Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues

Bone marrow-derived mesenchymal stem cells (BMSCs) are relatively accessible and exhibit a pluripotency suitable for cardiovascular applications such as tissue-engineered heart valves (TEHVs). Recently, Sutherland et al. [From stem cells to viable autologous semilunar heart valve. Circulation 2005; 111(21): 2783-91] demonstrated that BMSC-seeded TEHV can successfully function as pulmonary valve substitutes in juvenile sheep for at least 8 months. Toward determining appropriate mechanical stimuli for use in BMSC-seeded TEHV cultivation, we investigated the independent and coupled effects of two mechanical stimuli physiologically relevant to heart valves-cyclic flexure and laminar flow (i.e. fluid shear stress)-on BMSC-mediated tissue formation. BMSC isolated from juvenile sheep were expanded and seeded onto rectangular strips of nonwoven 50:50 blend poly(glycolic acid) (PGA) and poly(l-lactic acid) (PLLA) scaffolds. Following 4 days static culture, BMSC-seeded scaffolds were loaded into a novel flex-stretch-flow (FSF) bioreactor and incubated under static (n=12), cyclic flexure (n=12), laminar flow (avg. wall shear stress=1.1505 dyne/cm(2); n=12) and combined flex-flow (n=12) conditions for 1 (n=6) and 3 (n=6) weeks. By 3 weeks, the flex-flow group exhibited dramatically accelerated tissue formation compared with all other groups, including a 75% higher collagen content of 844+/-278 microg/g wet weight (p<0.05), and an effective stiffness (E) value of 948+/-233 kPa. Importantly, collagen and E values were not significantly different from values measured for vascular smooth muscle cell (SMC) -seeded scaffolds incubated under conditions of flexure alone [Engelmayr et al. The independent role of cyclic flexure in the early in vitro development of an engineered heart valve tissue. Biomaterials 2005; 26(2): 175-87], suggesting that BMSC-seeded TEHV can be optimized to yield results comparable to SMC-seeded TEHV. We thus demonstrated that cyclic flexure and laminar flow can synergistically accelerate BMSC-mediated tissue formation, providing a basis for the rational design of in vitro conditioning regimens for BMSC-seeded TEHV.     

Estimation of the Shear Stress on the Surface of an Aortic Valve Leaflet

The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves. A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix. An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization. Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design. To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve. Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle. The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet. The maximum shear stress recorded was 79 dyne/cm², in agreement with boundary layer theory and previous experimental and computational studies. This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct. © 1999 Biomedical Engineering Society. PAC99: 8719Rr, 8768+z, 8719Hh, 4262Be, 4727Nz, 0630Gv     

A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials

Dynamic flexure is a major mode of deformation in the native heart valve cusp, and may effect the mechanical and biological development of tissue engineered heart valves (TEHV). To explore this hypothesis, a novel bioreactor was developed to study the effect of dynamic flexural stimulation on TEHV biomaterials. It was implemented in a study to compare the effect of uni-directional cyclic flexure on the effective stiffness of two candidate TEHV scaffolds: a non-woven mesh of polyglycolic acid (PGA) fibers, and a non-woven mesh of PGA and poly L-lactic acid (PLLA) fibers, both coated with poly 4-hydroxybutyrate (P4HB). The bioreactor has the capacity to dynamically flex 12 rectangular samples (25 x 7.5 x 2mm) under sterile conditions in a cell culture incubator. Sterility was maintained in the bioreactor for at least 5 weeks of incubation. Flexure tests to measure the effective stiffness in the "with-flexure" (WF) and opposing "against-flexure" (AF) directions indicated that dynamically flexed PGA/PLLA/P4HB scaffolds were approximately 72% (3 weeks) and 76% (5 weeks) less stiff than static controls (p<0.01), and that they developed directional anisotropy by 3 weeks of incubation (stiffer AF, p<0.01). In contrast, both dynamically flexed and static PGA/P4HB scaffolds exhibited a trend of decreased stiffness with incubation, with no development of directional anisotropy. Dynamically flexed PGA/P4HB scaffolds were significantly less stiff than static controls at 3 weeks (p<0.05). Scanning electron microscopy revealed signs of heterogeneous P4HB coating and fiber disruption, suggesting possible explanations for the observed mechanical properties. These results indicate that dynamic flexure can produce quantitative and qualitative changes in the mechanical properties of TEHV scaffolds, and suggest that these differences need to be accounted for when comparing the effects of mechanical stimulation on the development of cell-seeded TEHV constructs.     

