American Journal of Otolaryngology - Head and Neck Medicine and Surgery
Volume 24, Issue 1 , Pages 14-18, January 2003

Mastoid obliteration by BMP-2/collagen composites: An experimental study using tissue engineering☆

From the Departments of *Otolaryngology–Head and Neck Surgery, †Oral Pathology and Medicine, and ‡Pathological Research, Okayama University Graduate Schools, Okayama, Japan

Article Outline

Abstract 

Purpose: Several materials have been used in the application of mastoid cavity obliteration during surgery for cholesteatoma; however, nothing has won universal acceptance. Through the advancement of tissue engineering, bone morphogenetic protein–2 (BMP-2)/collagen composites have been elucidated as inducers of heterogenic bone formation. This study was performed to investigate whether these composites are potentially obliteration materials for use in the mastoid cavity by using an animal experimental study. Materials and Methods: The composites were implanted in the rat mastoid to investigate whether new bone would be tissue engineered in the mastoid and, if so, whether the newly formed bone was stable. The composites were examined histologically over a 24-week period. Results: The composites implanted in the rat mastoid were able to tissue engineer new bone, and the newly formed bone was stable as assessed histologically, with almost normal bone structure, that was not resorbed during the 24-week period. Adverse immunological reactions were not found during our observation. Conclusions: Bone that was tissue engineered by the BMP-2/collagen composites was stable as assessed by histological examination and persisted in the rat mastoid. The present study shows that the composites have the potential to become real materials for use in mastoid obliteration. (Am J Otolaryngol 2003;24:14-18. Copyright 2003, Elsevier Science (USA). All rights reserved.)

 

The prevention of recurrent cholesteatoma through surgical intervention has been a controversial issue among otologists. The canal down method is certainly advantageous in this point, but the mastoid problem remains to be solved. As one of the answers to this problem, mastoid obliteration has often been performed. Historically, many obliteration materials have been adopted and abandoned because of unacceptable results such as necrosis and infection.1 At present, soft-tissue flaps,2 bone plate,3 cartilage,4 and biocompatible materials5, 6 continue to be used clinically for this purpose. However, each of them has disadvantages and thus competes for first place.7 Soft-tissue flaps, like muscular flaps, atrophy with age, and a recavity is often formed. Cartilage is a material of limited supply. Biocompatible materials like hydroxyapatite sometimes cause a foreign body reaction to the surrounding tissue and subsequently extrude. Bone plate may be the current preferred material but may lack enough strength to prevent recurrent cholesteatoma because of its fragility and resorption.8 Bone plate also has a disadvantage of limited supply in cases previously operated on using the canal down technique. Thus, the real winner has not been decided yet.

Recent advancements in tissue engineering provide a new concept to cope with diseases, and their clinical application is just about to be considered. BMP and insoluble bone matrix (mainly consisting of collagen) composites are reported to induce heterotropic bone formation.9 We used bovine collagen as a carrier of BMP-2 instead of insoluble bone matrix because of its easy availability. BMP-2/bovine collagen composites were then embedded in the rat mastoid to investigate whether these composites are appropriate as obliteration material for the mastoid cavity or not.

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Materials and methods 

Thirty-six four-week-old healthy male Wister rats were used in this experiment. Animals used in the present study were housed, supervised, and handled according to the Okayama University Medical School Guidelines for the Care and Use of Laboratory Animals. The animals were randomly divided into an experimental group and a control group. Under general anesthesia with diethyl ether, the temporal bone of each animal was unilaterally exposed via postauricular incision, and recombinant human BMP-2 (rhBMP-2)/bovine collagen composites (the experimental group) or bovine collagen only (the control group), was implanted there with a pellet of 2 × 2 × 2 mm. rhBMP-2 was kindly provided by Yamanouchi Pharmaceutical Co, Ltd (Tokyo, Japan). As the carrier of rhBMP-2, bovine collagen (Atelocollagen, Koken Co Ltd, Tokyo, Japan) was used. One microgram of rhBMP-2 was added to 10 mg of bovine collagen.

Histological analysis 

At 1, 2, 4, 8, 12, and 24 weeks after surgery, animals (n = 4 at each week from the experimental group, n = 2 from the control group) were decapitated under overdose of diethyl ether, and the ear was removed and fixed with formalin. The specimens were decalcified in ethylenediaminetetraacetic acid for 2 weeks, dehydrated with a graded alcohol series, and embedded in paraffin. Consecutive 6-μm thick sections were cut with a horizontal plane.

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Results 

Experimental group 

Histological analysis 

From 1 to 2 weeks, lymphocytes, histiocytes, and fibroblasts infiltrated the tissue around the implanted collagen (Fig 1).

As the implanted collagen was being absorbed, these cells disappeared (Fig 2). Angiogenesis occurred and woven bone was formed by endochondral and membranous osteogenesis (Fig. 1, Fig. 2). Chondrocytes were seen in the woven bone, whereas osteoblasts and osteoclasts were seen around the woven bone. Only at 1 week was endochondral osteogenesis seen, whereas membranous osteogenesis was seen during the whole 24-week period. At 4 weeks, osteocytes were seen in the newly formed bone, and bone marrow with adipose tissue and hematopoetic cells were formed in the bony trabeculae (Fig 3). Newly formed bone matured during this period and had a laminar pattern similar to the normal bone by 4 weeks (Fig 4). At 24 weeks, the proportion of adipose tissue in the bone marrow was increasing (Fig 5).
  • View full-size image.
  • Fig. 5. 

