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Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone
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CERAMICS INTERNATIONAL
Ceramics International ] (]]]]) ]]]–]]] www.elsevier.com/locate/ceramint
Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone S.M. Nagaa,n, H.F. El-Maghrabya, E.M. Mahmouda, M.S. Talaatb, A.M. Ibrhimc a
National Research Centre, Ceramics Department, 12622 El-Bohouth Street, Dokki, Cairo, Egypt b Ain Shams University, Physics Department, Cairo Egypt c Ain Shams University, College of Women (Arts, Science and Education) Physics Department, Cairo Egypt Received 17 July 2015; received in revised form 7 August 2015; accepted 12 August 2015
Abstract The present study aims at the preparation of highly porous 3D scaffolds from the biological resource; fish bone skeletons. To the best of the authors' knowledge, few works were reported for preparation of highly porous scaffolds from biogenic hydroxyapatite. The preparation method includes two essential steps: the first is the extraction of pure hydroxyapatite powder having an average particle size of 50–80 nm from fish bone skeletons. The second is the formation of highly porous hydroxyapatite scaffolds prepared from fish bone skeletons via polymeric sponge method. Such scaffolds are highly bioactive as they are able to attract calcium and phosphorous ions to the scaffold surface in a very short time; less than one hour. The produced hydroxyapatite powder was investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR), while the formed porous scaffolds were evaluated for their physical and chemical properties in addition to their pore size, microstructure and bioactivity. The results revealed that the porosity of the obtained 3D scaffolds is 85 70.4 and the median pore diameter ranging between about 1 and 3 μm. The compressive and bending strengths of the scaffolds are 0.137 0.007 MPa and 1.72 7 0.02 MPa respectively, which is very near to the strength of trabecular bone. & 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: Fish bone; Biogenic hydroxyapatite; Porous scaffolds; In vitro
1. Introduction Ideal scaffolds must mimic the extracellular matrix of the tissue, which it will replace. The scaffolds have to act as a 3D template onto which cells can attach, multiply, migrate and function. The most important criteria for ideal scaffolds used for bone regeneration are: biocompatibility, interconnected micro porous network, the surface texture that promotes cell adhesion and sufficient mechanical properties [1–3]. Various microstructural features such as, mean size and total volume of pores; pore size must be between 100 and 400 mm; size and amount of interconnections and walls microstructure are the main factors that influence the quality of both the scaffold and the bone integration [4–6]. Many authors tackled the preparation of porous scaffold n
Corresponding author. Fax: þ20 2 33371718. E-mail address: [email protected] (S.M. Naga).
and the influence of preparation methods on the properties of the prepared scaffolds [7–12]. Owing to its remarkable biocompatibility, hydroxyapatite (HA) in particular biological apatites is used for biomedical applications. Natural apatites contain cationic (i.e. Na þ , Mg2 þ , K þ , Sr2 þ , Zn2 þ , Ba2 þ , Al3 þ ….) or anionic (e.g. F , Cl , SiO24 and CO23 ) or both of them [13]. Such composition is close to that of bone mineral, which makes biological apatites an attractive material for biomedical applications [14]. Biological hydroxyapatite can be easily obtained from natural sources such as bovine, cattle bones, pig bones and fish bones. Thermal treatment method, the traditional route for obtaining hydroxyapatite from fish bone was used by many groups [15–20]. The effect of the calcination temperature and time on the isolation and characterization of hydroxyapatite produced from fish bone was studied by Lima et al. [21] and Venkatesan and Kim [22]. Their results revealed that the
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Please cite this article as: S.M. Naga, et al., Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone, Ceramics International (2015), http://dx.doi.org/10.1016/j.ceramint.2015.08.057
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optimum calcination temperature is between 600 and 900 1C. It also, showed that the purity of HA increases with the increase in the calcination temperature as well as its grain size. Calcination time has an influence on the grain size of the produced HA powder. According to their study, the optimum calcination temperature and calcination time are 900 1C and 8 h respectively. Alkaline hydrothermal analysis method was used by Venkatesan et al. [23], Kongsri et al. [24] and Rocha et al. [25] to extract HA from fish bone. They claimed that via such method it is easy to obtain nano-scale HA powder. The obtained nano powder is thermally stable up to 1350 1C. There are several techniques for synthesizing nano-particle HA, e.g. microwave irradiation [26], laser ablation in de-ionized water [27], and enzymatic hydrolysis [28]. The objective of the present study is the extraction of HA powder from inexpensive and eco-friendly source by thermal treatment technique. The preparation of highly porous 3D scaffolds with relatively moderate mechanical strength via the polymeric sponge method is another goal of the study. The invitro tests were conducted to address these scaffolds on bone tissue formation. The utilization of fish bones as a biomaterial for biomedical applications will add a great value to such biowaste material. 