Patients and methods

This study was approved by the institutional ethics committee (School of Dentistry, University of São Paulo). Healthy patients aged between 30 and 39 years, of both sexes, with systolic blood pressure ≤140 mmHg and diastolic pressure ≤90 mmHg and an average body temperature between 36 and 37 °C, were included in the present study. The following patients were excluded: pregnant and/or lactating women, smokers, women in menopause, and those undergoing radiotherapy in the head and neck region or presenting any type of immune deficiency, acute infection (e.g., periodontal abscess), or metabolic diseases, such as diabetes and osteoporosis. Patients were also excluded if any type of complication occurred during surgery, such as bleeding or operative difficulties, and if the duration of surgery was >90 min. In these patients, antibiotics were prescribed after surgery, along with the care necessary for their needs, and they were not considered in the final study sample.

Local inclusion criteria were the following: patients with an indication for lower molar extraction and posterior dental implant placement; the fresh socket should present most gaps between the implant platform and the alveolar bone in any direction of >4 mm following surgery, with the absence of suppuration and presence of all alveolar bone walls around the sockets; other clinical parameters appropriate for the future installation of implants to be feasible.

Thirty-two out of 236 patients evaluated fulfilled the inclusion criteria and did not meet the exclusion criteria. These patients were randomized to the study groups as follows: after clinical examination, the name of each patient who met the necessary criteria was placed in an envelope until a number of 8 was reached. Then, another envelope was filled with patient names, and so on, until four envelopes containing eight names in each were obtained. These unlabeled envelopes were closed and in a blinded fashion two of them were labelled ‘laser’ and the other two ‘control’. Twelve patients were not able to undergo all of the steps of the study and were therefore excluded. Thus, data from 20 out of the 32 patients were analyzed by micro-CT and histomorphometry. A description of the two groups is given in Table 1 .

All patients underwent clinical examinations and radiographic examination by means of panoramic and peri-apical radiographs. Tooth extractions were performed by the same operator, following a simple extraction technique. Patients were anaesthetized by pterygomandibular injection of 2% mepivacaine with 1:100,000 adrenalin. After tooth extraction, the inter-radicular septum was removed using an osteotome. The fresh extraction socket was curetted slightly using a number 85 Lucas curette and irrigated with sterile physiological saline solution. The thickness of the fresh dental socket was measured using a millimetre probe and it was then closed with a mucous flap covering the whole fresh socket to achieve a first intention healing process. Only fresh sockets presenting most gaps between the dental implant platform and the alveolar bone in any direction of >4 mm were included in the study. Flaps were performed using a 15C blade (Suzhou Kyuan Medical Apparatus Co., Ltd, Suzhou, Jiangsu, China), and two relaxing incisions were made, both mesial and distal to the fresh socket over the alveolar ridge and up to the buccal sulcus (quadrangular incision), followed by mucoperiosteal flap reflection using a Molt periosteal elevator. The socket was sutured using 4–0 Ethicon silk thread (Johnson & Johnson, São Paulo, SP, Brazil) with total approximation of the surgical wound edges. All sockets were filled only with blood clot.

In this randomized controlled clinical trial, the patients were divided randomly into two experimental groups, as follows: laser group ( n = 10): patients underwent LPT as described below; control group ( n = 10): patients were submitted to the same procedures as in the laser group, but with the laser device unpowered. LPT was applied using a GaAlAs diode laser device in continuous wave mode (wavelength 808 nm, Twin Flex; MMOptics Ltda, São Carlos, SP, Brazil). The spot size was 0.04 cm ² , power was 100 mW, energy density was 75 J/cm ² , and energy was 3 J. Irradiations were punctual and in contact mode. Each socket was irradiated at five points (two buccal, two lingual, and one occlusal) for 30 s each at the following times: during the surgical procedure, immediately after the procedure, and at 24 h, 48 h, 72 h, 96 h, 7 days (session when sutures were removed), and 15 days after the procedure ( Fig. 1 ). Forty days later, samples of the neoformed tissue inside the extraction sockets were collected from the patients in both groups using a trephine stainless steel bur (3 mm internal diameter, 4 mm external diameter). The site for bone tissue sample removal was standardized as the mid-point of the edentulous space left by tooth extraction. All collected samples were immediately fixed in 10% neutral buffered formalin. After these procedures, patients underwent implant placement.

Photographs illustrating the application of LPT in one of the patients. Irradiation being applied in the punctual and contact mode at one buccal point (A) and one lingual point (B).

Material was first submitted to micro-CT analysis and then processed for histomorphometric analysis. A SkyScan 1176 high-resolution X-ray micro-CT in vivo device (Bruker micro-CT, Kontich, Belgium) was used for the micro-CT analyses. Samples on cotton wetted with 10% formaldehyde solution were transferred to an Eppendorf tube. These tubes were placed in a polystyrene cot to maintain stability in the micro-CT device. The micro-CT scan was performed using the scanning parameters described in Table 2 .

After scanning, images were rebuilt by a trained technician. Data were evaluated using CT-analyser computer software (SkyScan; Bruker, Antwerp, Belgium) for analysis of the trabecular microstructure starting from the region of interest (ROI) in the binary images ( Fig. 2 A) . The maximum threshold in the grey-scale was 255; the minimum was defined regarding trabecular density, based on non-binary radiographs of each sample. With this established threshold, the software program (Adaptive, CT-analyser; SkyScan) analyzed the following bone morphometric data: number, thickness, and separation of the bone trabeculae, as well as the relative bone formation using the ratio between bone volume (BV) and total tissue volume (TV) (BV/TV). These data could also be used to build 3D images, as shown in Fig. 2 B.

Illustration of micro-CT images. (A) Binary image of a sample from the laser group; (B) 3D image of a sample from the laser group built after the acquisition of the bone morphometric data.

After micro-CT imaging, samples were processed for histological analysis in tissue slices stained with haematoxylin and eosin (HE) dye solution. The histomorphometric analysis was carried out using an image analyzing program (NIS–Elements Advanced Research microscope imaging software; Nikon Instruments, Tokyo, Japan). The relative bone formation was estimated by the ratio between areas filled with bone trabeculae (bone area, BA) and the total histological field (100× magnification histological field; total area, TA) (BA/TA).

Student’s t -test was used to compare the data of the two groups for both the micro-CT and histomorphometry analysis. Data for the different micro-CT parameters in the same group were also submitted to Pearson’s correlation test. The level of significance for both statistical tests was 5% ( P < 0.05).

Results