Maxillary sinus floor augmentation (MSFA) is the surgical intervention that raises the maxillary sinus floor in the posterior maxilla.1 Sinus floor elevation may be obtained after various surgical procedures, such as the use of a lateral or crestal approach, the timing of implant insertion in relation to the grafting, the type of grafting materials used, and the type of implant.2 Several grafting materials, such as autogenous bone (AB), allografts, xenografts, and alloplastic materials, have been used in MSFA.3–5 MSFA is commonly performed with a combination of AB grafts and other biomaterials to provide increased volume and reduced resorption.3 Several articles have studied the effect of graft materials in the quality of the newly formed bone in MSFA.2,3,5,6 A systematic review and meta-analysis of long-term studies (5 or more years) assessing MSFA concluded that AB graft, a mixture of AB graft and bone substitutes, or bone substitutes alone enhance the alveolar bone height of the posterior part of the maxilla and has a high long-term implant survival rate.7 Porous hydroxyapatite (HA) is suitable when mixed with AB because it enhances bone formation and bone-to-implant contact in augmented sinuses.7
The use of low-level laser therapy (LLLT) as a biomodulation therapy has the advantages of promoting biomodulation in the tissue to be repaired because laser therapy may increase mitochondrial respiration and adenosine triphosphate synthesis,8 favor the repair process,9 and increase cell division10 and collagen synthesis.11 In bone tissue, it can accelerate new bone formation, promoting increased osteoblastic activity9 and increased vascularization.12 The use of LLLT has been presented as an adjutant therapy on bone repair process. Several studies have demonstrated that LLLT promotes acceleration of tissue repair in either normal or compromised conditions, as well as promoting bone repair acceleration.13,14 Recently, some studies have evaluated the low-level laser effect on repairing bone defects filled with biomaterials13,15 or with morphogenetic proteins.5 However, scientific proofs that evaluate the association of bone graft to low-level laser irradiation in sinus cavity repair are still scarce in the literature. The purpose of this study was to evaluate the effect of LLLT in combination with the use of AB graft mixed with bovine HA in sinus lifting procedures.
Materials and Methods
This was a single-center, randomized, parallel-design trial of 6 months' duration. The protocol was approved by Ethical Committee for Human Research of the University Centre of the Educational Foundation of Barretos, São Paulo, Brazil (# 02/2009). Research was conducted according to the principles outlined in the Declaration of Helsinki on experimentation involving human subjects.
The patients were selected from the postgraduation clinic of the University Centre of the Educational Foundation of Barretos, Barretos, São Paulo, Brazil, after their medical histories and dental examinations were evaluated, including standard radiographs. Twelve patients between 20 and 60 years of age (mean age: 48.12 ± 6.24 years) with bone height requiring a MSFA to place dental implants were included in this study, and 26 patients were excluded (Fig. 1).16 A crestal bone height of 4 mm between the sinus floor and the alveolar ridge, along with a thickness of 5 mm, were prerequisites. Systemic and local exclusion criteria were defined as any factor interfering with implant surgery. Patients were excluded if they presented with uncontrolled systemic problems or local problems such as uncontrolled periodontitis or sinus pathology. Smokers and patients who had received radiation treatment in the head and neck region were also excluded.
The number of the samples for each group was determined by statistical power test realized in the web site http://www.lee.dante.br. The difference to be detected was 0.75, a SD of 0.5, a significance level of 5%, power test of 85% in a one-tail hypothesis test. All patients gave their informed consent, which was approved by Ethical Committee for Human Research of the University Centre of the Educational Foundation of Barretos, São Paulo, Brazil. The 12 patients were randomly divided into 2 groups using an online randomizer (www.sealedenvelope.com). Envelopes with the treatment distribution were prepared by a person independent of the clinical examiner (C.E.S.), and the envelopes were instructed to be opened only after received bone graft treatment. The patients were randomly allocated to 2 groups by a computer-generated table. The first group was the AB/HA group, which consisted of 6 patients who received AB graft and bovine HA (proportion 1:1) for sinus grafting; and the second group was the AB/HA-LLLT group, which consisted of 6 patients who received AB graft and bovine HA (proportion 1:1) for sinus grafting, followed by LLLT.
