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Evaluation of bone regeneration with biphasic calcium phosphate substitute implanted with bone morphogenetic protein 2 and mesenchymal stem cells in a rabbit calvarial defect model Beom-Su Kim, PhD,a Moon-Ki Choi, DDS, PhD,b Jung-Hoon Yoon, DDS, PhD,c and Jun Lee, DDS, PhDd Objective. The aim of this study was to evaluate the in vivo osteogenic potential of biphasic calcium phosphate (BCP), bone morphogenetic protein 2 (BMP-2), and/or mesenchymal stem cell (MSC) composites by using a rabbit calvarial defect model. Study Design. Bone formation was assessed by using three different kinds of implants in rabbit calvarial defects, BCP alone, BCP/recombinant human (rh) BMP-2, and BCP/rhBMP-2/MSCs composite. The implants were harvested after 2 or 8 weeks, and the area of new bone formation was quantified by microecomputed tomography (micro-CT) and histologic studies. Results. The highest bone formation was achieved with the BCP/rhBMP-2/MSCs treatment, and it was significantly higher than that achieved with the empty or BCP-alone treatment. The quantity of new bone at 8 weeks was greater than at 4 weeks in each group. The relative density of osteocalcin immunoreactivity also increased during this interval. Conclusions. These results indicate that the combination of BCP, rhBMP-2, and MSCs synergistically enhances osteogenic potential during the early healing period and could be used as a bone graft substitute. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;-:1-8)
Calcium phosphate ceramics have been widely applied as bone substitutes because of their resemblance to bone in orthopedic, maxillofacial, and dental surgery. Biphasic calcium phosphate (BCP) is composed of various concentrations of hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP). BCP shows efficient incorporation into bone tissue; its biocompatibility, degradability, and porous structure facilitate intergrowth through newly formed bone. However, the osteoinductive properties of synthetic ceramics, including BCP, are insufficient for the healing of extensive bone defects.1 To overcome the problem, several growth factors, such as bone morphogenetic proteins (BMPs), fibroblast growth factors, and vascular endothelial growth factors have been used for bone tissue engineering.2 Although many growth factors have shown potential for use in bone regeneration and repair, BMPs are clearly the most investigated osteoinductive factors described to date.3 BMP-2 belongs to the tumor growth factor b (TGF-b) superfamily and has been This research was part of the project titled “Development of a bone substitute and scaffold using cuttlefish bone,” funded by the Ministry of Oceans and Fisheries, Korea (20120265). a Wonkwang Bone Regeneration Research Institute, Wonkwang University, Daejeon Korea; Bonecell Biotech Inc., Daejeon, Korea. b Department of Oral and Maxillofacial Surgery, Wonkwang University, Iksan, Korea. c Department of Oral and Maxillofacial Pathology, Daejeon Dental Hospital, Wonkwang University, Daejeon, Korea. d Department of Oral and Maxillofacial Surgery, Daejeon Dental Hospital, Wonkwang University, Daejeon, Korea. Received for publication Oct 17, 2014; accepted for publication Feb 12, 2015. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2015.02.017
shown to regulate osteoblast differentiation. Moreover, BMP-2 is a promising therapeutic agent promoting bone regeneration when delivered locally in combination with bone substitute. For example, Notodihardjo et al.4 reported a higher level of bone induction in the group that received BMP-2/HA composite compared with a group implanted with only HA. In addition, Jang et al.5 showed that bone healing was significantly greater in the BMP-2/BCP combination at either 2 or 8 weeks in a rat calvarial defect model than in a group implanted with only BCP. These reports indicate that locally delivered BMP-2 combined with a bone substitute can synergistically enhance bone formation. The bone-forming ability of the bone substitute/BMP-2 composite is insufficient because of the time required for osteoblast differentiation and for recruiting mesenchymal stem cells (MSCs) to the injury site. To overcome this problem, recent studies have reported cell-based therapy using MSCs. The cell source is an important factor for successful bone tissue regeneration.6 MSCs are isolated from several tissues, such as bone marrow and adipose tissue, and can differentiate into several cell types, such as osteoblast, chondrocytes, and adipocyte cells under proper conditions.7 Therefore, the aim of this study was to evaluate the osteogenic therapeutic efficacy of BCP, BMP-2, and MSCs in combination by using a rabbit calvarial defect model. The osteogenic capacity was evaluated by a microecomputed tomography (micro-CT) analysis and histologic studies.
