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A comparative study of calcium phosphate formation on bioceramics in vitro and in vivo
ARTICLE IN PRESS
Biomaterials 26 (2005) 6477–6486 www.elsevier.com/locate/biomaterials
A comparative study of calcium phosphate formation on bioceramics in vitro and in vivo Renlong Xina, Yang Lenga,, Jiyong Chenb, Qiyi Zhangb a
Department of Mechanical Engineering, Hong Kong University of Science and Technology, Hong Kong, China b Engineering Research Center in Biomaterials, Sichuan University, Chengdu, China Received 30 November 2004; accepted 3 April 2005 Available online 29 June 2005
Abstract Formation of calcium phosphate (Ca-P) on various bioceramic surfaces in simulated body fluid (SBF) and in rabbit muscle sites was investigated. The bioceramics were sintered porous solids, including bioglass, glass-ceramics, hydroxyapatite, a-tricalcium phosphate and b-tricalcium phosphate. The ability of inducing Ca-P formation was compared among the bioceramics. The Ca-P crystal structures were identified using single-crystal diffraction patterns in transmission electron microscopy. The examination results show that ability of inducing Ca-P formation in SBF was similar among bioceramics, but considerably varied among bioceramics in vivo. Sintered b-tricalcium phosphate exhibited a poor ability of inducing Ca-P formation both in vitro and in vivo. Octacalcium phosphate (OCP) formed on the surfaces of bioglass, A-W, hydroxyapatite and a-tricalcium phosphate in vitro and in vivo. Apatite formation in physiological environments cannot be confirmed as a common feature of bioceramics. r 2005 Elsevier Ltd. All rights reserved. Keywords: Calcium phosphate; Bioceramics; Simulated body fluid; Bioactivity; Transmission electron microscopy
1. Introduction Bioceramics, such as bioactive glass, glass-ceramics and calcium phosphates (Ca-Ps) have been widely studied for orthopedic and dental applications due to their good ability of osteoconduction. It has been commonly accepted that the bioactivity of bioceramics relies on their ability to induce hydroxyapatite (HA) formation in the physiological environment. Thus, the ability of apatite formation in simulated body fluid (SBF) or in animal model has been regarded as the evidence of bioactivity for bioceramics, and even for other types of orthopedic materials [1–4]. This judgment however is not free from controversy because two observations attract our attention: (1) HA is not the only phase of calcium phosphate which may form in the physiological environment [5–7]; and (2) other calcium Corresponding author. Fax: +86 852 23581543.
E-mail address: [email protected] (Y. Leng). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.04.028
phosphate phases, such as octacalcium phosphate (OCP) and dicalcium phosphate dihydrate (DCPD), may have been misidentified as HA on the bioactive ceramic or metallic surfaces [8–10]. We note that the stable thermodynamic structure of apatite does not ensure that HA is the most favorable precipitation phase from supersaturated calcium and phosphorous solutions, because the OCP and DCPD are kinetically more favorable [11–14]. In fact, OCP or DCPD has been considered as a precursor phase for HA formation [7]. Theoretical analysis based on nucleation kinetics indicates that the OCP nucleation rates could be much faster than that of HA in the physiological environment [14]. We also note that there is a possibility of misidentifying OCP as HA when using powder X-ray diffraction (XRD) because of structural similarity between HA and OCP [9]. The patterns of single-crystal diffraction obtained by transmission electron microscopy (TEM) provided a more reliable means to identify the
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precipitation phases of Ca-Ps. Using electron diffraction in TEM, we have found that OCP is more likely to be formed on HA/TCP biphasic ceramics in SBF and in living body than HA [8]. Apparently, it is necessary to examine the formation of apatite or other calcium phosphate on various bioceramics under well-controlled conditions in vitro and in vivo. Side-by-side comparison among bioceramics would provide information that possibly leads us to reveal the relationship between apatite formation and bioactivity of bioceramic materials. In this study, we examined the porous ceramic samples, which are commonly believed to be bioactive, after being immersed in simulated body fluid and implanted in rabbit body. Formation of Ca-P on the surfaces of bioceramics has been compared. The phases of formed Ca-Ps on bioceramics have been identified by single-crystal diffraction.
2. Experimental 2.1. Specimen preparation Six types of bioceramics were examined in this study; including sintered Bioglasss (BG), A-W glass ceramics (AW), hydroxyapatite (HA), a-tricalcium phosphate (a-TCP) and b-tricalcium phosphate (b-TCP) and bi-phasic hydroxyapatite/ a-tricalcium phosphate (HA/TCP). All bioceramic specimens were in the form of porous solids for comparative study. Porous A-W ceramic specimens were provided by Professor Tadashi Kokubo in Japan. Porous b-TCP ceramic specimens, provided by the Biomedical Materials Centre at Wuhan University of Technology in China, were made by mixing micro-sized b-TCP particles with pore-forming rosin and a CaP glass (80–65 wt% P2O5, 18–6 wt% Na2O, 15–10 wt% CaO, 5–1 wt% MgO, 3–0 wt% Al2O3). The mixtures were foamed and sintered in air at 850 1C for 2 h and then cooled down to ambient temperature in furnace. Porous ceramic Bioglasss (BG) was made from the Bioglasss 45S5 particulates with diameters in the range of 250–600 mm, provided by the USBiomaterials Corporation in USA. The particulates were ball-milled for 4 h and further sifted through a 200-mesh sieve. The sifted powders were foamed by 5–10% H2O2 solution to produce porous body. After drying in an oven at 70–80 1C, the green body was sintered at 1000 1C for 2 h and cooled in furnace. Porous solids of HA, a-TCP and HA/TCP were made by the same foaming method with H2O2 solution. HA/TCP ceramics was made by mixing HA and TCP powder with weight ratio of 7:3 and sintering at 1200 1C and cooling in furnace. HA was also sintered at 1200 1C and cooled in furnace. a-TCP was sintered at 1100 1C and quenched subsequently.
