Toxicologic pathology is the art of assessment of potential adverse effects at the tissue level in pre-clinical studies. In the case of biomaterials and medical devices, the toxicologic pathologists assess the safety (biocompatibility) and efficacy (conditions of the use) of the implantable materials. Proper assessment of biocompatibility of biomaterials is of utmost importance, since it helps to determine their safety after implantation in humans. Biomaterial-related toxicity can be attributed to several factors, including for example leachable compounds from the material leading to thrombosis or carcinogenesis, or biodegradation of the material causing changes in its physical and compatibility properties. Evaluation of biocompatibility and biofunctionality involves assessment of cytotoxicity, allergic responses, irritation, inflammation and systemic and chronic toxicity. In many of these assessments, the toxicologic pathologist has an important role in determining product safety and potential toxicity. In this article, we review the special needs for proper toxicologic pathology assessment of biomaterials and degradable polymers. We review common adverse effects expected with biomaterials and describe their pathological picture and their clinical relevance. We also introduce a novel compact MR imaging technology as a tool for assessing biocompatibility and efficacy of implanted biodegradable materials, since it allows for the longitudinal imaging and quantification of inflammation in vivo caused by the device implantation, and enabling general inspection of shape, location and integrity of the device in vivo. Since the MR imaging technique is non-invasive, the effects of the implantable device can be monitored longitudinally in the same animal without perturbation of the pathology.
toxicologic pathology; histopathology; MRI; safety assessment
Toxicologic pathology involves evaluation of biological tissues for adverse effects, and is especially important in pre-clinical studies. When biomaterials and medical devices are implanted in tissues, the toxicologic pathologists are responsible for evaluating the safety (biocompatibility) and efficacy (conditions of the use) of the implantable materials. The term “biocompatibility” encompasses a broad spectrum of biological notions. In addition to referring to the fact that the biomaterial should lack cytotoxicity, it also relates to its biofunctionality, or its ability to support cell–biomaterial interactions in the tissue milieu where the biomaterial is applied.
The perfect biomaterial preferentially should be prepared from a polymer which has the following characteristics:
• Does not cause an inflammatory reaction
• Is metabolized without leaving traces after accomplishing its goal
• Processed with ease to its final form
• Has long shelf life
• Sterilized easily
Appropriate inspection of biocompatibility of biomaterials is of utmost significance, since it allows to estimate their safety and function after implantation in humans. This is especially true for biomaterials, considering the fact that they are implanted in humans for long periods and reside in close contact with biological tissues. Biocompatibility of biomaterials depends on various parameters, which can be divided into internal (the material itself) and external (the host) parameters. These factors which define biocompatibility are summarized in Table 1.
Many factors can lead to biomaterial-related toxicity, which include for example thrombosis or carcinogenesis induced by leachable compounds from the material, or biodegradation of the material which alters its physical and compatibility characteristics. Additional factors such as immunologic reactions of the host or accidental introduction of biofilms also play a major role in toxicity of biomaterials.
Unfortunately, toxicity evaluation of biomaterials is a complex process, which involves both in vitro and in vivo methods. In vitro cell culture tests, usually involving permanent cell lines, are often applied to screen for the biocompatibility of a compound. The International Organization of Standardization (ISO) 10993 provides a series of standards for assessing biocompatibility of medical devices. These standards are presented in 20 different parts, which can be accessed in the following web address: http://www.iso.org/iso/home.
The process of biocompatibility and biofunctionality evaluation usually involves assessment of cytotoxicity, allergic responses, irritation, inflammation and systemic and chronic toxicity. In many of these assessments, the toxicologic pathologist has an important role.
Toxicologic Evaluation Of Biomaterials
An important difference between the toxicologic pathology evaluation of drugs and biomaterials is that biomaterials always cause tissue reaction, due to their close interaction with the tissue for a long time. It is also for this reason that the response to biomaterials is often time dependent and not dose dependent. Other factors that should be taken into consideration are the implantation procedure, which often involves tissue trauma and the placement of sutures. Additionally, secondary infection can result from the operative procedure or from contamination of the implanted device.
The toxicologic pathologist is often asked to evaluate implantation tests, which are intended for determining the local effect of devices on tissues or body fluids. These tests are routinely directed at paravertebral or hind-limb muscle of rabbits or less frequently rats. These tests often pose a special challenge to the toxicologic pathologist due to unique confounders and problems, which are summarized in Table 2. Species-related differences in response to implanted material should also be taken into account, and it is our recommendation that in comparable circumstances when relative severe host reaction is seen in one species (e.g. pig), then another animal species should be tested for safety, in order to exclude potential particular sensitivity in one species. In addition, until this aspect is clarified, it is recommended that no clinical testing with that compound will be performed.
The histopathology evaluation form for implanted materials can be found in the paper by Schuh, which represents a modification of the historical evaluation form. When evaluating pathology of biomaterials, special emphasis should be given for providing a detailed summary of pathology results in addition to semi-quantitative scores, since many parameters are not included in this scoring system, including for example hemorrhage, edema and the different cell populations in the infiltrate. Additionally, it is obligatory that the pathologist will also add to the semi-quantitative scoring a detailed interpretative assessment, in order to have a clear understanding of the characteristic and potential significance of the findings. Detailed and annotated photographic documentation should be encouraged, especially when dealing with unusual findings.
Upon histopathologic evaluation, it is important that the pathologist will adopt the terminology currently recommend by the International Harmonization of Nomenclature and Diagnostic (INHAND) criteria for lesions. Also, according to the “Best Practices for Reporting Pathology Interpretations within GLP Toxicology Studies,” published by the Society of Toxicologic Pathology (STP), when evaluating the histological slides from a study, the study pathologist should have access to the study protocol and all protocol amendments, all study data including the intended pharmacologic target and mechanism of action, in-life study data, clinical pathology data, organ weight data, necropsy findings, toxicokinetic information and (when possible) data from previous studies with the same test article.
The importance of host immune response to the implanted material has gained much attention in recent years. Therefore, immunotoxicology of biomaterials should be part of the histopathological evaluation. To address this need, evaluation of hematopoietic and lymphoid tissues should be performed.
When designing the toxicology protocol, special issues that should be taken into consideration include scheduling interim sacrifices, using concurrent untreated samples, and histopathological examination of organs that may suggest potential systemic toxicity, such as liver, kidneys, spleen, lungs, regional lymph nodes and heart.[3,10] Scheduled interim sacrifices will allow the toxicologic pathologist to determine whether there is time-related decrease in the inflammatory reaction. When assessing the longterm biocompatibility, an example of interim sacrifice sche