2. Materials and methods 2.1. Materials Materials used in the present study are; fish bone skeleton (Thickness Perch), Polyvinyl alcohol, PVA (M.Wt. ¼ 125,000) Product no. 39791, BOISAR 401506, Batch no.1197/297/ 091113,S.d.AfINE-CHEM Ltd., Doflex, and high-density polyethylene sponge. 2.2. Methods 2.2.1. Extraction of hydroxyapatite powder from fish bone skeletons The collected fish bone skeletons (Thickness Perch) were cleaned by boiling with water for 8 h; to remove all organic matter; washed by distilled water, then dried at temperature of 105 1C over three nights. The dried bone skeletons were stored frozen till the calcination step. The calcination was carried out at temperatures of 800, 850, 900, 950, 1000 and 1050 1C with a heating rate of 10 1C/min for 3 h. The calcined bone was grinded using porcelain mortar and pestle [29]. 2.2.2. Formation of highly porous hydroxyapatite scaffolds using polymeric sponge method A set of cubic specimens with dimensions of 1 cmx1 cmx1 cm were cut from high-density polyethylene sponge. HA slurry was prepared by dissolving 1.5 g of PVA in 28.5 g of distilled water, and then 0.25 g of Doflex and 20 g of HA (calcined at 900 1C) were added to the mixture. The mixture was vigorously stirred for 1 h to form suspended slurry. The sponge specimens were immersed in the HA slurry under vacuum for 3 h to form porous HA scaffolds after firing. The soaked
samples were dried slowly at 50 1C for 6 h, then at 80 1C for 6 h and finally at 110 1C overnight. To accelerate the oxidation of the formed carbon produced by the sponge fiber and to sweep the gases formed, the samples were fired up to 600 1C under static air. A slow heating rate was adopted at low temperature to prevent bodies cracking during the burnout process, i.e., a rate of 1 1C/min from room temperature to 600 1C followed by a higher rate of 5 1C/min to the sintering temperature. The samples were fired at 1250 1C with a soaking time of 3 h. Fig. 1 shows the morphology of the prepared porous scaffolds. 2.2.3. Characterization Bulk density and apparent porosity of the fired specimens were evaluated using the Archimedes method (ASTM C-20). The phase composition of the samples was evaluated by X-ray diffraction (XRD) using monochromated Cu-Kα radiation over an angular range of 5–701, a step size of 0.02, a scan speed of 4 1 m 1 at a 40 kV voltage and a 30 mA current (D500, Siemens, Mannheim, Germany). The microstructure of gold plated samples was investigated via scanning electron microscopy (SEM; Model XL 30, Philips, Eindhoven, Netherlands). Bending strength was measured using a three-point bending test universal testing machine (Model 4204, Instron Corp., Danvers, MA) at a crosshead speed of 0.5 mm/min. At least 10 specimens with nominal dimensions of 3 cm 1 cm 1 cm were measured for each data point. Calcined scaffolds compressive strength was measured using LR/OK plus 10 KN (2248 16F apparatus) with a crosshead travel of 950 mm and speed range of 0.01 mm/min. Mercury porosimetry (Model pore sizer 9320, Micromerities, USA) was used to measure the samples average pore size. The apparent density of HA powder was determined by Quantachrome instrument (Corporation Upyc 1200 eV 5.03). The grain size of calcined HA powder was investigated by transmission electron microscope (TEM), JEOL, JEM-2100-HR, Japan. The HA powder sample was dispersed in ethyl alcohol by ultrasonic for 15 min before testing on a carbon grid. FTIR analysis of HA powder was carried out using FT/IR6100 Fourier transform Infrared Spectrometer from JASCO. Thermal analysis of as prepared HA powder was carried out
Fig. 1. Prepared scaffolds morphology.
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with Q-600 thermo gravimetric analyzer (sample heated from room temperature up to 1000 1C at heating rate of 5 1C). Simulated body fluid (SBF) was prepared using the method described by Kokubo [30]. The HA scaffolds were immersed in the SBF and kept at 37 1C for four weeks. Aliuots of 1.5 cc were collected from the solution every day to be analyzed for Ca2 þ and PO34 . Determination of variations in the Ca2 þ and PO34 ions was carried out using Inductively Coupled Plasma Spectrometer (ICP), Model Ultima-2, JY 2000-2, France. 3. Results and discussion 3.1. Characterization of the hydroxyapatite powder XRD patterns of the HA powders obtained at different firing temperatures are shown in Fig. 2. Bones transformed to a pure and well crystalline HA phase [JCPDS (76-0694) standard data] when they heat-treated at 8001 up to 1050 1C. The intensity and sharpness of HA peaks increase with the increase of the calcination temperature up to 950 1C, which indicates that the crystallinity is enhanced with the increase in calcination temperature. The figure also, indicates that the HA phase is stable up to 1050 1C as whitelockite phase is completely absent. TEM micrograph for fish bones calcined at 900 1C for 3 h is given in Fig. 3. It shows many agglomerated particles together with fine particles with a particle size ranging between 50 and 80 nm. The presented particles are of sphere-shape. The obtained particle size is less than or close to that obtained by different authors [31,32]. The FT-IR spectra of the fish bone calcined at 900 1C is represented in Fig. 4. The bands present at 603 and 569 cm 1are corresponding to the bending mode of phosphate ions (v4). While the bands at 1048 and 1089 cm 1 are corresponding to (v3) stretching vibration mode of the phosphate ion [16]. The hydroxyl group (–OH) present in the lattice structure is assigned by the bands at 3443, 3570 and 631 cm 1 [28]. The band at 1624 cm 1 is the bonding mode band of absorbed H2O. The abovementioned band together with highenergy region bands of O–H group suggest that the intermolecular hydrogen bonding of water molecules is not proper [17]. 700
. .. . . . ... ... . . ....... . .. .