Design of a Modular Bioreactor to Incorporate Both Perfusion Flow and Hydrostatic Compression for Tissue Engineering Applications

Physiological models have demonstrated that cells undergo a cyclic regimen of hydrostatic compression and fluid shear stress within the lacunar-canalicular porosity of bone. A new modular bioreactor was designed to incorporate both perfusion fluid flow and hydrostatic compression in an effort to more accurately simulate the mechanical loading and stress found in natural bone in vivo. The bioreactor design incorporated custom and off-the-shelf components to produce levels of mechanical stimuli relevant to the physiologic range, including hydrostatic compression exceeding 300 kPa and perfusion shear stress of 0.7 dyne/cm². Preliminary findings indicated that the novel system facilitated the viable growth of cells on discrete tissue engineering scaffolds. The bioreactor has established an experimental platform for ongoing investigation of the interactive effect of perfusion fluid flow and hydrostatic compression on multiple cell types.     

High-Shear Stress Sensitizes Platelets to Subsequent Low-Shear Conditions

Individuals with mechanical heart valve implants are plagued by flow-induced thromboembolic complications, which are undoubtedly caused by platelet activation. Flow fields in or around the affected regions involve brief exposure to pathologically high-shear stresses on the order of 100 to 1000 dyne/cm². Although high shear is known to activate platelets directly, their subsequent behavior is not known. We hypothesize that the post-high-shear activation behavior of platelets is particularly relevant in understanding the increased thrombotic risk associated with blood-recirculating prosthetic cardiovascular devices. Purified platelets were exposed to brief (5–40 s) periods of high-shear stress, and then exposed to longer periods (15–60 min) of low shear. Their activation state was measured using a prothrombinase-based assay. Platelets briefly exposed to an initial high-shear stress (e.g., 60 dyne/cm² for 40 s) activate a little, but this study shows that they are now sensitized, and when exposed to subsequent low shear stress, they activate at least 20-fold faster than platelets not initially exposed to high shear. The results show that platelets in vitro exposed beyond a threshold of high-shear stress are primed for subsequent activation under normal cardiovascular circulation conditions, and they do not recover from the initial high-shear insult.     

Hemodynamic Regulation of Inflammation at the Endothelial–Neutrophil Interface

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm² blocked by >80% the induction by 5 ng/mL TNFα of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-l-arginine methylester, l-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm². Also, neutrophil rolling lengths at 1 dyne/cm² were longer when TNFα-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.     

Physiologic Pulsatile Flow Bioreactor Conditioning of Poly(ethylene glycol)-based Tissue Engineered Vascular Grafts

Mechanical conditioning represents a potential means to enhance the biochemical and biomechanical properties of tissue engineered vascular grafts (TEVGs). A pulsatile flow bioreactor was developed to allow shear and pulsatile stimulation of TEVGs. Physiological 120 mmHg/80 mmHg peak-to-trough pressure waveforms can be produced at both fetal and adult heart rates. Flow rates of 2 mL/sec, representative of flow through small diameter blood vessels, can be generated, resulting in a mean wall shear stress of ∼6 dynes/cm² within the 3 mm ID constructs. When combined with non-thrombogenic poly(ethylene glycol) (PEG)-based hydrogels, which have tunable mechanical properties and tailorable biofunctionality, the bioreactor represents a flexible platform for exploring the impact of controlled biochemical and biomechanical stimuli on vascular graft cells. In the present study, the utility of this combined approach for improving TEVG outcome was investigated by encapsulating 10T-1/2 mouse smooth muscle progenitor cells within PEG-based hydrogels containing an adhesive ligand (RGDS) and a collagenase degradable sequence (LGPA). Constructs subjected to 7 weeks of biomechanical conditioning had significantly higher collagen levels and improved moduli relative to those grown under static conditions. These authors contributed equally to this work     

A Validated System for Simulating Common Carotid Arterial Flow In Vitro: Alteration of Endothelial Cell Response