Sinus Floor Augmentation Technique
All patients were treated with the same surgical technique consisting of MSFA.3 After infiltrative anesthesia (Articaine 100; DFL, São Paulo, Brazil) and elevation of the mucogingival flap, the MSFA was performed using a round bur (no. 2; 3i Implant Innovations, Inc., Palm Beach, FL) in a straight handpiece at 1.500 rpm, under copious saline solution irrigation, to outline a large buccal window at the maxillary sinus lateral wall. A delicate dissection using blunt sinus curettes (SIN, Sistema de Implante Nacional Ltd.; São Paulo, Brazil) was performed to push the sinus membrane inward and upward. The membrane was released and an adequate compartment for the bone graft was created.
AB grafts from the mandible ramus were obtained with a bone scraper (SIN, Sistema de Implante Nacional Ltd.) after an intraoral incision at the posterior mandible region. The corticocancellous particles of bone were stored in physiological saline solution until they were mixed with HA (SIN, Sistema de Implante Nacional Ltd.) at a proportion of 1:1. The graft material was placed against the medial aspect of the compartment in the sinus cavity under meticulous condensation, and the mucoperiosteal flap was closed with interrupted sutures (nylon 4, Shalon; Montes Belos, Brazil).
Patients received postoperative and systemic medication that consisted of an antibiotic (Amoxicillin 500 mg, Hexal do Brazil Ltd., São Paulo, Brazil) for 10 days, a nonsteroidal anti-inflammatory drug (Nimesulide 100 mg; Pharmacia Ltd., São Paulo, Brazil) for 5 days, and an analgesic (Paracetamol Drops 200 mg; Hortolândia, Brazil) for 3 days. The topical medication prescribed was a rinse with 0.12% chlorhexidine gluconate twice a day 30 minutes after the patients' oral hygiene routine. The sutures were removed after 10 days, and the operated site received no direct influence of load throughout the monitoring period.
Low-Level Laser Therapy
The low-intensity laser used in this study was a gallium–aluminium–arsenide laser (GaAlAs; Laser Bio Wave; Kondortech Equipament Ltd., São Carlos, Brazil) with wavelength of 830 nm, power of 40 mW, and spot size of 0.07 cm2. The irradiation was performed in continuous mode at 4 points around the maxillary sinus cavity (mesial, distal, upper, and lower) before the placement of the graft and also at a central point over the graft in a scanning movement. The laser energy was 5.32 J/point for 133 s/point with irradiance of 0.57 W/cm2.
Obtaining and Processing of Samples
The implant placements were performed 6 months after the MSFA. At this time, bone biopsies from the alveolar crest were obtained with a trephine bur of 2.0 × 8.0 mm (Trephine Burr; 3i Implant Innovations, Inc.).3 The biopsies were removed and fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 hours. The samples were demineralized (10% ethylenediamine tetraacetic acid), conventionally processed, embedded in paraffin, and sectioned longitudinally. Histological sections (4 μm) were subjected to staining with hematoxylin and eosin for histologic analysis, Masson trichrome for histometric analysis, or immunohistochemistry. The indirect immunoperoxidase method was used for detection of tartrate-resistant acid phosphatase (TRAP), an osteoclast biomarker; and runt-related transcription factor 2 (RUNX2), a transcription factor that shows osteoblastic differentiation. Samples from each of the groups were divided into 2 batches, and each batch was incubated with one of the following primary antibodies: goat anti-TRAP (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-RUNX2 (1:180) (Santa Cruz Biotechnology). All stages of the immunohistochemical reactions were conducted following the protocol established by Theodoro et al.17
The histological analyses were performed by a certified histologist (E.E.) blinded to the treatment rendered. The following parameters were evaluated: presence and extent of inflammation; structuring pattern of the extracellular matrix of bone, connective and hematopoietic tissues; cellularity pattern of bone, connective and hematopoietic tissues; maturation pattern of bone tissue; presence and pattern of incorporation of the graft material; presence and extent of remnants of the graft material.
The images of the histological sections were obtained by a digital camera connected to a light microscope (Jung Supercut 2065; LEICA Instruments GmbH, Heidelberg, Germany) and image analysis software (ImageLab 2000 Software; Diracon Bio Informática Ltd., Vargem Grande do Sul, Brazil). The histometric analyses were performed by a calibrated examiner blinded to the treatment rendered (G.S.R.). Four equidistant sections representing the most central portion of the specimens were selected. In each section, an area of 14.66 mm2 located at its center was subjected to quantification of vital bone (VB), bone marrow or fibrous tissue (BMFT), and remaining biomaterial (RB). These measurements were expressed in mm2 and also as a percentage of the total area.3
A certified histologist (E.E.), blinded to the treatment, conducted the data analysis. The number of immunoreactive cells in an area measuring 1 mm2 within the central portion of each specimen was determined at ×200 magnification. Quantitative analysis was performed for TRAP and RUNX2 using 3 sections from each specimen. These measurements were expressed in immunoreactive cells per mm2.
