Biomaterials and Biocompatibility Biological response Biocompatibility tests Lenka Martinová

1. Biomaterials and Biocompatibility Biological response Biocompatibility tests Lenka Martinová

2. A thorough biocompatibility safety testing program will typically comprise in vivo studies supplemented by select in vitro assays. Photo: Northview Biosciences, Inc.

3. Table I. Listing of individual parts of ISO 10993, Biological Evaluation of Medical Devices.

4. The original definition of biocompatible materials (BM): • BM is inert toward the physiological environment, has been modified to include materials having minimal interaction with the environment.It is unlikely that an absolutely inert biomaterial can be found, but physiological response to any biomaterial be kept within acceptable bounds. • Specification of biocompatibility must include the conditions of use end evaluation • Screening and animal testing before clinical trials are imperative. • Alteration(změna) in biomaterilas by the physiological environment is biodegradation. • In vitro and in vivo test are valuable in the study of materials properties • Acceptable biological response to the material and absence of physiologicaly induces damage • At presence: standardizes tests of biological performance – physiochemical evaluation of biomedical polymers and evaluation of biomaterials exposed to blood.

5. Biocompatibility • Arises from differences between living and non-living materials • Bio-implants trigger(spouštět) inflammation or foreign body response • New biomaterials must be tested prior to implantation according to FDA (Food and Drug Administration) regulation

6. Biomaterial-Tissue Interactions

7. Definition • Neutrophil- common leucocyte of the blood- short-lived phagocytic cell • Lymphocyte- small cell in blood- recirculates through tissues and back through lymph --polices body for non-self material-- recognizes antigens through surface receptors • Antigen- produces antibody- stimulate adaptive immune response • Antibody- Serum globulins with wide range of specificity for different antigens-- bind to surface • Monocyte- largest nucleated cell of blood-develops into macrophage when it migrates to tissues • Macrophage- phagocyte of tissues • Lysozyme- enzyme secreted by macrophages- attack cell wall of bacteria “natural antibiotic” • Mast Cell- large tissue cell which releases inflammatory mediators-- increases vascular permeability-- allows complement to enter tissues from blood • Complement- a series of enzymes in blood- when activated produce inflammatory effects

8. Figure 1. Flowchart(postupový diagram) illustrating steps in the biological evaluation of medical devices according to the ISO 10993-1 standard.

9. Leukocytes activity

10. Response to implantation • Inflammation • Acute inflammation • Chronic inflammation • Granulation tissue • Foreign Body Reaction • Fibrosis and Encapsulation

11. Inflammation • Inflammation (zánět) and wound healing The inflammatory response is a normal physiological reaction to trauma and the invasion of foreign substance. After an initial acute response a chronic inflamatory response is seen when the injury or foreign substance persists(trvale působí).

12. Inflammation is the reaction of vascularized living tissue to injury. • Its function is to control, neutralize or isolate the damaging agent. • The inflammation process includes a sequence of events that can heal the implant site. • This is done through the generation of new tissue via native parenchymal cells or the formation of fibroblastic scar tissue.

13. Acute inflammation • Acute inflammationis short term (minutesdays) • Characterized by exudation of fluid, plasma, proteins,andleukocytes (neutrophils). • Once at the injury site, phagocytosis and enzymatic release occurs. • This is followed by the activation of neutrophils and macrophages whose purpose is to digest foreign materials. This involves recognition, attachment, engulfment(pohlcení) and degradation.

14. Chronical inflammation • Chronic inflammation is long term (≥≥ days). • Accompanied by the proliferation(rozšíření, bujení) of blood vessels and connective tissue. • Lymphocytes and plasma cells are involved in immune reactions- mediate antibody production. • Characterized by the presence of macrophages, monocytes, and mononuclear cells including lymphocytes and plasma cells. • Macrophages process and deliver antigen to immuno-competent cells— mediate immune reactions.

15. Foreign body reaction • The foreign body reaction is indicated by the presence of foreign body giant cells and the components of granulation tissue (macrophages, fibroblasts, and capillaries in varying amounts) • observed in silicone breast implants • surface of the biomaterial will often determine the composition of the foreign body response.

16. Surface structure important for biocompatibility Even in “biocompatible” materials, the high surface to volume ratio of fabrics, porous structures, etc. will result in higher ratios of macrophages and foreign body giant cells than a smooth component made of the identical material.

