BME Experiment 2010 Summer Ming-Long Yeh 8/25/2010

1. BME Experiment2010 SummerMing-Long Yeh8/25/2010 Biological Testing of Biomaterials From: Biomaterials Science by Ratner

2. 5. Biological Testing of Biomaterials 5.1 INTRODUCTION TO TESTING BIOMATERIALS 5.2 IN VITRO ASSESSMENT OF TISSUE COMPATIBILITY 5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY 5.4 EVALUATION OF BLOOD-MATERIALS INTERACTIONS 5.5 LARGE ANIMAL MODELS IN CARDIAC AND VASCULAR BIOMATERIALS RESEARCH AND TESTING 5.6 MICROSCOPY FOR BIOMATERIALS SCIENCE

3. 5.1 INTRODUCTION TO TESTING BIOMATERIALS

4. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • How can biomaterials be evaluated to determine if they are biocompatible and will function in a biologically appropriate manner in the in vivo environment? • This introduction: common to all biomaterials biological testing.

5. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • Evaluation under in vitro (literally "in glass") conditions: rapid and inexpensive data on biological interaction (Chapter 5. 2). • The question: will the in vitro test measure parameters relevant to what will occur in vivo environment?

6. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • For example, tissue culture polystyrene, a surface modified polymer, will readily attach and grow most cells in culture. • Untreated polystyrene will neither attach nor grow mammalian cells. • Yet when implanted in vivo, both materials heal almost indistinguishably with a thin foreign body capsule. • Thus, the results of the in vitro test do not provide information relevant to the implant situation.

7. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • In vitro tests minimize the use of animals in research, a desirable goal. • Also, in vitro testing is required by most regulatory agencies in the device approval process for clinical application. • When appropriately used, in vitro testing provides useful insights that can dictate whether a device need be further evaluated in expensive in vivo experimental models.

8. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • Animals are used for testing biomaterials to model the environment that might be encountered in humans (Chapter 5. 3). • However, there is great range in animal anatomy, physiology, and biochemistry. • Will the animal model provide data useful for predicting how a device performs in humans? • Without validation to human clinical studies, it is often difficult to draw strong conclusions from performance in animals.

9. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • The first step in designing animal testing procedures is to choose an animal model that offers a reasonable parallel anatomically or biochemically to the situation in humans. • Experiments designed • to minimize the number of animals needed, • ensure that the animals are treated humanely (e. g., NIH guidelines for the use of laboratory animals), and • maximize the relevant information generated by the testing procedure are essential.

10. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • Some biomaterials fulfill their intended function in seconds. • Others are implanted for a lifetime (10 years ? 70 years?). • Are 6-month implantation times useful to learn about a device intended for 3-minute insertion? • Will six months implantation in a test model provide adequate information to draw conclusions about the performance of a device intended for lifetime implantation?

11. 5.1 INTRODUCTION TO TESTING BIOMATERIALS • Assistance in the design of many biomaterials tests is available through national and international standards-organizations. • Thus, the American Society for Testing Materials (ASTM) and the International Standards Organization (IS0) can often provide detailed protocols for widely accepted, carefully thought out testing procedures. • Other testing protocols are available through government agencies (e. g., the FDA) and through commercial testing laboratories.

12. 5.2 IN VITRO ASSESSMENT OF TISSUE COMPATIBILITY

13. 5.2 IN VITRO ASSESSMENT OF TISSUE COMPATIBILITY • “Cytotoxicity”: to cause toxic effects (death, alterations in cellular membrane permeability, enzymatic inhibition, etc.) at the cellular level. • It is distinctly different from physical factors that affect cellular adhesion (surface charge of a material, hydrophobicity, hydrophilicity, etc.). • Evaluation of biomaterials by methods that use isolated, adherent cells in culture to measure cytotoxicity and biological compatibility.

14. HISTORICAL REVIEW • Cell culture methods: more than two decades (Northup, 1986). • Most often today the cells used for culture are from established cell lines purchased from biological suppliers or cell banks. • Primary cells are seldom used • less assay repeatability, reproducibility, efficiency, and, in some cases, availability.