The values of VB, BMFT, and RB for each patient were represented by the mean of percentage of the 4 histological sections. The hypothesis that there were no differences in VB, bone marrow, or fibrous tissue; remaining particles and number of TRAP immunoreactivity (TRAP-IR) and RUNX2 immunoreactivity (RUNX2-IR) cells between groups was tested by software (Bioestat 5.0; Sonopress Brazilian Industry, Manaus, Brazil). After histometric data normality analysis by Shapiro–Wilk test, the intergroup analysis was carried out by Student t test (α = 5%). After immunohistochemical data normality analysis by Shapiro–Wilk test, the intergroup analysis was carried out by Student t test (α = 5%).
The MSFA procedure showed uneventful healing in all cases after 10 days posttreatment. It was observed that the patients treated with laser had a faster healing and less postoperative discomfort, but these data were not measured. The implant placements were performed 6 months after the MSFA. A total of 22 implants (S.I.N. Sistema de Implante Nacional) were placed in the grafted areas, regular neck, with diameter of 4.1 mm and a length of 8.5 to 13 mm, 10 in AB/HA group, and 12 in AB/HA-LLLT group. No severe postoperative complications occurred. After 6 months of implant placement, it was observed through clinical and radiographic analysis at the time of reopening that all implants were osseointegrated without complications.
Gross histology in both groups was similar. The histological findings regarding the 6-month period demonstrated the formation of a trabecular bone structure pattern, with irregular arrangement of variable size, in general, with ample places and coalescence trabeculae (Fig. 2, A and B). In these structures, it was possible to observe incremental lines in a regular arrangement with variable gaps between them, being sometimes interrupted by other irregular lines, characterized as reversal (Fig. 2, A–D). They also showed randomly arranged osteoplasts, most of them containing osteocytes, as well as the presence of an osteoblast layer in the bone surface, where there was a prevalence of spindle-shaped osteoblasts and some polyhedral ones with hyperchromatic nuclei. The marrow spaces of variable amplitude are filled with BMFT (Fig. 3, A and B); however, a moderate proportion of graft material retention may partially or fully occupy these spaces (Fig. 3, C and D), which is presented as a mixture of small amphophilic bone segments of variable irregular amplitude with other basophilic amorphous micro-fragmented segments, with a smaller amount in the AB/HA-LLLT group. In some situations, the graft presented as juxtaposed to and integrated with the new bone formed (Fig. 3, A and B); in others, the graft material was found to be free and/or surrounded by delicate fibrous encapsulation (Fig. 3, C and D). Eventually, it was possible to observe macrophage activity exerted by both mononuclear phagocytes and inflammatory multinucleated giant cells over hemosiderotic pigments and also over the graft material. Moreover, osteoclasts and osteoblasts on the surface of bone trabeculae are sporadically shown to be involved in remodeling.
The histometric data are shown in Table 1. Statistical intragroup analysis revealed no significant difference in VB presence (P = 0.64) when the AB/HA (5.20 ± 0.58 mm2) and AB/HA-LLLT (4.70 ± 2.02 mm2) groups were compared. There was no statistically significant reduction in BMFT (P = 0.78) in the AB/HA group (8.25 ± 1.15 mm2) when compared to the AB/HA-LLLT group (9.48 ± 2.17 mm2). There was statistically significant decrease in the RB (P = 0.0081) in AB/HA-LLLT group (0.48 ± 0.29 mm2) when compared to the AB/HA group (1.21 ± 0.66 mm2).
The immunohistochemical method used to detect TRAP and RUNX2 was highly specific for the detection of these proteins, as evidenced by the total absence of IR in the negative control. Immunolabeling seemed as a brownish color that was confined to the cytosolic compartment in the case of TRAP (Fig. 4, A and B) and to the nuclear compartment in the case of RUNX2 (Fig. 4, C and D). The TRAP-IR cells often presented multinucleated, and they were located on the surface of the bone (Fig. 4, A and B). RUNX2-IR cells were located on the surface of the bone near osteoblasts (Fig. 4, C and D). There were no statistically significant differences in the amount of TRAP-IR cells (P = 0.26) between the AB/HA (22.75 ± 7.80) and AB/HA-LLLT (33.33 ± 14.57) groups. Similar result was found in RUNX2-IR cells (P = 0.51) when the AB/HA (60.75 ± 9.88) and AB/HA-LLLT (66.67 ± 12.66) groups were compared. The immunohistochemical data are shown in Table 2.
