Hard Tissues and Materials
Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge
Journal of Biomaterials Applications 2015, Vol. 30(2) 193–200 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215575763 jba.sagepub.com
Goichi Matsumoto1,4, Takayo Ueda2, Yoshihiko Sugita3, Katsutoshi Kubo3, Megumi Mizoguchi2, Eiji Kotani2, Naoki Oda2, Shin Kawamata5, Natsuki Segami4 and Hajime Mori2
Abstract Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 107 BMP-2 polyhedra, and half were treated with sponges containing 3.6 106 BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 mg of recombinant human BMP-2 (rhBMP-2) or 1.8 107 empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development. Keywords BMP-2, polyhedra, alveolar ridge, bone regeneration, atelocollagen sponge
Introduction Autologous bone has been the preferred transplant material to use in treating a bony defect in the oral and maxillofacial region that must be augmented before placing a dental implant. However, bone for an autologous bone graft must be harvested from a donor site, which introduces the risk of infection and hemorrhage and has other significant disadvantages. Bone tissue engineering has the potential to identify bone regeneration techniques that overcome these disadvantages. In order to facilitate bone healing and regeneration, more than one healing factor is desirable. Marginal healing conditions should be augmented by including an adequate osteosynthetic device, growth factors, and progenitor cells. The basic strategy incorporates one or more of the elements that are needed to regenerate bone tissue into the target site such as
scaffolding, growth factors, and osteogenic progenitor cells, which then induce bone formation and guide the
1 Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, Kanagawa-ku, Yokohama, Japan 2 Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan 3 Department of Oral Pathology, School of Dentistry, Aichi-Gakuin University, Chikusa-ku, Nagoya, Japan 4 Department of Oral and Maxillofacial Surgery, Kanazawa Medical University, Uchinada, Ishikawa, Japan 5 Basic Research Group for Regenerative Medicine, Foundation for Biomedical Research and Innovation, Kobe, Japan
Corresponding author: Goichi Matsumoto, Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, 3-31-6 Tsuruya-cho, Kanagawa-ku, Yokohama 221-0835, Japan. Email: [email protected]
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
194
Journal of Biomaterials Applications 30(2)
tissue repair function of the living body by encouraging osteogenic cells to migrate, proliferate, and differentiate.1 Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b superfamily and are disulfide-linked homodimers that induce bone and cartilage formation. Among these, BMP-2 has been shown to be a strong inducer of new bone formation in animal models and is now well recognized as one of the key osteogenic factors in the field of bone tissue engineering.2–4 BMP-2 induces bone regeneration by activating a set of cellular events including chemotaxis of uncommitted mesenchymal cells and differentiation of these cells into osteoblasts.5,6 BMP-2 has been approved for limited clinical use in the form of recombinant human BMP-2 (rhBMP-2).7 However, therapeutic use of rhBMP-2 has been hampered by the lack of an efficacious way to deliver this protein to the site of the regenerating bone. Despite its limited commercial use, there remain problems with the systems used to deliver rhBMP-2 and there remains uncertainty about the most effective dose.8–12 We have thus sought to identify an appropriate carrier to facilitate retention of rhBMP-2 at the healing site in order to reduce the required dose. A good way to deliver rhBMP-2 continuously to the healing site is via a carrier material. The preferred carrier is a scaffold that is biocompatible, biodegradable, nontoxic, and is immunologically inert in order to avoid tissue rejection. However, the most important requirement for a carrier is that it controls the release of the growth factor it delivers. The carrier materials for BMP-2 that are currently clinically available are not ideal because large amounts of BMP-2 are required to achieve the desired osteogenesis.13,14 Despite the ready availability of rhBMP-2 for clinical use, the dilemma facing clinicians and the biotechnology industry is how to achieve an optimal delivery system that can decrease the dose of BMP-2, maintain a more sustained release pattern, and effectively promote osteoconduction.15 Bone formation in large animals requires that BMP-2 is present for long periods of time for solid bone formation.11,16 Several insect viruses produce polyhedral microcrystals consisting of a protein called polyhedrin. In these polyhedra, many progeny virus particles are occluded and protected from hostile environmental conditions. These polyhedral microcrystals are the main vectors that transport virus particles from one insect to another. They are highly resistant to both nonionic and ionic detergents and they are soluble at neutral pH.17 In our previous studies, using both in vitro and in vivo rat calvarial bone defect models, polyhedra containing full-length BMP-2 had a similar biological
activity to BMP-2 protein and prolonged the period of BMP-2 release to induce new bone more effectively than conventional atelocollagen–BMP-2 protein complexes.18 Our previous data suggest that therapy with BMP-2 polyhedra improves bone regeneration. However, to date there have been no studies of the usefulness of BMP-2 polyhedra to facilitate the healing of defects in the mandibular alveolar ridge in large animals. Therefore, the aim of the present study is to investigate the effectiveness of BMP-2 polyhedra for alveolar ridge augmentation of surgically created defects in the canine mandible.
Materials and methods Preparation of atelocollagen sponges impregnated with BMP-2 polyhedra or rhBMP-2 We have already reported the encapsulation of human full-length BMP-2 into Bombyx mori Cypovirus (BmCPV) polyhedra by use of the H1-tag (BMP-2 polyhedra) where the average mass of BMP-2 encapsulated in a polyhedral microcrystal (one cube) was 2.8 105 ng (the average volume of a microcrystal is approximately 5 5 5 mm3) (Figure 1(a)).18 We have estimated that 3.6 107 BMP-2 polyhedra contain 1 mg of BMP-2 protein. In this study, we mixed either 1.8 107 or 3.6 106 BMP-2 polyhedra with 200 ml Phosphate Buffered Saline (PBS) and added it dropwise onto a 20 mm 10 mm 2 mm PelnacÕ atelocollagen sponge (Gunze, Kyoto, Japan), taking care to avoid any runoff of excess liquid. The atelocollagen sponge was used to retain polyhedral cubes and as a scaffold for bone regeneration (Figure 1(a)). Atelocollagen sponges were prepared in two additional ways to serve as controls. One type contained 1.8 107 CP-H polyhedra (empty polyhedra). In the other, a solution containing 5 mg of rhBMP-2 (Yamanouchi Pharmaceutical, Tokyo, Japan) in PBS was added dropwise onto the atelocollagen sponge. Again, care was exercised so as to avoid runoff of excess liquid. The impregnated atelocollagen sponges were left to stand for 12 h at 4 C before being implanted into the experimental bone defects.
