Osteoblasts engulf apoptotic bodies during alveolar bone formation in the rat maxilla

During bone formation, as in other tissues and organs, intense cellular proliferation and differentiation are usually observed. It has been described that programmed cell death, i.e., apoptosis, takes place in the control of the cellular population by removing of the excessive and damaged cells. Although it is generally accepted that apoptotic bodies are engulfed by professional phagocytes, the neighboring cells can also take part in the removal of apoptotic bodies. In the present study, regions of initial alveolar bone formation of rat molars were examined with the aim to verify whether osteoblasts are capable of engulfing apoptotic bodies, such as professional phagocytes. Rats aged 11-19 days were sacrificed and the maxillary fragments containing the first molar were removed and immersed in the fixative solution. The specimens fixed in glutaraldehyde-formaldehyde were processed for light microscopy and transmission electron microscopy. For the detection of apoptosis, the specimens were fixed in formaldehyde, embedded in paraffin, and submitted to the TUNEL method. The results revealed round/ovoid structures containing dense bodies on the bone surface in close contact to osteoblasts and in conspicuous osteoblast vacuoles. These round/ovoid structures showed also positivity to the TUNEL method, indicating that bone cells on the bone surface are undergoing apoptosis. Ultrathin sections showed images of apoptotic bodies being engulfed by osteoblasts. Occasionally, the osteoblasts exhibited large vacuoles containing blocks of condensed chromatin and remnants of organelles. Thus, these images suggest that osteoblasts are able to engulf and degrade apoptotic bodies. (c) 2005 Wiley-Liss, Inc.

Periosteum-Derived Cells as an Alternative to Bone Marrow Cells for Bone Tissue Engineering Around Dental Implants. A Histomorphometric Study in Beagle Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alveolar process preservation at implants installed immediately into extraction sockets using deproteinized bovine bone mineral - an experimental study in dogs

Aim To evaluate the soft tissue and the dimensional changes of the alveolar bony crest at sites where deproteinized bovine bone mineral (DBBM) particles, concomitantly with the placement of a collagen membrane, were used at implants installed into sockets immediately after tooth extraction. Material and methods The pulp tissue of the mesial roots of 3P3 was removed in six Labrador dogs, and the root canals were filled. Flaps were elevated bilaterally, the premolars hemi-sectioned, and the distal roots removed. Recipient sites were prepared in the distal alveolus, and implants were placed. At the test sites, DBBM particles were placed in the residual marginal defects concomitantly with the placement of a collagen membrane. No treatment augmentation was performed at the control sites. A non-submerged healing was allowed. Impressions were obtained at baseline and at the time of sacrifice performed 4 months after surgery. The cast models obtained were analyzed using an optical system to evaluate dimensional variations. Block sections of the implant sites were obtained for histological processing and soft tissue assessments. Results After 4 months of healing, no differences in soft tissue dimensions were found between the test and control sites based on the histological assessments. The location of the soft tissue at the buccal aspect was, however, more coronal at the test compared with the control sites (1.8 +/- 0.8 and 0.9 +/- 0.8 mm, respectively). At the three-dimensional evaluation, the margin of the soft tissues at the buccal aspect appeared to be located more apically and lingually. The vertical dislocation was 1 +/- 0.6 and 2.7 +/- 0.5 mm at the test and control sites, respectively. The area of the buccal shrinkage of the alveolar crest was significantly smaller at the test sites (5.9 +/- 2.4 mm2) compared with the control sites (11.5 +/- 1.7 mm2). Conclusion The use of DBBM particles concomitantly with the application of a collagen membrane used at implants placed into sockets immediately after tooth extraction contributed to the preservation of the alveolar process.

Influence of implants with different sizes and configurations installed immediately into extraction sockets on peri-implant hard and soft tissues: an experimental study in dogs

Aim: To study the influence on the healing of soft and hard peri-implant tissues when implants of different sizes and configurations were installed into sockets immediately after tooth extraction.Material and methods: Transmucosal cylindrical implants, 3.3 mm in diameter in the control sites, and conical 5 mm in diameter in the test sites, were installed into the distal socket of the fourth mandibular premolars in dogs immediately after tooth extraction. After 4 months, the hard and soft tissue healing was evaluated histologically. Results: All implants were integrated in mineralized mature bone. Both at the test and control sites, the alveolar crest underwent resorption. The buccal bony surface at the implant test sites (conical; 3.8 mm) was more resorbed compared with the control sites (cylindrical; 1.6 mm). The soft tissue dimensions were similar in both groups. However, in relation to the implant shoulder, the peri-implant mucosa was located more apically at the test compared with the control sites.Conclusion: The present study confirmed that the distance between the implant surface and the outer contour of the buccal alveolar bony crest influenced the degree of resorption of the buccal bone plate. Consequently, in relation to the implant shoulder, the peri-implant mucosa will be established at a more apical level, if the distance between the implant surface and the outer contour of the alveolar crest is small.

