Evaluation of a double-setting alpha-tricalcium phosphate cement in eviscerated rabbit eyes

Purpose: To evaluate the macroscopy, microstructure, and tissue reaction of a double-setting a-tricalcium phosphate bone cement used as an intraocular implant in rabbits.Methods: the internal and external surface of the double-setting a-tricalcium phosphate implant was analyzed macroscopically and by scanning electron microscopy. Twelve New Zealand rabbits received 12-mm implants made of double-setting alpha-tricalcium phosphate cement after unilateral evisceration. Clinical evaluation was performed daily for the first 15 days after surgery and at 15-day intervals until the end of the study period. For histopathologic analysis, 3 animals per experimental period were submitted to enucleation at 15, 45, 90, and 180 days.Results: on gross inspection, the external surface of the implant was solid, smooth, and compact. The microarchitecture was characterized by the formation of columns of hexagonal crystals with interconnecting channels forming micropores. No wound dehiscence, signs of infection, or implant extrusion were observed in any animal throughout the study period. Histologic examination revealed the formation of fibrovascular tissue surrounding the implants, and there were signs of minimal integration of the surface limiting the fibrocellular cap with the space previously occupied by the implant.Conclusions: the double-setting alpha-tricalcium phosphate implant behaved as an inert and nonintegratable material. The lack of incorporation of this material by fibrovascular tissue is related to its characteristics of compactness and high resistance.

Influence of creatine supplementation on bone quality in the ovariectomized rat model: an FT-Raman spectroscopy study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Application of impedance spectroscopy to evaluate the effect of different setting accelerators on the developed microstructures of calcium phosphate cements

The main goal of the present study was to evaluate the effect of different setting accelerator agents on the developed microstructures of calcium phosphate cements (CPCs) by employing the impedance spectroscopy (IS) technique. Six compositions of CPCs were prepared from mixtures of commercial dicalcium phosphate anhydrous (DCPA) and synthesized tetracalcium phosphate (TTCP) as the solid phases. Two TTCP/DCPA molar ratios (1/1 and 1/2) and three liquid phases (aqueous solutions of Na(2)HPO(4), tartaric acid (TA) and oxalic acid (OA), 5% volume fraction) were employed. Initial (I) and final (F) setting times of the cement pastes were determined with Gillmore needles (ASTM standard C266-99). The hardened samples were characterized by X-ray powder diffraction (XRD), Fourier transformed infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and apparent density measurements. The IS technique was employed as a non-destructive tool to obtain information related to porosity, tortuosity and homogeneity of the cement microstructures. The formulation prepared from a TTCP/DCPA equimolar mixture and OA as the liquid phase presented the shortest I and F (12 and 20 min, respectively) in comparison to the other studied systems. XRD analyses revealed the formation of low-crystallinity hydroxyapatite (HA) (as the main phase) as well as the presence of little amounts of unreacted DCPA and TTCP after 24 h hardening in 100% relative humidity. This was related to the proposed mechanisms of dissolution of the reactants. The bands observed by FTIR allowed identifying the presence of calcium tartrate and calcium oxalate in the samples prepared from TA and OA, in addition to the characteristic bands of HA. High degree of entanglement of the formed crystals was observed by SEM in samples containing OA. SEM images were also correlated to the apparent densities of the hardened cements. Changes in porosity, tortuosity and microstructural homogeneity were determined in all samples, from IS results, when the TTCP/DCPA ratio was changed from 1/1 to 1/2. The cement formulated from an equimolar mixture of TTCP/DCPA and OA as the liquid phase presented setting times, degree of conversion to low-crystallinity HA and microstructural features suitable to be used as potential bone cement in clinical applications. The IS technique was shown to be a very sensitive and non-destructive tool to relate the paste composition to the developed microstructures. This approach could be very useful to develop calcium phosphate bone cements for specific clinical demands.

