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Zinc alginate–hydroxyapatite composite microspheres for bone repair
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CERAMICS INTERNATIONAL
Ceramics International 40 (2014) 11369–11375 www.elsevier.com/locate/ceramint
Short communication
Zinc alginate–hydroxyapatite composite microspheres for bone repair Renan Costa Cuozzoa, Maria Helena Miguez da Rocha Leãob, Luciano de Andrade Gobboc, Daniel Navarro da Rochaa, Nadia Mohammed Elmassalami Ayada, Willian Trindadea, Andrea Machado Costaa, Marcelo Henrique Prado da Silvaa,n b
a Instituto Militar de Engenharia—IME, Rio de Janeiro, RJ, Brazil PANalytical Spectris do Brasil Instr. Eletrônicos Ltda., São Paulo, SP, Brazil c Universidade Federal do Rio de Janeiro—UFRJ, Rio de Janeiro, RJ, Brazil
Received 29 August 2013; received in revised form 22 February 2014; accepted 24 February 2014 Available online 12 March 2014
Abstract This paper presents a novel method for producing composite and ceramic microspheres containing zinc for bone regeneration. The microspheres were prepared from a mixture of sodium alginate and hydroxyapatite that was extruded into a zinc nitrate solution. After 24 h in the solution, Na þ exchange with Zn2 þ ions produced zinc alginate–hydroxyapatite composite microspheres. After calcination, Zn-containing betaTCP ceramic microspheres were produced. & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: B. Composite; Alginate; Biospheres; Hydroxyapatite; Zinc
1. Introduction The gold standard for bone repair of damaged or lost tissue sites is autologous bone, i.e., from the same individual. However, the limited amount of available tissue, along with the inconvenience of creating another defect, suggests the need for other classes of grafts. Xenografts and allografts are options, but since these grafts come from other individuals, the question of infection arises. In this aspect, artificial grafts, i.e. can be a good alternative to biomaterials of biological origin [1], since they minimize the risk of bacterial and viral contamination, and are commercially available [2].
n
Corresponding author. E-mail addresses: [email protected] (R. Costa Cuozzo), [email protected] (M. Helena Miguez da Rocha Leão), [email protected] (L. de Andrade Gobbo), [email protected] (D. Navarro da Rocha), [email protected] (N. Mohammed Elmassalami Ayad), [email protected] (W. Trindade), [email protected] (A. Machado Costa), [email protected] (M. Henrique Prado da Silva). http://dx.doi.org/10.1016/j.ceramint.2014.02.107 0272-8842 & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Although hydroxyapatite shows composition and structure similar to those of biological apatites, bioactivity and bio-resorption rate can be relatively limited [3]. Several studies have focused on cationic and anionic substitutions, aiming to mimic biological apatite properties. These mimicked biomaterials are more similar to human bone than pure and stoichiometric hydroxyapatite, Ca10 (PO4)6(OH)2 [4,5]. Ionic substitutions in hydroxyapatite structure induce changes in morphology, crystallinity, thermal stability, and resorption rate. Several ions present in the mineral phase of bone can be used in experimental studies on apatite synthesis [6]. Zinc is a relevant metallic element in the human body, since it is essential for bone structure and metabolism [7]. Mineral bone presents the greatest amount of this element, about 160– 300 ppm [8]. Zinc is important in cell differentiation, growth and metabolism of proteins, carbohydrates and lipids. It also has a role on the hormonal structure and genetic transcription, and participates in the synthesis and degradation of nucleic acids, carbohydrates, lipids, and proteins, and many other vital reactions. Zinc deficiency can lower bone metabolism, reducing growth, weight and development, affecting bone tissue health [9,10]. Studies have shown the role of zinc in
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stimulating bone growth and mineralization in vivo and in vitro, and decreasing bone resorption, since it inhibits osteoclasts activity and stimulates alkaline phosphatase activity [11,12]. Besides, Yamagushi et al. (1987) proposed that zinc stimulated collagen formation in rat calvaria. In other studies [13], zinc showed to stimulate proliferation and cell differentiation as well as synthesis of bone-growth proteins, such as osteocalcin, IGF-1, TGR-f and aminoacyl-tRNA. Alginate is an anionic polysaccharide, which has a linear structure formed by glycosidic units of β-D-mannuronic acid and α-L-guluronic [14]. It can perform many exchanges in the presence of divalent ions by a cross-linking process. These exchanges occur between sodium ions and the divalent ions, and gelling the alginate solution. When sodium ion from guluronic acid exchanges with divalent cations, a structure known as “egg box” is created by cross-linking, which leads to the possibility of incorporating or entrapping desired substances. Divalent cations bind to blocks of α-L-guluronic acid, enabling the association with more than 20 monomers. Each chain of alginate may dimerize to create links with many other chains, resulting in a reticulated gel [15]. Due to this gelling property, alginate can be used as a entrapping matrix for biological molecules such as proteins, cells, and drugs. In the surgical field, it can be used in the repair of cellular tissues, and as treatment for restricting capillaries bleeding and skin burns [16]. Tissue engineering aims to develop three-dimensional structural biomaterials to be used as a physical support for cells. These scaffolds should also stimulate cell migration and differentiation [17]. In vitro culture on tissue engineering scaffolds is supposed to produce a set of differentiated cells reproducing the tissue to be reconstructed. This strategy represents one step towards the transition from restorative to regenerative medicine.
