Tissue Engineering for Otorhinolaryngology–Head and Neck Surgery

SYMPOSIUM ON REGENERATIVE MEDICINE

Tissue Engineering for Otorhinolaryngologye Head and Neck Surgery David G. Lott, MD, and Jeffrey R. Janus, MD CME Activity From the Division of Otorhinolaryngologye Head and Neck Surgery, Mayo Clinic College of Medicine, Phoenix, AZ.

Target Audience: The target audience for Mayo Clinic Proceedings is primarily internal medicine physicians and other clinicians who wish to advance their current knowledge of clinical medicine and who wish to stay abreast of advances in medical research. Statement of Need: General internists and primary care physicians must maintain an extensive knowledge base on a wide variety of topics covering all body systems as well as common and uncommon disorders. Mayo Clinic Proceedings aims to leverage the expertise of its authors to help physicians understand best practices in diagnosis and management of conditions encountered in the clinical setting. Accreditation: Mayo Clinic College of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit Statement: Mayo Clinic College of Medicine designates this journalbased CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s).TM Physicians should claim only the credit commensurate with the extent of their participation in the activity. Learning Objectives: On completion of this article, you should be able to (1) summarize the role of regenerative medicine in treating the anatomical and functional deficits that affect the head and neck region, (2) differentiate the benefits and drawbacks of various tissue engineering techniques when applied to distinct head and neck subsites, and (3) recognize areas in need of further investigation prior to clinical application. Disclosures: As a provider accredited by ACCME, Mayo Clinic College of Medicine (Mayo School of Continuous Professional Development) must ensure balance, independence, objectivity, and scientific rigor in its educational activities. Course Director(s), Planning Committee members, Faculty, and all others who are in a position to control the content of, this educational activity

are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of, the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off-label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of this information will be published in course materials so that those participants in the activity may formulate their own judgments regarding the presentation. In their editorial and administrative roles, William L. Lanier Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry. The authors report no competing interests. Method of Participation: In order to claim credit, participants must complete the following: 1. Read the activity. 2. Complete the online CME Test and Evaluation. Participants must achieve a score of 80% on the CME Test. One retake is allowed. Visit www.mayoclinicproceedings.com, select CME, and then select CME articles to locate this article online to access the online process. On successful completion of the online test and evaluation, you can instantly download and print your certificate of credit. Estimated Time: The estimated time to complete each article is approximately 1 hour. Hardware/Software: PC or MAC with Internet access. Date of Release: 12/01/2014 Expiration Date: 11/30/2016 (Credit can no longer be offered after it has passed the expiration date.) Privacy Policy: http://www.mayoclinic.org/global/privacy.html Questions? Contact [email protected].

Abstract Tissue regeneration in otorhinolaryngologyehead and neck surgery is a diverse area filled with specialized tissues and functions. Head and neck structures govern many of the 5 senses, swallowing, breathing, communication, facial animation, and aesthetics. Loss of these functions can have a severe negative effect on patient quality of life. Regenerative medicine techniques have the potential to restore these functions while minimizing the risks associated with traditional reconstruction techniques. This article serves as a review and update on some of the regenerative medicine research in this field. A description of the predominant clinical problems is presented, followed by a discussion of some of the most promising research working toward a solution. There are many noteworthy findings appropriate for inclusion, but limitations preclude mention of them all. This article focuses on laryngeal surgery, craniofacial reconstruction and plastic surgery, and otology and hearing. ª 2014 Mayo Foundation for Medical Education and Research

he field of otorhinolaryngologyehead and neck surgery (Oto-HNS) spans many different tissue types and specialized functions. Some of these vital functions include hearing, balance, air filtration and humidification, facial animation, deglutition, breathing, and speaking. These vast functional tissue differences make tissue regeneration in the head and neck challenging and exciting.

