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Tutorial |
The University of Memphis, Memphis, TN
Lafayette Otolaryngology Associates, Lafayette, IN
University of Pittsburgh, Pittsburgh, PA
Contact author: Joel C. Kahane, School of Audiology and Speech-Language Pathology, The University of Memphis, 807 Jefferson Avenue, Memphis, TN 38105. Email: jckahane{at}memphis.edu
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Abstract |
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KEY WORDS: extracellular matrix (ECM), tissue engineering, tissue grafting, hemilaryngectomy repair, laryngeal anatomy, morphology, histology
The field of tissue engineering/regenerative medicine combines the quantitative principles of engineering with the principles of the life sciences toward the goal of reconstituting structurally and functionally normal tissues and organs. Techniques and concepts that are fundamental to regenerative medicine may provide clinicians with a valuable tool for improving surgical outcomes in terms of function, cosmetic appearance, improved healing, and a faster return to a more normal life. The use of biologic matrices, growth factors, and signal proteins has become reality in several clinical applications as part of regenerative and restorative approaches to human organ repair (Petrungaro, 2002). Unlike research where cells that have been cultured outside of the body are then reimplanted to induce development of tissues within the body, researchers in tissue engineering have directed efforts toward developing substrates capable of orchestrating the replication of fully functional tissues and organs.
The reconstruction of a tissue or organ by the principles of tissue engineering typically is based on one of three approaches: a cell-based approach, a scaffold-based approach, or a bioactive factor–based approach. Eventually, all three of these components must self-assemble into an appropriately organized and functional structure that is biocompatible with the host. In theory and in practice, most tissue engineering approaches involve a combination of one or more of the above. The starting point is what typically varies among different approaches. Each approach has its advantages and disadvantages. The use of cells for organ and tissue reconstruction typically requires an autologous source because of the immune-mediated rejection problems associated with nonautologous cells. Scaffold-based approaches have been successful in many clinical applications of tissue engineering, particularly the use of biologic scaffolds composed of extracellular matrix (ECM). However, the eventual involvement of cells is obviously very important. The source of cells that participate in the remodeling of biologic scaffolds is a question of immense interest and has important implications for the practical application to the scaffold-based approach to tissue and organ regeneration. Bioactive factors, specifically growth factors such as vascular endothelial cell growth factor and bone morphogenetic protein, are presently in clinical use. However, the problems associated with controlled delivery of these potent factors have limited their clinical utility at the present time.
The application of these regenerative medicine principles to the reconstitution of laryngeal tissues is thus far a relatively unexplored area of practice. There is reason however to believe that cell-based and scaffold-based approaches to the reconstitution of oral-laryngeal structures can be successfully employed for the reconstitution of many head and neck structures, including the larynx and other speech and voice organs. The precedents from other organ structure remodeling is there, existing surgical protocols are applicable, materials are available (Badylak, Lantz, Coffey, & Geddes, 1989; Cook, Tomlinson, Kreeger, & Cook, 1999; Kropp, 1998; Prevel et al., 1995; Robotin-Johnson, Swanson, Johnson, Schuessler, & Cox, 1998; Sandusky, Lantz, & Badylak, 1995), and patients' needs and expectations for such surgical management are likely to grow quickly.
The purpose of this article is to introduce voice/speech scientists and practicing clinicians to this newly emerging area of medical technology and to provide an example of an ongoing research project involving the use of a biological scaffold in the repair of a canine larynx. We believe that scientists and practitioners in the voice and speech production disciplines must be aware of developments in tissue engineering if for no other reason than that they are likely to be called on to evaluate the functional capacity of organs so repaired and to treat patients who have been so surgically managed. From a more basic science perspective, it is anticipated that a line of research exploring the use of tissue engineering approaches for the remodeling of the diseased or injured larynx will provide insights into fundamental processes of laryngeal development and voice production.