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis.Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05).Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam.Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Biomechanical effect of one session of low-level laser on the bone-titanium implant interface

Low-level laser (LLL) has been used on peri-implant tissues for accelerating bone formation. However, the effect of one session of LLL in the strength of bone-implant interface during early healing process remains unclear. The present study aims to evaluate the removal torque of titanium implants irradiated with LLL during surgical preparation of implant bed, in comparison to non-irradiation. Sixty-four Wistar rats were used. Half of the animals were included in LLL group, while the other half remained as control. All animals had the tibia prepared with a 2 mm drill, and a titanium implant (2.2 × 4 mm) was inserted. Animals from LLL group were irradiated with laser (gallium aluminum arsenide), with a wavelength of 808 nm, a measured power output of 50 mW, to emit radiation in collimated beams (0.4 cm2), for 1 min and 23 s, and an energy density of 11 J/cm2. Two applications (22 J/cm 2) were performed immediately after bed preparation for implant installation. Flaps were sutured, and animals from both groups were sacrificed 7, 15, 30, and 45 days after implant installation, when load necessary for removing implant from bone was evaluated by using a torquimeter. In both groups, torque values tended to increase overtime; and at 30 and 45 days periods, values were statistically higher for LLL group in comparison to control (ANOVA test, p < 0.0001). Thus, it could be suggested that a single session of irradiation with LLL was beneficial to improve bone-implant interface strength, contributing to the osseointegration process. © 2012 Springer-Verlag London Ltd.

Influence of buccal bony crest width on marginal dimensions of peri-implant hard and soft tissues after implant installation. An experimental study in dogs

Aim: To evaluate the influence of the width of the buccal bony wall on hard and soft tissue dimensions following implant installation. Material and methods: Mandibular premolars and first molars of six Labrador dogs were extracted bilaterally. After 3 months of healing, two recipient sites, one on each side of the mandible, were prepared in such a way as to obtain a buccal bony ridge width of about 2 mm in the right (control) and 1 mm in the left sides (test), respectively. Implants were installed with the coronal margin flush with the buccal alveolar bony crest. Abutments were placed and the flaps were sutured to allow a non-submerged healing. After 3 months, the animals were euthanized and ground sections obtained. Results: All implants were completely osseointegrated. In respect to the coronal rough margin of the implant, the most coronal bone-to-implant contact was apically located 1.04 ± 0.91 and 0.94 ± 0.87 mm at the test and control sites, respectively, whereas the top of the bony crest was located 0.30 ± 0.40 mm at the test and 0.57 ± 0.49 mm at the control sites. No statistically significant differences were found. A larger horizontal bone resorption, however, evaluated 1 mm apically to the rough margin, was found at the control (1.1 ± 0.7 mm) compared to the test (0.3 ± 0.3 mm) sites, the difference being statistically significant. A thin peri-implant mucosa (2.4-2.6 mm) was found at implant installation while, after 3 months of healing, a biological width of 3.90-4.40 mm was observed with no statistically significant differences between control and test sites. Conclusions: A width of the buccal bony wall of 1or 2 mm at implant sites yielded similar results after 3 months of healing in relation of hard tissue and soft tissues dimensions after implant installation. © 2012 John Wiley & Sons A/S.

Tissue engineering: Using collagen type i matrix for bone healing of bone defects

Among the many tissues in the human body, bone has been considered as a powerful marker for regeneration and its formation serves as a prototype model for tissue engineering based on morphogenesis. Therefore, collagen type I is one of the most useful biomaterials used in tissue engineering as extracellular matrix components capable to promote bone healing. The literature reveals excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenicity, making collagen type I the primary resource in medical applications. Thus, it was also used for tissue engineering including skin replacement, bone substitutes, and artificial blood vessels and valves. The authors describe the treatment of an abscessed apical periodontal cyst and show good outcomes of bone healing, using tissue engineering, as collagen type I matrix. © 2013 by Mutaz B. Habal, MD.

Orthodontic force increases interleukin-1β and tumor necrosis factor-α expression and alveolar bone loss in periodontitis

Background: The present study aims to evaluate the effects of orthodontic movement (OM) on the periodontal tissues of rats with ligature-induced periodontal disease. Methods: Eighty-eight rats were divided into four groups: 1) negative control (sham operated); 2) periodontal disease; 3) OM; and 4) periodontal disease followed by OM (OMP). Rats were sacrificed 3 hours or 1, 3, or 7 days after OM commencement. Bone volume fraction (BVF) and bone mineral density (BMD) were assessed in hemimaxillae by microcomputed tomography analysis. Expression of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated in gingival samples by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, and in the furcation region by immunohistochemistry analysis (IHC). Results: The OMP group had lower BVF and BMD levels compared to the other groups at day 7 (P <0.05). Maximum messenger ribonucleic acid expression of both cytokines was observed in the OMP group at day 1 (P <0.05). In the same period, all proteins were expressed in high levels for all test groups compared to the control group. The number of cells positive for IL-1β and TNF-α by IHC was highest in the OMP group at day 1, with progressive reduction thereafter. Conclusion: The results suggest that OM acts synergistically with periodontal disease in periodontal breakdown through upregulation of proinflammatory cytokines.

