Effect of different calcium hydroxide formulations used during regenerative endodontic procedures on the viability, mineralization, and dentino/cemento/osteogenic differentiation potential of human periodontal ligament stem cells. An in-vitro study.

Aim: To evaluate the effect of CH and NCH on the viability, attachment, osteogenic, cementogenic and dentinogenic differentiation potential of hPDLSCs in-vitro. Materials and Methods: Calcium Hydroxide (CH) and nano calcium hydroxide (NCH) powders were initially characterized using transmission electron microscope (TEM), X-ray Diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and a zeta sizer instrument. Standard dentin discs were prepared, irrigated according to the Regenerative Endodontic Procedures (REP) protocol, then treated with 1 mg/ml of CH or NCH pastes, and incubated for 1 week. Untreated discs served as control. Then, they were washed off, and all discs were treated with 17% EDTA before seeding of human periodontal ligament stem cells (hPDLSCs) . Cell viability was assessed using MTT assay. Mineralized nodules and alkaline phosphatase (ALP) activity were determined to assess cell differentiation. Cell dentino/cementogenic and osteogenic potential were assessed through monitoring gene expression levels Dentin sialophosphoprotein (DSSP), Cementum protein 1 (CEMP1), Runt-related transcription factor 2 (RUNX2), Osteoprotegrin (OG), and Osteocalcin (OC) using RT-qPCR. Results: TEM examination of NCH powder particles revealed a regular pattern and hexagonal shape with side dimension up to 60 ±10 nm. XRD analysis of NCH powder demonstrated its highly crystalline nature. FTIR spectroscopy confirmed that CH and NCH consisted mainly of pure calcium hydroxide. The zeta sizer instrument showed that NCH is positively charged with an average zeta potential of +27.8 mV. hPDLSCs seeded on NCH-treated discs showed significantly higher cell survival , more mineralization, higher alkaline phosphatase activity and increased expression of DSPP, CEMP1, RUNX2, OPG and OC markers. Conclusion: NCH can be an alternative ICM during REP.