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In vitro response of retinal pigment epithelial cells exposed to chitosan materials prepared with different cross-linkers.

Lai JY, Li YT, Wang TP - Int J Mol Sci (2010)

Bottom Line: The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples.The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated.In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, 33302, Taiwan.

ABSTRACT
The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

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Apoptotic index of ARPE-19 cells exposed to various indicated chitosan membranes (5 mg) as determined by the TUNEL assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).
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f9-ijms-11-05256: Apoptotic index of ARPE-19 cells exposed to various indicated chitosan membranes (5 mg) as determined by the TUNEL assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).

Mentions: Figure 8 shows TUNEL analysis of the ARPE-19 cells after exposure to various chitosan membranes for 24 h. While TUNEL-positive cells were observed in the DNase-treated cultures (positive controls), no apoptotic activity was detectable in the negative controls (data not shown). Representative fluorescence microscopic images of the control, Chi and GP-chi groups demonstrated total cell nuclei stained with DAPI in blue (Figure 8A–C). In contrast, numerous TUNEL-labeled cells with fragmented DNA (green) could be visualized within the cultures from the GTA-chi groups (Figure 8D). The apoptotic index determined by TUNEL assay is shown in Figure 9. There was no statistically significant difference in the apoptotic index between the control, Chi, and GP-chi groups (p > 0.05). In the case of Chi and GP-chi groups, only approximately 1% of the cells were positively labeled with TUNEL. These results indicate that the non-cross-linked and GP cross-linked chitosan materials do not cause apoptosis in RPE cells. The proportion of apoptotic cells was significantly higher in cultures treated with GTA cross-linked chitosan membranes compared with those exposed to GP cross-linked samples (p < 0.05), suggesting that cross-linking of biomaterials with GTA may be relevant for apoptosis induction.


In vitro response of retinal pigment epithelial cells exposed to chitosan materials prepared with different cross-linkers.

Lai JY, Li YT, Wang TP - Int J Mol Sci (2010)

Apoptotic index of ARPE-19 cells exposed to various indicated chitosan membranes (5 mg) as determined by the TUNEL assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100825&req=5

f9-ijms-11-05256: Apoptotic index of ARPE-19 cells exposed to various indicated chitosan membranes (5 mg) as determined by the TUNEL assay. An asterisk indicates statistically significant differences (*p < 0.05; n = 3) as compared to controls (without materials).
Mentions: Figure 8 shows TUNEL analysis of the ARPE-19 cells after exposure to various chitosan membranes for 24 h. While TUNEL-positive cells were observed in the DNase-treated cultures (positive controls), no apoptotic activity was detectable in the negative controls (data not shown). Representative fluorescence microscopic images of the control, Chi and GP-chi groups demonstrated total cell nuclei stained with DAPI in blue (Figure 8A–C). In contrast, numerous TUNEL-labeled cells with fragmented DNA (green) could be visualized within the cultures from the GTA-chi groups (Figure 8D). The apoptotic index determined by TUNEL assay is shown in Figure 9. There was no statistically significant difference in the apoptotic index between the control, Chi, and GP-chi groups (p > 0.05). In the case of Chi and GP-chi groups, only approximately 1% of the cells were positively labeled with TUNEL. These results indicate that the non-cross-linked and GP cross-linked chitosan materials do not cause apoptosis in RPE cells. The proportion of apoptotic cells was significantly higher in cultures treated with GTA cross-linked chitosan membranes compared with those exposed to GP cross-linked samples (p < 0.05), suggesting that cross-linking of biomaterials with GTA may be relevant for apoptosis induction.

Bottom Line: The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples.The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated.In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, 33302, Taiwan.

ABSTRACT
The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

Show MeSH
Related in: MedlinePlus