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

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.

Electrospun hyaluronate-collagen nanofibrous matrix and the effects of varying the concentration of hyaluronate on the characteristics of foreskin fibroblast cells.

In this study we propose a novel electrospinning fabrication process for the production of a nanofibrous matrix composed of collagen and hyaluronate. This procedure utilized 1,1,1,3,3,3-hexafluoro-2-propanol and formic acid as a mixed solvent. Fluorescence microscopy photographs revealed that the resulting electrospun nanofibers contained both collagen and hyaluronate. The mean diameter of the composite nanofibrous matrix (as observed using scanning electron micrographs) was approximately 200nm; this dimension is similar to that of native fibrous protein within the extracellular matrix. The expression of proteinases (e.g. matrix metalloproteinases, MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in epidermal repair during wound healing. Moreover, the characteristics of scarless wounds are known to be related to a decreased ratio of TIMP to MMP expression. In the present study the ratio of expression of TIMP1 to MMP1 was lower in foreskin fibroblast cells that were cultured on a hyaluronate-collagen composite nanofibrous matrix than in those cultured on an exclusively collagen nanofibrous matrix. This indicates that the hyaluronate-collagen composite nanofibrous matrix could potentially be used as a wound dressing for the regeneration of scarless skin.