15. BACKGROUND CONCEPT Toxicity • A toxic material is defined as a material that releases a chemical in sufficient quantities to kill cells either directly or indirectly through inhibition of key metabolic pathways. • The number of cells that are affected is an indication of the dose and potency效力of the chemical. • Although a variety of factors affect the toxicity of a chemical (e.g., compound, temperature, test system), the most important is the dose or amount of chemical delivered to the individual cell.

16. BACKGROUND CONCEPT Delivered and Exposure Doses • Delivered dose: the dose that is actually absorbed by the cell. • Exposure dose: the amount applied to a test system. • For example, if an animal is exposed to an atmosphere containing a noxious有毒的substance(exposure dose), only a small portion of the inhaled substance will be absorbed and delivered to the internal organs and cells (delivered dose).

17. Delivered and Exposure Doses • The cells that are most sensitive are referred to as the target cells. • Cell culture methods: evaluate target cell toxicity by using delivered doses of the test substance. • This distinguishes cell culture methods from whole- animal studies, which evaluate the exposure dose and do not determine the target cell dose of the test substance.

18. Safety Factors • A highly sensitive test system is desirable for evaluating the potential hazards of biomaterials because the inherent characteristics of the materials often do not allow the dose to be exaggerated. • There is a great deal of uncertainty in extrapolating from one test system to another, such as from animals to humans. • To allow for this, toxicologists have used the concept of safety factors to take into account intra- and interspecies variation.

19. Safety Factors • This practice requires being able to exaggerate the anticipated預期human clinical dosage in the nonhuman test system. • Local toxicity model in animals: there is ample opportunity for reducing the target cell dose by distribution, diffusion, metabolism, and changes in the number of exposed cells (because of the inflammatory response). • Cell culture models: the variables of metabolism, distribution, and absorption are minimized, the dosage per cell is maximized to produce a highly sensitive test system.

20. ASSAY METHODS

21. ASSAY METHODS • Three primary cell culture assays are used for evaluating biocompatibility: • direct contact, • agar diffusion, • elution (also known as extract dilution). • These are morphological assays, meaning that the outcome is measured by observations of changes in the morphology of the cells.

22. cell culture assays • http://en.wikipedia.org/wiki/Cell_Culture_Assays • direct contact, • agar diffusion, • elution

23. Direct contact method • A near confluent layer of fibroblasts are prepared in a culture plate • Old cell culture media (agar generally) is removed • Fresh media is added • Material being tested is placed onto the cultures, which are incubated for 24 hours at 37 degrees Celsius • The material is removed • The culture media is removed • The remaining cells are fixed and stained, dead cells are lost during fixation and only the live cells are stained • The toxicity of the material is indicated by the absence of stained cells around the material

24. Agar diffusion method • A near confluent layer of fibroblasts are prepared in a culture plate • Old cell culture media is removed • The cells are covered with a solution of 2% agar, which often contains red vital stain • When the agar solidifies the cells will have dispersed throughout its volume • The material is then placed on the surface of the agar and incubated for 24 hours at 37 degrees Celsius • Live cells take up the vital stain and retain it, dead cells do not • The toxicity of the material is evaluated by the loss of vital stain under and around the material • Surface microscopy is also needed to evaluate the material-cell interfacae

25. Elution method • A near confluent layer of fibroblasts are prepared in a culture plate • An extract of the material which is being tested is prepared using physiological saline or serum free media (the latter is generally preferred) • Extraction conditions are used which are appropriate for the type of exposure which the cells would receive in the in vivo environment if the material were to be implanted • The extract is placed on the cells and incubated for 48 hours at 37 degrees Celsius • After 48 hours the toxicity is evaluated using either a histochemical or vital stain

26. ASSAY METHODS • To standardize the methods and compare the results of these assays, the variables • number of cells, • growth phase of the cells (period of frequent cell replication), • cell type, • duration of exposure, • test sample size (e.g., geometry, density, shape, thickness), and • surface area of test sample

27. ASSAY METHODS • In general, cell lines that have been developed for growth in vitro are preferred to primary cells that are freshly harvested from live organisms because the cell lines improve the reproducibility of the assays and reduce the variability among laboratories. • That is, a cell line is the in vitro counterpart of inbred近親交配的animal strains used for in vivo studies. • Cell lines maintain their genetic and morphological characteristics throughout a long (sometimes called infinite) life span.

