HemoLens: An Open-Source Pressure Myography and Cardiac Simulator Platform

Introduction: : Ex-vivo characterization of blood vessel mechanics allows for tightly controlled environments to discriminate between cellular and extracellular remodeling processes. Many traditional mechanical testing methods have been applied to both native and engineered blood vessels to test various mechanical characteristics. Yet, pressure and wire myography remain the most ubiquitous methods for assessing vascular mechanics and graft compatibility for clinical use by measuring maximum burst pressure and vascular tone. Despite their ubiquity, however, commercial myograph systems can cost upwards of $40,000, are limited in throughput, and lack capabilities for customization. Recent open-source myography systems, such as “Vasotracker”, provide excellent software packages for monitoring vessel mechanics and a more affordable hardware platform, but still require custom CNC manufacturing and standalone microscopic imaging systems that drive costs above $6000. Moreover, no current system allows for experimentation in dynamic pressure environments that mimic physiologic cyclic pressure changes. Thus, there is an emerging need for a low-cost biomechanical myography platform specifically designed for testing small diameter ( < 1 mm) arteries and resistance vessels that use affordable manufacturing processes, integrated and accessible imaging systems, and can evaluate both conventional burst pressure and functional vasoreactivity under physiological conditions. To this end, we designed a modular open-source pressure myography system built from primarily 3D-printed and low-cost linear motion components called HemoLens. In this report, HemoLens was developed and validated by characterizing all mechanical aspects of the system and implemented to delineate mechanical differences of native vessels and FRESH printed acellular scaffolds.

Materials and

Methods: : HemoLens was modeled in Fusion360 (Autodesk). The primary structure of its components was 3D printed from Polylactic acid (PLA) and Polyethylene Terephthalate Glycol (PETG) with a BambuLab X1C. Commercial components, including screws, linear rails, and nuts, were added as needed. To measure micromanipulator precision, a digital indicator (Mitutoyo model# S112S) was zeroed and placed perpendicular to the back of the micromanipulator on HemoLens. The X, Y, and Z-axes were manually turned and linear travel displayed on the digital indicator was recorded. Native vessels were excised from twenty-week-old C57BL/6J wildtype (WT) and Sickle Townes knock-in mice (SCA) that were bred and maintained at the University of Pittsburgh. The tested engineered scaffolds were FRESH printed from Collagen I (ABM) using FRESH v2.0 support bath and 4% Paraformaldehyde (PFA) was used as the crosslinker. For both static and pulsatile pressure experiments, HemoLens bath was filled with 30 mL of 1x PBS and each sample was mounted to two 30-gauge dispensing needles (JensonGlobal #JG30-0.5HPX) and sutured with 7-0 silk sutures (Braintree Scientific #SUT-S103). After mounting, vessels were allowed to rest for at least 30 minutes to allow the bath to reach physiological temperature. Additionally, we used a computational biomechanical framework to characterize the mechanical behavior of samples to demonstrate the usability of data gathered with the HemoLens platform. A four-fiber family-based (4FF) stored energy density function, extensively used to simulate mechanical behavior of vascular tissue, was used for this work.

Results, Conclusions, and Discussions:: HemoLens’ (Figure 1A) micromanipulators achieved linear travel as low as 250 µm (Figure 1B). The incremental pressure ratchet system (iPRS) created discrete pressure increases at various syringe sizes (Figure 1C). Our designed Regular Adjustment of Modulated Pressure (RAMP) system produces physiological cyclic pressure waves of 80 to 120 mmHg, averaging 48.16 mmHg per pulse with a frequency of 1.03 Hz (Figure 1D). In pressure myography, wildtype and sickle cell anemic mouse vessels distended similarly in increasing intraluminal pressure until 125 mmHg where SCD vessels plateaued, compared to WT vessels’ plateau at 200 mmHg (Figure 1E). Under physiological pressure via RAMP, WT vessels showed an average distention of 124.3 µm for outer diameter and 129 µm for inner diameter. In contrast, SCA vessels only dilated to an average of 43.07 µm OD and 40.62 µm ID. The calculated dynamic compliance for carotid arteries during pulsatile flow is significantly higher in WT vessels (4.445-3) compared to SCA vessels (1.160-3) (Figure 1F). Chemical crosslinking of our FRESH bioprinted scaffolds more than doubled the burst pressure, from 218 mmHg to 464 mmHg. Under physiological pulsatile pressure, uncrosslinked vessels distended 11.60 µm; however, to achieve similar distention, crosslinked vessels required pathological pressure of 180+ mmHg to achieve an average distention of 15.67 µm. Since higher pulsatile pressure was required to achieve similar vascular wall distention, the calculated dynamic compliance of our crosslinked vessel was significantly lower than the non-crosslinked vessels, 6.23-5 and 3.04-4 (Figure 1G),respectively. Following advanced computational analysis, we determined that SCA vessel's stiffness is substantially elevated compared to the WT vessel, as reflected by the sharp increase in slope of the stress vs stretch plot at higher values of stretch (Figure 1H). Similarly, there was a leftward shift in the stress vs stretch curve of our bioprinted crosslinked vessel that was indicative of stiffening at comparable levels of stretch (Figure 1I). As a characterization platform, HemoLens integrates static and cyclic pressure, vessel manipulation, and varifocal imaging for comprehensive vascular assessment and significantly decreases the barrier of entry in vascular research by providing a rapidly deployable, low-cost myography system.

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