DOI: 10.1177/0003489414523710 Annals of Otology, Rhinology & Laryngology 123(2):135-140. © The Author(s) 2014
Ovine Model for Auricular Reconstruction: Porous Polyethylene Implants Marc H. Hohman, MD; Robin W. Lindsay, MD; Irina Pomerantseva, MD, PhD; David A. Bichara, MD; Xing Zhao, MD; Matthew Johnson; Katherine M. Kulig; Cathryn A. Sundback, ScD; Mark A. Randolph, MAS; Joseph P. Vacanti, MD; Mack L. Cheney, MD; Theresa A. Hadlock, MD Objectives: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model. Methods: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infraauricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented. Results: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks. Conclusions: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs. Key Words: microtia, porous polyethylene implant, sheep.
disadvantages. Costal cartilage autografting requires the greatest degree of skill. Costal cartilage is harvested and carved into several pieces that are assembled into a complex, 3-dimensional ear framework and typically implanted subcutaneously; the overlying soft tissue can stress the implanted framework and distort the structural elements. Auricular reconstruction with a carved cartilage framework requires 2 to 4 operations over the course of several months to a year. Multiple donor sites are common, for example, the chest wall for cartilage, the groin or the scalp for skin grafts, and the contralateral ear for full-thickness skin grafts and/or composite skin-cartilage grafts. Often, additional revision procedures are necessary, including hair removal from
Introduction
Complete auricular reconstruction remains challenging for today’s surgeon. The most common indication for reconstruction is congenital microtia, with an incidence of approximately 2 per 10,000 live births in the United States; traumatic avulsions and burns also require similar reparative procedures.1 Current alternatives for complete auricular reconstruction include carved costal cartilage autografts, synthetic high-density porous polyethylene (HDPPE) implants, and external auricular prostheses anchored to osseo-integrated implants. All approaches provide satisfactory to excellent results in experienced hands, but each method has
From Massachusetts Eye and Ear Infirmary (Hohman, Lindsay, Cheney, Hadlock); Harvard Medical School (Hohman, Lindsay, Pomerantseva, Bichara, Zhao, Sundback, Randolph, Vacanti, Cheney, Hadlock); and the Department of Surgery, Division of Pediatric Surgery (Pomerantseva, Kulig, Sundback, Vacanti), and the Plastic Surgery Research Laboratories (Bichara, Zhao, Johnson, Randolph), Massachusetts General Hospital; Boston, Massachusetts; and the Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands (Zhao). This research was sponsored by the Armed Forces Institute of Regenerative Medicine award number W81XWH-08-2-0034. The US Army Medical Research Acquisition Activity, Fort Detrick, Maryland, is the awarding and administering acquisition office. Dr Hohman’s effort was also supported by the US Army Medical Command. The content of the manuscript does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital. Correspondence: Joseph P. Vacanti, MD, Dept of Pediatric Surgery, 55 Fruit St, Warren 1151, Boston, MA 02114.
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A
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B
Fig 1. Gross images demonstrate good aesthetic outcome of high-density porous polyethylene (HDPPE) auricle-shaped frameworks implanted in ovine infraauricular area. Images were obtained from same animal A) immediately after implantation and B) at explantation 17 weeks later.
