Biocompatibility Testing

Abstract

The insertion of a foreign material into the body induces a cascade of events, basically at the interface between the implanted material and the tissue, which results in the recognition of the material as foreign matter. The degree of this physiological response depends on the location of implantation and the composition and mechanical properties of the material. Thus, the body’s response to an implanted material is affected by a number of different factors. To evaluate and reduce the risk for unexpected or unwanted side effects, biocompatibility testing is used to examine new biomaterials and biomedical devices destined for implantation. The biological evaluation of the material’s safety is a complex task since it requires knowledge in the disciplines of medicine, biology, pathology, engineering and materials science.

13.1 Introduction

The insertion of a foreign material into the body induces a cascade of events, basically at the interface between the implanted material and the tissue, which results in the recognition of the material as foreign matter. The degree of this physiological response depends on the location of implantation and the composition and mechanical properties of the material. Thus, the body’s response to an implanted material is affected by a number of different factors. To evaluate and reduce the risk for unexpected or unwanted side effects, biocompatibility testing is used to examine new biomaterials and biomedical devices destined for implantation. The biological evaluation of the material’s safety is a complex task since it requires knowledge in the disciplines of medicine, biology, pathology, engineering and materials science.

The word “biocompatibility” can be defined as the compatibility between a material and the biological system. Probably the best definition of biocompatibility is that agreed upon at the Consensus Conference on Definitions in Biomaterials in 1986 (European Society for Biomaterials), namely, “the ability of a material to perform with an appropriate host response in a specific application” [1]. The corollary of this is that the type of testing method will depend on the intended function of the biomaterial being used. It then becomes obvious that the testing strategy must take into account the biological situation in which the biomaterial will find itself. The biological situation is influenced by the type of affected tissue or organ, physico-chemical features of the material, and the implant duration period.

The biocompatibility concept includes two principal elements. First there is the absence of a cytotoxic effect, and second, there is the aspect of functionality. Cytotoxicity deals mainly with the survival of cells and the maintenance of specific cellular functions under the influence of a material and/or its degradation products. Functionality includes the integration of the biomaterial into a biological system (the tissue or organ). Functionality also assumes the absence of impairment of cellular function and requires that the mechanical, chemical, and physical features of the material are sufficient for the performance of cell-specific functions. Since biomaterial-induced modifications of cellular functions might also accumulate or act synergistically, cell-material interactions could lead to deleterious effects with respect to implant acceptance and long-term stability.

The protocols for the evaluation of the safety of biomaterials planned for use as a medical device are guided by specific organizations. In the United States, the Food and Drug Administration (FDA), which is a government body, is responsible for the determination of standards in testing biocompatibility of medical devices. In Europe, the International Organization for Standardization (ISO), a commercial organization, is the authorized body. The definitions for medical devices are similar in Europe and the United States. In Europe the testing of medical devices is guided by an ISO standard (ISO 10993). The US standards (Quality System Regulation/QRS) are mostly comparable to the ISO standard. This chapter refers generally to the ISO standard when accredited test systems are mentioned. Currently, 20 parts of the ISO 10993 standard are either accepted or under preparation. Tests that may be used in the assessment of medical device biocompatibility include procedures for cytotoxicity, sensitization, irritation, acute systemic toxicity, subchronic toxicity, mutagenicity, genotoxicity, hemocompatibility, etc. The testing procedures can be performed in vitro as well as in vivo (mainly in animal studies).

The main aspects of biocompatibility testing are discussed in the following sections. It must be pointed out that due to the widely facetted aspects that require to be taken into account one cannot deal with the topic exhaustively. Since the preparation of the samples is an important point in biocompatibility, testing we will start this chapter with a short overview of the issue of sample preparation.

13.2 Sample Preparation

The preparation and processing of the test specimens is a fundamental aspect in biocompatibility testing. Both ISO and FDA standards address sample preparation and reference materials (e.g., [2].). The types of test samples, suitable extraction medium and conditions, and appropriate reference materials to be used as controls are covered. The standards recommend testing medical devices in their final product form and composition whenever possible. If finished devices are not available for testing, the evaluation of representative subcomponents of the device is also acceptable. If the device is too large or cannot be tested as a whole, each different material component that has the potential of coming into contact with body tissues should be represented in the same proportion in the test sample as it is in the final product.

Since biomaterials are normally insoluble and the methods for biocompatibility testing are often designed for soluble compounds, the biocompatibility tests can be modified for the evaluation of liquid extracts. The selection of the appropriate extraction medium depends on the test system of choice. For example, bacterial test systems are frequently exposed to physiological osmolarity solutions (0.9% sodium chloride) or either ethanol or dimethyl sulfoxide extracts. Since in vitro mammalian cell culture–based test systems require media that induce cell growth, cell culture medium is often employed as an extraction medium. In vivo test models frequently employ aqueous and nonaqueous extraction fluids that are capable of extracting both water-soluble and lipid-soluble chemicals.

However, one has to keep in mind that the problems often lie in the details since the materials can undergo a wide range of annealing, passivation, electropolishing, and cleaning processes which may lead to extensive changes of their character. Thus, although the underlying material can clearly be identified, the exact surface chemistry, surface corrosion, and durability of the device over a prolonged time period can differ significantly depending on the specific processing and finishing steps.

13.3 Mammalian Cell Culture

A basic requirement in biocompatibility testing is the reduction and replacement of animal studies wherever possible. Biocompatibility testing in mammalian cell cultures in vitro offers an excellent screening method. Due to detailed cell isolation protocols, the availability of different recombinant growth factors, and highly defined cell culture media, a large number of different mammalian cell types can be more or less easily propagated in vitro (Fig. 13.1). These cell types originate from different tissues and are characterized by unique functions, in accordance with the functions they exhibit within the original organ, some of which are retained during cultivation in vitro. These cell type–specific functions are, e.g., pro-inflammatory cytokine production in the case of activation (inflammation in vitro), in vitro capillary formation by endothelial cells after stimulation with blood vessel-inducing growth factors (blood vessel formation in vitro), mineralization after long-term cultivation of osteoblasts (bone formation in vitro), or the production of large amounts of extracellular matrix by fibroblasts in vitro.

Fig. 13.1

Exemplary isolation protocol for cells from fresh tissues. First, tissues must be mechanically minced into small pieces. This is generally followed by an enzymatic digestion. The type of enzyme utilized (dispase, collagenase, etc.) depends on various characteristics of the tissue. For example, when the tissue has a significant amount of collagen fibers, the more appropriate enzyme for the disintegration of the tissue might be a collagenase; when the tissue is characterized by large amounts of elastin fibers, the appropriate enzyme is most likely an elastase. Therefore, the isolation methods for different cell types show large variations. The enzymatic digestion is again followed by a mechanical treatment to disconnect the loosened cells from the disintegrated tissues. The isolated cells are placed into cell culture flasks that are, dependent on the cell type, coated with specific adhesion factors (e.g., collagen, fibronectin) to initiate a rapid attachment of the cells. The cell culture media are supplemented with growth factors, amino acids, carbohydrates, vitamins, etc. These cell cultures deriving from fresh tissues are referred to as primary cultures. After growing to confluence (i.e., all available surface area is covered by cells), the cells can be subcultivated. This subcultivation leads to cell propagation that is accompanied by cell proliferation. The subcultivation step marks the transition from the primary culture to cell strain

There are two large subgroups of cells that can be cultured, and these can be grossly divided into the so-called primary cells and permanent cell lines. Primary cells are directly isolated from fresh mammalian tissues. A large number of cell types are able to adapt to cell culture conditions and may be subcultivated for a few passages (cell strains). The primary cells and their resulting cell strains normally have high similarity to the respective cell type in vivo. The preparation of primary cells as isolated single cell type cultures is labor intensive. Primary cells are characterized by an individual heterogeneity due to the genetic heterogeneity of the donor. This heterogeneity may have effects on the reproducibility of results. Primary cells and the resultant cell strains can be maintained in vitro only for a limited period of time. Depending on the cell type, primary cells become senescent after a limited number of cell divisions. This senescence is observed as a reduction in proliferation rate and the loss of cell-specific functions [3].

