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Reconstruction of auricular cartilage using tissue-engineering techniques
Operative Techniques in Otolaryngology (2008) 19, 278-284
Reconstruction of auricular cartilage using tissue-engineering techniques Nicole Rotter, MD,a Alexander Steiner, MD, DMD,b and Marc Scheithauer, MDa From the aDepartment of Otorhinolaryngology, Head and Neck Surgery, Ulm University, Ulm, Germany; and the b Department of Maxillofacial Surgery, Ruppiner Clinics, Neuruppin, Germany. KEYWORDS Tissue engineering; Cartilage; Auricle; Plastic-reconstructive surgery
Reconstructive surgery of the nose and the auricle frequently requires grafting from different sites of cartilage as donor material. Typically, grafts for nasal reconstruction are obtained from within the nose whenever possible; alternatively, cartilage can be obtained from the auricle or the rib. Auricular reconstruction procedures usually involve the harvesting of rib cartilage when large parts of the auricle have to be reconstructed. However depending on the underlying disease, harvesting might not be possible to a sufficient degree, eg, after multiple reconstruction efforts or in burn or malformation surgery. Also a severe donor site morbidity has to be taken into account in the case of harvesting rib cartilage. Tissue engineering is an evolving area of research, with the aim of growing tissue in vitro that can be used for reconstructive purposes. This article reviews the current state of the art of tissue engineering procedures of cartilage for reconstruction of the auricle and is determined to answer the question why the technique has not yet found its way into daily clinical routine in otolaryngology in contrast to its performance in orthopaedic surgery. © 2008 Elsevier Inc. All rights reserved.
Defects of nasal or auricular cartilage frequently are congential or caused by trauma or tumor. The surgery of extensive defects is complex and, often, a multistage procedure is required. Even then, functional and cosmetic results are not always satisfactory. Because cartilage lacks an intrinsic healing property, defects need to be treated either by grafting autologous tissue or by implantation of alloplastic material. Alloplastic materials comprise the possible risk of chronic infection and extrusion. Because they are readily available “off the shelf” in the desired shape, some authors favor their use to the use of autologous grafting.1 The use of autologous cartilage as grafting material is considered the gold standard by many authors today.2,3 This tissue can be harvested from the patient in the same operative procedure. The use of autologous cartilage minimizes the risk of infection and extrusion. However, harvesting of suitable donor tissue might not be possible at all or only to a very limited extent, especially if patients have suffered from severe burn injury or have already undergone multiple surgical procedures. A possible future al-
Address reprint requests and correspondence: Nicole Rotter, MD, Department of Otorhinolaryngology, Ulm University, Frauensteige 12, 89075 Ulm, Germany. E-mail address: [email protected]. 1043-1810/$ -see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.otot.2008.10.004
ternative to obtain tissue for reconstructive purposes in head and neck surgery is to in vitro cultivate autologous cartilage by means of tissue engineering. The basic concept of tissue engineering is to isolate autologous cells and to grow them in vitro on a 3-dimensional scaffold in a suitable culture system to construct tissue for transplantation. However, several hurdles have to be cleared before this technology will be ready for routine clinical use in auricular reconstruction.
History and presence of auricular reconstruction As early as 600 BC, physicians have been dealing with the problem of treating structural defects of the auricle. Since about 1890, detailed descriptions of options for surgical therapy are available.4 Around 1930, the necessity to use mechanically stable scaffolds was advocated.5 Since then, the search for an optimal scaffold has been ongoing. Just to name a few, autologous6 and allogeneic7 rib cartilage has been used as well as nasal septal cartilage8 or xenogenic cartilage.9 Furthermore alloplastic materials like ivory,10 silicon,11 Teflon,12 or polyurethane1 have been proposed for auricular reconstruction. In parallel, a multitude of operative
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techniques have been discussed. Today, the surgical methods most frequently applied for total auricular reconstruction are based on descriptions of Brent,13,14 Firmin,15 and Nagata.16-19 Although Brent described a 4-step technique, Nagata summarized the surgical steps in 2 operations. However, modifications might have to be used depending on the individual surgical situation. An alternative treatment option to surgical reconstruction is the use of bone-anchored prostheses.20 In certain cases, such as in aged patients with high comorbidity, this option might be chosen to reduce possible complications related to staged, time-consuming operations. However, there is evidence that surgical rehabilitation enhances psychosocial development in children.21 In surgical reconstruction, rib cartilage nowadays is used most frequently as scaffold. The harvesting of rib cartilage, however, might cause severe comorbidity,22,23 such as pneumothorax, deformities of the thorax, and considerable postoperative pain. Also, in certain cases, eg, after unsuccessful surgical reconstruction, there might not be enough cartilage available to perform the desired reconstruction. In the following section, we will review techniques for reconstruction of traumatic auricular defects in detail.
Reconstruction of traumatic auricular defects An appropriate operation strategy can be determined if both the localization of the defect and its amount of tissue loss is analyzed thoroughly in the preoperative assessment.
279 It is also essential to investigate the quality of the surrounding skin and check for scars, evidence of previous surgery and the hair-line. Central skin defects of the concha and anthelix In case of an intact perichondrium, a full-thickness skin graft is the method of choice. It is harvested in the postauricular region, because of the similar color and texture of the transplanted skin. The cosmetic result is usually very favorable. Central two-layer defects of the concha and anthelix If the defect consists of skin as well as cartilage an island flap, which is pedicled in the postauricular subcutaneous region can be used. Involving the posterior auricular artery the flap might be pedicled caudally or cranially and reaches any location in the concha or anthelix without stressing the vessels. The safety of this flap is remarkable and it heals reliably (Figure 1). Small peripheral defects of the cranial third of the auricle A cutaneous defect can be reconstructed by with the use of a postauricular pedicled transposition flap. In this case, the pedicle has to be detached after 3 weeks, which makes it a 2-stage procedure. If the defect consists of the cartilaginous part of the helix and the overlying skin, a combination of a conchal cartilage transplant and a cranially pedicled
Figure 1 (A) This central 2-layer defect of the concha was caused by resection of a basalioma. (B) The intraoperative view of the reconstructing island flap from the retroauricular region is demonstrated. (Color version of figure is available online.)