28. ASSAY METHODS • Cell lines from other tissues or species may also be used. • Selection of a cell line is based upon the type of assay, the investigator’s experience, measurement endpoints (viability, enzymatic activity, species specific receptors, etc.), and various other factors. • It is not necessary to use human cell lines for this testing because, by definition, these cells have undergone some dedifferentiation and lost receptors and metabolic pathways in the process of becoming cell lines.

29. ASSAY METHODS • Positive and negative controls are often included in the assays to ensure the operation and suitability of the test system. • The negative control of choice: high-density polyethylene material. • The positive controls:low-molecular-weight organotin-stabilized poly (vinyl chloride), gum rubber, and dilute solutions of toxic chemicals, such as phenol and benzalkonium chloride.

31. CLINICAL USE • The in vitro cytotoxicity assays are the primary biocompatibility screening tests for a wide variety of elastomeric, polymeric, and other materials used in medical devices. • After the cytotoxicity profile of a material has been determined, then more application specific tests are performed to assess the biocompatibility of the material.

32. CLINICAL USE • Current experience indicates that a material that is judged to be nontoxic in vitro will be nontoxic in in vivo assays. • This does not necessarily mean that materials that are toxic in vitro could not be used in a given clinical application. • The clinical acceptability of a material depends on many different factors, of which target cell toxicity is but one. • For example, glutaraldehyde-fixed porcine valves produce adverse effects in vitro owing to low residues of glutaraldehyde; however, this material has the greatest clinical efficacy for its unique application.

33. 5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY

34. 5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY INTRODUCTION • The goal of in vivo assessment of tissue compatibility of a biomaterial, prosthesis, or medical device is to determine the biocompatibility or safety of the biomaterial, prosthesis, or medical device in a biological environment.

35. INTRODUCTION • Biocompatibility has been defined as the ability of a medical device to perform with an appropriate host response in a specific application, and • biocompatibility assessment is considered to be a measurement of the magnitude and duration of the adverse alterations in homeostatic mechanisms that determine the host response.

36. INTRODUCTION • 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. • Thus, the goal of the in vivo assessment of tissue compatibility is to predict whether a medical device presents potential harm to the patient or user by evaluations under conditions simulating clinical use.

37. INTRODUCTION • Recently, extensive efforts have been made by government agencies, i. e., FDA, and regulatory bodies, i.e., ASTM, IS0, and USP, to provide procedures, protocols, guidelines, and standards that may be used in the in vivo assessment of the tissue compatibility of medical devices. • This chapter draws heavily on the IS0 10993 standard, Biological Evaluation of Medical Devices, in presenting a systematic approach to the in vivo assessment of tissue compatibility of medical devices.

38. List of the standards in the 10993 series ISO 10993-1:2003 Biological evaluation of medical devices Part 1: Evaluation and testing ISO 10993-2:2006 Biological evaluation of medical devices Part 2: Animal welfare requirements ISO 10993-3:2003 Biological evaluation of medical devices Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity ISO 10993-4:2002/Amd 1:2006 Biological evaluation of medical devices Part 4: Selection of tests for interactions with blood ISO 10993-5:1999 Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity

