Calcium phosphate biomaterials from marine algae. Hydrothermal synthesis and characterisation

Quimica nova

Felício-Fernandes, G.¹  Laranjeira, Mauro C. M.¹ 

[1] Universidade Federal de Sta. Catarina, Florianópolis

关键词: hydroxyapatite;    calcium phosphate ceramics;    algae.  ;     ;    INTRODUCTION Research on the synthesis of calcium phosphate compounds such as hydroxyapatite (HAp) (Ca10(PO4)6(OH) 2) took on great momentum when it was observed that this substance is present in substantial amounts in the mineralised tissue of the vertebrates _ 60-70% of the mineral phase of the human bone1. In order to propose the biosynthetic processes of the bone;    the synthesis of HAp was developed in the laboratory. This calcium phosphate compound was chemically similar to that found in bone and was found to have a great potential for use as an auxiliary in bone regeneration2-6. In recent years bioceramics based on calcium phosphate salts have received attention as bone substitutes7. These materials may be resorbable (tricalcium phosphate);    bioactive (hydroxyapatite and bioactive glasses);    porous for tissue ingrowth (hydroxyapatite-coated metals)8 or composites (stainless-steel-fibre-reinforced bioglass)9-11. The human bone is formed basically by an organic phase and other minerals. In the organic phase;    the fibres of collagen serve as a matrix for the precipitation of HAp;    determining the organisation of the crystals. The collagen gives the bone its elastic resistance. The mineral phase is formed by HAp. The microstructure of the mineral phase of the bone is directly linked to the mechanical requirements of the location in the skeleton12. Two different types of bone tissue are observed: the compact or cortical and the trabecular or spongy bone tissue4: The trabecular bone forms a homogeneous porous three-dimensional structure with interconnected pores of characteristic diameters. Both types of bone tissue are anisotropic;    a typical characteristic of crystalline bodies. To produce a material with similar microstructure is of great interest in the areas of biomedicine linked to the development of prostheses for bone reconstitution or substitution. This is due to the unique property of bone tissue to regenerate;    forming new healthy tissue that grows in the direction of the damaged area;    filling in with functional bone. If a physical and chemically similar material is placed in contact with this healthy bone;    then the growth process is accelerated and the material is filled in with new bone and reabsorbed13. HAp that integrates the mineral phase of the bone is said to be non-stoichiometric and calcium-deficient with a relationship of Ca/P <;    1.67. However;    some difficulties exist in the study of this HAp that occurs in the bones in the form of small crystals without defined orientation and with a great amount of organic matter and other bound or adsorbed ions at the surface14. Nevertheless;    the substitution of several ions such as CO32- with 4-6% of substitution is observable12. When the non-stoichiometric HAp is synthesised;    other intermediary products can be formed. An example is octacalcium phosphate (OCP);    which forms a mixture of two intercalated layers of HAp/OCP/HAp15-17. Table 1 introduces some calcium phosphates that can appear as by-products of the synthesis of HAp and whose Ca/P ratios are quite close to one another. ;    To use a porous material of natural origin as a basic raw material for the synthesis of calcium phosphates;    simulating bone and stimulating its development;    is an idea that was put into practice some time ago4;    13;    20;    21. When its aim is to simulate human bone;    e.g.;    to synthesise HAp with similar microstructure as that found in the bone tissue;    hydrothermal synthesis is used;    since this method allows the synthesis of a material chemically similar to the bone;    presenting good crystalline quality;    physiological stability and the maintenance of the morphological characteristics of the initial material;    the calcium carbonate of natural origin. Several processes4;    5 are used to produce HAp of wide application as a temporary substitute for the human bone5;    10;    11;    20;    21. HAp behaves as a temporary substitute by acting as an auxiliary agent in the bone regeneration in such a way that it can be reabsorbed later by the organism22. Thus;    the use of HAp in the areas of orthopaedics20-25;    and dentistry26-28 emerges as one of the most important applications of this material. Hydrothermal synthesis is characterised by the reaction of aqueous solutions in closed recipients under controlled temperature and/or pressure. The temperature can be elevated above the boiling point