2. Materials and methods 2.1. HA Production Hydroxyapatite was produced and characterized in the Ceramics Laboratory of the Military Institute of Engineering —IME (Rio de Janeiro, Brazil). The synthesis was done using an aqueous precipitation method with start solution composed by a 0.5 M calcium hydroxide [Ca(OH)2] (Merck, Darmstadt, Germany) suspension, 1 M lactic acid (C3H6O3) (Vetec, Rio de Janeiro, Brazil) and 0.3 M ortho-phosphoric acid (H3PO4) (Merck, Darmstadt, Germany). After homogenization, the solution pH was adjusted up to 12 by the addition of 1 M KOH solution. The resulting precipitates were aged for 24 h and the solution was then filtered under negative pressure by using a vacuum pump system (Edwards, Neuberger, Germany). The filtered cake was resuspended in ultrapure water in order to remove remaining KOH, up to pH neutralization. The filtrate was lyophilized (Freezone 1—Labconco, USA) for 48 h in the Biomaterials Laboratory of the Brazilian Centre for
Research in Physics—CBPF (Rio de Janeiro, Brazil). Then the powders were de-agglomerated and sieved. 2.2. Spheres production and characterization A solution of 1% sodium alginate (SigmaAldrich) in ultrapure water was prepared by mixing in magnetic stirrer under a heat plate (Warmer–Quimis, São Paulo, Brazil), until the formation of a homogeneous paste without the presence of visible particles. Hydroxyapatite was weighed in a ratio of 1:15 alginate/hydroxyapatite. HA was added to the alginate solution and mixed until a homogeneous slurry was formed. The mixture was then extruded with the aid of a 10 ml disposable syringe (BD Plastipak) with 0.7 mm diameter needle (BD Precisionglide) into two different solutions: 0.3 M zinc nitrate (Vetec, Rio de Janeiro) and 0.3 M calcium chloride (Vetec, Rio de Janeiro). The spheres were immediately produced as the drops came into both solutions due to ion exchange. In contact with zinc nitrate and calcium chloride solutions, sodium alginate gelled due to exchanges with sodium ions present in the slurry, allowing the formation of Zn-alginate–HA and Ca-alginate– HA composite biospheres. The spheres remained in the solution for 24 h in order to allow complete ion exchange. The spheres were then removed from the solution, washed three times with ultrapure water and placed pel on a paper filter, where they remained for 24 h until complete drying. The samples were heat-treated at 900 1C and 1250 1C. The investigation of the phases present in the samples was performed in the X-ray Diffraction Laboratory of the Brazilian Centre for Research in Physics (CBPF), in a brand PANalytical diffractometer, model X'Pert PRO MPD, operating with a copper source, current 40 mA and voltage of 40 kV. The scan was performed in the range 51 o 2θ o 801, with a collecting step of 1 s. The phases identification was performed with the program X'Pert High Score Plus. The percentage of phases at different temperatures was assessed by Rietveld refinement analyses. The biospheres morphology was evaluated by scanning electron microscopy (SEM) on a field emission gun SEM (FEG–SEM) FEI Quanta 250FEG with energy-dispersive X-ray spectroscopy (EDS) to identify the chemical elements present in the biospheres. The samples were coated with gold, deposited by a sputter Emitech K550X at a current of 50 mA for 2 min. The spheres were measured under 100 magnification, by SEM. Diameters of spheres were measured before and after heat treatment and mean values with standard deviations were obtained for five spheres on each condition. BET analyses were performed in both samples, calcium and zinc, calcined at 900 1C. Specific area and mean diameter measurements were determined, in desorption, by an automatic physical adsorption analyzer (Micrometrics, model ASAP 2010). 3. Results and discussion The XRD analysis of the HA powder identified the presence of a poorly crystalline hydroxyapatite (Fig. 1). The XRD
R. Costa Cuozzo et al. / Ceramics International 40 (2014) 11369–11375
patterns shown in Fig. 1 also shows that HA is the only present phase in the composite biospheres after ion exchange in calcium chloride and zinc nitride solutions. After calcination at 900 1C, spheres showed higher crystallinity when compared to the corresponding green samples, as shown in Figs. 2 and 3. However, the zinc alginate spheres showed the presence of β-TCP as the major phase (Fig. 3), while HA was maintained as the only phase in the Ca-alginate spheres. In fact, Rietveld refinement showed 100 wt% HA in
Fig. 1. XRD patterns confirming hydroxyapatite as the only phase present in HA green powder in Ca-alginate–HA and in Zn-alginate–HA biospheres.