17. Fibrosis and Encapsulation The final stage of the foreign body response and healing process is the development of a fibrous encapsulation (porous structures may be excluded from this stage due to tissue ingrowth). Repair involves two separate processes: replacement of tissue by parenchymal cells of the same type or replacement by connective tissue that constitute the fibrous capsule. These processes are controlled by the growth capacity of the cells in the tissue receiving the implant, the persistence of the tissue framework and degree of injury.

18. Response to the inflammatory challenge • Decreased tissue mass and formation of new tissue through granulation • Collagen and other molecules are synthesized • Formation of scar (jizva) tissue • Remodeling(přestavbový)process differs for various tissues

19. Implant Factors • Bulk properties: chemical composition, structure, purity and presence of leachables(vyluhovatelná substance) • Surface properties: smoothness, geometry, hydrophilicity, surface charge • Mechanical properties: match properties of component being replaced, such as elastic modulus,stability and fixation • Long-term structural integrity: design for fatigue(únava) and fractureloading(lomové zatížení), wear(opotřebení), creep, and stress corrosion cracking

20. Implant can affect local tissue Systemic: Embolization (occlusion of blood vessels) Hypersensitivity Implant elements in blood Lymphatic Particle Transport Locally: Blood-material Protein adsorption Platelet (krevní destička)Adhesion Complement activation Leukocyte adhesion/activation Toxicity Encapsulation Foreign body reaction Scarring(zjizvení) Infection Tumorogenesis

21. Host can affect the implant • Physically • Abrasive, adhesive, delamination wear • Fatigue and Fracture • Stress Corrosion cracking • General corrosion • Biologically • Absorption of substances from the tissues • Enzymatic degradation • Calcification

22. Host Factors • Age and health status • Immunological/metabolic status • Choice of surgeon: minimize tissue damage and contamination, proper implantation

23. Biomedical polymers (BP) – design parameters- understanding of the physiological function and conditions under which the device must operate. • Mechanical properties • Purity– polymer must be available in reproducible pure form, quality control in biopolymers is more critical than in other fields, especially for materials that will have direct contact with body tissue and fluids • Fabrication- must be fabricated into the desired form without being degradated or adversely affected, reproducible polymer morphology and properties • Stability - BP should not be adversely affected by the normal physiological environment, no changes  for example crystallization, embrittlement(zkřehnutí) or plasticizing mayresult form oxidation, absorption of biological compounds (proteins,lipids),  deposition of inorganic materials or ingrowth (vrůstání) of tissue, resulting in impaired function, the physiological temperature (37C may accelerate these effects over what is seen at room temperature  Interaction with cells, as are leukocytes, enzymes (proteins with specific catalytic function) – they can attack foreign substances, including implants • Tolerabilty – should not exhibit toxic or irritant qualities, or elicit adverse (vyvolat nepříznivou)physiological responses locally or systematically.

24. Adverse biological responses to an implanted biomedical polymer include excessive (nadměrnou) foreign-body response, thrombogenicity and immunogenetic, antileukotactic (predisposition toward infection), and mutagenic or carcinogenic responses. • Biocompatibility testing • Characterization and evaluation - structure –responce relationships and reproducibility of results • Cell toxicity • Thromobogenecity • Inflammatory response • Animal tests • Clinical trials • FDA regulations • ASTM/ISO standards

25. Test methods (compiled in the NIH publication, Guidelines(pravidla) for Physicochemical Characterization of Biomaterials (1)  Level-I bulk- characterization tests identify the polymer and confirm it meets specification, consists of simple tests for surface topography, cleanliness, chemical structure and composition, toxicity tests  Level-II – more detailed information of specific elements of structural and functional behaviour that may relate to performance, surface chemistry and morphology and interfacial properties

