© 2003 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003

15.2.1.5 Contemporary Biocompatibility Test Methods

As pointed out by Jonathan Black [234], biological performance of an implant includes consideration of the host response and the material response to implantation. The traditional approach [237, 241, 6038] has been to define biological performance in terms of host response – biocompatibility – and then to observe evidence of material degradation that arises during in vitro and in vivo testing. Black [234] sets forth a 5-part strategy for materials qualification studies:

(1) select material based on engineering properties and previous host response information;

(2) determine experimentally if host response is acceptable for the intended application;

(3) acquire evidence of unacceptable material response during host response studies;

(4) verify satisfactory material response during long-term in vivo implant studies; and

(5) monitor clinical reports detailing changes in material response during actual service.

There are two general classes of in vitro screening methods: tissue culture tests and blood contact tests (in blood contact applications [6052]).

According to Black [234], tissue culture tests involve maintaining portions of living tissue in a viable state in vitro by any of three generic methods: (1) Cell Culture – the growth of initially matrix-free, dissociated cells, usually in monolayers; cells may be grown in solution, on agar, or on other media substrates, and are exposed to biomaterials by direct contact with bulk materials, by diffusional contact through an intermediate layer, or by contact with particles or eluants from biomaterials in the culture media. (2) Tissue Culture – the growth of portions of intact tissue without prior cellular dissociation, usually on a substrate rather than in free suspension, with exposure to the biomaterial as in cell culture. (3) Organ Culture – the growth of intact organs in vitro, varying from fetal bone extracts that can survive without external support to whole adult perfused organs such as kidney or heart. Tissue culture tests are used to study various aspects of host responses including cell survival (toxicity, organelle/membrane integrity), cell reproduction (growth inhibition), metabolic activity (energetics, synthesis, catabolism), cell activities (inhibition of chemotaxis, locomotion, or phagocytosis, and alteration of cell size and shape), cell damage (chromosomal aberration, carcinogenicity [5845], mutagenicity [5846]), and specific immune system response (delayed hypersensitivity) [5847, 5848]. “Tissue culture techniques for screening materials may use one or more normal mammalian cell lines such as murine macrophages, abnormal cells such as HeLa or lymphoma cells, or bacterial cell lines such as Staphylococcus aureus or Escherichia coli.” Each test that has been developed uses selected cells suitable for the particular questions posed, the utility of which depends on its correlation with in vivo host response [5849-5851].

According to Black [234], blood contact tests involve examining either coagulation times or hemolysis rates in either static or dynamic systems during or after contact with foreign materials. Because these responses are not only intrinsic to materials but are also influence by implant device functioning, the presence of interfaces, flow rates and turbulence conditions, etc., three sequential phases of testing of a new biomaterial are often employed: (1) in vitro static tests (e.g., clotting time relative to a reference surface [5852, 5853]), (2) ex vivo dynamic tests (platelet adherence under controlled flow conditions [5854, 5855], exposure to whole blood, erythrocyte damage rates, etc.), and (3) in vivo dynamic tests. Despite their flaws, in vitro tests are widely used for screening because they are relatively inexpensive and are not known to yield false negatives (e.g., a material that tests poorly will not be a good implant in cardiovascular applications) [234].

After showing satisfactory results during in vitro screening, a new biomaterial is then tested using extended-time whole-animal studies [5856] that expose the biomaterial to systemic physiological processes, prior to human clinical testing. Initial nonfunctional testing is usually in soft tissue because cytotoxic effects “have a generality of action and because soft tissue sites can be approached in animals with relatively minor surgery.” [234] The most popular sites include subcutaneous, intramuscular, intraperitoneal [5857], transcortical (e.g., femur, cranium), and intramedullary (e.g., femur, tibia). Tests are of two types: nonfunctional and functional. In nonfunctional tests, the implant has a carefully selected and standardized shape and floats passively in the tissue site [5858]. Nonfunctional tests focus on the interaction between the implant material and the biochemical implant environment, and are of short to intermediate duration (e.g., 0.5-24 months) [234]. Adequate experimental controls must be provided to include effects of relative tissue-implant motion and electrical charge density at the implant-tissue interface which can influence the observed host response. Functional tests require that the implanted material be placed in the functional mode that it would experience in actual service as a human implant [234]. This allows the study of tissue ingrowth into porous materials for fixation purposes [5859, 5860]; the effects of mechanical forces during actual use [5861]; formation of neointima, degree of thrombosis and patency [5862, 5863], and effects of mechanical loading [5864] in vascular processes; and production of wear particles in load-bearing devices (and clinically relevant tissue responses to them). Functional tests are more costly (>$1000/animal) and complex than nonfunctional ones [234], and test animals have shorter life spans and higher metabolic rates than humans [5865], introducing additional uncertainty into the results.

“In the final analysis,” notes Black [234], “clinical testing is the only technique by which the true biological performance of any implantable biomaterials can be determined. [However,] any human clinical experiment must provide a potential benefit to the patients involved, [which] essentially prevents the use of humans as test subjects for biomaterials.” Further discussion of clinical testing procedures for medical nanodevices is deferred to Chapter 17.

Last updated on 30 April 2004