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these samples (Fig. 2). The Zn-alginate spheres calcined at 900 1C showed Witlockita (β-TCP), and zincite (zinc oxide) phases. These XRD results strongly suggest the role of Zn as a substitutional element in the HA lattice. The excess of zinc present in the sample may account for the presence of zinc oxide. Rietveld refinement indicated 86.9 wt% whitlockite and 13.1 wt% ZnO in these samples. The Ca-alginate spheres sintered at 1250 1C were composed mainly by hydroxyapatite (80.6 wt%), CaO (1.4 wt%) and β-TCP (18 wt%) phases (Fig. 4). The biospheres produced in zinc nitrate and sintered at 1250 1C showed the formation of β-TCP as the main phase (85.6 wt%) and ZnO (14.4 wt%). The excess of zinc provided the formation of ZnO, as shown in the XRD pattern of Fig. 5. Table 1 shows the resulting mean diameters of the spheres before (Do) and after (Df) heat treatment with respective standard deviations. After heat treatment, the spheres underwent a shrinkage and this was assessed by SEM–FEG. Diameter measurements were performed, as shown in Fig. 6. The spheres produced in the zinc nitrate solution were smaller than calcium alginate ones. This characteristic was observed immediately after dripping the zinc nitrate solution. This finding was attributed to the zinc ionic radius (74 pm), smaller than the calcium ionic radius (100 pm) in a single coordination number [18]. The spheres dripped in a zinc nitrate solution have a more compact “egg box” structure than that created by calcium alginate. Thus, small zinc ions occupy a smaller volume allowing a closer approximation to the HA particles. In addition, zinc has an affinity for guluronic and manuronic acids, whereas calcium binds only to guluronic acid chains. This more
Fig. 2. XRD pattern of the Ca-alginate–HA spheres calcined at 900 1C showing HA as the only present phase.
Fig. 3. XRD pattern of the Zn-alginate–HA spheres calcined at 900 1C, showing the presence of β-TCP as the major phase and ZnO.
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Fig. 4. XRD pattern of Ca-alginate samples sintered at 1250 1C, showing the presence of hydroxyapatite as the major phase, and β-TCP and CaO minor phases.
Fig. 5. XRD pattern of Zn-alginate samples sintered at 1250 1C, showing the presence of β-TCP as the major phase and ZnO minor phase.
general affinity of zinc also may account for the higher retraction of zinc alginate spheres. By the SEM–FEG analyses of the samples, some morphological differences between zinc and calcium spheres could be observed and the first one refers to particles morphology. Calcium spheres calcined at 900 1C showed aggregates of fine particles, as shown in Fig. 7a, whereas calcined zinc spheres presented a more homogeneous distribution of bigger particles (Fig. 7b). After sintering at 1250 1C, there was no marked difference between the grain sizes of hydroxyapatite (Fig. 8a) and β-TCP (Fig. 8b). However, the β-TCP grains were strongly bound, suggesting complete sintering. In addition, an intergranular phase was observed, suggesting that the sintering mechanism was liquid-phase sintering. EDS analyses on the grains of Fig. 8a and b are shown in Fig. 8c and d, respectively. Fig. 8c shows the EDS result of a square area encompassing several grains, whereas Fig. 8d shows the result of an analysis performed on the intergranular phase, confirming the presence of zinc. This findings agree with the Rietveld analysis that determined the presence of 11.5 wt% ZnO in these samples. Fig. 9 shows, in a higher magnification, the grain boundary region from where EDS analyses were performed. BET analyses in the spheres calcined at 900 1C confirmed the SEM findings: Zn spheres showed smaller mean pore diameters and porosity when compared to HA spheres, as shown in Table 2. HA spheres showed higher specific area, 11.5470.08 m2/g, when
Table 1 Mean diameter and standard deviation of the biospheres before (Do) and after (Df) heat treatment. HA_CaCl2_900 HA_Zn_900 HA_CaCl2_1250 HA_Zn_1250
Mean diameter (mm) Standard deviation
Do
Df
Do
Df
Do
Df
Do
Df
1.44
1.22
1.2
1.13
1.53
0.97
1.21
0.68
0.07
0.07
0.07
0.04
0.07
0.07
0.08
0.04
compared to Zn spheres, 6.6970.01m2/g. Specific area, porosity and mean pore diameter results agree with SEM observations, that revealed a more homogeneous pore and particle size for the Zn spheres, when compared to HA. In fact, experimental data showed that both spheres showed macro- and meso-pores. However, the curve obtained for the HA samples is closer to the macropores fitting, according to equation p/v(p0 p) p/p0, whose slope is (c 1)/vmc, where
v is the total adsorbed gas volume; vm is the adsorbed gas volume when the solid surface is fully coated by a monolayer; c is the BET constant;
R. Costa Cuozzo et al. / Ceramics International 40 (2014) 11369–11375
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Fig. 6. Spheres shrinkage observed by SEM–FEG: (a) Ca-alginate–HA calcined at 900 1C, (b) Zn-alginate–HA calcined at 900 1C, (c) Ca-alginate–HA sintered at 1250 1C, and (d) Zn-alginate–HA sintered at 1250 1C.
