Reproducing human and cross-species drug toxicities using a Liver-Chip.

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three-dimensionally woven scaffolds.

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three-dimensionally woven poly(ε-caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations-PCL alone, PCL-bone, "point-of-care" seeded MSC-PCL-bone, and chondrogenically precultured Ch-MSC-PCL-bone constructs-were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch-MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.

Organ-on-Chip Recapitulates Thrombosis Induced by an anti-CD154 Monoclonal Antibody: Translational Potential of Advanced Microengineered Systems.

Clinical development of Hu5c8, a monoclonal antibody against CD40L intended for treatment of autoimmune disorders, was terminated due to unexpected thrombotic complications. These life-threatening side effects were not discovered during preclinical testing due to the lack of predictive models. In the present study, we describe the development of a microengineered system lined by human endothelium perfused with human whole blood, a "Vessel-Chip." The Vessel-Chip allowed us to evaluate key parameters in thrombosis, such as endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and thrombin anti-thrombin complexes in the Chip-effluent in response to Hu5c8 in the presence of soluble CD40L. Importantly, the observed prothrombotic effects were not observed with Hu5c8-IgG2σ designed with an Fc domain that does not bind the FcγRIIa receptor, suggesting that this approach may have a low potential risk for thrombosis. Our results demonstrate the translational potential of Organs-on-Chips, as advanced microengineered systems to better predict human response.

Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model.

Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well-defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose-dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose-dependent increase in secretion of IL-6, IL-8, TNF-α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well-characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner.

Biomimetic scaffold combined with electrical stimulation and growth factor promotes tissue engineered cardiac development.

Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T. IGF-1 reduced apoptosis, promoted cell-to-cell connectivity, and lowered excitation threshold, an index of electrophysiological activity. ES promoted formation of tissue-like bundles oriented in parallel to the electrical field and a more than ten-fold increase in matrix metalloprotease-2 (MMP-2) gene expression. The combination of IGF-1 and ES increased 2D projection length, an index of overall contraction strength, and enhanced expression of the gap junction protein connexin-43 and sarcomere development. This culture environment, designed to combine cardiac-like scaffold architecture and biomechanics with molecular and biophysical signals, enabled functional assembly of engineered heart muscle from dissociated cells and could serve as a template for future studies on the hierarchy of various signaling domains relative to cardiac tissue development.

A biodegradable microvessel scaffold as a framework to enable vascular support of engineered tissues.

A biodegradable microvessel scaffold comprised of distinct parenchymal and vascular compartments separated by a permeable membrane interface was conceptualized, fabricated, cellularized, and implanted. The device was designed with perfusable microfluidic channels on the order of 100 µm to mimic small blood vessels, and high interfacial area to an adjacent parenchymal space to enable transport between the compartments. Poly(glycerol sebacate) (PGS) elastomer was used to construct the microvessel framework, and various assembly methods were evaluated to ensure robust mechanical integrity. In vitro studies demonstrated the differentiation of human skeletal muscle cells cultured in the parenchymal space, a 90% reduction in muscle cell viability due to trans-membrane transport of a myotoxic drug from the perfusate, and microvessel seeding with human endothelial cells. In vivo studies of scaffolds implanted subcutaneously and intraperitoneally, without or with exogenous cells, into nude rats demonstrated biodegradation of the membrane interface and host blood cell infiltration of the microvessels. This modular, implantable scaffold could serve as a basis for building tissue constructs of increasing scale and clinical relevance.

3D structural patterns in scalable, elastomeric scaffolds guide engineered tissue architecture.

Microfabricated elastomeric scaffolds with 3D structural patterns are created by semiautomated layer-by-layer assembly of planar polymer sheets with through-pores. The mesoscale interconnected pore architectures governed by the relative alignment of layers are shown to direct cell and muscle-like fiber orientation in both skeletal and cardiac muscle, enabling scale up of tissue constructs towards clinically relevant dimensions.

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.

Optimization of electrical stimulation parameters for cardiac tissue engineering.