Development, characterization and clinical use of a biodegradable composite scaffold for bone engineering in oro-maxillo-facial surgery

We have developed a biodegradable composite scaffold for bone tissue engineering applications with a pore size and interconnecting macroporosity similar to those of human trabecular bone. The scaffold is fabricated by a process of particle leaching and phase inversion from poly(lactide-co-glycolide) (PLGA) and two calcium phosphate (CaP) phases both of which are resorbable by osteoclasts; the first a particulate within the polymer structure and the second a thin ubiquitous coating. The 3-5 mu m thick osteoconductive surface CaP abrogates the putative foreign body giant cell response to the underlying polymer, while the internal CaP phase provides dimensional stability in an otherwise highly compliant structure. The scaffold may be used as a biomaterial alone, as a carrier for cells or a three-phase drug delivery device. Due to the highly interconnected macroporosity ranging from 81% to 91%, with macropores of 0.8 similar to 1.8 mm, and an ability to wick up blood, the scaffold acts as both a clot-retention device and an osteoconductive support for host bone growth. As a cell delivery vehicle, the scaffold can be first seeded with human mesenchymal cells which can then contribute to bone formation in orthotopic implantation sites, as we show in immune-compromised animal hosts. We have also employed this scaffold in both lithomorph and particulate forms in human patients to maintain alveolar bone height following tooth extraction, and augment alveolar bone height through standard sinus lift approaches. We provide a clinical case report of both of these applications; and we show that the scaffold served to regenerate sufficient bone tissue in the wound site to provide a sound foundation for dental implant placement. At the time of writing, such implants have been in occlusal function for periods of up to 3 years in sites regenerated through the use of the scaffold.

Comparative therapeutic use of Risedronate and Calcarea phosphorica - Allopathy versus homeopathy - In bone repair in castrated rats

Osteoporosis, a disease characterized by progressive bone loss, has been the target of several studies in the past few years. It results in a much higher risk for fractures and might cause slower bone lesion healing. The aim of this work was to study the effects of Risedronate (allopathic medicine) and Calcarea phosphorica 6CH (homeopathic medicine) on the repair of bone lesions in male rats with osteoporosis induced by castration. Eighty-four three-month-old rats were used divided into four groups of twenty-one animals each. Three groups where castrated and one group was submitted to Sham surgery. One month later, cortical lesions were made in all animals' tibiae and, after one day, the different experimental treatments began according to the following groups: CR - castrated/Risedronate (1 mg/kg/day); CCp - castrated/ Calcarea phosphorica 6CH (3 drops/day); CP - castrated/placebo and SP - Sham/placebo. The animals were sacrificed at seven, fourteen and twenty-eight days after the beginning of the treatments and had their tibiae removed. Digital radiographs of the tibiae were taken and analyzed in order to evaluate the optical density of the defect area. Then, they were decalcified and processed for histological and histomorphometrical analysis. The data were submitted to ANOVA, and to the Tukey and Dunnett tests (5%). The allopathic and homeopathic treatments led to different bone formation as regards remodeling and maturation aspects. Further research is necessary to access the resistance and quality of the newly formed bone.

In vivo biological behavior of calcium phosphate cements with different Ca/P ratio

Bioceramics with different Ca/P ratio were prepared from a mechanical mixture of NaPO3, CaCO3, Ca(OH)2 and phosphate buffer solution and implanted in rats subcutaneous tissues. The cements were characterized by Thermo gravimetric analysis (TG-TDA), X-ray diffraction and 31P-NMR. The implant sites were excised after 1, 4 and 16 weeks, fixed, dehydrated, included in paraffin wax for serial cutting and examined under the light transmitted microscope. They were biocompatible and biodegradable when implanted in rat subcutaneous. None of the materials induced ectopic osteogenesis. According to the results, the studied materials seem to be able for manufacturing reabsorbable bone implants.