Fig. 7. SEM micrographs of the samples calcined at 900 1C: (a) Ca-alginate–HA and (b) Zn-alginate–HA.
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Fig. 8. SEM micrographs and EDS spectra of the samples sintered at 1250 1C: (a) hydroxyapatite grains of Ca-alginate–HA sample, (b) β–TCP grains of Znalginate–HA, (c) EDS analysis of grains on Ca-alginate–HA sample and (d) EDS analysis of the intergranular phase of Zn-alginate–HA sample, showing Zn-rich phase on the grain boundaries.
Table 2 BET results for the spheres calcined at 900 1C. Sample
Specific area (m2/g)
Mean pore diameter (̊)
Pore volume (cm3/g)
HA Zn–HA
11.5470.08 6.6970.01
291.87 69.11
0.35 0.008
p is the measured pressure in the equilibrium; p0 is the initial pressure of the system.
4. Conclusion
Fig. 9. Detailed location from where EDS shown in Fig. 8d is performed.
The preparation of hydroxyapatite–alginate spheres in a solution rich in zinc ions produced Zn-alginate–hydroxyapatite composite
R. Costa Cuozzo et al. / Ceramics International 40 (2014) 11369–11375
spheres. During calcinations, zinc ions entered the hydroxyapatite structure and induced its decomposition to β-TCP. Zinc induced the formation of smaller biospheres when compared to the ones produced in the calcium ions solution. This finding is associated with the smaller zinc atomic radius and also with the fact that zinc has affinity for both guluronic and manuronic acids, whereas calcium binds only to guluronic acid chains. This broader affinity favors the higher retraction of zinc alginate spheres. Acknowledgments The authors thank CAPES, CNPq and the institutions IME and CBPF for their cooperation and support for the paper. References [1] D. Kaigler, D. Mooney, Tissue engineering‘s impact on dentistry, J. Dent. Educ. 65 (5) (2001) 456–462. [2] D.F. Williams, Definitions in Biomaterials, Elsevier, New York, 1987. [3] L.L. Hench, Biomaterials: a forecast for the future, Biomaterials 19 (16) (1998) 1419–1423. [4] M. Vallet-Regi, D. Arcos, Silicon substituted hydroxyapatites, a method to upgrade calcium phosphate based implants, J. Mater. Chem. 15 (15) (2005) 1509–1516. [5] D.N. Da Rocha, L.A. Gobbo, M.H.P. Da Silva, Production and characterization of niobate apatite, J. Mater. Res. Technol. 2 (1) (2013) 24–29. [6] M. Palard, E. Champion, S. Foucaud, Synthesis of silicated hydroxyapatite Ca10(PO4)6 x(SiO4)x(OH)2 x, J. Solid State Chem. 181 (2008) 1950–1960.
