J Periodont Res 2016; 51: 77–85 All rights reserved

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12285

Ridge augmentation using recombinant human fibroblast growth factor-2 with biodegradable gelatin sponges incorporating b-tricalcium phosphate: a preclinical study in dogs

S. Hoshi1, T. Akizuki1,2 T. Matsuura1, T. Ikawa1, A. Kinoshita3, S. Oda4, Y. Tabata5 M. Matsui5, Y. Izumi1 1 Department of Periodontology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan, 2 Division of Periodontology, Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, Kanagawa, Japan, 3Department of Educational Media Development, Institute for Library and Media Information Technology, Tokyo Medical and Dental University, Tokyo, Japan, 4Oral Diagnosis and General Dentistry, University Hospital of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan and 5Department of Biomaterials, Kyoto University, Kyoto, Japan

Hoshi S, Akizuki T, Matsuura T, Ikawa T, Kinoshita A, Oda S, Tabata Y, Matsui M, Izumi Y. Ridge augmentation using recombinant human fibroblast growth factor-2 with biodegradable gelatin sponges incorporating b-tricalcium phosphate: a preclinical study in dogs. J Periodont Res 2016; 51: 77–85. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Fibroblast growth factor-2 (FGF-2) regulates the proliferation and differentiation of osteogenic cells, resulting in the promotion of bone formation. Biodegradable gelatin sponges incorporating b-tricalcium phosphate (b-TCP) have been reported as a scaffold, which has the ability to control growth factor release, offering sufficient mechanical strength and efficient migration of mesenchymal cells. In this study, we evaluated the effects of the combined use of recombinant human FGF-2 (rhFGF-2) and gelatin/b-TCP sponge on ridge augmentation in dogs. Material and Methods: Six male beagle dogs were used in this study. Twelve wk after tooth extraction, bilateral 10 9 5 mm (width 9 depth) saddle-type defects were created 3 mm apart from the mesial side of the maxillary canine. At the experimental sites, the defects were filled with gelatin/b-TCP sponge infiltrated with 0.3% rhFGF-2, whereas gelatin/b-TCP sponge infiltrated with saline was applied to the control sites. Eight wk after surgery, qualitative and quantitative analyses were performed. Results: There were no signs of clinical inflammation at 8 wk after surgery. Histometric measurements revealed that new bone height at the experimental sites (2.98  0.65 mm) was significantly greater than that at the control sites (1.56  0.66 mm; p = 0.004). The total tissue height was greater at the experimental sites (6.62  0.66 mm) than that at the control sites (5.95  0.74 mm), although there was no statistical significant difference (p = 0.051). Cast model measurements revealed that the residual defect height at the experimental sites (2.31  0.50 mm) was significantly smaller than that at the control sites (3.51  0.78 mm; p = 0.012).

Tatsuya Akizuki, DDS, PhD, Department of Periodontology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Tel: +81 3 5803 5488 Fax: +81 3 5803 0196 e-mail: [email protected] Key words: alveolar ridge augmentation; beta-

tricalcium phosphate; fibroblast growth factor-2; gelatin Accepted for publication March 23, 2015

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Conclusion: The combined use of rhFGF-2 and gelatin/b-TCP sponge promotes ridge augmentation in canine saddle-type bone defects.

Periodontal disease, trauma and tooth extraction often lead to an alveolar ridge of extremely reduced height and width causing both esthetic and functional disorder (1,2). To achieve good esthetic and functional results, hard tissue augmentation, soft tissue augmentation or their combination is necessary depending on the case (3). Recently, the ability of hard tissue augmentation to create sufficient bone has been investigated by several methods (4–6). Autografts are considered as the gold standard for bone grafting, but may cause additional morbidity, as a second surgical site is necessary (7). Thus, alloplastic materials such as hydroxyapatite and b-tricalcium phosphate (b-TCP) are used as alternatives (8). It has been suggested that b-TCP is suitable for bone regeneration because of its osteoconductivity, biocompatibility, biodegradability and bioresorbability (9). Subepithelial connective tissue grafts and free gingival grafts are most commonly used for soft tissue regeneration and achieving good clinical results (10,11). The main postoperative complication of using autogenous tissue is the morbidity of the donor site, including prolonged bleeding and continuous pain (12–14). To overcome these problems, alternative materials have been developed (15,16). In animal studies, collagenbased matrices have been evaluated in soft tissue augmentation and showed similar outcomes to connective tissue grafts (17,18). The results of preclinical studies and multicenter clinical trials have attributed the effect of fibroblast growth factor-2 (FGF-2) on periodontal regeneration (19–21). FGF-2 induces strong angiogenic activity and stimulates the proliferation of undifferentiated mesenchymal cells (22). These functions appear effective for periodontal soft and hard tissue regeneration. In addition, FGF-2 is known to regulate the proliferation