was prepared by dissolving 5.403 g NaCl, 0.736 g NaHCO3, 2.036 g Na2CO3, 0.225 g KCl, 0.182 g K2HPO4, 0.310 g MgCl2 6H2O, 11.928 g 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethane surfonic acid (HEPES), 0.293 g CaCl2, 0.072 g Na2SO4 and 1.5 ml 1 mol L1 NaOH into double distilled water in sequence. HEPES and NaOH serve as buffers to keep the pH value at 7.4. The specimen of bioceramics was cut into a cube of 5 5 5 mm. Ten specimens of each type of bioceramics were used for the SBF immersion. In each experiment, only two specimens of the same type of bioceramics were placed in a 250 ml glass beaker filled with 200 ml SBF. The SBF temperature was kept at 37 1C using a water bath. After SBF immersion, the specimens were removed from the SBF and washed gently with distilled water and ethanol, and then dried at room temperature. 2.3. In vivo experiments The specimens for in vivo experiments, with the same shape and size as those of the in vitro ones, were implanted in the dorsal muscles of New Zealand white rabbits. We chose muscle site of rabbits for implantation so that possible Ca-P formation on bioceramic surfaces could be examined after extracting the specimens. The body fluid in the rabbit muscle sites, at least, provided a real physiological environment, even though the environment is not totally identical to that of the bone site. Eight specimens of each type of bioceramics were implanted. In order to eliminate possible variations in individual rabbits, each rabbit accommodated a whole set of six different bioceramic specimens. Surgery was performed under general anesthesia and sterile conditions. The specimens were sterilized with ethylene oxide gas before implantation. The specimens were harvested after 2 to 6 week implantation by scarificing the rabbits. The implanted specimens were extracted with the surrounding tissues and washed with phosphate-buffered saline (PBS). The attached tissues were cleaned with a mixture of PBS (90 wt%) and pepsin (10 wt%) solutions, and then the implants were dried at 50 1C in an oven. 2.4. Characterization The surface morphologies of specimens after in vitro and in vivo experiments were examined using SEM (JEOL 6300 and 6300F) after gold coating. Crystal morphology and structure of Ca-P precipitates were examined using TEM (Philip CM20 and JEOL 2010). Samples of Ca-P precipitates on bioceramic surfaces were carefully extracted in ethanol using ultrasound vibration, and then were picked up using copper meshes with carbon film coating. Bright-field images and electron diffraction patterns of single crystalline Ca-P were obtained in TEM.
3. Results 3.1. Ca-P formation
2.2. In vitro experiments In vitro experiments were conducted in SBF with the same ionic composition as human plasma [15]. Every liter of SBF
3.1.1. In vitro Ca-P precipitation was occurred on all bioceramic specimens after immersion in SBF, except on the b-TCP.
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The morphology of precipitation is quite similar among the bioceramic surfaces. Fig. 1 shows the typical surface morphology of six types of specimens before immersion in SBF. Fig. 2 shows the typical features of precipitation on specimens of BG, A-W, HA, a-TCP and HA/TCP after immersing in SBF for 1 day. Precipitation starts at individual granules and the granules gradually grow together to form a dense layer on the specimen surface. High-magnification SEM images further reveal that each Ca-P granule consists of a large number of tiny flake-like crystals (Fig. 3). Ca-P precipitations with similar morphology have been reported in previous studies [16,17]. We note that the A-W, HA and HA/TCP specimens exhibited a better ability of precipitation than that of aTCP, and in turn, a-TCP exhibited a better ability than that of BG. The ability of precipitation can be judged by the amount and size of granules as shown in Fig. 2. The size of granules on BG is much smaller than that on
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other bioceramics (Fig. 2b). The morphology on BG actually reveals an early stage of Ca-P precipitation. The granules on a-TCP indicate that Ca-P precipitation prefers to start on the surface sites with sharp curvature. With increasing immersion, the Ca-P granules on a-TCP and BG grew, and eventually covered the whole surface as it occurred on the other bioceramic specimens. To our surprise, the b-TCP specimens did not show visible Ca-P precipitation on their surfaces (Fig. 2f). We could not detect Ca-P formation on b-TCP, even after immersing the specimens in SBF for 5 days. 3.1.2. In vivo Ca-P formation in vivo apparently was more difficult than in vitro. After being implanted in the muscle of rabbits for weeks, the precipitation on bioceramic specimens are much less than those immersed in SBF for 1 or 2 days. Also, the morphology and amount of precipitation varies with the type of bioceramics. Fig. 4
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. SEM micrographs of bioceramic surfaces before immersion in SBF: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2. SEM micrographs of bioceramic surfaces after 1 day of immersion in SBF: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
Fig. 3. High magnification micrograph of Ca-P granules formed after 1 day of immersion in SBF.