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[7] H. Tapiero, K.D. Tew, Trace elements in human physiology and pathology: zinc and metallothioneins, Biomed. Pharmacother. 57 (2003) 399–411. [8] W.R. Holloway, F.M. Collier, R.E. Herbst, J.M. Hodge, G.C. Nicholson, Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number, Bone 19 (2) (1996) 137–142 (August). [9] H.S. Hsieh, J.M. Navia, Zinc deficiency and bone formation in guinea pig alveolar implants, J. Nutr. 110 (1980) 1581–1588. [10] G. Oner, B. Bhaumick, R.M. Bala, Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats, Endocrinology 114 (1984) 1860–1863. [11] M. Yamaguchi, R. Yamaguchi, Action of zinc on bone metabolism in rats: increases in alkaline phosphatase activity and DNA content, Biochem. Pharmacol. 35 (1986) 773–777. [12] S.L. Hall, H.P. Dimai, J.R. Farley, Effects of zinc on human skeletal alkaline phosphatase activity in vitro, Calcif. Tissue Int. 64 (1999) 163–172. [13] R.T. Franceschi, B.S. Iyer, Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells, J. Bone Miner. Res. 7 (1992) 235–246. [14] F.J. Zhang, G.X. Cheng, X.G. Ying, Emulsion and macromolecules templated alginate based polymer microspheres, Reactive Funct. Polym. 66 (2006) 712–719. [15] W.R. Gombotz, S.F. Wee, Protein release from alginate matrices, Adv. Drug Deliv. Rev. 31 (1998) 267–285. [16] C. Liu, Z. Han, J.T. Czernuska, Gradient collagen/nanohydroxyapatite composite scaffold: development and characterization, Acta Biomater. 5 (2) (2009) 661–669. [17] M.G. Blaine, Experimental observations on absorbable alginate products in surgery, Ann. Surg. 125 (1947) 102–114. [18] J.A. Wasastjerna, On the radii of ions, Commun. Phys.-Math. Soc. Sci. Fenn. 1 (38) (1923) 1–25.
CERAMICS INTERNATIONAL
Ceramics International 40 (2014) 11369–11375 www.elsevier.com/locate/ceramint
Short communication
Zinc alginate–hydroxyapatite composite microspheres for bone repair Renan Costa Cuozzoa, Maria Helena Miguez da Rocha Leãob, Luciano de Andrade Gobboc, Daniel Navarro da Rochaa, Nadia Mohammed Elmassalami Ayada, Willian Trindadea, Andrea Machado Costaa, Marcelo Henrique Prado da Silvaa,n b
a Instituto Militar de Engenharia—IME, Rio de Janeiro, RJ, Brazil PANalytical Spectris do Brasil Instr. Eletrônicos Ltda., São Paulo, SP, Brazil c Universidade Federal do Rio de Janeiro—UFRJ, Rio de Janeiro, RJ, Brazil
Received 29 August 2013; received in revised form 22 February 2014; accepted 24 February 2014 Available online 12 March 2014
Abstract This paper presents a novel method for producing composite and ceramic microspheres containing zinc for bone regeneration. The microspheres were prepared from a mixture of sodium alginate and hydroxyapatite that was extruded into a zinc nitrate solution. After 24 h in the solution, Na þ exchange with Zn2 þ ions produced zinc alginate–hydroxyapatite composite microspheres. After calcination, Zn-containing betaTCP ceramic microspheres were produced. & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: B. Composite; Alginate; Biospheres; Hydroxyapatite; Zinc
1. Introduction The gold standard for bone repair of damaged or lost tissue sites is autologous bone, i.e., from the same individual. However, the limited amount of available tissue, along with the inconvenience of creating another defect, suggests the need for other classes of grafts. Xenografts and allografts are options, but since these grafts come from other individuals, the question of infection arises. In this aspect, artificial grafts, i.e. can be a good alternative to biomaterials of biological origin [1], since they minimize the risk of bacterial and viral contamination, and are commercially available [2].
n
Corresponding author. E-mail addresses: [email protected] (R. Costa Cuozzo), [email protected] (M. Helena Miguez da Rocha Leão), [email protected] (L. de Andrade Gobbo), [email protected] (D. Navarro da Rocha), [email protected] (N. Mohammed Elmassalami Ayad), [email protected] (W. Trindade), [email protected] (A. Machado Costa), [email protected] (M. Henrique Prado da Silva). http://dx.doi.org/10.1016/j.ceramint.2014.02.107 0272-8842 & 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Although hydroxyapatite shows composition and structure similar to those of biological apatites, bioactivity and bio-resorption rate can be relatively limited [3]. Several studies have focused on cationic and anionic substitutions, aiming to mimic biological apatite properties. These mimicked biomaterials are more similar to human bone than pure and stoichiometric hydroxyapatite, Ca10 (PO4)6(OH)2 [4,5]. Ionic substitutions in hydroxyapatite structure induce changes in morphology, crystallinity, thermal stability, and resorption rate. Several ions present in the mineral phase of bone can be used in experimental studies on apatite synthesis [6]. Zinc is a relevant metallic element in the human body, since it is essential for bone structure and metabolism [7]. Mineral bone presents the greatest amount of this element, about 160– 300 ppm [8]. Zinc is important in cell differentiation, growth and metabolism of proteins, carbohydrates and lipids. It also has a role on the hormonal structure and genetic transcription, and participates in the synthesis and degradation of nucleic acids, carbohydrates, lipids, and proteins, and many other vital reactions. Zinc deficiency can lower bone metabolism, reducing growth, weight and development, affecting bone tissue health [9,10]. Studies have shown the role of zinc in