Hydroxyapatite
Intenstiy (a.u)
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1050 c
1000 c
400 300
950 c
200 900 c
100
850 c 800 c
0
10
20
30
40
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70
2θ degree
Fig. 2. XRD patterns for fish bone powder heated at 8001 up to 1050 1C for 3 h.
Fig. 3. TEM micrograph for fish bone skeletons calcined at 900 1C for 3 h.
Fig. 4. FT-IR spectra of fish bone skeletons calcined at 900 1C for 3 h.
Thermal behavior of the fish bone powder is shown in Fig. 5. The thermogravimetric degradation is observed in three steps. The first step occurred between room temperature and E350 1C (5.56%). It is due to the evaporation of entrapped water. The second step is observed between 350 1C and 550 1C (0.91%). It is resulted from the decomposition of organic material in the fish bone. The final step is between 500 1C and 1000 1C (2.15%). It could be due to the dehydroxylation and the formation of (PO4) 3 ions [33]. The total weight loss at 1000 1C is 8.15%. On the other hand the differential curve shows two broad exothermic peaks at 231.10 1C and 406.60 1C. These two peaks accompanied the first and second steps of the weight loss respectively. Fig. 6a and b shows the SEM micrograph of both the crude fish bone and the fish bone calcined at 900 1C respectively. It is easy to observe that crude fish bone is composed of oriented aggregates of HA crystals regularly arranged within the collagen. The SEM micrograph of calcined fish bone; Fig. 6b shows a peculiar microstructure pseudomorphous to the native fish bone structure.
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Fig. 5. DTA and TGA analysis of fish bone skeletons calcined at 900 1C for 3 h.
Fig. 6. (a). SEM micrograph of the crude fish bone. (b). SEM micrograph of fish bone calcined at 900 1C for 3 h.
Such phenomenon is occurred because the sintering of fish bone takes place through the burning-out of the collagen matrix; accordingly the resultant structure is able to form crystallized particles replicate of the raw fish bone aggregates. 3.2. Characterization of the HA scaffolds XRD pattern of the HA scaffolds calcined at 1250 1C for 3 h showed a well- crystallized hydroxyapatite, Fig. 7. It indicates that HA phase is thermally stable up to 1250 1C. The prepared scaffolds are of highly porous nature, (apparent porosity ¼ 85 7 0.4%). Their bulk density is 0.577 0.01 g/ cm3, while their specific surface area is 178.47 m2/g, Table 1. Fig. 8 indicates the homogenous distribution of the macropores of the calcined scaffolds, which maintains the initial
sponge structure. Kim and Mooney [34] reported that porous 3D scaffolds with interconnected pore network must be used for tissue engineering, so that new tissue can be grown and the nutrients and metabolic wastes flow can be transported. It worthy to conclude that the HA scaffolds calcined at 1250 1C for 3 h are satisfying the required demands needed for biocompatible scaffolds. To affirm that the obtained product is hydroxyapatite EDS analysis (Fig. 9) has been performed. The results indicate that the formed film is composed of calcium phosphate, and the calcium to phosphorous molar ratio is approximately 1.67, which lies within the acceptable range for the hydroxyapatite. Fig. 10 illustrates the FT-IR spectra of the calcined HA scaffolds. It is easy to recognize the characteristic bands of HA assigned to the (PO4)3 (1040, 600 and 566.1 cm 1 -v4, 469.7 cm 1 -v2, and E 1000 cm 1 -v1).
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700 600
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500 400 300 200 100 0 -100
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2θ degree
Fig. 7. XRD pattern of the HA scaffolds calcined at 1250 1C for 3 h.
Fig. 9. EDS spectra of the HA scaffold surface calcined at 1250 1C for 3 h.
Table 1 Compressive strength, bending strength, apparent porosity, bulk density and surface area of the porous HA scaffolds calcined at 1250 1C for 3 h.
0.137 0.007
Bending strength, MPa
95
Specific Apparent Bulk porosity, % density, g/ surface area, m2/g cm3
1.7270.467 8570.96%
0.577 0.04 178.47
absorbed H2O
90
T ( %)
Compressive strength, MPa
100
(CO3)
OH
-2
-
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80
75
OH
(PO4 ) 500
-
-3
(PO4 ) 1000
-3
1500
2000
2500
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3500
4000
-1
wave number [cm ]
Fig. 10. FT-IR spectra of the HA scaffolds calcined at 1250 1C for 3 h.
Fig. 8. SEM micrograph of the HA scaffolds surface.
