Bioengineered tissues: the science, the technology, and the industry.

OBJECTIVE: The bioengineering of tissues and organs, sometimes called tissue engineering and at other times regenerative medicine, is emerging as a science, as a technology, and as an industry. The goal is the repair, replacement, and/or the regeneration of tissues and organs. The objective of this paper is to identify and discuss the major issues that have become apparent. RESULTS: One of the critical issues is that of cell source, i.e. what will be the source of the cells to be employed? Another critical issue is the development of approaches for the fabrication of substitute tissues/organs and/or vehicles for the delivery of biological active molecules for use in the repair/regeneration of tissues. A third critical issue, one very much related to cell source, is that of immune acceptance. In addition, there are technological hurdles; there are additional issues such as the scale-up of manufacturing processes and the preservation of living-cell products for off-the-shelf availability. Although the initial products have been superficially applied skin substitutes, as this fledgling industry continues to evolve, it is beginning to focus on a wider range of more invasive and complicated products. From a public health perspective, the real opportunity may be in addressing chronic diseases, as well as the transplantation crisis (i.e. the tremendous disparity between patient need for vital organs and donor availability) and, equally important is the challenge of neural repair. CONCLUSION: These are the grand challenges, and the scientific community, business/private sector, and federal government must mobilize itself together in this emerging area to translate the benchtop science to the patient bedside.

Endothelial cell monolayer formation: effect of substrate and fluid shear stress.

The formation of an endothelial cell (EC) monolayer is one critical factor in the development of a tissue engineered vascular graft. One potential method of endothelialization is the migration of native ECs from the surrounding blood vessel onto the newly implanted graft. In the present study, in vitro experiments were performed to investigate the potential of ECs to migrate on a tissue-engineered blood vessel wall model (TEWM) and form a new monolayer. The TEWM was composed of a three-dimensional, type I collagen matrix seeded with smooth muscle cells. The potential of ECs to form new monolayer was evaluated in the absence and presence of fluid shear stress (10 dynes/cm(2)). The monolayer formation on the TEWM was compared to a control, two-dimensional substrate of glass coated with type I collagen. Results from these studies showed that EC monolayer formation was inhibited on the TEWM in comparison to glass coated with collagen. This correlated with an inhibition of proliferation on the TEWM. The application of shear stress to the EC on the glass coated with collagen also caused an inhibition of monolayer formation, with a corresponding inhibition of proliferation. Furthermore, blocking proliferation by incubation with mitomycin C caused a dose-response inhibition of monolayer formation. In contrast, stimulating proliferation with basic fibro- blast growth factor (bFGF) did not further increase monolayer formation on glass coated with collagen. These results suggest that proliferation is one necessary factor for monolayer formation, although not the only factor, where EC proliferation is regulated by its environment, including both substrate and the local shear stress. Continued investigation into the mechanism and stimulation of EC proliferation on the TEWM may lead to developing new strategies for the endothelialization of a tissue engineered vascular graft.

Modeling of cryopreservation of engineered tissues with one-dimensional geometry.

Long-term storage of engineered bio-artificial tissues is required to ensure the off-the-shelf availability to clinicians due to their long production cycle. Cryopreservation is likely the choice for long-term preservation. Although the cryopreservation of cells is well established for many cell types, cryopreservation of tissues is far more complicated. Cells at different locations in the tissue could experience very different local environmental changes, i.e., the change of concentration of cryoprotecting chemicals (CPA) and temperature, during the addition/removal of CPA and cooling/warming, which leads to nonuniformity in cell survival in the tissue. This is due to the limitation of mass and heat transfer within the tissue. A specific aim of cryopreservation of tissue is to ensure a maximum recovery of cells and their functionality throughout a tissue. Cells at all locations should be protected adequately by the CPA and frozen at rates conducive to survival. It is hence highly desirable to know the cell transient and final states during cryopreservation within the whole tissue, which can be best studied by mathematical modeling. In this work, a model framework for cryopreservation of one-dimensional artificial tissues is developed on the basis of solving the coupled equations to describe the mass and heat transfer within the tissue and osmotic transport through the cell membrane. Using an artificial pancreas as an example, we carried out a simulation to examine the temperature history, cell volume, solute redistribution, and other state parameters during the freezing of the spherical heterogeneous construct (a single bead). It is found that the parameters affecting the mass transfer of CPA in tissue and through the cell membrane and the freezing rate play dominant roles in affecting the cell volume transient and extracellular ice formation. Thermal conductivity and extracellular ice formation kinetics, on the other hand, have little effect on cell transient and final states, as the heat transfer rate is much faster than mass diffusion. The outcome of such a model study can be used to evaluate the construct design on its survivability during cryopreservation and to select a cryopreservation protocol to achieve maximum cell survival.

