Zafer Evis

A direct role of collagen glycation in bone fracture

Non-enzymatic glycation (NEG) is an age-related process accelerated by diseases like diabetes, and causes the accumulation of advanced glycation end-products (AGEs). NEG-mediated modification of bones organic matrix, principally collagen type-I, has been implicated in impairing skeletal physiology and mechanics. Here, we present evidence, from in vitro and in vivo models, and establish a causal relationship between collagen glycation and alterations in bone fracture at multiple length scales. Through atomic force spectroscopy, we established that NEG impairs collagens ability to dissipate energy. Mechanical testing of in vitro glycated human bone specimen revealed that AGE accumulation due to NEG dramatically reduces the capacity of organic and mineralized matrix to creep and caused bone to fracture under impact at low levels of strain (3000-5000 μstrain) typically associated with fall. Fracture mechanics tests of NEG modified human cortical bone of varying ages, and their age-matched controls revealed that NEG disrupted microcracking based toughening mechanisms and reduced bone propagation and initiation fracture toughness across all age groups. A comprehensive mechanistic model, based on experimental and modeling data, was developed to explain how NEG and AGEs are causal to, and predictive of bone fragility. Furthermore, fracture mechanics and indentation testing on diabetic mice bones revealed that diabetes mediated NEG severely disrupts bone matrix quality in vivo. Finally, we show that AGEs are predictive of bone quality in aging humans and have diagnostic applications in fracture risk.

Nanosize hydroxyapatite: doping with various ions

Abstract Abstract Natural crystal sizes of bone minerals are present in the nanoscale regime (specifically less than 100 nm in at least one direction). Therefore, research on the synthesis and characterisation of nanosize hydroxyapatite has gained significant importance in numerous biomedical applications. This is because currently used pure micrometre sized hydroxyapatite has poor mechanical properties, which limits it use in non-load bearing applications. For these reasons, various ions could be easily substituted into nanostructured hydroxyapatite to alter its biocompatibility, sinterability and mechanical properties. In this study, the synthesis methods, biocompatibility, physical, microstructural and nanostructural characteristics of nanocrystalline hydroxyapatite are reviewed. Compared to pure micrometre structured hydroxyapatite, numerous properties (most notably, biocompatibility properties pertinent for orthopedic applications) are improved for nanostructured hydroxyapatite doped with various ions. Such studies demonstrated that the mechanical properties and phase stability of nanohydroxyapatite doped with various ions after sintering at high temperatures should be investigated in more detail.

Microstructural, mechanical, and osteocompatibility properties of Mg2+/F−-doped nanophase hydroxyapatite

Pure as well as Mg(2+)- and F(-)-doped nanophase (i.e., grain sizes in the nanometer regime in at least one dimension) hydroxyapatite (HA) samples were synthesized by a precipitation method followed by sintering at 1100 degrees C for 1 h to determine their microstructural, mechanical, and osteoblast (bone-forming cell) adhesion properties pertinent for orthopedic applications. Different amounts of Mg(2+) and F(-) ions (specifically from 0 to 7.5 mol %) were doped into the HA samples. X-ray diffraction was used to identify the presence of crystalline phases, lattice parameters, and crystal volumes of the samples. Fourier transform infrared (FTIR) was further used to chemically characterize HA, and thus FTIR patterns revealed the characteristic absorption bands of HA. Microhardness measurements were also performed to assess mechanical properties of the novel formulations. Results of this study showed an improvement in sample density for some of the samples, which was a consequence of the molar percentage variation of the dopants. Moreover, in most of the samples doped with Mg, beta-tricalcium phosphate was observed as a second phase to HA. In addition, 1% Mg- and 2.5% F-doped HA had the highest microhardness values. Lastly, results demonstrated the highest osteoblast densities when the HA samples were doped with 2.5-7.5% Mg(2+) and F(-). Thus, the results of this study suggest that decreasing the grain size of HA into the nanometer regime and doping HA with Mg(2+) and F(-) can potentially increase the efficacy of HA for orthopedic applications.

