Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Oxytocin promotes bone formation during the alveolar healing process in old acyclic female rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fresh-frozen human bone allograft in vertical ridge augmentation: clinical and tomographic evaluation of bone formation and resorption

The aim of the current study is to evaluate fresh-frozen human bone allografts (FHBAs) used in vertical ridge augmentation clinically and by computed tomography, and to analyze the resulting bone formation and graft resorption. Sixteen FHBAs were grafted in the maxillae and mandibles of 9 patients. The FHBAs, which were provided by the Musculoskeletal Tissue Bank of Marilia Hospital (Unioss), were frozen at -80A degrees C. After 7 months, dental implants were placed and bone parameters were evaluated. Vertical bone formation was measured by computerized tomography before (T0) and at 7 months (T1) after the surgical procedure. Bone graft resorption was measured clinically from a landmark screw head using a periodontal probe. The results were analyzed by Student's t-test. Significant differences existed in the bone formation values at T0 and T1, with an average change of 4.03 +/- A 1.69 mm. Bone graft resorption values were 1.0 +/- A 0.82 mm (20%). Implants were placed with varying insertion torque values (35-45 Ncm), and achieved primary stability. This study demonstrates that FHBAs promote satisfactory vertical bone formation with a low resorption rates, good density, and primary implant stability.

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.

Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic rats

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

Use of BioGran and Calcitite in bone defects: Histologic study in monkeys (Cebus apella)

The present study compares the biologic behavior of BioGran and Calcitite as fillers for surgical cavities in the mandibles of 4 adult monkeys (Cebus apella). The surgical cavities were prepared through both mandibular cortices, with a diameter of 5 mm, in the angle region. Two cavities were prepared on the right side and 1 on the left and divided into 3 groups: R-1 sites were filled with bioglass (BioGran), R, sites were not filled, and L sites were filled with hydroxyapatite (Calcitite). After 180 days the animals were sacrificed and the specimens were removed for histologic processing. Results showed no bone formation in group R, (empty cavities). BioGran-treated sites showed bone formation and total repair of the bone defect, and the bioglass particles were almost totally resorbed and substituted by bone. The few remaining crystals were in intimate contact with newly formed bone. Calcitite did not allow bone formation, and granules inside the cavities were involved by connective tissue. Based upon those results, the authors concluded that bioglass resulted in total obliteration of the surgical cavity with bone and hydroxyapatite was present in a large amount and involved by connective tissue, without bone formation.

TGF-β and new bone formation

The aim of this work is to present, by a literature review, the principal characteristics of TGF-beta, in the regulation and new bone formation. © 2007 Sociedad Chilena de Anatomía.

Treatment with tacrolimus enhances alveolar bone formation and decreases osteoclast number in the maxillae: A histomorphometric and ultrastructural study in rats

Recent studies have suggested that tacrolimus monotherapy is a beneficial therapeutic alternative for the normalization of cyclosporin- induced bone loss in animal models and humans. The mechanism accounting for this action is unclear at present. In the present study, we attempted to determine the effect of tacrolimus monotherapy on alveolar bone using histological, histomorphometrical and transmission electron microscopy (TEM).Groups of rats (n= 10 each) were treated with either tacrolimus (1mg/ kg/ day, s.c.) or drug vehicle for 60 days. Fragments containing maxillary molars were processed for light microscopy to investigate the alveolar bone volume, trabecular separation, number of osteoclasts and osteoblasts, and transmission electron microscopy to investigate their ultrastructural basic phenotype.Treatment with tacrolimus monotherapy during 60 days may induce increases in alveolar bone volume (BV/ TV,%; P < 0.05) and a non- significant decrease in trabecular separation (Tb. Sp, mm; P > 0.05), represented by a decrease in osteoclast number (N. Oc/ BS; P < 0.05) and maintenance of osteoblast number (N. Ob/ BS; P > 0.05). Osteoblasts were often observed as a continuous layer of active cells on the bone surface. Osteoclasts appeared to be detached from the resorbed bone surface, which was often filled by active osteoblasts and collagen- rich matrix. Moreover, osteoclasts in the treated group were frequently observed as inactive cells (without ruffled border, clear zone and detached from the bone surface).Within the limits of the present study, we conclude that tacrolimus leads to an increase in alveolar bone formation, which probably exerts action on osteoclasts. Tacrolimus could, therefore, play a crucial role in the control of both early osteoclast differentiations from precursors, as well as in functional activation.