MATERIALS AND METHODS Preparation of mesenchymal stem cell Bone marrow cells were isolated and cultured according to a previously reported method.8 Briefly, rabbit 1
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bone marrow was collected from the iliac after anesthesia by using 18-gauge needles. The extracted bone marrow was cultured in a-Minimum Essential Medium (Gibco, Gaithersburg, MD) containing 20% fetal bovine serum (Gibco) and 1% antibiotics (10,000 units/mL penicillin G and 25 mg/mL amphotericin B; Gibco), in a humidified carbon dioxide incubator at 37 C for 1 week. After the cells became confluent, they were released from the culture dish with TrypLE Express (Invitrogen, Carlsbad, CA) and reseeded in a new culture dish and expanded for 2 weeks. Before the in vivo transplantation experiment, cells were dissociated with TrypLE Express (Invitrogen), washed with PBS, and collected. The collected cells were resuspended at a concentration of 1 107 cells/mL and used for in vivo study. Implantation of BCP ceramic BCP was purchased from Dio Implant (Busan, Korea; HA: b-TCP ¼ 70:30 and particle size ranging from 400 to 1000 mm). The recombinant human BMP-2 (rhBMP-2) supplied by the Korea Bone Bank (Seoul, Korea) was dissolved in a buffer at a concentration of 1.5 mg/mL. Then, 10 mL of the rhBMP-2 solution was soaked with 30 mg of BCP and freezedried for 48 hours. Thus, 15 mg of rhBMP-2 was loaded into each specimen. To transplant the MSC, 30 mg BCP or BCP/BMP-2 was soaked with 10 mL of prepared MSC suspension solution (at 1 107 cells/ mL). The BCP ceramic was easily and sufficiently soaked in 10 mL of the cell suspension, and thus approximately 1 106 cells were loaded in each specimen. Loading of BCP ceramics with BMP, MSCs, or both The animal experiments were performed according to the protocols approved by the Institutional Animal Care Committee of Wonkwang University. To determine the osteogenic potential in a calvarial defect model, we prepared four experimental groups of implants as follows: empty (nonimplanted), BCP, BCP/rhBMP-2, and BCP/rhBMP-2/MSCs. The rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg; Yuhan, Seoul, Korea) and xylazine (5 mg/kg; Bayer Korea, Seoul, Korea), following the application of a local anesthetic (2% lidocaine solution). Four separate circular calvarial defects were created by using a trephine that was 8 mm in diameter. Four bicortical defects were created in each animal: 1 defect remained unfilled, and the remaining 3 defects were filled with 30 mg BCP, BCP/BMP-2, or BCP/BMP-2/MSCs composite. According to the established animal care protocol, the rabbits were killed with an overdose of pentobarbital (Choongwae Pharm Corp, Seoul, Korea)
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at 2 and 8 weeks. After the death of the animals, bicortical bony blocks, including the original surgical defects, were harvested and fixed in a 4% paraformaldehyde solution buffered with 0.1 M phosphate solution (pH 7.2) for 3 to 5 days. Micro-CT analysis To determine the regenerated bone volume, the samples were scanned using micro-CT (Sky-Scan 1172 TM; Skyscan, Kontich, Belgium) under an x-ray source set at a voltage of 60 kV and a current of 167 mA using a 0.5-mm aluminum filter. The scanned data were reconstructed by using image analysis software (CT-analyzer; Skyscan). The bone volumes percentiles were calculated as the ratio of radiopaque regions of interest to the total defect volume, and residual BCP volumes were also measured from the micro-CT data. Histologic examination After micro-CT scanning, the harvested samples were dehydrated in a graded series of alcohol solutions (80%e100%), decalcified in a 10% ethylenediaminetetraacetic acid (EDTA) solution, and embedded in paraffin. Using a rotary microtome (HM 325; Microm, Walldorf, Germany), 5-mm sections of the samples were cut. At least five samples per group were stained with hematoxylin and eosin and Goldner’s Masson trichrome and randomly selected for histologic observation under a light microscope (DMR; Leica, Nussloch, Germany) equipped with a digital camera (DFC-480; Leica). After histologic preparation, the slides were evaluated for new bone formation by three blind examiners qualified in pathology. Immunohistochemical analysis Immunohistochemistry was performed on paraffin sections by using an avidin-biotinylated peroxidase enzyme complex-based kit (ABC Elite Kit, Vector, Burlingame, CA). Briefly, tissues were incubated with antiosteocalcin (1:200, Abcam, Cambridge, MA), then incubated with biotinylated antirabbit immunoglobulin G secondary antibody (ABC Elite Kit, Vector) and developed with a Diaminobenzidine (DAB) Kit (Sigma-Aldrich, St. Louis, MO) until the desired color intensity was reached. Statistical analysis The statistically significant differences in mean values among experimental groups were analyzed by one-way analysis of variance followed by a t test. P < .05 was considered statistically significant.
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Fig. 1. The two-dimensional reconstructed image of calvarial defects 8 weeks after implantation, using a micro-CT analysis.
Fig. 2. Quantification of the newly formed bone volume after 8 weeks. Each column represents the mean standard deviation (SD) (*P < .05 vs. empty group, #P < .05 vs. the BCP/ BMP-2/MSCs-implanted group).
RESULTS Animals All animals had an uneventful recovery. Histopathologic features of graft-versus-host disease or immune rejection were not observed in any of the treatment groups. Micro-CT evaluation Figure 1 shows the two-dimensional reconstruction images of new bone formation and bone in-growth within the defects after 8 weeks as analyzed by micro-CT. The micro-CT images show that bone ingrowth occurred from the side of the dura and the defect border in all experimental groups. In particular, new bone formed in the central area of the defect in the treatments containing BCP. In these groups, remaining BCP granules were observed, as was ingrowth of newly formed bone into the space between granules. To quantify the newly formed bone, the percentage of bone volume was calculated from the micro-CT data. Figure 2 shows the bone volume percentage in the
residual material fraction of BCP. Two weeks after implantation, the most dramatic bone regeneration results were observed in the BCP/BMP-2/MSCsimplanted defects. The bone volume was significantly higher in defects containing BCP (11.73 1.37%), BCP/BMP-2 (13.09 0.40%), and BCP/BMP-2/ MSCs (16.47 1.29%) than untreated defects (4.64 0.70%) (P < .05). In addition, the percentage bone volume observed for the BCP/BMP-2/MSCs treatment was remarkably higher than those of BCPand BCP/BMP-2-implanted defects. Furthermore, 8 weeks after implantation, the percentage bone volume for the BCP/BMP-2/MSCs treatment (24.20 1.47%) was remarkably higher than that of the BCP (17.91 0.94%) and BCP/BMP-2 (21.16 1.03%) treatments (P < .05). The residual BCP volume determined by micro-CT at 8 weeks for the BCP group was 63.33 4.04 mm3. However, the residual BCP volume was 52.00 3.00 mm3 and 39.33 5.13 mm3 in the BCP/ rhBMP-2 and BCP/rhBMP-2/MSCs groups, respectively. These results suggested that BMP-2 and BMP-2/ MSCs may have influenced the BCP resorption rate (Figure 3). Histologic analysis In this study, neither foreign body reaction nor necrosis was observed in any of the samples during the experimental period. Figure 4 shows the hematoxylin and eosin stained histologic image of new bone covering the entire area of the bone defect 8 weeks after implantation. In the empty group, some newly formed bone occurred from the defect side to the center site, but only connective tissue was observed in central area. Otherwise, newly formed bone was observed in the central region as well as at marginal sites in the BCP, BCP/BMP-2, and BCP/BMP-2/MSCs groups and the defect coalesced by newly formed bone. Figures 5 and 6 show the higher magnification of the bone generated in the marginal bone defect after implantation for 2 weeks and 8 weeks, respectively. At 2 weeks, all groups showed inflammatory cells in the
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Fig. 3. Image of the residual BCP granules (A) and quantification of the volume (B) at 8 weeks, calculated from micro-CT data. The image of residual BCP granules was false-colored with red and the residual volume was calculated using ImageJ (NIH, Bethesda, MD, USA).