shows typical SEM images of the surfaces of bioceramic samples after 4–6 week implantation. Comparing the implanted surfaces under the same magnification as
shown in Fig. 4, we note that the HA/TCP and a-TCP samples exhibited the best Ca-P precipitation ability in vivo among the bioceramics. The surfaces of HA/TCP and a-TCP were covered with a large number of Ca-P crystals with planar dimension of 1 mm, which is similar to the OCP crystals obtained by electrochemical method or formed in a membrane system [18–21]. The good ability of Ca-P precipitation in vivo is probably attributed to the presence of a-TCP component. As a high-temperature Ca-P phase, a-TCP has been considered as a favorable Ca-P for osteoinduction because of the amount of Ca2+ and PO3 4 ions which were released into the local physiological environment [17]. The surfaces of HA, A-W and BG specimens also exhibited Ca-P precipitation with much smaller size than those on HA/TCP and a-TCP. The Ca-P precipitates on HA are granular in shape similar to that formed in SBF (Fig. 4c). The Ca-P granules on A-W (Fig. 4a) and BG (Fig. 4b) are less and smaller than on HA. Again, b-TCP specimens exhibit poor Ca-P precipitation in vivo as compared to that in vitro. No sign of Ca-P formation
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(a)
(b)
(c)
(d)
(e)
(f)
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Fig. 4. SEM micrographs of bioceramic surfaces after implantation in rabbit muscle for 4 weeks: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
was observed on their surfaces even after implantation in rabbit muscles for 6 weeks (Fig. 4f). The abilities of Ca-P formation or Ca-P induction in vitro and in vivo are semi-quantitatively compared by SEM examinations. The percentage of areas covered by Ca-P precipitation was evaluated from ten SEM micrographs for each type of sample. Table 1 summarizes the comparison among bioceramics, even though a quantitative evaluation is not practical. Apparently, the evaluation results in SBF are not fully consistent with those in in vivo evaluations. However, we note a general trend of Ca-P formation capability: poor on b-TCP and excellent on HA/TCP. 3.2. Ca-P phases The single diffraction patterns of Ca-P precipitates reveal the OCP phase on every type of bioceramics,
Table 1 Ability of Ca-P formation on bioceramics Bioceramics
In SBF
In rabbit muscle
A-W BG HA HA/TCP a-TCP b-TCP
||| || ||| ||| ||
| | || ||| |||
except the b-TCP, either from immersing in SBF or from implantation in rabbit. Fig. 5 shows the typical TEM images and diffraction patterns of OCP crystals extracted from the bioceramics surfaces after immersion in SBF. Fig. 6 shows the typical TEM images and diffraction patterns of OCP extracted from the bioceramics surfaces after implantation in rabbits. Also, we
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(a)
(b)
(c)
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Fig. 5. TEM bright-field images and corresponding single diffraction patterns of OCP crystals extracted from bioceramic surfaces after immersion in SBF: (a) 7 days on A-W, B ¼ ½1 1 0; (b) 1 day on BG, B ¼ ½1 1 2; (c) 1 day on HA, B ¼ ½1 1 0; (d) 1 day on HA/TCP, B ¼ ½3 2 0 and (e) 1 day on aTCP, B ¼ ½1 1¯ 0.
obtained the OCP diffraction patterns in various orientations (represented by electron beam direction, B). The most typical OCP pattern is that of B ¼ ½1 1 0 shown in Figs. 5c and 6e. This is consistent with our previous finding on HA/TCP that the OCP crystals exhibit (1 1 0) planar surfaces [8]. The apatite structure was also identified in precipitation on surfaces of bioceramic specimens in vitro and in vivo, even though it was not found on every
type of bioceramic specimen. Fig. 7 shows the typical TEM images and diffraction patterns of apatite crystals extracted from the BG and HA/TCP surfaces after immersion in SBF. Fig. 8 shows the typical TEM images and diffraction patterns of apatite crystals extracted from the A-W and BG surfaces after being implanted in rabbit. Fig. 9 shows both the HA and OCP crystals found on the surface of A-W by carefully measuring the angles and distance between diffraction
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(a)
(b)
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(e)
Fig. 6. TEM bright-field images and corresponding single diffraction patterns of OCP crystals extracted from bioceramic surfaces after 4 weeks of ¯ (c) on HA, B ¼ ½1 1 0; (d) on HA/TCP, B ¼ ½1 1 1 ¯ and (e) on a-TCP, implantation in rabbit muscle: (a) on A-W, B ¼ [2 1 5]; (b) on BG, B ¼ ½3 2 6; B ¼ ½1 1 0.
spots. Table 2 summarized the 87 diffraction patterns which were identified on various specimens after immersion in SBF and implantation in rabbit. HA was mainly identified on A-W and BG, while OCP was identified in very type of bioceramics. Noting that HA exists in A-W and HA/TCP specimens originally, we cannot totally rule out the possibility that some identified HA crystals are from substrates of these specimens.
4. Discussion One of the important findings in this study is that OCP formation is a common feature of bioceramics in either simulated or real physiological environment; even though apatite is identified on some types of bioceramics. Apparently, this finding does not agree with the common belief that bioactivity relies on the ability of apatite formation.
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(b)
(a)
Fig. 7. TEM bright-field images and corresponding single diffraction patterns of HA crystals extracted from bioceramic surfaces after immersion in SBF: (a) 2 days on BG, B ¼ ½2¯ 1 0 and (b) 1 day on HA/TCP, B ¼ ½1 1¯ 1.
(a)
(b)
Fig. 8. TEM bright-field images and corresponding single diffraction patterns of HA crystals extracted from bioceramic surfaces after 4 weeks of implantation in rabbit muscle: (a) on A-W, B ¼ ½0 1 0 and (b) on BG, B ¼ ½0 1 0.
Table 2 Crystal structures of Ca-Ps formed on bioceramics Bioceramics
In SBF
In rabbit muscle
OCP
HA
OCP
3 4 10 13 5 — 35
4 4 5 2 — — 15
5 2 5 3 6 — 21
HA
OCP A-W BG HA HA/TCP a-TCP b-TCP Total
HA
Fig. 9. TEM bright-field images of Ca-P formed on A-W after 6 weeks of implantation in rabbit muscle. The single-crystal diffraction reveals the HA and OCP in one image field.
