Design and testing of a cyclic stretch and flexure bioreactor for evaluating engineered heart valve tissues based on poly(glycerol sebacate) scaffolds.

Cyclic flexure and stretch are essential to the function of semilunar heart valves and have demonstrated utility in mechanically conditioning tissue-engineered heart valves. In this study, a cyclic stretch and flexure bioreactor was designed and tested in the context of the bioresorbable elastomer poly(glycerol sebacate). Solid poly(glycerol sebacate) membranes were subjected to cyclic stretch, and micromolded poly(glycerol sebacate) scaffolds seeded with porcine aortic valvular interstitial cells were subjected to cyclic stretch and flexure. The results demonstrated significant effects of cyclic stretch on poly(glycerol sebacate) mechanical properties, including significant decreases in effective stiffness versus controls. In valvular interstitial cell-seeded scaffolds, cyclic stretch elicited significant increases in DNA and collagen content that paralleled maintenance of effective stiffness. This work provides a basis for investigating the roles of mechanical loading in the formation of tissue-engineered heart valves based on elastomeric scaffolds.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Valvular interstitial cell seeded poly(glycerol sebacate) scaffolds: toward a biomimetic in vitro model for heart valve tissue engineering.

Tissue engineered replacement heart valves may be capable of overcoming the lack of growth potential intrinsic to current non-viable prosthetics, and thus could potentially serve as permanent replacements in the surgical repair of pediatric valvular lesions. However, the evaluation of candidate combinations of cells and scaffolds lacks a biomimetic in vitro model with broadly tunable, anisotropic and elastomeric structural-mechanical properties. Toward establishing such an in vitro model, in the current study, porcine aortic and pulmonary valvular interstitial cells (i.e. biomimetic cells) were cultivated on anisotropic, micromolded poly(glycerol sebacate) scaffolds (i.e. biomimetic scaffolds). Following 14 and 28 days of static culture, cell-seeded scaffolds and unseeded controls were assessed for their mechanical properties, and cell-seeded scaffolds were further characterized by confocal fluorescence and scanning electron microscopy, and by collagen and DNA assays. Poly(glycerol sebacate) micromolding yielded scaffolds with anisotropic stiffnesses resembling those of native valvular tissues in the low stress-strain ranges characteristic of physiologic valvular function. Scaffold anisotropy was largely retained upon cultivation with valvular interstitial cells; while the mechanical properties of unseeded scaffolds progressively diminished, cell-seeded scaffolds either retained or exceeded initial mechanical properties. Retention of mechanical properties in cell-seeded scaffolds paralleled the accretion of collagen, which increased significantly from 14 to 28 days. This study demonstrates that valvular interstitial cells can be cultivated on anisotropic poly(glycerol sebacate) scaffolds to yield biomimetic in vitro models with which clinically relevant cells and future scaffold designs can be evaluated.

Laser microfabricated poly(glycerol sebacate) scaffolds for heart valve tissue engineering.

Microfabricated poly(glycerol sebacate) (PGS) scaffolds may be applicable to tissue engineering heart valve leaflets by virtue of their controllable microstructure, stiffness, and elasticity. In this study, PGS scaffolds were computationally designed and microfabricated by laser ablation to match the anisotropy and peak tangent moduli of native bovine aortic heart valve leaflets. Finite element simulations predicted PGS curing conditions, scaffold pore shape, and strut width capable of matching the scaffold effective stiffnesses to the leaflet peak tangent moduli. On the basis of simulation predicted effective stiffnesses of 1.041 and 0.208 MPa for the scaffold preferred (PD) and orthogonal, cross-preferred (XD) material directions, scaffolds with diamond-shaped pores were microfabricated by laser ablation of PGS cured 12 h at 160°C. Effective stiffnesses measured for the scaffold PD (0.83 ± 0.13 MPa) and XD (0.21 ± 0.03 MPa) were similar to both predicted values and peak tangent moduli measured for bovine aortic valve leaflets in the circumferential (1.00 ± 0.16 MPa) and radial (0.26 ± 0.03 MPa) directions. Scaffolds cultivated with fibroblasts for 3 weeks accumulated collagen (736 ± 193 µg/g wet weight) and DNA (17 ± 4 µg/g wet weight). This study provides a basis for the computational design of biomimetic microfabricated PGS scaffolds for tissue-engineered heart valves.