the reconstructed pinna.2 Delaying autogenous cartilage grafting until the patient is at least 10 years of age avoids chest wall deformation related to costal cartilage harvest and increases the likelihood that a sufficient amount of cartilage will be available for harvest. In contrast, patients undergoing HDPPE implantation are typically 3 to 5 years of age.2,3 The HDPPE framework is usually implanted under a temporoparietal fascia flap (TPFF) based on the superficial temporal vessels. Reconstruction with a HDPPE framework can be completed in 1 or 2 stages, but the rate of infection and extrusion over a patient’s lifetime is substantially higher than that reported for autologous costal cartilage implants.1,4-6 However, the aesthetic results with these implants are more consistent for less experienced surgeons.1 Lastly, auricular prostheses can have a natural appearance and require the least complicated surgical procedure, but these prostheses wear out, change color with age, and must be periodically replaced. In many areas of the world, use of different springsummer and autumn-winter prostheses is necessary to match seasonal changes in patients’ skin pigmentation. A prosthesis held in place by magnets may be dislodged during physical activities and must be removed during sleep. Additionally, whereas a patient frequently considers a reconstructed auricle to be a part of his or her own body, the same is seldom felt about a prosthetic auricle.7 A cartilage framework engineered from a patient’s own chondrocytes has the potential to combine the best characteristics of the existing reconstructive approaches while minimizing the disadvantages.8 An engineered cartilage framework would decrease the operative time and would eliminate inconsistencies related to carving costal cartilage. The avoidance of harvesting large costochondral grafts from the chest
wall would decrease postoperative pain, speed recovery, and eliminate the risk of pneumothorax. The finished engineered product would possess the advantages of both costal cartilage and synthetic implant approaches, providing a custom-sized auricle with defined 3-dimensional contours. This engineered product would have a low risk of infection or extrusion and would ideally mimic the mechanical properties of auricular cartilage. Although neocartilage does tend to be less resilient than native cartilage, addition of an internal coiled titanium wire scaffold can greatly improve resistance to deformation.9 Our team and others have made significant progress in engineering cartilage in the shape of the human ear in laboratory animals.8,9 An essential step toward bringing the technique of cartilage engineering into clinical practice is the development of a large animal model. We selected a sheep model on the basis of the similarity of its facial vascular anatomy to that of humans — an aspect vital to translating the results of large animal studies to humans. As the development of ovine engineered auricular cartilage is under way, controls are needed to compare ovine-based results with conventional reconstructive methods. We established a surgical model based on HDPPE auricular implants in sheep; these implants were subcutaneously implanted without the use of a vascularized flap to mimic the expected reconstruction approach for engineered cartilage frameworks. Methods
All protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. Nine Polypay sheep of both sexes, 3.4 ± 0.7 months of age and weighing 30.2 ±
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A
B
Fig 2. Small nonhealing exposure of antihelix of HDPPE framework occurred during first 2 weeks after operation in 1 animal. Wound remained stable in size A) at 8 weeks and B) at 17 weeks.
3.9 kg, each received bilateral implants in the infraauricular area. Before the induction of general anesthesia, the animals were premedicated with an intramuscular injection of a combination of xylazine hydrochloride 0.4 mg/kg and Telazol 2 mg/kg (tiletamine hydrochloride–zolazepam hydrochloride; Fort Dodge Animal Health, Fort Dodge, Iowa). Anesthesia was A
then induced with 4% to 4.5% isoflurane and maintained at 2% to 3% via endotracheal tube throughout the surgical procedure. Buprenorphine hydrochloride 0.01 mg/kg subcutaneous injections and cefazolin sodium 40 mg/kg intramuscular injections were administered for pain and infection prophylaxis, respectively. The skin on the side of the neck was shaved, prepared with alcohol and povidone-iodine solution, and draped in sterile fashion. B
Fig 3. Significant nonhealing exposure of antihelix of HDPPE framework occurred in 2 separate animals. Both exposures occurred during first 2 weeks after operation and progressed, and implants were removed at 7 weeks. A) Appearance at 2 weeks. B) Appearance at 7 weeks.
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A
Fig 4. Representative histologic section through HDPPE implant harvested at 17 weeks (hematoxylin-eosin stain). A) Fibrovascular growth into pores occurred throughout implant. Scale bar — 1 mm. B) Fibroblasts and small blood vessels were observed within pores. Scale bar — 200 μm. C) Occasional inflammatory cellular infiltrate was also discerned within pores, and giant cells lined pores (arrows). Scale bar — 200 μm.
