MG63 Osteoblast-Like Cells Exhibit Different Behavior when Grown on Electrospun Collagen Matrix versus Electrospun Gelatin Matrix

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.

Calcium Supplement Derived from Gallus gallus domesticus Promotes BMP-2/RUNX2/SMAD5 and Suppresses TRAP/RANK Expression through MAPK Signaling Activation

“…Previous studies have shown that the ERK signaling pathway plays a pivotal role in mediating the collagen or collagen-related peptide-induced osteogenic differentiation of osteoblast precursors or mesenchymal stem cells [45,46,47,48]. The present study demonstrated that the 1% Ca-supplemented diet promoted the activation of p-ERK and p-JNK signaling.…”

Periodic Mechanical Stress Stimulates the FAK Mitogenic Signal in Rat Chondrocytes Through ERK1/2 Activity

“…Tyrosine phosphorylation at different sites within FAK is catalyzed in distinct ways and affects various cellular functions. Previous studies regarding the FAK activation state in a variety of non-chondrocytic cell types under mechanical stress have mainly concentrated on the autophosphorylation site [25,26,27]. In a study on fibroblasts, Tyr 576/577 residues of FAK were shown to be required for the mechanical stress-induced cellular response [13,28].…”

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

“…Medium was replaced every 2–3 days. Mineralization was visualized using von Kossa phosphate staining [21], Alizarin Red S (Sigma-Aldrich) staining [22], or OsteoImage Mineralization Assay Kit (Lonza). Mineralization was quantified using a microplate reader for Alizarin Red S staining (405nm absorbance) and Lonza staining (492/520nm excitation/emission fluorescence).…”