ISSN 00125008, Doklady Chemistry, 2016, Vol. 468, Part 1, pp. 159–161. © Pleiades Publishing, Ltd., 2016. Original Russian Text © I.V. Fadeeva, I.I. Selezneva, G.A. Davydova, A.S. Fomin, O.S. Antonova, Ya.Yu. Filippov, S.M. Barinov, 2016, published in Doklady Akademii Nauk, 2016, Vol. 468, No. 2, pp. 171–174.
CHEMICAL TECHNOLOGY
IronSubstituted Tricalcium Phosphate Ceramics I. V. Fadeevaa, I. I. Seleznevab, G. A. Davydovab, A. S. Fomina, O. S. Antonovaa, Ya. Yu. Filippovc, and Corresponding Member of the RAS S. M. Barinova Received November 19, 2015
Abstract—Porous ironsubstituted tricalcium phosphate (FeTCP) ceramics with a Fe content of 0.49 and 1.09% has been developed. The hydrostatically estimated ceramics porosity is 40–45%. The solubility of ceramics in an isotonic solution has been studied. The solubility rate of FeTCP ceramics is slightly higher as compared with ironfree ceramics. Based on the results of in vitro tests of FeTCP ceramics on cultured fibro blasts, these materials are believed to be biocompatible. The developed materials can be recommended for use in medicine in the treatment of diseases associated with bone lesions. DOI: 10.1134/S0012500816050049
For bone recovery after surgery, osteoconductive materials capable of being carriers for osteoinductive cells and proteins that promote the de novo bone for mation are used. This approach has been referred to as bone tissue engineering [1]. Major requirements for such materials are biocompatibility with tissues, the lack of an adverse reaction of organisms, and the pres ence of pores of different size necessary for protein adsorption and cell and vessel sprouting. Hydroxyapa tite (HA) and tricalcium phosphate (TCP) are cur rently used as such materials [2]. Isomorphous substi tution of iron for calcium in the TCP structure is a promising strategy for producing new materials: as a result of resorption of such materials in the body, iron ions will be released into surrounding tissues. The bio logical role of iron ions in organism is, first of all, oxy gen delivery to all organs, which is due to the capacity of iron(III) to reversibly bind oxygen [3]. In addition, iron is incorporated in enzymes and ironbinding pro teins contained in intracellular fluid. Ironsubstituted TCP ceramics has not been studied so far. This study deals with the development of porous ironsubstituted TCP ceramics and their physicochemical and biologi cal properties.
a
Baikov Institute of Metallurgy and Materials Science, Russian Academy of Sciences, Leninskii pr. 49, Moscow, 119991 Russia email:
[email protected] b Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia c Research Institute of Mechanics, Moscow State University, Michurinskii pr. 1, Moscow, 119992 Russia
EXPERIMENTAL Powders of Fesubstituted TCPs containing 1.0 wt % and 0.5 mol % Fe (FeTCP1 and FeTCP2, respectively) were synthesized as described in [4]. For crystal struc ture formation, the powders were heat treated at 400°C for 1 h. Fesubstituted TCP ceramics was fabri cated by sintering the samples obtained by bilateral uniaxial pressing the Fesubstituted TCPs at a pressure of 100 MPa. Sintering was carried out for 2 h at 1100°C in a chamber furnace with silicon carbide heaters The ceramics porosity was determined by hydrostatic weighing [5]. The microstructure of ceramic materials was studied by a Tescan Vega II scanning electron microscope. The solubility of Fesubstituted TCPs in an iso tonic solution was studied at 37°C by measuring the calcium concentration. The calcium ion concentra tion in an isotonic solution, as well as the concentra tion of iron ions in ceramics, was determined on an Ultima II inductively coupled plasma atomic emission spectrometer. Cytotoxicity tests were carried out for extracts and materials according to the requirements [6, 7]. Cul ture medium DMEM/F12 supplemented with 100 U/mL penicillin/streptomycin was used a model one for the preparation of extracts. NCTC L929 fibro blasts were used in the experiments. The cytotoxicity tests of the extracts of materials were carried out using the MTT assay based on the reduction of a colorless tetrazolium salt (3[4,5dimethylthiazol2yl]2,5 diphenyltetrazolium bromide, MTT) with mitochon drial and cytoplasmic dehydrogenases of living, meta bolically active cells to form blue crystals of formazan soluble in dimethyl sulfoxide. Statistical processing of results was performed with the Origin program. The rootmeansquare deviation from the average value
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Fig. 2. Metabolic activity of NCTCL929 cells as deter mined by MTT assay after incubation for 48 h in three day extracts of materials (1) TCP 1100, (2) FeTCP1 1100, (3) FeTCP2 1100, (4) control, and (5) DMSO. Statisti cally significant deviations from the control according to the Mann–Whitney Utest (p < 0.05) are asterisked.