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transposition flap is recommended.24 Again, the pedicle needs detachment after some weeks. This technique seems reliable if the defect is not bigger than 2.0 cm in length.
Subtotal or total defects Defects larger than 2.0 cm mostly require a timely-staged reconstruction using autologous rib cartilage. An alternative method is the use of a temporoparietal fascial flap (fan-flap) pedicled on the superficial temporal artery. Its advantage is that it is a one-stage-procedure.25 In case of severely damaged skin in the proximity of the defect, an implantable skin expander helps to enlarge the area of vital skin, thus ensuring safe coverage of the rib cartilage. Approximately 10 to 12 weeks later, the first step of reconstruction can be performed. Subtotal defects. Subtotal defects of the upper two-thirds of the auricle can be reconstructed by means of an axial pattern fan-flap, rib cartilage, and skin grafts (Figure 2). The superficial temporal artery is easily localized preoperatively with a continuous-wave Doppler and marked on the skin. The flap is then harvested in the needed size and rotated caudally. The cartilaginous scaffold is wrapped into the flap and covered with full-thickness grafts. These are taken from
the contralateral postauricular region and from the inguinal area. Total defects. If the auricle is missing completely, a 3-stage procedure is required in most cases.15,26 A template of the contralateral auricle is made preoperatively. According to this template, the cartilaginous scaffold is carved form the explanted sixth, seventh, and eighth rib. The cartilaginous part of the ninth rib is used additionally to rebuild the helix. It is recommended that the scaffold should be created about 3.0 mm smaller than the template in all dimensions. This is important, because the thickness of the overlying skin otherwise produces a rather plump and unnatural looking esthetic result. The scaffold is then placed in a skin pocket. The incision is made cranially to the defect in the hair bearing site. The carved model is connected to the existing ear cartilage with absorbable sutures. A drain is located close to the reconstruction. Its suction molds the skin to the underlying scaffold. In the second stage the auricle is detached from the mastoid skin creating a postauricular groove. For stabilization of the groove a piece of carved cartilage is wrapped into a fascial flap and sutured to the scaffold. A full-thickness skin graft is used to close the skin defect in the mastoid region. During the third stage, minor corrections of the skin and cartilage are performed if necessary.
Figure 2 (A) A subtotal defect due to resection of a basalioma is partially covered with Epigard until final histology is obtained and reconstruction can be performed. (B) Reconstruction was performed using an axial pattern fan-flap, rib cartilage and a free skin graft. Four weeks postoperatively. (Color version of figure is available online.)
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Clinically and economically even more relevant, autologous cartilage grafting is performed in orthopedic and trauma surgery. Because the amount of cartilage donor tissue available for these procedures is limited, cartilage has become one main target in tissue engineering research. In this context, tissue engineering has also been proposed as an option to engineer cartilage in vitro for the use in auricular reconstruction. This option would decrease the surgical comorbidity on one hand and would simplify and shorten the surgical procedures on the other hand. Extensive efforts have been undertaken to create engineered cartilage in the shape of human ears. In the following section, these efforts will be reviewed and own results will be presented, furthermore it will be discussed, why tissue engineered auricles have not yet entered the clinical realm.
Engineering of auricular cartilage in the shape of a human auricle The basic ingredients of cartilage tissue engineering are cells, scaffolds, and a suitable in vitro culture system. The shape of an engineered implant is determined by the shape of the biomaterial provided to host the cells as a scaffold. Most strategies for cartilage tissue engineering are based on resorbable biomaterials as temporary scaffolds for chondrocytes or precursor cells.27 Cells are amplified in vitro, seeded onto the scaffold and then transplanted. Differentiated cells are producing cartilage specific matrix components in vivo. The developing tissue should resemble native cartilage with regard to specific function and morphology. With increasing production of newly synthesized matrix the biomaterials should degrade thus avoiding chronic foreign body reactions and inflammation.
Cellular basis for cartilage tissue engineering Chondrocytes can be isolated from cartilage in an anatomic localization away from the area to be reconstructed, eg, cartilage from the nasal septum, the rib or joints for the reconstruction of an auricular defect. Another option is to isolate precursor cells from a different type of tissue, such as the peripheral blood, fat tissue, or bone marrow. However, these cells need to be cultured under specific conditions to differentiate into the desired cell type.
Cell and tissue culture Successful amplification of human cells and tissue in vitro, determination of differentiation, and maintenance of specific functions are basic requirements for the clinical application of tissue engineering.28 Nutrition of tissue in vivo is guaranteed by the capillary system. Conventional cell culture methods only mimic these conditions to a limited extent, which results in the partial loss of cell- and tissue-specific function.29 Perfusion culture systems have been developed to guarantee a constant supply of fresh nutrients while draining used culture media and toxic metabolites at the same time.30 Perfusion culture systems can be operated in cell incubators at 5% CO2 or at room con-
281 ditions using pH-stabilizing media. Other types of culture systems, like rotating bioreactors, have been developed to promote matrix synthesis and cell differentiation in vitro.31-33 Mechanisms of matrix accumulation and differentiation are still a matter of research.
Choice of cell type When attempting to transfer the clinical routine that uses hyaline rib cartilage for the reconstruction of elastic auricular cartilage, we face the situation that the key question which type of chondrocyte is most suitable for auricular cartilage tissue engineering has not been answered yet. So far, engineering of auricular cartilage has been performed in a few studies only. Successful production of flexible cartilage in the shape of a human ear has been reported with the use of swine auricular chondrocytes and perichondrium34 in nude mice but also in immunocompetent porcine models.35,36 Bovine articular chondrocytes have been used for the same purpose,37 as well as human nasal chondrocytes.38 Comparative studies of chondrocytes of different origin have been performed in porcine models39,40 and a bovine model41; however, they are not intended specifically for auricular cartilage repair. Still, it can be concluded that auricular chondrocytes are a valuable source for cartilage tissue engineering procedures, as they are able to produce matrix with mechanical properties similar or even superior to articular and costal chondrocytes. Although the comparative studies have not been performed with human cells, we tend to conclude that auricular chondrocytes are most likely, whenever available, the cells of choice for auricular reconstruction. On the other hand, clinical conditions that require cell amplification to obtain large transplants from small biopsies have not been taken into account in these studies, thus preventing a clear statement based on these studies. Strong in vitro cell amplification results in the loss of cell specific functions, a process called dedifferentiation.42 The engineering of functional tissue however requires the maintenance of specific functions. These can be maintained by a three-dimensional arrangement of the cells under specific culture conditions.
