Pharmaceutical applications of chitosan in skin regeneration: A review.

Skin regeneration is the process that restores damaged tissues. When the body experiences trauma or surgical incisions, the skin and tissues on the wound surface become damaged. The body repairs this damage through complex physiological processes to restore the original structural and functional states of the affected tissues. Chitosan, a degradable natural bioactive polysaccharide, has attracted widespread attention partly owing to its excellent biocompatibility and antimicrobial properties; additionally, a modified form of this compound has been shown to promote skin regeneration. This review evaluates the recent research progress in the application of chitosan to promote skin regeneration. First, we discuss the basic principles of the extraction and preparation processes of chitosan from its source. Subsequently, we describe the functional properties of chitosan and the optimization of these properties through modification. We then focus on the existing chitosan-based biomaterials developed for clinical applications and their corresponding effects on skin regeneration, particularly in cases of diabetic and burn wounds. Finally, we explore the challenges and prospects associated with the use of chitosan in skin regeneration. Overall, this review provides a reference for related research and contributes to the further development of chitosan-based products in cutaneous skin regeneration.

miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.

MicroRNAs play an important role in the adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). How miR-100-3p influences such adipogenesis, however, remains uncertain. In this study, hMSC adipogenic differentiation was associated with miR-100-3p downregulation, and overexpressing this miRNA inhibited adipogenesis and the expression of adipogenic marker genes. Through bioinformatics approaches, miR-100-3p can bind the 3'-untranslated region (3'-UTR) of the mRNA encoding phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) such that miR-100-3p overexpression resulted in significant reductions in PIK3R1 expression. Importantly, overexpressing PIK3R1 was sufficient to reverse the anti-adipogenic effects of miR-100-3p overexpression. PIK3R1 is a critical component of the PI3K/AKT signaling pathway, and miR-100-3p overexpression resulted in reduced AKT phosphorylation in the context of adipogenesis. In addition, the adipogenic differentiation of hMSCs in which miR-100-3p was overexpressed was further enhanced upon treatment with the PI3K/AKT agonist 740Y-P relative to miR-100-3p overexpression alone. Taken together, these findings provide evidence that miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates.

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

A microwell pattern for C17.2 cell aggregate formation with concave cylindrical surface induced cell peeling.

We have developed a polydimethylsiloxane (PDMS) pattern with arrays of microwells for the formation of multicellular aggregates by C17.2 neural stem cells. Upon interfacing with the patterns, the neural stem cells would firstly attach to the microwell sidewalls, forming cellular strips on day 1 after plating. For channel connected microwells, cellular strips on the concave semi-cylindrical sidewall surfaces continued among wells and through channels, followed by strip peeling due to prestress arising from actin filaments and assembly of suspending cellular aggregates within the microwells in the following 1-2 days. Our results also suggested that a small microwell diameter of 80 and 100 µm and a narrow channel width of 20 µm would facilitate the aggregate formation among the structural dimensions tested. Finite element method (FEM) simulation revealed that cellular strips on the semi-cylindrical sidewall surfaces peeled under significantly smaller prestresses (critical peeling prestress, CPP), than cells on flat substrates. However, the CPP by itself failed to fully account for the difference in aggregate inducing capability among the patterns addressed, suggesting cell growth behaviors might play a role. This study thus justified the current patterning method as a unique and practical approach for establishing 3D neural stem cell-based assay platforms.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid).

INTRODUCTION: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). METHODS AND MATERIALS: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. RESULTS: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. CONCLUSION: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

[Secretion and expression of vascular endothelial growth factor and interleukin-8 by SH-SY5Y human neuroblastoma cells].

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.