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43 and the best-developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering.

Assessment of canine vocal fold function after injection of a new biomaterial designed to treat phonatory mucosal scarring.

OBJECTIVES: Most cases of irresolvable hoarseness are due to deficiencies in the pliability and volume of the superficial lamina propria of the phonatory mucosa. By using a US Food and Drug Administration-approved polymer, polyethylene glycol (PEG), we created a novel hydrogel (PEG30) and investigated its effects on multiple vocal fold structural and functional parameters. METHODS: We injected PEG30 unilaterally into 16 normal canine vocal folds with survival times of 1 to 4 months. High-speed videos of vocal fold vibration, induced by intratracheal airflow, and phonation threshold pressures were recorded at 4 time points per subject. Three-dimensional reconstruction analysis of 11.7 T magnetic resonance images and histologic analysis identified 3 cases wherein PEG30 injections were the most superficial, so as to maximally impact vibratory function. These cases were subjected to in-depth analyses. RESULTS: High-speed video analysis of the 3 selected cases showed minimal to no reduction in the maximum vibratory amplitudes of vocal folds injected with PEG30 compared to the non-injected, contralateral vocal fold. All PEG30-injected vocal folds displayed mucosal wave activity with low average phonation threshold pressures. No significant inflammation was observed on microlaryngoscopic examination. Magnetic resonance imaging and histologic analyses revealed time-dependent resorption of the PEG30 hydrogel by phagocytosis with minimal tissue reaction or fibrosis. CONCLUSIONS: The PEG30 hydrogel is a promising biocompatible candidate biomaterial to restore form and function to deficient phonatory mucosa, while not mechanically impeding residual endogenous superficial lamina propria.

Application of a dense gas technique for sterilizing soft biomaterials.

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.

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.

Chondrogenesis and mineralization during in vitro culture of human mesenchymal stem cells on three-dimensional woven scaffolds.

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

Combined technologies for microfabricating elastomeric cardiac tissue engineering scaffolds.

Polymer scaffolds that direct elongation and orientation of cultured cells can enable tissue engineered muscle to act as a mechanically functional unit. We combined micromolding and microablation technologies to create muscle tissue engineering scaffolds from the biodegradable elastomer poly(glycerol sebacate). These scaffolds exhibited well defined surface patterns and pores and robust elastomeric tensile mechanical properties. Cultured C2C12 muscle cells penetrated the pores to form spatially controlled engineered tissues. Scanning electron and confocal microscopy revealed muscle cell orientation in a preferential direction, parallel to micromolded gratings and long axes of microablated anisotropic pores, with significant individual and interactive effects of gratings and pore design.

Three-dimensional hydrogel model using adipose-derived stem cells for vocal fold augmentation.

Adipose-derived stem cells (ASCs) may provide a clinical option for rebuilding damaged superficial lamina propria of the vocal fold. We investigated the effects of five hydrogels (hyaluronic acid [HA], collagen, fibrin, and cogels of fibrin-collagen and fibrin-HA) on the differentiation of ASCs, with the long-term goal of establishing the conditions necessary for controlling the differentiation of ASC into the functional equivalent of superficial lamina propria fibroblasts. Human ASCs were isolated and characterized by fluorescence-activated cell sorting and real-time polymerase chain reaction. According to fluorescence-activated cell sorting and gene analysis, over 90% of isolated ASCs expressed adult stem cell surface markers and expressed adult stem cell genes. Scaffold-specific gene expression and morphology were assessed by culturing the ASCs in three-dimensional hydrogels. Twofold higher amounts of total DNA were detected in fibrin and cogel cultures than in collagen and HA cultures. Elastin expression was significantly higher in cells grown in fibrin-based gels than in cells grown in other gels. Cells grown in the cogels showed elongated morphology, expressed decorin marker, and exhibited glycosaminoglycan synthesis, which indicate ASC differentiation. Our data suggest that it may be possible to control the differentiation of ASCs using scaffolds appropriate for vocal fold tissue engineering applications. In particular, cogels of HA or collagen with fibrin enhanced proliferation, differentiation, and elastin expression.