39. ISO 10993-6:1994 Biological evaluation of medical devices Part 6: Tests for local effects after implantation ISO 10993-7:1995 Biological evaluation of medical devices Part 7: Ethylene oxide sterilization residuals ISO 10993-8:2001 Biological evaluation of medical devices. Part 8: Selection and qualification of reference materials for biological tests ISO 10993-9:1999 Biological evaluation of medical devices Part 9: Framework for identification and quantification of potential degradation products ISO 10993-10:2002/Amd 1:2006 Biological evaluation of medical devices Part 10: Tests for irritation and delayed-type hypersensitivity

40. ISO 10993-11:2006 Biological evaluation of medical devices Part 11: Tests for systemic toxicity ISO 10993-12:2002 Biological evaluation of medical devices Part 12: Sample preparation and reference materials (available in English only) ISO 10993-13:1998 Biological evaluation of medical devices Part 13: Identification and quantification of degradation products from polymeric medical devices ISO 10993-14:2001 Biological evaluation of medical devices Part 14: Identification and quantification of degradation products from ceramics ISO 10993-15:2000 Biological evaluation of medical devices Part 15: Identification and quantification of degradation products from metals and alloys

41. ISO 10993-16:1997 Biological evaluation of medical devices Part 16: Toxicokinetic study design for degradation products and leachables ISO 10993-17:2002 Biological evaluation of medical devices Part 17: Establishment of allowable limits for leachable substances ISO 10993-18:2005 Biological evaluation of medical devices Part 18: Chemical characterization of materials ISO/TS 10993-19:2006 Biological evaluation of medical devices Part 19: Physico-chemical, morphological and topographical characterization of materials ISO/TS 10993-20:2006 Biological evaluation of medical devices Part 20: Principles and methods for immunotoxicology testing of medical device

42. INTRODUCTION

43. BIOMATERIALS AND DEVICE PERSPECTIVES IN IN VIVO TESTINGS

44. BIOMATERIALS AND DEVICE PERSPECTIVES IN IN VIVO TESTINGS

45. Sensitization, irritation刺激, and intracutaneous (intradermal) Reactivity • Exposure to or contact with even minute amounts of potential leachables from medical devices or biomaterials can result in allergic or sensitization reactions. • Sensitization tests estimate the potential for contact sensitization to medical devices, materials, and/or their extracts. • Symptoms of sensitization are often seen in skin and tests are often carried out topically in guinea pigs.

46. Sensitization, irritation, and intracutaneous (intradermal) Reactivity • The most severely irritating chemical leachables may be discovered prior to in vivo studies by careful material characterization and in vitro cytotoxicity tests. • 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 are not appropriate. • Albino rabbits are most commonly used.

47. Systemic Toxicity: Acute, Subacute, and Subchronic Toxicity • 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. Peritoneal 腹膜的

48. Systemic Toxicity: Acute, Subacute, and Subchronic Toxicity • Acute toxicity is considered to be the adverse effects that occur after administration of a single dose or multiple doses of a test sample given within 24 hours. • Subacute toxicity (repeat-dose toxicity) focuses on adverse effects occurring after administration of a single dose or multiple doses of a test sample per day during a period of from 14 to 28 days. • Sub-chronic toxicity is considered to be the adverse effects occurring after administration of a single dose or multiple doses of a test sample per day given during a part of the life span, usually 90 days but not exceeding 10 % of the life span of the animal.

49. Systemic Toxicity: Acute, Subacute, and Subchronic Toxicity • Pyrogenicity tests are also included in the systemic toxicity category to detect material-mediated fever-causing reactions to extracts of medical devices or materials. • It is noteworthy that no single test can differentiate pyrogenic reactions that are material-mediated from those due to endotoxin contamination Pyrogenicity: producing or produced by heat or fever

50. Genotoxicity • In vivo genotoxicity tests are carried out if indicated by the chemistry and/or composition of the biomaterial (see Table 1) or if in vitro test results indicate potential genotoxicity [changes in deoxyribonucleic acid (DNA)]. • Initially, at least three in vitro assays should be used and two of these assays should utilize mammalian cells. • The initial in vitro assays should cover the three levels of genotoxic effects: • DNA destruction, • gene mutations, and • chromosomal aberrations .