Cytotherapy, 2014; 16: 1441e1448

Optimal condition of heparin-conjugated fibrin with bone morphogenetic protein-2 for spinal fusion in a rabbit model

JAE-YOUNG HONG1, SUN-WOONG KANG2, JUNG-WOOK KIM1, SEUNG-WOO SUH3, YOU-JIN KO1 & JUNG-HO PARK1 1

Department of Orthopedics, Korea University Ansan Hospital, Ansan, South Korea, 2Next-Generation Pharmaceutical Research Center, Korea Institute of Toxicology, Daejeon, Republic of Korea, and 3Scoliosis Research Institute, Department of Orthopedics, Korea University Guro Hospital, Seoul, South Korea Abstract Background aims. Heparin-conjugated fibrin (HCF) is a carrier for long-term release of bone morphogenetic protein-2 (BMP-2) and has been shown to promote bone formation in animal models. We performed an experimental study to determine the optimal dose of BMP-2 with an HCF carrier that promotes bone formation comparable to that of autograft while minimizing complications in spinal fusion. Methods. Twenty-four rabbits underwent posterolateral fusion of the L5e6 spinal segments. Different concentrations of HCF BMP-2 (1/10, 1/20, 1/30 or 1/40) were implanted in the spines of experimental rabbits, and autograft or INFUSE was implanted in the spines of control animals. Eight weeks after treatment, spinal fusion efficacy was evaluated by plain radiography, micro-computed tomography (micro-CT), mechanical testing and histomorphometry. Results. Similar to autograft, the 1/40 HCF BMP-2 showed significant bone formation on micro-CT and histomorphometry with mechanical stability. However, the other HCF BMP-2 concentrations did not show significant bone formation compared with autograft. Although conventional BMP-2 (INFUSE) led to higher bone formation and stability, it also led to excessive ectopic bone and fibrous tissue formation. Conclusions. This study suggests the optimal concentration of BMP-2 using HCF for spinal fusion, which may decrease the complications of high-dose conventional BMP-2. Key Words: autograft, BMP-2, heparin-conjugated fibrin, spinal fusion

Introduction Bone morphogenetic proteins (BMPs) are osteoinductive growth factors that offer significant therapeutic promise for bone regeneration. Two BMPs (BMP-2 and BMP-7) are currently available for clinical use in lumbar fusion surgery (1,2). However, high doses of BMPs that are administered to bone defects can cause side effects, such as ectopic bone formation and various immune reactions (3). Overcoming these problems requires identifying the appropriate delivery system that minimizes the BMP dose while promoting functional improvement. Recently, heparin-conjugated fibrin (HCF) gel has been highlighted as an alternative carrier for BMP-2 delivery (4e8). Several authors have reported that HCF gel can achieve a sustained release of BMP-2 and promote the osteogenic efficacy of BMP-2 in various animal models. However, no studies have compared how different doses of BMP-2 affect the

therapeutic outcomes, and questions remain regarding protein dose-response relationships. Therefore, the aim of the present study was to characterize and evaluate the dose-response of BMP2 in an HCF delivery system on bone regeneration for lumbar spine fusion and to assess whether the results were comparable to those of auto-graft. Methods Subjects Twelve-week-old New Zealand white rabbits weighing 2.5e3.0 kg were used for this study. The rabbits were divided into four groups of four rabbits each, and each group received a different dose of heparinconjugated BMP-2. An additional eight rabbits served as controls and had either autogenous iliac chip bone grafting or conventional BMP-2 grafting (INFUSE, Medtronics, Memphis, TN, USA;

J.-Y. Hong and S.-W. Kang contributed equally to this article. Correspondence: Jung-Ho Park, MD, Department of Orthopedics, Korea University Ansan Hospital, Gojan Dong, Danwon Gu, Ansan 425-707, South Korea. E-mail: [email protected] (Received 1 November 2013; accepted 5 April 2014) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2014.04.005

