RECONSTRUCTION OF THE TEMPORAL BONE WITH BONE-MORPHOGENETIC PROTEIN 2 (BMP-2)

Akira Sugimoto, Natsumi Sakamoto, Kana Matsushima, Naoko Kikkawa, Heizaburo Yamamoto, Kouichi Kobayashi, Yoshitaka Okamoto

Otorhinolaryngology and Head & Neck Surgery, Chiba University Graduate School of Medicine, Chiba, Japan

Introduction

Until now, reconstruction of temporal bone defects after tympanoplasty or mastoidectomy has been performed using cartilage, fascia, bone chips, bone putty, and other reconstruction materials. However, each material has its own problems, and there is currently no established method for reconstructing temporal bone. Moreover, current reconstruction methods do not result in physiologically normal bone with remodeling abilities such as bone tissue ossification and absorption. Reconstruction with physiological bone tissue that is capable of bone remodeling would resolve these problems and be the ideal solution.

Bone tissue reconstruction using tissue-engineering techniques is currently receiving attention. The following substances have strong potential for bone formation:

Bone-morphogenetic protein (BMP)

Platelet-derived growth factor (PDGF)

β-tricalcium phosphate (β-TCP)

These bone-forming substances were used in our previous experiment. Of these substances, BMP has the highest ability to form new bone, which is why we used BMP in this experiment.

Experimental method

The test animal is a six-week-old male Hartley white guinea pig weighing 400 to 450 g with normal tympanic membranes. The carrier used was type-I atelocollagen. The animal was injected with BMP. From among the roughly 20 types of BMP, BMP-2, which has the strongest bone-forming power, was used. The BMP-2 concentration was 1.5 mg/cm3, which in previous studies has been shown to be the optimal concentration. Guinea pig was anesthetized with a pentobarbital injection into the abdominal cavity, and a diamond bur was used to drill a hole in the middle-ear otic capsule of the temporal bone on one side of the face. The carriers were impregnated with the injection drug and embedded, the incision was closed, and the animal was returned to its rearing place. The site where the incision was created was examined with CT scans after certain periods of time. The animal was given isoflurane inhalation anesthesia for CT scan.

CT study

A CT scan was made with a Micro CT for animals that allows scans to be taken of living animal and permits changes to be assessed over time in a single specimen. The LaTheta LCT-200 (Hitachi Aloka Medical, Ltd. Tokyo, Japan) used, enables sensitive imaging with a pixel size of 24 μm. Analysis by 3D software can be used to view any cross-section. Bone-mineral density can be measured directly, and bone-tissue composition can be analyzed.

Results

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Fig. 1. 3D-CT image of the animal two weeks after BMP injection. Post-incision bone-tissue regeneration identified on the surface of the treatment site.

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Fig. 2. 3D-CT image of control model (normal saline injection), two weeks after the procedure. There is no post-incision bone regeneration in the treatment site.

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Fig. 3.* CT images of the bone two weeks after the procedure (3D cross-section, axial image, bone composition analysis image). Observation of this part with an axial view shows that bone-mineral density only increases in the perimeter, and there is almost no increase in bone-mineral content in the center at this point in time. Bone-composition analysis was performed. Green spots show cortical bone and yellow spots show cancellous bone. Green cortical bone is seen in the bone-regeneration site, but no regeneration of bone tissue is seen in the center.

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Fig. 4.* CT images of the bone one month after the procedure (3D lateral view, 3D cross-section, axial image, bone composition analysis image). Further post-incision bone regeneration is seen in the treatment site. Axial views and bone-composition analysis show an increase in bone-mineral density in the center.

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Fig. 5*. Six months after the procedure. Bone mineral content has increased and the bone tissue is maturing. Pneumatization in the center of the bone-regeneration site in axial views and bone-composition analysis is starting.

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Fig. 6.* One year after the procedure. Mature-bone-tissue regeneration is identified. Axial views and bone composition analysis show further increase in pneumatization in the center of the bone regeneration site, indicating development of normal temporal bone.

Table 1. Increase of bone-mineral density in the bone regeneration site.

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Temporal changes in bone regeneration from BMP-2 treatment were observed, and the increase in bone-mineral density was measured by CT scan in the bone-regeneration site and displayed in a graph. The graph shows that bone-mineral density gradually increases with time. It appears to be approaching 1180 mg/cm3, which is the density in the untreated area on the other side.