Permanent cell lines evolve from primary cells after genetic transformation. This transformation can occur in vivo (i.e., malignant transformation in tumor cells) or in vitro. Genetic transformations develop spontaneously or by the influence of chemical or physical mutagens (e.g., Co-60 gamma rays, nickel chloride, 4-nitroquinoline 1-oxide). The development of genetically transformed cells is a multi-step process involving accumulation of multiple genetic derangements over an extended time-period. One step found in nearly all permanent cell lines is the acquisition of an unlimited proliferative potential that requires overcoming the senescence barrier that is typical for primary cells. Permanent cell lines are clonal since they arise from one single cell, although genomic alterations also take place subsequently in vitro to give a certain degree of population heterogeneity.

Figure 13.2 shows a number of different human cell types in vitro. These cells differ in their origin (i.e., endothelial, epithelial, fibroblastic, monocytic) and their transformation status (i.e., primary or permanent type). Whereas the different endothelial cells, epithelial cells, and fibroblasts require adherence for growth, the monocytic cell line normally grows in suspension. All adherent growing cells are shown in the confluent state. This is the stage reached when the cells have covered the entire cultivation-area available. The same cell type derived from different tissues/organs of the same species shows different phenotypes (Fig. 13.2a: primary human umbilical vein endothelial cells, HUVEC, b: primary human dermal microvascular endothelial cells, HDMEC, c: a permanent cell line of human pulmonary microvascular endothelial cells, HPMEC-ST1). Most endothelial cells in culture develop a so-called cobblestone morphology. This morphology is variable and is dependent on the origin of the cells (type of tissue) and the transformation status. Such phenotypic differences are also observed in different permanent epithelial cell lines (Fig. 13.2d: A549, e: 16HBE140-, f: Calu-3). Phenotypic differences refer to differences in the expression status of the cells which are induced by varying gene activation.

Fig. 13.2

Different primary and permanent cell types in vitro. (a) HUVEC (primaryculture, passage 0), (b) HDMEC (primary culture/cell strain, passage 2), (c) HPMEC-ST1 (lung microvascular endothelial cell line), (d) A549 (alveolar epithelial cell line), (e) 16HBE140- (human bronchial epithelial cell line), (f) Calu-3 (airway epithelial cell line), (g) human dermal fibroblasts (primary culture/cell strain, passage 15), (h) U937 (cell line of a human histiocytic lymphoma, non-stimulated), (i) U937 (phorbolester-stimulated) (magnification 10x)

In comparison to both endothelial and epithelial cells, human fibroblast cultures (Fig. 13.2g) show large differences in phenotype in vitro. The intercellular contacts of fibroblasts are not as pronounced and the growth pattern of fibroblasts is not as regular as for the other two cell types. The difference between the appearance of fibroblasts and endothelial/epithelial cells is due to their arrangement in the tissue: endothelial and epithelial cells are responsible for barrier formation (e.g., between the tissue and the blood or between the air and the tissue) and thus show a polarized growth pattern as monolayers where they have an apical and a basal surface. Fibroblasts on the other hand are three-dimensionally embedded within tissues without an apical/basal polarization.

Most cells derived from solid tissues are dependent on substrate adhesion. However, a few cell lines are able to replicate while floating in the culture medium (suspension culture). Anchorage-independent growth is usually an indication of genetic transformation. This of growth often reflects the origin of the cell type from which they are derived, e.g., from leukemia or lymphomas (e.g., U937 cells derived from a human histiocytic lymphoma, Fig. 13.2h). Interestingly, these cells are also able to attach to the cultivation surface after a specific stimulation (Fig. 13.2i, phorbolester-activated U937, most cells attach to the cultivation surface). This attachment after stimulation is mediated by the expression of different cell surface adhesion molecules [4]. However, the spreading of the cell body onto the surface does not occur. On the other hand, many types of adhesion-dependent cells undergo cell death (i.e., the so-called programmed cell death or apoptosis) if they are prevented from attaching to an appropriate substrate.

Due to the almost limitless expansion potential of permanent cell lines , these are often preferred for testing cytotoxicity instead of primary cells. However, one has to keep in mind that permanent cell lines have often lost a significant part of their cell type-specific functions and may be less sensitive to modulating effects of biomaterials than primary cell cultures. Also, the examination of cell cycle–modulating aspects by biomaterials might be questionable in permanent cell lines, since these cell lines have often lost important cell cycle–regulating abilities. In addition to the choice between permanent cell lines or primary cells, there is the choice of species. Various comparative studies demonstrate that human cells react in a very different manner compared to cells from other mammals. Since biomaterials are intended for clinical use, in the authors’ laboratory only cells of human origin are utilized.

In addition to the fact that in vitro testing leads to the reduction of animal experimentation, cell culture methodologies are generally more economical. However, it must not be forgotten that cell culture represents a reduction of the complexity of the entire organism generally to a single cell type. Thus, the buffering capacity of complex cellular and humoral systems in the intact organism is missing, so that a biomaterial may not perform well in the in vitro test, but be biocompatible in vivo. Nevertheless, this is a risk which is inherent to the system.

The work with mammalian cells in culture requires specific laboratory equipment and technical experience. For example, a cell culture laboratory should be segregated from often-utilized rooms; and a laminar air flow cabinet, an inverted microscope, and a carbon dioxide-incubator are part of the basic requirements (Fig. 13.3). The primary reason for the safety precautions for cell cultivation is to avoid any hazard to individuals and the environment stemming from the cell cultures (this is especially true for human and primate cell cultures). In addition, an undesired contamination of the cell cultures by bacteria, fungi, viruses, and mycoplasma needs to be prevented [5]. Furthermore, experience in cell cultivation is needed since different cell types possess unique demands and can exhibit large variations in phenotype induced by the cultivation methods and handling. Thus, a basic knowledge in cell biology is considered a prerequisite for working with cell cultures in biocompatibility testing and is essential for ensuring reproducibility of results.

Fig. 13.3

Basic equipment for cell culture–based experiments. (a) Laminar air flow cabinet (Heraeus, Germany), (b) inverted microscope (Leica Microsystems, Germany), (c) cell counter/analyzer (Schärfe System GmbH, Germany), (d) multiwell plate-washer (Amersham, Germany), (e) spectrophotometer for nucleic acid quantification (NanoDrop Technologies, USA), (f) UV-source for nucleic acid analysis (Bachofer, Germany)

The utilization of human cells is regulated by the specific organizations, depending on the national laws. A number of key guidelines need to be followed: e.g., the donor must be informed and has to sign a consent form whenever a sample is extracted (donors should understand what the sample is used for and how the results might impact scientific problems). Furthermore, the extraction of human tissue/samples must exclude any possibility of harm for the donor. All human tissues/samples extracted for research studies must be approved by an ethics committee. In most cases sample anonymity is adequate. Depending on the type of extracted tissue or cells, there may be formalities which need to be clarified with the local responsible authorities.

13.3.1 Cytotoxicity Testing

As mentioned above, the absence of cytotoxicity (a toxic effect on cellular functions) is a prerequisite for the biocompatibility of a material. For the assessment of risk of a compound to human health, the testing of toxicity at the cellular level in vitro has been very useful. The intention is to reduce the complexity of the entire organism generally to a single cell type. Nowadays, the testing of cytotoxic effects of biomaterials or their degradation products in cells in vitro is a major part of the development of new biomaterials.

