The application of VP-ESEM in microstructure analysis of ceramic macroporous scaffolds for bone tissue engineering

Transcription

1 IOP Conference Series: Materials Science and Engineering The application of VP-ESEM in microstructure analysis of ceramic macroporous scaffolds for bone tissue engineering To cite this article: A M Janus and M Faryna 2012 IOP Conf. Ser.: Mater. Sci. Eng View the article online for updates and enhancements. Related content - Microstructure and mechanical properties of nanocrystalline Ni-Mo protective coatings A Bigos, E Beltowska-Lehman and P Indyka - Microstructural characterisation of electrodeposited coatings of metal matrix composite with alumina nanoparticles P Indyka, E Beltowska-Lehman and A Bigos - Orientation mapping applied to the study of ferroelectric ceramic K Berent, M Faryna and M Poska This content was downloaded from IP address on 09/03/2019 at 00:10

2 The application of VP-ESEM in microstructure analysis of ceramic macroporous scaffolds for bone tissue engineering A M Janus 1 and M Faryna Polish Academy of Sciences, Institute of Metallurgy and Materials Science, 25 ul. Reymonta, PL Krakow, Poland annamariajanus@yahoo.com Abstract. Macroporous ceramic scaffolds are widely used in the therapy of bone defects. Adaptation of such materials inside the human body depends strongly on their microstructure, particularly on the size of pores and the degree of pore interconnections. In the presented research a novel macroporous ceramic was prepared by means of the polymer sponge method from mixtures of hydroxyapatite of porcine origin and 30 or 50 wt% of biocompatible and bioresorbable phosphate glass. The materials were sintered at 700, 800, and 900 ºC and at 700 and 800 ºC, respectively. A unique set of VP-ESEM micrographs of ceramics microstructure was recorded. Based on direct measurements taken on these images, histograms representing macropore size distribution and distribution of size of macropore interconnections were plotted for each sample. The obtained data was used to determine average macropore size and average size of macropore interconnections for each material. The average macropore size varied from 532 to 761 µm while the average size of macropore interconnections ranged from 145 to 369 µm. 1. Introduction A development of materials designed for therapy of bone tissue is of great importance due to the fact that human organism is not able to heal larger bone defects [1], which leads to immobilisation that creates a substantial burden to the society. A large number of materials have been already commercialized and many of them are based on calcium phosphate ceramics [2]. Very often such materials are designed to act as scaffolds, i.e., porous structures of desired geometry which stimulate regeneration of bone, as in this form they can be used to treat load bearing sites which is impossible in case of dense phosphate ceramics [3]. At present, investigations of macroporous ceramics focus on improvement of biocompatibility and bioresorption. Enhancement of biocompatibility can be achieved through the use of materials of biological origin or synthesis of biomimetic materials [4-7] while resorbability can be influenced through the introduction of new phases [8]. Presented research was focused on preparation of resorbable calcium phosphate scaffolds from hydroxyapatite derived from porcine bones. The application of porcine hydroxyapatite was driven by the fact that this material showed better performance in in vitro biocompatibility tests compared to stoichiometric synthetic hydroxyapatites [9]. In order to improve resorption of the material, a biocompatibile and resorbable phosphate glass described in [10] was introduced into the material. 1 To whom any correspondence should be addressed. Published under licence by IOP Publishing Ltd 1

3 As microstructure is one of the key parameters determining the usefulness of macroporous ceramics in bone tissue therapy [11], it was thoroughly examined and characterized using scanning electron microscopy and image analysis techniques. 2. Experimental 2.1. Starting materials Hydroxyapatite of porcine origin (NAT) was prepared from long porcine bones by means of leaching out of organic matter with 4 M aqueous sodium hydroxide solution. Details of the procedure are given in [9]. Reference materials (synthetic hydroxyapatites) were purchased from Plasma Biotal Ltd. (Captal, UK) and Chema-Elektromet (HA Biocer, PL). As modifier specular phosphate was used (composition in mol%: 3 - SiO 2 ; 45 - P 2 O 5 ; 26 - CaO; 15 - Na 2 O; 7 - MgO; 4 - K 2 O). Commercially available polyurethane foam was used as the template for replication. Microstructure of the foam, macropore size distribution and distribution of size of macropore interconnections determined in accordance with procedure described in section 2.3 are shown in figure 1. Figure 1. VP-ESEM micrograph (a), pore size distribution (b) and distribution of pore interconnection size (c) of polyurethane sponge template Preparation of macroporous ceramics Macroporous ceramics were prepared using polymer sponge method. In the first step aqueous slurry of hydroxyapatite, glass and polyvinyl alcohol was prepared through mixing of solid components (36 wt%) with distilled water. In the next steps, shapes of dimensions 1.5 x 1.5 x 1.5 cm cut from polyurethane sponge were soaked with the slurry, the excess of water was removed by means of three-axial pressing and resulting material was dried at ambient temperature in air. The soaking was performed three times. Dried shapes were sintered in air at 700, 800 and 900 C (ceramics modified with 30 wt% of glass) and 700 and 800 C (ceramics modified with 50 wt% of glass). Detailed description of preparation of macroporous scaffolds can be found in [12] Microscopy examination and image analysis Microstructure of obtained macroporous scaffolds was examined using FEI scanning electron microscope (SEM) Quanta 3D FEG. Instead of sputter coating the samples were investigated in the environmental mode. Micrographs were recorded under the following operating conditions: accelerating voltage: 20 kv; spot: 4.0; pressure: 90 Pa; working distance (WD): 5.5 to 9.9 mm; detector: low vacuum secondary electron detector (LVSED); chamber gas: water vapour. 2