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R. Costa Cuozzo et al. / Ceramics International 40 (2014) 11369–11375
stimulating bone growth and mineralization in vivo and in vitro, and decreasing bone resorption, since it inhibits osteoclasts activity and stimulates alkaline phosphatase activity [11,12]. Besides, Yamagushi et al. (1987) proposed that zinc stimulated collagen formation in rat calvaria. In other studies [13], zinc showed to stimulate proliferation and cell differentiation as well as synthesis of bone-growth proteins, such as osteocalcin, IGF-1, TGR-f and aminoacyl-tRNA. Alginate is an anionic polysaccharide, which has a linear structure formed by glycosidic units of β-D-mannuronic acid and α-L-guluronic [14]. It can perform many exchanges in the presence of divalent ions by a cross-linking process. These exchanges occur between sodium ions and the divalent ions, and gelling the alginate solution. When sodium ion from guluronic acid exchanges with divalent cations, a structure known as “egg box” is created by cross-linking, which leads to the possibility of incorporating or entrapping desired substances. Divalent cations bind to blocks of α-L-guluronic acid, enabling the association with more than 20 monomers. Each chain of alginate may dimerize to create links with many other chains, resulting in a reticulated gel [15]. Due to this gelling property, alginate can be used as a entrapping matrix for biological molecules such as proteins, cells, and drugs. In the surgical field, it can be used in the repair of cellular tissues, and as treatment for restricting capillaries bleeding and skin burns [16]. Tissue engineering aims to develop three-dimensional structural biomaterials to be used as a physical support for cells. These scaffolds should also stimulate cell migration and differentiation [17]. In vitro culture on tissue engineering scaffolds is supposed to produce a set of differentiated cells reproducing the tissue to be reconstructed. This strategy represents one step towards the transition from restorative to regenerative medicine.
2. Materials and methods 2.1. HA Production Hydroxyapatite was produced and characterized in the Ceramics Laboratory of the Military Institute of Engineering —IME (Rio de Janeiro, Brazil). The synthesis was done using an aqueous precipitation method with start solution composed by a 0.5 M calcium hydroxide [Ca(OH)2] (Merck, Darmstadt, Germany) suspension, 1 M lactic acid (C3H6O3) (Vetec, Rio de Janeiro, Brazil) and 0.3 M ortho-phosphoric acid (H3PO4) (Merck, Darmstadt, Germany). After homogenization, the solution pH was adjusted up to 12 by the addition of 1 M KOH solution. The resulting precipitates were aged for 24 h and the solution was then filtered under negative pressure by using a vacuum pump system (Edwards, Neuberger, Germany). The filtered cake was resuspended in ultrapure water in order to remove remaining KOH, up to pH neutralization. The filtrate was lyophilized (Freezone 1—Labconco, USA) for 48 h in the Biomaterials Laboratory of the Brazilian Centre for
Research in Physics—CBPF (Rio de Janeiro, Brazil). Then the powders were de-agglomerated and sieved. 2.2. Spheres production and characterization A solution of 1% sodium alginate (SigmaAldrich) in ultrapure water was prepared by mixing in magnetic stirrer under a heat plate (Warmer–Quimis, São Paulo, Brazil), until the formation of a homogeneous paste without the presence of visible particles. Hydroxyapatite was weighed in a ratio of 1:15 alginate/hydroxyapatite. HA was added to the alginate solution and mixed until a homogeneous slurry was formed. The mixture was then extruded with the aid of a 10 ml disposable syringe (BD Plastipak) with 0.7 mm diameter needle (BD Precisionglide) into two different solutions: 0.3 M zinc nitrate (Vetec, Rio de Janeiro) and 0.3 M calcium chloride (Vetec, Rio de Janeiro). The spheres were immediately produced as the drops came into both solutions due to ion exchange. In contact with zinc nitrate and calcium chloride solutions, sodium alginate gelled due to exchanges with sodium ions present in the slurry, allowing the formation of Zn-alginate–HA and Ca-alginate– HA composite biospheres. The spheres remained in the solution