9 6 1 — — 16
It is possible that some of the reported apatite formation resulted from misidentifying OCP as HA in a XRD spectrum. In the XRD spectrum, two main diffraction peak positions of OCP and HA in the spectra obtained by the XRD are so close that conventional XRD cannot distinguish each other; the 2y of (0 0 2)HA ( ¼ 25.881) is slightly lower than (0 0 2)OCP ( ¼ 26.081) and the 2y of (2 1 1)HA ( ¼ 31.771) is slightly higher than (4 0 2)OCP ( ¼ 31.661). The main differences between HA
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and OCP spectra are in the low 2y range. The OCP spectrum has a unique (1 0 0) peak near 51 and ð1 1¯ 0Þ and (2 0 0) peaks near 101. Unfortunately, such lowangle peaks often either have been ignored or are difficult to be detected. We often see the published XRD data only in the 2y range of 201 to 401. The difficulties of detecting low-angle peaks might result from the preferential orientation of OCP crystals on surfaces [10] and/or small size in directions of the (hk0) planes [22]. Also, we note that OCP might be mistakenly identified as HA from the ring pattern of electron diffraction in TEM [8]. OCP rings of (1 0 0) and ð1 1¯ 0Þ are too close to the central beam spot in the diffraction. The high brightness of the central spot makes those rings invisible. Thus, distinguishing the OCP ring pattern from that of HA becomes difficult. The diffraction patterns of single crystals obtained in TEM exhibit a unique merit in distinguishing OCP and HA, because diffraction spots of the OCP low-index planes, such as (2 0 0) andð1 1¯ 0Þ, can clearly reveal its crystal structure identity [8,10]. Since the examined bioceramics are widely considered as bioactive, OCP formation in vitro and in vivo should not imply their poor ability of osteoconduction. Thus, we might have to reconsider whether apatite formation is the criterion to judge bioactivity of orthopedic materials. Actually, we are forced to reconsider whether formation of any bioactive Ca-P is a necessary condition for osteoconduction, because there was no Ca-P formation found on b-TCP which has also been widely considered to be bioactive. The b-TCP specimens used in this study were sintered at a relatively low temperature (900 1C) with a sintering aid. The same b-TCP ceramic has been implanted in cancellous bone in rabbit tibia. TEM examination revealed the direct bonding between the b-TCP ceramic and bone tissue after 2-month implantation as shown in Fig. 10. Thus, its osteoconduction should be beyond any doubt. We also implanted another b-TCP specimens, which has been processed and sintered following a conventional route, in rabbit muscle for 6 weeks. Fig. 11 shows that no obvious Ca-P formation was found on its surfaces, similar to what we previously found in the b-TCP sintered at a lower temperature. Thus, the effects of b-TCP specimen processing on Ca-P formation in vivo should be ruled out.
5. Conclusions Examination of calcium phosphate (Ca-P) formation on bioceramics under the same simulated and real physiological conditions generated the following results. (1) OCP formation ubiquitously occurs on all types of bioceramic surfaces in vitro and in vivo, except on
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B T
Fig. 10. TEM bright-field image of b-TCP implanted in a cavity of cancellous bone of rabbit femur for 8 weeks. B—bone tissue; T— b-TCP.
Fig. 11. SEM micrograph of b-TCP (sintered at 1100 1C) surfaces after implantation in rabbit muscles for 6 weeks.
b-TCP. (2) Apatite formation does not occur on every type of bioceramic surface; it is less likely to occur on the surfaces of HA and a-TCP. (3) Ca-P formation on bioceramic surfaces is more difficult in vivo than in vitro. (4) Difference in Ca-P formation among bioceramic surfaces is less in vitro than in vivo. (5) The b-TCP ceramics which have a good ability of osteointegration show poor ability of Ca-P formation both in vitro as well as in vivo.
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Acknowledgements This work was financially supported by the Research Grant Council of Hong Kong (Grant no. HKUST6191/ 03E), a research fund at The Hong Kong University of Science & Technology (HIA01/02.EG08), and The Key Basic Research Project of China (no. G1999064760). We thank the USBiomaterials Corp. in USA for providing Bioglasss particulates; Professor T. Kokubo in Japan for providing A-W glass-ceramics; and Professors S.P. Li and H.L. Dai at Wuhan University of Technology in China for providing b-TCP ceramics and the implanted b-TCP samples. References [1] Ra´mila A, Vallet-Regı´ M. Static and dynamic in vitro study of a sol–gel glass bioactivity. Biomaterials 2001;22:2301–6. [2] Fujibayashi S, Neo M, Kim HM, Kokubo T, Nakamura T. A comparative study between in vivo bone ingrowth and in vitro apatite formation on Na2O–CaO–SiO2 glasses. Biomaterials 2003; 24:1349–56. [3] Siriphannon P, Kameshima Y, Yasumori A, Okada K, Hayashi S. Comparative study of the formation of hydroxyapatite in simulated body fluid under static and flowing system. J Biomed Mater Res 2002;60:175–85. [4] Ragel CV, Vallet-Regı´ M, Rodrı´ guez-Lorenzo LM. Preparation and in vitro bioactivity of hydroxyapatite/sol–gel glass biphasic material. Biomaterials 2002;23:1865–72. [5] Brown WE, Eidelman N, Tomazic B. Octacalcium phosphate as precursor in biomineral formation. Adv Dent Res 1987;1:306–13. [6] LeGeros RZ. Apatites in biological systems. Prog Cryst Growth Charact Mater 1981;4:1–45. [7] Elliott JC. Structure and chemistry of the apatites and other calcium phosphates. Amsterdam: Elsevier; 1994. [8] Leng Y, Chen JY, Qu SX. TEM examinations of calcium phosphate precipitation on HA/TCP. Biomaterials 2003;24: 2125–31.