of the water;    reaching the pressure of vapour saturation. One specific method of hydrothermal synthesis consists of submitting an aqueous solution containing Ca2+ and PO43-;    to high temperatures (200oC-500oC). Thus;    a calcium phosphate compound is obtained which is able to maintain the morphological structure of the original material (e.g.;    CaCO3 of corals) and which possesses physical-chemical characteristics very similar to human bones;    is relatively stable in a physiological medium;    and also originates a pure crystalline product when it is used as a synthetic reagent17;    29-33. The hydrothermal method has been widely5;    13 used for the preparation of materials for prosthetic purposes. It is believed that the use of natural raw materials for the synthesis of HAp enables it to be accepted better by the organism because of its similar physical-chemical characteristics27;    28;    35. Calcium carbonate for the synthesis of calcium phosphates similar to bone can be obtained from several natural sources. Only the calcium carbonate originating from marine algae and corals shows characteristic porosity and interconnectivity that makes it like human bone. To use these calcium carbonates as starting material for the synthesis of HAp;    they cannot be pulverised. These materials should be used;    as taken out;    in fragments in the case of algae and in shaped blocks in the case of corals. Thus;    the morphological structure of the natural calcium carbonate with macropore and interconnections will be maintained. The way to do this is through hydrothermal synthesis;    which allows ionic exchange;    without modification of the morphological structure of HAp in order to use it as an auxiliary in bone regeneration4. The use of corals as the only source of CaCO3 for this synthesis has the disadvantage of destroying the banks of corals;    which form a delicate and important ecosystem for the maintenance of marine life. The use of marine algae appears then as a less aggressive alternative;    since the banks of algae can be managed with relative simplicity without destroying the seabed. The exploitation of banks of algae in Brazil is viable;    since they are very frequently found on the Brazilian coast;    the available stock being of approximately 2 x 1011 tons36. The use of phycogenic calcium carbonate in the synthesis of HAp has already shown promise in relation to human bone substitution and regeneration27;    28. In recent years;    research has been demonstrating that the usefulness of HAp also extends to other areas of great interest. Its use as a support of proteins in column chromatography is already established as a method that unites good performance with high affinity in the separation of several proteins such as g-globulin;    lysozyme and human albumin;    among others37;    38. In general;    awareness of the importance of protecting the environment has grown greatly in recent years;    and the products and processes that aid in the removal of substances harmful to nature are more welcome than hitherto: thus;    HAp will have a contribution to make in this field. Several papers39-43 have demonstrated that this material presents very convenient qualities for the removal of toxic metals such as Cd;    Pb;    As;    Mn;    Al and Ni. The facility of producing ionic exchanges of hydroxyapatite;    allied to its application over a great surface area;    makes it a material with great potential in the treatment of industrial effluents. The possibility of HAp being used as catalyst for the treatment of toxic gases before they reach the atmosphere is also being researched44;    45. The present work describes the synthesis and characterisation of a calcium phosphate compound such as HAp;    which has calcium carbonate from marine algae as starting material.  ;    MATERIAL AND METHODS Starting material The biogenic material was obtained from algae of the Rodophycophyta division collected in the coastal area of Santa Catarina Island. These algae are characterised by a high content of CaCO3 in their vegetative structure. The collected algae were selected in the laboratory for removal of greater impurities such as mollusk shells or small marine organisms. This material was washed with tap water and than dried in an oven at 80oC for 48 hours. After drying a new selection was made and the dry weight was measured. The organic matter present was digested through treatment with a dilute aqueous solution (10%) of sodium hypochlorite. The material was then washed until its pH was close to that of physiological pH. This white material formed by particles of 3mm length on average;    was dried in the oven at 80oC for 30 hours and then it was stocked for later analyses