Three-dimensional elastomeric scaffolds designed with cardiac-mimetic structural and mechanical features.

Tissue-engineered constructs, at the interface of material science, biology, engineering, and medicine, have the capacity to improve outcomes for cardiac patients by providing living cells and degradable biomaterials that can regenerate the native myocardium. With an ultimate goal of both delivering cells and providing mechanical support to the healing heart, we designed three-dimensional (3D) elastomeric scaffolds with (1) stiffnesses and anisotropy mimicking explanted myocardial specimens as predicted by finite-element (FE) modeling, (2) systematically varied combinations of rectangular pore pattern, pore aspect ratio, and strut width, and (3) structural features approaching tissue scale. Based on predicted mechanical properties, three scaffold designs were selected from eight candidates for fabrication from poly(glycerol sebacate) by micromolding from silicon wafers. Large 20×20 mm scaffolds with high aspect ratio features (5:1 strut height:strut width) were reproducibly cast, cured, and demolded at a relatively high throughput. Empirically measured mechanical properties demonstrated that scaffolds were cardiac mimetic and validated FE model predictions. Two-layered scaffolds providing fully interconnected pore networks were fabricated by layer-by-layer assembly. C2C12 myoblasts cultured on one-layered scaffolds exhibited specific patterns of cell elongation and interconnectivity that appeared to be guided by the scaffold pore pattern. Neonatal rat heart cells cultured on two-layered scaffolds for 1 week were contractile, both spontaneously and in response to electrical stimulation, and expressed sarcomeric α-actinin, a cardiac biomarker. This work not only demonstrated several scaffold designs that promoted functional assembly of rat heart cells, but also provided the foundation for further computational and empirical investigations of 3D elastomeric scaffolds for cardiac tissue engineering.

Anisotropic collagen fibrillogenesis within microfabricated scaffolds: implications for biomimetic tissue engineering.

Anisotropic collagen fibrillogenesis is demonstrated within the pores of an accordion-like honeycomb poly(glycerol sebacate) tissue engineering scaffold. Confocal reflectance microscopy and image analysis demonstrate increased fibril distribution order, fibril density, and alignment in accordion-like honeycomb pores compared with collagen gelled unconstrained. Finite element modeling predicts how collagen gel and scaffold mechanics couple in matching native heart muscle stiffness and anisotropy.

The significance of pore microarchitecture in a multi-layered elastomeric scaffold for contractile cardiac muscle constructs.

Multi-layered poly(glycerol-sebacate) (PGS) scaffolds with controlled pore microarchitectures were fabricated, combined with heart cells, and cultured with perfusion to engineer contractile cardiac muscle constructs. First, one-layered (1L) scaffolds with accordion-like honeycomb shaped pores and elastomeric mechanical properties were fabricated by laser microablation of PGS membranes. Second, two-layered (2L) scaffolds with fully interconnected three dimensional pore networks were fabricated by oxygen plasma treatment of 1L scaffolds followed by stacking with off-set laminae to produce a tightly bonded composite. Third, heart cells were cultured on scaffolds with or without interstitial perfusion for 7 days. The laser-microablated PGS scaffolds exhibited ultimate tensile strength and strain-to-failure higher than normal adult rat left ventricular myocardium, and effective stiffnesses ranging from 220 to 290 kPa. The 7-day constructs contracted in response to electrical field stimulation. Excitation thresholds were unaffected by scaffold scale up from 1L to 2L. The 2L constructs exhibited reduced apoptosis, increased expression of connexin-43 (Cx-43) and matrix metalloprotease-2 (MMP-2) genes, and increased Cx-43 and cardiac troponin-I proteins when cultured with perfusion as compared to static controls. Together, these findings suggest that multi-layered, microfabricated PGS scaffolds may be applicable to myocardial repair applications requiring mechanical support, cell delivery and active implant contractility.

Finite element analysis of an accordion-like honeycomb scaffold for cardiac tissue engineering.

