Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Bone healing in critical-size defects treated with either bone graft, membrane, or a combination of both materials: a histological and histometric study in rat tibiae

Objectives: The aim of the present investigation was to histologically analyze the effect of using lyophilized bovine bone (GenOx (R) organic matrix) with (or without) guided tissue regeneration (using a decalcified cortical osseous membrane [GenDerm (R)]) on bone healing in surgically created critical-size defects created in rat tibia.Material and methods: Surgical critical-size bone defects were created in 64 animals that were randomly divided into four groups: group I (control); group II (defect filled with GenOx (R)); group III (defect covered by GenDerm (R)); group IV (defect filled with GenOx (R) and covered by GenDerm (R)). Animals were killed at 30 or 90 days post-surgery. The specimens were embedded in paraffin, serially cut, and stained with hematoxylin and eosin for analysis under light microscopy. The formation of new bone in the cortical area of the defect was histomorphometrically evaluated.Results: All experimental groups demonstrated superior bone healing compared with the control group. However, group IV samples showed evidence of more advanced healing at both 30 and 90 days post-surgery as compared with the other experimental groups.Conclusions: The bovine organic bone graft GenOx (R) associated with GenDerm (R) this produced the best treatment results in the case of critical-size defects in rat tibia.

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cartical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs

AimTo compare the influence of autologous or deproteinized bovine bone mineral as grafting material on healing of buccal dehiscence defects at implants installed immediately into the maxillary second incisor extraction socket in dogs.Material and methodsIn the maxillary second incisor sockets of 12 Labrador dogs, implants were installed immediately following tooth extraction. A standardized buccal defect was created and autologous bone particles or deproteinized bovine bone mineral were used to fill the defects. A collagen membrane was placed to cover the graft material, and the flaps were sutured to fully submerge the experimental areas. Six animals were sacrificed after 2 months, and six after 4 months of healing. Ground sections were obtained for histological evaluation.ResultsAfter 2 months of healing, all implants were osseointegrated. All buccal dehiscence defects were completely filled after 2 months irrespective of the augmentation material (autologous bone or Bio-Oss (R)) applied. Bone-to-implant contact (BIC) on the denuded implant surfaces was within a normal range of 30-40%. However, the newly formed tissue at 2 months was partially resorbed (> 50% of the area measurements) after 4 months.ConclusionsApplying either autologous bone or deproteinized bovine bone mineral to dehiscences at implants installed immediately into extraction sockets resulted in high degree of regeneration of the defects with satisfactory BIC on the denuded implant surface.To cite this article:De Santis E, Botticelli D, Pantani F, Pereira FP, Beolchini M, Lang NP. Bone regeneration at implants placed into extraction sockets of maxillary incisors in dogs.Clin. Oral Impl. Res. 22, 2011; 430-437.

Avaliação radiográfica, densitométrica e histológica do uso de perfurações ósseas para o estímulo de consolidação de fraturas do terço distal do rádio de cães

To stimulate the bone callus development process from distal third of radius, 24 adult mongrel dogs used were from both sexes. These dogs were separated in two experimental groups of 12 animals each, named control and treated, divided in 4 moments (M1=15 days; M2=30 days; M3=45 days; M4=60 days), who underwent were performed surgical fractures. In treated group, it was performed bone perforations on proximal and distal edges, craniolateral and mediolateral to the fracture site. At the end of each moment, control and treated animals were evaluated by radiography, histology, and bone mineral densitometry (BMD) was determined on fracture site. According to the radiographic data of treated dogs, it was verified on days 15 and 30 more intense bone regeneration than control group. During M3 and M4, it wasn't detected any difference in bone reparation process betweencontrol and treated groups. In densitometric study, BMD values were greater in treated animals than in control dogs. Histological studies revealed at 15 and 30 days chondrocyte hyperplasia and initial endochondral ossification on drilled limbs; control group showed sustainment connective tissue and initial chondrocyte hiperplasia. At M3 and M4 of the treated group, were verified development and remodeling of periosteal callus in more advanced phases when comparing with limbs from control group. It can be concluded that using perforations enhances blood flow supply and activation of osteogenic cells on fracture site, stimulating the beginning of fracture consolidation process.

