Trial design

A randomized controlled clinical split-mouth trial was conducted, in which the experimental side received the alveolar preservation treatment with L-PRF membranes and the other side served as a control. The criteria used to report the results were derived from the Consolidated Standards of Reporting Trials ^{, , ,} guidelines and the Cochrane risk of bias tool for assessing the methodological quality of randomized trials.

Participants, eligibility criteria, and settings

The inclusion criteria were as follows: (1) a minimum age of 20 years and requiring orthodontic treatment and (2) indication of extraction of the maxillary first premolars for treatment. All patients signed an informed consent before treatment initiation.

The exclusion criteria were as follows: (1) the presence of autoimmune diseases, (2) pregnancy or lactation, (3) use of long-term medications (antibiotics, antihistamines, cortisone, or hormones) that could interfere with the inflammatory response process or exert an adverse effect on the periodontal ligament in the 6 months before the initiation of the study, and (4) the presence of systemic diseases.

The sample consisted of 21 healthy young adult patients diagnosed with Class I or Class II Division 1 malocclusion. All patients needed orthodontic treatment with extraction of the maxillary first premolars.

Interventions

All patients were treated with a 0.022-in preadjusted MBT bracket slot system (Abzil Kirium, 3M, São Paulo, Brazil). The alignment and the leveling phases were performed using the following sequence: 0.014- and 0.016-in nickel titanium arches, and 0.018- and 0.020-in stainless steel arches. On the day of the fitting of the 0.020-in stainless steel arch in the maxillary arch ( Fig 1 ), a referral was made for the removal of the maxillary first premolars by the orthodontist at the Department of Periodontics. On the same day, the first computed cone-beam computed tomography (CBCT) corresponding to the beginning of the retraction phase (T1) was also requested of the patient. A CBCT at T1 was performed a few days before the surgical intervention and was delivered to the orthodontist. All CBCTs were performed with a Planmeca ProMax 3D Max (Planmeca, Helsinki, Finland), with a field of view of Ø 23 × 26 cm, exposure time of 18-30 seconds, voxel size of 0.200 mm ³ , reconstruction time of 30-150 seconds, and radiation of 101-252 mSv.

Intrabuccal images of the alignment and leveling phases.

On the day of the surgical procedure, 10 mm of blood were collected before the extraction. BD Vacutainer Safety-Lok blood collection sets (Becton, Dickinson and Company, Franklin Lakes, New Jersey) and scalp vein sets were directly connected to the blood collection tubes. No disposable syringes were used because they could promote blood coagulation or decrease the quality of fibrin-rich plasma when transferring blood from the syringe into the tube. ^{, , ,}

L-PRF membranes were obtained using the L-PRF IntraSpin centrifuge system following the manufacturer’s protocol (Intra-Lock International, Boca Raton, Fla). In brief, blood was centrifuged for 14 minutes at 2700 rpm. Subsequently, the formed fibrin clot was extracted from the test tube and pressed between 2 glass plates covered with saline-impregnated compresses for 30 seconds to remove any excess and obtain uniformity. The experimental alveolus was subsequently treated with L-PRF membranes. The alveoli were sutured with a 4-0 nylon suture (Mononylon; Ethicon, Johnson & Johnson, São Paulo, Brazil) to approximate the edges, thus promoting healing. This intervention sequence was performed to decrease the exposure time of the wells and to place the plasma on the treated side as soon as possible. Premolar luxation was performed during the surgical procedure with the use of Piezotome (Satelec, Acteon, Merignac, France) to allow for the preservation of the alveolar bridge, a smoother extraction procedure, and the reduction of postoperative complications.

Fifteen days after the extractions were performed, the initial distance from the maxillary midline to the mesial surface of both canines was recorded using a flexible ruler before initiating canine distalization. An 0.008-in ligature wire was placed from the first molar to the second premolar as an anchor unit. In the distal wing of the canine brackets, another 0.008-in ligature wire was placed to prevent rotation. The distalization of the canines was performed using the 0.020-in stainless steel archwire. All the arches had an omega mesially to the second molar and were tied back. The maxillary incisors were tied together to avoid the formation of drifting and opening spaces. Elastic chains (Memory Orthodontics; American Orthodontics, Sheboygan, Wis) were used to apply a force of 150 g ( Fig 2 ) according to Burrow and Mezomo et al. Elastic chains were placed between the second premolar and the canine. The amount of force was measured with the CORREX tensiometer (50-250 g) (Haag Streit, Bern, Switzerland). Activations were performed once a month, every 4 weeks, for 5 months. During the study, the patients did not receive any additional interventions, and no elastics or anchorage devices were used.

Use of tensiometer and beginning retraction of maxillary canines 15 days after extractions.

All patients were compliant with the treatment and attended their scheduled appointments.

Outcomes (primary and secondary)

The main objective of this study was to evaluate the rate of distalization and inclination of the maxillary canines in alveoli preserved with L-PRF membranes compared with the control side. The monthly distalization rate was the primary outcome. The 5-month distalization period lasted from T1 until the end of the fifth month (T2). Every month, the distalization rate was measured using a flexible ruler placed at the dental midline under the brackets from the maxillary central incisors to the mesial surface of the canines. The measurements were repeated 4 times during the same appointment, and the mean was recorded on the datasheet. Subsequently, the measured distance was divided by the number of days (28), which yielded the amount of movement per month.

Canine inclination was considered as the secondary outcome of the study. The canine inclination was evaluated using the initial (T1, predistalization) and final (T2, completed 5 months) CBCT scans. To measure the inclination of the canines, the sagittal slice was obtained at the angle formed by the plane of the longer axis of the canine and the horizontal plane drawn from the same canine apex to the most prominent and visible end of the posterior nasal spine ( Fig 3 ). Measurements were performed using the Planmeca Romexis Viewer software (Planmeca).

Assessment of canine inclination.

Sample size calculation

The sample size was calculated using the formula presented by Pandis assuming a canine retraction movement of 1 mm per month and a difference between sides of 0.5 mm, a standard deviation of the difference (σ) = 0.7 mm at a 5% significance level with 80% power. The formula is presented as

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