Abstract
Tissue Engineering Regenerative Medical (TERM) products are a new technology currently in human clinical testing for a variety of unmet medical needs involving tissue and organ dysfunction and failure. Safety evaluation of TERM products overlaps 3 established product paradigms: pharmaceuticals (biologically active substances), transplantation (cells or tissue), and devices (biomaterials). As TERM products recapitulate organ or tissue structure and function with unique biological activity and characteristics, they require new preclinical paradigms to bring TERM products through to clinical trials. Establishing TERM-product safety programs requires broad-based knowledge of tissue and organ homeostasis, regenerative biology, and translational medicine to design new preclinical paradigms. Therefore, toxicologic pathologists have a compelling scientific role in evaluating TERM products, characterizing tissue responses, and helping distinguish optimal (regeneration) from deficient or incomplete outcomes indicative of substandard functionality (repair). As new-tissue engineering and regenerative medical technologies develop for tissue and organ regeneration, the toxicologic pathologist will be asked to develop novel testing, reevaluate established toxicologic diagnostic criteria, and reinterpret tissue responses that may extend beyond current standards.
Keywords
Historical Background
In 1987, the National Science Foundation Workshop defined
Regenerative medical products represent the next evolution of medical treatments, and the U.S. Government has proposed an initiative to meet the needs expected in the next 20 years. Although few regenerative medical products are currently marketed (e.g., skin and cartilage), $4 billion has been invested to date in the United States in the burgeoning field of regenerative and tissue-engineering product research and development. One reason for the paucity of products, despite robust investment, is a gap in the basic knowledge necessary to serve as building blocks for translational medicine and clinical application (Interagency Federal Working Group on Regenerative Medicine, 2006). As a consequence, preclinical testing and development strategies for Tissue Engineering Regenerative Medical (TERM) products still face complex scientific questions and regulatory hurdles. A role for toxicologic pathologists in the safety evaluation of current biotechnology products has been established for recombinant proteins, gene therapeutics, and nucleic acid–based biopharmaceutical products (Pilling, 1999). From a toxicologic pathology perspective, these products laid the groundwork and built awareness that a relevant testing program for TERM products needs to go further than traditional safety tests. Toxicological pathologists’ established leadership and pioneering work in the development of TERM products can provide the leadership needed to help this emerging industry, and the Food and Drug Administration (FDA) set appropriate regulatory guidelines.
TERM Products
The feasibility of developing products that partially augment or totally replace diseased organs with laboratory-grown, tissue-engineered neo-organs has been robustly demonstrated in both clinical and preclinical studies (Atala et al., 2006; Oberpenning et al., 1999; Stocum, 2002). By converging biomaterial sciences, tissue engineering, and basic regenerative biology, products can be developed that harness the known regenerative capabilities of the human body. A neo-organ product is defined as “a product composed of synthetic or natural biodegradable materials, with or without living cells and/or cellular products (biologicals) that is implanted in the body to incorporate, replace, and/or regenerate damaged cells, tissues, and/or organs” (Russell and Bertram, 2007, p. 15).
Merging medical devices with pharmaceutical chemicals (e.g., controlled delivery systems) or biologics (cell and/or cell products) into combination products has made regenerative medicine a reality using tissue-engineering technologies. These products have also blurred traditional regulatory pathways and defined new roles for toxicologic pathologists. In response to these complex biomedical advances, the FDA established the Office of Combination Products as a point of entry into the regulatory review process for these new technologies. However, there are relatively few product examples to follow. Thus, regulatory agencies and scientists are engaged in pioneering science to establish regulatory and product-development guidelines for the evaluation of neo-tissue and neo-organ product safety. Today, there is no single established reference that can comprehensively guide toxicologic pathologists in assessing the safety of tissue-engineered or regenerative medical products. Consequently, the toxicologic pathologist must use strong scientific rationale and understanding of basic tissue and cellular responses to establish development strategies that will comply with available regulatory guidelines.
Neo-organ products are regulated under section 351 of the Public Health Services (PHS) Act, and regulations that apply to investigational new drug (IND)/biological license application (BLA), IND/new drug application (NDA), or investigative device exemption (IDE)/pre-marketing authorization (PMA) also apply to the development of a TERM product. All preclinical studies are regulated by 21 CFR (Code of Federal Regulations) part 58 Good Laboratory Practices (GLP). The production of components and assembling of neo-organ products are regulated by 21 CFR part 210 Good Manufacturing Practices (GMP). Cellular components of neo-organ products are regulated by 21 CFR parts 600, 1270, and 1271, addressing biologics and tissues. The scaffold components of neo-organ products are regulated by 21 CFR part 800, addressing devices. Lastly, before clinical trials, filing an IND (21 CFR part 312) or IDE (21 CFR part 812) is required to allow the sponsor to study a neo-organ product in a human clinical setting. Within the IND or IDE, the FDA expects the sponsor to address both safety and functionality of TERM products in preclinical animal models that emulate the human condition so that the regenerative potential and biological activity of these products can be evaluated before placing them into clinical trials.
