The Spine Journal
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Basic Science
The bone morphogenetic protein-2/7 heterodimer is a stronger inducer of bone regeneration than the individual homodimers in a rat spinal fusion model Tokimitsu Morimoto, MDa, Takashi Kaito, MD, PhDa,*, Yohei Matsuo, MDb, Tsuyoshi Sugiura, MDb, Masafumi Kashii, MD, PhDa, Takahiro Makino, MDa, Motoki Iwasaki, MDa, Hideki Yoshikawa, MD, PhDa a Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan Department of Orthopedic Biomaterial Science, Graduate School of Medicine, Osaka University, 2-2 Yamadaok, Suita, Osaka 565–0871, Japan Received 8 September 2014; revised 26 December 2014; accepted 18 February 2015
b
Abstract
BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are a group of dimeric growth factors that belong to the transforming growth factor super family and are capable of eliciting new bone formation. Previous studies have suggested that the coexpression of two different BMP genes in a cell can result in the production of BMP heterodimers that are more potent than homodimers. However, because of the difficulty in optimizing the level of BMP gene expression, the coexpression of two different BMP genes also produces BMP homodimers as a by-product. These homodimers could, in theory, interact with the heterodimers. PURPOSE: To elucidate the effects of a BMP-2/7 heterodimer, which were investigated in depth using purified BMP-2/7 heterodimers, BMP-2 homodimers, and BMP-7 homodimers in a rat spinal fusion model. METHODS: Bilateral posterolateral fusion at L4–L5 was performed in four different groups: control group animals were implanted with collagen carriers alone; BMP-7 group animals with collagen carriersþ1 mg of BMP-7 homodimer; BMP-2 group animals with collagen carriersþ1 mg of BMP-2 homodimer; and BMP-2/7 group animals with collagen carriersþ1 mg of the BMP-2/7 heterodimer. The following assessments were performed: bone microstructural analysis of the fusion mass and tissue volume (TV) with microcomputed tomography (micro-CT); fusion assessment with manual palpation testing and three-dimensional CT images; and bone histomorphometrical analysis of the fusion mass. RESULTS: The fusion scores, as determined by radiography, and the TV of the newly formed bone, as determined by micro-CT, were significantly higher in the BMP-2/7 heterodimer group than the other groups (p!.0001). The microstructural indices of the newly formed bone did not differ between the groups. Moreover, histologic analysis of the fused spines revealed that the formation of the trabecular bone bridging the transverse process was the highest in this group. CONCLUSIONS: This study demonstrated that BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-2 or -7 homodimers. The use of a purified BMP-2/7 heterodimer may represent an efficient alternative to the current clinical use of BMP-2 or -7 homodimers. Further studies as to the side effects of BMP-2/7 heterodimer are required. Ó 2015 Elsevier Inc. All rights reserved.
Keywords:
Bone morphogenetic protein; BMP heterodimer; BMP homodimer; Cytokine therapy; Spinal fusion; Bone regeneration
FDA device/drug status: Not applicable. Author disclosures: TM: Nothing to disclose. TK: Grant: Japan Society for the Promotion of Science (No. 25861313, D, Paid directly to institution). YM: Nothing to disclose. TS: Nothing to disclose. MK: Nothing to disclose. TM: Nothing to disclose. MI: Nothing to disclose. HY: Nothing to disclose. http://dx.doi.org/10.1016/j.spinee.2015.02.034 1529-9430/Ó 2015 Elsevier Inc. All rights reserved.
The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan. Tel.: (81) 668-793-552; fax: (81) 668-793-559. E-mail address: [email protected] (T. Kaito)
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Introduction
Materials and methods
Autogenous bone grafting is the current gold standard for achieving spinal fusion. However, the effectiveness of this technique is limited by the amount of bone available and the associated donor site morbidity. Moreover, the reported rate of pseudoarthrosis ranges from 5% to 43% [1,2], thus necessitating a search for alternative methods to induce bone formation. One alternative to autogenous grafting is the use of bone morphogenetic proteins (BMPs), which are a group of dimeric growth factors that belong to the transforming growth factor superfamily and are capable of eliciting new bone formation [3–5]. Although the use of BMP homodimers in bone repair has been approved by the Food and Drug Administration, the dose required to induce adequate bone repair is very high because of the burst release and diffusion over a short period [6]. Accordingly, there are concerns regarding a substantial economic burden [7,8], but also a series of potential side-effects, including inflammation, soft tissue edema, and unintended bone formation [9,10]. Plenty of drug delivery systems are invented to decrease the required dose of BMP(s) and control their release kinetics (Table 1). One alternative approach to overcome these problems is to enhance the potency of the BMPs used. Previous studies have suggested that the coexpression of two different BMP genes can result in the production of BMP heterodimers that are more potent than their constituent homodimers in inducing in vitro osteoblastic differentiation [18–20] and in vivo osteogenesis [21–23]. In a previous study in our lab [24], combined BMP-2 and -7 ex vivo gene transfer was significantly more effective than individual BMP transfer in inducing new bone formation. However, the interpretation of our results was limited by the fact that the coexpression of two different BMP genes also produces BMP homodimers as a by-product because of the difficulties in optimizing the expression level of each BMP gene. Therefore, it was possible that interactions between BMP homodimers or between BMP homodimers and BMP heterodimers may have contributed to the improved bone induction. To overcome these limitations, the present study was designed to compare the effects of BMP-2/7 heterodimer with those of their constituent homodimers using purified cytokines instead of ex vivo gene expression in a rat spinal fusion model.
Experimental design A total of 38 male 8-week-old Sprague-Dawley rats (weight range, 280–330 g) were used in the present study. Because of the high effect size based on the results of the preliminary experiment and the significance level in this study (p!.0083), the number of animals required for the experiment was calculated as more than 32 (eight animals for each group). The ages of the animals were decided with reference to previous reports [25]. The animals were allowed to acclimate to the facility for 4 to 5 days before the operation. The animals were operated with bilateral posterolateral fusion at L4–L5 and divided into four different treatment groups: Group A received collagen carriers only (control, n510); Group B received collagen carriers with 1 mg of the recombinant human (rh) BMP-7 homodimer (BMP-7, n59); Group C received collagen carriers with 1 mg of the rhBMP-2 homodimer (BMP-2, n59); and Group D received collagen carriers with 1 mg of the rhBMP-2/7 heterodimer (BMP-2/7, n510). Preparation of a collagen carrier vehicle A commercially available absorbable collagen sponge (CollaCote; Zimmer Dental, Inc., Carlsbad, CA, USA) was cut into 510-mm fragments and placed in a sterile tube. Thereafter, the rhBMPs (rhBMP-7 homodimer, rhBMP-2 homodimer, or rhBMP-2/7 heterodimer; R&D System, Inc., Minneapolis, MN, USA) were dissolved in a sterile 4 mM hydrogen chloride (HCl) solution (rhBMP concentration: 10 mg/mL) and applied to the carrier just before implantation. The control group carriers were treated with the 4 mM HCl vehicle alone. All BMPs were applied to the collagen just before implantation. L4–L5 posterolateral spine fusion procedure All the animal procedures were conducted in accordance with the Regulations on Animal Experimentation at Osaka University. The rats were anesthetized using a combination of 0.15 mg/kg medetomidine (Domitol; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 2 mg/kg
Table 1 BMP dosages and fusion rates in previous reports using a rat spinal fusion model Animals
Age (wk)
Carrier
Dose (mg)
Assessment
FR
BMP-7
SD rat SD rat Lewis rat
— — 8
ICBM ICBM ACS
10 30 10
BMP-2
Lewis rat Athymic rats SD rat Lewis rat
— — 8 8
ACS ACS ACS ACS
10 5 50 5
X ray X ray X ray Micro-CT X ray X ray X ray X ray
100% 83.3% 100% 100% 100% 100% 88% 100%
BMP used
Reference (8/8) (5/6) (10/10) (10/10) (10/10) (8/8) (7/8) (8/8)
[11] [12] [13] [14] [15] [16] [17]
BMP, bone morphogenetic protein; SD, Sprague-Dawley; FR, fusion rate; ICBM, insoluble collagen bone matrix; ACS, absorbable collagen sponge; micro-CT, microcomputed tomography.
