## 2 Materials and Methods
### Subject Eligibility  
Subjects qualifying for the study met the following inclusion criteria:  
* Over 25 years of age;
* No known systemic pathology;
* No known allergy;
* Not alcohol or vitamin consumers;
* No periodontal treatment in the last 6 months. Patients were excluded for one of the following conditions:  
* Smoking;
* Pregnancy;
* Antibiotic intake within 8 weeks of the baseline examination. The initial study group included 88 subjects, divided into two groups of 44 subjects each (group I--test and group II--control). Because of the reduced sample size, the simple randomization method was used with computer-generated numbers assigned initially for every subject, then the numbers were associated randomly, either within the test or control groups. Group I (test) received Alflutop(r) after scaling, root planning, and debridement (SRP), and group II (control) only received SRP therapy. After SRP, group I cases received infiltrations with Alflutop(r) solution into the mobile mucosa of the affected sites, or, in case of generalized periodontitis, into the mobile mucosa of the canine-premolar area, 2-3 times per week until all 10 vials were finished (approximately 3-4 weeks). A topical anesthetic was previously administered. Periodontal status was assessed for both cases and controls, before and after one month of periodontal therapy, by a single-blinded examiner who did not discuss with the patients the type of periodontal treatment they received. The evolution of inflammation indices, tooth mobility, and maximum PD were recorded in the database for all subjects. Appointments were made for all patients, for every treatment visit, as well as one month after the last infiltration with Alflutop(r) (test group). Patients from the control group were scheduled for the second periodontal examination one month after SRP treatment. Both groups of patients received oral hygiene instructions, and the use of antiseptic solution or antimicrobial treatment was forbidden until the final clinical examination. The active ingredients of Alflutop(r) are based on anchovy (_Engraulis encrassichous ponticus_), sprot (_Sprattus sprattus sprattus_), and puzanka (_Rizaefca nordamanni_) extracts, purified and deproteinated. The chemical composition of this bioactive small marine fish concentrate is extremely complex, represented by glycosaminoglycans (GAG), proteoglycans, glycoproteins, hyaluronic acid (C\({}_{14}\)H\({}_{21}\)NO\({}_{11}\)), chondroite 6-sulfate (C\({}_{28}\)H\({}_{42}\)N\({}_{2}\)O\({}_{85}\)), chondroite 4-sulfate (C\({}_{13}\)H\({}_{21}\)NO\({}_{15}\)S), dermatan sulfate (C\({}_{14}\)H\({}_{21}\)NO\({}_{15}\)S), keratan sulfate (C\({}_{28}\)H\({}_{48}\)N\({}_{2}\)O\({}_{32}\)S\({}_{4}\)), low-molecular-weight polypeptides, free amino acids, myoinositol, glycerol-phospholipids, nitrogen, sulfur, phosphorus and microdelements (Na, K, Fe, Ca, Mg, Cu, Mn, Zn), and distilled water and phenol used as excipients [20]. From the test group, only 35 patients could finalize the research. The other 9 patients did not finalize the protocol for other reasons which were unrelated to the therapy results (2 patients did not present in time and could not finish all 10 infiltrations, and 7 patients finished the infiltrations, but they did not present for the final assessment one month after therapy). Among the test group, after SRP therapy, 14 patients did not come for the final clinical examination. One month after therapy, 9 patients did not come, and 5 patients presented some days later than scheduled, so their data were not included in the evaluation (Figure 1). The final study groups are group I (test) with 35 cases and control group II with 30 patients. The statistical analysis was performed using IBM SPSS statistics software version 20. Data are presented as mean \(\pm\) standard deviation (SD) or percentage for categorical variables. Descriptive statistics were performed within the two groups of cases and controls before and after therapy.