Biomaterial Testing Flashcards
Determine the required testing for a biomaterial based on the application.
- Exogenous chemicals found inside the body are referred to as xenobiotics. Any chemical leaching from a medical device or material falls under this definition. Thus, assessing the cellular and tissue compatibility of a device/material often involves examination of the potential xenobiotic chemicals that it may release.
The compatibility of such xenobiotics depends on whether or not it elicits undesirable cell responses or damages cellular structures.
Xenobiotics that possess potentially harmful or adverse properties are referred to as toxicants. The response to a toxicant coming from a device/material is greatly influenced not only by the material itself, but also by the type of exposure.
Exposure characteristics include:
(1) the route and site of exposure (e.g., brain tissue vs. subcutaneous tissue, or skin contact vs. vascular tissue contact, direct or indirect circulating blood contact)
(2) duration of exposure (e.g., minutes to hours vs. permanent implantation),
and
(3) the potential frequency of exposure over the treatment period or lifetime of the host.
(4) Chemical reactivity of a toxicant is an additional key factor that will impact the degree and type of response in the host. A particular reaction may occur because of an agent’s chemical reactivity, or it may be due to a more specific recognition or reactivity toward a molecular structure in the biological environment.
- These factors influence the required testing for a biomaterial and are ultimately based on the device application.
- Challenges have led to the development of international standards on device or test material evaluation and sample preparation methods, taking a simplifying premise in that compatibility is most readily verified by focusing on the impact of chemical constituents that may potentially be released, or actually are released as intended in the case of biodegradable materials, from the device/material in vivo.
- To facilitate the selection of appropriate tests, medical devices with their component biomaterials can be categorized by the nature of tissue contact of the medical device, and by its duration of contact.
Four impact factors of Biocompatibility
- Toxicology (the measurement and study of the effects
of material leaching from biomaterials). - Reactions related to products from extrinsic microbiologic organisms colonizing the biomaterial (for example, endotoxin contamination).
- Mechanical effects such as rubbing, irritation, compression, stress concentrations caused by high aspect ratio shapes, corners and modulus mismatch, and mechanical failure. Closely related are size-related effects, for example, if the implant is much larger than a macrophage, comparable in size to a macrophage, and capable of being phagocytosed, or much smaller than a macrophage, i.e., nano-size.
- A broad range of interactions with the surrounding environment, including proteins, other soluble molecules and ions in extracellular fluid and blood, live microorganisms, and cells, inducing cell–biomaterial interactions (and tissue–biomaterial interactions) that might affect and, indeed, in some cases dominate longer term in vivo outcomes.
Points 1, 2, and 3, above, are well understood, and
often applied in the design of biocompatible biomaterials—we understand the principles, have the ability to mea- sure their impact, and we can design devices using clearly defined principles to achieve good outcomes. Point 4 is less well-developed.
Biocompatibility as we understand it today
- The biological reaction we call biocompatibility is neg- atively impacted by leachables, products of extrinsic organism surface contamination, and micromotion.
- As long as leachables, extrinsic organism surface contami- nation, and micromotion are not impacting the reaction, all materials will give an approximately similar bioreac- tion in vivo, referred to as the “normal” (i.e., expected, low-level) FBR, composed of a thin fibrous capsule and minimal ongoing inflammation.
- When the foreign body capsule is thin and the reaction site, after approximately 1month or longer, is relatively quiescent, this is an acceptable (and indeed, usually unavoidable) FBR and we call the implant “biocompat- ible (or inert).”
- The favorable long-term interface between a biomaterial and the surrounding tissues in most medical devices is characterized by a thin, dense, collagenous capsule that isolates the biomaterial implant from the body, and has a minimal, if any, inflammatory component.
Fundamental elements of IN VITRO CYTOTOXICITY evaluation of biomaterials (ISO 10993-5:2009)
- The fundamental question sought to be answered by in vitro tests for cytotoxicity is ‘Does the material, or any component of the material, result in cell damage or cell death?’.
The most commonly used in vitro tests to assess medical devices or their component materials for cytotoxicity: - (see week 7 ch 2.2.3)
- Three main categories (see notes):
- EXTRACT TEST (Will cytotoxic substances leach out?)
- DIRECT CONTACT TEST [MATERIAL-CELLS] (Will cells survive?)
- INDIRECT CONTACT TEST [AGAR DIFFUSION] (Indicative of systemic cytotoxicity?)
- It should be noted that these tests detect general toxicity pathways and mechanisms.
- Thus, the nature of the tests is not inclusive of all possible toxicity pathways.