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midazolam (Dormicum; Astellas Pharma, Inc., Tokyo, Japan), and 2.5 mg/kg butorphanol (Vetorphale; Meiji Seika, Ltd., Tokyo, Japan). In addition, preoperative antibiotics (20,000 U/kg penicillin G; Meiji Seika, Ltd., Tokyo, Japan) were administered subcutaneously. Posterolateral lumbar fusion in rats has been well established as an acceptable model for measuring bone growth. Following the standard method [24,26], a posterior midline skin incision was made, followed by two separate paramedian incisions in the lumbar fascia 3 mm from the midline, through which the transverse processes were exposed. The L4 and L5 transverse processes were decorticated using a high-speed diamond burr of 3 mm in diameter until the bleeding from the transverse processes could be observed. Subsequently, a collagen sponge containing 1 mg of each rhBMP dissolved in 100 ml of 4 mM HCL solution or 100 ml of 4 mM HCL solution without rhBMP in the control group was implanted on each side. The fascia and skin incisions were closed using a 4-0 absorbable suture. The surgeons TK and TM performed all the operations. The rats were housed in separate cages and allowed access to food and water ad libitum, with daily monitoring of their condition. Euthanasia and tissue harvesting The rats were euthanized using an overdose of anesthetics 6 weeks after surgery. The spinal segments were harvested and fixed with 10% formalin for further assessments. Radiographic analysis Fusion between L4 and L5 was evaluated using plainfilm radiographs obtained using an MX-20 Specimen Radiography System (Faxitron X-ray Corp., Wheeling, IL, USA) under consistent conditions (35 kV, 300 mA, 300 seconds). These films were evaluated in a blinded manner by three independent observers. Bony fusion at L4–L5 was quantified using plain radiographs based on a 5point scale (0, no bone formation; 1, poor new bone formation; 2, moderate new bone formation; 3, good new bone formation and probable fusion; and 4, definite fusion) bilaterally as described previously [25] (Table 2). Definite fusion was considered when there was clear evidence of new bone formation and bony bridging with cortical continuity between the L4 and L5 transverse processes. Table 2 Scoring system to evaluate posterolateral spinal fusion Description
Score
No bone Poor new bone formation Moderate new bone formation Good new bone formation and probable fusion Definite fusion
0 1 2 3 4
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Manual assessment of fusion The explanted lumbar spines were manually tested for intersegmental motion. Any motion detected on either side between the facets or between the transverse processes was considered to be a failure of fusion. The absence of motion (both in the anteroposterior and left-right direction) was considered an indication of successful fusion [24]. The spines were scored as either fused or not fused independently by three examiners. The L4–L5 segments were considered to be fused only when a unanimous agreement of all three observers was obtained. Microcomputed tomography analysis The spines were scanned using high-resolution microcomputed tomography (micro-CT) (R_mCT; Rigaku Mechatronics, Tokyo, Japan). The micro-CT data were collected at 90 kV and 200 mA. Visualization and data reconstruction were performed using TRI/3D-BON (RATOC System Engineering, Tokyo, Japan). Sequential microstructural analysis of the fused spine segments in vivo: to compare the volume of the newly formed bone mass in each group, the total volume of L4– L5 fusion segments (from bottom of the L5 transverse process cranially to the top of L4 end plate) was measured in vivo at a 59 mm resolution at various time points (Day 0, 1 week, 2 weeks, 4 weeks, and 6 weeks). Assessment of spinal fusion using micro-CT: coronal and sagittal images of L4–L5 at a resolution of 50 mm per voxel were evaluated in a blinded manner by three independent observers based on a 5-point scale as mentioned previously [27], and fusion was considered to be clear evidence of formation of a bridging bone with cortical continuity between the L4 and L5 transverse processes. Analysis of the microstructural indices of the newly formed fusion mass of the fused spine segments at 6 weeks after operation: after manual evaluation of the explanted lumbar spines, the quality and quantity of the newly formed fusion mass between the intertransverse processes were analyzed as described previously [16,28]. Scanning of the fusion mass was initiated from the lower end plate level of L4 vertebral body and continued cranially in 1.5 mm increments (75 slices) at a resolution of 20 mm per voxel. Bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), thickness of cortical bone (Ct), and cortical bone ration (Cv/Av) were estimated. Bone volume/TV corresponds to bone volume to fusion mass volume ratio. In addition, the TV and BV values of the total fusion mass (15 mm cranially from the bottom of L5 transverse process [300 slices]) were measured at a resolution of 50 mm per voxel. Histologic analysis The dissected and formalin-fixed spines were demineralized with 50% formic acid and 10% sodium citrate,
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dehydrated through an ethanol series, and embedded in paraffin wax. Coronal sections at the level of anterior one-third of vertebral body were used for the evaluation. Serial coronal sections (thickness, 5 mm) of the dissected segments were cut and stained with hematoxylin and eosin. Three independent observers blindly scored histologic findings. Histologic fusion was defined as bony trabeculae bridging from one transverse process to the next. The extent of new bone formation was scored using the following scoring criteria: 0, empty cleft; 1, slight bump within the fibrocartilage tissue (filling less than 25% gap area); 2, some gap within the fibrocartilage tissue (filling 25%–50% of the gap area); 3, small gaps within the fibrocartilage and bone tissue (filling 75%– 99% of the gap area); 4, bridged with bone tissue, however, the fusion masses were composed of thin trabecular bone; and 5, completely bridged with abundant mature bone tissue [29]. Immunohistochemistry was performed as described previously [30] using anti-alkaline phosphatase (Ab108337, dilution 1:500; Abcam, Cambridge, UK) and anti-cathepsin K (dilution 1:200; Abcam, Cambridge, UK). Immune complexes were visualized with a goat polyclonal anti-rabbit IgG antibody coupled to biotin (426051; Nichirei Biosciences, Inc., Tokyo, Japan). 3-amino-9-ethylcarbazole was used as a substrate.
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Statistical analysis The computer software program PASW (version 18; SPSS, Chicago, IL, USA) was used for all the analyses. Mann-Whitney U test was used for the comparison (TV). Chi-squared test was used for the comparison of assessment of fusion. The level of significance was set at p!.05 for the comparison in the same group and p!.0083 for the comparison between the four groups (Bonferroni method). Intraand interobserver interclass correlation coefficients (ICCs) for radiographic fusion scores (plain radiography and micro-CT) and histologic scores were calculated. Only interobserver ICC was calculated for manual assessment of fusion.
Results Radiographic fusion analysis The average radiographic fusion scores were the highest in the BMP-2/7 group (7.6360.06), compared with values of 0.7060.17 in the control group, 2.8560.51 in the BMP-7 group, and 4.8960.40 in the BMP-2 group, and these differences were statistically significant (p!.0001 vs. control, p!.0001 vs. BMP-7, and p5.0007 vs. BMP-2) (Figs. 1 and 2). Intra- and interobserver ICCs were 0.932 (95% confidence
Fig. 1. Radiographs of the lumbar spine 6 weeks post-surgery. (A) Minimal new bone formation was found in the controls. (B) In the bone morphogenetic protein (BMP)-7 group, the newly developed bone formed a bridge between transverse processes, but gaps were evident. (C) In the BMP-2 group, the gap between the transverse process on the left side was occupied by the newly formed bone, but the clear line and discontinuous trabecular bone is suggestive of nonunion. (D) In the BMP-2/7 group, the new bone bridge between transverse processes and continuous trabecular bone suggests osseous fusion.
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Fig. 2. Fusion scores based on plain radiography. The BMP-2/7 group shows significantly higher fusion scores. Dotted line (score58) indicates complete fusion in all specimens. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.*p!.01; **p!.001.
interval [CI], 0.894–0.956) and 0.905 (95% CI, 0.865–0.935), respectively, for the radiographical fusion scoring. Manual palpation All spines were examined by manual palpation to determine fusion. The fusion rate was strongly increased in the BMP-2/7 group (90%; 9 of 10), compared with 0% (0 of 10) in the control group, 11.1% (1 of 9) in the BMP-7 group, and 22.2% in the BMP-2 group, and these differences were statistically significant (p!.0001 vs. control, p!.0001 vs. BMP-7, p!.0001 vs. BMP-2) (Table 3). Interobserver ICC was 0.783 (95% CI, 0.663–0.872).
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however, the increase was not as pronounced as that in the BMP-2/7 group. The change in the TV in the BMP-7 group was almost parallel to that in the control group. The average TV values 6 weeks after surgery were 67.766.0 mm3 in the control group, 66.067.0 mm3 in the BMP-7 group, 88.1626.2 mm3 in the BMP-2 group, and 104.3625.1 mm3 in the BMP-2/7 group. The TV in the BMP-2/7 group was significantly higher than that in the control and BMP-7 groups (p!.001 vs. control, p!.004 vs. BMP-7), but the difference in TV between the BMP-2/7 and -2 groups was not statistically significant (p5.21) (Figs. 3 and 4). We also assessed spinal fusion using micro-CT. The average micro-CT fusion scores were 1.3060.06 in the control group, 3.5260.26 in the BMP-7 group, 4.8160.32 in the BMP-2 group, and 7.6060.10 in the BMP-2/7 group. The micro-CT fusion score was significantly higher in the BMP-2/7 group compared with the other groups (p!.0000001 vs. control, p!.000001 vs. BMP-7; p!.0008 vs. BMP-2) (Figs. 5 and 6). Intra- and interobserver ICCs were 0.964 (95% CI, 0.944–0.977) and 0.939 (95% CI, 0.913–0.959), respectively. Analysis of the microstructural indices of the newly formed bone revealed that the TV of the newly formed bone was 0.5360.32 mm3 in the control group, 1.4361.29 mm3 in the BMP-7 group, 2.1461.26 mm3 in the BMP-2 group, and 8.1763.20 mm3 in the BMP-2/7 group, with the values in the BMP-2/7 heterodimer group being significantly higher than that of the other groups (p!.00001 vs. control, p!.00001 vs. BMP-7, p!.0001 vs. BMP-2) (Fig. 7).
