Growth of Calcium Phosphate Using Chemically Treated Titanium Oxide Nanotubes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In situ evaluation of the remineralizing capacity of pit and fissure sealants containing amorphous calcium phosphate and/or fluoride

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Osteoconductive Properties of beta-Tricalcium Phosphate Matrix, Polylactic and Polyglycolic Acid Gel, and Calcium Phosphate Cement in Bone Defects

Extensive bone defects in maxillofacial region can be corrected with autogenous grafts; otherwise, the disadvantages of the therapeutics modality take the research for new bone substitutes. The aim of the study was to evaluate and compare the osteoconductive properties of 3 commercial available biomaterials. A total of 30 calvarial defects (5-mm diameter) were randomly divided into 5 treatment groups, with a total of 6 defects per treatment group (n = 6). The treatment groups were as follows: 500 to 1000 Km beta-tricalcium phosphate (beta-TCP), polylactic and polyglycolic acid (PL/PG) gel, calcium phosphate cement, untreated control, and autograft control. The evaluations were based on histomorphometric analysis at 60 postoperative days. The results have shown that beta-TCP and autograft control supported bone formation at 60 postoperative days. beta-Tricalcium phosphate showed the highest amount of mineralized area per total area and statistically significant compared with PL/PG, calcium phosphate cement, and untreated control groups. The PL/PG gel does not have osteoconductive properties and performed similar to empty control. Calcium phosphate cement showed higher number of multinucleated giant cells around the sites of the biomaterial and showed newly formed bone only at the edges of the biomaterial, without bone formation within the biomaterial. The findings presented herein indicate that bone formation reached a maximum level when rat calvarial defects were filled with beta-TCP at 60 postoperative days. Further studies should be conducted with beta-TCP to understand the potential of this biomaterial in bone regeneration.

Biomimetic calcium phosphate coating on Ti-7.5Mo alloy for dental application

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

Cytotoxicity of two novel formulations of calcium phosphate cements: A comparative in vitro study

The purpose was to evaluate the cytotoxicity of two novel formulations (alpha and beta) of calcium phosphate cements. Positive control, represented by a commercial hydroxyapatite cement, and negative control were included for comparative purposes. A continuous lineage of fibroblastic cells was used, and the effect of the tested materials on both cell proliferation and viability was assessed by counting cell number on hemocytometer and by the trypan blue exclusion test, respectively. Study design attempted to simulate clinical use by allowing direct and indirect contact of cells and cements. Results were analyzed by the Kruskal-Wallis test and indicated that the beta formulation was extremely cytotoxic (P < 0.001), because this material induced the greatest reduction on cell proliferation and viability. The alpha formulation behaved similarly to the positive control regarding its effect on cell proliferation and viability. Thus, it is concluded that alpha formulation has promise for further evaluation of its behavior in vivo.

Utilization of autogenous bone, bioactive glasses, and calcium phosphate cement in surgical mandibular bone defects in Cebus apella monkeys

Purpose: the purpose of the present study was to evaluate the histologic results of bone cavities that were surgically created in the mandibles of Cebus apella monkeys and filled with autogenous bone, PerioGlas, FillerBone, or Bone Source. Materials and Methods: Surgical cavities 5 mm in diameter were prepared through both mandibular cortices in the mandibular angle region. The cavities were randomly filled, and the animals were divided into groups according to the material employed: Group 1 cavities were filled with autogenous corticocancellous bone; group 2 cavities were filled with calcium phosphate cement (BoneSource); and group 3 and group 4 cavities were filled with bioactive glass (FillerBone and PerioGlas, respectively). After 180 days the animals were sacrificed, and specimens were prepared following routine laboratory procedures for hematoxylin/eosin staining and histologic evaluation. Results: the histologic analysis showed that autogenous bone allowed total repair of the bone defects; bioactive glasses (FillerBone and PerioGlas) allowed total repair of the defects with intimate contact of the remaining granules and newly formed bone; and the cavities filled with calcium phosphate cement (BoneSource) were generally filled by connective fibrous tissue, and the material was almost totally resorbed. Discussion: the autogenous bone, FillerBone, and PerioGlas provided results similar to those in the current literature, showing that autogenous bone is the best Choice for filling critical-size defects. Synthetic implanted materials demonstrated biocompatibility, but the bioglasses demonstrated osteoconductive activity that did not occur with calcium phosphate (BoneSource). Conclusion: According to the methodology used in this study, it can be concluded that the utilization of autogenous bone and bioactive glasses permitted the repair of surgically created critical-size defects by newly formed bone; the synthetic implanted materials demonstrated biocompatibility, and the bioactive glasses demonstrated osteoconductive activity. The PerioGlas was mostly resorbed and replaced by bone and the remaining granules were in close contact with bone; the FillerBone showed many granules in contact with the newly formed bone; BoneSource did not permit repair of the critical-size defects, and the defects were generally filled by connective fibrous tissue.

