Bone Induction of Human Tooth and Bone Crushed by Newly Developed Automatic Mill

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 completely 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.

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. a. cart age between DDM, b. d rect bone ormat on on (0.420.85 mm) were superior to super fine (under 0.42 mm) DDM, c. cartilage in crack of DBM, d. bone and marrow between DBM. in ectopic bone and cartilage induction, because super fine granules were phagocyted by giant cells, and digested by enzymes.10) In the present study, human coarse DDM and DBM 3. Results (0.51.0 mm) induced bone and cartilage at 4 weeks in mouse 3.1 Size distribution of tooth- and/or bone-derived (Fig. 6). As the resource of recycled teeth are limited, there are granules after pulverization clinical needs that bone autograft must be accomplished with Pulverized tooth and bone granules were sieved to discrete DDM autograft for enough bone formation. In such cases, sizes offine (0.51.0 mm), moderate (1.02.0 mm), and large simultaneous crush of both tooth and bone should be needed. An (2.05.0 mm). The results summarized in Table1.Briefly, ina immediate application of DDM and DBM with BMP activity will human wisdom tooth (wet volume: mean 1.84 g), the distribution be achieved by using the automaticmill.Webelieve, therefore, offine granules, moderate, and large was 0.38, 0.45, and 0.08 g, that autograftof DDM may spread as an innovative dental respectively in dry weight. The granular distribution of a human therapy for patients, using the novel ZrO2 mill for bone bone block (wet volume: mean 2.23 g, 1.0 cm3) was fine (0.64 g), regeneration. moderate (0.42 g), and large (0.74 g) in dry weight. In the case of awisdom tooth and a cortical bone block (Fig. 5a), the 5. Conclusion distribution of dry granules was fine (1.91 g), moderate (1.33 g), The innovative ZrO2 mill for teeth and bone has been and large (0.31 g), respectively(Fig. 5b). developed first in the world. DDM and DBM prepared by the In 5 wisdom teeth (total wet volume: 10.0 g), the distribution new automaticmill induced bone and cartilage in mouse of the freeze-dried DDM granules was fine (0.27 g), moderate subcutaneous tissues. The automaticmill should be essential (0.44 g), and large (0.64 g). for the immediate autograftof dentin and bone for local bone engineering. The autograftof recycled DDM should make up for 3.2 Bioassay of lyophilized DDM or DBM in bone transplant and biomaterialimplant. mouse subcutaneous tissues DDM induced bone and cartilage independently at 4 weeks Acknowledgments This work was supported by grants of 2004- (Fig. 6a,b). Cartilage formation occurred in narrow spaces 5 METI, 2006-7 JST, and 2006-8 Hokkaido, Japan. We would like to between DDM granules. Induced bone was found on DDM thank Mr. Kumagai (Manager, NOASTEC), and Dr. Takoshima surfaces. Cell proliferation and differentiation occurred in spaces (Coordinator, JST) very much for the advice and manegements of l between DDM granules and on DDM surfaces. Almost DDM our research and deve opment. granules were not absorbed, while several giant cells were observed on DDM surfaces. i il References DBM nduced cart age, bone and marrow at 4 weeks 1) G. Bang and M. R. Urist, Arch. Surg., 94, 781789 (1967). i il f i f (F g. 6c,d). Cart age was ound n the crack-spaces o the 2) J. D. Yeomans and M. R. Urist, Arch. Oral Biol., 12, 9991008 granule. Active bone formation occurred between DBM gran- (1967). ules. DBM were partially replaced by induced bone, which 3) M. R. Urist, Science, 150, 893899 (1965). revealed mosaic-like structure. 4) M. Murata, D. Sato, T. Akazawa et al., J. Hard Tissue Biol., 11,

436 Journal of the Ceramic Society of Japan 118 [6] 434-437 2010 JCS-Japan

110114 (2003). 228232 (1977). 5) M. Murata, T. Akazawa, J. Hino et al., Arch. BioCeram. Res., 8) T. Kawai and M. R. Urist, J. Dent. Res., 68, 10691074 (1989). 5, 178181 (2005). 9) K. Ito, T. Arakawa, M. Murata et al., J. Hard Tissue Biol., 16, 6) M. Murata, J. Hino, T. Akazawa et al., Key Eng. Mater., 361– 199204 (2007). 363, 13271330 (2008). 10) A. H. Reddi and C. B. Huggins, Proc. Soc. Exp. Biol. Med., 43, 7) W. T. Butler, A. Mikulski and M. R. Urist, J. Dent. Res., 56, 634637 (1973).

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