Biomimetic apatite formation on Ultra-High Molecular Weight Polyethylene (UHMWPE) using modified biomimetic solution

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

Surface characterization of Ti-7.5Mo alloy modified by biomimetic method

Titanium and its alloys have been used in dentistry due to their excellent corrosion resistance and biocompatibility. It was shown that even a pure titanium metal and its alloys spontaneously form a bone-like apatite layer on their surfaces within a living body. The purpose of this work was to evaluate the growth of calcium phosphates at the surface of the experimental alloy Ti-7.5Mo. We produced ingots from pure titanium and molybdenum using an arc-melting furnace We then submitted these Ingots to heat treatment at 1100 degrees C for one hour, cooled the samples in water, and cold-worked the cooled material by swaging and machining. We measured the media roughness (Ra) with a roughness meter (1.3 and 2.6 mu m) and cut discs (13 mm in diameter and 4 mm in thickness) from each sample group. The samples were treated by biomimetic methods for 7 or 14 days to form an apatite coating on the surface. We then characterized the surfaces with an optical profilometer, a scanning electron microscope and contact angle measurements. The results of this study indicate that apatite can form on the surface of a Ti-7.5Mo alloy, and that a more complete apatite layer formed on the Ra = 2 6 mu m material. This Increased apatite formation resulted in a lower contact angle (C) 2010 Elsevier B.V. All rights reserved

Wood as a model material for medical biomaterials. In vivo and in vitro studies with bone and Betula pubescens Ehrh

Novel biomaterials are needed to fill the demand of tailored bone substitutes required by an ever‐expanding array of surgical procedures and techniques. Wood, a natural fiber composite, modified with heat treatment to alter its composition, may provide a novel approach to the further development of hierarchically structured biomaterials. The suitability of wood as a model biomaterial as well as the effects of heat treatment on the osteoconductivity of wood was studied by placing untreated and heat‐treated (at 220 C , 200 degrees and 140 degrees for 2 h) birch implants (size 4 x 7mm) into drill cavities in the distal femur of rabbits. The follow‐up period was 4, 8 and 20 weeks in all in vivo experiments. The flexural properties of wood as well as dimensional changes and hydroxyl apatite formation on the surface of wood (untreated, 140 degrees C and 200 degrees C heat‐treated wood) were tested using 3‐point bending and compression tests and immersion in simulated body fluid. The effect of premeasurement grinding and the effect of heat treatment on the surface roughness and contour of wood were tested with contact stylus and non‐contact profilometry. The effects of heat treatment of wood on its interactions with biological fluids was assessed using two different test media and real human blood in liquid penetration tests. The results of the in vivo experiments showed implanted wood to be well tolerated, with no implants rejected due to foreign body reactions. Heat treatment had significant effects on the biocompatibility of wood, allowing host bone to grow into tight contact with the implant, with occasional bone ingrowth into the channels of the wood implant. The results of the liquid immersion experiments showed hydroxyl apatite formation only in the most extensively heat‐treated wood specimens, which supported the results of the in vivo experiments. Parallel conclusions could be drawn based on the results of the liquid penetration test where human blood had the most favorable interaction with the most extensively heat‐treated wood of the compared materials (untreated, 140 degrees C and 200 degrees C heat‐treated wood). The increasing biocompatibility was inferred to result mainly from changes in the chemical composition of wood induced by the heat treatment, namely the altered arrangement and concentrations of functional chemical groups. However, the influence of microscopic changes in the cell walls, surface roughness and contour cannot be totally excluded. The heat treatment was hypothesized to produce a functional change in the liquid distribution within wood, which could have biological relevance. It was concluded that the highly evolved hierarchical anatomy of wood could yield information for the future development of bulk bone substitutes according to the ideology of bioinspiration. Furthermore, the results of the biomechanical tests established that heat treatment alters various biologically relevant mechanical properties of wood, thus expanding the possibilities of wood as a model material, which could include e.g. scaffold applications, bulk bone applications and serving as a tool for both mechanical testing and for further development of synthetic fiber reinforced composites.