Stem cell-based tissue engineering Recently the successful isolation of human stem cells from bone marrow, periosteum, and fat tissue was established by different groups. These cells are highly proliferative and are capable of differentiating into different types of tissue. Today, it is possible to amplify mesenchymal stem cells from an adult human for more than 30 passages in vitro thus obtaining more than 1 billion of cells. During amplification, cells maintain their phenotypic characteristics without losing their differentiation capacity, eg, the osteogenic or chondrogenic potential. However, growth factors need to be added to obtain chondrogenic differentiation, thus making the entire process more complex and expensive. Concerning growth factor application, it is not known yet whether this could have any unfavorable or even harmful effects, such as unlimited cell proliferation or tumor
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induction, after in vivo application. Stem cells are a tempting option for cartilage tissue engineering; however, currently differentiated chondrocytes seem the favorable option whenever available. Only broader and more detailed knowledge on stem cell biology might make these cells valuable alternative candidates for cartilage tissue engineering procedures.
showed that alginates are the materials of choice, as pressurefree application is possible in a relatively short time and at low cost and we were able to construct detailed molds (Figure 4) without causing discomfort to the patients. These molds can be used to produce biomaterials and cell-biomaterial constructs in the shape of the respective contralateral ear for tissue engineering purposes.
Shaping of tissue-engineered cartilage
Three-dimensional data acquisition for rapid prototyping techniques
The shape of the engineered cartilage is determined by the shape of the biomaterial. So far, there have been no reports about increase of cartilage volume or shape as compared with the original implant, however significant reduction in volume has been observed, as well as complete resorption of implants. Thus it is necessary either to prevent resorption to a significant degree or to anticipate volume reduction by using implants of larger shape and volume. However, these issues have not been clarified yet. Prevention of resorption can be performed by different strategies, eg, immunoisolation.
Production of individual auricular molds The first step, which will determine the quality in the production of an engineered auricle, is a precise molding and modeling of the individual contralateral auricle. It is necessary to apply the molding material in a precise and pressure-free mode, to take into account the flexible consistency of the auricle. Pressure will lead to distortion of the auricle, which will in turn result in an insufficient model. This is comparable with the situation in mucosa-fixed total dental prosthesis. We developed a simple method to mold the auricle by producing a series of molding shapes (Figure 3). They can be applied in an ambulatory setting on a sitting patient in a short period of time. The application of different materials, such as plaster (Snow White; Kerr, Bioggio, Switzerland), polyether (Impregum; 3M ESPE, Neuss, Germany), polysulfide (Permlastic; Kerr), silicone (EUROSIL and VPS by Henry Schein International, Langen, Germany; Xantopren; Heraeus Kulzer, Dormagen, Germany), hydrocolloids (Hydrocolloid Impression Material by Schein), and alginates (Alginate Type I and II and Alginate Plus by Schein), clearly
Figure 3 Auricular molding shapes in different sizes suitable for various sizes of auricles. (Color version of figure is available online.)
The individual production of molds is time consuming and the quality is closely related to the experience of the applying person. Using touch-free laseroptic scanning of the auricle is an interesting alternative to the aforementioned method. We applied the Vivid 700™ Non-Contact 3D Digitizer (Minolta, Hannover, Germany). A laser with a wavelength of 685 nm and radiation power of 25mW scans the desired object within 0,6 seconds and creates a digital model within 2 seconds. However, this digitizer is not applicable for direct scanning of the auricle of a patient because the object to be digitized needs to be placed on a rotating plate. Direct scanning is of course possible using different types of scanners. A disadvantage is the currently high cost of these scanners. However, the use of centralized systems could solve this problem. The obtained data might be used for the production of 3-dimensional models or scaffolds for tissue engineering with the help of rapid prototyping techniques. These techniques are used in automobile and machine tool engineering routinely and allow the production of materials and models based on digital data. They enable among others to control shape, porosity, pore size, and permeability. Different types of production technologies such as 3-dimensional printing and fused deposition modeling are currently available and seem to be applicable to biomaterials as well. However reports on biomaterials produced by this technology today focus on scaffolds for bone repair.43
Biomaterials for auricular reconstruction An optimal scaffold for tissue engineering procedures should support cell adhesion and cell function44; at the same time, it should degrade into nontoxic substances.45 The mechanical properties should provide initial mechanical strength, degradation should be adjusted to production of newly synthesized matrix by the transplanted chondrocytes. A large variety of biomaterials have been under investigation for cartilage tissue-engineering purposes. Biodegradable polymers in the shape of woven meshes, nonwoven fleeces, or foams composed of polyglycolic or polylactide acid and naturally derived polymers like collagen or hyaluronic acid are used frequently. The use of resorbable materials for rapid-prototyping technologies has been described.46 Nonresorbable materials produced by rapid-prototyping have been proposed for auricular reconstruction.47 Some authors favor the use of internal or external stenting34 to provide initial and mid-term mechanical strength. In summary, neither an optimal material nor a production technology nor an implantation tech-
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Figure 4 (A) The patients auricle was used to determine an optimal molding material. (B) The fabricated model was created using an alginate mold. It precisely reflects the original shape. (Color version of figure is available online.)
nique has been determined for use in auricular reconstruction yet.
