Fabrication and mechanical evaluation of anatomically-inspired quasilaminate hydrogel structures with layer-specific formulations.

A major tissue engineering challenge is the creation of multilaminate scaffolds with layer-specific mechanical properties representative of native tissues, such as heart valve leaflets, blood vessels, and cartilage. For this purpose, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are attractive materials due to their tunable mechanical and biological properties. This study explored the fabrication of trilayer hydrogel quasilaminates. A novel sandwich method was devised to create quasilaminates with layers of varying stiffnesses. The trilayer structure was comprised of two "stiff" outer layers and one "soft" inner layer. Tensile testing of bilayer quasilaminates demonstrated that these scaffolds do not fail at the interface. Flexural testing showed that the bending modulus of acellular quasilaminates fell between the bending moduli of the "stiff" and "soft" hydrogel layers. The bending modulus and swelling of trilayer scaffolds with the same formulations were not significantly different than single layer gels of the same formulation. The encapsulation of cells and the addition of phenol red within the hydrogel layers decreased bending modulus of the trilayer scaffolds. The data presented demonstrates that this fabrication method can make quasilaminates with robust interfaces, integrating layers of different mechanical properties and biofunctionalization, and thus forming the foundation for a multilaminate scaffold that more accurately represents native tissue.

Flexural characterization of cell encapsulated PEGDA hydrogels with applications for tissue engineered heart valves.

The limitations of the current clinical options for valve replacements have inspired the development of enabling technologies to create a tissue engineered heart valve (TEHV). Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds permit greater biological and biomechanical customization than do non-woven mesh scaffold technologies. However, the material characterization of PEGDA hydrogels has been predominantly limited to compression and tension, as opposed to bending. Since large flexural deformations result in points of maximum stress in native valves as well as TEHVs, it is crucial to evaluate any potential scaffold material in this mode. The effect of formulation parameters on the bending mechanics of cell-seeded PEGDA hydrogels were investigated with a custom designed bending tester. Three molecular weights (3.4, 6, and 8 kDa) and three weight fractions (5%, 10%, and 15%, w/v) were subjected to three-point bending tests and the flexural stiffness was calculated. Manipulating the composition of the hydrogels resulted in flexural stiffnesses comparable with native tissues (15-220 kPa) with varied mesh sizes and swelling ratios. Hydrogels containing encapsulated valve cells, methacrylated heparin (Hep-MA), or both were substantially less stiff than acellular hydrogels. In conclusion, PEGDA hydrogels are an attractive potential scaffold system for TEHVs because they are not only cytocompatible and modifiable but can also withstand bending deformations. These studies are the first to explore the encapsulation of valvular interstitial cells in pure PEGDA hydrogels as well as to investigate the bending properties of PEGDA gels.

Mitral valvular interstitial cell responses to substrate stiffness depend on age and anatomic region.

The material properties of heart valves depend on the subject's age, the state of the disease and the complex valvular microarchitecture. Furthermore, valvular interstitial cells (VICs) are mechanosensitive, and their synthesis of extracellular matrix not only determines the valve's material properties but also provides an adhesive substrate for VICs. However, the interrelationship between substrate stiffness and VIC phenotype and synthetic properties is poorly understood. Given that the local mechanical environment (substrate stiffness) surrounding VICs differs among different age groups and different anatomic regions of the valve, it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness. Therefore, 6-week-, 6-month- and 6-year-old porcine VICs from the center of the mitral valve anterior leaflet (MVAC) and posterior leaflet (PML) were seeded onto poly(ethylene) glycol hydrogels of different stiffnesses and stained for markers of VIC activation (smooth muscle alpha-actin (SMaA)) and collagen synthesis (heat shock protein-47 (HSP47), prolyl 4-hydroxylase (P4H)). Six-week-old MVAC demonstrated decreased SMaA, P4H and HSP47 on stiffer gels, while 6-week-old PML only demonstrated decreased HSP47. Six-month-old MVAC demonstrated no difference between substrates, while 6-month-old PML demonstrated decreased SMaA, P4H and HSP47. Six-year-old MVAC demonstrated decreased P4H and HSP47, while 6-year-old PML demonstrated decreased P4H and increased HSP47. In conclusion, the age-specific and valve-region-specific responses of VICs to substrate stiffness link VIC phenotype to the leaflet regional matrix in which the VICs reside. These data provide further rationale for investigating the role of substrate stiffness in VIC remodeling within diseased and tissue engineered valves.

Design and validation of a novel splashing bioreactor system for use in mitral valve organ culture.

