Efficacy of rhBMP-2 during distraction osteogenesis

J Orthop Sci (2005) 10:529–533 DOI 10.1007/s00776-005-0934-4

Original article Efficacy of rhBMP-2 during distraction osteogenesis Yoshihisa Nunotani, Muneaki Abe, Hisaya Shirai, and Hisashi Otsuka Department of Orthopedic Surgery, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki 569-8686, Japan

Abstract Background. Osteoinductive ability of bone morphogenetic protein-2 (BMP-2) has been studied in recent reports. In this study, we evaluated the efficacy of BMP-2 during distraction osteogenesis. Methods. A group of 24 Japanese white rabbits were divided into four groups randomly and underwent lengthening of the right femur. Distraction was performed for 2 weeks (1.0 mm/ day). Group A began distraction following a 7-day waiting period after surgery. For all other groups (B–D), distraction was started immediately after surgery. Groups A and B did not undergo any surgical intervention at the osteotomy site, as a control. The delivery system for rhBMP-2 used a polymercoated gelatin sponge (PGS). Buffer/PGS was implanted into the osteotomy site in group C, and group D received rhBMP2/PGS. Callus was evaluated radiographically at 1, 2, 4, and 6 weeks after the surgery, and all rabbits were killed at 6 weeks. One rabbit from each group was examined histologically; the remaining five rabbits underwent biomechanical testing. Results. A significant increase in callus formation was evident in group D compared with that in other groups. In group D, regenerative changes were evident during the earlier phase. Formation of bone cortex and bone marrow cavity was almost complete in group D, and the cortex was thicker than that in the other groups. Torsional strength values (10-2 Nm) of each group were as follows: A, 118.7 ± 52.4; B, 160.0 ± 40.7; C, 106.8 ± 8.1; D, 266.1 ± 93.1. Stiffness values (10-3 Nm/degree) were as follows: A, 390.2 ± 137.2; B, 391.8 ± 158.4; C, 183.1 ± 78.5; D, 624.4 ± 214.2. Group D exhibited the highest values for both torsional strength and stiffness. Conclusions. Acceleration of the regenerative changes during the early phase occurred in the BMP-2-treated group. The efficacy of BMP-2 in distraction osteogenesis was recognized radiographically, histologically, and by biomechanical testing (torsional strength and stiffness).

Offprint requests to: Y. Nunotani Received: December 20, 2004 / Accepted: June 7, 2005

Introduction Distraction osteogenesis is a useful technique for limb lengthening, treating fracture nonunion or malunion and bone defects.1–6 With the callotasis method (generally used as a term of distraction osteogenesis), it is necessary to wait for callus formation to occur in the regenerated site before initiating distraction.1 A waiting period of 1–2 weeks is often required clinically. Subsequently, several weeks of distraction at approximately 1 mm/day and several months for bone consolidation are also required. During this whole period, however, the morbid condition is unavoidable in these patients, and daily activities are often limited. Therefore, to accelerate osteogenic changes and to enhance bone consolidation would be necessary to shorten the morbid period for patients. In 1965, Urist reported that new bone formation occurred when demineralized bone was implanted intramuscularly.7 He proposed the existence of bone morphogenetic protein based on this phenomenon. In recent studies, several growth factors, hormones, and cytokines have been discovered that support tissue reconstruction.8–12 Some of these cytokines, the bone morphogenetic proteins (BMPs), exhibit osteoinductive abilities that can enhance bone formation or consolidation for bone fractures or defects. Among these proteins, BMP-2 is said to exhibit the strongest osteoinductive ability.9,13,14 BMP-2 belongs to the transforming growth factor-b (TGFb) superfamily. In a recent study, BMP-2 was found to be effective for treating bone fractures or bone defects in an animal model,14–17 and it has already begun to be used clinically in other countries.18 However, few studies have examined distraction osteogenesis using BMP-2 with proper biomechanical testing.16,17,19 The purpose of this study was to investigate the efficacy of BMP-2 in distraction osteogenesis with biomechanical testing. This knowledge can aid in shortening the duration of bone lengthening.