TERM Products and Toxicologic Pathologists
The published scientific literature related to TERM products is rapidly growing. In 2000, there were 403 publications that used the term
An electronic search of literature published as of May 31, 2007, using the PubMed database confirms the rapid growth of literature with the keywords
Toxicological pathologists possess training and experience in regulatory preclinical study planning, design, and implementation that are directly applicable to the safety evaluation of these breakthrough medical products and the necessary skills to assess the structural and functional characteristics of the regenerated organ. Veterinary medical training imparts medical and surgical proficiencies that complement the skills of tissue engineers and regenerative medical professionals for integrating basic scientific knowledge of mammalian homeostatic mechanisms and regenerative biology. Through specialized training in pathology, toxicologic pathologists bring an understanding of molecular, cellular, tissue, and organ pathobiology that is necessary to investigate the regenerative potential within the context of various disease states. Furthermore, toxicologic pathologists understand issues related to experimental design, biostatistical methods, and cellular and molecular research required for establishing appropriate endpoints and clinical translation of preclinical information for TERM product development.
TERM Products Preclinical Safety Paradigm
Traditional preclinical development safety-assessment paradigms are becoming constrained in their capacity for commercializing TERM products. Safety data for an IND reported to the FDA (21 CFR 312.23:
Figure 2 shows a “traditional” experimental design used in pharmaceutical and chemical regulatory toxicology. Regardless of study duration, most pharmaceutical and biopharmaceutical toxicology studies are designed to compare cell, tissue, and organ-based endpoints between various dose groups (e.g., control, low, medium, and high dose) and to demonstrate product safety; these studies are not conducted in animal models of disease, nor are the products’ functionality evaluated in the same study. Animals are healthy at the start of a study, and those assigned to the control group are expected to remain within physiologically normal ranges for the duration of the study. Tested doses are used to evaluate morbidity and mortality, clinical observations, and tissue and organ-system toxicity and to identify a margin of safety for human exposure.
Cell- or genetherapy products also have established pre-clinical safety evaluation approaches similar to large- or small-molecule pharmaceuticals. With cell or gene therapies, safety testing focuses on the biomolecule used to restore a particular deficiency (e.g., insulin production for diabetes). Implanted cells are the source of the biologically active molecule. These cells are derived from a source that is most suited to the production of the particular biological molecule of interest and are highly characterized in advance to eliminate the potential for adverse effects. Some cellular therapeutics require that cells be implanted within a nondegradable, semipermeable casing. In these products, the toxicologic pathologist’s role is to evaluate the host’s inflammatory and immunogenic response to the biomaterial (U.S. Food and Drug Administration, 1998).
In contrast to these traditional approaches of safety evaluation, TERM products are often combination products with both cellular and biomaterial components requiring a comprehensive assessment of each component. Cell source (autologous, allogenic, or xenogenic; homologous or nonhomologous) may affect the biological potential and final outcome of regeneration (Jayo et al., 2007). Inappropriate cells or inadequate cell numbers may lead to a suboptimal healing response that is more akin to repair with fibrosis and scarring than the optimal outcome of regeneration. Of the various stem cells available for potential regenerative medical product use, autologous adult stem cells tend to generate fewer safety concerns and have higher potential of promoting an optimal healing outcome by regeneration of native or native-like tissue rather than simply repair, fibrosis, or a scar. Importantly, autologous adult cells are not expected to elicit an immunogenic response, although not all cell types have equivalent immunogenic privilege.
TERM products composed of novel biomaterials are evaluated for biocompatibility (i.e., require ISO-10993/USP 71), and cellular components may require the same safety assessment as a product composed only of cellular elements. Autologous homologous cells (from the same individual and same organic tissue) intrinsically share embryologic, morphologic, and phenotypic correspondence to the tissue to be regenerated. These characteristics make autologous homologous cells a preferred resource for facilitating optimal healing and regeneration in TERM products. Safety assessment of products using autologous homologous adult cells involves the lowest degree of extrapolation from established regulatory paradigms. In contrast, assessing safety for products that use allogenic or xenogenic cell sources or products that implant cells into nonhomologous tissues requires more robust immunotoxicologic evaluations, while at the same time, it has more tenuous established regulatory paradigms for assessments such as cell fate. Toxicologic pathologists experienced in evaluating graft vs. host reactions and nonmatched organ transplantation can make valuable contributions to the safety assessment of TERM products that incorporate allogenic or xenogenic cells or implant cells into nonhomologous sites in the body.
