Table 1.

In vitro studies that observed the behavior of EMD on titanium surface.

Authors	Year	Tested Cells	Titanium Surface	Methods	Outcomes and Observation Periods	Results
Schwarz et al. [13]	2004	Human osteoblast-like cells	SLA	Cells placed on titanium discs with medium alone or +EMD at different concentrations (25, 50, 100, and 200 μg/mL); the medium was changed after 3 days without EMD	Adhesion: 1, 3, and 6 days Proliferation, viability: 1, 3, and 6 days Cell morphology: 1, 3, and 6 days	Concentration-dependent increase in proliferation; statistically significant higher increase in viability with 100 and 200 μg/mL EMD at day 6 as compared to control
Dacy et al. [14]	2007	Rat osteoblasts	Phosphated and non-phosphated	Cells placed on titanium discs with medium alone or +EMD (180 μg); the medium was changed every 2 days for 28 days	Adhesion: every 2 days up to 28 days nodule formation and mineralization: every 2 days up to 28 days TGF-β1 and IL-1β production: every 2 days up to 28 days	Increased production of TGF-β1 for up to 8 days
Miron et al. [15]	2010	Rat calvarial osteoblasts	Smooth and SLA	Cells placed on titanium discs with medium alone or +EMD, cultured from 1 h to 4 weeks	Adhesion: 2, 4, and 8 h Cell spreading: 30 min, 2 and 4 h Proliferation: 1, 3, 5, and 7 days ALP activity: 1, 2, and 3 weeks Gene expression (Runx2, BSP, OC): 1, 7, 14, 21, and 28 days	Increased cell spreading after 2 and 4 h and increased proliferation after 3 to 7 days; increased ALP activity; increased levels of mRNA encoding bone sialoprotein and osteocalcin
Qu et al. [16]	2011	osteoblast-like MG-63 cells	SLA	Cells cultured on titanium discs for 7 days; the medium was changed every 2 days; EMD addition in test groups at different concentrations (12.5, 25, 50, and 100 μg/mL) at day 7	Proliferation, viability: 48 h ALP activity, OC production: 48 h Gene expression (OPG, RANKL): 48 h	Dose-dependent decrease in proliferation and viability at 50 and 100 μg/mL; increased ALP activity, osteocalcin production, and mRNA expression level of OPG at 50 and 100 μg/mL
Wang et al. [18]	2016	Human primary gingival fibroblasts	Smooth and SLA	Cells placed on titanium discs with medium alone or +EMD	Adhesion: 2, 4, and 8 h Proliferation: 1, 3, and 5 days Morphology: 2, 4, 8, and 24 h Gene expression (COL1A1, VEGF-A, FN1): 5 days Synthesis of collagen matrix (collagen type I staining): 7 and 14 days	Increased cell spreading and proliferation; increased mRNA levels of VEGF-A and fibronectin, increased extracellular matrix synthesis of collagen type I
Shi et al. [19]	2017	HUVECs	smooth, acid-etched or SLA	Cells cultured on titanium discs for 24 h; EMD addition (50 μg/mL) after 24 h	Proliferation, viability: 48 h Angiogenic activity (expression of genes ICAM-1, EPCR, E-selectin, vWF, Ang-2): 2 days	Decrease in proliferation/viability on acid-etched and SLA titanium; increased ICAM-1, EPCR, E-selectin, and vWF on SLA surface
Ramenzoni et al. [17]	2020	MC3T3 osteoblast-like cells	SLA	Cells cultured on titanium discs alone or with EMD or DMD at 100 μg/mL, 1, 10, and 30 mg/mL	Adhesion: 2, 4, and 8 h Proliferation, viability: 24, 48, and 72 h Cell migration (assessed with scratch wound healing model): 24 h Gene expression for osteoblast differentiation and bone formation (Runx2, COL1A2, ALP, BSP): 24 h	Increased osteoblasts proliferation and viability both with EMD and DMD; increased gene expression with EMD or DMD concentrations ≥10 mg/mL

ALP: Alkaline phosphatase; Ang-2: Angopoietin-2; BSP: Bone sialoprotein; COL1A1: Collagen 1a1; COL1A2: Collagen 1a2; DMD: Dentin matrix derivative; EMD: Enamel matrix derivative; EPCR: Endothelial protein C receptor; FN1: Fibronectin-1; HUVECs: Human umbilical vein endothelial cells; ICAM-1: Intercellular adhesion molecule-1; LM: Light microscopy; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor κB ligand; SEM: Scanning electron microscopy; SLA: Sand-blasted, large grit, acid-etched; VEGF-A: Vascular endothelial growth factor-A; vWF: Von Willebrand Factor.