Animals Six one-year-old female beagle dogs (approximately 10 kg body weight) were used for the experiment. The Animal Care Committee of Kanagawa Dental University approved animal management and the surgical protocol. The animals had access to a standard laboratory diet and water until the beginning of the study. Postoperatively, the animals were kept in cages for six months with free access to water and food that
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
195 both sides of the mandible. Teeth were sectioned using a diamond-coated separating bur before surgically extracting them using an elevator. Subsequently, two bone defects were prepared on each side of the mandible by surgically removing parts of the buccal bone plate with a fissure bur. Each defect was 10 mm long and 5 mm high. The lingual cortical bone wall was left intact. The anterior bone defect was separated by 5 mm from the posterior bone defect (Figure 1). The four lateral bone defects were symmetrically created on left and right in the region of the extracted teeth. Atelocollagen sponges were used as carriers for the experimental substances, which included BMP-2 polyhedra, CP-H polyhedra, and free rhBMP-2. The sponges also acted as scaffolding to guide new bone generation. We implanted the sponges in the bone defects (Figure 1) in the following manner: Left side:
(a)
ACS
(b)
Group 1: 1.8 107 BMP-2 polyhedra (n ¼ 6) Group 2: 3.6 106 BMP-2 polyhedra (n ¼ 6) Right side:
(c)
Group 3: 5 mg of rhBMP-2 (n ¼ 6) Group 4: 1.8 107 CP-H polyhedra (n ¼ 6) ACS
ACS
Figure 1. Clinical appearance of the surgically created bone defects and atelocollagen sponge placement. Scanning electron micrograph (SEM) image of recombinant polyhedra encapsulating BMP-2/atelocollagen sponge (ACS). Some polyhedral cubes retained on the surface of atelocollagen sponge (a). Intraoperative view of the two lateral bone defects created in the mandible before and after implanting atelocollagen sponges impregnated with BMP-2 polyhedra, CP-H polyhedra, or rhBMP-2 (b and c).
was moist, soft, and nutritionally balanced. All animals were given antibiotics (Viccillin, Meiji Co., Tokyo, Japan) for three days after surgery. All experiments were performed in accordance with relevant guidelines and regulations.
Procedures used to create bone defects and to treat them All animals were anesthetized by intravenously injecting SomnopentyleÕ at 25 mg/kg (Kyoritsu Seiyaku, Tokyo, Japan). Routine dental infiltration anesthesia was used at the surgical sites. We extracted all first molars and all second, third, and fourth premolars on
Because healing characteristics of anterior defect sites may be different than those of posterior defect sites, half of each of the four groups was implanted in an anterior defect, and the other half was implanted in a posterior defect. The right-side defects in the mandible were treated as control sites, and the left-side sites as experimental sites.
Micro-CT All animals were killed six months after surgery with an overdose of SomnopentyleÕ by intravenous injection. Block sections from both the right and left sides of the mandible were removed separately and placed in a solution of 70% ethanol for five days, after which the muscle around the mandible was removed. Each mandible was treated and examined as follows. The bone sample was positioned on the stage of a Micro X-ray CT System (Rigaku, Tokyo, Japan) so that the shaft was oriented vertically. The X-ray generator was operated at an accelerating potential of 90 kV with a beam current of 50 mA. Each slice consisted of 480 480 pixels; slice thickness was 0.8 mm. The volume and density of newly generated bone in the defect were measured using Mimics software. The volume of interest (VOI) of the alveolar ridge in the defect site was calculated. The VOI was drawn with a slice-based method starting from the first slice containing the crown of the
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
196
Journal of Biomaterials Applications 30(2)
first premolar and moving dorsally in the area between the distal wall of the first premolar and the mesial wall of the second molar. On the original three-dimensional (3D) micro-CT image, micro-structural indices were calculated directly from the digitized VOI. The VOI of the bone between the distal wall of the first premolar and the mesial wall of the second molar was measured. The newly regenerated bone was delineated by erasing the lingual cortical bone on each coronal slice image in the defect site. The density of newly generated bone was computed relative to neighboring normal cortical bone.
(a)
(b)
Histological analysis Each bone sample was sectioned perpendicular to its axis at the middle of each bone defect. The bones at all defect sites were undecalcified and dehydrated in 70% ethanol, followed by immersion in Villanueva bone stain solution for 14 days. The bones were again dehydrated through gradient ethanols and embedded in methyl methacrylate resin (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Specimens 20 mm thick were sectioned using a band saw perpendicular to the buccolingual plane. Surfaces of the sections were polished with diamond paper and observed under a light microscope.
(c)
(d)
(e)
(f)
Statistical analysis P values were computed using Student’s t-tests. A P value less than 0.01 was considered statistically significant.
Results Clinical observations All animals tolerated the surgical procedures without clinical signs of adverse reactions and were healthy during the entire experimental period.
Micro-CT observation The healing of lateral bone defects in each group was clearly visible in the 3D micro-CT reconstructions, which were used to calculate the volume of new bone. Bone defects appeared to be healed almost completely in the groups treated with BMP-2 polyhedra in which full-length BMP-2 was immobilized and encapsulated by the H1 tag (Figure 2(a), (c), and (d))). In contrast, there was almost no regenerated bone in the bone defects in the groups treated with rhBMP-2 or empty polyhedra (CP-H polyhedra) (Figure 2(b), (e), and (f)). Cross-sectional views show that there was almost complete healing of bone defects only in the group treated
Figure 2. Three-dimensional micro-CT reconstructed appearance of bone regeneration in the mandibular bone defect six months after surgery. The lateral bone defects on the left side of the mandible were implanted with atelocollagen sponges impregnated with 1.8 107 BMP-2 polyhedra (front side) and 3.6 106 BMP-2 polyhedra (back side) (a). Bone defects on the right side of the mandible were implanted with atelocollagen sponges impregnated with rhBMP-2 (front side) and 1.8 107 CP-H polyhedra (empty polyhedra) (back side) (b). Micro-CT images of the frontal sections of mandibular bone defects in the 1.8 107 BMP-2 polyhedra group (c), the 3.6 106 BMP-2 polyhedra group (d), the 1.8 107 CP-H polyhedra group (e), and the rhBMP-2 group (f). Newly generated alveolar bone in each treatment group is shown with dotted squares.
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
197
(a) 350
*
Bone volume (mm3)
300
defect sites in the group treated with CP-H polyhedra. However, the mean bone density at defect sites in the CP-H polyhedra-treated, BMP-2 polyhedra-treated, and rhBMP-2-treated groups was not significantly different than that of the neighboring normal cortical bone. For both the BMP-2 polyhedra-treated group and the rhBMP-2-treated group, the mean bone density at defect sites was not different than that of the neighboring normal cortical bone (Figure 3(b)).
*
250 200 150 100 50
Histological observation a dr
2
he
P-
6
10
ly po H
rh
1.
3.
8
6
x
x
BM
7
10
0
C
P-
BMP-2 polyhedra
(b) 1.4
Relative bone density
1.2 1 0.8 0.6 0.4 0.2
a po
rh
ly
BM
he
P-
dr
2
6
10 x 6 3.
x 8 1.