Microstructure, microhardness, and biocompatibility characteristics of yttrium hydroxyapatite doped with fluoride

The current study focused on doping of hydroxyapatite (HA) with constant yttrium (Y(3+) ) and varying fluoride (F(-) ) compositions to investigate its microstructure, microhardness, and biocompatibility. HA was synthesized by precipitation method and sintered at 1100°C for 1 h. Y(3+) and F(-) ion dopings resulted in changes in densities. In x-ray diffraction analysis, no secondary phase formation was observed. Lattice parameters decreased upon ion substitutions. Scanning electron microscopy (SEM) results showed that ion addition resulted in smaller grains. In Fourier transform infrared spectroscopy analysis, F(-) ion substitution was confirmed. HA doped with 2.5% Y(3+) and 1% F(-) exhibited the highest microhardness. Y(3+) and F(-) ions improved Saos-2 cell proliferation on discs in Methylthiazolyldiphenyl-tetrazolium (MTT) assay. In SEM analysis, cells attached and proliferated on all disc surfaces. Alkaline phosphatase (ALP) assay showed that cell differentiation on the discs was improved by doping HA with an optimum F(-) amount. Dissolution tests revealed that structural stability of HA was improved with F(-) ion incorporation. The dissolution behavior of fluoridated samples exhibited a parallel pattern with the cell proliferation and differentiation behavior on these samples. Overall, this work shows that fluoride and yttrium cosubstitution into HA HA2.5Y1F was the most promising material for biomedical applications.

Fe3+/SeO42− dual doped nano hydroxyapatite: A novel material for biomedical applications

Dual ions substituted hydroxyapatite (HA) received attention from scientists and researchers in the biomedical field owing to their excellent biological properties. This paper presents a novel biomaterial, which holds potential for bone tissue applications. Herein, we have successfully incorporated ferric (Fe3+ )/selenate ( SeO42-) ions into the HA structure (Ca10-x-y Fey (PO4 )6-x (SeO4 )x (OH)2-x-y Oy ) (Fe-SeHA) through a microwave refluxing process. The Fe-SeHA materials were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and field emission scanning electron microscopy (FESEM). XRD and FTIR analyses revealed that Fe-SeHA samples were phase pure at 900°C. FESEM images showed that formation of rod-like shaped particles was inhibited dramatically with increasing Fe3+ amount. The Vickers hardness (HV) test showed that hardness values increased with increasing Fe3+ concentrations. Optical spectra of Fe-SeHA materials contained broadband over (200-600) nm. In vitro degradation and bioactivity tests were conducted in simulated body fluid (SBF). The incorporation of Fe3+ / SeO42- ions into the HA structure resulted in a remarkably higher degradation rate along with intense growth of apatite granules on the surface of the Fe-SeHA discs with Ca/P ratio of 1.35-1.47. In vitro protein adsorption assay was conducted in fetal bovine serum (FBS) and it was observed that the adsorption of serum proteins on Fe-SeHA samples significantly increased with increasing Fe3+ concentration. In vitro cytotoxicity tests were performed with human fetal osteoblast (hFOB) cell line and the results demonstrated that hFOB cells attached and proliferated faster on the Fe-SeHA materials compared to pure HA showing that Fe-SeHA materials were cytocompatible. ALP activity and intracellular calcium of hFOB cells on 1Fe-SeHA discs were statistically higher than pure HA, suggesting that presence of Fe3+ ion supported osteogenic differentiation of hFOB cells. Our results suggest that 1Fe-SeHA (0.2M Fe3+ /0.5M SeO42- co-doped HA) material could be considered as a promising candidate material for orthopedic applications.

Investigation of tensile strength of hydroxyapatite with various porosities by diametral strength test

Abstract It is appropriate to administer the diametral test to biomedical materials used in dental applications because stresses formed on dental implants are similar to those that formed in this test. To show this similarity, an experimental study of diametral strength testing of hydroxyapatite was performed. The influence of porosity on hydroxyapatite was investigated experimentally to determine how the diametral strength was affected. Hydroxyapatite was air sintered at 1100°C for 1 h with porosities ranging from 1 to 32%. The results indicated that hydroxyapatite with improved densification had higher diametral strength values. X-ray diffraction analysis showed that sintered samples were pure hydroxyapatite.

