JBJS | Recombinant Human Bone Morphogenetic Protein-2 Accelerates Healing in a Rabbit Ulnar Osteotomy Model

The Journal of Bone & Joint Surgery, Volume 83, Issue 8

Articles | August 01, 2001

Recombinant Human Bone Morphogenetic Protein-2 Accelerates Healing in a Rabbit Ulnar Osteotomy Model

M. L. Bouxsein, PhD; T. J. Turek, BS; C. A. Blake, BS; D. D’Augusta, BS; X. Li, MD; M. Stevens, BS; H. J. Seeherman, PhD, VMD; J. M. Wozney, PhD

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Investigation performed at Musculoskeletal Sciences, Genetics Institute/Wyeth-Ayerst Research, Cambridge, Massachusetts
M.L. Bouxsein, PhD
T.J. Turek, BS
C.A. Blake, BS
D. D’Augusta, BS
X. Li, MD
M. Stevens, BS
H.J. Seeherman, PhD, VMD
J.M. Wozney, PhD
Musculoskeletal Sciences, Genetics Institute/Wyeth-Ayerst Research, 87 Cambridge Park Drive, Cambridge, MA 02140. E-mail address for M.L. Bouxsein: mbouxsein@genetics.com

The authors did not receive grants or outside funding in support of their research or preparation of this manuscript. One or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Genetics Institute). No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Read in part at the Annual Meeting of the Orthopaedic Research Society, Anaheim, California, February 2, 1999.

The Journal of Bone & Joint Surgery. 2001; 83:1219-1230

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Abstract

Background: Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously.

Methods: Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of ¹²⁵I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically.

Results: Gamma scintigraphy of ¹²⁵I-recombinant human bone morphogenetic protein-2 showed that 73% ± 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% ± 10% of the initial dose remained at the site one week after surgery, and 8% ± 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points.

Conclusions and Clinical Relevance: These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Of the 6.2 million fractures that occur annually in the United States, approximately 5% to 20% have delayed or impaired healing^1-6. The factors that contribute to impaired healing include the severity and location of the injury, the treatment method, and the demographic and lifestyle characteristics of the patient^4,7,8. With the exception of the treatment method, the factors that influence the success of fracture repair are not under the control of the individual treating the fracture. Therefore, new therapies that accelerate fracture-healing, that ensure successful union, and that could be used in conjunction with traditional treatment methods are clinically desirable.

Approaches for enhancing fracture-healing can be broadly divided into two categories: mechanical or physical interventions^9-11, and biological interventions^12,13. One rationale for the use of biological agents to enhance fracture repair is that they are involved in both embryological bone formation and fracture-healing. Thus, these factors have the potential to interact therapeutically in a process in which they are naturally involved^8,14. Numerous growth factors and cytokines—including transforming growth factor-beta (TGF-β), several bone morphogenetic proteins (BMPs), fibroblast growth factors (FGF)-1 and 2, insulin-like growth factor-1 (IGF-1), and platelet-derived growth factor (PDGF)—have been identified in healing fractures at different time-points^15-23.

Among the factors identified in healing fractures, BMPs have been implicated as key elements in the cascade of molecular events required for skeletal development and repair^15,21,24-26. The present study focuses on one member of this protein family: recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro, the primary effect of rhBMP-2 is to induce the differentiation of mesenchymal cells into cells expressing a chondroblastic and/or osteoblastic phenotype^27,28. In vivo studies have shown that rhBMP-2 induces ectopic bone formation²⁹ and promotes the healing of critical-sized defects^30-34 and spinal fusion sites^35,36. However, less is known about the ability of rhBMP-2 to promote the healing of non-critical-sized defects, such as those used in fracture-healing models. Bax et al. found that rhBMP-2, delivered by means of a collagen gel or buffer injection, had a limited effect on the healing of mechanically stable tibial fractures in rabbits, but it enhanced the healing of unstable fractures³⁷. In comparison, implantation of rhBMP-2 with use of an absorbable collagen sponge at the time of fracture enhanced the healing of tibial fractures in goats³⁸. Neither of those studies evaluated the time-course of the healing induced by rhBMP-2.

The overall goal of the present study was to test whether rhBMP-2, implanted with an absorbable collagen sponge, would accelerate healing of non-critical-sized ulnar osteotomies in rabbits. This model of spontaneous healing was chosen to reflect what is commonly seen with fracture-healing in humans. In addition, we studied the time-course of healing and evaluated the in vivo retention of rhBMP-2 using ¹²⁵I gamma scintigraphy.

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Fig. 1:: In vivo (left panel) anteroposterior and mediolateral (middle panel) radiographs and representative histological section (right panel) showing the site of the ulnar osteotomy. The osteotomy and the placement of the absorbable collagen sponge were performed through a palmar approach. The plane used for histological analysis was parallel to that of the lateral radiograph (shown by the box in the middle panel) and perpendicular to that of the anteroposterior radiograph.

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Fig. 2:: Graph depicting the retention of rhBMP-2 at the ulnar osteotomy site, expressed as a percentage of the initial dose delivered (left axis) and as the absolute amount of protein remaining (right axis). The error bars represent one standard deviation.

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Fig. 3:: Graphs showing the effect of absorbable collagen sponges (ACS) containing rhBMP-2 on failure torque (A), torsional stiffness (B), and energy absorbed to failure (C), expressed as percentages of the values for an intact limb. An asterisk indicates that the value for the ulnae treated with an absorbable collagen sponge containing rhBMP-2 was significantly greater than that for the ulnae treated with a sponge containing buffer and that for the untreated ulnae (p < 0.01 for all).