and differentiation of osteogenic cells, resulting in the promotion of bone formation (23,24). FGF-2 administered in solution form diffuse rapidly after being applied to the defective sites. Thus, carrier/delivery systems are necessary for the binding and controlled release of growth factors. A biodegradable hydrogel comprising acidic gelatin enabled FGF-2 to be released at therapeutic sites for an extended time period and significantly enhanced bone regeneration at rabbit skull defects in contrast to free FGF-2 of the same dose (25). The acidic gelatin forms a polyion complex with FGF-2, which is released from the gelatin into the body due to the biodegradation of gelatin (26). Biodegradable gelatin sponge incorporating b-TCP reportedly acts as a scaffold with the ability to control the release of growth factors, offering sufficient mechanical strength and allowing the migration of mesenchymal cells (27,28). In a rabbit model, FGF-2 complexed with gelatin/b-TCP composites enabled the controlled release of FGF-2 and yielded significant ulna bone regeneration (29). However, to date, ridge augmentation using FGF-2 and gelatin/b-TCP sponges has not been reported. In this study, we evaluated the effect of the combined use of recombinant human FGF-2 (rhFGF-2) and gelatin/b-TCP sponges on ridge augmentation in canine saddle-type bone defects.

Material and methods Preparation of gelatin/b-tricalcium phosphate sponges

Biodegradable gelatin sponges incorporating b-TCP used in this study was made as previously described (27). Considering the basic electrical nature of rhFGF-2, acidic gelatin (Nitta Gelatin, Osaka, Japan) is isolated from porcine skin with an

isoelectric point of 5.0 instead of 9.0. An aqueous solution of gelatin and 50 wt% b-TCP granules (average diameter 2 lm; Taihei Chemical Industries, Nara, Japan) were mixed at 5000 rpm at 37°C for 3 min using a homogenizer (ED-12; Nihonseiki, Tokyo, Japan). After 0.16 wt% of glutaraldehyde aqueous solution was added to the mixed solution, the b-TCP dispersed in the gelatin and the solution was further mixed for 15 s using the homogenizer. The mixed solution was cast into a polypropylene dish, and left at 4°C for 12 h to allow gelatin cross-linking. The cross-linked sponges were then placed into 100 mM aqueous glycine solution at 37°C for 1 h to block the residual aldehyde groups of glutaraldehyde. After washing with doubledistilled water, the sponges were freeze-dried. Each composite was viewed by a scanning electron microscope (SEM; SU3500; Hitachi, Tokyo, Japan) and the average pore size was measured from the SEM image. Surgical procedures

All procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Tokyo Medical and Dental University, Tokyo, Japan (0130249A). Six healthy beagle dogs (1-year-old males) were used in this study. All surgical procedures were performed under general and local anesthesia. Medetomidine hydrochloride (0.05 mL/kg, DomitorÒ; Orion Corporation, Espoo, Finland) was administered intramuscularly as a premedication. General anesthesia was induced by intravenous injection of sodium thiopental (0.005 mL/kg, Ravonal; Mitsubishi Tanabe Seiyaku Co., Osaka, Japan) and spontaneous breathing was maintained. Local anesthesia was administered using lidocaine hydrochloride (2% with 1 : 80,000 epinephrine, xylocaine;