26. The predominant feature of the foreign-body reaction is the encapsulation of the implant with fibrous tissue, which forms a lesion called a granulona. Persistent granuloma can undergo caseous necrosis(odumření) if the avascular inner layer of phagocytic cell dies. Calcification, liquefaction (zkapalnění) and hyperplasia are also possible. • Blood-clotting system- the main function of the blood-clootting (srážení, hrudkovatění) system is to prevent blood loss from the vascular system and reestablish perfusion (obnovit promývání) by restoring the function of the vessel ( many aspects of blood coagulation are still poorly undesrtood).  Kinin system (bradykinin- type of plasma hormon) – the kinin cause dilation of blood vessels, pain and increased permeability of blood vessels. They are controlled by circulation peptidases and broken down by kininases Immune system – the imune system is two defense systems: humoral and cellular. Cellular and humoral immune systems represent an integrated effort to identify, neutralize and remove tumorcells, bacteria, viruses and foreign tissue as well as foreign macromolecular substances, so that these invaders do not compromise the host. The immune system works in cooperation with the complement system and the phagocytic cells, especially the macrophages.

27. Complement System– the serum complement system contains nine protein component. Complement is required for the cytolytic destruction of cellular antigens • Important activity of the complement complex is that of opsonization (vazba specifických látek na povrch bakterií) – modification of the antigen- antibody complex – so it can be more readily identified for phagocytosis • Biological Responses to Implants - the evaluation should determine any adverse reaction and short and long-term effects of the physiological enviroment on the implant – complex relationships exist between materials properties,design and implantation parameters, and the biological response • Blood–materials Interactions – correlation between biomaterial surface and blood compatibility has been much studied. Several approaches to non-trombogenic surface have emerged, each emphasizing certain surface properties: surface texture, electrostatic effects, interfacial properties (critical surface tension) and polar-apolar ratio, adsorption of plasma proteins, „natural“ surface, „modified“ surface, etc.- a recent review has identified over 50 significant variables.

28. The adsorption of plasma proteins, including the coagulation proteins, is the firs event in blood-surface interactions and is crucial in understanding surface-induced thrombosis. • Immunity and Infection – foreign materials may disturb responses associated with the immune and component system, especially with ragard to infection The immune system identifies foreign materials and elicits responses to neutralize and remove the substances. Synthetic polymers are weakly antigenic because of their highly regular structure, chemical inertness and relative stability – they are not likely to elicit an immune response except if contaminated, e.g. by a residual monomer. Many monomers and additives are cytotoxic and are capable of causing prolonged inflammation.

29. The presence of foreign material increases the risk of infection at an impant site. Mutagenesis and Carcinogenesis – in rodents and other experimental animals, synthetic polymer and other materials impanted subcutaneously (podkožně) cause sarcomas. Factors that influence tumor frequency and length of the latency period include: genetic differences between various test strain, sexual differences, nutritional state of the test animals ( high protein decreased tumor frequency and increased the latency period), implant size and shape (tumoral frequency was proportional to total surface area). Inflamantory and Wound-healing Response – acute inflamation defends the body in the case of trauma and reestablish a physiological environment. The chronic inflammatory response to animplant is affected by its chemical , mechanical and surface characteristics and usually associated with encapsulation in fibrous tissue. Soft tissue response is sensitive to surface texture.

30. Biocompatibility Testing • No one material will be appropriate for all medical device application • The material, its composition and degradation products may affect host cells and tissues • The host environment may also affect material properties and device performance IN VITRO TESTING Types of in - Vitro tests for estimating biocompatibility Cytotoxicity – Elution or extract test Agar or agarose overly test Direct contact test Hemocompatibility - Hemolysis assay Clotting and complement activation Mutagenecity – Ames test Hypersensitivity – Lymphocyte trasnsformation test Leukocyte migration inhibition test

31. Cytotoxicity Tests • Cytotoxicity - ability to cause death or damage at the cellular level by direct cell lysis (desintegrace, rozpad) or by fatally altering cellular metabolism. Inhibition of enzyme activity, changes in cell membrane permeability and other sublethal effects. Special tests protocols may be found in ISO, ASTM, NIH • Elution test – the aim : to determine toxic doses and changes in cell growth or proliferation, to compare to non-treated cells over 24-78-hpurs period (hemocytometer or electronic cell counter) • Agar overlay – material are placed on an agar or agarose layer covering the cells for 24 hours, components from test material are allowed to diffuse through the agar or agarose to the cells, cytotoxicity of the diffusible components is determined by staining the cells with a viability dye and then measuring the zone of dead cells surrounding the test materials. • Direct contact tests – changes in cell growth or proliferation are measured in similar manner as in the elution tests

