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Effect of nano-hydroxyapatite on bone morphogenetic protein-2-induced hard tissue formation and dentin resorption on a dentin surface
Applied Surface Science 262 (2012) 140–145
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Effect of nano-hydroxyapatite on bone morphogenetic protein-2-induced hard tissue formation and dentin resorption on a dentin surface Hiroki Tamagawa, Taichi Tenkumo, Tsutomu Sugaya ∗ , Masamitsu Kawanami Department of Periodontology and Endodontology, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan
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Article history: Available online 28 March 2012 Keywords: Nano-hydroxyapatite rhBMP-2 Hard tissue formation Dentin resorption
a b s t r a c t Aim: The purpose of this study was to evaluate the effects of the addition of nano-hydroxyapatite to a collagen membrane-carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) on hard tissue formation and dentin resorption on dentin surfaces in vivo. Materials and methods: Nano-hydroxyapatite collagen composite (nHAC) membranes or collagen (C) membranes were each immersed in either 100 or 400 g/ml rhBMP-2 and placed on dentin chips that were implanted into rat thigh muscle. The implants were analyzed at 2 or 4 weeks after surgery by histological observation and histomorphometric analysis. Results: The percentage of the hard tissue formed by each nHAC group was significantly higher than that formed by any of the C groups, except for that formed by the group loaded with 400 g/ml rhBMP2 at 4 weeks after implantation. No significant differences were observed in the percentage of dentin resorption between the nHAC groups and C groups at any stage or at any rhBMP-2 concentration. Conclusion: These findings showed that addition of nano-hydroxyapatite to a collagen membrane accelerated the formation of hard tissue induced by a low dose of rhBMP-2 on dentin surfaces at an early stage after implantation into rat thigh muscle, without increasing dentin resorption. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The periodontium consists of the supporting tissues of the tooth (cementum, alveolar bone, and periodontal ligament (PDL)) and gingiva. The root cementum is the thin calcified tissue that covers the root dentin surfaces and has many features in common with bone tissue. The periodontal ligament joins the root cementum with the alveolar bone through collagen fiber bundles. The periodontium attaches the tooth to the alveolar bone and transmits occlusal forces to the bone, and its main function is to provide resistance to the impact of occlusal force. The periodontium is broken in periodontal disease, after bacteria have formed a biofilm on the cementum surface. Periodontal therapy to obtain a healthy periodontium removes these biofilms and the infected cementum by mechanical means. However, following periodontal therapy, the dentin surface of the tooth root is exposed and periodontium is not regenerated. A critical step toward achieving periodontal regeneration is the new attachment of connective tissue fibers to the tooth root surface. Root cementum plays an important role in this process, because it securely attaches the periodontal ligament fibers to the root
∗ Corresponding author. Tel.: +81 11 706 4264; fax: +811 11 706 4334. E-mail address: [email protected] (T. Sugaya). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.03.103
surface. Therefore the formation of cementum on dentin surfaces of the tooth root is necessary for regeneration of the periodontium [1]. However treatment that can successfully and efficiently induce the regeneration of cementum has not yet been developed. Application of the bone morphogenetic protein-2 (BMP-2) to the periodontium is known to induce bone and cementum formation. We previously reported that recombinant human BMP-2 (rhBMP2) application to dentin surfaces also enhanced new cementum-like tissue formation on the dentin surfaces in palatal connective tissue that had low ability to form hard tissue [2]. However application of rhBMP-2 also resulted in concurrent dentin resorption [2–6]. We reported that rhBMP-2 application to dentin surfaces of a beagle induced not only new cementum formation but also dentin resorption and ankylosis of dentin surfaces [4,5]. Furthermore, we reported that the ratio of dentin resorption and cementum-like tissue formation was influenced by the concentration of rhBMP-2; a high concentration of applied BMP-2 not only induced more hard tissue formation but also more dentin resorption than that of a low concentration [2,3,6]. Therefore, inhibition of dentin resorption and the formation of new cementum on dentin surface are important challenges that need to be overcome to achieve periodontal regeneration. Hydroxyapatite is the main mineral component of bones and teeth, and is known to have high capacity for protein adsorption as well as high biocompatibility and osteoconductivity [7–10].