Ridge augmentation using rhFGF-2 Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan). Twelve wk before the experimental surgery, all second and third incisors in the maxilla were extracted to ensure abundant space for the experimental defects. After 12 wk of healing, a semilunar incision was performed from the distal angle of the canine to the distal angle of the first incisor beyond the mucogingival junction and a mucoperiosteal flap was elevated (Fig. 1A). Bilateral saddle-type bone defects (5 mm high and 10 mm wide) were surgically created 3 mm mesial to the canine, according to a previous report (4) with slight modification (Fig. 1B). Bilateral defects were randomly designated as experimental or control sites by coin toss. Gelatin/b-TCP sponges were trimmed using a surgical knife to fit and fill the defects. At the experimental sites, the defects were filled with gelatin/b-TCP sponges infiltrated with 0.3% rhFGF-2 (Kaken Pharmaceutical Co. Ltd, Tokyo, Japan), whereas gelatin/b-TCP sponges infiltrated with saline were applied to the control sites. Finally, the flap was repositioned and sutured (Gore-TexÒ CV-5 Suture; W.L. Gore & Associates, Inc., Newark, DE, USA). Sutures were removed 2 wk after the surgery. For postsurgical management, the surgical sites were rinsed with a 2% solution of chlorhexidine (HiBiTaneÒ concentrate; Sumitomo Seiyaku Co., Ltd., Osaka, Japan) three times a week for 8 wk. Eight wk after surgery, all animals were killed by injecting them with a sodium thiopental overdose. All surgical sites were dissected en

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were selected (experimental sites: n = 6, control sites: n = 6; one per each site). The center of the defects was designated as 8 mm from the mesial-most point of the canine. Histological observations were performed by light microscopy (Eclipse Ni-U, Nikon Corporation, Tokyo, Japan; and DP70, Olympus Corporation, Tokyo, Japan). Histometric analyses were performed using a computerized image system, consisting of a highdefinition color camera head (DS-Fi2; Nikon Corporation) and a microscope camera control unit (DS-L3; Nikon Corporation) equipped with a measurement function. The histological measurement method used was based on a previous report (4). The mean values of each histometric parameters were calculated for each site. The following parameters were measured by a blinded experienced examiner (Fig. 2A):

bloc and prepared for histological observation. Micro-computed tomography

Block specimens were fixed in 10% neutral-buffered formalin (MildformÒ 10N; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Before decalcification, micro-computed tomography (micro-CT) scanning was performed on all biopsy specimens. Each specimen was fixed on a cylindrical holder and scanned using a micro-CT system (inspeXio SMX-100CT; Shimadzu Corporation, Kyoto, Japan) at a voltage of 85 kV and an electric current of 77 mA. The resolution of one CT tomogram slice was set at 512 9 512 pixels. To obtain three-dimensional (3-D) images, scanned data were converted into digital images using 3-D image reconstruction software (TRI/ 3D-BON; Ratoc System Engineering Co., Ltd., Tokyo, Japan). To visualize the inside of the filled defects, twodimensional (2-D) images at the most central part (mesiodistal and buccolingual) of the defects were obtained.

•

• Histological observation and histometric analysis

Block specimens were decalcified in K-CX solution (Falma Co., Tokyo, Japan) for approximately 1 mo, and then trimmed, dehydrated and embedded in paraffin. The specimens were sectioned in the buccolingual plane at a thickness of 6 lm and stained with hematoxylin and eosin. Sections of the most central part of the defects

B

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New bone height, defined as the distance between the top of the new bone and line connecting the buccal and lingual tops of the host bone; and Total tissue height, defined as the distance between the top of the soft tissue and line connecting the buccal and lingual tops of the host bone.

Cast model measurement

All surgical sites were impressed by silicone (Exahiflex; GC, Tokyo, Japan) for cast model measurement at the point of death. The cast models

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Fig. 1. A mucoperiosteal flap was elevated (A) and a saddle-type bone defect (10 9 5 mm in mesiodistal width and depth, respectively; B) was surgically created. The defect was filled with gelatin/b-tricalcium phosphate sponge infiltrated with 0.3% recombinant human fibroblast growth factor-2 at the experimental site (C).

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B

Fig. 2. Histometric analysis. The new bone height was defined as the distance between the top of the NB and line connecting the buccal and lingual tops of the HB. The total tissue height was defined as the distance between the top of the soft tissue and line connecting the buccal and lingual tops of HB (A). Cast model measurement. The residual defect height was defined as the distance from the base of the cast model perpendicular to the line connecting the mesial-most point of the gingival margin of the canine to the distal-most point of the gingival margin of the first incisor (B). HB, host bone; NB, new bone.

were made from extra-hard plaster (New Fujirock; GC, Tokyo, Japan). The cast model measurement method used was based on a previous report (30). Three points of vertical height, at 7 mm, 8 mm and 9 mm mesial to the canine of the experimental defect, were measured. These points represented the most central point of the experimental defect and 1 mm away from the most central point mesially and distally. The residual defect height was defined as the distance from the soft tissue profile of the augmented crest of the cast model perpendicular to the line connecting the mesial-most point of the gingival margin of the canine to the distal-most point of the gingival margin of the first incisor (Fig. 2B). The mean of the values measured at these three points was calculated and designated

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the representative data of each cast model.

study, the coefficient ranged between 0.97 and 0.99. The values were close to 1, indicating high reliability.

