Transplants Made to Order

The possibility that we might engineer replacements for worn out tissues - from the simple slips of cartilage that cushion joints to fully differentiated, functional grafts in a ready-to-use format - is increasingly plausible. The need is obvious. With advances in medicine, we are outliving the functional life of our organs. Roughly one in five people reaching the age of 65 will benefit from some kind of tissue replacement or transplant in their lifetime,¹ but due to poor availability, many will not. While visions of a healthy, shrink-wrapped heart ready to drop in the chest cavity of a needy patient are pure fantasy for now, tissue engineering is remarkably close to producing biological grafts that can reestablish normal tissue structure and function across different size scales, on a long term, and with the ability to remodel in response to environmental factors, growth, and aging.

Only living, biosynthetically active cells can regenerate a functional tissue structure; thus, tissue engineering invariably involves recapitulation of native development. Mimicking the complexity of living tissue is no small feat. Tissues emerge from coordinated sequences of cell renewal, specialization, and assembly. Embryonic development involves dynamic patterns of multiple signaling cascades, with the positional and time profiles of molecular and physical factors that regulate cell propagation, differentiation, and functional assembly. A fundamental question of developmental biology is: How can cells differentiate in exactly the right place, at the right time, and into the right phenotype to create the right tissues? If we understand how tissues develop, we might also understand how to better engineer their functional equivalents.²

This paradigm holds true in reverse as well. Engineered tissues are being increasingly used as high-fidelity models for quantitative research in biology and medicine, for example in the evaluation of cell and tissue responses to genetic alterations, drugs, hypoxia, and physical stimuli. Tissue engineers are rapidly developing a growing set of tools that may change the way we conduct biological experiments, and eliminate the "flat biology" of Petri dishes in favor of more relevant, yet controllable, three-dimensional models.³

The cell is at the center of the developmental world. Truth be told, we cannot, as tissue engineers, actually claim to engineer tissues. We can only engineer an environment for cells that might induce, enhance, or mediate their developmental processes. But progress has been buoyed by biomimetics - lifting recipes from nature for the design of tissue engineering systems.

Concepts that are intrinsic for developmental biology are now becoming essential for the new generation of tissue engineering: complex signaling, "niche"development, and physical regulatory factors. Thus, to direct biophysical regulation of the cells, we will need to integrate developmental and systems biology into the design of our tissue engineering systems. We know today that this context includes not only regulatory molecules but also physical factors, and that the spatial distribution and dynamics of regulatory factors are as important as the factors themselves. Recent advances in the design and implementation of scaffolds and bioreactors will enable us to establish a high-fidelity niche for controlling cell differentiation and tissue assembly.

HEART STRUCTURE AND FUNCTION Three key features define the approach to developing a strategy for cardiac repair: At the top, a dense vascular network that supports efficient supply of oxygen and nutrients and removal of metabolic products. In the middle, a high density of metabolically active cells. And at the bottom, well orchestrated excitation-contraction coupling at the cellular level that provides a basis for the pumping action of the heart. Middle & Bottom Photos: ©2000 J Wiley & Sons

Many of the advances our group has made have been in trying to recapitulate the complex and highly organized environment of heart tissue. Nearly eight million people in the United States have had myocardial infarction, with 800,000 new cases occurring each year.⁴ Myocardial infarction results in the substantial death of cardiomyocytes in the infarct zone followed by pathological remodeling that involves cardiac dilation, wall thinning, and severe deterioration of contractile function, leading to congestive heart failure in more than 500,000 patients each year.4 Conventional therapies are limited by the inability of myocardium to regenerate after injury, and the shortage of organs available for transplantation. Although transplantation of exogenous cells into damaged myocardium was reported to improve myocardial function and vascular supply, the extent and mechanisms of myocardial repair are not well known, and several reports raise doubts about the actual differentiation of stem cells into cardiac phenotype.^5-7

While the application of cells alone is being pursued vigorously for small-scale injuries, tissue engineering may provide functional cell-based substitutes of the native tissue to treat large defects. Compared to the transplantation of cells alone, synchronously contracting cardiac constructs would offer the advantage of immediate functionality, essentially operating as a viable, functional patch designed to rescue damaged tissue.

The heart itself is a marvel of engineering (see the figure at left). The tissue is composed of tightly packed myocytes and fibroblasts with a dense supporting vasculature and collagen-based extracellular matrix. The myocytes occupy 80% to 90% of the heart volume and form a three-dimensional syncytium that enables propagation of electrical signals across specialized intracellular junctions to produce coordinated mechanical contractions that pump blood forward. An adult heart contains about three billion cells packed at a density of roughly 500 million cells/cm3.