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Materials and methods A group of 24 Japanese white rabbits (male; 24–30 weeks of age; body weight 2.0–2.8 kg) underwent lengthening of the right femur with an M-100 fixator (Orthofix, Bussolengo-Verona, Italy). Distraction was conducted at a rate of 1.0 mm/day once a day for 2 weeks. The rabbits were randomly divided into four groups (A–D), with each group consisting of six rabbits. Groups A and B did not undergo any surgical intervention at the osteotomy site and were the controls. Group A began distraction following a 7-day waiting period after surgery. For all other groups (B–D), distraction was started immediately after surgery. The delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2; Astellas Pharma, Tokyo, Japan) used a polymer-coated gelatin sponge (PGS) (Astellas Pharma). Buffer or rhBMP-2 (80 mg) was soaked in the PGS (10 ¥ 10 ¥ 1.0 mm) before implantation. Buffer/PGS was implanted in the osteotomy site in animals from group C, and group D received rhBMP2/PGS. Osteotomy was undertaken using a bone saw; then, about a 1 mm wide space was created in the osteotomy site. Once the femur was lengthened a few millimeters, and its circumferential discontinuity confirmed, PGS was inserted into the extended space. Subsequently, lengthening was restored to its original position. Radiographic evaluation was performed 1, 2, 4, and 6 weeks after the surgery, and all rabbits were killed at 6 weeks. One rabbit from each group was examined histologically, and the remaining five rabbits underwent biomechanical testing. Radiographic evaluation Radiographs were obtained at 1, 2, 4, and 6 weeks using a Softex type CMB X-ray machine (Softex, Tokyo, Japan) at 60 kV, 40 mA, and 80 s. Callus formation and bone consolidation at the distraction site were evaluated. Histological examination The femurs were harvested and fixed in 10% formaldehyde solution over several days. The specimens were demineralized by soaking in Plank-Rychlo’s solution (aluminum chloride, hydrochloride, formic acid, distilled water) for 2 days. Blocks were cut using a microtome and stained with hematoxylin and eosin (H&E). Qualitative evaluation of the osteogenesis was performed by examining the formation of new cortex and bone marrow cavity at the regenerated site.

Y. Nunotani et al.: Osteoinductive ability of rhBMP-2

Biomechanical test The rabbit femur has a unique three-dimensional curvature anatomically. The ends of the femur were cut off, and the diaphysis including the distraction osteogenesis site was harvested as a biomechanical specimen. These specimens were mounted on the Autograph testing machine (model AG-50kN I; Shimadzu, Kyoto, Japan) and loaded to failure at a rotation of 1°/s. Torsional strength and stiffness were measured and recorded. Statistical analysis Biomechanical data from each group were compared using one-way analysis of variance (ANOVA). P < 0.05 indicated a significant difference. All animal experiment procedures were approved and licensed by the guidelines on animal experiments in Osaka Medical College. Results Radiographic findings. All rabbits exhibited callus formation 6 weeks after surgery, although in three in group C there was poor callus formation. Although the rabbits in group D were submitted to immediate distraction after surgery, they exhibited good callus formation at 2 weeks; in fact, some of them exhibited good callus formation after only 1 week. A significant increase in callus formation was evident in group D compared with the other groups. In group D, the regenerative changes seemed to occur during the early phase (Fig. 1). Histological findings. Continuity of bone cortex and bone marrow cavity at the distraction site was poor in groups A and B, as was the thickness of the cortex. In group C, callus formation seemed to be interrupted by the presence of PGS. The bone cortex and bone marrow cavity were not continuous as a result of obstruction by the PGS. In group D, formation of bone cortex and bone marrow cavity was almost complete, and the cortex was thicker than that in the other groups (Fig. 2). Biomechanical findings and statistical analysis. Torsional strength and stiffness values are shown in Figs. 3 and 4. Torsional strength values (10-2 Nm) of each group were as follows: A, 118.7 ± 52.4; B, 160.0 ± 40.7; C, 106.8 ± 8.1; D, 266.1 ± 93.1. Stiffness values (10-3 Nm/degree) were as follows: A, 390.2 ± 137.2; B, 391.8 ± 158.4; C, 183.1 ± 78.5; D, 624.4 ± 214.2. Group D exhibited the highest values for both torsional strength and stiffness (P < 0.05 compared to each group).

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Fig. 1. Radiographic findings. All patients exhibited callus formation at 6 weeks after surgery. A significant increase in callus formation was evident in group D compared with other groups

Fig. 2. Histological findings. Formation of bone cortex and bone marrow cavity was poor in groups A and B. In group C, they were not continuous by the polymercoated gelatin sponge (PGS). In group D, they were almost complete. Arrows indicate the ends of the osteotomy site

