Development of biomimetic antimicrobial platelet-like particles comprised of microgel nanogold composites.

A blood clot is formed in response to bleeding by platelet aggregation and adherence to fibrin fibers. Platelets contract over time, stabilizing the clot, which contributes to wound healing. We have developed platelet-like particles (PLPs) that augment clotting and induce clot retraction by mimicking the fibrin-binding capabilities and morphology of native platelets. Wound repair following hemostasis can be complicated by infection; therefore, we aim to augment wound healing by combining PLPs with antimicrobial gold to develop nanogold composites (NGCs). PLPs were synthesized with N-isopropylacrylamide (NIPAm)/co-acrylic acid in a precipitation polymerization reaction and conjugated to a fibrin-specific antibody. Two methods were employed to create NGCs: 1) noncovalent swelling with aqueous gold nanospheres, and 2) covalent seeding and growth. Since the ability of PLPs to mimic platelet morphology and clot retraction requires a high degree of particle deformability, we investigated how PLPs created from NGCs affected these properties. Cryogenic Scanning Electron Microscopy (cryoSEM) and atomic force microscopy (AFM) demonstrated that particle deformability, platelet-mimetic morphology and clot retraction were maintained in NGC-based PLPs. The effect of NGCs on bacterial adhesion and growth was assessed with antimicrobial assays. These results demonstrate NGCs fabricated through noncovalent and covalent methods retain deformability necessary for clot collapse and exhibit some antimicrobial potential. Therefore, NGCs are promising materials for preventing hemorrhage and infection following trauma.

Platelet-like particles dynamically stiffen fibrin matrices and improve wound healing outcomes.

Native platelets perform several critical functions within the context of wound healing, including participating in initial hemostasis and interacting with fibrin at the wound site to induce clot retraction. Platelet depletion or dysfunction due to trauma or disease can inhibit robust wound healing responses. There has been a focus recently on developing synthetic, non-immunogenic platelet mimetic technologies for the purpose of augmenting hemostatic responses in cases of deficient native platelet functionality. Here we describe the application of synthetic platelet-like particles (PLPs), capable of recapitulating the deformable platelet body and fibrin specificity found in native platelets, to enhance healing outcomes. We first demonstrate PLPs mimic activated platelet morphology and induce fibrin clot retraction. During clot retraction, native platelets generate forces within a fibrin network to stiffen the fibrin matrix; therefore, we hypothesized that our PLPs will likewise be able to stiffen provisional fibrin matrices. Due to previous studies indicating that increased matrix stiffness is linked to increased cellular migration, we further hypothesize that PLP-mediated fibrin stiffening will enhance cell migration and improve healing outcomes within in vitro and in vivo models of wound healing. PLPs were found to enhance fibroblast migration in in vitro models of early wound healing and enhance healing outcomes in an in vivo murine model of wound healing. These studies demonstrate the utility of PLPs for enhancing wound repair and also provide insight into the role of native platelet-mediated clot retraction in wound healing.

Fibrin Nanoparticles Coupled with Keratinocyte Growth Factor Enhance the Dermal Wound-Healing Rate.

Expediting the wound-healing process is critical for patients chronically ill from nonhealing wounds and diseases such as hemophilia or diabetes or who have suffered trauma including easily infected open wounds. FDA-approved external tissue sealants include the topical application of fibrin gels, which can be 500 times denser than natural fibrin clots. With lower clot porosity and higher polymerization rates than physiologically formed fibrin clots, the commercial gels quickly stop blood loss but impede the later clot degradation kinetics and thus retard tissue-healing rates. The fibrin nanoparticles (FBNs) described here are constructed from physiologically relevant fibrin concentrations that support new tissue and dermal wound scaffold formation when coupled with growth factors. The FBNs, synthesized in a microfluidic droplet generator, support cell adhesion and traction generation, and when coupled to keratinocyte growth factor (KGF), support cell migration and in vivo wound healing. The FBN-KGF particles enhance cell migration in vitro greater than FBN alone or free KGF and also improve healing outcomes in a murine full thickness injury model compared to saline, bulk fibrin sealant, free KGF, or bulk fibrin mixed with KGF treatments. Furthermore, FBN can be potentially administered with other tissue-healing factors and inflammatory mediators to improve wound-healing outcomes.