The response of endothelial cells to fluid shear stress using a co-culture model of the arterial wall.

An endothelial cell (EC) smooth muscle cell (SMC) co-culture model of the arterial wall was used to study the effect of fluid shear stress on EC behavior. This model, in addition to being a more realistic tissue analogue, is a valuable research tool for studying the effects of mechanical stimulation upon the behavior of both SMCs and ECs. In the present study, a 10% cyclic strain was used to alter the characteristics of an SMC-seeded collagen gel. This form of strain preconditioning resulted in a rearrangement of the vessel wall that yielded circumferentially oriented cells and collagen fibrils. The preconditioned collagen gel was subsequently seeded with ECs and exposed to fluid-induced shear stress (10 dynes/cm2) for 48 hr. In the absence of flow, ECs seeded on slab constructs were oriented with the underlying collagen fibrils. Sheared constructs exhibited ECs oriented in the flow direction. Shear stress also affected EC proliferation, reducing the total number of dividing ECs by as much as 48 percent compared to unsheared constructs. The shear-induced reduction in proliferation was further enhanced when constructs were first strain-preconditioned (64% reduction). Moreover, conditioned media from shear stress experiments inhibited proliferation of ECs seeded on tissue culture plastic. These results suggest that EC response to fluid shear stress in a collagen co-culture model is influenced by the underlying substrate, and one that in this study is modified by strain preconditioning.

Endothelial cellular response to altered shear stress.

Endothelial cells are normally exposed constantly to mechanical forces that significantly influence their phenotype. This symposium presented recent information concerning endothelial cell responses to shear stress associated with blood flow. Endothelial cell shear stress mechanosensors that have been proposed include membrane receptor kinases, integrins, G proteins, ion channels, intercellular junction proteins, membrane lipids (e.g., those associated with caveolae), and the cytoskeleton. These sensors are linked to signaling cascades that interact with or result in generation of reactive oxygen species, nitric oxide, and various transcription factors among other responses. Endothelial cells adapt to sustained shear stress, and either an increase or decrease from normal shear leads to signaling events. In vitro models for the study of endothelial cell responses must consider the pattern of shear stress (e.g., steady vs. oscillatory flow), the scaffold for cell growth (e.g., basement membrane or other cell types such as smooth muscle cells), and the extent of flow adaptation. These cellular responses have major relevance for understanding the pathophysiological effects of increased shear stress associated with hypertension or decreased shear stress associated with thrombotic occlusion.

Vascular tissue engineering.

The development of a tissue-engineered blood vessel substitute has motivated much of the research in the area of cardiovascular tissue engineering over the past 20 years. Several methodologies have emerged for constructing blood vessel replacements with biological functionality. These include cell-seeded collagen gels, cell-seeded biodegradable synthetic polymer scaffolds, cell self-assembly, and acellular techniques. This review details the most recent developments, with a focus on core technologies and construct development. Specific examples are discussed to illustrate both the benefits and shortcomings of each methodology, as well as to underline common themes. Finally, a brief perspective on challenges for the future is presented.

The role of matrix metalloproteinase-2 in the remodeling of cell-seeded vascular constructs subjected to cyclic strain.

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes.

Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro.

Dynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs.