Statistical analysis

Results

Means and standard deviations for each parameter were calculated for the experimental and control groups. Differences between the experimental and control groups were analyzed using the Student’s paired t-test with one-tailed distribution (Microsoft Excel 2011; Microsoft Corporation, Redmond, WA, USA) (n = 6). p < 0.05 was considered statistically significant. To minimize intraexaminer errors, the blinded experienced examiner (T.A.) conducted the measurements again, at least 4 wk apart, and reproducibility was evaluated using the concordance correlation coefficient (31,32). Within the context of this

B

Characterization of gelatin/btricalcium phosphate sponge

Gross view and SEM image of gelatin/b-TCP sponge are shown in Fig. 3. Gelatin/b-TCP sponge had an interconnected porous structure with a pore size range of 150–200 lm. bTCP granules were homogeneously localized in the gelatin walls of the sponges. Clinical observations

The initial inflammation immediately after surgery was similar in both the

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Fig. 3. Macrostructure and microstructure of gelatin/b-TCP sponge. Gross view of gelatin/b-TCP sponge (A). Scanning electron microscopy images show that gelatin/b-TCP sponge had an interconnected porous structure with a pore size range of 150–200 lm (B). b-TCP granules were homogeneously localized in the gelatin walls of the sponges (C). Scale bars indicate 500 lm (B), 50 lm (C). b-TCP, b-tricalcium phosphate.

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Fig. 4. Micro-CT images. 3-D micro-CT images revealed new bone-like radiopacity at the experimental sites (A). 2-D images of mesiodistal sections revealed that the new bone-like radiopacity was present not only near the surface of the original host bone but also far from the host bone in the defects at the experimental sites (B). 2-D images of buccolingual sections showed the large height of the new bonelike radiopacity at the experimental sites (C). 3-D micro-CT images revealed any remaining large bony defects at the control sites (D). 2D images of mesiodistal sections revealed that the new bone-like radiopacity was generated by the surface of the original host bone (E). 2D images of buccolingual sections showed that the height of the bone-like radiopacity was slightly higher at the control sites (F). Scale bars indicate 10 mm. micro-CT, micro-computed tomography.

experimental and control groups. No signs of excessive swelling or suppuration occurred at any site. A small dehiscence of a flap in the apical region away from the bone defect was observed in one of six sites in both the experimental and control groups at 1 wk after the surgery. However, the exposure of the applied material was not observed and additional treatment was not performed. Both the experimental and control sites had healed equally well 8 wk after surgery, but the residual defect volume of the experimental sites was smaller than that of the control sites. Micro-computed tomography observations

3-D micro-CT images revealed new bone-like radiopacity at the

experimental sites (Fig. 4A). 2-D images of mesiodistal sections of the central part of the defects revealed new bone-like radiopacity present not only near the surface of the original host bone but also far from the host bone in the defects at the experimental sites (Fig. 4B). 2-D images of buccolingual sections of the central part of the defects showed the large height of the new bone-like radiopacity at the experimental sites (Fig. 4C). 3-D micro-CT images revealed the remaining large bony defects at the control sites (Fig. 4D). 2-D images of mesiodistal sections of the central part of the defects revealed that the new bone-like radiopacity was generated by the surface of the original host bone (Fig. 4E). 2-D images of buccolingual sections of the central part of the

defects showed the height of the new bone-like radiopacity was low at the control sites (Fig. 4F). Histological observations

Histological observations showed that both the experimental and control sites healed well, without any sign of infection or acute inflammation such as bacterial infiltration or polymorphonuclear neutrophils. At the experimental sites, a large amount of new bone formation continuous with the host bone was evident (Fig. 5A). In the higher magnification image of the area framed in Fig. 5A, lamellar bone incorporating vessels and woven bone were observed in the upper portion of the new bone (Fig. 5B). In the central portion of the new bone, features of woven bone with vessels were evident

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A

E

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D

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Fig. 5. Hematoxylin and eosin staining; overview. A large amount of new bone formation, continuous with the host bone, was observed at the experimental site (A). Higher magnification image of the area framed in (A). LB incorporating vessel and WB were observed (B). WB with vessels was observed (C). WB continuous with original LB was observed (D). New bone formation was limited and fibrous connective tissue covered the edentulous alveolar ridge at the control site (E). Higher magnification image of the area framed in (E). Dense connective tissue was observed (F). WB with vessels was observed (G). WB continuous with original LB was observed (H). Scale bars indicate 1 mm (A and E) and 100 lm (B, C, D, F, G, and H). LB, lamellar bone; WB, woven bone.