Biology of bone tissue: structure, function, and factors that influence bone cells

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

Bone stress and strain after use of a miniplate for molar protraction and uprighting: a 3-dimensional finite element analysis

The aim of this study was to use the finite element method to evaluate the distribution of stresses and strains on the local bone tissue adjacent to the miniplate used for anchorage of orthodontic forces. Methods: A 3-dimensional model composed of a hemimandible and teeth was constructed using dental computed tomographic images, in which we assembled a miniplate with fixation screws. The uprighting and mesial movements of the mandibular second molar that was anchored with the miniplate were simulated. The miniplate was loaded with horizontal forces of 2, 5, and 15 N. A moment of 11.77 N.mm was also applied. The stress and strain distributions were analyzed, and their correlations with the bone remodeling criteria and miniplate stability were assessed. Results: When orthodontic loads were applied, peak bone strain remained within the range of bone homeostasis (100-1500 mu m strain) with a balance between bone formation and resorption. The maximum deformation was found to be 1035 mu m strain with a force of 5 N. At a force of 15 N, bone resorption was observed in the region of the screws. Conclusions: We observed more stress concentration around the screws than in the cancellous bone. The levels of stress and strain increased when the force was increased but remained within physiologic levels. The anchorage system of miniplate and screws could withstand the orthodontic forces, which did not affect the stability of the miniplate.

Effects of the Er,Cr:YSGG laser on bone and soft tissue in a rat model

The purpose of this study was to investigate the histological changes that occur in rat soft and hard tissues after Er,Cr:YSGG laser surgery. Each of 20 rats was submitted to four procedures which were randomly distributed to the right and left sides of the animal: procedure 1 dorsal incision with a scalpel; procedure 2 dorsal incision with a 2.0-W Er,Cr:YSGG laser; procedure 3 skull defect created with a diamond bur; procedure 4 skull defect created with a 3.0-W Er,Cr:YSGG laser. The animals were killed 3, 7, 15 and 30 days after surgery, and histological examinations were performed. The histometric analysis of the bone defects was evaluated using an unpaired t-test. Initially, the dorsum showed more histological signs of repair following procedure 1, although similar healing responses following procedures 1 and 2 were seen on day 30 after surgery. By day 30 the bone formation observed following procedure 4 was much more evident than following procedure 3. The unpaired t-test identified significant differences in bone formation on day 30 (p = 0.01), whereas a greater bone percentage was seen following procedure 4 than following procedure 3 (79.96 +/- 10.30% and 58.23 +/- 9.99%, respectively). Thus, histological repair of the Er,Cr:YSGG laser wounds was similar to that of the scalpel wounds. However, skull defects created with the Er,Cr:YSGG laser showed greater bone formation than defects created with the bur. Within the limitations of this study, we can conclude that the Er,Cr:YSGG laser is a promising surgical instrument in vivo, particularly for bone surgery.

Evaluation of the tissue response to MTA and MBPC: Microscopic analysis of implants in alveolar bone of rats

The aim of this study was to evaluate and compare the quantitative and qualitative inflammatory responses and bone formation potential after implantation of polyethylene tubes filled with a new calcium hydroxide containing sealer (MBPc) and Prolloot mineral trioxide aggregate (MIA). There were 48 Wistar rats divided in three groups: Group I (control group) empty polyethylene tubes were implanted in the extraction site; group II and III, polyethylene tubes were implanted filled with ProRoot mineral trioxide aggregate (MIA) and MBPc, respectively. At 7, 15, and 30 days after tube implantation, the animals were killed, the hemi-maxillas were removed and prepared to light microscopic analyses. The scores obtained were submitted to Kruskal-Wallis statistical test (p < 0.05). Significant differences between the materials were not observed. The results showed that both materials had similar biological response.