Biological response to wollastonite doped α-tricalcium phosphate implants in hard and soft tissues in rats

The biological response following subcutaneous and bone implantation of β-wollastonite(β-W)-doped α-tricalcium phosphate bioceramics in rats was evaluated. Tested materials were: tricalcium phosphate (TCP), consisting of a mixture of α- and β-polymorphs; TCP doped with 5 wt. % of β-W (TCP5W), composed of α-TCP as only crystalline phase; and TCP doped with 15 wt. % of β-W (TCP 15), containing crystalline α-TCP and β-W. Cylinders of 2×1 mm were implanted in tibiae and backs of adult male Rattus norvegicus, Holtzman rats. After 7, 30 and 120 days, animals were sacrificed and the tissue blocks containing the implants were excised, fixed and processed for histological examination. TCP, TCP5W and TCP15W implants were biocompatible but neither bioactive nor biodegradable in rat subcutaneous tissue. They were not osteoinductive in connective tissue either. However, in rat bone tissue β-W-doped α-TCP implants (TCP5W and TCP 15W) were bioactive, biodegradable and osteoconductive. The rates of biodegradation and new bone formation observed for TCP5W and TCP15W implants in rat bone tissue were greater than for non-doped TCP.

Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration

The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA) nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl2 followed by Na2HPO4. BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40-50 of the total weight. Spectroscopy, electronic microscopy/energy dispersive X-ray analyses, and X-ray diffraction showed formation of HA crystals on BC nanofibres. Low crystallinity HA crystals presented Ca/P a molar ratio of 1.5 (calcium-deficient HA), similar to physiological bone. Fourier transformed infrared spectroscopy analysis showed bands assigned to phosphate and carbonate ions. In vivo tests showed no inflammatory reaction after 1 week. After 4 weeks, defects were observed to be completely filled in by new bone tissue. The BC-HA membranes were effective for bone regeneration. © 2011 S. Saska et al.

Dispersed hydroxyapatite bioglass 45S5 composites: Comparative evaluation of the use of bovine bone and synthetic hydroxyapatite

The bovine bone and sintetic hydroxyapatite (HA) bioceramics are reference materials to employment as a bone substitute, however, their slow rate of degradation and its low rate of bioactivity index (Ib) are presented as limiting factors for application as bone graft. In contrast, the bioglass is a resorbable and osteoinductive material. the present work objective the development of composites of dispersed bovine bone or sintetic HA in silicate-phosphate bioglass, seeking to obtain a biomaterial with properties suitable for application as bone grafts. The composites were prepared by mixing between the powder components followed by sintering for 1h. Were used HA and bioglass (45S5) with particle size <240μm. The tested proportions of HA/45S5 were 20/80, 30/70 and 40/60 (wt%). The composites characterization was made employing scanning electron microscopy, Infra-Red Spectrometry and hydrolytic resistance test. The test results indicate the potential use of the materials developed for applications such as bone graft. © (2012) Trans Tech Publications, Switzerland.

Comparisons between Bio-Oss® and Straumann® Bone Ceramic in immediate and staged implant placement in dogs mandible bone defects

Objective: To compare immediate and staged approach implant placement in circumferential defects treated with deproteinized bovine bone mineral (DBBM); hidroxyapatite/tricalcium phosphate (HA/TP); autogenous bone (Ab); and coagulum (Cg); upon implant stability, osseointegration and alveolar crest maintenance. Materials and methods: Six dogs underwent extractions of lower premolars, bilaterally. Twelve weeks later four bone defects (6 mm wide/4 mm long) were drilled at one side and randomly filled with DBBM; HA/TP; Ab; and Cg, respectively, and left to heal (staged approach). Eight weeks later one implant (Osseospeed™, AstraTech) was placed in experimental sites. At the same session four defects were drilled on contra-lateral side and implants were inserted immediately after biomaterials grafting (immediate approach). Animals were euthanized 8 weeks later. Implant stability was measured by resonance frequency analysis (RFA) at installation and after sacrifice. Ground sections were prepared for bone contact (BIC); bone area (BA); distance implant shoulder-bone crest (IS-C); distance implant shoulder first bone contact (IS-B); and areas occupied by soft tissue. Results: The BA and BIC were superior in the staged approach. The Cg exhibited higher BIC and BA as compared with other materials at the total implant body (P = 0.004 and 0.012, respectively). The DBBM, HA/TP and Ab groups rendered similar BA and BIC. The immediate approach resulted in less crest resorption compared to staged approach. The biomaterials did not affect the IS-C and IS-B measurements. Particles area tended to be higher in DBBM group than HA/TP (P = 0.15), while soft tissue infiltrate was higher in DBBM group when used in the immediate approach (P = 0.04). The RFA indicated gain in stability in the staged approach (P = 0.002). The correlation test between RFA vs. BIC and BA demonstrated inferior stability for DBBM group in immediate approach (P = 0.01). Conclusions: Implants placed in healed defects resulted in better stability as a consequence of higher BIC and BA. The Cg alone rendered increased BIC compared to other materials in both approaches. Immediate approach should be preferable to staged approach in terms of alveolar crest maintenance. The BIC and BA values did not vary between micro and macro-threads in this experimental model. Implants installed in sites filled with DBBM in immediate approach were less stable. © 2011 John Wiley & Sons A/S.