(Fig. 5C). At the margin between the new and host bones, woven bone continuous with the original lamellar bone was observed (Fig. 5D). In the control group, new bone formation was limited, and fibrous connective tissue covered the edentulous alveolar ridge (Fig. 5E). In the higher magnification image of the area indicated in Fig. 5E, dense connective tissue was evident in the upper portion of the newly formed tissue (Fig. 5F). In the central portion of the new bone, features of woven bone with vessels were present (Fig. 5G). At the margin between the new and host bones, woven bone continuous with the original lamellar bone was observed (Fig. 5H). Gelatin/bTCP sponge has been completely absorbed at both the experimental and control sites at 8 wk. Histometric analyses

The results of the histometric analyses are shown in Table 1. New bone height at the experimental sites (2.98  0.65 mm) was significantly greater than that at the control sites (1.56  0.66 mm; p = 0.004). The total tissue height was greater at the experimental sites (6.62  0.66 mm)

Table 1. Histometric analysis of tissue formation Parameters

Experimental

Control

New bone height (mm)* Total tissue height (mm)

2.98  0.65 6.62  0.66

1.56  0.66 5.95  0.74

Student’s t-test was used for paired observations. Results are given as mean  standard deviation. *Significant difference (p < 0.05); n = 6.

than that at the control sites (5.95  0.74 mm), although there was no statistical significant difference (p = 0.051). Cast model measurement

The results of the cast model measurements are shown in Table 2. The residual defect height at the experimental sites (2.31  0.50 mm) was significantly smaller than that at the control sites (3.51  0.78 mm; p = 0.012).

Discussion In this study, the combined use of rhFGF-2 and gelatin/b-TCP sponge yielded a gain in alveolar ridge height within 8 wk of surgery. The results of the cast model measurements showed that residual defect height was significantly smaller at the experimental sites (2.31  0.50 mm) than that at the control sites (3.51  0.78 mm; p = 0.012). This result reflects the clinical observation that the concavity of the alveolar ridge was greater at

Table 2. Cast model measurement of total tissue volume Parameters

Experimental

Control

Residual defect height (mm)*

2.31  0.50

3.51  0.78

Student’s t-test was used for paired observations. Results are given as mean  standard deviation. *Significant difference (p < 0.05); n = 6.

Ridge augmentation using rhFGF-2 the control sites than at the experimental sites. Histological measurements revealed that the total height of the newly formed tissue was greater at the experimental sites (6.62  0.66 mm) than at the control sites (5.95  0.74 mm), although the difference was not statistically significant (p = 0.051). The greater degree of ridge augmentation evident in the experimental group was because of a large amount of new bone formation under the soft tissue. The height of the new bone at the experimental sites (2.98  0.65 mm) was significantly greater than at the control sites (1.56  0.66 mm; p = 0.004). In this study, because of the limitations of the surgical sites, we were unable to create a sham operation site as a control. However, when compared with a previous study (4), albeit with some differences in defect size (8 mm high and 7 mm wide), the height of new bone formed at sham operation sites (0.6  0.3 mm) was much less than at our control sites. The new bone formation observed with gelatin/b-TCP sponge alone may reflect a function of b-TCP particles. b-TCP incorporation increases the compression modulus of gelatin/bTCP sponge without changing the pore structure adjusted at 150– 200 lm; this pore structure is suitable for vascularization and the infiltration of cells into the sponge (27,33). The incorporation of biodegradable bTCP particles strengthens the mechanical properties of the gelatin scaffold, allowing space for tissue regeneration and stimulating osteogenic differentiation (27). In this study, we were unable to compare gelatin/b-TCP sponge with gelatin sponges lacking b-TCP particles. A previous in vivo study using rats reported the osteogenic effect of gelatin/b-TCP sponge compared with a gelatin scaffold (34). Gelatin/b-TCP sponges and gelatin sponges lacking b-TCP and were implanted into the backs of rats and evaluated using micro-CT. While implanted gelatin/b-TCP sponges showed an increase in the intensity of newly formed bone after implantation, implanted gelatin sponges

