental differences between two frequently used skin substitutes and discuss how these differences may impact in vivo performance. Methods: Polyurethane- and collagen-based matrices were characterized in vitro for pore size via scanning electron microscopy, hydrophobicity via liquid contact angle, conformability via bending angle, and biocompatibility via fibroblast and keratinocyte adhesion and proliferation. These matrices were then evaluated in a full-thickness excisional pig wound study followed by histological analysis. Statistical analysis was performed using t-tests or one-way analysis of variances with Tukey's multiple post hoc comparisons, where appropriate. Results: Average pore diameter in the tested polyurethane matrix was over four times larger than that of the collagen matrix (589 ± 297 µm vs. 132 ± 91 µm). Through liquid contact angle measurement, the collagen matrix (not measurable) was found to be hydrophilic compared to the hydrophobic polyurethane matrix (>90°). The collagen matrix was significantly more conformable than the polyurethane matrix (9 ± 2° vs. 84 ± 5° bending angle, respectively). Fibroblast and keratinocyte adhesion and proliferation assays elucidated a significantly greater ability of both cell types to attach and proliferate on collagen versus polyurethane. While the porcine study showed minimal contraction of either matrix material, histological findings between the two treatments were markedly different. Collagen matrices were associated with early fibroblast infiltration and fibroplasia, whereas polyurethane matrices elicited a strong multinucleated giant cell response and produced a network of comparatively aligned collagen fibrils. Conclusions: The more favorable in vitro properties of the collagen matrix led to less inflammation and better overall tissue response in vivo. Overall, our findings demonstrate how the choice of biomaterial and its design directly translate to differing in vivo mechanisms of action and overall tissue quality.

Biophysical Characterization of a Novel Tri-Layer Placental Allograft Membrane.

Objective: Placental tissues, including membranes composed of amnion and chorion, are promising options for the treatment of chronic wounds. Amnion and chorion contain multiple extracellular matrix (ECM) proteins and a multitude of growth factors and cytokines that, when used clinically, assist in the progression of difficult to heal wounds through restoration of a normal healing process. The objective of this study was to characterize the in vitro physical and biological properties of a dehydrated tri-layer placental allograft membrane (TPAM) consisting of a chorion layer sandwiched between two layers of amnion. Approach: Mechanical properties were evaluated by mechanical strength and enzyme degradation assays. The ECM composition of TPAM membranes was evaluated by histological staining while growth factors and cytokine presence was evaluated by a multiplex enzyme-linked immunosorbent assay. Proliferation, migration, and ECM secretion assays were performed with fibroblasts. Immunomodulatory properties were assessed by a pro-inflammatory cytokine reduction assay while the macrophage phenotype was determined by quantifying the ratio of M1 versus M2 secreted factors. Results: The unique three-layer construction improves mechanical handling properties over single- and bi-layer membranes. Results demonstrate that TPAM is rich in ECM proteins, growth factors, cytokines, and tissue inhibitors of metalloproteinases, and favorably influences fibroblast migration, proliferation, and ECM secretion when compared to negative controls. Furthermore, after processing and preservation, these membranes maintain their intrinsic immunomodulatory properties with the ability to suppress pro-inflammatory processes and modulate the M1 and M2 macrophage phenotype toward a pro-regenerative profile when compared to a negative control. Innovation: This is the first study to characterize both the biophysical and biological properties of a tri-layer placental membrane. Conclusion: This work demonstrates that TPAM has improved handling characteristics over single- and bi-layer membranes, stimulates pro-healing cellular responses, and advantageously modulates inflammatory responses, altogether making this scaffold a promising option for treating wounds, especially those that are complex or difficult to heal.

Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion.

The extracellular matrix (ECM) microenvironment is increasingly implicated in the instruction of pathologically relevant cell behaviors, from aberrant transdifferentation to invasion and beyond. Indeed, pathologic ECMs possess a panoply of alterations that provide deleterious instructions to resident cells. Here we demonstrate the precise manner in which the ECM protein fibronectin (FN) undergoes the posttranslational modification citrullination in response to peptidyl-arginine deiminase (PAD), an enzyme associated with innate immune cell activity and implicated in systemic ECM-centric diseases, like cancer, fibrosis and rheumatoid arthritis. FN can be citrullinated in at least 24 locations, 5 of which reside in FN's primary cell-binding domain. Citrullination of FN alters integrin clustering and focal adhesion stability with a concomitant enhancement in force-triggered integrin signaling along the FAK-Src and ILK-Parvin pathways within fibroblasts. In vitro migration and in vivo wound healing studies demonstrate the ability of citrullinated FN to support a more migratory/invasive phenotype that enables more rapid wound closure. These findings highlight the potential of ECM, particularly FN, to "record" inflammatory insults via post-translational modification by inflammation-associated enzymes that are subsequently "read" by resident tissue fibroblasts, establishing a direct link between inflammation and tissue homeostasis and pathogenesis through the matrix.

The evolution of fibrin-specific targeting strategies.

Fibrin-specific targeting capabilities have been highly sought for over 50 years due to their implications for bio-molecule delivery, diagnostics, and regenerative medicine. Yet only recently has our full knowledge of fibrin's complex polymerization dynamics and biological interactions begun to be fully exploited in pursuit of this goal. This highlight will discuss the range of rapidly changing strategies for specifically targeting fibrin over the precursor fibrinogen and the advantages and disadvantages of these approaches for various applications.