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Vital Bone Formation After Grafting of Autogenous Bone and Biphasic Calcium Phosphate Bioceramic in Extraction Sockets of Rats

Histological, Histometric, and Immunohistochemical Evaluation

Macedo, Rander Moreira, PhD*; Lacerda, Suzie Aparecida, PhD; Okamoto, Roberta, PhD; Shahdad, Shakeel, PhD§; Brentegani, Luiz Guilherme, PhD

doi: 10.1097/ID.0000000000000815
Basic and Clinical Research

Purpose: The study aimed to investigate through histology, histometry, and immunohistochemistry the vital bone formation after grafting of biphasic calcium phosphate bioceramic (BC) in combination with calvarial autogenous bone into the dental sockets of rats.

Materials and Methods: Forty-five male rats were submitted to upper right incisor extraction and divided according to the grafted material in: control, bioceramic (BC), and bioceramic + autogenous bone (BC + AB). The animals were killed 7, 21, and 42 days after surgery for histological, histometric, and immunohistochemistry analysis.

Results: Histomorphometric results demonstrated, for BC + AB group, formation of trabecular bone between the particles of BCs and autogenous bone, connecting them, as well as higher percentage of vital bone in comparison with BC. Immunohistochemical reactions showed intense labeling for Runx2-positive cells in the group BC + AB.

Conclusions: Autogenous bone was able to stimulate bone turnover enabling a larger amount of vital bone synthesis and can be recommended as a viable grafting material in combination with synthetic biphasic BC.

*Associate Professor, Department of Dentistry, Positivo University, Curitiba, PR, Brazil.

Associate Professor, Department of Stomatology, Public Health and Forensic Dentistry, School of Dentistry of Ribeirão Preto, USP—University of São Paulo, Ribeirão Preto, SP, Brazil.

Associate Professor, Department of Basic Sciences, School of Dentistry of Aracatuba, UNESP, Universidade Estadual Paulista, Araçatuba, SP, Brazil.

§Consultant at The Royal London Dental Hospital, Department of Restorative Dentistry, Barts and The London NHS Trust; Honorary Senior Clinical Lecturer at Queen Mary University of London, Barts and The London School of Medicine and Dentistry; Chairman of the International Team for Implantology (ITI) UK & Ireland Section.

Professor, Department of Stomatology, Public Health and Forensic Dentistry, School of Dentistry of Ribeirão Preto, USP—University of São Paulo, Ribeirão Preto, SP, Brazil.

Reprint requests and correspondence to: Rander Moreira Macedo, PhD, Departamento de Odontologia, Universidade Positivo, Av. Prof. Pedro Viriato Parigot de Souza, 5300, Curitiba, PR, CEP 81280-330, Brazil, Phone: +55-41-3317-3180, Fax: +55-41-3317-3082, E-mail: mmrander@hotmail.com

The healing process after dental extraction initiates a series of cellular and tissue-related events with an objective to restore the homeostasis of the area.1 After tooth extraction, the body aims to fill the cavity (alveolar socket) with new bone and the repair is completed when the alveolar bone trabeculae is thick, the marrow spaces tiny and alveolar crest is completely remodeled.2

After the loss of a natural tooth, 3-dimensional reduction of the height and width of the alveolar bone3 inevitably occurs in the first 6 months,4 primarily at the expense of the buccal bone wall.5 In some cases, this may not only compromise the aesthetic outcome but also prosthodontic treatment in the posterior areas.

Various treatment protocols have been described for maintenance and reconstruction of alveolar bone after extraction, using different biomaterials as well as the use of resorbable and nonresorbable membranes to cover the socket site.6 Human and animals studies have shown better bone quality and quantity after grafting the alveolar socket with autogenous,7 allogenic,8 xenogenic,9–11 and synthetic bone substitutes.10,12,13 Araú’jo et al9 demonstrated that sockets grafted with BioOss Collagen (Geistlich), a deproteinized bovine bone mineral, markedly counteracted the reduction in hard tissue component when compared to nongrafted sites after 4 months.

