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Tissue engineering and cartilage regeneration for auricular reconstruction
International Journal of Pediatric Otorhinolaryngology (2006) 70, 1507—1515
www.elsevier.com/locate/ijporl
REVIEW ARTICLE
Tissue engineering and cartilage regeneration for auricular reconstruction Andrea Ciorba, Alessandro Martini * Audiology Department, University Hospital of Ferrara, C.so Giovecca 203, Ferrara, Italy Received 14 February 2006; received in revised form 16 March 2006; accepted 21 March 2006
KEYWORDS Regenerative medicine; Stem cells; Auricolar reconstruction; Anotia and microtia; Cartilage; Bio-scaffolds
Summary Objective: This paper will provide (a) a review on current status of auricular reconstruction (b) particularly focusing on the current data about pinna reconstruction using stem cells in combination with tissue engineering. Methods: The paper is divided into two sections. The first section presents a brief overview of the current status of auricular reconstruction. In the second section, the authors review the aspects and the current status of stem cells and tissue engineering researches related to cartilage regeneration. Conclusions: Total auricular reconstruction represents one of the greatest challenges for the ENT and Facial Plastic surgeon. The matter of auricular cartilage reconstruction is complex, and progresses in material designs as well as in stem cells field are essential. Even if this bio-technology field is promising, the progresses still are not adequate as patient expectations remain high. # 2006 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of microtia and anotia . . . . . . . . . . . . . . . . An overview of the current status of auricolar reconstruction 3.1. Costal cartilage graft reconstruction . . . . . . . . . . . . 3.2. Temporoparietal fascia flap . . . . . . . . . . . . . . . . . . 3.3. Temporoparietal fascia flap and alloplastic implants. . . 3.4. Prosthetic aids . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue engineering and stem cells . . . . . . . . . . . . . . . . . . 4.1. Possible cell sources . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Isolated human chondrocytes (IHC) . . . . . . . . 4.1.3. Perichondrial cells . . . . . . . . . . . . . . . . . . . 4.1.4. Periosteum . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +39 0532237451; fax: +39 0532237886. E-mail address: [email protected] (A. Martini). 0165-5876/$ — see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2006.03.013
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A. Ciorba, A. Martini 4.2. Bioartificial tissue scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Scaffold properties and neocartilage and matrix production after 4.2.2. Suitable animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Possible concerns of tissue-engineered reconstruction in humans Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Every year, more than one million patients in the developed world undergo some kind of procedure involving cartilage reconstruction [1]. Adult human cartilage shows a poor capacity for repair and regeneration. Explanations for this include the limited potential for chondrocyte proliferation, the capacity of chondrocytes to become catabolic in response to pathological mediators, and the avascular nature of the tissue. Various surgical procedures have been developed for repairing cartilage defects, but they are highly dependent upon technique and are limited to small lesions. Total auricular reconstruction for congenital microtia or auricular traumatic amputation remains one of the greatest challenges for the ENT surgeon. Although the auricle represents only a minority of the total body surface area, it is one of the most complex three-dimensional structures of the external body. The ability to construct a fully satisfactory complete external ear has been an elusive goal for many years. With the advances both in surgical technique and biotechnology, an expanding range of options is available to the surgeon and the most promising field, with the hope of eventual clinical utility is represented by the bio-engineered cultured cartilage. In fact, so far no perfect materials have been found to substitute the shapely elastic cartilage normally present in the ear. In this paper, the aspects concerning the surgical approach and particularly the problem of the skin flap, will not be discussed. We focused on the reconstruction of the skeleton of the pinna.
2. Epidemiology of microtia and anotia Microtia, a smaller than normal and usually malformed auricle, has a prevalence at birth that varies significantly in different parts of the world between 0.76 per 10,000 births in the French registry to 5.5 in the Southern American. In Northern America and across most of the EU countries, the prevalence of anotia/microtia has an average rate of about 2 per 10,000 births. It has also been reported that there is a racial variability with lower values among whites than with Hispanics and Asians. Also the proportion
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of anotia and microtia varies between races, with the lowest percentage of anotia in whites. Furthermore, anotia and microtia are often associated with other malformations such as facial clefts and cardiac defects (30%), anophthalmia or microphthalmia (14%) and renal malformations (11%). Microtia or anotia are often bilateral and in unilateral cases the right side is more frequently malformed. There is a male excess, most pronounced in isolated forms [2—5]. There is no universally accepted classification system for microtia. One widely adopted system, originally described by Tanzer, and then modified and simplified by Aguilar, assigns a grade from I to III based on the severity of the deformity [6]. Grade I depicts a slightly smaller than normal ear with essentially normal features. Grade II represents an auricle that is rudimentary and malformed but contains some recognizable components. Grade III includes the classic ‘‘peanut’’ ear, which is severely attenuated with a small lump of deformed tissue, and anotia.
3. An overview of the current status of auricolar reconstruction 3.1. Costal cartilage graft reconstruction For many years the most suitable substitute for the auricular skeleton has been the cartilage from some other source. So far, the main body site from which cartilage can be obtained, in necessary quantity and suitable integrity, is the anterior region of the ribs (Fig. 1). It was in 1971 that Tanzer advocated a plan for the pinna total reconstruction, that required four stages for completion, using costal cartilage as a skeletal scaffold. Since that time, many different authors have described modifications of this concept; an excellent description and comparison of the techniques by Tanzer and other authors has been reported by Walton and Breham in 2002, in the second of a twopart review on auricular reconstruction for microtia [7,8]. Also Nagata from Japan, described in a large cohort of patients superb results with autogenous rib reconstruction [9,10]. As a result of these reports, and particularly by the large series by Brent [11,12] (who further refined Tanzer technique), autogenous reconstruction using cartilage frameworks has
Tissue engineering and cartilage regeneration for auricular reconstruction
Fig. 1
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Optimal use of the rib cartilage.
become a standard procedure of pinna reconstructions in US and most European countries. These methods are chosen for their quality, reproducibility and patient satisfaction, but unfortunately are not without drawbacks. Multiple surgical procedures, with the associated anaesthetic risk, are required to complete the reconstruction. Even in experienced hands, results are not always consistent. Donor site morbidity related to the harvest of the rib cartilage includes the risk of pneumothorax, significant initial postoperative pain, scarring, and a visible chest-wall deformity in most patients. These shortcomings have compelled surgeons to continually refine and modify techniques used for microtia reconstruction [13].
3.2. Temporoparietal fascia flap The temporoparietal fascia flap is a dense fascial layer that lies immediately beneath the subcutaneous fat in the temporoparietal region and it is considered an extension of the galea aponeurotica into this area. It may actually be used in a variety of different ways and in combination with other adjacent tissues to accomplish a number of tasks in this region of the head. A series of precautions are necessary with the use of this flap. Considering its proximity to the skin, care must be taken during dissection to minimize trauma and devascularization to the scalp hair
bulbs, thus avoiding some degree of alopecia. Care must also be taken to guarantee that there is no kinking of the pedicle or pressure applied to the vessels. Finally, it is important to remember that the temporal branch of the facial nerve courses beneath the temporoparietal fascia as it crosses over the zygomatic arch [14,45].
3.3. Temporoparietal fascia flap and alloplastic implants In some instances, because of the multiple difficulties associated with collecting, constructing and placing costal grafts and because of the technical difficulties in modulating an adequate temporoparietal fascial flap, a growing number of surgeons is currently working with alloplastic implants as skeletal support for total auricular reconstruction. While silicone implants were initially the most frequently used, they have gradually lost favour due to unacceptably high rates of infection and extrusion. Presently, porous polyethylene is the most widely used implant material; it is relatively easy to shape and its structure is resistant to collapse. Furthermore, porous polyethylene causes minimal tissue reaction, and its porosity allows soft-tissue ingrowths, thereby providing greater stability. Romo and coworkers have been using this method for auricular reconstruction for many years [15,16]. It has been reported that complications with the use
1510 of these implants are relatively rare and exposed polyethylene parts can be managed conservatively, with skin flaps or even secondary intention healing [16].
