(I-335) Mechanical stimulation of tissue engineered blood vessels affects their structural and mechanical properties

Approximately 90% of drugs that enter clinical trials fail.¹ This is primarily because the preclinical models (e.g., 2-dimensional in vitro and animal models) used to evaluate drugs do not reproducibly mimic human anatomy and physiology, nor do they predict drug safety and efficacy in humans. Thus, we need more complex, human-derived models that recapitulate human systems and can be used to accurately and rapidly screen therapeutic compounds. For models related to the human vascular network, tissue engineered blood vessels (TEBVs) have shown promising results since they can mimic the 3-dimensional geometry of native human blood vessels and can be fabricated within a few weeks.² However, most TEBVs are engineered using a scaffold, which dominates the TEBV mechanical properties and limits the cell-cell interactions that are crucial for proper tissue function.³ Here, we demonstrate that a scaffold-free TEBV fabricated from human bone marrow-derived mesenchymal stem cells (hMSCs) and endothelial cells (ECs) forms a cohesive, tubular structure and expresses the contractile smooth muscle protein – smooth muscle alpha actin (SMA). To enhance their structural and mechanical properties, TEBVs were subjected to mechanical stimulation, similar to Syedain et al.⁴ We hypothesized that mechanical stimulation would increase TEBV wall thickness, expression of SMA, and mechanical properties.

TEBVs were fabricated using a previously described method.⁵ Briefly, hMSCs and ECs were seeded into non-adhesive agarose molds to form tissue rings. These rings were then stacked onto a silicone tube mandrel and allowed to fuse for four days in differentiation medium containing 5ng/mL transforming growth factor beta-1 (TGFb-1) and 50ug/mL ascorbic acid to differentiate the hMSCs into smooth muscle cells (SMCs). After four days, tissue rings were fully fused into a cohesive TEBV. TEBVs were then mounted onto a custom bioreactor, modified from Piola et al and subjected to mechanical stimulation via circumferential cyclic distension (CD) for six days.⁶ Two types of mechanical stimulation were used: a gradual increase that started at 1.1% CD and increased by 0.6% CD every two days for a final CD of 2.3%, and a constant CD of 2.3% for the entire six days. As a control, an unstimulated TEBV group was left unstimulated for six days. TEBVs were fixed and stained for hematoxylin and eosin (H&E) to visualize overall tissue morphology, picrosirius red/fast green (PRFG) to visualize collagen, as well as SMA and CD31 via immunohistochemistry (IHC) to visualize SMCs and ECs, respectively. TEBV wall thicknesses and the thickness of cells expressing SMA (“SMA thickness”) were calculated from stained tissue sections using ImageJ. To evaluate TEBV mechanics, TEBVs were cut into ring segments, mounted onto a uniaxial testing system, and pulled to failure. Maximum tangent modulus (MTM) and ultimate tensile strength (UTS) were calculated using a MATLAB script from the stress-strain curves.

Mechanical stimulation significantly affected TEBV wall thickness, SMA thickness, and TEBV mechanical properties. Both mechanically stimulated groups exhibited increased wall thicknesses, with mean values of 64.3 ± 3.51 mm, 109 ± 8.66 mm, and 167 ± 6.20 mm for unstimulated, gradually, and constantly stimulated TEBVs, respectively (mean±SEM). Figure 1A-C displays representative images of TEBVs stained for H&E, while Figure 1D-F displays representative images of TEBVs stained for PRFG. Similarly, mechanically stimulated TEBVs exhibited increased SMA thicknesses, with mean values of 15.0 ± 3.34 mm, 30.6 ± 2.20 mm, and 49.7 ± 14.6 mm for unstimulated, gradually, and constantly stimulated TEBVs, respectively (mean±SEM). Lastly, mechanical testing of TEBV ring segments suggested that gradually stimulated TEBVs possessed significantly greater MTM than unstimulated TEBVs, with mean values of 60.6 ± 6.88 kPa, 119 ± 18.1 kPa, and 145 ± 49.7 kPa for unstimulated, gradually, and constantly stimulated TEBVs, respectively (mean±SEM). Additionally, gradually and constantly stimulated TEBVs exhibited significantly greater UTS than unstimulated TEBVs, with mean values of 55.3 ± 1.33 kPa, 135 ± 18.5 kPa, and 98.6 ± 13.7 kPa for unstimulated, gradually, and constantly stimulated TEBVs, respectively (mean±SEM). Importantly, gradually stimulated TEBVs exhibited a mean UTS that is similar to previously published data for fibrin-based TEBVs mechanically stimulated for two weeks.⁴

Interestingly, for all TEBV groups, the H&E and PRFG stains demonstrate that the region of cells farthest away from the silicone tube (i.e., closest to the differentiation media) appears more organized with circumferentially aligned cells and elongated nuclei. In contrast, the region of cells closest to the silicone tube appears less organized with rounder nuclei. These observations are consistent with the IHC staining for SMA, where the region of cells expressing SMA is isolated to the outer portion of the TEBV wall. These results may be due to the diffusion limitations of the media, since the inner portion of the TEBVs is adjacent to the silicone tube, through which media does not diffuse. Future work aims to perfuse the TEBV lumens with differentiation media after mechanical stimulation to overcome these limitations and achieve luminal shear stresses similar to physiological conditions.

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