**2.2 Cell proliferation**

Cell proliferation refers to the rate at which cells divide and increase in number. Assessing cell proliferation provides insights into how toxic agents affect cell division and growth. Inhibition of cell proliferation can indicate the potential anti-cancer effects of a drug, while excessive inhibition may indicate general toxicity. Cell proliferation is a crucial parameter in evaluating cytotoxicity. The cytotoxic effects of a biomaterial can threaten cell viability by compromising its structural or metabolic integrity and affecting its regenerative capacity [6]. Toxic agents can inhibit cell proliferation, which can be assessed using assays such as BrdU (bromodeoxyuridine) incorporation, EdU (5-ethynyl-2′-deoxyuridine) incorporation, or Ki-67 staining. A simple approach to measure cell proliferation involves comparing cell counts in cultures exposed to test material extracts for varying durations with control cultures. This method typically requires trypsinizing the cell culture and counting individual cells with a microscope or electronic cell counter [7]. Evaluating the protein content of cell cultures is a practical test for assessing the toxicity of biomaterials [8]. Cell proliferation assays can be used to determine the optimal concentration of a drug or compound for further testing.

### **2.3 Cytotoxicity**

Cytotoxicity is a measure of cell damage or death caused by toxic agents. Cytotoxicity assays focus on detecting the extent of cell damage or death caused by toxic agents. These assays provide information on the direct effects of a substance on cells, which can be useful for identifying potential therapeutic targets or understanding the mechanisms underlying toxicity. Common cytotoxicity assays include LDH (lactate dehydrogenase) release assay, which measures the release of LDH from damaged cells, and the trypan blue dye exclusion assay, which measures membrane integrity. Cytotoxicity assays can also be used to determine the selectivity of a drug or compound for different cell types.

#### **2.4 Apoptosis and necrosis**

Apoptosis (programmed cell death) and necrosis (uncontrolled cell death) are distinct mechanisms of cell death that can be triggered by toxic agents.: Understanding the mechanisms of cell death induced by toxic agents is essential for developing targeted therapies and identifying potential side effects. Apoptosis and necrosis are distinct types of cell death, and differentiating between them can provide insights into the mode of action of a toxic agent and its potential therapeutic value. Assays such as annexin V/propidium iodide staining, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and caspase activity assays can be used to assess apoptosis and necrosis.

#### **2.5 Oxidative stress**

Toxic agents can cause oxidative stress by inducing the production of reactive oxygen species (ROS) and/or impairing cellular antioxidant defenses. Assays such as DCFDA (2′,7′-dichlorofluorescin diacetate) fluorescence, lipid peroxidation assays, and glutathione assays can be used to evaluate oxidative stress. Evaluating oxidative stress is crucial for understanding how toxic agents affect cellular redox balance, which plays a vital role in maintaining cellular homeostasis. Oxidative stress can lead to cellular damage, dysfunction, and eventually cell death. Identifying agents that induce or prevent oxidative stress can help in the development of novel therapeutic strategies.

#### **2.6 Genotoxicity**

Genotoxic agents can cause DNA damage, which may lead to mutations, chromosomal aberrations, or DNA strand breaks. Genotoxicity assays assess the potential of a substance to damage DNA, which can lead to mutations, chromosomal aberrations, or other genomic changes. These assays can help identify potential carcinogens, mutagens, or teratogens and provide insights into the mechanisms of DNA damage and repair. Genotoxicity can be assessed using assays such as the comet assay (singlecell gel electrophoresis), micronucleus assay, and γ-H2AX (phosphorylated histone H2AX) staining.

### **2.7 Cellular morphology**

Toxic agents can induce changes in cellular morphology, such as cell shrinkage, membrane blebbing, or cytoplasmic vacuolization. Examining cellular morphology provides a visual assessment of the effects of toxic agents on cell structure and organization. Changes in cellular morphology can indicate alterations in cellular functions, such as cell adhesion, migration, or differentiation, which can help elucidate the mechanisms underlying toxicity. These morphological changes can be visualized using light microscopy, phase-contrast microscopy, or fluorescence microscopy.

Studies examining morphological changes caused by cell adhesion to compatible and incompatible material surfaces have shown that human fibroblasts proliferate extensively on glass, but experience inhibition on hydrophobic biomaterials. Furthermore, cell rounding, detachment, and decreased proliferation have been observed [9]. This incompatibility of the biomaterial surface is known as intrinsic toxicity. The rounding of cells and other morphological alterations often occur before the loss of cell viability, which is accompanied by the disconnection of cells from the substrate [10]. Another characteristic of cell morphological changes is the increased vacuolation of the cytoplasm, often involving the formation of autophagosomes. Cytoplasmic vacuolation has been recognized as a dependable indicator of toxicity [11].

#### **2.8 Protein synthesis and enzyme activity**

Toxic agents can affect protein synthesis or the activity of specific enzymes, which can be assessed using assays such as western blotting, enzyme-linked immunosorbent assay (ELISA), or enzymatic activity assays. Assessing protein synthesis and enzyme activity can reveal how toxic agents affect specific cellular processes or signaling pathways. Identifying the proteins or enzymes affected by a toxic agent can help in understanding its mode of action and potential therapeutic applications.