An approximate procedure for the evaluation of biomaterials in vitro has been developed by ISO (International Organization for Standardization) and is documented in ISO 10993-5 (Biological evaluation of medical devices – Part 5: Tests for cytotoxicity: in vitro methods) [6]. Due to the general applicability of testing cytotoxicity in vitro and the widespread utilization to evaluate large amounts of medical devices and biomaterials, this part of ISO 10993 does not consist of a fixed test protocol. ISO 10993-5 guidelines serve as a starting point for the determination of a scheme that requires incremental decisions and leads to the most appropriate cytotoxicity test. Thus, a basic knowledge about the different test systems and of their specific attributes is absolutely necessary.

ISO 10993-5 deals with three testing categories: (1) testing of extracts (conditions for the extraction are specified), (2) testing by direct cell/material contact, (3) testing by indirect cell/material contact. The choice of one or more of these testing categories depends on the character of the tested material, the area of application within the body, and the task that it has to fulfill.

The cell type selected for cytotoxicity testing is important. The origin of the cells (human vs. other mammalian cells, tissue type from which the cells are isolated, differentiation status) as well as the transformation status (primary vs. permanent cell lines) is dependent on the application. Whereas the choice of the cell origin (e.g., osteoblasts, fibroblasts, endothelial cells etc.) depends on the future application of the tested biomaterial, the choice of the transformation status of the cells depends on the availability of fresh tissue (for primary cultures) and suitable and reproducible cell isolation procedures (see above). In ISO 10993-5 a preference is made for permanent cell lines that come from accredited suppliers (e.g., American Type Culture Collection/ATCC, German Collection of Microorganisms and Cell Cultures/DSMZ). However, ISO 10993-5 also recommends the utilization of primary cells/cell strains and organ-typic cultures if a specific sensitivity is needed and if reproducibility and accuracy can be assured. This is an important point since permanent cell lines often suffer from low sensitivity and non-congruent behavior compared to the respective primary cell culture.

An important factor in cytotoxicity testing is choosing between qualitative or quantitative assays (Table 13.1). A qualitative assay evaluates morphological changes microscopically (morphological changes, vacuolization, detachment, etc.). An example for the validation of morphological changes is given in Fig. 13.4. Here, the f-actin cytoskeleton of human endothelial cells is stained by the fluorescent-labeled actin-binding molecule phalloidin. Whereas non-treated endothelial cells show a peripheral actin ring (negative control) (Fig. 13.4a), cobalt ion (Co²⁺)-treated endothelial cells exhibit a number of f-actin stress fibers and loosened intercellular contacts (Fig. 13.4b). These cytotoxicity validation methods often provide impressive images and may also indicate a number of possible pathomechanisms [7]. However, the significance of these validation methods depends on the cell type, the interpretation of results requires experienced personnel and the results cannot be exactly or easily quantified.

Table 13.1 Aspects of testing cytotoxicity in vitro

Fig. 13.4

F-actin cytoskeleton in human endothelial cells: (a) non-treated endothelial cells and (b) Co²⁺-treated endothelial cells; fluorescence microscopy, magnification 40x

Quantitative cytotoxicity assays on the other hand are mostly indirect methods for the detection of destroyed cellular structures, impairment of cell proliferation, or impaired metabolic cell functions (Table 13.1). Within these categories, there are different possibilities for measurement, and most important is the use of appropriate negative and positive control materials that exhibit the reproducibility of the test system. The negative control should be a material that does not induce a cytotoxic reaction (e.g., cell culture polystyrene). This control is used to show the basal reaction of the cell culture and is necessary since all cells in culture show a variable fraction of dying cells. A positive control must also be used to evaluate the sensitivity of the test system. Therefore, a material or compound should be utilized that shows a reproducible cytotoxic response in the exposed cell culture.

The range of commercially available and accredited cytotoxicity assays is large (Table 13.2). An example for the biochemical quantification of destroyed cellular structures (Table 13.2, item: membrane integrity) is the detection of the cytoplasmic, soluble lactate dehydrogenase (LDH) that is released into the cell culture supernatant when the cell membrane is disrupted. A disruption of the cell membrane generally correlates with an increase in cell death. Therefore, the activity of LDH detected in the cell culture supernatant can be used as a quantitative measurement of cell death in vitro [8]. LDH activity catalyzes the oxidation of lactate to pyruvate in a reversible manner by reducing NAD⁺ to NADH+H⁺.

$$ {\mathrm{NAD}}^{+}+\mathrm{lactate}\overset{\mathrm{LDH}}{\to}\mathrm{pyruvate}+\mathrm{NADH}+{\mathrm{H}}^{+} $$

(13.1)

Table 13.2 Classification of accredited cell viability/cytotoxicity assays (the list is not exhaustive)

The development of reduction equivalents by LDH is used for the LDH-activity assay: the developed NADH+H⁺ reduces (by means of the added enzyme diaphorase) the light yellow tetrazolium salt INT (2-[4-iodophenyl]-3- [4-nitrophenyl]-5-phenyl-2H-tetrazolium-chloride), that is added to the test system, to a red formazan salt. This conversion can be measured photometrically at 492 nm.

$$ \mathrm{NADH}+{\mathrm{H}}^{+}+\mathrm{INT}\kern0.28em \left(\mathrm{yellow}\right)\overset{\mathrm{diaphorase}}{\to }{\mathrm{NAD}}^{+}+\mathrm{formazan}\kern0.28em \left(\mathrm{red}\right) $$

(13.2)

Thus, an increase in the number of dead cells results in an increase of LDH activity in the cell culture supernatant, which is reflected in an increase of absorption at 492 nm after the addition of the tetrazolium salt INT and the enzyme diaphorase.

Other cytotoxicity assays also utilize the color changes of tetrazolium salts to formazan by reduction. Very prominent representatives of these are the tetrazolium salt assays (e.g., MTT, MTS, WST-1, XTT) that indicate the energy metabolic state of the cells. The tetrazolium salts are membrane-permeable. When the salts are added to the cell culture medium, they permeate into the living cells. Here, the light yellow tetrazolium is reduced by the physiologically developing NADH+H⁺ to (mostly) water-soluble, membrane-permeable red or orange formazan products [9, 10] that are released into the cell culture supernatant (since the MTT reagent leads to a water-insoluble product, a further solubilization step is necessary). As in the LDH release assay, this color change can be detected photometrically. In contrast to the conclusion of the LDH assay, an absorbance increase in the tetrazolium salt assays refers to an increase in metabolic activity and thus indirectly in cell viability, a decrease of absorbance is normally an indirect measurement for an impairment of cell viability.

A further important parameter in cytotoxicity testing is the evaluation of the proliferative state, i.e., the capacity of the cells to divide and propagate. In this case, a number of different assays are also available (Table 13.2). The most direct way to obtain information about proliferation is to measure the cell number. This can be done very classically using the Neubauer cell counting chamber. More progressive is the method of cell counting using an electronic cell counter that works by the resistance measurement principle. The advantage of the electronic cell counting systems is that additional information is provided since cell size, volume, etc., are also analyzed and the recorded data are saved electronically. However, both methods are relatively time consuming for adherent cells since the cells have to initially be detached from the cultivation surface primarily by an enzymatic digestion.

Crystal violet staining can be used as an indirect method for the quantification of relative cell number. Crystal violet is a DNA-binding dye. The staining of cells by crystal violet can be performed in a microplate format and analyzed spectrophotometrically. These data indicate the relative number so that the absorbance of the relevant samples can be compared to that of the non-treated controls (i.e., negative control) [11]. A further method of proliferation quantification is the Ki67 assay. The Ki67 antigen is only expressed in the nucleus of cells, actively present in the cell cycle. The detection of this antigen can be used to quantify proliferating cells using an enzyme-linked immunoassay based on a peroxidase staining reaction and subsequent dye quantification by a microplate-spectrophotometer [12].