Optimizing the function of tissue engineered cardiac muscle is becoming more feasible with the development of microfabricated scaffolds amenable to mathematical modeling. In the current study, the elastic behavior of a recently developed poly(glycerol sebacate) (PGS) accordion-like honeycomb (ALH) scaffold [Engelmayr et al., 2008. Nature Materials 7 (12), 1003-1010] was analyzed. Specifically, 2D finite element (FE) models of the ALH unit cell (periodic boundary conditions) and tessellations (kinematic uniform boundary conditions) were utilized to determine a representative volume element (RVE) and to retrospectively predict the elastic effective stiffnesses. An RVE of 90 ALH unit cells (≃3.18×4.03mm) was found, indicating that previous experimental uni-axial test samples were mechanically representative. For ALH scaffolds microfabricated from PGS cured 7.5h at 160°C, FE predicted effective stiffnesses in the two orthogonal material directions (0.081±0.012 and 0.033±0.005MPa) matched published experimental data (0.083±0.004 and 0.031±0.002MPa) within 2.4% and 6.4%. Of potential use as a design criterion, model predicted global strain amplifications were lower in ALH (0.54 and 0.34) versus rectangular honeycomb (1.19 and 0.74) scaffolds, appearing to be inversely correlated with previously measured strains-to-failure. Important in matching the anisotropic mechanical properties of native cardiac muscle, FE predicted ALH scaffolds with 50µm wide PGS struts to be maximally anisotropic. The FE model will thus be useful in designing future variants of the ALH pore geometry that simultaneously provide proper cardiac anisotropy and reduced stiffness to enhance heart cell-mediated contractility.

Zero-order controlled release of ciprofloxacin-HCl from a reservoir-based, bioresorbable and elastomeric device.

A reservoir-based device constructed of a completely biodegradable elastomer can enable several new implantation and insertion options for localized drug therapy, particularly in the case of urological therapies. We performed an in vitro performance evaluation of an implantable, bio-resorbable device that supplies short-term controlled release of ciprofloxacin-HCl (CIP). The proposed device functions through a combination of osmosis and diffusion mechanisms to release CIP for short-term therapies of a few weeks duration. Poly(glycerol-co-sebacic acid) (PGS) was cast in a tubular geometry with solid drug powder packed into its core and a micro-machined release orifice drilled through its wall. Drug release experiments were performed to determine the effective release rate from a single orifice and the range of orifice sizes in which controlled zero-order release was the main form of drug expulsion from the device. It is demonstrated that PGS is sufficiently permeable to water to allow the design of an elementary osmotic pump for drug delivery. Indeed, PGS's water permeability is several orders of magnitude larger than commonly used cellulose acetate for elementary osmotic pumps.

In vivo monitoring of function of autologous engineered pulmonary valve.

OBJECTIVES: Clinical translation of tissue-engineered heart valves requires valve competency and lack of stenosis in the short and long term. Early studies of engineered valves showed promise, although lacked complete definition of valve function. Building on prior experiments, we sought to define the in vivo changes in structure and function of autologous engineered pulmonary valved conduits. METHODS: Mesenchymal stem cells were isolated from neonatal sheep bone marrow and seeded onto a bioresorbable scaffold. After 4 weeks of culture, valved conduits were implanted. Valve function, cusp, and conduit dimensions were evaluated at implantation (echocardiography), at the experimental midpoint (magnetic resonance imaging), and at explant, at 1 day, and 1, 6, 12, or 20 weeks postoperatively (direct measurement, echocardiography). Histologic evaluation was performed. RESULTS: Nineteen animals underwent autologous tissue-engineered valved conduit replacement. At implantation, valved conduit function was excellent; maximum transvalvular pressure gradient by Doppler echocardiography was 17 mm Hg; most valved conduits showed trivial pulmonary regurgitation. At 6 postoperative weeks, valve cusps appeared less mobile; pulmonary regurgitation was mild to moderate. At 12 weeks or more, valved conduit cusps were increasingly attenuated and regurgitant. Valved conduit diameter remained unchanged over 20 weeks. Dimensional measurements by magnetic resonance imaging correlated with direct measurement at explant. CONCLUSIONS: We demonstrate autologous engineered tissue valved conduits that function well at implantation, with subsequent monitoring of dimensions and function in real time by magnetic resonance imaging. In vivo valves undergo structural and functional remodeling without stenosis, but with worsening pulmonary regurgitation after 6 weeks. Insights into mechanisms of in vivo remodeling are valuable for future iterations of engineered heart valves.

Endothelial progenitor cells as a sole source for ex vivo seeding of tissue-engineered heart valves.

