Micro-computed tomography and histomorphometric analysis of human alveolar bone repair induced by laser phototherapy: a pilot study

Abstract

Immediate dental implant placement in the molar region is critical, because of the high amount of bone loss and the discrepancy between alveolar crest thickness and the implant platform. Laser phototherapy (LPT) improves bone repair. The aim of this study was to evaluate the human alveolar bone repair 40 days after molar extraction in patients submitted to LPT. Twenty patients were selected for this randomized controlled clinical trial; 10 underwent LPT (laser group) with a GaAlAs diode laser (808 nm, 100 mW, 0.04 cm 2 , 75 J/cm 2 , 30 s per point, 3 J per point, at five points). The control group patients ( n = 10) were not irradiated. Forty days later, the tissue formed inside the sockets was analyzed by micro-computed tomography and histomorphometry. Data from the two groups were compared with Student’s t -test and Pearson’s correlation test. The relative bone volume was significantly higher in the laser group ( P < 0.0001). The control group showed negative correlations ( P < 0.01) between number and thickness, and between number and separation of trabeculae, and a positive correlation between thickness and separation of trabeculae. The laser group showed a significant negative correlation between the number and thickness of trabeculae ( P < 0.01). The results suggest that LPT is able to accelerate alveolar bone repair after molar extraction, leading to a more homogeneous trabecular configuration represented by thin and close trabeculae.

Implant placement in sockets following recent tooth extraction presents many limitations that may affect osseointegration. Clinical criteria for adequate implant placement following tooth extraction should be observed, such as extraction technique, alveolar structure, contiguous structures, periodontal disease, peri-apical lesions, socket size, and bone quality. Molar areas represent a major problem for immediate implant placement as the alveolar thickness leaves gaps between the implant surface and the bone walls, which may lead to implant failure due to tissue invagination, even when grafting is performed. There is evidence that these invaginations are more frequent in gaps larger than 3 mm. Specific surgery techniques, as well as the use of biological grafting and biological membrane placement, can minimize these gaps, but make the socket more prone to the development of infections, resulting in treatment failure.

Laser phototherapy (LPT) is a therapy that might be useful to accelerate the alveolar bone healing process, since it enhances bone repair and osteoblast cell proliferation and differentiation, and increases bone matrix formation. There are articles in the literature reporting an accelerated healing process, altered cell behaviour in the healing process, and increased vascular neoformation and collagen production, as well as proliferation of fibroblasts and epithelial cells. The mechanism of action of LPT is based on the increase in adenosine triphosphate (ATP), thus accelerating mitosis and enhancing tissue repair . LPT with a gallium–aluminium–arsenide (GaAlAs) laser has demonstrated promising results on the acceleration of bone repair; however, most studies have been performed in vitro , or in vivo in laboratory animals, using different application protocols.

Histomorphometric analyses are considered the gold standard for the evaluation of trabeculae and the cortical bone crest. They may provide information on cellularity and dynamic indices of bone remodelling, but also present limitations in the evaluation of bone microstructure, since they are derived from two-dimensional stereological analysis (2D). On the other hand, micro-computed tomography (micro-CT) is a computed tomography analysis capable of providing quantitative data through reconstruction of virtual transversal sections of tissue samples; this constitutes a three-dimensional structure analysis (3D). Micro-CT is a method with high reproduction potential capability and non-destructive characteristics. Moreover, the mineral content can be mapped by reconstructing micro-CT scans showing bone morphology alterations.

In order to obtain clinical data on LPT in bone repair, the aim of this study was to evaluate the histomorphology of the neoformed tissue inside the bone sockets using micro-CT and histomorphometric analyses, at 40 days after human molar extraction in patients submitted or not to LPT initiated during the surgical procedure.

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 .