OH group band is found as a small absorption band at 3569.7 cm 1. It is associated with the stretching mode of hydroxyl group. The band at 633 cm 1 is also assigned for the OH group [28]. The vibrational frequencies for (CO3)2 can be detected around 1400 and at 1630.9 cm 1 for v2 [25]. We believe that the carbonate group exists as the polymeric template residue. The compressive strength of the calcined HA scaffolds is 0.137 0.007 MPa, while their bending strength is 1.7270.02 MPa, Table 1. The low mechanical strength figures displayed by the HA scaffolds are due to the influence of the porosity on the mechanical properties [35]. Although the mechanical properties are seemed to be low, but they are sufficient to maintain the scaffolds
Ions concentrations (mg/l)
12 11
Ca
10
+3
P
9
+2
8 7 6 5 4 3 2 1 0 0
5
10
15
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Fig. 11. The mean value of Ca2 þ and P3 þ concentrations (mg/l) as a function of incubation time in SBF.
until the formed new bone support itself during the process of osteoblasts growth, proliferation and differentiation [35]. The prepared scaffolds compressive strength is 0.2370.007 MPa in comparison to the standard compressive strength of spongy bone (not the strut), which is in the range of (0.2 4 MPa) [36,37].
Please cite this article as: S.M. Naga, et al., Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone, Ceramics International (2015), http://dx.doi.org/10.1016/j.ceramint.2015.08.057
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Fig. 11 illustrates the mean value of Ca2þ and P3 þ concentrations (mg/l) as a function of immersion time in SBF. It shows that Ca 2 þ ions concentration decreases sharply at the first week. From the
Fig. 12. Thin film XRD pattern for hydroxyapatite scaffolds calcined at 1250 1C for 3 h after immersion in SBF for 4 weeks.
second week to the third week, the concentration-decreasing rate is reduced, and then it becomes constant. P3 þ ions behave similar to Ca 2þ ions, but their concentration decrease is higher. It is to be stated that Ca2þ and P3þ ions are consumed in the formation of apatite. The hydrated surfaces (OH)- of HA scaffolds induce the nucleation of the apatite. On the other hand, the increased ionic activity product of the apatite accelerates its nucleation [36,37]. Once the apatite nuclei are formed, they grow spontaneously by consuming the Ca2þ and P3 þ ions from SBF. XRD pattern of the HA scaffold surface; Fig. 12 confirms the deposition of a hydroxyapatite layer on the surface of the scaffolds. The indexed d- spacing of the XRD peaks was found to be very close to the values obtained from the JCPDS (76-0694) standard data. Fig. 13(a) and (b) indicates the difference between the HA scaffold surface before and after immersion in SBF for four weeks respectively. Fig. 13(b) depicts SEM image of calcium phosphate thick film deposited on the surface of the HA scaffolds. It shows a uniform microstructure with small globule–like grains distributed all over the scaffold surface. Meanwhile, EDS analysis (Fig. 13c) of the formed region indicated that it is composed of calcium phosphate thick layer.
Fig. 13. (a). SEM micrograph of HA scaffold before immersion in SBF for 4 weeks at magnification 2000.(b). SEM micrograph of HA scaffold after immersion in SBF for 4 weeks at magnification 2000. (c). EDS spectra of immersed HA scaffold surface, immersed in SBF for 4 weeks. Please cite this article as: S.M. Naga, et al., Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone, Ceramics International (2015), http://dx.doi.org/10.1016/j.ceramint.2015.08.057
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4. Conclusions 1- Porous hydroxyapatite scaffolds are prepared from biogenic hydroxyapatite powder and polymeric sponge template via heat treatment at 1250 1C for 3 h. 2- The calcined fish bone maintains the microstructural features of the crude fish bones. 3- In spite of the high porosity of the prepared scaffolds they possess mechanical properties sufficient for the application as bone graft materials. 4- Thin layer XRD examination of the calcium phosphate layer deposited on the surface of the scaffolds after immersing in the SBF showed a pure hydroxyapatite phase identical to the standard one. References [1] R.J. Jones, L.L. Hench, Regeneration of trabecular bone using porous ceramics, Curr. Opin. Sol. St. Mater. Sci. 7 (2003) 301–307. [2] T.M. Freyman, I.V. Yannas, L.J. Gibson, Cellular materials as porous scaffolds for tissue engineering, Prog. Mater. Sci. 46 (2001) 273–282. [3] L.L. Hench, Sol–gel materials for bioceramic applications, Curr. Opin. Sol. St. Mater. Sci. 2 (1997) 604–610. [4] F. Barrere, T.A. Mahmood, K. de Groot, C.A. van Blitter-Swijk, Advanced biomaterials for skeletal tissue regeneration: instructive and smart functions, Mater. Sci. Eng. R 59 (2008) 38–71. [5] D.W. Hutmacher, J.T. Schantz, C.X.F. Lam, K.C. Tan, T.C. Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective, J. Tissue Eng. Regen. Med. 1 (2007) 245–260. [6] K.A. Hing, Bioceramic bone graft substitution: influence of porosity and chemistry, Int. Appl. Ceram. Tech. 2 (2005) 184–199. [7] S. Jinawath, P. Sujaridworakum, Fabrication of porous calcium phosphates, Mater. Sci. Eng. C 22 (2002) 41–46. [8] X. Wang, H. Fan, Y. Xiao, X. Zhang, Fabrication and characterization of porous hydroxyapatite/β-tricalcium phosphate ceramics by microwave sintering, Mater. Lett. 60 (2006) 455–458. [9] S. Teixeira, M.A. Rodriguez, P. Pena, A.H. De Aza, S. De Aza, M.P. Ferraz, F.J. Monteiro, Physical characterization of hydroxyapatite porous scaffolds for tissue engineering, Mater. Sci. Eng. C 29 (5) (2009) 1510–1514. [10] M. Lombardi, P. Palmero, K. Haberko, W. Pyda, L. Montanaro, Processing of a natural hydroxyapatite powder: from powder optimization to porous bodies development, J. Eur. Ceram. Soc. 31 (2001) 2153–2518. [11] N. Monmaturapoj, C. Yatongchai, Influence of preparation method on hydroxyapatite porous scaffolds, Bull. Mater. Sci. 34 (7) (2011) 1733–1737. [12] H.F. El-Maghraby, M. Awaad, A. Al-Wassil, S.M. Naga, Preparation and characterization of highly porous ceramic substrate suitable for biomedical application, Interceram 61 (1-2) (2012) 43–46. [13] J. Elliott, Structure and chemistry of the apatites and other calcium orthophosphates, Elsevier Science, Amsterdam, 1994. [14] J.P. Lafon, E. Champion, D. B-Assollant, Processing of AB-type carbonated hydroxyapatite Ca 10 x (PO4) 6 x (CO3)x (OH)2 x 2y (CO3)y ceramics with controlled composition, J. Eur. Ceramic. Soc. 29 (2008) 139–147. [15] J. Zhou, X. Zhang, J. Chen, S. Zeng, K. Groot, High temperature characteristics of synthetic hydroxyapatite, J. Mater. Sci. Mater. Med. 4 (1993) 83–85. [16] M. Boutinguiza, J. Pou, R. Comesaña, F. Lusquiños, A. de Carlos, B. León, Biological hydroxyapatite obtained from fish bones, Mater. Sci. Eng. C 32 (2012) 478–486. [17] K. Prabakaran, S. Rajeswari, Development of hydroxyapatite from natural fish bone through heat treatment, Trends Biomater. Artif. Organs 20 (2006) 20–23.
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CERAMICS INTERNATIONAL
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Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone S.M. Nagaa,n, H.F. El-Maghrabya, E.M. Mahmouda, M.S. Talaatb, A.M. Ibrhimc a
National Research Centre, Ceramics Department, 12622 El-Bohouth Street, Dokki, Cairo, Egypt b Ain Shams University, Physics Department, Cairo Egypt c Ain Shams University, College of Women (Arts, Science and Education) Physics Department, Cairo Egypt Received 17 July 2015; received in revised form 7 August 2015; accepted 12 August 2015
Abstract The present study aims at the preparation of highly porous 3D scaffolds from the biological resource; fish bone skeletons. To the best of the authors' knowledge, few works were reported for preparation of highly porous scaffolds from biogenic hydroxyapatite. The preparation method includes two essential steps: the first is the extraction of pure hydroxyapatite powder having an average particle size of 50–80 nm from fish bone skeletons. The second is the formation of highly porous hydroxyapatite scaffolds prepared from fish bone skeletons via polymeric sponge method. Such scaffolds are highly bioactive as they are able to attract calcium and phosphorous ions to the scaffold surface in a very short time; less than one hour. The produced hydroxyapatite powder was investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR), while the formed porous scaffolds were evaluated for their physical and chemical properties in addition to their pore size, microstructure and bioactivity. The results revealed that the porosity of the obtained 3D scaffolds is 85 70.4 and the median pore diameter ranging between about 1 and 3 μm. The compressive and bending strengths of the scaffolds are 0.137 0.007 MPa and 1.72 7 0.02 MPa respectively, which is very near to the strength of trabecular bone. & 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Keywords: Fish bone; Biogenic hydroxyapatite; Porous scaffolds; In vitro
1. Introduction Ideal scaffolds must mimic the extracellular matrix of the tissue, which it will replace. The scaffolds have to act as a 3D template onto which cells can attach, multiply, migrate and function. The most important criteria for ideal scaffolds used for bone regeneration are: biocompatibility, interconnected micro porous network, the surface texture that promotes cell adhesion and sufficient mechanical properties [1–3]. Various microstructural features such as, mean size and total volume of pores; pore size must be between 100 and 400 mm; size and amount of interconnections and walls microstructure are the main factors that influence the quality of both the scaffold and the bone integration [4–6]. Many authors tackled the preparation of porous scaffold n
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and the influence of preparation methods on the properties of the prepared scaffolds [7–12]. Owing to its remarkable biocompatibility, hydroxyapatite (HA) in particular biological apatites is used for biomedical applications. Natural apatites contain cationic (i.e. Na þ , Mg2 þ , K þ , Sr2 þ , Zn2 þ , Ba2 þ , Al3 þ ….) or anionic (e.g. F , Cl , SiO24 and CO23 ) or both of them [13]. Such composition is close to that of bone mineral, which makes biological apatites an attractive material for biomedical applications [14]. Biological hydroxyapatite can be easily obtained from natural sources such as bovine, cattle bones, pig bones and fish bones. Thermal treatment method, the traditional route for obtaining hydroxyapatite from fish bone was used by many groups [15–20]. The effect of the calcination temperature and time on the isolation and characterization of hydroxyapatite produced from fish bone was studied by Lima et al. [21] and Venkatesan and Kim [22]. Their results revealed that the
