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.

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.

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

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

Avaliação microscópica da propriedade biológica e da biocompatibilidade do osso composto e do osso integral de origem bovina

The aim of this study was to evaluate the biological properties and biocompatibility of bovine non-demineralized lyophilized and composite bones implanted in tibiae bone cavities and at the subcutaneous level. Twenty-four rats were used and sacrificed 15 and 45 days later. At the subcutaneous level, after 15 days an inflammatory reaction was seen around biomaterial particles with the presence of giant cells and at 45 days fibrous connective tissue had also developed. No signs of ectopic bone formation were observed at tibiae regions; more bone neoformation was observed at the control group (15 days) with 42.8% of the outer cortex layer against 22.6% at Orthogen and 25% at GenMix groups. At 45 days, correspondent values for bone neoformation were 62.5% at control, 26% at Orthogen, and 35% at GenMix groups, respectively. It can be concluded that both materials tested were biocompatible aiming to bone neoformation by their osteoconductive properties with no ectopic formation sites observed.

Ectopic ossification presenting as osteoid metaplasia in a salivary mucocele in a Shih Tzu dog

Background: Salivary mucocele is an accumulation of saliva in a single or multiloculated cavity lined by connective tissue that is contiguous to a salivary gland-duct complex and is the most common condition affecting the salivary glands in dogs. Occasionally, different types of metaplastic lesions, such as squamous and osseous metaplasia - which are rare lesions in animals - can be observed in association with salivary mucocele.Case presentation: A right facial enlargement was suddenly observed in a 4-year-old non-spayed female Shih-Tzu dog. The lesion presented itself as a soft and fluctuant mass located in the right side of the face near to the neck. Histologically, the mass consisted of a cavitary formation without an epithelial lining. Additionally, microscopic examination revealed the presence of osteoid-producing cells which gave rise to areas of bone formation, probably induced by irritation due to the presence sialoliths. Such cells and bone formations were also present in the cavity wall, consequently leading us to classify the condition as a salivary mucocele with osseous metaplasia.Conclusions: In the present case, the pathogenesis was probably associated with the presence of sialoliths, which can behave as etiological agents for the metaplastic lesion. The occurrence of osteoid metaplasia is a rare peculiar condition in the canine salivar y gland, and due to the rarity and lack of information about this specific disease, no clinical data can yet be associated with the development of salivary mucocele with osseous metaplasia in dogs.

Impact of skeletal maturation on bone metabolism biomarkers and bone mineral density in healthy Brazilian male adolescents

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

Use of platelet-rich plasma under autogenous onlay bone grafts

Objectives: To analyze the healing of autogenous onlay bone grafts in three different situations, focusing on the interface area.Material and methods: Sixteen rabbits underwent autogenous bone graft surgeries in the calvaria. The block bone grafts were positioned in three different situations: direct contact between bone graft and receptor bed, graft interposed by particulate bone, and graft interposed by platelet-rich plasma (PRP). After 7, 15, 30, and 60 days, the specimens were retrieved for histological and morphometric evaluation.Results: All groups healed uneventfully and presented incorporation of the grafts after 30 days. A slightly more evident new bone formation could be observed in the PRP group in the first analyzed period, and an earlier maturation of bone in the last period, although no statistically significant differences were achieved.Conclusion: the use of additional material between the bone graft and the receptor bed when using the onlay technique must be carefully considered, taking into account the size of the reconstruction and the cost/benefit relation. The addition of PRP in between autogenous bone blocks and the receptor bed did not confer significant benefit for the new bone formation and healing on the calvaria of bone of rabbits.

Bone metabolism biomarkers, body weight, and bone age in healthy Brazilian male adolescents

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

Histological and radiological changes in cranial bone in the presence of bone wax

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

Bone healing in critical-size defects treated with platelet-rich plasma activated by two different methods. A histologic and histometric study in rat calvaria

The purpose of this study was to analyze histologically the influence of platelet-rich plasma (PRP) coagulated with two different activators on bone healing in surgically created critical-size defects (CSD) in rat calvaria.Forty-eight rats were divided into three groups: C, PRP-C and PRP-T. An 8 mm diameter CSD was created in the calvarium of each animal. In group C, the defect was filled by a blood clot only. In groups PRP-C and PRP-T, the defect was filled with PRP activated with either calcium chloride or thromboplastin solution, respectively. Each group was divided into two subgroups (n = 8 per subgroup) and killed at either 4 or 12 weeks postoperatively. Histologic and histometric analyses were performed. The amount of new bone formed was calculated as a percentage of the total area of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance, Tukey's post hoc test, p < 0.05).No defect completely regenerated with bone. Group PRP-C had a statistically greater amount of bone formation than groups C and PRP-T at both time points of analysis. No statistically significant differences were observed between groups C and PRP-T.It can be concluded that the type of activator used to initiate PRP clot formation influences its biological effect on bone healing in CSD in rat calvaria.

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.