and utilisation. The obtained material was considered as being entirely free of organic matter46. The use of CaCO3 from the algae was evaluated as 80% of the dry weight;    a remarkable value considering the natural origin of the material. The calcium carbonate from the algae was characterised as calcitic according to X-Ray powder diffractometry and FTIR spectroscopy47. The porous microstructure of the material was analysed by gas adsorption;    showing that the material was mesoporous. Photomicrographs by scanning electron microscopy also confirmed the presence of macropores and interconnections. The porous structure of this phycogenic calcium carbonate;    allied to its surface morphology without edges;    makes it a promising material as a precursor for biomedical use. Hydrothermal synthesis Two different methodologies were used in the synthesis;    both involving hydrothermal reactions: 1. (NH4)2HPO4 was dissolved in distilled water in the desired proportions. This solution was placed in the pressure vessel on top of the phycogenic CaCO3. 2. (NH4)2HPO4 was dissolved in an NH4F (20ppm) aqueous solution;    and this solution alone was later run onto the phycogenic CaCO3 for the synthesis. The pH was controlled prior to the reactions in the range of 8.5 - 9.0 with use of NH4OH when necessary. Next;    the solutions were placed in the pressure vessels;    which were then closed and put into the oven. The temperature was controlled at 200oC. The time of permanence in the oven varied between 24 and 48 hours. After the withdrawal of the samples from the vessel;    they were measured for pH and washed until all excess of HPO42- had been removed and the pH was close to that of the physiological pH. The samples as prepared were fully dried in an oven at 80oC for 30 hours. The prepared material with particles not bigger than 3 mm was stocked for analysis. From the many proportions of the main reagents that were tested;    two are presented in this paper because they showed the closest similarity with non-stoichiometric HAp;    like bone. The reaction conditions for the hydrothermal synthesis are presented in Table 2. ;    X-ray diffraction analysis (XRD) Samples of the synthesised material were pulverised and examined in a Phillips diffractometer model APD15 and Seiffert model 300TT;    the XRD patterns being recorded in the range of 5o £ 2q £ 50o using CuKa radiation (l=1.54 nm);    with 0.05o 2q step size and step scanning 1 s per 2q value. To determine the peaks from HAp or other phases that might occur;    the diffractograms were compared with the ICDD cards - for HAp (9-432);    a_ TCP;    9 _ 348;    b _ TCP ( 9-169). The x-ray diffraction patterns from commercial calcium phosphate prepared by Aldrich (Milwaukee;    USA);    catalogue number 12167-74-7;    were also recorded as reference material. Fourier transform-infrared spectrometry (FTIR) After crushing;    the samples were prepared in KBr discs and analysed in Mattson Galaxy 2030 and Nicolet DXB Fourier transform IR spectrometers in the range of 4000cm-1 and 400cm-1. The obtained spectra were compared with that of the commercial material from Aldrich;    used as reference;    and with the literature19-20. Thermogravimetry (TG) Thermogravimetric analysis in air up to 1000oC was performed at a heating rate of 10oCmin-1 using a Linseis L81 thermobalance operated by the Lynseis Acquisition Program. For this analysis;    the samples were not crushed. A platinum crucible was used as reference. Scanning Electron Microscopy (SEM) Fragments of the synthesised material were analysed employing scanning electron microscopy (SEM - ZEISS DSM962). For SEM analyses;    the fragments were not crushed so that the material was very similar to the starting raw material for the synthesis. Gas adsorption analysis (BET) For a verification of the morphology of the microstructure of the obtained material;    gas adsorption analysis was made;    using N2 at boiling point (Micrometrics ASAP 2110 and Quantachrome Autosorb MGS). The synthesised material was not crushed so results could be obtained from the material without morphological alteration. Chemical analysis The phosphorus content was analysed colorimetrically. The complexation titration method was used for the calcium determination. In order to verify the presence of other elements contaminating the material;    energy-dispersive electron probe X-ray analysis (EDX-PHILIPS) was used. Some trace elements;    which might have been connected with the biocompatibility;    were detected by spectrochemical analysis.  ;    RESULTS AND DISCUSSION;   

DOI  :  10.1590/S0100-40422000000400002

学科分类：化学（综合）

来源: Sociedade Brasileira de Quimica

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