Although autogenous bone graft is still considered the gold standard in bone repair and regeneration14 due to its osteogenicity, osteoinductivity, and osteoconduction, nevertheless, harvesting it requires an additional surgical site resulting in increased patient morbidity.15 Among many other biomaterials, synthetic bioceramics (BCs) have been investigated as an alternative in maxillofacial bone surgery and implantology.11,15,16

The biphasic calcium phosphate synthetic BC (Straumann BoneCeramic) is composed of 60% hydroxyapatite (HA; 100% crystalline) and 40% β-tricalcium phosphate (β-TCP) sintered at temperatures of 1100°C to 1500°C. It is 90% porous with interconnected pores of 100 to 500 microns in diameter.17 The HA is less soluble with the ability to maintain volume, whereas the β-TCP is resorbed faster leaving viable gaps to invasion of osteoblasts and bone formation, providing good osteoconductive characteristics.18

Several studies have reported favorable clinical and histological outcomes for BCs.10,11,15,19 Cordaro et al20 compared the Straumann BoneCeramic with BioOss (Geistlich) in a multicenter clinical trial and showed histomorphometrically that both biomaterials produced similar amounts of newly formed bone (vital bone), with similar histological appearance, indicating that they are suitable in sinus augmentation for the placement of dental implants. Similar outcomes have been demonstrated when comparing the 2 biomaterials in alveolar ridge preservation procedures.10,19 Histologically, Macedo et al12 reported good integration ability of biphasic bone BC with simultaneous graft resorption and synthesis of newly formed bone.

An important aspect in bone metabolism is the osteoblast differentiation response, which demonstrates that the bone turnover is activated by the grafting of different biomaterials in the created cavity. Immunohistochemical methods allow detection of some transcription factors that determine the degree of maturation of cells in the osteoblast lineage,21–23 which include the Cbfa1/Runx2, a specific gene whose early expression appears in every future osteoblast independently of its embryonic origin.21,24 Therefore, the presence of a transcription factor like Runx2, which labels preosteoblasts, is an important feature of the new bone formed within the extraction socket.

The aim of the this study was to investigate through histology, histometry, and immunohistochemistry the vital bone formation after grafting of biphasic calcium phosphate BC in combination with calvarial autogenous bone into the dental sockets of rats.

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Materials and Methods

Forty-five male Wistar albino rats, weighing 250 to 300 g, were used in this study. All the procedures were conducted in accordance with ethical principles for animal research, as approved by Committee of Ethics for use of Animals of Campus of Ribeirão Preto, University of São Paulo, Brazil, and received a vermifuge for animal use (Systamex; Shering of Brazil, São Paulo, SP, Brazil) during 3 days.

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Dental Extraction

All the 45 rats were anesthetized with intraperitoneal injection of 2.2.2-Tribromoethanol (Sigma-Aldrich, St. Louis, MO), with a dose of 25 mg/100 g body weight. Under sterile conditions, the upper right incisors were extracted using forceps, after disconnection of the surrounding gingiva and luxation using an enamel hatchet with a cutting edge. The animals were divided into 3 groups according to the biomaterial grafted in the extraction sockets: control, BC, and bioceramic + autogenous bone (BC + AB). Straumann Bone Ceramic was used as particulate bone graft substitute.

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Harvesting of Autogenous Bone

For obtaining autogenous bone graft from the skull, trichotomy was performed, and antisepsis of the surgical area was carried out using a solution of 10% polyvinylpyrrolidone iodine. An incision in the sagittal plane of the skull with 15C blade, respecting the anatomical planes of the scalpel, skin, and periosteum, followed by the detachment of a total thickness flap in the right side of the zygomatic arch, was performed, to visualize the donor site. Using a 5-mm-diameter trephine (Neodent, Curitiba, Parana', Brazil), osteotomy was performed in the parietal bone under 0.9% NaCl irrigation, and the fragment was removed with Micro Ochsenbein chisel No. 01 (Millennium Golgran, Pirituba, SP, Brazil). The bone fragments were immersed in physiologic solution, and the surgical wound was sutured with nylon monofilament (Ethicon, São José dos Campos, SP, Brazil).

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Grafting of Biomaterials inside the Dental Socket

For the control group, the extraction socket was filled only with the blood clot followed by the soft tissue suture with Mononylon 4.0 (Ethicon).

