中華民國牙體復形學會

I. Objective
Extracellular matrix (ECM) protein plays an important role in directing the cellular behavior. In this context, the current work seeks to compare the in vitro effects of two major types of ECM: fibronectin (Fn) and type I collagen.

II. Materials & Methods
MDPC-23, a rat odontoblast-like cell line was used. Various concentrations of fibronectin (Fn) (0.1, 1, 10, 20, 30, 40, 50µg/mL, 33016-015, Gibco) and porcine skin type I collagen (PCOL300) (300µg/mL, Nitta gelatin) were coated into non-treated polystyrene wells (96 well plate; 24 well plate; 12 well plate, IWAKI). Wells coated in equal volume of phosphate buffered saline (PBS) were taken to be control. The cells were inoculated into those wells at the concentration of either 5000/mL or 10000/mL in dulbecco’s modified eagle medium (DMEM, D5796, Sigma) supplemented with 5% fetal bovine serum (FBS, 10270-098, Gibco). Cell morphology was observed under light microscopy (Olympus). F-actin was visualized by staining with phalloidin conjugated to Alexa Fluor® 568 (A12380, Invitrogen). Nucleus was stained with DAPI (D9542, Sigma) and the cells were documented using EVOS® FLoid® Cell Imaging Station (Advanced Microscopy Group). Cell proliferation activity was evaluated by CCK-8 assay (347-07621, Dojindo); Cell differentiation and mineralization capacity were investigated using ALPase activity assay (Wako), real time RT-PCR (6402712 FastStart Essential DNA Green Master, Roche), and Alizarin red staining (Wako).

III. Results
Cells maintained a high proliferation activity on Fn and PCOL even at a lower concentration (5000/mL). The proliferation activity of cells on Fn increases in a concentration dependent manner while it reached a plateau after 10µg/mL. Cells adopted long, thin and spindle shape on Fn and PCOL. Parallel actin filaments were observed in MDPC-23 cultured on Fn and PCOL. Gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01 fold; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24 fold; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates. In accordance with the differentiation data, calcific deposition in cells cultured on Fn(10-50) and PCOL300 was observed as well.

IV. Conclusion
Fn significantly promoted the spreading, proliferation, differentiation and mineralization of MDPC-23 at a concentration range markedly lower than that of type I collagen. Despite of the limitation of this work, it is suggested that the minimum concentration for coating of Fn on polystyrene is 10µg/mL.