<b>Supplemental Material - Hierarchically porous surface of HA-sandblasted Ti implant screw using the plasma electrolytic oxidation: Physical characterization and biological responses</b>

Supplemental Material for Hierarchically porous surface of HA-sandblasted Ti implant screw using the plasma electrolytic oxidation: Physical characterization and biological responses by Young-Eun Choe, Cheng Ji Li, Dong-Hyeon Yeo, Yu-Jin Kim, Jung-Hwan Lee and Hae-Hyoung Lee in Journal of Biomaterials Applications.</p

Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways

<div><p>Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by <i>in vitro</i> wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.</p></div

Effects of magnetic scaffolds on the migration of HDPCs; (a) cell images before and after migration for 12 hours, and (b) migration rate measured.

<p>*: statistically significant difference compared with 0%MNP (p<0.05, n = 3).</p

Characteristics of magnetic scaffolds: (a-f) SEM images at different magnifications, (g) EDS atomic signal, (h) XRD phase analysis and (i) FT-IR chemical groups.

<p>Low (a-c, 150 X) and high (d-f, 1000 X) SEM morphologies of PCL, PCL–MNPs 5%, and PCL–MNPs 10% scaffolds showing a highly porous structure, and their EDS results showing existence of MNPs (Fe) on the surface, which was also confirmed by XRD and FT-IR spectrum.</p

Activation of intracellular integrin downstream pathways: (a) MAPK and (b, c) NF-κB.

<p>Cells were cultured for (a) 45 minutes and (b, c) 60 minutes. Signaling pathways were assessed via (a, b) Western blotting and (c) immunofluorescence staining. A change in the color of the nucleus from red to yellow (due to co-localization of green FITC fluorescence and red propidium iodide fluorescence, arrows) was indicative of NF-κB translocation in the cells. These data are representative of three independent experiments.</p

Effects of magnetic scaffolds on the odontogenic differentiation of HDPCs.

<p>Differentiation was determined by (a) alkaline phosphatase (ALP) activity, (b) mRNA expression of genes (ALP, OPN, OCN, DSPP, and DMP-1) by RT-PCR, and (c) mineralization nodule formation by Alizarin red staining. *: statistically significant difference compared with 0%MNP (p<0.05, n = 3).</p

(a) Gene expressions of integrin subsets as analyzed by RT-PCR at 60 min and 180 min and (b) the protein expressions involved in integrin downstream pathways as assessed by Western blotting at 60 min.

<p>These data are representative of three independent experiments.</p