Micro-CT analysis Sequential analysis of the fused spine segments in vivo There were no significant differences in the average TV values among the groups at Day 0 (just before surgery). The TV of the fused spine segments in the BMP-2/7 group sharply increased between 1 and 2 weeks after surgery (1 week, 38.366.3 mm3; 2 weeks, 81.9628.5 mm3, p!.001) and continued to increase until 6 weeks after the operation. The TV in the BMP-2 group also significantly increased between 1 and 2 weeks after surgery (1 week, 47.666.2 mm3; 2 weeks, 68.0615.3 mm3, p!.01); Table 3 Assessment of spinal fusion by manual palpation Groups
Control
BMP-7
BMP-2
BMP-2/7
Numbers assessed 0% (0/10) 11.1% (1/9) 22.2% (2/9) 90.0% (9/10)*, as bilaterally **, *** fused Chi-square test. *p!.0001 vs. control. **p!.0001 vs. BMP-7. ***p5.0001 vs. BMP-2.
Fig. 3. Change in TV of the fused spine segments by in vivo microcomputed tomography. The time course showing the change in the TV of the fused segments demonstrates a significant increase in the TV in the BMP-2/7 group between the first and second postoperative weeks, which was sustained until the sixth week. The BMP-2 group also showed a significant increase in the TV of the fused segment between the first and second postoperative week, but the increase was moderate compared with that in the BMP-2/7 group. BMP, bone morphogenetic protein; TV, tissue volume; CTL, control.
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Fig. 4. Sequential coronal reconstructed images of the fused spinal segments by in vivo microcomputed tomography. There was no apparent difference between groups in the first week after surgery. After 14 days, discontinuous faint newly formed bone was detected in the BMP-7- and BMP-2-treated groups. In the BMP-2/7 heterodimer group, the faint newly induced bone was continuous between the transverse processes. After 42 days, the newly formed bone was remodeled to give mature bone through shaping of the cortex bone and trabecular bone structure in the BMP-treated groups; however, the discontinuity of the newly formed bone at 14 days continued in the BMP-7 and -2 groups. BMP, bone morphogenetic protein.
However, the other microstructural indices used to evaluate new bone formation (BV/TV, Tb.Th, Tb.N, and Tb.Sp) did not differ between the four groups (Table 4). Histology Microscopic evaluation of the coronal sections of the treated spinal segments showed that the groups treated
without BMP (controls) showed minimal evidence of new bone formation. Rats treated with BMP-7 or -2 showed bone formation, but the fusion mass between the L4–L5 transverse processes was discontinuous or separated by cartilage. In contrast, the animals treated with the BMP-2/7 heterodimer demonstrated abundant new bone formation with bridging between the transverse processes (Fig. 8). The average histologic scores were
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Fig. 5. Microcomputed tomography 3D and 2D reconstructive images of the fused segments. The control group showed little new bone formation. The BMP-7 group showed some new bone formation, but the gap between transverse processes was apparent. In the BMP-2 group, although the volume of new bone formation was unilaterally sufficient, trabecular bone was discontinuous (indicating nonunion). The BMP-2/7 groups demonstrated continuous new bone formation with an abundant filling of the trabecular bone. Axial slices were cut at the L4–L5 disc level. BMP, bone morphogenetic protein; 2D, two-dimensional; 3D, three-dimensional.
0.4760.13 in the control group, 1.3360.20 in the BMP-7 group, 2.6560.18 in the BMP-2 group, and 4.4860.13 in the BMP-2/7 group. The histologic score was significantly higher in the BMP-2/7 group compared with the other groups (p!.0001 vs. control, BMP-7, BMP-2) (Fig. 9). Intra- and interobserver ICCs were 0.924 (95% CI, 0.891–0.949) and 0.949 (95% CI, 0.920–0.967), respectively. The immunohistochemistry demonstrated the existence of abundant osteoblastic cells on the surface trabeculae at both the newly formed bones and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups. However, the quantitative comparison was difficult because of the difference in the volume of the newly formed bone among the groups (Fig. 10). The cathepsin-K positive multinuclear osteoclasts were also observed to be dotted at both the newly formed bones and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of osteoclasts
were also not apparently different between the groups and the sites (Fig. 11).
Discussion A previous study from our lab established the efficacy of BMP-2/7 heterodimers over homodimers of either type in inducing bone formation, but as these experiments involved ex vivo gene transfer, it was possible that interactions between BMP homodimers or between BMP homodimers and heterodimers may have contributed to the high bone induction in the cotransfected group [24]. In this study, using purified recombinant BMP proteins, we show that the BMP-2/7 heterodimer significantly increased the fusion rate and newly formed bone TV by comparing its effects with that of its constituent BMP homodimers in a spinal fusion model. The dose for acquiring 100% spinal fusion is reported to be 10 mg for BMP-2 homodimer [29,31] and
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Table 4 Microstructural indices of fused site Indices
BMP-7
BMP-2
BMP-2/7
BV/TV (%) Tb.Th (mm) Tb.N (1/mm) Tb.Sp (mm)
18.866.7 109.1612.1 1.160.4 168.0653.4
17.968.3 110.3622.6 1.060.4 193.2660.5
22.667.5 n.s. 120.7617.5 n.s. 1.160.3 n.s. 196.8632.8 n.s.
Mean6SD; Mann-Whitney U test (Bonferroni method) was used for the statistical analysis. BMP, bone morphogenetic protein; BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; SD, standard deviation.
Fig. 6. Fusion scores based on microcomputed tomography evaluation. The BMP-2/7 group showed significantly higher fusion scores. The dotted line (score 8) indicates complete fusion in all specimens. *p!.001; **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
$10 mg for BMP-7 homodimer [11,32]. Therefore, the almost 100% fusion achieved using 1 mg of BMP heterodimer in this study denotes a 10-fold reduction in the required dose, clearly indicating that the BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-7 or -2 homodimers. Recombinant BMP homodimers are now clinically available and have been successfully used for the treatment of patients undergoing spinal fusion. Although the US Food and Drug Administration has approved the use of BMPs only for anterior lumbar interbody fusion, the high fusion rate provided by BMPs has led to ‘‘off-label’’ uses in primary and revision posterior lumbar surgery as well. However, there are concerns regarding the large doses of BMPs required to induce sufficient fusion and the limited effects achieved because of the degradation and rapid dilution (‘‘burst release’’) of these cytokines in the tissues [9,10]. Our finding that BMP-2/7 significantly increases the TV
Fig. 7. The tissue volume (TV) of the newly formed fusion mass. The TV of the fused segments in the BMP-2/7 group were significantly higher than those in the other groups. **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
of the newly formed bone but not that of the fused segments has great significance because this indicates that the use of the heterodimer enables spatial control of bone formation by efficient bone induction at the intended site. Indeed, the BMP-2/7 heterodimer might play a more important role than homodimers in not only in the spatial control of dorsoventral patterning [33], but also in the spatial control of fracture healing process. Although it has been known for many years that heterodimeric BMPs have an increased biological activity compared with homodimeric BMPs [18–23], the mechanism underlying this increased activity has not yet been fully elucidated. Bone morphogenetic proteins use a common
Fig. 8. Histology of the treated spinal segments. Low-power photomicrographs (0.5). Coronal sections of the L4–L5 transverse processes of the spines of rats from the (A) control demonstrate no evidence of bone formation between the transverse processes. Cross-section of the transverse processes of rats from the (B) bone morphogenetic protein (BMP)-7 and (C) BMP-2 groups showed fibrocartilaginous union (arrows). In the (D) BMP2/7 group, new bone formation bridging the processes is demonstrated.
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Fig. 9. Histologic scores of the treated spinal segments. The histologic score of the fused segments in the BMP-2/7 group were significantly higher than those in the other groups. **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
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signal transduction pathway involving Type-I (BMPRI) and II (BMPRII) receptors [33]. Affinities of the different BMPs to these receptors differ: BMP-2 has a high affinity for BMPRIa, but a low affinity for BMPRII; conversely, BMP-7 has a low affinity for BMPRIa, but a high affinity for BMPRII [34–36]. Therefore, BMP-2 and -7 belong to different subtypes and exhibit different receptor binding properties. One possible explanation for the increased bioactivity of the BMP-2/7 heterodimer is that it has a higher affinity for both BMPRI (BMPRIa) and BMPRII (ActRIIb), compared with the constituent homodimers. Isaacs et al. [37] reported the affinity of BMP-2 and -6 homodimers and BMP-2/6 heterodimer to BMPRIa and BMPRII (ActRIIb) by surface plasmon resonance. Lower value in the binding constant (KD: the dissociation rate [Koff]/the association rate [Kon]) means high affinity. In the report, BMP-2 has a KD of 1.31 nM for BMPRI (BMPRIa) and a weaker KD of 38.5 nM for BMPRII (ActRIIb). Conversely, BMP-6 displays a relatively weak affinity for
Fig. 10. Immunohistochemistry for alkaline phosphatase (ALP) of the fused spine. Low-power photomicrographs (0.5) of the fused segments (Top figures) and high-power photomicrographs (100) of the newly formed bone and vertebral bodies (Middle and Bottom figures). The ALP-positive osteoblastic cells are visualized on the surface of trabeculae at both the newly formed bone and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups. Arrows indicate ALP-positive osteoblastic cells. BMP, bone morphogenetic protein.