Histologic Characterization of Engineered Tissues in the Canal Space of Closed-apex Teeth with Apical Periodontitis

Introduction: The aim of this study was to investigate the capacity of endodontic regenerative procedures combining an induced blood clot, platelet-rich plasma (PRP), and bone marrow aspirate (BMA) to regenerate dental pulp in canine closed-apex necrotic teeth. Methods: Apical periodontitis was induced in 20 upper and lower premolars of 2 dogs. After biomechanical preparation, enlargement to a #60 file, and disinfection with a triantibiotic paste for 28 days, the roots were randomly assigned to 4 treatment groups: blood clot (BC), BC + PRP gel, BC + BMA gel, and BC + BMA/PRP gel. Negative controls were also included. After a 3-month follow-up period, the animals were killed. Results: Histologic analysis showed the presence of newly formed vital tissues (connective, cement-like, and bone-like tissue) in 23 of the 32 treated roots (71.87%). There was no statistically significant difference between the treatment groups. Conclusions: New vital tissues were formed and characterized as connective, cementum-like, or bone-like, but not as pulp-like tissue; PRP and/or BMA did not improve the tissue ingrowth. © 2013 American Association of Endodontists.

Stress analysis in bone tissue around single implants with different diameters and veneering materials: A 3-D finite element study

The aimof this study was to evaluate the stress distribution on bone tissue with a single prosthesis supported by implants of large and conventional diameter and presenting different veneering materials using the 3-D finite elementmethod. Sixteenmodels were fabricated to reproduce a bone block with implants, using two diameters (3.75 × 10 mmand 5.00 × 10 mm), four different veneering materials (composite resin, acrylic resin, porcelain, and NiCr crown), and two loads (axial (200 N) and oblique (100 N)). For data analysis, the maximum principal stress and vonMises criterion were used. For the axial load, the cortical bone in allmodels did not exhibit significant differences, and the trabecular bone presented higher tensile stresswith reduced implant diameter. For the oblique load, the cortical bone presented a significant increase in tensile stress on the same side as the loading for smaller implant diameters. The trabecular bone showed a similar but more discreet trend. There was no difference in bone tissue with different veneering materials. The veneering material did not influence the stress distribution in the supporting tissues of single implant-supported prostheses. The large-diameter implants improved the transference of occlusal loads to bone tissue and decreased stress mainly under oblique loads.Oblique loading was more detrimental to distribution stresses than axial loading. © 2013 Elsevier B.V. All rights reserved.

Comparison of changes in dental and bone radiographic densities in the presence of different soft-tissue simulators using pixel intensity and digital subtraction analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oral squamous carcinoma cells secrete RANKL directly supporting osteolytic bone loss

Objective: Local invasion of bone is a frequent complication of oral squamous cell carcinoma (OSCC). Development of these osteolytic lesions is mediated by osteoclasts. Receptor activation of NF-kappa B ligand (RANKL) signaling, counteracted by osteoprotegerin (OPG), regulates osteoclastogenesis. Previous studies in rodent models have demonstrated that inhibition of RANKL decreases tumor growth and lesions within bone. However, the contributory role of OSCC cells to this disease process has yet to be defined.Methods: RANKL expression was assessed in a panel of OSCC cell lines by qPCR, flow cytometry, and ELISA. Induction of osteoclastogenesis was assessed by co-culture with macrophages or with OSCC-derived conditioned medium. In an animal model of bone invasion, nude mice were injected intratibially with UMSCC-11B cells expressing a RANKL luciferase promoter to detect tumor-derived RANKL activity. Osteolytic lesions were analyzed by X-ray, micro-CT, and histological methods. RANKL expression was assessed in human OSCC tissues by immunohistochemistry.Results: We demonstrated that OSCCs express varied levels of all RANKL isoforms, both membrane-bound and soluble RANKL. Both co-culture and treatment with OSCC-conditioned media induced osteoclastogenesis. In mice, we demonstrated human RANKL promoter activity during bone invasion. Over the course of the experiment, animals suffered osteolytic lesions as RANKL-driven luciferase expression increased with time. After 8 weeks, human-derived RANKL was detected in areas of bone resorption by immunohistochemistry. Similar epithelial RANKL expression was detected in human OSCC tissues.Conclusion: These data demonstrate the ability of OSCCs to produce RANKL, directly altering the tumor microenvironment to increase osteoclastogenesis and mediate local bone invasion. (C) 2012 Elsevier Ltd. All rights reserved.

Bone-implant contact around crestal and subcrestal dental implants submitted to immediate and conventional loading

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Periodontal disease-associated compensatory expression of osteoprotegerin is lost in type 1 diabetes mellitus and correlates with alveolar bone destruction by regulating osteoclastogenesis

Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD.