Effect of Intracanal Medications on Cell Adhesion, Viability, and Differentiation During Regenerative Endodontic Procedures: A Systematic Review of In Vitro Studies

INTRODUCTION: Intracanal medications used during regenerative endodontic procedures may influence the biological behavior of stem cells through changes in the dentin microenvironment. Understanding these effects is essential for optimizing regenerative outcomes. OBJECTIVE: This systematic review aimed to answer the following PICO (Population, Intervention, Comparison, Outcome, and Study design) question: Does dentin conditioning with different intracanal medications alter the adhesion, migration, and differentiation of undifferentiated cells during regenerative endodontic therapy? METHODS: A comprehensive search was performed in PubMed, Embase, Scopus, LILACS, and Web of Science up to May 2025. Gray literature was screened through Google Scholar and ProQuest. In vitro studies evaluating the effects of dentin conditioning with intracanal medications on cell behavior (proliferation, adhesion, migration, and differentiation) were included. Two reviewers independently conducted study selection, data extraction, and risk of bias assessment using the RoBDEMAT tool. Data were qualitatively synthesized. RESULTS: From 2620 retrieved records, 12 studies fulfilled the eligibility criteria. Most studies used human root dentin as a substrate and tested calcium hydroxide-based formulations, triple antibiotic paste, or double antibiotic paste; others evaluated silver nanoparticles, calcium hypochlorite, Bio-C Temp, or antibiotic-containing fibers. Dental pulp stem cells were most frequently used, followed by apical papilla, deciduous tooth, and periodontal ligament stem cells. Conditioning times ranged from 7 to 28 days. Calcium hydroxide showed the most favorable outcomes for cell adhesion, morphology, and proliferation. Lower antibiotic concentrations (1 mg/mL) demonstrated acceptable biocompatibility. Only one study assessed cell differentiation, showing increased dentin matrix protein-1 and DSPP expression after calcium hydroxide conditioning; none evaluated cell migration. CONCLUSIONS: Calcium hydroxide exhibited the most consistent and favorable biological responses. Triple antibiotic paste and double antibiotic paste showed concentration-dependent cytotoxicity; however, more robust evidence is needed to clarify how intracanal medications influence cell behavior on conditioned dentin.