Minimally Invasive Non- Surgical Periodontal Therapy of Intrabony Defects: Healing Patterns and Smoking Impact

The aim of this study is to define the possible differences of molecular healing response between MINST and cNSPT and, also, between smokers and non-smokers after MINST and to examine whether smoking is associated with inferior clinical outcomes after MINST.

The specific objectives of our study are:

1. To compare the response of non-smokers after MINST and cNSPT in terms of inflammation and healing GCF biomarkers,
2. To compare the response of non-smokers and smokers after MINST, in terms of clinical and radiographic outcomes,
3. To compare the response of non-smokers and smokers after MINST, in terms of regeneration GCF biomarkers.
4. Research implementation
5. Randomized controlled clinical trial (RCT) study A statistical power analysis program (GPower 3.1, Franz Faul, Universität Kiel, Kiel, Germany) was used for the sample size calculation. Since there were no prior studies comparing MINST and cNSPT in terms of their GCF biomarker levels, the effect size was be defined based on previous research reporting significantly different gingival crevicular fluid levels of biomarkers between periodontitis and health. Firstly a parallel-group, single centre, examiner-blind randomized controlled trial will be conducted. Eighty systematically healthy, non-smoker patients with Periodontitis stage III or IV, grades B to C will be included. Each patient will have to present one intrabony defect site ( site with Probing Depth (PD)>5mm and radiographic defect depth > or =3mm). The patients will be randomized into one of the following periodontal treatment modality groups regarding the intrabony defect:

• Group A: cNSPT
• Group B: MINST The patients will receive periodontal cause related therapy, including comprehensive oral hygiene instructions and full- mouth conventional NSPT, namely Phase I therapy with piezoelectric devices (EMS, Switzerland) and hand instruments (Gracey, Hu Friedy) under local anaesthesia. Specifically in the intrabony defects included, the intervention will be performed according to each patient's group allocation. At 5 days and 3 months following intervention, GCF samples will be collected from the intrabony defects in order to assess the molecular healing patterns through intergroup comparisons of inflammation and healing biomarkers.

1. Prospective cohort studies Subsequently patients of Group B will be matched with forty smoker patients with Periodontitis stage III or IV, grades B to C for age and sex, with each of them presenting one intrabony defect site ( site with Probing Depth (PD)>5mm and radiographic defect depth > or =3mm). These patients (Group C) will receive the same full-mouth periodontal treatment management as Groups A and B and regarding the intrabony defects included, MINST interventions will be performed. A prospective cohort study will be conducted to assess the differences in treatment outcomes after MINST in smokers and in non-smokers, by comparing clinical and radiographic data obtained by clinical and radiographic examinations performed in both groups' intrabony defects pre-treatment and 6 months following the intervention. Also, a prospective cohort study will be conducted to compare the molecular healing patterns in smokers and non-smokers after MINST, via assessment of the differences in regeneration biomarkers in GCF samples obtained from the intrabony defects 5 days and 3 months post-treatment.
2. Participants All patients who will be included in the study will be periodontal patients seeking treatment at the Department of Periodontology, 251 Hellenic Airforce General Hospital, Athens, Greece. All participants should be free of any systemic or oral conditions that could affect disease progression or treatment outcomes. Thus reasons for exclusion will be: medical history including diabetes, hepatic or renal disease, history of conditions requiring prophylactic antibiotics, anti-inflammatory or anticoagulant therapy the preceding month, systemic antibiotic therapy the preceding 3 months, history of alcohol or drug abuse, self-reported pregnancy or lactation, other severe acute or chronic medical or psychiatric condition, periodontal treatment within the last 12 months and presence of drug-induced gingival overgrowth. As smokers will be defined patients who are current smokers and smoke at least 10 cigarettes per day. As non-smokers will be considered patients who have never smoked or who have quit smoking for 5 years at least.
3. Ethical Considerations These studies will be conducted in full accordance with the guidelines of the Declaration of Helsinki. Ethical approval will be requested from the Ethics and Research Committee of the 251 Hellenic Airforce General Hospital, Athens, Greece (date of application 09/2024).
4. Clinical Intra-oral Examination Plaque index (PI), gingival index (GI), bleeding on probing (BoP), probing depth (PD), gingival recession (REC) and clinical attachment level (CAL) will be recorded at the involved tooth site and also full mouth to establish a diagnosis by a single-blinded, calibrated examiner, using a computerized pressure sensitive probe (Florida Probe).
5. Radiographic examination Periapical digital radiographs will be taken using the parallel cone technique with a customized radiographic stent. Radiographic measurements will be made using a software programme (ImageJ, Oracle Corporation, San Jose, California) by a single-blinded, calibrated examiner and will include: the distance between the tooth cementoenamel junction (CEJ) and the bottom of the intrabony defect, the distance between the CEJ and the alveolar bone crest, the depth of the intrabony defect and the angle of the intrabony defect (defined by the lines of the tooth root surface and the defect's bone wall)21.
6. GCF Collection and analysis GCF sampling will be performed for 30s using Periopaper (OraFlow Inc., New York) placed at the entrance of the sulcus. Volumes of GCF samples will be determined with a Periotron (OraFlow Inc., New York) device immediately after the sample collection. Afterwards, the samples will be stored dry in Eppendorf tubes at -80°C s until the day of analyze. At the day of analyze, GCF will be eluted in 200μl of PBS (with 0.1% BSA). Biomarker levels in GCF samples will be determined by Luminex method using commercial kits.
7. MINST and cNSPT treatment interventions in the intrabony defects MINST treatment protocol will include: local anaesthesia by infiltration without adrenaline, thorough debridement of the root surface through a subpapillary access using exclusively piezoelectric devices with specific thin and delicate tips (EMS, Switzerland) and 4X magnification loupes, taking care to minimize the soft tissue trauma and to stimulate the formation of a stable blood clot22. cNSPT treatment protocol will include thorough debridement of the root surface under local anaesthesia, using conventional piezoelectric devices (EMS, Switzerland) and hand instruments (Gracey, Hu Friedy).
8. Statistical analysis Following the detection of the data distribution with the Kolmogorov-Smirnov test, repeated t-test or ANOVA in case of normal distribution will be used in the statistical analysis, and the Mann-Whitney U or Kruskal Wallis tests in case of non-normal distribution. Repeated Measures ANOVA and Linear Mixed Models will be used to compare parameters between visits. In determining the correlation between chemokine concentrations and clinical parameters, the parametric Pearson test or the non-parametric Spearman test will be used according to the distribution.