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We previously reported that incorporation of nano-hydroxyapatite (nHap) into a collagen membrane rhBMP-2 carrier enhanced ectopic bone formation when implanted in rat thigh muscle [11]. Based on this result, we assumed that the combination of a lowconcentration of rhBMP-2 with this nano-hydroxyapatite collagen composite membrane could form a cementum-like hard tissue on dentin surfaces. Therefore, in this study, we investigated the effect of nHap incorporation into a collagen membrane rhBMP-2 carrier on cementum-like tissue formation and dentin resorption on dentin surfaces in rat thigh muscle by histological observation and histomorphometric analysis. 2. Materials and methods 2.1. Preparation of mineralized nano-hydroxyapatite composite membranes and collagen membranes The nHap-collagen composite (nHAC) membrane was prepared from biomimetically mineralized collagen type I according to a method described by Gelinsky et al. [12] Briefly, 1 mg/ml purified type I atelocollagen gel (2 wt.%, Koken Co., Tokyo, Japan) was dissolved in 0.01 M HCl at a temperature of 4 ◦ C and mineralized in KH2 PO4 /K2 HPO4 and Tris buffer in the presence of CaCl2 . The reaction mixture was incubated for 12 h at 37 ◦ C, resulting in a white precipitate of mineralized collagen that was collected by centrifugation. The collagen (C) membrane was prepared similarly but without CaCl2 in the reaction. The collagen was dissolved at a concentration of 0.4 mg/ml in 0.01 M HCl at a temperature of 4 ◦ C. Fibril formation was induced by mixing with an equal amount of neutralization buffer. A membrane-like material was obtained through vacuum filtration and a cross-linking process using an 1% aqueous solution of 1-ethyl-3-(3-dimethylamiopropyl) carbodiimide hydrochloride (EDC; Kanto Chemical, Tokyo, Japan) for 1 h. The remaining activated carboxy groups were quenched with glycine. Following extensive washing with distilled water, the materials were freeze-dried. nHAC and C membranes were analyzed by SEM (S-4000; Hitachi, Tokyo, Japan) observation. The membranes used in the animal study were sterilized using ultraviolet ray irradiation. 2.2. Histological analysis of an rhBMP-2-loaded collagen composite with or without nHap 2.2.1. Application of rhBMP-2 to membranes After washing with phosphate-buffer saline (PBS), nHAC and C membranes (2 mm × 2 mm × 0.1 mm) were immersed in PBS containing 1000 U/ml penicillin and 1000 g/ml streptomycin for 24 h. Subsequently the nHAC and C membranes were rinsed with PBS and desiccated. The nHAC and C membranes were then each immersed in 100 or 400 g/ml rhBMP-2 (R&D, USA) for 10 min, as described by Tenkumo et al. [13]. The rhBMP-2 doses used were based on previous study [3]. The prepared membranes were classified into 4 groups: C100 (C membrane with 100 g/ml rhBMP-2), C400 (C membrane with 400 g/ml rhBMP-2), H100 (nHAC membrane with 100 g/ml rhBMP-2), and H400 (nHAC membrane with 400 g/ml rhBMP-2). 2.2.2. Preparation of dentin chips Dentin chips were extracted from rat tooth roots and the cementum was removed. Sixty-four flat dentin chips were cut into pieces of 2 mm × 2 mm × 0.3 mm in size. These flat dentin chips were treated for 3 min with 24% EDTA (pH 7.0), and were then incubated in 1000 U/ml penicillin and 1000 g/ml streptomycin overnight, as described in previous studies.
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2.2.3. Surgical procedures Thirty-two Wistar male rats (10 weeks old) were used for this experiment in accordance with the guide for the care and use of laboratory animals of Hokkaido University. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (7.0 × 10−2 ml/kg body weight; Kyoritsu Seiyaku Co., Tokyo, Japan). The prepared dentin chips on which the nHAC or C membranes had been placed were implanted into both the left and the right thigh muscles of the rats. 2.2.4. Histological observation and histomorphometric analysis The rats were sacrificed using an overdose of sodium pentobarbital at 2 or 4 weeks after implantation, and the tissue surrounding the implants was excised from the subcutaneous tissue in the left and right thigh muscle of the rats, as described by Tenkumo et al. [13]. The tissue was fixed in 10% formalin, decalcified for 4 weeks in 10% EDTA (pH 7.0), and then embedded in paraffin. The tissue sections (5 m thick) were stained with hematoxylin and eosin (H&E) and their histology was examined under a light microscope. 2.2.5. Statistical analysis Three sections were stained with H&E; one section was obtained from the center of the dentin chip and the other two sections were obtained at a distance of 150 m from either side of the center. After the length of newly formed hard tissue on dentin surfaces was measured by using ImageJ (NIH, Bethesda, MD, USA), the percentage of the new hard tissue that had formed was calculated as a percentage of newly formed hard tissue to the length of the surface on which the nHAC or C membranes had been placed. The percentage of resorbed dentin surfaces was similarly measured and calculated. Statistical differences between each group were analyzed using Mann–Whitney U-tests with SPSS 10.0J® (IBM, Tokyo, Japan). 3. Results 3.1. SEM observation of nHAC and C membranes SEM analysis of nHAC membranes indicated a porous structure with a pore size of 1.5–2.5 m. A large amount of apatite crystals that formed needle- or plate like-shapes 50–150 nm in size on collagen fibrils was observed (Fig. 1a). Analysis of the C membranes indicated a dense structure with a small number of pores. The topography of the collagen surface was flatter than that of the nHAC surface (Fig. 1b). 3.2. Histological observation of implanted membranes Two and 4 weeks after implantation of the rhBMP-2-loaded membranes on dentin chips into a rat thigh muscle, the implants and surrounding tissue were excised and histologically observed. Severe inflammatory changes such as necrosis and infections were not detected in any of the groups. The wound had healed well, and invasion of epithelial cells and wound dehiscence were not observed. 3.2.1. Analysis of membranes loaded with 100 g/ml rhBMP-2 In the C100 group at 2 weeks, most of the dentin surface was surrounded by fibrous connective tissue which contained a few blood vessels. A small amount of cementum-like hard tissue formation including cementocyte-like cells was observed (Fig. 2a). A few cuboidal or oval cells that stained with hematoxylin were observed on newly formed cementum-like hard tissue and on the dentin surface. A little dentin resorption was observed, and multinuclear giant cells were often observed at dentin resorption lacunas. Most of the
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Fig. 1. SEM images of the microstructure of nHAC membranes (a) and C membranes (b).
C membranes had been degraded, but some membrane fragments persisted. In the C100 group at 4 weeks, thin cementum-like hard tissue formation and dentin resorption were discontinuously observed on the dentin surface (Fig. 2b). Dentin resorption lacunas and newly formed cementum-like hard tissue was surrounded by fibrous tissue that was enriched with flat cells. No C membrane remnants were observed. In the H100 group at 2 weeks, most of the dentin surface was surrounded by fibrous cell-rich tissue. Extensive, newly formed cementum-like hard tissue including cementocyte-like cells was observed on the dentin surface, which displayed a continuous, long, thin shape of a uniform thickness (Fig. 2c). Cuboidal cells that stained with hematoxylin were often observed on the newly formed cementum-like hard tissue and on the dentin surface. A few multinuclear giant cells were observed on dentin resorption lacunas. nHAC was degraded into fragments, and numerous multinuclear giant cells were observed around these fragments. In the H100 group at 4 weeks, the dentin surface was covered with newly formed thin cementum-like hard tissue of a uniform thickness, and cuboidal cells stained with hematoxylin were observed on the newly formed cementum-like hard tissue (Fig. 2d).
