Journal of the Ceramic Society of Japan 118 [6] 434-437 2010 Paper Bone induction of human tooth and bone crushed by newly developed automatic mill Masaru MURATA,³ Toshiyuki AKAZAWA,* Masahiko TAKAHATA,** Manabu ITO,** Junichi TAZAKI,*** Jun HINO,*** Katsuo NAKAMURA,* Norimasa IWASAKI,** Takanori SHIBATA*** and Makoto ARISUE Oral and Maxillofacial Surgery, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, Hokkaido 061–0293 *Materials Technology, Hokkaido Industrial Research Institute, Kita 19 Nishi 11, Kita-ku, Sapporo, Hokkaido 060–0819 **Orthopaedic Surgery, Hokkaido University Graduate School of Medicine, Kita 14 Nishi 7, Kita-ku, Sapporo, Hokkaido 060–8586 ***Reconstructive Surgery for Oral and Maxillofacial Region, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, Hokkaido 061-0293 A novel automaticmill of teeth and bone has been developed for bone engineering. A human frozen-tooth and/or a human frozen-bone block were put into the Zirconium oxide (ZrO2) ceramics vessel of the machine, and crushed for 1 minwith 20 saline- 3 ice blocks (1 © 1 © 1cm /block) at 12000 rpm of ZrO2 blade. The crushed granules were demineralized completely in2% HNO3 solution for 20 min, and rinsed incoldsaline. We named each biomaterial after the acid treatment and washing, demineralized dentin matrices (DDM), demineralized bone matrices (DBM). Five wisdom teeth (total wet volume: 10.0 g) were crushed, decalcified, and lyophilized. The distribution offreeze-dried DDM granules was fine granules (0.5­1.0 mm: 0.27 g), moderate (1.0­2.0 mm: 0.46 g), and large (2.0­5.0 mm: 0.64 g). The fine granules of human DDM or DBM were implanted into the subcutaneous tissue of 4 week-old nude mice, and theirtissue-inductive properties were estimated at 4 weeks after implantation histologically. The explanted samples were demineralized, embedded in paraffin, and sectioned. The specimens were stained with hematoxylin and eosin. We confirmed that DDM induced bone and cartilage independently, and DBM induced cartilage, bone and marrow at 4 weeks in the back skinof nude mice. These results indicated that our material preparation system by the novel mill with a vessel and a blade of ZrO2 under ice-cooling maintained the bone-inducing activity of human dentin and bone. ©2010 The Ceramic Society of Japan. All rights reserved. Key-words : Human, Dentin, Bone, Demineralization, Bone induction, Crush [Received February 9, 2010; Accepted April 15, 2010] 1. Introduction 2. Experimental procedures Bone-inducing property in rabbit dentin was confirmed in the 2.1 Conventional hand-operated method using intramuscular pockets in 1967,1),2) after the discovery of bone order-made stainless steel apparatus induction by rabbit demineralized bone matrices (DBM).3) The We designed a hand-operated apparatus for crushing tooth rabbit studies reported that demineralized dentin matrics (DDM) and especially ordered to make a stainless steal vessel and a induced bone at 4 weeks, while non-demineralized dentin bar.4) Frozen tooth was crushed by hammer in liquidnitrogen (so-called, calcified dentin) induced bone at 8­12 weeks after (Fig. 1). implantation.2) We confirmed histologically that human com- pletely demineralized dentin granules by hand-made method using hammer induced bone and cartilage independentlyat4 weeks,4),5) and have obtained successful results inclinical studies of the autograftof DDM for bone regeneration.6) The preparation of DDM granules by the conventionally hand-made method needs a very complicated process and take one day. Until now, there isnomill that isclinically available for teeth. For standardization of DDM autograft, the automaticmill will be needed in the clinicalfields. The purposes of this study are to develop an automaticmill for teeth and bone, and to estimate the bone-inducing capability of human tooth-derived dentin and human bone-derived granules after automatic crushing with our newly developed mill, histologically. ³ Corresponding author: M. Murata; E-mail: murata@hoku-iryo-u. Fig. 1. Stainless steel apparatus. a. parts of apparatus, b. aspect, ac.jp c. frozen tooth, d. adding ofliquidN2, e. tooth-derived granules. 