C
BMP-2 polyhedra
P-
H
N
or
m
al
bo
10
7
ne
0
Figure 3. Newly generated alveolar bone volume and density from three-dimensional micro-CT analysis in the 1.8 107 BMP-2 polyhedra, 3.6 106 BMP-2 polyhedra, 5 mg rhBMP-2, and 1.8 107 CP-H polyhedra groups. The alveolar bone volume in the area of the bone defect with atelocollagen sponge implantation at six months postoperatively. *P < 0.01 versus 1.8 107 CP-H polyhedra and rhBMP-2 (a). The relative density of new bone six months postoperatively. Data were normalized with respect to the density of the neighboring normal cortical bone (b).
with BMP-2 polyhedra. The volume of new bone was calculated from 3D micro-CT images. The volume of newly generated bone at defect sites in the group treated with BMP-2 polyhedra was significantly greater than that formed at defect sites in groups treated with rhBMP-2 or CP-H polyhedra. However, there was no significant difference between groups treated with 3.6 106 BMP-2 polyhedra and 1.8 107 BMP-2 polyhedra (Figure 3(a)). In addition, there was no significant difference between anterior and posterior sites treated with BMP-2 polyhedra (data not shown). Next, we measured the bone density in the newly regenerated bone. The greatest density of new bone was at defect sites in the group treated with 1.8 107 BMP-2 polyhedra. The least density of new bone was at
Six months postoperatively, atelocollagen sponge was not observed at any of the healing sites. There was very little osseous regeneration at sites treated with rhBMP2 or CP-H polyhedra. New bone formation was limited, resulting in a less adequate ridge profile with dents on the buccal aspect (Figure 4(a) and (b)). In contrast, in the newly generated bone induced by BMP-2 polyhedra, concentric layers of bone tissue were observed around Haversian canals, and well-defined lamellar bone exhibiting characteristics of mature bone was observed (Figure 4(e) and (f)). Inflammatory cell was not observed in the group treated with BMP-2 polyhedra. A trabecular pattern of regenerated bone was observed in direct contact to original bone. The bony regenerate had fully filled the defect space, and defect sites in general demonstrated well-maintained ridge contours (Figure 4(c) and (d)). These findings suggest that newly generated bone in the groups treated with BMP-2 polyhedra develops into mature bone tissue. Regenerated bone in both anterior and posterior sites treated with BMP-2 polyhedra consisted of mature bone tissue (data not shown).
Discussion Many studies have demonstrated extensive bone formation using BMP-2 with different carriers in various animal models.13–15 A local delivery system for BMP2 may be important for those applications in which an optimal dose of BMP-2 is required.19,20 The concentration of BMP-2 necessary to promote highly effective bone formation is still unclear. Our hypothesis is that long-term delivery of BMP-2 protein induces greater in vivo bone formation that does short-term delivery at an equivalent dose. For this reason, we have developed a superior carrier system with sustained release of BMP-2 using polyhedra, and we continue to investigate its performance. Polyhedra were prepared to serve as the new carriers for rhBMP-2. Exogenously delivered BMP-2 has a short half-life and it is rapidly degraded.21 The disadvantages of therapies using BMP-2 include its rapid degradation and diffusion in vivo and the cost and side effects of
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
198
Journal of Biomaterials Applications 30(2)
(a)
(b)
NB
NB (d)
NB
NB
(e)
1.0 mm
(c)
(f)
*
* Figure 4. Histological microphotographs six months after implantation. Villanueva bone staining: 1.8 107 CP-H polyhedra group (a); rhBMP-2 group (b); 1.8 107 BMP-2 polyhedra group (c); 3.6 106 BMP-2 polyhedra group (d). Inserts of (c) and (d) shown at high magnification (e) and (f). Newly generated bone (NB) was entirely replaced by trabecular bone with hematopoietic bone marrow (*). Arrows: the boundary between new bone and preexisting bone.
the high doses of rhBMP-2 required for efficacy.22,23 To be maximally efficacious in regenerating bone, rhBMP2 must be combined with biomaterials. Current clinical strategies involving the absorbable collagen sponge matrix containing rhBMP-2 promise to greatly enhance bone regeneration therapy.24–27 Although these delivery systems have been shown to promote bone regeneration, they do not adequately control the release rate, often resulting in a high initial release of growth factor.28,29 Consequently, milligram doses are often necessary for protein-based therapies, which makes
therapy expensive and carries a risk of serious complications including edema and cancer.22,30–32 The main role of a delivery system for BMP-2 is to retain the growth factors at the bone defect site for a prolonged time, providing an initial support on which cells can attach and regenerate bone.33 In our experiments, six months after implantation, micro-CT images indicated greater improvements in bone volume for those defects treated with atelocollagen sponge impregnated with BMP-2 polyhedra than for those treated with atelocollagen sponge impregnated with free rhBMP-2. Although we have not determined the optimal therapeutic dose of BMP-2 polyhedra for this application, we estimate that 3.6 107 cubes of BMP-2 contain the equivalent 1 mg of BMP-2 protein. We found that there was greater bone regeneration at sites treated with 3.6 106 or 1.8 107 cubes of BMP-2 polyhedra than at control sites. BMP-2 activity from 3.6 106 or 1.8 107 cubes of BMP-2 polyhedra was estimated to be equivalent to the activity of 0.1 mg or 0.5 mg rhBMP-2, respectively. Statistical analysis shows that a quantity of 3.6 106 BMP-2 polyhedra has the same effect on bone regeneration as 1.8 107 BMP-2 polyhedra. In this experiment, the effect of BMP-2 delivered with polyhedral microcrystals was not dose dependent. This contrast with our previous experiments, in which a dose of 1.8 107 BMP-2 polyhedra induced more bone regeneration than did a dose of 3.6 106 BMP-2 polyhedra in the rat calvarial bone regeneration model. This discrepancy might be due to the lower relative dose of polyhedra used in our experiments with dogs. In any case, we have not made any efforts to determine the optimal dosage of BMP-2. We suspect that more careful determination of BMP-2 dosage will reveal the effects of dose on bone regeneration. Atelocollagen is considered to be the best carrier because of its high biocompatibility and low immunogenicity. However, because rhBMP-2 has little natural affinity for collagen, this approach requires the use of a very large dose of BMP-2. Geiger et al.34 showed that incorporation of rhBMP-2 into the collagen matrix reached levels of more than 90% especially at lower rhBMP-2 concentration. Protein incorporation could be slightly increased by extending the waiting time after impregnation. In our study, we soaked collagen sponge with 5 mg of rhBMP-2 for 12 h, the amount of incorporated rhBMP-2 in collagen sponge might exceed more than 90%. Consequently, loss of rhBMP-2 solution due to mechanical manipulation during implantation as well as potential effect of a matrix on in vivo retention has to be considered. On the other hand, polyhedral particles can easily be immobilized onto the surface of atelocollagen sponge.35,36 Our scanning electron micrograph images confirm that polyhedral
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
199
particles are retained on the surface of atelocollagen sponge. In our previous experiments, polyhedral cubes were present in the tissue 10 weeks after implantation, therefore polyhedral particles were absorbed and BMP-2 protein was secreted for 10 or more weeks.18 These polyhedra with their sequestered proteins are slowly degraded by extracellular proteases, and the proteins are gradually released into the target tissue in vivo. BMP-2 polyhedra induce complete healing of the mandibular alveolar bone ridge for the development of implant sites. The goal of delivering BMP-2 from a scaffold over an extended time period is achieved by this novel strategy.
