## 2 Materials and Methods
### Clinical Pilot Study  
The protocol of the study, informed consents, and the subject's information sheets were approved by the local research ethic committee (Bioethical Commission at the Medical University of Wroclaw) (decision number KB 98/2016): ClinicalTrials.gov Identifier: NCT03791099. Clinical assessment was performed on the selected matrix based on hydroxyethyl cellulose (HEC2) according to the protocol (Figure 1). Twenty-three generally healthy patients were qualified for the study (14 women and 9 men, average age of 51). Enrollment started in April 2018, lasted three months, and finished in July 2018. Patients were divided into two groups--the test group (8 women and 4 men) and control group (6 women and 5 men). The patients had been treated at Wroclaw University Department of Periodontology for periodontitis (stadium 2 or 3, grade B) [30]. In each group, fifteen periodontal pockets were examined. The selected patients were nonsmokers and had not taken any antibiotics (applied generally or locally) for the past 6 months. The patients had \(\geq\)2 interproximal sites with CAL \(\geq\) 6 mm (not on same tooth) and \(\geq\)1 interproximal site with PD \(\geq\) 5 mm. In order to rate the oral cavity hygiene, Approximal Plaque Index (API by [PERSON] et al. (1977)) was used and plaque presence in interproximal sites was evaluated [31]. To assess the gum inflammation, the Bleeding on Probing (BOP by [PERSON] and [PERSON] (1975)) index was used [32]. Gum inflammation intensity was assessed with the use of the Papilla Bleeding Index (PBI, [PERSON] (1975)), [33]. Periodontal tissue degradation was assessed by the measurement of periodontal pockets depth (PPD, probing pocket depth) and by the measurement of clinical attachment level (CAL). The probing was carried out with the use of a calibrated 1-mm periodontal probe (PCP UNC-15; Hu-Friedy, Chicago, IL, USA). Six measurement points were marked: meso-buccal, buccal (i.e., mid-buccal), disto-buccal, meso-palatal, palatal (i.e., mid-palatal), disto-palatal (upper jaw), meso-lingual, lingual (i.e., mid-lingual), and disto-lingual (lower jaw). Periodontal examination was performed by one trained and calibrated periodontitis (M.S.) at baseline and at follow-up time points. Calibration was performed on 10 patients who were not recruited to the study. Examiner reliability was high with agreement in assessment on all clinical parameters above 85%. The clinical examination was performed three times. On the first (qualification) visit, the output indicators were defined. During the first visit, a subgingival ultrasonic scaling was performed. For every patient, the deepest periodontal pocket was identified and, in the test group, metronidazole in polymer matrix was applied to the selected periodontal pocket, as shown in Figure 2. There was no application of the matrix in the control group. Figure 1: Structure flowchart of the clinical assessment of metronidazole-loaded matrix (HEC2). Two control tests of periodontal indices were carried out one week and one month following application [34; 35]. During each visit the samples from selected, deepest periodontal pockets were collected (one in every patient) in order to check the existence of Porphyromonas gingivalis DNA, the bacteria of which is known as major periodotopathic bacterium [36]. After drying the site and isolation from saliva, a sterile paper point was inserted into the pocket for 10 s, then transferred to a sterile Eppendorf tube, and sent to the laboratory. Molecular biology was carried out in the Laboratory of Scientific Research of Molecular Biology, Department of Pathology, Oral Faculty of Dentistry. Genomic DNA from clinical specimens was obtained by a modified etrimonium bromide (CTAB) method [37; 38]. Every paper point taken from the gingival pockets was stored at -20 \({}^{\circ}\)C. During DNA isolation, they were placed in a 1.5-mL Eppendorf tube and 100 \(\mu\)L of sterile water was added and stirred in a vortex apparatus.