434 ©2010 The Ceramic Society of Japan Journal of the Ceramic Society of Japan 118 [6] 434-437 2010 JCS-Japan Fig. 2. Automaticmill. a. aspect, b. ZrO2 vessel and blade, c. tooth i i i l i l f i w th ces n vesse , d. sett ng, e. granu es a ter crush ng. Fig. 4. Preparation protocol of DDM and DBM. Fig. 3. Tooth-derived granules before and after acid-demineralization. Fig. 5. Granules distribution of tooth and bone crushed simultaneously a. tooth-derived granules before demineralization, b. freeze-dried DDM, in new mill. a. tooth and bone with 20 ices before crushing, b. dry c. SEM of DDM, d. Higher magnification of c. granules (total weight: 3.55 g) after crushing and sieving, c. SEM of DBM, d. Higher magnification of c. 2.2 Development of automatic mill machinery and Sciences University of Hokkaido. Human bone was obtained new speedy method from a patient who underwent total hipjoint replacement surgery. Zirconium oxide (ZrO2) ceramics was selected as a material The resected femoral head was kept in the freezer at ¹80°C until of vessel and blade, because ZrO2 have gained the approval use. Cartilage and other softtissues were completely removed of Food and Drug Administration (FDA) for human use. from the sample, and spongy bone was mainly used for the We have been developing an automaticmill (International experiment. Patient was informed and gave consent, and the application No. PCT/JP2007/053321, International published experimental protocol was approved by Hokkaido University No. WO2007/099861 A1), (Fig. 2). Briefly, vessel and blade Hospitalinstitutional review board. were made in ZrO2 (Fig. 2b), and the ZrO2 ceramics were The human, adultthird molar tooth (wet volume: mean 1.84 g) fabricated by sintering at 1400°C for 2 h after the slip casting of and/or bone (wet volume: mean 2.23 g) were crushed for 1 minat the mixture of ZrO2 powder and distilled water. As the results 12000 rpm with 20 saline-ice blocks, using the automaticmill, of characteristics analyses of ZrO2 objects, the contraction rate, and analyzed the granule-size distribution. After demineraliza- the relative density, and the bending strength were 21%,99%, tion of tooth- or bone-derived granules in2% HNO3 solution and 400 MPa, respectively. The automaticmill could crush a (pH 1.0) for 20 min, lyophilization and sieving (Fig. 4), we tooth and/or a cortical bone block (1 © 1 © 1cm3) for 1 minat named each demineralized matrics, DDM (Fig. 3c,d) and DBM 12000 rpm under the condition of cooling by 20 saline ice blocks (Fig. 5c,d). (1 © 1 © 1cm3/block) (Figs. 2, 3, 4, 5). 2.4 Bioassay 2.3 Preparation of DDM and DBM granules Twenty mg of the lyophilized DDM or DBM granules (0.5­ Human third molar teeth were obtained from adult patients 1.0 mm) were implanted into the subcutaneous tissues of 4 week- who underwent tooth extraction. Extracted teeth were kept in the old nude mice, and then removed at 4 weeks after the implanta- freezer at ¹80°C until use. Patients were informed and gave tion. All procedures were followed the Guidelines in Health consent, and the experimental protocol was approved by Health Sciences University of Hokkaido for Experiments on Animals. 435 JCS-Japan Murata et al.: Bone induction of human tooth and bone crushed by newly developed automatic mill Table 1. Size distributlon of granules 4. Discussion Dry weight (g) Sample l lli ill il l l f <0.5 0.5­1.0 1.0­2.0 2.0­5.0 >5.0 mm Severa meta cm s have been ava ab eony or bone, not f ill i i or teeth. The bone m s are hand-made type, not automat c. We 1wsdom tooth N.D 0.38 0.45 0.08 0.0 fi i ll il l ill f li i l 5wisdom teeth N.D 0.27 0.44 0.64 0.0 con rmed that the commerc a y ava ab e bone m s or c n ca l il l l 1 bone block N.D 0.64 0.42 0.74 0.0 use cou d not crush a human tooth. Unt our deve opmenta 1 tooth and l bone N.D 1.91 1.33 0.31 0.0 researches, there was no idea for clinical use of human teeth, especially dentin. As ZrO have been approved medical material Value = mean, N = 3­5, N.D: not determined 2 for human use in Japan and FDA, ZrO2 have been used as ceramic material widely in the dental and orthopedic fields. ZrO2 is very strong against corrosion and destruction. In this study, the automatic ZrO2 mill for teeth and bone was first developed in the world. In addition, we confirmed human DDM and DBM induced bone and cartilage (Fig. 6). The histological results indicated that DDM and DBM maintained the hard tissue-inducing capability even after the several preparation processes with the developed machine. DDM and DBM can be defined as acid-insolublecollagen binding bone morphogenetic proteins (BMPs), which are member of transforming growth factor-beta (TGF-¢) super-family. BMPs were discovered from bone and had bone-inducing property.3) Dentin BMPs were identified from rat incisor7) and bovine tooth.8) We reported that both mature and immature types of BMPs existed in human dental pulps.9) It is noteworthy that proper granules (size 0.5­2.0 mm) for bone induction couldbemainlygained after pulverization Fig. 6. Histologicalfindings of DDM and DBM at 4 weeks after using our mill (Table 1). It’s known that coarse DBM granules i l i il i f i mp antat on.