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.
Highlights
Controlling cell behavior is of critical importance for tissue engineering, regenerative medicine, and the study of cellular molecules
Hypothesizing that the nanotopographic features of designed substrates are important for modulating cellular behavior, we previously showed that varying the nanotopography of a collagen matrix with and without the Dperiod affects the behavior of osteoblasts [5]
The electrospun collagen (EC) exhibited a circular dichroism spectrum indicating a massive loss of triple-helical collagen and suggesting random coils similar to those seen in electrospun gelatin (EG)
Summary
Controlling cell behavior is of critical importance for tissue engineering, regenerative medicine, and the study of cellular molecules. Considerable efforts have been made to develop scaffolds for tissue engineering. The ideal scaffold should be both biodegradable and bioactive, and it should mimic the structure and biological function of the native extracellular matrix as much as possible in terms of both chemical composition and physical structure. The native extracellular matrix contains structural protein fibrils such as collagen and elastin that range from tens of nanometers to micrometers in scale. These nanofibrils entangle with each other and form an organized structural matrix that guides tissue morphogenesis and remodeling in vivo. The matrix fibrils serve as a reservoir for growth factors and cytokines that regulate cell migration, proliferation, and differentiation [1]
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