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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Schwann cells play a critical role in the repair of the peripheral nerve. The goal of this study was to fabricate electrospun gelatin (Gel) and hyaluronan-gelatin (HA-Gel) composite nanofibers to provide a suitable growth environment for Schwann cells. The fiber diameters of Gel, 0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm, respectively. The biological performance of Gel and HA-Gel was evaluated using an in vitro culture of RT4-D6P2T rat Schwann cells. We found that the cell attachment and proliferation rates were not significantly different on these matrices. However, the Schwann cells displayed better organized F-actin on HA-Gel than on Gel. Moreover, the expression levels of several genes, including Nrg1, GFAP, and P0, were significantly higher on HA-Gel than on Gel. In addition, the levels of Nrg1 and P0 protein expression were also higher on the HA-Gel than on Gel. These results indicate that the hyaluronan-gelatin composite nanofibrous matrix could potentially be used in peripheral nerve repair.

Numerous studies about bone matrix fabrication focus on how the species and concentrations of components affect the cellular response. However, there are few studies that investigate how the related spatial arrangement of the components influences cellular activity. The aim of this work was to develop a novel method to biomimetically manufacture a three-dimensional mineral bone matrix and study the effect of apatite-collagen-chondroitin sulfate (CS) microspheres on the adhesion rate and activity of osteoblast-like cells. Although previous studies used a crosslinking agent or lyophilized methods to fabricated three-dimensional collagen microspheres, we produced beads composed of collagen and CS under mild reaction conditions. This process not only maintains collagen self-assembly into fibrils with a D-periodic pattern ability but also simultaneously introduces two major native bone matrix elements, collagen and CS, into the beads. Furthermore, we mimic the native in vivo bone matrix formation process by the direct nucleation and growth of apatite crystals on collagen fibrils. The apatite crystals are similar in composition to human bone mineral via X-ray diffraction and energy-dispersive X-ray spectrometric analysis. The cellular attachment rate of MG63 osteoblast-like cells is significantly higher for collagen-CS-apatite gel beads than for collagen-CS gel beads. In addition, with regard to the osteoblast bioactivity, we observed that alkaline phosphatase activity of MG63 cells on the collagen-CS-apatite gel beads higher than on the collagen-CS gel beads on day 14.

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