B
C
A 4-cm skin incision was made from the base of the auricle to the caudal border of the parotid gland, and after careful hemostasis, a subdermal pocket was bluntly dissected. A 2-piece Medpor ear implant (helical rim 37 × 62 mm and ear base 55 × 28 × 13 mm; Porex Surgical, Inc, Newnan, Georgia) was assembled and placed inside the pocket. Two continuous closed suction drains (TLS Max-Glide Facial Drainage kit, Porex Surgical, Inc) were placed between the implant and the skin and left in place for 48 hours in all 9 animals. The skin was closed with 3-0 polyglactin sutures. After the operation, the animals were monitored for signs of discomfort or complications twice daily for 3 days. The wounds were then monitored 3 times per week for infection, dehiscence, and implant exposure. Pain relief was provided with flunixin meg lumine 1 mg/kg intramuscular injections daily for 3 days after operation and then administered as needed. The animals were painlessly euthanized 17 weeks after implantation. The implants were then removed
along with a cuff of surrounding tissue. Gross photographs were taken; the implants were fixed in 10% buffered formalin and embedded in paraffin. The embedded tissue was cut into 5-μm sections and stained with hematoxylin-eosin and Masson’s trichrome (American MasterTech Scientific, Inc, Lodi, California) for microscopic analysis. Results
There were no perioperative anesthetic complications or deaths. Overall, the implants retained an easily recognizable auricular appearance throughout the study (Fig 1). All implants were firmly fixed to the surrounding tissue. Several implants were associated with transient alopecia. There were 8 instances of surgical complications in the 18 implants. In 2 cases, drain failures resulted in seromas in the early postoperative period; these seromas were drained uneventfully 1 week after the operation. The aesthetic appearance of these implants remained acceptable throughout the study. Infection occurred in 1 implant, which was removed 2
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A
B
Fig 5. Collagen fibers in capsule and within pores of implant harvested at 17 weeks (Masson’s trichrome stain). A) Collagen fibers were denser in capsule (arrows) than within pores. Scale bar — 500 μm. B) Collagen fibers were located throughout pores. Scale bar — 200 μm.
weeks after implantation. None of the implants was completely extruded. Implant exposure occurred in 5 cases, beginning during the first postoperative week; all exposures developed at the most prominent part of the antihelix. In 1 case, the exposure resolved spontaneously and the wound healing led to an acceptable appearance at the time of explantation at 17 weeks. In 2 cases, moderate exposure occurred and was stabilized; the wound did not heal, but the implant was maintained until the end of the study (Fig 2). In 2 cases, significant nonhealing, progressing exposure developed; these implants were removed at 7 weeks on the recommendation of the attending veterinarian (Fig 3). The explants and surrounding tissue were examined histologically after explantation at 17 weeks. Each implant was surrounded by a thin, moderately cellular, fibrovascular capsule composed of spindleshaped fibroblasts and small blood vessels oriented along the capsule. The inflammatory response in the capsule was minimal at 17 weeks. The overlying dermis was adherent to the implant. Robust tissue ingrowth was noted in the interconnecting pores of the implants (Fig 4), which contained spindleshaped fibroblasts and small blood vessels. A foreign body response was evidenced by a moderate number of giant cells and macrophages located adjacent to the implant material. The collagen deposition within the pores consisted of organized loose fibers seen on trichrome-stained sections (Fig 5); the collagen fibers were more mature and dense in the capsule that surrounded the implant. Discussion
An auricular reconstruction surgical model was developed in sheep, with the ultimate goal of im-
plantation of engineered cartilage frameworks. The HDPPE frameworks were subcutaneously implanted without the use of a highly vascular TPFF; we expect that engineered cartilage constructs will be implanted in the same manner as costal cartilage grafts and that TPFF coverage will not be required. In our ovine model, 10 of 18 implants remained in place until 17 weeks without any complications. One implant (5%) became infected and had to be removed, 5 implants (28%) suffered various degrees of exposure, and 2 of the exposed implants (11%) were removed before the completion of the study, resulting in the premature removal of a total of 3 implants (17%). This complication rate is higher than that reported for Medpor auricular reconstructions in humans, which had only a 7% exposure rate.6 The HDPPE exposures were likely caused by ischemia and excessive stress on the thin skin overlying the implants, as we did not use vascularized tissue coverage. When HDPPE is used for microtia reconstruction in humans, it is customarily placed under a TPFF based on the superficial temporal vessels; this practice is particularly important when suction drains or pressure dressings appose the skin tightly to the underlying implant. However, we implanted the HDPPE frameworks without additional vascularized tissue coverage in order to mimic the ultimate reconstruction model expected for engineered cartilage frameworks. Early reports of HDPPE implantation in humans with microtia without the use of a TPFF described high complication rates — up to 44%.6 Histologically, robust fibrovascular tissue ingrowth was demonstrated within the pores of the implant. The HDPPE has a pore size of approximately 40 to 200 μm. The porous structure facilitates the
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ingrowth of soft tissue into the material, anchoring it in position, integrating it into the host, improving its resistance to infection, and preventing its total extrusion.10,11 The extent of fibrovascular ingrowth into HDPPE implants was correlated with resistance to infection and the ability to support overlying skin grafts in experimental animals.12,13 The thin and flexible capsule with a minimal inflammatory response in our sheep model was similar to that described by others.14 An occasional inflammatory reaction was noted within the pores of the implants, with foreign body giant cells located adjacent to the HDPPE material, and it remained unresolved after 17 weeks of implantation. Overall, the ovine reaction to porous HDPPE implants was similar to that observed in other animal models and in humans.10,15
Conclusions
In our series of 9 animals, surgical complications and histologic changes were demonstrated that were similar to those seen after HDPPE implantation in humans. The ovine tissue model possesses sufficient gross and microscopic similarities to human tissue to suggest that it is a promising translational animal model for this application. Inadequate healing was demonstrated with skin-only coverage of a rigid synthetic implant; however, we anticipate that improved healing will occur when engineered cartilage is subcutaneously implanted in a way similar to that observed in implantation of a carved costal cartilage framework. In future studies, engineered ear frameworks will be implanted in this surgical model.
