## Materials and Methods  
### Study Sample  
From a private clinic in Bergamo, Italy, 20 patients (10 male and 10 female, mean age 55) were randomly selected according to the following inclusion and exclusion criteria:  
All patients met the inclusion criteria described below. The inclusion criteria were:  
* Informed consent signed;
* Both male and female sexes;
* Ages between 18 and 65;
* Stage III periodontitis, according to the new classification of periodontal diseases [1];
* Good general health. The exclusion criteria were the following:  
* Pregnancy;
* Systemic diseases: liver disease, collagen disease, osteoporosis, chronic inflammatory disease, thyroid disease, diabetes;
* Medical condition or medical history that required antibiotic prophylaxis prior to treatment;
* Use of anti-inflammatory drugs or antibiotics within the past 6 months;
* Heavy smokers (more than 10 cigarettes per day);
* Periodontal surgical treatment in the last 12 months. They were treated between February 2019 and January 2020, and all of them signed the appropriate informed consent. The study population was randomly divided into 2 groups of 10 subjects each:  
* Group 1 (5 male, 5 female, mean age 53): patients treated following the LANAP protocol [7; 9; 11];
* Group 2 (5 male, 5 female, mean age: 57): patients treated following the OHLLT protocol [29; 30]. All procedures were performed by the same clinician, a periodontist with 30 years of clinical experience in both periodontal surgery and laser dentistry and post-graduate training in both fields. Patients belonging to Group 1 underwent a nonsurgical periodontal treatment session with LANAP protocol, while patients belonging to Group 2 had an OHLLT protocol, according to the operative protocols described below. After the first session of nonsurgical periodontal therapy, all patients were asked to refer to pain perception using a Numerical scale rate (NRS), ranging from 0 to 10, with specific intervals of time:  
- T0: 0 h;  
- T1 6 h;  
- T2: 12 h;  
- T3: 24 h;  
- T4: 48 h;  
- T5: 7 days. All patients were given verbal instructions on how to correctly assess the level of perceived pain to indicate on the scale a value from 0 or no pain to 10 or maximum imaginable pain. None of the analyzed patients used analgesics or anti-inflammatory during the postoperative phase. The collection, processing, and archiving of data took place through electronic questionnaires, which can be filled out online and made accessible to each participating researcher. The data were recorded electronically and analyzed. Furthermore, a microbiological test of the periodontal bacterial load was performed on all patients with real-time PCR tests before treatment (T0) and after 7 days (T5) (Figures 1 and 2). The subgingival plaque was removed from each patient at 2 separate sites in the oral cavity. The first sample was taken before the laser-assisted nonsurgical periodontal treatment (LANAP or OHLLT protocol), and the second took place 1 week after the end of the treatments. Figure 1: Subgingival plaque sampling with sterile paper cones. Figure 2: A sample of PCR Real-Time test results in one patient at T0 (in Italian language). The subgingival plaque collection was carried out following the following protocol: removal of the supragingival plaque, isolation of the operating field, insertion of a sterile paper cone in the periodontal pockets, insertion of the cones in the appropriate test tubes, fast shipment by courier and delivery of the sample to the laboratory of microbiology in good time. Each plaque sample arrived at the microbiology laboratory with a card containing information about the patient, including age, sex, date of collection, number of teeth, collection site, depth of periodontal pocket (PPD), and presence of bleeding or pus at the time of examination or collection. Oligonucleotide primers and probes were designed based on 16S rRNA gene sequences (845 entries in total) from the Human Oral Microbiome Database (HOMD 16S rRNA RefSeq Version 10.1, Forsyth Institute, Cambridge, MA, USA).