Pulsations in blood flow alter gene and protein expressions in endothelial cells (EC). A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier–Stokes equation solution. Following 24 h of steady flow (SF: 13 dyne/cm²), pulsatile arterial flow (AF: average=13 dyne/cm², range=7–25 dyne/cm²) or static conditions, heme oxygenase-1 (HO-1) and prostaglandin H synthase-2 (PGHS-2) mRNA and protein expressions from human umbilical vein endothelial cells were measured. Relative to steady flow, pulsatile arterial flow significantly attenuated mRNA upregulation of HO-1 (SF: 7.26±2.70-fold over static, AF: 4.84±0.37-fold over static; p < 0.01) and PGHS-2 (SF: 6.11±1.79-fold over static, AF: 3.54±0.79-fold over static; p < 0.001). Pulsatile arterial flow (4.57±0.81-fold over static, p < 0.01) also significantly reduced the steady-flow-induced HO-1 protein upregulation (7.99±1.29-fold over static). These findings reveal that EC can discriminate between different flow patterns of the same average magnitude and respond at the molecular level.     

Hemodynamic modulation of monocytic cell adherence to vascular endothelium

Hemodynamic shear stress is hypothesized to contribute to the localization of atherosclerotic plaques to certain arterial sites. Monocyte recruitment to these sites is an early event in atherogenesis. To determine the possible mechanisms by which shear stress modulates monocyte adhesionin vivo, studies of human monocytic cell adherence to endothelium were conducted under different shear conditions in a parallel-plate flow chamber. The number of monocytes capable of developing firm adhesive contacts with endothelium decreased as shear stress-induced drag forces increased over the range of 0.5 to 30 dynes/cm². The number of adherent monocytic cells at a given shear stress was highly dependent on the activation state of the endothelium. To test the direct effect of shear stress on endothelial cell adhesivity, endothelial cells were presheared for 2 to 6 hr at 2, 10, or 30 dynes/cm², and monocytic cell adherence was quantified at 1 dyne/cm². Adherence increased 330% or 370% when endothelial cells were presheared for 2 hr at 2 or 10 dynes/cm², respectively, as compared to unsheared endothelium. In contrast, when endothelial cells were presheared at 30 dynes/cm², monocytic cell adherence at 1 dyne/cm² was not significantly different from unsheared controls. Increased monocytic cell adherence to presheared endothelium was via a vasuclar cell adhesion molecule 1 (VCAM-1)α₄β₁ mechanism, and enzyme-linked immunosorbent assay studies showed that preshearing at 2 dynes/cm² for 2 hr increased endothelial VCAM-1 expression by 38%. These data demonstrated that low levels of shear stress induce enthelial VCAM-1 expression and increase monocytic cell adherence via a VCAM-1/α₄β₁ mechanism. Thus, shear stress can modulate monocyte adherence to vascular endothelium through drag forces that affect the establishment and maintenance of adhesive bonds and by directly modulating the expression of endothelial VCAM-1. This dual effect of shear stress produces the most favorable conditions for adhesion at low-shear regions, where drag forces are low and induction of VCAM-1 is likely. The preferential adherence of monocytes to these regions may contribute to the localization of atherosclerotic plaques to low-shear regions of the arterial circulationin vivo.     

Effect of Zinc and Nitric Oxide on Monocyte Adhesion to Endothelial Cells under Shear Stress

This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm² or 1 dyne/cm²) or reversing shear stress (time average 1 dyne/cm²) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike other shear stress regimes, reversing shear stress alone enhanced monocyte adhesion, which may be associated with increased H₂O₂ and superoxide together with relatively low levels of nitric oxide (NO) production. L-N^G-Nitroarginine methyl ester (L-NAME) treatment increased monocyte adhesion under 15 dynes/cm² and under reversing shear stress. After reversing shear stress, monocyte adhesion dramatically increased with heparinase III treatment followed by a zinc scavenger. Static culture experiments supported the reduction of monocyte adhesion by zinc following endothelial cell cytokine activation. These results suggest that endothelial cell zinc levels are important for the inhibition of monocyte adhesion to endothelial cells, and may be one of the key factors in the early stages of atherogenesis.     

Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation

The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm² for 1 min at 37°C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin α_IIbβ₃) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm², whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin α_vβ₃) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm²; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same β₃ subunit as GP IIb-IIIa, plays no role in SIPA. On the basis of these results, the assessment of platelet size distributions provides a sensitive and quantitative measurement for the study of SIPA.     