Table 1
Overall description of the two study groups.
Patients Sex Age, years Tooth Tooth extraction rationale
Control group
1 M 36 36 Furcation lesion
2 F 30 47 Tooth fracture
3 M 37 47 Caries lesion
4 M 33 37 Caries lesion
5 F 31 36 Periodontitis
6 F 35 36 Advanced periodontitis
7 M 36 47 Caries lesion
8 F 39 37 Caries lesion
9 F 37 46 Advanced periodontitis
10 M 36 36 Caries lesion
Mean ± SEM 35 ± 0.89
Laser group
1 M 36 46 Caries lesion
2 F 38 36 Furcation lesion
3 F 36 36 Furcation lesion
4 F 35 46 Caries lesion
5 F 32 47 Advanced periodontitis
6 M 38 46 Advanced periodontitis
7 F 38 46 Caries lesion
8 M 31 36 Advanced periodontitis
9 M 37 37 Tooth extrusion
10 F 36 36 Furcation lesion
Mean ± SEM 35.7 ± 0.77

SEM, standard error of the mean.

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.

Fig. 1
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 .

Table 2
Micro-CT scanning parameters.
Scanning parameters SkyScan 1176
Filter – Al 0.2
Resolution 9 μm
Voltage 40 kV
Amperage 500 μA
Rotation angle 360°
Rotation step 0.30
Frames 3

Micro-CT, micro-computed tomography.

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.

Fig. 2
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

Micro-CT

The data obtained from micro-CT of the surgical specimens are presented in Tables 3 and 4 . The relative percentage of bone volume in relation to the total volume (BV/TV) of specimens in the laser group was significantly greater than that in the control group ( Table 3 ; P < 0.0001). The other parameters assessed, related to the morphology and arrangement of bone trabeculae in the specimens, showed similar results for the two experimental groups ( Table 4 ).

Table 3
Bone volume/total volume (%) data obtained by micro-CT for each patient.
Patient Bone volume/total volume (%)
Control group Laser group
1 65.72 93.01
2 77.17 82.72
3 66.62 98.9
4 68.21 91.88
5 65.45 88.86
6 66.99 81.82
7 64.72 96.02
8 62.32 90.41
9 63.62 77.72
10 74.48 84.22
Mean ± SEM 67.54 ± 1.50 a 88.55 ± 2.14 b
Micro-CT, micro-computed tomography; SEM, standard error of the mean.
a,b Different letters indicate a statistically significant difference ( P < 0.0001).

Table 4
Results of micro-CT analysis (mean ± SEM).
Parameters Control group Laser group P -value
Bone volume/total volume (%) 67.54 ± 1.50 88.55 ± 2.14 <0.0001
Thickness of trabeculae (μm) 272.65 ± 26.63 255.73 ± 47.38 0.35
Number of trabeculae 3.64 ± 0.41 4.23 ± 0.57 0.20
Separation of trabeculae (μm) 96.00 ± 12.49 82.09 ± 9.72 0.17
Micro-CT, micro-computed tomography; SEM, standard error of the mean.

Pearson’s correlation analysis for specimens in both groups showed no significant linear correlation between the values of the relative volume of bone formed (BV/TV) and the values of all other parameters analyzed ( P > 0.05) ( Tables 5 and 6 ).

Table 5
Results of the Pearson’s linear correlation test for the control group.
Relative bone volume vs. thickness of trabeculae (μm) Relative bone volume vs. number of trabeculae Relative bone volume vs. separation of trabeculae (μm) Thickness (μm) vs. number of trabeculae Thickness (μm) vs. separation of trabeculae (μm) Number vs. separation of trabeculae (μm)
N 10 10 10 10 10 10
r −0.22 0.40 −0.17 −0.89 0.90 −0.85
95% CI −0.75 to 0.47 −0.30 to 0.83 −0.73 to 0.51 −0.98 to −0.62 0.64 to 0.98 −0.97 to −0.49
99% CI −0.84 to 0.63 −0.49 to 0.89 −0.82 to 0.66 −0.98 to −0.45 0.48 to 0.99 −0.98 to −0.29
r 2 0.0524 0.1666 0.0315 0.8074 0.8199 0.7317
t −0.6651 1.2647 −0.5099 −5.7917 6.0345 −4.6708
FD 8 8 8 8 8 8
P -value 0.5247 0.2415 0.6239 0.0004 0.0003 0.0016
Power 0.05 0.1508 0.3091 0.1189 0.9871 0.9901 0.9582
Power 0.01 0.0284 0.1170 0.0079 0.9394 0.9503 0.8531
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Jan 17, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Micro-computed tomography and histomorphometric analysis of human alveolar bone repair induced by laser phototherapy: a pilot study
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