[9] Leng Y, Lu X, Chen JY. Identifying calcium phosphates formed in simulated body fluid by electron diffraction. Key Eng Mater 2004;254–256:339–42. [10] Lu X, Leng Y. TEM study of calcium phosphate precipitation on bioactive titanium surfaces. Biomaterials 2004;25:1779–86. [11] Boistelle R, Lopez-Valero I. Growth units and nucleation: the case of calcium phosphates. J Cryst Growth 1990;102L:609–17. [12] Heughebaert JC, Nancollas GH. Kinetics of crystallization of octacalcium phosphate. J Phys Chem 1984;88:2478–81. [13] Kanzaki N, Onuma K, Treboux G, Ito A. Dissolution kinetics of dicalcium phosphate dehydrate under pseudophysiological conditions. J Cryst Growth 2002;235:465–70. [14] Lu X, Leng Y. Theoretical analysis of calcium phosphate precipitation in simulated body fluid. Biomaterials 2005;26: 1097–108. [15] Kim HM, Miyazaki T, Kokubo T, Nakamura T. Revised simulated body fluid. Key Eng Mater 2001;192–195:47–50. [16] Zhang YL, Mizuno M, Yanagisawa M, Takadama H. Bioactive behaviors of porous apatite- and wollastonite-containing glassceramic in two kinds of simulated body fluid. J Mater Res 2003; 18(2):433–41. [17] Guo LH, Huang M, Leng Y, Davies JE, Zhang XD. Structure and composition comparison of bone mineral and apatite layers formed in vitro. Key Eng Mater 2001;192–195:187–90. [18] Zhang QY, Leng Y, Xin RL. A comparative study of electrochemical deposition and biomimetic deposition of calcium phosphate on porous titanium. Biomaterials 2005;26:2857–65. [19] Mao CB, Li HD, Cui FZ, Ma CL, Feng QL. Oriented growth of phosphates on polycrystalline titanium in a process mimicking biomineralization. J Cryst Growth 1999;206:308–21. [20] Iijima M, Moriwaki Y. Effects of ionic inflow and organic matrix on crystal growth of octacalcium phosphate; relevant to tooth enamel formation. J Cryst Growth 1999;198/199:670–6. [21] Iijima M, Moriwaki Y. Lengthwise and oriented growth of octacalcium phosphate crystal in polyacrylamide gel in a model system of tooth enamel apatite formation. J Cryst Growth 1998; 194:125–32. [22] Iijima M, Kamemizu H, Wakamatsu N, Goto T, Doi Y, Moriwaki Y. Effects of CO3 2 ion on the formation of octacalcium phosphate at pH 7.4 and 371C. J Cryst Growth 1994;135: 229–434.
Biomaterials 26 (2005) 6477–6486 www.elsevier.com/locate/biomaterials
A comparative study of calcium phosphate formation on bioceramics in vitro and in vivo Renlong Xina, Yang Lenga,, Jiyong Chenb, Qiyi Zhangb a
Department of Mechanical Engineering, Hong Kong University of Science and Technology, Hong Kong, China b Engineering Research Center in Biomaterials, Sichuan University, Chengdu, China Received 30 November 2004; accepted 3 April 2005 Available online 29 June 2005
Abstract Formation of calcium phosphate (Ca-P) on various bioceramic surfaces in simulated body fluid (SBF) and in rabbit muscle sites was investigated. The bioceramics were sintered porous solids, including bioglass, glass-ceramics, hydroxyapatite, a-tricalcium phosphate and b-tricalcium phosphate. The ability of inducing Ca-P formation was compared among the bioceramics. The Ca-P crystal structures were identified using single-crystal diffraction patterns in transmission electron microscopy. The examination results show that ability of inducing Ca-P formation in SBF was similar among bioceramics, but considerably varied among bioceramics in vivo. Sintered b-tricalcium phosphate exhibited a poor ability of inducing Ca-P formation both in vitro and in vivo. Octacalcium phosphate (OCP) formed on the surfaces of bioglass, A-W, hydroxyapatite and a-tricalcium phosphate in vitro and in vivo. Apatite formation in physiological environments cannot be confirmed as a common feature of bioceramics. r 2005 Elsevier Ltd. All rights reserved. Keywords: Calcium phosphate; Bioceramics; Simulated body fluid; Bioactivity; Transmission electron microscopy
1. Introduction Bioceramics, such as bioactive glass, glass-ceramics and calcium phosphates (Ca-Ps) have been widely studied for orthopedic and dental applications due to their good ability of osteoconduction. It has been commonly accepted that the bioactivity of bioceramics relies on their ability to induce hydroxyapatite (HA) formation in the physiological environment. Thus, the ability of apatite formation in simulated body fluid (SBF) or in animal model has been regarded as the evidence of bioactivity for bioceramics, and even for other types of orthopedic materials [1–4]. This judgment however is not free from controversy because two observations attract our attention: (1) HA is not the only phase of calcium phosphate which may form in the physiological environment [5–7]; and (2) other calcium Corresponding author. Fax: +86 852 23581543.
E-mail address: [email protected] (Y. Leng). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.04.028
phosphate phases, such as octacalcium phosphate (OCP) and dicalcium phosphate dihydrate (DCPD), may have been misidentified as HA on the bioactive ceramic or metallic surfaces [8–10]. We note that the stable thermodynamic structure of apatite does not ensure that HA is the most favorable precipitation phase from supersaturated calcium and phosphorous solutions, because the OCP and DCPD are kinetically more favorable [11–14]. In fact, OCP or DCPD has been considered as a precursor phase for HA formation [7]. Theoretical analysis based on nucleation kinetics indicates that the OCP nucleation rates could be much faster than that of HA in the physiological environment [14]. We also note that there is a possibility of misidentifying OCP as HA when using powder X-ray diffraction (XRD) because of structural similarity between HA and OCP [9]. The patterns of single-crystal diffraction obtained by transmission electron microscopy (TEM) provided a more reliable means to identify the
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precipitation phases of Ca-Ps. Using electron diffraction in TEM, we have found that OCP is more likely to be formed on HA/TCP biphasic ceramics in SBF and in living body than HA [8]. Apparently, it is necessary to examine the formation of apatite or other calcium phosphate on various bioceramics under well-controlled conditions in vitro and in vivo. Side-by-side comparison among bioceramics would provide information that possibly leads us to reveal the relationship between apatite formation and bioactivity of bioceramic materials. In this study, we examined the porous ceramic samples, which are commonly believed to be bioactive, after being immersed in simulated body fluid and implanted in rabbit body. Formation of Ca-P on the surfaces of bioceramics has been compared. The phases of formed Ca-Ps on bioceramics have been identified by single-crystal diffraction.