3.4. Prosthetic aids Another option to consider, particularly in cases of traumatic loss of the pinna, consists of the application of a manufactured auricular prosthesis. As efficiently described by Thorne et al. in their review, a prosthesis minimizes the risks of a surgical procedure and it may give a quite good aesthetic result, if made with excellent shape [17]. The prosthetic pinna may be attached directly onto the skin using a variety of tissue adhesives, or it may be attached to a small metallic button fixed in the temporal bone. In the past, ear prostheses were not tolerated well because of difficulty with retention (especially in maintenance of a proper coloration), skin irritation and corrosion of the prosthesis caused by the chemical adhesive. Other problems could arise from incorrect positioning and insufficient patient’s compliance. Recently, Thorne et al. have outlined a list of relative indications for prosthetic microtia reconstruction [17]. These include (i) failed autogenous reconstruction, (ii) severe soft-tissue/skeletal hypoplasia, and (iii) a low or unfavourable hairline. One advantage of prosthetic reconstruction is a much less involved, usually single operative procedure at the outset of reconstruction.
4. Tissue engineering and stem cells The most exciting and promising progresses in auricular reconstruction come from stem cell research combined with the development of bio-engineered materials. Cartilage tissue has a very slow turnover at cellular and molecular levels and therefore a limited capacity for self-renewal and self-repair. Cartilage tissue is complex and consists of chondrocytes and cartilage specific extracellular matrix (which is mainly composed by collagens and proteoglycans). Elastic cartilage is a mesenchymal derivate and, considering the head and neck region, it can also be found in the epiglottis, in parts of the corniculate and cuneiform cartilages, as well as in the pinna, the external auditory canal and the Eustachian tube. For a successful cartilage tissue regeneration, two tools are necessary: 1. tissue specific cells, and 2. biocompatible carrier scaffolds, by which these cells are supported and can develop.
A. Ciorba, A. Martini The investments in this particular field of research rotate around the development of these two essential tips. On one hand, cells are important for the production of new tissue through their replication and through extracellular matrix synthesis. On the other hand, adequate scaffolds are among the most important prerequisites for a stable histotypic tissue development. To date, numerous synthetic and natural polymers have been tested. Scaffold materials provide short-term mechanical stability of the transplant and also provide a template for spatial growth of the developing tissue.
4.1. Possible cell sources Prerequisite for tissue engineering applications is the successful selection of cells; different biological preparations have been proposed for repair of cartilaginous defects (Table 1). The choice of autologous cells can be preferable to avoid the risks of transmittable disease and to prevent immunologic reactions. In theory, a variety of possible sources are available: 1. 2. 3. 4.
Stem cells (ESC, MSC, UCSC). Human chondrocytes. Perichondrial cells. Periosteum.
4.1.1. Stem cells Stem cells can be described at least by three main features: self-renewal, differentiation into the various cell lineages within that tissue, and then proliferation (the ability of in vivo reconstruction of a given tissue). Stem cells also own plasticity, which describes their property of differentiation into cells other than those expected. 4.1.1.1. Embryonic stem cells (ESC). ESC derive from the inner cell mass of the blastocysts. Because they are the precursors for all other embryonic cells, ESC have the greatest capacity of differentiation into multiple cell types. They give rise to mesoderm, Table 1
Sources of chondrogenic tissue
Stem cells Embryonic Mesenchymal Umbilical Freshly isolated or expanded human chondrocytes Perichondrium Periosteum Cartilage tissue autograft
Tissue engineering and cartilage regeneration for auricular reconstruction endoderm, ectoderm, and are therefore termed pluripotent. The generation of specific cell types by directing ESC differentiation hypothetically offers extensive resources for developing clinical applications to replace injured tissues. ESC lines from human fetal tissues were firstly established in 1998 [18]. The ESC use still represent one of the most controversial topic discussed in scientific symposia, and despite their great potentiality, practical and ethical questions currently delay much ESC research. There are no literature reports of ESC used for auricular cartilage reconstruction so far. 4.1.1.2. Mesenchimal stem cells (MSC). MSC are adult stem cells. They represent a subset of nonhematopoietic cells contained in the adult bone marrow stroma that have the capacity to undergo extensive replication. They also have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues including bone, fat, tendon, muscle, marrow stroma and cartilage (multipotency). These cells represent an attractive cell source for tissue engineering approaches. In vitro expanded MSC combined with scaffolds have been successfully implanted directly or after specific stimulation [19]. The potential advantage of using MSC includes low cell numbers required at the initial culture, relative simple and already clinically used procedures for bone marrow harvest. Moreover it has been reported that MSC from older donors also maintain high biological activity [19,20]. Further investigations, especially using animal models, have been advocated. 4.1.1.3. Umbilical cord stem cells (UCSC). Ovine mesenchimal progenitor cells also have been isolated from cord blood samples, expanded and seeded onto bio-scaffolds of polyglicolic acid. After 12 weeks of culture the constructs exhibited a chondrogenic differentiation revealed by both standard and matrix-specific staining. These results with UCSC are encouraging and more resources and studies have been claimed [21]. 4.1.2. Isolated human chondrocytes (IHC) The use of autologous chondrocytes is another promising potential field of research. The pioneering work by Vacanti and Vacant demonstrated that bovine chondrocytes seeded onto synthetic biodegradable scaffolds could produce new cartilage tissue after transplantation into athymic mice [22]. Further investigations verified the ability of human chondrocytes isolated from hyaline cartilage to replicate and to generate new matrix in vitro [23]. Major problems encountered with IHC cultures are represented by:
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Chondrocytes dedifferentiation. The small number of chondrocytes initially isolated from a cartilage biopsy must be firstly expanded. Using a monolayer culture medium, these cells loose their phenotype and turn into fibroblasts (dedifferentiation process). Collagen type I synthesis. As a consequence of the dedifferentiation process, in the culture medium there is a switch from collagen type II to collagen type I synthesis. New culture systems have already been developed; the results obtained by Naumann et al. using bio-artificial ‘‘three-dimensional’’ cultures are particularly interesting [24]. Serum factors and various growth factors have been shown to modulate cartilage matrix synthesis in 3D cultures, especially fibronectin [25], insulin-like growth factor-I (IGF-I; 26), transforming growth factor b (TGF-b [26]), and platelet-derived growth factor (PDGF [27]). The possible applications using IHC in vivo must be cleared [23] and more research is advocated; conditions that favour maintenance of the phenotype are not usually those that favour an increase in numbers [28]. As a result, there may be limitations in the numbers of suitable cells that can be grown in vitro for subsequent repair of cartilage defects. 4.1.3. Perichondrial cells Perichondral progenitor cells can be harvested from perichondrial tissues, isolated, cultured and guided into a transdifferentiation process using peculiar factors like TGF B2 and IGF 1. However, their yield rate is low and the amount of the new formed matrix is limited. There are reports of perichondrial grafts using a combination of different biomaterials [29—31]. Matsuda et al. [29] implanted a collagen sponge enwrapped in perichondrium subcutaneously in the back’s of rabbits, after 7 weeks showing new cartilage formation in 10 of 21 explants. Ruuskanen et al. [30] implanted a combined graft of polyglycolic acid and perichondrium in a muscular pouch demonstrating new cartilage formation in 7 of 8 implants. Ten Coppel et al. used de-mineralized bovin bone matrix enveloped by perichondrial cells for cartilage regeneration, showing a small amount of new cartilage generation when the site of implantation is well vascularized [31,32]. Further investigations have been advocated. 4.1.4. Periosteum Periosteum is more plentiful than perichondrium in adults; Ham’s classic studies in fracture healing pointed to the periosteum as the source of neochondrogenesis in callus tissue [33]. Transplanted
1512 autogenous periosteum produces an admixture of new bone and cartilage, probably depending upon vascularity of the microenvironment in the recipient bed [34]. Early attempts to resurface cartilaginous defects with transplanted periosteum have had little success so far [35].