Multinuclear giant cells were observed around the nHAC, but not, however, on dentin resorption lacunas. nHAC were degraded into smaller fragments than those observed at 2 weeks, and no hard tissue formation was observed around the nHAC.
3.2.2. Analysis of membranes loaded with 400 g/ml rhBMP-2 In the C400 group at 2 weeks, a small amount of new cementumlike hard tissue had formed on the dentin surface. Dentin resorption was frequently observed (Fig. 3a). Multinuclear giant cells were often observed at dentin resorption lacunas. Most of the dentin surfaces without cementum-like hard tissue formation or dentin resorption were lined by cuboidal cells that stained with hematoxylin, and were surrounded by connective tissue. In the C400 group at 4 weeks, extensive new cementum-like hard tissue and resorption were observed on the dentin surface (Fig. 3b). New thin cementum-like hard tissue, including cementocyte-like cells, was observed. Fibroblast-like flat cells that stained with hematoxylin were observed on newly formed cementum-like hard tissue and on the dentin surface. No C membrane remnants were observed. The dentin surface was surrounded by fibrous connective tissue.
Fig. 2. Tissue responses to C or nHAC membranes with 100 g/ml rhBMP-2. Tissue responses were analyzed by H&E staining. Responses to C membranes at 2 (a) or 4 (b) weeks after implantation or to nHAC membranes at 2 (c) or 4 (d) weeks after implantation are shown. In nHAC groups, newly formed cementum-like hard tissue was observed on the dentin surface, which displayed a continuous, long, thin shape of a uniform thickness. D: dentin, M: muscle, C: collagen membrane, N: nHAC membrane, arrow heads: new cementum-like hard tissue, *: dentin resorption. Bars indicate 50 m.
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Fig. 3. Tissue responses to C or nHAC membranes with 400 g/ml rhBMP-2. Responses to C membranes at 2 (a) or 4 (b) weeks after implantation or to nHAC membranes at 2 (c) or 4 (d) weeks after implantation are shown. Dentin resorption was frequently observed in both groups. In nHAC groups, newly formed hard tissue on the dentin surface exhibited irregular shape. D: dentin, M: muscle, N: nHAC membrane, BM: bone marrow-like tissue, arrow heads: new cementum-like hard tissue, arrows: new hard tissue, *: dentin resorption. Bars indicate 50 m.
In the H400 group at 2 weeks, extensive, newly formed hard tissue including osteocyte-like cells was observed on the dentin surface (Fig. 3c). The shape of this newly formed hard tissue on the dentin surface was different than that of cementum and was of a non-uniform thickness. Flat or cuboidal cells that stained with hematoxylin were observed on the newly formed hard tissue. Multinuclear giant cells were often observed on the dentin surface. nHAC was degraded into fragments, and numerous multinuclear giant cells were observed around these fragments. The dentin surface and the nHAC were surrounded by cell-rich tissue. In the H400 group at 4 weeks, newly formed hard tissue on the dentin surface exhibited a more irregular shape and some of this tissue included bone marrow-like tissue (Fig. 3d). Extensive dentin resorption was observed, and multinuclear giant cells were observed at resorption lacunas. nHAC was degraded into fragments that were smaller than those observed at 2 weeks. Furthermore, bone-like hard tissue was observed around these fragments, and some of this hard tissue had fused with the newly formed hard tissue on the dentin surface. The dentin surface and the nHAC were surrounded by cell-rich tissue.
3.3. Histomorphometric analysis The average percentage of newly formed hard tissue in the C100, H100, C400 and H400 groups was 6.5 ± 3.4%, 27.7 ± 6.8%, 2.3 ± 1.6% and 34.9 ± 5.2% at 2 weeks, and 12.8 ± 2.5%, 37.7 ± 6.5%, 32.3 ± 5.5% and 37.9 ± 7.7% at 4 weeks, respectively (Fig. 4a). The average percentage of new hard tissue that was formed in the H100 group was significantly higher than that of the C100 group (p < 0.05) at 2 and 4 weeks, respectively. The average percentage of new hard tissue that was formed in the H400 group was significantly higher than that of the C400 group (p < 0.05) at 2 weeks but not at 4 weeks. The average percentage of new hard tissue that was formed in the C100 (p < 0.05) and the C400 (p < 0.05) groups at 4 weeks was significantly higher than that of the C100 and C400 groups, respectively at 2 weeks. No significant difference in the average percentage of hard tissue that was formed was observed between the H100 and the H400 group at any time or at any concentration.