Bone spreading technique a case report

Bone spreading technique (BST) is a horizontal augmentation with minimal trauma for simultaneous implant placement and an alternative to Summer's osteotome technique both for its clinical use and for the armamentarium. The foremost advantage of the crest dilation technique is a substantially less invasive method; the buccal wall expands after the medullary bone is compressed against the cortical bone. The lateral dilation and compaction of medullary bone improved primary stability. The vital difference is that the BST used in this case report avoided discomfort of the patient, thus eliminating the need for malleting.

Bone structure, strength and microhardness resulting after hindlimb unloading in rats

Introduction: Bone strength is influenced by a number of different determinants, such as mass, size, geometry and also by the intrinsic material properties of the tissue. Aims: The structure and mechanical properties of the femur were analyzed in aged (14 mo-old) animals submitted to hindlimb unloading (HU) for 21 days. Methods: Twenty Wistar rats were randomly divided into Control and HU groups and the femur was submitted to dual X ray absorptiometry (DXA), DIGORA radiographic density, mechanical compression testing and Knoop microhardness analyse in cortical and cancellous bone. Results: Femurs from HU group presented significantly lower failure load, decreased bone mineral density (BMD)/bone mineral content (BMC) in whole bone; proximal/distal epiphysis and middiaphyseal cortical bone measured by DXA were similar in the two groups; radiographic density from areas in proximal epiphysis was significantly lower in HU group, and microhardness measured at periosteal and endosteal levels were also signifcantly diminished in HU compared with Control group. Conclusion: Disuse induced damage in the trabecular femoral bone architecture with decisive effect on the head and trochanteric fossa, which became weaker. Bone diaphyseal cortical hardness also suffered effect of unloading, probably related to osteocyte/osteoblast activity.

Healing outcomes at implants installed in sites augmented with particulate autologous bone and xenografts. An experimental study in dogs

Aim: To evaluate the integration of implants installed at the interface of pristine and grafted tissue augmented with particulate autologous bone or deproteinized bovine bone mineral (DBBM), concomitantly with a collagen membrane. Material and methods: In 6 Labrador dogs, the distal root of 3P3 and 4P4 was endodontically treated and hemi-sected, and the mesial roots extracted concomitantly with the extraction of 2P2. The buccal bony walls were removed, and two box-shaped defects, one larger and one smaller, were created. After 3 months, flaps were elevated, and the defects were filled with particulate autologous bone or DBBM in the right and left side of the mandible, respectively. Collagen membranes were used to cover the grafted areas. Three months later, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites at the interface between grafts and pristine bone. One implant was installed in each of the four defects. After 3 months, biopsies were harvested and ground sections prepared for histological evaluation. Results: The augmentation technique was effective at all sites and all the foreseen implants were installed. In the histological analysis, all implants were integrated in mature bone, at both the buccal and lingual aspects. The most coronal bone-to-implant contact and the top of the buccal bony crest were located at a similar distance between test and control implants. However, these distances were higher at the larger compared with the smaller defects. Especially in the large defect, residual particles of DBBM were found embedded into connective tissue and located outside the bony crest. Conclusions: Particulate autologous bone as well as DBBM particles used to augment horizontally the alveolar bony process allowed for the osseointegration of implants installed after 3 months of healing. © 2012 John Wiley & Sons A/S.

Análise densitométrica, histomorfométrica e biomecânica em fêmures de ratos submetidos à ausência de carga e atividade física em esteira

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

Estudo da atividade das enzimas envolvidas na ossificação de frangos de corte normais e acometidos por discondroplasia tibial

Pós-graduação em Biotecnologia - IQ

Clinical study on survival rate of short implants placed in the posterior mandibular region: resonance frequency analysis

Short implants are increasingly used, but there is doubt about their performance being similar to that of regular implants. The aim of this study was to compare the mechanical stability of short implants vs. regular implants placed in the edentulous posterior mandible. Twenty-three patients received a total of 48 short implants (5 × 5.5 mm and 5 × 7 mm) and 42 regular implants (4 × 10 mm and 4 × 11.5 mm) in the posterior mandible. Patients who received short implants had <10 mm of bone height measured from the bone crest to the outer wall of the mandibular canal. Resonance frequency analysis (RFA) was performed at time intervals T0 (immediately after implant placement), T1 (after 15 days), T2 (after 30 days), T3 (after 60 days), and T4 (after 90 days). The survival rate after 90 days was 87.5% for the short implants and 100% for regular implants (P < 0.05). There was no significant difference between the implants in time intervals T1, T2, T3, and T4. In T0, the RFA values of 5 × 5.5 implants were higher than values of 5 × 7 and 4 × 11.5 implants (P < 0.05). A total of six short implants that were placed in four patients were lost (three of 5 × 5.5 mm and three of 5 × 7 mm). Three lost implants started with high ISQ values, which progressively decreased. The other three lost implants started with a slightly lower ISQ value, which rose and then began to fall. Survival rate of short implants after 90 days was lower than that of regular implants. However, short implants may be considered a reasonable alternative for rehabilitation of severely resorbed mandibles with reduced height, to avoid performing bone reconstruction before implant placement. Patients need to be aware of the reduced survival rate compared with regular implants before implant placement to avoid disappointments.