4.2. Bioartificial tissue scaffolds 4.2.1. Scaffold properties and neocartilage and matrix production after seeding The development of adequate scaffolds is necessary for successful cartilage tissue regeneration; also the quality of cartilage tissue produced can be influenced by the carrier-materials, as the scaffold may regulate the seeded cell yield rate. In our opinion, in an ideal project, either in vitro or in vivo, the carrier material should provide several characteristics: Mechanical stability. Ensure a three-dimensional support for cell development and expansion. Biodegradability. After prolonged culture, as tissue develop, the newly formed extracellular matrix should firstly be substituted and then replaced by the artificial carrier. Shape. It is important that the transplants do not change their form and at the same show time elasticity as that of the normal ear. Biocompatibility. It is extremely important to control and manage the immunocompetent host response, after implantation of the bio-prosthesis. Other scaffold properties should be: Guarantee of uniform cell distribution. Avoid influence on seeded cell phenotype [24]. The development of 3D scaffolds have been particularly useful to define the space for the new tissue, and, potentially, to enhance the maturation and function of the regenerated tissue. Candidate scaffolds or matrices include natural polymeric materials, synthetic polymers, biodegradable polymers, and polymers with adsorbed proteins or immobilized functional groups. Details of the manufacturing and properties of the synthetic materials have been reviewed. Bio-materials Small intestinal submucosal graft ‘‘SurgiSIS’’. This is an acellular, freeze-dried soft tissue graft derived from porcine small intestinal submucosa. SIS has already been successfully used as an interpositional graft in the repair of nasal
A. Ciorba, A. Martini septal perforations. In the study by Pribitkin et al. this material have been interposed following excision of rabbit auricular cartilage, showing that some sort of cartilage regeneration was only possible in presence of adjacent host cartilage or perichondrium. These results justify further animal and then human studies [36]. De-mineralized bovine bone matrix (DBM). In vitro tests using cells seeded on DBM demonstrated islets of vital chondrocytes surrounded by newly generated cartilage matrix. More in vivo experiments are necessary, only fibrous tissue was detected when DBM had been implanted in the rabbit pinna [37]. Polymeric scaffolds Synthetic polymers have theoretical advantages regarding plentiful supply, precise control of composition and material properties, and possibilities of biocompatible and reabsorbable features. Polymers comprise two major categories: those that are permanent and those that are temporary because they are reabsorbed by the body. The key requirements of bioreabsorbable materials are that (i) their rates of degradation must be compatible with the intended use and (ii) the products of their degradation must be nontoxic. Of the synthetic materials, polyglycolic acid (PGA), is the most widely studied. Polyglycolic acid (PGA), poly-e-caprolactone (PCL), poly-4-hydroxybutyrate (P-4HB) are commonly used polymeric scaffolds. Prior to seeding, these scaffolds must be pre-treated by adding growth factors and then placing the constructs in suitable incubators (surface conditioning). Extracellular matrix formation with sparse chondrocytes have been reported. Particularly, after 8 weeks of in vitro culture the glycosaminoglycan content significantly increased and the type II collagen quote in the construct reached 40% [38]. Hyaluronic based biomaterials HYAFF 11. This is a polymer derived from the esterification of sodium hyaluronate. It is composed of nonwoven fibres and a membrane of hyaluronic acid benzyl ester. It has been reported that one of the advantages of this material is the good cell adhesiveness, even without prior surface conditioning; when combined with chondrocytes, the construct seems to acquire a stable structure and a firm consistency similar to that of cartilage, which confirmed that the cultured cells had retained their chondrogenic potential. The histochemical staining with azan-blue demonstrated the occurrence of collagen fibres between the scaffold fibres. These
Tissue engineering and cartilage regeneration for auricular reconstruction preliminary in vivo findings support the idea that the biomaterial did not negatively interfere, but on the contrary, had a relatively favourable effect on matrix production [38]. 4.2.1.1. Mechanisms of in vitro chondrogenesis. Several attempts have been made in order to understand the exact mechanism that regulate, especially at the very beginning, the in vitro cellular aggregation and the cellular interactions with bio-scaffold. It has been observed that during the in vitro engineering of cartilage (process also defined by the term of ‘‘artificial mesenchymal condensation’’) the seeded cells (SC or IHC) initially adhere to the bio-scaffold forming aggregates as an attempt to recreate the embryonic ‘‘mesenchymal condensation process’’ required for chondrogenesis [39,40]. Studying the cellular response to nano- and micrometric topographies, it has been observed that these cellular aggregates collided each other migrating along the direction of the bio-scaffold’s groove long axis [39,40]. Furthermore, it is already clear that several serum factors and various growth factors, especially fibronectin [25], insulin-like growth factor-I (IGF-I [26]), transforming growth factor b (TGF-b [26]), and platelet-derived growth factor (PDGF [27]) can modulate cartilage matrix synthesis in cultures. Also insoluble factors modulate cell behaviour in cultures; this understanding is founded on the appreciation among developmental biologists that extracellular matrix components influence the behaviour of the cells that secrete them and on the subsequent discovery of cell membrane receptors for extracellular matrix molecules. The integrin receptors for protein motifs (or ligands) are the best studied, but another category of proteoglycan receptors includes CD44, which mediates chondrocyte binding to hyaluronan [41]. More studies have been claimed in order to understand the molecular mechanisms that regulate the intra-cellular interactions and the neo-cartilage formation processes. 4.2.2. Suitable animal models The choice of an appropriate animal model is not so simple; it is important to match the model to the question being investigated. Several animal models have already been used to evaluate the remodelling process of tissue-engineered constructs in vivo, as well as the inflammatory and immunological responses by the host immune system. Mice are the favoured model, rabbit the second favoured: they both bear the advantages of small animal models, including low cost, ready availability and less ethical concerns compared to other animal (especially primate).
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Particularly nude mice have been used, as they represent an immunocompromized model of ideal choice for the evaluation of the in vivo remodelling process of the tissue-engineered auricle. Their skin is quite thin, pliable and hairless, mimicking the human auricular skin, presenting a fine three dimensional ear definition after implantation [20]. Possible reported drawbacks using nude mice came from [20]: The lack of functional T-cells to evaluate cellular responses. The animal is too small to implant the life-sized ear to mimick the construction against soft tissue contraction. The limited life length, that prevents the tissueengineered ear from long-term follow-up. Ten months is the longest follow-up ever reported so far with this model. It has been reported that several scaffolds progressively disappear in the follow-up observation after implantation. Still more studies are required for detecting the fine mechanisms of inflammatory reactions that might destroy the chondrocites against the scaffold, and subsequently deform the shape and impair the quality of the implanted tissue [20]. 4.2.3. Possible concerns of tissue-engineered reconstruction in humans It is important to remember that the clinical application of these bio-engineered construct still is faraway. Possible issues are represented by the following: Plasticity. It is represented by the difficulty in producing great variations of size and shape of the bio-scaffold, necessary to match the opposite, normal ear. At the same time the construct should be stiff enough to contrast the possible scaffold’s shape deformation over time by the taught human skin. Computer-aided designs have been proposed for the manufacturing of matching, suitable bioscaffolds before the cell-seeding phase (also see three-dimensional stereolithograpy [37]). Failure-extrusion/absorption. As mentioned above tissue-engineered frameworks should own an elevate biocompatibility. Uncontrolled cellular proliferation. Doubts regarding the possible application of constructs with stem cells came from the possible uncontrolled proliferation of stem cells, or from an abnormal cartilage matrix synthesis (stem cells are likely candidates for accumulating multiple mutations that can disrupt their tight control leading to tumorogenesis).