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optimum calcination temperature is between 600 and 900 1C. It also, showed that the purity of HA increases with the increase in the calcination temperature as well as its grain size. Calcination time has an influence on the grain size of the produced HA powder. According to their study, the optimum calcination temperature and calcination time are 900 1C and 8 h respectively. Alkaline hydrothermal analysis method was used by Venkatesan et al. [23], Kongsri et al. [24] and Rocha et al. [25] to extract HA from fish bone. They claimed that via such method it is easy to obtain nano-scale HA powder. The obtained nano powder is thermally stable up to 1350 1C. There are several techniques for synthesizing nano-particle HA, e.g. microwave irradiation [26], laser ablation in de-ionized water [27], and enzymatic hydrolysis [28]. The objective of the present study is the extraction of HA powder from inexpensive and eco-friendly source by thermal treatment technique. The preparation of highly porous 3D scaffolds with relatively moderate mechanical strength via the polymeric sponge method is another goal of the study. The invitro tests were conducted to address these scaffolds on bone tissue formation. The utilization of fish bones as a biomaterial for biomedical applications will add a great value to such biowaste material. 2. Materials and methods 2.1. Materials Materials used in the present study are; fish bone skeleton (Thickness Perch), Polyvinyl alcohol, PVA (M.Wt. ¼ 125,000) Product no. 39791, BOISAR 401506, Batch no.1197/297/ 091113,S.d.AfINE-CHEM Ltd., Doflex, and high-density polyethylene sponge. 2.2. Methods 2.2.1. Extraction of hydroxyapatite powder from fish bone skeletons The collected fish bone skeletons (Thickness Perch) were cleaned by boiling with water for 8 h; to remove all organic matter; washed by distilled water, then dried at temperature of 105 1C over three nights. The dried bone skeletons were stored frozen till the calcination step. The calcination was carried out at temperatures of 800, 850, 900, 950, 1000 and 1050 1C with a heating rate of 10 1C/min for 3 h. The calcined bone was grinded using porcelain mortar and pestle [29]. 2.2.2. Formation of highly porous hydroxyapatite scaffolds using polymeric sponge method A set of cubic specimens with dimensions of 1 cmx1 cmx1 cm were cut from high-density polyethylene sponge. HA slurry was prepared by dissolving 1.5 g of PVA in 28.5 g of distilled water, and then 0.25 g of Doflex and 20 g of HA (calcined at 900 1C) were added to the mixture. The mixture was vigorously stirred for 1 h to form suspended slurry. The sponge specimens were immersed in the HA slurry under vacuum for 3 h to form porous HA scaffolds after firing. The soaked
samples were dried slowly at 50 1C for 6 h, then at 80 1C for 6 h and finally at 110 1C overnight. To accelerate the oxidation of the formed carbon produced by the sponge fiber and to sweep the gases formed, the samples were fired up to 600 1C under static air. A slow heating rate was adopted at low temperature to prevent bodies cracking during the burnout process, i.e., a rate of 1 1C/min from room temperature to 600 1C followed by a higher rate of 5 1C/min to the sintering temperature. The samples were fired at 1250 1C with a soaking time of 3 h. Fig. 1 shows the morphology of the prepared porous scaffolds. 2.2.3. Characterization Bulk density and apparent porosity of the fired specimens were evaluated using the Archimedes method (ASTM C-20). The phase composition of the samples was evaluated by X-ray diffraction (XRD) using monochromated Cu-Kα radiation over an angular range of 5–701, a step size of 0.02, a scan speed of 4 1 m 1 at a 40 kV voltage and a 30 mA current (D500, Siemens, Mannheim, Germany). The microstructure of gold plated samples was investigated via scanning electron microscopy (SEM; Model XL 30, Philips, Eindhoven, Netherlands). Bending strength was measured using a three-point bending test universal testing machine (Model 4204, Instron Corp., Danvers, MA) at a crosshead speed of 0.5 mm/min. At least 10 specimens with nominal dimensions of 3 cm 1 cm 1 cm were measured for each data point. Calcined scaffolds compressive strength was measured using LR/OK plus 10 KN (2248 16F apparatus) with a crosshead travel of 950 mm and speed range of 0.01 mm/min. Mercury porosimetry (Model pore sizer 9320, Micromerities, USA) was used to measure the samples average pore size. The apparent density of HA powder was determined by Quantachrome instrument (Corporation Upyc 1200 eV 5.03). The grain size of calcined HA powder was investigated by transmission electron microscope (TEM), JEOL, JEM-2100-HR, Japan. The HA powder sample was dispersed in ethyl alcohol by ultrasonic for 15 min before testing on a carbon grid. FTIR analysis of HA powder was carried out using FT/IR6100 Fourier transform Infrared Spectrometer from JASCO. Thermal analysis of as prepared HA powder was carried out
Fig. 1. Prepared scaffolds morphology.