Surface characterization of Ti-7.5Mo alloy modified by biomimetic method

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

Influência dos íons K+ e Mg2+ na obtenção de apatitas biomiméticas

O crescimento da hidroxiapatita - HA, tanto no meio biológico quanto em soluções aquosas como a Synthetic Body Fluid - SBF, ocorre em meio contendo, além dos elementos Ca e P, elementos-traços essenciais tais como: Mg2+, HCO3-, K+ e Na+. Alguns destes elementos são conhecidos como inibidores do crescimento da HA, como Mg2+ e HCO3-. Neste trabalho, estudou-se a influência dos íons K+ e Mg2+ na formação de apatitas sobre substratos metálicos de Ti c.p. previamente tratados com NaOH 5M. Os efeitos destes íons no recobrimento obtidos, antes e após o tratamento térmico a 800ºC, foram analisados por microscopia eletrônica de varredura - MEV, espectroscopia de energia dispersiva de raios-X - EDX, difratometria de raios-X - DRX e espectroscopia no infravermelho - IV e mostraram que o efeito inibitório do Mg2+ na formação da HA se manifesta após o tratamento térmico. Diferentemente, o crescimento cristalino da HA não foi afetado pela presença do íon K+. Além disso, a formação de apatita carbonatada se deu também em soluções que não continham o íon CO3(2-) em sua composição.

Reparo ósseo em scaffolds de TI6AL4V sinterizados pela tecnologia de sinterização direta de metais a laser (DMLS) submetidos a tratamento de superfície associado à aplicação de ultrassom de baixa intensidade (LIPUS)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of Cal? minerals, whereas all the grafted membranes revealed the growth of Cal? minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic Cap minerals. Irreversibly adsorbed protein measured after a 1 h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro. (c) 2005 Elsevier Ltd. All rights reserved.

Mesoporous bioactive glasses. I. Synthesis and structural characterization

Highly ordered mesoporous bioactive glasses (MBGs) with different compositions have been synthesized by a combination of surfactant templating, sol-gel method and evaporation-induced self-assembly (EISA) processes. The texture properties and compositional homogeneity of MBGs have been characterized and compared with conventional bioactive glasses (BGs) synthesized in the absence of surfactants by evaporation method. The formation mechanism (pore - composition dependence) and compositional homogeneity in the case of MBG materials are different from those in conventional BGs. Unlike conventional sol-gel-derived BGs that shows a direct correlation between their composition and pore architecture, MBGs with different compositions may possess similar pore volume and uniformly distributed pore size when the same structure-directing agent is utilized. The framework of MBG is homogeneously distributed in composition at the nanoscale and the inorganic species generally exists in the form of amorphous phase. MBGs calcined at temperatures

The in-vitro bioactivity of mesoporous bioactive glasses

Ordered mesoporous bioactive glasses (MBGs) with different compositions were prepared by using nonionic block copolymer surfactants as structure-directing agents through an evaporation-induced self-assembly process. Their in-vitro bioactivities were studied in detail by electron microscopy, Fourier-transform infrared spectroscopy, and inductively coupled plasma (ICP) atomic emission spectroscopy. The ICP element analysis results were further calculated in terms of the total consumption of Ca and P, Delta[Ca]/Delta[P] ratios, and ionic activity product (IP) of hydroxyapatite. Through the above analysis, it is clear that MBGs show a different structure-bioactivity correlation compared to conventional sol-gel-derivcd BGs. The in vitro bioactivity of MBGs is dependent on the Si/Ca ratio in the network when the other material parameters such as the mesostructure and texture properties (pore size, pore volume) are controlled. MBG 80S15C with relatively lower calcium content exhibits the best in vitro bioactivity, in contrast to conventional sol-gel-derived BGs where usually higher calcium percentage BGs (e.g. 60S35C) show better bioactivity. Calcination temperature is another important factor that influences the in vitro bioactivity. According to our results, MBGs calcined at 973 K may possess the best in vitro bioactivity. The influences of the composition and calcination temperature upon bioactivity are explained in terms of the unique structures of MBGs. (c) 2006 Elsevier Ltd. All rights reserved.