Conclusion Although little bone regeneration was observed at bone defects treated by atelocollagen sponges containing rhBMP-2 alone, H1/fBMP-2 polyhedra-induced bone formation, almost entirely replacing the atelocollagen sponges and connecting the original bone with the regenerated bone. We conclude that use of polyhedra as a BMP-2 delivery system promises to advance the technology for bone regeneration.
Authors’ note Goichi Matsumoto and Hajime Mori contributed equally to this work.
Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding This work was supported in part by A-STEP exploratory research AS232Z00847F from JST and Grants-in-Aid for Scientific Research (C) 90237131 from JSPS.
References 1. Petite H, Viateau V, Bensaid W, et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000; 18: 959–963. 2. Reddi AH. Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage. J Cell Biochem 1994; 56: 192–195. 3. Reddi AH. BMPs: actions in flesh and bone. Nat Med 1997; 3: 837–839. 4. Schmitt JM, Hwang K, Winn SR, et al. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999; 17: 269–278. 5. Fiedler J, Roderer G, Gunther KP, et al. BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem 2002; 87: 305–312.
6. Mont MA, Ragland PS, Biggins B, et al. Use of bone morphogenetic proteins for musculoskeletal applications. An overview. J Bone Joint Surg Am 2004; 86-A: 41–55. 7. Bishop GB and Einhorn TA. Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery. Int Orthop 2007; 31: 721–727. 8. Carragee EJ, Hurwitz EL and Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J 2011; 11: 471–491. 9. Koo KH, Yeo do H, Ahn JM, et al. Lumbar posterolateral fusion using heparin-conjugated fibrin for sustained delivery of bone morphogenic protein-2 in a rabbit model. Artif Organs 2012; 36: 629–634. 10. La WG, Kang SW, Yang HS, et al. The efficacy of bone morphogenetic protein-2 depends on its mode of delivery. Artif Organs 2010; 34: 1150–1153. 11. Singh K, Smucker JD, Ugbo JL, et al. rhBMP-2 enhancement of posterolateral spinal fusion in a rabbit model in the presence of concurrently administered doxorubicin. Spine J 2007; 7: 326–331. 12. Vaccaro AR, Whang PG, Patel T, et al. The safety and efficacy of OP-1 (rhBMP-7) as a replacement for iliac crest autograft for posterolateral lumbar arthrodesis: minimum 4-year follow-up of a pilot study. Spine J 2008; 8: 457–465. 13. Jeppsson C, Bostrom M and Aspenberg P. Intraosseous BMP implants in rabbits. Inhibitory effect on bone formation. Acta Orthop Scand 1999; 70: 77–83. 14. Zabka AG, Pluhar GE, Edwards RB, 3rd, et al. Histomorphometric description of allograft bone remodeling and union in a canine segmental femoral defect model: a comparison of rhBMP-2, cancellous bone graft, and absorbable collagen sponge. J Orthop Res 2001; 19: 318–327. 15. Meikle MC. On the transplantation, regeneration and induction of bone: the path to bone morphogenetic proteins and other skeletal growth factors. Surgeon 2007; 5: 232–243. 16. Hsu WK and Wang JC. The use of bone morphogenetic protein in spine fusion. Spine J 2008; 8: 419–425. 17. Rohrmann GF. Polyhedrin structure. J Gen Virol 1986; 67: 1499–1513. 18. Matsumoto G, Ueda T, Shimoyama J, et al. Bone regeneration by polyhedral microcrystals from silkworm virus. Sci Rep 2012; 2: 935. 19. Choi Y, Yun JH, Kim CS, et al. Sinus augmentation using absorbable collagen sponge loaded with Escherichia coli-expressed recombinant human bone morphogenetic protein 2 in a standardized rabbit sinus model: a radiographic and histologic analysis. Clin Oral Implants Res 2011; 23: 682–689. 20. Lee J, Susin C, Rodriguez NA, et al. Sinus augmentation using rhBMP-2/ACS in a mini-pig model: relative efficacy of autogenous fresh particulate iliac bone grafts. Clin Oral Imp Res 2012; 24: 497–504. 21. Benglis D, Wang MY and Levi AD. A comprehensive review of the safety profile of bone morphogenetic
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
200
22.
23.
24.
25.
26.
27.
28.
Journal of Biomaterials Applications 30(2) protein in spine surgery. Neurosurgery 2008; 62: ONS423–431. (discussion ONS431). Cahill KS, Chi JH, Day A, et al. Prevalence, complications, and hospital charges associated with use of bonemorphogenetic proteins in spinal fusion procedures. JAMA 2009; 302: 58–66. Paramore CG, Lauryssen C, Rauzzino MJ, et al. The safety of OP-1 for lumbar fusion with decompression—a canine study. Neurosurgery 1999; 44: 1151–1155; discussion 1155–1156. Burkus JK, Gornet MF, Dickman CA, et al. Anterior lumbar interbody fusion using rhBMP-2 with tapered interbody cages. J Spinal Disord Tech 2002; 15: 337–349. Cochran DL, Jones AA, Lilly LC, et al. Evaluation of recombinant human bone morphogenetic protein-2 in oral applications including the use of endosseous implants: 3-year results of a pilot study in humans. J Periodontol 2000; 71: 1241–1257. Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002; 84-A: 2123–2134. Triplett RG, Nevins M, Marx RE, et al. Pivotal, randomized, parallel evaluation of recombinant human bone morphogenetic protein-2/absorbable collagen sponge and autogenous bone graft for maxillary sinus floor augmentation. J Oral Maxillofac Surg 2009; 67: 1947–1960. Helm GA, Sheehan JM, Sheehan JP, et al. Utilization of type I collagen gel, demineralized bone matrix, and bone
29.
30.
31.
32.
33.
34.
35.
36.
morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion. J Neurosurg 1997; 86: 93–100. Yang XB, Whitaker MJ, Sebald W, et al. Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds. Tissue Eng 2004; 10: 1037–1045. Argintar E, Edwards S and Delahay J. Bone morphogenetic proteins in orthopaedic trauma surgery. Injury 2010; 42: 730–734. Boraiah S, Paul O, Hawkes D, et al. Complications of recombinant human BMP-2 for treating complex tibial plateau fractures: a preliminary report. Clin Orthop Relat Res 2009; 467: 3257–3262. Shields LB, Raque GH, Glassman SD, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine (Phila Pa 1976) 2006; 31: 542–547. Seeherman H and Wozney JM. Delivery of bone morphogenetic proteins for orthopedic tissue regeneration. Cytokine Growth Factor Rev 2005; 16: 329–345. Geiger M, Li RH and Friess W. Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev 2003; 55: 1613–1629. Coulibaly F, Chiu E, Ikeda K, et al. The molecular organization of cypovirus polyhedra. Nature 2007; 446: 97–101. Matsushima K, Suyama T, Takenaka C, et al. Secreted frizzled related protein 4 reduces fibrosis scar size and ameliorates cardiac function after ischemic injury. Tissue Eng Part A 2010; 16: 3329–3341.
