Ability of Hydroxyapatite-Bone Morphologenetic Protein (BMP) Complex to Induce Dentin Formation in Dogs

Okajimas Folia Anat. Jpn., 70(5): 195-202, December, 1993 Ability of Hydroxyapatite-Bone Morphologenetic Protein (BMP) Complex to Induce Dentin Formation in Dogs By Fumihiko SUWA, Lianjia Yang, Yoshikuni OHTA, Yi-Ru FANG, Hiromi IKE and Taka-aki DEGUCHI Department of Anatomy, Osaka Dental University 5-31, Otemae 1-chome, Chuo-ku, Osaka 540, Japan Department of Oral Pathology, The Fourth Military Medical University, Faculty of Stomatology, Xian, China -Received for Publication, August 18, 1993- Key Words: Dentinogenesis, Osteogenesis, HAP, BMP, Dog Summary: This histological investigation examined the formation and differentiation of pulp cells under the influence of HAP/BMP complex. HAP/BMP complex was implanted in exposed pulp of the mandibular premolars and alveolar bone defect in the dog. Sequential changes in these areas were examined mainly under a light microscope and partly under a scanning electron microscope. Two weeks after the operation, fibroblast-like cells proliferated right beneath the implanted complex, and after 3 weeks, dentin including dentin tubules grew in the pulp. After four weeks a dentin bridge composed of osteoid dentin was found, and after 8 weeks this dentin calcified and covered the defective surface of the root and adhered to two types: tubular dentin and osteodentin. This complex exhibited outstanding ability to induce dentinogenesis and osteogenesis. In the embryo, dentinogenesis, which depends upon the differentiation of dental pulp cells, occurs after proliferation of the dental papilla and differentiation to odontoblasts. Cytogenetic and histogenetic influences are retained, and are capable of continuous dentin renewal throughout the postfetal life in a manner analogous to that of bone tissues (Cat 1989). There has been experimental evidence to show that mesenchymal cells are able to differentiate into odontoblasts without the influence of epithelial cells of the enamel organ when the dental pulp of the mature permanent tooth is injured. It has been suggested that some inductive factor(s) may exist in the epithelium of the enamel organ and the necrotic layer of the injured pulp (Chao-How 1978). However, the mechanisms of differentiation of dental pulp cells and the initiation of dentinogenesis have not been elucidated. In the past 20 years, knowledge about bone morphogenetic protein (BMP), including its purification, physiochemical characteristics and recombinant human BMP protein, has greatly increased (Wang 1988, Wozney et al. 1988, Wozney 1989). Dentinal BMP was extracted from human and bovine teeth by Kawai and Urist (1989) and Bessho et al. (1991). Their pioneering work demonstrated that BMP was clearly a protein stored in the dentin matrix and possibly in matrices of enamel and cement. However, much less progress has been made in the field of cellular differentiation in human and mammalian dental pulp cells. The effect of BMP complex on the differentiation of dental pulp cells has not been reported. The histological aspects of dentin formation and the differentiation of pulp cells developing under the influence of hydroxyapatite-bmp complex are described here. Materials and Methods 1. Dissociative extraction of BMP Partially purified BMP was obtained from a demineralized bovine bone which was prepared by sequential extractions, as shown in Table 1. The gelatinized insoluble bone matrix was chemically extracted with 4 M guanidine-hc1 (GuHC1)/0.5 M CaCl2, including 1.0 mm N-ethylmaleimide (NEM) for 24 hr at room temperature. This extract was dialyzed against distilled water for 3 days at 4 C, then the retentate was heated in the dialysis bags to 35 C each time gelling occurred, and centrifuged (40,000 x g, 20 min, at 4 C). The precipitate was redissolved in 4M GuHC1/0.05 CaC1,. The 4M GuHC1-soluble proteins were dialyzed in 0.25 M sodium citrate-citric acid buffer (ph 3.1). Following 195