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Table I. Implant assignment of the groups in the study. BMP-2 Ratio to Total Harvested Harvested concentration conventional Group grafts 4 weeks 8 weeks (mg/mL) BMP-2 1 2 3 4 5 6

8 8 8 8 8 8

2 2 2 2 2 2

6 6 6 6 6 6

— 0.15 0.075 0.05 0.037 1.5

Autograft 1:10 1:20 1:30 1:40 1:1 (INFUSE)

Table I). Posterolateral lumbar spinal fusion was then performed with the graft material in all 24 rabbits. This protocol, including animal care and use, was approved by the Institutional Committee for Animal Care and Experiments. Synthesis of heparin conjugated fibrin (HCF) HCF was fabricated as previously described (7). In brief, heparin (molecular weight 4000e6000 Da; Sigma-Aldrich, St. Louis, MO, USA) was covalently bonded to plasminogen-free fibrinogen (SigmaAldrich) by using standard carbodiimide chemistry.

Heparin (100 mg) was dissolved in a buffer solution (100 mL, pH 6) of 0.05 mol/L 2-morpholinoethanesulfonic acid (Sigma-Aldrich). N-hydroxysuccinimide (0.04 mmol/L; Sigma-Aldrich) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.08 mmol/L; Sigma-Aldrich) were added to the solution to activate the carboxylic acid groups of the heparin. After 12 h of reaction at 4  C, the solution was stirred to obtain a homogeneous solution, and the product was precipitated with excess anhydrous acetone and lyophilized. Fibrinogen (100 mg) was dissolved in phosphate-buffered saline (20 mL, pH 7.4) without bubbles at 4 C and reacted with activated carboxyl acid groups of the heparin (60 mg) under the same conditions for 3 h. The product was precipitated with a large excess of acetone and lyophilized. The resultant white powder was completely dissolved in phosphate-buffered saline and dialyzed through a porous membrane bag (12,000e14,000 Da molecular weight cutoff; Spectrum Lab, Rancho Dominguez, CA, USA) to remove residual heparin at 4  C for 24 h. Finally, HCF was lyophilized for 48 h.

Figure 1. (A) Representative three-dimensional CT images of posterolateral fusion at 8 weeks after surgery. The fusion masses of the 1/40 BMP-2 and INFUSE groups were well remodeled. (B) Radiological fusion scores of the 1/40 BMP-2 and INFUSE groups were higher than those of the other groups, and both scores were comparable to those of the autograft group. Values are mean  SD.

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(0.8e1 g per side) or INFUSE (a collagen membrane with 1500 mg of BMP-2 per side, Medtronics). The wounds were then closed with 3-0 nylon sutures and skin staples. Food and water were provided ad libitum to the rabbits before and after surgery. Assessment of fusion quality

Figure 2. The manual bending test revealed that the 1/40 BMP-2 and INFUSE groups showed greater stability than the autograft group, although this difference was not statistically significant. Values are mean  SD.

Preparation of the graft material with various doses of BMP-2 To prepare two implants to bridge the sides between the transverse processes of L5 and L6, HCF (40 mg/ mL) and various does of BMP-2 (75, 100, 150 or 300 mg) were mixed with 1 mL of aprotinin (1100 KIU/mL) solution. After 30 min, normal fibrinogen (60 mg, Greenplast; Greencross PD, Yongin, Korea) with factor XIII was added to the BMP-2 mixed solution. The thrombin (500 IU/mL) was mixed with 1 mL of calcium chloride (14 mg/mL). The HCF solution with various doses of BMP-2 was then mixed with the thrombin solution to form HCF with BMP-2. The weight ratios of heparin to BMP-2 were 22.4:1, 16.8:1, 11.2:1, 5.6:1 and 2.8:1, respectively.