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Fig. 4:: Representative histological sections of the ulnar osteotomy site at three weeks (top row), four weeks (middle row), and six weeks (bottom row). Ulnae treated with an absorbable collagen sponge (ACS) containing rhBMP-2 are shown in the left column, ulnae treated with a sponge containing buffer are shown in the middle column, and untreated ulnae are shown in the right column. The sections were stained with Goldner trichrome and were photographed at 0.5 times magnification.

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TABLE I: Effect of rhBMP-2 on the Properties of Mineralized Callus as Assessed by Peripheral Quantitative Computed Tomography*

*The values are given as the mean and the standard deviation. †The value is significantly greater than that for the ulnae treated with buffer and that for the untreated ulnae (p < 0.005 for both).

	rhBMP-2	Buffer	Untreated
Area of the callus (mm²)
2 wk	?9.3 ± 1.5	?8.0 ± 1.2	?8.2 ± 1.6
3 wk	?24.5 ± 5.6†	17.5 ± 4.4	15.1 ± 3.7
4 wk	?29.2 ± 4.4†	21.1 ± 5.4	20.2 ± 3.7
6 wk	24.3 ± 4.3	20.6 ± 4.7	20.4 ± 6.1
Mineral content (mg)
2 wk	?4.47 ± 0.69	?3.95 ± 0.54	?4.05 ± 0.74
3 wk	?11.83 ± 2.75†	?8.47 ± 2.11	?7.31 ± 1.79
4 wk	?14.90 ± 1.98†	10.99 ± 2.82	10.48 ± 2.05
6 wk	12.68 ± 2.38	11.04 ± 2.61	11.01 ± 3.49

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TABLE I: Effect of rhBMP-2 on the Properties of Mineralized Callus as Assessed by Peripheral Quantitative Computed Tomography*

*The values are given as the mean and the standard deviation. †The value is significantly greater than that for the ulnae treated with buffer and that for the untreated ulnae (p < 0.005 for both).

	rhBMP-2	Buffer	Untreated
Area of the callus (mm²)
2 wk	?9.3 ± 1.5	?8.0 ± 1.2	?8.2 ± 1.6
3 wk	?24.5 ± 5.6†	17.5 ± 4.4	15.1 ± 3.7
4 wk	?29.2 ± 4.4†	21.1 ± 5.4	20.2 ± 3.7
6 wk	24.3 ± 4.3	20.6 ± 4.7	20.4 ± 6.1
Mineral content (mg)
2 wk	?4.47 ± 0.69	?3.95 ± 0.54	?4.05 ± 0.74
3 wk	?11.83 ± 2.75†	?8.47 ± 2.11	?7.31 ± 1.79
4 wk	?14.90 ± 1.98†	10.99 ± 2.82	10.48 ± 2.05
6 wk	12.68 ± 2.38	11.04 ± 2.61	11.01 ± 3.49

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TABLE II: Effect of rhBMP-2 on Torsional Biomechanics*

*The values are given as the mean and the standard deviation. †The value is significantly greater than that for the ulnae treated with buffer (p < 0.005) and that for the untreated ulnae (p < 0.01).

	rhBMP-2	Buffer	Untreated
Failure torque (N-m)
2 wk	0.133 ± 0.050	0.112 ± 0.057	0.103 ± 0.057
3 wk	0.388 ± 0.140†	0.210 ± 0.103	0.202 ± 0.071
4 wk	0.633 ± 0.136†	0.317 ± 0.112	0.306 ± 0.119
6 wk	0.754 ± 0.132	0.664 ± 0.156	0.672 ± 0.226
Torsional stiffness (N-m/deg 100)
2 wk	0.482 ± 0.202	0.437 ± 0.235	0.399 ± 0.263
3 wk	1.909 ± 0.639†	1.022 ± 0.620	0.899 ± 0.396
4 wk	2.601 ± 0.741†	1.442 ± 0.655	1.389 ± 0.601
6 wk	3.419 ± 0.841	3.232 ± 1.119	3.096 ± 1.224
Energy absorbed to failure (N-m deg)
2 wk	?2.08 ± 0.91	?1.56 ± 1.06	?1.44 ± 0.92
3 wk	?4.85 ± 2.23†	?2.46 ± 1.21	?2.82 ± 0.78
4 wk	10.00 ± 3.67†	?4.27 ± 1.72	?4.26 ± 1.88
6 wk	10.05 ± 2.31	?8.39 ± 2.26	?8.55 ± 3.08

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TABLE II: Effect of rhBMP-2 on Torsional Biomechanics*

	rhBMP-2	Buffer	Untreated
Failure torque (N-m)
2 wk	0.133 ± 0.050	0.112 ± 0.057	0.103 ± 0.057
3 wk	0.388 ± 0.140†	0.210 ± 0.103	0.202 ± 0.071
4 wk	0.633 ± 0.136†	0.317 ± 0.112	0.306 ± 0.119
6 wk	0.754 ± 0.132	0.664 ± 0.156	0.672 ± 0.226
Torsional stiffness (N-m/deg 100)
2 wk	0.482 ± 0.202	0.437 ± 0.235	0.399 ± 0.263
3 wk	1.909 ± 0.639†	1.022 ± 0.620	0.899 ± 0.396
4 wk	2.601 ± 0.741†	1.442 ± 0.655	1.389 ± 0.601
6 wk	3.419 ± 0.841	3.232 ± 1.119	3.096 ± 1.224
Energy absorbed to failure (N-m deg)
2 wk	?2.08 ± 0.91	?1.56 ± 1.06	?1.44 ± 0.92
3 wk	?4.85 ± 2.23†	?2.46 ± 1.21	?2.82 ± 0.78
4 wk	10.00 ± 3.67†	?4.27 ± 1.72	?4.26 ± 1.88
6 wk	10.05 ± 2.31	?8.39 ± 2.26	?8.55 ± 3.08

