Biocompatibility and cytotoxicity

As mentioned before, synthetic biodegradable polymers have a leading position in use as medical devices material. For these purposes there are particularly important criteria they have to prove, the biocompatibility and the bioresorbability. A frequently published definition of biocompatibility according to Williams defines biocompatibility as “the ability of a material to perform with an appropriate host response in a specific application.”[86]. In other words the term biocompatibility includes the evaluation of ability of the material to elicit the response from tissue, namely toxic, inflammatory or infectious response [87]. These properties are usually clearly defined by relevant standard provided by International Organization for Standardization (ISO), namely ISO 10993, where are also references for testing methods. The ISO 10993 is a complex multi-part standard for evaluating effects of medical device material on the body and considers every aspect of biocompatibility. The first general part specifies the categories into which the devices are further classified. Based on this classification the number and manner of testing can be determined.

According to Table 1 biocompatibility matrix it is obvious that demands on

21

device testing grow with the length of device contact with body. For example the permanent implant in contact with blood for more than 30 days shall be subjected to test according to ISO 10993: part 3 (genotoxicity, carcinogenicity, reproductive toxicity), 4 ( interactions with blood) 5 (cytotoxicity), 6 (implantation), 10 (irritation, sensitization), 11 (systemic toxicity - acute and chronic). Additionally in case of polymeric materials it is necessary to identify and quantify degradation products from device - part 13. Deliberately degraded devices are further subjected to toxicokinetic study (part 16) and determination of leachates limits depending on health risks (part 17). Finally, for all materials the chemical, physicochemical and morphological properties characterization is obligation (part 18, 19). In the Czech legislation, the cornerstone of medical devices is the Act on Medical Devices (No. 268/2014) which has been amended with effect from 1 April 2015. The current form unifies previous legislative and takes into account the European legislative.

Appropriate biocompatibility testing is essential before any contact with human tissue of any kind in order to protect human being. Whole process comprises several stages starting from the less invasive, with the goal to eliminate animal tests. This means in practice that chemical analysis and characterization of material is firstly performed along with analysis of leachates in extracts obtained in vitro at elevated temperature usually of 37°C. Examples of standards dealing with regulations of testing in vitro are ISO 10993-4 and ISO 10993-5. Standards engaged with degradation of implantable materials are e.g. ISO 13781 – Poly(L-lactide) resins and fabricated forms for surgical implants - In vitro degradation testing ISO 15814 - Implants for surgery - Copolymers and blends based on polylactide - In vitro degradation testing.

Tissue permanent devices usually include in vivo tests. In vivo tests are supported by previous in vitro tests, due to which for example the duration of degradation of implant can be estimated. The comparison of in vitro and in vivo testing is reported in the study [88], which is furthermore dealing with development of a resorbable patch, based on poly(3-hydroxybutyrate).

Regarding the in vivo testing, it can be considered quite controversial, thus the very strict conditions are established by organizations and ethic committees. The test period, animals, surgery, testing conditions and test specimens are precisely defined in ISO 10993-6 and animal welfare requirements are described in ISO 10993-2.

Synthetic materials which come into direct contact with the human body also relate to the biomaterials, which defined Williams (2009) “A biomaterial is a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medicine.” From the viewpoint of tissue interaction and tissue response the biomaterials can be classified into: active, which positively affect

22

tissue [89]; inert, which almost do not elicit response [90] and degradable or bioresorbable [91], which are tissue integrated and after period of time slowly replaced by new cells of tissue.

Cytotoxicity

The cytotoxicity assays are used in screening the viability of cells in presence of chemical compounds or foreign material and its residues; in other words, the cytotoxicity tests can prove the biocompatibility. The cytotoxicity tests are reproducible and cost-effective, while providing sufficiently convincing and reliable results [92]. Cytotoxicity assessment is conducted both in vitro and in vivo and it is employed before clinical use, which eliminates potential biological damage. Nevertheless there is a difference between sensitivity in vitro and in vivo, because of the concentration of substance and intrinsic sensitivity of cells.

Moreover, the absorption of chemicals by cells in vivo is directly affected by other factors, such as distribution, biotransformation (metabolism), excretion and rate of absorption. Thus the in vitro cytotoxicity tests may appear to be more sensitive than in vivo and it can be difficult to extrapolate concentration which is toxic [93, 94].

In cytotoxicity assessment, there are many approaches how to estimate the viability of cells, which obviously also depends on the cell origin and nature [95]. Standard cytotoxicity (ISO) tests are: Direct Contact, Agar Overlay, minimum essential medium (MEM) Elution, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) Cytotoxicity Test and Colony Formation, where the last two are quantitative and preferred by regulation institutions [96].

23

Table 1 - Biocompatibility test matrix according to ISO 10993.

Device categories Biological effect Body contact

The table provides appropriate evaluation of the biocompatibility of devices for certain use.

• - Tests per ISO 10993

D - Additional tests that may be required in the U.S.

1 - Tissue includes tissue fluid and subcutaneous spaces E - For all devices used in extracorporeal circuits

24

1.5. Polymer characterization techniques