Tissue engineering: confronting the transplantation crisis.

Tissue engineering is the development of biological substitutes and/or the fostering of tissue regeneration/remodelling. It is emerging as a technology which has the potential to confront the crisis in transplantation caused by the shortage of donor tissues and organs. With the development of this technology, ther is emerging a new industry which is at the interface of biotechnology and the traditional medical implant field. For this technology and the associated industry to realize their full potential, there are core, enabling technologies that need to be developed. This is the focus of the Georgia Tech/Emory Center for the Engineering of Living Tissues, newly established in the United States, with an Engineering Research Center Award from the National Science Foundation. With the development of these core technologies, tissue engineering will evolve from an art form to a technology based on science and engineering.

Tissue engineering a blood vessel substitute: the role of biomechanics.

The engineering of a functional blood vessel substitute has for a quarter of a century been a "holy grail" within the cardiovascular research community. Such a substitute must exhibit long term patency, and the critical issues in this area in many ways are influenced by biomechanics. One of the requirements is that it must be non-thrombogenic, which requires an "endothelial-like" inner lining. It also must have mechanical strength, i.e. a burst pressure, sufficient to operate at arterial pressures. Ideally, however, it must be more than this. It also must have viscoelastic properties that match those of the native vessel being replaced. Finally, if it is to be able to adapt to changing blood flow conditions, it must exhibit vasoactivity, a function which in and of itself can be viewed as biomechanical in nature. To achieve this requires having, as part of the construct, vascular smooth muscle cells, which are contractile in nature and oriented in a circumferential direction. Only if an engineered blood vessel substitute possesses all of these functional characteristics, can one say that the functionality exhibited by a native vessel is being mimicked.

Pierre M. Galletti: a personal reflection.

Pierre Galletti, my friend and colleague, passed away on March 8, 1997, having left his mark on the emerging field of biomedical engineering. He was a pioneering researcher, making his impact in such fields as heart-lung bypass, artificial organs, and tissue engineering. He was a dedicated teacher and a mentor to many. He not only provided leadership in the establishment of the medical school at Brown University, but also helped start Morehouse School of Medicine in Atlanta. He was an entrepreneur and an individual who realized that ultimately basic science only impacts patient care when new technology is made available to the public. He served the bioengineering community in many ways, later in life becoming active in public policy, and as the second president of the American Institute for Medical and Biological Engineering, more than anyone focused this organization on its public policy role. He was the consummate biomedical engineer, a person of great vision, a man for all seasons.

The study of the influence of flow on vascular endothelial biology.

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.

Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase.

Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.

Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium.

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

A mathematical model of the cytosolic-free calcium response in endothelial cells to fluid shear stress.

Important among the responses of endothelial cells to flow stimuli are cytosolic-free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. A mathematical model has been developed describing the recognition and transduction of shear stress to the second messenger cytosolic calcium. Shear stress modulates the calcium response via at least two modalities. First, mass transfer of agonist to the cell surface is enhanced by perfusion and is thus related to shear stress. Second, the permeability of the cell membrane to extracellular calcium increases upon exposure to shear stress. A mass balance for agonist in the perfusate is coupled to a previously published calcium dynamics model. Computations indicate a flow region where the transient moves from transport limited to kinetically limited. Parametric studies indicate distinct contributions to the time course by each step in the process. These steps include the time to develop the concentration boundary layer of agonist, receptor activation, and the mobilization of calcium from intracellular stores. Exogenous calcium is presumed to enter the cell via shear stress-gated ion channels. The model predicts a sigmoidal dependence of calcium influx upon shear stress. The peak value of the transient is determined largely by the agonist pathway, whereas the plateau level is governed by calcium influx. The model predicts the modulation of the calcium transient in the physiologically relevant range of flow and the associated shear stress. This implies that hemodynamics is important in regulating endothelial biology.

Cyclic strain induces an oxidative stress in endothelial cells.