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Fig. 11. Immunohistochemistry for cathepsin-K (CTSK) of the fused spine. Low-power photomicrographs (0.5) of the fused segments (Top figures) and highpower photomicrographs (100) of the newly formed bone and vertebral bodies (Middle and Bottom figures). The CTSK-positive multinuclear osteoclasts were dotted on the surface of trabeculae at both the newly formed bone and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups and the sites. Arrows indicate CTSK-positive multinuclear osteoclasts. BMP, bone morphogenetic protein.
BMPIa with a KD of 62.8 nM and a higher affinity for ActRIIb with a KD of 6.68 nM. Bone morphogenetic protein2/6 heterodimer has a KD of 1.02 nM for BMPIa, which is comparable with that of BMP-2 homodimer (KD of 1.31 nM), and a KD of 6.52 nM for ActRIIb, which is comparable with that of BMP-6 homodimer (KD of 6.68 nM). In terms of the value of KD, BMP-2/6 heterodimer has higher affinity to BMRIa than BMPRII (ActRIIb) and causes a 10fold greater activation of a Smad-1-dependent reporter gene compared with BMP-2 or -6 homodimers. A second possibility is that the BMP-2/7 heterodimer can better upregulate the BMP receptor gene(s). Bone morphogenetic protein-2/6 has been reported to induce the expression of the BMPRII gene more effectively than the BMP-2 or -6 homodimers [38]. This may also contribute to the efficacy of the heterodimer. A third possibility is that hetero- and homodimeric BMPs differ in their ability to regulate the synthesis of BMP inhibitors and/or are differentially affected by these inhibitors. The BMP-2/7 heterodimer was
reported to be a weaker inducer of a BMP inhibitor, Noggin, compared with homodimeric BMPs and was also found to be resistant to Noggin inhibition [39]. One limitation of this study is that it does not examine whether the stronger bone induction induced by the heterodimer is accompanied by increased inflammation compared with the homodimers. Therefore, further studies on side effects are required before clinical application of BMP heterodimer. And the current results of a spinal fusion model with quadrupedal rodents cannot be directly extrapolated to spinal arthrodesis in humans because of the difference in biomechanics, biological reaction to the agents, and anatomical structures. The optimal dose may differ depending on drug delivery or timing of delivery, this also needs further investigation. The release kinetics of the BMPs can be different between the homodimers and heterodimer because of different molecular weight and affinity to the collagen. Further studies are required to clarify the release kinetics of BMP-2 and -7 homodimers and
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BMP-2/7 heterodimer. Age of animals (8 weeks old) used in this study is standard for the spinal fusion model; however, the efficacy in elder animals needs to be verified with a view to clinical condition in which elder people come to medical attention. In conclusion, this study, using purified recombinant proteins, demonstrates that BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-7 or -2 homodimers. Although further studies are required before clinical applications, our findings may contribute to the development of an efficient alternative strategy to the current clinical use of BMP-2 or -7 homodimers. References [1] Steinmann JC, Herkowitz HN. Pseudoarthrosis of the spine. Clin Orthop 1993;284:80–90. [2] Zdeblick TA. A prospective, randomized study of lumbar fusion. Spine 1993;18:983–91. [3] Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1998;242:1528–34. [4] Wang EA, Rosen V, D’Alessandro JS, Bauduy M, Cordes P, Harada T, et al. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A 1990;87:2220–4. [5] Urist MR. Bone formation by autoinduction. Science 1965;150:893–9. [6] Seeherman H, Wozney JM. Delivery of bone morphogenetic proteins for orthopedic tissue regeneration. Cytokine Growth Factor Rev 2005;16:329–45. [7] Ackerman SJ, Mafilios MS, Polly DW Jr. Economic evaluation of bone morphogenetic protein versus autogenous iliac crest bone graft in single-level anterior lumbar fusion: an evidence-based modeling approach. Spine 2002;27:94–9. [8] Cahill KS, Chi JH, Day A, Claus EB. Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures. JAMA 2009;302:58–66. [9] Shields LB, Raque GH, Glassman SD, Campbell M, Vitaz T, Harpring J, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine 2006;31:542–7. [10] Smucker JD, Rhee JM, Singh K, Yoon ST, Heller JG. Increased swelling complications associated with off-label usage of rhBMP-2 in the anterior cervical spine. Spine 2006;31:2813–9. [11] Salamon ML, Althausen PL, Gupta MC, Laubach J. The effects of BMP-7 in a rat posterolateral intertransverse process fusion model. J Spinal Disord Tech 2003;16:90–5. [12] Moazzaz P, Gupta MC, Gilotra MM, Gilotra MN, Maitra S, Theerajunyaporn T, et al. Estrogen-dependent actions of bone morphogenetic protein-7 on spine fusion in rats. Spine 2005;30:1706–11. [13] Taghavi CE, Lee KB, He W, Keorochana G, Murray SS, Brochmann EJ, et al. Bone morphogenetic protein binding peptide mechanism and enhancement of osteogenic protein-1 inducedbone healing. Spine 2010;35:2049–56. [14] Alanay A, Chen C, Lee S, Murray SS, Brochmann EJ, Miyazaki M, et al. The adjunctive effect of a binding peptide on bone morphogenetic protein enhanced bonehealing in a rodent model of spinal fusion. Spine 2008;33:1709–13. [15] Hsu WK, Polavarapu M, Riaz R, Roc GC, Stock SR, Glicksman ZS, et al. Nanocomposite therapy as a more efficacious and less inflammatory alternative to bone morphogenetic protein-2 in a rodent arthrodesis model. J Orthop Res 2011;29:1812–9. [16] Morimoto T, Kaito T, Kashii T, Matsuo Y, Sugiura T, Iwasaki M, et al. Effect of intermittent administration of Teriparatide (PTH134) on BMP-induced bone formation in a rat spinal fusion model. J Bone Joint Surg Am 2014;96:e107.
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[17] Montgomery SR, Nargizyan T, Meliton V, Nachtergaele S, Rohatgi R, Stappenbeck F, et al. A novel osteogenic osysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding. J Bone Miner Res 2014;29:1872–85. [18] Israel DI, Nove J, Kerns KM, Kaufman RJ, Roen V, Cox KA, et al. Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors 1996;13:291–300. [19] Aono A, Hazama M, Notaya K, Taketomi S, Yamasaki H, Tsukuda R, et al. Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 1995;210:670–7. [20] Hazama M, Aono A, Ueno N, Fujisawa Y. Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system. Biochem Biophys Res Commun 1995;209: 859–66. [21] Zhu W, Rawlins BA, Boachie-Adjei O, Myers ER, Arimizu J, Choi E, et al. Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J Bone Miner Res 2004;19:2021–32. [22] Koh JT, Zhao Z, Wang Z, Lewis IS, Krebsbach PH, Franceschi RT. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J Dent Res 2008;87:845–9. [23] Sun P, Wang J, Zheng Y, Fan Y, Gu Z. BMP2/7 heterodimer is a stronger inducer of bone regeneration in peri-implant bone defects model than BMP2 or BMP7 homodimer. Dent Mater J 2012;31:239–48. [24] Kaito T, Johnson J, Ellerman J, Tian H, Aydogan M, Chatsrinopkun M, et al. Synergistic effect of bone morphogenetic proteins 2 and 7 by ex vivo gene therapy in a rat spinal fusion model. J Bone Joint Surg Am 2013;95:1612–9. [25] Charan J, Kantharia ND. How to calculate sample size in animal studies? J Pharmacol Pharmacother 2013;4:303–6. [26] Miyazaki M, Zuk PA, Zou J, Yoon SH, Wei F, Morishita Y, et al. Comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model. Spine 2008;33:863–9. [27] Ming N, Cheng JT, Rui YF, Chan KM, Kuhstoss S, Ma YL, et al. Dose-dependent enhancement of spinal fusion in rats with teriparatide (PTH[1-34]). Spine 2012;37:1275–82. [28] Abe Y, Takahata M, Ito M, Irie K, Abumi K, Minami A. Enhancement of graft bone healing by intermittent administration of human parathyroid hormone (1-34) in a rat spinal arthrodesis model. Bone 2007;41:775–85. [29] Miyazaki M, Morishita Y, He W, Hu M, Sintuu C, Hymanson HJ, et al. A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats. Spine J 2009;9:22–30. [30] Horiki M, Imamura T, Okamoto M, Hayashi M, Murai J, Myoui A, et al. Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and caused dwarfism with osteopenica. J Cell Biol 2004;165:433–45. [31] Wang JC, Kanim LE, Yoo S, Campbell PA, Berk AJ, Lieberman JR. Effect of regional gene therapy with bone morphogenetic protein-2producing bone marrow cells on spinal fusion in rats. J Bone Joint Surg Am 2003;85-A:905–11. [32] Lu J, Bhargav D, Wei AQ, Diwan A. Posterolateral intertransverse spinal fusion possible in osteoporotic rats with BMP-7 in a higher dose delivered on composite carrier. Spine 2008;33:242–9. [33] Nohe A, Keating E, Knaus P, Petersen NO. Signal transduction of bone morphogenetic protein receptors. Cell Signal 2004;16:291–9. [34] ten Dijke P, Yamashita H, Sampath TK, Reddi AH, Estevez M, Riddle DL, et al. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J Biol Chem 1994;269:16985–8. [35] Nishitoh H, Ichijo H, Kimura M, Matsumoto T, Makishima F, Yamaguchi A, et al. Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. J Biol Chem 1996;271:21345–52.
