Otology & Neurotology 26:563566 2005, Otology & Neurotology, Inc.

Tympanic Membrane Perforation Repair with Acellular Porcine Submucosa

Jeffrey H. Spiegel and Joshua L. Kessler
Department of OtolaryngologyHead and Neck Surgery, Boston University School of Medicine, Boston, Massachusetts, U.S.A.

Objectives: To evaluate the efcacy of acellular porcine small intestine submucosa in the repair of chronic tympanic membrane perforations. Although tympanoplasty with autologous temporalis fascia and cartilage is common practice in the repair of chronic tympanic membrane perforations, these materials are associated with increased operative time and have variable availability and quality in individual patients. Recently, new materials for tympanoplasty have been explored, including acellular human dermis. Small intestine submucosa (Surgisis) is an inexpensive and readily available alternative to autologous and cadaveric grafts. In this study, we examined the use of small intestine submucosa in the repair of chronic tympanic membrane perforations in a chinchilla model. Study Design: Prospective pilot study using 10 adult chinchillas. Methods: Chronic tympanic membrane perforations were created in 10 adult chinchillas for a total of 20 perforations.

Each animal underwent observation in one ear and repair with either autologous cartilage or small intestine submucosa in the opposite ear with Type I tympanoplasty. Results: A total of 20 chronic membrane perforations were created, with zero healing spontaneously after 8 weeks. In tympanoplasties performed in ve chinchillas with small intestine submucosa, ve of ve (100%) remained healed 6 weeks postoperatively, whereas three of ve (60%) remained healed with cartilage repair. Histologic analysis was performed in both successful cartilage and small intestine submucosa repairs. Conclusion: These results suggest that small intestine submucosa is a viable alternative to autologous and cadaveric grafts in tympanoplasty. A larger randomized study in humans is indicated to evaluate this material in clinical practice. Key Words: ChinchillaSurgisisTympanoplastyXenograft. Otol Neurotol 26:563566, 2005.

Chronic tympanic membrane (TM) perforation is a common clinical entity in the otolaryngologists practice. It may lead to recurrent or chronic infection and conductive hearing loss. Perforations may arise from trauma, chronic or acute middle ear infections, or placement of ventilation tubes. Most acute TM perforations heal spontaneously; however, a chronic perforation may ensue as a result of failure of epithelial growth across the perforation. When a perforation persists, surgical repair in the form of myringoplasty or tympanoplasty is often indicated. Many materials have been used in the history of TM repair, including full-thickness or partial-thickness skin grafts, vein grafts, sclera, perichondrium, temporalis fascia, cartilage inlay, fat, and various papers including cigarette and rice paper (1,2). Recently, acellular human
Address correspondence and reprint requests to Jeffrey H. Spiegel, M.D., F.A.C.S., Department of OtolaryngologyHead and Neck Surgery, Boston University School of Medicine, 88 East Newton Street, D-6, Boston, MA, U.S.A.; E-mail: jeffrey.spiegel@bmc.org An unrestricted educational and research grant was received from Cook, Inc.

dermis (AlloDerm; LifeCell, Inc., The Woodlands, TX, U.S.A.) has shown promise in the repair of chronic TM perforations in both animal studies and human case reports (36). Acellular porcine small intestine submucosa (SIS) is an acellular biomaterial that functions as scaffolding for tissue repair and remodeling. SIS (Surgisis; Cook, Inc., Bloomington, IN, U.S.A.) has been used as a vascular graft; for skin graft donor sites; to cover and assist healing in complex wounds; and for the repair of defects in the bladder, dura, and abdominal wall (79). In all cases, the material has proven to be well tolerated and a useful product to effect successful soft-tissue coverage or bolstering. SIS may offer several advantages over both autografts and human acellular dermis in the repair of tympanic membrane perforations. Temporalis fascia is presently the most commonly used autograft in tympanoplasty because it may be harvested from the same surgical site and has a success rate of 88 to 95% (2). The disadvantages of autografting include donor-site morbidity, increased intraoperative time, the microsurgical skills of the surgeon, and the variability of the quality of autograft 563

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fashion. In the cartilage repair group, a small incision was created in the medial portion of the auricle, and mucoperichondrial aps raised on both sides of the cartilage, which was then excised. The cartilage was then placed medial to the drum in the same fashion as the SIS repairs. The skin was closed with a single 5-0 plain gut suture. Cartilage was selected for comparison because of the ability to harvest it locally and without the increased pain of a leg incision as would be necessary to obtain good fascia. In addition, cartilage is a more durable and substantial material that has proven to be effective in tympanoplasty. Although not expected to repair a TM with the mobility of a fascia graft repair, cartilage is expected to have a high success rate, as it is less likely to have the edges roll up or perforate. In all repairs, no gelatin sponge or other material was placed in the external auditory canal. Animals were checked daily for outward signs of infection.