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with Q-600 thermo gravimetric analyzer (sample heated from room temperature up to 1000 1C at heating rate of 5 1C). Simulated body fluid (SBF) was prepared using the method described by Kokubo [30]. The HA scaffolds were immersed in the SBF and kept at 37 1C for four weeks. Aliuots of 1.5 cc were collected from the solution every day to be analyzed for Ca2 þ and PO34 . Determination of variations in the Ca2 þ and PO34 ions was carried out using Inductively Coupled Plasma Spectrometer (ICP), Model Ultima-2, JY 2000-2, France. 3. Results and discussion 3.1. Characterization of the hydroxyapatite powder XRD patterns of the HA powders obtained at different firing temperatures are shown in Fig. 2. Bones transformed to a pure and well crystalline HA phase [JCPDS (76-0694) standard data] when they heat-treated at 8001 up to 1050 1C. The intensity and sharpness of HA peaks increase with the increase of the calcination temperature up to 950 1C, which indicates that the crystallinity is enhanced with the increase in calcination temperature. The figure also, indicates that the HA phase is stable up to 1050 1C as whitelockite phase is completely absent. TEM micrograph for fish bones calcined at 900 1C for 3 h is given in Fig. 3. It shows many agglomerated particles together with fine particles with a particle size ranging between 50 and 80 nm. The presented particles are of sphere-shape. The obtained particle size is less than or close to that obtained by different authors [31,32]. The FT-IR spectra of the fish bone calcined at 900 1C is represented in Fig. 4. The bands present at 603 and 569 cm 1are corresponding to the bending mode of phosphate ions (v4). While the bands at 1048 and 1089 cm 1 are corresponding to (v3) stretching vibration mode of the phosphate ion [16]. The hydroxyl group (–OH) present in the lattice structure is assigned by the bands at 3443, 3570 and 631 cm 1 [28]. The band at 1624 cm 1 is the bonding mode band of absorbed H2O. The abovementioned band together with highenergy region bands of O–H group suggest that the intermolecular hydrogen bonding of water molecules is not proper [17]. 700
. .. . . . ... ... . . ....... . .. .
Hydroxyapatite
Intenstiy (a.u)
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950 c
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Fig. 2. XRD patterns for fish bone powder heated at 8001 up to 1050 1C for 3 h.
Fig. 3. TEM micrograph for fish bone skeletons calcined at 900 1C for 3 h.
Fig. 4. FT-IR spectra of fish bone skeletons calcined at 900 1C for 3 h.
Thermal behavior of the fish bone powder is shown in Fig. 5. The thermogravimetric degradation is observed in three steps. The first step occurred between room temperature and E350 1C (5.56%). It is due to the evaporation of entrapped water. The second step is observed between 350 1C and 550 1C (0.91%). It is resulted from the decomposition of organic material in the fish bone. The final step is between 500 1C and 1000 1C (2.15%). It could be due to the dehydroxylation and the formation of (PO4) 3 ions [33]. The total weight loss at 1000 1C is 8.15%. On the other hand the differential curve shows two broad exothermic peaks at 231.10 1C and 406.60 1C. These two peaks accompanied the first and second steps of the weight loss respectively. Fig. 6a and b shows the SEM micrograph of both the crude fish bone and the fish bone calcined at 900 1C respectively. It is easy to observe that crude fish bone is composed of oriented aggregates of HA crystals regularly arranged within the collagen. The SEM micrograph of calcined fish bone; Fig. 6b shows a peculiar microstructure pseudomorphous to the native fish bone structure.
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Fig. 5. DTA and TGA analysis of fish bone skeletons calcined at 900 1C for 3 h.
Fig. 6. (a). SEM micrograph of the crude fish bone. (b). SEM micrograph of fish bone calcined at 900 1C for 3 h.
Such phenomenon is occurred because the sintering of fish bone takes place through the burning-out of the collagen matrix; accordingly the resultant structure is able to form crystallized particles replicate of the raw fish bone aggregates. 3.2. Characterization of the HA scaffolds XRD pattern of the HA scaffolds calcined at 1250 1C for 3 h showed a well- crystallized hydroxyapatite, Fig. 7. It indicates that HA phase is thermally stable up to 1250 1C. The prepared scaffolds are of highly porous nature, (apparent porosity ¼ 85 7 0.4%). Their bulk density is 0.577 0.01 g/ cm3, while their specific surface area is 178.47 m2/g, Table 1. Fig. 8 indicates the homogenous distribution of the macropores of the calcined scaffolds, which maintains the initial
sponge structure. Kim and Mooney [34] reported that porous 3D scaffolds with interconnected pore network must be used for tissue engineering, so that new tissue can be grown and the nutrients and metabolic wastes flow can be transported. It worthy to conclude that the HA scaffolds calcined at 1250 1C for 3 h are satisfying the required demands needed for biocompatible scaffolds. To affirm that the obtained product is hydroxyapatite EDS analysis (Fig. 9) has been performed. The results indicate that the formed film is composed of calcium phosphate, and the calcium to phosphorous molar ratio is approximately 1.67, which lies within the acceptable range for the hydroxyapatite. Fig. 10 illustrates the FT-IR spectra of the calcined HA scaffolds. It is easy to recognize the characteristic bands of HA assigned to the (PO4)3 (1040, 600 and 566.1 cm 1 -v4, 469.7 cm 1 -v2, and E 1000 cm 1 -v1).
