RESUMO
Scaffold engineering has attracted significant attention for three-dimensional (3D) growth, proliferation and differentiation of stem cells in vitro. Currently available scaffolds suffer from issues such as poor ability for cell adhesion, migration and proliferation. This paper addresses these issues with 3D porous chitosan scaffold, fabricated and functionalized with cysteine-terminated Arg-Gly-Asp (Cys-RGD) tri-peptide on their walls. The study reveals that the compressive moduli of the scaffold is independent to RGD functionalization but shows dependence on the applied freezing temperature (TM) during the fabrication process. The low freezing TM (-80°C) produces scaffold with high compressive moduli (14.64 ± 1.38 kPa) and high TM (-30°C) produces scaffold with low compressive moduli (5.6 ± 0.38 kPa). The Cys-RGD functionalized scaffolds lead to significant improvements in adhesion (150%) and proliferation (300%) of human mesenchymal stem cell (hMSC). The RGD-integrin coupling activates the focal adhesion signaling (Paxillin-FAK-ERK) pathways, as confirmed by the expression of p-Paxillin, p-FAK and p-ERK protein, and results in the observed improvement of cell adhesion and proliferation. The proliferation of hMSC on RGD functionalized surface was evaluated with scanning electron microscopy imaging and distribution though pore was confirmed by histochemistry of transversely sectioned scaffold. The hMSC adhesion and proliferation in scaffold with high compressive moduli showed a constant enhancement (with a slope value 9.97) of compressive strength throughout the experimental period of 28 days. The improved cell adhesion and proliferation with RGD functionalized chitosan scaffold, together with their mechanical stability, will enable new interesting avenues for 3D cell growth and differentiation in numerous applications including regenerative tissue implants.
RESUMO
This research demonstrates fluctuation of glutathione peroxidase1 (Gpx1) throughout cell cycle progression with significant decreased expression at mitosis of HeLa cell. This was achieved with western blot (WB) analysis of target proteins from each phase of synchronized cells. The synchronizations were performed with double thymidine (T/T) for G1/S arrest and thymidine followed by nocodazole (T/N) for G2/M arrest. The G1/S arrested cells were released in fresh medium for 3, 6, 9, 10, and 15h to obtain cell at each phase such as gap1 (G1), synthesis (S), gap2 (G2), mitosis (M), and gap1 (G1) phase, respectively, for investigating Gpx1 expression throughout a complete cycle. The synchronizations were confirmed using fluorescence activated cell sorting (FACS) and WB analysis of phase-specific markers. The fluctuations of Gpx1 expression were verified with universal protein actin and peroxiredoxin1 (Prx1) which are stable throughout the cell cycle. Intriguingly, immunoblots showed the level of Gpx1 decreases at mitosis phase and increased during mitotic exit to G1 phase in HeLa cells, while Prx1 protein level remained constant. The fractionation experiments reveal that only the cytosolic Gpx1 was decreased while their levels at mitochondria remain constant. The highest levels of mitochondrial ROS were measured in mitosis phase with FACS analysis using Mito sox indicating that antioxidant activity of Gpx1 for detoxifying excessive induced endogenous reactive oxygen species (ROS) in the mitosis phase could be the reason for such decreasing level. For unfolding the molecular mechanism of such decreased expression, the Gpx1 was investigated at transcriptional, translational, and proteosomal level. The results revealed that translational mechanism is involve in the decreased expression rather than transcriptional or proteosomal degradation at mitosis phase. This finding supports that Gpx1 is involved in the cell cycle progression through regulation of endogenous ROS. Based on this observation, further research could uncover their possible association with the infinitive division of a cancer cell.
