Evaluation of a microphysiological human placental barrier model for studying placental drug transfer.

Understanding drug transport across the placental barrier is important for assessing the potential fetal drug toxicity and birth defect risks. Current in vivo and in vitro models have structural and functional limitations in evaluating placental drug transfer and toxicity. Microphysiological systems (MPSs) offer more accurate and relevant physiological models of human tissues and organs on a miniature scale for drug development and toxicology testing. MPSs for the placental barrier have been recently explored to study placental drug transfer. We utilized a multilayered hydrogel membrane-based microphysiological model composed of human placental epithelial and endothelial cells to replicate the key structure and function of the human placental barrier. A macroscale human placental barrier model was created using a transwell to compare the results with the microphysiological model. Placental barrier models were characterized by assessing monolayer formation, intercellular junctions, barrier permeability, and their structural integrity. Three small-molecule drugs (glyburide, rifaximin, and caffeine) that are prescribed or taken during pregnancy were studied for their placental transfer. The results showed that all three drugs crossed the placental barrier, with transfer rates in the following order: glyburide (molecular weight, MW = 494 Da) < rifaximin (MW = 785.9 Da) < caffeine (MW = 194.19 Da). Using non-compartmental analysis, we estimated human pharmacokinetic characteristics based on in vitro data from both MPS and transwell models. While further research is needed, our findings suggest that MPS holds potential as an in vitro tool for studying placental drug transfer and predicting fetal exposure, offering insights into pharmacokinetics.

Effect of upstream priming on transient downstream platelet-substrate interactions.

Upstream exposure of platelets to activating proteins 'primes' platelets for increased downstream adhesion, though the mechanics of platelet translocation before permanently arresting are not well understood. To investigate platelet translocation on platelet-binding proteins, primed platelets' transient contacts with immobilized proteins were recorded and analyzed. Using a microfluidic channel, representative of a vascular graft, platelet-activating proteins were covalently attached to the upstream priming, center, and downstream capture positions. Image sequences of platelet interactions with the center protein were captured as platelet-rich plasma (PRP) was perfused through the channel. There was an increase in both platelet pause events and net platelet adhesion on von Willebrand factor, collagen, or fibrinogen following upstream exposure to the same protein. Upstream priming also caused a decrease in average platelet velocity. The duration of transient platelet arrests on the protein-coated surface and the distance that platelets travel between pause events depended on the protein with which they were interacting. The most significant increase in platelet pause events frequency and decrease in average velocity occurred on immobilized von Willebrand factor, compared to the control with no upstream priming. These results demonstrate that platelet priming increases downstream platelet-protein interactions prior to permanent adhesion.

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region. Whole blood with an antiplatelet agent was transiently exposed to an upstream high wall shear rate (either 4860 s-1 or 11 560 s-1), and subsequently flowed over the downstream capture region where the platelet adhesion was measured. Several antiplatelet agents (acetylsalicylic acid, tirofiban, eptifibatide, anti-vWf, and anti-GPIbα) were evaluated for their efficacy in attenuating downstream adhesion. Following antibody blocking of vWf or GPIbα, downstream platelet activation was also assessed in perfused blood by flow cytometry using two activation markers (active GPIIb/IIIa and P-selectin). Acetylsalicylic acid demonstrated its inability to diminish shear-induced platelet adhesion to all three binding proteins. GPIIb/IIIa inhibitors (tirofiban and eptifibatide) significantly reduced platelet adhesion to fibrinogen. Antibody blocking of vWf or GPIbα effectively diminished platelet adhesion to all three capture proteins as well as platelet activation in perfused blood, indicating an essential role of vWf-GPIbα interaction in mediating shear-induced platelet aggregation.

Effects of elapsed time on downstream platelet adhesion following transient exposure to elevated upstream shear forces.