Indirect low-intensity ultrasonic stimulation for tissue engineering.

Low-intensity ultrasound (LIUS) treatment has been shown to increase mass transport, which could benefit tissue grafts during the immediate postimplant period, when blood supply to the implanted tissue is suboptimal. In this in vitro study, we investigated effects of LIUS stimulation on dye diffusion, proliferation, metabolism, and tropomyosin expression of muscle cells (C2C12) and on tissue viability and gene expression of human adipose tissue organoids. We found that LIUS increased dye diffusion within adjacent tissue culture wells and caused anisotropic diffusion patterns. This effect was confirmed by a hydrophone measurement resulting in acoustic pressure 150-341 Pa in wells. Cellular studies showed that LIUS significantly increased proliferation, metabolic activity, and expression of tropomyosin. Adipose tissue treated with LIUS showed significantly increased metabolic activity and the cells had similar morphology to normal unilocular adipocytes. Gene analysis showed that tumor necrosis factor-alpha expression (a marker for tissue damage) was significantly lower for stimulated organoids than for control groups. Our data suggests that LIUS could be a useful modality for improving graft survival in vivo.

Modulation of vocal fold scar fibroblasts by adipose-derived stem/stromal cells.

OBJECTIVES/HYPOTHESIS: To explore whether adipose-derived stem/stromal cells (ASCs) have therapeutic potential for treating scarred superficial lamina propria through the effects of secreted hepatocyte growth factor (HGF) on scar fibroblasts. STUDY DESIGN: In vitro study using coculture system. METHODS: Scar fibroblasts (SFs) were isolated from ferret vocal folds electrocauterized 2 weeks previously. ASCs were isolated from ferret lipoaspirated subcutaneous abdominal fat. For coculture experiments, the two cell types were combined in Transwell plates for 6 days, followed by 1 or 3 days of monoculture after removing the upper chamber. Assays were then performed on cells and media from the bottom chamber. We measured: 1) the production of hyaluronic acid (HA), collagen and HGF via enzyme-linked immunosorbent assays, 2) the expression of alpha-smooth muscle actin (alpha-SMA), 3) cell proliferation, and 4) apoptosis of SFs (2, 3, and 4 via flow cytometry). Other experiments examined the effects of HGF on SFs and the effects of HGF neutralization in the coculture system. RESULTS: Coculture led to significant decreases in SF collagen production (P < .05), cell proliferation (P < .05), and alpha-SMA expression (P < .05), whereas HA production increased (P < .05). Coculture also increased HGF secretion from ASCs (P < .05). Neutralization of HGF abolished the inhibitory effects of ASCs on SF collagen synthesis (P < .05). CONCLUSIONS: ASCs influence SFs to adopt a less fibrotic profile. It appears that HGF is at least one of the soluble factors responsible for this effect. Implanted ASCs could potentially ameliorate vocal fold scar by acting as long-term, intrinsic sources of HGF.

