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1 Key Engineering Materials Vols (2005) pp Online available since 2005/Apr/15 at (2005) Trans Tech Publications, Switzerland doi: / Cathodoluminescence as a Method of Microstructure Characterization of Biphasic Ceramics Composed of Hydroxyapatite and β-tricalcium Phosphate Diggs, AB 1,2,a, Halloran,JW 2,b and Hollister,SJ 1,c 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI, USA a adiggs@engin.umich.edu, b peterjon@engin.umich.edu, c scottho@engin.umich.edu Keywords: Biphasic calcium phosphate, hydroxyapatite, β-tricalcium phosphate, cathodoluminescence, microstructure Abstract. This paper describes the investigation into the use of cathodoluminescence for distinguishing HA and β-tcp phases within the biphasic calcium phosphate ceramic microstructure. Polished samples were scanned using SEM-CL at an accelerating voltage in the range of kev with a beam current of 5-10 na. The grayscale images produced reveal distinctive patterns for each composition. EDS and EPMA suggest chemical differences among the contrasting regions. Image analysis of voxel values indicate that areas of bright contrast correspond to β-tcp grains with phase amounts confirmed by XRD. Introduction Characterizing the microstructure of the biphasic calcium phosphate ceramic (BPCP) construct will be instrumental in understanding its in vivo degradative properties, thereby making the changing design of a biodegradable scaffold of the same material predictable. Traditional methods of microstructure imaging, such as SEM using secondary electron and back scattered electron modes, have proven ineffective in ascertaining a spatial distribution of the two phases due to a lack of contrast. Electron Microprobe Analysis (EPMA) has also been ineffective in producing a reliable distinction between the two phases due to the similarity of the two starting materials: the C/P ratio is 1.67 and 1.5 for hydroxyapatite (HA) and β-tricalcium phosphate (β-tcp) respectively. An alternative method may be cathodoluminescence (CL). This method examines light emitted from features in the sample when excited by an electron beam. It has traditionally been used in the geosciences to detect and identify rare earth element impurities in apatites [1,2] as well as to determine information on rock formation and mineralization processes [2,3]. In the field of biomaterials, it has been investigated as a method of identifying the presence of amorphous phases in plasma-sprayed HA coatings [4], of differentiating the degree of carbonation in apatites [5], and in microstructural characterization of plasma-sprayed HA coatings [6]. In this study, we investigate the effectiveness of using cathodoluminescence in distinguishing between the two calcium phosphate phases. Materials and Methods HA and β-tcp powders (Plasma Biotal Ltd., Tideswell, UK) were used as received. As-fired samples of sintered BPCP (1100 o C for 5 hours) of varying compositions (0BPCP, 25BPCP, 50BPCP, 75BPCP, and 100BPCP: 0%, 25%, 50%, 75%, and 100% TCP respectively) were imbedded in an epoxy resin and polished to a flat finish. A panchromatic cathodoluminescence microscopy detector was attached to a Cameca SX100 Electron Microprobe Analyzer (EMPA). SEM-CL was performed using an accelerating voltage ranging from kev, with a beam current of 5-10 na. Back scattered electron images were used for comparison. EDS, EPMA, and image analysis using GEMS MicroView (GE Medical Systems) were employed to detect phase All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, (ID: /03/10,15:15:12)
2 334 Bioceramics 17 differences across the samples as well as to determine the effectiveness of CL in distinguishing between the phases of the calcium phosphate ceramic. Results and Discussion Back scattered SEM images for the various compositions are indistinguishable from one another and, furthermore, illustrate no distinction between the HA and β-tcp phases (Fig. 1). Conversely, CL images indicate visible differences in the biphasic microstructure for each of the compositions investigated, with differences seen in variations in gray-scale color (Fig. 2). HA (Fig. 2 A) exhibits a pattern best described as primarily dark with a distribution of light-colored specks scattered throughout; whereas β-tcp (Fig. 2 E) samples produce an image that is primarily light in color with patches of varying gray shades throughout. Biphasic samples produced images that lie between the two phases (Fig. 2, B-D). A: 100% HA B: 25% β-tcp C: 50% β-tcp D: 75% β-tcp E: 100% β-tcp void Fig. 1: Back-scattered SEM images of three biphasic compositions: 25BPCP(B), 50BPCP(C), 75BPCP(D) and their calcium phosphate components-- HA (A) and β-tcp (E). Images show no distinction across compositions and across phases. The lighter contrast of the 75BPCP composition is due to a change in imaging settings and not to compositional differences. All images are in a 600 micron field of view. Each CL image was considered to consist of a combination of black, white, light gray, and dark gray, with patterns in the arrangement consistent with the previous description. Preliminary elemental analysis with EDS shows that the white regions were low in calcium, the gray was intermediate, and the black regions had a higher Ca:P ratio. Quantitative elemental analysis with EPMA was performed at distinct locations within the biphasic construct to more accurately determine chemical differences within the grayscale contrast. The locations and compositions are shown in Fig. 3. Regions of lightest contrast (light gray) demonstrated a Ca:P Regions of darker contrast (dk. gray) exhibited a ratio of 1.54, and regions of darkest contrast (black) Both regions of light contrast are comparable in Ca:P to that of β-tcp (Ca:P = 1.50), suggesting that the grayscale contrast evident in the biphasic images correlate to the β-tcp phase within the construct.