for 24 h in order to allow complete ion exchange. The spheres were then removed from the solution, washed three times with ultrapure water and placed pel on a paper filter, where they remained for 24 h until complete drying. The samples were heat-treated at 900 1C and 1250 1C. The investigation of the phases present in the samples was performed in the X-ray Diffraction Laboratory of the Brazilian Centre for Research in Physics (CBPF), in a brand PANalytical diffractometer, model X'Pert PRO MPD, operating with a copper source, current 40 mA and voltage of 40 kV. The scan was performed in the range 51 o 2θ o 801, with a collecting step of 1 s. The phases identification was performed with the program X'Pert High Score Plus. The percentage of phases at different temperatures was assessed by Rietveld refinement analyses. The biospheres morphology was evaluated by scanning electron microscopy (SEM) on a field emission gun SEM (FEG–SEM) FEI Quanta 250FEG with energy-dispersive X-ray spectroscopy (EDS) to identify the chemical elements present in the biospheres. The samples were coated with gold, deposited by a sputter Emitech K550X at a current of 50 mA for 2 min. The spheres were measured under 100 magnification, by SEM. Diameters of spheres were measured before and after heat treatment and mean values with standard deviations were obtained for five spheres on each condition. BET analyses were performed in both samples, calcium and zinc, calcined at 900 1C. Specific area and mean diameter measurements were determined, in desorption, by an automatic physical adsorption analyzer (Micrometrics, model ASAP 2010). 3. Results and discussion The XRD analysis of the HA powder identified the presence of a poorly crystalline hydroxyapatite (Fig. 1). The XRD
R. Costa Cuozzo et al. / Ceramics International 40 (2014) 11369–11375
patterns shown in Fig. 1 also shows that HA is the only present phase in the composite biospheres after ion exchange in calcium chloride and zinc nitride solutions. After calcination at 900 1C, spheres showed higher crystallinity when compared to the corresponding green samples, as shown in Figs. 2 and 3. However, the zinc alginate spheres showed the presence of β-TCP as the major phase (Fig. 3), while HA was maintained as the only phase in the Ca-alginate spheres. In fact, Rietveld refinement showed 100 wt% HA in
Fig. 1. XRD patterns confirming hydroxyapatite as the only phase present in HA green powder in Ca-alginate–HA and in Zn-alginate–HA biospheres.
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these samples (Fig. 2). The Zn-alginate spheres calcined at 900 1C showed Witlockita (β-TCP), and zincite (zinc oxide) phases. These XRD results strongly suggest the role of Zn as a substitutional element in the HA lattice. The excess of zinc present in the sample may account for the presence of zinc oxide. Rietveld refinement indicated 86.9 wt% whitlockite and 13.1 wt% ZnO in these samples. The Ca-alginate spheres sintered at 1250 1C were composed mainly by hydroxyapatite (80.6 wt%), CaO (1.4 wt%) and β-TCP (18 wt%) phases (Fig. 4). The biospheres produced in zinc nitrate and sintered at 1250 1C showed the formation of β-TCP as the main phase (85.6 wt%) and ZnO (14.4 wt%). The excess of zinc provided the formation of ZnO, as shown in the XRD pattern of Fig. 5. Table 1 shows the resulting mean diameters of the spheres before (Do) and after (Df) heat treatment with respective standard deviations. After heat treatment, the spheres underwent a shrinkage and this was assessed by SEM–FEG. Diameter measurements were performed, as shown in Fig. 6. The spheres produced in the zinc nitrate solution were smaller than calcium alginate ones. This characteristic was observed immediately after dripping the zinc nitrate solution. This finding was attributed to the zinc ionic radius (74 pm), smaller than the calcium ionic radius (100 pm) in a single coordination number [18]. The spheres dripped in a zinc nitrate solution have a more compact “egg box” structure than that created by calcium alginate. Thus, small zinc ions occupy a smaller volume allowing a closer approximation to the HA particles. In addition, zinc has an affinity for guluronic and manuronic acids, whereas calcium binds only to guluronic acid chains. This more
Fig. 2. XRD pattern of the Ca-alginate–HA spheres calcined at 900 1C showing HA as the only present phase.
Fig. 3. XRD pattern of the Zn-alginate–HA spheres calcined at 900 1C, showing the presence of β-TCP as the major phase and ZnO.
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Fig. 4. XRD pattern of Ca-alginate samples sintered at 1250 1C, showing the presence of hydroxyapatite as the major phase, and β-TCP and CaO minor phases.
Fig. 5. XRD pattern of Zn-alginate samples sintered at 1250 1C, showing the presence of β-TCP as the major phase and ZnO minor phase.