11.7 Tesla magnetic resonance microimaging of laryngeal tissue architecture.

OBJECTIVES/HYPOTHESIS: High-resolution imaging of vocal folds that distinguishes vocal fold (VF) layered microstructure and VF implants would provide a key experimental tool for translational research investigating biomaterial-based interventions to treat vocal fold scar. To establish proof of concept, we studied whether 11.7 Tesla (T) magnetic resonance (MR) microimaging provides the needed resolution to resolve vocal fold tissue architecture. STUDY DESIGN: We performed ex vivo MR microimaging of fixed ferret and canine larynges to determine whether changes in the layered architecture can be detected in the presence of scar and subsequent to biomaterial injections into the vocal folds. Serial section histological analyses were done to corroborate MR microimaging findings. METHODS: Multiple axial and transverse/coronal 300-microm slices were obtained using an 11.7 T MR spectrometer/500 MHz for proton with gradient-recalled echo and rapid acquisition with relaxation enhancement imaging sequences. RESULTS: High-resolution (39 microm/pixel) MR microimages distinguished VF epithelium, lamina propria, muscle, and cartilage in ferret and canine larynges. In ferret scarred VFs (n = 25), collagen-rich dense scar tissue was distinguishable from contralateral nonscarred VFs and from normal ferret VFs (n = 25), as confirmed on histology. MR microimaging accurately detected injected autologous fat, hyaluronic acid-based and polyethylene glycol (PEG)-based implants injected into both ferret and canine VFs. Importantly, MRI accurately showed resorption of PEG implants in ferrets and canines, as confirmed on histology. Additionally, ex vivo MR spectroscopy distinguished fat from PEG-based implants. CONCLUSIONS: Ex vivo 11.7 T MR microimaging provided high-resolution images of ferret and canine laryngeal tissue microstructure, although the superficial lamina propria could not be distinguished. Histology confirmed MR microimaging findings, indicating utility of MR microimaging of modeled scar, implant residence time, and tissue responses, thus providing integrative insight relevant to translational research.

Crosstalk between adipose-derived stem/stromal cells and vocal fold fibroblasts in vitro.

OBJECTIVES/HYPOTHESIS: To explore adipose-derived stem cell/fibroblast interactions as a potential remodeling pathway for vocal fold scar. STUDY DESIGN: Fibroblasts and adipose-derived stem/stromal cells (ASCs) were cultured alone and in combination in a cell-contact-independent paracrine system. Analyses of cell proliferation, and the production of hyaluronic acid (HA) and collagen were performed on samples collected on days 1, 3, and 7. METHODS: Normal fibroblasts (NFs) were isolated bilaterally from the subepithelial lamina propria of two normal ferret vocal folds. Scar fibroblasts (SFs) were isolated from vocal folds that were electrocauterized 2 weeks before harvest. ASCs were isolated from lipoaspirated subcutaneous abdominal fat of two ferrets. A transwell cell-contact-independent cell communication culturing system was used for coculture experiments. Cells were seeded at 50,000/well in both monoculture and coculture experiments. RESULTS: In monoculture, SFs proliferated faster and produced less HA and more collagen than NFs at day 7 (P < .05). In SF/ASC coculture, SF proliferation was diminished and collagen production at day 7 decreased (P < .05). HA production did not differ between monoculture and coculture conditions. CONCLUSIONS: Normal and scar-tissue-derived vocal fold fibroblasts maintain phenotypic differences in culture, thus validating this in vitro scar model. In co-culture, contact-independent crosstalk occurs between SFs and ASCa, leading to less collagen secretion. The data support the hypothesis that ASCs can induce favorable remodeling of scarred vocal folds in vivo by their interactions with endogenous fibroblasts.

Optical mapping of impulse propagation in engineered cardiac tissue.

Cardiac tissue engineering has a potential to provide functional, synchronously contractile tissue constructs for heart repair, and for studies of development and disease using in vivo-like yet controllable in vitro settings. In both cases, the utilization of bioreactors capable of providing biomimetic culture environments is instrumental for supporting cell differentiation and functional assembly. In the present study, neonatal rat heart cells were cultured on highly porous collagen scaffolds in bioreactors with electrical field stimulation. A hallmark of excitable tissues such as myocardium is the ability to propagate electrical impulses. We utilized the method of optical mapping to measure the electrical impulse propagation. The average conduction velocity recorded for the stimulated constructs (14.4 +/- 4.1 cm/s) was significantly higher than that of the nonstimulated constructs (8.6 +/- 2.3 cm/s, p = 0.003). The measured electrical propagation properties correlated to the contractile behavior and the compositions of tissue constructs. Electrical stimulation during culture significantly improved amplitude of contractions, tissue morphology, and connexin-43 expression compared to the nonsimulated controls. These data provide evidence that electrical stimulation during bioreactor cultivation can improve electrical signal propagation in engineered cardiac constructs.