Jonathan T. Butcher

3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels.

Heart valve disease is a serious and growing public health problem for which prosthetic replacement is most commonly indicated. Current prosthetic devices are inadequate for younger adults and growing children. Tissue engineered living aortic valve conduits have potential for remodeling, regeneration, and growth, but fabricating natural anatomical complexity with cellular heterogeneity remain challenging. In the current study, we implement 3D bioprinting to fabricate living alginate/gelatin hydrogel valve conduits with anatomical architecture and direct incorporation of dual cell types in a regionally constrained manner. Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced modulus, ultimate strength, and peak strain reducing slightly over 7-day culture, while the tensile biomechanics of cell-laden hydrogels were maintained. Aortic valve conduits were successfully bioprinted with direct encapsulation of SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4 ± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated vimentin. These results demonstrate that anatomically complex, heterogeneously encapsulated aortic valve hydrogel conduits can be fabricated with 3D bioprinting.

Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds

The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12-22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.

Unique Morphology and Focal Adhesion Development of Valvular Endothelial Cells in Static and Fluid Flow Environments

Background—The influence of mechanical forces on cell function has been well documented for many different cell types. Endothelial cells native to the aortic valve may play an important role in mediating tissue responses to the complex fluid environment, and may therefore respond to fluid flow in a different manner than more characterized vascular endothelial cells. Methods and Results—Porcine endothelial cells of aortic and aortic valvular origin were subjected to 20 dynes/cm2 steady laminar shear stress for up to 48 hours, with static cultures serving as controls. The aortic valve endothelial cells were observed to align perpendicular to flow, in direct contrast to the aortic endothelial cells, which aligned parallel to flow. Focal adhesion complexes reorganized prominently at the ends of the long axis of aligned cells. Valvular endothelial cell alignment was dependent on Rho-kinase signaling, whereas vascular endothelial cell alignment was dependent on both Rho-kinase and phosphatidylinositol 3-kinase signal pathways. Conclusions—These differences in response to mechanical forces suggest a unique phenotype of valvular endothelial cells not mimicked by vascular endothelial cells, and could have implications for cardiovascular cell biology and cell-source considerations for tissue-engineered valvular substitutes.

Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences. Influence of Shear Stress

Objective—The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood. Methods and Results—Monolayers of porcine aortic endothelial cells (PAECs) or porcine aortic valve endothelial cells (PAVECs) were exposed to 20 dyne/cm2 steady laminar shear stress for 48 hours, with static cultures serving as controls. Multiple microarray comparisons were made using RNA from sheared and control batches of both cell types. More than 400 genes were significantly differentially expressed in each comparison group. The resulting profiles were validated at the transcription and protein level and expression patterns confirmed in vivo by immunohistochemistry. PAVECs were found to be less intrinsically inflammatory than PAECs, but both cell types expressed similar antioxidant and antiinflammatory genes in response to shear stress. PAVECs expressed more genes associated with chondrogenesis, whereas PAECs expressed osteogenic genes, and shear stress had a protective effect against calcification. Conclusions—Transcriptional differences between PAVECs and PAECs highlight the valvular endothelial cell as a distinct organ system and suggest more attention needs to be given to valvular cells to further our understanding of similarities and differences between valvular and vascular pathology.

Three-dimensional printed trileaflet valve conduits using biological hydrogels and human valve interstitial cells

Tissue engineering has great potential to provide a functional de novo living valve replacement, capable of integration with host tissue and growth. Among various valve conduit fabrication techniques, three-dimensional (3-D) bioprinting enables deposition of cells and hydrogels into 3-D constructs with anatomical geometry and heterogeneous mechanical properties. Successful translation of this approach, however, is constrained by the dearth of printable and biocompatible hydrogel materials. Furthermore, it is not known how human valve cells respond to these printed environments. In this study, 3-D printable formulations of hybrid hydrogels are developed, based on methacrylated hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel), and used to bioprint heart valve conduits containing encapsulated human aortic valvular interstitial cells (HAVIC). Increasing Me-Gel concentration resulted in lower stiffness and higher viscosity, facilitated cell spreading, and better maintained HAVIC fibroblastic phenotype. Bioprinting accuracy was dependent upon the relative concentrations of Me-Gel and Me-HA, but when optimized enabled the fabrication of a trileaflet valve shape accurate to the original design. HAVIC encapsulated within bioprinted heart valves maintained high viability, and remodeled the initial matrix by depositing collagen and glyosaminoglycans. These findings represent the first rational design of bioprinted trileaflet valve hydrogels that regulate encapsulated human VIC behavior. The use of anatomically accurate living valve scaffolds through bioprinting may accelerate understanding of physiological valve cell interactions and progress towards de novo living valve replacements.

Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation.

Aims Westernized countries face a growing burden of cardiovascular calcification and osteoporosis. Despite its vast clinical significance, the precise nature of this reciprocal relationship remains obscure. We hypothesize that cardiovascular calcification progresses with inflammation and inversely correlates with bone tissue mineral density (TMD). Methods and results Arterial, valvular, and bone metabolism were visualized using near-infrared fluorescence (NIRF) molecular imaging agents, targeting macrophages and osteogenesis. We detected significant arterial and aortic valve calcification in apoE−/− mice with or without chronic renal disease (CRD, 30 weeks old; n = 28), correlating with the severity of atherosclerosis. We demonstrated decreases in osteogenic activity in the femurs of apoE−/− mice when compared with WT mice, which was further reduced with CRD. Three-dimensional micro-computed tomography imaging of the cortical and cancellous regions of femurs quantified structural remodelling and reductions in TMD in apoE−/− and CRD apoE−/− mice. We established significant correlations between arterial and valvular calcification and loss of TMD (R2 = 0.67 and 0.71, respectively). Finally, we performed macrophage-targeted molecular imaging to explore a link between inflammation and osteoporosis in vivo. Although macrophage burden, visualized as uptake of NIRF-conjugated iron nanoparticles, was directly related to the degree of arterial and valvular inflammation and calcification, the same method inversely correlated inflammation with TMD (R2 = 0.73; 0.83; 0.75, respectively). Conclusion This study provides direct in vivo evidence that in arteries and aortic valves, macrophage burden and calcification associate with each other, whereas inflammation inversely correlates with bone mineralization. Thus, understanding inflammatory signalling mechanisms may offer insight into selective abrogation of divergent calcific phenomena.