The average percentage of the dentin surface that was resorbed in the C100, H100, C400 and H400 groups was 1.6 ± 1.0%, 4.6 ± 3.3%, 6.2 ± 4.1% and 4.6 ± 2.9% at 2 weeks, and 4.7 ± 3.2%, 4.7 ± 1.7%, 12.9 ± 3.8% and 10.8 ± 4.2% at 4 weeks, respectively (Fig. 4b). No significant differences were observed between the nHAC groups and the C groups at any stage or at any rhBMP-2 concentration. In the H400, C100 and C400 groups at 4 weeks, the average percentage of the resorbed dentin surface was significantly higher than that of the same group at 2 weeks. However, no significant difference in this percentage was observed for the H100 group between 2 weeks and 4 weeks. The average percentage of dentin resorption was significantly higher in the C400 group at 2 weeks than in the C100 group at 2 weeks (p < 0.05), but at 4 weeks the average percentage of dentin resorption of the C400 and H400 groups was significantly higher than that of the C100 (p < 0.05) and H100 (p < 0.05) groups, respectively. 4. Discussion SEM observation of the nHAC group indicated that crystals were present on the surface of the reassembled collagen fibrils. These crystals were identified as nanocrystalline hydroxyapatite in a previous study in which the same material was used. Therefore, the crystals observed in this study can be identified as nHap. In this study, using histological observation and histomorphometric analysis, we evaluated the effect of the addition of nHap to a collagen membrane-rhBMP-2 carrier on hard tissue formation and dentin resorption on dentin surfaces, following insertion into rat thigh muscle. We found that loading of the nHAC membrane with 100 g/ml BMP-2 enhanced new cementum-like hard tissue formation on the dentin surface at 2 weeks after implantation, which was at an earlier stage than when such tissue was formed in the C100 group. Ca and P ions have been reported to be released into the microenvironment from nano-hydroxyapatite microcrystals due to phagocytosis by osteoclasts or multinuclear giant cells [14–18]. Osteoblasts require Ca and P ions to form bone tissue [19,20]. In this study, degradation of nHAC was observed and multinuclear giant cells were observed in the areas surrounding this degraded nHAC. Thus, the concentration of Ca and P ions in the microenvironment
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Fig. 4. Hard tissue formation and dentin resorption. (a) Newly formed hard tissue on dentin surface. The percentage of newly formed hard tissue on the dentin surface in the nHAC group was significantly higher than that in the C group; *p < 0.05 and p < 0.05. (b) Resorbed dentin surface. No significant differences in the percentage of the dentin surface that was resorbed were observed between the nHAC groups and the C groups at any stage or at any rhBMP-2 concentration.
might have been increased due to the release of Ca and P ions from degraded nHAC, and these ions would stimulate the proliferation and differentiation of cementoblast- or osteoblast-like cells. Several studies have reported that BMP-2 may be preferentially deposited on nHap rather than on collagen [9,10]. Thus a larger amount of rhBMP-2 might have adhered to the nHAC membranes than to the C membranes of this study. During tissue regeneration, the release of BMP-2 from the carrier depends on degradation of the carrier in vivo [21]. In this study, a larger amount of rhBMP-2 might have been released from the nHAC membrane than from the C membrane when these membranes were degraded. The larger amount of rhBMP-2 released into the microenvironment from the dissolving nHAC membrane could increase the activity of cementoblast-like or osteoblast-like cells. Therefore, a higher level of released rhBMP-2 is one possible reason why a cementum-like hard tissue on the dentin surface was observed at an earlier time point in the nHAC group than in the C group. However, no significant differences in dentin resorption were observed between the C100 and the H100 group at any stage. Several studies have reported that the differentiation of osteoclasts depends on the dose of the applied BMP-2 [2,3,6]. Miyaji et al. [3] reported that a high concentration of BMP-2 induced dentin resorption and a relatively low concentration of BMP-2 promoted cementum-like hard tissue formation on a dentin surface. In the present study, dentin resorption was significantly enhanced in the C groups by a higher concentration of applied BMP-2 compared to a lower BMP-2 concentration. Therefore, it cannot be simply assumed that the different effects of the nHAC and C groups are due to a higher amount of rhBMP-2 released from the nHAC groups than from the C groups, because there was no significant difference in dentin resorption between the C groups and the nHAC groups, whereas there was a significant increase in hard tissue formation in the nHAC groups under the same conditions. We therefore considered that the reason why a cementum-like hard tissue was observed to form on a dentin surface earlier in the nHAC groups than in the C groups might be because nanohydroxyapatite could induce the proliferation and differentiation of cementoblast or osteoblast-like cells due to the increase in Ca and P ions that occurred when the nHAC was degraded. Tenkumo et al. [13] reported that, following implantation of collagen or nHAC membranes loaded with 100 g/ml rhBMP-2, but without dentin chips, into rat thigh muscle, no bone formation was observed around the collagen membranes, but bone formation was observed around the nHAC membranes. However in our study, cementum or bone-like hard tissue formation was observed
on the surface of dentin chips in both the C and the nHAC groups, but not on the C and nHAC carrier membranes. These results suggested that hard tissue was preferentially formed on the dentin surface rather than on the biodegradable carrier. Dentin is a composite of hydroxyapatite and collagen and decalcified dentin has been reported to be a useful carrier for BMP-2 [3,22]. Therefore, the rhBMP-2 that is released from degraded C or nHAC might not diffuse into the microenvironment resulting in an increase in the BMP-2 concentration in the microenvironment, but instead might adhere to and be retained on, the dentin surface, which is difficult to degrade. Cementoblasts or osteoblast-like cells might attach more easily to the dentin surface than to the biodegradable carriers, and the rhBMP-2 that had adhered to the dentin surface would then stimulate cell proliferation. Furthermore, a dentin matrix also contains a small amount of growth factors such as BMPs, which would also influence cell proliferation [23,24]. Hard tissue formation around the carrier was only observed in the H400 group. It is possible that the release of a higher dose of rhBMP-2 from this carrier might result not only in rhBMPadherence to the dentin surface but also in an increase in the concentration of rhBMP-2 in microenvironments around the nHAC. 5. Conclusion Implantation of an nHAC membrane that was loaded with 100 g/ml BMP-2 and placed on dentin chips, into rat thigh muscle, resulted in the formation of a long thin, cementum-like hard tissue on the dentin surface at an early stage after implantation without increasing dentin resorption. Acknowledgements This work is financially supported by Grants-in-Aid for Scientific Research (No. 22791917) from Japan Society for the Promotion of Science. We thank Dr. Gelinsky for information concerning preparation of the mineralized collagen membranes and Dr. Nakazawa and Dr. Nishio for their encouragement throughout the course of this work. References [1] D.D. Bosshardt, Are cementoblasts a subpopulation of osteoblasts or a unique phenotype? J. Dent. Res. 84 (2005) 390–406. [2] H. Miyaji, T. Sugaya, T. Miyamoto, K. Kato, H. Kato, Hard tissue formation on dentin surfaces applied with recombinant human bone morphogenetic