32. Hemocompatibility Tests • Are used to evaluate the effect of a material on blood coagulation processes, thrombus formation and hemolysis (destruction of red blood cells). • Materials or theirs extracts are incubated with red blood cells, isolated from rabits, mice, or rats for three hours with intermittent shaking to keep samples mixed and in contact with blood. The amount of hemoglobin released into the supernatant from the cells is determined spectrophotometrically and reported as percent hemolysis with respect to negative controls. • To evaluate the effect of materials and their surfaces on blood clotting(sraženina) , materials are exposed to whole blood serum. • Turbulent flow of blood may increase hemolysis and or clotting – the number of adherent platelets may be determined per unit area after exposure to whole blood.

33. Mutagenecity and Genotoxicity • Mutagenes – modify the genome of a host, so materials may be classified as genotoxic. It is widely accepted that carcinogenic behavior proceeds via a mutation in the genome. • Ames test – uses a mutant bacterial cell line (Salmonella typhimurium or Escherichia coli) that must be supplied with histidine to growth. The cells are cultured in histidine-free environment and only those material that mutate the cells back to state of histidine indipendence will allow the cells back to grow. • Hypersensitivity Tests – the leukocyte migration inhibition and lyphocyte transformation tests have been used as in-vitro models to estimate delayed hypersensitivity reaction to implant materials and their released components.

34. In- vivo testing for biocompatibility • In- vivo animal testing is necessary prior to human clinical testing. • Short-term implantation tests – subcutaneous, intramuscular and intraperitoneal implantation tests to evaluate general tissue necrosis, fibrosis and inflammation • Long-term functional tests – device or compositionally identical prototypes are implanted in appropriate animal models to replicate/simulate intended end-use in humans. Functionality of device and histopatological evaluation if tissues /organs are performed. • Sensitization - Guinea pig, Ocluded patch test, Open epicutaneous test • Irritation – Skin, Ocular, Mucosal • Other – Genotoxic, carcinogenic, reproductive,cerebrospinal, hemocompatible

35. Animal test I • Nonfunctional tests - the first study a interaction of the implant with physiological environment, nonspecific acute toxic or inflammatory reaction, specialized sites such as the cornea and cerebral cortex are used for materials. Histological analyses may be scored or graded based on degree of tissue necrosis/degeneration, fibrosis and types and amount of inflammatory • Functional testes - evaluation in soft tissues, much greater complexity, also specific in-vivo physiological assessment tests (genotoxicity, carcinogenityreproductive toxicity, etc) • Genotoxicity testing – alteration in DNA or chromosomal structure or other DNA or gene damage that result in permanent inheritable changes in cell function • Cancerogenity tests – determine, whether chemicals or compounds that may be released from biomaterials elicit sensitization reactions – result of immunologically medicated reaction resulting in redness(erythema) and swelling (edema).

36. Animal test II • Irritation test - a localized inflammatory response without involvement of an immunological mechanism Fresh extract is injected intracutaneously at multiple sites – tome 24, 48, 72 hours control. Ocular irritation – ophtalmological materials (rabbit) • Systemic effects – chemicals released from implant materials are distributed by the blood and lymphatic system and damage organs and tissues. Categories: acute (within 24 hr), sub-acute (in 14 – 28 days) sub-chronic ( 10% of an animal´s life span) and chronic (longer than 10% of an animal´s life span. The selection of evaluation in in-vivo depend on the animal used (mice, rabbit, dog, etc.) and the implant material

37. Clinical trials of biomedical implants • Standard practice – new drug to employ a double-blind study in which placebo is randomly administrated . • Implant testing – it is not possible to pair implanted patient which a placebo-treated patient, because it is not possible to conceal the implanted site from surgeon and or patient. • Clinical trials – divided into three phases Phase I (early trial) – biomaterial is tested on a small group people (ca 60-80 Phase II – large group (ca 100-300) Phase III – comparison of the effectiveness of the new treatment with a standard of management, (ca 1000 – 3000) • The reports of clinical trials should discuss the accuracy and precision of all measurements as well as define a minimum confidence level for all statistical measures of date (usually p<0,05).The reports must also include confidence intervals or other measures of significance associated with all derived parameters and must indicate the significance of any conclusion arrived at by analysis of the trial.