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iMatrix-511 is a novel integrin-binding fragment derived from laminin-511. Previous studies showed its superiority as a culture substrate for xeno-free culture and maintenance of pluripotency in stem cells. However, its effects in the dental field remain largely unknown. The aim of the present study was to unravel the in vitro effects of iMatrix-511 in comparison with vitronectin (VN).Biochemical assays were performed in vitro in MDPC-23 cells. The optimal coating density for 2 proteins was determined using the cell counting kit-8. To evaluate cell proliferation to both proteins, MDPC-23 cells were directly seeded onto the iMatrix-511 or VN-modified polystyrene and analyzed by the cell counting kit-8. The phenotype of cells seeded on iMatrix-511 and VN was characterized. Phenotypic characterization included real-time reverse-transcription polymerase chain reaction and alizarin red staining.The optimal coating density for iMatrix-511 and VN was determined to be 1 μg/cm2 and 0.25 μg/cm2, respectively. Cells cultured on iMatrix-511 showed higher cell proliferative activity than the noncoated control and VN on days 1, 2, and 4. Cell morphology observation revealed MDPC-23 cells attach preferentially to iMatrix-511 and start to spread as early as 1 hour after inoculation. MDPC-23 cells exhibited more potent odontogenic differentiation on iMatrix-511 than the control and VN as shown by the marked enhancement of dentin matrix protein 1 and dentin sialophosphoprotein messenger RNA expression. Although both proteins showed more mineralized nodule formation than the control, iMatrix-511 remained to be the one that elicited stronger calcific deposition.iMatrix-511 supported the proliferation and acquisition of odontogenic cell phenotype in vitro, rendering this novel material a potential candidate for dentin regeneration.