Challenges for Clinical Translation of Cardiovascular Tissue Engineering
Frederick J. Schoen, M.D., Ph.D.
President, ISACB Professor of Pathology and Health Sciences and Technology, Harvard Medical School;
Executive Vice-Chairman, Department of Pathology Brigham and Women’s Hospital Boston MA
Cardiovascular tissue engineering approaches to vascular, valvular and myocardial repair and replacement have exciting clinical potential. Engineered cardiovascular tissue will need to avoid the typical deleterious biomaterial-tissue interactions and complications that limit the safety and effectiveness of many contemporary cardiovascular medical devices, such as thrombosis, infection, inflammatory interactions and limitations to durability. However, tissue engineering and regenerative approaches will also encounter special challenges to safety and effectiveness, as tissue replacements that are living depend on complex biological interactions for their efficacy. This essay summarizes some critical technical challenges that will be encountered in translating the potential of cardiovascular tissue engineering from the laboratory to the clinical realm. These comments are relevant to the broad array of potential approaches under investigation for cardiovascular tissue engineering: 1) cell seeded biodegradable synthetic scaffolds, 2) cell seeded natural tissue-derived scaffolds, 3) substrates without prior in-vitro cell seeding that are designed to be repopulated by circulating endogenous cells, and 4) other cell-based therapies. A successful tissue engineered vascular graft, heart valve or myocardial replacement must be vital, dynamic, and composed of a complex mixture of specialized cells and extracellular matrix (ECM). It must have adequate mechanical and biological properties, often beginning at the instant of implantation and continuing indefinitely thereafter. Moreover, optimal long-term function may necessitate subsequent tissue remodeling to accommodate and thereafter adapt to injury and changes in local mechanical forces.
An agenda for translating cardiovascular tissue engineering from a constellation of extraordinarily interesting research endeavors to clinically useful surgical tools incorporates both research and clinical goals.1,2 Research goals include understanding mechanisms, defining animal models, developing biomarkers for tissue structure and function, developing assays/tools, and defining surrogate and true endpoints. Clinical goals include characterizing and assuring the quality of tissue constructs, both those grown in-vitro and those evolving in-vivo, accommodating patient-to-patient heterogeneity in inflammation and tissue remodeling, and predicting outcomes as early as possible in specific patients. A schema for the interrelationships among and challenges in tissue characterization for cardiovascular tissue engineering, originally formulated for heart valve tissue engineering, is summarized in (Figure 1).
Large animal models such as sheep and pigs are often used in the pre-clinical testing of innovative cardiovascular devices. However, there is considerable controversy over to what extent results from available animal models are relevant to human implantation, and the characteristics of the most suitable animal models for testing tissue engineered blood vessels, valves and myocardium have not yet been determined.3 For example, cardiovascular implants in sheep generally overgrow more rapidly with fibrous tissue than they do in humans, and likely overestimate the capacity for tissue formation and remodeling relative to humans.4,5 Owing to immunologic incompatibilities when cells are transplanted across individuals and species, the choice of an animal model for preclinical testing for allogenic or xenogeneic cell-based therapies presents unique challenges, and the extent and nature of certain types of interactions can be determined only by carefully designed human clinical studies.
Currently available cardiovascular medical devices such as vascular prostheses and heart valve replacement devices have largely predictable (albeit frequently suboptimal) behavior across large patient cohorts. Human variation impacts performance only in a minority of patients. For example, younger individuals calcify bioprosthetic heart valves more frequently and severely than older individuals6, and genetic and other causes of hypercoagulability correlate with the vulnerability to mechanical heart valve thrombosis.7 However, qualitative and quantitative variability among patients in inflammatory responses to and in-vivo remodeling behavior of tissue engineered blood vessels, heart valves and myocardium will likely contribute to outcome to a greater degree than with “conventional” prosthetics. This assertion derives from the increasingly recognized yet poorly characterized heterogeneity among individuals (both humans and experimental animals) in biological processes that are inherent to tissue engineered devices, including inflammatory and immunological responses, wound healing, and tissue remodeling potential.8, 9, 10,11 Such heterogeneity has been implicated in the pathogenesis of and susceptibility to human abdominal aortic aneurysms 12,13 and to variation among individuals in transplant rejection, susceptibility to infection and autoimmune diseases, and potentially atherosclerosis.14
Thus, owing to aberrant inflammation, immunological attack, or inadequate healing and tissue remodeling, some patients with tissue-engineered grafts, valves, and patches might develop inadequate strength or durability, which could lead to failure (Figure 2). Patient variability could result from mutations, single nucleotide polymorphisms or “structural” variation of genes that code key proteins central to inflammation, healing and remodeling. , , Indeed, as implants have become more interactive and integrative with the host tissues, there has arisen a corresponding need to understand and potentially control human variation in different facets of biomaterial-tissue interactions and the healing process, a field that might be termed “biomaterio-genomics”. This is analogous to the principles of pharmacogenomics, whereby investigators seek to understand and control the genetic basis of inter-individual variation in drug metabolism.18,19 Immune responses and teratoma formation will be potential risks with therapies using stem cells.20 Thus, the usual procedures for demonstrating pre-clinical safety and efficacy of medical devices and biologics may need to be altered for tissue engineered cardiovascular implants due to the inherent lesser predictability of interactions of the engineered tissue with the recipient’s native tissues.