PURPOSES: We investigated whether circulating endothelial progenitor cells (EPCs) can be used as a cell source for the creation of a tissue-engineered heart valve (TEHV). METHODS: Trileaflet valved conduits were fabricated using nonwoven polyglycolic acid/poly-4-hydroxybutyrate polymer. Ovine peripheral blood EPCs were dynamically seeded onto a valved conduit and incubated for 7, 14, and 21 days. RESULTS: Before seeding, EPCs were shown to express CD31(+), eNOS(+), and VE-Cadherin(+) but not alpha-smooth muscle actin. Histological analysis demonstrated relatively homogenous cellular ingrowth throughout the valved conduit. TEHV constructs revealed the presence of endothelial cell (EC) markers and alpha-smooth muscle actin(+) cells comparable with native valves. Protein levels were comparable with native valves and exceeded those in unseeded controls. EPC-TEHV demonstrated a temporal pattern of matrix metalloproteinases-2/9 expression and tissue inhibitors of metalloproteinase activities comparable to that of native valves. Mechanical properties of EPC-TEHV demonstrated significantly greater stiffness than that of the unseeded scaffolds and native valves. CONCLUSIONS: Circulating EPC appears to have the potential to provide both interstitial and endothelial functions and could potentially serve as a single-cell source for construction of autologous heart valves.

The role of organ level conditioning on the promotion of engineered heart valve tissue development in-vitro using mesenchymal stem cells.

We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.

Encapsulated pheochromocytoma cells secrete potent noncatecholamine factors.

Pheochromocytomas are widely believed to induce cardiomyopathy via hypersecretion of catecholamines, including norepinephrine (NE). NE can have direct cardiomyocyte toxicity and/or can stimulate myocardial remodeling secondary to the induction of hypertension. Yet, the development of cardiomyopathy is not entirely related to catecholamine dose or the extent of hypertension. To explore these effects, we engineered a polymeric encapsulation system to control PC12 cell kinetics and NE release in vitro and in vivo. Primary neonatal rat cardiomyocytes incubated with pheochromocytoma-conditioned media exhibited greater cytoskeletal changes than myocytes cultured with identical doses of NE alone, including more profound dose-dependent decreases in desmin, beta-tubulin, and vinculin and upregulation of dystrophin. Cardiomyocyte contractility was 29 +/- 6% greater at given levels of NE release. Agarose-encapsulated PC12 cells retain cell viability and structural integrity in vivo. These implants induce a 30% greater degree of cardiac enlargement as compared to pumps releasing equivalent doses of NE. Protein level alterations observed in vitro were mirrored in vivo after implantation of encapsulated cells or NE pumps for 28 days. Together, these data suggest that pheochromocytoma-induced cardiomyopathy is not solely a catecholamine-mediated event; rather, the pathogenesis of this dilated cardiomyopathy appears to be dependent upon secondary factors unexamined to date.

Insulin-like growth factor-I and slow, bi-directional perfusion enhance the formation of tissue-engineered cardiac grafts.

Biochemical and mechanical signals enabling cardiac regeneration can be elucidated using in vitro tissue-engineering models. We hypothesized that insulin-like growth factor-I (IGF) and slow, bi-directional perfusion could act independently and interactively to enhance the survival, differentiation, and contractile performance of tissue-engineered cardiac grafts. Heart cells were cultured on three-dimensional porous scaffolds in medium with or without supplemental IGF and in the presence or absence of slow, bi-directional perfusion that enhanced transport and provided shear stress. Structural, molecular, and electrophysiologic properties of the resulting grafts were quantified on culture day 8. IGF had independent, beneficial effects on apoptosis (p < 0.01), cellular viability (p < 0.01), contractile amplitude (p < 0.01), and excitation threshold (p < 0.01). Perfusion independently affected the four aforementioned parameters and also increased amounts of cardiac troponin-I (p < 0.01), connexin-43 (p < 0.05), and total protein (p < 0.01) in the grafts. Interactive effects of IGF and perfusion on apoptosis were also present (p < 0.01). Myofibrillogenesis and spontaneous contractility were present only in grafts cultured with perfusion, although contractility was inducible by electrical field stimulation of grafts from all groups. Our findings demonstrate that multi-factorial stimulation of tissue-engineered cardiac grafts using IGF and perfusion resulted in independent and interactive effects on heart cell survival, differentiation, and contractility.

Accordion-like honeycombs for tissue engineering of cardiac anisotropy.