Clinical experiences with tissue-engineered auricular cartilage One patient received an tissue engineered auricle at the Department of Otolaryngology at Charité University in Berlin, Germany48 in 1997. The engineered cartilage remained stable in shape for several weeks but was strongly resorbed afterward, leading to an unfavorable cosmetic result. In 2000, researchers at the University of Freiburg, Germany, reported the treatment of a partial traumatic ear defect with tissue engineered cartilage derived from costal chondrocytes. This effort also failed as the construct was completely resorbed after a few months. Until today, the clinical application of tissue-engineered cartilage for auricular reconstruction, in contrast to its orthopedic applications, has not yet been established. There have been case reports of using engineered cartilage for reconstructive nasal surgery.49,50 However, these results need to be confirmed and clarified. Most likely, the unique implantation site and requirements concerning shape and initial mechanical strength are responsible for the so far unfavorable results in the head and neck region. While in orthopedic surgery engineered constructs are placed in the somewhat “immunoprivileged” region of joints, which is not closely connected to the
vascular network, a subcutaneous transplant position in the head and neck region faces strong inflammatory reactions and resorptions. The shape of the transplants needs to be well defined before transplantation in auricular reconstruction with strong initial and long-term mechanical stability, whereas joints defects are preformed cavities to be filled by liquid or semisolid cell preparations, which can obtain mechanical stability during maturation in vivo.
Conclusion and perspectives Tissue engineering enables the fabrication of living and functional cartilage transplants for the nose and the auricle; therefore, it is a possible future alternative to surgical reconstruction techniques based on autologous rib cartilage, avoiding the disadvantages of surgically harvesting large quantities of cartilage. Before this technology will enter the clinical reality of reconstructive head and neck surgery, the optimization of scaffold materials as well as the development of novel scaffolds will be a key issue in reducing inflammatory reactions currently leading to resorption of such transplants in subcutaneous locations. Also, these materials have to be optimized with respect to their mechanical strength and degradation characteristics. A more profound knowledge in stem cell biology and genetic engineering might lead to the applicability of allogenic cells which could finally provide real “off the shelf” transplants.
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References 1. Romo T 3rd, Presti PM, Yalamanchili HR: Medpor alternative for microtia repair. Facial Plast Surg Clin North Am 14:129-136, 2006 2. Park SS: Reconstruction of nasal defects larger than 1.5 centimeters in diameter. Laryngoscope 110:1241-1250, 2000 3. Rettinger G: Autogenous and allogeneic cartilage transplants in head and neck surgery (excluding middle ear and trachea). Eur Arch Otorhinolaryngol Suppl 1:127-162, 1992 4. van Doorne JM: Extra-oral prosthetics: Past and present. J Invest Surg 7:267-274, 1994 5. Beck JC: The anatomy, psychology, diagnosis and treatment of congenital malformation and absence of the ear. Laryngoscope 35:813832, 1925 6. Herberhold C: Reconstruction of the auricle with preserved homologous rib cartilage. Facial Plast Surg 5:431-433, 1988 7. Pierce GW: Reconstruction of the external ear. Surg Gynecol Obstet 50:601-605, 1930 8. Graham HB: Reconstruction of the completely destroyed auricle: A case report. Calif West Med 53:518-519, 1927 9. Gillies H, Kristensen HK: Ox cartilage in plastic surgery. Br J Plast Surg 4:63-73, 1951 10. Eitner E: Ohrmuschelersatz. Dtsch Z Chir 242:797-801, 1934 11. Cronin TD: Use of a Silastic frame for total and subtotal reconstruction of the external ear: Preliminary report. Plast Reconstr Surg 37:399405, 1966 12. Edgerton MT: Ear construction in children with congenital atresia and stenosis. Plast Reconstr Surg 43:373-380, 1969 13. Brent B: The correction of microtia with autogenous cartilage grafts: I. The classic deformity? Plast Reconstr Surg 66:1-12, 1980 14. Brent B: The correction of microtia with autogenous cartilage grafts: II. Atypical and complex deformities. Plast Reconstr Surg 66:13-21, 1980 15. Firmin F: Ear reconstruction in cases of typical microtia. Personal experience based on 352 microtic ear corrections. Scand J Plast Reconstr Surg Hand Surg 32:35-47, 1998 16. Nagata S: Modification of the stages in total reconstruction of the auricle: Part I. Grafting the three-dimensional costal cartilage framework for lobule-type microtia. Plast Reconstr Surg 93:221-230; discussion 267-268, 1994 17. Nagata S: Modification of the stages in total reconstruction of the auricle: Part II. Grafting the three-dimensional costal cartilage framework for concha-type microtia. Plast Reconstr Surg 93:231-242; discussion 267-268, 1994 18. Nagata S: Modification of the stages in total reconstruction of the auricle: Part III. Grafting the three-dimensional costal cartilage framework for small concha-type microtia. Plast Reconstr Surg 93:243-253; discussion 267-268, 1994 19. Nagata S: Modification of the stages in total reconstruction of the auricle: Part IV. Ear elevation for the constructed auricle. Plast Reconstr Surg 93:254-266; discussion 267-268, 1994 20. Somers T, De Cubber J, Govaerts P, et al: Total auricular repair: Bone anchored prosthesis or plastic reconstruction? Acta Otorhinolaryngol Belg 52:317-327, 1998 21. Siegert R, Knolker U, Konrad E: [Psychosocial aspects in total external ear reconstruction in patients with severe microtia]. Laryngorhinootologie 76:155-161, 1997 22. Ohara K, Nakamura K, Ohta E: Chest wall deformities and thoracic scoliosis after costal cartilage graft harvesting. Plast Reconstr Surg 93:1030-1036, 1994 23. Thomson HG, Kim TY, Ein SH: Residual problems in chest donor sites after microtia reconstruction: A long-term study. Plast Reconstr Surg 95:961-968, 1995 24. Weerda H: Surgery of the auricle Stuttgart, Thieme, 2004. p. 62-67 25. Brent B, Byrd HS: Secondary ear reconstruction with cartilage grafts covered by axial, random, and free flaps of temporoparietal fascia. Plast Reconstr Surg 72:141-152, 1983 26. Nagata S: A new method of total reconstruction of the auricle for microtia. Plast Reconstr Surg 92:187-201, 1993