Experimental Calcification of HEMA-Based Hydrogels in the Presence of Albumin and a Comparison to the in Vivo Calcification

The precipitation patterns and characteristics of calcium phosphate (CaP) phases deposited on HEMA-based hydrogels upon incubation in simulated body fluid (SBF-2) containing a protein (human serum albumin) have been investigated in relation to the calcification in an organic-free medium (SBF-1) and to that occurring after subcutaneous implantation in rats. In SBF-2, the deposits occurred exclusively as a peripheral layer on the surface of the hydrogels and consisted mainly of precipitated hydroxyapatite, a species deficient in calcium and hydroxyl ions, similarly to the deposits formed on the implanted hydrogels, where the deposited layer was thicker. In SBF-1, the deposits were mainly of brushite type. There was no evidence that albumin penetrated the interstices of hydrogels. As the X-ray diffraction patterns of the CaP deposits generated in SBF-2 showed a similar nature with those formed on the implanted hydrogel, it was concluded that the calcification in SBF-2 can mimic to a reliable extent the calcification process taking place in a biological environment.

Synthesis of soluble phosphate polymers by RAFT and their in vitro mineralization

Soluble linear (non-cross-linked) poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were successfully synthesized through reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization and by keeping the molecular weight below 20 K. Above this molecular weight, insoluble (cross-linked) polymers were observed, postulated to be due to residual diene (cross-linkable) monomers formed during purification of the monomers, MOEP and MAEP. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy) ethyl methacrylate) (PAAEMA) were successfully prepared and were immobilized on aminated slides. Simulated body fluid studies revealed that calcium phosphate (CaP) minerals formed on both the soluble polymers and the cross-linked gels were very similar. Both the PMAEP polymers and the PMOEP gel showed a CaP layer most probably brushite or monetite based on the Ca/P ratios. A secondary CaP mineral growth with a typical hydroxyapatite (HAP) globular morphology was found on the PMOEP gel. The soluble PMOEP film formed carbonated HAP according to Fourier transform infrared (FTIR) spectroscopy. Block copolymers attached to aminated slides showed only patchy mineralization, possibly due to the ionic interaction of negatively charged phosphate groups and protonated amines.

In vitro bioactivity of titanium-doped bioglass

Previous studies have suggested that incorporating relatively small quantities of titanium dioxide into bioactive glasses may result in an increase in bioactivity and hydroxyapatite formation. The present work therefore investigated the in vitro bioactivity of a titanium doped bioglass and compared the results with 45S5 bioglass. Apatite formation was evaluated for bioglass and Ti-bioglass in the presence and absence of foetal calf serum. Scanning electron microscopy (SEM) images were used to evaluate the surface development and energy dispersive X-ray measurements provided information on the elemental ratios. X-ray diffraction spectra confirmed the presence of apatite formation. Cell viability was assessed for bone marrow stromal cells under direct and indirect contact conditions and cell adhesion was assessed using SEM. © 2014 Springer Science+Business Media.

Bioactive organic/inorganic hybrids with improved mechanical performance

New sol-gel functionalized poly-ethylene glycol (PEGM)/SiO2-CaO hybrids were prepared with interpenetrating networks of silica and PEGM through the formation of Si-O-Si bonds. Bioactive and mechanical properties were investigated for a series of hybrids containing varying organic/inorganic ratios and PEG molecular weights. In contrast to the unmodified PEG/SiO2-CaO hybrids, which rapidly dissolved and crumbled, the epoxy modified hybrids exhibited good mechanical properties and bioactivity. The compressive strength and Young's modulus were greater for higher molecular weight PEGM hybrids (PEGM600 compared to PEGM300). Compressive strengths of 138 MPa and 81 MPa were found for the 50: 50 and 60: 40 organic/inorganic hybrid samples respectively, which are comparable with cortical bone. Young's modulus values of ∼800 MPa were obtained for the 50 : 50 and 60 : 40 organic/inorganic hybrids. Bioactivity tests were conducted by immersing the hybrids into simulated body fluid and observing the formation of apatite. Apatite formation was observed within 24 hours of immersion. PEGM600 hybrids showed enhanced apatite formation compared to PEGM300 hybrids. Increased apatite formation was observed with increasing organic/inorganic ratio. 70 : 30 and 60 : 40 hybrids exhibited the greatest apatite formation. All PEGM hybrids samples had good cell viability and proliferation. The 60 : 40 PEGM600 hybrids displayed the optimal combination of bioactivity and mechanical strength. The bioactivity of these hybrids, combined with the enhanced mechanical properties, demonstrate that these materials have significant potential for bone regeneration applications.