Fig. 4. Histologic view of the bone generated 8 weeks after implantation. Arrows indicate the newly formed bone; Asterisks, BCP granule; arrowhead, marginal defect site. Scale bar ¼ 500 mm.
margins of the defect sites. Some loose connective tissue and weak new bone formation were observed in the empty group. In particular, in the BCP, BCP/BMP-2, and BCP/BMP-2/MSCs groups, new bone formation was observed in the margins of the defect site, and weak new bone formation occurred over the outer surfaces of the BCP granules. This bone formation was markedly higher in BCP/BMP-2/MSCs-implanted defects. Eight weeks after implantation, the boundary between host bone and newly formed bone could not be
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distinguished in the BCP, BCP/BMP-2, and BCP/BMP2/MSCs groups. BCP granules were surrounded by new bone, mature bone was further developed, and a bone marrow cavityelike morphology was observed. The mature new bone was significantly thicker in the BCP/ BMP-2/MSCs-implanted group than in other groups. Goldner’s Masson trichrome staining 2 weeks after implantation revealed the presence of loose connective tissue instead of obvious bonelike tissue at the defect sites. After 8 weeks, all of the experimental groups, except for the empty group, showed newly formed bone in the central region as well as at the marginal sites. Newly formed bone in the BCP group formed over the outer surface of the BCP, and the central area of all defect sites were filled with pronounced bone. In addition, a significantly larger amount of mature bone formed in the BCP/BMP-2/MSCs group than in the other groups (Figure 7). Biochemical findings To quantify the osteogenic responses, the biochemical parameters of the osteocalcin content of the implants were analyzed. Figure 8 shows positive staining of osteocalcin for all treatments. However, the expression level of osteocalcin was higher in the group implanted with BCP/BMP-2/MSCs than in the empty, BCP, and BCP/BMP-2 groups. These results suggest that BMP-2 significantly enhanced bone regeneration when combined with BCP. Furthermore, the MSCs synergistically increased the osteogenic potential of BCP/BMP-2.
DISCUSSION Although autograft, allograft, xenograft, and synthetic materials have been used as bone substitutes for a long time, each has limitations. Recently, to improve the effectiveness of bone regeneration, several growth factors, such as BMP-2, have been used with various bone substitutes. A successful candidate bone substitute material should be reproducible, biocompatible, and bioabsorbable.9 BCP ceramics composed of HA and bTCP at various ratios are considered safe, biocompatible, and effective for new bone formation.10 The degradation rate of BCP ceramics in vivo can be accelerated by increasing the proportion of b-TCP and mechanical support depends on the proportion of HA.11 Jensen et al.12 tested the resorption rate for several different HA/b-TCP ratios and reported that HA/b-TCP 20/80 has a high resorption rate but that HA/b-TCP 80/20 and 60/40 have low resorption rates. The BCP ceramics used in the present study were a mixture of HA and b-TCP at a ratio of 70:30, and the highest resorption of BCP
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Fig. 5. Hematoxylin and eosin staining at a higher magnification for the bone generated after implantation for 2 weeks, in the marginal defect. Mature bone observed from around the BCP granule (asterisk), which has a lack of fibrous tissue interface (arrow), in the BCP, BCP/rhBMP-2, and BCP/rhBMP-2/MSCs groups. Arrow: new bone near a BCP granule; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
was observed in the group implanted with BCP/BMP-2/ MSCs, at 8 weeks. These results suggest that there is a relationship between the degree of BCP resorption and the addition of BMP-2/MSCs. Generally, in vivo resorption of calcium phosphate ceramics, such as BCP, mainly occurs by osteoblastic activation.13 Our microCT results showed not only the most bone formation but also the highest BCP resorption in the BCP/BMP-2/ MSCs-implanted group. This suggested that osteoblastic differentiation was related to the BCP resorption rate. Jang et al.5 reported that when BCP is implanted in combination with rhBMP-2 in rats, the quantity of remaining BCP granules did not differ significantly between the groups treated with BCP alone or with rhBMP-2. However, several studies have reported a relationship between osteoblast differentiation and osteoclast activation. The BCP degradation mechanism is based on the detection of resorption lacunae on the BCP surface, and this might contribute to osteoblastic bone formation and osteoclastic activation on the surface of the b-TCP.14 Udagawa et al.15 reported that osteoblast cells stimulate osteoclast activation via the expression of osteoclast differentiation factor and that
osteoblastic cells induce activation of osteoclasts through a mechanism independent of macrophage colony-stimulating factor.16 Furthermore, according to Jensen et al.,17 BMP-2 directly enhances osteoclast differentiation. In addition, Kaneko et al.18 reported that BMP-2 stimulates osteoclastic bone resorption via the expression of BMP receptors in mature osteoclasts.18 Therefore, although we did not directly evaluate the effects of BMP-2 on osteoclast activity, these previous studies suggested that BMP-2 also influences BCP resorption. BMPs are considered potential therapeutic agents to enhance bone regeneration. The mechanism of osteogenesis enhanced by BMPs is related to their effect on BMP target cells. In particular, rhBMP-2 plays an important role in cell growth and in stimulating the differentiation of MSCs. It also possesses strong osteoinductive properties, and a number of preclinical studies have assessed the efficacy of rhBMP-2 in the healing of bone defects.19,20 For example, Burdick et al.21 reported that bone mineralization is approximately 2.7-fold increased when BMP-2 is locally delivered into defect sites.