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TABLE III: Location of Fractures Following Torsion-Testing*

*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

Time-Point (wk)	Fracture Site	rhBMP-2	Buffer	Untreated
2	Osteotomy	12/12	12/12	12/12
	Osteotomy/host bone
	Host bone
3	Osteotomy	?9/10	12/12	?9/10
	Osteotomy/host bone	?1/10		?1/10
	Host bone
4	Osteotomy	?3/12	11/12	11/12
	Osteotomy/host bone	?2/12	?1/12	?1/12
	Host bone	?7/12
6	Osteotomy	?1/11	?1/11	?6/12
	Osteotomy/host bone	?3/11	?6/11	?2/12
	Host bone	?7/11	?4/11	?4/12
*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

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TABLE III: Location of Fractures Following Torsion-Testing*

*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

Time-Point (wk)	Fracture Site	rhBMP-2	Buffer	Untreated
2	Osteotomy	12/12	12/12	12/12
	Osteotomy/host bone
	Host bone
3	Osteotomy	?9/10	12/12	?9/10
	Osteotomy/host bone	?1/10		?1/10
	Host bone
4	Osteotomy	?3/12	11/12	11/12
	Osteotomy/host bone	?2/12	?1/12	?1/12
	Host bone	?7/12
6	Osteotomy	?1/11	?1/11	?6/12
	Osteotomy/host bone	?3/11	?6/11	?2/12
	Host bone	?7/11	?4/11	?4/12
*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Experimental Design

Ninety-six skeletally mature male New Zealand White rabbits (age, eight to nine months; weight, 3.4 to 4.0 kg) were used in this study. Bilateral ulnar osteotomies were surgically created in seventy-two rabbits (Fig. 1). The ulnae of twenty-four rabbits that were not operated on served as intact controls. The rabbits with bilateral osteotomies were assigned to one of three treatment groups (with twenty-four animals per group): those treated with an absorbable collagen sponge containing rhBMP-2 on one side and a sponge containing buffer on the contralateral side; those treated with a sponge containing rhBMP-2 on one side, with the contralateral side left untreated; and those treated with a sponge containing buffer on one side, with the contralateral side left untreated. Six rabbits from each treatment group were killed at two, three, four, or six weeks after the surgery. The intact ulnae from the control rabbits were used to compare the properties of a healing limb with those of an intact limb. The housing and care of the animals and the study protocol were approved by the Institutional Animal Care and Use Committee of Genetics Institute. All procedures were carried out according to the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

Surgical Procedure

The rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg), and a bland opthalmic ointment was applied to both eyes to prevent corneal dryness. Both forelimbs were then shaved and prepared aseptically for surgery. Following surgical preparation, halothane was administered through a facemask to maintain general anesthesia. A posterior longitudinal incision was made over the ulna and the underlying muscles were retracted, exposing the mid-diaphysis of both the radius and the ulna. No attempt was made to resect the periosteum around the osteotomy site. However, on the palmar surface, the fibrous periosteum was most likely retracted along with the overlying muscle. An osteotome was used to mark the osteotomy site approximately 45 to 50 mm distal to the olecranon process. A high-speed oscillating saw was used to create a blade-width (0.5-to-1-mm) defect in the ulna. The osteotomy site was then lavaged with 0.9% sodium chloride containing gentamicin sulfate (0.2 mg/mL). For the animals assigned to receive treatment, an 8 20-mm strip of absorbable collagen sponge (Helistat; Integra LifeSciences, Plainsboro, New Jersey) was wrapped around the osteotomy site in a single layer. The implanted sponge surrounded approximately two-thirds of the ulna, including the anterior, lateral, and posterior surfaces. Each strip of dry sponge was wetted with either 200 L of buffer or 200 L of rhBMP-2 solution (0.2 mg/mL, for a total dose of 40 g of rhBMP-2) at least fifteen minutes before implantation. The rhBMP-2 dose and the time-point at which the animals were killed were chosen on the basis of previous reports on the use of rhBMP-2 in a rabbit segmental defect model^30,34 and on a pilot study in which several concentrations of rhBMP-2 were tested in a rabbit osteotomy model (unpublished data). The deep muscle layer and the skin were closed, and the limbs were bandaged and splinted for four days. After four days, the splints were removed and the animals were allowed to move freely. Analgesics (Buprenex [buprenorphine hydrochloride], 0.03 mg/kg) were administered twice on the day of surgery and for up to two days after surgery as needed. The general health of the rabbits was monitored daily throughout the study.

Anteroposterior and lateral radiographs were made immediately after surgery and weekly thereafter with use of Kodak Ektascan M film (Eastman Kodak, Rochester, New York) and a standard technique (40-cm focal distance, 40 kV, 200 mA, and 0.2 s). Before they were killed, the rabbits were sedated with an intramuscular injection of acepromazine (0.5 mL) and given a lethal dose of euthanasia solution (Euthasia V, 0.22 mL/kg). Both forelimbs were carefully harvested, cleaned of soft tissue, wrapped in saline-soaked gauze, and frozen (at —20°C) in airtight containers until further testing.