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[36] Yamashita H, ten Dijke P, Huylebroeck D, Sampath TK, Andries M, Smith JC, et al. Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J Cell Biol 1995;130:217–26. [37] Isaacs MJ, Kawakami Y, Allendorph GP, Yoon BH, Izpisua Belmonte JC, Choe S. Bone morphogenetic protei-2 and -6 heterodimer illustrates the nature of ligand-receptor assembly. Mol Endocrinol 2010;24:1469–77.
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[38] Valera E, Isaacs MJ, Kawakami Y, Izpisua Belmonte JC, Choe S. BMP-2/6 heterodimer is more effective than BMP-2 or BMP-6 homodimers as inductor of differentiation of human embryonic stem cells. PLoS One 2010;5:e11167. [39] Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, et al. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006;39:61–71.
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Basic Science
The bone morphogenetic protein-2/7 heterodimer is a stronger inducer of bone regeneration than the individual homodimers in a rat spinal fusion model Tokimitsu Morimoto, MDa, Takashi Kaito, MD, PhDa,*, Yohei Matsuo, MDb, Tsuyoshi Sugiura, MDb, Masafumi Kashii, MD, PhDa, Takahiro Makino, MDa, Motoki Iwasaki, MDa, Hideki Yoshikawa, MD, PhDa a Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan Department of Orthopedic Biomaterial Science, Graduate School of Medicine, Osaka University, 2-2 Yamadaok, Suita, Osaka 565–0871, Japan Received 8 September 2014; revised 26 December 2014; accepted 18 February 2015
b
Abstract
BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are a group of dimeric growth factors that belong to the transforming growth factor super family and are capable of eliciting new bone formation. Previous studies have suggested that the coexpression of two different BMP genes in a cell can result in the production of BMP heterodimers that are more potent than homodimers. However, because of the difficulty in optimizing the level of BMP gene expression, the coexpression of two different BMP genes also produces BMP homodimers as a by-product. These homodimers could, in theory, interact with the heterodimers. PURPOSE: To elucidate the effects of a BMP-2/7 heterodimer, which were investigated in depth using purified BMP-2/7 heterodimers, BMP-2 homodimers, and BMP-7 homodimers in a rat spinal fusion model. METHODS: Bilateral posterolateral fusion at L4–L5 was performed in four different groups: control group animals were implanted with collagen carriers alone; BMP-7 group animals with collagen carriersþ1 mg of BMP-7 homodimer; BMP-2 group animals with collagen carriersþ1 mg of BMP-2 homodimer; and BMP-2/7 group animals with collagen carriersþ1 mg of the BMP-2/7 heterodimer. The following assessments were performed: bone microstructural analysis of the fusion mass and tissue volume (TV) with microcomputed tomography (micro-CT); fusion assessment with manual palpation testing and three-dimensional CT images; and bone histomorphometrical analysis of the fusion mass. RESULTS: The fusion scores, as determined by radiography, and the TV of the newly formed bone, as determined by micro-CT, were significantly higher in the BMP-2/7 heterodimer group than the other groups (p!.0001). The microstructural indices of the newly formed bone did not differ between the groups. Moreover, histologic analysis of the fused spines revealed that the formation of the trabecular bone bridging the transverse process was the highest in this group. CONCLUSIONS: This study demonstrated that BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-2 or -7 homodimers. The use of a purified BMP-2/7 heterodimer may represent an efficient alternative to the current clinical use of BMP-2 or -7 homodimers. Further studies as to the side effects of BMP-2/7 heterodimer are required. Ó 2015 Elsevier Inc. All rights reserved.
Keywords:
Bone morphogenetic protein; BMP heterodimer; BMP homodimer; Cytokine therapy; Spinal fusion; Bone regeneration
FDA device/drug status: Not applicable. Author disclosures: TM: Nothing to disclose. TK: Grant: Japan Society for the Promotion of Science (No. 25861313, D, Paid directly to institution). YM: Nothing to disclose. TS: Nothing to disclose. MK: Nothing to disclose. TM: Nothing to disclose. MI: Nothing to disclose. HY: Nothing to disclose. http://dx.doi.org/10.1016/j.spinee.2015.02.034 1529-9430/Ó 2015 Elsevier Inc. All rights reserved.
The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Orthopedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 5650871, Japan. Tel.: (81) 668-793-552; fax: (81) 668-793-559. E-mail address: [email protected] (T. Kaito)
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Introduction
Materials and methods
Autogenous bone grafting is the current gold standard for achieving spinal fusion. However, the effectiveness of this technique is limited by the amount of bone available and the associated donor site morbidity. Moreover, the reported rate of pseudoarthrosis ranges from 5% to 43% [1,2], thus necessitating a search for alternative methods to induce bone formation. One alternative to autogenous grafting is the use of bone morphogenetic proteins (BMPs), which are a group of dimeric growth factors that belong to the transforming growth factor superfamily and are capable of eliciting new bone formation [3–5]. Although the use of BMP homodimers in bone repair has been approved by the Food and Drug Administration, the dose required to induce adequate bone repair is very high because of the burst release and diffusion over a short period [6]. Accordingly, there are concerns regarding a substantial economic burden [7,8], but also a series of potential side-effects, including inflammation, soft tissue edema, and unintended bone formation [9,10]. Plenty of drug delivery systems are invented to decrease the required dose of BMP(s) and control their release kinetics (Table 1). One alternative approach to overcome these problems is to enhance the potency of the BMPs used. Previous studies have suggested that the coexpression of two different BMP genes can result in the production of BMP heterodimers that are more potent than their constituent homodimers in inducing in vitro osteoblastic differentiation [18–20] and in vivo osteogenesis [21–23]. In a previous study in our lab [24], combined BMP-2 and -7 ex vivo gene transfer was significantly more effective than individual BMP transfer in inducing new bone formation. However, the interpretation of our results was limited by the fact that the coexpression of two different BMP genes also produces BMP homodimers as a by-product because of the difficulties in optimizing the expression level of each BMP gene. Therefore, it was possible that interactions between BMP homodimers or between BMP homodimers and BMP heterodimers may have contributed to the improved bone induction. To overcome these limitations, the present study was designed to compare the effects of BMP-2/7 heterodimer with those of their constituent homodimers using purified cytokines instead of ex vivo gene expression in a rat spinal fusion model.
Experimental design A total of 38 male 8-week-old Sprague-Dawley rats (weight range, 280–330 g) were used in the present study. Because of the high effect size based on the results of the preliminary experiment and the significance level in this study (p!.0083), the number of animals required for the experiment was calculated as more than 32 (eight animals for each group). The ages of the animals were decided with reference to previous reports [25]. The animals were allowed to acclimate to the facility for 4 to 5 days before the operation. The animals were operated with bilateral posterolateral fusion at L4–L5 and divided into four different treatment groups: Group A received collagen carriers only (control, n510); Group B received collagen carriers with 1 mg of the recombinant human (rh) BMP-7 homodimer (BMP-7, n59); Group C received collagen carriers with 1 mg of the rhBMP-2 homodimer (BMP-2, n59); and Group D received collagen carriers with 1 mg of the rhBMP-2/7 heterodimer (BMP-2/7, n510). Preparation of a collagen carrier vehicle A commercially available absorbable collagen sponge (CollaCote; Zimmer Dental, Inc., Carlsbad, CA, USA) was cut into 510-mm fragments and placed in a sterile tube. Thereafter, the rhBMPs (rhBMP-7 homodimer, rhBMP-2 homodimer, or rhBMP-2/7 heterodimer; R&D System, Inc., Minneapolis, MN, USA) were dissolved in a sterile 4 mM hydrogen chloride (HCl) solution (rhBMP concentration: 10 mg/mL) and applied to the carrier just before implantation. The control group carriers were treated with the 4 mM HCl vehicle alone. All BMPs were applied to the collagen just before implantation. L4–L5 posterolateral spine fusion procedure All the animal procedures were conducted in accordance with the Regulations on Animal Experimentation at Osaka University. The rats were anesthetized using a combination of 0.15 mg/kg medetomidine (Domitol; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 2 mg/kg
Table 1 BMP dosages and fusion rates in previous reports using a rat spinal fusion model Animals
Age (wk)
Carrier
Dose (mg)
Assessment
FR
BMP-7
SD rat SD rat Lewis rat
— — 8
ICBM ICBM ACS
10 30 10
BMP-2
Lewis rat Athymic rats SD rat Lewis rat
— — 8 8
ACS ACS ACS ACS
10 5 50 5
X ray X ray X ray Micro-CT X ray X ray X ray X ray
100% 83.3% 100% 100% 100% 100% 88% 100%
BMP used
Reference (8/8) (5/6) (10/10) (10/10) (10/10) (8/8) (7/8) (8/8)
[11] [12] [13] [14] [15] [16] [17]
BMP, bone morphogenetic protein; SD, Sprague-Dawley; FR, fusion rate; ICBM, insoluble collagen bone matrix; ACS, absorbable collagen sponge; micro-CT, microcomputed tomography.