Figure 3 illustrates how TERM-product safety and functionality are evaluated over time and how a potential cellular “dose” is identified, especially for TERM products that have a metabolic cell function as the primary mode of action. An animal-model analogue of disease is the optimal model for TERM-product safety testing. Product safety is tested concomitantly with functionality, and restoration of native tissue structure and function serves as the indicator of the product’s utility. Such an approach challenges the toxicologic pathologist to distinguish tissue alterations leading to regeneration from similar alterations that traditionally signaled safety issues. For example, inflammation appearing at any time during a traditional drug toxicology study might be considered an adverse event. However, for TERM products that are implanted via a surgical procedure, a transient inflammatory response is expected and only becomes an adverse event if it does not resolve. Similarly, compounds generated during scaffold biodegradation can recruit inflammatory cells to the implant site. Thus, the temporal context of toxicologic pathology evaluation has increased importance in TERM-product safety assessment.
Preclinical Testing of TERM Product
The objective of a preclinical study to evaluate a TERM product is to determine both the safety of the product and the structure and function of the regenerated tissue or organ. The cellular and biomaterial scaffold elements of the product are combined to form the final product or “construct.” Importantly, this construct is not equivalent to the native organ but serves only as a regenerative template for the final regenerated organ. Constructs may be implanted at various stages of maturation. They can range from a biomaterial scaffold with or without a biological element (active molecules or cells) to a highly engineered scaffold, designed with near-native biomechanical and functional properties.
A complete safety evaluation of any TERM product includes in-life observations and clinically relevant examinations (e.g., imaging via MRI, ultrasound, etc.) of the regenerated tissue or organ. Such endpoints provide a holistic assessment of the neo-organ’s contribution to sustaining homeostasis. Clinically relevant assessments also allow translation of certain structural and functional information that can only be acquired in animal studies (e.g., histological and in situ analyses) to be extrapolated to the information expected during a clinical trial. Organ-system–specific clinical pathology assays also form a basis for evaluating organ-system function and product safety for direct translation to clinical trials.
As in standard toxicological studies, comprehensive necropsy and tissue collection are used for detecting organ-specific and systemic changes as well as for evaluating healing processes elicited by the TERM construct. Special methods of fixation, quantitative morphology, and immunohistochemical procedures are used to characterize the process of regeneration and to evaluate any potential safety signals. Macroscopic findings provide evidence that an organ was regenerated in situ and that it did not displace or adversely affect adjacent visceral or tissue elements. In contrast to pharmaceutical toxicologic evaluations, the presence of proliferating tissues (hyperplasia) is a necessary and expected part of the healing process. Although proliferative tissue and cellular response are normal parts of the healing process, these observations should be transient and diminish over time as the neo-tissue and neo-organ are regenerated.
To confirm biomaterial safety, biodegradable scaffold fibers should also be evaluated for elimination over time and appropriate degradation-associated inflammatory cells (macrophages and giant cells). Tissue-layer thickness (histomorphometry) and assessment of correct type and orientation of microanatomical layers in the regenerated tissue is an expected structural outcome to achieve baseline functional values as well as normal tissue composition. Necrosis, mineralization, excessive fibrosis, osseous metaplasia, and prolonged inflammation indicate poor outcomes that would not lead to proper function or normal healing if these changes predominate or persist during the regenerative process (Cain et al., 2000; Jokinen, 1990; Walker et al., 2002).
Summary
There is a compelling scientific role for toxicologic pathologists in the evaluation of TERM products. A comprehensive ability to integrate molecular, cellular, tissue, and organ assessments into an understanding of regenerative outcomes allows the toxicologic pathologist to distinguish between optimal (regeneration) and suboptimal outcomes (e.g., repair), establish the mechanism by which these different outcomes occur, and translate the identified safety risks of the product into a risk assessment for entry into clinical trials and subsequent commercialization. As new tissue engineering and regenerative technologies develop into medically advanced neo-organs, there will be a continued demand for novel testing paradigms, appropriate diagnostic criteria, sensitive in situ assays of cellular and molecular events, and scientists who can integrate information from complex biological systems. The toxicologic pathologist is at the forefront of product evaluation by having a broad-based understanding of biological mechanisms involved in safety evaluation, healing, and regeneration. Toxicologic pathology provides the foundation for safety assessment, risk management, and development-study planning for the safety and functional assessments of new TERM products.