In Vitro Wound Healing Assay

A linear scratch on confluent MC3T3-E1 cells was performed by a sterile pipette. We removed the cellular debris by washing with PBS, and then we incubated the cells in DMEM 1% FBS alone or in the presence of various treatments for 24 h (see [41 (link)] for reference). We took the photographs immediately after the scratch (t0) and at various times after treatment (t18, t24, and t48) using an Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam Icc1 camera at a 4× magnification. We analyzed the images using ImageJ software (NIH, Bethesda, MD, USA) as previously reported [42 (link)]. The covered area percentage was calculated for each experimental group by measuring the wound size at different times from treatment compared with the initial (t0) wound size considered as 100%.