27. Vacanti CA, Langer R, Schloo B, et al: Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plast Reconstr Surg 88:753-759, 1991 28. Minuth WW, Sittinger M, Kloth S: Tissue engineering: Generation of differentiated artificial tissues for biomedical applications. Cell Tissue Res 291:1-11, 1998 29. Kloth S, Eckert E, Klein SJ, et al: Gastric epithelium under organotypic perfusion culture. In Vitro Cell Dev Biol Anim 34:515-517, 1998 30. Minuth WW, Stockl G, Kloth S, et al: Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions. Eur J Cell Biol 57:132-137, 1992 31. Freed LE, Langer R, Martin I, et al: Tissue engineering of cartilage in space. Proc Natl Acad Sci U S A 94:13885-13890, 1997 32. Freed LE, Hollander AP, Martin I, et al: Chondrogenesis in a cellpolymer-bioreactor system. Exp Cell Res 240:58-65, 1998 33. Vunjak-Novakovic G, Martin I, Obradovic B, et al: Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 17:130-138, 1999 34. Xu JW, Johnson TS, Motarjem PM, et al: Tissue-engineered flexible ear-shaped cartilage. Plast Reconstr Surg 115:1633-1641, 2005 35. Kamil SH, Vacanti MP, Aminuddin BS, et al: Tissue engineering of a human sized and shaped auricle using a mold. Laryngoscope 114:867870, 2004 36. Saim AB, Cao Y, Weng Y, et al: Engineering autogenous cartilage in the shape of a helix using an injectable hydrogel scaffold. Laryngoscope 110:1694-1697, 2000 37. Kamil SH, Kojima K, Vacanti MP, et al: In vitro tissue engineering to generate a human-sized auricle and nasal tip. Laryngoscope 113:90-94, 2003 38. Haisch A, Klaring S, Groger A, et al: A tissue-engineering model for the manufacture of auricular-shaped cartilage implants. Eur Arch Otorhinolaryngol 259:316-321, 2002 39. Xu JW, Zaporojan V, Peretti GM, et al: Injectable tissue-engineered cartilage with different chondrocyte sources. Plast Reconstr Surg 113: 1361-1371, 2004 40. Johnson TS, Xu JW, Zaporojan VV, et al: Integrative repair of cartilage with articular and nonarticular chondrocytes. Tissue Eng 10:13081315, 2004 41. Isogai N, Kusuhara H, Ikada Y, et al: Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue Eng 12:691-703, 2006 42. von der Mark K, Gauss B, von der Mark H, et al: Relationship between shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature 267:531-532, 1977 43. Habibovic P, Gbureck U, Doillon CJ, et al: Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants. Biomaterials 29:944-953, 2008 44. Atala A, Vacanti JP, Peters CA, et al: Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol 148:658-662, 1992 45. Cohen S, Bano MC, Cima LG, et al: Design of synthetic polymeric structures for cell transplantation and tissue engineering. Clin Mater 13:3-10, 1993 46. Weigel T, Schinkel G, Lendlein A: Design and preparation of polymeric scaffolds for tissue engineering. Expert Rev Med Devices 3:835851, 2006 47. Chetty A, Steynberg T, Moolman S, et al: Hydroxyapatite-coated polyurethane for auricular cartilage replacement: an in vitro study. J Biomed Mater Res A 84:475-482, 2008 48. Rotter N, Haisch A, Bucheler M: Cartilage and bone tissue engineering for reconstructive head and neck surgery. Eur Arch Otorhinolaryngol 262:539-545, 2005 49. Yanaga H, Yanaga K, Imai K, et al: Clinical application of cultured autologous human auricular chondrocytes with autologous serum for craniofacial or nasal augmentation and repair. Plast Reconstr Surg 117:2019-2030; discussion 2031-2032, 2006 50. Yanaga H, Koga M, Imai K, et al: Clinical application of biotechnically cultured autologous chondrocytes as novel graft material for nasal augmentation. Aesthetic Plast Surg 28:212-221, 2004
Reconstruction of auricular cartilage using tissue-engineering techniques Nicole Rotter, MD,a Alexander Steiner, MD, DMD,b and Marc Scheithauer, MDa From the aDepartment of Otorhinolaryngology, Head and Neck Surgery, Ulm University, Ulm, Germany; and the b Department of Maxillofacial Surgery, Ruppiner Clinics, Neuruppin, Germany. KEYWORDS Tissue engineering; Cartilage; Auricle; Plastic-reconstructive surgery
Reconstructive surgery of the nose and the auricle frequently requires grafting from different sites of cartilage as donor material. Typically, grafts for nasal reconstruction are obtained from within the nose whenever possible; alternatively, cartilage can be obtained from the auricle or the rib. Auricular reconstruction procedures usually involve the harvesting of rib cartilage when large parts of the auricle have to be reconstructed. However depending on the underlying disease, harvesting might not be possible to a sufficient degree, eg, after multiple reconstruction efforts or in burn or malformation surgery. Also a severe donor site morbidity has to be taken into account in the case of harvesting rib cartilage. Tissue engineering is an evolving area of research, with the aim of growing tissue in vitro that can be used for reconstructive purposes. This article reviews the current state of the art of tissue engineering procedures of cartilage for reconstruction of the auricle and is determined to answer the question why the technique has not yet found its way into daily clinical routine in otolaryngology in contrast to its performance in orthopaedic surgery. © 2008 Elsevier Inc. All rights reserved.
