Chitosan delaying human fibroblast senescence through downregulation of TGF-β signaling pathway

<p>This study evaluated the effect of chitosan, poly vinyl alcohol (PVA) and poly (2-hydroxyethyl methacrylate) (pHEMA) on delaying the human fibroblast senescence. Cells could form suspending multicellular spheroids on these biomaterials, but only chitosan was capable of decreasing the SA β-gal activity and increasing the proliferation ability of senescent fibroblasts. Therefore, in addition to the structure of multicellular spheroids, chitosan itself should play an important role in delaying fibroblast senescence. The main difference of senescence-related protein expressions for cells cultured on chitosan, PVA and pHEMA occurred on the TGF-β signaling pathway. In addition to the intracellular TGF-β expression, the extracellular TGF-β expression was also downregulated. Chitosan with cationic amino structure was assumed to bind with anionic TGF-β by forming polyelectrolyte complexes. This assumption was demonstrated by directly adding chitosan into the medium to downregulate the cell TGF-β expression and further to delay cell senescence, indicating TGF-β signaling pathway was involved in the chitosan-mediating fibroblast senescence process. Finally, the delaying cell senescence ability of chitosan increased with increasing the amount of amino groups in chitosan and its ionization degree. In summary, these results provide important information for considering the application of chitosan in the future cell therapy and regeneration medicine.</p

Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains

<div><p>Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.</p></div

Fibroblasts sorted by FSC and FITC.

<p>(a) The gating range of cell sorting. P1: PD45 (+, +) population. P2: PD45 (-, -) population. (b) Viability of PD45 (+, +) cells determined by PI uptake and annexin-V-FITC labeling. (c) SA-β-gal staining of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells. Scale bar = 200 μm (d) The percentage of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells stained for SA-β-gal. At least 400 cells were calculated from ten randomly selected fields for each case. **p<0.01. (e) BrdU incorporation assay of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells (n = 4). *p<0.05.</p

Fibroblast spheroids were reseeded on TCPS after chitosan treatment.

<p>(a) SA-β-gal staining of PD48-Cd3-PD2 cells. Scale bar = 200 μm (b) The percentage of PD50 and PD48-Cd3-PD2 cells stained for SA-β-gal. At least 400 cells were calculated from ten randomly selected fields for each case. **p<0.01. (c) BrdU incorporation assay of PD50 and PD48-Cd3-PD2 cells (n = 4). *p<0.05. (d) Cell cycle (PI staining) analysis of PD52 and PD48-Cd3-PD2 cells. (e) The PD curve with and without chitosan treatment (n = 3). Red arrows indicate PD48 (1st treatment) and PD70 (2nd treatment) cells were seeded on chitosan for 3 days and reseeded on TCPS for serial passages.</p

Effect of chitosan treatment on scratch wound assay.

<p>(a) Both wounds were gradually filled with PD50 and PD48-Cd3-PD2 cells. (b) The percentage of wound closure by PD50 and PD48-Cd3-PD2 cells (n = 5). **p<0.01.</p

The characteristics of fibroblasts after chitosan treatment.

<p>(a) Western blot results of senescence-associated protein pRB, p16, p21 and p53 expression in PD20, PD50, and PD48-Cd3-PD2 cells. (b) Colony formation of HeLa and PD45-Cd3-PD2 cells by soft agar assay.</p

The characteristics of fibroblast spheroids after chitosan treatment.

<p>(a) Western blot results of proteins pRB, p16, p21, p53, TGF-β, IL-1β, IL-6 and IL-8 expression in PD48 and PD48-Cd3 cells. (b) The relative amount of protein pRB, p16, p21, p53, TGF-β, IL-1β, IL-6 and IL-8 expression in PD48 and PD48-Cd3 cells. (*p<0.05 and **p<0.01, n = 5). (c) BrdU incorporation assay of PD10, PD48, PD48-Cd3, and H₂O₂-treated cells (**p<0.01, n = 5).</p