2. Experimental 2.1. Specimen preparation Six types of bioceramics were examined in this study; including sintered Bioglasss (BG), A-W glass ceramics (AW), hydroxyapatite (HA), a-tricalcium phosphate (a-TCP) and b-tricalcium phosphate (b-TCP) and bi-phasic hydroxyapatite/ a-tricalcium phosphate (HA/TCP). All bioceramic specimens were in the form of porous solids for comparative study. Porous A-W ceramic specimens were provided by Professor Tadashi Kokubo in Japan. Porous b-TCP ceramic specimens, provided by the Biomedical Materials Centre at Wuhan University of Technology in China, were made by mixing micro-sized b-TCP particles with pore-forming rosin and a CaP glass (80–65 wt% P2O5, 18–6 wt% Na2O, 15–10 wt% CaO, 5–1 wt% MgO, 3–0 wt% Al2O3). The mixtures were foamed and sintered in air at 850 1C for 2 h and then cooled down to ambient temperature in furnace. Porous ceramic Bioglasss (BG) was made from the Bioglasss 45S5 particulates with diameters in the range of 250–600 mm, provided by the USBiomaterials Corporation in USA. The particulates were ball-milled for 4 h and further sifted through a 200-mesh sieve. The sifted powders were foamed by 5–10% H2O2 solution to produce porous body. After drying in an oven at 70–80 1C, the green body was sintered at 1000 1C for 2 h and cooled in furnace. Porous solids of HA, a-TCP and HA/TCP were made by the same foaming method with H2O2 solution. HA/TCP ceramics was made by mixing HA and TCP powder with weight ratio of 7:3 and sintering at 1200 1C and cooling in furnace. HA was also sintered at 1200 1C and cooled in furnace. a-TCP was sintered at 1100 1C and quenched subsequently.
was prepared by dissolving 5.403 g NaCl, 0.736 g NaHCO3, 2.036 g Na2CO3, 0.225 g KCl, 0.182 g K2HPO4, 0.310 g MgCl2 6H2O, 11.928 g 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethane surfonic acid (HEPES), 0.293 g CaCl2, 0.072 g Na2SO4 and 1.5 ml 1 mol L1 NaOH into double distilled water in sequence. HEPES and NaOH serve as buffers to keep the pH value at 7.4. The specimen of bioceramics was cut into a cube of 5 5 5 mm. Ten specimens of each type of bioceramics were used for the SBF immersion. In each experiment, only two specimens of the same type of bioceramics were placed in a 250 ml glass beaker filled with 200 ml SBF. The SBF temperature was kept at 37 1C using a water bath. After SBF immersion, the specimens were removed from the SBF and washed gently with distilled water and ethanol, and then dried at room temperature. 2.3. In vivo experiments The specimens for in vivo experiments, with the same shape and size as those of the in vitro ones, were implanted in the dorsal muscles of New Zealand white rabbits. We chose muscle site of rabbits for implantation so that possible Ca-P formation on bioceramic surfaces could be examined after extracting the specimens. The body fluid in the rabbit muscle sites, at least, provided a real physiological environment, even though the environment is not totally identical to that of the bone site. Eight specimens of each type of bioceramics were implanted. In order to eliminate possible variations in individual rabbits, each rabbit accommodated a whole set of six different bioceramic specimens. Surgery was performed under general anesthesia and sterile conditions. The specimens were sterilized with ethylene oxide gas before implantation. The specimens were harvested after 2 to 6 week implantation by scarificing the rabbits. The implanted specimens were extracted with the surrounding tissues and washed with phosphate-buffered saline (PBS). The attached tissues were cleaned with a mixture of PBS (90 wt%) and pepsin (10 wt%) solutions, and then the implants were dried at 50 1C in an oven. 2.4. Characterization The surface morphologies of specimens after in vitro and in vivo experiments were examined using SEM (JEOL 6300 and 6300F) after gold coating. Crystal morphology and structure of Ca-P precipitates were examined using TEM (Philip CM20 and JEOL 2010). Samples of Ca-P precipitates on bioceramic surfaces were carefully extracted in ethanol using ultrasound vibration, and then were picked up using copper meshes with carbon film coating. Bright-field images and electron diffraction patterns of single crystalline Ca-P were obtained in TEM.
3. Results 3.1. Ca-P formation
2.2. In vitro experiments In vitro experiments were conducted in SBF with the same ionic composition as human plasma [15]. Every liter of SBF
3.1.1. In vitro Ca-P precipitation was occurred on all bioceramic specimens after immersion in SBF, except on the b-TCP.
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The morphology of precipitation is quite similar among the bioceramic surfaces. Fig. 1 shows the typical surface morphology of six types of specimens before immersion in SBF. Fig. 2 shows the typical features of precipitation on specimens of BG, A-W, HA, a-TCP and HA/TCP after immersing in SBF for 1 day. Precipitation starts at individual granules and the granules gradually grow together to form a dense layer on the specimen surface. High-magnification SEM images further reveal that each Ca-P granule consists of a large number of tiny flake-like crystals (Fig. 3). Ca-P precipitations with similar morphology have been reported in previous studies [16,17]. We note that the A-W, HA and HA/TCP specimens exhibited a better ability of precipitation than that of aTCP, and in turn, a-TCP exhibited a better ability than that of BG. The ability of precipitation can be judged by the amount and size of granules as shown in Fig. 2. The size of granules on BG is much smaller than that on
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other bioceramics (Fig. 2b). The morphology on BG actually reveals an early stage of Ca-P precipitation. The granules on a-TCP indicate that Ca-P precipitation prefers to start on the surface sites with sharp curvature. With increasing immersion, the Ca-P granules on a-TCP and BG grew, and eventually covered the whole surface as it occurred on the other bioceramic specimens. To our surprise, the b-TCP specimens did not show visible Ca-P precipitation on their surfaces (Fig. 2f). We could not detect Ca-P formation on b-TCP, even after immersing the specimens in SBF for 5 days. 3.1.2. In vivo Ca-P formation in vivo apparently was more difficult than in vitro. After being implanted in the muscle of rabbits for weeks, the precipitation on bioceramic specimens are much less than those immersed in SBF for 1 or 2 days. Also, the morphology and amount of precipitation varies with the type of bioceramics. Fig. 4
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. SEM micrographs of bioceramic surfaces before immersion in SBF: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
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Fig. 2. SEM micrographs of bioceramic surfaces after 1 day of immersion in SBF: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
Fig. 3. High magnification micrograph of Ca-P granules formed after 1 day of immersion in SBF.