For the group BC, a mini amalgam carrier (Millennium Golgran) calibrated to carry approximately 20 mg of BC (BoneCeramic; Straumann AG, Basel, Switzerland) was introduced in the middle and cervical third of the extraction socket. A medium size amalgam condenser (Millennium Golgran) was used to gently pack the material within the socket. The soft tissue was sutured with Mononylon 4.0 (Ethicon).

For the group AB + BC, the autogenous bone was cut in approximately 4 2 × 2-mm fragments and inserted in the middle and cervical third of the extraction socket mixed with the BC, and the soft tissue was sutured.

A single 0.2 mL intramuscular dose of antibiotic (Veterinary Pentabiotic; Fontoura-Wyeth, São Bernardo do Campo, SP, Brazil) was administered to each rat after surgical procedure.

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Killing and Histological Processing

The animals were killed by an overdose of the same anesthetic on the 7th, 21st, and 42nd days after the extraction (n = 5 per group). The mandibles were separated from the maxillae, and the right hemi-maxilla was separated from the left one by a sagittal incision along the intermaxillary suture. The maxillary halves were fixed in buffered (pH 7) 10% formaldehyde solution for 24 hours.

For light microscopy, the specimens were decalcified by a solution of 20% sodium citrate and 30% formic acid for 6 days. This solution was replaced every 2 days and neutralized with 5% sodium sulfate. The specimens were then dehydrated by increasingly concentrated ethanol solution: 70% overnight, then 80%, 85%, 90%, and 95% for 2 hours each, until 100%. The specimens were then processed with xylol and embedded in paraffin. Longitudinal semi-serial sections (5 µm thick) were stained with hematoxylin and eosin.

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Histological and Histometric Analyses of the Graft Periphery

Histological analysis consisted of direct observations in the area around the graft with optical microscope for the presence of immune reactions, inflammatory foreign body reactions, and the quality of bone graft union to the newly formed bone (vital bone) in 7, 21, and 42 days postsurgery.

For histometric analysis, the degree of new bone formation (vital bone), residual BC, and soft tissue was estimated at 7, 21, and 42 days after the surgery. A Leica DM LB2 optical microscope (Leica MicrosystemsWetzlar GmbH, Wetzlar, Germany) with a Leica DFC 280 digital video camera (Leica Microsystems Imaging AG, Cambridge, United Kingdom) were used to capture the images that were processed using the Leica Qwin program (Leica Imaging Systems Ltd., Cambridge, United Kingdom). A grid containing 100 equidistant points were superimposed on microscopic image (final magnification, ×250) of alveolar components according to a modification of the method described by Schroeder and Münzel-Pedrazzoli.25 A total of 500 points were counted in 5 histological sections in the middle third per alveoli (n = 5). The percentage of points lying of bone trabeculae (vital bone), BC, and soft tissue (connective tissue + blood clot) was estimated by a differential point counting method performed by a masked (with respect to time and group division), trained, and calibrated examiner. Differences among groups were analyzed by the nonparametric Mann-Whitney and Kruskal-Wallis tests (P ≤ 0.01 for statistical significance) using the GMC statistical software package (available from www.forp.usp.br/restauradora/gmc/gmc.html).

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Immunohistochemical Analysis

Slices obtained after the microtomy were separated to perform the immunolabeling experiments. The immunoperoxidase method was used with the primary polyclonal antibody against Runx2 (Santa Cruz Biotechnology, Dallas, TX), a transcription factor that label preosteoblasts. As secondary antibody, biotinylated rabbit anti-goat antibody (Pierce Biotechnology, Rockford, IL) was used. The avidin and biotin system (Vector Laboratories, Burlingame, CA) was used to amplify the signal of the reaction, and the diaminobenzidine (Dako Laboratories, Carpinteria, CA) was the chromogen. At the end of the immunolabeling reaction, the slices were counterstained with Harry hematoxylin (Merck, Kenilworth, NJ).

