Biomechanical Characterization of 3D Bioprinted Collagen-based Small Diameter Resistance Vessels

Introduction: Hypertension is a vascular disease affecting ~116 million Americans per year with healthcare costs of $131 billion/year. Considerable effort has been made toward developing pharmaceutical therapeutics for patients with hypertension, but we are increasingly learning that human specific genomic variations alter drug efficacy and treatment [3]. For this reason, mouse models are becoming less relevant in predicting clinical outcomes. Addressing this challenge requires tissue engineered blood vessel (TEBV) systems capable of incorporating both the complex 3D microenvironment and human specific physiology. Worthwhile progress has been made toward generating larger (> 1 mm) vascular scaffolds via folding 2D sheets, electrospinning, casting, and 3D bioprinting; however, these methods have difficulty in generating small diameter arteries from ECM biomaterials. In hypertension, the major functional units regulating vascular resistance are small diameter resistance arteries ( < 400 µm ID). An ideal small diameter TEBV to study hypertension would comprise biomaterials that can be mechanically tunned to mimic both the native and diseased states of the vasculature, and amenable to remodeling during culture with vascular cells to investigate ECM deposition and measure vascular reactivity. Using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting, we printed an all ECM-based small diameter resistance artery scaffold capable of connecting to a perfusion bioreactor to study how ECM composition, endothelization, and fluid flow contribute to the development of native and hypertensive disease like small diameter resistance arteries.

Materials and

Methods: Single and multi-channel perusable 3D vascular models featuring inner diameters between 300 to 1000 µm suspended within a frame were designed using 3D CAD software (FUSION 360). FRESH gelatin support bath and collagen bioinks used were prepared as previously described. Briefly, 3% w/v Gelatin Type B (Fisher Chemical), 0.3% w/v gum arabic (Sigma-Aldrich), and 0.125% w/v Pluronic F-127 (Sigma-Aldrich) were dissolved in a 50% w/v warm ethanol/water solution to produce 30 micron gelatin particles. Prior to printing, the support is washed with 50 mM HEPES solution (Sigma-Aldrich) followed by centrifugation at 1800xg for 5 mins. The collagen bioink was prepared by diluting LifeInk200 (Advanced Biomatrix) 35 mg/mL to ~23 mg/mL with 0.8 M acetic acid. For printing, the bioink was transferred into a gastight syringe (Hamilton) and attached to our custom bioprinter. Biomechanical evaluations of printed vessels were conducted using pressure and wire myography (DMT-620M Systems) according to the manufacture protocol. Each myography well was filled with 1x Phosphate Buffered Saline (PBS, Sigma-Aldrich) and warmed to 37ºC for 30 minutes. Printed vessels were cut into ~2 mm sections and mounted, either onto 200 µm pins, 40 µm tungsten wires, or pulled glass pipettes. Vessels were acclimatized for 15 minutes prior to testing. For wire myography, jaws were moved apart 5 µm every 30 seconds. Tension was recorded using LabChart8 (ADInstruments). For pressure myography, the pressure was increased by 10 mmHg every 30 seconds until bursting confirmed by visual tearing and a loss in pressure.

Results, Conclusions, and Discussions: In this study, we employed FRESH bioprinting to fabricate small-diameter vessels with similar mechanical properties to mouse arteries to serve as a platform for studying vascular diseases. The FRESH printed collagen-I vessels (Figure 1A) are comprised of two sections. An outer supporting frame that features inlets/outlets to interface with custom perfusion bioreactors and a central suspended vessel with a 300 µm inner lumen. The patency of our vessels was confirmed with the perfusion of a blue dye through the vessel lumen (Figure 1B). To further recapitulate the internal structure of native blood vessels, we designed and FRESH printed a 3D model containing the three major vascular layers, adventitia (blue), media (green), and intima (yellow) to highlight our multi-material fabrication capabilities (Figure 1C). Pressure and wire myography (Figure 1D-E) was employed to evaluate burst pressure and peak tensions of our printed vessels. With a peak burst pressure of >140mmHg, our printed vessels are comparable to mouse mesenteric arteries (Figure 1F). Furthermore, wire myography results yielded an average peak tension of 600mg, again similar to mouse mesenteric arteries (Figure 1G). To demonstrate that our FRESH printed vessels can be scaled for larger artery-like models, we designed and printed vessels with ID >1mm. The resultant myography data for the 1 mm collagen vessels demonstrated peak tension of ~3600mg with no significant difference compared to decellularized mouse aortas (Figure 1H). This work highlights the exciting potential of applying FRESH bioprinting of ECM-based perfusable scaffolds to generate physiological relevant human resistance artery models that are responsive to both vasodilation and constriction. We demonstrate our ability to alter the vessel’s mechanical properties by changing model dimensions, ECM composition, or by fixation of scaffolds. Future work will extend these acellular studies into a fully cellularized resistance artery scaffold to create microphysiological models of healthy and hypertensive diseased arteries. Additionally, beyond vascular biology, these printed collagen vessel models can be applied to study cell proliferation, metastasis, fluid transport, and ECM remodeling in large vessels and perfusable conduits found in many organ systems.

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