References 1. Romo T III, Reitzen SD. Aesthetic microtia reconstruction with Medpor. Facial Plast Surg 2008;24:120-8. 2. Firmin F. Ear reconstruction in cases of typical microtia. Personal experience based on 352 microtic ear corrections. Scand J Plast Reconstr Surg Hand Surg 1998;32:35-47. 3. Berghaus A, Stelter K, Naumann A, Hempel JM. Ear reconstruction with porous polyethylene implants. Adv Otorhinolaryngol 2010;68:53-64. 4. Brent B. Technical advances in ear reconstruction with autogenous rib cartilage grafts: personal experience with 1200 cases. Plast Reconstr Surg 1999;104:319-38. 5. Nagata S. A new method of total reconstruction of the auricle for microtia. Plast Reconstr Surg 1993;92:187-201. 6. Reinisch JF, Lewin S. Ear reconstruction using a porous polyethylene framework and a temporoparietal fascia flap. Facial Plast Surg 2009;25:181-9. 7. Thorne CH, Brecht LE, Bradley JP, Levine JP, Hammerschlag P, Longaker MT. Auricular reconstruction: indications for autogenous and prosthetic techniques. Plast Reconstr Surg 2001;107:1241-52. 8. Bichara DA, O’Sullivan NA, Pomerantseva I, et al. The tissue-engineered auricle: past, present, and future. Tissue Eng
Part B Rev 2012;18:51-61. 9. Zhou L, Pomerantseva I, Bassett EK, et al. Engineering ear constructs with a composite scaffold to maintain dimensions. Tissue Eng Part A 2011;17:1573-81. 10. Sclafani AP, Romo T III, Silver L. Clinical and histologic behavior of exposed porous high-density polyethylene implants. Plast Reconstr Surg 1997;99:41-50. 11. Berghaus A. Implants for reconstructive surgery of the nose and ears. GMS Curr Top Otorhinolaryngol Head Neck Surg 2007;6:Doc06. Epub 2008 Mar 14. 12. Sclafani AP, Thomas JR, Cox AJ, Cooper MH. Clinical and histologic response of subcutaneous expanded polytetrafluo roethylene (Gore-Tex) and porous high-density polyethylene (Medpor) implants to acute and early infection. Arch Otolaryngol Head Neck Surg 1997;123:328-36. 13. Williams JD, Romo T III, Sclafani AP, Cho H. Porous high-density polyethylene implants in auricular reconstruction. Arch Otolaryngol Head Neck Surg 1997;123:578-83. 14. Shanbhag A, Friedman HI, Augustine J, von Recum AF. Evaluation of porous polyethylene for external ear reconstruction. Ann Plast Surg 1990;24:32-9. 15. Neel HB III. Implants of Gore-Tex. Arch Otolaryngol 1983;109:427-33.
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Ovine Model for Auricular Reconstruction: Porous Polyethylene Implants Marc H. Hohman, MD; Robin W. Lindsay, MD; Irina Pomerantseva, MD, PhD; David A. Bichara, MD; Xing Zhao, MD; Matthew Johnson; Katherine M. Kulig; Cathryn A. Sundback, ScD; Mark A. Randolph, MAS; Joseph P. Vacanti, MD; Mack L. Cheney, MD; Theresa A. Hadlock, MD Objectives: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model. Methods: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infraauricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented. Results: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks. Conclusions: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs. Key Words: microtia, porous polyethylene implant, sheep.