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700 600
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Fig. 7. XRD pattern of the HA scaffolds calcined at 1250 1C for 3 h.
Fig. 9. EDS spectra of the HA scaffold surface calcined at 1250 1C for 3 h.
Table 1 Compressive strength, bending strength, apparent porosity, bulk density and surface area of the porous HA scaffolds calcined at 1250 1C for 3 h.
0.137 0.007
Bending strength, MPa
95
Specific Apparent Bulk porosity, % density, g/ surface area, m2/g cm3
1.7270.467 8570.96%
0.577 0.04 178.47
absorbed H2O
90
T ( %)
Compressive strength, MPa
100
(CO3)
OH
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OH
(PO4 ) 500
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-3
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wave number [cm ]
Fig. 10. FT-IR spectra of the HA scaffolds calcined at 1250 1C for 3 h.
Fig. 8. SEM micrograph of the HA scaffolds surface.
OH group band is found as a small absorption band at 3569.7 cm 1. It is associated with the stretching mode of hydroxyl group. The band at 633 cm 1 is also assigned for the OH group [28]. The vibrational frequencies for (CO3)2 can be detected around 1400 and at 1630.9 cm 1 for v2 [25]. We believe that the carbonate group exists as the polymeric template residue. The compressive strength of the calcined HA scaffolds is 0.137 0.007 MPa, while their bending strength is 1.7270.02 MPa, Table 1. The low mechanical strength figures displayed by the HA scaffolds are due to the influence of the porosity on the mechanical properties [35]. Although the mechanical properties are seemed to be low, but they are sufficient to maintain the scaffolds
Ions concentrations (mg/l)
12 11
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10
+3
P
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+2
8 7 6 5 4 3 2 1 0 0
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Fig. 11. The mean value of Ca2 þ and P3 þ concentrations (mg/l) as a function of incubation time in SBF.
until the formed new bone support itself during the process of osteoblasts growth, proliferation and differentiation [35]. The prepared scaffolds compressive strength is 0.2370.007 MPa in comparison to the standard compressive strength of spongy bone (not the strut), which is in the range of (0.2 4 MPa) [36,37].
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Fig. 11 illustrates the mean value of Ca2þ and P3 þ concentrations (mg/l) as a function of immersion time in SBF. It shows that Ca 2 þ ions concentration decreases sharply at the first week. From the
Fig. 12. Thin film XRD pattern for hydroxyapatite scaffolds calcined at 1250 1C for 3 h after immersion in SBF for 4 weeks.
second week to the third week, the concentration-decreasing rate is reduced, and then it becomes constant. P3 þ ions behave similar to Ca 2þ ions, but their concentration decrease is higher. It is to be stated that Ca2þ and P3þ ions are consumed in the formation of apatite. The hydrated surfaces (OH)- of HA scaffolds induce the nucleation of the apatite. On the other hand, the increased ionic activity product of the apatite accelerates its nucleation [36,37]. Once the apatite nuclei are formed, they grow spontaneously by consuming the Ca2þ and P3 þ ions from SBF. XRD pattern of the HA scaffold surface; Fig. 12 confirms the deposition of a hydroxyapatite layer on the surface of the scaffolds. The indexed d- spacing of the XRD peaks was found to be very close to the values obtained from the JCPDS (76-0694) standard data. Fig. 13(a) and (b) indicates the difference between the HA scaffold surface before and after immersion in SBF for four weeks respectively. Fig. 13(b) depicts SEM image of calcium phosphate thick film deposited on the surface of the HA scaffolds. It shows a uniform microstructure with small globule–like grains distributed all over the scaffold surface. Meanwhile, EDS analysis (Fig. 13c) of the formed region indicated that it is composed of calcium phosphate thick layer.
Fig. 13. (a). SEM micrograph of HA scaffold before immersion in SBF for 4 weeks at magnification 2000.(b). SEM micrograph of HA scaffold after immersion in SBF for 4 weeks at magnification 2000. (c). EDS spectra of immersed HA scaffold surface, immersed in SBF for 4 weeks. Please cite this article as: S.M. Naga, et al., Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone, Ceramics International (2015), http://dx.doi.org/10.1016/j.ceramint.2015.08.057
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Please cite this article as: S.M. Naga, et al., Preparation and characterization of highly porous ceramic scaffolds based on thermally treated fish bone, Ceramics International (2015), http://dx.doi.org/10.1016/j.ceramint.2015.08.057