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge
Journal of Biomaterials Applications 2015, Vol. 30(2) 193–200 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215575763 jba.sagepub.com
Goichi Matsumoto1,4, Takayo Ueda2, Yoshihiko Sugita3, Katsutoshi Kubo3, Megumi Mizoguchi2, Eiji Kotani2, Naoki Oda2, Shin Kawamata5, Natsuki Segami4 and Hajime Mori2
Abstract Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 107 BMP-2 polyhedra, and half were treated with sponges containing 3.6 106 BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 mg of recombinant human BMP-2 (rhBMP-2) or 1.8 107 empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development. Keywords BMP-2, polyhedra, alveolar ridge, bone regeneration, atelocollagen sponge
Introduction Autologous bone has been the preferred transplant material to use in treating a bony defect in the oral and maxillofacial region that must be augmented before placing a dental implant. However, bone for an autologous bone graft must be harvested from a donor site, which introduces the risk of infection and hemorrhage and has other significant disadvantages. Bone tissue engineering has the potential to identify bone regeneration techniques that overcome these disadvantages. In order to facilitate bone healing and regeneration, more than one healing factor is desirable. Marginal healing conditions should be augmented by including an adequate osteosynthetic device, growth factors, and progenitor cells. The basic strategy incorporates one or more of the elements that are needed to regenerate bone tissue into the target site such as
scaffolding, growth factors, and osteogenic progenitor cells, which then induce bone formation and guide the
1 Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, Kanagawa-ku, Yokohama, Japan 2 Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan 3 Department of Oral Pathology, School of Dentistry, Aichi-Gakuin University, Chikusa-ku, Nagoya, Japan 4 Department of Oral and Maxillofacial Surgery, Kanazawa Medical University, Uchinada, Ishikawa, Japan 5 Basic Research Group for Regenerative Medicine, Foundation for Biomedical Research and Innovation, Kobe, Japan
Corresponding author: Goichi Matsumoto, Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, 3-31-6 Tsuruya-cho, Kanagawa-ku, Yokohama 221-0835, Japan. Email: [email protected]
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
194
Journal of Biomaterials Applications 30(2)
tissue repair function of the living body by encouraging osteogenic cells to migrate, proliferate, and differentiate.1 Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b superfamily and are disulfide-linked homodimers that induce bone and cartilage formation. Among these, BMP-2 has been shown to be a strong inducer of new bone formation in animal models and is now well recognized as one of the key osteogenic factors in the field of bone tissue engineering.2–4 BMP-2 induces bone regeneration by activating a set of cellular events including chemotaxis of uncommitted mesenchymal cells and differentiation of these cells into osteoblasts.5,6 BMP-2 has been approved for limited clinical use in the form of recombinant human BMP-2 (rhBMP-2).7 However, therapeutic use of rhBMP-2 has been hampered by the lack of an efficacious way to deliver this protein to the site of the regenerating bone. Despite its limited commercial use, there remain problems with the systems used to deliver rhBMP-2 and there remains uncertainty about the most effective dose.8–12 We have thus sought to identify an appropriate carrier to facilitate retention of rhBMP-2 at the healing site in order to reduce the required dose. A good way to deliver rhBMP-2 continuously to the healing site is via a carrier material. The preferred carrier is a scaffold that is biocompatible, biodegradable, nontoxic, and is immunologically inert in order to avoid tissue rejection. However, the most important requirement for a carrier is that it controls the release of the growth factor it delivers. The carrier materials for BMP-2 that are currently clinically available are not ideal because large amounts of BMP-2 are required to achieve the desired osteogenesis.13,14 Despite the ready availability of rhBMP-2 for clinical use, the dilemma facing clinicians and the biotechnology industry is how to achieve an optimal delivery system that can decrease the dose of BMP-2, maintain a more sustained release pattern, and effectively promote osteoconduction.15 Bone formation in large animals requires that BMP-2 is present for long periods of time for solid bone formation.11,16 Several insect viruses produce polyhedral microcrystals consisting of a protein called polyhedrin. In these polyhedra, many progeny virus particles are occluded and protected from hostile environmental conditions. These polyhedral microcrystals are the main vectors that transport virus particles from one insect to another. They are highly resistant to both nonionic and ionic detergents and they are soluble at neutral pH.17 In our previous studies, using both in vitro and in vivo rat calvarial bone defect models, polyhedra containing full-length BMP-2 had a similar biological
activity to BMP-2 protein and prolonged the period of BMP-2 release to induce new bone more effectively than conventional atelocollagen–BMP-2 protein complexes.18 Our previous data suggest that therapy with BMP-2 polyhedra improves bone regeneration. However, to date there have been no studies of the usefulness of BMP-2 polyhedra to facilitate the healing of defects in the mandibular alveolar ridge in large animals. Therefore, the aim of the present study is to investigate the effectiveness of BMP-2 polyhedra for alveolar ridge augmentation of surgically created defects in the canine mandible.
Materials and methods Preparation of atelocollagen sponges impregnated with BMP-2 polyhedra or rhBMP-2 We have already reported the encapsulation of human full-length BMP-2 into Bombyx mori Cypovirus (BmCPV) polyhedra by use of the H1-tag (BMP-2 polyhedra) where the average mass of BMP-2 encapsulated in a polyhedral microcrystal (one cube) was 2.8 105 ng (the average volume of a microcrystal is approximately 5 5 5 mm3) (Figure 1(a)).18 We have estimated that 3.6 107 BMP-2 polyhedra contain 1 mg of BMP-2 protein. In this study, we mixed either 1.8 107 or 3.6 106 BMP-2 polyhedra with 200 ml Phosphate Buffered Saline (PBS) and added it dropwise onto a 20 mm 10 mm 2 mm PelnacÕ atelocollagen sponge (Gunze, Kyoto, Japan), taking care to avoid any runoff of excess liquid. The atelocollagen sponge was used to retain polyhedral cubes and as a scaffold for bone regeneration (Figure 1(a)). Atelocollagen sponges were prepared in two additional ways to serve as controls. One type contained 1.8 107 CP-H polyhedra (empty polyhedra). In the other, a solution containing 5 mg of rhBMP-2 (Yamanouchi Pharmaceutical, Tokyo, Japan) in PBS was added dropwise onto the atelocollagen sponge. Again, care was exercised so as to avoid runoff of excess liquid. The impregnated atelocollagen sponges were left to stand for 12 h at 4 C before being implanted into the experimental bone defects.