Controlling Fibrin Network Morphology, Polymerization, and Degradation Dynamics in Fibrin Gels for Promoting Tissue Repair.

Fibrin is an integral part of the clotting cascade and is formed by polymerization of the soluble plasma protein fibrinogen. Following stimulation of the coagulation cascade, thrombin activates fibrinogen, which binds to adjacent fibrin(ogen) molecules resulting in the formation of an insoluble fibrin matrix. This fibrin network is the primary protein component in clots and subsequently provides a scaffold for infiltrating cells during tissue repair. Due to its role in hemostasis and tissue repair, fibrin has been used extensively as a tissue sealant. Clinically used fibrin tissue sealants require supraphysiological concentrations of fibrinogen and thrombin to achieve fast polymerization kinetics, which results in extremely dense fibrin networks that are inhibitory to cell infiltration. Therefore, there is much interest in developing fibrin-modifying strategies to achieve rapid polymerization dynamics while maintaining a network structure that promotes cell infiltration. The properties of fibrin-based materials can be finely controlled through techniques that modulate fibrin polymerization dynamics or through the inclusion of fibrin-modifying biomaterials. Here, we describe methods for characterizing fibrin network morphology, polymerization, and degradation (fibrinolysis) dynamics in fibrin constructs for achieving fast polymerization dynamics while promoting cell infiltration.

Biomimetic microgels with controllable deformability improve healing outcomes.

Platelets mediate hemostasis by aggregating and binding to fibrin to promote clotting. Over time, platelets contract the fibrin network to induce clot retraction, which contributes to wound healing outcomes by increasing clot stability and improving blood flow to ischemic tissue. In this study, we describe the development of hollow platelet-like particles (PLPs) that mimic the native platelet function of clot retraction in a controlled manner and demonstrate that clot retraction-inducing PLPs promote healing in vivo. PLPs are created by coupling fibrin-binding antibodies to CoreShell (CS) or hollow N-isopropylacrylamide (NIPAm) microgels with varying degrees of shell crosslinking. We demonstrate that hollow microgels with loosely crosslinked shells display a high degree of deformability and mimic activated platelet morphology, while intact CS microgels and hollow microgels with increased crosslinking in the shell do not. When coupled to a fibrin-binding antibody to create PLPs, hollow particles with low degrees of shell crosslinking cause fibrin clot collapse in vitro, recapitulating the clot retraction function of platelets, while other particle types do not. Furthermore, hollow PLPs with low degrees of shell crosslinking improve some wound healing outcomes in vivo.

Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes.

Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening and macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In this study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in rat ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5-fold. Gene ontology analysis showed that components of the extracellular matrix were the most significantly affected by V3 expression. In addition, genes regulating the formation of the cytoskeleton, which also serve as markers of contractile smooth muscle cells (SMCs), were significantly up-regulated. In contrast, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most down-regulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile SMC phenotype, was greatly increased, and the proinflammatory transcription factors NFκB1 and CCAAT/enhancer-binding protein ß were significantly attenuated in V3-expressing SMCs. Overall, these findings demonstrate that V3 expression reprograms ASMCs promoting differentiated and anti-inflammatory phenotypes.

A cytokine axis regulates elastin formation and degradation.

Underlying the dynamic regulation of tropoelastin expression and elastin formation in development and disease are transcriptional and post-transcriptional mechanisms that have been the focus of much research. Of particular importance is the cytokine-governed elastin regulatory axis in which the pro-elastogenic activities of transforming growth factor ß-1 (TGFß1) and insulin-like growth factor-I (IGF-I) are opposed by anti-elastogenic activities of basic fibroblast growth factor (bFGF/FGF-2), heparin-binding epidermal growth factor-like growth factor (HB-EGF), EGF, PDGF-BB, TGFα, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß and noncanonical TGFß1 signaling. A key mechanistic feature of the regulatory axis is that cytokines influence elastin formation through effects on the cell cycle involving control of cyclin-cyclin dependent kinase complexes and activation of the Ras/MEK/ERK signaling pathway. In this article we provide an overview of the major cytokines/growth factors that modulate elastogenesis and describe the underlying molecular mechanisms for their action on elastin production.