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midazolam (Dormicum; Astellas Pharma, Inc., Tokyo, Japan), and 2.5 mg/kg butorphanol (Vetorphale; Meiji Seika, Ltd., Tokyo, Japan). In addition, preoperative antibiotics (20,000 U/kg penicillin G; Meiji Seika, Ltd., Tokyo, Japan) were administered subcutaneously. Posterolateral lumbar fusion in rats has been well established as an acceptable model for measuring bone growth. Following the standard method [24,26], a posterior midline skin incision was made, followed by two separate paramedian incisions in the lumbar fascia 3 mm from the midline, through which the transverse processes were exposed. The L4 and L5 transverse processes were decorticated using a high-speed diamond burr of 3 mm in diameter until the bleeding from the transverse processes could be observed. Subsequently, a collagen sponge containing 1 mg of each rhBMP dissolved in 100 ml of 4 mM HCL solution or 100 ml of 4 mM HCL solution without rhBMP in the control group was implanted on each side. The fascia and skin incisions were closed using a 4-0 absorbable suture. The surgeons TK and TM performed all the operations. The rats were housed in separate cages and allowed access to food and water ad libitum, with daily monitoring of their condition. Euthanasia and tissue harvesting The rats were euthanized using an overdose of anesthetics 6 weeks after surgery. The spinal segments were harvested and fixed with 10% formalin for further assessments. Radiographic analysis Fusion between L4 and L5 was evaluated using plainfilm radiographs obtained using an MX-20 Specimen Radiography System (Faxitron X-ray Corp., Wheeling, IL, USA) under consistent conditions (35 kV, 300 mA, 300 seconds). These films were evaluated in a blinded manner by three independent observers. Bony fusion at L4–L5 was quantified using plain radiographs based on a 5point scale (0, no bone formation; 1, poor new bone formation; 2, moderate new bone formation; 3, good new bone formation and probable fusion; and 4, definite fusion) bilaterally as described previously [25] (Table 2). Definite fusion was considered when there was clear evidence of new bone formation and bony bridging with cortical continuity between the L4 and L5 transverse processes. Table 2 Scoring system to evaluate posterolateral spinal fusion Description
Score
No bone Poor new bone formation Moderate new bone formation Good new bone formation and probable fusion Definite fusion
0 1 2 3 4
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Manual assessment of fusion The explanted lumbar spines were manually tested for intersegmental motion. Any motion detected on either side between the facets or between the transverse processes was considered to be a failure of fusion. The absence of motion (both in the anteroposterior and left-right direction) was considered an indication of successful fusion [24]. The spines were scored as either fused or not fused independently by three examiners. The L4–L5 segments were considered to be fused only when a unanimous agreement of all three observers was obtained. Microcomputed tomography analysis The spines were scanned using high-resolution microcomputed tomography (micro-CT) (R_mCT; Rigaku Mechatronics, Tokyo, Japan). The micro-CT data were collected at 90 kV and 200 mA. Visualization and data reconstruction were performed using TRI/3D-BON (RATOC System Engineering, Tokyo, Japan). Sequential microstructural analysis of the fused spine segments in vivo: to compare the volume of the newly formed bone mass in each group, the total volume of L4– L5 fusion segments (from bottom of the L5 transverse process cranially to the top of L4 end plate) was measured in vivo at a 59 mm resolution at various time points (Day 0, 1 week, 2 weeks, 4 weeks, and 6 weeks). Assessment of spinal fusion using micro-CT: coronal and sagittal images of L4–L5 at a resolution of 50 mm per voxel were evaluated in a blinded manner by three independent observers based on a 5-point scale as mentioned previously [27], and fusion was considered to be clear evidence of formation of a bridging bone with cortical continuity between the L4 and L5 transverse processes. Analysis of the microstructural indices of the newly formed fusion mass of the fused spine segments at 6 weeks after operation: after manual evaluation of the explanted lumbar spines, the quality and quantity of the newly formed fusion mass between the intertransverse processes were analyzed as described previously [16,28]. Scanning of the fusion mass was initiated from the lower end plate level of L4 vertebral body and continued cranially in 1.5 mm increments (75 slices) at a resolution of 20 mm per voxel. Bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), thickness of cortical bone (Ct), and cortical bone ration (Cv/Av) were estimated. Bone volume/TV corresponds to bone volume to fusion mass volume ratio. In addition, the TV and BV values of the total fusion mass (15 mm cranially from the bottom of L5 transverse process [300 slices]) were measured at a resolution of 50 mm per voxel. Histologic analysis The dissected and formalin-fixed spines were demineralized with 50% formic acid and 10% sodium citrate,
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dehydrated through an ethanol series, and embedded in paraffin wax. Coronal sections at the level of anterior one-third of vertebral body were used for the evaluation. Serial coronal sections (thickness, 5 mm) of the dissected segments were cut and stained with hematoxylin and eosin. Three independent observers blindly scored histologic findings. Histologic fusion was defined as bony trabeculae bridging from one transverse process to the next. The extent of new bone formation was scored using the following scoring criteria: 0, empty cleft; 1, slight bump within the fibrocartilage tissue (filling less than 25% gap area); 2, some gap within the fibrocartilage tissue (filling 25%–50% of the gap area); 3, small gaps within the fibrocartilage and bone tissue (filling 75%– 99% of the gap area); 4, bridged with bone tissue, however, the fusion masses were composed of thin trabecular bone; and 5, completely bridged with abundant mature bone tissue [29]. Immunohistochemistry was performed as described previously [30] using anti-alkaline phosphatase (Ab108337, dilution 1:500; Abcam, Cambridge, UK) and anti-cathepsin K (dilution 1:200; Abcam, Cambridge, UK). Immune complexes were visualized with a goat polyclonal anti-rabbit IgG antibody coupled to biotin (426051; Nichirei Biosciences, Inc., Tokyo, Japan). 3-amino-9-ethylcarbazole was used as a substrate.
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Statistical analysis The computer software program PASW (version 18; SPSS, Chicago, IL, USA) was used for all the analyses. Mann-Whitney U test was used for the comparison (TV). Chi-squared test was used for the comparison of assessment of fusion. The level of significance was set at p!.05 for the comparison in the same group and p!.0083 for the comparison between the four groups (Bonferroni method). Intraand interobserver interclass correlation coefficients (ICCs) for radiographic fusion scores (plain radiography and micro-CT) and histologic scores were calculated. Only interobserver ICC was calculated for manual assessment of fusion.
Results Radiographic fusion analysis The average radiographic fusion scores were the highest in the BMP-2/7 group (7.6360.06), compared with values of 0.7060.17 in the control group, 2.8560.51 in the BMP-7 group, and 4.8960.40 in the BMP-2 group, and these differences were statistically significant (p!.0001 vs. control, p!.0001 vs. BMP-7, and p5.0007 vs. BMP-2) (Figs. 1 and 2). Intra- and interobserver ICCs were 0.932 (95% confidence
Fig. 1. Radiographs of the lumbar spine 6 weeks post-surgery. (A) Minimal new bone formation was found in the controls. (B) In the bone morphogenetic protein (BMP)-7 group, the newly developed bone formed a bridge between transverse processes, but gaps were evident. (C) In the BMP-2 group, the gap between the transverse process on the left side was occupied by the newly formed bone, but the clear line and discontinuous trabecular bone is suggestive of nonunion. (D) In the BMP-2/7 group, the new bone bridge between transverse processes and continuous trabecular bone suggests osseous fusion.