Bone repair investigation using rhBMP-2 and angiogenic protein extracted from latex

Background: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP-2) and P-1 application, using 5 and 10 mu g of each, combined to a material carrier, in critical bone defects. Methods: It was used 70 Wistar rats (male, similar to 250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 mu g of pure P-1, 5 mu g of pure rhBMP-2, 5 mu g of P-1/monoolein gel, 5 mu g of rhBMP-2/monoolein gel, 10 mu g of pure P-1, 10 mu g of pure rhBMP-2, 10 mu g of P-1/monoolein gel, 10 mu g of rhBMP-2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. Results: Animals treated with pure P-1 protein, in both situations with 5 mu g and 10 mu g, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 mu g were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 mu g rhBMP-2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. Conclusion: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP-2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent. Microsc. Res. Tech., 2011.(c) 2011 Wiley Periodicals, Inc.

Conditional and transgenic mouse models as tools to study the role of TGFbeta, BMP signaling in skeletal development

Die TGFbeta/BMP Signaltransduktionskaskade ist wichtig für viele Entwicklungsprozesse fast aller embryonaler sowie extraembryonaler Gewebe und sie ist ebenso essentiell bei der Aufrechterhaltung der Homöostase im adulten Organismus. In vielen Mausmodellen und Zellkulturversuchen wurde gezeigt, dass Liganden dieses Signalweges in verschiedene Stadien der Knorpel- und Knochenentwicklung involviert sind. BMPs sind beispielsweise maßgeblich an der frühen Kondensation und Bildung des Knorpels und später an Proliferation und Hypertrophie der Chondrozyten beteiligt. BMPs können ektopisch Knochenbildung auslösen und das Expressionsmuster der Liganden und spezifischen Rezeptoren in der Wachstumsfuge lässt auf eine wichtige Rolle der BMPs in der Wachstumsfuge schließen. Der gezielte knock out der BMP-Rezeptoren Bmpr1a und Bmpr1b in proliferierenden Chondrozyten führt zur Ausbildung einer generellen Chondrodysplasie. Smad1, Smad5 und Smad8 sind die Mediatoren der BMP-Signalkaskade. Im Rahmen der vorliegenden Arbeit sollte die Rolle und Funktion der Smad1- und Smad5-Proteine in der Wachstumsfuge untersucht werden. Hierzu wurden konditionale Smad1-knock out-Mäuse mit einer transgenen Mauslinie gekreuzt, die die Cre-Rekombinase spezifisch in proliferierenden Chondrozyten exprimiert. Diese Mäuse wurden mit und ohne heterozygotem Smad5-Hintergrund charakterisiert. Bei einem knock out von Smad1 allein konnte ein leichte Verkürzung der Wachstumsfuge beobachtet werden, wobei prähypertrophe und hypertrophe Zone gleichermaßen betroffen waren. Dieser Phänotyp war verstärkt in Mäusen mit zusätzlichem heterozygotem Smad5-Hintergrund. Eine Verringerung der Proliferationsrate konnte zusammen mit einer verminderten Ihh-Expression nachgewiesen werden. Zusätzlich konnte anhand von Röntgenaufnahmen eine Dysorganisation der nasalen Region und ein fehlendes nasales Septum beobachtet werden. Produktion und Mineralisation der extrazellulären Matrix waren nicht beeinträchtigt. Um die Rolle der BMP- und TGFbeta-Signalkaskaden während der endochondralen Ossifikation zu vergleichen, wurden transgene Mäuse generiert, in denen die TGFbeta-Signalkaskade spezifisch in proliferierenden Chondrozyten gestört war. Zwei Mauslinien, die ähnliche Phänotypen zeigten, wurden untersucht. Esl1 ist ein TGFbeta-bindendes Protein, von dem man annimmt, dass es die TGFbeta-Signalkaskade inhibieren kann. Esl1-knock out-Mäuse sind kleiner als Wildtypmäuse und die Überexpression von Esl1 in proliferierenden Chondrozyten führt zu einer Verlängerung der Wachstumsfuge und einer verstärkten Proliferationsrate. Knorpelmarker, wie Col2a1 und Sox9 sind in diesen Mäusen herunterreguliert, während Col10a1 und Ihh als Marker für die hypertrophe und prähypertrophe Zone herunterreguliert waren. Dies führt zu der Annahme, dass mehr Zellen in die terminale Differenzierung eintreten. Bei transgenen Mäusen, in denen ein dominant-negativer (dn) TGFbeta-Rezeptor in proliferierenden Chondrozyten überexprimiert wurde, konnte eine verlängerte prähypertrophe Zone, eine erhöhte Ihh-Expression, sowie eine verstärkte Proliferationsrate beobachtet werden. Zusätzlich konnte in homozygoten Tieren ein craniofacialer Phänotyp beschrieben werden, der zu Problemen bei der Nahrungsaufnahme und damit zu einer starken Wachstumsbeeinträchtigung führte. Die BMP- und TGFbeta-Signalkaskaden haben möglicherweise antagonistische Effekte in der Wachstumsfuge. Während der Ausfall von BMP in proliferierenden Chondrozyten aufgrund einer gesunkenen Proliferationsrate zu einer Verkürzung der Wachstumsfuge führte, kann man in Mäusen mit einer Störung der TGFbeta-Signalkaskade eine verstärkte Proliferation in einer daher verlängerten Wachstumsfuge beobachten. Ein weiteres Ziel dieser Arbeit war die Generation einer transgenen Mauslinie, die die Cre-Rekombinase spezifisch in hypertrophen Chondrozyten exprimiert. Promoterstudien mit transgenen Mäusen weisen darauf hin, dass ein putatives AP1-Element, etwa 4 kb vor dem ersten Exon des Col10a1 gelegen, wichtig für die spezifische Expression in hypertrophen Chondrozyten ist. Ein Konstrukt, dass vier Kopien dieses Elements und den basalen Promoter enthält, wurde benutzt, um die Cre-Rekombinase spezifisch zu exprimieren. Diese Mauslinie befindet sich in der Testphase und erste Daten deuten auf eine spezifische Expression der Cre-Rekombinase in hypertrophen Chondrozyten hin.