Discussion

Tympanoplasty and mastoidectomy often result in bone destruction due to middle-ear lesions. Alternatively, incision into the bone during surgery often leaves defects in the mastoid cortical bone or ear canal posterior wall. The ossicles may become eroded, leading to a defect and hearing impairment. Until now, temporal bone defects have not received any type of treatment and have been left untreated. However, mastoid cortical bone defects can result in postauricular skin depression after surgery, leaving the site dysmorphic. In addition, many patients may suffer complications of cavity problems for a long time after surgery due to external exposure of the mastoid cavity. The cavity problems are a result of continued otorrhea or excessive earwax in the middle ear cavity that has become immunocompromised. Many such patients must continue receiving periodic treatment from an otolaryngologist for the rest of their lives following middle-ear surgery. Resolving these problems requires some method of reconstruction of bone defects in the posterior ear canal wall and the ossicular chain following cleaning after middle-ear surgery. While there have been reports of using cartilage and bone putty to reconstruct the posterior ear canal wall, both have collection limitations and cannot necessarily be used for all cases. Artificial materials (such as hydroxyapatite) are often used in ossicular-chain reconstruction, but these often cause a foreign-body reaction, and the ossicular prosthesis is naturally expelled. When cartilage is used, sound conduction has been reported to be inefficient. There is therefore a need to develop a new method for reconstructing temporal bone.

In 1965, Urist1 reported ectopic bone induction in rats by subcutaneous and intramuscular decalcified bone matrix grafting. He suggested that a bone-forming protein factor existed within the bone matrix, and he called it bone morphogenetic protein (BMP). After this discovery, numerous researchers attempted to purify BMP, but it remained unidentified for a long time. In 1988, Wozney et al.2 of the Genetics Institute successfully cloned the DNA of four types of human BMP. The structure of BMP was revealed for the first time, and it can now be artificially produced. About 20 proteins belonging to the BMP family have been found to work not only on bone tissue, but are also involved in morphogenesis of various organs, differentiation, apopto-sis, and tissue regeneration. In addition to bone and cartilage tissue, BMPs work on a wide range of organs including nerves, the cardiovascular system, the kidneys, and the digestive tract. Today, BMP is ranked as a cytokine that functions as a signaling protein for morphogenesis, and it has been confirmed to be a protein of the TGF-β superfamily. BMP-2 has the strongest bone-forming power among proteins in the BMP family, and, when placed in the body with a carrier that locally retains BMP-2 and releases it at an appropriate rate, mesenchymal stem cells are stimulated to differentiate into osteoblasts, and the increasing number of osteoblasts results in rapid formation of bone tissue. Normal bone capable of bone remodeling is then regenerated. Past studies on bone regeneration using BMP-2 have been reported, including a study by Gerhart et al.3 on bone formation of the sheep femur, a study by Mushuler et al.4 on dog spinal fusion surgery, and a study by Seto et al.5 on mandible bone defect repair in monkeys. It is also being clinically used for some spinal diseases in Western countries.

We therefore thought that it may be possible to easily regenerate the ossicles, reconstruct the ossicular chain, and reconstruct mastoid cortical bone defects using BMP-2. Temporal bone is morphologically classified as pneumatic bone because it has an internal air sinus and a special structure with many hollow cavities and cellulae. Since the interior surface of the middle-ear cavity does not come into contact with skin or muscle tissues, undifferentiated mesenchymal cells cannot migrate easily, and bone formation by BMP-2 action is thought to be unfavorable. There are hardly any reports on the question of BMP efficacy in the temporal bone, and this question remained unanswered. We previously reported measurement by soft X-rays after euthanizing the animal, but this method resulted in measurement errors between experiments and subjects. Using a micro-CT device for animals in the present study, inter-experimental and inter-subject errors were removed. This is the first report to describe the use of BMP-2 on the temporal bone, a pneumatic space with an internal sensory organ, and the ability of BMP-2 to promote remodeling of an incision made in the temporal bone so that it once again became a pneumatic space. These results demonstrate the potential usefulness of BMP-2 in otologic surgery.

Conclusions

This study demonstrated that micro-CT devices for animals are very effective for observing bone regeneration.

BMP-2 was shown to have a strong ability to reconstruct bone tissue in temporal bone, and construction of a pneumatic space was confirmed for the first time. These results suggest the potential for clinical use of BMP-2 in otologic surgery.

References

1.Urist MR. Bone formation by autoinduction. Science 150: 893–899, 1965

2.Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534, 1988

3.Gerhart TN, Kirker-Head CA, Kriz MJ, Holtrop ME, Hennig GE, Hipp J, et al. Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin Orthop 293:317–326, 1993

4.Muschler GF, Hyodo A, Manning T, Kambic H, Easley K. Evaluation of human bone morphogenetic protein 2 in a canine spinal fusion model. Clin Orthop 308:229–240, 1994

5.Seto I, Asahina I, Oda M, Enomoto S. Reconstruction of primate mandible by the combination graft of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bone marrow. J Oral Maxillofacial Surg 59:53–61, 2001

Address for correspondence: Akira Sugimoto, Otorhinolaryngology and Head & Neck Surgery Chiba University Graduate School of Medicine, 1–8-1 Inohana Chuo-ku, Chiba, Japan. sugimoto@faculty.chiba-u.jp

Cholesteatoma and Ear Surgery - An Update, pp. 303–308

Edited by Haruo Takahashi

2013 © Kugler Publications, Amsterdam, The Netherlands

*Figures 3 to 6: Bone formation gradually progresses and bone tissue continues to mature. Moreover, composition analysis of the bone tissue shows formation of a pneumatic space in the inner cavity, and an almost normal pneumatic space has been formed in the site one year after the procedure.