material among patients. For revision cases, temporalis fascia may not be available and a second incision must be made for harvesting. The potential advantages of SIS over autografting include its ready availability, ease of use, decreased intraoperative time, and uniform thickness. Acellular human dermis has been studied recently in tympanoplasty and appears to be effective (36). The use of cadaver tissue is limited by the shortage of suitable human donors (i.e., those with a low risk of transmission of infection). Xenografting offers a reasonable alternative to human cadaver harvesting in that there is a plethora of available tissue that is unlikely to pose an infectious risk. As a result, xenograft materials tend to be much less expensive and more readily available than cadaveric tissue while posing less of a risk of transmission of an infectious agent. The utility of xenografting has been proven time and time again, with the most obvious example in the use of porcine xenografts for cardiac valve replacements. Although there has been anecdotal evidence regarding the use of SIS in ofce myringoplasty, to our knowledge there have been no animal studies exploring the histology and efcacy of the material. In light of these factors, we conducted an animal-based pilot study to explore the efcacy of SIS in the repair of chronic tympanic membrane perforations. MATERIALS AND METHODS
Ten adult chinchillas (Moulton Chinchilla Ranch, Rochester, MN, U.S.A.) were included in the study, each weighing from 350 to 500 g.

Evaluation of Repair
After 6 weeks, each animal was again anesthetized with i.p. xylazine and ketamine and each ear was examined under the operating microscope. Each ear was recorded as healed or not healed. Representative drums recorded as healed were harvested via a postauricular incision and dissection with an otologic drill and xed in formalin for histologic analysis after the animal was killed with 1 ml of 26% sodium pentobarbital injected i.p. These specimens were later mounted in parafn, stained with hematoxylin and eosin, and examined by a staff pathologist. Six of 10 animals were adopted out to private homes.

RESULTS Evaluation of Creating and Repairing Chronic Perforations Of the 20 ears in which chronic perforations were created, all (100%) remained perforated after 8 weeks, with no evidence of infection or signicant healing. Of those 10 that were left unrepaired as controls, 100% remained perforated after the additional 6-week course it took to complete the study. All ve of ve perforations (100%) repaired with SIS were completely healed 6 weeks after initial repair, whereas three of ve perforations (60%) repaired with cartilage were completely healed. Of those two perforations that persisted in the cartilage repair group, both had large perforations with little evidence of healing. This was a pilot study to assess the ability of SIS to be used for TM repair and so qualitative observations were desired and expected; as a result, in this trial, statistical calculations are not used. Gross and Histologic Analysis On gross inspection of both groups of successfully repaired drums, there was complete closure of the perforation with neovascularization. On the drums repaired with SIS, there appeared to be a translucent appearance to the repaired surface, with evidence of mild calcication (Fig. 1). In contrast, those drums repaired with cartilage demonstrated complete opacication in the area where the graft was placed. This likely represented the persistence of cartilage medial to the repaired drum. In those areas not repaired, the drum appeared normal in all specimens.

Creation of Perforations
Each animal was anesthetized with a solution of 30 mg/kg ketamine and 5 mg/kg xylazine injected intraperitoneally (i.p.). Under microscopic vision, a 3-mm otologic speculum was used to expose the TM. A myringotomy knife was used to create a perforation in the anterior half of the TM and a right-angled hook was used to fold the free edges of the drum medially. This is a modication of the method originally described by Amoils et al. (11) to create chronic TM perforations in the chinchilla. Each animal was examined daily for outward signs of infection; however, none required topical or enteral antibiotics. After 2 weeks and then every 3 weeks for a total of 8 weeks, each animal was examined under anesthesia for signs of healing; however, no perforation healed spontaneously.

Tympanoplasties
All 10 animals were selected randomly to undergo tympanoplasty with autologous cartilage or with SIS. Once divided into the two arms, tympanoplasties were randomly conducted in the left or right ear. The opposite ear was left unrepaired as a control. Each animal was anesthetized with ketamine and xylazine as above. Under the operating microscope, the edges of the perforation were roughened with a Rosen needle and the middle ear space packed with absorbable gelatin squares (Gelfoam; The Upjohn Co., Kalamazoo, MI, U.S.A.) up to the rim of the perforation. In those animals that underwent repair with SIS, a small rectangular piece of SIS (approximately 0.1 mm thick) was placed medial to the drum and over the gelatin sponge in a punch-through underlay
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FIG. 1. In vivo telescopic photograph of a healed TM repair with porcine small intestine submucosa demonstrating a translucent repaired site with surrounding calcication and minimal scarring.

FIG. 3. SIS-grafted TM photomicrograph demonstrating trilaminar TM with incorporated graft material (lled arrowhead marks the junction of normal and grafted TM) and moderate inammation. Open arrowhead shows out-pouching of drum from histologic processing (hematoxylin and eosin stain; original magnication, 3100).