Tissue-engineered grafts may be useful in myocardial repair; however, previous scaffolds have been structurally incompatible with recapitulating cardiac anisotropy. Here, we use microfabrication techniques to create an accordion-like honeycomb microstructure in poly(glycerol sebacate), which yields porous, elastomeric three-dimensional (3D) scaffolds with controllable stiffness and anisotropy. Accordion-like honeycomb scaffolds with cultured neonatal rat heart cells demonstrated utility through: (1) closely matched mechanical properties compared to native adult rat right ventricular myocardium, with stiffnesses controlled by polymer curing time; (2) heart cell contractility inducible by electric field stimulation with directionally dependent electrical excitation thresholds (p<0.05); and (3) greater heart cell alignment (p<0.0001) than isotropic control scaffolds. Prototype bilaminar scaffolds with 3D interconnected pore networks yielded electrically excitable grafts with multi-layered neonatal rat heart cells. Accordion-like honeycombs can thus overcome principal structural-mechanical limitations of previous scaffolds, promoting the formation of grafts with aligned heart cells and mechanical properties more closely resembling native myocardium.

Co-culture induces alignment in engineered cardiac constructs via MMP-2 expression.

Cardiac tissue engineering has been limited by the inability to recreate native myocardial structural features. We hypothesized that heart cell elongation and alignment in 3D engineered cardiac constructs would be enhanced by using physiologic ratios of cardiomyocytes (CM) and cardiac fibroblasts (CF) via matrix metalloprotease (MMP)-dependent mechanisms. Co-cultured CM and CF constructs were compared to CM-enriched constructs using either basal media or media with a general MMP inhibitor for 8 days. Co-cultured constructs exhibited significantly increased cell alignment (p<0.0002), which was eliminated by MMP inhibition. Co-cultured constructs expressed substantial active MMP-2 protein that was not present in CM-enriched constructs, increased pro-MMP-2 (p<0.001), and reduced pro-MMP-9 (p<0.001) expression. Apoptosis was decreased by co-culture (p<0.05), independent of MMP inhibition. These results demonstrated that co-culture of CF in physiologic ratios within engineered cardiac constructs improved cell elongation and alignment via increased MMP-2 expression and activation, and also improved viability independent of MMP activity.

A novel flex-stretch-flow bioreactor for the study of engineered heart valve tissue mechanobiology.

Tissue engineered heart valves (TEHV) have been observed to respond to mechanical conditioning in vitro by expression of activated myofibroblast phenotypes followed by improvements in tissue maturation. In separate studies, cyclic flexure, stretch, and flow (FSF) have been demonstrated to exhibit both independent and coupled stimulatory effects. Synthesis of these observations into a rational framework for TEHV mechanical conditioning has been limited, however, due to the functional complexity of tri-leaflet valves and the inherent differences of separate bioreactor systems. Toward quantifying the effects of individual mechanical stimuli similar to those that occur during normal valve function, a novel bioreactor was developed in which FSF mechanical stimuli can be applied to engineered heart valve tissues independently or in combination. The FSF bioreactor consists of two identically equipped chambers, each having the capacity to hold up to 12 rectangular tissue specimens (25 x 7.5 x 1 mm) via a novel "spiral-bound" technique. Specimens can be subjected to changes-in-curvature up to 50 mm(-1) and uniaxial tensile strains up to 75%. Steady laminar flow can be applied by a magnetically coupled paddlewheel system. Computational fluid dynamic (CFD) simulations were conducted and experimentally validated by particle image velocimetry (PIV). Tissue specimen wall shear stress profiles were predicted as a function of paddlewheel speed, culture medium viscosity, and the quasi-static state of specimen deformation (i.e., either undeformed or completely flexed). Velocity profiles predicted by 2D CFD simulations of the paddlewheel mechanism compared well with PIV measurements, and were used to determine boundary conditions in localized 3D simulations. For undeformed specimens, predicted inter-specimen variations in wall shear stress were on average +/-7%, with an average wall shear stress of 1.145 dyne/cm(2) predicted at a paddlewheel speed of 2000 rpm and standard culture conditions. In contrast, while the average wall shear stress predicted for specimens in the quasi-static flexed state was approximately 59% higher (1.821 dyne/cm(2)), flexed specimens exhibited a broad intra-specimen wall shear stress distribution between the convex and concave sides that correlated with specimen curvature, with peak wall shear stresses of approximately 10 dyne/cm(2). This result suggests that by utilizing simple flexed geometric configurations, the present system can also be used to study the effects of spatially varying shear stresses. We conclude that the present design provides a robust tool for the study of mechanical stimuli on in vitro engineered heart valve tissue formation.