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Fig. 6. Hematoxylin and eosin stained higher magnification images at 8 weeks after implantation, in the marginal defect. The matured bone is significantly generated, and the defects tended to coalesce with new bone. Arrow, new bone near a BCP granule; arrowhead, lamella of mature bone; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
Fig. 7. Histologic view of the bone generated 2 and 8 weeks after implantation, in the central area of bone defects. Two weeks after implantation, immature bone was observed in the fibrous connective tissue in the BCP/rhBMP/MSCs group. After 8 weeks, there was more newly formed mature bone in the BCP/rhBMP/MSCs group than in the BCP/rhBMP group. Arrow, new bone near a bTCP/HA granule; CT, connective tissue; asterisk, b-TCP/HA granule; Goldner’s trichrome stain. Scale bar ¼ 250 mm.
Although various factors, such as carrier type, animal model, and experimental site, can influence bone formation capacity, BMP-2 does can also be important. Several studies have reported that there is no correlation between bone formation and the dose of rhBMP-2.22,23 However, other studies have found a
marked dose-dependent response.24,25 Cowan et al.25 reported that the highest bone regeneration in a rat calvarial defect model was induced by using 240 ng/ mm3 BMP-2, as in our study. We used 15 mg per defect (200 ng/mm3 per dose), and group with the additional BMP-2 treatment had significantly more
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Fig. 8. The immunohistochemical localization of osteocalcin in the margin of bone defects 8 weeks after implantation. Osteocytes and the lamellar bone matrix show markedly positive reactions to osteocalcin. OC immunoreactivity indicated that the active osteoblast-like cells rarely contributed around the hard tissues. Arrow, osteoblast-like cells surrounding lamellar bone matrix; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
new bone formation than the group treated with BCP alone. The osteogenic potential of MSCs has been defined by the amount of bone formation following the transplantation of MSCs in vivo.26 Kruyt et al.27 prepared BCP ceramic scaffolds and found significantly greater bone formation in the treatment that included MSCs in goat. Hou et al.28 implanted the composites of coral bone substitute/rhBMP-2/ MSCs and compared the bone formation capacity with that of iliac autografts; the coral/rhBMP/MSCs composite may be an alternative to autologous bone grafts. In the present study, new bone formation increased synergistically when MSCs were incorporated into the treatment. Osteocalcin is a bone-specific noncollagenous protein used as a marker for mature osteoblast activity. The protein is important for bone turnover, new bone formation, and bone mineralization.29 In the present study, the immunoreactivity of osteocalcin was highest in the BCP/BMP-2/MSCs-implanted group. This confirmed the rapid osteogenic activities resulting in new bone formation in the BCP/BMP-2/MSCs composite implant group.
CONCLUSIONS This study provided histologic evidence that the combination of BCP, rhBMP-2, and MSCs could synergistically enhance BCP resorption rate and new bone formation in rabbit calvarial defects.
REFERENCES 1. Bruder SP, Jaiswal N, Ricalton NS, Mosca JD, Kraus KH, Kadiyala S. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res. 1998;355: S247-S256. 2. Aravamudhan A, Ramos DM, Nip J, et al. Osteoinductive small molecules: growth factor alternatives for bone tissue engineering. Curr Pharm Des. 2013;19:3420-3428. 3. Chin M, Ng T, Tom WK, Carstens M. Repair of alveolar clefts with recombinant human bone morphogenetic protein (rhBMP-2) in patients with clefts. J Craniofac Surg. 2005;16: 778-789. 4. Notodihardjo FZ, Kakudo N, Kushida S, Suzuki K, Kusumoto K. Bone regeneration with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J Craniomaxillofac Surg. 2012;40: 287-291. 5. Jang JW, Yun JH, Lee KI, et al. Osteoinductive activity of biphasic calcium phosphate with different rhBMP-2 doses in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2012;113: 480-487.
ORAL AND MAXILLOFACIAL SURGERY 8 Kim et al. 6. Schaefer DJ, Klemt C, Zhang XH, Stark GB. Tissue engineering with mesenchymal stem cells for cartilage and bone regeneration [translation]. Chirurg. 2000;71:1001-1008. 7. Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Handb Exp Pharmacol. 2006;174:249-282. 8. Park H, Temenoff JS, Tabata Y, Caplan AI, Mikos AG. Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials. 2007;28:3217-3227. 9. Ignatius AA, Schmidt C, Kaspar D, Claes LE. In vitro biocompatibility of resorbable experimental glass ceramics for bone substitutes. J Biomed Mater Res. 2001;55:285-294. 10. Schopper C, Ziya-Ghazvini F, Goriwoda W, et al. HA/TCP compounding of a porous CaP biomaterial improves bone formation and scaffold degradationda long-term histologic study. J Biomed Mater Res B. 2005;74:458-467. 11. Daculsi G. Biphasic calcium phosphate concept applied to artificial bone, implant coating and injectable bone substitute. Biomaterials. 1998;19:1473-1478. 12. Jensen SS, Bornstein MM, Dard M, Bosshardt DD, Buser D. Comparative study of biphasic calcium phosphates with different HA/TCP ratios in mandibular bone defects. A long-term histomorphometric study in minipigs. J Biomed Mater Res B. 2009;90: 171-181. 13. Leeuwenburgh S, Layrolle P, Barrere F, et al. Osteoclastic resorption of biomimetic calcium phosphate coatings in vitro. J Biomed Mater Res. 2001;56:208-215. 14. Yamada S, Heymann D, Bouler JM, Daculsi G. Osteoclastic resorption of biphasic calcium phosphate ceramic in vitro. J Biomed Mater Res. 1997;37:346-352. 15. Udagawa N, Takahashi N, Jimi E, et al. Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colonystimulating factor: receptor activator of NF-kappa B ligand. Bone. 1999;25:517-523. 16. Takami M, Woo JT, Nagai K. Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production. Cell Tissue Res. 1999;298:327-334. 17. Jensen ED, Pham L, Billington CJ Jr, et al. Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors. J Cell Biochem. 2010;109:672-682. 18. Kaneko H, Arakawa T, Mano H, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone. 2000;27:479-486. 19. Tuominen T, Jamsa T, Tuukkanen J, et al. Native bovine bone morphogenetic protein improves the potential of biocoral to heal segmental canine ulnar defects. Int Orthop. 2000;24:289-294.
