In vivo Comparative Study of Chelate-setting Calcium-phosphate Cements with Various Bioresorbability Using Rabbit Model

+1Konishi, T; 1, 2Takahashi, S; 1Mizumoto, M, 1, 3Sato, S; 1Honda, M; 2Kida, K; 3Horiguchi, Y; 3Oribe, K;

1, 4Ishii, K; 4Morisue, K; 4Toyama, Y; 1, 4Matsumoto, M; 1, 2Aizawa, M +1Kanagawa Academy of Science and Technology (KAST), Kawasaki, JPN, 2Meiji University, Kawasaki, JPN,

3SHOWA IKA KOHGYO co., ltd., Toyahashi, JPN, 4Keio University, Tokyo, JPN ap-konishi@newkast.or.jp

ABSTRACT INTRODUCTION: Hydroxyapatite (Ca10(PO4)6(OH)2; HAp) and tricalcium phosphate (Ca3(PO4)2; TCP) have been currently used as bioceramics for bone grafting. They are known to be biocompatible and osteoconductive. The HAp is clinically applied as following morphologies: blocks, granules, dense ceramics, porous ceramics and cements. Among these, apatite cement can form a desired shape during surgery operation. In general, apatite cements consist of two kinds of calcium-phosphate powders: acidic dicalcium phosphate dihydrate (CaHPO4∙2H2O) and basic tetracalcium phosphate (Ca4O(PO4)2) on the basis of the previous study reported by Brown and Chow [1]. However, apatite cements may cause inflammation of surrounding tissues due to the setting reaction based on acid-base reaction [2]. Thus, we have developed novel apatite cement without acid-base reactions, “chelate-setting apatite cements” [3, 4]. The cements can be simply fabricated by mixing the apatite particle surface-modified with inositol phosphate (IP6) and deionized water. On the other hand, the apatite cements have a disadvantage in the clinical performance that is slow rate of resorption. Thus, we developed novel chelate-setting cement using α-TCP and/or β-TCP instead of the HAp as starting materials [5, 6]. These cements, IP6-HAp, IP6-α-TCP and IP6-β-TCP cement, have a different bioresorbability on the basis of the chemical composition as starting materials. Our aim in the present work is to examine the biocompatibility and bioresorbability of these cements in vivo using rabbit model. The above-mentioned three kinds of cements and commercially available cement (Biopex®-R, HOYA, Japan) were implanted into tibiae of Japanese rabbits for 4, 8 and 24 weeks. The tissue reaction around the cements was histologically evaluated after desired implantation period. METHODS: Preparation of cement powder The starting HAp (HAp-100, Taihei Chemical, Japan) powders were added into the IP6 solution, and then stirred at 400 rpm for 5 h. After that, the slurry was freeze-dried for 24 h to prepare the HAp powders surface-modified with IP6 (IP6-HAp). The starting α-TCP (α-TCP-A, Taihei Chemical, Japan) and β-TCP (β-TCP-100, Taihei Chemical, Japan) powders were prepared by ball-milling for 120 min and 4 h respectively using ZrO2 pod and beads with a diameter of 10 mmφ. Each of obtained α-TCP and β-TCP slurry was added into the IP6 solution, and then stirred at 400 rpm for 24 h. After that, the slurry was freeze-dried for 24 h to prepare the α-TCP and β-TCP powders surface-modified with IP6 (IP6-α-TCP and IP6-β-TCP). The changes of the powder property before and after surface-modification were determined by X-ray diffractometry (XRD). Fabrication of chelate-setting cement Three kinds of cement powders, IP6-HAp, IP6-α-TCP and IP6-β-TCP, were mixed with the deionized water at the desired powder-to-liquid ratio (w/v). The resulting cements were set in the cylindrical stainless mold and kept under room temperature in air for 24 h. Cylindrical cement specimen (4.2 mm in diameter and of 7 mm height) was fabricated for in vivo study. Commercially-available cement (Biopex®-R) was also prepared according to the protocol recommended by manufacturer instruction for use (4.2 mm in diameter and of 7 mm height). In vivo evaluation The above-mentioned three kinds of cements and Biopex®-R as a control were implanted into tibiae of Japanese rabbits (average weight, 3 kg) for 4, 8 and 24 weeks. After the implantation, the rabbits were sacrificed to retrieve the specimens with the surrounding bone, and then decalcified and undecalcified sections were prepared for histological evaluation. The decalcified sections were stained with tartrate-resistant acid phosphatase (TRAP) and the undecalcified sections were stained