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Fig. 1. Microstructure of ceramics (a) FeTCP1 and (b) FeTCP2.
was taken to be the error, and differences were thought of as significant according to the Mann–Whitney Utest at p < 0.5. For studying the adhesive characteristics of the materials and their cell cytotoxicity, the direct contact method was used. The separation of the mixed primary culture was carried out using the difference in duration of adhesion of different types of cells to the culture surface [8]. RESULTS AND DISCUSSION According to Xray diffraction, the major crystal line phase of the synthesized powders is whitlockite. Up to 3 wt % αFe2O3 is identified as an impurity phase. The coherent scattering length (CSL) calcu lated for FeTCP is 10.6 nm. This value is somewhat lower as compared with the TCP ceramics (12 nm), which is evidence that the introduction of iron leads to a decrease in crystallite size. The decrease in crystallite
size is caused by crystal lattice distortions that accom pany the substitution of iron atoms for calcium atoms of smaller ionic radius. According to elemental analysis, the iron content is 1.09 wt % for FeTCP1 and 0.49 wt % for FeTCP2, which corresponds to the iron amount introduces in the course of synthesis The microstructure of FeTCP1 is heterogeneous. There are two types of crystals: large, wellfaceted crystals with fused edges 1–2 µm in size and tiny crys tals less than 0.1 µm in size (Fig. 1). The microstruc ture of ceramics FeTCP2 is more homogeneous with crystallites 1–3 µm in size. The heterogeneity of the FeTCP1 structure is due to the fact that, at larger iron content, iron oxide is released as a separate αFe2O3 phase. According to EDX analysis, iron is uniformly distributed in the bulk of ceramics FeTCP1 and FeTCP2. Sintering has led to ceramic discs with porosity 40– 45%. The introduction of iron into the TCP structure has almost no effect on the ceramics solubility. The metabolic activity of NCTC L929 cells in the presence of extracts of the materials has been studied using the MTT assay [11]. The MTT assay is based on the ability of mitochon drial dehydrogenases to convert watersoluble 3(4,5 dimethylthiazol2yl)2,5diphenyl2Htetrazolium bromide (MTT) into formazan which is crystallized in a cell. Only the cells with living mitochondria can be involved in this reaction; hence, the color intensity is directly related to the content of intact mitochondria. DOKLADY CHEMISTRY
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The results of studying the characteristics of mate rials via direct contact with human cells are shown in Fig. 3. Our study showed no significant differences between the control and TCP material and an insignif icant decrease in cell activity not related to the cell death for materials FeTCP1 and FeTCP2. Based on in vitro tests, these materials can be considered biocom patible. Thus, a method of fabrication of ironsubstituted tricalcium phosphates containing 0.5–1.1 wt % Fe and the corresponding porous ceramics has been developed. According to in vitro biotests, the fabri cated materials are biocompatible. The solubility rate of porous ironsubstituted tricalcium phosphate ceramics is higher than the solubility rate of analogous TCP ceramics. The developed materials can be rec ommended for use in medicine use in medicine in the treatment of diseases associated with bone lesions. REFERENCES
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Fig. 3. Appearance of human fibroblasts incubated on material (a) FeTCP1 and (b) FeTCP2.
This assay is useful for revealing common cytotoxic compounds as well as the agents for which mitochon dria are specific targets. Statistical analysis of the results was performed using the Mann–Whitney Utest—a nonparametric test used to compare two independent samples on the basis of some quantified property. Study of the meta bolic activity of NCTC L929 cell using the MTT assay showed the absence of significant differences between the experiment and control only for sample 1, which is evidence of the absence of the toxic effect of threeday extracts. In the presence of extracts of materials 2 and 3, the cell viability was slightly reduced (Fig. 2). However, inoculation of the cells on the surface of materials and study of their viability (direct contact method) made it possible to prove the lack of the toxic effect of the ironcontaining materials.
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Translated by G. Kirakosyan