*3.2. Cytotoxicity Test*

As a surgical implant, it is important to verify the biocompatible properties of the ES PLA95/β-TCP membrane. In a previous study, the results indicated that the MC-3T3-E1 cells could adhere and proliferate on the surface of ES PLA95/β-TCP membrane [22]. Therefore, we alternatively evaluated the cytotoxicity of the ES PLA95/β-TCP membrane to ensure the safety of the manufacturing process as well as the PLA95 material. In this study, the MEM elution assay was used to verify the cytotoxicity of the ES PLA95/β-TCP membrane in accordance with the ISO-10993-5 guideline. The results revealed no cell lysis in the membrane extract, reagent blank, and the negative control extracts under light microscope. Thus, it was inferred that the ES PLA95/β-TCP membrane did not exhibit cytotoxic reactivity (Table 1).


**Table 1.** Cytotoxicity assay of ES PLA95/β-TCP membrane by indirect method.

Note: (Grade 0: no cell lysis or reduction of cell growth, Grade 1: not more than 20% of the cells are round, Grade 2: not more than 50% of the cells are round and devoid of intra-cytoplasmic granules, Grade 3: not more than 70% of the cell layers contain rounded cells or are lysed, Grade 4: nearly complete or complete destruction of the cell layers).