Defects of nasal or auricular cartilage frequently are congential or caused by trauma or tumor. The surgery of extensive defects is complex and, often, a multistage procedure is required. Even then, functional and cosmetic results are not always satisfactory. Because cartilage lacks an intrinsic healing property, defects need to be treated either by grafting autologous tissue or by implantation of alloplastic material. Alloplastic materials comprise the possible risk of chronic infection and extrusion. Because they are readily available “off the shelf” in the desired shape, some authors favor their use to the use of autologous grafting.1 The use of autologous cartilage as grafting material is considered the gold standard by many authors today.2,3 This tissue can be harvested from the patient in the same operative procedure. The use of autologous cartilage minimizes the risk of infection and extrusion. However, harvesting of suitable donor tissue might not be possible at all or only to a very limited extent, especially if patients have suffered from severe burn injury or have already undergone multiple surgical procedures. A possible future al-
Address reprint requests and correspondence: Nicole Rotter, MD, Department of Otorhinolaryngology, Ulm University, Frauensteige 12, 89075 Ulm, Germany. E-mail address: [email protected]. 1043-1810/$ -see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.otot.2008.10.004
ternative to obtain tissue for reconstructive purposes in head and neck surgery is to in vitro cultivate autologous cartilage by means of tissue engineering. The basic concept of tissue engineering is to isolate autologous cells and to grow them in vitro on a 3-dimensional scaffold in a suitable culture system to construct tissue for transplantation. However, several hurdles have to be cleared before this technology will be ready for routine clinical use in auricular reconstruction.
History and presence of auricular reconstruction As early as 600 BC, physicians have been dealing with the problem of treating structural defects of the auricle. Since about 1890, detailed descriptions of options for surgical therapy are available.4 Around 1930, the necessity to use mechanically stable scaffolds was advocated.5 Since then, the search for an optimal scaffold has been ongoing. Just to name a few, autologous6 and allogeneic7 rib cartilage has been used as well as nasal septal cartilage8 or xenogenic cartilage.9 Furthermore alloplastic materials like ivory,10 silicon,11 Teflon,12 or polyurethane1 have been proposed for auricular reconstruction. In parallel, a multitude of operative
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techniques have been discussed. Today, the surgical methods most frequently applied for total auricular reconstruction are based on descriptions of Brent,13,14 Firmin,15 and Nagata.16-19 Although Brent described a 4-step technique, Nagata summarized the surgical steps in 2 operations. However, modifications might have to be used depending on the individual surgical situation. An alternative treatment option to surgical reconstruction is the use of bone-anchored prostheses.20 In certain cases, such as in aged patients with high comorbidity, this option might be chosen to reduce possible complications related to staged, time-consuming operations. However, there is evidence that surgical rehabilitation enhances psychosocial development in children.21 In surgical reconstruction, rib cartilage nowadays is used most frequently as scaffold. The harvesting of rib cartilage, however, might cause severe comorbidity,22,23 such as pneumothorax, deformities of the thorax, and considerable postoperative pain. Also, in certain cases, eg, after unsuccessful surgical reconstruction, there might not be enough cartilage available to perform the desired reconstruction. In the following section, we will review techniques for reconstruction of traumatic auricular defects in detail.
Reconstruction of traumatic auricular defects An appropriate operation strategy can be determined if both the localization of the defect and its amount of tissue loss is analyzed thoroughly in the preoperative assessment.
279 It is also essential to investigate the quality of the surrounding skin and check for scars, evidence of previous surgery and the hair-line. Central skin defects of the concha and anthelix In case of an intact perichondrium, a full-thickness skin graft is the method of choice. It is harvested in the postauricular region, because of the similar color and texture of the transplanted skin. The cosmetic result is usually very favorable. Central two-layer defects of the concha and anthelix If the defect consists of skin as well as cartilage an island flap, which is pedicled in the postauricular subcutaneous region can be used. Involving the posterior auricular artery the flap might be pedicled caudally or cranially and reaches any location in the concha or anthelix without stressing the vessels. The safety of this flap is remarkable and it heals reliably (Figure 1). Small peripheral defects of the cranial third of the auricle A cutaneous defect can be reconstructed by with the use of a postauricular pedicled transposition flap. In this case, the pedicle has to be detached after 3 weeks, which makes it a 2-stage procedure. If the defect consists of the cartilaginous part of the helix and the overlying skin, a combination of a conchal cartilage transplant and a cranially pedicled
Figure 1 (A) This central 2-layer defect of the concha was caused by resection of a basalioma. (B) The intraoperative view of the reconstructing island flap from the retroauricular region is demonstrated. (Color version of figure is available online.)
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transposition flap is recommended.24 Again, the pedicle needs detachment after some weeks. This technique seems reliable if the defect is not bigger than 2.0 cm in length.
Subtotal or total defects Defects larger than 2.0 cm mostly require a timely-staged reconstruction using autologous rib cartilage. An alternative method is the use of a temporoparietal fascial flap (fan-flap) pedicled on the superficial temporal artery. Its advantage is that it is a one-stage-procedure.25 In case of severely damaged skin in the proximity of the defect, an implantable skin expander helps to enlarge the area of vital skin, thus ensuring safe coverage of the rib cartilage. Approximately 10 to 12 weeks later, the first step of reconstruction can be performed. Subtotal defects. Subtotal defects of the upper two-thirds of the auricle can be reconstructed by means of an axial pattern fan-flap, rib cartilage, and skin grafts (Figure 2). The superficial temporal artery is easily localized preoperatively with a continuous-wave Doppler and marked on the skin. The flap is then harvested in the needed size and rotated caudally. The cartilaginous scaffold is wrapped into the flap and covered with full-thickness grafts. These are taken from
the contralateral postauricular region and from the inguinal area. Total defects. If the auricle is missing completely, a 3-stage procedure is required in most cases.15,26 A template of the contralateral auricle is made preoperatively. According to this template, the cartilaginous scaffold is carved form the explanted sixth, seventh, and eighth rib. The cartilaginous part of the ninth rib is used additionally to rebuild the helix. It is recommended that the scaffold should be created about 3.0 mm smaller than the template in all dimensions. This is important, because the thickness of the overlying skin otherwise produces a rather plump and unnatural looking esthetic result. The scaffold is then placed in a skin pocket. The incision is made cranially to the defect in the hair bearing site. The carved model is connected to the existing ear cartilage with absorbable sutures. A drain is located close to the reconstruction. Its suction molds the skin to the underlying scaffold. In the second stage the auricle is detached from the mastoid skin creating a postauricular groove. For stabilization of the groove a piece of carved cartilage is wrapped into a fascial flap and sutured to the scaffold. A full-thickness skin graft is used to close the skin defect in the mastoid region. During the third stage, minor corrections of the skin and cartilage are performed if necessary.
