Assessing the Biocompatibility of Tannic Acid-Based Biomaterials: Addressing Challenges in Standard Cytotoxic Assays

Abstract

In this comprehensive study, we delve into the intricate binding properties of tannic acid (TA) and examine their dual role in the realm of biomaterial development. While TA's properties can enhance the functionality and performance of biomaterials, they also raise concerns regarding potential biases in in vitro biocompatibility assessments. We focus on the relevance and constraints of several widely employed cell viability assays, namely the DNA-based PicoGreen assay, the PrestoBlue assay, and the Live/Dead staining technique utilizing fluorescein diacetate (FDA) and propidium iodide (PI). We investigate how these assays perform when applied to TA-coated scaffolds and cell sheets. Through a detailed presentation of our experimental findings, we juxtapose them through a critical review of the existing literature, allowing us to identify and elucidate the limitations these assays face when assessing TA-based biomaterials. In doing so, we aim not only to enhance the understanding of these potential assay biases but also to provide actionable recommendations for accurately evaluating the biocompatibility of TA-modified substances. This dual approach, combining empirical research with literature analysis, offers vital insights for the research community, ensuring that the assessment of TA-coated biomaterials is scientifically sound and reproducible.

Keywords: DNA; PrestoBlue; biocompatibility; cytotoxicity; tannic acid.

Figure 1

A schematic representation of the study design. (A) Tannic acid (TA) can interfere with biocompatibility assays by binding DNA via hydrophobic interactions and hydrogen bonding, non-specifically reducing compounds such as resazurin, and by quenching the fluorescence of proteins and fluorophores. (B) The 3D-printed mPCL scaffolds used to evaluate TA assay interference. (i) Scaffolds including uncoated controls and scaffolds coated with 1% or 10% TA. Scale bars: scaffold = 1 mm; insets = 50 µm. (ii) The TA release profiles from coated scaffolds [7]. (C) Biocompatibility testing using cell sheets. A timeline is shown of cell sheet maturation and scaffold testing, including an indirect assay, where TA-coated scaffolds are not in direct contact with cell sheets and TA diffuses into the culture medium, and a direct assay, where cell sheets are wrapped around TA-coated scaffolds, leading to direct cell–TA interaction.

Figure 2

Biocompatibility assessment of uncoated and TA-coated scaffolds. Experimental approaches exposing TA-coated scaffolds to mature cell sheets: (A) Indirect assay: Cell sheets remained attached to the bottom of the well, exposing them to TA released from the scaffold in a diluted form, minimizing potential accumulation and toxicity. Red arrows indicate cell sheets attached to the well surface (i). (B) Direct assay: Cell sheets were wrapped around the coated scaffolds, allowing for direct cell–TA interactions and the potential accumulation of TA within the cell sheet. Biocompatibility was assessed quantitatively by measuring metabolic activity via a PrestoBlue assay at 3 and 7 days in culture (ii), and the DNA content after 7 days in culture (iii); red dotted lines represent the metabolic activity and DNA content of cell sheets at day 0, prior to the assays. All measurements are reported as the average ± standard deviation (SD). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, (n = 6).