## 4 Results  
The viability and proliferation assessments of hGFs from the migration assay were evaluated using AlamarBlue(r). All membrane groups (BioMend and CopiOs Pericardium) exhibited increased fluorescence intensities (Table 2), with significantly higher results in the smoker group membranes compared to those of the non-smoker group (\(p<0.05\)) (Figure 2). A higher activity was observed in all pericardium membrane models compared to BioMend membranes, regardless of the cell source and treatment induction. The Wilcoxon signed-rank test showed a statistically significant increase in the mean viability of cells cultured on BioMend collagen membranes after treatment with vitamin C (Bio-C) 6411.0 \(\pm\) 401.1 when compared to the mean before treatment with vitamin C (Bio-N) 6055.2 \(\pm\) 339.6 (\(p\) = 0.028; \(p\) value \(<\) 0.05) among smokers. However, there was no statistically significant difference among the non-smoker BioMend group after treatment with vitamin C (Bio-C) (\(p\) = 0.917). \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{2}{c}{**Group**} & **n** & **Mean \(\pm\) (SD)** & \(p\)**-Value \({}^{*}\)** \\ \hline \multirow{8}{*}{Non-smokers} & Assay control & 6 & 4173.5 \(\pm\) (532.8) & - \\ \cline{2-5}  & Bio-N-N & 6 & 4422.5 \(\pm\) (1394.1) & \multirow{2}{*}{0.917} \\  & Bio-N-C & 6 & 4719.8 \(\pm\) (515.6) & \\ \cline{2-5}  & Peri-N-N & 6 & 5659.0 \(\pm\) (600.4) & \multirow{2}{*}{0.028} \\  & Peri-N-C & 6 & 6329.8 \(\pm\) (593.0) & \\ \hline \multirow{8}{*}{Smokers} & Assay control & 6 & 4399.5 \(\pm\) (896.4) & - \\ \cline{2-5}  & Bio-S-N & 6 & 6055.2 \(\pm\) (339.6) & \multirow{2}{*}{0.028} \\ \cline{1-1}  & Bio-S-C & 6 & 6411.0 \(\pm\) (401.1) & \\ \cline{1-1} \cline{2-5}  & Peri-S-N & 6 & 8012.8 \(\pm\) (112.9) & \multirow{2}{*}{0.028} \\ \cline{1-1}  & Peri-S-C & 6 & 7349.0 \(\pm\) (360.7) & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparison of native (BioMend) and crosslinked (CopiOs) membranes before and after treatment with vitamin C. \({}^{*}\) Using Wilcoxon signed-rank test, statistical significance (\(p\)-value) set at 0.05; Bio = BioMend crosslinked membrane; Peri = CopiOs Pericardium membrane; N = without vitamin C treatment; C = with vitamin C treatment. SEM images revealed the crosslinked pattern of the BioMend membrane and the non-crosslinked natural tissue structure of the CopiOs Pericardium membrane (Figure 3A,B). The original microscopy patterns of both membranes' structures did not change, regardless of whether the cultured smoker or non-smoker hGFs were treated with vitamin C. Small numbers of attached hGFs in membrane matrices of all samples for all models, mainly in the peripheries, were observed. There were no significant differences in the number of attached fibroblasts between samples from smokers and non-smokers after vitamin C treatment. Additionally, SEM analysis of BioMend and pericardium membranes did not demonstrate a marked increase in the number of hGFs on the surface (Figure 4A-D and Figure 5A-D). Although SEM images of pericardium membranes revealed fewer attached hGFs than images of BioMend membranes, the results were not statistically significant. All models showed limited numbers of flat polyhedral cells with long cytoplasmic extensions attached to the membrane surfaces. Figure 3: Scanning electron microscopy (SEM) of non-smoker hGFs on different membranes before treatment with vitamin C. (**A**) Representative SEM image of BioMend membrane cultured with untreated non-smoker hGFs (magnification \(\times\)160 at 100 \(\upmu\)m scale). (**B**) Representative SEM image of CopiOs Pericardium membrane cultured with untreated non-smoker hGFs (magnification \(\times\)22 at 1 mm scale). Figure 2: Absorption rate of experimental smoking and non-smoking groups before and after treatment with vitamin C as measured by fluorescent rate (\(y\)-axis). Mann–Whitney U test was used for independent group comparisons.