general affinity of zinc also may account for the higher retraction of zinc alginate spheres. By the SEM–FEG analyses of the samples, some morphological differences between zinc and calcium spheres could be observed and the first one refers to particles morphology. Calcium spheres calcined at 900 1C showed aggregates of fine particles, as shown in Fig. 7a, whereas calcined zinc spheres presented a more homogeneous distribution of bigger particles (Fig. 7b). After sintering at 1250 1C, there was no marked difference between the grain sizes of hydroxyapatite (Fig. 8a) and β-TCP (Fig. 8b). However, the β-TCP grains were strongly bound, suggesting complete sintering. In addition, an intergranular phase was observed, suggesting that the sintering mechanism was liquid-phase sintering. EDS analyses on the grains of Fig. 8a and b are shown in Fig. 8c and d, respectively. Fig. 8c shows the EDS result of a square area encompassing several grains, whereas Fig. 8d shows the result of an analysis performed on the intergranular phase, confirming the presence of zinc. This findings agree with the Rietveld analysis that determined the presence of 11.5 wt% ZnO in these samples. Fig. 9 shows, in a higher magnification, the grain boundary region from where EDS analyses were performed. BET analyses in the spheres calcined at 900 1C confirmed the SEM findings: Zn spheres showed smaller mean pore diameters and porosity when compared to HA spheres, as shown in Table 2. HA spheres showed higher specific area, 11.5470.08 m2/g, when
Table 1 Mean diameter and standard deviation of the biospheres before (Do) and after (Df) heat treatment. HA_CaCl2_900 HA_Zn_900 HA_CaCl2_1250 HA_Zn_1250
Mean diameter (mm) Standard deviation
Do
Df
Do
Df
Do
Df
Do
Df
1.44
1.22
1.2
1.13
1.53
0.97
1.21
0.68
0.07
0.07
0.07
0.04
0.07
0.07
0.08
0.04
compared to Zn spheres, 6.6970.01m2/g. Specific area, porosity and mean pore diameter results agree with SEM observations, that revealed a more homogeneous pore and particle size for the Zn spheres, when compared to HA. In fact, experimental data showed that both spheres showed macro- and meso-pores. However, the curve obtained for the HA samples is closer to the macropores fitting, according to equation p/v(p0 p) p/p0, whose slope is (c 1)/vmc, where
v is the total adsorbed gas volume; vm is the adsorbed gas volume when the solid surface is fully coated by a monolayer; c is the BET constant;
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Fig. 6. Spheres shrinkage observed by SEM–FEG: (a) Ca-alginate–HA calcined at 900 1C, (b) Zn-alginate–HA calcined at 900 1C, (c) Ca-alginate–HA sintered at 1250 1C, and (d) Zn-alginate–HA sintered at 1250 1C.
Fig. 7. SEM micrographs of the samples calcined at 900 1C: (a) Ca-alginate–HA and (b) Zn-alginate–HA.
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Fig. 8. SEM micrographs and EDS spectra of the samples sintered at 1250 1C: (a) hydroxyapatite grains of Ca-alginate–HA sample, (b) β–TCP grains of Znalginate–HA, (c) EDS analysis of grains on Ca-alginate–HA sample and (d) EDS analysis of the intergranular phase of Zn-alginate–HA sample, showing Zn-rich phase on the grain boundaries.
Table 2 BET results for the spheres calcined at 900 1C. Sample
Specific area (m2/g)
Mean pore diameter (̊)
Pore volume (cm3/g)
HA Zn–HA
11.5470.08 6.6970.01
291.87 69.11
0.35 0.008
p is the measured pressure in the equilibrium; p0 is the initial pressure of the system.
4. Conclusion
Fig. 9. Detailed location from where EDS shown in Fig. 8d is performed.
The preparation of hydroxyapatite–alginate spheres in a solution rich in zinc ions produced Zn-alginate–hydroxyapatite composite
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spheres. During calcinations, zinc ions entered the hydroxyapatite structure and induced its decomposition to β-TCP. Zinc induced the formation of smaller biospheres when compared to the ones produced in the calcium ions solution. This finding is associated with the smaller zinc atomic radius and also with the fact that zinc has affinity for both guluronic and manuronic acids, whereas calcium binds only to guluronic acid chains. This broader affinity favors the higher retraction of zinc alginate spheres. Acknowledgments The authors thank CAPES, CNPq and the institutions IME and CBPF for their cooperation and support for the paper. References [1] D. Kaigler, D. Mooney, Tissue engineering‘s impact on dentistry, J. Dent. Educ. 65 (5) (2001) 456–462. [2] D.F. Williams, Definitions in Biomaterials, Elsevier, New York, 1987. [3] L.L. Hench, Biomaterials: a forecast for the future, Biomaterials 19 (16) (1998) 1419–1423. [4] M. Vallet-Regi, D. Arcos, Silicon substituted hydroxyapatites, a method to upgrade calcium phosphate based implants, J. Mater. Chem. 15 (15) (2005) 1509–1516. [5] D.N. Da Rocha, L.A. Gobbo, M.H.P. Da Silva, Production and characterization of niobate apatite, J. Mater. Res. Technol. 2 (1) (2013) 24–29. [6] M. Palard, E. Champion, S. Foucaud, Synthesis of silicated hydroxyapatite Ca10(PO4)6 x(SiO4)x(OH)2 x, J. Solid State Chem. 181 (2008) 1950–1960.
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