196 F. Suwa et al. Table 1. Method of purifying BMP from decalcified bovine bones thick, and stained with hematoxylin and eosin for light microscopic examinations. In numerical order hr C Remarks Results dialysis, the retentate was heated to 40 C, and centrifuged (40,000 x g, 60 min, at 35 C). The precipitate was washed in distilled water and 1:1 chloroformmethanol, and lyophilized at 4 C. 2. Preparation of hydroxyapatite (HAP)/BMP complex BMP was redissolved in 4 M GuHC1, to which porous HAP (pores: 60-120iLtm in diameter) was added. This complex was dialyzed against distilled water 24 hr at 4 C and lyophilized. The dried HAP/ BMP complex was examined under a scanning electron microscope (accelerating voltage 20 kv). 3. Bioassay The HAP combined with BMP by dialysis was implanted in the interfascicular tissue (pouch) of a femoral muscle of the mouse. HAP alone was implanted in the muscle as a control. After 1, 2, 3 and 4 weeks the implanted region was dissected out with surrounding tissues, and immediately fixed in 10% formalin for histological examinations. 4. Dentin inductive activity of HAP/BMP complex in dogs Four male adult dogs (average body weight 22 kg) were empolyed for this experiment. After the dogs were anesthetized by an intramuscular injection of sodium pentobarbital (Nembutal 0.5 ml/kg), the unilateral mandibular premolars were cut at the cemento-enamel junction. Alveolar ridges were reduced 2-3 mm in height. The HAP/BMP complex was implanted in the exposed pulp and bone defect, and the wound was closed by gingival ligation. HAP alone was implanted in defects of the opposite mandibular premolars as a control. Two, 3, 4 and 8 weeks after operation, the dogs were killed and the implanted areas were dissected out, fixed in 10% formalin, embedded in paraffin, sectioned at 5 [inn 1. Osteoinductive activity of HAP/BMP complex Scanning electron microscopy revealed that the surface of the complex was covered with BMP. Pores of HAP were occupied by a large amount of BMP (Fig. 1). Seven days after implantation of the complex into the interfascicular tissue, many mesenchymal cells proliferated around the granules of the complex. A few lymphocytes could be found. At 14 days, no new bone formation was found, but the mesenchymal cells had begun to differentiate into osteoblasts. By three weeks, a thin layer of new bone rimming the HAP granules was found. By four weeks, the new bone had become thickened, and new bone formation was also seen in the center of the granules (Fig. 2). In the control group, new bone formation was not found at this time (Fig. 3). 2. HAP/BMP-induced dentin formation in dogs One week after implantation, dental pulps were completely capped with HAP/BMP complex and were free of inflammation. A thin, necrotic layer was found on the surface of the exposed pulp. By two weeks, fibroblast-like cells were proliferating in a wide area just beneath the HAP/BMP complex, but a dentin bridge had not formed. By three weeks, regular dentin was induced in the pulp, and many dentin tubules in the shape of normal dentin were observed (Fig. 4). Odontoblasts were observed around the newly formed dentin. From the fourth week after the operation, the rate of dentin formation increased, and a dentin bridge composed of osteodentin without any dentin tubules was formed (Fig. 5). By eight weeks, the osteodentin bridge had calcified. Newly formed bone covered the surface of the dentin defect of the root and adhered to the new bone rimming the HAP granules (Fig. 6). In the control group with HAP alone, a dentin bridge was not found by eight weeks. The granules of the complex were surrounded by a fibrous tissue (Fig. 7). Discussion It is well-known that BMP is an efficient boneinducing protein, and may induce the differentiation of mesenchymal cells into chondro- and osteocytes in soft tissues and bone defect (Urist et al. 1983). Implantation of BMP in a thigh muscle pouch of the mouse consistently induces new cartilage formation