Retention of 125I-rhBMP-2 at the Osteotomy Site

In eight rabbits, gamma scintigraphy of ¹²⁵I-radiolabeled rhBMP-2 was used to determine the retention of rhBMP-2 at the osteotomy site according to previously described methods^39,40. Iodination of the rhBMP-2 was performed with use of the Iodogen reagent (Pierce, Rockford, Illinois). The iodinated protein was combined with noniodinated protein at a ratio of approximately 1:26 to 1:106. The radiolabeled rhBMP-2 was applied to the absorbable collagen sponge in a manner identical to that described above. After wetting, the baseline radioactivity of the sponge was measured with a gamma counter (CRC-15R; Capintec, Ramsey, New Jersey). The sponge containing rhBMP-2 was then implanted at the osteotomy site. A scintigraphic image of the defect site was made with a large field-of-view gamma camera (ZLC 7500 Orbital; Siemans Medical Solutions Group, Hoffman Estates, Illinois). A low-energy, high-resolution collimator was used to minimize scatter and optimize the spatial resolution. Scintigraphic imaging was repeated at three hours and at one, two, three, four, seven, ten, and fourteen days after surgery. At each time-point, a phantom containing a known amount of ¹²⁵I-radioisotope (Ci/mg of rhBMP-2) was imaged along with the defect site to allow quantification of the in vivo scintigraphic images. Imaging of the phantom was also used to correct for normal radioactive decay. The regions of interest were drawn separately around the osteotomy site and the phantom, and the ¹²⁵I activity was calculated for each region in counts per minute. The activity of the phantom was determined with use of the Capintec gamma counter and was used to convert the ¹²⁵I activity in counts per minute to the amount of rhBMP-2 in milligrams. Image analysis was performed with Odyssey software (Marconi Medical Systems, Cleveland, Ohio). The rate of rhBMP-2 loss from the osteotomy site and the predicted half-life were computed with a noncompartmental model provided by a commercial software program (WinNonlin; Pharsight, Mountain View, California). Uludag et al. showed that the protein is stable following the iodination process and, furthermore, that the measured radioactivity is protein-bound and not a result of free ¹²⁵I in circulation^39,41.

Morphology of the Mineralized Callus

The excised forelimbs were scanned with use of peripheral quantitative computed tomography (XCT3000; Norland Medical Systems, White Plains, New York) to determine the area and mineral content of the mineralized callus. Contiguous images (1-mm separation) perpendicular to the diaphyseal axis were made, starting at the distal extent of the fracture callus and extending proximally for 15 mm. The slice closest to the middle of the osteotomy site was identified. The cross-sectional area (in square millimeters) and the total bone mineral content (in grams) of the mineralized portion of the fracture callus were calculated for this middle slice and for two slices proximal and two slices distal to it. The mean area and bone mineral content of the five slices were used for subsequent comparisons.

Biomechanical Testing

The ulnae were thawed to room temperature, and the ends of each limb were embedded in square molds with use of polymethylmethacrylate. A scalpel blade was then placed between the radius and the ulna at the proximal and distal ends, and a high-speed saw was used to cut through the radius at those points. The radius was then gently pried away from the ulna. After they had been embedded, the ulnae were tested quasi-statically to failure in torsion (1.5°/sec) with use of a servo-hydraulic materials-testing system (model 8500; Instron, Canton, Massachusetts). The testing was stopped when the specimen broke or when 35° of rotation was reached, whichever occurred first. The torque-rotation data were used to calculate the maximum torque to failure, torsional stiffness, and energy absorbed to failure. After testing, radiographs of the limbs were made (Faxitron X-ray, Wheeling, Illinois) and used to identify the location of the fractures. Using a variation of the classification system of White et al.⁴², an orthopaedic surgeon (H.J.S.) who was blinded to the time-point and the treatment group classified the fractures as occurring through the osteotomy site only, through both the osteotomy site and the intact bone, or through the intact bone only.

Histological Evaluation

After mechanical testing, the specimens were placed in 70% ethanol, dehydrated in graded concentrations of alcohol, and embedded (undecalcified) in methylmethacrylate. During embedding, the positioning of the ulna was standardized in an attempt to ensure that the same region of the callus was evaluated in all specimens. Five-micrometer-thick sections were cut in the coronal plane and were stained with Goldner trichrome. The slides were evaluated qualitatively for callus composition and maturity. Bridging of the osteotomy site and cortical continuity were recorded for each ulna. The total callus area and the area of cartilage, bone, and absorbable collagen sponge were measured by tracing the corresponding regions within the callus.

Data Analysis

Descriptive statistics were generated for all outcome variables. A two-factor analysis of variance, with the time-point and the type of treatment as the grouping variables, was used to evaluate the effect of rhBMP-2 treatment on the healing of the osteotomy. When there was a significant interaction between the type of treatment and the time-point, the treatment effect was evaluated with a one-factor analysis of variance at each time-point. Post hoc multiple comparisons between groups were conducted with Fisher’s protected least-square-difference method. Differences were considered significant at p < 0.05.

Results

Introduction | Materials and Methods | Results | Discussion | References

The surgical procedure was well tolerated. Rabbits were weight-bearing two to three days after surgery and were moving freely after the splint was removed on the fourth postoperative day. Three rabbits (two from the three-week group and one from the six-week group) were killed because a radial fracture was observed. Thus, the final sample size included sixty-nine rabbits with bilateral ulnar osteotomies and twenty-four rabbits with intact limbs.

Retention of 125I-rhBMP at the Osteotomy Site

Gamma scintigraphy of ¹²⁵I-rhBMP-2 indicated that 73% ± 6% (mean and standard deviation) of the dose administered three hours after the procedure was initially retained at the fracture site. Approximately 37% ± 10% and 8% ± 7% of this initially administered dose remained at the site seven and fourteen days after surgery, respectively (Fig. 2). These values correspond to 29.2 ± 2.3 g rhBMP-2 initially retained at the defect site, with 12.8 ± 2.4 and 2.4 ± 2.4 g rhBMP-2 retained at one and two weeks, respectively. The area under the curve was 164.7 ± 35.3 g-days (mean and standard deviation), and the predicted half-life of rhBMP-2 was 3.76 ± 1.38 days (mean and standard deviation).