Osteogenesis-inducing calcium phosphate nanoparticle precursors applied to titanium surfaces

This study investigated the effects of the morphology and physicochemical properties of calcium phosphate (CaP) nanoparticles on osteogenesis. Two types of CaP nanoparticles were compared, namely amorphous calcium phosphate (ACP) nano-spheres (diameter: 9-13 nm) and poorly crystalline apatite (PCA) nano-needles (30-50 nm x 2-4 nm) that closely resemble bone apatite. CaP particles were spin-coated onto titanium discs and implants; they were evaluated in cultured mouse calvarial osteoblasts, as well as after implantation in rabbit femurs. A significant dependence of CaP coatings was observed in osteoblast-related gene expression (Runx2, Col1a1 and Spp1). Specifically, the PCA group presented an up-regulation of the osteospecific genes, while the ACP group suppressed the Runx2 and Col1a1 expression when compared to blank titanium substrates. Both the ACP and PCA groups presented a more than three-fold increase of calcium deposition, as suggested by Alizarin red staining. The removal torque results implied a slight tendency in favour of the PCA group. Different forms of CaP nanostructures presented different biologic differences; the obtained information can be used to optimize surface coatings on biomaterials. © 2013 IOP Publishing Ltd.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Increased osseointegration effect of bone morphogenetic protein 2 on dental implants: An in vivo study

Application of recombinant human bone morphogenetic protein 2 (rhBMP-2) to implant surfaces has been of great interest due to its osteoinductive potential. However, the optimal coating methodology has not been clarified. The objective of the study was to determine whether the application of rhBMP-2 onto plasma-sprayed hydroxyapatite implant surfaces by immersion in protein solution before implant installation would result in significantly improved bone apposition. Using a sheep iliac model, titanium (Ti) and plasma-sprayed calcium-phosphate (PSCaP)-coated implants uncoated and coated with rhBMP-2 were assessed for their osteogenic effects in the peri-implant area over time in terms of osseointegration and de novo bone formation. After 3 and 6 weeks postoperatively, the samples were retrieved and were subjected to bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) evaluation. When rhBMP-2 was applied to the PSCaP surface, significant increases in BIC and BAFO were observed at 3 weeks in vivo, whereas when adsorbed directly onto the titanium implant surface, rhBMP-2 did not as effectively improve the bone response (although significantly higher than control Ti). The outcomes of the present study suggested that the combination of plasma-sprayed calcium-phosphate surface and rhBMP-2 coating significantly enhanced osseointegration, which validated the postulated hypothesis. © 2013 Wiley Periodicals, Inc.

The role of cytokines in inflammatory bone loss

Chronic inflammatory processes close to bone often lead to loss of bone in diseases such as rheumatoid arthritis, periodontitis, loosened joint prosthesis and tooth implants. This is mainly due to local formation of bone resorbing osteoclasts which degrade bone without any subsequent coupling to new bone formation. Crucial for osteoclastogenesis is stimulation of mononuclear osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) which induces their differentiation along the osteoclastic lineage and the fusion to mature, multinucleated osteoclasts. M-CSF and RANKL are produced by osteoblasts/ osteocytes and by synovial and periodontal fibroblasts and the expression is regulated by pro- and anti-inflammatory cytokines. These cytokines also regulate osteoclastic differentiation by direct effects on the progenitor cells. In the present overview, we introduce the basic concepts of osteoclast progenitor cell differentiation and summarize the current knowledge on cytokines stimulating and inhibiting osteoclastogenesis by direct and indirect mechanisms. © Informa Healthcare USA, Inc.