    Twenty-four weeks after implantation. A row of osteoclasts (arrows) in the bone marrow is still surrounding the newly formed bone. Osteocytes (arrowheads) are shown.

Control group 

New bone formation was not observed histologically. Only early histological (at 1-4 weeks) examination revealed a portion of the collagen had implanted.

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Discussion 

BMP is one of the most remarkable growth factors in orthopedics10 and dentistry.11 The presence of BMP in decalcified bone matrix was predicted in 1965,12 and the clones of 3 gene subtypes of BMP were determined in 1988.13 Now BMP families (subtypes 1-15) are known to be involved not only in bone morphogenesis and reorganization, as in repair of bone fractures,14, 15 but also in development.16, 17 BMP-2 is one of the most capable BMP members in inducing cartilage and bone.

Like other growth factors, BMP needs a carrier to be released continuously over a long enough period to induce heterotropic bone formation.18, 19 In this experiment, collagen was used as a carrier. rhBMP-2 delivered gradually from collagen is thought to interact with neighboring mesenchymal stem cells and induce them to differentiate into chondroblasts or osteoblasts and subsequently form new tissue-engineered bone in the mastoid. Operative manipulations reached the bone marrow of the temporal bone and easily dislodged mesenchymal stem cells from the bone marrow, releasing them into the microenvironment in which the rhBMP-2/bovine collagen composites were implanted.

In mastoid obliteration, a cavity surgically created in the mastoid should be tightly filled with obliteration materials. For this purpose, plastic materials or small particles like biocompatible materials are suitable. In the present experiment, the contour of the newly formed bone was similar to the contour of collagen used. Collagen is easily processed in a desirable form so that it can fit the variable osseous contour of a surgically created mastoid cavity. This shows the possibility that the rhBMP-2/bovine collagen composites can be used in other applications like tissue-engineered ossicles, as well as for mastoid obliteration materials.

Materials used for obliteration of the mastoid cavity should be neither resorbable nor lead to atrophy. rhBMP and collagen composites do not work as permanent materials,20 differing from other obliteration materials. Although the composites themselves are resorbed, new bone that is tissue engineered by the composites obliterates the mastoid. In the rat, the mastoid is underdeveloped, and instead the tympanic bulla is developed. It may not be appropriate to use rats to study the recurrence of cholesteatoma. However, histological observation revealed that tissue-engineered bone in the rat mastoid was stable as determined by histological examination for a long time and not resorbed. Therefore, this newly formed bone is also thought to have enough strength to prevent recurrent cholesteatoma.

Materials clinically used for obliteration should not induce adverse immunological tissue reactions. rhBMP-2 is made in accordance with the human amino acids sequence by genetic engineering. Therefore, rhBMP-2 seldom causes adverse immunological reactions. On the contrary, collagen is a xenogenic protein made from bovine bone. Clinical applications of bovine collagen for tissue defects such as a paralyzed vocal cord, rarely lead to delayed allergic reactions or inflammation.21 In the experimental group, bovine collagen was absorbed rapidly and replaced by connective tissue and subsequent bone, whereas in the control group it was present longer. However, no adverse immunological reactions were found during our observation period in either group.

As mentioned previously, rhBMP-2/bovine collagen composites are thought to be excellent materials for mastoid obliteration. However, problems remain to be solved before clinical use. The most important consideration would be adverse immunological reactions. Surely, they were not found in the rat during our observation, but it may happen in humans. A large amount of human collagen is easily created using animals transfected with the genes coding for human collagen. The practical use of human collagen in genetic engineering has advantages not only in immunological tolerance but also in escaping troubles associated with bovine spongiform encephalopathy. Although further preclinical and clinical works should be performed to establish the safety of rhBMP-2/human collagen composites before they are adopted for widespread clinical use, in the near future this combination may qualify as the preferred obliteration material for the mastoid cavity.

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Conclusions 

In the canal down technique of cholesteatoma surgery, the obliteration method has been used worldwide to solve mastoid problems. Several materials have been adopted as obliteration materials; however, the winner is undecided. The recent advancement of tissue engineering provides new clues to diseases. In this study, we investigated whether rhBMP-2/collagen composites are potentially useful obliteration materials or not. New bone tissue engineered in the rat mastoid by these composites was persistently stable as assessed by histological examination. rhBMP-2/collagen composites are potentially an ideal implant for mastoid obliteration.

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References 

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☆ Address correspondence to: Kazunori Nishizaki, MD, Department of Otolaryngology–Head and Neck Surgery, Okayama University Graduate School, 700-8558 Japan. E-mail: nishizak@cc.okayama-u.ac.jp.

PII: S0196-0709(02)32415-3

doi:10.1053/ajot.2003.13

American Journal of Otolaryngology - Head and Neck Medicine and Surgery
Volume 24, Issue 1 , Pages 14-18, January 2003

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