1514 Infections and cutaneous dehiscences. Once incorporated tissue-engineered frameworks should be quite resistant to trauma and infections. Lacerations that expose the implant or infections should respond to antibiotic treatments. Instead, alloplastic implants for example, can be easily dislodged or even lost in case of flogosis [12]. Nature of tissue-engineered frameworks. Auricular tissue-engineered reconstruction once completed should not require further interventions. In contrast, costal cartilage graft reconstruction require multiple surgical procedures and also auricular reconstruction with alloplastic implants needs prosthesis replacement every 3—5 years [13]. Morbidity. In the case of an auricular tissue-engineered reconstruction, only a small piece of cartilage (i.e. obtained from a small biopsy) would be necessary for chondrocytes extraction (even if no biopsy could be required in case of stem cells use). Consequently much more elevated would be the morbidity associated with the care of an alloplastic implant reconstruction or with an autogenous costal cartilage graft reconstruction considering the multiple surgical procedures (including the thorax harvest) [12]. Possible bio-ethical limitations. Stem cells and their applications are exciting to physicians, scientists and patients because of their potential and their possible use to treat large numbers of patients with a variety of diseases. Even if this science is still at a very early stage, legislators around the world are attempting to enact legislation to govern the development of stem cell research and its potential applications. They are doing this in a worried and evolving global regulatory environment concerning stem cell research (embryonic SC particularly) that varies from the permissive (United Kingdom), to the restrictive (Germany, Italy), to the apparently conservative (United States). The formulation of a policy and a legislation in this area requires a careful understanding of this science because the moral considerations involved are complex and delicate. A further challenge is that the common terms used in this field (embryonic, stem cells or regenerative medicine) have strong emotional associations linked to the debate about the beginning of life [42—44].
5. Conclusions and future directions It is evident that the matter of cartilage reconstruction remains complex: although promising, microtia reconstruction using a tissue-engineered,
A. Ciorba, A. Martini prefabricated framework is not yet a practical option. It is conceivable that in a near future genetically modified cells or stem cells (embryonic or adult) could be grown on a biocompatible scaffold with internal signals for programmed histogenesis. Advances in material design may generate ‘‘smart’’ scaffolds that will control tissue topology and have surface modifications to stimulate and control cell adhesion, differentiation, and growth. Composite engineered tissues or organs, like a bio-engineered ear, look as a future goal. Cross-disciplinary interactions are likely to accelerate progress and to mediate application of advances made in other fields to consistently successful in vitro engineering of cartilage for all clinical needs.
References [1] S. Chang, G. Tobias, A. Roy, C. Vacanti, L. Bonassar, Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding, Plast. Reconstr. Surg. 112 (3) (2003) 793—799. [2] G.M. Shaw, S.L. Carmichael, Z. Kaidarova, A. Harris, Epidemiologic characteristics of anotia and microtia in California, 1989—1997, Birth Defects Res., Part A 70 (2004) 472—475. [3] J. Harris, B. Kallen, E. Robert, The epidemiology of anotia and microtia, J. Med. Gen. 33 (10) (1996) 809—813. [4] P. Mastroiacovo, C. Corchia, L.D. Botto, R. Lanni, G. Zampino, D. Fusco, Epidemiology and genetics of microtia— anotia: a registry based study on over one million births, J. Med. Gen. 32 (6) (1995) 453—457. [5] O. Sancez, J.R. Mendez, E. Gomez, D. Guerra, Clinical-epidemiologic study of microta, Invest. Clin. 38 (4) (1997) 203—217. [6] E.F. Aguilar, Auricular reconstruction in congenital anomalies of the ear, Facial Plast. Surg. Clin. North Am. 9 (2001) 159—169. [7] R. Walton, E. Beham, Auricolar reconstruction for microtia. Part I. Anatomy, embryology and clinical evaluation, Plast. Reconstr. Surg. 109 (2002) 2473—2482. [8] R. Walton, E. Beham, Auricolar reconstruction for microtia. Part II. Surgical techniques, Plast. Reconstr. Surg. 110 (2002) 234—249. [9] S. Nagata, 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 (1994) 221—230. [10] S. Nagata, 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 (1994) 231—242. [11] B. Brent, Microtia repair with rib cartilage grafts: a review of personal experience with 1000 cases, Clin. Plast. Surg. 29 (2002) 257—271. [12] B. Brent, Technical advances in ear reconstruction with autogenous rib cartilage grafts: personal experience with 1200 cases, Plast. Recon. Surg. 104 (1999) 319—334. [13] S.A. Zim, Microtia reconstruction: an update, Curr. Opin. Otol. Head Neck Surg. 11 (2003) 275—281. [14] K. Olson, S. Manolidis, The pedicle superficial temporalis fascial flap: a new method for reconstruction in otologic surgery, Otolaryngol. Head Neck Surg. 126 (2002) 538—547.
Tissue engineering and cartilage regeneration for auricular reconstruction [15] T. Romo, M. Fozo, A. Sclafani, Microtia reconstruction using a porous polyethylene framework, Facial Plast. Surg. 126 (2000) 538—547. [16] W. James, T. Romo, A. Sclafani, H. Cho, Porous high-density polyethylene implants in auricular reconstruction, Arch. Otolaryngol. Head Neck. 123 (1997) 578—583. [17] C.H. Thorne, L.E. Brecht, J.P. Bradley, Auricular reconstruction: indications for autogenous and prosthetic techniques, Plast. Reconstr. Surg. 107 (2001) 1241—1252. [18] J.A. Thomson, J. Itskovits-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, Embryonic stem cell lines derived from human blastocyst, Science 282 (1998) 1145—1147. [19] J. Ringe, C. Kaps Gerd-Rudiger, M. Sittinger, Stem cells for regenerative medicine: advances in the engineering of tissues and organs, Naturwissenschaften 89 (2002) 338—351. [20] S.J. Shieh, S. Terada, J.P. Vacanti, Tissue engineering auricolar reconstruction: in vitro and in vivo studies, Biomaterials (2004) 1545—1557. [21] J.R. Fuchs, D. Hannouche, S. Terada, S. Zand, J.P. Vacanti, D.O. Fauza, Cartilage engineered from ovine umbilical cord blood mesenchymal progenitor cells, Stem Cells 23 (7) (2005) 958—964. [22] C.A. Vacanti, J.P. Vacant, Bone and cartilage reconstruction with tissue engineering approaches, Otolaryngol. Clin. North Am. 24 (1994) 263—276. [23] A. Rodriguez, Y.L. Cao, C. Ibarra, S. Pap, M. Vacanti, R. Eavey, C. Vacanti, Characteristics of cartilage engineered from human pediatric auricular cartilage, Plast. Reconstr. Surg. 103 (1999) 1111—1119. [24] A. Naumann, J.E. Dennis, J. Aigner, J. Coticchia, J. Arnold, A. Berghaus, E.R. Kastenbauer, A.I. Caplan, Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system, Tiss. Eng. 10 (11) (2004) 1695—1706. [25] J.A. Martin, J.A. Buckwalter, Effects of fibronectin on articular cartilage chondrocytes proteoglycan synthesis and response to insulin-like growth factor I, J. Orthop. Res. 16 (1998) 752—757. [26] G.J. van Osch, W.B. van den Berg, E.B. Hunziker, H.J. Hauselmann, Differential effects of IGF-I and TGF beta-2 on the assembly of proteoglycans in pericellular and territorial matrix by cultured bovine articular chondrocytes, Osteoarthritis Cartilage 6 (1998) 187—195. [27] L. Weiser, M. Bhargava, E. Attia, P.A. Torzilli, Effect of serum and platelet-derived growth factor on chondrocytes grown in collagen gels, Tiss. Eng. 5 (1999) 533—544. [28] J. Glowacki, E. Trepman, J. Folkman, Cell shape and phenotypic expression in chondrocytes, Proc. Soc. Exp. Biol. Med. 172 (1983) 93—98. [29] K. Matsuda, N. Nagasawa, S. Suzuki, N. Isshiki, Y. Ikada, In vivo chondrogenesis in collagen sponge sandwiched by perichondrium, J. Biomater. Sci. Polym. Ed. 7 (1995) 221. [30] M. Ruuskanen, M.K. Virtanen, H. Tuominen, P. Tormala, T. Waris, Generation of cartilage from auricolar and rib free perichondrial grafts around self-reinforced polyglycolic acid mould in rabbits, Scand. J. Plast. Reconstr. Surg. 28 (1994) 81.