Area and Mineral Content of the Mineralized Callus

The area of the mineralized callus was similar in all ulnae at two weeks. However, at three, four, and six weeks the callus area was approximately 20% to 60% greater in the ulnae treated with an absorbable collagen sponge containing rhBMP-2 than it was in either the ulnae treated with a sponge containing buffer or the untreated ulnae (Table I). For example, at three weeks, the mineralized callus area in the ulnae treated with rhBMP-2 was 40% greater than in the ulnae treated with buffer and 62% greater than in the untreated ulnae (p < 0.001 for both). At four weeks, the callus area in the ulnae treated with rhBMP-2 was 38% and 45% greater than that of the control (buffer-treated and untreated) ulnae. At six weeks, the callus in the ulnae treated with rhBMP-2 had begun to remodel and, although its area was still approximately 20% greater than that of the callus in the control groups, this difference did not reach significance. A similar pattern was observed for the mineral content of the callus, with no differences between the groups at two weeks, approximately 40% to 60% greater mineral content in the ulnae treated with rhBMP-2 at three and four weeks, and no significant differences between the groups at six weeks (Table I).

Torsional Biomechanics

Torsional biomechanical properties were similar in all of the treatment groups at two weeks (Table II). However, at three and four weeks, failure torque, torsional stiffness, and energy absorbed to failure of the ulnae treated with an absorbable collagen sponge containing rhBMP-2 were each at least 80% greater than the corresponding measurements for either the ulnae treated with buffer (p < 0.005) or those left untreated (p < 0.01) (Table II). Furthermore, at four weeks, the mean failure torque of the ulnae treated with rhBMP-2 was equivalent to that of the intact ulnae, whereas the mean failure torque of the ulnae treated with buffer and that of the untreated ulnae were only approximately 45% of that of the intact ulnae (Fig. 3). Torsional stiffness and energy absorbed to failure also returned to intact levels quicker in the ulnae treated with rhBMP-2 (Fig. 3). At six weeks, the torsional properties were similar in all treatment groups and were equivalent to those of the intact ulnae. The mechanical properties of the ulnae treated with buffer and those left untreated were similar at all time-points (Fig. 3 and Table II).

The locations of the fractures resulting from destructive torsion-testing were similar in all groups at two and three weeks, with 90% to 100% of the limbs fracturing through the original osteotomy site (Table III). At four weeks, 25% of the limbs treated with rhBMP-2 fractured through the original osteotomy site, whereas 58% fractured through the host bone. In comparison, 92% of the limbs treated with buffer and 92% of the untreated limbs fractured through the original osteotomy site. At six weeks, 64% of the ulnae treated with rhBMP-2 fractured through the host bone. In contrast, only 36% of the ulnae treated with buffer and 30% of the untreated ulnae fractured through the host bone at six weeks.

Histological Evaluation

Different healing patterns were observed on the palmar and dorsal surfaces of the ulna, presumably because of the nature of the surgical approach and placement of the absorbable collagen sponge implant (Fig. 1). On the side of the surgical approach (the palmar surface of the ulna), the muscles were retracted and the fibrous periosteum was elevated and likely disrupted. The absorbable collagen sponge was then placed directly on this surface and was wrapped circumferentially around two-thirds of the ulna. In contrast, on the surface opposite that of the surgical approach (the dorsal surface of the ulna), the disruption of the periosteum was variable and the absorbable collagen sponge was not in direct contact with the bone. In the untreated ulnae, the callus formation was asymmetric, with a larger callus on the dorsal surface than on the palmar surface (Fig. 4). This asymmetry in callus formation was less pronounced in the ulnae treated with a sponge containing rhBMP-2, as callus formed on both surfaces, with a slightly larger callus on the palmar surface (the side of the implanted sponge containing rhBMP-2) compared with the dorsal surface.

The healing pattern on the palmar surface showed that, two weeks after surgery, little callus had formed in the ulnae treated with buffer and in the untreated ulnae. In contrast, callus formation was evident in the ulnae treated with rhBMP-2, with most of the callus located on the surface of the sponge that was facing the muscle and with minimal response from the bone surface itself. At three weeks, the ulnae treated with rhBMP-2 had a fully developed callus, primarily composed of bone with isolated islands of cartilage. In most instances, the fracture gap was bridged with bone. In the ulnae treated with buffer and in the untreated ulnae, a small callus had formed and the fracture gap was bridged with either fibrous tissue or cartilage. At four weeks, the callus in the ulnae treated with rhBMP-2 was composed completely of bone and all of the osteotomy sites were fully bridged with bone. In comparison, in both the ulnae treated with buffer and the untreated ulnae, the callus remained small and was composed of bone and cartilage. Still, most of the limbs in the control groups showed bone bridging the fracture gap at this point. At six weeks, the osteotomy was completely bridged with bone in all groups.

Overall, in each of the groups, callus formation on the dorsal surface proceeded through the conventional stages of endochondral bone formation that is associated with secondary, or indirect, fracture-healing. However, at all time-points, the composition of the callus in the ulnae treated with rhBMP-2 was more advanced than it was in the control groups (Fig. 4). For example, at the time-points when the callus in the control limbs was primarily composed of fibrous tissue, the callus in the ulnae treated with rhBMP-2 was primarily composed of fibrocartilage or cartilage. At the time-points when the callus in the control limbs was primarily composed of cartilage, the callus in the ulnae treated with rhBMP-2 was composed of bone or a combination of cartilage and bone.

