Growth of engineered human myocardium with mechanical loading and vascular coculture.

RATIONALE: The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. OBJECTIVE: We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. METHODS AND RESULTS: Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. CONCLUSIONS: Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Proangiogenic scaffolds as functional templates for cardiac tissue engineering.

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30-40 microm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering.

OBJECTIVE: Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. METHODS AND RESULTS: To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. CONCLUSIONS: VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.

Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts.

BACKGROUND: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration. METHODS: We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation. RESULTS: Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint. CONCLUSION: Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

Photo-patterning of porous hydrogels for tissue engineering.

Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360+/-25 to 730+/-70 microm were patterned into approximately 700 microm thick hydrogels with pore diameters of 62+/-8 or 147+/-15 microm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147 microm pores and 730 microm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications.

Chitosan-alginate hybrid scaffolds for bone tissue engineering.

A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan-alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan-alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid-solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.

A model for studying epithelial attachment and morphology at the interface between skin and percutaneous devices.

Percutaneous devices are indispensable in modern medicine, yet complications from their use result in significant morbidity, mortality, and cost. Bacterial biofilm at the device exit site accounts for most infections in short-term devices. We hypothesize that advanced biomaterials can be developed that facilitate attachment of skin cells to percutaneous devices, forming a seal to preclude bacterial invasion. To study the skin/biomaterial interface systematically, we first identified biomaterials with physical properties compatible with histological processing of skin. Second, we developed an organ culture system to study skin response to implants. Organ cultures implanted with porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] or polytetrafluoroethylene (PTFE) could easily be evaluated histologically with preservation of the skin/biomaterial interface. Epithelial cells migrated down the cut edges of the biomaterial in a pattern seen in marsupialization of percutaneous devices in vivo. This in vitro model maintains skin viability and allows histologic evaluation of the skin/biomaterial interface, making this a useful, inexpensive test-bed for studies of epidermal attachment to modified biomaterials.

Linear chains and chain-like fractals from electrostatic heteroaggregation.

The internal structure of materials prepared by aggregation of oppositely charged polystyrene spheres (electrostatic heteroaggregation) is investigated by static light scattering, optical microscopy, and Brownian dynamics simulation. Light scattering indicates ultralow mass fractal dimensions, as low as 1.2. Such low fractal dimensions, approaching the theoretical limit of a linear object, imply a chaining mechanism. Optical micrographs reveal linear chains with the particle charge alternating down the chains. Brownian dynamics simulation gives additional support for a chaining mechanism. For the polystyrene system (120-nm primary particle diameters), the fractal dimension is found to increase from 1.2 to 1.7 as the background electrolyte is increased. In terms of electrostatic screening, the results match those reported recently for larger polystyrene spheres. The low fractal dimensions appear to represent a crossover from linear chains to a structure of diffusion-limited aggregates; however, experiments under density-neutral conditions imply that sedimentation plays an important role in the formation of ultralow fractal dimensions. The practical implication is that microcomposites with a locally uniform distribution of starting materials and almost any degree of branching can be prepared from oppositely charged particles.

Identification and Imaging of 15N Labeled Cells with ToF-SIMS.

Stable isotope labeling may provide a novel method for tracking stem cells once they have been injected into a human or animal host. Here we present a simple pilot study to determine the potential for using ToF-SIMS to detect and localize 15N labeled cells in tissue biopsies for use in cell therapy studies. For this pilot study, 3T3 fibroblasts were grown in normal media and in two different media containing 15N labeled amino acids. Samples containing a mixture of 15N labeled and unlabeled cells were prepared, fixed and dried for analysis and were then imaged using a bunched Bi3+ primary ion source. The cells containing 15N labeled amino acids could be readily distinguished using nitrogen containing peaks which have been previously associated with the labeled amino acids. Contrast was sufficient to allow easy identification of labeled cells in both sparsely and densely plated cultures. Multivariate analysis showed that the image contrast could be improved by including peaks originating from characteristic fragments of the labeled amino acids as well as lower mass NH4+ and CH4N+ peaks. Additional work is being pursued to determine and improve the longevity of the label.

Quantifying the effect of pore size and surface treatment on epidermal incorporation into percutaneously implanted sphere-templated porous biomaterials in mice.

The sinus between skin and a percutaneous medical device is often a portal for infection. Epidermal integration into an optimized porous biomaterial could seal this sinus. In this study, we measured epithelial ingrowth into rods of sphere-templated porous poly(2-hydroxyethyl methacrylate) implanted percutaneously in mice. The rods contained spherical 20-, 40-, or 60-µm pores with and without surface modification. Epithelial migration was measured 3, 7, and 14 days post-implantation utilizing immunohistochemistry for pankeratins and image analysis. Our global results showed average keratinocyte migration distances of 81 ± 16.85 µm (SD). Migration was shorter through 20-µm pores (69.32 ± 21.73) compared with 40 and 60 µm (87.04 ± 13.38 µm and 86.63 ± 8.31 µm, respectively). Migration was unaffected by 1,1' carbonyldiimidazole surface modification without considering factors of pore size and healing duration. Epithelial integration occurred quickly showing an average migration distance of 74.13 ± 12.54 µm after 3 days without significant progression over time. These data show that the epidermis closes the sinus within 3 days, migrates into the biomaterial (an average of 11% of total rod diameter), and stops. This process forms an integrated epithelial collar without evidence of marsupialization or permigration.