It was not possible to observe a significant increase in bone formation by applying LLLT to an AB transplant mixed with a bovine HA in the present study. With respect to the variable and irregular amplitude of the medullar lacuna, the percentage of new bone formation found is in conformity with the results of other studies, which evaluated similar experimental conditions in the formation of bone tissue in the maxillary sinus cavity3,18,19; that is, the homogeneous mix represented by the association of this substitute bone with autogenic bone zest was sufficient to conduct the formation of new bone tissue with adequate quality and quantity to receive an osseointegrated implant.
In a study by Boëck-Neto et al,3 the maxillary sinus histometrical analysis presented an average bone formation of 50.46% when demineralized freeze-dried bone allograft was used in association with autogenic bone, and 46.79% when HA was used in association with autogenic bone; remaining material was still present for a period of 10 months. A systematic review has reported high short-term survival rates of suprastructures and implants after MSFA with different mixtures of AB graft and bone substitutes or bone substitutes alone.20 Others, assessing histomorphometric variables, concluded that AB grafts result in the highest amount of newly formed bone in comparison with various bone substitutes.21,22 Moreover, bone substitute materials might delay the physiological ossification process at the early stage of healing.23
In the present study, it was possible to observe 35.5% of bone neoformation in the grafted area, whereas the area irradiated with infrared laser presented 32% of bone formation as well as remaining inserted material in a 6-month period. In some locations, the authors found that HA remnant particles started to become degraded with the presence of osteoclasts on the surface, which corroborated the findings of another study.19 Animal studies revealed improvement of bone repair by HA and suggest that HA may be used successfully as material for bone repair when a critical-sized defect was induced.24 Other study demonstrated that bovine HA is an effective and safe material that can be used as a space filler, allowing high survival rates of implants placed in the posterior maxilla.25
In the present study, the fragments of autogenic graft used were collected from the retromolar region with a bone scraper, which generated less trauma and greater postoperative comfort to patients (data not indicated in the present study). The histological analysis of the results showed that substitute bone associated with autogenic bone grafts in the proportion established for the present research was capable of inducing bone neoformation, although there was immature bone characterized by a huge amount of osteocytes in a 6-month period in the laser-irradiated group as well as in the nonirradiated area of the maxillary sinus cavities. Another important finding is that the new bone apposition was not constant on the inserted material in both treatments. The material acted as an osteoconductor for new bone depositions, and portions of this material were incorporated into the bone. The presence of incremental lines and interposed reversion lines showed HA's potential to induce new bone formation when combined with AB obtained from the retromolar areas.
Nevertheless, considering the parameters of the present study, low-level laser irradiation was not capable of inducing greater bone neoformation when compared to a nonirradiated area in a 6-month evaluation period (P = 0.64). However, there was a significantly lower percentage of substitute bone in the irradiated areas than in the nonirradiated areas (P = 0.0081). This fact can be related to the ability of LLLT to promote angiogenesis and local vasodilatation,26 favor inflammatory cell migration,11 and increase the proliferation of cells and stimulate their metabolism, thus accelerating the inserted material resorption process.13 However, there was no difference in the number of TRAP-IR cells (P = 0.26), an osteoclast marker, or of RUNX2-IR cells (P = 0.51), a transcription factor that indicates osteoblastic differentiation cells, between groups.
Several authors have focussed their studies on LLLT related to bone reshuffle, aiming to provide greater postoperative comfort as well as to accelerate bone repair process.27–29 In addition to an in vitro study that demonstrated osteoblast stimulation after low-level laser irradiation,30 some authors have recently demonstrated in several experimental animal models that infrared low-level laser promotes acceleration of the repair process in the area grafted with osteoconductive materials.13,26,31,32 It is unclear whether biomodulation of LLLT on bone formation is a consequence of stimulation of mesenchymal cells or a direct stimulation of osteoblasts.33
Lopes et al34 demonstrated through an animal study that LLLT was effective in improving bone healing as a result of an increased deposition of HA. A recent study showed that LLLT may contribute to bone repair in association with AB grafts by its biostimulatory effects on the suppression of inflammation.35 Jakse et al36 investigated whether LLLT enhanced bone regeneration in a sinus graft model. The sites were irradiated 3 times during the first week (75 mW, 680 nm, 3–4 J/cm2), and the experimental animal model did not confirm a positive effect on bone regeneration within a sinus graft, as found in the present study. LLLT can accelerate bone formation,12,37 and it can also accelerate either resorption or formation activities depending on the phase of bone repair.38,39 Most studies have evaluated the effect of infrared laser light on bone healing13,15,29 because of its deeper penetration of tissues.