Synthesis, phase transitions and cellular biocompatibility of nanophase alumina–hydroxyapatite composites

Abstract Abstract Nanophase alpha-alumina and hydroxyapatite (HA) composites with and without CaF2 were prepared and sintered at 1100°C for 1 h to investigate their densification, structural and biocompatibility properties. X-ray diffraction method was performed to examine the second phases in the materials. It was observed that HA slightly decomposed into tricalcium phosphate and CaO with the addition of CaF2. The addition of CaF2 also resulted in an increase in the density of the composites. The composites were then evaluated for their biocompatibility using cytotoxicity tests. Saos-2 cells were seeded on composite discs in order to investigate the cellular responses to materials in terms of morphology, attachment and proliferation using scanning electron microscopy and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium-bromide viability assays. Cell culture studies showed that the nano-alumina and HA composite discs with or without CaF2 additions were biocompatible. CaF2 addition into the composites improved cell attachment and proliferation after 3 days of culture.

Prediction of hexagonal lattice parameters of various apatites by artificial neural networks

In this study, the hexagonal lattice parameters of apatite compounds, M10(TO4)6X2, where M is Na+, Ca2+, Ba2+, Cd2+, Pb2+, Sr2+, Mn2+, Zn2+, Eu2+, Nd3+, La3+ or Y3+, T is As+5, Cr+5, P5+, V5+ or Si+4, and X is F−, Cl−, OH− or Br−, were predicted from their ionic radii by artificial neural networks. A multilayer perceptron network was used for training and the best results were obtained with a Bayesian regularization method. Four neurons were used in the hidden layer, utilizing a tangent sigmoid activation function, while one neuron was used in the output layer with a pure linear function. The results of the training showed that the correlation coefficients for the hexagonal lattice parameters were 0.991 for the training data set, which is very close to unity, demonstrating that the learning process was successful. In addition, the average errors of the predicted lattice parameters were less than 1% for the data set prepared with single ions at the M, T and X sites, as well as for apatites with coupled substitutions involving up to three different ions at each site. Simple mathematical formulae were derived for the prediction of lattice parameters using average ionic radii as independent variables.

Structural and mechanical characteristics of nanohydroxyapatite doped with zinc and chloride

Abstract Abstract In this study, hydroxylapatite (HA) doped with Zn2+ and/or Cl− ions was synthesised by precipitation method and sintered at 1100°C for 1 h. Densities of the samples were measured by the Archimedes method. Zn2+ addition increased the density significantly, while Cl− increased the density insignificantly. Hydroxylapatite phase and inconsiderable amount of CaO phase were detected in some samples according to the X-ray diffraction results. Cl− added samples (2·5 mol.-%) increased the hexagonal unit cell volume of HA. Characteristic PO3−4 and OH− bands of HA were detected in Fourier transform infrared spectroscopy. Cl− related band was also observed at a wavenumber of 3497 cm−1. Grain sizes of the samples decreased with Cl− addition and increased with Zn2+ addition according to the SEM images. Zn2+ and Cl− addition improved the microhardness of pure HA. Fracture toughness of the samples decreased with Cl− and Zn2+ addition. When compared with other compositions, 2Zn2·5ClHA produced the best results in terms of mechanical properties.

Early Alterations in Bone Characteristics of Type I Diabetic Rat Femur: A Fourier Transform Infrared (FT-IR) Imaging Study.

Alterations in microstructure and mineral features can affect the mechanical and chemical properties of bones and their capacity to resist mechanical forces. Controversial results on diabetic bone mineral content have been reported and little is known about the structural alterations in collagen, maturation of apatite crystals, and carbonate content in diabetic bone. This current study is the first to report the mineral and organic properties of cortical, trabecular, and growth plate regions of diabetic rat femurs using Fourier transform infrared (FT-IR) microspectroscopy and the Vickers microhardness test. Femurs of type I diabetic rats were embedded into polymethylmethacrylate blocks, which were used for FT-IR imaging and microhardness studies. A lower mineral content and microhardness, a higher carbonate content especially labile type carbonate content, and an increase in size and maturation of hydroxyapatite crystals were observed in diabetic femurs, which indicate that diabetes has detrimental effects on bone just like osteoporosis. There was a decrease in the level of collagen maturity in diabetic femurs, implying a decrease in bone collagen quality that may contribute to the decrease in tensile strength and bone fragility. Taken together, the findings revealed alterations in structure and composition of mineral and matrix components, and an altered quality and mechanical strength of rat femurs in an early stage of type I diabetes. The results contribute to the knowledge of structure–function relationship of mineral and matrix components in diabetic bone disorder and can further be used for diagnostic or therapeutic purposes.