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Effect of Cerebrospinal Fluid on Fibroblasts Concerning Epidural Fibrosis: An In Vitro Study

Doğa Gürkanlar
Doğa Gürkanlar
, 
Sevda Lafcı Fahrioğlu
Sevda Lafcı Fahrioğlu
 et 
Umut Fahrioğlu
Umut Fahrioğlu
   | 24 juil. 2021
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Article Category: Research Article - Medicine And Biotechnology
Publié en ligne: 24 juil. 2021
Pages: 100 - 108
DOI: https://doi.org/10.2478/ebtj-2021-0019
Mots clés
© 2021 Doğa Gürkanlar, Sevda Lafcı Fahrioğlu, Umut Fahrioğlu, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

CTG Assay results using PCS-201-012 cells at 24h,48h and 72h with 0%, 25%, 50%, 75% and 100% CSF. Three different CSFs were used in the experiments. All experiments were performed in quadruplicate. CSF 3 caused the highest amount of growth out of all the CSFs across all hours and all CSF percentages. There is inhibition of growth with the 75% and 100% but it is more prominent with 100% CSF. This can be seen in all of the time series. Two Way RM ANOVA with Holm-Sidak multiple comparison test was used for statistical analysis.
CTG Assay results using PCS-201-012 cells at 24h,48h and 72h with 0%, 25%, 50%, 75% and 100% CSF. Three different CSFs were used in the experiments. All experiments were performed in quadruplicate. CSF 3 caused the highest amount of growth out of all the CSFs across all hours and all CSF percentages. There is inhibition of growth with the 75% and 100% but it is more prominent with 100% CSF. This can be seen in all of the time series. Two Way RM ANOVA with Holm-Sidak multiple comparison test was used for statistical analysis.

Figure 2

All the CSF results were combined and presented as one CSF. Variation in cell number can be seen as a variation in luminescence (RLU). The cell number goes up from 24h to 72h but in 100% CSF proliferation is inhibited. Two Way RM ANOVA with Holm-Sidak multiple comparison test was used for statistical analysis.
All the CSF results were combined and presented as one CSF. Variation in cell number can be seen as a variation in luminescence (RLU). The cell number goes up from 24h to 72h but in 100% CSF proliferation is inhibited. Two Way RM ANOVA with Holm-Sidak multiple comparison test was used for statistical analysis.

Figure 3

Wound healing assay where an 80% confluent well was scratched with a pipet tip to create the “scar”. Cells are then allowed to close the scar under different CSF percentages. 0 hours and 96 hours after scar formation is shown above for 0%, 25% and 100% CSF. 25% CSF almost completely closes the wound. 0% CSF mostly closes the wound but patches of unclosed sections are still apparent. 100% CSF fails to close the wound. Some cells can be seen reaching into the wound.
Wound healing assay where an 80% confluent well was scratched with a pipet tip to create the “scar”. Cells are then allowed to close the scar under different CSF percentages. 0 hours and 96 hours after scar formation is shown above for 0%, 25% and 100% CSF. 25% CSF almost completely closes the wound. 0% CSF mostly closes the wound but patches of unclosed sections are still apparent. 100% CSF fails to close the wound. Some cells can be seen reaching into the wound.

For each given hour different CSF percentages were compared to each other. Mean difference of each comparison and its statistical significance is indicated. The degree of significance is indicated with the number of *, higher the number of * stronger the significance.

Holm-Sidak’s multiple comparison test Mean Diff. Significant? Summary
24 Hours
0 vs. 25 -1496 Yes ***
0 vs. 50 -1238 Yes ***
0 vs. 75 -516.8 No ns
0 vs. 100 392 No ns
25 vs. 50 258.1 No ns
25 vs. 75 979.5 Yes **
25 vs. 100 1888 Yes ****
50 vs. 75 721.4 Yes *
50 vs. 100 1630 Yes ****
75 vs. 100 908.8 Yes **
48 Hours
0 vs. 25 -1260 Yes ***
0 vs. 50 -1338 Yes ***
0 vs. 75 -496.3 No ns
0 vs. 100 908.9 Yes **
25 vs. 50 -78.19 No ns
25 vs. 75 763.9 Yes *
25 vs. 100 2169 Yes ****
50 vs. 75 842.1 Yes *
50 vs. 100 2247 Yes ****
75 vs. 100 1405 Yes ***
72 Hours
0 vs. 25 -1159 Yes **
0 vs. 50 -1301 Yes ***
0 vs. 75 -369.5 No ns
0 vs. 100 1307 Yes ***
25 vs. 50 -142.1 No ns
25 vs. 75 789 Yes *
25 vs. 100 2466 Yes ****
50 vs. 75 931.1 Yes **
50 vs. 100 2608 Yes ****
75 vs. 100 1677 Yes ****

Relative Luminescence Unit (RLU) values are shown for different CSF concentrations and different hours. Same letters in the columns indicate no statistically significant difference (p>0.05). Same symbols in the rows indicate no statistically significant difference (p>0.05). The variability due to the CSF percentages was statistically significant. Two Way RM ANOVA with Holm-Sidak multiple comparison test was performed for this analysis.

TIME
24 Hours 48 Hours 72 Hours
Mean ± SEM Mean ± SEM Mean ± SEM
CSF Concentration (%) 0 7237.93 ± 1536.22 a,d,# 8324.95 ± 1982.12 a,& 9103.60 ± 1653.54 a,*
25 8734.22 ± 1678.76 b,c,# 9585.13 ± 1942.57 b,c,& 10262.11 ± 1698.34 b,c,*
50 8476.16 ± 1509.59 c,# 9663.32 ± 2168.09 c,& 10404.24 ± 1916.68 c,*
75 7754.74 ± 1708.62 a,# 8821.20 ± 2048.36 a,& 9473.12 ± 1802.25 a,*
100 6845.98 ± 1568.58 d,# 7416.05 ± 1501.35 d,#,& 7796.31 ± 1660.38 d,&

Comparisons of different time points within different CSF percentages. Mean difference of each comparison and its statistical significance is indicated. The degree of significance is indicated with the number of *, higher the number of * stronger the significance.

Holm-Sidak’s multiple comparisons test Mean Diff. Significant? Summary
0% CSF
24 Hours vs. 48 Hours -1087 Yes ***
24 Hours vs. 72 Hours -1866 Yes ****
48 Hours vs. 72 Hours -778.6 Yes **
25% CSF
24 Hours vs. 48 Hours -850.9 Yes **
24 Hours vs. 72 Hours -1528 Yes ****
48 Hours vs. 72 Hours -677 Yes *
50% CSF
24 Hours vs. 48 Hours -1187 Yes ***
24 Hours vs. 72 Hours -1928 Yes ****
48 Hours vs. 72 Hours -740.9 Yes **
75% CSF
24 Hours vs. 48 Hours -1066 Yes ***
24 Hours vs. 72 Hours -1718 Yes ****
48 Hours vs. 72 Hours -651.9 Yes *
100% CSF
24 Hours vs. 48 Hours -570.1 No ns
24 Hours vs. 72 Hours -950.3 Yes **
48 Hours vs. 72 Hours -380.3 No ns
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