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[31] P.J. Ten Coppel, G.J. van Osch, C.D. Verwored, H.L. Verwoerd-Verhoef, Efficacy of perichondrium and trabecular demineralized bone matrix for generating cartilage, Plast. Reconstr. Surg. 102 (6) (1998) 2012—2020. [32] P.J. Ten Coppel, G.J. van Osch, C.D. Verwored, H.L. Verwoerd-Verhoef, A new in vivo model for testing cartilage grafts and biomaterials: the ‘‘rabbit pinna punch-hole’’ model, Biomaterials 22 (2001) 1407—1414. [33] A.W. Ham, A histological study of the early phases of bone repair, J. Bone Joint Surg. 12 (1930) 827—844. [34] J. Cohen, P. Lacroix, Bone and cartilage formation by periosteum: assay of experimental autogenous grafts, J. Bone Joint Surg. 37A (1955) 717—730. [35] J.M. Ruback, Reconstruction of articular cartilage defects with free periosteal grafts: an experimental study, Acta Orthop. Scand. 53 (1982) 175—180. [36] E.A. Pribitkin, B.T. Ambro, E. Bloeden, B.J. O’Hara, Rabbit ear cartilage regeneration with a small intestinal submucosa graft, Laryngoscope 114 (Suppl.) (2004) 1—19. [37] A. Naumann, J. Aigner, et al., Clinical aspects and strategy for biomaterial engineering of an auricle based on threedimensional stereolithography, Eur. Arch. Otorhinolaryngol. 260 (2003) 568—575. [38] J. Aigner, J. Tegeler, P. Hutzler, D. Campoccia, A. Pavesio, C. Hammer, E. Kastenbauer, A. Naumann, Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic benzyl ester, J. Biomed. Mater. Res. 42 (2) (1998) 172—181. [39] D.W. Hamilton, M.O. Riehle, W. Monaghan, A.S. Curtis, Chondrocyte aggregation on micrometric surface topography: a time-lapse study, Tiss. Eng. 12 (2006) 1—11. [40] V. Vunjak-Novakovic, B. Obradovic, I. Martin, P.M. Bursac, R. Langer, L.E. Freed, Dynamic cell seeding of polymer scaffold for cartilage tissue engineering, Biotechnol. Prog. 14 (1998) 193—199. [41] G. Vunjak-Novakovic, I. Martin, B. Obradovic, S. Treppo, A.J. Grodzinsky, R. Langer, et al., Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage, J. Orthop. Res. 17 (1999) 130—138. [42] A.S. Daar, L. Sheremeta, The science of stem cells: ethical, legal and social issues, Exp. Clin. Transplant. 1 (2) (2003) 139—146. [43] National Academies Committee, Biological and Biomedical Application of Stem Cell Research, Stem Cells and the Future of Regenerative Medicine, The National Academic Press, Washington, 2002 http://books.nap.edu/books/ 0309076307/html/8.html. [44] Study Commission on Law and Ethics in Modern Medicine, Summary Report, Supplement to the interim report on stem cell research focussing on importation problems, Research in imported human embryonic stem cells, November 12, 2001. Online version: http://www.bundestag.de/gremien/medi/ medi_2zwisch.html. Last visited March 2, 2004. [45] G. Renner, L. Robert, Auricular reconstruction: an update, Curr. Opin. Otol. Head Neck Surg. 12 (2004) 277—280.
www.elsevier.com/locate/ijporl
REVIEW ARTICLE
Tissue engineering and cartilage regeneration for auricular reconstruction Andrea Ciorba, Alessandro Martini * Audiology Department, University Hospital of Ferrara, C.so Giovecca 203, Ferrara, Italy Received 14 February 2006; received in revised form 16 March 2006; accepted 21 March 2006
KEYWORDS Regenerative medicine; Stem cells; Auricolar reconstruction; Anotia and microtia; Cartilage; Bio-scaffolds
Summary Objective: This paper will provide (a) a review on current status of auricular reconstruction (b) particularly focusing on the current data about pinna reconstruction using stem cells in combination with tissue engineering. Methods: The paper is divided into two sections. The first section presents a brief overview of the current status of auricular reconstruction. In the second section, the authors review the aspects and the current status of stem cells and tissue engineering researches related to cartilage regeneration. Conclusions: Total auricular reconstruction represents one of the greatest challenges for the ENT and Facial Plastic surgeon. The matter of auricular cartilage reconstruction is complex, and progresses in material designs as well as in stem cells field are essential. Even if this bio-technology field is promising, the progresses still are not adequate as patient expectations remain high. # 2006 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of microtia and anotia . . . . . . . . . . . . . . . . An overview of the current status of auricolar reconstruction 3.1. Costal cartilage graft reconstruction . . . . . . . . . . . . 3.2. Temporoparietal fascia flap . . . . . . . . . . . . . . . . . . 3.3. Temporoparietal fascia flap and alloplastic implants. . . 3.4. Prosthetic aids . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue engineering and stem cells . . . . . . . . . . . . . . . . . . 4.1. Possible cell sources . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Isolated human chondrocytes (IHC) . . . . . . . . 4.1.3. Perichondrial cells . . . . . . . . . . . . . . . . . . . 4.1.4. Periosteum . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +39 0532237451; fax: +39 0532237886. E-mail address: [email protected] (A. Martini). 0165-5876/$ — see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2006.03.013
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A. Ciorba, A. Martini 4.2. Bioartificial tissue scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Scaffold properties and neocartilage and matrix production after 4.2.2. Suitable animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Possible concerns of tissue-engineered reconstruction in humans Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Every year, more than one million patients in the developed world undergo some kind of procedure involving cartilage reconstruction [1]. Adult human cartilage shows a poor capacity for repair and regeneration. Explanations for this include the limited potential for chondrocyte proliferation, the capacity of chondrocytes to become catabolic in response to pathological mediators, and the avascular nature of the tissue. Various surgical procedures have been developed for repairing cartilage defects, but they are highly dependent upon technique and are limited to small lesions. Total auricular reconstruction for congenital microtia or auricular traumatic amputation remains one of the greatest challenges for the ENT surgeon. Although the auricle represents only a minority of the total body surface area, it is one of the most complex three-dimensional structures of the external body. The ability to construct a fully satisfactory complete external ear has been an elusive goal for many years. With the advances both in surgical technique and biotechnology, an expanding range of options is available to the surgeon and the most promising field, with the hope of eventual clinical utility is represented by the bio-engineered cultured cartilage. In fact, so far no perfect materials have been found to substitute the shapely elastic cartilage normally present in the ear. In this paper, the aspects concerning the surgical approach and particularly the problem of the skin flap, will not be discussed. We focused on the reconstruction of the skeleton of the pinna.