Tissue Engineered Products
Tissue engineered products in current use or under development include isolated cells used for implantation (e.g., Carticel, Genzyme Corporation); cells combined with biomaterials (e.g., Apligraf, trademark of Novartis and produced by Organogenesis, Inc.); biomaterial-based scaffolds (e.g., OaSIS, Cook Biotech Inc.; and Restore, DePuy/J&J); and the administration of bioactive molecules to affect endogenous tissue. Naturally occurring biomaterials of cells used in tissue engineering applications are often categorized by their source. Autogenous materials come from the same individual as that to which they will be reimplanted. Allogeneic substance comes from another individual of the same species. Xenogeneic transplants come from another species. Autologous cells and tissues have virtually no problems with rejection and pathogen transmission; however, they are often not available for harvesting. Allogeneic materials are available in limited quantities, but concerns of disease transmission accompany the use of such materials.
Some materials are synthetic (e.g., polymers and gels), others are naturally derived (e.g., whole or partial organs, ECMs, collagen and polysaccharides), while still other materials may be a combination of differently derived substances. In some instances, a mechanical system may be substituted for an organ in failure.
The search for an acceptable bone substitute in the tissue engineering field has been of consuming interest for many years. Bone grafting is a surgical procedure by which new bone or a replacement material is placed into spaces between or around broken or gapped bone to aid healing. Bone grafting is now used frequently to repair maxillo-facial defects in boney structures caused by congenital flaws such as cleft palate and acquired conditions of traumatic injury, bone cancer, or the sequela of surgical damage.
The ideal bone graft for any surgical procedure is a viable implant of autogenous bone, which contains both the inorganic and organic components necessary for bone repair. In the absence of material of such origin, bone substitute products typically derived from a bovine source have provided grafting material with the necessary characteristics for repair. The clinical effectiveness of these types of materials has been extensively documented and, until recently, seemed to be the best alternative and/or complement to autogenous bone grafts. The year 2000 however brought forth the first tissue engineered graft material to mimic both the inorganic and organic components of autogenous bone (Krauser, 2001).
In all instances of soft and bone tissue engineering, the materials that are implanted are expected to enhance wound site structural properties, deliver essential bioactive factors, facilitate the delivery of vital cell nutrients, and exert certain mechanical and biological influences to modify the behavior of subsequent cellular growth. The critical components of this process are the presence of a scaffold structure or matrix, the migration to the wound site of undifferentiated cells, and an increase in the concentration of bioactive proteins to act as a catalyst to accelerate the wound healing process. The bioactive proteins act to replace, repair, and regenerate tissue or to stimulate other tissues to facilitate healing.
A particular type of tissue repair material provides a scaffold on which newly grown cells may affix themselves and thus proliferate in an orderly and well-structured fashion. This material is called an ECM and is of particular interest to the research reported in the present communication. Recent studies have suggested that an important component of the host tissue response is mediated by biologically active degradation products of the graft material itself (Badylak, 2004; Li et al., 2004). Previous research has found that growth factors and other structural and functional molecules exist within this scaffolding material (Hodde, Badylak, Record, & Liang, 2001; McPherson & Badylak, 1998; Voytik-Harbin, Brightman, Kraine, Waisner, & Badylak, 1997). In addition to the biologic effects of the growth factors and structural proteins that are known constituents of the intact ECM (e.g., vascular endothelial growth factor, basic fibroblast growth factor, and fibronectin), there are also biologic effects that are spatially and temporally localized to scaffold degradation products. Peptides with antibacterial properties (Sarikaya et al., 2002) and chemoattractant properties for marrow-derived cells (Li et al., 2004) have been identified within the products of enzymatically digested ECM. In studies that have used an ECM as a scaffolding structure, it has been reported that the materials are rapidly and completely degraded in vivo and exert their remodeling effects in part by angiogenesis and the recruitment of host cells, including marrow-derived cells, to the scaffold remodeling site (Badylak, Park, Peppas, McCabe, & Yodoer, 2001; Li et al., 2004).
It is believed that the presence of ECM tissue at the wound site may modify the default wound healing mechanism of adults (i.e., scarring) such that the host mounts a biologic response that includes vascularization, cellular infiltration, and constructive remodeling. Further site and function customization of the remodeled tissue is thought to result from the effects of local environmental stressors such as adjacent tissue pH, oxygen tension, performance demands, and mechanical loading.