Posterolateral fusion model in rabbits Each rabbit underwent bilateral single-level posterolateral intertransverse process fusion at L5eL6. We anesthetized the animals with an intramuscular injection of ketamine (50 mg/kg). Gentamicin was administered intramuscularly as a prophylactic antibiotic. We then made a dorsal midline skin incision followed by two paramedian fascial incisions on both sides. The intermuscular plane between the multifidus and longissimus muscles was retracted to expose both transverse processes of L5eL6 and the intertransverse membranes. We used an electric burr to decorticate the posterior cortex of the transverse processes, and we implanted the transplant materials on both sides, one implant per side. Each implant contained HCF with 37.5 (1/40), 50 (1/30), 75 (1/20) or 150 (1/10) mg of BMP-2, depending on the experimental group. For the controls, we prepared autogenous bone chips harvested from the iliac crest

Four weeks after surgery, one rabbit from each group was sacrificed in a CO2 chamber, and their L5eL6 lumbar spines were harvested and processed. At 8 weeks after surgery, the remaining animals were sacrificed and processed in the same fashion. Plain radiographs and computed tomography (CT): For each rabbit, the fusion mass of the lumbar spine was assessed by anteroposterior plain radiography and CT (SKYSCAN, Kartuizersweg, Belgium) at 1-mm slice thickness. Fusion was evaluated on the sagittal CT view at the transverse process area. Two independent observers (JYH, JWK) categorized the fusion mass on the plain radiograms and CT scans as follows: 0, no bone formation; 1, some bone formation between the transverse processes but no bridging; and 2, continuous bone formation between the transverse processes with bridging (9). Both observers agreed on all observations. Mechanical testing: To evaluate the solidity of the L5eL6 fusion site, mechanical testing was performed with a manual bending test. Each fused segment was assessed by manual palpation in a blinded fashion by two observers (J-YH, J-WK). Flexion and extension were assessed at the operated lumbar segment, as described previously. Clinical fusion was considered to be “no movement” at the L5eL6 level. This method has been shown to correlate closely with the results of biomechanical testing and is more accurate than plain radiographs (10e13). If any motion was observed, the area was deemed not solid. To facilitate statistical analysis, we categorized the solidity of the fusion site as follows: 0, significant movement similar to that of the nonfused vertebrae; 1, minimal movement; and 2, no movement. Histomorphometric examination: 8 weeks after surgery, each specimen was fixed in 10% neutralbuffered formalin, decalcified with Calci-Clear Rapid (National Diagnostics, East Riding, UK) for 1 week, dehydrated in a graded ethanol series and embedded in paraffin for histologic examination. Sagittal sections at the intertransverse process were cut at 4-mm thickness and stained with hematoxylin and eosin. Stained slides were scanned, and individual sections were analyzed for each sample. New bone formation was quantified with the “absolute bone matrix area” (mm2) and “percent bone matrix value” (bone matrix area divided by total area; %) using Adobe Photoshop CS2 (Adobe, San Jose, CA, USA).

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Figure 3. Representative histology sections of the fusion mass of each group 8 weeks after surgery. (A) Autograft, (B) 1/10, (C) 1/20, (D) 1/30, (E) 1/40 and (F) INFUSE (hematoxylin and eosin stain). The newly formed bone mass between the transverse processes of the 1/30, 1/40 and INFUSE groups showed continuous cortical bone, similar to the autogenous bone graft control group. Although the INFUSE group showed a significantly larger bone regeneration area, this group also showed heterotrophic ossification involving the vertebral body and the spinous process with a high proportion of fibrous tissue.

Statistical analysis We used Mann-Whitney U-tests to compare the radiographic scores, manual bending load and histomorphometric measures between groups. SPSS 13.0 (SPSS, Chicago, IL, USA) was used for all analyses. Results Initially, we included two animals in the 4-week group as controls. However, we could not find significant bone formation in the 4-week group, so we excluded this group from statistical analysis. Thus,

the statistical analysis included only the 8-week group.

