The Preliminary Assessment of New Biomaterials Necessitates a Comparison of Direct and Indirect Cytotoxicity Methodological Approaches

Abstract

Background: Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. Methods: Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. Results: The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. Conclusions: Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

Evaluation of tensile, thermal, and biological properties of natural rubber‐based biocomposite with biosilicate and 45S5‐K bioglass

“…The results suggested NR/BioS (90/10) and the neat NR as the best candidates to be used in vivo, as both specimens presented higher cell proliferation. Chraniuk et al 72 observed similar behavior in their study of the preliminary assessment of new biomaterials, which necessitates a comparison between direct and indirect cytotoxicity methodologies. A biological study was conducted on chitosan‐BG composites loaded with different bioglasses (P5, P5Sr2, P5Zn2) at a ratio of 1:0.5; 1:1; 1:2 72 .…”

“…Chraniuk et al 72 observed similar behavior in their study of the preliminary assessment of new biomaterials, which necessitates a comparison between direct and indirect cytotoxicity methodologies. A biological study was conducted on chitosan‐BG composites loaded with different bioglasses (P5, P5Sr2, P5Zn2) at a ratio of 1:0.5; 1:1; 1:2 72 . Proliferation results obtained after incubation with extracts (the indirect method) and composites (the direct method) indicated statistically significant differences between the test methods for each composite, as observed in the authors' comparison study.…”

“…Proliferation results obtained after incubation with extracts (the indirect method) and composites (the direct method) indicated statistically significant differences between the test methods for each composite, as observed in the authors' comparison study. In particular, in the direct contact test, specimens with the highest amounts of bioglass revealed the least cell proliferation (less than 50%) 72 . Vergnol et al 73 evaluated the ability of a poly(l,dl‐lactide)‐bioglass® (P(l,dl)LA‐BG 45S5) composite to be used as a bone fixation device.…”

“…Direct methods involve seeding cells directly onto a material's surface and they are especially important when materials and products are intended to be used in a longer contact with tissues 72,74 . This method is used to examine both the influence of substances secreted by the product, as well as the interaction of cells with the material surface 72,74 .…”

“…However, in vitro tests are limited by the fact that they involve monolayer cells. On the other hand, direct contact with the material can affect the body's immune response in vivo because the device interacts with multilayered tissues 72,75 . Direct methods also include consideration of locally increased concentrations of substances released from the material, which can affect cytotoxicity and cell proliferation 72,75 …”