Immunohistochemical results were evaluated in a semiquantitative manner through the scores attribution that varied from “−” for absence of immunolabeling to “+, ++, and +++” for mild, moderate, and intense immunolabeling, respectively, characterized for the cells (pre osteoblasts) with brown staining because of the diaminobenzidine reaction.26,27

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Results

Histological Analysis

The alveolar sockets in control group at 7 days were partially filled with immature bone trabeculae surrounded by osteoblasts, interspersed in blood clot mass and granulation tissue (Fig. 1, A). After 21 days, the apical and medium thirds showed thicker and more mineralized bone when compared to 7th day specimens (Fig. 1, B). After 42 days, the sockets were completely filled by compact bone with small marrow spaces constituted by connective tissue and blood vessels (Fig. 1, C).

Fig. 1

Fig. 1

After 7 days, the BC group revealed immature trabecular bone adjacent to the BC granules without any signs of foreign body reaction (Fig. 2, A). After 21 days, the new bone showed more maturity and thickness of trabeculae, but some areas were still in contact with connective tissue (Fig. 2, C). After 42 days, thick and compact bone tissue directly attached to the BC surface and filling the spaces between the particles (Fig. 2, E) was noted, indicating osseointegration of the particles.

Fig. 2

Fig. 2

After 7 days, the BC graft in the AB + BC group revealed trabecular bone formation between the particles of BCs and autogenous bone, and connecting the 2 graft materials with new bone tissue developing from the surface of biomaterials to the remainder of the alveoli, thereby demonstrating the contact osteogenesis process (Fig. 2, B). At 21 days, the bone trabeculae were more mature showing integration of the granules by the newly synthetized bone (Fig. 2, D). The autogenous bone was almost totally surrounded with some areas already demonstrating signs of bone remodeling through the resorption activity of osteoclasts inside a Howship lacunae (Fig. 3). The analysis at 42 days showed a well-organized and mature trabecular bone present between the BC granules and autogenous bone joining them. The interface of biomaterials/bone tissue demonstrated biocompatibility and effective osseointegration with no interfacial soft tissue for both grafts implanted in the alveolus (Fig. 2, F). The autogenous bone particles showed remodeling through resorption and deposition of new bone, which made it difficult to distinguish the interface between the new and the grafted bone (Fig. 4).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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Histometric Analysis

The quantitative assessment showed the volumetric percentage of newly formed bone tissue (vital bone), soft tissue (coagulum and connective tissue), and BC in the dental alveoli at 7, 21, and 42 days after surgery (Fig. 5). The control group demonstrated the highest vital bone percentage (green area) when compared with BC and BC + AB groups (statistically significant at 1%) because no biomaterial was graft. There was no statistically significant difference in the percentage of BCs between BC and BC + AB groups (gray area). The ceramic material had undergone 7.76% absorption between 7 and 42 days in both groups, whereas in parallel with it, new vital bone deposition could be observed in both the groups. The deposition of new vital bone was significantly higher (statistically significant at 1%) in BC + AB group when compared to BC at all periods.

Fig. 5

Fig. 5

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Immunohistochemical Results

The immunolabeling analyses were performed to characterize the presence of pre-osteoblasts at 21 and 42 days after tooth extraction (Fig. 6). Runx2 transcription factor was present in the preosteoblasts, close to the bone trabeculae after 21 days, especially in the AB + BC group (Fig. 6, B). After 42 days, the AB + BC group showed high Runx2-positive cells in comparison to the BC group (Fig. 6, C and D). The scores obtained from the semiquantitative analysis showed a moderate immunolabeling at 21 days and mild at 42 days for the BC group, whereas the BC + AB group showed intense labeling for both periods (Table 1).

Fig. 6

Fig. 6

Table 1

Table 1

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Discussion

The bone substitutes in various bone regeneration procedures in implantology including grafting of alveolar sockets should preferably be biocompatible, integrate with the new bone, and maintain the volume of the regenerated area by slow resorption and concomitant bone deposition, and whenever possible it should accelerate the repair process.28,29 The purpose of this study was to evaluate the vital bone formation in alveolar sockets of rats grafted with a biphasic calcium phosphate BC alone or in combination with autogenous bone harvested from the skull.