Sediment geochemistry PASOM research cruise (2009) - Murray Ridge region, northern Arabian Sea

In this study, we investigate phosphorus (P) and iron (Fe) cycling in sediments along a depth transect from within to well below the oxygen minimum zone (OMZ) in the northern Arabian Sea (Murray Ridge). Pore-water and solid-phase analyses show that authigenic formation of calcium phosphate minerals (Ca-P) is largely restricted to where the OMZ intersects the seafloor topography, likely due to higher depositional fluxes of reactive P. Nonetheless, increased ratios of organic carbon to organic P (Corg/Porg) and to total reactive P (Corg/Preactive) in surface sediments indicate that the overall burial efficiency of P relative to Corg decreases under the low bottom water oxygen concentrations (BWO) in the OMZ. The relatively constant Fe/Al ratio in surface sediments along the depth transect suggest that corresponding changes in Fe burial are limited. Sedimentary pyrite contents are low throughout the ~25 cm sediment cores at most stations, as commonly observed in the Arabian Sea OMZ. However, pyrite is an important sink for reactive Fe at one station in the OMZ. A reactive transport model (RTM) was applied to quantitatively investigate P and Fe diagenesis at an intermediate station at the lower boundary of the OMZ (bottom water O2: ~14 µmol/L). The RTM results contrast with earlier findings in showing that Fe redox cycling can control authigenic apatite formation and P burial in Arabian Sea sediment. In addition, results suggest that a large fraction of the sedimentary Ca-P is not authigenic, but is instead deposited from the water column and buried. Dust is likely a major source of this Ca-P. Inclusion of the unreactive Ca-P pool in the Corg/P ratio leads to an overestimation of the burial efficiency of reactive P relative to Corg along the depth transect. Moreover, the unreactive Ca-P accounts for ~85% of total Ca-P burial. In general, our results reveal large differences in P and Fe chemistry between stations in the OMZ, indicating dynamic sedimentary conditions under these oxygen-depleted waters.

(Figure 1 and 2) Phosphorus pools in the investigated sediments quantified by SEDEX sequential extraction and distribution of recovered 33P spike between sedimentary P pools after incubation

Phosphorus is an essential nutrient for life. In the ocean, phosphorus burial regulates marine primary production**1, 2. Phosphorus is removed from the ocean by sedimentation of organic matter, and the subsequent conversion of organic phosphorus to phosphate minerals such as apatite, and ultimately phosphorite deposits**3, 4. Bacteria are thought to mediate these processes**5, but the mechanism of sequestration has remained unclear. Here, we present results from laboratory incubations in which we labelled organic-rich sediments from the Benguela upwelling system, Namibia, with a 33P-radiotracer, and tracked the fate of the phosphorus. We show that under both anoxic and oxic conditions, large sulphide-oxidizing bacteria accumulate 33P in their cells, and catalyse the nearly instantaneous conversion of phosphate to apatite. Apatite formation was greatest under anoxic conditions. Nutrient analyses of Namibian upwelling waters and sediments suggest that the rate of phosphate-to-apatite conversion beneath anoxic bottom waters exceeds the rate of phosphorus release during organic matter mineralization in the upper sediment layers. We suggest that bacterial apatite formation is a significant phosphorus sink under anoxic bottom-water conditions. Expanding oxygen minimum zones are projected in simulations of future climate change**6, potentially increasing sequestration of marine phosphate, and restricting marine productivity.