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effect of nano-hydroxyapatite on bone morphogenetic protein-2-induced hard tissue formation and dentin resorption on a dentin surface Hiroki Tamagawa, Taichi Tenkumo, Tsutomu Sugaya ∗ , Masamitsu Kawanami Department of Periodontology and Endodontology, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan
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Article history: Available online 28 March 2012 Keywords: Nano-hydroxyapatite rhBMP-2 Hard tissue formation Dentin resorption
a b s t r a c t Aim: The purpose of this study was to evaluate the effects of the addition of nano-hydroxyapatite to a collagen membrane-carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) on hard tissue formation and dentin resorption on dentin surfaces in vivo. Materials and methods: Nano-hydroxyapatite collagen composite (nHAC) membranes or collagen (C) membranes were each immersed in either 100 or 400 g/ml rhBMP-2 and placed on dentin chips that were implanted into rat thigh muscle. The implants were analyzed at 2 or 4 weeks after surgery by histological observation and histomorphometric analysis. Results: The percentage of the hard tissue formed by each nHAC group was significantly higher than that formed by any of the C groups, except for that formed by the group loaded with 400 g/ml rhBMP2 at 4 weeks after implantation. No significant differences were observed in the percentage of dentin resorption between the nHAC groups and C groups at any stage or at any rhBMP-2 concentration. Conclusion: These findings showed that addition of nano-hydroxyapatite to a collagen membrane accelerated the formation of hard tissue induced by a low dose of rhBMP-2 on dentin surfaces at an early stage after implantation into rat thigh muscle, without increasing dentin resorption. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The periodontium consists of the supporting tissues of the tooth (cementum, alveolar bone, and periodontal ligament (PDL)) and gingiva. The root cementum is the thin calcified tissue that covers the root dentin surfaces and has many features in common with bone tissue. The periodontal ligament joins the root cementum with the alveolar bone through collagen fiber bundles. The periodontium attaches the tooth to the alveolar bone and transmits occlusal forces to the bone, and its main function is to provide resistance to the impact of occlusal force. The periodontium is broken in periodontal disease, after bacteria have formed a biofilm on the cementum surface. Periodontal therapy to obtain a healthy periodontium removes these biofilms and the infected cementum by mechanical means. However, following periodontal therapy, the dentin surface of the tooth root is exposed and periodontium is not regenerated. A critical step toward achieving periodontal regeneration is the new attachment of connective tissue fibers to the tooth root surface. Root cementum plays an important role in this process, because it securely attaches the periodontal ligament fibers to the root
∗ Corresponding author. Tel.: +81 11 706 4264; fax: +811 11 706 4334. E-mail address: [email protected] (T. Sugaya). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.03.103
surface. Therefore the formation of cementum on dentin surfaces of the tooth root is necessary for regeneration of the periodontium [1]. However treatment that can successfully and efficiently induce the regeneration of cementum has not yet been developed. Application of the bone morphogenetic protein-2 (BMP-2) to the periodontium is known to induce bone and cementum formation. We previously reported that recombinant human BMP-2 (rhBMP2) application to dentin surfaces also enhanced new cementum-like tissue formation on the dentin surfaces in palatal connective tissue that had low ability to form hard tissue [2]. However application of rhBMP-2 also resulted in concurrent dentin resorption [2–6]. We reported that rhBMP-2 application to dentin surfaces of a beagle induced not only new cementum formation but also dentin resorption and ankylosis of dentin surfaces [4,5]. Furthermore, we reported that the ratio of dentin resorption and cementum-like tissue formation was influenced by the concentration of rhBMP-2; a high concentration of applied BMP-2 not only induced more hard tissue formation but also more dentin resorption than that of a low concentration [2,3,6]. Therefore, inhibition of dentin resorption and the formation of new cementum on dentin surface are important challenges that need to be overcome to achieve periodontal regeneration. Hydroxyapatite is the main mineral component of bones and teeth, and is known to have high capacity for protein adsorption as well as high biocompatibility and osteoconductivity [7–10].
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We previously reported that incorporation of nano-hydroxyapatite (nHap) into a collagen membrane rhBMP-2 carrier enhanced ectopic bone formation when implanted in rat thigh muscle [11]. Based on this result, we assumed that the combination of a lowconcentration of rhBMP-2 with this nano-hydroxyapatite collagen composite membrane could form a cementum-like hard tissue on dentin surfaces. Therefore, in this study, we investigated the effect of nHap incorporation into a collagen membrane rhBMP-2 carrier on cementum-like tissue formation and dentin resorption on dentin surfaces in rat thigh muscle by histological observation and histomorphometric analysis. 2. Materials and methods 2.1. Preparation of mineralized nano-hydroxyapatite composite membranes and collagen membranes The nHap-collagen composite (nHAC) membrane was prepared from biomimetically mineralized collagen type I according to a method described by Gelinsky et al. [12] Briefly, 1 mg/ml purified type I atelocollagen gel (2 wt.