OOOO Month 2015 20. Barboza E, Caula A, Machado F. Potential of recombinant human bone morphogenetic protein-2 in bone regeneration. Implant Dent. 1999;8:360-367. 21. Burdick JA, Frankel D, Dernell WS, Anseth KS. An initial investigation of photocurable three-dimensional lactic acid based scaffolds in a critical-sized cranial defect. Biomaterials. 2003;24: 1613-1620. 22. Wikesjo UM, Guglielmoni P, Promsudthi A, et al. Periodontal repair in dogs: effect of rhBMP-2 concentration on regeneration of alveolar bone and periodontal attachment. J Clin Periodontol. 1999;26:392-400. 23. Lee JH, Kim CS, Choi KH, et al. The induction of bone formation in rat calvarial defects and subcutaneous tissues by recombinant human BMP-2, produced in Escherichia Coli. Biomaterials. 2010;31:3512-3519. 24. Kenley R, Marden L, Turek T, Jin L, Ron E, Hollinger JO. Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). J Biomed Mater Res. 1994;28:1139-1147. 25. Cowan CM, Aghaloo T, Chou YF, et al. MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects. Tissue Eng. 2007;13:501-512. 26. Derubeis AR, Cancedda R. Bone marrow stromal cells (BMSCs) in bone engineering: limitations and recent advances. Ann Biomed Eng. 2004;32:160-165. 27. Kruyt MC, Dhert WJ, Yuan H, et al. Bone tissue engineering in a critical size defect compared to ectopic implantations in the goat. J Orthop Res. 2004;22:544-551. 28. Hou R, Chen F, Yang Y, et al. Comparative study between coralmesenchymal stem cells-rhBMP-2 composite and auto-bone-graft in rabbit critical-sized cranial defect model. J Biomed Mater Res A. 2007;80:85-93. 29. Ferron M, Lacombe J. Regulation of energy metabolism by the skeleton: osteocalcin and beyond. Arch Biochem Biophys. 2014;561:137-146.
Reprint requests: Jun Lee, DDS, PhD Department of Oral and Maxillofacial Surgery College of Dentistry Wonkwang University 77 Dunsan-ro Seo-gu Daejeon Korea. [email protected]
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Evaluation of bone regeneration with biphasic calcium phosphate substitute implanted with bone morphogenetic protein 2 and mesenchymal stem cells in a rabbit calvarial defect model Beom-Su Kim, PhD,a Moon-Ki Choi, DDS, PhD,b Jung-Hoon Yoon, DDS, PhD,c and Jun Lee, DDS, PhDd Objective. The aim of this study was to evaluate the in vivo osteogenic potential of biphasic calcium phosphate (BCP), bone morphogenetic protein 2 (BMP-2), and/or mesenchymal stem cell (MSC) composites by using a rabbit calvarial defect model. Study Design. Bone formation was assessed by using three different kinds of implants in rabbit calvarial defects, BCP alone, BCP/recombinant human (rh) BMP-2, and BCP/rhBMP-2/MSCs composite. The implants were harvested after 2 or 8 weeks, and the area of new bone formation was quantified by microecomputed tomography (micro-CT) and histologic studies. Results. The highest bone formation was achieved with the BCP/rhBMP-2/MSCs treatment, and it was significantly higher than that achieved with the empty or BCP-alone treatment. The quantity of new bone at 8 weeks was greater than at 4 weeks in each group. The relative density of osteocalcin immunoreactivity also increased during this interval. Conclusions. These results indicate that the combination of BCP, rhBMP-2, and MSCs synergistically enhances osteogenic potential during the early healing period and could be used as a bone graft substitute. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;-:1-8)
Calcium phosphate ceramics have been widely applied as bone substitutes because of their resemblance to bone in orthopedic, maxillofacial, and dental surgery. Biphasic calcium phosphate (BCP) is composed of various concentrations of hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP). BCP shows efficient incorporation into bone tissue; its biocompatibility, degradability, and porous structure facilitate intergrowth through newly formed bone. However, the osteoinductive properties of synthetic ceramics, including BCP, are insufficient for the healing of extensive bone defects.1 To overcome the problem, several growth factors, such as bone morphogenetic proteins (BMPs), fibroblast growth factors, and vascular endothelial growth factors have been used for bone tissue engineering.2 Although many growth factors have shown potential for use in bone regeneration and repair, BMPs are clearly the most investigated osteoinductive factors described to date.3 BMP-2 belongs to the tumor growth factor b (TGF-b) superfamily and has been This research was part of the project titled “Development of a bone substitute and scaffold using cuttlefish bone,” funded by the Ministry of Oceans and Fisheries, Korea (20120265). a Wonkwang Bone Regeneration Research Institute, Wonkwang University, Daejeon Korea; Bonecell Biotech Inc., Daejeon, Korea. b Department of Oral and Maxillofacial Surgery, Wonkwang University, Iksan, Korea. c Department of Oral and Maxillofacial Pathology, Daejeon Dental Hospital, Wonkwang University, Daejeon, Korea. d Department of Oral and Maxillofacial Surgery, Daejeon Dental Hospital, Wonkwang University, Daejeon, Korea. Received for publication Oct 17, 2014; accepted for publication Feb 12, 2015. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2015.02.017
shown to regulate osteoblast differentiation. Moreover, BMP-2 is a promising therapeutic agent promoting bone regeneration when delivered locally in combination with bone substitute. For example, Notodihardjo et al.4 reported a higher level of bone induction in the group that received BMP-2/HA composite compared with a group implanted with only HA. In addition, Jang et al.5 showed that bone healing was significantly greater in the BMP-2/BCP combination at either 2 or 8 weeks in a rat calvarial defect model than in a group implanted with only BCP. These reports indicate that locally delivered BMP-2 combined with a bone substitute can synergistically enhance bone formation. The bone-forming ability of the bone substitute/BMP-2 composite is insufficient because of the time required for osteoblast differentiation and for recruiting mesenchymal stem cells (MSCs) to the injury site. To overcome this problem, recent studies have reported cell-based therapy using MSCs. The cell source is an important factor for successful bone tissue regeneration.6 MSCs are isolated from several tissues, such as bone marrow and adipose tissue, and can differentiate into several cell types, such as osteoblast, chondrocytes, and adipocyte cells under proper conditions.7 Therefore, the aim of this study was to evaluate the osteogenic therapeutic efficacy of BCP, BMP-2, and MSCs in combination by using a rabbit calvarial defect model. The osteogenic capacity was evaluated by a microecomputed tomography (micro-CT) analysis and histologic studies.