Much research and application of the principles and practices of tissue engineering have also been represented under such research discipline titles as reparative or regenerative medicine, cell therapy, gene therapy, and developmental biology. Indeed, while the requirements for each type of individual tissue may be drastically different, the desired outcome of tissue engineering treatment is to develop tissue that demonstrates certain specific structure-function properties. For example, bone must have strength and load-bearing capacity; cartilage must absorb impact and resist wear/tear; muscle must show elasticity and contractile force while at work; blood vessels must be pulsatile and nonthrombogenic and exhibit high levels of burst strength; and nerve tissue must be capable of handling high speed conduction with appropriate insulation characteristics during the process of signal propagation.
The feasibility of using tissue engineering technology for the repair of absent or damaged human organs has been established in preclinical studies and in human clinical applications. As an example, in an approach that uses a biologic matrix material as an inductive scaffold for tissue reconstruction, there have already been more than 300,000 human patients treated in the past 5 years (Badylak, 2004). This estimate of use/impact is exceedingly conservative since Badylak's work describes but only one of many kinds of tissue source origins. Tissue engineering technology is now accepted in many instances as a viable, effective, and often superior approach for tissue repair and reconstruction. The evolution of this approach to surgical repair, in comparison with the options of whole organ transplants or insertion of mechanical devices, has been much influenced by favorable responses to such considerations as the patient's postsurgical overall health recovery, the lack of availability of donor organs, issues of tissue rejection, lower cost, lesser need for expensive equipment, and specialized hospital facilities (Niklason & Langer, 2001).
Reconstruction of the Larynx
The primary goals for reconstructive surgery of the larynx are the prevention of aspiration, maintenance of an adequate airway, and the production of a functional voice. Most surgical approaches to laryngeal repair have used autologous or synthetically derived materials. Andrews et al. (1997) and colleagues evaluated four reconstruction techniques in canines and concluded that an autologous hemilarynx transplantation provided the most efficient phonation of the techniques they studied. Hicks (2000) reported on a human-to-human full larynx transplant. At 16 months postsurgery, the patient was described as making excellent progress in breathing, swallowing, and voice production.
Recently there was a report of the successful use of a porcine-derived xenogeneic extracellular matrix for the reconstruction of the larynx in adult dogs after a modified hemilaryngectomy procedure (Huber, Spievack, Simmons-Byrd, Ringel, & Badylak, 2003). Histologic examination of the reconstructed tissue 3 months after surgery showed the presence of a simple squamous epithelial lining, organized glandular structures deep to the epithelial layer, reconstructed thyroid cartilage, vasculature, and bundles of skeletal muscle. This report suggests that ECM scaffolds are promising templates for constructive remodeling of laryngeal tissue.
This current article, which involves two investigators who were coauthors with Huber, seeks to provide a more extensive discussion of the morphology and histology of the repaired larynx along with thoughts about the possible mechanisms of action which may facilitate the successful use of biologic scaffolds. It is also believed that observations of the remodeling process for postoperative time longer than 3 months will yield important new insights into the progression of the remodeling process. Further, we wish to study a different surgical procedure for the ECM repair than had been used previously in the hope that it might result in an improved healing site. Finally, at this time in our ongoing research program, we felt it would be helpful to offer further support for the belief that the remodeled tissue has many of the essential characteristics of the original in vivo tissue. The importance of this observation is critical to reconstruction of functional tissue that is capable of producing an acceptable voice. It is well established that such physical aspects of voice as intensity, fundamental frequency, glottal source characteristics, signal periodicity, and harmonic-to-noise ratio are dependent on the composition and intactness of tissues that serve as the sound generator. Andrews et al. (1997) have hypothesized that vocal function will improve as the layered structure of the vocal folds is more accurately replicated in a reconstructed hemilarynx.
Support for the view that certain characteristics of laryngeal tissue are essential for laryngeal function has been provided on numerous occasions (M. Hirano, 1975, 1981; Kurita & Hirano, 1983; Mihashi et al., 1981; Nakagawa, Fukuda, Kawaida, Shiotani, & Kanzaki, 1998). Additionally, Cooper and Titze (1983) emphasized that laryngeal muscular activity is dependant on the efficiency of vascular perfusion, which is critical for optimal oxygen consumption and heat dissipation in tissue substance during phonation. Also, Jiang, Verdolini, Aquino, Ng, and Hanson (2000) made a strong case for adequate tissue hydration as a major determinant of quality phonatory performance.