Tri-layered elastomeric scaffolds for engineering heart valve leaflets

Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.     

Spatial Distribution of Wall Shear Stress in Common Carotid Artery by Color Doppler Flow Imaging

The purpose of this study is to provide a novel approach for measuring the spatial distribution of wall shear stress (WSS) in common carotid artery in vivo. WSS distributions were determined by digital image processing from color Doppler flow imaging (CDFI) in 50 healthy volunteers. In order to evaluate the feasibility of the spatial distribution, the mean values of WSS distribution were compared to the results of conventional WSS calculating method (Hagen–Poiseuille formula). In our study, the mean value of WSS distribution from 50 healthy volunteers was (6.91?±?1.20) dyne/cm², while it was (7.13?±?1.24) dyne/cm² by Hagen–Poiseuille approach. The difference was not statistically significant (t?=??0.864, p?=?0.604). Hence, the feasibility of the spatial distribution of WSS was proved. Moreover, this novel approach could provide three-dimensional distribution of shear stress and fusion image of shear stress with ultrasonic image for each volunteer, which made WSS “visible”. In conclusion, the spatial distribution of WSS could be used for WSS calculation in vivo. Moreover, it could provide more detailed values of WSS distribution than those of Hagen–Poiseuille formula.     

Anin vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation

A novelin vitro system was developed to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device (CSID) is a parallel disk viscometer that applies fluid shear stress with variable onset rate. The CSID was used in conjunction with microscopy and biochemical techniques to obtain a quantitative expression of the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows. A significant portion of the shear stress was directed in ther-direction during start-up, and therefore the full Navier-Stokes equation was necessary to accurately describe the transient shear stress. When shear stress was applied at a high rate (800 dyne cm⁻² sec⁻¹) to cultured neurons, a range of cell membrane strains (0.01 to 0.53) was obtained, suggesting inhomogeneity in cellular response. Functionally, cytosolic calcium and extracellular lactate dehydrogenase levels increased in response to high strain rate (>1 sec⁻¹) loading, compared with quasistatic (<1 sec⁻¹) loading. In addition, a subpopulation of the culture subjected to rapid deformation subsequently died. These strain rates are relevant to those shown to occur in traumatic injury, and as such, the CSID is an appropriate model for studying the biomechanics and pathophysiology of neuronal injury.     

Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach

The aortic heart valve is constantly subjected to pulsatile flow and pressure gradients which, associated with cardiovascular risk factors and abnormal hemodynamics (i.e. altered wall shear stress), can cause stenosis and calcification of the leaflets and result in valve malfunction and impaired circulation. Available options for valve replacement include homograft, allogenic or xenogenic graft as well as the implantation of a mechanical valve. A tissue-engineered heart valve containing living autologous cells would represent an alternative option, particularly for pediatric patients, but still needs to be developed. The present study was designed to demonstrate the feasibility of using a living tissue sheet produced by the self-assembly method, to replace the bovine pericardium currently used for the reconstruction of a stented human heart valve. In this study, human fibroblasts were cultured in the presence of sodium ascorbate to produce tissue sheets. These sheets were superimposed to create a thick construct. Tissue pieces were cut from these constructs and assembled together on a stent, based on techniques used for commercially available replacement valves. Histology and transmission electron microscopy analysis showed that the fibroblasts were embedded in a dense extracellular matrix produced in vitro. The mechanical properties measured were consistent with the fact that the engineered tissue was resistant and could be cut, sutured and assembled on a wire frame typically used in bioprosthetic valve assembly. After a culture period in vitro, the construct was cohesive and did not disrupt or disassemble. The tissue engineered heart valve was stimulated in a pulsatile flow bioreactor and was able to sustain multiple duty cycles. This prototype of a tissue-engineered heart valve containing cells embedded in their own extracellular matrix and sewn on a wire frame has the potential to be strong enough to support physiological stress. The next step will be to test this valve extensively in a bioreactor and at a later date, in a large animal model in order to assess in vivo patency of the graft.     

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-l-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm²) to physiological (15 dyne/cm²). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.     