Osteoconductivity of modified fluorcanasite glass-ceramics for bone tissue augmentation and repair

Modified fluorcanasite glasses were fabricated by either altering the molar ratios of Na 2O and CaO or by adding P 2O 5 to the parent stoichiometric glass compositions. Glasses were converted to glass-ceramics by a controlled two-stage heat treatment process. Rods (2 mm x 4 mm) were produced using the conventional lost-wax casting technique. Osteoconductive 45S5 bioglass was used as a reference material. Biocompatibility and osteoconductivity were investigated by implantation into healing defects (2 mm) in the midshaft of rabbit femora. Tissue response was investigated using conventional histology and scanning electron microscopy. Histological and histomorphometric evaluation of specimens after 12 weeks implantation showed significantly more bone contact with the surface of 45S5 bioglass implants when compared with other test materials. When the bone contact for each material was compared between experimental time points, the Glass-Ceramic 2 (CaO rich) group showed significant difference (p = 0.027) at 4 weeks, but no direct contact at 12 weeks. Histology and backscattered electron photomicrographs showed that modified fluorcanasite glass-ceramic implants had greater osteoconductivity than the parent stoichiometric composition. Of the new materials, fluorcanasite glass-ceramic implants modified by the addition of P 2O 5 showed the greatest stimulation of new mineralized bone tissue formation adjacent to the implants after 4 and 12 weeks implantation. © 2010 Wiley Periodicals, Inc.

Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing

This study evaluated the influence of bone marrow aspirate (BMA), low-level laser therapy (LLLT) and their combination on bone healing in surgically created critical-size defects (CSDs) in rat calvaria. 40 rats were divided into four groups: C (control), BMA, LLLT and BMA/LLLT. A 5 mm diameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group BMA, the defect was filled with BMA. In groups LLLT and BMA/LLLT, the defect received laser irradiation (InGaAlP laser), was filled with blood clot or BMA respectively, and irradiated again. Animals were euthanized 30 days postoperatively. Histomorphometric and immunohistochemical analyses were performed. Newly formed bone area (NFBA) was calculated as percentage of the total area of the original defect. Proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) immunohistochemical staining were performed. PCNA-positive, Runx2-positive and OCN-positive cells were quantified. Data were statistically analyzed. Group BMA/LLLT had significantly greater NFBA than groups C, BMA or LLLT. Group BMA presented significantly greater NFBA than control, while group LLLT did not. Group BMA/LLLT presented a significantly higher number of PCNA-positive and OCN-positive cells than any of the other groups. Groups BMA/LLLT and BMA showed a significantly lower number of Runx2-positive cells than groups C or LLLT. The combination of BMA/LLLT yielded significantly greater bone formation in surgically created CSD in rat calvaria when compared to control, or either treatment alone. © 2013 Elsevier B.V. All rights reserved.

Lack of influence of the Schneiderian membrane in forming new bone apical to implants simultaneously installed with sinus floor elevation: an experimental study in monkeys

Aim: To describe the early healing processes around the implants installed after elevation of the sinus mucosa applying the lateral access technique without the use of grafting material.Material and methods: Immediately after the elevation of the maxillary sinus Schneiderian membrane by the lateral approach in eight monkeys, implants were installed without the use of grafting material. The healing of the tissue around the implants was evaluated after 4, 10, 20 and 30 days. Ground sections were prepared and analyzed histologically.Results: After 4 days of healing, the formation of coagulum and provisional matrix was documented within the elevated area. At 10-day interval, sprouts of woven bone were in continuity with the parent bone, and partly in contact with the implant surface at the base of the augmented area. While bone-to-implant contact increased after 20 and 30 days, the area underneath the Schneiderian membrane appeared reduced in volume and condensed toward the apex of the implants. The sinus mucosa was to some extent collapsed onto the implant surface and on the newly formed bone.Conclusions: The void initially occupied by the coagulum after sinus membrane elevation shrank substantially during the observation period. A lack of influence of the Schneiderian membrane in bone formation apical to implants was documented in the early phase of healing.