Figure 2 (A) A subtotal defect due to resection of a basalioma is partially covered with Epigard until final histology is obtained and reconstruction can be performed. (B) Reconstruction was performed using an axial pattern fan-flap, rib cartilage and a free skin graft. Four weeks postoperatively. (Color version of figure is available online.)
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Clinically and economically even more relevant, autologous cartilage grafting is performed in orthopedic and trauma surgery. Because the amount of cartilage donor tissue available for these procedures is limited, cartilage has become one main target in tissue engineering research. In this context, tissue engineering has also been proposed as an option to engineer cartilage in vitro for the use in auricular reconstruction. This option would decrease the surgical comorbidity on one hand and would simplify and shorten the surgical procedures on the other hand. Extensive efforts have been undertaken to create engineered cartilage in the shape of human ears. In the following section, these efforts will be reviewed and own results will be presented, furthermore it will be discussed, why tissue engineered auricles have not yet entered the clinical realm.
Engineering of auricular cartilage in the shape of a human auricle The basic ingredients of cartilage tissue engineering are cells, scaffolds, and a suitable in vitro culture system. The shape of an engineered implant is determined by the shape of the biomaterial provided to host the cells as a scaffold. Most strategies for cartilage tissue engineering are based on resorbable biomaterials as temporary scaffolds for chondrocytes or precursor cells.27 Cells are amplified in vitro, seeded onto the scaffold and then transplanted. Differentiated cells are producing cartilage specific matrix components in vivo. The developing tissue should resemble native cartilage with regard to specific function and morphology. With increasing production of newly synthesized matrix the biomaterials should degrade thus avoiding chronic foreign body reactions and inflammation.
Cellular basis for cartilage tissue engineering Chondrocytes can be isolated from cartilage in an anatomic localization away from the area to be reconstructed, eg, cartilage from the nasal septum, the rib or joints for the reconstruction of an auricular defect. Another option is to isolate precursor cells from a different type of tissue, such as the peripheral blood, fat tissue, or bone marrow. However, these cells need to be cultured under specific conditions to differentiate into the desired cell type.
Cell and tissue culture Successful amplification of human cells and tissue in vitro, determination of differentiation, and maintenance of specific functions are basic requirements for the clinical application of tissue engineering.28 Nutrition of tissue in vivo is guaranteed by the capillary system. Conventional cell culture methods only mimic these conditions to a limited extent, which results in the partial loss of cell- and tissue-specific function.29 Perfusion culture systems have been developed to guarantee a constant supply of fresh nutrients while draining used culture media and toxic metabolites at the same time.30 Perfusion culture systems can be operated in cell incubators at 5% CO2 or at room con-
281 ditions using pH-stabilizing media. Other types of culture systems, like rotating bioreactors, have been developed to promote matrix synthesis and cell differentiation in vitro.31-33 Mechanisms of matrix accumulation and differentiation are still a matter of research.
Choice of cell type When attempting to transfer the clinical routine that uses hyaline rib cartilage for the reconstruction of elastic auricular cartilage, we face the situation that the key question which type of chondrocyte is most suitable for auricular cartilage tissue engineering has not been answered yet. So far, engineering of auricular cartilage has been performed in a few studies only. Successful production of flexible cartilage in the shape of a human ear has been reported with the use of swine auricular chondrocytes and perichondrium34 in nude mice but also in immunocompetent porcine models.35,36 Bovine articular chondrocytes have been used for the same purpose,37 as well as human nasal chondrocytes.38 Comparative studies of chondrocytes of different origin have been performed in porcine models39,40 and a bovine model41; however, they are not intended specifically for auricular cartilage repair. Still, it can be concluded that auricular chondrocytes are a valuable source for cartilage tissue engineering procedures, as they are able to produce matrix with mechanical properties similar or even superior to articular and costal chondrocytes. Although the comparative studies have not been performed with human cells, we tend to conclude that auricular chondrocytes are most likely, whenever available, the cells of choice for auricular reconstruction. On the other hand, clinical conditions that require cell amplification to obtain large transplants from small biopsies have not been taken into account in these studies, thus preventing a clear statement based on these studies. Strong in vitro cell amplification results in the loss of cell specific functions, a process called dedifferentiation.42 The engineering of functional tissue however requires the maintenance of specific functions. These can be maintained by a three-dimensional arrangement of the cells under specific culture conditions.
Stem cell-based tissue engineering Recently the successful isolation of human stem cells from bone marrow, periosteum, and fat tissue was established by different groups. These cells are highly proliferative and are capable of differentiating into different types of tissue. Today, it is possible to amplify mesenchymal stem cells from an adult human for more than 30 passages in vitro thus obtaining more than 1 billion of cells. During amplification, cells maintain their phenotypic characteristics without losing their differentiation capacity, eg, the osteogenic or chondrogenic potential. However, growth factors need to be added to obtain chondrogenic differentiation, thus making the entire process more complex and expensive. Concerning growth factor application, it is not known yet whether this could have any unfavorable or even harmful effects, such as unlimited cell proliferation or tumor
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induction, after in vivo application. Stem cells are a tempting option for cartilage tissue engineering; however, currently differentiated chondrocytes seem the favorable option whenever available. Only broader and more detailed knowledge on stem cell biology might make these cells valuable alternative candidates for cartilage tissue engineering procedures.
showed that alginates are the materials of choice, as pressurefree application is possible in a relatively short time and at low cost and we were able to construct detailed molds (Figure 4) without causing discomfort to the patients. These molds can be used to produce biomaterials and cell-biomaterial constructs in the shape of the respective contralateral ear for tissue engineering purposes.