Plain radiograms and micro-CT The 1/40 and INFUSE groups showed significantly greater bone formation than the 1/10 and 1/20 groups (P < 0.05), and all four groups showed greater bone formation than the autograft group (Figure 1A,B). The amount of bone formation in the 1/30 group was similar to that of the autograft group. The INFUSE group showed the largest regeneration

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Figure 3. (continued).

area on the three-dimensional CT scans and radiographs; however, every case also showed significant ectopic bone formation involving the dorsal surface of the vertebra.

Mechanical testing The 1/40 and INFUSE groups showed greater stability than the autograft group, and the remaining groups showed lower stability than the autograft group; however, none of these differences were statistically significant (Figure 2). The results from the mechanical testing were similar to those from the plain radiograms and CT scans.

Histomorphologic examination The conjugated heparin appeared to be replaced by a calcified mass with a flat surface in a timedependent fashion in 4e8 weeks. The 1/30, 1/40 and INFUSE groups achieved a fusion mass with peripheral cortical bone bridging the transverse processes, and these results were comparable to those of the autograft group (Figure 3AeF). However, the other groups showed little bone formation; instead, the fibrous tissue remained without bone bridging. Although the INFUSE group had a significantly larger bone regeneration area, heterotrophic ossification involving the vertebral body and spinous process with a high proportion of fibrous

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Figure 3. (continued).

tissue was evident. Regarding the absolute bone matrix area, the INFUSE group showed significantly greater bone formation than the other groups (P < 0.05). The 1/40 group showed significantly greater bone formation than the other groups, although the amount of bone formation was lower than that of the INFUSE group (Figure 4A). Regarding the percent bone matrix value, the 1/30 and 1/40 groups had a significantly higher percent bone mass than the other groups (P < 0.05). The percent bone mass was similar between the INFUSE group and the other groups (P > 0.05; Figure 4B).

Discussion Since BMP was developed in the early 1970s, it has gained popularity for its ability to promote bone formation (3). BMP is now used in 52% of spinal surgeries in the United States, and many published studies have supported its use (12,14). However, high doses of BMP-2 can lead to side effects, such as ectopic bone formation and immune responses. Thus, a novel means of delivering BMP that promotes bone formation while minimizing side effects is needed. Previous studies in various animal models have shown that delivery of BMP-2 with heparin-

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Figure 4. (A) The INFUSE group had a significantly higher absolute bone matrix area than the other groups (P < 0.05). In addition, the 1/40 group had significantly greater bone formation than the other groups, although the degree of bone formation was lower than that of the INFUSE group. (B) The 1/30 and 1/40 groups had a significantly higher percent bone matrix value than the other groups (P < 0.05). However, the INFUSE group showed a percent bone mass similar to the other groups (P > 0.05).

based carriers offers significant therapeutic promise for bone regeneration (4e9,15e21). Because of its continuous release of BMP-2, HCF may decrease the therapeutic dose of BMP-2 and thus decrease side effects. Animals receiving BMP-2-loaded HCF after experimental injury show accelerated bone regeneration, increased bone density and improved quality of regenerated bone (4e8). However, the optimal dose of BMP-2 in a heparin-based carrier system has not yet been determined. This study explored the dose-response relationships for BMP-2 delivered in an HCF hydrogel compared with the currently used BMP-2 graft. In addition, the therapeutic efficacy of HCF with BMP-2 was compared with that of autogenous iliac crest bone graft in a rabbit posterolateral fusion model. In this study, the 1/40 HCF BMP-2 group and the autograft group showed a similar degree of bone formation. However, the 1/10 and 1/20 groups showed no significant bone formation in radiographic, mechanical or histologic examinations, even though the dose of BMP was higher. Moreover, in the 1/10 and 1/20 groups, fibrous tissue was observed in the histologic examination, and bone formation was limited. It appears that HCF applied with a high dose of BMP-2 does not produce effective bony fusion. Importantly, spinal fusion efficacy did not improve with BMP-2 doses higher than 1/30 of the conventional dose with HCF. Thus, the optimal dose of BMP-2 was appears to be no higher than 50 mg because of a plateau effect. In addition, higher doses of BMP-2 were associated with increased local inflammatory and immunogenic responses. The delivery system used in this study allows binding with BMP-2 via electrostatic interactions between the negatively charged sulfate groups of heparin and the positively charged amino acid groups of BMP-2 (16). The weak ionic bonding enables the sustained release