At two weeks, a fibrocartilaginous callus had formed in the osteotomy gap and there was a periosteal bone response at the edges of the callus in all groups (Fig. 4). The callus was composed primarily of cartilage in ten of the twelve ulnae treated with rhBMP-2 compared with six of the twelve ulnae treated with buffer and one of the twelve untreated ulnae. At three weeks, the callus was composed of either cartilage or a combination of cartilage and bone in all of the ulnae treated with rhBMP-2. In contrast, the callus was still composed of either fibrous or fibrocartilaginous tissue in five of the twelve ulnae treated with buffer and four of the ten ulnae left untreated. At four weeks, all of the ulnae treated with rhBMP-2 were at the most advanced stages of healing (with the callus composed of either cartilage and bone or bone only), whereas only one (4%) of the twenty-four ulnae in the control groups had progressed to this stage of healing. Moreover, at four weeks, the osteotomy site was bridged by bone in eleven of the twelve ulnae treated with rhBMP-2 compared with only two of the twelve ulnae treated with buffer and one of the twelve untreated ulnae. At six weeks, the callus was composed primarily of bone and the osteotomy site was bridged by bone in most of the ulnae in all groups.

The absorbable collagen sponge was resorbed more quickly in the ulnae treated with rhBMP-2 than in those treated with buffer. At two weeks, the sponge was visible both in the limbs treated with rhBMP-2 and in those treated with buffer (Fig. 4). However, the area occupied by the sponge was much smaller in the ulnae treated with rhBMP-2 (2.42 mm²) than it was in those treated with buffer (10.08 mm²). At three weeks, the sponge had been completely resorbed in the ulnae treated with rhBMP-2, whereas small amounts of sponge were still visible in a few of the ulnae treated with buffer. At four weeks, the sponge was no longer visible in the ulnae treated with buffer.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

In the current study, we determined the ability of rhBMP-2 to enhance healing of non-critical-sized ulnar defects in rabbits. Osteotomy sites treated with rhBMP-2 healed approximately 33% faster than did osteotomy sites either treated with buffer or left untreated. At three and four weeks, the mechanical properties of the healing osteotomy sites were as much as twofold greater in the rhBMP-2 group than in either control group. Healing patterns in the ulnae treated with the carrier alone (buffer) were similar to those of the untreated ulnae, demonstrating that the carrier itself did not enhance healing.

The primary mechanisms for enhanced healing in the ulnae treated with rhBMP-2 appeared to be more rapid and greater initial callus formation, as well as more advanced callus maturation and remodeling, compared with the control ulnae at each time-point. The presence of a larger and more mature callus is consistent with the increased biomechanical properties of the ulnae treated with rhBMP-2. These histological and biomechanical observations support the reported ability of rhBMP-2 to induce the differentiation of uncommitted mesenchymal precursor cells to the osteoblast lineage^43,44. In addition to inducing differentiation, BMPs have also been shown to stimulate transcription of the gene encoding an osteoblast-specific transcription factor, Cbfa1, that appears to be essential for osteoblast development^45,46. Although the precise cellular targets for rhBMP-2 treatment during fracture-healing are not known, it is reasonable to assume that precursor cells found in the periosteum, bone marrow, and surrounding muscle (including vascular pericytes) may be affected by exogenous rhBMP-2. This assumption is supported by the observation that, during fracture-healing, the expression of BMP receptors is substantially increased in osteogenic cells of the periosteum near the ends of the fracture, in osteoblasts, and in fibroblast-like spindle cells and chondrocytes participating in endochondral ossification^26,47.

It is interesting to note that, in all groups at all time-points, less cartilage had formed on the palmar surface than on the dorsal surface of the ulnae, although healing occurred by endochondral bone formation on both surfaces. One factor that may have contributed to this healing pattern is the extent of periosteal disruption^48-50. On the palmar surface, it is likely that the periosteum was completely disrupted by the surgical intervention, whereas on the dorsal surface the periosteum was disrupted to varying degrees. The periosteum is believed to be a source of mesenchymal precursor cells, although its precise role as a source of chondrogenic versus osteogenic precursor cells is not known. Therefore, the degree to which the periosteum was disrupted may have contributed to the observed asymmetric healing pattern.

In addition to observing enhanced callus formation in the limbs treated with rhBMP-2, we also noted that the absorbable collagen sponge was resorbed more quickly in these limbs than in those treated with buffer. In the limbs treated with buffer, the sponge was resorbed by processes associated with a typical foreign-body reaction, including infiltration of neutrophils, monocytes, and macrophage or macrophage-like cells, as well as by direct degradation by collagenases. Compared with the ulnae treated with buffer, the ulnae treated with rhBMP-2 showed an overall increase in cellularity, including a dramatic increase in the number of macrophage or macrophage-like cells seen resorbing the sponge at early time-points. Moreover, in contrast to the findings in the limbs treated with buffer, the cell population participating in resorption of the sponge in the limbs treated with rhBMP-2 included giant cells. The mechanism or mechanisms by which rhBMP-2 accelerated the resorption of the absorbable collagen sponges are not fully known.

Another mechanism by which rhBMP-2 may enhance healing is through an interaction with mechanical loading¹⁰. If rhBMP-2 treatment led to more rapid healing and stabilization of the osteotomy site, the rabbits in the treatment group may have been able to increase weight-bearing on the ulna to a greater extent than those in the control groups. Increased weight-bearing would likely promote healing⁵¹ and thereby accentuate the positive effect of rhBMP-2. Force-plate measurements of limb-loading during the healing period would be useful for addressing this hypothesis.