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Fig. 2. Fusion scores based on plain radiography. The BMP-2/7 group shows significantly higher fusion scores. Dotted line (score58) indicates complete fusion in all specimens. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.*p!.01; **p!.001.
interval [CI], 0.894–0.956) and 0.905 (95% CI, 0.865–0.935), respectively, for the radiographical fusion scoring. Manual palpation All spines were examined by manual palpation to determine fusion. The fusion rate was strongly increased in the BMP-2/7 group (90%; 9 of 10), compared with 0% (0 of 10) in the control group, 11.1% (1 of 9) in the BMP-7 group, and 22.2% in the BMP-2 group, and these differences were statistically significant (p!.0001 vs. control, p!.0001 vs. BMP-7, p!.0001 vs. BMP-2) (Table 3). Interobserver ICC was 0.783 (95% CI, 0.663–0.872).
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however, the increase was not as pronounced as that in the BMP-2/7 group. The change in the TV in the BMP-7 group was almost parallel to that in the control group. The average TV values 6 weeks after surgery were 67.766.0 mm3 in the control group, 66.067.0 mm3 in the BMP-7 group, 88.1626.2 mm3 in the BMP-2 group, and 104.3625.1 mm3 in the BMP-2/7 group. The TV in the BMP-2/7 group was significantly higher than that in the control and BMP-7 groups (p!.001 vs. control, p!.004 vs. BMP-7), but the difference in TV between the BMP-2/7 and -2 groups was not statistically significant (p5.21) (Figs. 3 and 4). We also assessed spinal fusion using micro-CT. The average micro-CT fusion scores were 1.3060.06 in the control group, 3.5260.26 in the BMP-7 group, 4.8160.32 in the BMP-2 group, and 7.6060.10 in the BMP-2/7 group. The micro-CT fusion score was significantly higher in the BMP-2/7 group compared with the other groups (p!.0000001 vs. control, p!.000001 vs. BMP-7; p!.0008 vs. BMP-2) (Figs. 5 and 6). Intra- and interobserver ICCs were 0.964 (95% CI, 0.944–0.977) and 0.939 (95% CI, 0.913–0.959), respectively. Analysis of the microstructural indices of the newly formed bone revealed that the TV of the newly formed bone was 0.5360.32 mm3 in the control group, 1.4361.29 mm3 in the BMP-7 group, 2.1461.26 mm3 in the BMP-2 group, and 8.1763.20 mm3 in the BMP-2/7 group, with the values in the BMP-2/7 heterodimer group being significantly higher than that of the other groups (p!.00001 vs. control, p!.00001 vs. BMP-7, p!.0001 vs. BMP-2) (Fig. 7).
Micro-CT analysis Sequential analysis of the fused spine segments in vivo There were no significant differences in the average TV values among the groups at Day 0 (just before surgery). The TV of the fused spine segments in the BMP-2/7 group sharply increased between 1 and 2 weeks after surgery (1 week, 38.366.3 mm3; 2 weeks, 81.9628.5 mm3, p!.001) and continued to increase until 6 weeks after the operation. The TV in the BMP-2 group also significantly increased between 1 and 2 weeks after surgery (1 week, 47.666.2 mm3; 2 weeks, 68.0615.3 mm3, p!.01); Table 3 Assessment of spinal fusion by manual palpation Groups
Control
BMP-7
BMP-2
BMP-2/7
Numbers assessed 0% (0/10) 11.1% (1/9) 22.2% (2/9) 90.0% (9/10)*, as bilaterally **, *** fused Chi-square test. *p!.0001 vs. control. **p!.0001 vs. BMP-7. ***p5.0001 vs. BMP-2.
Fig. 3. Change in TV of the fused spine segments by in vivo microcomputed tomography. The time course showing the change in the TV of the fused segments demonstrates a significant increase in the TV in the BMP-2/7 group between the first and second postoperative weeks, which was sustained until the sixth week. The BMP-2 group also showed a significant increase in the TV of the fused segment between the first and second postoperative week, but the increase was moderate compared with that in the BMP-2/7 group. BMP, bone morphogenetic protein; TV, tissue volume; CTL, control.
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Fig. 4. Sequential coronal reconstructed images of the fused spinal segments by in vivo microcomputed tomography. There was no apparent difference between groups in the first week after surgery. After 14 days, discontinuous faint newly formed bone was detected in the BMP-7- and BMP-2-treated groups. In the BMP-2/7 heterodimer group, the faint newly induced bone was continuous between the transverse processes. After 42 days, the newly formed bone was remodeled to give mature bone through shaping of the cortex bone and trabecular bone structure in the BMP-treated groups; however, the discontinuity of the newly formed bone at 14 days continued in the BMP-7 and -2 groups. BMP, bone morphogenetic protein.
However, the other microstructural indices used to evaluate new bone formation (BV/TV, Tb.Th, Tb.N, and Tb.Sp) did not differ between the four groups (Table 4). Histology Microscopic evaluation of the coronal sections of the treated spinal segments showed that the groups treated
without BMP (controls) showed minimal evidence of new bone formation. Rats treated with BMP-7 or -2 showed bone formation, but the fusion mass between the L4–L5 transverse processes was discontinuous or separated by cartilage. In contrast, the animals treated with the BMP-2/7 heterodimer demonstrated abundant new bone formation with bridging between the transverse processes (Fig. 8). The average histologic scores were
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Fig. 5. Microcomputed tomography 3D and 2D reconstructive images of the fused segments. The control group showed little new bone formation. The BMP-7 group showed some new bone formation, but the gap between transverse processes was apparent. In the BMP-2 group, although the volume of new bone formation was unilaterally sufficient, trabecular bone was discontinuous (indicating nonunion). The BMP-2/7 groups demonstrated continuous new bone formation with an abundant filling of the trabecular bone. Axial slices were cut at the L4–L5 disc level. BMP, bone morphogenetic protein; 2D, two-dimensional; 3D, three-dimensional.
0.4760.13 in the control group, 1.3360.20 in the BMP-7 group, 2.6560.18 in the BMP-2 group, and 4.4860.13 in the BMP-2/7 group. The histologic score was significantly higher in the BMP-2/7 group compared with the other groups (p!.0001 vs. control, BMP-7, BMP-2) (Fig. 9). Intra- and interobserver ICCs were 0.924 (95% CI, 0.891–0.949) and 0.949 (95% CI, 0.920–0.967), respectively. The immunohistochemistry demonstrated the existence of abundant osteoblastic cells on the surface trabeculae at both the newly formed bones and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups. However, the quantitative comparison was difficult because of the difference in the volume of the newly formed bone among the groups (Fig. 10). The cathepsin-K positive multinuclear osteoclasts were also observed to be dotted at both the newly formed bones and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of osteoclasts
were also not apparently different between the groups and the sites (Fig. 11).
Discussion A previous study from our lab established the efficacy of BMP-2/7 heterodimers over homodimers of either type in inducing bone formation, but as these experiments involved ex vivo gene transfer, it was possible that interactions between BMP homodimers or between BMP homodimers and heterodimers may have contributed to the high bone induction in the cotransfected group [24]. In this study, using purified recombinant BMP proteins, we show that the BMP-2/7 heterodimer significantly increased the fusion rate and newly formed bone TV by comparing its effects with that of its constituent BMP homodimers in a spinal fusion model. The dose for acquiring 100% spinal fusion is reported to be 10 mg for BMP-2 homodimer [29,31] and
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Table 4 Microstructural indices of fused site Indices
BMP-7
BMP-2
BMP-2/7
BV/TV (%) Tb.Th (mm) Tb.N (1/mm) Tb.Sp (mm)
18.866.7 109.1612.1 1.160.4 168.0653.4
17.968.3 110.3622.6 1.060.4 193.2660.5
22.667.5 n.s. 120.7617.5 n.s. 1.160.3 n.s. 196.8632.8 n.s.
Mean6SD; Mann-Whitney U test (Bonferroni method) was used for the statistical analysis. BMP, bone morphogenetic protein; BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; SD, standard deviation.
Fig. 6. Fusion scores based on microcomputed tomography evaluation. The BMP-2/7 group showed significantly higher fusion scores. The dotted line (score 8) indicates complete fusion in all specimens. *p!.001; **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
$10 mg for BMP-7 homodimer [11,32]. Therefore, the almost 100% fusion achieved using 1 mg of BMP heterodimer in this study denotes a 10-fold reduction in the required dose, clearly indicating that the BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-7 or -2 homodimers. Recombinant BMP homodimers are now clinically available and have been successfully used for the treatment of patients undergoing spinal fusion. Although the US Food and Drug Administration has approved the use of BMPs only for anterior lumbar interbody fusion, the high fusion rate provided by BMPs has led to ‘‘off-label’’ uses in primary and revision posterior lumbar surgery as well. However, there are concerns regarding the large doses of BMPs required to induce sufficient fusion and the limited effects achieved because of the degradation and rapid dilution (‘‘burst release’’) of these cytokines in the tissues [9,10]. Our finding that BMP-2/7 significantly increases the TV
Fig. 7. The tissue volume (TV) of the newly formed fusion mass. The TV of the fused segments in the BMP-2/7 group were significantly higher than those in the other groups. **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
of the newly formed bone but not that of the fused segments has great significance because this indicates that the use of the heterodimer enables spatial control of bone formation by efficient bone induction at the intended site. Indeed, the BMP-2/7 heterodimer might play a more important role than homodimers in not only in the spatial control of dorsoventral patterning [33], but also in the spatial control of fracture healing process. Although it has been known for many years that heterodimeric BMPs have an increased biological activity compared with homodimeric BMPs [18–23], the mechanism underlying this increased activity has not yet been fully elucidated. Bone morphogenetic proteins use a common
Fig. 8. Histology of the treated spinal segments. Low-power photomicrographs (0.5). Coronal sections of the L4–L5 transverse processes of the spines of rats from the (A) control demonstrate no evidence of bone formation between the transverse processes. Cross-section of the transverse processes of rats from the (B) bone morphogenetic protein (BMP)-7 and (C) BMP-2 groups showed fibrocartilaginous union (arrows). In the (D) BMP2/7 group, new bone formation bridging the processes is demonstrated.