of BMP-2. Furthermore, heparin-conjugated polymers immersed in or mixed with BMP-2 solution can bond BMP-2 to the polymer-conjugated heparin (9,16e18). Thus, the ratio of heparin-conjugated polymers to BMP-2 is an important factor. In previous studies, the weight ratio of heparin-conjugated polymers to BMP-2 was more than 50:1 for successful ionic bonding between heparin and BMP-2 (7,8). In this study, we used various doses of BMP-2 (37.5, 50, 75 or 150 mg/site), and the weight ratio of heparin to BMP-2 was 22.4:1, 16.8:1, 11.2:1, 5.6:1 and 2.8:1, respectively. The weight ratio of heparin to BMP-2 influences the release of BMP-2 from HCF and the bioactivity of BMP-2. The results demonstrate that 37.5 (1/40) mg of BMP-2 (low dose) induced the maximum bone formation, whereas higher doses (75 [1/20] and 150 [1/10] mg) were inhibitory and induced an inflammatory response. These findings suggest that there is a limited range of BMP-2 doses that can induce bone formation when HCF is used as the carrier. The results of the current study demonstrate that 1/40 of the conventional BMP-2 dose applied with HCF has comparable efficacy to that of autograft. The HCF complex can decrease the BMP dose while promoting effective bone formation, which may decrease the side effects associated with a high dose of BMP. Koo et al. reported a poor result with 20 mg of rhBMP-2 but a good result with 100 mg of rhBMP-2 at each spinal level (4,5). In our study, we found a good fusion rate with 75 or 100 mg of rhBMP-2 at each level, similar to the results of Koo et al. However, the optimal concentration of BMP may vary according to the animal model used. A previous study estimated that the BMP-2 dose required to regenerate 1 cm3 of new bone was approximately 48 mg (21). However, human host tissue is approximately 15e30 times less responsive

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than that of rodents, so the optimal concentration of BMP-2 required to elicit 1 cm3 of new bone would be approximately 720e1440 mg. Therefore, before clinical utilization, large animal models should be used to test an HCF system with various doses of BMP-2 to arrive at an optimal concentration of BMP-2 in humans. In addition, our study design was limited in its capacity to compare BMP-2 with other substitutions. Bone regeneration after BMP-2loaded HCF transplantation should be compared with that of different carriers, such as plain fibrin, collagen sponge and hydroxyapatite with equal doses of BMP-2. Consequently, further study is needed to confirm the dose of HCF-BMP capable of inducing sufficient bone formation after surgery in humans. In conclusion, this study investigated the dose of HCF-BMP that would produce comparable results to those of autograft in spinal fusion. We found that a 1/40 dose of commercial BMP-2 applied in an HCF carrier induced sufficient bone formation and reduced side effects, such as ectopic bone formation, in a rabbit spinal fusion model. Acknowledgments This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ0099562014), Rural Development Administration, Republic of Korea. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Deyo RA, Ching A, Matsen L, Martin BI, Kreuter W, Jarvik JG, et al. Use of bone morphogenetic proteins in spinal fusion surgery for older adults with lumbar stenosis: trends, complications, repeat surgery, and charges. Spine. 2012;37:222e30. 2. Sheehan JP, Sheehan JM, Seeherman H, Quigg M, Helm GA. The safety and utility of recombinant human bone morphogenetic protein-2 for cranial procedures in a nonhuman primate model. J Neurosurg. 2003;98:125e30. 3. Harwood PJ, Giannoudis PV. Application of bone morphogenetic proteins in orthopaedic practice: their efficacy and side effects. Expert Opin Drug Saf. 2005;4:75e89. 4. Koo KH, Lee JM, Ahn JM, Kim BS, La WG, Kim CS, Im GI. Controlled delivery of low-dose bone morphogenetic protein2 using heparin-conjugated fibrin in the posterolateral lumbar fusion of rabbits. Artif Organs. 2013;37:487e94. 5. Koo KH, Yeo do H, Ahn JM, Kim BS, Kim CS, Im GI. Lumbar posterolateral fusion using heparin-conjugated fibrin for sustained delivery of bone morphogenic protein-2 in a rabbit model. Artif Organs. 2012;36:629e34. 6. La WG, Kwon SH, Lee TJ, Yang HS, Park J, Kim BS. The effect of the delivery carrier on the quality of bone formed via bone morphogenetic protein-2. Artif Organs. 2012;36:642e7.