4 Recorded micrographs were used as a source of input data in analysis of size of macropores and size of macropore interconnections. For each material maximum Feret diameter was measured for 150 completely visible macropores (figure 2a - measurement on the left) and 150 macropore interconnections (figure 2a - measurement on the right). The measured Feret diameter was a diameter of the flat object visible in the micrograph and not of the actually existing pore or pore interconnection as it is impossible to take into account various angles at which object are situated with regards to the observation plane. This effect of non normal incidence was considered negligible. Based on data obtained during measurements, histograms representing pore size distribution and distribution of size of pore interconnections were plotted for each sample. Obtained histograms were used to determine average pore size and average size of pore interconnections for each material through fit of normal distribution and subsequent determination of the maximum of the normal distribution. Figure 2. VP-ESEM micrograph (a), macropore size distribution (b) and distribution of macropore interconnection size (c) of NAT ceramics. 3. Results and discussion All compositions were successfully sintered apart from the mixture of hydroxyapatite UK and 30 wt% of glass sintered at 700 C which did not retain structural integrity. Thus, data for material UK are not presented. Figures 2 to 4 show representative micrographs, macropore size distributions and distributions of macropore interconnection size of ceramics based on natural origin hydroxyapatite modified with 30 wt% of glass. Figures 5 to 9 show micrographs of the remaining materials (including ones modified with 50 wt% of glass). Table 1 summarizes data on macropore size and macropore interconnection size and their distributions. All produced materials exhibited an internal structure of large number of interconnected macropores. Investigations showed that in obtained ceramics the average macropore size and the average size of macropore interconnections ranged from 532 to 761 µm and 145 to 369 µm, respectively. According to literature the minimum macropore size in macroporous ceramics for bone tissue therapy is 100 µm [13-15] and the optimum macropore size is of several hundreds of µm [14, 15]. The size of macropore interconnections in macroporous ceramics for bone tissue engineering should exceed 50 µm [14, 15]. That means all macroporous ceramics produced in the presented the work showed morphology desired for such materials. Conclusions The application of polymer sponge method for preparation of ceramics from porcine origin hydroxyapatite leads to macroporous materials; such materials show desired structure of large number of open and interconnected macropores. From the morphology point of view, macroporous ceramics produced with the use of polymer sponge method from porcine origin hydroxyapatite and phosphate glass can be used as scaffolds in bone tissue engineering. Presented combination of scanning electron 3

5 Figure 3. VP-ESEM micrograph (a), macropore size distribution (b) and distribution of macropore interconnection size (c) of NAT ceramics. Figure 4. VP-ESEM micrograph (a), macropore size distribution (b) and distribution of macropore interconnection size (c) of NAT ceramics. Figure 5. VP-ESEM micrographs of (a) UK and (b) UK ceramics. microscopy and image analysis techniques allows to characterize in detail macroporous structures using quantitative measure. Described approach can be used in analyses of SEM micrographs to characterize any visible morphology feature such as e.g., pores, pore interconnections, grains, crystallites. That makes SEM micrographs a valuable source of quantitative data. 4