%, Koken Co., Tokyo, Japan) was dissolved in 0.01 M HCl at a temperature of 4 ◦ C and mineralized in KH2 PO4 /K2 HPO4 and Tris buffer in the presence of CaCl2 . The reaction mixture was incubated for 12 h at 37 ◦ C, resulting in a white precipitate of mineralized collagen that was collected by centrifugation. The collagen (C) membrane was prepared similarly but without CaCl2 in the reaction. The collagen was dissolved at a concentration of 0.4 mg/ml in 0.01 M HCl at a temperature of 4 ◦ C. Fibril formation was induced by mixing with an equal amount of neutralization buffer. A membrane-like material was obtained through vacuum filtration and a cross-linking process using an 1% aqueous solution of 1-ethyl-3-(3-dimethylamiopropyl) carbodiimide hydrochloride (EDC; Kanto Chemical, Tokyo, Japan) for 1 h. The remaining activated carboxy groups were quenched with glycine. Following extensive washing with distilled water, the materials were freeze-dried. nHAC and C membranes were analyzed by SEM (S-4000; Hitachi, Tokyo, Japan) observation. The membranes used in the animal study were sterilized using ultraviolet ray irradiation. 2.2. Histological analysis of an rhBMP-2-loaded collagen composite with or without nHap 2.2.1. Application of rhBMP-2 to membranes After washing with phosphate-buffer saline (PBS), nHAC and C membranes (2 mm × 2 mm × 0.1 mm) were immersed in PBS containing 1000 U/ml penicillin and 1000 g/ml streptomycin for 24 h. Subsequently the nHAC and C membranes were rinsed with PBS and desiccated. The nHAC and C membranes were then each immersed in 100 or 400 g/ml rhBMP-2 (R&D, USA) for 10 min, as described by Tenkumo et al. [13]. The rhBMP-2 doses used were based on previous study [3]. The prepared membranes were classified into 4 groups: C100 (C membrane with 100 g/ml rhBMP-2), C400 (C membrane with 400 g/ml rhBMP-2), H100 (nHAC membrane with 100 g/ml rhBMP-2), and H400 (nHAC membrane with 400 g/ml rhBMP-2). 2.2.2. Preparation of dentin chips Dentin chips were extracted from rat tooth roots and the cementum was removed. Sixty-four flat dentin chips were cut into pieces of 2 mm × 2 mm × 0.3 mm in size. These flat dentin chips were treated for 3 min with 24% EDTA (pH 7.0), and were then incubated in 1000 U/ml penicillin and 1000 g/ml streptomycin overnight, as described in previous studies.
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2.2.3. Surgical procedures Thirty-two Wistar male rats (10 weeks old) were used for this experiment in accordance with the guide for the care and use of laboratory animals of Hokkaido University. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (7.0 × 10−2 ml/kg body weight; Kyoritsu Seiyaku Co., Tokyo, Japan). The prepared dentin chips on which the nHAC or C membranes had been placed were implanted into both the left and the right thigh muscles of the rats. 2.2.4. Histological observation and histomorphometric analysis The rats were sacrificed using an overdose of sodium pentobarbital at 2 or 4 weeks after implantation, and the tissue surrounding the implants was excised from the subcutaneous tissue in the left and right thigh muscle of the rats, as described by Tenkumo et al. [13]. The tissue was fixed in 10% formalin, decalcified for 4 weeks in 10% EDTA (pH 7.0), and then embedded in paraffin. The tissue sections (5 m thick) were stained with hematoxylin and eosin (H&E) and their histology was examined under a light microscope. 2.2.5. Statistical analysis Three sections were stained with H&E; one section was obtained from the center of the dentin chip and the other two sections were obtained at a distance of 150 m from either side of the center. After the length of newly formed hard tissue on dentin surfaces was measured by using ImageJ (NIH, Bethesda, MD, USA), the percentage of the new hard tissue that had formed was calculated as a percentage of newly formed hard tissue to the length of the surface on which the nHAC or C membranes had been placed. The percentage of resorbed dentin surfaces was similarly measured and calculated. Statistical differences between each group were analyzed using Mann–Whitney U-tests with SPSS 10.0J® (IBM, Tokyo, Japan). 3. Results 3.1. SEM observation of nHAC and C membranes SEM analysis of nHAC membranes indicated a porous structure with a pore size of 1.5–2.5 m. A large amount of apatite crystals that formed needle- or plate like-shapes 50–150 nm in size on collagen fibrils was observed (Fig. 1a). Analysis of the C membranes indicated a dense structure with a small number of pores. The topography of the collagen surface was flatter than that of the nHAC surface (Fig. 1b). 3.2. Histological observation of implanted membranes Two and 4 weeks after implantation of the rhBMP-2-loaded membranes on dentin chips into a rat thigh muscle, the implants and surrounding tissue were excised and histologically observed. Severe inflammatory changes such as necrosis and infections were not detected in any of the groups. The wound had healed well, and invasion of epithelial cells and wound dehiscence were not observed. 3.2.1. Analysis of membranes loaded with 100 g/ml rhBMP-2 In the C100 group at 2 weeks, most of the dentin surface was surrounded by fibrous connective tissue which contained a few blood vessels. A small amount of cementum-like hard tissue formation including cementocyte-like cells was observed (Fig. 2a). A few cuboidal or oval cells that stained with hematoxylin were observed on newly formed cementum-like hard tissue and on the dentin surface. A little dentin resorption was observed, and multinuclear giant cells were often observed at dentin resorption lacunas. Most of the
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Fig. 1. SEM images of the microstructure of nHAC membranes (a) and C membranes (b).
C membranes had been degraded, but some membrane fragments persisted. In the C100 group at 4 weeks, thin cementum-like hard tissue formation and dentin resorption were discontinuously observed on the dentin surface (Fig. 2b). Dentin resorption lacunas and newly formed cementum-like hard tissue was surrounded by fibrous tissue that was enriched with flat cells. No C membrane remnants were observed. In the H100 group at 2 weeks, most of the dentin surface was surrounded by fibrous cell-rich tissue. Extensive, newly formed cementum-like hard tissue including cementocyte-like cells was observed on the dentin surface, which displayed a continuous, long, thin shape of a uniform thickness (Fig. 2c). Cuboidal cells that stained with hematoxylin were often observed on the newly formed cementum-like hard tissue and on the dentin surface. A few multinuclear giant cells were observed on dentin resorption lacunas. nHAC was degraded into fragments, and numerous multinuclear giant cells were observed around these fragments. In the H100 group at 4 weeks, the dentin surface was covered with newly formed thin cementum-like hard tissue of a uniform thickness, and cuboidal cells stained with hematoxylin were observed on the newly formed cementum-like hard tissue (Fig. 2d).
