Characterization of cardiovascular tissue geometry and mechanical properties of large animal models is essential when developing cardiovascular devices such as heart valve replacements. These datasets are especially critical when designing devices for pediatric patient populations, as there is often limited data for guidance. Here, we present a previously unavailable dataset capturing anatomical measurements and mechanical properties of juvenile Yorkshire (YO) and Yucatan (YU) porcine main pulmonary artery (PA) and pulmonary valve (PV) tissue regions that will inform pediatric heart valve design requirements for preclinical animal studies. In addition, we developed a novel radial balloon catheter-based method to measure tissue stiffness and validated it against a traditional uniaxial tensile testing method. YU piglets, which were significantly lower weight than YO counterparts despite similar age, had smaller PA and PV diameters (7.6-9.9 mm vs. 10.1-12.8 mm). Young's modulus (stiffness) was measured for the PA and the PV region using both the radial and uniaxial testing methods. There was no significant difference between the two breeds for Young's modulus measured in the elastic (YU PA 84.7 ± 37.3 kPa, YO PA 79.3 ± 15.7 kPa) and fibrous regimes (YU PA 308.6 ± 59.4 kPa, YO PA 355.7 ± 68.9 kPa) of the stress-strain curves. The two testing techniques also produced similar stiffness measurements for the PA and PV region, although PV data showed greater variation between techniques. Overall, YU and YO piglets had similar PA and PV diameters and tissue stiffness to previously reported infant pediatric patients. These results provide a previously unavailable age-specific juvenile porcine tissue geometry and stiffness dataset critical to the development of pediatric cardiovascular prostheses. Additionally, the data demonstrates the efficacy of a novel balloon catheter-based technique that could be adapted to non-destructively measure tissue stiffness
Vascular disease, such as atherosclerosis, is accompanied by changes in the mechanical properties of the vessel wall. Although altered mechanics is thought to contribute to disease progression, the molecular mechanisms whereby vessel wall stiffening could promote vascular occlusive disease remain unclear. It is well known that platelet-derived growth factor (PDGF) is a major stimulus for the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) and contributes critically to vascular disease. Here we used engineered substrates with tunable mechanical properties to explore the effect of tissue stiffness on PDGF signaling in VSMCs as a potential mechanism whereby vessel wall stiffening could promote vascular disease. We found that substrate stiffness significantly enhanced PDGFR activity and VSMC proliferation. After ligand binding, PDGFR followed distinct routes of activation in cells cultured on stiff versus soft substrates, as demonstrated by differences in its intensity and duration of activation, sensitivity to cholesterol extracting agent, and plasma membrane localization. Our results suggest that stiffening of the vessel wall could actively promote pathogenesis of vascular disease by enhancing PDGFR signaling to drive VSMC growth and survival.
Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.
Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFβ1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFβ1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFβ1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.
In pediatric patients requiring vascular reconstruction, the development of a cell-based tissue-engineered vascular patch (TEVP) has great potential to overcome current issues with nonliving graft materials. Determining the optimal cell source is especially critical to TEVP success. In this study, we compared the ability of human aortic smooth muscle cells (HuAoSMCs) and human mesenchymal stem cells (hMSCs) to form cell sheets on thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) substrates. hMSCs treated with transforming growth factor beta 1 (TGFβ1) and ascorbic acid (AA) had higher expression of SMC-specific proteins compared to HuAoSMCs. hMSCs also had larger cell area and grew to confluence more quickly on PIPAAm than did HuAoSMCs. hMSCs typically formed cell sheets in 2–3 weeks and had greater wet tissue weight and collagen content compared with HuAoSMC sheets, which generally required growth for up to 5 weeks. Assays for calcification and alkaline phosphatase activity revealed that the osteogenic potential of TGFβ1+AA-treated hMSCs was low; however, Alcian Blue staining suggested high chondrogenic behavior of TGFβ1+AA-treated hMSCs. Although hMSCs are promising for cell-based TEVPs in their ability to form robust tissue with significant extracellular matrix content, improved control over hMSC behavior will be required for long-term TEVP success.
Stimulating or maintaining the proliferative capacity of postnatal mammalian cardiomyocytes is a major challenge to cardiac regeneration. Previously, it is found that fetal cardiac extracellular matrix (ECM) can promote neonatal rat cardiomyocyte proliferation in vitro better than neonatal or adult ECM. It is hypothesized that partial digestion of adult ECM (PD‐ECM) would liberate less crosslinked components that promote cardiomyocyte proliferation, similar to fetal ECM. Neonatal rat cardiac cells are seeded onto substrates coated with adult rat cardiac ECM that has been solubilized in pepsin‐HCl for 1, 3, 6, 12, 24, or 48 h. Cardiomyocyte proliferation and fold‐change in numbers from 1 to 5 d are highest on 1 and 3 h PD‐ECM compared to other conditions. Sarcomeres tend to mature on 24 and 48 h PD‐ECM where low proliferation is observed. 3 h PD‐ECM is primarily composed of Fibrillin‐1, Fibrinogen, and Laminins while 48 h PD‐ECM is dominated by Collagen I. Our results suggest that adult ECM retains regenerative cues that may be masked by more abundant, mature ECM components. PD‐ECM provides a simple yet powerful approach to promoting cardiomyocyte proliferation.