-These tests have, however, demonstrated appropriate usefulness in human health risk assessment, which is the main objective of the ISO 10993 standards.
IN VITRO evaluation of biomaterials
While in vitro evaluation methods unavoidably utilize simplified systems as compared to the complex in vivo milieu, they are valuable because they provide insight into potential in vivo tissue and cellular responses.
- Exogenous chemicals found inside the body are referred to as xenobiotics. Any chemical leaching from a medical device or material falls under this definition. Thus, assessing the cellular and tissue compatibility of a device/material often involves examination of the potential xenobiotic chemicals that it may release.
Testing the potential toxicity of a material.
The potential toxicity of a material is determined by a toxicological risk assessment. Such assessments involve: (1) identifying hazardous chemicals present in medical devices;
(2) assessing the extent of a patient’s potential exposure to these chemicals;
(3) determining the dose–response relationship
of each chemical; and
(4) combining this information to characterize the risk.
- A well-thought-out and thorough risk assessment may negate the need for expensive and time- consuming in vitro and in vivo biological testing.
Preparing In vitro samples
- Detailed guidance for preparing in vitro testing samples is provided throughout ISO 10993 Biological evaluation of medical devices, Parts 1, 3, 4, 5, and 12 (ISO 10993- 1:2018; ISO 10993-3:2014 Annex D, 10993-4:2017; 10993-5:2009; 10993-12:2012).
- Sample preparation for in vitro biological tests requires careful attention due to each test’s volume and extractable percentage requirements.
- Extraction procedures should:
(1) exaggerate the clinical conditions of use;
(2) take into account the chemical characteristics of the test and control samples;
(3) not cause significant changes in the physicochemical properties of the sample (e.g., degrade or dissolve the material);
(4) test the device or material in its intended clinical use state (e.g., identically packaged and sterilized);
(5) consider use of both a polar and a nonpolar extraction solvent to simulate the body’s aqueous and lipophilic environments.
(6) understand potential impact to the in vitro test system; (7) be performed in chemically inert sterile closed containers;
(8) be conducted for appropriate duration; and
(9) be carried out at a suitable temperature, particularly when using cell culture media as an extrac- tion vehicle.
Fundamentals of IN VITRO Tests for Interactions with Blood: ISO 10993-4
- Given the importance of this interface, and the range of blood-contacting device applications, this area of testing has a separate ISO 10993 standard (ISO 10993- 4:2017).
The commonly applied in vitro tests by the key categories of concern in material/device interaction with blood:
- Thrombosis (Thrombus assessment)
- Coagulation (Assays for coagulation proteins (examples include TAT and FPA)
- Platelets and platelet function (Platelet counting and assays that assess platelet degranulation)
- Hematology (Material-induced hemolysis, Mechanical-induced hemolysis, Differential cell blood count (CBC) analysis)
- Immunology (Complement pathway activation
via C3a and/or SC5b9 protein assessment)
- These assays focus on evaluating the device or material for potential to bring about red blood cell damage and lysis, cause thrombosis, interact with platelets, and stimulate alternative pathway immune reactions (i.e., complement pathway activation).
- Just as there are a host of unique blood-contacting considerations associated with each device application, in vitro testing for blood interaction requires unique models and carefully designed and controlled experiments.
- Models must be designed to mimic the conditions expected in clinical use.
- e.g: temperature, fresh/not aged blood, blood flow, test material surface area:blood ratio (cm2/mL blood), anticoagulant conditions, and contact duration.
- Despite the availability of various in vitro assays and models for characterization of materials with blood, lack of validation and questions on relevance to chronic in vivo responses have made such testing more common in acute blood exposure applications and feasibility studies, rather than as standard regulatory requirements
IN VIVO evaluation of biomaterials
The goal of in vivo assessment of tissue compatibility of a biomaterial, prosthesis, or medical device is to determine the biocompatibility (as a key component of safety) of the biomaterial, prosthesis, or medical device in a biological environment reflective of its intended use.
- From a practical perspective, the in vivo assessment of tissue compatibility of medical devices is carried out to determine that the device performs as intended, and presents no significant harm to the patient or user simulating clinical use.
- As it is recognized that biomaterial–tissue interaction may vary with the anatomic site and duration of exposure, and application-specific conditions, in vivo tests for assessment of tissue compatibility are chosen to simulate end-use applications.
Fundamentals of ISO 10993 testing
ISO 10993 standards advise that the biological
evaluation of all medical device materials include testing for cytotoxicity, sensitization, and irritation. (Cytotoxicity tests are considered in vitro tests.) Beyond these fundamentals, the selection of further tests for in vivo biocompatibility assessment is based on the characteristics and end-use appli- cation of the device or biomaterial under consideration.