Multiscale Characterization of Acrylic Bone Cement Modified with Functionalized Mesoporous Silica Nanoparticles

Acrylic bone cement is widely used to anchor orthopedic implants to bone and mechanical failure of the cement mantle surrounding an implant can contribute to aseptic loosening. In an effort to enhance the mechanical properties of bone cement, a variety of nanoparticles and fibers can be incorporated into the cement matrix. Mesoporous silica nanoparticles (MSNs) are a class of particles that display high potential for use as reinforcement within bone cement. Therefore, the purpose of this study was to quantify the impact of modifying an acrylic cement with various low-loadings of mesoporous silica. Three types of MSNs (one plain variety and two modified with functional groups) at two loading ratios (0.1 and 0.2 wt/wt) were incorporated into a commercially available bone cement. The mechanical properties were characterized using four-point bending, microindentation and nanoindentation (static, stress relaxation, and creep) while material properties were assessed through dynamic mechanical analysis, differential scanning calorimetry, thermogravimetric analysis, FTIR spectroscopy, and scanning electron microscopy. Four-point flexural testing and nanoindentation revealed minimal impact on the properties of the cements, except for several changes in the nano-level static mechanical properties. Conversely, microindentation testing demonstrated that the addition of MSNs significantly increased the microhardness. The stress relaxation and creep properties of the cements measured with nanoindentation displayed no effect resulting from the addition of MSNs. The measured material properties were consistent among all cements. Analysis of scanning electron micrographs images revealed that surface functionalization enhanced particle dispersion within the cement matrix and resulted in fewer particle agglomerates. These results suggest that the loading ratios of mesoporous silica used in this study were not an effective reinforcement material. Future work should be conducted to determine the impact of higher MSN loading ratios and alternative functional groups. (C) 2014 Elsevier Ltd. All rights reserved.