Spatial Variations in Shear Stress in a 3-D Bifurcation Model at Low Reynolds Numbers

Real-time wall shear stress is difficult to monitor precisely because it varies in space and time. Microelectromechanical systems sensor provides high spatial resolution to resolve variations in shear stress in a 3-D bifurcation model for small-scaled hemodynamics. At low Reynolds numbers from 1.34 to 6.7 skin friction coefficients (C_f) varied circumferentially by a factor of two or more within the bifurcation. At a Reynolds number of 6.7, the C_f value at the lateral wall of the bifurcation along the 270^∘ plane was 7.1, corresponding to a shear stress value of 0.0061 dyn/cm². Along the 180^∘ plane, C_f was 13 or 0.0079 dyn/cm², and at the medial wall along the 90^∘ plane, C_f was 10.3 or 0.0091 dyn/cm². The experimental skin friction coefficients correlated with values derived from the Navier–Stokes solutions.     

Three-component laser doppler velocimetry measurements in the regurgitant flow region of a björk-shiley monostrut mitral valve

Three-dimensional laser Doppler velocimetry measurements were acquired in a mock-circulatory loop proximal to a Björk-Shiley monostrut valve in the mitral position, and synchronous ensemble-averaging was applied to form an “average” beat. Two axial locations in the regurgitant flow region of the valve (in the minor orifice) were mapped, and maximum Reynolds shear stresses were calculated. A large spike in regurgitant flow was noted at the beginning of systole, which may be thesqueeze flow phenomenon computed by other researchers. A region of sustained regurgitant flow 50 msec later was the focus of this study. Maximum velocities of ～3.7 mps were noted, and maximum Reynolds shear stresses of ～10,000 dyne/cm² were calculated. Comparisons were made of two-dimensional (ignoring tangential component)versus three-dimensional shear stresses, and, in this case, in regions of high stress, the differences were insignificant. This suggests that the tangential component of velocity can probably be ignored in similar measurements where the tangential velocity is likely to be small.     

Design of a Mechanobioreactor to Apply Anisotropic,Biaxial Strain to Large Thin Biomaterials for Tissue Engineered Heart Valve Applications

Repair and replacement solutions for congenitally diseased heart valves capable of post-surgery growth and adaptation have remained elusive. Tissue engineered heart valves (TEHVs) offer a potential biological solution that addresses the drawbacks of existing valve replacements. Typically, TEHVs are made from thin, fibrous biomaterials that either become cell populated in vitro or in situ. Often, TEHV designs poorly mimic the anisotropic mechanical properties of healthy native valves leading to inadequate biomechanical function. Mechanical conditioning of engineered tissues with anisotropic strain application can induce extracellular matrix remodelling to alter the anisotropic mechanical properties of a construct, but implementation has been limited to small-scale set-ups. To address this limitation for TEHV applications, we designed and built a mechanobioreactor capable of modulating biaxial strain anisotropy applied to large, thin, biomaterial sheets in vitro. The bioreactor can independently control two orthogonal stretch axes to modulate applied strain anisotropy on biomaterial sheets from 13 × 13 mm² to 70 × 40 mm². A design of experiments was performed using experimentally validated finite element (FE) models and demonstrated that biaxial strain was applied uniformly over a larger percentage of the cell seeded area for larger sheets (13 × 13 mm²: 58% of sheet area vs. 52 × 31 mm²: 86% of sheet area). Furthermore, bioreactor prototypes demonstrated that over 70% of the cell seeding area remained uniformly strained under different prescribed protocols: equibiaxial amplitudes between 5 to 40%, cyclic frequencies between 0.1 to 2.5 Hz and anisotropic strain ratios between 0:1 (constrained uniaxial) to 2:1. Lastly, proof-of-concept experiments were conducted where we applied equibiaxial (ε_x = ε_y = 8.75%) and anisotropic (ε_x = 12.5%, ε_y = 5%) strain protocols to cell-seeded, electrospun scaffolds. Cell nuclei and F-actin aligned to the vector-sum strain direction of each prescribed protocol (nuclei alignment: equibiaxial: 43.2° ± 1.8°, anisotropic: 17.5° ± 1.7°; p < 0.001). The abilities of this bioreactor to prescribe different strain amplitude, frequency and strain anisotropy protocols to cell-seeded scaffolds will enable future studies into the effects of anisotropic loading protocols on mechanically conditioned TEHVs and other engineered planar connective tissues.