MATERIALS AND METHODS Preparation of mesenchymal stem cell Bone marrow cells were isolated and cultured according to a previously reported method.8 Briefly, rabbit 1
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bone marrow was collected from the iliac after anesthesia by using 18-gauge needles. The extracted bone marrow was cultured in a-Minimum Essential Medium (Gibco, Gaithersburg, MD) containing 20% fetal bovine serum (Gibco) and 1% antibiotics (10,000 units/mL penicillin G and 25 mg/mL amphotericin B; Gibco), in a humidified carbon dioxide incubator at 37 C for 1 week. After the cells became confluent, they were released from the culture dish with TrypLE Express (Invitrogen, Carlsbad, CA) and reseeded in a new culture dish and expanded for 2 weeks. Before the in vivo transplantation experiment, cells were dissociated with TrypLE Express (Invitrogen), washed with PBS, and collected. The collected cells were resuspended at a concentration of 1 107 cells/mL and used for in vivo study. Implantation of BCP ceramic BCP was purchased from Dio Implant (Busan, Korea; HA: b-TCP ¼ 70:30 and particle size ranging from 400 to 1000 mm). The recombinant human BMP-2 (rhBMP-2) supplied by the Korea Bone Bank (Seoul, Korea) was dissolved in a buffer at a concentration of 1.5 mg/mL. Then, 10 mL of the rhBMP-2 solution was soaked with 30 mg of BCP and freezedried for 48 hours. Thus, 15 mg of rhBMP-2 was loaded into each specimen. To transplant the MSC, 30 mg BCP or BCP/BMP-2 was soaked with 10 mL of prepared MSC suspension solution (at 1 107 cells/ mL). The BCP ceramic was easily and sufficiently soaked in 10 mL of the cell suspension, and thus approximately 1 106 cells were loaded in each specimen. Loading of BCP ceramics with BMP, MSCs, or both The animal experiments were performed according to the protocols approved by the Institutional Animal Care Committee of Wonkwang University. To determine the osteogenic potential in a calvarial defect model, we prepared four experimental groups of implants as follows: empty (nonimplanted), BCP, BCP/rhBMP-2, and BCP/rhBMP-2/MSCs. The rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg; Yuhan, Seoul, Korea) and xylazine (5 mg/kg; Bayer Korea, Seoul, Korea), following the application of a local anesthetic (2% lidocaine solution). Four separate circular calvarial defects were created by using a trephine that was 8 mm in diameter. Four bicortical defects were created in each animal: 1 defect remained unfilled, and the remaining 3 defects were filled with 30 mg BCP, BCP/BMP-2, or BCP/BMP-2/MSCs composite. According to the established animal care protocol, the rabbits were killed with an overdose of pentobarbital (Choongwae Pharm Corp, Seoul, Korea)
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at 2 and 8 weeks. After the death of the animals, bicortical bony blocks, including the original surgical defects, were harvested and fixed in a 4% paraformaldehyde solution buffered with 0.1 M phosphate solution (pH 7.2) for 3 to 5 days. Micro-CT analysis To determine the regenerated bone volume, the samples were scanned using micro-CT (Sky-Scan 1172 TM; Skyscan, Kontich, Belgium) under an x-ray source set at a voltage of 60 kV and a current of 167 mA using a 0.5-mm aluminum filter. The scanned data were reconstructed by using image analysis software (CT-analyzer; Skyscan). The bone volumes percentiles were calculated as the ratio of radiopaque regions of interest to the total defect volume, and residual BCP volumes were also measured from the micro-CT data. Histologic examination After micro-CT scanning, the harvested samples were dehydrated in a graded series of alcohol solutions (80%e100%), decalcified in a 10% ethylenediaminetetraacetic acid (EDTA) solution, and embedded in paraffin. Using a rotary microtome (HM 325; Microm, Walldorf, Germany), 5-mm sections of the samples were cut. At least five samples per group were stained with hematoxylin and eosin and Goldner’s Masson trichrome and randomly selected for histologic observation under a light microscope (DMR; Leica, Nussloch, Germany) equipped with a digital camera (DFC-480; Leica). After histologic preparation, the slides were evaluated for new bone formation by three blind examiners qualified in pathology. Immunohistochemical analysis Immunohistochemistry was performed on paraffin sections by using an avidin-biotinylated peroxidase enzyme complex-based kit (ABC Elite Kit, Vector, Burlingame, CA). Briefly, tissues were incubated with antiosteocalcin (1:200, Abcam, Cambridge, MA), then incubated with biotinylated antirabbit immunoglobulin G secondary antibody (ABC Elite Kit, Vector) and developed with a Diaminobenzidine (DAB) Kit (Sigma-Aldrich, St. Louis, MO) until the desired color intensity was reached. Statistical analysis The statistically significant differences in mean values among experimental groups were analyzed by one-way analysis of variance followed by a t test. P < .05 was considered statistically significant.