2. Epidemiology of microtia and anotia Microtia, a smaller than normal and usually malformed auricle, has a prevalence at birth that varies significantly in different parts of the world between 0.76 per 10,000 births in the French registry to 5.5 in the Southern American. In Northern America and across most of the EU countries, the prevalence of anotia/microtia has an average rate of about 2 per 10,000 births. It has also been reported that there is a racial variability with lower values among whites than with Hispanics and Asians. Also the proportion
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of anotia and microtia varies between races, with the lowest percentage of anotia in whites. Furthermore, anotia and microtia are often associated with other malformations such as facial clefts and cardiac defects (30%), anophthalmia or microphthalmia (14%) and renal malformations (11%). Microtia or anotia are often bilateral and in unilateral cases the right side is more frequently malformed. There is a male excess, most pronounced in isolated forms [2—5]. There is no universally accepted classification system for microtia. One widely adopted system, originally described by Tanzer, and then modified and simplified by Aguilar, assigns a grade from I to III based on the severity of the deformity [6]. Grade I depicts a slightly smaller than normal ear with essentially normal features. Grade II represents an auricle that is rudimentary and malformed but contains some recognizable components. Grade III includes the classic ‘‘peanut’’ ear, which is severely attenuated with a small lump of deformed tissue, and anotia.
3. An overview of the current status of auricolar reconstruction 3.1. Costal cartilage graft reconstruction For many years the most suitable substitute for the auricular skeleton has been the cartilage from some other source. So far, the main body site from which cartilage can be obtained, in necessary quantity and suitable integrity, is the anterior region of the ribs (Fig. 1). It was in 1971 that Tanzer advocated a plan for the pinna total reconstruction, that required four stages for completion, using costal cartilage as a skeletal scaffold. Since that time, many different authors have described modifications of this concept; an excellent description and comparison of the techniques by Tanzer and other authors has been reported by Walton and Breham in 2002, in the second of a twopart review on auricular reconstruction for microtia [7,8]. Also Nagata from Japan, described in a large cohort of patients superb results with autogenous rib reconstruction [9,10]. As a result of these reports, and particularly by the large series by Brent [11,12] (who further refined Tanzer technique), autogenous reconstruction using cartilage frameworks has
Tissue engineering and cartilage regeneration for auricular reconstruction
Fig. 1
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Optimal use of the rib cartilage.
become a standard procedure of pinna reconstructions in US and most European countries. These methods are chosen for their quality, reproducibility and patient satisfaction, but unfortunately are not without drawbacks. Multiple surgical procedures, with the associated anaesthetic risk, are required to complete the reconstruction. Even in experienced hands, results are not always consistent. Donor site morbidity related to the harvest of the rib cartilage includes the risk of pneumothorax, significant initial postoperative pain, scarring, and a visible chest-wall deformity in most patients. These shortcomings have compelled surgeons to continually refine and modify techniques used for microtia reconstruction [13].
3.2. Temporoparietal fascia flap The temporoparietal fascia flap is a dense fascial layer that lies immediately beneath the subcutaneous fat in the temporoparietal region and it is considered an extension of the galea aponeurotica into this area. It may actually be used in a variety of different ways and in combination with other adjacent tissues to accomplish a number of tasks in this region of the head. A series of precautions are necessary with the use of this flap. Considering its proximity to the skin, care must be taken during dissection to minimize trauma and devascularization to the scalp hair
bulbs, thus avoiding some degree of alopecia. Care must also be taken to guarantee that there is no kinking of the pedicle or pressure applied to the vessels. Finally, it is important to remember that the temporal branch of the facial nerve courses beneath the temporoparietal fascia as it crosses over the zygomatic arch [14,45].
3.3. Temporoparietal fascia flap and alloplastic implants In some instances, because of the multiple difficulties associated with collecting, constructing and placing costal grafts and because of the technical difficulties in modulating an adequate temporoparietal fascial flap, a growing number of surgeons is currently working with alloplastic implants as skeletal support for total auricular reconstruction. While silicone implants were initially the most frequently used, they have gradually lost favour due to unacceptably high rates of infection and extrusion. Presently, porous polyethylene is the most widely used implant material; it is relatively easy to shape and its structure is resistant to collapse. Furthermore, porous polyethylene causes minimal tissue reaction, and its porosity allows soft-tissue ingrowths, thereby providing greater stability. Romo and coworkers have been using this method for auricular reconstruction for many years [15,16]. It has been reported that complications with the use
1510 of these implants are relatively rare and exposed polyethylene parts can be managed conservatively, with skin flaps or even secondary intention healing [16].
3.4. Prosthetic aids Another option to consider, particularly in cases of traumatic loss of the pinna, consists of the application of a manufactured auricular prosthesis. As efficiently described by Thorne et al. in their review, a prosthesis minimizes the risks of a surgical procedure and it may give a quite good aesthetic result, if made with excellent shape [17]. The prosthetic pinna may be attached directly onto the skin using a variety of tissue adhesives, or it may be attached to a small metallic button fixed in the temporal bone. In the past, ear prostheses were not tolerated well because of difficulty with retention (especially in maintenance of a proper coloration), skin irritation and corrosion of the prosthesis caused by the chemical adhesive. Other problems could arise from incorrect positioning and insufficient patient’s compliance. Recently, Thorne et al. have outlined a list of relative indications for prosthetic microtia reconstruction [17]. These include (i) failed autogenous reconstruction, (ii) severe soft-tissue/skeletal hypoplasia, and (iii) a low or unfavourable hairline. One advantage of prosthetic reconstruction is a much less involved, usually single operative procedure at the outset of reconstruction.
4. Tissue engineering and stem cells The most exciting and promising progresses in auricular reconstruction come from stem cell research combined with the development of bio-engineered materials. Cartilage tissue has a very slow turnover at cellular and molecular levels and therefore a limited capacity for self-renewal and self-repair. Cartilage tissue is complex and consists of chondrocytes and cartilage specific extracellular matrix (which is mainly composed by collagens and proteoglycans). Elastic cartilage is a mesenchymal derivate and, considering the head and neck region, it can also be found in the epiglottis, in parts of the corniculate and cuneiform cartilages, as well as in the pinna, the external auditory canal and the Eustachian tube. For a successful cartilage tissue regeneration, two tools are necessary: 1. tissue specific cells, and 2. biocompatible carrier scaffolds, by which these cells are supported and can develop.
A. Ciorba, A. Martini The investments in this particular field of research rotate around the development of these two essential tips. On one hand, cells are important for the production of new tissue through their replication and through extracellular matrix synthesis. On the other hand, adequate scaffolds are among the most important prerequisites for a stable histotypic tissue development. To date, numerous synthetic and natural polymers have been tested. Scaffold materials provide short-term mechanical stability of the transplant and also provide a template for spatial growth of the developing tissue.
4.1. Possible cell sources Prerequisite for tissue engineering applications is the successful selection of cells; different biological preparations have been proposed for repair of cartilaginous defects (Table 1). The choice of autologous cells can be preferable to avoid the risks of transmittable disease and to prevent immunologic reactions. In theory, a variety of possible sources are available: 1. 2. 3. 4.
Stem cells (ESC, MSC, UCSC). Human chondrocytes. Perichondrial cells. Periosteum.