shows typical SEM images of the surfaces of bioceramic samples after 4–6 week implantation. Comparing the implanted surfaces under the same magnification as
shown in Fig. 4, we note that the HA/TCP and a-TCP samples exhibited the best Ca-P precipitation ability in vivo among the bioceramics. The surfaces of HA/TCP and a-TCP were covered with a large number of Ca-P crystals with planar dimension of 1 mm, which is similar to the OCP crystals obtained by electrochemical method or formed in a membrane system [18–21]. The good ability of Ca-P precipitation in vivo is probably attributed to the presence of a-TCP component. As a high-temperature Ca-P phase, a-TCP has been considered as a favorable Ca-P for osteoinduction because of the amount of Ca2+ and PO3 4 ions which were released into the local physiological environment [17]. The surfaces of HA, A-W and BG specimens also exhibited Ca-P precipitation with much smaller size than those on HA/TCP and a-TCP. The Ca-P precipitates on HA are granular in shape similar to that formed in SBF (Fig. 4c). The Ca-P granules on A-W (Fig. 4a) and BG (Fig. 4b) are less and smaller than on HA. Again, b-TCP specimens exhibit poor Ca-P precipitation in vivo as compared to that in vitro. No sign of Ca-P formation
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(a)
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Fig. 4. SEM micrographs of bioceramic surfaces after implantation in rabbit muscle for 4 weeks: (a) A-W; (b) BG; (c) HA; (d) HA/TCP; (e) a-TCP and (f) b-TCP.
was observed on their surfaces even after implantation in rabbit muscles for 6 weeks (Fig. 4f). The abilities of Ca-P formation or Ca-P induction in vitro and in vivo are semi-quantitatively compared by SEM examinations. The percentage of areas covered by Ca-P precipitation was evaluated from ten SEM micrographs for each type of sample. Table 1 summarizes the comparison among bioceramics, even though a quantitative evaluation is not practical. Apparently, the evaluation results in SBF are not fully consistent with those in in vivo evaluations. However, we note a general trend of Ca-P formation capability: poor on b-TCP and excellent on HA/TCP. 3.2. Ca-P phases The single diffraction patterns of Ca-P precipitates reveal the OCP phase on every type of bioceramics,
Table 1 Ability of Ca-P formation on bioceramics Bioceramics
In SBF
In rabbit muscle
A-W BG HA HA/TCP a-TCP b-TCP
||| || ||| ||| ||
| | || ||| |||
except the b-TCP, either from immersing in SBF or from implantation in rabbit. Fig. 5 shows the typical TEM images and diffraction patterns of OCP crystals extracted from the bioceramics surfaces after immersion in SBF. Fig. 6 shows the typical TEM images and diffraction patterns of OCP extracted from the bioceramics surfaces after implantation in rabbits. Also, we
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Fig. 5. TEM bright-field images and corresponding single diffraction patterns of OCP crystals extracted from bioceramic surfaces after immersion in SBF: (a) 7 days on A-W, B ¼ ½1 1 0; (b) 1 day on BG, B ¼ ½1 1 2; (c) 1 day on HA, B ¼ ½1 1 0; (d) 1 day on HA/TCP, B ¼ ½3 2 0 and (e) 1 day on aTCP, B ¼ ½1 1¯ 0.
obtained the OCP diffraction patterns in various orientations (represented by electron beam direction, B). The most typical OCP pattern is that of B ¼ ½1 1 0 shown in Figs. 5c and 6e. This is consistent with our previous finding on HA/TCP that the OCP crystals exhibit (1 1 0) planar surfaces [8]. The apatite structure was also identified in precipitation on surfaces of bioceramic specimens in vitro and in vivo, even though it was not found on every
type of bioceramic specimen. Fig. 7 shows the typical TEM images and diffraction patterns of apatite crystals extracted from the BG and HA/TCP surfaces after immersion in SBF. Fig. 8 shows the typical TEM images and diffraction patterns of apatite crystals extracted from the A-W and BG surfaces after being implanted in rabbit. Fig. 9 shows both the HA and OCP crystals found on the surface of A-W by carefully measuring the angles and distance between diffraction
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Fig. 6. TEM bright-field images and corresponding single diffraction patterns of OCP crystals extracted from bioceramic surfaces after 4 weeks of ¯ (c) on HA, B ¼ ½1 1 0; (d) on HA/TCP, B ¼ ½1 1 1 ¯ and (e) on a-TCP, implantation in rabbit muscle: (a) on A-W, B ¼ [2 1 5]; (b) on BG, B ¼ ½3 2 6; B ¼ ½1 1 0.
spots. Table 2 summarized the 87 diffraction patterns which were identified on various specimens after immersion in SBF and implantation in rabbit. HA was mainly identified on A-W and BG, while OCP was identified in very type of bioceramics. Noting that HA exists in A-W and HA/TCP specimens originally, we cannot totally rule out the possibility that some identified HA crystals are from substrates of these specimens.
4. Discussion One of the important findings in this study is that OCP formation is a common feature of bioceramics in either simulated or real physiological environment; even though apatite is identified on some types of bioceramics. Apparently, this finding does not agree with the common belief that bioactivity relies on the ability of apatite formation.
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(b)
(a)
Fig. 7. TEM bright-field images and corresponding single diffraction patterns of HA crystals extracted from bioceramic surfaces after immersion in SBF: (a) 2 days on BG, B ¼ ½2¯ 1 0 and (b) 1 day on HA/TCP, B ¼ ½1 1¯ 1.
(a)
(b)
Fig. 8. TEM bright-field images and corresponding single diffraction patterns of HA crystals extracted from bioceramic surfaces after 4 weeks of implantation in rabbit muscle: (a) on A-W, B ¼ ½0 1 0 and (b) on BG, B ¼ ½0 1 0.
Table 2 Crystal structures of Ca-Ps formed on bioceramics Bioceramics
In SBF
In rabbit muscle
OCP
HA
OCP
3 4 10 13 5 — 35
4 4 5 2 — — 15
5 2 5 3 6 — 21
HA
OCP A-W BG HA HA/TCP a-TCP b-TCP Total
HA
Fig. 9. TEM bright-field images of Ca-P formed on A-W after 6 weeks of implantation in rabbit muscle. The single-crystal diffraction reveals the HA and OCP in one image field.