Multinuclear giant cells were observed around the nHAC, but not, however, on dentin resorption lacunas. nHAC were degraded into smaller fragments than those observed at 2 weeks, and no hard tissue formation was observed around the nHAC.
3.2.2. Analysis of membranes loaded with 400 g/ml rhBMP-2 In the C400 group at 2 weeks, a small amount of new cementumlike hard tissue had formed on the dentin surface. Dentin resorption was frequently observed (Fig. 3a). Multinuclear giant cells were often observed at dentin resorption lacunas. Most of the dentin surfaces without cementum-like hard tissue formation or dentin resorption were lined by cuboidal cells that stained with hematoxylin, and were surrounded by connective tissue. In the C400 group at 4 weeks, extensive new cementum-like hard tissue and resorption were observed on the dentin surface (Fig. 3b). New thin cementum-like hard tissue, including cementocyte-like cells, was observed. Fibroblast-like flat cells that stained with hematoxylin were observed on newly formed cementum-like hard tissue and on the dentin surface. No C membrane remnants were observed. The dentin surface was surrounded by fibrous connective tissue.
Fig. 2. Tissue responses to C or nHAC membranes with 100 g/ml rhBMP-2. Tissue responses were analyzed by H&E staining. Responses to C membranes at 2 (a) or 4 (b) weeks after implantation or to nHAC membranes at 2 (c) or 4 (d) weeks after implantation are shown. In nHAC groups, newly formed cementum-like hard tissue was observed on the dentin surface, which displayed a continuous, long, thin shape of a uniform thickness. D: dentin, M: muscle, C: collagen membrane, N: nHAC membrane, arrow heads: new cementum-like hard tissue, *: dentin resorption. Bars indicate 50 m.
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Fig. 3. Tissue responses to C or nHAC membranes with 400 g/ml rhBMP-2. Responses to C membranes at 2 (a) or 4 (b) weeks after implantation or to nHAC membranes at 2 (c) or 4 (d) weeks after implantation are shown. Dentin resorption was frequently observed in both groups. In nHAC groups, newly formed hard tissue on the dentin surface exhibited irregular shape. D: dentin, M: muscle, N: nHAC membrane, BM: bone marrow-like tissue, arrow heads: new cementum-like hard tissue, arrows: new hard tissue, *: dentin resorption. Bars indicate 50 m.
In the H400 group at 2 weeks, extensive, newly formed hard tissue including osteocyte-like cells was observed on the dentin surface (Fig. 3c). The shape of this newly formed hard tissue on the dentin surface was different than that of cementum and was of a non-uniform thickness. Flat or cuboidal cells that stained with hematoxylin were observed on the newly formed hard tissue. Multinuclear giant cells were often observed on the dentin surface. nHAC was degraded into fragments, and numerous multinuclear giant cells were observed around these fragments. The dentin surface and the nHAC were surrounded by cell-rich tissue. In the H400 group at 4 weeks, newly formed hard tissue on the dentin surface exhibited a more irregular shape and some of this tissue included bone marrow-like tissue (Fig. 3d). Extensive dentin resorption was observed, and multinuclear giant cells were observed at resorption lacunas. nHAC was degraded into fragments that were smaller than those observed at 2 weeks. Furthermore, bone-like hard tissue was observed around these fragments, and some of this hard tissue had fused with the newly formed hard tissue on the dentin surface. The dentin surface and the nHAC were surrounded by cell-rich tissue.
3.3. Histomorphometric analysis The average percentage of newly formed hard tissue in the C100, H100, C400 and H400 groups was 6.5 ± 3.4%, 27.7 ± 6.8%, 2.3 ± 1.6% and 34.9 ± 5.2% at 2 weeks, and 12.8 ± 2.5%, 37.7 ± 6.5%, 32.3 ± 5.5% and 37.9 ± 7.7% at 4 weeks, respectively (Fig. 4a). The average percentage of new hard tissue that was formed in the H100 group was significantly higher than that of the C100 group (p < 0.05) at 2 and 4 weeks, respectively. The average percentage of new hard tissue that was formed in the H400 group was significantly higher than that of the C400 group (p < 0.05) at 2 weeks but not at 4 weeks. The average percentage of new hard tissue that was formed in the C100 (p < 0.05) and the C400 (p < 0.05) groups at 4 weeks was significantly higher than that of the C100 and C400 groups, respectively at 2 weeks. No significant difference in the average percentage of hard tissue that was formed was observed between the H100 and the H400 group at any time or at any concentration.