7. Yang HS, La WG, Bhang SH, Jeon JY, Lee JH, Kim BS. Heparin-conjugated fibrin as an injectable system for sustained delivery of bone morphogenetic protein-2. Tissue Eng Part A. 2010;16:1225e33. 8. Yang HS, La WG, Cho YM, Shin W, Yeo GD, Kim BS. Comparison between heparin-conjugated fibrin and collagen sponge as bone morphogenetic protein-2 carriers for bone regeneration. Exp Mol Med. 2012;44:350e5. 9. Jeon O, Song SJ, Kang SW, Putnam AJ, Kim BS. Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(Llactic-co-glycolic acid) scaffold. Biomaterials. 2007;28: 2763e71. 10. Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine. 1995;20:412e20. 11. Grauer JN, Patel TC, Erulkar JS, Troiano NW, Panjabi MM, Friedlaeder GE. 2000 Young Investigator Research Award winner. Evaluation of OP-1 as a graft substitute for intertransverse process lumbar fusion. Spine. 2001;26:127e33. 12. 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 a composite carrier. Spine. 2008;33:242e9. 13. 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;30:1706e11. 14. Lad SP, Nathan JK, Boakye M. Trends in the use of bone morphogenetic protein as a substitute to autologous iliac crest bone grafting for spinal fusion procedures in the United States. Spine. 2011;36:E274e81. 15. Docherty SA, Engstrand T. Bone morphogenetic proteins in cranial reconstructions: clinical evaluation of heparin-chitosan as a carrier for BMP-2. Plast Reconstr Surg. 2009;123: 192ee3e. 16. Jeon O, Powell C, Solorio LD, Krebs MD, Alsberg E. Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels. J Control Release. 2011;154:258e66. 17. Jeon O, Song SJ, Yang HS, Bhang SH, Kang SW, Sung MA, et al. Long-term delivery enhances in vivo osteogenic efficacy of bone morphogenetic protein-2 compared to short-term delivery. Biochem Biophys Res Commun. 2008;369: 774e80. 18. Kim SE, Jeon O, Lee JB, Bae MS, Chun HJ, Moon SH, Kwon IK. Enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated PLGA nanoparticles with transplantation of bone marrowderived mesenchymal stem cells. J Biomed Sci. 2008;15: 771e7. 19. Lee JW, Lee S, Lee SH, Yang HS, Im GI, Kim CS, et al. Improved spinal fusion efficacy by long-term delivery of bone morphogenetic protein-2 in a rabbit model. Acta Orthop. 2011;82:756e60. 20. Zhao B, Katagiri T, Toyoda H, Takada T, Yanai T, Fukuda T, et al. Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem. 2006;281:23246e53. 21. Kato M, Toyoda H, Namikawa T, Hoshino M, Terai H, Miyamoto S, Takaoka K. Optimized use of a biodegradable polymer as a carrier material for the local delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2). Biomaterials. 2006;27:2035e41.

Optimal condition of heparin-conjugated fibrin with bone morphogenetic protein-2 for spinal fusion in a rabbit model.

Heparin-conjugated fibrin (HCF) is a carrier for long-term release of bone morphogenetic protein-2 (BMP-2) and has been shown to promote bone formatio...
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