9 6 1 — — 16
It is possible that some of the reported apatite formation resulted from misidentifying OCP as HA in a XRD spectrum. In the XRD spectrum, two main diffraction peak positions of OCP and HA in the spectra obtained by the XRD are so close that conventional XRD cannot distinguish each other; the 2y of (0 0 2)HA ( ¼ 25.881) is slightly lower than (0 0 2)OCP ( ¼ 26.081) and the 2y of (2 1 1)HA ( ¼ 31.771) is slightly higher than (4 0 2)OCP ( ¼ 31.661). The main differences between HA
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and OCP spectra are in the low 2y range. The OCP spectrum has a unique (1 0 0) peak near 51 and ð1 1¯ 0Þ and (2 0 0) peaks near 101. Unfortunately, such lowangle peaks often either have been ignored or are difficult to be detected. We often see the published XRD data only in the 2y range of 201 to 401. The difficulties of detecting low-angle peaks might result from the preferential orientation of OCP crystals on surfaces [10] and/or small size in directions of the (hk0) planes [22]. Also, we note that OCP might be mistakenly identified as HA from the ring pattern of electron diffraction in TEM [8]. OCP rings of (1 0 0) and ð1 1¯ 0Þ are too close to the central beam spot in the diffraction. The high brightness of the central spot makes those rings invisible. Thus, distinguishing the OCP ring pattern from that of HA becomes difficult. The diffraction patterns of single crystals obtained in TEM exhibit a unique merit in distinguishing OCP and HA, because diffraction spots of the OCP low-index planes, such as (2 0 0) andð1 1¯ 0Þ, can clearly reveal its crystal structure identity [8,10]. Since the examined bioceramics are widely considered as bioactive, OCP formation in vitro and in vivo should not imply their poor ability of osteoconduction. Thus, we might have to reconsider whether apatite formation is the criterion to judge bioactivity of orthopedic materials. Actually, we are forced to reconsider whether formation of any bioactive Ca-P is a necessary condition for osteoconduction, because there was no Ca-P formation found on b-TCP which has also been widely considered to be bioactive. The b-TCP specimens used in this study were sintered at a relatively low temperature (900 1C) with a sintering aid. The same b-TCP ceramic has been implanted in cancellous bone in rabbit tibia. TEM examination revealed the direct bonding between the b-TCP ceramic and bone tissue after 2-month implantation as shown in Fig. 10. Thus, its osteoconduction should be beyond any doubt. We also implanted another b-TCP specimens, which has been processed and sintered following a conventional route, in rabbit muscle for 6 weeks. Fig. 11 shows that no obvious Ca-P formation was found on its surfaces, similar to what we previously found in the b-TCP sintered at a lower temperature. Thus, the effects of b-TCP specimen processing on Ca-P formation in vivo should be ruled out.
5. Conclusions Examination of calcium phosphate (Ca-P) formation on bioceramics under the same simulated and real physiological conditions generated the following results. (1) OCP formation ubiquitously occurs on all types of bioceramic surfaces in vitro and in vivo, except on
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B T
Fig. 10. TEM bright-field image of b-TCP implanted in a cavity of cancellous bone of rabbit femur for 8 weeks. B—bone tissue; T— b-TCP.
Fig. 11. SEM micrograph of b-TCP (sintered at 1100 1C) surfaces after implantation in rabbit muscles for 6 weeks.
b-TCP. (2) Apatite formation does not occur on every type of bioceramic surface; it is less likely to occur on the surfaces of HA and a-TCP. (3) Ca-P formation on bioceramic surfaces is more difficult in vivo than in vitro. (4) Difference in Ca-P formation among bioceramic surfaces is less in vitro than in vivo. (5) The b-TCP ceramics which have a good ability of osteointegration show poor ability of Ca-P formation both in vitro as well as in vivo.
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Acknowledgements This work was financially supported by the Research Grant Council of Hong Kong (Grant no. HKUST6191/ 03E), a research fund at The Hong Kong University of Science & Technology (HIA01/02.EG08), and The Key Basic Research Project of China (no. G1999064760). We thank the USBiomaterials Corp. in USA for providing Bioglasss particulates; Professor T. Kokubo in Japan for providing A-W glass-ceramics; and Professors S.P. Li and H.L. Dai at Wuhan University of Technology in China for providing b-TCP ceramics and the implanted b-TCP samples. References [1] Ra´mila A, Vallet-Regı´ M. Static and dynamic in vitro study of a sol–gel glass bioactivity. Biomaterials 2001;22:2301–6. [2] Fujibayashi S, Neo M, Kim HM, Kokubo T, Nakamura T. A comparative study between in vivo bone ingrowth and in vitro apatite formation on Na2O–CaO–SiO2 glasses. Biomaterials 2003; 24:1349–56. [3] Siriphannon P, Kameshima Y, Yasumori A, Okada K, Hayashi S. Comparative study of the formation of hydroxyapatite in simulated body fluid under static and flowing system. J Biomed Mater Res 2002;60:175–85. [4] Ragel CV, Vallet-Regı´ M, Rodrı´ guez-Lorenzo LM. Preparation and in vitro bioactivity of hydroxyapatite/sol–gel glass biphasic material. Biomaterials 2002;23:1865–72. [5] Brown WE, Eidelman N, Tomazic B. Octacalcium phosphate as precursor in biomineral formation. Adv Dent Res 1987;1:306–13. [6] LeGeros RZ. Apatites in biological systems. Prog Cryst Growth Charact Mater 1981;4:1–45. [7] Elliott JC. Structure and chemistry of the apatites and other calcium phosphates. Amsterdam: Elsevier; 1994. [8] Leng Y, Chen JY, Qu SX. TEM examinations of calcium phosphate precipitation on HA/TCP. Biomaterials 2003;24: 2125–31.
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