Proliferation can also be quantified by labeling the newly synthesized DNA by [³H]-thymidine or bromodeoxyuridine (BrdU). Both compounds are incorporated into newly synthesized DNA strands of actively proliferating cells during the S-phase of the cell cycle. Whereas the radioactive thymidine-incorporation is measured by scintillation counting of autoradiography, the BrdU-incorporation is detected immunocytochemically. The [³H]-thymidine-incorporation assay has several limitations, primarily in the handling and disposal of radioisotopes and the necessity for specialized equipment.

A comparison of two assays that reflect the proliferative state is shown in Fig. 13.5. Human endothelial cells were exposed to different concentrations of the metal ion salt CoCl₂ (Co²⁺ ions are corrosion products of CoCr-alloys). Low concentrations (0.1 mM) induced only slight changes of the relative cell number detected by crystal violet staining; higher concentrations of CoCl₂ (0.7 mM) induced a reduction of more than 20%. However, the results of the proliferation-inhibiting effects of CoCl₂ are more pronounced, when the proliferative state is examined by the Ki67 assay (0.1 mM: more than 10%; 0.7 mM: nearly 55%). The differences between these assays occur since different parameters of proliferation are being examined; crystal violet stains the cell nuclei since it binds to DNA and is thus an indirect method for the quantification of the relative cell number, whereas the Ki67 assay detects a protein that is only expressed during the active cell cycle. Thus, the Ki67 assay reflects a parameter that is mechanistically ranged ahead of the cell propagation and might therefore be more sensitive than the crystal violet staining . Since crystal violet staining is much easier to perform than the complex Ki67 assay, the selection of the assay depends on the individual case and has to be carefully considered.

Fig. 13.5

Detection of the proliferative state in human endothelial cells after exposure to metal ions (crystal violet staining vs. Ki67-staining, exposure 24 h, untreated control – negative control – set as 100%)

There are different mechanisms of cell death which generally can be divided into necrosis and apoptosis. Whereas necrosis is also called the “accidental” cell death, apoptosis is referred to as “programmed” cell death. This original classification depends on the different morphological, biochemical, and molecular hallmarks that are expressed during cellular breakdown.

Necrosis is caused by extreme conditions (toxins, heat, radiation, trauma, oxygen deficiency, etc.). These physical or chemical stimuli can lead to the lethal disruption of cell structure and generally affect a number of cells simultaneously. In necrosis the cells swell, the plasma membrane disintegrates, and intracellular compounds are released. The release of intracellular material leads to pro-inflammatory processes surrounding the dying cells.

In contrast to necrosis, apoptotic cells are characterized by the condensation of the cytoplasm and the nuclei, so that affected cells shrink. In the course of apoptosis nuclear fragmentation and cleavage of chromosomal DNA into internucleosomal fragments occurs. The degradation of DNA in the nuclei of apoptotic cells occurs by a number of ways following the activation of executioner enzymes, the caspases. Characteristic is also the packaging of the deceased cells into apoptotic bodies without plasma membrane breakdown. These apoptotic bodies can be recognized and removed by phagocytic cells. Therefore, apoptosis is also notable for the absence of inflammation around the dying cells. Currently this original classification of necrosis (as the accidental cell death) and apoptosis (as programmed cell death) is extended to a number of alternative cell death styles. There is growing evidence that, besides apoptosis, autophagic and necrotic forms of cell degeneration may be programmed [13].

Apoptosis-typical hallmarks can be analyzed in vitro and a number of different test systems are available (see Table 13.2). The nuclear condensation and fragmentation is easily detected by nuclear staining (e.g., by Hoechst 33342, DAPI). An example of different stages of apoptosis-typical nuclear break-up is shown in Fig. 13.6a. Non-affected nuclei of endothelial cells possess an oval, regular shape (1); during the course of apoptosis the nuclei condense (2) and are fragmented (3); at a late stage of apoptosis the nuclear fragments come apart (4).

Fig. 13.6

Detection methods of apoptosis. (a) Nuclear staining of H₂O₂-treated human endothelial cells (0.7 mM, 24 h, staining with Hoechst 33342; different stages of the nuclear degradation: (1) non-affected nucleus, (2) nuclear condensation, (3) nuclear fragmentation, (4) nuclear break-up); (b) DNA ladder formation in human endothelial cells after metal ion-treatment (agarose electrophoretic separated nucleic acid preparations of: lane 1 – non-treated control cells, lane 2 – non-treated control cells after RNase-treatment, lane 3 – Co²⁺-treated cells, lane 4 – Co²⁺-treated cells after RNase-treatment; arrowheads mark the distinct “steps of the ladder”); (c) TUNEL staining in sporadically occurring apoptosis in human endothelial cells (TUNEL positive, nuclear staining, digital overlay of the TUNEL and the nuclear staining, fluorescence microscopy); (d) detection of active caspase-3 in cobalt-particle-treated endothelial cells (digital overlay of caspase-3 and nuclear staining, fluorescence microscopy)

A further method in the detection of apoptosis is the analysis of fragments of chromosomal DNA. Characteristic in apoptosis is a specific form of DNA degradation in which the genome is cleaved at internucleosomal sites, generating a “ladder” of discretely sized DNA fragments when analyzed by agarose gel electrophoresis (Fig. 13.6b). The nucleic acid preparations contain RNA in addition to the DNA of interest. Since the RNA masks the DNA-ladder (Fig. 13.6b, lane 1 and 3), removal of RNA with a specified enzyme (RNase) is necessary (Fig. 13.6b, lanes 2 and 4). After the treatment of endothelial cells with high concentrations of Co²⁺, a DNA-ladder is detectable (Fig. 13.6b, lane 4). This apoptosis-typical fragmentation can also be detected by the so-called TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) . Therefore, the low-molecular-weight DNA fragments as well as single strand breaks (“nicks”) in high-molecular-weight DNA are labeled at the apoptosis-typical breaks with modified nucleotides in an enzymatic reaction. Incorporated nucleotides are detected by fluorescence or light microscopy depending on the modifications of the incorporated nucleotides (Fig. 13.6c: fluorescently labeled). The TUNEL-positive endothelial cell nucleus (green) in Fig. 13.6c shows simultaneously fragmented nuclear material.

The core effectors for the cellular breakdown during apoptosis are a family of proteolytic enzymes, the caspases. Caspases exist as latent precursors in most nucleated mammalian cells. The caspase precursors are activated by cleavage or proximity-induced dimerization. One of the executioner caspases is caspase-3, which mediates the apoptotic cascade. The active enzyme can be detected in the apoptotic cells by immunostaining (Fig. 13.6d). Here, the specific binding of an antibody against the active caspase-3 (not the precursor) is detected in cobalt-particle-treated endothelial cells by labeling with a fluorescent secondary antibody and a subsequent fluorescence microscopic analysis.

Thus, there are numerous possibilities for testing cytotoxicity in vitro. The significance of these assays often depends on the choice of the assay and the mechanisms that are influenced by the tested compound. Therefore, the results achieved by a special cytotoxicity test have to be evaluated critically. To exclude misinterpretations of the assays, the utilization of at least two different tests that are based on different test methods is highly recommended. Thus, if the cytotoxicity assays are utilized correctly, these in vitro test systems might be helpful for the pre-selection of compounds and materials and lead to the reduction of animal studies.

13.3.2 Hemocompatibility

Hemocompatibility, the ability of a material to perform in contact with blood, is one of the most complex fields in biocompatibility testing. This is due to the large number of interacting cell types and factors which are addressed during the cascade of activation steps (see below). All materials implanted come into direct contact with tissues and thus into direct contact with blood. ISO 10993 requires an evaluation of hemocompatibility for any medical device that has contact with circulating blood, directly or indirectly, during routine use. In other words, testing of hemocompatibility is relevant for all devices and implants that come into large-area contact with the circulatory system. Thus, in addition to heart valves, vascular prostheses, and vascular stents, most forms of extracorporeal devices involve a blood-biomaterial interaction. Guidelines for such evaluations and the respective devices are found in ISO 10993-4 (Biological evaluation of medical devices – Part 4: Selection of tests for interactions with blood) [14].