Hypertension imposes an oxidant stress on the aorta and also causes mechanical deformation of the aortic wall. To assess whether deformation causes an oxidative stress, isolated porcine aortic endothelial cells (PAEC) were subjected to cyclic strain, and the cumulative amount of thiobarbituric acid reactive substances (TBARS, an index of lipid peroxidation) and H2O2 (a reactive oxygen species) was measured in the eluent at 2, 6, and 24 h. TBARS were increased by 40.5 +/- 9.2% after 24 h in cells exposed to cyclic strain vs. static controls (P < 0.05). No difference was seen at 2 and 6 h. H2O2 release was increased after 6 and 24 h of cyclic strain by 22.0 +/- 8.0 and 57.6 +/- 11.1 nmol H2O2/mg, respectively (P < 0.005), but was not increased after 2 h of strain. In vascular smooth muscle cells, TBARS were not observed and H2O2 release was not increased by cyclic strain. To investigate a potential source of H2O2 induced by strain, the activity of NADH/NADPH oxidase, a superoxide-generating enzyme, was measured by chemiluminescence. After 2 h, cells exposed to cyclic strain had greater activity than static controls (531.0 +/- 68.4 vs. 448.3 +/- 54.2 pmol O2- x mg(-1) x s(-1), respectively, when incubated with NADH, P < 0.005; 85.8 +/- 8.9 vs. 71.6 +/- 3.8 pmol O2- x mg(-1) x s(-1) when incubated with NADPH, P < 0.05). No effect on NADH/NADPH oxidase activity was seen after 6 or 24 h. The following conclusions were made: 1) cyclic strain induces an oxidant stress in PAEC monolayers as measured by TBARS formation and H2O2 release, 2) NADH/NADPH oxidase is a potential source of H2O2 release in cyclically strained cells, and 3) mechanical deformation of endothelial cells may play a critical role in the generation of oxidative stress within the vessel wall.

Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress.

Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.

Pulsatile and steady flow-induced calcium oscillations in single cultured endothelial cells.

The influence of flow-imposed shear stress on the intracellular calcium concentration ([Ca2+]i) of cultured endothelial cells (ECs) remains incompletely understood. In the present study, we measured [Ca2+]i in single bovine aortic ECs, using fluorescence ratiometric image analysis. The effects of several flow patterns were analysed: steady shear stress (5-70 dyn/cm2), 1-Hz pulsatile shear stress (nonreversing 40 +/- 20 dyn/cm2, reversing 20 +/- 40 dyn/cm2, or purely oscillatory 0 +/- 20 dyn/cm2), or changing shear stress levels. Under all flow conditions, single-cell analyses revealed flow-induced asynchronous [Ca2+]i oscillations, which occurred randomly over the monolayer and which were not seen in the average [Ca2+]i signal corresponding to the monolayer response. The number of single-cell [Ca2+]i oscillations and the corresponding oscillation frequency rose as the shear stress associated with the steady flow increased: 0.06 +/- 0.02 min-1 at 5 dyn/cm2, 0.19 +/- 0.03 min-1 at 20 dyn/cm2, and 0.28 +/- 0.02 min-1 at 70 dyn/cm2 (means +/- SD). Also, the number of oscillations was greater for any type of pulsatile flow (0.53 +/- 0.07 min-1 at 40 +/- 20 dyn/cm2, 0.54 +/- 0.08 min-1 at 20 +/- 40 dyn/cm2, and 0.39 +/- 0.07 min-1 at 0 +/- 20 dyn/cm2), as compared to any level of steady flow. The most dramatic finding was that purely oscillatory flow induced numerous single-cell [Ca2+]i oscillations, yet the average [Ca2+]i response for the monolayer did not change. Furthermore, an EC monolayer switched from low to high (or from high to low) steady flow consistently showed an increase (or a decrease) in the number of single-cell [Ca2+]i oscillations. These experiments show that ECs respond to different flow conditions by varying single-cell [Ca2+]i oscillatory activity. This may have important implications in the endothelium-dependent control of vascular physiology, such as the release of vasoactive substances.