## 5 Discussion
When comparing smokers to non-smokers before and after treatment with vitamin C, the Mann-Whitney U test showed that cells from smokers had statistically significantly higher results on both collagen membranes. However, the addition of vitamin C was markedly greater in smoker hGFs with the BioMend membrane compared to non-smokers' cells, which indicated the positive effect of vitamin C addition on the efficacy of the membrane in the smoker environment. The current findings illustrate the promising potential of using collagen membranes as carriers for vitamin C.  
The current study focused on the in vitro behavior of hGFs extracted from healthy gingiva of smoker patients compared to non-smokers, and then seeded on two types of collagen membranes (i.e., BioMend and CopiOs). In terms of hGF attachment to the membranes, SEM analysis revealed that both membranes' topographies supported cell adhesion. The limited number of flat polyhedral cells with long cytoplasmic extensions attached to the membrane surfaces may have been caused by the short incubation duration, which was 3 days in this experiment; another reason may be that cells were lost in the process of preparation and fixation. Similar results were observed in another experiment that involved a crosslinked membrane [35]. The AlamarBlue viability assay was used to assess the viability and proliferation of the cultured cells on the collagen membranes using a migration assay assembly. Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) is the active constituent in the AlamarBlue assay. It is stable in culture medium, non-toxic, and water-soluble. It can also permeate cell membranes. Therefore, it is possible to monitor cultured cells in a continuous manner [36]. Cells can change the dye from an oxidized to a reduced state through many enzymes, such as mitochondrial enzymes. The inability of the cells to reduce the dye may signify an impairment of cellular metabolism [36]. This in vitro study has several limitations. The short incubation period limited our assessment of hGF behavior to a single point in time. Cell growth, proliferation, attachment, and adhesion are better understood if we examine them at different time points to reveal their behavior toward different membrane surfaces and to explore the potential effect of vitamin C on these processes. Secondly, the SEM images did not show the attachment of hGF to the membranes. This could be avoided by using Osmium tetroxide in the fixation protocol of SEM. However, we could not follow this protocol for logistical and safety reasons in our laboratory. Another limitation is the use of the Resazurin reduction assay (AlamarBlue(r)) to assess cell viability and proliferation. The use of another cell viability assay that is more sensitive, less prone to artifacts, and faster, like the ATP assay, would yield more precise results for this study. Lastly, this in vitro study examined one type of cell line, fibroblasts, and evaluated only their migration and proliferation in association with two kinds of membranes. Evaluating other cell types involved in periodontal regeneration and other aspects of their behavior during wound healing like differentiation and interaction with other cells will add better insight to this research.