The cardiac myocyte is the most physically energetic cell in the body, contracting more than three billion times in an average human lifespan. To support the high metabolic activity of these cells, the heart has a very dense vasculature with capillaries spaced only about 20 µm apart. The contractile apparatus of cardiac myocytes consists of sarcomeres arranged in parallel myofibrils. Specialized intercellular gap junctions provide electrical signal propagation. The control of heart contractions is almost entirely self-contained. Groups of specialized cardiac myocytes (pace makers), the fastest of which are located in the sinoatrial node, drive periodic contractions of the heart as the majority of myocytes follow along.

The complexity of the structural and functional hierarchy of the heart, the function of which is driven by highly specialized cells, inspired a biomimetics approach to tissue engineering. The general tissue engineering principles outlined above are translated into the set of design criteria for cardiac tissue engineering in a way that enables the cells to drive the functional assembly of highly differentiated, contractile cardiac constructs.

To enable engineering of a functional cardiac patch under laboratory conditions, it is necessary to mimic the structure of native myocardium accurately over several different length scales⁸-from centimeter to nanometer:

• At the centimeter scale, tissue engineering should yield a mechanically stable construct of clinically relevant thickness, comparable to the thickness of the human myocardium (from millimeters to a centimeter). This requirement is hampered by the diffusional limitations of oxygen supply encountered in most tissue-culture vessels, coupled with the high metabolic demand of cardiomyocytes for oxygen.
• At the millimeter scale, the tissue should consist of elongated myofibers aligned in parallel and capable of synchronous contractions, a requirement that is hampered by the lack of appropriate electromechanical stimulation in conventional cultures.
• At the micrometer scale, cells in the engineered cardiac tissue must be coupled by functional gap junctions and be capable of electrical impulse propagation in order to prevent arrhythmia upon implantation.
• At the nanometer scale, functional excitation-contraction machinery of individual cardiomyocytes needs to be established.

Due to the high density and metabolic activity of the cells, myocardium consumes large amounts of oxygen and cannot tolerate hypoxia for long. In native myocardium, oxygen is supplied to the cells through a rich vasculature. The solubility of oxygen in plasma is low, but hemoglobin increases total oxygen content of blood by two orders of magnitude, and thereby increases the mass of tissue that can be supported in a single pass of blood through capillary network. The average oxygen concentration in arterial blood is 130 mM; in venous blood it is 54 mM.

In Petri dishes, oxygen transport into the construct interior occurs only by diffusion, which can support cell viability and function only in thin, superficial layers. To provide oxygen supply to the cells at levels necessary to maintain their viability, we developed a technique of seeding that involves rapid cell inoculation into three-dimensional scaffolds followed by immediate establishment of the interstitial flow of culture medium through the scaffold. Constructs seeded in dishes had most cells located within a ~100 µm thick layer at the top surface, and only a small number of cells penetrated the entire construct depth. Constructs seeded and cultured with medium perfusion had high and spatially uniform cell density throughout the construct.⁹ It is clear that medium perfusion is key for engineering millimeters-thick constructs with high cell densities.

MIMICKING MYOCARDIUM The key features of native tissue are listed on the left; the corresponding methods to these same features for cultivation of engineered tissue are listed on the right.

Nevertheless, most cells were round and mononucleated, a situation that was likely due to the exposure of cardiac myocytes to hydrodynamic shear. In contrast to the native heart muscle where blood is confined within the capillary bed, cells in our constructs were in direct contact with the fluid flow. This motivated the design of tissue engineering systems that provide an in vivo-like oxygen supply. To mimic the capillary network, heart cells were cultured on a scaffold with an array of parallel channels that were perfused with culture medium, (see figure below). To mimic oxygen supply by hemoglobin, culture medium was supplemented by 10% v/v perfluorocarbon (PFC) emulsion that substantially increases the amount of oxygen in culture medium.⁹

As the medium flowed through the channel array, oxygen was depleted by diffusion into the construct space where it was used for cell respiration. Oxygen depletion in the aqueous phase acted as a driving force for the diffusion of dissolved oxygen from the PFC particles, thereby contributing to the maintenance of higher oxygen concentrations in the medium. After only three days in vitro, the cell subpopulations on scaffolds formed constructs that contracted synchronously in response to electrical stimulation. The scaffold pores remained open, and the pressure drop measured across the construct was as low as 0.1 kPa/mm construct thickness.¹⁰

Contraction of the cardiac muscle is driven by the pacing cell-generated waves of electrical excitation that spread rapidly along the membranes of adjoining cardiac myocytes and trigger calcium release, which in turn stimulates myofibril contraction. Electromechanical coupling of the myocytes is crucial for their synchronous response.