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Fig. 1. The two-dimensional reconstructed image of calvarial defects 8 weeks after implantation, using a micro-CT analysis.
Fig. 2. Quantification of the newly formed bone volume after 8 weeks. Each column represents the mean standard deviation (SD) (*P < .05 vs. empty group, #P < .05 vs. the BCP/ BMP-2/MSCs-implanted group).
RESULTS Animals All animals had an uneventful recovery. Histopathologic features of graft-versus-host disease or immune rejection were not observed in any of the treatment groups. Micro-CT evaluation Figure 1 shows the two-dimensional reconstruction images of new bone formation and bone in-growth within the defects after 8 weeks as analyzed by micro-CT. The micro-CT images show that bone ingrowth occurred from the side of the dura and the defect border in all experimental groups. In particular, new bone formed in the central area of the defect in the treatments containing BCP. In these groups, remaining BCP granules were observed, as was ingrowth of newly formed bone into the space between granules. To quantify the newly formed bone, the percentage of bone volume was calculated from the micro-CT data. Figure 2 shows the bone volume percentage in the
residual material fraction of BCP. Two weeks after implantation, the most dramatic bone regeneration results were observed in the BCP/BMP-2/MSCsimplanted defects. The bone volume was significantly higher in defects containing BCP (11.73 1.37%), BCP/BMP-2 (13.09 0.40%), and BCP/BMP-2/ MSCs (16.47 1.29%) than untreated defects (4.64 0.70%) (P < .05). In addition, the percentage bone volume observed for the BCP/BMP-2/MSCs treatment was remarkably higher than those of BCPand BCP/BMP-2-implanted defects. Furthermore, 8 weeks after implantation, the percentage bone volume for the BCP/BMP-2/MSCs treatment (24.20 1.47%) was remarkably higher than that of the BCP (17.91 0.94%) and BCP/BMP-2 (21.16 1.03%) treatments (P < .05). The residual BCP volume determined by micro-CT at 8 weeks for the BCP group was 63.33 4.04 mm3. However, the residual BCP volume was 52.00 3.00 mm3 and 39.33 5.13 mm3 in the BCP/ rhBMP-2 and BCP/rhBMP-2/MSCs groups, respectively. These results suggested that BMP-2 and BMP-2/ MSCs may have influenced the BCP resorption rate (Figure 3). Histologic analysis In this study, neither foreign body reaction nor necrosis was observed in any of the samples during the experimental period. Figure 4 shows the hematoxylin and eosin stained histologic image of new bone covering the entire area of the bone defect 8 weeks after implantation. In the empty group, some newly formed bone occurred from the defect side to the center site, but only connective tissue was observed in central area. Otherwise, newly formed bone was observed in the central region as well as at marginal sites in the BCP, BCP/BMP-2, and BCP/BMP-2/MSCs groups and the defect coalesced by newly formed bone. Figures 5 and 6 show the higher magnification of the bone generated in the marginal bone defect after implantation for 2 weeks and 8 weeks, respectively. At 2 weeks, all groups showed inflammatory cells in the
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Fig. 3. Image of the residual BCP granules (A) and quantification of the volume (B) at 8 weeks, calculated from micro-CT data. The image of residual BCP granules was false-colored with red and the residual volume was calculated using ImageJ (NIH, Bethesda, MD, USA).
Fig. 4. Histologic view of the bone generated 8 weeks after implantation. Arrows indicate the newly formed bone; Asterisks, BCP granule; arrowhead, marginal defect site. Scale bar ¼ 500 mm.
margins of the defect sites. Some loose connective tissue and weak new bone formation were observed in the empty group. In particular, in the BCP, BCP/BMP-2, and BCP/BMP-2/MSCs groups, new bone formation was observed in the margins of the defect site, and weak new bone formation occurred over the outer surfaces of the BCP granules. This bone formation was markedly higher in BCP/BMP-2/MSCs-implanted defects. Eight weeks after implantation, the boundary between host bone and newly formed bone could not be
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distinguished in the BCP, BCP/BMP-2, and BCP/BMP2/MSCs groups. BCP granules were surrounded by new bone, mature bone was further developed, and a bone marrow cavityelike morphology was observed. The mature new bone was significantly thicker in the BCP/ BMP-2/MSCs-implanted group than in other groups. Goldner’s Masson trichrome staining 2 weeks after implantation revealed the presence of loose connective tissue instead of obvious bonelike tissue at the defect sites. After 8 weeks, all of the experimental groups, except for the empty group, showed newly formed bone in the central region as well as at the marginal sites. Newly formed bone in the BCP group formed over the outer surface of the BCP, and the central area of all defect sites were filled with pronounced bone. In addition, a significantly larger amount of mature bone formed in the BCP/BMP-2/MSCs group than in the other groups (Figure 7). Biochemical findings To quantify the osteogenic responses, the biochemical parameters of the osteocalcin content of the implants were analyzed. Figure 8 shows positive staining of osteocalcin for all treatments. However, the expression level of osteocalcin was higher in the group implanted with BCP/BMP-2/MSCs than in the empty, BCP, and BCP/BMP-2 groups. These results suggest that BMP-2 significantly enhanced bone regeneration when combined with BCP. Furthermore, the MSCs synergistically increased the osteogenic potential of BCP/BMP-2.