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Fig. 9. Histologic scores of the treated spinal segments. The histologic score of the fused segments in the BMP-2/7 group were significantly higher than those in the other groups. **p!.0001. Data are presented as the mean6standard deviation. BMP, bone morphogenetic protein.
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signal transduction pathway involving Type-I (BMPRI) and II (BMPRII) receptors [33]. Affinities of the different BMPs to these receptors differ: BMP-2 has a high affinity for BMPRIa, but a low affinity for BMPRII; conversely, BMP-7 has a low affinity for BMPRIa, but a high affinity for BMPRII [34–36]. Therefore, BMP-2 and -7 belong to different subtypes and exhibit different receptor binding properties. One possible explanation for the increased bioactivity of the BMP-2/7 heterodimer is that it has a higher affinity for both BMPRI (BMPRIa) and BMPRII (ActRIIb), compared with the constituent homodimers. Isaacs et al. [37] reported the affinity of BMP-2 and -6 homodimers and BMP-2/6 heterodimer to BMPRIa and BMPRII (ActRIIb) by surface plasmon resonance. Lower value in the binding constant (KD: the dissociation rate [Koff]/the association rate [Kon]) means high affinity. In the report, BMP-2 has a KD of 1.31 nM for BMPRI (BMPRIa) and a weaker KD of 38.5 nM for BMPRII (ActRIIb). Conversely, BMP-6 displays a relatively weak affinity for
Fig. 10. Immunohistochemistry for alkaline phosphatase (ALP) of the fused spine. Low-power photomicrographs (0.5) of the fused segments (Top figures) and high-power photomicrographs (100) of the newly formed bone and vertebral bodies (Middle and Bottom figures). The ALP-positive osteoblastic cells are visualized on the surface of trabeculae at both the newly formed bone and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups. Arrows indicate ALP-positive osteoblastic cells. BMP, bone morphogenetic protein.
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Fig. 11. Immunohistochemistry for cathepsin-K (CTSK) of the fused spine. Low-power photomicrographs (0.5) of the fused segments (Top figures) and highpower photomicrographs (100) of the newly formed bone and vertebral bodies (Middle and Bottom figures). The CTSK-positive multinuclear osteoclasts were dotted on the surface of trabeculae at both the newly formed bone and the vertebral bodies in all the BMP-2, -7, and -2/7 groups. The numbers of the cells were not apparently different among the groups and the sites. Arrows indicate CTSK-positive multinuclear osteoclasts. BMP, bone morphogenetic protein.
BMPIa with a KD of 62.8 nM and a higher affinity for ActRIIb with a KD of 6.68 nM. Bone morphogenetic protein2/6 heterodimer has a KD of 1.02 nM for BMPIa, which is comparable with that of BMP-2 homodimer (KD of 1.31 nM), and a KD of 6.52 nM for ActRIIb, which is comparable with that of BMP-6 homodimer (KD of 6.68 nM). In terms of the value of KD, BMP-2/6 heterodimer has higher affinity to BMRIa than BMPRII (ActRIIb) and causes a 10fold greater activation of a Smad-1-dependent reporter gene compared with BMP-2 or -6 homodimers. A second possibility is that the BMP-2/7 heterodimer can better upregulate the BMP receptor gene(s). Bone morphogenetic protein-2/6 has been reported to induce the expression of the BMPRII gene more effectively than the BMP-2 or -6 homodimers [38]. This may also contribute to the efficacy of the heterodimer. A third possibility is that hetero- and homodimeric BMPs differ in their ability to regulate the synthesis of BMP inhibitors and/or are differentially affected by these inhibitors. The BMP-2/7 heterodimer was
reported to be a weaker inducer of a BMP inhibitor, Noggin, compared with homodimeric BMPs and was also found to be resistant to Noggin inhibition [39]. One limitation of this study is that it does not examine whether the stronger bone induction induced by the heterodimer is accompanied by increased inflammation compared with the homodimers. Therefore, further studies on side effects are required before clinical application of BMP heterodimer. And the current results of a spinal fusion model with quadrupedal rodents cannot be directly extrapolated to spinal arthrodesis in humans because of the difference in biomechanics, biological reaction to the agents, and anatomical structures. The optimal dose may differ depending on drug delivery or timing of delivery, this also needs further investigation. The release kinetics of the BMPs can be different between the homodimers and heterodimer because of different molecular weight and affinity to the collagen. Further studies are required to clarify the release kinetics of BMP-2 and -7 homodimers and
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BMP-2/7 heterodimer. Age of animals (8 weeks old) used in this study is standard for the spinal fusion model; however, the efficacy in elder animals needs to be verified with a view to clinical condition in which elder people come to medical attention. In conclusion, this study, using purified recombinant proteins, demonstrates that BMP-2/7 heterodimer is a stronger inducer of bone regeneration than BMP-7 or -2 homodimers. Although further studies are required before clinical applications, our findings may contribute to the development of an efficient alternative strategy to the current clinical use of BMP-2 or -7 homodimers. References [1] Steinmann JC, Herkowitz HN. Pseudoarthrosis of the spine. Clin Orthop 1993;284:80–90. [2] Zdeblick TA. A prospective, randomized study of lumbar fusion. Spine 1993;18:983–91. [3] Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 1998;242:1528–34. [4] Wang EA, Rosen V, D’Alessandro JS, Bauduy M, Cordes P, Harada T, et al. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A 1990;87:2220–4. [5] Urist MR. Bone formation by autoinduction. Science 1965;150:893–9. [6] Seeherman H, Wozney JM. Delivery of bone morphogenetic proteins for orthopedic tissue regeneration. Cytokine Growth Factor Rev 2005;16:329–45. [7] Ackerman SJ, Mafilios MS, Polly DW Jr. Economic evaluation of bone morphogenetic protein versus autogenous iliac crest bone graft in single-level anterior lumbar fusion: an evidence-based modeling approach. Spine 2002;27:94–9. [8] Cahill KS, Chi JH, Day A, Claus EB. Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures. JAMA 2009;302:58–66. [9] Shields LB, Raque GH, Glassman SD, Campbell M, Vitaz T, Harpring J, et al. Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine 2006;31:542–7. [10] Smucker JD, Rhee JM, Singh K, Yoon ST, Heller JG. Increased swelling complications associated with off-label usage of rhBMP-2 in the anterior cervical spine. Spine 2006;31:2813–9. [11] Salamon ML, Althausen PL, Gupta MC, Laubach J. The effects of BMP-7 in a rat posterolateral intertransverse process fusion model. J Spinal Disord Tech 2003;16:90–5. [12] Moazzaz P, Gupta MC, Gilotra MM, Gilotra MN, Maitra S, Theerajunyaporn T, et al. Estrogen-dependent actions of bone morphogenetic protein-7 on spine fusion in rats. Spine 2005;30:1706–11. [13] Taghavi CE, Lee KB, He W, Keorochana G, Murray SS, Brochmann EJ, et al. Bone morphogenetic protein binding peptide mechanism and enhancement of osteogenic protein-1 inducedbone healing. Spine 2010;35:2049–56. [14] Alanay A, Chen C, Lee S, Murray SS, Brochmann EJ, Miyazaki M, et al. The adjunctive effect of a binding peptide on bone morphogenetic protein enhanced bonehealing in a rodent model of spinal fusion. Spine 2008;33:1709–13. [15] Hsu WK, Polavarapu M, Riaz R, Roc GC, Stock SR, Glicksman ZS, et al. Nanocomposite therapy as a more efficacious and less inflammatory alternative to bone morphogenetic protein-2 in a rodent arthrodesis model. J Orthop Res 2011;29:1812–9. [16] Morimoto T, Kaito T, Kashii T, Matsuo Y, Sugiura T, Iwasaki M, et al. Effect of intermittent administration of Teriparatide (PTH134) on BMP-induced bone formation in a rat spinal fusion model. J Bone Joint Surg Am 2014;96:e107.
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