We hypothesized that applying electrical signals designed to induce synchronous contractions during cultivation would enhance cell differentiation and functional assembly of engineered tissue via physiologically relevant mechanisms. So we prepared cardiac constructs by seeding collagen sponges with neonatal rat ventricular cells and stimulated them using suprathreshold square biphasic pulses mimicking those that drive contractions of the heart. Stimulation was initiated one to five days after scaffold seeding (to allow the cells to lay down gap junctional and contractile proteins) and applied for up to eight days (to stimulate cell coupling and functional assembly).¹¹

During just eight days of cultivation, electrical field stimulation had induced cell alignment and coupling and increased the amplitude of synchronous contractions by a factor of seven. The stimulated constructs demonstrated a remarkable level of ultrastructural differentiation, comparable in several respects to that of native myocardium (see figure above). Myofibers aligned in the direction of the electrical field lines, possibly in an attempt to decrease the voltage threshold for inducing contractile behavior in response to pacing.

Photos:© Mary Ann Liebert

OXYGEN SUPPLY Medium flow (0.1 ml/min) was provided by a multi-channel peristaltic pump (A) and gas exchange was provided by a coil of thin silicone tubing (B). Channeled elastomer scaffolds (C) 5mm in diameter and 2mm thick were seeded with heart cells at physiologic density (~100 million cells/ml) At left see a scanning electron micrograph of the biorubber scaffold with a parallel channel array and a single channel shown at the beginning and after seven days of cultivation. (Adapted from reference 9.)

In contrast, cells in nonstimulated constructs stayed round and expressed relatively low levels of cardiac markers. Most importantly, electrical stimulation induced the development of long, well-aligned registers of sarcomeres that closely resembled those in native myocardium and that represent a hallmark of maturing cardiomyocytes. The volume fraction of sarcomeres and the frequency of gap junctions and Z lines in the constructs were indistinguishable from those measured for neonatal ventricles.¹¹

On a molecular level, electrical stimulation elevated the levels of all measured cardiac proteins and enhanced the expression of the corresponding genes, without causing pathological cell hypertrophy. With time in culture, the ratio of mature and immature forms of myosin heavy chain (a-MHC and b-MHC, respectively) decreased in nonstimulated constructs and increased in stimulated constructs, suggesting that the maturation of cardiomyocytes depended both on culture duration and electrical stimulation. These studies suggest that electrical stimulation of construct contractions during cultivation progressively enhanced the excitation-contraction coupling and improved the molecular, cellular, and functional properties of engineered myocardium.¹¹

Much progress has been made in understanding the conditions required for directing cell differentiation and assembly into functional tissue structures. Nevertheless, most of the work has been done using animal cells. One major challenge ahead of us is to extend the application of technologically advanced, biologically inspired tissue engineering systems to the cultivation of functional tissue grafts based on human cells. For cardiac repair, various cell sources are currently under consideration, including primitive cells from the adult heart that are capable of dividing and developing into mature heart and vascular cells. A recent study revealed relatively unspecialized cells that can both divide while maintaining their own population and give rise to functional heart cells.¹² Human embryonic stem cells represent another potential source of cardiac cells. For each of these cell sources, the exact factors that need to be applied in vitro to induce and support cardiac cell differentiation and subsequent tissue development remain to be determined.¹³

Photos:©2004 PNAS

CLOSE MATCH Electrical field stimulation resulted in remarkably well developed ultrastructure of engineered cardiac constructs (at left) over only eight days of cultivation. The structures of sarcomeres and gap junctions in stimulated constructs after eight days of cultivation were remarkably similar to neonatal ventricles (at right). Samples stained green for myosin heavy chain, the most abundant contractile protein in the heart. Cell nuclei are shown in blue. (Adapted from reference 11.)

Another challenge is to further advance the capabilities of our in vitro culture systems. We now have prototypes of bioreactors that can provide local microenvironmental control of oxygen and pH (via medium perfusion), simultaneously with the application of physical stimuli (via electrical stimulation). In this way, the cultured cells can be subjected to multiple signals present in the native heart. This system could also enable a new approach to studies of cardiac development and function.

The growing interactions between the fields of tissue engineering and developmental biology are expected to facilitate translation of biological principles into our engineering designs. Using engineered tissues in regenerative medicine requires the demonstration of their safety and efficacy in animal models. To make this happen, we need to continue to move the science and practice of tissue engineering from observational to mechanistic, from serendipitous to rational, and from the limited current products to many more that will improve people's lives.

Gordana Vunjak-Novakovic is a professor at Columbia University's Department for Biomedical Engineering. She is chairing the forthcoming Keystone Symposium on Tissue Engineering and Developmental Biology in April 2007.