The dental socket after extraction offers an appropriate model to study the alveolar bone formation and has been used as a way to analyze systemic and/or local factors, which include the bone substitute materials, what in some cases can disturb or improve the bone deposition process.7,12,26 In the present study, the harvesting of bone from calvaria and its grafting into the alveolus had no intention to demonstrate that this procedure is viable and can be performed clinically in patients as a routine, but only to analyze the influence of the autogenous bone when mixed with a synthetic biomaterial. As the study was conducted in rats, it is quite difficult to harvest bone intraorally in enough quantity due to mouth size, what has brought our option to collect it from skull because of the good amount available and the similarities of it with those from the oral cavity.

Despite the feasible use of bone substitutes in implantology for reconstructive procedures, some studies have reported that the presence of the biomaterial by itself is able to impair the reparative dynamics, including healing of the alveolar socket,30,31 by delaying the deposition of new bone (vital bone). This finding was corroborated in our study as evidenced by histometry, with a smaller percentage of vital bone (statistically significant at 1%) present in the groups, which were grafted with BC when compared to the control group (Fig. 5). Nonetheless, several other studies have shown that the use of osteoconductive biomaterials with slow substitution rate may be clinically beneficial, especially in the aesthetic zone to maintain the crestal bone levels over time, as they reduce the degree of buccal bone wall resorption after dental extraction.9,28,30,32 Autogenous bone has been associated with faster deposition of new bone matrix (vital bone) when combined with bone biomaterials possibly due to increased cell source and reduced healing time.28,33

Quantitative analysis demonstrated an average resorption rate of 7.75% for the bioceramic (BC and BC + AB groups) over 42 days, with concomitant new osteoid tissue synthesis (vital bone) (Fig. 5). In the BC + AB group, a more rapid progression of healing process compared to the BC group was seen. At 7 days, several areas of the alveolar socket demonstrated contact osteogenesis, in that the new bone formation was seen from the surface of the graft particles and autogenous bone to the connective tissue. Many of these trabeculae, albeit still immature, were joined through the marrow area creating a link between graft particles and autogenous bone (Fig. 2, B). This characteristic was also observed in the BC group, but comparatively, the osteoid matrix demonstrated lower maturity and less organization, with some areas still intermingled in blood clot debris and granulation tissue (Fig. 2, A).

This osteogenesis by contact on the surface of BC is most likely due to its properties such as optimal macro/micro porosity, interconnectivity, the pore diameter, and chemical composition. The biphasic calcium phosphate ceramic with high porosity favors the adsorption of plasma proteins on its surface, forming a temporary matrix which is able to recruit and modulate the cellular activities, thereby promoting osteoblast adhesion.34–36 This whole integration process of BC composed of HA and β-TCP to bone, from the initial stages to the late ones was previously evaluated by our research group, which provided evidence for osteogenesis by contact by using the light and scanning electron microscopy.12 The addition of autogenous bone as grafting material (BC + AB group) seems to have enhanced this process probably due to its osteogenic capacity, in which osteoblasts and osteocytes from the bone fragments provided an additional source of cells during the initial process of osteosynthesis. Histometrically, this is confirmed by the fact that 23.24% vital bone was present after 7 days in the BC + AB group compared to 16.11% in the BC group (P < 0.01) (Fig. 5). The autogenous bone graft from skull is considered a material with good clinical and experimental outcome,37,38 as its intramembranous embryological origin is similar to the bones from oral and maxillofacial region, thereby improving its integration to the recipient bed and apposition of the new bone when compared to grafts of endochondral origin, such as the iliac crest.39,40

