2.3. Prearation of Clumps of Periodontal MSCs (C-MSCs)

This study used the method developed by Kittaka et al. to create clumps of MSCs [9]. Briefly, the P4 PDLSCs were seeded at a density of 7 × 10⁴ in 24-well plates and cultured with a growth medium supplemented with 50 µgm/mL of L-ascorbic acid (Sigma) for seven days. To obtain C-MSCs, confluent cells that had formed as a cellular sheet, consisting of the extracellular matrix produced by MSCs, were scratched with a micropipette tip and then were torn off. The MSC/ECM complex was then detached from the plate’s bottom in a rolled sheet shape and incubated for a day.

LPS treatment for the C-MSCs: The formed clumps of MSCs were carefully transferred into a 24-well ultra-low binding plate (Corning, New York, USA) and cultured in a regular culture medium added with different concentrations (5, 10 or 20 µgm/mL) of E. coli derived LPS for 3 and 5 days. The medium was replenished every third day. All experiments were performed in triplicate.