Animals Six one-year-old female beagle dogs (approximately 10 kg body weight) were used for the experiment. The Animal Care Committee of Kanagawa Dental University approved animal management and the surgical protocol. The animals had access to a standard laboratory diet and water until the beginning of the study. Postoperatively, the animals were kept in cages for six months with free access to water and food that
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
195 both sides of the mandible. Teeth were sectioned using a diamond-coated separating bur before surgically extracting them using an elevator. Subsequently, two bone defects were prepared on each side of the mandible by surgically removing parts of the buccal bone plate with a fissure bur. Each defect was 10 mm long and 5 mm high. The lingual cortical bone wall was left intact. The anterior bone defect was separated by 5 mm from the posterior bone defect (Figure 1). The four lateral bone defects were symmetrically created on left and right in the region of the extracted teeth. Atelocollagen sponges were used as carriers for the experimental substances, which included BMP-2 polyhedra, CP-H polyhedra, and free rhBMP-2. The sponges also acted as scaffolding to guide new bone generation. We implanted the sponges in the bone defects (Figure 1) in the following manner: Left side:
(a)
ACS
(b)
Group 1: 1.8 107 BMP-2 polyhedra (n ¼ 6) Group 2: 3.6 106 BMP-2 polyhedra (n ¼ 6) Right side:
(c)
Group 3: 5 mg of rhBMP-2 (n ¼ 6) Group 4: 1.8 107 CP-H polyhedra (n ¼ 6) ACS
ACS
Figure 1. Clinical appearance of the surgically created bone defects and atelocollagen sponge placement. Scanning electron micrograph (SEM) image of recombinant polyhedra encapsulating BMP-2/atelocollagen sponge (ACS). Some polyhedral cubes retained on the surface of atelocollagen sponge (a). Intraoperative view of the two lateral bone defects created in the mandible before and after implanting atelocollagen sponges impregnated with BMP-2 polyhedra, CP-H polyhedra, or rhBMP-2 (b and c).
was moist, soft, and nutritionally balanced. All animals were given antibiotics (Viccillin, Meiji Co., Tokyo, Japan) for three days after surgery. All experiments were performed in accordance with relevant guidelines and regulations.
Procedures used to create bone defects and to treat them All animals were anesthetized by intravenously injecting SomnopentyleÕ at 25 mg/kg (Kyoritsu Seiyaku, Tokyo, Japan). Routine dental infiltration anesthesia was used at the surgical sites. We extracted all first molars and all second, third, and fourth premolars on
Because healing characteristics of anterior defect sites may be different than those of posterior defect sites, half of each of the four groups was implanted in an anterior defect, and the other half was implanted in a posterior defect. The right-side defects in the mandible were treated as control sites, and the left-side sites as experimental sites.
Micro-CT All animals were killed six months after surgery with an overdose of SomnopentyleÕ by intravenous injection. Block sections from both the right and left sides of the mandible were removed separately and placed in a solution of 70% ethanol for five days, after which the muscle around the mandible was removed. Each mandible was treated and examined as follows. The bone sample was positioned on the stage of a Micro X-ray CT System (Rigaku, Tokyo, Japan) so that the shaft was oriented vertically. The X-ray generator was operated at an accelerating potential of 90 kV with a beam current of 50 mA. Each slice consisted of 480 480 pixels; slice thickness was 0.8 mm. The volume and density of newly generated bone in the defect were measured using Mimics software. The volume of interest (VOI) of the alveolar ridge in the defect site was calculated. The VOI was drawn with a slice-based method starting from the first slice containing the crown of the
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
196
Journal of Biomaterials Applications 30(2)
first premolar and moving dorsally in the area between the distal wall of the first premolar and the mesial wall of the second molar. On the original three-dimensional (3D) micro-CT image, micro-structural indices were calculated directly from the digitized VOI. The VOI of the bone between the distal wall of the first premolar and the mesial wall of the second molar was measured. The newly regenerated bone was delineated by erasing the lingual cortical bone on each coronal slice image in the defect site. The density of newly generated bone was computed relative to neighboring normal cortical bone.
(a)
(b)
Histological analysis Each bone sample was sectioned perpendicular to its axis at the middle of each bone defect. The bones at all defect sites were undecalcified and dehydrated in 70% ethanol, followed by immersion in Villanueva bone stain solution for 14 days. The bones were again dehydrated through gradient ethanols and embedded in methyl methacrylate resin (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Specimens 20 mm thick were sectioned using a band saw perpendicular to the buccolingual plane. Surfaces of the sections were polished with diamond paper and observed under a light microscope.
(c)
(d)
(e)
(f)
Statistical analysis P values were computed using Student’s t-tests. A P value less than 0.01 was considered statistically significant.
Results Clinical observations All animals tolerated the surgical procedures without clinical signs of adverse reactions and were healthy during the entire experimental period.
Micro-CT observation The healing of lateral bone defects in each group was clearly visible in the 3D micro-CT reconstructions, which were used to calculate the volume of new bone. Bone defects appeared to be healed almost completely in the groups treated with BMP-2 polyhedra in which full-length BMP-2 was immobilized and encapsulated by the H1 tag (Figure 2(a), (c), and (d))). In contrast, there was almost no regenerated bone in the bone defects in the groups treated with rhBMP-2 or empty polyhedra (CP-H polyhedra) (Figure 2(b), (e), and (f)). Cross-sectional views show that there was almost complete healing of bone defects only in the group treated
Figure 2. Three-dimensional micro-CT reconstructed appearance of bone regeneration in the mandibular bone defect six months after surgery. The lateral bone defects on the left side of the mandible were implanted with atelocollagen sponges impregnated with 1.8 107 BMP-2 polyhedra (front side) and 3.6 106 BMP-2 polyhedra (back side) (a). Bone defects on the right side of the mandible were implanted with atelocollagen sponges impregnated with rhBMP-2 (front side) and 1.8 107 CP-H polyhedra (empty polyhedra) (back side) (b). Micro-CT images of the frontal sections of mandibular bone defects in the 1.8 107 BMP-2 polyhedra group (c), the 3.6 106 BMP-2 polyhedra group (d), the 1.8 107 CP-H polyhedra group (e), and the rhBMP-2 group (f). Newly generated alveolar bone in each treatment group is shown with dotted squares.
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
197
(a) 350
*
Bone volume (mm3)
300
defect sites in the group treated with CP-H polyhedra. However, the mean bone density at defect sites in the CP-H polyhedra-treated, BMP-2 polyhedra-treated, and rhBMP-2-treated groups was not significantly different than that of the neighboring normal cortical bone. For both the BMP-2 polyhedra-treated group and the rhBMP-2-treated group, the mean bone density at defect sites was not different than that of the neighboring normal cortical bone (Figure 3(b)).
*
250 200 150 100 50
Histological observation a dr
2
he
P-
6
10
ly po H
rh
1.
3.
8
6
x
x
BM
7
10
0
C
P-
BMP-2 polyhedra
(b) 1.4
Relative bone density
1.2 1 0.8 0.6 0.4 0.2
a po
rh
ly
BM
he
P-
dr
2
6
10 x 6 3.
x 8 1.