The biodistribution data indicated that approximately 70% (approximately 28 g) of the administered dose of rhBMP-2 was initially retained at the fracture site, with 10% (approximately 4 g) remaining at two weeks after implantation. This retention profile is similar to that observed following the implantation of an absorbable collagen sponge containing rhBMP-2 in full-thickness cartilage defects in rabbits⁴⁰. Thus, in both the previous and the current study gamma scintigraphy was used to characterize an rhBMP-2 retention profile in vivo that was associated with improved healing. Although an ideal retention profile leading to enhanced fracture-healing has not been formally established, these data provide a benchmark against which new rhBMP-2/matrix combinations can be compared.

Despite differences in animal models, dosing regimens, and outcome assessments, our findings are consistent with those of previous studies showing enhanced fracture-healing following rhBMP-2 treatment^37,38. For example, Welch et al., in a tibial fracture model in goats, reported that limbs that had been treated with an absorbable collagen sponge containing rhBMP-2 had increased callus formation, radiographic healing scores, and torsional stiffness compared with the contralateral limb that had been treated with a sponge containing buffer³⁸. Dynamic histomorphometric analysis indicated that the enhanced callus formation associated with rhBMP-2 treatment was due to increased recruitment of osteoprogenitor cells rather than to stimulation of cellular activity³⁸.

In addition to the BMPs, other growth and differentiation factors, including TGF-β, FGFs, and PDGF, have been evaluated for their ability to enhance bone-healing when delivered locally^22,52-54. In contrast to rhBMP-2, many of these other factors require continuous infusion or multiple injections to induce a positive effect on healing. Furthermore, it is likely that these factors act through different mechanisms to affect the fracture-healing process.

For instance, in contrast to rhBMP-2 (which acts as a differentiation factor), the pleiotropic growth factor TGF-β is thought to act mainly as a mitogen by stimulating the proliferation of preosteoblasts and chondrocytes as well as by enhancing the production of extracellular matrix²². When given as a continuous infusion or as multiple injections, TGF-β enhances fracture-healing in rats and rabbits^55,56. However, a single injection of TGF-β at the time of fracture does not improve healing^57-59. Taken together, these findings indicate that, in contrast to rhBMP-2, TGF-β appears to require continuous dosing at high concentrations to achieve consistent effects on healing²².

The FGFs are also believed to act as mitogens on several types of cells involved in fracture-healing, including osteoblasts, chondrocytes, and fibroblasts. In addition, FGF-2 is thought to stimulate angiogenesis. A single injection of FGF-2 at the time of fracture has been shown to enhance fracture-healing^60-62. One of the primary mechanisms by which FGFs appear to enhance healing is by stimulating mesenchymal cell proliferation in the periosteum⁶¹. In contrast, Bland et al. reported that neither FGF-1 nor FGF-2 enhanced the healing of rabbit tibial fractures when delivered as a single injection four days after the fracture⁶³. In total, these studies suggest that the timing of FGF delivery to the fracture site may be critical and, therefore, the opportunity for intervention may be restricted to the immediate post-fracture period.

There were several potential limitations associated with the current study. First, the study involved an osteotomy defect rather than a fracture. Although the osteotomy model provides a well-controlled surrogate fracture, the extent of soft-tissue damage and vascular disruption may be different from that observed in fracture models⁶⁴. If rhBMP-2 relies on induction and differentiation of mesenchymal precursor cells, extensive soft-tissue damage may decrease its ability to enhance healing. On the other hand, in cases with extensive soft-tissue damage, rhBMP-2 may have an even more dramatic effect on healing relative to the poor outcome that could be anticipated for untreated fractures.

Second, this study involved the use of a rabbit model, in which healing occurs quickly (six weeks). The number of cells available to respond to rhBMP-2 and the extent to which the cells can respond may be greater in rabbits than in humans, who heal more slowly. However, the fact that rabbits heal relatively quickly would have made it more difficult to demonstrate accelerated healing, thereby reinforcing the potential of rhBMP-2 to enhance healing.

In conclusion, our study demonstrates that a one-time application of an absorbable collagen sponge containing rhBMP-2 consistently accelerates healing of an ulnar osteotomy in rabbits by inducing more rapid and more extensive callus formation. The in vivo pharmacokinetic data show that rhBMP-2 is delivered to and retained at the fracture site. The marked acceleration of healing following treatment with rhBMP-2 provides a strong rationale for testing the ability of an absorbable collagen sponge containing rhBMP-2 to enhance healing in patients with fractures requiring open surgical management.

Note: The authors thank Russ Palmer, Cynthia Luppen, Anne Despotopolous, Joshua Seeherman, and Camille Francois for their assistance with the technical aspects of this study. They also thank Dr. Gerard Riedel for his insightful review of the manuscript.

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TABLE I: Effect of rhBMP-2 on the Properties of Mineralized Callus as Assessed by Peripheral Quantitative Computed Tomography*

*The values are given as the mean and the standard deviation. †The value is significantly greater than that for the ulnae treated with buffer and that for the untreated ulnae (p < 0.005 for both).

	rhBMP-2	Buffer	Untreated
Area of the callus (mm²)
2 wk	?9.3 ± 1.5	?8.0 ± 1.2	?8.2 ± 1.6
3 wk	?24.5 ± 5.6†	17.5 ± 4.4	15.1 ± 3.7
4 wk	?29.2 ± 4.4†	21.1 ± 5.4	20.2 ± 3.7
6 wk	24.3 ± 4.3	20.6 ± 4.7	20.4 ± 6.1
Mineral content (mg)
2 wk	?4.47 ± 0.69	?3.95 ± 0.54	?4.05 ± 0.74
3 wk	?11.83 ± 2.75†	?8.47 ± 2.11	?7.31 ± 1.79
4 wk	?14.90 ± 1.98†	10.99 ± 2.82	10.48 ± 2.05
6 wk	12.68 ± 2.38	11.04 ± 2.61	11.01 ± 3.49