The reader of this tutorial is respectfully urged not to consider this article as a comprehensive coverage of all types of tissue engineering approaches that have or may in the future be applied to the repair of speech or voice organ repair. We have thus far only collected a limited amount of information about one type of restorative surgery. There is still much to learn and much research to be done about how the larynx might be reconstructed and how it will function.
Tissue Harvesting for ECM Scaffolds
All ECM bioscaffold materials used in the present study were provided by ACell, Inc. Preparation of the ECM was conducted as follows. The urinary bladder was harvested from market weight (240–260 lb) pigs immediately after commercial slaughter. The bladders were placed in cold 1.0 N saline, which effectively lifted the transitional epithelium from the underlying basement membrane, leaving the basement membrane intact and preserving the hydrated state of the material.
The specimens were mechanically stripped of the abluminal layers, including the tunica serosa, the tunica muscularis externa, and the tunica submucosa. The remaining ECM material consisted of the basement membrane and the subjacent tunica propria. The material was treated with 0.1% peracetic acid, which lysed cell membranes and nuclear membranes. Cellular remnants were removed from the ECM by a series of rinses in buffered saline and deionized water. The single-layer sheet material was then terminally sterilized with e-beam irradiation (2.4 Mrad).
Multilaminate sheets (four layers) were prepared by vacuum pressing four single-layer sheets together, with each sheet oriented such that the basement membrane surface faced the same direction. The adjacent sheets were placed at 90° angles, thus accounting for an isotropic four-layer sheet. These sheets were rehydrated for a minimum of 5 min in sterile water immediately before use.
When the construct was used in the surgical procedures described below, the basement membrane surface of the urinary bladder ECM was positioned toward the lumen of the larynx. This positioning provided an intact native surface for the subsequent proliferation and differentiation of laryngeal mucosal epithelial cells. The opposite surface that consisted of the more coarsely textured tunica propria was in direct contact with host tissue, providing an excellent conduit for vascularization and host cell migration into the ECM scaffold.
Animal Subjects
The use of animal larynges to study vocal fold morphology and details of phonatory dynamics is well documented. (Baer, 1975; Hast, 1983; M. Hirano, 1975, 1981; Kurita & Hirano, 1983; Mihashi et al., 1981), and the dog has been the experimental animal of choice (Tayama, Chan, & Kaga, & Titze, 2001). These data have been enormously useful in constructing models of laryngeal functioning during phonation. Often these findings have been extrapolated to explain human laryngeal function (M. Hirano, 1975, 1981; Jiang et al., 2000; Perlman & Cooper, 1984; Rousseau et al., 2004). Even while acknowledging that laryngeal morphology and aerodynamic characteristics of dog phonation differ in some important ways from human voicing, it seems reasonable that the experimental nature of the current animal research must be considered as an antecedent to any human application.
Ten dogs were original subjects in this study, but technical and experimental design limitations and personnel relocation resulted in usable data from only 2 animals. Two adult female dogs, each weighing about 21 kg, are thus the subjects reported on in this study. Each dog was subjected to a hemilaryngectomy; 1 dog was the recipient of an ECM tissue laryngeal reconstruction implant procedure while the other dog's larynx was repaired using a standard strap-muscle (STM) closure protocol. The animals were sacrificed at 24 weeks postsurgery. By this postoperative time, both dogs had fully resumed barking behavior, although to a trained listener their vocalizations appeared to be less intense and more noisy and the sound frequency ranges were somewhat restricted.
Surgical Technique
Each dog was anesthetized and orally intubated while hemodynamic and respiratory parameters were monitored during the surgical procedure. The neck was extended, shaved, prepped, and draped in sterile fashion. An incision was made from the level of the hyoid bone to the cricoid cartilage. Sharp dissection was taken through the skin and platysmal layers. Subplatysmal plane flaps were elevated and retracted. The strap muscles were separated at the midline and retracted laterally. The thyroid cartilage was exposed. An incision was made in the thyroid cartilage down to the cricothyroid membrane, lateral to midline on the contralateral side of the planned hemilaryngectomy. The thyroid cartilage was carefully reflected laterally to a point just anterior to the cricothyroid joint. All soft tissues were removed along with the aryepiglottic fold, vestibular fold, and the true vocal fold. The tip of the vocal process of the arytenoid cartilage was removed with the vocal fold, but care was taken to retain the remainder of the arytenoid cartilage and the conus elasticus in continuity. The surgical procedure up to this point created a rectangular defect which was to be reconstructed as described below.