In a recent study by our team, we reported the influence of LLLT with a wavelength of 660 nm and a dose of 24.7 J/cm2. It could be concluded that the association of AB grafts and LLLT (660 nm) improved bone healing in immunosuppressed rats.40 It is known that the wavelength, the total delivered energy, the emission frequency, and the dose are directly related to an effective cell response to laser therapy.10 However, with the parameters established for one single irradiation in the present study, it was not possible to demonstrate greater bone neoformation in maxillary sinus cavities that had been irradiated with LLLT. Coombe et al38 showed that single irradiation had no effect on the newly formed mineralized bone. Other studies demonstrated a significant difference in the area of mineralized bone between irradiated and nonirradiated subjects during the early stages of healing, although it has not been observed in advanced stages of healing.32,39 In the present study, the graft was analyzed only in advanced stages of bone healing (6 months).
In relation to the percentage rates of medullar spaces, significantly greater values were observed on low-intensity laser irradiated areas when compared to the nonirradiated ones. This result is probably related to the capacity of LLLT to promote a greater resorption of the grafted material41; thus, the spaces formerly filled by this material were replaced by a more organized connective tissue matrix, which represented bone neoformation area in some specimens. Cosso et al6 showed that in human maxillary sinus augmentation, the mixture of HA and AB graft showed lower degree of resorption and higher dimensional stability when compared with AB graft alone, at least at 180 days of healing. Moreover, further studies are necessary to evaluate the behavior of this graft tissue after the installation of the dental implant. It is difficult to compare studies that use LLLT on bone regeneration because of different experimental models and treatment durations. In this context, it is necessary to conduct in vivo studies that would evaluate and develop other protocols of area irradiation of maxillary sinus grafting with low-level laser, inasmuch as not only physical irradiation parameters (wavelength, power, irradiation time, energy, and energy density) but also clinical parameters (irradiation frequency, irradiation intervals, and application mode) can influence bone tissue repair.
Within the limitations of the present study, it was possible to conclude that the association of AB graft with the substitute bone proved to be effective in the formation of new bone integrated with the receptor site in the sinus cavity. It was also evident that LLLT, in parameters used in this study, did not increase the formation of new bone but accelerated the bone remodeling process.
The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
Leticia Helena Theodoro, Gustavo Spina Rocha, Valmir Lucas Ribeiro Junior, Celso Eduardo Sakakura, João Martins de Mello Neto, Valdir Gouveia Garcia, Edilson Ervolino, and Elcio Marcantonio Junior contributed equally to this work.
All patients gave their informed consent, which was approved by Ethical Committee for Human Research of the University Centre of the Educational Foundation of Barretos, São Paulo, Brazil (#02/2009).
Roles/Contributions by Authors
L. H. Theodoro: Substantially contributed to conception or design, contributed to acquisition, analysis, or interpretation of data, drafted the manuscript, critically revised the manuscript for important intellectual content, and gave final approval. G. S. Rocha: Substantially contributed to conception or design, contributed to clinical procedure and acquisition, analysis, or interpretation of data, and gave final approval. V. L. Ribeiro Junior: Substantially contributed to conception or design, contributed to clinical procedure and acquisition, analysis, or interpretation of data, and gave final approval. C. E. Sakakura: Substantially contributed to conception or design, contributed to clinical procedure and acquisition, analysis, or interpretation of data, drafted the manuscript, and gave final approval. J. M. de Mello Neto: Contributed to acquisition, analysis, or interpretation of data, critically revised the manuscript for important intellectual content, and gave final approval. V. G. Garcia: Substantially contributed to conception or design, contributed to acquisition, analysis, or interpretation of data, drafted the manuscript, critically revised the manuscript for important intellectual content, and gave final approval. E. Ervolino: Contributed to acquisition, analysis, or interpretation of histological data, drafted the manuscript, critically revised the manuscript for important intellectual content, and gave final approval. E. M. Junior: Substantially contributed to conception or design, contributed to acquisition, analysis, or interpretation of data, drafted the manuscript, critically revised the manuscript for important intellectual content, and gave final approval.
The authors thank SIN (Sistema de Implante Nacional Ltd.) for funding this study.
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