Aortic valve disease and treatment: The need for naturally engineered solutions☆

The aortic valve regulates unidirectional flow of oxygenated blood to the myocardium and arterial system. The natural anatomical geometry and microstructural complexity ensures biomechanically and hemodynamically efficient function. The compliant cusps are populated with unique cell phenotypes that continually remodel tissue for long-term durability within an extremely demanding mechanical environment. Alteration from normal valve homeostasis arises from genetic and microenvironmental (mechanical) sources, which lead to congenital and/or premature structural degeneration. Aortic valve stenosis pathobiology shares some features of atherosclerosis, but its final calcification endpoint is distinct. Despite its broad and significant clinical significance, very little is known about the mechanisms of normal valve mechanobiology and mechanisms of disease. This is reflected in the paucity of predictive diagnostic tools, early stage interventional strategies, and stagnation in regenerative medicine innovation. Tissue engineering has unique potential for aortic valve disease therapy, but overcoming current design pitfalls will require even more multidisciplinary effort. This review summarizes the latest advancements in aortic valve research and highlights important future directions.

Valvulogenesis: the moving target

Valvulogenesis is an extremely complex process by which a fragile gelatinous matrix is populated and remodelled during embryonic development into thin fibrous leaflets capable of maintaining unidirectional flow over a lifetime. This process occurs during exposure to constantly changing haemodynamic forces, with a success rate of approximately 99%. Defective valvulogenesis results in impaired cardiac function and lifelong complications. This review integrates what is known about the roles of genetics and mechanics in the development of valves and how changes in either result in impaired morphogenesis. It is hoped that appropriate developmental cues and phenotypic endpoints could help engineers and clinicians in their efforts to regenerate living valve alternatives.

Transitions in Early Embryonic Atrioventricular Valvular Function Correspond With Changes in Cushion Biomechanics That Are Predictable by Tissue Composition

Endocardial cushions are critical to maintain unidirectional blood flow under constantly increasing hemodynamic forces, but the interrelationship between endocardial cushion structure and the mechanics of atrioventricular junction function is poorly understood. Atrioventricular (AV) canal motions and blood velocities of embryonic chicks at Hamburger and Hamilton (HH) stages 17, 21, and 25 were quantified using ultrasonography. Similar to the embryonic zebrafish heart, the HH17 AV segment functions like a suction pump, with the cushions expanding in a wave during peak myocardial contraction and becoming undetectable during the relaxation phase. By HH25, the AV canal contributes almost nothing to the piston-like propulsion of blood, but the cushions function as stoppers apposing blood flow with near constant thickness. Using a custom built mesomechanical testing system, we quantified the nonlinear pseudoelastic biomechanics of developing AV cushions, and found that both AV cushions increased in effective modulus between HH17 and HH25. Enzymatic digestion of major structural constituent collagens or glycosaminoglycans resulted in distinctly different stress-strain curves suggestive of their individual contributions. Mixture theory using histologically determined volume fractions of cells, collagen, and glycosaminoglycans showed good prediction of cushion material properties regardless of stage and cushion position. These results have important implications in valvular development, as biomechanics may play a larger role in stimulating valvulogenic events than previously thought.

Neonatal and adult cardiovascular pathophysiological remodeling and repair: developmental role of periostin.

The neonatal heart undergoes normal hypertrophy or compensation to complete development and adapt to increased systolic pressures. Hypertrophy and increased neonatal wall stiffness are associated with a doubling of the number of fibroblasts and de novo formation of collagen. Normal postnatal remodeling is completed within 3–4 weeks after birth but can be rekindled in adult life in response to environmental signals that lead to pathological hypertrophy, fibrosis, and heart failure. The signals that trigger fibroblast and collagen formation (fibrosis) as well as the origin and differentiation of the cardiac fibroblast lineage are not well understood. Using mice studies and a single‐cell engraftment model, we have shown that cardiac fibroblasts are derived from two extracardiac sources: the embryonic proepicardial organ and the recruitment of circulating bone marrow cells of hematopoietic stem cell origin. Periostin, a matricellular protein, is normally expressed in differentiating fibroblasts but its expression is elevated several fold in pathological remodeling and heart failure. Our hypothesis that periostin is profibrogenic (i.e., it promotes differentiation of progenitor mesenchymal cells into fibroblasts and their secretion and compaction of collagen) was tested using isolated and cultured embryonic, neonatal, and adult wild‐type and periostin‐null, nonmyocyte populations. Our findings indicate that abrogation of periostin by targeted gene deletion inhibits differentiation of nonmyocyte progenitor cells or permits misdirection into a cardiomyocyte lineage. However, if cultured with periostin or forced to express periostin, they became fibroblasts. Periostin plays a significant role in promoting fibrogenesis residual stress, and tensile testings indicated that periostin played an essential regulatory role in maintaining the biomechanical properties of the adult myocardium. These findings indicate that periostin is a profibrogenic matricellular protein that promotes collagen fibrogenesis, inhibits differentiation of progenitor cells into cardiomyocytes, and is essential for maintaining the biomechanical properties of the adult myocardium.