The human blood consists of cellular components (40–45%) and the plasma (55–60%). Red blood cells (erythrocytes), white blood cells (leukocytes), and the platelets (thrombocytes) constitute the cellular part of the blood (Fig. 13.7a). The blood circulates within the vascular system and fulfils a number of different functions, including immune responses and tissue repair as well as oxygen and nutrient supply and the disposal of metabolites. Because of the broad range and the critical nature of these functions, any source of cytotoxicity to blood cells can cause significant harm. For example, the breakdown of erythrocytes (called hemolysis), which can be induced by a material or a toxin or might result from mechanical damage, directly impairs the ability of the circulatory system to provide the body’s tissues with oxygen. Moreover, adverse interactions with leukocytes can impair the body’s ability to repel invading pathogens efficiently. Furthermore, blood contains several soluble multi-component protein systems that systematically interact in various ways to perform critical functions. For example, the complement system participates in inflammatory reactions and facilitates removal of invading pathogens. The clotting cascade operates to initiate blood coagulation, thereby preventing excessive loss of fluid, and facilitates tissue repair (Fig. 13.7b). The impairment of and other cascade systems (e.g., kinin system, fibrinolytic system) can have significant adverse effects on the body.

Fig. 13.7

Human blood. (a) Composition and function of blood components in humans (staining of blood cells: panoptical staining according to Pappenheim), (b) blood coagulation, human blood cells entrapped in a fibrin network (scanning electron microscopy)

The above-mentioned clotting cascade consists of two sub-pathways that lead to the formation of a fibrin clot: the intrinsic and the extrinsic system. These systems are initiated by distinct mechanisms but work together for a common result, the blood clot. In Fig. 13.8, a highly simplified depiction of the cascade is shown. Only the major components are shown; a large number of interacting factors are not included. Beside the process of blood clotting, the subsequent dissolution of the clot following repair of the injured tissue is also an important aspect (for more information, see: http://www.labtestsonline.org/images/coag_cascade.pdf).

Fig. 13.8

Diagram of the clotting cascade: the intrinsic system is initiated when contact is made between blood and the cell-denuded vessel wall. The extrinsic system is initiated by the release of the so-called tissue factor by injured cells. The two pathways converge at the activation of factor X. The active factor X (called Xa) activates prothrombin to thrombin. Thrombin ultimately converts fibrinogen to fibrin and activates a fibrin-cross-linking factor (factor XIII). The developing fibrin polymers are weaved into the thrombocyte aggregations, forming a stable fibrin clot

There are three different approaches for testing hemocompatibility: (a) in vivo (in animals), (b) ex vivo (test systems that shunt blood directly from a human subject or test animal into a test chamber that is located outside the body), and (c) in vitro (test systems with fresh, usually anticoagulant-treated blood samples). The types of tests required by ISO 10993-4 depend on the blood contact category of the device or material. In vivo experiments cannot focus on a single parameter of hemocompatibility as the different blood cells/components and their versatile functions act together. These complex interactions cannot be significantly mimicked in vitro. On the other hand, the in vitro experiments allow a detailed examination of single factors involved in hemocompatibility.

The methods used for testing hemocompatibility are classified into different categories. These categories are (1) thrombosis, (2) coagulation, (3) platelets, (4) hematology, and (5) immunology. This classification is based on the different aspects and detection methods for testing hemocompatibility.

• 1. Thrombosis: Thrombosis is an in vivo phenomenon resulting in the partial or complete occlusion of the vessel or device following blood coagulation. A detached and thus moving thrombus is called an embolus. When an embolus becomes lodged in a blood vessel, it is called embolism. Depending on the affected tissue, an embolism might induce a severe life-threatening situation, such as in the lungs or brain.

  In most applications, non-thrombogenic behavior of the materials is required. The characterization of the thrombogenicity of a material includes the microscopic analysis (light, fluorescence, or electron microscopic) of adherent platelets, leukocytes, aggregates, erythrocytes, fibrin, etc., by in vitro studies. The adsorption of proteins to the material surface and the interactions between different proteins and the material surface has a crucial effect on the thrombogenicity of the material. Materials that do not induce adhesion of thrombocytes by protein adsorption of plasma proteins or by interaction of blood compounds on contacting the material are referred to as non-thrombogenic .

• 2. Coagulation : A number of different parameters are involved in the induction of coagulation in vivo. A simple method to examine hemocompatibility of a material is the blood coagulation test: Blood is brought into contact with the surface and the coagulation time is determined. The time frame for coagulation determines if a material activates or impairs coagulation. Also, different parameters of the coagulation cascade can be detected in vitro and can be quantified. Among these are the detection of TAT (thrombin-antithrombin III-complex), and some key enzymes of the coagulation cascade such as Xa (activated factor X), XIIa (activated factor XII) utilized for the evaluation of the coagulative capacity of a material. The so-called “partial thromboplastin time” (PTT) is used to assess the extent of activation of the intrinsic coagulation pathway. Therefore quickly thawed, pooled, citrated, fresh human plasma is used and the material is brought into contact with this plasma (37 °C). The plasma is immediately removed from the material samples and placed on ice. Afterward the plasma is incubated with CaCl₂ and cephalin (phospholipids derived from rabbit brain). The re-calcification together with the addition of cephalin induces the clotting. The plasma samples are monitored until clot formation is seen. The time that is needed for clot formation is recorded and gives the PTT.

• 3. Platelets : The activation of platelets can be confirmed by the determination of the number of material-adherent platelets and their degree of spreading. Furthermore, the surface expression or secretion of the platelet activation markers CD62, CD63, and platelet factor 4 (PF4) can be useful for the evaluation of the activation status.

• 4. Hematology : Testing hemolysis (i.e., measurement of erythrocyte fragility) in contact with materials or their extraction products is a significant screening method used in hematology. The hemolysis tests are performed using anticoagulated blood that is brought into direct contact with the materials or co-incubated with sample extracts. After incubation at 37 °C, the blood cells are removed by centrifugation and the supernatant is analyzed spectroscopically at 540 nm for the release of hemoglobin. These data are compared to the corresponding positive (e.g., deionized water that will cause bursting of all erythrocytes) and negative controls (e.g., phosphate-buffered saline solution). The higher the absorption at 540 nm, the higher the hemolytic properties of a material or its extraction products. A further aspect in the hematology category is the evaluation of leukocyte total number. Leukocyte counting is carried out on samples of whole blood after in vitro contact with the material.

• 5. Immunology : The complement system is part of the immune response in the blood. It consists of a series of approximately 25 proteins that work to “complement” the activity of antibodies in destroying bacteria. Therefore, the unintended activation of this system by a material could induce a chronic triggering of complement. Complement proteins circulate in the blood in an inactive form. When the first complement member is triggered, a cascade of activation steps is initiated. Generally the activation of the complement system is possible by two different pathways: (1) the so-called classical pathway that is triggered by bacteria to which antibodies are bound and (2) the alternative pathway, triggered by bacterial components, other microorganisms and material surfaces without the assistance of an antibody-binding (Fig. 13.9).

Fig. 13.9

A schematic drawing of the complement system (highly simplified): The classical pathway is induced by bacteria to which antibodies have bound. The alternative pathway is activated by bacterial components, microorganisms, and material surfaces without antibody-binding

A panel of complement proteins is available that can be utilized for the detection of complement activation in hemocompatibility testing. These different proteins encompass the various complement activation pathways. Among these are the proteins C3a, C5a, TCC (terminal complement complex), C4d, and SC5b-9. Elevated levels of these complement components indicate the activation of the complement system. Usually these factors are detected and quantified by enzyme immunoassays.

