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.

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.

Protein precoating of elastomeric tissue-engineering scaffolds increased cellularity, enhanced extracellular matrix protein production, and differentially regulated the phenotypes of circulating endothelial progenitor cells.

BACKGROUND: Optimal cell sources and scaffold-cell interactions remain unanswered questions for tissue engineering of heart valves. We assessed the effect of different protein precoatings on a single scaffold type (elastomeric poly (glycerol sebacate)) with a single cell source (endothelial progenitor cells). METHODS AND RESULTS: Elastomeric poly (glycerol sebacate) scaffolds were precoated with laminin, fibronectin, fibrin, collagen types I/III, or elastin. Characterized ovine peripheral blood endothelial progenitor cells were seeded onto scaffolds for 3 days followed by 14 days incubation. Endothelial progenitor cells were CD31+, vWF+, and alpha-SMA- before seeding confirmed by immunohistochemistry and immunoblotting. Both precoated and uncoated scaffolds demonstrated surface expression of CD31+ and vWF+, alpha-SMA+ cells and were found in the "interstitium" of the scaffold. Protein precoating of elastomeric poly (glycerol sebacate) scaffolds revealed significantly increased cellularity and altered the phenotypes of endothelial progenitor cells, which resulted in changes in cellular behavior and extracellular matrix production. Moreover, mechanical flexure testing demonstrated decreased effective stiffness of the seeded scaffolds compared with unseeded controls. CONCLUSIONS: Scaffold precoating with extracellular matrix proteins can allow more precise "engineering" of cellular behavior in the development of tissue engineering of heart valves constructs by altering extracellular matrix production and cell phenotype.

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.

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.

Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds.

BACKGROUND: Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-beta1. Method and Results- Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2 x 10(6) cells/cm2, scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-beta1. After seeding at 15 x 10(6) cells/cm2, scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-beta1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31+, von Willebrand factor+, and alpha-smooth muscle actin (SMA)-) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31+ and alpha-SMA+) in response to TGF-beta1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-beta1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-beta1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-beta1-mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-beta1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-beta1-stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls. CONCLUSIONS: Stimulation of EPC-seeded tissue engineering scaffolds with TGF-beta1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-beta1 could offer new strategies to guide tissue formation in engineered cardiac valves.

Effects of regulatory factors on engineered cardiac tissue in vitro.

We tested the hypothesis that supplemental regulatory factors can improve the contractile properties and viability of cardiac tissue constructs cultured in vitro. Neonatal rat heart cells were cultured on porous collagen sponges for up to 8 days in basal medium or medium supplemented with insulin-like growth factor-I (IGF), insulin-transferrin-selenium (ITS), platelet-derived growth factor-BB (PDGF), or angiopoietin-1 (ANG). IGF and ITS enhanced contractile properties of the 8-day constructs significantly more than with unsupplemented controls according to contractile amplitude and excitation threshold, and IGF also significantly increased the amount of cardiac troponin-I and enhanced cell viability according to different assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and terminal deoxynucleotidyl transferase biotin-2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)). PDGF significantly increased the contractile amplitude of 4-day constructs and enhanced cell viability according to MTT, LDH, and TUNEL; ANG enhanced cell viability according to the LDH assay. Our results demonstrate that supplemental regulatory molecules can differentially enhance properties of cardiac tissue constructs and imply that these constructs can provide a platform for systematic in vitro studies of the effects of complex stimuli that occur in vivo to improve our basic understanding of cardiogenesis and identify underlying mechanisms that can potentially be exploited to enhance myocardial regeneration.

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.

From stem cells to viable autologous semilunar heart valve.

BACKGROUND: An estimated 275,000 patients undergo heart valve replacement each year. However, existing solutions for valve replacement are complicated by the morbidity associated with lifelong anticoagulation of mechanical valves and the limited durability of bioprostheses. Recent advances in tissue engineering and our understanding of stem cell biology may provide a lifelong solution to these problems. METHODS AND RESULTS: Mesenchymal stem cells were isolated from ovine bone marrow and characterized by their morphology and antigen expression through immunocytochemistry, flow cytometry, and capacity to differentiate into multiple cell lineages. A biodegradable scaffold was developed and characterized by its tensile strength and stiffness as a function of time in cell-conditioned medium. Autologous semilunar heart valves were then created in vitro using mesenchymal stem cells and the biodegradable scaffold and were implanted into the pulmonary position of sheep on cardiopulmonary bypass. The valves were evaluated by echocardiography at implantation and after 4 months in vivo. Valves were explanted at 4 and 8 months and examined by histology and immunohistochemistry. Valves displayed a maximum instantaneous gradient of 17.2+/-1.33 mm Hg, a mean gradient of 9.7+/-1.3 mm Hg, an effective orifice area of 1.35+/-0.17 cm2, and trivial or mild regurgitation at implantation. Gradients changed little over 4 months of follow-up. Histology showed disposition of extracellular matrix and distribution of cell phenotypes in the engineered valves reminiscent of that in native pulmonary valves. CONCLUSIONS: Stem-cell tissue-engineered heart valves can be created from mesenchymal stem cells in combination with a biodegradable scaffold and function satisfactorily in vivo for periods of >4 months. Furthermore, such valves undergo extensive remodeling in vivo to resemble native heart valves.

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.

Guidance of engineered tissue collagen orientation by large-scale scaffold microstructures.

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale ( approximately 200 microm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 microm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds (n=6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124-127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.

The independent role of cyclic flexure in the early in vitro development of an engineered heart valve tissue.

Tissue engineered heart valves (TEHV) are being investigated as an alternative to current non-viable prosthetic valves and valved conduits. Studies suggest that pulse duplicator bioreactors can stimulate TEHV development. In the current study, a model system was used to determine if cyclic flexure, a major mode of heart valve deformation, has independent effects on TEHV cell and extracellular matrix (ECM) development. Ovine vascular smooth muscle cells (SMC) were seeded for 30 h onto strips of non-woven 50:50 polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffold. After 4 days of incubation, SMC-seeded and unseeded scaffolds were either maintained under static conditions (static group), or subjected to unidirectional cyclic three-point flexure at a physiological frequency and amplitude in a bioreactor (flex group) for 3 weeks. After seeding or incubation, the effective stiffness (E) was measured, with SMC-seeded scaffolds further characterized by DNA, collagen, sulfated glycosaminoglycan (S-GAG), and elastin content, as well as by histology. The seeding period was over 90% efficient, with a significant accumulation of S-GAG, no significant change in E, and no collagen detected. Following 3 weeks of incubation, unseeded scaffolds exhibited no significant change in E in the flex or static groups. In contrast, E of SMC-seeded scaffolds increased 429% in the flex group (p<0.01) and 351% in the static group (p<0.01), with a trend of increased E, a 63% increase in collagen (p<0.05), increased vimentin expression, and a more homogenous transmural cell distribution in the flex versus static group. Moreover, a positive linear relationship (r2=0.996) was found between the mean E and mean collagen concentration. These results show that cyclic flexure can have independent effects on TEHV cell and ECM development, and may be useful in predicting the mechanical properties of TEHV constructed using novel scaffold materials.

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 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.

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.