In the dog receiving the ECM implant, a single-layer sheet of acellular ECM was fashioned to drape as a fold tissue from the anterior commissure to the vocal process. This was tacked in place with 5-0 Prolene. A second sheet of four-layer ECM was then fashioned to the dimensions of the hemilaryngectomy defect. A running suture of 5-0 Prolene was used to create an airtight closure of this rectangular defect. The grafted material was sutured directly to the thyrohyoid membrane, cricothyroid membrane, and the perichondrium of the remaining thyroid cartilage ala. The strap muscles were then reunited, and platysmal skin layers were closed. In the STM-repaired dog, the reconstruction was achieved by reappropriation of soft tissue and STMs into the defect. These tissues were sutured to the remaining cricothyroid membrane, thyrohyoid membrane, and contralateral thyroid ala periochondrium to obtain an airtight closure. After the respective surgeries, the dogs were awakened from anesthesia, observed until they were responsive and breathing independently, and then returned to their kennels. Recovery from surgery for both dogs was unremarkable.
Preparation of Tissue Samples for Anatomic and Histological Examination
The histologic methods used to examine the cells and tissues composing the organs of the body provide great insight into how these organs function. Metabolic functions of cells and biomechanical properties of tissues can be explained or illuminated by detailed study of their structure. The analysis, however, requires specialized techniques which selectively disclose features of cells based on their affinity for chemical reactions with selective chemical agents (staining). Before reactions can take place, the tissues must be prepared to allow for uptake of these stains for subsequent study via the microscope. In the majority of cases, tissues are cut into ultra-thin slices (sections), often only hundredths of an inch in thickness, to permit the dyes or other agents contained within a stain to bond with the cells within the tissue. The thinness of the section permits light to be transmitted through them, so that details can be resolved and magnified by the optics of the microscope. Before observations can be made, the individual tissue sections, which are quite fragile, must be reinforced with purified wax or plastic and mounted on glass slides to prevent them from tearing or becoming distorted by handling.
Immediately on animal sacrifice, the larynx was excised and studied for details of postoperative morphology. Before any chemical fixation, morphologic observations were made regarding the color and texture of the epithelial linings, form and symmetry of the laryngeal cavity, vocal and vestibular folds, and the laryngeal skeleton. Special attention was made to the comparisons between the native and the ECM- or STM-remodeled halves of the larynx. Color photographs were made of the intact remodeled larynges and of these specimens after they were split along the sagittal plane. When so prepared, a clear view could be obtained of the vocal folds, ventricles, arytenoids, and other major cartilages and musculature of the excised larynges (see Figure 1).
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It has been well documented that histological studies of the larynx provide significant insights into fundamental properties of the vocal folds and how tissue changes in the larynx and vocal folds resulting from postnatal maturation and aging of the larynx contribute to the evolution and involution of the voice. For example, definitive information on the structure of the vocal folds obtained from elegant histological studies by M. Hirano (1974, 1981) has set the standard for interpretation of vibration of the vocal folds and its alteration in various disease states. Other studies have illuminated the nature of developmental changes in the vocal folds (M. Hirano, Kurita, & Nakashima, 1983, Kahane, 1983c), and in the involutional effects in the larynx caused by aging (Kahane, 1983b, 1988, Kahane & Hammons, 1987). Histologic studies of the larynx, using serial sectioning techniques, have revealed information about the patterns of arterial supply within the larynx (Kahane, 1983a). Special staining and tissue clearing techniques have enabled the complexities of neural innervation of the larynx to be better understood (Sanders, Wu, Mu, Li, & Biller, 1993). Recent studies using enzyme histochemical techniques have shed light on the influences of proteoglycans in the vocal folds and their influence on biomechanics (Gray, Titze, Chan, & Hammond, 1999). Regeneration of aged vocal folds using basic fibroblast growth factor has been studied by S. Hirano et al. (2005) to understand changes in collagen density in the lamina propria of the vocal fold. Similarly, recent investigation on vocal fold scarring resulting from phonotrauma, surgery, or healing has been studied with new histological techniques that have been quite useful in describing the mechanisms of scar formation in the vocal folds (Thibeault, Rousseau, Welham, Hirano, & Bless, 2004). Histological analysis provides a fertile resource for exploring structural details and metabolic properties of laryngeal tissues in growth and repair and also provides an empirical basis for designing biomechanical studies to examine tissue properties within the larynx. These may aid in explaining their biology more completely as well as their capacity to contribute to voice production and protection of the airway.
