MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix - PubMed (original) (raw)

MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix

Shiao-Wen Tsai et al. PLoS One. 2012.

Abstract

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Electron microscopic images of electrospun collagen matrix (EC) and electrospun gelatin matrix (EG).

Scanning electron microscopic images of (A) electrospun collagen matrix (EC), and (B) electrospun gelatin matrix (EG) at 2,500× magnification. The EC nanofibers had an average diameter of 692±214 nm, and the EG nanofibers had an average diameter of 573±368 nm. Scale bar is 20 µm. Transmission electron microscopic images of (C) electrospun collagen matrix, (D) electrospun gelatin matrix and (E) self-assembled collagen matrix. Scale bar is 0.2 µm. EC nanofibers did not exhibit the characteristic D-periods pattern that was apparent for the native collagen molecules self-assembled into collagen fibrils. Self-assembled collagen was formed by dialyzed collagen solution (in acetic acid) against 0.02 M phosphate buffered-saline (PBS, pH 7) at 4°C. Fibril formation (self-assembled) was initiated by warming the mixture to 37°C for 4 hours.

Figure 2

Figure 2. CD spectra of the different matrix preparations.

Circular dichroism spectra of the acid-solubilized materials plotted as mean residue ellipticity (mdeg×cm2×dmol−1)] vs. wavelength (nm). The triple-helical structure of native collagen have a characteristic positive peak at around 220 nm. The EC curve indicated a massive loss of triple-helical structure and similar to EG.

Figure 3

Figure 3. The tensile stress-strain curve of an electrospun collagen matrix and an electrospun gelatin matrix.

The Young's modulus of the EC and EG were 94.29±15.18 and 71.88±21.10 MPa, respectively. The ultimate tensile stresses the EC and EG were 1.93±0.37 and 0.93±0.46 MPa, respectively.

Figure 4

Figure 4. Quantification of MG63 osteoblast-like cells on EC and EG matrices.

WST assay quantifying cell attachment and proliferation on electrospun scaffolds of collagen and gelatin. (A) The attachment of MG63 osteoblast-like cells on various matrices after culturing for up to 4 hours. (B) The viability of MG63 osteoblast-like cells on various matrices after culturing for up to14 days. Data are presented as mean ± SD, n = 4. Statistical analysis was compared between EC and EG. There were no significant differences in cellular attachment or proliferation between EC and EG (_p_>0.05).

Figure 5

Figure 5. Different cellular morphology of cells on EC and EG matrices.

Fluorescent microscopic micrographs of MG63 osteoblast-like cells cultured on matrix for 4 hours. (A) electrospun collagen matrix. (B) electrospun gelatin matrix. Cells cultured on EC showed well-organized F-actin stress fibers. On the other hand, cells cultured on EG showed with numerous, very thin and long filopodia. Cytoskeletal F-actin is stained green with FITC and cell nuclei are stained blue with DAPI. (white arrow: filopodia, Scale bar = 10 µm)

Figure 6

Figure 6. Analysis of alkaline phosphatase activity of cells on EC and EG matrices.

The alkaline phosphatase activity of cells was found to be significantly higher on EC than on EG on day 21. Data are presented as mean ± SD, n = 4. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (p<0.05).

Figure 7

Figure 7. Matrices regulate the bone-associated genes expressed by MG63 osteoblast-like cells.

Real–time PCR analyses of bone-associated genes expressed by MG63 osteoblast-like cells on various matrix. (A) β-actin levels, (B) type I collagen levels, (C) OPN levels and (D) OCN levels after normalization to 18S ribosomal RNA levels. Data are shown as the fold change relative to the electrospun collagen matrix after 7 days of incubation. On day 7, the cells showed significantly higher expression levels of β-actin, type I collagen, OPN and OCN when grown on EC. And, the expression levels of β-actin, OPN and OCN were significantly higher on day 21 when grown on EC. Data are presented as mean ± SD, n = 4. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (p<0.05).

Figure 8

Figure 8. Effects of matrix on FAK and phospho-FAK and ERK1/2 activation in MG63 osteoblasts-like cell.

Densitometric quantitation of the protein bands was performed after normalizing to nucleophosmin B23 levels. (Figure S1 shown the images of full western blots). Data are shown as the fold change of the corresponding 2 h of incubation on the EC. The total FAK levels expressed by MG63 osteoblast-like cells were significant higher in cells grown on EC than in those grown on EG. Moreover, the level of FAK phosphorylation at tyrosine 397 was lower in cells grown on EG than in those grown on EC. Data are presented as mean ± SD, n = 3. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (p<0.05).

Figure 9

Figure 9. Effects of matrix on BSP and OPN protein expression in MG63 osteoblasts-like cell.

Western blot analysis for BSP and OPN proteins in cells cultured for (A) 14 and (B) 21 days. Densitometric quantitation of the OPN and BSP protein band were performed after normalizing to nucleophosmin B23 levels (Figure S2 shown the images of full western blots). Data are shown as the fold change of the corresponding on the electrospun collagen matrix. The expression level of BSP and OPN proteins showed a significant increase in cells grown on EC at 21 days. Data represent mean ± SD, n = 3. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (p<0.05).

Figure 10

Figure 10. Effects of matrix on bone mineralization in MG63 osteoblasts-like cell.

Optical images of ARS staining for mineralization in MG63 osteoblast-like cells for day 28 (A) EC, (B) EG. (C) EC with 10 µM Y27632, (D) EG with 10 µM Y27632. Quantification of mineral deposition by Alizarin Red-S staining. Data represent mean ± SD, n = 4. Statistical analysis was compared between EC and EG with or without Y27632. Different letters represent significance at p<0.05 by non-parametric Mann-Whitney U test.

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