DISCUSSION Although autograft, allograft, xenograft, and synthetic materials have been used as bone substitutes for a long time, each has limitations. Recently, to improve the effectiveness of bone regeneration, several growth factors, such as BMP-2, have been used with various bone substitutes. A successful candidate bone substitute material should be reproducible, biocompatible, and bioabsorbable.9 BCP ceramics composed of HA and bTCP at various ratios are considered safe, biocompatible, and effective for new bone formation.10 The degradation rate of BCP ceramics in vivo can be accelerated by increasing the proportion of b-TCP and mechanical support depends on the proportion of HA.11 Jensen et al.12 tested the resorption rate for several different HA/b-TCP ratios and reported that HA/b-TCP 20/80 has a high resorption rate but that HA/b-TCP 80/20 and 60/40 have low resorption rates. The BCP ceramics used in the present study were a mixture of HA and b-TCP at a ratio of 70:30, and the highest resorption of BCP
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Fig. 5. Hematoxylin and eosin staining at a higher magnification for the bone generated after implantation for 2 weeks, in the marginal defect. Mature bone observed from around the BCP granule (asterisk), which has a lack of fibrous tissue interface (arrow), in the BCP, BCP/rhBMP-2, and BCP/rhBMP-2/MSCs groups. Arrow: new bone near a BCP granule; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
was observed in the group implanted with BCP/BMP-2/ MSCs, at 8 weeks. These results suggest that there is a relationship between the degree of BCP resorption and the addition of BMP-2/MSCs. Generally, in vivo resorption of calcium phosphate ceramics, such as BCP, mainly occurs by osteoblastic activation.13 Our microCT results showed not only the most bone formation but also the highest BCP resorption in the BCP/BMP-2/ MSCs-implanted group. This suggested that osteoblastic differentiation was related to the BCP resorption rate. Jang et al.5 reported that when BCP is implanted in combination with rhBMP-2 in rats, the quantity of remaining BCP granules did not differ significantly between the groups treated with BCP alone or with rhBMP-2. However, several studies have reported a relationship between osteoblast differentiation and osteoclast activation. The BCP degradation mechanism is based on the detection of resorption lacunae on the BCP surface, and this might contribute to osteoblastic bone formation and osteoclastic activation on the surface of the b-TCP.14 Udagawa et al.15 reported that osteoblast cells stimulate osteoclast activation via the expression of osteoclast differentiation factor and that
osteoblastic cells induce activation of osteoclasts through a mechanism independent of macrophage colony-stimulating factor.16 Furthermore, according to Jensen et al.,17 BMP-2 directly enhances osteoclast differentiation. In addition, Kaneko et al.18 reported that BMP-2 stimulates osteoclastic bone resorption via the expression of BMP receptors in mature osteoclasts.18 Therefore, although we did not directly evaluate the effects of BMP-2 on osteoclast activity, these previous studies suggested that BMP-2 also influences BCP resorption. BMPs are considered potential therapeutic agents to enhance bone regeneration. The mechanism of osteogenesis enhanced by BMPs is related to their effect on BMP target cells. In particular, rhBMP-2 plays an important role in cell growth and in stimulating the differentiation of MSCs. It also possesses strong osteoinductive properties, and a number of preclinical studies have assessed the efficacy of rhBMP-2 in the healing of bone defects.19,20 For example, Burdick et al.21 reported that bone mineralization is approximately 2.7-fold increased when BMP-2 is locally delivered into defect sites.
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Fig. 6. Hematoxylin and eosin stained higher magnification images at 8 weeks after implantation, in the marginal defect. The matured bone is significantly generated, and the defects tended to coalesce with new bone. Arrow, new bone near a BCP granule; arrowhead, lamella of mature bone; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
Fig. 7. Histologic view of the bone generated 2 and 8 weeks after implantation, in the central area of bone defects. Two weeks after implantation, immature bone was observed in the fibrous connective tissue in the BCP/rhBMP/MSCs group. After 8 weeks, there was more newly formed mature bone in the BCP/rhBMP/MSCs group than in the BCP/rhBMP group. Arrow, new bone near a bTCP/HA granule; CT, connective tissue; asterisk, b-TCP/HA granule; Goldner’s trichrome stain. Scale bar ¼ 250 mm.
Although various factors, such as carrier type, animal model, and experimental site, can influence bone formation capacity, BMP-2 does can also be important. Several studies have reported that there is no correlation between bone formation and the dose of rhBMP-2.22,23 However, other studies have found a
marked dose-dependent response.24,25 Cowan et al.25 reported that the highest bone regeneration in a rat calvarial defect model was induced by using 240 ng/ mm3 BMP-2, as in our study. We used 15 mg per defect (200 ng/mm3 per dose), and group with the additional BMP-2 treatment had significantly more
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Fig. 8. The immunohistochemical localization of osteocalcin in the margin of bone defects 8 weeks after implantation. Osteocytes and the lamellar bone matrix show markedly positive reactions to osteocalcin. OC immunoreactivity indicated that the active osteoblast-like cells rarely contributed around the hard tissues. Arrow, osteoblast-like cells surrounding lamellar bone matrix; asterisk, BCP granule; HB, host bone; NB, new bone. Scale bar ¼ 250 mm.
new bone formation than the group treated with BCP alone. The osteogenic potential of MSCs has been defined by the amount of bone formation following the transplantation of MSCs in vivo.26 Kruyt et al.27 prepared BCP ceramic scaffolds and found significantly greater bone formation in the treatment that included MSCs in goat. Hou et al.28 implanted the composites of coral bone substitute/rhBMP-2/ MSCs and compared the bone formation capacity with that of iliac autografts; the coral/rhBMP/MSCs composite may be an alternative to autologous bone grafts. In the present study, new bone formation increased synergistically when MSCs were incorporated into the treatment. Osteocalcin is a bone-specific noncollagenous protein used as a marker for mature osteoblast activity. The protein is important for bone turnover, new bone formation, and bone mineralization.29 In the present study, the immunoreactivity of osteocalcin was highest in the BCP/BMP-2/MSCs-implanted group. This confirmed the rapid osteogenic activities resulting in new bone formation in the BCP/BMP-2/MSCs composite implant group.
CONCLUSIONS This study provided histologic evidence that the combination of BCP, rhBMP-2, and MSCs could synergistically enhance BCP resorption rate and new bone formation in rabbit calvarial defects.
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Reprint requests: Jun Lee, DDS, PhD Department of Oral and Maxillofacial Surgery College of Dentistry Wonkwang University 77 Dunsan-ro Seo-gu Daejeon Korea. [email protected]