General Morphologic Influences of ECM and STM Implants
The ECM implant procedure resulted in better remodeling of the hemilarynx than the STM repair approach. ECM remodeled tissue appeared to reflect a yet incomplete state of maturation, as reflected by neovascularization and basal cell hyperplasia in the epithelium, suggesting continuing growth or remodeling. These morphologic findings were not observed in STM remodeled tissue of comparable age. Both ECM and STM implants appeared to have the capacity to induce tissue differentiation, including neural and muscular tissues at the site of remodeling. Both ECM- and STM-remodeled larynges exhibited asymmetry; however, the ECM specimens were relatively more symmetric and muscles were less hypoplastic than STM-remodeled tissues. Also, the differences between remodeled and unoperated tissues were less profound in the ECM implants compared with corresponding tissues from the STM procedure. The ECM-remodeled tissues appeared qualitatively to have a more classic textbook appearance (see Figure 2).
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Endochondral ossification of hyaline cartilage appears to be part of a normal maturational process, as evidenced by its presence on the unoperated thyroid cartilages in both STM and ECM specimens. The STM implant appeared to exaggerate this process by accelerating the amount and extent of cartilage degradation and replacement by bone. Perhaps this process is derived from marrow cells from ossified thyroid cartilage to which the STM implant was sutured. The STM may have facilitated the spreading of osteogenic material into the surgical site. While the ECM implant was also attached to the sectioned thyroid cartilage anteriorly, a strong regulatory effect on the process may have been exerted by the remnant of arytenoid cartilage, which was retained within the surgical site and attached onto the ECM implant. The extensive remodeling of cartilage resulting from the STM procedure was not anticipated. However, when STM and ECM remodeling was compared, STM remodeling did not replicate unoperated morphology as faithfully as did the ECM procedure. Further discussion of this observation is offered later in this article.
Laryngeal cavity. Both ECM and STM implants imperfectly reconstructed the laryngeal tissue and laryngeal cavity. All regions were present in each, without evidence of atresia or stenosis. The ECM implant, however, produced better reconstruction than the STM implant, as evidenced by less dysmorphic vestibular fold, ventricle of Morgagni, and vocal fold. Both implants produced a glottal region that was irregular in geometry owing to misshaped vestibular and true folds. In the STM implant, the vestibular folds were truncated, attaching onto the wall of the ventricle rather than onto the anterior aspect of the reconstituted thyroid cartilage. The true folds in both implants were located on a lower horizontal plane than on the unoperated side. Thus, complete planer apposition of the vocal folds and subsequent closure of the glottal chink did not appear to be morphologically possible. The edges of the folds were irregular, and the folds themselves only slightly protruded from the glottal wall (particularly in the STM specimen), owing to a shallow ventricle of Morgagni and intrinsic deficiencies of the folds (to be described later). The aggregate of these anatomical deficiencies resulted in irregular geometry of the transglottal airway and the rima glottides. This dysmorphology was more marked in the STM than in the ECM hemilarynx (see Figure 4).
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Gross morphology of the vocal folds. On visual inspection, the vocal folds of both implants were located on a lower level plane than their unoperated counterparts, but the extent of morphologic asymmetry was greater in the STM than ECM implant. The irregularities in surface morphology were also more evident in the STM-remodeled fold than the ECM fold. When the intrinsic histologic structure of the vocal folds was examined at low power, the ECM vocal fold was less hypoplastic compared with its unoperated pair than the STM fold was to its counterpart. In addition, the ECM remodeling displayed a greater organization within muscle fibers and fascicles than did the STM counterparts (see Figure 5).