Histomorphometric evaluation of human sinus floor augmentation healing responses to placement of calcium phosphate or Ricinus communis polymer associated with autogenous bone

Background: Prosthetic rehabilitation of the posterior maxilla with dental implants is often difficult because of proximity to the maxillary sinus and insufficient bone height. Maxillary sinus floor augmentation procedures aim to obtain enough bone with an association between biomaterials and autogenous bone.Purpose: the purpose of this study was to evaluate histomorphometrically two grafting materials (calcium phosphate and Ricinus communis polymer) used in maxillary sinus floor augmentation associated with autogenous bone.Materials and Methods: Biopsies were taken from 10 consecutive subjects (mean age 45 years) 10 months after maxillary sinus floor augmentation. The sinus lift was performed with a mixture of autogenous bone and R. communis polymer or calcium phosphate in a 1:2 proportion. Routine histologic processing and staining with hernatoxylin and eosin were performed.Results: the histomorphometric analysis indicated satisfactory regenerative results in both groups for a mean of bone tissue in the grafted area (44.24 +/- 13.79% for the calcium phosphate group and 38.77 +/- 12.85% for the polymer group). Histologic evaluation revealed the presence of an inflammatory infiltrate of mononuclear prevalence that, on average, was nonsignificant. The histologic sections depicted mature bone with compact and cancellous areas in both groups.Conclusion: the results indicated that both graft materials associated with the autogenous bone were biocompatible, although both were still present after 10 months.

Genetic Responses to Nanostructured Calcium-phosphate-coated Implants

Nanostructured calcium phosphate (CaP) has been histologically and biomechanically proven to enhance osseointegration of implants; however, conventional techniques were not sufficiently sensitive to capture its biological effects fully. Here, we compared the conventional removal torque (RTQ) evaluation and gene expression in tissues around nanostructured CaP-coated implants, using real-time RT-PCR, with those of uncoated implants, in a rabbit model. At 2 wks, RTQ values were significantly higher, alkaline phosphatase (ALP) expression was significantly higher, and runt-related transcription factor 2 and tumor necrosis factor-alpha expressions were significantly lower in the coated than in the uncoated implants. This indicates that inflammatory responses were suppressed and osteoprogenitor activity increased around the CaP-coated surface. At 4 wks, although RTQ values did not significantly differ between the 2 groups, ALP and osteocalcin (OCN) were significantly up-regulated in the coated group, indicating progressive mineralization of the bone around the implant. Moreover, an osteoclast marker, adenosine triphosphatase, which indicates acidification of the resorption lacunae, was significantly higher for the coated implants, suggesting gradual resorption of the CaP coating. This study reveals detailed genetic responses to nanostructured CaP-coated implants and provides evidence that the effect of nanotopography is significant during the osseointegration cascade.

Histological and three-dimensional evaluation of osseointegration to nanostructured calcium phosphate-coated implants

Nanostructures on implant surfaces have been shown to enhance osseointegration; however, commonly used evaluation techniques are probably not sufficiently sensitive to fully determine the effects of this process. This study aimed to observe the osseointegration properties of nanostructured calcium phosphate (CaP)-coated implants, by using a combination of three-dimensional imaging and conventional histology. Titanium implants were coated with stable CaP nanoparticles using an immersion technique followed by heat treatment. Uncoated implants were used as the control. After topographical and chemical characterizations, implants were inserted into the rabbit femur. After 2 and 4 weeks, the samples were retrieved for micro-computed tomography and histomorphometric evaluation. Scanning electron microscopy evaluation indicated that the implant surface was modified at the nanoscale by CaP to obtain surface textured with rod-shaped structures. Relative to the control, the bone-to-implant contact for the CaP-coated implant was significantly higher at 4 weeks after the implant surgery. Further, corresponding 3-D images showed active bone formation surrounding the implant. 3-D quantification and 2-D histology demonstrated statistical correlation; moreover, 3-D quantification indicated a statistical decrease in bone density in the non-coated control implant group between 2 and 4 weeks after the surgery. The application of 3-D evaluation further clarified the temporal characteristics and biological reaction of implants in bone. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

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.

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.

Growth of calcium phosphate coating on Ti-7.5Mo alloy after anodic oxidation

Titanium and its alloys are widely used as biomaterials due to their mechanical, chemical and biological properties. To enhance the biocompatibility of titanium alloys, various surface treatments have been proposed. In particular, the formation of titanium oxide nanotubes layers has been extensively examined. Among the various materials for implants, calcium phosphates and hydroxyapatite are widely used clinically. In this work, titanium nanotubes were fabricated on the surface of Ti-7.5Mo alloy by anodization. The samples were anodized for 20 V in an electrolyte containing glycerol in combination with ammonium fluoride (NH4F, 0.25%), and the anodization time was 24 h. After being anodized, specimens were heat treated at 450 °C and 600°C for 1 h to crystallize the amorphous TiO2 nanotubes and then treated with NaOH solution to make them bioactive, to induce growth of calcium phosphate in a simulated body fluid. Surface morphology and coating chemistry were obtained respectively using, field-emission scanning electron microscopy (FEG-SEM), AFM and X-ray diffraction (XRD). It was shown that the presence of titanium nanotubes induces the growth of a sodium titanate nanolayer. During the subsequent invitro immersion in a simulated body fluid, the sodium titanate nanolayer induced the nucleation and growth of nano-dimensioned calcium phosphate. It was possible to observe the formation of TiO2 nanotubes on the surface of Ti-7.5Mo. Calcium phosphate coating was greater in the samples with larger nanotube diameter. These findings represent a simple surface treatment for Ti-7.5Mo alloy that has high potential for biomedical applications. © (2013) Trans Tech Publications, Switzerland.