Gum disease, including gingivitis and more advanced periodontitis, is a common condition that damages the tissues supporting the teeth. This study looks at how gum disease affects specific proteins inside gum tissue cells that help control how cells communicate between their internal structures and clean out damaged material (a process called autophagy). Researchers will collect small gum tissue samples from people with healthy gums, gingivitis, or periodontitis during dental procedures they are already having. These samples will be tested to measure the levels of three proteins (called PTPIP51, VAPB, and LC3B) and calcium. In a separate laboratory experiment, gum cells will be exposed to a substance made by bacteria commonly linked to gum disease, to see how these same proteins respond. The goal of this study is to better understand the biological changes that happen in gum tissue as gum disease develops, which may help guide future research into new treatments.

Periodontal disease encompasses a spectrum of inflammatory conditions ranging from gingivitis to periodontitis, both of which are driven by the host immune response to periodontopathogenic bacteria such as Porphyromonas gingivalis. Mitochondria-associated endoplasmic reticulum membranes (MAMs) are physical contact sites between the endoplasmic reticulum and mitochondria that regulate calcium exchange, lipid metabolism, and autophagy. The VAPB-PTPIP51 tethering complex is a key molecular determinant of MAM formation, and prior research has shown that alterations in this complex affect autophagosome formation and intracellular calcium homeostasis. However, the role of the VAPB-PTPIP51 complex and autophagy-related protein LC3B in periodontal disease has not been previously characterized.

This study includes two components. First, an observational, cross-sectional investigation was conducted in 60 participants classified into three groups (periodontal health, gingivitis, and periodontitis) according to the 2017 World Workshop classification criteria, based on clinical parameters including plaque index, gingival index, probing depth, clinical attachment level, and bleeding on probing. Gingival tissue biopsies were obtained from participants undergoing clinically indicated periodontal procedures (crown lengthening, gingivectomy, or flap surgery). Tissue samples were analyzed for gene expression (RT-PCR) and protein levels (Western blot) of PTPIP51, VAPB, and LC3B, as well as tissue calcium concentration.

Second, an in vitro experimental component was performed using a commercially available human gingival fibroblast cell line (HGF-1) treated with Porphyromonas gingivalis lipopolysaccharide (LPS) at 5 μg/mL and 10 μg/mL for 24 hours, to evaluate dose-dependent changes in PTPIP51, VAPB, and LC3B gene expression in response to a periodontopathogenic stimulus.

The findings from this study aim to clarify the involvement of the VAPB-PTPIP51 tethering complex and autophagy-related signaling in periodontal tissue destruction, providing mechanistic insight that may inform future therapeutic approaches targeting mitochondria-endoplasmic reticulum communication in periodontal disease.

Inclusion Criteria:

• Inclusion criteria for the Healthy Control group were: presence of ≥ 20 teeth in the mouth, probing pocket depth (PPD) ≤ 3 mm, the percentage of bleeding sites for the whole mouth < 10%, and, on radiographic examination, a distance of ≤ 3 mm between the cemento-enamel junction and the alveolar bone crest in 95% of all teeth. Inclusion criteria for the Gingivitis group were: presence of ≥ 20 teeth in the mouth, PPD ≤ 3 mm, % of bleeding sites for the whole mouth ≥ 10%, and, on radiographic examination, a distance of ≤ 3 mm between the cemento-enamel junction and the alveolar bone crest in 95% of all teeth. Inclusion criteria for the Stage 3 Periodontitis group were: presence of ≥ 15 teeth in the mouth, more than 30% of teeth affected by periodontal disease, affected teeth exhibiting probing depths of 6 mm or more and clinical attachment loss (CAL) of ≥ 5 mm, vertical bone loss of 3 mm or more, class 2 or 3 furcation involvement, and radiographic evidence of alveolar bone loss extending to the middle third and beyond (33%) in the relevant teeth

Exclusion Criteria:

Exclusion criteria for all groups were defined as: presence of any systemic disease, regular use of any medication, smoking, pregnancy or lactation, periodontal treatment within the last 6 months, antibiotic use within the last 6 months, and the presence of prosthetic restorations on the teeth to be sampled

Adults aged 18 to 60 years attending the periodontology clinic were enrolled and classified into three groups based on clinical periodontal parameters according to the 2017 World Workshop classification: periodontal health, gingivitis, and periodontitis (Stage III). Exclusion criteria included any systemic disease, regular medication use, smoking, pregnancy or lactation, periodontal treatment within the past 6 months, antibiotic use within the past 6 months, and presence of prosthetic restorations on the teeth to be sampled. A total of 60 participants (20 per group; 30 male, 30 female) were included.

De-identified individual participant data will be shared, including participant age, sex, and clinical periodontal parameters (plaque index, gingival index, probing depth, clinical attachment level, bleeding on probing). Participants will be identified only by coded/numbered case report forms; no names, gender identity beyond biological sex, or other identifying information will be shared.

Beginning 6 months and ending 24 months following article publication

Data will be made available to researchers who provide a methodologically sound proposal, subject to approval by the corresponding author, for the purpose of achieving the aims outlined in the approved proposal.