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The intrinsic musculature of the 24-week ECM preparation appeared to be less hypoplastic and more symmetrical than counterparts of the STM preparation. Infiltration of intrinsic laryngeal muscles by newly sprouting neurons was already prominent at 24 weeks in the ECM specimen. We have also observed regrowth of laryngeal tissue accompanied by significant differentiation of tissue types. The extent of the latter was dependent on the surgical procedure applied, but clearly the ECM procedure produced the more robust reconstruction.
Even while acknowledging that our observations of ECM- versus STM-remodeling effectiveness is based on the study of very limited tissue samples (Huber et al., 2003, and the current report), we suggest that the ECM remodeling appears to be superior because it created a potentially more naturalistic biological environment for laryngeal functioning. This was suggested by the quality of reengineered tissue and the faithfulness with which the reengineered hemilarynx mirrored the morphology of the unoperated side. Although it is well known that there is not a direct correlation between form and function, it is well accepted that functional integrity of a structure is strongly influenced by the intactness and biomechanical responsiveness of the tissues that compose it. Details of the histologic differences between the two surgical methods have been presented earlier, but several points of comparison are noteworthy and illustrate potential functional consequences of each type of surgical reconstruction.
EMC appeared to establish the characteristics of the laryngeal skeleton more accurately than STM. This was reflected in the remodeled thyroid cartilage, when it was compared with the unoperated side, which served as a control. As previously shown, the ECM produced hyaline cartilage that was more comparable in its distribution of unossified, calcified/ossified areas than in STM cartilage, which was more ossified than its unoperated counterpart. The functional significance of this may be a greater potential of the ECM cartilage compared with STM cartilage, to exhibit flexibility and resiliency than STM. This appears to be important, by affording the larynx the mechanical advantage (via compression effected by extralaryngeal muscle forces) to produce folding and compression of soft tissue within glottal area and in reestablishing glottal geometry through elastic recoil of the cartilage (Fink, 1974a, 1974b).
Another example of the superiority of ECM over STM remodeling was evident in reconstitution of the laryngeal cavity. The ECM glottal region exhibited significantly less distortion, that is, more faithful reconstruction of the spatial relationships of the transglottal region than STM. This resulted greatly from the superior ECM reconstruction of the intrinsic properties of the vestibular and true vocal folds. The ECM procedure appears to offer greater potential than STM for providing the functional morphology for reestablishment of effective transglottal impedance, necessary for both vocalization and protection of the airway.
Protection of the laryngeal airway afforded by the epithelial lining also appears to be more effectively produced by ECM remodeling. The ECM neo-epithelialization of squamous epithelium was relatively thick and contained well-defined and differentiated cell layers. In comparision, the epithelium derived from STM was thinner and less cellular. Additionally, ECM respiratory epithelium had numerous cilia. In STM respiratory epithelium, the cilia were less plentiful and routinely dysmorphic. The functional significance of this observation is that ECM epithelia in the laryngeal cavity appear to provide greater potential for protection against compressive and shearing forces and better filtering functioning than STM counterparts. The ECM tissues may also have enhanced capacity over STM tissues for repair and/or adaptation, as evidenced by ECM's better potential for cellular replacement, suggested by its well-established basal cell layer.
Finally, ECM-reengineered connective tissues appeared to be superior to those of STM. This was best exemplified by the manner in which the lamina propria was developed and in the integrity of the collagenous and elastic fibers in the perimysium of the vocalis muscle. Unlike the lamina propria in STM, which was disordered, the ECM folds exhibited no scarring and had a layering of connective tissue fibers similar to the unoperated side. Organization of the coupling of the vocalis muscle to the lamina propria in the ECM vocal fold suggests that it may have better biomechanical potential and viscoelastic properties than STM-reengineered folds. The latter has the potential to directly influence mechanics of vocal fold vibration, glottal closure, and transglottal aerodynamics, all factors that may impact substantially in voice quality and vocal efficiency.