Transient exposure to elevated shear forces is known to prime platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. In the present study, the platelet capture regions, prepared by immobilizing fibrinogen, collagen, or von Willebrand factor, were placed at three different distances from the upstream stenotic region to vary the elapsed time of circulating platelets downstream. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s-1 to 11,560 s-1 for all three downstream proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and the downstream capture region. In contrast, platelet adhesion to downstream collagen remained essentially independent on the distance and the adhesion to von Willebrand factor marginally increased with the distance after transient platelet exposure to upstream wall shear rates of 2145 s-1 and 11,560 s-1. The results implied that the activation of fibrinogen receptor GPIIb/IIIa by transient exposure to high upstream wall shear rates progresses in a time-dependent manner during the downstream flow of platelets. The highly elevated upstream wall shear rate of 11,560 s-1 altered the morphology of many platelets adhered to downstream fibrinogen from their native ellipsoidal to spread circular form. The platelet shape analysis showed that longer periods of post-stenotic flow increased the surface coverage fraction of ellipsoidal platelet population and decreased the surface coverage fraction of fully spread platelets on fibrinogen for both transiently elevated upstream wall shear rates.

Downstream platelet adhesion and activation under highly elevated upstream shear forces.

Elevated shear force caused by an anastomotic stenosis is a common complication at the blood vessel-vascular implant interface. Although elevated shear forces were found to cause platelet aggregation around a stenotic region, transient platelet exposure to elevated shear forces and subsequent downstream events occurring under lower shear force were not extensively studied. We hypothesize that effects of elevated shear forces on pre-activation of platelets for downstream adhesion and activation are relevant in understanding the increased thrombotic risk associated with blood-contacting devices. We designed a microfluidic flow system to mimic the hemodynamic environment of vasculature with an upstream anastomotic stenosis with five wall shear strain rates ranging from 1620 s-1 to 11560 s-1. Under shear flow conditions, transient exposure of whole blood to elevated shear forces resulted in higher downstream platelet adhesion onto three different immobilized platelet agonists: fibrinogen, collagen, or von Willebrand factor. Platelet expression of four activation markers (P-selectin, GPIIb/IIIa, lysosomal glycoprotein, and phosphatidylserine) significantly increased after transient exposure to higher upstream wall shear strain rates of 2975-11560 s-1. A significant lysis was observed when platelets were primed by upstream wall shear strain rate of 11560 s-1. These experimental results could be helpful to understand how altered hemodynamics around an anastomotic stenosis promotes thrombus formation downstream. STATEMENT OF SIGNIFICANCE: Studying the downstream response of platelets following transient exposure to an upstream agonist is important because of significant clinical implications to the implantation of vascular devices. Due to intimal fibrous hyperplasia, vascular biomaterials such as synthetic small-diameter vascular grafts sometimes become stenotic (narrow), leading to transient platelet exposure to elevated shear forces. In this study, a microfluidic flow system was developed to mimic a stenosed vascular graft and to investigate how highly elevated, transient upstream shear forces, typically found in severe stenosis, results in the pre-activation of platelets for downstream adhesion and activation. The findings of the present study have implications for optimizing the design of blood-contacting biomaterials in order to minimize thrombotic risk associated with transiently elevated shear forces. The findings also provide additional insights into the mechanisms of thrombus formation at the post-stenotic regions of vascular implants.

Effects of upstream shear forces on priming of platelets for downstream adhesion and activation.