However, although a number of different test systems are available, the evaluation of hemocompatibility is unique in that the design of the final device can determine its performance. Thus one needs to consider specific surface properties, stiffness, shape, etc., which may cause deleterious effects to the blood elements in addition to the accepted hemocompatibility tests.

13.3.3 Hypersensitivity/Allergic Responses

Allergies are the most common disorder of immunity , affecting more than 25% of the Western World population. The term “allergy” is used to describe the body’s response to a substance which is harmless in itself but results in an immune response and a reaction that causes symptoms and disease in a predisposed person. The symptoms of an allergy may include everything from a runny nose, itchy eyes, and skin rash to life-threatening complications such as asthmatic attacks and anaphylactic shock.

Allergic reactions are usually classified by the type of tissue damage that they cause. Some allergic reactions produce more than one type of tissue damage, and other reactions involve antigen-specific lymphocytes (a subclass of the white blood cells/leukocytes) rather than antibodies. There are four recognized types of allergic reactions based on different subclasses of antibodies (or immunoglobulins, Ig) and cell types that interact with each other (see also Table 13.3): (1) Type I: Anaphylaxis (immediate-type hypersensitivity) – anaphylaxis occurs when a specific allergen combines and cross-links antibodies (i.e., the Ig-subclass IgE) affixed to so-called mast cells; anaphylactic disturbances occur only if the body had previous contact to the allergen. Shortly after re-exposure to the allergen the reaction occurs (within minutes, e.g., hay fever), (2) Type II: Cytotoxic Reactions – These are antigen-antibody reactions usually mediated by IgG and IgM antibodies at cell surfaces that result in the lysis of blood cells (red cells, white cells, and platelets) due to the activation of complement (well-known incidences in this category are blood transfusion reactions), (3) Type III: Immune complex reactions – These are antigen-antibody reactions mediated by IgG and IgM. The reactions produce toxic antigen-immunoglobulin complexes that can circulate in the blood. In these cases, the complexes cause damage by adhering to blood vessel walls and initiating an inflammatory response (such as vasculitis), (4) Type IV: Delayed-type hypersensitivity or cell-mediated immunity reactions – These are reactions between antigens and sensitized antigen-specific T lymphocytes (subclass of leukocytes), but not antibodies. The reaction subsequently releases inflammatory and toxic substances, and lymphokines that attract other leukocytes. The induction of hypersensitivity is called sensitization. Clinical examples include transplant rejection, and contact dermatitis in response to metals such as chromium, cobalt and nickel. Type I and Type IV reactions are the most common.

Table 13.3 Types of allergic response

Most allergens are proteins. However, non-protein “allergens” (in quotes since this term is usually reserved for the inducers of IgE reactions, therefore better: sensitizer) also exist. These non-protein sensitizers include nickel, chromium, cobalt, gold, palladium ions, and other metals and metal-containing substances [15]. Due to their small size these metals referred to as “haptens,” in that they must bind to a protein in order to become allergenic [16]. Furthermore, acrylic compounds [17], epoxy moieties [18], and Teflon implants, and a few mineral substances are shown to induce hypersensitivity [19]. Most of these non-protein compounds do not cause Ig reactions. Thus, most allergic reactions induced by implanted artificial materials are Type IV reactions, which belong to the cell-mediated immunity reactions and do not involve Ig.

In the early 1970s, it was observed that metallic materials used in bone surgery could cause the development or an increase in contact dermatitis [20, 21]. From this it was concluded that contact hypersensitivity to metals might explain some orthopedic complications. This was supported by the fact that in patients developing such complications after implantation of metallic devices, a positive contact dermatitis (detected by the so-called patch test) with metals was more frequent than in other patients [22]. Hypersensitivity reactions usually occur as a result of repeated or prolonged contact with sensitizing compounds (such as with an implant). Biomaterials and devices that cause sensitization reactions do so by means of the extractable compounds.

To exclude a hypersensitivity-inducing potential of materials, different test systems have been developed. Methods for performing such tests are described in ISO 10993-10 (Biological evaluation of medical devices – Part 10: Tests for irritation and delayed-type hypersensitivity) [23]. Due to the complexity of reactions in sensitization, appropriate in vitro methods are not available. However, there are different methods for testing the hypersensitivity/sensitization potential of materials in animals. Most of those reactions to biomaterials are Type IV reactions (cell-mediated immunity) and are tested on the skin of laboratory animals. Dermal hypersensitivity reactions in laboratory animals are marked by redness and swelling. Biomaterials and other device materials are tested for the presence of sensitizing chemicals using guinea pigs, a species known to be nearly as responsive to dermal sensitizers as humans.

The repeated-patch test method (also called Buehler-test) involves exposing the skin of guinea pigs directly to the test material under occlusive dressings for a minimum of 6 h [24]. This procedure is repeated three times a week for 3 weeks (induction phase). Following a two-week rest to allow for the development of a delayed response (recovery phase), the animals are exposed to a patch of the biomaterial (final exposure). The repeated-patch model is used primarily for topical devices such as dermal electrodes, since this method of applying test materials to the animals simulates clinical use.

A further test system that is based on guinea pigs is the maximization test method (called guinea pig maximization test/GPMT, described by Magnusson and Kligman) [25]. In this case extracts of the test material are prepared in saline and vegetable oil and guinea pigs are exposed repeatedly to the extracts. The guinea pigs are first injected with an extract along with an adjuvant intended to enhance an immune response, and then receive a topical application. Following a two-week recovery phase, the animals are covered with a topical patch containing the extract. Generally considered more sensitive than the repeated-patch model, the maximization test is used for device materials that will contact areas other than skin. The use of both a saline extract and an oil extract simulates extraction by body fluids and by intravenous liquids and other pharmaceutical products that first contact the device and then the patient. Both guinea pig sensitization tests require nearly 2 months and thus take the longest time to complete of all the biocompatibility tests described in the 10,993 standards. In both techniques, the area of the challenge patches is examined for reactions (redness and swelling) that are not present in negative-control animals. In addition, known sensitizing chemicals are used periodically to validate the methods.

Human allergens generally sensitize guinea pigs although this animal is known to be less sensitive than the human. However, some substances known to be human sensitizers have failed to sensitize guinea pigs. These include some metals, drugs, latex-based products, and macromolecular proteins such as collagen [26]. Thus, these methods are not perfect in their ability to detect weak sensitizers or chemicals, and they do not detect chemicals that act as adjuvant, enhancing an immune response to other chemicals to which a patient might be exposed. To overcome these problems, additional test methods have been developed. One of these tests is the local lymph node assay (LLNA) in mice [27]. The LLNA has been validated for pharmaceutical and agricultural chemicals. In contrast to the classical sensitization tests, no complete sensitization has to be caused in the animal. In the LLNA, the ears of mice are treated with the extract/chemical, and then the surrounding lymph nodes are examined for a lymphocyte proliferative response as demonstrated by the accumulation of radio-labeled DNA constituents (uptake of the labeled constituent if there is a lymphocyte activity induced by the tested compound). This testing method is also mentioned in the Annex of ISO 10993-10 [23]. However, one should keep in mind that no single test method will adequately identify all substances with a potential for sensitizing human skin and which is relevant for all substances. Much effort has also been devoted to developing in vitro alternatives to the guinea pig sensitization models, but thus far no suitable replacements have been identified.

13.3.4 Genotoxicity

A further striking issue of biocompatibility testing is the detection of genotoxicity. Genotoxicity is defined as any toxic modification of the structure or function of the genetic material, the DNA. DNA contains the instructions for all cellular activities. These instructions are encoded within sections of DNA devoted to each specific function, the genes. Specific units consisting of many genes are called chromosomes. The number of chromosomes is defined for each species (e.g., 46 chromosomes in humans, 78 in dogs). An alteration in any part of the DNA that results in permanent changes in cell function is called a mutation. The agents that cause such mutations are known as genotoxins. The rationale for the testing of a biomaterial’s genotoxic potential is the concern for the ability of a biomaterial to induce cell death based on genetic mutation or to initiate a malignant transformation (i.e., cancer). DNA modifications can have permanent inheritable effects on genes and on chromosomal structure or number (chromosomal aberration). DNA single- or double-strand breaks, base modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions which can lead to cell death or result in mutational events which in turn may initiate cancer [28].

