Periodontists

The horizontal alveolar bone resorption (ABR) area and the presence of periodontal intrabony defects were measured by histomorphometry as described previously [37] (link), . Briefly, the maxilla and mandible (n = 10−15) were immersed in 3% (vol/vol) hydrogen peroxide overnight after autoclaving and defleshing the maxillae and mandibles and stained in an aqueous solution of 0.1% methylene blue to delineate the cemento-enamel junction (CEJ) [37] (link), [38] (link), [39] (link). Digital images of both buccal and lingual root surfaces of all molar teeth were captured under a 10×stereo dissecting microscope (SteReo Discovery V8; Carl Zeiss Microimaging, Inc, Thornwood, NY), after superimposition of buccal and lingual cusps to ensure reproducibility and consistency. The line tool was used to measure the horizontal alveolar bone resorption from the CEJ to the alveolar bone crest (ABC). The surface perimeters of CEJ and ABC were traced using the calibrated line tool (AxioVision LE 29A software version 4.6.3.). Two blinded examiners performed all measurements twice at separate times. The means of the measurements were obtained for each of the two quadrants. Periodontal intrabony defects were detected under a 10×stereo dissecting microscope (SteReo Discovery V8) by an experienced periodontist (JL). The maxillae and mandibles were tilted and stabilized with dental wax to verify the presence of the intrabony defects in buccal and lingual surfaces. Only the presence or absence of intrabony defects was detected because the crevasses in the mouse jaw are too small to measure depth and width.

For this study, newly admitted consecutive patients (≥18 years of age) at the Academic Centre for Dentistry Amsterdam (ACTA) were recruited while attending for the first time the outpatient clinic. There was no (recent) dental or medical information available about these patients beforehand. The study was approved by the medical ethical committee of the Vrije Universiteit Medical Centre (2014.585 [A2016.155]). All patients with at least one natural tooth were eligible; edentulous patients, with or without full dentures, were not considered (regardless of dental implant support). First, the SROH questionnaire was conducted, followed by the oral rinse sampling. Next, a complete clinical periodontal examination was performed by one of two calibrated periodontists (SB and WJT). Finally, demographic data, such as age, sex and smoking status were derived from the electronic health records.

Tissue samples were obtained during surgical procedure. Cases had the following criteria: chronic periodontitis (Stage II to IV) with at least two remaining periodontal pockets in each sextant after nonsurgical periodontal treatment, probing depth of 5 mm or greater, bleeding on probing (BOP), and at least 3 mm of attachment loss needing surgical periodontal treatment [23 ]. Moreover, they were older than 18 years and had at least 16 teeth. Exclusion criteria were smoking, systemic diseases, history of consumption of antibiotic or anti-inflammatory drugs 3 months prior to surgical procedures, pregnancy and breastfeeding. Diagnosis of periodontitis was based on the clinical and radiographic examinations performed by a periodontist. Control samples were obtained from BOP sites of patients who underwent crown lengthening. The sites were examined by a periodontist and sites with no BOP and less than 3 mm probing depths were included. The study protocol was approved by ethical committee of Shahid Beheshti University of Medical Sciences (Ethic Code: IR.SBMU.DRC.REC.1398.086).

Dental plaque samples were collected by board-certified periodontists using sterile paper points at baseline prior to any dental treatment and labeled in numbers according to sampling sequences. The paper points were placed in the mesiobuccal sulci of the first molar in each quadrant for 1 minute and then immersed immediately in an Eppendorf tube containing 0.5 ml of Tris-EDTA (TE) buffer (pH 7.5) (Wang et al., 2009 (link)). Oral bacteria were harvested by centrifugation, and the bacterial pellet was resuspended in 100 µl TE buffer. Bacterial chromosomal DNA was released by two cycles of freezing at -80^°C overnight and boiling for 10 minutes.

Experimental and control regenerative surgeries were performed by a periodontist (BG) in a single surgical appointment. Depending on the width of the interdental space at the intra-bony defect site, either the modified papilla preservation technique (MPPT > 2 mm) or the simplified papilla preservation flap (SPPF when 2 mm or less) was used [14 (link),15 ,16 (link)]. After applying the appropriate infiltrative local anesthesia, a full thickness mucoperiosteal flap was raised, exposing the intra-bony lesion, which was thoroughly debrided from and the affected teeth carefully root planned. Then, randomization and treatment allocation were carried out by opening sealed envelopes containing the assigned treatment modality. Randomization was prepared using a computer-generated randomization list by an independent investigator and was concealed throughout the study from the clinical and radiographic examiners.
Once the experimental and control treatments were allocated, the surgeon filled the intra-bony lesion with either a frozen, radiation sterilized, allogenic, bone granules (FRSABG) in the test sites, or deproteinized bovine bone mineral granules (DBBM (Bio-Oss^®, Geistlich Biomaterials, Princeton, NJ, USA) in the control sites. Patients were blind to the treatment mode. In both sites, a porcine-derived bioabsorbable collagen membrane (Bio-Gide^®, Geistlich Biomaterials) was trimmed, closely filling the bone replacement graft and adapted to the anatomy of the defect. Flaps were then coronally repositioned without any tension (periosteum was released when necessary) to fully cover the regenerated site and secured with a combination of horizontal mattress sutures (Seralon 5/0 15 mm 3/8, Serag-Wiessner GmbH & Co., Naila, Germany) and a single vertical mattress suture positioned vertically in the inter-dental areas (Seralon 6/0 12 mm 3/8). Periodontal dressing was not used in any case.
Post-surgically, the patients were prescribed the use of anti-inflammatory medication (ibuprofen 600 mg twice a day for 2 days) and systemic antibiotic therapy (amoxicillin clavulanic acid (1 g) twice daily for 7 days). Patients also received postoperative instructions to avoid brushing or chewing for two weeks and to use an antiseptic rinse (0.2% chlorhexidine) for 3 weeks. Sutures were removed at 2 weeks when patients resumed careful brushing with a soft toothbrush. Patients were recalled every 2 weeks during the first 3 months postoperatively, and every 3 months for 1 year afterward.

All patients fulfilling the above eligibility criteria were subject to adjunctive orthodontic treatment using pre-adjusted fixed appliance (0.022-inch slot system, MBT, 3M Unitek, Monrovia, CA, USA), which was provided by the same experienced operator (Y.Y.). Individual treatment plans and goals were made via discussion between orthodontists and periodontists and then finalized with patients’ approval. Accordingly, orthodontic forces were delivered to teeth for designated movement. A standardized archwire protocol at the initial treatment stage was employed considering its effect on orthodontic pain [22 (link),35 (link),43 (link)], specifically, with a 0.014-inch thermal nickel–titanium (NiTi) wire (G&H Orthodontics, Franklin, IN, USA) for the first two months, followed by a 0.016-inch thermal NiTi wire (G&H Orthodontics, Franklin, IN, USA) for one month. By then, most teeth had achieved a preliminary alignment. Subsequently, 0.018-inch, 0.017 × 0.025-inch NiTi archwires (G&H Orthodontics, Franklin, IN, USA), and 0.017 × 0.025-inch stainless steel archwires (G&H Orthodontics, Franklin, IN, USA) were sequentially administered to the patients depending on individual circumstances. After anticipated treatment outcomes were achieved, fixed appliances were removed, and orthodontic treatment entered the retention phase with immediate delivery of polyvinyl chloride retainers and fixed-lingual retainers (0.0215-inch multistranded wire) attached to the anterior teeth.