After 21 days, histological analysis showed higher maturity and mineralization of bone trabeculae between the granules of the BC in the BC + AB group (Fig. 2, C and D), and Howship lacunae in the surface of autogenous bone fragments containing osteoclasts in resorptive function (Fig. 3). Through histometry, this group demonstrated 11.3% more vital bone in comparison to the BC group. In this intermediate period, we believe that the best performance was led by the synergistic action of 3 factors: (1) the osteogenic capacity of the calvarial bone graft; (2) due to the resorption/remodeling process of autogenous bone graft with the release of bone morphogenetic protein promoting an osteoinductive effect on the cells around the surface of the BC and autogenous bone41; and (3) by the ratio of HA and β-TCP (60/40) in the BC used in this study because the gradual dissolution of their phase components, especially the β-TCP leaves osteoconductive micro lacunae18,20,42 rich in calcium and phosphate ions, which assist in the process of bone formation and mineralization.43–45 This observation was confirmed by the immunohistochemical findings that showed an intense labeling “+++” of Runx2 in an intermediate period of the bone repair process. This was not expected as several studies have demonstrated a strong expression of such protein in the early periods of osteosynthesis when the osteoblast differentiation rate is high.46,47 However, the BC group presented moderate labeling “++” at 21 days compared to the BC + AB group (Fig. 6, A and B; Table 1).

After 42 days, the autogenous bone fragments were nearly indistinguishable from the newly formed bone (Fig. 2, F). During this process, the autogenous fragments are likely to have undergone reportion while releasing bone morphogenetic protein and thereby promoting osteoinduction. In association with the osteoconductive potential of the biphasic calcium phosphate ceramic, a synergistic effect could have resulted in an increased amount of vital bone formation (49%) for BC + AB when compared to BC (41.7%), thereby corroborating with the results of Galindo-Moreno et al.33

It is worth mentioning that the osteoinductive response, represented by the positive staining for transcription factor Runx2 at 42 days, had a high score “+++” demonstrating that the cells were still differentiating, that is, there was activity of the transcription factor that was transforming preosteoblasts into osteoblasts, feature not observed in the group containing BC only, as a light labeling “+” was measured (Fig. 6, C and D; Table 1). The presence of autogenous bone seems to have led to a constant stimulus in the osteogenesis process even in the late stages of analysis (42 days), which is fairly advantageous due to the synergistic action through their osteogenic and osteoinductive activity, combined with the osteoconductive properties of BCs leading to the restructuring of intra-alveolar bone matrix quickly and more effectively.

A perfect and coordinated chronological activity of biomaterial resorption simultaneously with deposition of new bone matrix is one of the key factors for the success of bone reconstructive procedures. Materials with excessively fast resorption rates, such as TCP BCs, lead to a considerable decrease in bone volume at the end of the reparative process.42 Synthetic bone substitutes with a controlled degree of resorption, as in HA/β-TCP biphasic BCs, can over time promote a larger volumetric stability of the newly synthesized bone, which is especially desirable for postextraction sites in the aesthetic zone.48,49 Although we observed a low resorption rate of BCs and stimulatory activity of autogenous bone, the results of this study did not allow us to make any assertion about the maintenance of alveolar dimensions during the healing process. We would recommend further studies quantifying the volume to clarify this issue.

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Conclusions

Given the limitations of this study, it can be concluded that the autogenous bone was able to stimulate bone turnover enabling a larger amount of vital bone synthesis and can be recommended as a viable grafting material in combination with synthetic biphasic BC.

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Disclosure

The authors claim to have no financial interest, directly or indirectly, in any entity that is commercially related to the products mentioned in this article.

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Approval

All the procedures were conducted in accordance with ethical principles for animal research, as approved by Committee of Ethics for use of Animals of Campus of Ribeirão Preto, University of São Paulo, Brazil, and received a vermifuge for animal use (Systamex; Shering of Brazil, São Paulo, SP, Brazil) during 3 days.

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Roles/Contributions by Authors

R. M. Macedo: Researcher and PhD student at the time of the development of this study; executioner of all the procedures with animals, histomorphometrical analysis, and writing of the article. S. A. Lacerda: Vice coordinator and co-supervisor of the study; responsible for statistical analysis of the research. R. Okamoto: Collaborating researcher; responsible for all the immunohistochemical analysis. S. Shahdad: Collaborating researcher; responsible for scientific revision and correction of English language. L. G. Brentegani: Coordinator and supervisor of the entire research developed.

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Acknowledgments

The authors would like to thank Ms. Adriana M. G. Silva, Ms. Edna A. S. Moraes, Mr. Gilberto Andre´ e Silva, and Mr. Kleber Augusto Loureiro for technical assistance.

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Keywords:

extraction socket; bone graft; bone substitutes; animal study

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