with hematoxylin and eosin (HE) and toluidine blue (TB). The stained sections were observed by light microscopy. RESULTS SECTION: The changes of the crystalline phase of the starting powders before and after surface-modification could not be detected by XRD (Fig. 1) and FT-IR. The three kinds of cement specimens were fabricated from the above-mentioned powders. As for the IP6-HAp, IP6-α-TCP and IP6-β-TCP cement, cement specimens were set with maintenance of the crystalline phase of their starting powders from XRD pattern. The obtained three kinds of cements and Biopex®-R were implanted into tibiae of Japanese rabbits for 4, 8 and 24 weeks. The newly-formed bones were directly bonded with both IP6-HAp and Biopex®-R cements (Figs. 2 a, b). As for the IP6-α-TCP and IP6-β-TCP cements, newly-formed bones increased time-dependently around the cement specimens (Figs. 2 c, d). The results of TRAP stain indicated that osteoclasts were also present around the present cement specimen. The rates of bioresorbability for the IP6-α-TCP, IP6-β-TCP, Biopex®-R and IP6-HAp cements at 24 weeks after implantation were of 16.2, 13.7, 6.7 and 5.0%. DISCUSSION: The four kinds of cements were implanted into tibiae of Japanese rabbits for 4, 8 and 24 weeks. The newly-formed bones were directly bonded with both IP6-HAp and Biopex®-R cements. As for the IP6-α-TCP and IP6-β-TCP cements, newly-formed bones increased time-dependently around the cement specimens. The rates of bioresorbability for the IP6-α-TCP, IP6-β-TCP, Biopex®-R and IP6-HAp cements at 24 weeks after implantation were of 16.2, 13.7, 6.7 and 5.0%. We conclude that chelate-setting cements with various bioresorbability are useful as novel calcium-phosphate cements. ACKNOWLEDGMENT: A part of this work was supported by JSPS KAKENHI (21591905). REFERENCES: [1] Brown, W.E and Chow, L.C, Dental restorative cement pastes (US Patent No. 4518430, 1985). [2] Miyamoto, Y, Ishikawa, K, Takechi, M, Toh, T, Yuasa, T, Nagayama, M and Suzuki, K, J. Biomed. Mater. Res., 48, 36-42 (1999). [3] Aizawa, M, Haruta, Y and Okada, I, Arch. BioCeram. Res. 3, 134-138 (2003). [4] Horiguchi, Y, Yoshikawa. A, Oribe, K and Aizawa, M, J. Ceram. Soc. Jpn. 116, 50-55 (2008). [5] Konishi, T, Horiguchi, Y, Oribe, K, Matsumoto, M, Morisue H, Toyama, Y and Aizawa M, Arch. BioCeram. Res., 8, 126-129 (2008). [6] Nishiyama, K, Takahashi, S, Mizumoto, M, Oribe, K, Matsumoto, M, Morisue, H, Toyama, Y and Aizawa, M, Bioceramics, 22, 871-874 (2009).

Fig. 2 Histological evaluation of four kinds of cements with HE stain (24 weeks implantation). a) Biopex®-R, b) IP6-HAp, c) IP6-α-TCP, d) IP6-β-TCP, I: Implant, N: Newly-formed bone

Fig. 1 XRD patterns of starting powders after surface modification with IP6 a) IP6-HAp, b) IP6-α-TCP, c) IP6-β-TCP ●: HAp, ■: α-TCP, ▲: β-TCP

Poster No. 1834 • ORS 2011 Annual Meeting