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Use of Cross‐linked Polypeptide Multilayer‐coated Electrodes to Monitor Osteogenic Differentiation of Human Dental Pulp Stem Cells

Polypeptide multilayer films made of synthetic peptides are non‐cytotoxic and biocompatible. In addition, this material allows for modulation of thickness, stiffness, and adhesiveness. Previous study suggested that mesenchymal stem cells cultured on gel substrates with ~34 kPa Young's modulus demonstrated good cell spreading and osteogenic differentiation as a consequence of increased contractility of the actomyosin cytoskeleton. In this study, human dental pulp stem cells (hDPSCs) derived from teeth were cultured on polypeptide multilayer films fabricated by the alternate deposition of cationic poly‐L‐lysine and anionic poly‐L‐glutamic acid, cross‐linked with EDC/sulfo‐NHS, and coated with type‐I collagen. Osteogenic differentiation of hDPSCs was evaluated by calcium deposition staining, q‐PCR analysis of osteogenic specific genes, such as ALP (alkaline phosphatase), OCN (osteocalcin), and OPN (osteopontin), and also monitored by cross‐linked polypeptide multilayer‐coated electrodes using electric cell‐substrate impedance sensing (ECIS). The results showed that hDPSCs demonstrated better cell attachment and spreading on even number layer of films. Furthermore, hDPSCs showed a significantly higher osteogenic differentiation rate on cross‐linked films than on culture dishes or cover glasses. In the ECIS monitoring of osteogenic differentiation, hDPSCs were seeded into electrode wells and allowed to form confluent cell layers. The medium was then changed with osteogenic induction medium and the impedance time courses of the cell‐covered electrodes throughout the osteogenic induction process were measured up to 21 days at 11 frequencies ranging from 62.5 Hz to 64 kHz. Compared with the data without osteogenic induction medium, significantly lower impedance time courses were observed for hDPSCs treated with induction medium. In addition, these two distinct time course profiles were distinguished as early as 4 hours after induction. To detect subtle changes in cell morphology in the early stage of osteogenic differentiation, the frequency‐dependent impedance data were analyzed with a theoretical cell‐electrode model. The results showed that the decrease of measured impedance of differentiated cells was mainly due to a decrease of the junctional resistance between cells (Rb) and an increase of the cell‐substrate separation (h). Thus, we described a strategic approach for the real‐time monitoring of osteogenic differentiation using cross‐linked polypeptide multilayer‐coated electrodes.