Fundamentals of IN VIVO sensitisation evaluation of biomaterials. (ISO 10993-10)
- Sensitisation tests estimate the potential for contact sensitisation to medical devices, materials, and/or their extracts.
- Symptoms of sensitisation are often seen in skin, and tests are often carried out topically in guinea pigs.
- Test design should reflect the intended route (skin, eye, mucosa) and nature, degree, frequency, duration, and conditions of exposure of the bio- material in its intended clinical use.
- Critical to the conduct of these tests is that the preparation of the test material and/or extract solution should be chosen to include testing for both water-soluble and fat-soluble leachables.
Fundamentals of IN VIVO irritation evaluation of biomaterials. (ISO 10993-10)
- While sensitisation reactions are immune system responses to contact with chemical substances, ISO guidelines suggest irritation to be a local tissue inflammation response to chemicals, without a systemic immunological component.
- Irritant tests emphasize utilization of extracts of the biomaterials to determine the irritant effects of potential leachables.
- Intracutaneous (intradermal) reactivity tests determine the localized reaction of tissue to intracutaneous injection of extracts of medical devices, biomaterials, or prostheses in the final product form. Intracutaneous tests may be applicable where determination of irritation by dermal or mucosal tests is not appropriate.
- Albino rabbits are most commonly used, however newer ISO guidelines refer to determination of skin irritation using reconstructed human epidermis as an alternative to animal testing (ISO 10933-23).
- Critical to the conduct of these tests is that the preparation of the test material and/or extract solution should be chosen to include testing for both water-soluble and fat-soluble leachables.
Fundamentals of IN VIVO Tests for Interactions with Blood (Hemocompatbility) (ISO 10993-4)
- Hemocompatibility tests evaluate effects on blood and/or blood components by blood-contacting medical devices or materials.
- In vivo hemocompatibility tests are usually designed to simulate the geometry, contact conditions, and flow dynamics of the device or material in its clinical application.
- Thus, in vivo testing in ani- mals may be convenient, but anatomic differences among species and species-related differences in blood reactivity must be considered, and these may limit the predictability of any given test in the human clinical situation.
- While blood values and reactivity between humans and nonhuman primates are very similar, European Community law prohibits the use of nonhuman primates for blood compatibility and medical device testing.
- Hemocompatibility evaluation in animals is complicated by the lack of appropriate and adequate test materials, for example, appropriate antibodies for immunoassays.
- Use of human blood in hemocompatibility evaluation implies in vitro testing, which usually requires the use of anticoagulants that are not generally present with the device in the clinical situation, except for perhaps the earliest implantation period.
- Although species differences may complicate hemocompatibility evaluation, the utilization of animals in short- and long-term testing is considered to be appropriate for evaluating thrombosis and tissue interaction.
- From the ISO standards perspective, five test categories are indicated for hemocompatibility evaluation: thrombosis; coagulation; platelets; hematology; and immunology (complement and leukocytes).
- Two levels of evaluation are indicated: Level 1 (required); and Level 2 (optional).
- Regardless of blood contact duration, hemocompatibility testing is indicated for three categories of medical devices: (1) external communicating devices—blood path indirect; (2) external communicating devices—circulating blood; and (3) blood- contacting implant devices.
Selection of Animal Models for In Vivo Tests
- Preclinical testing in animal models is an important part of the regulatory process, used to determine the safety and efficacy of devices prior to human clinical trials.
- The choice of the animal model and the selection of in vitro tests should be made according to the intended use of the respective medical device, prosthesis, or biomaterial.
- (see diagram in notes or text book (week 7 in vivo) for list of animal models for respective device classification).
- A single test animal may not assess all pertinent clinically important complications.
- The in vivo assessment of tissue responses to vascular graft materials is an example in which animal models present a particularly misleading picture of what generally occurs in humans (use of multiple animal types - see textbook).
Fundamentals of IN VIVO Tests for systemic toxicity (ISO 10993-11)
- Systemic toxicity tests estimate the potential harmful effects in vivo on target tissues and organs away from the point of contact (i.e., site of implantation) with either single or multiple exposures to medical devices, biomaterials, and/or their extracts.
- These tests evaluate the systemic toxicity potential of medical devices that release constituents into the body.
- These tests also include pyrogenicity testing, which assesses the induction of a systemic inflammatory response, often measured as fever.
- Mice, rats, or rabbits are the usual animals of choice for the conduct of these tests, and oral, dermal, inhalation, intravenous, intraperitoneal, or subcutaneous application of the test substance may be used, depending on the intended application of the biomaterial.