Controlled release of dexamethasone from subcutaneously-implanted biosensors in pigs: localized anti-inflammatory benefit without systemic effects.

Chronically implanted biosensors typically lose sensitivity 1-2 months after implantation, due in large part to the development of a collagen-rich capsule that prevents analytes of interest from reaching the biosensor. Corticosteroids are likely candidates for reducing collagen deposition but these compounds have many serious side effects when given over a prolonged period. One method of assessing whether or not locally released corticosteroids have a systemic effect is to measure cortisol concentrations in venous serum. We hypothesized that a very low release rate of the potent corticosteroid, dexamethasone, would lead to a localized anti-inflammatory effect without systemic effects. We found that reduction in subcutaneous granulocytes (primarily eosinophils), and to a lesser extent, reduction of macrophages served as a good local indicator of the steroid effect. When released over a 28-day period, a total dexamethasone dose of < or =0.1 mg/kg led to a consistent reduction in the number of granulocytes and macrophages found in the local vicinity of the implant without a reduction of these cells at distant tissue locations. The lack of suppression of serum cortisol with these doses confirmed that low-release rates of dexamethasone can lead to consistent local anti-inflammatory effects without distant, systemic effects. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Cardiac applications for human pluripotent stem cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can self-renew indefinitely, while maintaining the capacity to differentiate into useful somatic cell types, including cardiomyocytes. As such, these stem cell types represent an essentially inexhaustible source of committed human cardiomyocytes of potential use in cell-based cardiac therapies, high-throughput screening and safety testing of new drugs, and modeling human heart development. These stem cell-derived cardiomyocytes have an unambiguous cardiac phenotype and proliferate robustly both in vitro and in vivo. Recent transplantation studies in preclinical models have provided exciting proof-of-principle for their use in infarct repair and in the formation of a "biological pacemaker". While these successes give reason for cautious optimism, major challenges remain to the successful application of hESCs (or hiPSCs) to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. In this review, we describe the phenotype of hESC- and hiPSC-derived cardiomyocytes, the state of preclinical transplantation studies with these cells, and potential approaches to overcome the aforementioned hurdles.

Human embryonic stem cells and cardiac repair.

The muscle lost after a myocardial infarction is replaced with noncontractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. Human embryonic stem cell-derived cardiomyocytes exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain in the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-derived cardiomyocytes and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges.

Effect of adsorbed von Willebrand factor and fibrinogen on platelet interactions with synthetic materials under flow conditions.

The effect of adsorbed fibrinogen (Fg) and von Willebrand factor (vWf) on platelet adhesion at low or high shear rate to several materials was studied. The materials studied were polyethylene terephthalate (PET), polystyrene (PS), glass, and tetraglyme-coated PET. The materials were preadsorbed with normal plasma, serum, and Fg-deficient plasma replenished with various amounts of Fg, and vWf-deficient plasma with or without added vWf. Platelet adhesion to PET preadsorbed with Fg-deficient plasma or serum was low at either low or high shear rate, but increased as Fg was added to the preadsorption media. However, the effect of added Fg on adhesion at the higher shear rate was much greater on surfaces preadsorbed with plasma than for serum, probably due to the much lower vWf concentration in serum in comparison to plasma. Platelet adhesion to either polystyrene or glass preadsorbed with normal plasma was much higher at high shear than low shear, but when vWf-deficient plasma was used to preadsorb these surfaces, adhesion was much less at the higher shear rate than at low shear rate. Platelet adhesion to polystyrene preadsorbed with vWf-deficient plasma to which vWf was added was higher at high shear rate than low shear rate. These results show that under high shear rate, both Fg and vWf are required for platelet adhesion on synthetic biomaterials. The results suggest that developing surfaces that adsorb low amounts of vWf is a good approach to improving the blood compatibility of biomaterials.

Characterization of an in vitro model for evaluating the interface between skin and percutaneous biomaterials.

Percutaneous devices play an essential role in medicine; however, they are often associated with a significant risk of infection. One approach to circumvent infection would be to heal the wound around the devices by promoting skin cell attachment. We used two in vitro assay models to evaluate cutaneous response to poly(2-hydoxyethyl methacrylate) (poly(HEMA)). One approach was to use a cell adhesion assay to test the effects of surface modification of poly(HEMA), and the second used an organ culture system of newborn foreskin biopsies implanted with porous poly(HEMA) rods (20 microm pores) to evaluate the skin/poly(HEMA) interface. Surface modification of poly(HEMA) using 1,1'-carbonyldiimidazole (CDI) enhanced keratinocyte, fibroblast, and endothelial cell adhesion. Keratinocytes in the organ culture model not only remained functionally and structurally viable as observed by immunohistochemistry and electron microscopy, but migrated into the pores of CDI-modified poly(HEMA) rods. No biointegration was seen in the non-CDI-modified poly(HEMA). Laminin 5 immunostaining was seen along the poly(HEMA)/skin interface in a pattern resembling the junctional epithelium of the tooth, the unique natural interface between the skin and tooth that serves as a barrier to bacteria. In vitro systematic evaluation of biomaterials for use in animal implant studies is both cost effective and time efficient.