Getting a good view: in vitro imaging of platelets under flow.

As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.

Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease.

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.

Platelet Mechanotransduction.

The vasculature is a dynamic environment in which blood platelets constantly survey the endothelium for sites of vessel damage. The formation of a mechanically coherent hemostatic plug to prevent blood loss relies on a coordinated series of ligand-receptor interactions governing the recruitment, activation, and aggregation of platelets. The physical biology of each step is distinct in that the recruitment of platelets depends on the mechanosensing of the platelet receptor glycoprotein Ib for the adhesive protein von Willebrand factor, whereas platelet activation and aggregation are responsive to the mechanical forces sensed at adhesive junctions between platelets and at the platelet-matrix interface. Herein we take a biophysical perspective to discuss the current understanding of platelet mechanotransduction as well as the measurement techniques used to quantify the physical biology of platelets in the context of thrombus formation under flow.

Extracellular fluid tonicity impacts sickle red blood cell deformability and adhesion.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.

Clinical Implications of Single-Cell Microfluidic Devices for Hematological Disorders.

Single-cell microfluidic devices are poised to substantially impact the hematology field by providing a high-throughput and rapid device to analyze disease-mediated biophysical cellular changes in the clinical setting in order to diagnose patients and monitor disease prognosis. In this Feature, we cover recent advances of single-cell microfluidic devices for studying and diagnosing hematological dysfunctions and the clinical impact made possible by these advances.

Platelet-Microcapsule Hybrids Leverage Contractile Force for Targeted Delivery of Hemostatic Agents.

We report a cell-mediated, targeted drug delivery system utilizing polyelectrolyte multilayer capsules that hybridize with the patient's own platelets upon intravenous administration. The hybridized platelets function as the sensor and actuator for targeted drug delivery and controlled release in our system. These capsules are biochemically and mechanically tuned to enable platelet adhesion and capsule rupture upon platelet activation and contraction, enabling the targeted and controlled "burst" release of an encapsulated biotherapeutic. As platelets are the "first responders" in the blood clot formation process, this platelet-hybridized system is ideal for the targeted delivery of clot-augmenting biotherapeutics wherein immediate therapeutic efficacy is required. As proof-of-concept, we tailored this system to deliver the pro-clotting biotherapeutic factor VIII for hemophilia A patients that have developed inhibitory antifactor VIII antibodies. The polyelectrolyte multilayer capsules physically shield the encapsulated factor VIII from the patient's inhibitors during circulation, preserving its bioactivity until it is delivered at the target site via platelet contractile force. Using an in vitro microfluidic vascular injury model with factor VIII-inhibited blood, we demonstrate a 3.8× increase in induced fibrin formation using capsules loaded with factor VIII at a concentration an order of magnitude lower than that used in systemic delivery. We further demonstrate that clot formation occurs 18 min faster when factor VIII loaded capsules are used compared to systemic delivery at the same concentration. Because platelets are integral in the pathophysiology of thrombotic disorders, cancer, and innate immunity, this paradigm-shifting smart drug delivery system can be similarly applied to these diseases.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.

Ultrasoft, highly deformable microgels.

Microgels are colloidally stable, hydrogel microparticles that have previously been used in a range of (soft) material applications due to their tunable mechanical and chemical properties. Most commonly, thermo and pH-responsive poly(N-isopropylacrylamide) (pNIPAm) microgels can be fabricated by precipitation polymerization in the presence of the co-monomer acrylic acid (AAc). Traditionally pNIPAm microgels are synthesized in the presence of a crosslinking agent, such as N,N'-methylenebisacrylamide (BIS), however, microgels can also be synthesized under 'crosslinker free' conditions. The resulting particles have extremely low (<0.5%), core-localized crosslinking resulting from rare chain transfer reactions. AFM nanoindentation of these ultralow crosslinked (ULC) particles indicate that they are soft relative to crosslinked microgels, with a Young's modulus of ∼10 kPa. Furthermore, ULC microgels are highly deformable as indicated by a high degree of spreading on glass surfaces and the ability to translocate through nanopores significantly smaller than the hydrodynamic diameter of the particles. The size and charge of ULCs can be easily modulated by altering reaction conditions, such as temperature, monomer, surfactant and initiator concentrations, and through the addition of co-monomers. Microgels based on the widely utilized, biocompatible polymer polyethylene glycol (PEG) can also be synthesized under crosslinker free conditions. Due to their softness and deformability, ULC microgels are a unique base material for a wide variety of biomedical applications including biomaterials for drug delivery and regenerative medicine.

Microgel mechanics in biomaterial design.

The field of polymeric biomaterials has received much attention in recent years due to its potential for enhancing the biocompatibility of systems and devices applied to drug delivery and tissue engineering. Such applications continually push the definition of biocompatibility from relatively straightforward issues such as cytotoxicity to significantly more complex processes such as reducing foreign body responses or even promoting/recapitulating natural body functions. Hydrogels and their colloidal analogues, microgels, have been and continue to be heavily investigated as viable materials for biological applications because they offer numerous, facile avenues in tailoring chemical and physical properties to approach biologically harmonious integration. Mechanical properties in particular are recently coming into focus as an important manner in which biological responses can be altered. In this Account, we trace how mechanical properties of microgels have moved into the spotlight of research efforts with the realization of their potential impact in biologically integrative systems. We discuss early experiments in our lab and in others focused on synthetic modulation of particle structure at a rudimentary level for fundamental drug delivery studies. These experiments elucidated that microgel mechanics are a consequence of polymer network distribution, which can be controlled by chemical composition or particle architecture. The degree of deformability designed into the microgel allows for a defined response to an imposed external force. We have studied deformation in packed colloidal phases and in translocation events through confined pores; in all circumstances, microgels exhibit impressive deformability in response to their environmental constraints. Microgels further translate their mechanical properties when assembled in films to the properties of the bulk material. In particular, microgel films have been a large focus in our lab as building blocks for self-healing materials. We have shown that their ability to heal after damage arises from polymer mobility during hydration. Furthermore, we have shown film mobility dictates cell adhesion and spreading in a manner that is fundamentally different from previous work on mechanotransduction. In total, we hope that this Account presents a broad introduction to microgel research that intersects polymer chemistry, physics, and regenerative medicine. We expect that research intersection will continue to expand as we fill the knowledge gaps associated with soft materials in biological milieu.