Platelets in flowing blood are sometimes exposed to elevated shear forces caused by anastomotic stenosis at the blood vessel-vascular implant interface. The objective of this study was to determine how effective upstream shear forces are in priming platelets for downstream adhesion and activation. Flow chambers with upstream stenotic regions (shear rates of 400-1000 s-1) were manufactured by relief molding of polydimethylsiloxane. Downstream from the stenotic regions, microcontact printing was used to covalently immobilize three different proteins (fibrinogen, collagen, or von Willebrand factor) to serve as platelet capture agents. Anticoagulated whole blood was perfused through the flow chambers and platelet adhesion to the downstream capture region was quantified. It was found that transient exposure of platelets to increased shear forces resulted in higher platelet adhesion on all three proteins. The duration of the platelet exposure to elevated shear forces was varied by changing the length of the stenotic regions. The results indicated that, in addition to the magnitude of shear forces, the duration of exposure to these forces was also an important factor in priming platelets. The effect of upstream shear forces on platelet activation was assessed by quantifying P-selectin, integrin αIIbß3, lysosomal glycoprotein, and phosphatidylserine exposure using flow cytometry. The results suggested that increased shear forces were capable of increasing the priming of platelets for downstream activation. This study implicates the anastomotic region(s) of vascular implants as a locus of platelet pre-activation that may lead to thrombus formation downstream. STATEMENT OF SIGNIFICANCE: A synthetic small-diameter vascular graft can often become stenotic due to intimal fibrous hyperplasia, either generally along the inside of the graft or at the anastomotic regions, leading to an increased shear force on flowing platelets. Our lab is studying how the upstream platelet preactivation (aka "priming") in flowing blood affects their downstream adhesion and activation. This manuscript describes a study in which priming of platelets is achieved by upstream stenotic narrowing in a microfluidic flow chamber. Such experimental design was intended to mimic a vascular implant with stenotic upstream anastomosis and downstream exposed platelet protein agonists. Understanding how the pre-activated platelets respond to imperfect vascular implant surfaces downstream is an important factor in designing better vascular implants.

Fabrication and Characterization of Magnesium Ferrite-Based PCL/Aloe Vera Nanofibers.

Composite nanofibers of biopolymers and inorganic materials have been widely explored as tissue engineering scaffolds because of their superior structural, mechanical and biological properties. In this study, magnesium ferrite (Mg-ferrite) based composite nanofibers were synthesized using an electrospinning technique. Mg-ferrite nanoparticles were first synthesized using the reverse micelle method, and then blended in a mixture of polycaprolactone (PCL), a synthetic polymer, and Aloe vera, a natural polymer, to create magnetic nanofibers by electrospinning. The morphology, structural and magnetic properties, and cellular compatibility of the magnetic nanofibers were analyzed. Mg-ferrite/PCL/Aloe vera nanofibers showed good uniformity in fiber morphology, retained their structural integrity, and displayed magnetic strength. Experimental results, using cell viability assay and scanning electron microscopy imaging showed that magnetic nanofibers supported 3T3 cell viability. We believe that the new composite nanofibrous membranes developed in this study have the ability to mimic the physical structure and function of tissue extracellular matrix, as well as provide the magnetic and soluble metal ion attributes in the scaffolds with enhanced cell attachment, and thus improve tissue regeneration.

Aloe Vera for Tissue Engineering Applications.

Aloe vera, also referred as Aloe barbadensis Miller, is a succulent plant widely used for biomedical, pharmaceutical and cosmetic applications. Aloe vera has been used for thousands of years. However, recent significant advances have been made in the development of aloe vera for tissue engineering applications. Aloe vera has received considerable attention in tissue engineering due to its biodegradability, biocompatibility, and low toxicity properties. Aloe vera has been reported to have many biologically active components. The bioactive components of aloe vera have effective antibacterial, anti-inflammatory, antioxidant, and immune-modulatory effects that promote both tissue regeneration and growth. The aloe vera plant, its bioactive components, extraction and processing, and tissue engineering prospects are reviewed in this article. The use of aloe vera as tissue engineering scaffolds, gels, and films is discussed, with a special focus on electrospun nanofibers.

Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application.

Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva. Commercial GTR membranes are typically composed of synthetic polymers that have had mild clinical success mostly because of their lack of proper bioactivity and appropriate degradation profile. In this study, a natural polymer, aloe vera was blended with polycaprolactone (PCL) to create nanofibrous GTR membranes by electrospinning. Aloe vera has proven anti-inflammatory properties and enhances the regeneration of periodontium tissues. PCL, a synthetic polymer, is well known to produce miscible polyblends nanofibers with natural polymers. Nanofibrous membranes with varying composition of PCL to aloe vera were fabricated, and several physicochemical and biological properties, such as fiber morphology, wettability, chemical structure, mechanical strength, and cellular compatibility of the membranes were analyzed. PCL/aloe vera membranes with ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in aqueous solutions. Experimental results, using cell viability assay and cell attachment observation, showed that the nanofibrous membranes support 3T3 cell viability and could be a potential candidate for GTR therapy.