3 Key Engineering Materials Vols A: 100% HA B: 25% β-tcp C: 50% β-tcp D: 75% β-tcp E: 100% β-tcp Fig. 2: Corresponding CL images for the BSE images depicted in Figure 1: 0BPCP (A), 25BPCP (B), 50BPCP (C), 75BPCP (D), AND 100BPCP (E). CL images reveal variation in grayscale contrast across compositions. Images have a 600 micron field of view. CL images were subject to image analysis using GEMS MicroView, in which a bimodal histogram depicting the range of voxel values present within each image was generated; with one mode representing HA and the other, β-tcp (Fig. 4B). Voxel values corresponding to the β-tcp phase were highlighted on the histogram (Fig. 4D), and corresponding contrast highlighted red in the new image (Fig. 4C). For the image depicted here, the volume fraction of the highlighted voxel values was This trend was consistent across all compositions, with highlighted volume fraction 50% β-tcp Known Standard Hydroxyapatite (HA) β-tricalcium Phosphate (β- TCP) Ca: P = Ca: P = 1.50 Point # (Region color) Amount Ca (atomic %) Amount P (atomic %) Ca: P 3 1 (Lt. Gray) 2 (Dk. Gray) (Black) 4 (Lt. Gray) 5 (Dk. Gray) Fig. 3: EPMA analysis at various grayscale regions within the 50% β-tcp CL image.
4 336 Bioceramics 17 A: 25% β-tcp B C D Fig. 4: CL image of biphasic calcium phosphate containing 25% β- TCP (A) and corresponding bimodal histogram of voxel values (B). Values corresponding to β- TCP grains are highlighted in the histogram (D) (voxel value vs. frequency) and colored red in the CL image (C). Image analysis reveals the volume fraction of the red region (β-tcp) is 0.25 and is confirmed by x-ray diffraction. corresponding to the known phase amount of β-tcp as confirmed by x-ray diffraction. Conclusion The results of this work indicate that cathodoluminescence is an effective technique in distinguishing HA and β-tcp in biphasic microstructures. CL images indicate differences in the biphasic microstructure for each of the compositions investigated, with differences seen in variations in gray-scale contrast. Bright and dark regions in the CL images of the biphasic samples reveal which grains are HA and which are β-tcp, with quantitative fraction in bright contrast proportional to β-tcp phase fraction by quantitative x-ray diffraction. Acknowledgement The authors gratefully acknowledge the expertise of Dr. Eric Essene and Carl Henderson. This work was supported in part by NIH BRP R01 DE 13416, NIH BRP DE 13608, NSF Grant EAR and the National Physical Science Consortium. References [1] J. Barbarand and M. Pagel: American Mineralogist Vol. 86 (2001), p.473 [2] U. Kempe and J. Goetz: Mineralogical Magazine Vol. 66 (2002), p.151 [3] J. Leichmann, I. Broska, and K. Zachovalova: Terra Nova Vol. 15 (2003),p. 104 [4] K.A. Gross and M.R. Phillips: J Mat Sci Mat Med Vol. 9 (1998), p.797 [5] K.A. Gross, M.R. Phillips,and Y.Suetsugu: Key Engin Mater Vol (2001), p.179 [6] J. Gotze, R.B Heimann, H. Hildebrandt, and U. Gburek: Mat.-wiss. U. Werkstoffteh Vol. 32 (2001), p.130
5 Bioceramics 17 doi: / Cathodoluminescence as a Method of Microstructure Characterization of Biphasic Ceramics Composed of Hydroxyapatite and β-tricalcium Phosphate doi: /
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