Prediction of extracellular matrix stiffness in engineered heart valve tissues based on nonwoven scaffolds.

The in vitro development of tissue engineered heart valves (TEHV) exhibiting appropriate structural and mechanical characteristics remains a significant challenge. An important step yet to be addressed is establishing the relationship between scaffold and extracellular matrix (ECM) mechanical properties. In the present study, a composite beam model accounting for nonwoven scaffold-ECM coupling and the transmural collagen concentration distribution was developed, and utilized to retrospectively estimate the ECM effective stiffness in TEHV specimens incubated under static and cyclic flexure conditions (Engelmayr Jr et~al. in Biomaterials 26(2):175-187 2005). The ECM effective stiffness was expressed as the product of the local collagen concentration and the collagen specific stiffness (i.e., stiffness/concentration), and was related to the overall TEHV effective stiffness via an empirically determined scaffold-ECM coupling parameter and measured transmural collagen concentration distributions. The scaffold-ECM coupling parameter was determined by flexural mechanical testing of polyacrylamide gels (i.e., ECM analogs) of variable stiffness and associated scaffold-polyacrylamide gel composites (i.e., engineered tissue analogs). The transmural collagen concentration distributions were quantified from fluorescence micrographs of picro-sirius red stained TEHV sections. As suggested by a previous structural model of the nonwoven scaffold (Engelmayr Jr and Sacks in J Biomech Eng 128(4):610-622, 2006), nonwoven scaffold-ECM composites did not follow a traditional rule of mixtures. The present study provided further evidence that the primary mode of reinforcement in nonwoven scaffold-ECM composites is an increase in the number fiber-fiber bonds with a concomitant increase in the effective stiffness of the spring-like fiber segments. Simulations of potential ECM deposition scenarios using the current model indicated that the present approach is sensitive to the specific time course of tissue deposition, and is thus very suitable for studies of ECM formation in engineered heart valve tissues.

Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast.

BACKGROUND: Phenotypically, aortic valve interstitial cells are dynamic myofibroblasts, appearing contractile and activated in times of development, disease, and remodeling. The precise mechanism of phenotypic modulation is unclear, but it is speculated that both biomechanical and biochemical factors are influential. Therefore, we hypothesized that isolated and combined treatments of cyclic tension and transforming growth factor-beta1 would alter the phenotype and subsequent collagen biosynthesis of aortic valve interstitial cells in situ. METHODS AND RESULTS: Porcine aortic valve leaflets received 7- and 14-day treatments of 15% cyclic stretch (Tension); 0.5 ng/ml transforming growth factor-beta1 (TGF); 15% cyclic stretch and 0.5 ng/ml transforming growth factor-beta1 (Tension+TGF); or neither mechanical nor cytokine stimuli (Null). Tissues were homogenized and assayed for aortic valve interstitial cell phenotype (smooth muscle alpha-actin) and collagen biosynthesis (via heat shock protein 47, which was further verified with type I collagen C-terminal propeptide). At both 7 and 14 days, smooth muscle alpha-actin, heat shock protein 47, and type I collagen C-terminal propeptide quantities were significantly greater (P<.001) in the Tension+TGF group than in all other groups. Additionally, Tension alone appeared to maintain smooth muscle alpha-actin and heat shock protein 47 levels that were measured on Day 0, while TGF alone elicited an increase in smooth muscle alpha-actin and heat shock protein 47 compared to Day 0 levels. Null treatment revealed diminished proteins at both time points. CONCLUSIONS: Elevated transforming growth factor-beta1 levels, in the presence of cyclic mechanical tension, resulted in synergistic increases in contractile and biosynthetic proteins in aortic valve interstitial cells. Since cyclic mechanical stimuli can never be relieved in vivo, the presence of transforming growth factor-beta1 (possibly from infiltrating macrophages) may result in overly biosynthetic aortic valve interstitial cells, leading to altered extracellular matrix architecture, compromised valve function, and, ultimately, degenerative valvular disease.