C
BMP-2 polyhedra
P-
H
N
or
m
al
bo
10
7
ne
0
Figure 3. Newly generated alveolar bone volume and density from three-dimensional micro-CT analysis in the 1.8 107 BMP-2 polyhedra, 3.6 106 BMP-2 polyhedra, 5 mg rhBMP-2, and 1.8 107 CP-H polyhedra groups. The alveolar bone volume in the area of the bone defect with atelocollagen sponge implantation at six months postoperatively. *P < 0.01 versus 1.8 107 CP-H polyhedra and rhBMP-2 (a). The relative density of new bone six months postoperatively. Data were normalized with respect to the density of the neighboring normal cortical bone (b).
with BMP-2 polyhedra. The volume of new bone was calculated from 3D micro-CT images. The volume of newly generated bone at defect sites in the group treated with BMP-2 polyhedra was significantly greater than that formed at defect sites in groups treated with rhBMP-2 or CP-H polyhedra. However, there was no significant difference between groups treated with 3.6 106 BMP-2 polyhedra and 1.8 107 BMP-2 polyhedra (Figure 3(a)). In addition, there was no significant difference between anterior and posterior sites treated with BMP-2 polyhedra (data not shown). Next, we measured the bone density in the newly regenerated bone. The greatest density of new bone was at defect sites in the group treated with 1.8 107 BMP-2 polyhedra. The least density of new bone was at
Six months postoperatively, atelocollagen sponge was not observed at any of the healing sites. There was very little osseous regeneration at sites treated with rhBMP2 or CP-H polyhedra. New bone formation was limited, resulting in a less adequate ridge profile with dents on the buccal aspect (Figure 4(a) and (b)). In contrast, in the newly generated bone induced by BMP-2 polyhedra, concentric layers of bone tissue were observed around Haversian canals, and well-defined lamellar bone exhibiting characteristics of mature bone was observed (Figure 4(e) and (f)). Inflammatory cell was not observed in the group treated with BMP-2 polyhedra. A trabecular pattern of regenerated bone was observed in direct contact to original bone. The bony regenerate had fully filled the defect space, and defect sites in general demonstrated well-maintained ridge contours (Figure 4(c) and (d)). These findings suggest that newly generated bone in the groups treated with BMP-2 polyhedra develops into mature bone tissue. Regenerated bone in both anterior and posterior sites treated with BMP-2 polyhedra consisted of mature bone tissue (data not shown).
Discussion Many studies have demonstrated extensive bone formation using BMP-2 with different carriers in various animal models.13–15 A local delivery system for BMP2 may be important for those applications in which an optimal dose of BMP-2 is required.19,20 The concentration of BMP-2 necessary to promote highly effective bone formation is still unclear. Our hypothesis is that long-term delivery of BMP-2 protein induces greater in vivo bone formation that does short-term delivery at an equivalent dose. For this reason, we have developed a superior carrier system with sustained release of BMP-2 using polyhedra, and we continue to investigate its performance. Polyhedra were prepared to serve as the new carriers for rhBMP-2. Exogenously delivered BMP-2 has a short half-life and it is rapidly degraded.21 The disadvantages of therapies using BMP-2 include its rapid degradation and diffusion in vivo and the cost and side effects of
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
198
Journal of Biomaterials Applications 30(2)
(a)
(b)
NB
NB (d)
NB
NB
(e)
1.0 mm
(c)
(f)
*
* Figure 4. Histological microphotographs six months after implantation. Villanueva bone staining: 1.8 107 CP-H polyhedra group (a); rhBMP-2 group (b); 1.8 107 BMP-2 polyhedra group (c); 3.6 106 BMP-2 polyhedra group (d). Inserts of (c) and (d) shown at high magnification (e) and (f). Newly generated bone (NB) was entirely replaced by trabecular bone with hematopoietic bone marrow (*). Arrows: the boundary between new bone and preexisting bone.
the high doses of rhBMP-2 required for efficacy.22,23 To be maximally efficacious in regenerating bone, rhBMP2 must be combined with biomaterials. Current clinical strategies involving the absorbable collagen sponge matrix containing rhBMP-2 promise to greatly enhance bone regeneration therapy.24–27 Although these delivery systems have been shown to promote bone regeneration, they do not adequately control the release rate, often resulting in a high initial release of growth factor.28,29 Consequently, milligram doses are often necessary for protein-based therapies, which makes
therapy expensive and carries a risk of serious complications including edema and cancer.22,30–32 The main role of a delivery system for BMP-2 is to retain the growth factors at the bone defect site for a prolonged time, providing an initial support on which cells can attach and regenerate bone.33 In our experiments, six months after implantation, micro-CT images indicated greater improvements in bone volume for those defects treated with atelocollagen sponge impregnated with BMP-2 polyhedra than for those treated with atelocollagen sponge impregnated with free rhBMP-2. Although we have not determined the optimal therapeutic dose of BMP-2 polyhedra for this application, we estimate that 3.6 107 cubes of BMP-2 contain the equivalent 1 mg of BMP-2 protein. We found that there was greater bone regeneration at sites treated with 3.6 106 or 1.8 107 cubes of BMP-2 polyhedra than at control sites. BMP-2 activity from 3.6 106 or 1.8 107 cubes of BMP-2 polyhedra was estimated to be equivalent to the activity of 0.1 mg or 0.5 mg rhBMP-2, respectively. Statistical analysis shows that a quantity of 3.6 106 BMP-2 polyhedra has the same effect on bone regeneration as 1.8 107 BMP-2 polyhedra. In this experiment, the effect of BMP-2 delivered with polyhedral microcrystals was not dose dependent. This contrast with our previous experiments, in which a dose of 1.8 107 BMP-2 polyhedra induced more bone regeneration than did a dose of 3.6 106 BMP-2 polyhedra in the rat calvarial bone regeneration model. This discrepancy might be due to the lower relative dose of polyhedra used in our experiments with dogs. In any case, we have not made any efforts to determine the optimal dosage of BMP-2. We suspect that more careful determination of BMP-2 dosage will reveal the effects of dose on bone regeneration. Atelocollagen is considered to be the best carrier because of its high biocompatibility and low immunogenicity. However, because rhBMP-2 has little natural affinity for collagen, this approach requires the use of a very large dose of BMP-2. Geiger et al.34 showed that incorporation of rhBMP-2 into the collagen matrix reached levels of more than 90% especially at lower rhBMP-2 concentration. Protein incorporation could be slightly increased by extending the waiting time after impregnation. In our study, we soaked collagen sponge with 5 mg of rhBMP-2 for 12 h, the amount of incorporated rhBMP-2 in collagen sponge might exceed more than 90%. Consequently, loss of rhBMP-2 solution due to mechanical manipulation during implantation as well as potential effect of a matrix on in vivo retention has to be considered. On the other hand, polyhedral particles can easily be immobilized onto the surface of atelocollagen sponge.35,36 Our scanning electron micrograph images confirm that polyhedral
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
Matsumoto et al.
199
particles are retained on the surface of atelocollagen sponge. In our previous experiments, polyhedral cubes were present in the tissue 10 weeks after implantation, therefore polyhedral particles were absorbed and BMP-2 protein was secreted for 10 or more weeks.18 These polyhedra with their sequestered proteins are slowly degraded by extracellular proteases, and the proteins are gradually released into the target tissue in vivo. BMP-2 polyhedra induce complete healing of the mandibular alveolar bone ridge for the development of implant sites. The goal of delivering BMP-2 from a scaffold over an extended time period is achieved by this novel strategy.
