Odontogenic differentiation of stem cell from apical papilla (SCAP) using a dual bloactive molecule releasing core-shell nanopolymeric system

摘要

Introduction: Regenerative procedures are a biologically based procedure that utilizes the principles of tissue engineering, intended to replace damaged, diseased, or missing structures, such as dentin and the cells of dentin-pulp complex. Various bioactive molecules (BMs) are known to be involved in different stages of odontogenesis. The spatial and temporal control in the release of BMs at different stages of odontogenic differentiation remains to be one of the major challenges in regenerative procedures. The current study aims to synthesize and characterize a novel chitosan-based dual bioactive molecules releasing system for dentin-pulp regeneration. Materials and Methods: Core-shell nanopolymeric system containing transforming growth factor (TGF)-β1 in alginate shell and dexamethasone (Dex) in CSnp core was synthesized. The physicochemical characterization of TGF-β1 and Dex releasing core-shell nanopolymeric system (TD-NS) was assessed by Transmission electron microscopy (TEM), Zetasizer and Fourier Transform Infrared (FTIR) spectroscopy. The TGF-β1 and Dex release kinetics was determined by enzyme-linked immunosorbent assay (ELISA) and spectrophotometry respectively. Previously characterized stem cells from apical papilla (SCAP) cell line was cultured in presence of TD-NS. SCAP viability was determined by resazurin assay, and morphology was examined by phase contrast and fluorescence microscopy. Biomineralization potential was determined by alizarin red staining (AR-S) and alkaline phosphatase (ALP) activity assay. Immunofluorescence was performed to determine the effect on odontogenic protein expression, while alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) gene expressions were analyzed by Real-time reverse-transcription polymerase chain reaction (qRT-PCR). Results and Discussion: The TD-NS showed reversal of charge confirming the formation of alginate shell on CSnp core. TD-NS were noncytotoxic, and promoted proliferation of SCAP. They simultaneously released TGF-b1 and Dex. ALP activity at 1 week was significantly higher (p ＜ 0.05) in TD-NS group compared to control or group containing free BMs. After 2 weeks culture in vitro, SCAP showed significantly higher degree of biomineralization (p ＜ 0.05) and expression levels of DSPP and DMP-1 than control or free BMs groups. The qRT-PCR analysis revealed that the expression of ALP, DSPP and DMP-1 was significantly higher than in control or free BMs groups (p ＜ 0.05). Conclusion: Collectively, the findings from the current study suggest that TD-NS enhanced odontogenic differentiation of SCAP. These findings highlight the potential of controlled delivery of multiple BMs from a single carrier to direct the spatial and temporal controlled odontogenic differentiation of dental stem cells.