There are different levels of genetic variations that can occur as specified above. These different levels can be detected in vitro and in vivo (Table 13.4). Again, no single assay is capable of detecting all types of DNA damage; therefore, the utilization of different test systems is generally recommended. In vitro tests in each category can be conducted using microorganisms or mammalian cells and in vivo tests systems (mostly with rodents) are also available which are usually performed subsequently to the in vitro assays. In the following, a number of established tests for genotoxicity detection are described.

Table 13.4 In vitro and in/ex vivo test systems in genotoxicity detection approximately assigned to the induced defect and the class of test method

A gene mutation test can be used for the detection of so-called point mutations. Point mutations are characterized by minor alterations of the DNA molecule (e.g., substitution, addition or deletion of base pairs) and these mutations can be detected by the so-called reverse mutation test (previously known as the Ames test) [29, 30]. This assay is commonly employed as an initial screening for genotoxic activity and, in particular, for point mutation-inducing activity. The principle of the test system is based on genetically modified bacterial cells that are deficient in histidine biosynthesis. These bacterial cells do not have the ability to grow without the supplementation of histidine. If these bacteria are exposed to the test substance and regain the possibility to grow without the addition of histidine, this indicates that a material−/compound-induced genetic transformation most likely occurred.

A test method that can be utilized in vitro as well as ex vivo is the so-called unscheduled DNA-synthesis test (UDS). UDS is the term used to describe the replication of DNA during the repair of DNA damage and as such is distinct from the DNA replication which is confined to cell proliferation. The test is based on the incorporation of radioactive-labeled thymidine into the DNA of non-dividing mammalian cells [31]. Thus, the higher the amount of incorporated radiolabeled thymidine, the higher the DNA repair activity and consequently the higher the genotoxicity of the tested compound. A further assay that detects DNA strand breaks is the Comet assay (also known as single cell gel electrophoresis). In case of a severe damage of DNA, an increase of DNA fragments occur. These DNA fragments can be detected and visualized. The name “Comet assay” is based on the appearance of a characteristic streak similar to the tail of a comet. This assay can be utilized in vitro as well as in vivo [32].

Further detection methods for mutagenesis are the in vitro mammalian cell gene mutation tests (to which the well-known mouse lymphoma assay/MLA belongs). The assays of this category work by a comparable principle in that heterozygous deficient cell lines (e.g., the mouse lymphoma cell line L5178Y) are utilized. These cell lines have heterozygous deficiencies in specific enzymes due to mutations. For example, L5178Y cells have deficiency in thymidine kinase (TK, the heterozygous mutated L5178Y cell line is denoted as tk^+/−). The principle of this test method is based on the fact that tk^+/+ and tk^+/− cells arrest their growth totally in the presence of the pyrimidine analogue trifluorothymidine (TFT), whereas the tk^−/− cells are not susceptible to the cytotoxic effects of TFT. Thus, homozygous mutant cells are able to survive and proliferate in the presence of the selective agent TFT, whereas non-mutant and heterozygous cells (tk^+/+ and tk^+/−), which contain TK, are not [33]. For testing the mutagenic activity of a compound or its extract, respectively, known numbers of the heterozygous tk^+/− cells are exposed for a suitable period of time (with and without the selective agent TFT) and subcultivated to determine cytotoxicity and to allow phenotypic expression of potentially induced mutations. After approximately 2 weeks colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium. Thus, the higher the colony formation of the tk^+/− cell strain in the presence of the test compound and the selective agent, the higher the mutagenicity of the compound. The range of genetic mutations detected with the MLA is broad: the MLA measures either gene mutations or heritable chromosomal events, including genetic events associated with carcinogenesis [34].

A further possibility for testing genotoxicity in vitro is the chromosome aberration test (CAT) . The purpose of this method is to identify compounds that cause structural chromosome aberrations in cultured mammalian (human) cells. Chromosome aberrations and related events appear to cause alterations in genes regulating the cell cycle of somatic cells and are thus involved in the induction of cancer in humans and experimental animals. The test principle is based on the exposure of accepted cell cultures (e.g., cell lines, strains, or primary cells may be used) to the test compound. At predetermined intervals the cells are treated with a cell cycle-arresting substance (i.e., colchicin, a poison that does not allow the last steps of the cell cycle to proceed; colchicin arrests the cells in metaphase). Since the chromosomes are highly condensed in the metaphase, a microscopic analysis for the occurrence of chromosome aberrations can be performed.

Interestingly, both the CAT and the MLA often yield similar results. Therefore the utilization of both in the course of biomaterial testing may not be reasonable. Besides these above-mentioned in vitro test systems, a number of other assays are utilized to analyze genotoxic and/or mutagenic effects. One example is the so-called sister chromatid exchange assay (SCE) [35]. This assay examines the reciprocal exchanges between two sister chromatids of a duplicating chromosome. Through the use of special staining techniques, the sister chromatids display differential staining in case of an exchange, permitting enumeration of SCE. The molecular basis of these mutations remains unknown, although it is presumed to involve complex DNA breakage and reunion processes [36]. The SCE assay is usually performed on human peripheral blood lymphocytes.

A genotoxicity testing method that is intended for in vivo application is the mammalian erythrocyte micronucleus test. Micronuclei are small nuclei, separated from and additional to the main nuclei of the cells (1/5 to 1/20 the size of the nucleus). The micronuclei develop during cell division by lagging chromosome fragments or whole chromosomes by chromosome breaks or dysfunction of the spindle apparatus. For this test method mammalian erythrocytes are utilized. When the erythrocyte progenitors (the erythroblasts) develop into polychromatic erythrocytes (intermediate stage in the development of erythrocytes), the main nucleus is extruded but the already present micronuclei remain. Thus, an increase in the number of micronucleated erythrocytes is an indication for a genotoxic potential of the compound tested [37]. This method is especially relevant for assessing the mutagenic hazard of the test compound. Cells derived from bone marrow or peripheral blood can be used for this analysis. Usually mice are used for testing the effects in peripheral blood. For testing in bone marrow both mice and rats are recommended.

13.3.5 Tissue-Specific Aspects of Biocompatibility Testing

The potentially broad applicability of biomaterials requires that the construction of the medical device and the testing scheme has to be adapted to the intended implantation site. As we have seen before, the problems in hemocompatibility testing deviate strongly from those of tissue compatibility for example. Therefore, as example, materials that are intended for utilization as a vascular implant must be tested by different biocompatibility tests than a material that is produced for a metallic hip prosthesis. The reasons for this are due to the different cell types and protein subsets that the material comes into contact with. However, different tissues also show large discrepancies regarding their requirements, for example soft tissue and hard tissue (bone) implants: soft tissue-implants destined for utilization in extensive injuries such as deep burns and lacerations need to permit filling of large defects and allow the ingrowth of adequate cells and blood vessel formation. In contrast to this, the routinely utilized metallic implants are not intended for cellular ingrowth. Furthermore, materials for soft tissue implants have to be soft and flexible, similar to the tissue that is to be replaced, whereas the materials for hard tissue replacement must fulfill the mechanical tasks of bones and withstand high mechanical forces. Mechanical forces are an important aspect in the field of biocompatibility, and due to the intensity of these forces especially in the field of osteocompatibility and odontocompatibility (i.e., the material’s compatibility to perform with bone and tooth tissues).