6 EMAS 2011: 12th European Workshop on Modern Developments in Microbeam Analysis IOP Publishing IOP Conf. Series: Materials Science and Engineering 32 (2012) doi: / x/32/1/ Figure 6. VP-ESEM micrographs of (a) PL30 700, (b) PL30 800, and (c) PL ceramics. Figure 7. VP-ESEM micrographs of (a) NAT and (b) NAT ceramics. Figure 8. VP-ESEM micrographs of (a) UK and (b) UK ceramics. Figure 9. VP-ESEM micrographs of (a) PL and (b) PL ceramics. 5

7 Table 1. Average macropore and macropore interconnection size and distribution width of these two parameters for hydroxyapatite ceramics modified with 30 and 50 wt% of phosphate glass. Material Average macropore size [µm] Width of macropore size distribution [µm] Average macropore interconnection size [µm] Width of macropore interconnection size distribution [µm] NAT ± ± ± ± 32 NAT ± ± ± ± 16 NAT ± ± ± ± 30 UK ± ± ± ± 54 UK ± ± ± ± 39 PL ± ± ± ± 21 PL ± ± ± ± 80 PL ± ± ± ± 24 NAT ± ± ± ± 10 NAT ± ± ± ± 14 UK ± ± ± ± 38 UK ± ± ± ± 31 PL ± ± ± ± 27 PL ± ± ± ± 64 Acknowledgements The research was realized in part at the Materials Science and Chemical Engineering Department of Politecnico di Torino within the framework of a research grant under KMM-NoE programme. The authors would like to thank Ms. Chiara Vitale-Brovarone and Ms. Enrica Verné for their help. References [1] Joschek S, Nies B, Krotz R and Göpferich A 2000 Chemical and physicochemical characterization of porous hydroxyapatite ceramics made of natural bone. Biomaterials [2] Legeros R Z and Legeros J P 2008 Bioceramics and their clinical applications. ed, Kokubo T (Cambridge: Woodhead Publishing Limited) chapters 15-20, pp [3] Baino F and Vitale-Brovarone C 2011 Three-dimensional glass-derived scaffolds for bone tissue engineering: current trends and forecasts for the future. J. Biomed. Mat. Res. A 97A [4] Ruksudjatir A, Pengpat K, Rujijanagul G and Tunkasiri T 2008 Synthesis and characterization of nanocrystalline hydroxyapatite from natural bovine bone. Current Appl. Phys [5] Haberko K, Bućko M M, Mozgawa W, Pyda A, Brzezińska-Miecznik J and Carpentier J 2009 Behaviour of bone origin hydroxyapatite at elevated temperatures and in O 2 and CO 2 atmospheres. Ceramics Int [6] Landi E, Logroscino G, Proietti L, Tampieri A, Sandri M and Sprio S 2008 Biomimetic Mg-substituted hydroxyapatite: from synthesis to in vivo behaviour. J. Mater. Sci.: Mat. Medic [7] Komlev V S, Fadeeva I V, Gurin A N, Kovaleva E S, Smirnov V V, Gurin N A and Barinov S M 2009 Effect of the concentration of carbonate groups in a carbonate hydroxyapatite ceramic on its in vivo behavior. Inorg. Mater [8] Banerjee S S, Bandyopadhyay A and Bose S 2010 Biphasic resorbable calcium phosphate ceramic for bone implants and local alendronate delivery. Adv. Engng. Mater. 12 B

8 [9] Janus A M, Faryna M, Haberko K, Rakowska A and Panz T 2008 Chemical and microstructural characterization of natural hydroxyapatite derived from pig bones. Microchim. Acta [10] Vitale-Brovarone C, Baino F, Bretcanu O and Verne E 2009 Foam-like scaffolds for bone tissue engineering based on a novel couple of silicate-phosphate specular glasses: synthesis and properties. J. Mater. Sci.: Mater. Medic [11] Klawitter J J, Bagwell J G, Weinstein A M, Sauer B W and Pruitt J R 1976 An evaluation of bone growth into porous high density polyethylene. J. Biomed. Mater. Res [12] Janus A M, Faryna M, Verné E and Vitale-Brovarone C 2007 Preparation of phosphate glass reinforced hydroxyapatite scaffolds for tissue engineering. Inżynieria Materiałowa XXVIII [13] Kim H W, Lee S Y and Bae C J 2003 Porous ZrO 2 bone scaffold coated with hydroxyapatite with fluorapatite intermediate layer. Biomaterials [14] Baino F, Vitale-Brovarone C 2011 Three-dimensional glass-derived scaffolds for bone tissue engineering: current trends and forecasts for the future. J. Biomed. Mater. Res. 97A [15] Karageorgiou V and Kaplan D 2005 Porosity of 3D biomaterial scaffolds and osteogenesis. Biomater