showed low intensity. Reportedly, porous b-TCP particles are completely resorbed at 12–24 mo (35). However, gelatin/b-TCP sponges were completely resorbed at both the experimental and control sites by 8 wk in this study. Because the average diameter of the b-TCP granules in gelatin/ b-TCP sponge was approximately 2 lm, rapid resorbance occurred. Gelatin/b-TCP sponge not only provides a 3-D structure for bone progenitors but also functions as a carrier for growth factors. The acidic gelatin forms a polyion complex with rhFGF-2, which is released from the gelatin into the body due to the biodegradation of gelatin/b-TCP sponge (26). Gelatin/b-TCP sponge was also easily applied and caused no adverse effects in this clinical situation. Gelatin/b-TCP sponge can be trimmed using a surgical knife and scissors to fit any shape of bone defect. Other graft materials used in alveolar ridge augmentation, such as hydroxyapatite or b-TCP were supplied as particles and did not easily maintain the shape of the defect. New bone formation was significantly greater at the experimental sites than at the control sites. Reportedly, rhFGF-2 affects osteogenesis. In vitro studies found that FGF-2 stimulates the proliferation of bone-derived cells (23). A mineralized bone-like tissue, formed using rat bone marrow stem cells in the presence of dexamethasone, b-glycerophosphate and ascorbic acid, showed a biphasic sequence when FGF-2 was added (24). After FGF-2 stimulation, cell proliferation and matrix accumulation were observed in the early stage. Following cell growth, a progressive increase in alkaline phosphatase activity, mineral deposition and osteocalcin expression was observed in the later stage. Based on these studies, several studies using animal models reported osteogenic properties of FGF-2 in bone defects (25,29). Likewise, human clinical trials of rhFGF-2 for the treatment of bone fractures showed earlier repair compared with that for traditional treatment (36). Bone morphogenetic protein-2 (BMP-2) has been also investigated as

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a candidate cytokine useful for bone regeneration (37). BMP-2 has osteogenic properties and was shown to cause effective alveolar ridge augmentation in a canine model (38). In a previous study using BMP-2 with a poly(D,L-lactic-co-glycolic acid) copolymer/gelatin sponge scaffold for alveolar ridge augmentation, although there were differences in defect size and position (mandibular; 6 mm high and 30 mm wide), the height of new bone from the original bone at the experimental sites (reported as about 72%; 4.3 mm/6 mm) was slightly greater than that at our experimental sites (60%; 2.98 mm/5 mm). Although BMP-2 is known to stimulate new bone formation, its use is associated with adverse effects, such as swelling, seroma and dose-dependent cancer risk (39,40). A study (41) describing the treatment of alveolar clefts with BMP2 and a hyaluronanbased hydrogel showed good bone quantity, comparable to autologous bone grafts, although severe gingival swelling occurred. In this study, clinical observations showed that the initial inflammation immediately after surgery was similar between the experimental and control groups, and no severe swelling or suppuration occurred in either group. rhFGF-2 induces strong angiogenic activity and stimulates the proliferation of fibroblast cells. These functions appear to be effective for alveolar ridge augmentation; therefore, rhFGF-2 is worthy of further investigation, particularly for esthetic reasons because of its effects on both soft and hard tissue formation (22). This study indicates that the combined use of rhFGF-2 and gelatin/bTCP sponge is effective for alveolar ridge augmentation. Further studies of longer duration, examining the regenerative process, and clinical investigations comparing this material with other known techniques are necessary to confirm the effectiveness of these materials for clinical therapy.

Conclusion The results of this study indicate that the combined use of rhFGF-2 and

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gelatin/b-TCP sponges promote alveolar ridge augmentation in canine saddle-type bone defects in dogs. The additional use of rhFGF-2 resulted in a significant amount of new bone formation underneath the connective tissue. Within the limits of this study, gelatin/b-TCP sponges with rhFGF-2 may be suitable for alveolar ridge augmentation.

Acknowledgements The authors would like to thank Dr. Shogo Takeuchi for surgical assistance. The authors are also grateful to Dr. Wataru Ono and Kiichi Maruyama for their help in completing the manuscript. This study was supported by JSPS KAKENHI grant no. 23792465. The authors report no conflicts of interest related to this study.

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