4.1.1. Stem cells Stem cells can be described at least by three main features: self-renewal, differentiation into the various cell lineages within that tissue, and then proliferation (the ability of in vivo reconstruction of a given tissue). Stem cells also own plasticity, which describes their property of differentiation into cells other than those expected. 4.1.1.1. Embryonic stem cells (ESC). ESC derive from the inner cell mass of the blastocysts. Because they are the precursors for all other embryonic cells, ESC have the greatest capacity of differentiation into multiple cell types. They give rise to mesoderm, Table 1
Sources of chondrogenic tissue
Stem cells Embryonic Mesenchymal Umbilical Freshly isolated or expanded human chondrocytes Perichondrium Periosteum Cartilage tissue autograft
Tissue engineering and cartilage regeneration for auricular reconstruction endoderm, ectoderm, and are therefore termed pluripotent. The generation of specific cell types by directing ESC differentiation hypothetically offers extensive resources for developing clinical applications to replace injured tissues. ESC lines from human fetal tissues were firstly established in 1998 [18]. The ESC use still represent one of the most controversial topic discussed in scientific symposia, and despite their great potentiality, practical and ethical questions currently delay much ESC research. There are no literature reports of ESC used for auricular cartilage reconstruction so far. 4.1.1.2. Mesenchimal stem cells (MSC). MSC are adult stem cells. They represent a subset of nonhematopoietic cells contained in the adult bone marrow stroma that have the capacity to undergo extensive replication. They also have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues including bone, fat, tendon, muscle, marrow stroma and cartilage (multipotency). These cells represent an attractive cell source for tissue engineering approaches. In vitro expanded MSC combined with scaffolds have been successfully implanted directly or after specific stimulation [19]. The potential advantage of using MSC includes low cell numbers required at the initial culture, relative simple and already clinically used procedures for bone marrow harvest. Moreover it has been reported that MSC from older donors also maintain high biological activity [19,20]. Further investigations, especially using animal models, have been advocated. 4.1.1.3. Umbilical cord stem cells (UCSC). Ovine mesenchimal progenitor cells also have been isolated from cord blood samples, expanded and seeded onto bio-scaffolds of polyglicolic acid. After 12 weeks of culture the constructs exhibited a chondrogenic differentiation revealed by both standard and matrix-specific staining. These results with UCSC are encouraging and more resources and studies have been claimed [21]. 4.1.2. Isolated human chondrocytes (IHC) The use of autologous chondrocytes is another promising potential field of research. The pioneering work by Vacanti and Vacant demonstrated that bovine chondrocytes seeded onto synthetic biodegradable scaffolds could produce new cartilage tissue after transplantation into athymic mice [22]. Further investigations verified the ability of human chondrocytes isolated from hyaline cartilage to replicate and to generate new matrix in vitro [23]. Major problems encountered with IHC cultures are represented by:
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Chondrocytes dedifferentiation. The small number of chondrocytes initially isolated from a cartilage biopsy must be firstly expanded. Using a monolayer culture medium, these cells loose their phenotype and turn into fibroblasts (dedifferentiation process). Collagen type I synthesis. As a consequence of the dedifferentiation process, in the culture medium there is a switch from collagen type II to collagen type I synthesis. New culture systems have already been developed; the results obtained by Naumann et al. using bio-artificial ‘‘three-dimensional’’ cultures are particularly interesting [24]. Serum factors and various growth factors have been shown to modulate cartilage matrix synthesis in 3D cultures, especially fibronectin [25], insulin-like growth factor-I (IGF-I; 26), transforming growth factor b (TGF-b [26]), and platelet-derived growth factor (PDGF [27]). The possible applications using IHC in vivo must be cleared [23] and more research is advocated; conditions that favour maintenance of the phenotype are not usually those that favour an increase in numbers [28]. As a result, there may be limitations in the numbers of suitable cells that can be grown in vitro for subsequent repair of cartilage defects. 4.1.3. Perichondrial cells Perichondral progenitor cells can be harvested from perichondrial tissues, isolated, cultured and guided into a transdifferentiation process using peculiar factors like TGF B2 and IGF 1. However, their yield rate is low and the amount of the new formed matrix is limited. There are reports of perichondrial grafts using a combination of different biomaterials [29—31]. Matsuda et al. [29] implanted a collagen sponge enwrapped in perichondrium subcutaneously in the back’s of rabbits, after 7 weeks showing new cartilage formation in 10 of 21 explants. Ruuskanen et al. [30] implanted a combined graft of polyglycolic acid and perichondrium in a muscular pouch demonstrating new cartilage formation in 7 of 8 implants. Ten Coppel et al. used de-mineralized bovin bone matrix enveloped by perichondrial cells for cartilage regeneration, showing a small amount of new cartilage generation when the site of implantation is well vascularized [31,32]. Further investigations have been advocated. 4.1.4. Periosteum Periosteum is more plentiful than perichondrium in adults; Ham’s classic studies in fracture healing pointed to the periosteum as the source of neochondrogenesis in callus tissue [33]. Transplanted
1512 autogenous periosteum produces an admixture of new bone and cartilage, probably depending upon vascularity of the microenvironment in the recipient bed [34]. Early attempts to resurface cartilaginous defects with transplanted periosteum have had little success so far [35].
4.2. Bioartificial tissue scaffolds 4.2.1. Scaffold properties and neocartilage and matrix production after seeding The development of adequate scaffolds is necessary for successful cartilage tissue regeneration; also the quality of cartilage tissue produced can be influenced by the carrier-materials, as the scaffold may regulate the seeded cell yield rate. In our opinion, in an ideal project, either in vitro or in vivo, the carrier material should provide several characteristics: Mechanical stability. Ensure a three-dimensional support for cell development and expansion. Biodegradability. After prolonged culture, as tissue develop, the newly formed extracellular matrix should firstly be substituted and then replaced by the artificial carrier. Shape. It is important that the transplants do not change their form and at the same show time elasticity as that of the normal ear. Biocompatibility. It is extremely important to control and manage the immunocompetent host response, after implantation of the bio-prosthesis. Other scaffold properties should be: Guarantee of uniform cell distribution. Avoid influence on seeded cell phenotype [24]. The development of 3D scaffolds have been particularly useful to define the space for the new tissue, and, potentially, to enhance the maturation and function of the regenerated tissue. Candidate scaffolds or matrices include natural polymeric materials, synthetic polymers, biodegradable polymers, and polymers with adsorbed proteins or immobilized functional groups. Details of the manufacturing and properties of the synthetic materials have been reviewed. Bio-materials Small intestinal submucosal graft ‘‘SurgiSIS’’. This is an acellular, freeze-dried soft tissue graft derived from porcine small intestinal submucosa. SIS has already been successfully used as an interpositional graft in the repair of nasal
A. Ciorba, A. Martini septal perforations. In the study by Pribitkin et al. this material have been interposed following excision of rabbit auricular cartilage, showing that some sort of cartilage regeneration was only possible in presence of adjacent host cartilage or perichondrium. These results justify further animal and then human studies [36]. De-mineralized bovine bone matrix (DBM). In vitro tests using cells seeded on DBM demonstrated islets of vital chondrocytes surrounded by newly generated cartilage matrix. More in vivo experiments are necessary, only fibrous tissue was detected when DBM had been implanted in the rabbit pinna [37]. Polymeric scaffolds Synthetic polymers have theoretical advantages regarding plentiful supply, precise control of composition and material properties, and possibilities of biocompatible and reabsorbable features. Polymers comprise two major categories: those that are permanent and those that are temporary because they are reabsorbed by the body. The key requirements of bioreabsorbable materials are that (i) their rates of degradation must be compatible with the intended use and (ii) the products of their degradation must be nontoxic. Of the synthetic materials, polyglycolic acid (PGA), is the most widely studied. Polyglycolic acid (PGA), poly-e-caprolactone (PCL), poly-4-hydroxybutyrate (P-4HB) are commonly used polymeric scaffolds. Prior to seeding, these scaffolds must be pre-treated by adding growth factors and then placing the constructs in suitable incubators (surface conditioning). Extracellular matrix formation with sparse chondrocytes have been reported. Particularly, after 8 weeks of in vitro culture the glycosaminoglycan content significantly increased and the type II collagen quote in the construct reached 40% [38]. Hyaluronic based biomaterials HYAFF 11. This is a polymer derived from the esterification of sodium hyaluronate. It is composed of nonwoven fibres and a membrane of hyaluronic acid benzyl ester. It has been reported that one of the advantages of this material is the good cell adhesiveness, even without prior surface conditioning; when combined with chondrocytes, the construct seems to acquire a stable structure and a firm consistency similar to that of cartilage, which confirmed that the cultured cells had retained their chondrogenic potential. The histochemical staining with azan-blue demonstrated the occurrence of collagen fibres between the scaffold fibres. These