Some similarities in the response of an implant are exhibited by all tissue types. The initial response after the insertion of any biomaterial leads to an injury that is accompanied by blood clotting, inflammation, the recruitment of inflammation-involved cells and afterward by the development of the provisional tissue (the so-called granulation tissue). These first steps occur regardless of the materials and the implantation site. However, the character and the course of the following repair processes depend once again on the type of tissue and the localization of the implant.

13.4 Animal Experimentation

Animal studies are used to determine the in vivo compatibility of biomaterials/medical devices and are performed prior to human testing to help predict the human response. Animal studies should only be performed when the information required is essential to characterize the test material and when no suitable scientifically validated in vitro test method is available. Furthermore, animal experiments shall not be performed before appropriate in vitro tests have been carried out, and the results have been evaluated.

In the course of the already-described biocompatibility issues, different in vivo assays have also been mentioned. This includes tests for sensitization, irritation, intracutaneous reactivity, subacute, acute and chronic toxicity, hemocompatibility, genotoxicity, implantation, carcinogenicity, reproductive and developmental toxicity, and biodegradation. The selection and design of animal tests should be appropriate and address the specific scientific objectives of the study. Furthermore, minimizing the pain, suffering, distress, or lasting harm that might be inflicted on the test animals is a fundamental requirement of all regulating organizations (e.g., [38]). These fundamental requirements have to be applied and are regulated by respective supervisory authorities. The type of authority and the specific needs for the application of animal studies depends on each country. The responsible investigator has to verify that animal care is appropriate and medical care is guaranteed prior to, during, and after the experiments.

The use of appropriate animal models is an important consideration in the safety evaluation of medical devices. Different animal models are typically used for specific applications. Examples for subject-bound animal models are the sensitization test in guinea pigs (see Sect. 13.3.3), the testing of irritation and intracutaneous reactivity, intramuscular implantation and pyrogenicity in rabbits, and subacute, acute, and chronic toxicity in mice and rats. In addition, the in vivo assessment of the functionality of medical devices is also being performed in sheep, dogs, pigs, and calves. Sheep are commonly used for the evaluation of heart valves since the sizes are nearly proportional to human dimensions. Furthermore, pigs are often used for the in vivo testing of vascular grafts and artificial hearts are mainly tested in calves. Because of the homology to humans, the most appropriate animal models are primates. However, ethical concerns regarding the utilization of intelligent animals and the costs of purchasing and maintaining such primate colonies are substantial.

The acquisition of the animal experimental data depends on the type of test: for example the sensitization in guinea pigs can be detected on the skin by redness and swelling, the pyrogenicity (fever inducing capacity) of a material is usually detected by rectal temperature control in rabbits. The analyses of body tissues that are exposed to materials are more complex and must be analyzed by histological means. For example, the tissues are analyzed for the occurrence of inflammatory cell populations. It should be noted, however, that whereas a positive response to a material (i.e., a pathological reaction in the animal) can be interpreted relatively easily, the absence of a response in the animal does not necessarily prove compatibility in humans. In conclusion, animal experiments are expensive, complex, and difficult to interpret and require experienced personnel and expertise. However, if performed properly, they are useful and necessary prior to human trials.

13.5 Alternatives to Animal Experimentation

As mentioned above, the demand for the utilization of alternatives to animal studies is a high priority. All in vitro tests addressed in the previous sections are approved alternatives to animal testing since they give information about possible cellular impairment induced by the material or its extraction product prior to animal testing. However, these in vitro test systems are based on the utilization of one cell type (bacterial or mammalian) and do not reflect the complexity of the multicellular/multitissue-organism, the human body.

More recently, more complex in vitro test systems working with multiple (human) cell types have been added as alternatives. These are, to a degree, under consideration and partially validated and accepted by different regulating organizations. Examples for these are the human skin equivalent tests (e.g., EpiDerm™, EpiSkin™). These tests consist of normal, human skin cells, which are cultured to form a multi-layered model of human skin. The significance of these human skin equivalent tests has been established through validation studies by the European Centre for the Validation of Alternative Methods (ECVAM) in Ispra, Italy. These three-dimensional models allow the application of the test material directly to the “artificial skin” surface and permit the assessment of dermal toxicity via different parameters (cytotoxicity, histological examination, detection of inflammatory mediators, etc.) [39].

Medical devices that come into direct or indirect contact with blood must be examined for pyrogens. Pyrogens are defined as fever-inducing substances. Pyrogens can be components of the cell wall of Gram-negative bacteria (endotoxins) or chemical compounds. In the past, pyrogen testing was always performed in rabbits (see Sect. 13.4). Fluid extracts of the test materials were administered intravenously to rabbits, and afterward the animals´ rectal temperature was monitored over the course of several hours. A significant rise in temperature indicated the presence of pyrogens. However, this test is subjected to well-documented drawbacks, including marked species and strain differences in sensitivity [40]. In addition to this conventional pyrogen test system , alternative methods are currently available. Alternatively to the rabbit test, the limulus amoebocyte lysate (LAL) test is used. The LAL test is based on the blood cells of the Horseshoe crab Limulus polyphemus. It has been shown that the blood lysates of Limulus exhibit clotting after contact to the above-mentioned endotoxin. Thus, the LAL test detects only one specific pyrogen, the endotoxin. As a replacement method for pyrogen animal studies, the so-called human whole-blood test (also in vitro pyrogen test (IPT)) has been developed and validated. The materials to be examined are incubated in human whole blood containing a number of different, relevant cell types (see Sect. 13.3.2). The blood is then examined for its pro-inflammatory response, the release of the pro-inflammatory cytokine IL-1β by an enzyme-linked immuno-assay (ELISA). Beside the aspect of replacement of animal studies, the test method possesses further advantages: i.e., that the system is of human origin and can be used for all potential pyrogens (not only bacterial cell wall components like in the LAL test) [40, 41].

Other complex in vitro models utilizing human cells are accepted for research purposes but have not yet been certified for the testing of medical devices. An example of such a model is shown in Fig. 13.10. This in vitro model examines the capability of human endothelial cells (the cells that line the blood vessels) to form new blood vessels, a process that is called angiogenesis. Angiogenesis is a fundamental requirement in wound healing. This model system of angiogenesis in vitro makes use of human endothelial cells suspended in a matrix of extracellular proteins (fibrin and collagen) resembling the wound healing matrix. After the addition of specific angiogenesis-inducing factors in vitro capillaries evolve within a few days (Fig. 13.10a). If performed on a titanium surface (commercial pure titanium/cpTi), the cells develop the normal capillary phenotype (Fig. 13.10b). Cells on a Co28Cr6Mo-alloy exhibit striking deviations compared to the other two surfaces: the formation of in vitro capillaries is completely inhibited, whereas the cell viability is not impaired (Fig. 13.10c). Since angiogenesis is fundamental in wound healing, the impairment of the angiogenic capacity of endothelial cells by a material could lead to the disturbance of wound healing and aseptic implant loosening [42].

Fig. 13.10

A model of angiogenesis for use in vitro for biocompatibility studies. (a) Human endothelial cells embedded into a three-dimensional matrix consisting of fibrin and collagen. After stimulation with angiogenesis-inducing factors, the cells form in vitro capillaries within 5--7 days; (b) angiogenic-stimulated endothelial cells on a cpTi-surface; (c) endothelial cells on a Co28Cr6Mo-alloy exhibit striking deviations compared to the other two surfaces (digital overlay of a vital and a nuclear staining)

Other complex in vitro models are also currently under validation for approval in the biocompatibility safety testing program. With increasing knowledge in this field and concerted, stringent validation processes the number of relevant approved alternatives to animal testing should increase within the next years. In conclusion, these alternative methods are beneficial for the replacement and reduction of animal testing and are often superior to animal studies. However, despite progress and considerable effort in this direction, currently no satisfactory in vitro test system has been devised to completely eliminate the requirement for in vivo testing.