disadvantages. Costal cartilage autografting requires the greatest degree of skill. Costal cartilage is harvested and carved into several pieces that are assembled into a complex, 3-dimensional ear framework and typically implanted subcutaneously; the overlying soft tissue can stress the implanted framework and distort the structural elements. Auricular reconstruction with a carved cartilage framework requires 2 to 4 operations over the course of several months to a year. Multiple donor sites are common, for example, the chest wall for cartilage, the groin or the scalp for skin grafts, and the contralateral ear for full-thickness skin grafts and/or composite skin-cartilage grafts. Often, additional revision procedures are necessary, including hair removal from
Introduction
Complete auricular reconstruction remains challenging for today’s surgeon. The most common indication for reconstruction is congenital microtia, with an incidence of approximately 2 per 10,000 live births in the United States; traumatic avulsions and burns also require similar reparative procedures.1 Current alternatives for complete auricular reconstruction include carved costal cartilage autografts, synthetic high-density porous polyethylene (HDPPE) implants, and external auricular prostheses anchored to osseo-integrated implants. All approaches provide satisfactory to excellent results in experienced hands, but each method has
From Massachusetts Eye and Ear Infirmary (Hohman, Lindsay, Cheney, Hadlock); Harvard Medical School (Hohman, Lindsay, Pomerantseva, Bichara, Zhao, Sundback, Randolph, Vacanti, Cheney, Hadlock); and the Department of Surgery, Division of Pediatric Surgery (Pomerantseva, Kulig, Sundback, Vacanti), and the Plastic Surgery Research Laboratories (Bichara, Zhao, Johnson, Randolph), Massachusetts General Hospital; Boston, Massachusetts; and the Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands (Zhao). This research was sponsored by the Armed Forces Institute of Regenerative Medicine award number W81XWH-08-2-0034. The US Army Medical Research Acquisition Activity, Fort Detrick, Maryland, is the awarding and administering acquisition office. Dr Hohman’s effort was also supported by the US Army Medical Command. The content of the manuscript does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital. Correspondence: Joseph P. Vacanti, MD, Dept of Pediatric Surgery, 55 Fruit St, Warren 1151, Boston, MA 02114.
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B
Fig 1. Gross images demonstrate good aesthetic outcome of high-density porous polyethylene (HDPPE) auricle-shaped frameworks implanted in ovine infraauricular area. Images were obtained from same animal A) immediately after implantation and B) at explantation 17 weeks later.
the reconstructed pinna.2 Delaying autogenous cartilage grafting until the patient is at least 10 years of age avoids chest wall deformation related to costal cartilage harvest and increases the likelihood that a sufficient amount of cartilage will be available for harvest. In contrast, patients undergoing HDPPE implantation are typically 3 to 5 years of age.2,3 The HDPPE framework is usually implanted under a temporoparietal fascia flap (TPFF) based on the superficial temporal vessels. Reconstruction with a HDPPE framework can be completed in 1 or 2 stages, but the rate of infection and extrusion over a patient’s lifetime is substantially higher than that reported for autologous costal cartilage implants.1,4-6 However, the aesthetic results with these implants are more consistent for less experienced surgeons.1 Lastly, auricular prostheses can have a natural appearance and require the least complicated surgical procedure, but these prostheses wear out, change color with age, and must be periodically replaced. In many areas of the world, use of different springsummer and autumn-winter prostheses is necessary to match seasonal changes in patients’ skin pigmentation. A prosthesis held in place by magnets may be dislodged during physical activities and must be removed during sleep. Additionally, whereas a patient frequently considers a reconstructed auricle to be a part of his or her own body, the same is seldom felt about a prosthetic auricle.7 A cartilage framework engineered from a patient’s own chondrocytes has the potential to combine the best characteristics of the existing reconstructive approaches while minimizing the disadvantages.8 An engineered cartilage framework would decrease the operative time and would eliminate inconsistencies related to carving costal cartilage. The avoidance of harvesting large costochondral grafts from the chest
wall would decrease postoperative pain, speed recovery, and eliminate the risk of pneumothorax. The finished engineered product would possess the advantages of both costal cartilage and synthetic implant approaches, providing a custom-sized auricle with defined 3-dimensional contours. This engineered product would have a low risk of infection or extrusion and would ideally mimic the mechanical properties of auricular cartilage. Although neocartilage does tend to be less resilient than native cartilage, addition of an internal coiled titanium wire scaffold can greatly improve resistance to deformation.9 Our team and others have made significant progress in engineering cartilage in the shape of the human ear in laboratory animals.8,9 An essential step toward bringing the technique of cartilage engineering into clinical practice is the development of a large animal model. We selected a sheep model on the basis of the similarity of its facial vascular anatomy to that of humans — an aspect vital to translating the results of large animal studies to humans. As the development of ovine engineered auricular cartilage is under way, controls are needed to compare ovine-based results with conventional reconstructive methods. We established a surgical model based on HDPPE auricular implants in sheep; these implants were subcutaneously implanted without the use of a vascularized flap to mimic the expected reconstruction approach for engineered cartilage frameworks. Methods
All protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. Nine Polypay sheep of both sexes, 3.4 ± 0.7 months of age and weighing 30.2 ±
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Hohman et al, Polyethylene Auricular Reconstruction 137
A
B
Fig 2. Small nonhealing exposure of antihelix of HDPPE framework occurred during first 2 weeks after operation in 1 animal. Wound remained stable in size A) at 8 weeks and B) at 17 weeks.
