Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 μM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen

Development and Evaluation of a Nanometer-Scale Hemocompatible and Antithrombotic Coating Technology Platform for Commercial Intracranial Stents and Flow Diverters

An intracranial aneurysm is a local dilation of an artery in the cerebral circulation and can be endovascularly treated with two types of medical devices known as intracranial stents or flow diverters–both are metallic devices that help redirect blood from the diseased arterial segment; yet the placement of intracranial devices in the cerebral circulation mandates the adjunctive administration of dual antiplatelet pharmaceuticals to the patient to minimize thromboembolic events, despite being associated with increased patient risk. We present a new multilayer, nanometer-scale coating technology platform suitable for commercial intracranial flow diverters to minimize the use of dual antiplatelet therapy in the elective setting and expand the use of intracranial devices in the acute setting of ruptured intracranial aneurysms. A combination of qualitative and quantitative characterization techniques including scanning electron microscopy, ellipsometry, confocal microscopy, X-ray photoelectron spectroscopy, and focused ion beam milling coupled with scanning electron microscopy were used to assess the composition, uniformity, and thickness of each coating layer on commercially available flow diverting devices. Overall, the coating was found to be relatively uniform, less than 50 nm thick, and conformal to device microwires. X-ray photoelectron spectroscopy data further indicates the developed nanoscale coating technology can be modified for use as a platform for the attachment of human recombinant thrombomodulin, a naturally occurring glycoprotein with antithrombotic functionality. The in vitro thrombin generation capacity of commercial intracranial flow diverters coated with the technology was assessed using the calibrated automated thrombogram assay; further, platelet and fibrin deposition on coated commercial flow diverters was assessed ex vivo via a primate arteriovenous shunt model. The in vitro and ex vivo test results suggest potential hemocompatible and antithrombotic properties