Conclusion Although little bone regeneration was observed at bone defects treated by atelocollagen sponges containing rhBMP-2 alone, H1/fBMP-2 polyhedra-induced bone formation, almost entirely replacing the atelocollagen sponges and connecting the original bone with the regenerated bone. We conclude that use of polyhedra as a BMP-2 delivery system promises to advance the technology for bone regeneration.
Authors’ note Goichi Matsumoto and Hajime Mori contributed equally to this work.
Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding This work was supported in part by A-STEP exploratory research AS232Z00847F from JST and Grants-in-Aid for Scientific Research (C) 90237131 from JSPS.
References 1. Petite H, Viateau V, Bensaid W, et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000; 18: 959–963. 2. Reddi AH. Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage. J Cell Biochem 1994; 56: 192–195. 3. Reddi AH. BMPs: actions in flesh and bone. Nat Med 1997; 3: 837–839. 4. Schmitt JM, Hwang K, Winn SR, et al. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999; 17: 269–278. 5. Fiedler J, Roderer G, Gunther KP, et al. BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem 2002; 87: 305–312.
6. Mont MA, Ragland PS, Biggins B, et al. Use of bone morphogenetic proteins for musculoskeletal applications. An overview. J Bone Joint Surg Am 2004; 86-A: 41–55. 7. Bishop GB and Einhorn TA. Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery. Int Orthop 2007; 31: 721–727. 8. Carragee EJ, Hurwitz EL and Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J 2011; 11: 471–491. 9. Koo KH, Yeo do H, Ahn JM, et al. Lumbar posterolateral fusion using heparin-conjugated fibrin for sustained delivery of bone morphogenic protein-2 in a rabbit model. Artif Organs 2012; 36: 629–634. 10. La WG, Kang SW, Yang HS, et al. The efficacy of bone morphogenetic protein-2 depends on its mode of delivery. Artif Organs 2010; 34: 1150–1153. 11. Singh K, Smucker JD, Ugbo JL, et al. rhBMP-2 enhancement of posterolateral spinal fusion in a rabbit model in the presence of concurrently administered doxorubicin. Spine J 2007; 7: 326–331. 12. Vaccaro AR, Whang PG, Patel T, et al. The safety and efficacy of OP-1 (rhBMP-7) as a replacement for iliac crest autograft for posterolateral lumbar arthrodesis: minimum 4-year follow-up of a pilot study. Spine J 2008; 8: 457–465. 13. Jeppsson C, Bostrom M and Aspenberg P. Intraosseous BMP implants in rabbits. Inhibitory effect on bone formation. Acta Orthop Scand 1999; 70: 77–83. 14. Zabka AG, Pluhar GE, Edwards RB, 3rd, et al. Histomorphometric description of allograft bone remodeling and union in a canine segmental femoral defect model: a comparison of rhBMP-2, cancellous bone graft, and absorbable collagen sponge. J Orthop Res 2001; 19: 318–327. 15. Meikle MC. On the transplantation, regeneration and induction of bone: the path to bone morphogenetic proteins and other skeletal growth factors. Surgeon 2007; 5: 232–243. 16. Hsu WK and Wang JC. The use of bone morphogenetic protein in spine fusion. Spine J 2008; 8: 419–425. 17. Rohrmann GF. Polyhedrin structure. J Gen Virol 1986; 67: 1499–1513. 18. Matsumoto G, Ueda T, Shimoyama J, et al. Bone regeneration by polyhedral microcrystals from silkworm virus. Sci Rep 2012; 2: 935. 19. Choi Y, Yun JH, Kim CS, et al. Sinus augmentation using absorbable collagen sponge loaded with Escherichia coli-expressed recombinant human bone morphogenetic protein 2 in a standardized rabbit sinus model: a radiographic and histologic analysis. Clin Oral Implants Res 2011; 23: 682–689. 20. Lee J, Susin C, Rodriguez NA, et al. Sinus augmentation using rhBMP-2/ACS in a mini-pig model: relative efficacy of autogenous fresh particulate iliac bone grafts. Clin Oral Imp Res 2012; 24: 497–504. 21. Benglis D, Wang MY and Levi AD. A comprehensive review of the safety profile of bone morphogenetic
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
200
22.
23.
24.
25.
26.
27.
28.
Journal of Biomaterials Applications 30(2) protein in spine surgery. Neurosurgery 2008; 62: ONS423–431. (discussion ONS431). Cahill KS, Chi JH, Day A, et al. Prevalence, complications, and hospital charges associated with use of bonemorphogenetic proteins in spinal fusion procedures. JAMA 2009; 302: 58–66. Paramore CG, Lauryssen C, Rauzzino MJ, et al. The safety of OP-1 for lumbar fusion with decompression—a canine study. Neurosurgery 1999; 44: 1151–1155; discussion 1155–1156. Burkus JK, Gornet MF, Dickman CA, et al. Anterior lumbar interbody fusion using rhBMP-2 with tapered interbody cages. J Spinal Disord Tech 2002; 15: 337–349. Cochran DL, Jones AA, Lilly LC, et al. Evaluation of recombinant human bone morphogenetic protein-2 in oral applications including the use of endosseous implants: 3-year results of a pilot study in humans. J Periodontol 2000; 71: 1241–1257. Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002; 84-A: 2123–2134. Triplett RG, Nevins M, Marx RE, et al. Pivotal, randomized, parallel evaluation of recombinant human bone morphogenetic protein-2/absorbable collagen sponge and autogenous bone graft for maxillary sinus floor augmentation. J Oral Maxillofac Surg 2009; 67: 1947–1960. Helm GA, Sheehan JM, Sheehan JP, et al. Utilization of type I collagen gel, demineralized bone matrix, and bone
29.
30.
31.
32.
33.
34.
35.
36.
morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion. J Neurosurg 1997; 86: 93–100. Yang XB, Whitaker MJ, Sebald W, et al. Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds. Tissue Eng 2004; 10: 1037–1045. Argintar E, Edwards S and Delahay J. Bone morphogenetic proteins in orthopaedic trauma surgery. Injury 2010; 42: 730–734. Boraiah S, Paul O, Hawkes D, et al. Complications of recombinant human BMP-2 for treating complex tibial plateau fractures: a preliminary report. Clin Orthop Relat Res 2009; 467: 3257–3262. Shields LB, Raque GH, Glassman SD, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine (Phila Pa 1976) 2006; 31: 542–547. Seeherman H and Wozney JM. Delivery of bone morphogenetic proteins for orthopedic tissue regeneration. Cytokine Growth Factor Rev 2005; 16: 329–345. Geiger M, Li RH and Friess W. Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev 2003; 55: 1613–1629. Coulibaly F, Chiu E, Ikeda K, et al. The molecular organization of cypovirus polyhedra. Nature 2007; 446: 97–101. Matsushima K, Suyama T, Takenaka C, et al. Secreted frizzled related protein 4 reduces fibrosis scar size and ameliorates cardiac function after ischemic injury. Tissue Eng Part A 2010; 16: 3329–3341.
Downloaded from jba.sagepub.com at Purdue University on June 4, 2016