3.9 kg, each received bilateral implants in the infraauricular area. Before the induction of general anesthesia, the animals were premedicated with an intramuscular injection of a combination of xylazine hydrochloride 0.4 mg/kg and Telazol 2 mg/kg (tiletamine hydrochloride–zolazepam hydrochloride; Fort Dodge Animal Health, Fort Dodge, Iowa). Anesthesia was A
then induced with 4% to 4.5% isoflurane and maintained at 2% to 3% via endotracheal tube throughout the surgical procedure. Buprenorphine hydrochloride 0.01 mg/kg subcutaneous injections and cefazolin sodium 40 mg/kg intramuscular injections were administered for pain and infection prophylaxis, respectively. The skin on the side of the neck was shaved, prepared with alcohol and povidone-iodine solution, and draped in sterile fashion. B
Fig 3. Significant nonhealing exposure of antihelix of HDPPE framework occurred in 2 separate animals. Both exposures occurred during first 2 weeks after operation and progressed, and implants were removed at 7 weeks. A) Appearance at 2 weeks. B) Appearance at 7 weeks.
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A
Fig 4. Representative histologic section through HDPPE implant harvested at 17 weeks (hematoxylin-eosin stain). A) Fibrovascular growth into pores occurred throughout implant. Scale bar — 1 mm. B) Fibroblasts and small blood vessels were observed within pores. Scale bar — 200 μm. C) Occasional inflammatory cellular infiltrate was also discerned within pores, and giant cells lined pores (arrows). Scale bar — 200 μm.
B
C
A 4-cm skin incision was made from the base of the auricle to the caudal border of the parotid gland, and after careful hemostasis, a subdermal pocket was bluntly dissected. A 2-piece Medpor ear implant (helical rim 37 × 62 mm and ear base 55 × 28 × 13 mm; Porex Surgical, Inc, Newnan, Georgia) was assembled and placed inside the pocket. Two continuous closed suction drains (TLS Max-Glide Facial Drainage kit, Porex Surgical, Inc) were placed between the implant and the skin and left in place for 48 hours in all 9 animals. The skin was closed with 3-0 polyglactin sutures. After the operation, the animals were monitored for signs of discomfort or complications twice daily for 3 days. The wounds were then monitored 3 times per week for infection, dehiscence, and implant exposure. Pain relief was provided with flunixin meg lumine 1 mg/kg intramuscular injections daily for 3 days after operation and then administered as needed. The animals were painlessly euthanized 17 weeks after implantation. The implants were then removed
along with a cuff of surrounding tissue. Gross photographs were taken; the implants were fixed in 10% buffered formalin and embedded in paraffin. The embedded tissue was cut into 5-μm sections and stained with hematoxylin-eosin and Masson’s trichrome (American MasterTech Scientific, Inc, Lodi, California) for microscopic analysis. Results
There were no perioperative anesthetic complications or deaths. Overall, the implants retained an easily recognizable auricular appearance throughout the study (Fig 1). All implants were firmly fixed to the surrounding tissue. Several implants were associated with transient alopecia. There were 8 instances of surgical complications in the 18 implants. In 2 cases, drain failures resulted in seromas in the early postoperative period; these seromas were drained uneventfully 1 week after the operation. The aesthetic appearance of these implants remained acceptable throughout the study. Infection occurred in 1 implant, which was removed 2
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A
B
Fig 5. Collagen fibers in capsule and within pores of implant harvested at 17 weeks (Masson’s trichrome stain). A) Collagen fibers were denser in capsule (arrows) than within pores. Scale bar — 500 μm. B) Collagen fibers were located throughout pores. Scale bar — 200 μm.
