Hyoungshin Park

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.

The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation–contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.

Tissue Engineering by Self-Assembly of Cells Printed into Topologically Defined Structures

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.

A NOVEL COMPOSITE SCAFFOLD FOR CARDIAC TISSUE ENGINEERING

SummaryOne approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large intereconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(Dl-lactide-co-caprolactone), poly(Dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80±5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35×108 cells/cm3) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam⋆m) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.

Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors.

Gene delivery to stem cells holds great potential for tissue regeneration and delivery of therapeutic proteins. The major barrier is the lack of safe and efficient delivery methods. Here, we report enhanced gene delivery systems for human stem cells using biodegradable polymeric vectors. A library of poly (β-amino esters) end-modified derivatives was developed and optimized for high transfection efficiency and low cytotoxicity for three human stem cell lines including human mesenchymal stem cells (hMSCs), human adipose-derived stem cells (hADSCs) and human embryonic stem cell-derived cells (hESCds). In the presence of 10% serum, leading end-modified C32 polymeric vectors exhibited significantly high transfection efficiency in hMSCs (27±2%), hADSCs (24±3%) and hESCds (56±11%), with high cell viability (87–97%) achieved in all cell types. Our results show that poly(β-amino esters) as a class, and end-modified versions of C32 in particular, are efficient polymeric vectors for gene delivery to both adult and embryonic-derived stem cells.

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. Copyright

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.

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.

Effects of electrical stimulation in C2C12 muscle constructs.

Electrical stimulation affects the deposition of extracellular matrices and cellular differentiation. Type I collagen is one of the most abundant extracellular matrix proteins; however, not much is known about the effects of electrical stimulation on collagen type I deposition in C2C12 cells. Thus, we studied the effects of electrical voltage and stimulation frequency in 3D cultured C2C12 muscle cells in terms of metabolic activity, type I collagen deposition and cell morphology. Electrically excitable C2C12 muscle cells were seeded in collagen scaffolds and stimulated with rectangular signals of voltage (2, 5, 7 V) and frequency (1, 2 Hz), using parallel carbon electrodes spaced 1 cm apart. Metabolic activity was quantified by the glucose:lactate concentration ratio in the medium. Apoptotic activity was assessed by TUNEL staining and changes in collagen deposition were identified by immunohistology. The ultrastructure of the tissue was examined by TEM. Glucose and lactate analysis indicated that all groups had similar metabolic activity. TUNEL stain showed no significant difference in apoptotic damage induced by electrical stimulation compared to the control. Samples stimulated at 2 Hz exhibited reduced collagen deposition compared to the control and 1 Hz stimulated samples. Muscle‐protein marker desmin was highly expressed in constructs stimulated with 1 Hz/5 V sample. TEM revealed that the stimulated samples developed highly organized sarcomeres, which coincided with improved contractile properties in the 1 Hz/5 V‐ and 2 Hz/5 V‐stimulated groups. Our data implicate that a specific electrical frequency may modulate type I collagen accumulation and a specific voltage may affect the differentiation of muscle sarcomeres in excitable cells. Copyright

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.

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.