Previous research in our lab suggested that heart valve tissues cultured without mechanical stimulation do not retain their in vivo microstructure, i.e., cell density decreased within the deep tissue layers and increased at the periphery. In this study, a splashing rotating bioreactor was designed to apply mechanical stimulation to a mitral valve leaflet segment. Porcine valve segments (n = 9-10 per group) were cultured in the bioreactor for 2 weeks (dynamic culture), negative controls were cultured without mechanical stimulation (static culture), and baseline controls were fresh uncultured samples. Overall changes in cellularity and extracellular matrix (ECM) structure were assessed by H&E and Movat pentachrome stains. Tissues were also immunostained for multiple ECM components and turnover mediators. After 2 weeks of culture, proliferating cells were distributed throughout the tissue in segments cultured in the bioreactor, in contrast to segments cultured without mechanical stimulation. Most ECM components, especially collagen types I and III, better maintained normal expression patterns and magnitudes (as found in baseline controls) over 2 weeks of dynamic organ culture compared to static culture. Lack of mechanical stimulation changed several aspects of the tissue microstructure, including the cell distribution and ECM locations. In conclusion, mechanical stimulation by the bioreactor maintained tissue integrity, which will enable future in vitro investigation of mitral valve remodeling.

Design and physical characterization of a synchronous multivalve aortic valve culture system.

For many tissues, cyclic mechanical stimulation is considered necessary to maintain the normal morphology in vitro. The aim of this study was to design and evaluate a simple bioreactor system capable of medium-term (more than 2 weeks) culture of native and engineered aortic valves. The system consists of three pistons in separate cylindrical chambers that are simultaneously driven through the culture medium by a crank and cam assembly. The faces of these pistons have unidirectional valves mounted in opposing orientations that permit flow from one side of the face to the other. A custom designed stent was employed to secure either native or engineered tri-leaflet valves to the pistons. Computational fluid dynamics and finite element modeling was used to assist selection of materials and components in the system. Finally, sterility testing using base culture medium was performed to verify the ability of the system to retain sterile conditions. The current design permits the cyclic opening and closing of three aortic valves, however this device can be modified to accommodate up to 12 valves simultaneously. This new bioreactor system has applications not only for development of tissue-engineered valves, but for also studying disease models in the aortic valve.

Age-related changes in material behavior of porcine mitral and aortic valves and correlation to matrix composition.

Recent studies showing significant changes in valvular matrix composition with age offer design criteria for age-specific tissue-engineered heart valves. However, knowledge regarding aging-related changes in valvular material properties is limited. Therefore, 6-week, 6-month, and 6-year-old porcine aortic valves (AV) and mitral valves (MV) were subjected to uniaxial tensile testing. In addition to standard material parameters, the radius of transition curvature (RTC) was measured to assess the acuteness of the transition region of the tension-strain curve. Radially, the MV had greater stiffness and a smaller RTC compared with the AV. Circumferentially, the center of the MV anterior leaflet (MVAC) had the highest stiffness (MVAC > AV > MV free edge [MVF]), greater stress relaxation (MVAC > MVF/AV), lowest extensibility (MVAC < AV < MVF), and smaller RTC compared with MVF (AV < MVAC < MVF). AV and MV radial strips had a larger RTC compared with circumferential strips. Aging elevated stiffness for MV and AV radial and circumferential strips, elevated stress relaxation in AV and MVF circumferential strips, and increased RTC for MV radial and MVF circumferential strips. In conclusion, there are significant age-related differences in the material properties of heart valves, which parallel differences in tissue composition and structure, likely impact valve function, and highlight the need for age-specific design goals for tissue-engineered heart valves.

FUNCTIONAL COUPLING OF VALVULAR INTERSTITIAL CELLS AND COLLAGEN VIA α2ß1 INTEGRINS IN THE MITRAL LEAFLET.

Once considered passive flaps, we now understand that mitral leaflets are dynamic structures with their own vasculature and innervation that actively remodel and even generate force in response to their environments. Valvular interstitial cells (VICs) are contractile and could underlie mitral leaflet force generation, but the exact mechanisms for VICs in mitral leaflet force generation are not understood. This study tested the hypothesis that actin-mediated VIC force generation coupled to collagen via alpha2beta1 integrins is necessary for force generation in the mitral leaflet. High magnification fluorescent imaging of freshly excised porcine mitral leaflets revealed VIC cytoplasm tightly conforming to collagen fibers, with actin within VIC cytoplasmic processes appearing to attach to the collagen fibers. Functional studies of isometric force development demonstrated that while control samples developed force in response to KCl, either blocking alpha2beta1 integrins or blocking actin polymerization via cytochalasin abolished KCl-induced force development (p<0.001). These results strongly suggest that VIC-collagen coupling, mediated by alpha2beta1 integrins, is necessary for KCl-induced force generation in the mitral leaflet. This functional coupling between collagen and VICs via alpha2beta1 integrins may play a role for in vivo mitral valve function.