The average percentage of the dentin surface that was resorbed in the C100, H100, C400 and H400 groups was 1.6 ± 1.0%, 4.6 ± 3.3%, 6.2 ± 4.1% and 4.6 ± 2.9% at 2 weeks, and 4.7 ± 3.2%, 4.7 ± 1.7%, 12.9 ± 3.8% and 10.8 ± 4.2% at 4 weeks, respectively (Fig. 4b). No significant differences were observed between the nHAC groups and the C groups at any stage or at any rhBMP-2 concentration. In the H400, C100 and C400 groups at 4 weeks, the average percentage of the resorbed dentin surface was significantly higher than that of the same group at 2 weeks. However, no significant difference in this percentage was observed for the H100 group between 2 weeks and 4 weeks. The average percentage of dentin resorption was significantly higher in the C400 group at 2 weeks than in the C100 group at 2 weeks (p < 0.05), but at 4 weeks the average percentage of dentin resorption of the C400 and H400 groups was significantly higher than that of the C100 (p < 0.05) and H100 (p < 0.05) groups, respectively. 4. Discussion SEM observation of the nHAC group indicated that crystals were present on the surface of the reassembled collagen fibrils. These crystals were identified as nanocrystalline hydroxyapatite in a previous study in which the same material was used. Therefore, the crystals observed in this study can be identified as nHap. In this study, using histological observation and histomorphometric analysis, we evaluated the effect of the addition of nHap to a collagen membrane-rhBMP-2 carrier on hard tissue formation and dentin resorption on dentin surfaces, following insertion into rat thigh muscle. We found that loading of the nHAC membrane with 100 g/ml BMP-2 enhanced new cementum-like hard tissue formation on the dentin surface at 2 weeks after implantation, which was at an earlier stage than when such tissue was formed in the C100 group. Ca and P ions have been reported to be released into the microenvironment from nano-hydroxyapatite microcrystals due to phagocytosis by osteoclasts or multinuclear giant cells [14–18]. Osteoblasts require Ca and P ions to form bone tissue [19,20]. In this study, degradation of nHAC was observed and multinuclear giant cells were observed in the areas surrounding this degraded nHAC. Thus, the concentration of Ca and P ions in the microenvironment
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Fig. 4. Hard tissue formation and dentin resorption. (a) Newly formed hard tissue on dentin surface. The percentage of newly formed hard tissue on the dentin surface in the nHAC group was significantly higher than that in the C group; *p < 0.05 and p < 0.05. (b) Resorbed dentin surface. No significant differences in the percentage of the dentin surface that was resorbed were observed between the nHAC groups and the C groups at any stage or at any rhBMP-2 concentration.
might have been increased due to the release of Ca and P ions from degraded nHAC, and these ions would stimulate the proliferation and differentiation of cementoblast- or osteoblast-like cells. Several studies have reported that BMP-2 may be preferentially deposited on nHap rather than on collagen [9,10]. Thus a larger amount of rhBMP-2 might have adhered to the nHAC membranes than to the C membranes of this study. During tissue regeneration, the release of BMP-2 from the carrier depends on degradation of the carrier in vivo [21]. In this study, a larger amount of rhBMP-2 might have been released from the nHAC membrane than from the C membrane when these membranes were degraded. The larger amount of rhBMP-2 released into the microenvironment from the dissolving nHAC membrane could increase the activity of cementoblast-like or osteoblast-like cells. Therefore, a higher level of released rhBMP-2 is one possible reason why a cementum-like hard tissue on the dentin surface was observed at an earlier time point in the nHAC group than in the C group. However, no significant differences in dentin resorption were observed between the C100 and the H100 group at any stage. Several studies have reported that the differentiation of osteoclasts depends on the dose of the applied BMP-2 [2,3,6]. Miyaji et al. [3] reported that a high concentration of BMP-2 induced dentin resorption and a relatively low concentration of BMP-2 promoted cementum-like hard tissue formation on a dentin surface. In the present study, dentin resorption was significantly enhanced in the C groups by a higher concentration of applied BMP-2 compared to a lower BMP-2 concentration. Therefore, it cannot be simply assumed that the different effects of the nHAC and C groups are due to a higher amount of rhBMP-2 released from the nHAC groups than from the C groups, because there was no significant difference in dentin resorption between the C groups and the nHAC groups, whereas there was a significant increase in hard tissue formation in the nHAC groups under the same conditions. We therefore considered that the reason why a cementum-like hard tissue was observed to form on a dentin surface earlier in the nHAC groups than in the C groups might be because nanohydroxyapatite could induce the proliferation and differentiation of cementoblast or osteoblast-like cells due to the increase in Ca and P ions that occurred when the nHAC was degraded. Tenkumo et al. [13] reported that, following implantation of collagen or nHAC membranes loaded with 100 g/ml rhBMP-2, but without dentin chips, into rat thigh muscle, no bone formation was observed around the collagen membranes, but bone formation was observed around the nHAC membranes. However in our study, cementum or bone-like hard tissue formation was observed
on the surface of dentin chips in both the C and the nHAC groups, but not on the C and nHAC carrier membranes. These results suggested that hard tissue was preferentially formed on the dentin surface rather than on the biodegradable carrier. Dentin is a composite of hydroxyapatite and collagen and decalcified dentin has been reported to be a useful carrier for BMP-2 [3,22]. Therefore, the rhBMP-2 that is released from degraded C or nHAC might not diffuse into the microenvironment resulting in an increase in the BMP-2 concentration in the microenvironment, but instead might adhere to and be retained on, the dentin surface, which is difficult to degrade. Cementoblasts or osteoblast-like cells might attach more easily to the dentin surface than to the biodegradable carriers, and the rhBMP-2 that had adhered to the dentin surface would then stimulate cell proliferation. Furthermore, a dentin matrix also contains a small amount of growth factors such as BMPs, which would also influence cell proliferation [23,24]. Hard tissue formation around the carrier was only observed in the H400 group. It is possible that the release of a higher dose of rhBMP-2 from this carrier might result not only in rhBMPadherence to the dentin surface but also in an increase in the concentration of rhBMP-2 in microenvironments around the nHAC. 5. Conclusion Implantation of an nHAC membrane that was loaded with 100 g/ml BMP-2 and placed on dentin chips, into rat thigh muscle, resulted in the formation of a long thin, cementum-like hard tissue on the dentin surface at an early stage after implantation without increasing dentin resorption. Acknowledgements This work is financially supported by Grants-in-Aid for Scientific Research (No. 22791917) from Japan Society for the Promotion of Science. We thank Dr. Gelinsky for information concerning preparation of the mineralized collagen membranes and Dr. Nakazawa and Dr. Nishio for their encouragement throughout the course of this work. References [1] D.D. Bosshardt, Are cementoblasts a subpopulation of osteoblasts or a unique phenotype? J. Dent. Res. 84 (2005) 390–406. [2] H. Miyaji, T. Sugaya, T. Miyamoto, K. Kato, H. Kato, Hard tissue formation on dentin surfaces applied with recombinant human bone morphogenetic
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