Shaping of tissue-engineered cartilage
Three-dimensional data acquisition for rapid prototyping techniques
The shape of the engineered cartilage is determined by the shape of the biomaterial. So far, there have been no reports about increase of cartilage volume or shape as compared with the original implant, however significant reduction in volume has been observed, as well as complete resorption of implants. Thus it is necessary either to prevent resorption to a significant degree or to anticipate volume reduction by using implants of larger shape and volume. However, these issues have not been clarified yet. Prevention of resorption can be performed by different strategies, eg, immunoisolation.
Production of individual auricular molds The first step, which will determine the quality in the production of an engineered auricle, is a precise molding and modeling of the individual contralateral auricle. It is necessary to apply the molding material in a precise and pressure-free mode, to take into account the flexible consistency of the auricle. Pressure will lead to distortion of the auricle, which will in turn result in an insufficient model. This is comparable with the situation in mucosa-fixed total dental prosthesis. We developed a simple method to mold the auricle by producing a series of molding shapes (Figure 3). They can be applied in an ambulatory setting on a sitting patient in a short period of time. The application of different materials, such as plaster (Snow White; Kerr, Bioggio, Switzerland), polyether (Impregum; 3M ESPE, Neuss, Germany), polysulfide (Permlastic; Kerr), silicone (EUROSIL and VPS by Henry Schein International, Langen, Germany; Xantopren; Heraeus Kulzer, Dormagen, Germany), hydrocolloids (Hydrocolloid Impression Material by Schein), and alginates (Alginate Type I and II and Alginate Plus by Schein), clearly
Figure 3 Auricular molding shapes in different sizes suitable for various sizes of auricles. (Color version of figure is available online.)
The individual production of molds is time consuming and the quality is closely related to the experience of the applying person. Using touch-free laseroptic scanning of the auricle is an interesting alternative to the aforementioned method. We applied the Vivid 700™ Non-Contact 3D Digitizer (Minolta, Hannover, Germany). A laser with a wavelength of 685 nm and radiation power of 25mW scans the desired object within 0,6 seconds and creates a digital model within 2 seconds. However, this digitizer is not applicable for direct scanning of the auricle of a patient because the object to be digitized needs to be placed on a rotating plate. Direct scanning is of course possible using different types of scanners. A disadvantage is the currently high cost of these scanners. However, the use of centralized systems could solve this problem. The obtained data might be used for the production of 3-dimensional models or scaffolds for tissue engineering with the help of rapid prototyping techniques. These techniques are used in automobile and machine tool engineering routinely and allow the production of materials and models based on digital data. They enable among others to control shape, porosity, pore size, and permeability. Different types of production technologies such as 3-dimensional printing and fused deposition modeling are currently available and seem to be applicable to biomaterials as well. However reports on biomaterials produced by this technology today focus on scaffolds for bone repair.43
Biomaterials for auricular reconstruction An optimal scaffold for tissue engineering procedures should support cell adhesion and cell function44; at the same time, it should degrade into nontoxic substances.45 The mechanical properties should provide initial mechanical strength, degradation should be adjusted to production of newly synthesized matrix by the transplanted chondrocytes. A large variety of biomaterials have been under investigation for cartilage tissue-engineering purposes. Biodegradable polymers in the shape of woven meshes, nonwoven fleeces, or foams composed of polyglycolic or polylactide acid and naturally derived polymers like collagen or hyaluronic acid are used frequently. The use of resorbable materials for rapid-prototyping technologies has been described.46 Nonresorbable materials produced by rapid-prototyping have been proposed for auricular reconstruction.47 Some authors favor the use of internal or external stenting34 to provide initial and mid-term mechanical strength. In summary, neither an optimal material nor a production technology nor an implantation tech-
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Figure 4 (A) The patients auricle was used to determine an optimal molding material. (B) The fabricated model was created using an alginate mold. It precisely reflects the original shape. (Color version of figure is available online.)
nique has been determined for use in auricular reconstruction yet.
Clinical experiences with tissue-engineered auricular cartilage One patient received an tissue engineered auricle at the Department of Otolaryngology at Charité University in Berlin, Germany48 in 1997. The engineered cartilage remained stable in shape for several weeks but was strongly resorbed afterward, leading to an unfavorable cosmetic result. In 2000, researchers at the University of Freiburg, Germany, reported the treatment of a partial traumatic ear defect with tissue engineered cartilage derived from costal chondrocytes. This effort also failed as the construct was completely resorbed after a few months. Until today, the clinical application of tissue-engineered cartilage for auricular reconstruction, in contrast to its orthopedic applications, has not yet been established. There have been case reports of using engineered cartilage for reconstructive nasal surgery.49,50 However, these results need to be confirmed and clarified. Most likely, the unique implantation site and requirements concerning shape and initial mechanical strength are responsible for the so far unfavorable results in the head and neck region. While in orthopedic surgery engineered constructs are placed in the somewhat “immunoprivileged” region of joints, which is not closely connected to the
vascular network, a subcutaneous transplant position in the head and neck region faces strong inflammatory reactions and resorptions. The shape of the transplants needs to be well defined before transplantation in auricular reconstruction with strong initial and long-term mechanical stability, whereas joints defects are preformed cavities to be filled by liquid or semisolid cell preparations, which can obtain mechanical stability during maturation in vivo.
Conclusion and perspectives Tissue engineering enables the fabrication of living and functional cartilage transplants for the nose and the auricle; therefore, it is a possible future alternative to surgical reconstruction techniques based on autologous rib cartilage, avoiding the disadvantages of surgically harvesting large quantities of cartilage. Before this technology will enter the clinical reality of reconstructive head and neck surgery, the optimization of scaffold materials as well as the development of novel scaffolds will be a key issue in reducing inflammatory reactions currently leading to resorption of such transplants in subcutaneous locations. Also, these materials have to be optimized with respect to their mechanical strength and degradation characteristics. A more profound knowledge in stem cell biology and genetic engineering might lead to the applicability of allogenic cells which could finally provide real “off the shelf” transplants.
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