Human plasma fibronectin promotes proliferation and differentiation of odontoblast

Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; 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; 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 as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.


Introduction Fibronectin (Fn) is a dimeric multi-domain
glycoprotein (about 450kDa per dimer) that is found in circulation or tissue extracellular matrix (ECM).
Two major types of Fn are present in vertebrates arising from alternative splicing of its pre-mRNA: soluble plasma Fn (corresponds to the aforementioned CIg) and insoluble cellular Fn. Both of them contain various adhesive domains for cells and other proteins 9,21 , the synergic site of RGD: 18 9 , Arg-Glu-Asp-Val (REDV) 10 15 , and 17 . The plasma form of Fn (pFn) is predominantly synthesized by hepatocytes, circulates in blood and deposits rapidly upon tissue injury to initiate hemostasis, this deposition process is independent of other hemostasis factors, such as platelet 30 . Cellular Fn (cFn) is mainly produced by epithelial cell 25 , macrophage 2 , and endothelial cells 20 .
cFn contributes to support the extracellular structure framework by actively binding with cells and other matrix proteins as mentioned above.
Previous histological localization study in tooth germ revealed that Fn, which is present in the mesenchymal tissue, basement membrane, and predentine, was not detected in late pre-dentine and mineralized dentine. Further, epithelial tissues of tooth germ were negative for Fn except in the stellate reticulum 12,28 . Tooth development or odontogenesis is a complex process, which needs reciprocal interaction between epithelium and mesenchyme. Various growth factors, paracrine signal molecules 27 and extracellular matrix (ECM) proteins 6 are believed to be essential for this process. Among those, the importance of ECM proteins is becoming increasingly apparent, since serious changes were noted in oral cancer as compared with normal tissue because of the alteration of ECM 26 . As a ubiquitous ECM, it is reported that Fn is required for calvarial osteoblast differentiation and mineralization 16 . Since Fn is expressed in early pre-dentine and disappeared in mineralized mature dentine, it is thus reasonable to conceive that Fn might play some roles in the differentiation of dental mesenchyme into dentine-forming odontoblast.
However, the precise effect of Fn on cells of dental mesenchymal origin is still unclear to date. Hence, the current experiment seeks to uncover the potential interaction between Fn and odontoblast-like cells.
To better imitate the real scenario that cells are surrounded by extracellular matrix proteins in situ, we examined the cell proliferation, differentiation and mineralization behavior in Fn-coated substrate using porcine type I collagen as a comparison ECM.  Primers were generated from Invitrogen.

Alizarin red staining
In the same manner, cells were cultured in 12-well plates to day 10 at the concentration of 5×10 3 /mL (Low concentration) and 1×10 4 /mL (High concentration).
(Wako) for 20 minutes, then the cell monolayer was   Cells started to spread merely one hour after inoculation in Fn (10-50) (coated in either non-treated  ITGB1, also known as CD29, is able to associate with a number of alpha integrin to form various types of heterogeneous integrin dimers, a recent paper ITGB1 is essential for ameloblast differentiation and enamel formation 22 . In particular, among the genes tested, BSP and OCN were found to be enhanced by a much higher fold change on Fn(10-50) than PCOL300, which indicated that Fn possesses a stronger capacity of initiating odontogenic differentiation as compared with PCOL300. Indeed, a previous study using dog dental to exhibit odontoblastic phenotype in response to absence of exogenous inductive molecules 29 . The

Conclusion
The proliferation of MDPC-23 cells cultured on polystyrene preadsorbed with Fn was promoted.
Further, an early differentiation marker of odontoblast, grown on Fn-treated polystyrene. The expression of odontogenesis' markers during the differentiation and mineralization phase was increased by coated Fn.

Finally, the mineralization of cells was facilitated by
Fn as well. The results suggest that, a surface coating of polystyrene with Fn at the concentration of 10 μg/ mL or more effectively supported the proliferation, differentiation and mineralization of odontoblastlike cells; moreover, this inductive effects of Fn was achieved at a coating concentration markedly lower than type I collagen (300 μg/mL).