Moreover, it follows from the above discussion that patient-to-patient differences in inflammation, wound healing and tissue remodeling capability in-vivo will need to be understood, monitored and potentially controlled. Toward this end, conventional and innovative invasive and/or non-invasive anatomic and functional imaging modalities will be important tools to assess success and failure in and evolution of structure and function in tissue engineered cardiovascular devices. Gene and protein “biomarkers” that correlate with and potentially predict implant outcomes must be identified and validated by assessing tissue healing and remodeling during in-vitro and in-vivo experiments.21,22 Moreover, the most suitable biomarkers will need to be followed in-vivo, possibly via chemical assays in the serum or urine or via molecular imaging (see below). Indeed, researchers are currently working to identify serum-specific protein biomarkers of ECM remodeling such as MMPs in acute coronary syndromes 23,24, and urine-based ECM biomarkers correlate with the presence of metastases in several common cancers 25, 26, 27, and other diseases.28 Biomarkers measurable through laboratory assays or visible on imaging that correlate directly with success and failure may be applicable as “surrogate endpoints”, which are events that reflect the mechanism of a significant clinical event or characteristic (such as degradative dilation, intimal hyperplasia, regurgitation, stenosis, thromboembolism, calcification, infection, or death). Validated surrogate endpoints could be assessed in an individual patient, in order to predict outcome of tissue engineering therapy as early as possible in the patient’s course and potentially to stimulate a specific change in management. A preimplant test that would assay the blood levels or activities of key mediators of biological function (and thereby predict the outcome of inflammation, healing, or remodeling pathways in order to predict a particular patient’s capacity to appropriately integrate a tissue engineered vascular graft, heart valve, or myocardial patch) would be particularly useful.
In-vivo monitoring through imaging of biomarkers is highly desirable and potentially useful. The evolving technique “molecular imaging” has particularly exciting promise to probe tissue composition, collagen and elastin ECM, the fate, gene expression and phenotype of cells seeded in-vitro and the endogenous local and circulating cells that may be recruited to the implant in-vivo.29,30 Various studies have demonstrated the potential of this approach. In-vivo molecular imaging has been used to demonstrate key enzymatic and cellular events in atherosclerosis and thrombosis. For example, inflammatory markers such as proteases (cathepsins and MMPs), activated macrophages (expressing iron oxide), and activated endothelium (intercellular and vascular adhesion molecules) have been demonstrated in atherosclerotic mice.31,32 Early heart valve calcification has been assessed experimentally in-vivo. Moreover, molecular imaging has been used to probe polymorphisms of ECM gene expression in in-vivo in models of wound healing and cardiovascular disease , , and can potentially be translated to perform real-time in-vivo characterization of scaffold matrices implanted in animal models. Furthermore, molecular resonance imaging (MRI) of magnetically labeled mesenchymal stem cells injected into porcine myocardium has been performed in-vivo. Molecular imaging could be utilized to track the presence, migration, proliferation, and function of bone marrow derived endothelial and other progenitor cells used to seed scaffolds in-vitro. Investigators have followed magnetically labeled mesenchymal stem cells injected into porcine myocardium by in-vivo molecular resonance imaging (MRI) , and demonstrated hematopoietic derivation of cardiac valve interstitial cells. Other imaging modalities such as optical coherence tomography (OCT) , and intravascular ultrasound (IVUS) have been used to assess collagen content of coronary atherosclerotic plaque; allowing real-time in vivo analysis without tissue sampling. Quality assurance of an engineered tissue implant before in-vivo use will also be critical. Guidelines are needed for science-based approaches to the characterization of the structure and properties of fabricated/manufactured engineered tissue products. This characterization may include: 1) dynamic assessment/measurement of the structure and mechanical properties of the scaffold and the evolving tissue-scaffold complex in-vitro, 2) dynamic assessment of cell phenotypes and ECM components, 3) understanding the progressive evolution of the final manufactured product over time before implantation, including shelf-life, stability and shipping considerations, as well as, 4) maintenance of sterility. In addition, important biological changes that could potentially accrue during the shelf life of the product will need to be understood, assessed and controlled as appropriate. The above considerations suggest that the benefit/risk profiles of engineered tissue will likely be less predictable than those of well-accepted and existing technologies, especially during early phases. Moreover, demonstration of acceptable long-term efficacy (implantability, functionality, long-term performance) and safety (biocompatibility, durability, modes of failure, ease of monitoring) of tissue engineered cardiovascular products in humans will be critical. As a result of these uncertainties, it may be particularly challenging to gain acceptance of tissue engineering approaches by the surgical community, especially in clinical areas where problems with conventional technology exist but benefit to risk is already high. For example, owing to the predictable outcomes of conventional heart valve replacement in adults, a leading cardiologist suggested that surgeons consider the use of a novel valve replacement only when appropriately controlled clinical research can demonstrate that the 15-year lifetime of conventional valve substitutes can be exceeded. This is particularly difficult for the key pathobiologic process widely encountered with substitute heart valves and other cardiovascular devices, in which investigations can only be assessed “real time”. However, for subgroups of patients in whom conventional valve substitutes currently perform less well (i.e., children with complex congenital heart disease who need valves), the enhanced benefit that could accrue through use of tissue engineered devices (including the potential ability of the valve replacement to grow with the recipient) may justify their early use. Such particularly needy subgroups will likely be the focus of early clinical studies in other areas as well. Conclusions The goal of cardiovascular tissue engineering is to regenerate functional structures containing cells and extracellular matrix that function structurally and biomechanically in the intended application. Despite exciting potential for tissue engineered cardiovascular products, significant technical barriers must be overcome before widespread clinical acceptance of application of these cardiovascular devices will be feasible. Nevertheless, we hope that the interest and efforts in this field will spawn a host of novel testing strategies, methods, including in-vitro safety studies, investigation of the mechanisms and regulation of novel host-tissue interactions, ex-vivo performance characterization in functional testing in devices akin to bioreactors, in-vivo preclinical studies, and novel methodologies applied to explant retrieval studies.