Tissue engineering and cartilage regeneration for auricular reconstruction preliminary in vivo findings support the idea that the biomaterial did not negatively interfere, but on the contrary, had a relatively favourable effect on matrix production [38]. 4.2.1.1. Mechanisms of in vitro chondrogenesis. Several attempts have been made in order to understand the exact mechanism that regulate, especially at the very beginning, the in vitro cellular aggregation and the cellular interactions with bio-scaffold. It has been observed that during the in vitro engineering of cartilage (process also defined by the term of ‘‘artificial mesenchymal condensation’’) the seeded cells (SC or IHC) initially adhere to the bio-scaffold forming aggregates as an attempt to recreate the embryonic ‘‘mesenchymal condensation process’’ required for chondrogenesis [39,40]. Studying the cellular response to nano- and micrometric topographies, it has been observed that these cellular aggregates collided each other migrating along the direction of the bio-scaffold’s groove long axis [39,40]. Furthermore, it is already clear that several serum factors and various growth factors, especially fibronectin [25], insulin-like growth factor-I (IGF-I [26]), transforming growth factor b (TGF-b [26]), and platelet-derived growth factor (PDGF [27]) can modulate cartilage matrix synthesis in cultures. Also insoluble factors modulate cell behaviour in cultures; this understanding is founded on the appreciation among developmental biologists that extracellular matrix components influence the behaviour of the cells that secrete them and on the subsequent discovery of cell membrane receptors for extracellular matrix molecules. The integrin receptors for protein motifs (or ligands) are the best studied, but another category of proteoglycan receptors includes CD44, which mediates chondrocyte binding to hyaluronan [41]. More studies have been claimed in order to understand the molecular mechanisms that regulate the intra-cellular interactions and the neo-cartilage formation processes. 4.2.2. Suitable animal models The choice of an appropriate animal model is not so simple; it is important to match the model to the question being investigated. Several animal models have already been used to evaluate the remodelling process of tissue-engineered constructs in vivo, as well as the inflammatory and immunological responses by the host immune system. Mice are the favoured model, rabbit the second favoured: they both bear the advantages of small animal models, including low cost, ready availability and less ethical concerns compared to other animal (especially primate).
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Particularly nude mice have been used, as they represent an immunocompromized model of ideal choice for the evaluation of the in vivo remodelling process of the tissue-engineered auricle. Their skin is quite thin, pliable and hairless, mimicking the human auricular skin, presenting a fine three dimensional ear definition after implantation [20]. Possible reported drawbacks using nude mice came from [20]: The lack of functional T-cells to evaluate cellular responses. The animal is too small to implant the life-sized ear to mimick the construction against soft tissue contraction. The limited life length, that prevents the tissueengineered ear from long-term follow-up. Ten months is the longest follow-up ever reported so far with this model. It has been reported that several scaffolds progressively disappear in the follow-up observation after implantation. Still more studies are required for detecting the fine mechanisms of inflammatory reactions that might destroy the chondrocites against the scaffold, and subsequently deform the shape and impair the quality of the implanted tissue [20]. 4.2.3. Possible concerns of tissue-engineered reconstruction in humans It is important to remember that the clinical application of these bio-engineered construct still is faraway. Possible issues are represented by the following: Plasticity. It is represented by the difficulty in producing great variations of size and shape of the bio-scaffold, necessary to match the opposite, normal ear. At the same time the construct should be stiff enough to contrast the possible scaffold’s shape deformation over time by the taught human skin. Computer-aided designs have been proposed for the manufacturing of matching, suitable bioscaffolds before the cell-seeding phase (also see three-dimensional stereolithograpy [37]). Failure-extrusion/absorption. As mentioned above tissue-engineered frameworks should own an elevate biocompatibility. Uncontrolled cellular proliferation. Doubts regarding the possible application of constructs with stem cells came from the possible uncontrolled proliferation of stem cells, or from an abnormal cartilage matrix synthesis (stem cells are likely candidates for accumulating multiple mutations that can disrupt their tight control leading to tumorogenesis).
1514 Infections and cutaneous dehiscences. Once incorporated tissue-engineered frameworks should be quite resistant to trauma and infections. Lacerations that expose the implant or infections should respond to antibiotic treatments. Instead, alloplastic implants for example, can be easily dislodged or even lost in case of flogosis [12]. Nature of tissue-engineered frameworks. Auricular tissue-engineered reconstruction once completed should not require further interventions. In contrast, costal cartilage graft reconstruction require multiple surgical procedures and also auricular reconstruction with alloplastic implants needs prosthesis replacement every 3—5 years [13]. Morbidity. In the case of an auricular tissue-engineered reconstruction, only a small piece of cartilage (i.e. obtained from a small biopsy) would be necessary for chondrocytes extraction (even if no biopsy could be required in case of stem cells use). Consequently much more elevated would be the morbidity associated with the care of an alloplastic implant reconstruction or with an autogenous costal cartilage graft reconstruction considering the multiple surgical procedures (including the thorax harvest) [12]. Possible bio-ethical limitations. Stem cells and their applications are exciting to physicians, scientists and patients because of their potential and their possible use to treat large numbers of patients with a variety of diseases. Even if this science is still at a very early stage, legislators around the world are attempting to enact legislation to govern the development of stem cell research and its potential applications. They are doing this in a worried and evolving global regulatory environment concerning stem cell research (embryonic SC particularly) that varies from the permissive (United Kingdom), to the restrictive (Germany, Italy), to the apparently conservative (United States). The formulation of a policy and a legislation in this area requires a careful understanding of this science because the moral considerations involved are complex and delicate. A further challenge is that the common terms used in this field (embryonic, stem cells or regenerative medicine) have strong emotional associations linked to the debate about the beginning of life [42—44].
5. Conclusions and future directions It is evident that the matter of cartilage reconstruction remains complex: although promising, microtia reconstruction using a tissue-engineered,
A. Ciorba, A. Martini prefabricated framework is not yet a practical option. It is conceivable that in a near future genetically modified cells or stem cells (embryonic or adult) could be grown on a biocompatible scaffold with internal signals for programmed histogenesis. Advances in material design may generate ‘‘smart’’ scaffolds that will control tissue topology and have surface modifications to stimulate and control cell adhesion, differentiation, and growth. Composite engineered tissues or organs, like a bio-engineered ear, look as a future goal. Cross-disciplinary interactions are likely to accelerate progress and to mediate application of advances made in other fields to consistently successful in vitro engineering of cartilage for all clinical needs.
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