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TABLE I: Effect of rhBMP-2 on the Properties of Mineralized Callus as Assessed by Peripheral Quantitative Computed Tomography*

*The values are given as the mean and the standard deviation. †The value is significantly greater than that for the ulnae treated with buffer and that for the untreated ulnae (p < 0.005 for both).

	rhBMP-2	Buffer	Untreated
Area of the callus (mm²)
2 wk	?9.3 ± 1.5	?8.0 ± 1.2	?8.2 ± 1.6
3 wk	?24.5 ± 5.6†	17.5 ± 4.4	15.1 ± 3.7
4 wk	?29.2 ± 4.4†	21.1 ± 5.4	20.2 ± 3.7
6 wk	24.3 ± 4.3	20.6 ± 4.7	20.4 ± 6.1
Mineral content (mg)
2 wk	?4.47 ± 0.69	?3.95 ± 0.54	?4.05 ± 0.74
3 wk	?11.83 ± 2.75†	?8.47 ± 2.11	?7.31 ± 1.79
4 wk	?14.90 ± 1.98†	10.99 ± 2.82	10.48 ± 2.05
6 wk	12.68 ± 2.38	11.04 ± 2.61	11.01 ± 3.49

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TABLE II: Effect of rhBMP-2 on Torsional Biomechanics*

	rhBMP-2	Buffer	Untreated
Failure torque (N-m)
2 wk	0.133 ± 0.050	0.112 ± 0.057	0.103 ± 0.057
3 wk	0.388 ± 0.140†	0.210 ± 0.103	0.202 ± 0.071
4 wk	0.633 ± 0.136†	0.317 ± 0.112	0.306 ± 0.119
6 wk	0.754 ± 0.132	0.664 ± 0.156	0.672 ± 0.226
Torsional stiffness (N-m/deg 100)
2 wk	0.482 ± 0.202	0.437 ± 0.235	0.399 ± 0.263
3 wk	1.909 ± 0.639†	1.022 ± 0.620	0.899 ± 0.396
4 wk	2.601 ± 0.741†	1.442 ± 0.655	1.389 ± 0.601
6 wk	3.419 ± 0.841	3.232 ± 1.119	3.096 ± 1.224
Energy absorbed to failure (N-m deg)
2 wk	?2.08 ± 0.91	?1.56 ± 1.06	?1.44 ± 0.92
3 wk	?4.85 ± 2.23†	?2.46 ± 1.21	?2.82 ± 0.78
4 wk	10.00 ± 3.67†	?4.27 ± 1.72	?4.26 ± 1.88
6 wk	10.05 ± 2.31	?8.39 ± 2.26	?8.55 ± 3.08

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TABLE II: Effect of rhBMP-2 on Torsional Biomechanics*

	rhBMP-2	Buffer	Untreated
Failure torque (N-m)
2 wk	0.133 ± 0.050	0.112 ± 0.057	0.103 ± 0.057
3 wk	0.388 ± 0.140†	0.210 ± 0.103	0.202 ± 0.071
4 wk	0.633 ± 0.136†	0.317 ± 0.112	0.306 ± 0.119
6 wk	0.754 ± 0.132	0.664 ± 0.156	0.672 ± 0.226
Torsional stiffness (N-m/deg 100)
2 wk	0.482 ± 0.202	0.437 ± 0.235	0.399 ± 0.263
3 wk	1.909 ± 0.639†	1.022 ± 0.620	0.899 ± 0.396
4 wk	2.601 ± 0.741†	1.442 ± 0.655	1.389 ± 0.601
6 wk	3.419 ± 0.841	3.232 ± 1.119	3.096 ± 1.224
Energy absorbed to failure (N-m deg)
2 wk	?2.08 ± 0.91	?1.56 ± 1.06	?1.44 ± 0.92
3 wk	?4.85 ± 2.23†	?2.46 ± 1.21	?2.82 ± 0.78
4 wk	10.00 ± 3.67†	?4.27 ± 1.72	?4.26 ± 1.88
6 wk	10.05 ± 2.31	?8.39 ± 2.26	?8.55 ± 3.08

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TABLE III: Location of Fractures Following Torsion-Testing*

*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

Time-Point (wk)	Fracture Site	rhBMP-2	Buffer	Untreated
2	Osteotomy	12/12	12/12	12/12
	Osteotomy/host bone
	Host bone
3	Osteotomy	?9/10	12/12	?9/10
	Osteotomy/host bone	?1/10		?1/10
	Host bone
4	Osteotomy	?3/12	11/12	11/12
	Osteotomy/host bone	?2/12	?1/12	?1/12
	Host bone	?7/12
6	Osteotomy	?1/11	?1/11	?6/12
	Osteotomy/host bone	?3/11	?6/11	?2/12
	Host bone	?7/11	?4/11	?4/12
*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

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TABLE III: Location of Fractures Following Torsion-Testing*

*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.

Time-Point (wk)	Fracture Site	rhBMP-2	Buffer	Untreated
2	Osteotomy	12/12	12/12	12/12
	Osteotomy/host bone
	Host bone
3	Osteotomy	?9/10	12/12	?9/10
	Osteotomy/host bone	?1/10		?1/10
	Host bone
4	Osteotomy	?3/12	11/12	11/12
	Osteotomy/host bone	?2/12	?1/12	?1/12
	Host bone	?7/12
6	Osteotomy	?1/11	?1/11	?6/12
	Osteotomy/host bone	?3/11	?6/11	?2/12
	Host bone	?7/11	?4/11	?4/12
*The values are given as the number of ulnae that had a fracture at each site/the number of ulnae that were tested.