weeks after implantation. None of the implants was completely extruded. Implant exposure occurred in 5 cases, beginning during the first postoperative week; all exposures developed at the most prominent part of the antihelix. In 1 case, the exposure resolved spontaneously and the wound healing led to an acceptable appearance at the time of explantation at 17 weeks. In 2 cases, moderate exposure occurred and was stabilized; the wound did not heal, but the implant was maintained until the end of the study (Fig 2). In 2 cases, significant nonhealing, progressing exposure developed; these implants were removed at 7 weeks on the recommendation of the attending veterinarian (Fig 3). The explants and surrounding tissue were examined histologically after explantation at 17 weeks. Each implant was surrounded by a thin, moderately cellular, fibrovascular capsule composed of spindleshaped fibroblasts and small blood vessels oriented along the capsule. The inflammatory response in the capsule was minimal at 17 weeks. The overlying dermis was adherent to the implant. Robust tissue ingrowth was noted in the interconnecting pores of the implants (Fig 4), which contained spindleshaped fibroblasts and small blood vessels. A foreign body response was evidenced by a moderate number of giant cells and macrophages located adjacent to the implant material. The collagen deposition within the pores consisted of organized loose fibers seen on trichrome-stained sections (Fig 5); the collagen fibers were more mature and dense in the capsule that surrounded the implant. Discussion
An auricular reconstruction surgical model was developed in sheep, with the ultimate goal of im-
plantation of engineered cartilage frameworks. The HDPPE frameworks were subcutaneously implanted without the use of a highly vascular TPFF; we expect that engineered cartilage constructs will be implanted in the same manner as costal cartilage grafts and that TPFF coverage will not be required. In our ovine model, 10 of 18 implants remained in place until 17 weeks without any complications. One implant (5%) became infected and had to be removed, 5 implants (28%) suffered various degrees of exposure, and 2 of the exposed implants (11%) were removed before the completion of the study, resulting in the premature removal of a total of 3 implants (17%). This complication rate is higher than that reported for Medpor auricular reconstructions in humans, which had only a 7% exposure rate.6 The HDPPE exposures were likely caused by ischemia and excessive stress on the thin skin overlying the implants, as we did not use vascularized tissue coverage. When HDPPE is used for microtia reconstruction in humans, it is customarily placed under a TPFF based on the superficial temporal vessels; this practice is particularly important when suction drains or pressure dressings appose the skin tightly to the underlying implant. However, we implanted the HDPPE frameworks without additional vascularized tissue coverage in order to mimic the ultimate reconstruction model expected for engineered cartilage frameworks. Early reports of HDPPE implantation in humans with microtia without the use of a TPFF described high complication rates — up to 44%.6 Histologically, robust fibrovascular tissue ingrowth was demonstrated within the pores of the implant. The HDPPE has a pore size of approximately 40 to 200 μm. The porous structure facilitates the
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ingrowth of soft tissue into the material, anchoring it in position, integrating it into the host, improving its resistance to infection, and preventing its total extrusion.10,11 The extent of fibrovascular ingrowth into HDPPE implants was correlated with resistance to infection and the ability to support overlying skin grafts in experimental animals.12,13 The thin and flexible capsule with a minimal inflammatory response in our sheep model was similar to that described by others.14 An occasional inflammatory reaction was noted within the pores of the implants, with foreign body giant cells located adjacent to the HDPPE material, and it remained unresolved after 17 weeks of implantation. Overall, the ovine reaction to porous HDPPE implants was similar to that observed in other animal models and in humans.10,15
Conclusions
In our series of 9 animals, surgical complications and histologic changes were demonstrated that were similar to those seen after HDPPE implantation in humans. The ovine tissue model possesses sufficient gross and microscopic similarities to human tissue to suggest that it is a promising translational animal model for this application. Inadequate healing was demonstrated with skin-only coverage of a rigid synthetic implant; however, we anticipate that improved healing will occur when engineered cartilage is subcutaneously implanted in a way similar to that observed in implantation of a carved costal cartilage framework. In future studies, engineered ear frameworks will be implanted in this surgical model.
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