Vascular smooth muscle cells can be circumferentially aligned inside a channel using tunable gelatin microribbons

Introduction: Cardiovascular disease (CVD), a leading global cause of morbidity and mortality, is thought to initiate with endothelial cell (EC) dysfunction, which is characterized by a reduction in nitric oxide (NO) and disruption in vascular tone. Vascular tone, which is the ability of vessels to contract and dilate, is essential for regulating blood pressure and blood flow. In a healthy blood vessel, NO produced by ECs in the inner vessel layer regulates the ability of vascular smooth muscle cells (SMCs) in the middle vessel layer to relax. For SMCs to change the blood vessel diameter when they contract and relax, the SMCs must be circumferentially aligned around the vessel lumen. While vascular contractility can be studied in humans in vivo and animals ex vivo, there are significant challenges. Human studies require skilled technicians and the stimuli that can be applied are limited, and ex vivo pressure myography of animal arteries have limited applicability towards humans. Therefore, it is essential to develop a relevant human in vitro model to emulate blood vessel contractility. Although in vitro microfluidic vascular systems have been created, these systems do not have circumferentially aligned SMCs, which limit their contractile and relaxation responses and reduce their ability to replicate physiological function. To address this deficiency, we developed a method to circumferentially align SMCs within a cylindrical channel.

Materials and

Methods: Gelatin microribbons with tunable elastic moduli (soft or stiff) and thickness (thin or thick) were synthesized by injecting dissolved gelatin into a hollow, heat-sealed polycaprolactone (PCL) tube, and subsequently stretching, folding, and twisting the tube. The PCL was dissolved, and microribbons were treated with 0.53% succinic acid and 0.2% glutaraldehyde with varying percentages of methacrylate anhydride to modulate elastic moduli. Microribbon mechanical properties were determined via atomic force microscopy (AFM). Microribbons were washed, dialyzed, lyophilized, and stored at -20°C until use. Rehydrated microribbons were circumferentially wrapped around a needle and crosslinked in a 10% gelatin methacryloyl (GelMA) hydrogel within a 3D-printed device. The needle was removed to form a microribbon-lined channel within the GelMA hydrogel. Coronary artery smooth muscle cells (SMCs) were cultured in the channel for 24 hours and analyzed for cell viability and alignment along the microribbons. SMCs in microribbon-lined channels were compared to SMCs on individual microribbons, GelMA-encapsulated microribbons, and GelMA channels. Cell viability was observed using a Live/Dead viability assay with calcein AM and ethidium homodimer-1, and imaging was completed with a confocal fluorescent microscope. Cell viability was quantified by measuring maximum intensity projections and comparing the number of cells from both live and dead images to the total cells. Alignment was quantified using MatFiber software. Briefly, the microribbon major axis was determined from images. Microribbon sub-regions were defined, and cell vectors were generated based on intensity projections. Cell vector angles were then calculated with respect to the microribbon major axis, where 0° is ideal alignment.

Results, Conclusions, and Discussions: Gelatin microribbons were created of varying thickness (thin:10.60 ± 2.54 µm, thick: 33.85 ± 15.3 µm) and elastic moduli (soft: 6.59 ± 3.02 kPa, stiff: 35.08 ± 23.1 kPa). SMCs seeded on individual microribbons had an average viability greater than 80%, with the greatest viability (87%) and spread area (20 µm2) on thick microribbons. SMC were more aligned on soft microribbons (average 18°). Pre-seeded microribbons that were wrapped around needles had decreased cell viability (51%), area (7 µm2), and alignment (average 40°). Seeding cells after wrapping microribbons around needles maintained cell viability (84%), area (16 µm2), and alignment (average 24°). When microribbons were encapsulated in GelMA, SMC initially adhered and were viable on both uncoated and fibronectin-coated microribbons. However, after 24 hours fibronectin-coated microribbons had higher cell viability (84%) and alignment (average 22°) than uncoated microribbons. Soft, thick microribbons were coated with fibronectin and used to line the inside of the GelMA channel, and SMCs were then seeded inside the channel. SMCs in the microribbon-lined channels maintained cell viability (84%) and had higher circumferential alignment (average 33°) compared to SMCs inside a channel without microribbons (average 45°). We developed a method for circumferentially aligning microribbons within a hydrogel channel to achieve SMC alignment. The channel diameter can be changed by using needles with different diameters and potentially can be layered to create concentric microribbon-lined channels to mimic the multiple SMCs layers seen in blood vessels. The gelatin microribbon thickness can be tailored to the size of the cell. The microribbon elastic moduli can also be designed to mimic the native or pathological tissue mechanics. While we used the softer thicker microribbons, stiffer microribbons could be utilized to mimic atherosclerosis. Limitations to the device are variability in microribbon layer thickness within the channel and the channel can only be scaled down to the size of the needle used. This technique enables us to mimic SMC native structure and hence contractile function. Future work entails tuning microribbons to maximize SMC contractility, measuring contractility with traction force microscopy, and introducing an EC inner layer to create a relevant human in vitro model.

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