Can Vitamin C Improve Proliferation and Viability of Smokers’ Gingival Fibroblasts on Collagen Membranes? An In Vitro Study

Abstract

Periodontal regeneration using a barrier membrane can be affected by several factors, including patient-related factors (such as smoking habits), surgical techniques, and type of barrier membrane. Smoking exposure has a negative impact on the periodontium due to its direct inhibition of gingival fibroblast function. Vitamin C is widely recognized as an antioxidant that can be used to mitigate the detrimental impact of smoking products on periodontal cells. This study aimed to investigate whether vitamin C could improve the proliferation and viability of gingival fibroblasts extracted from smoking and non-smoking donors and then cultured on non-crosslinked (CopiOs Pericardium) and crosslinked (BioMend) collagen membranes. To address this aim, human gingival fibroblasts were extracted from healthy periodontium of smoker patients (Group 1) and non-smoker patients (Group 2). The cells were cultivated and subsequently subcultured in a growth medium supplemented with the required nutrients. Subsequently, the medium at passage six was supplemented with vitamin C, i.e., at the start of the experiment. An evaluation of cell proliferation and viability was carried out using cell migration assays and AlamarBlue® assays for cells grown on BioMend and CopiOs Pericardium collagen membranes. Assessment of the morphology and attachment of gingival fibroblasts to the experimental collagen membranes was conducted using scanning electron microscopy (SEM). The viability and proliferation assessments of hGFs from the migration assay were evaluated using AlamarBlue®. The results exhibited significant fluorescent intensity of gingival fibroblasts on both membrane groups (BioMend and CopiOs Pericardium) in the smoker group compared to the non-smoker group (p < 0.05), which was interpreted to be the result of hGF metabolic activity and the exclusion of any cytotoxic effects, particularly from vitamin C addition. Vitamin C positively affected cells from the smoker group with statistically significant results in the BioMend group (Wilcoxon signed-rank test of p value < 0.05; p = 0.028). SEM images revealed the crosslinking pattern of the BioMend membrane and the non-crosslinked natural tissue structure of the CopiOs Pericardium membrane, which 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 in all samples, mainly in the peripheries, were observed. It can be concluded that the addition of vitamin C to collagen membranes in vitro seems to combat the adverse effects of smoking products on gingival fibroblasts.