## 5 Discussion  
This study aimed to investigate if vitamin C can improve the functional ability of human gingival fibroblasts (hGFs) extracted from smoking and non-smoking donors and then cultured on non-crosslinked (CopiOs) and crosslinked (BioMend) collagen membranes. The findings indicate that vitamin C has the potential to mitigate the detrimental impact of smoking products on gingival fibroblasts when cultivated in vitro on collagen membranes. Periodontal tissue regeneration using a barrier collagen membrane, called guided tissue regeneration (GTR), is a common clinical periodontal procedure. The principle of GTR is based on using a physical barrier to shield the affected area from undesirable tissue ingrowth, while guiding the repopulation of periodontal and bone cells [2]. Importantly, gingival fibroblasts have a vital role in periodontal regeneration. According to [PERSON] et al. [27], the utilization of an appropriate collagen membrane material has the potential to expedite the process of tissue repair by facilitating the attachment and growth of gingival fibroblasts. Collagen membranes are manufactured for clinical applications in two forms: native (non-crosslinked) or chemically processed (crosslinked). The chemical processing involves the introduction of covalent bonds to enhance the strength of collagen fibers [4; 9; 11; 28; 29]. Nevertheless, there has been limited research conducted on the impact of crosslinking collagen membranes on the bioactivity of gingival fibroblasts. Therefore, the present in vitro investigation examined the vitality and proliferation of gingival fibroblasts on a native collagen membrane (CopiOs) in comparison to a crosslinked collagen membrane (BioMend). The findings of our study revealed a notable increase in the fluorescence intensity of gingival fibroblasts when exposed to both BioMend and CopiOs collagen membranes. Furthermore, scanning electron microscopy (SEM) pictures revealed that gingival fibroblasts adhered to both membranes, corroborating the findings of the fluorescent test. It can be inferred that crosslinking does not inherently lead to a decrease in the proliferation of gingival fibroblasts. In the literature, several clinical studies have confirmed the negative role of nicotine, one of the particulate components of tobacco smoke, in periodontal health [30]. Smoking is a risk factor that increases the severity of periodontal tissue destruction and decreases the function of regenerative cells (e.g., gingival fibroblasts) [31]. Cigarettes are comprised of many toxic chemicals, including reactive oxygen species, which have the potential to directly impede the functioning of gingival fibroblasts [31]. In fact, the period of smoking exposure can differently inhibit the growth of hGFs. [PERSON] (1998) found that exposure for more than 9 weeks causes a decrease of 23% in hGFs growth. Therefore, the absence of correlation observed in the vitality of smokers' human gingival fibroblasts (hGFs) may be attributed to the intracellular impact of nicotine. It can be noted that nicotine acts on lysosomes by lowering the inner pH [32]. Additionally, hGFs appear to be differently sensitive to nicotine. This can be due to their high rate of replication or to the lower metabolic activity. In our in vitro work, gingival fibroblasts from smokers have different growth properties compared to fibroblasts from non-smokers, independent of the collagen membrane. Vitamin C is acknowledged as an antioxidant supplement that can combat the adverse effects of smoking products on periodontal cells [33; 34]. Our results showed that hGFs exhibited significant fluorescent intensities when seeded on a collagen membrane and before or after the addition of vitamin C. Furthermore, this can be interpreted as the result of hGFs' metabolic activity integrity and the exclusion of any cytotoxic effects, particularly from vitamin C addition.