Tissue engineering appears to provide great potential for learning about the properties of tissues and the physiological stressors that condition their development and attainment of functional competency. These issues have been acknowledged by Chan and Titze in several studies (Chan, Gray, & Titze, 2001; Chan & Titze, 1998, 1999a, 1999b) as essential for understanding vocal fold behavior in response to mechanical and environmental stressors encountered during phonation. The influence of specific proteins in the laminar propria also provides an important mechanical advantage to the structure of the vocal folds by enhancing their responsiveness to aerodynamic forces (Gray et al., 1999).
The value of using tissue engineering procedures to explicate design features of the vocal folds and to evaluate its clinical effectiveness in reconstituting vocal functioning must await systematic studies that correlate histological features of regenerated (remodeled) vocal folds with acoustic, aerodynamic, and biomechanical data obtained during voicing. Such cross-modality studies may make it possible to better understand how such features of the vocal folds as induction and distribution of its interstitial proteins and the establishment of its connective tissue and vascular architecture result in improved sound production capabilities.
The Possible Mechanisms of Action
It is recognized that all tissues have their own local reserve cell (progenitor cell) population. In the larynx, this may reside in the arytenoid cartilage-conus elasticus complex as inferred from descriptions by Frazer (1932). Frazer noted that an inner mesodermal condensation or cell mass gave rise to the arytenoid cartilage, the thyroarytenoid muscle, and the other intrinsic muscles except for cricothyroid, the conus elasticus, and the upper portion of the cricoid arch. Of particular relevance to the current study is Frazier's description of the formation of the vocal folds, which is derived from two mesodermal condensations—precordal nodule anteriorly and condensed mesencyme posteriorly, which is attached to the arytenoids. The precordal nodule disintegrates, and the intervening material thins and becomes drawn out between the forming thyroid and arytenoid cartilages to form the vocal fold. Other material, from the same primordial source, course laterally toward the cricoid arch and form the conus elasticus. Thus, it appears that the vocal folds, the conus elasticus, and the arytenoid cartilages share a common embryological origin and have functional dependency on one another. The availability and extent to which these cells participate in response to tissue injury vary.
Based on this insight into the embryological development of the larynx, we may speculate about the potential of the arytenoid area to organize the glottal region, which includes the vocal folds. This leads us to consider that the arytenoids and the attached conus elasticus may retain their ability to serve as a progenitor site postsurgically, in the presence of appropriate inductor tissue such as the ECM implant. At this point, it is important to recall that in both surgical procedures used in this study, a rudimentary piece of arytenoid cartilage was left in place and became a point of attachment in the tissue repair process.
Little is known about these possible arytenoid progenitor cells. A review of the literature revealed no studies that attempted to characterize or isolate the cells that may be the source of the newly formed, differentiated laryngeal tissues observed in our study. Most of the published works studying the regeneration of injured larynges took different approaches than ours. To regenerate injured vocal folds, Kanemaru et al. (2003) applied the concept that a combination of three distinct elements—cell, scaffold, and regulation factor—under the appropriate conditions is needed to regenerate tissues and organs. To examine this hypothesis, they injected autologous mesenchymal stem cells from the bone marrow—the cell component of the triad—into the previously injured canine vocal folds and assessed the regeneration. This approach, however, differs from our work in that the multipotent cell is harvested from a different tissue rather than stimulating the progenitor cell, which already resides in the organ in question. S. Hirano and colleagues (2004) used the equivalent of the regulating factor used by Kanemaru et al. to study prevention and treatment of scarring in previously injured vocal folds. They found that the hepatocyte growth factor—the regulation factor of the triad—when injected into the previously injured vocal folds stimulated the fibroblasts of the lamina propria to produce hyaluronic acid while suppressing the collagen production, a major constituent of the scar. In this approach, the fibroblasts were definitive cells, with inducting capabilities, rather than an undifferentiated, pluripotential substrate (the acellular ECM implant). In the current study, only the ECM scaffold was implanted into the injured site, and it served to potentiate laryngeal tissue growth through cytochemical induction and recruitment of other cells into the neolaryngeal substrate.
Our work is thus far preliminary, but the results of this study have been greatly encouraging. We are optimistic that we have found a means by which to study the developmental morphology of the larynx and have also identified an approach that may be useful in the repair of damaged laryngeal tissue.
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Acknowledgments |
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Received August 18, 2005
Accepted September 7, 2005
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