Discussion When discontinuity of bone occurs, as with a fracture or an osteotomy of distraction osteogenesis, bone healing occurs by intramembranous or endochondral ossification (or both). The regenerative changes during distraction osteogenesis are considered the same as those that occur during bone fracture gap healing. The rate of bone callus formation with distraction osteogenesis is said about twice that of fracture gap healing. During intramembranous ossification the osteoblasts are stimulated and differentiate into osteocytes. During endochondral ossification the chondroblasts are stimulated and differentiate into the osteoblasts with the invasion of vessels from the intramedullary tissue. These

osteoblasts around the new vessels differentiate into osteocytes, and calcification occurs, ultimately resulting in ossification. These reactions are regulated by cytokines. When a fracture occurs, the cytokine TGFb is released from platelets in the hematoma around the fracture site. TGFb immediately interacts with the periosteal cells and stimulates BMPs, which subsequently interact with preosteoblasts, osteoblasts, chondroblasts, and pericytes.20–22 In a recent study, 20 subtypes of BMPs were described, and each exhibited characteristic roles in tissue repair.11,12 For instance, vessels, nerves, tendons, skin, cartilage, and bone could be induced. Li et al.16 reported the efficacy of rh-BMP2 in a rabbit model of distraction osteogenesis. Rabbit femurs were lengthened a total of 20 mm at rate of 2 mm/day after a

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Fig. 3. Torsional strength values (10-2 Nm) for each group were A 118.7 ± 52.4; B 160.0 ± 40.7; C 106.8 ± 8.1, D 266.1 ± 93.1. The highest values were exhibited in group D, and there was a significant difference statistically compared to each group. *P < 0.05

Fig. 4. Stiffness values (10-3 Nm/degree) were A 390.2 ± 137.2; B 391.8 ± 158.4; C 183.1 ± 78.5; D 624.4 ± 214.2. Group D exhibited the highest values, and there was a significant difference statistically compared to each group. *P < 0.05

7 day waiting period. RhBMP-2 was administered to the osteogenesis site by surgical implantation with absorbable collagen sponge or a percutaneous injection at the end of the lengthening phase. Acceleration of the regenerative changes during the early phase occurred in the rhBMP-2-treated groups. Their waiting period was established in accordance with a method of callotasis that was described by De Bastiani et al.,1 who stated that the distraction should be initiated after the appearance of callus formation. Clinically, a 1- to 2-week waiting period is usually required between the osteotomy and initiating distraction. In the present investigation, however, we did not utilize a waiting period in the

Y. Nunotani et al.: Osteoinductive ability of rhBMP-2

group using rhBMP-2 in anticipation of its osteoinductive ability. Rhinelander23 reported that after osteotomy only 3–4 days were necessary for recovery of the periosteum and less than 1 week for that of the intramedullary blood supply. During this early phase, the high concentration of rhBMP-2 at the site of regeneration might accelerate callus formation, which was the hypothesis of the present study. In another study, hemangioendothelial cells or pericytes of the new vessels, which differentiated into osteoblasts or preosteoblasts during the early phase of regeneration, played an important role in osteogenesis.20,21 These reactions were induced and controlled by the cytokines, resulting in a transient increase in the blood flow in the distraction site (10 times maximal blood flow), followed by a sustained increased blood flow (3 times maximal blood flow) for about 17 weeks after osteotomy.24,25 The concentration of rhBMP-2 with PGS has been shown to be gradually reduced to half for 3–4 days, to 10% for 10 days, and to 0.5% for the subsequent 3 weeks after osteotomy. Thus, rhBMP-2 with the PGS exhibited sustained release for up to 3 weeks.26 In another study, rhBMP-2 that was injected locally without any delivery system remained in place for about 7 days.18 Histologically, distraction osteogenesis exhibits mainly intramembranous ossification, which occurs in the central portion called the fibrous interzone.6,27 Type I collagen, consisting of the same elements as PGS, are evident primarily in this zone. By contrast, endochondral ossification occurs at either end of the distraction site after the invasion of vessels from the intramedullary tissue. Type II collagen is evident only at the surface of the periosteal membrane at either end. Yasui et al. found that intramembranous ossification occurred mainly during distraction osteogenesis, but that endochondral ossification occurred only during the early phase.6,27 The high concentration of rhBMP-2 during the early phase might interact with both intramembranous and endochondral ossification. Among all of the cytokines, BMP-2 has been demonstrated to exhibit the strongest osteoinductive ability. The results in the present study demonstrated that, consistent with previous reports, osteogenic changes were accelerated in the rhBMP-2-treated group, as seen by radiographic, histological, and biomechanical evaluation.16–18 Bone fracture repair in rodent or rabbit models is more rapid than that in primate models or in clinical practice. Although this was not a clinical study, administration of rhBMP-2 during the early phase was so effective during distraction osteogenesis that we propose that rhBMP-2 might be able to shorten the morbid period for patients.

Y. Nunotani et al.: Osteoinductive ability of rhBMP-2 No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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