Histologic analysis was conducted on representative drums repaired with both cartilage and SIS (Figs. 2 and 3). The drum repaired with cartilage revealed a monomeric layer composed of squamous epithelium and the cartilage graft situated medially to the repaired drum. There were small areas of repair where a bilaminar repair was present consisting of epithelium and mucosa, both lateral to the cartilage. There was evidence of mild inammation and areas of calcication within the cartilage. SIS-repaired drum sections demonstrated a trilaminar membrane composed of squamous epithelium, graft material within the drum in place of a brous layer, and a medial cuboidal mucosa. Compared with the cartilage graft, there was more inammation present.

DISCUSSION Multiple studies have demonstrated the results of various materials in the repair of tympanic membrane perforations (16,10). The normal anatomy of the human and chinchilla TM consists of a thin lateral squamous epithelium, a middle brous layer, and a medial cuboidal mucosal layer. In a chinchilla model of chronic tympanic membrane perforations, Amoils et al. (11) explored the histologic results of creating perforations in the tympanic membrane. The drums that maintained a chronic perforation with no evidence of healing displayed marginal strands of dense connective tissue covered with a thin layer of squamous epithelium, and at the edge of the perforation the squamous layer extended medially to contact the medial mucosal surface. Both the squamous layer and the brous layer demonstrated hyperplasia and hypertrophy. In those drums that demonstrated spontaneous healing, a thin layer of squamous epithelium covering the original site of the perforation was present. A recent study comparing rice paper to acellular human dermis (AlloDerm) in the repair of chronic perforations in the chinchilla demonstrated the formation of a bilaminar membrane in perforations repaired with rice paper and a trilaminar membrane in the AlloDerm repair group (5). The trilaminar repair was felt to signify a more desirable result. This study yielded a successful repair in 78% of drums repaired with AlloDerm using a medial graft technique and 66% repaired with rice paper in an onlay myringoplasty technique. Another signicant nding revealed a much larger average thickness of drums repaired with AlloDerm compared with normal drum thickness and those repaired with rice paper. The clinical signicance of this nding is unknown. Our study explored the use of acellular porcine small intestine submucosa (Surgisis) in the repair of chronic
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FIG. 2. Cartilage-grafted TM photomicrograph showing cartilage medial to a monomeric squamous epithelium (arrowhead). Note the adjacent normal trilaminar TM (hematoxylin and eosin stain; original magnication, 3100).

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J. H. SPIEGEL AND J. L. KESSLER CONCLUSIONS The results of this pilot study suggest that SIS is effective in the repair of chronic TM perforations. We chose SIS as a material to explore in tympanoplasty because it is characterized by many of the qualities sought in grafting materials: it is readily available, inexpensive, and easy to work with; it decreases the time spent harvesting a native graft and requires no second incision in transcanal procedures; it is not antigenic; and it carries no risk of transmission of human disease. These benets suggest that SIS is an excellent choice for graft material in myringoplasty and tympanoplasty.
Acknowledgment: The authors would like to thank Dr. Charles Allam for help with the preparation and interpretation of the histologic slides.

membrane perforations compared with controls of no repair and also with the use of endogenous cartilage using the same technique of punch-through transcanal tympanoplasty with the graft placed medial to the drum. We found an excellent repair rate compared with controls. Our results were evaluated qualitatively rather than statistically in this pilot study to avoid subjecting the large number of animals necessary for statistical power to surgery until the method was tested in a smaller population. Nonetheless, the results strongly suggest that SIS is as effective if not superior to cartilage in the repair of chronic TM perforations. In all TMs repaired with SIS, an excellent result was obtained. Given that none of our nonrepaired controls healed spontaneously, it is also suggested that SIS is a viable candidate for use in tympanoplasty. On the basis of power studies before the beginning of our study, we would have required the use of over 100 animals to prove that SIS is, in fact, statistically equal or superior to cartilage grafting. The histologic analysis coincides with that of other studies. We found in repair with SIS the creation of a trilaminar membrane with the grafting material imbedded in the brous layer of the drum. This was located between a lateral layer of squamous epithelium and a medial layer of cuboidal mucosa. Interestingly, the cartilage repairs revealed a predominantly monolaminar membrane, with the cartilage evident medial to a squamous layer. The middle brous layer was absent and there were areas where mucosa was present in a bilaminar membrane lateral to the cartilage. This suggests that although the cartilage served as a conduit for the migration of the healing drum, the SIS was actually incorporated into the drum itself. It has been suggested that the formation of a trilaminar repair is superior to that of a dimeric or monomeric repair, in that it represents a more durable drum and more closely resembles the native drum. Although there was evidence of inammation present in both types of repair, there was no evidence of rejection of either the cartilage or the SIS. This was an expected nding, as the cartilage was an autograft and the SIS had been stripped of its antigenic properties during its preparation.

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