Ability of HAP-BMP Complex to Induce Dentin Formation 197 by the 7th day, woven bone by the 14th day, and lamellar bone by the 21st day (Urist et al. 1984). But BMP extracted from bone matrix by chemical methods is limited in amount. After implantation of BMP alone, it dissolves readily in the tissue fluid. In recent studies on the development of new implantable biomaterials as bone substitutes in the reparation of bone defect, BMP bound to 3-TCP, HAP and human fibrin have been investigated (Urist et al. 1979, Kawamura et al. 1988, Mieki 1990). Mieki (1990) reported that the osteoinductive activity of HAP/BMP complex was higher than that of BMP alone. A porous HAP combined with BMP by dialysis was used for our experiment. The results revealed that the complex induced new bone formation around the granules of the material by the 21st day. Proliferation and differentiation of mesenchymal cells were observed before new bone formation. Since new bone is induced in the center of the granules after three weeks, the complex retains higher osteoinductive activity. Because the differentiation and development of odontoblasts, osteo- and chondrocytes are essential to both new dentin and bone formation, investigators have been looking for a substance or factor that can induce the differentiation of odontoblasts. Some researchers (Sampath et al. 1983, Urist et al. 1984) found that BMP was present not only in the bone matrix of rats, pigs and humans, but also in the dentin of rabbits and humans. Yang and Yan (1990) demonstrated that BMP might be found in human tooth anlages such as human predentin, and cells of the outer and inner enamel epithelium. It may be possible that BMP synthesized by the inner enamel epithelium is secreted into the pulp and thus induces the mesenchymal cells of the pulp to differentiate into odontoblasts, which produce the dentin matrix. There has been experimental evidence to indicate that crude BMP may induce the differentiation of pulp cells into odontoblasts in vivo and in vitro (Nakashima 1992). The dentins induced by HAP/BMP complex in the present study were of two types: osteodentin and regular tubular dentin. This feature may be related to the characteristics of the BMP that penetrates into the pulp tissue. Each particle of BMP may become the center of differentiation of dental pulp cells. Those cells adjacent to the BMP acquire sufficient stimulation for differentiation into typical odontoblasts, which secrete regular tubular dentin. But many pulp cells are induced by the complex to differentiate into immature odontoblasts, which produce the osteodentin. In the present experiment, the osteodentin bridge induced by HAP/BMP complex was produced over a time period that was more rapid than in the control group. Therefore, it can be said that this complex could be used as a substitute for the existing commonly used capping agents. On the other hand, the osteodentin bridge induced by HAP/BMP complex repairs not only pulp but also the dentin defect of roots (Fig. 6). The development of alveolar bone depends upon the growth of the tooth root. Alveolar atrophy occurs without sufficient stimulation of the root, as suggested by Cat (1989). This experiment provides further evidence that the dental pulp and dentin defect of the root are repaired by osteodentin, and thereby the living root can be retained in the alveolar bone. It, therefore, is possible to prevent the atrophy of alveolar bone. Literature cited 1) Bessho, K., Tanaka, N., Matsumoto, J., Tagawa, T. & Murata, M.: Human dentin matrix-derived bone morphogenetic protein. J. Dent. Res., 1991;70(3):171-175. 2) Cat, A. R. T.: Oral Histology. Development, Structure and Function. 3rd ed. C. V. Mosby Co., 59-79, 139-156. 1989. 3) Chao-How, C.: An experimental autoradiographic study on healing of pulp wound following pulpotomy in dogs with tritiated thymidine. Shikwa Gakuho 1978;78:287-310. (in Japanese) 4) Kawamura, M., Urist, M. R.: Human fibrin is a physiologic delivery system for bone morphogenetic protein. Clin. Orthop., 1988;235:302-310. 5) Kawai, T., Urist, M. R.: Bovine tooth-derived bone morphogenetic protein. J. Dent. Res., 1989;68:1069-1074. 6) Mieki, A.: Bone inductive activity of13-tricalcium phosphatebone morphogenetic protein complex. Aichi Gakuin Daigaku Shigakkai Shi, 1990:28:43-58. (in Japanese) 7) Nakashima, M.: Mitogenetic and dentin-inductive effects of crude bone morphogenetic protein from bone and dentin in primary adult pulp cell culture. Oral Surg. Oral Med. Oral Athol., 1992;73(4) :484-489. 8) Sampath, T. K., Reddi, A. H.: Homology of bone-inductive protein from human, monkey, bovine, and rat extracellular matrix. Proc. Natl. Acad. Sci. USA, 1983;80:6591-6595. 9) Urist, M. R., Mukulski, A., Lietze, A.: A solubilized and insolubilized bone morphgenetic protein. Proc. Natl. Acad. Sci. USA, 1979;76:1828-1832. 10) Urist, M. R., Lietze, A., Dawson, E.: P-tricalcium phosphate delivery system for bone morphogenetic protein. Clin. Orthop., 1984;187:277-280. 11) Urist, M. R., Huo, Y. K., Brownell, A. G., Hohl, W. M.. Buyske, J., Lietze, A., Tempst, P., Hunkapillcr, M.. Delange, R.J.: Purification of bovine bone morphogenctic protein by hydroxyapatite chromatography. Proc. Natl. Acad. Sci. USA, 1983;81:371-375. 12) Wang, E. A., Rosen, V., Cordes, P., Hewick, R. M., Kriz, M. J., Luxenberg, D. P., Sibley, B. S., Wozney, J. M.: Purification and characterization of other distinct bone-inducing factors. Proc. Natl. Acad. Sci. USA, 1988:85:9484-9488. 13) Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M., Wang, F. A.: Novel regulators of bone formation: Molecular clones and activities. Science, 1988;242:1528-1534. 14) Wozney, J. M.: Bone morphogenetic proteinṣ Prog. Growth Factor Res., 1989;1(0267-280. 15) Yang, L., Yan, J.: Immunohistochemical observations

198 F. Suwa et al. on bone morphogenetic protein in normal and abnormal conditions. Clin. Orthop., 1990257:249-256. Explanation of Figures Plate I Fig. la. Fig. lb. Porous HAP. Surfaces of HAP are combined with BMP. Pores (GB) are occupied by BMP. Fig. 2. Fig. 3. Four weeks after implantion of HAP/BMP complex, new bone (arrows) is rimming the granules of the complex. HE staining x 66. Four weeks after implantion of the granules of HAP, as a control group, the granules of HAP are covered with fibrous tissue (arrow). HE staining x 66.

Ability of HAP-BMP Complex to Induce Dentin Formation 199 Plate I

200 F. Suwa et al. Plate II Fig. 4. Three weeks after implantation of HAP/BMP complex in the dog pulp, regular dentin (arrows) is observed. HE staining x 13. Fig. 5. Fig. 6. A dentin bridge composed of osteodentin (0) is formed by 4 weeks after the operation. D: normal dentin, H: HAP granules. HE staining x 66. By 8 weeks, the osteodentin bridge (B) is calcified. New bone covers the cutting surface of the root dentin (D) and continues to new bone (arrows) of HAP granules (H). HE staining x 33. Fig. 7. In the control group, new bone formation is not observed at 8 weeks. H: HAP granules HE staining x 33.

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