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Regenerative medicine techniques are being studied to manage the many etiologies of head and neck disorders. However, most research in Oto-HNS is focused on functional reconstruction of cancer defects. Cancers in this region are associated with high morbidity and mortality. The biggest contributing factor to these poor outcomes is the deficit that remains after surgical resection or radiotherapy. For some patients, loss of these functional tissues may result

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in deafness, a permanent tracheotomy tube or tracheostoma, or a permanent percutaneous feeding tube. Many patients succumb to their disease only because the anticipated functional deficits prevent treatment. In addition to the functional deficits, the aesthetic and social embarrassment that accompanies these defects can lead to social isolation and depression. Many patients choose to perish from the disease rather than experience this loss of quality of life. Transplantation of head and neck structures can overcome these functional and aesthetic concerns. However, the requisite immunosuppression prevents the use of traditional transplantation techniques. Transplantation of bioengineered tissues, however, bypasses the need for immunosuppression and has the potential to vastly improve the morbidity and mortality associated with head and neck cancers. Although regenerative medicine is a relatively new field, there have been many exciting discoveries in all areas of the head and neck that promise to benefit numerous Oto-HNS patients. This article focuses on the head and neck subsites that have had the largest depth of study and the most significant findings to date: laryngeal surgery, craniofacial reconstruction and plastic surgery, and otology and hearing. APPLICATIONS IN LARYNGEAL SURGERY The larynx is a true biomechanical structured it is both a living organ and a machine. Laryngeal mechanics necessitate multiplanar movement of the vocal folds. Open vocal folds facilitate breathing. Closing the vocal folds serves as a protective valve for the airway during deglutition. Also during deglutition, the entire larynx must move superiorly and anteriorly to open the esophageal inlet. Forced exhalation of air against closed vocal folds enables phonation. Pitch variation is achieved through shortening and lengthening of the vocal folds. The larynx is composed of multiple different tissue types. The supraglottis and subglottis consist of respiratory mucosa. The specialized layered microstructure of the vocal folds consists of squamous epithelium, lamina propria (superficial, middle, and deep layers), and muscle (Figure 1). The superficial layer of the lamina propria (SLP) plays the most important role in phonation. Restoration of laryngeal anatomy and function is difficult given the complex nature of Mayo Clin Proc. n December 2014;89(12):1722-1733 www.mayoclinicproceedings.org

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this organ. Procedures can be performed to individually improve airway, voice, or swallowing. However, improvement of one function is usually at the sacrifice of another. There is currently nothing that can be done to improve all laryngeal functions simultaneously. Tissue engineering of new functional laryngeal tissue can help millions of patients, from those with scarred vocal folds to laryngectomy. Laryngeal regeneration research is currently focused on vocal fold microstructure restoration and on laryngeal superstructure regeneration. Vocal Folds The vocal folds are the functional unit of the larynx. Their function depends on neuromuscular activity for gross movement and on pliability for phonation. Loss of pliability from scar formation can result from longterm vocal misuse, trauma, or surgery. The present therapeutic materials (most commonly collagen and fat) do not have viscoelastic properties similar to the natural lamina propria and do not promote tissue regeneration or repair. Vocal fold regeneration would have widespread benefit to patient care. Reduction in scarring can create a normal voice in a patient who was previously aphonic or may allow a professional singer to prolong a career. Regeneration of an entire vocal fold may allow speech in a patient after a cancer resection. This is also a paramount step in the regeneration of a total larynx. Regenerative therapies for scar essentially encompass 2 main strategies. The first strategy is to inhibit or repair the process of scarring and fibrosis. The second aim is to rebuild the native extracellular matrix (ECM). Generally, tissue engineering consists of 3 interconnected approaches (Figure 2): (1) development and implementation of scaffolds, (2) use of bioactive factors, and (3) use of cell therapy. Scaffolds. Decellularized organ matrix, biological polymers, synthetic biomimetic hydrogels, and synthetic polymers have all been described as scaffolds for 3-dimensional lamina propria replacement.1 Injectable scaffolds have the advantages of ease of application, biomechanical properties similar to native lamina propria, and the ability to deliver cells and bioactive factors.

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FIGURE 1. Vocal fold layers: superior view (A), cross section (B), and individual layers (C). The specialized structure of the vocal folds is in large part created through vibration. The superficial layer of the lamina propria is vital to phonation. BMZ ¼ basement membrane zone.

They have the ability to modulate the inflammatory response and direct ECM remodeling.2 Experimental hydrogel scaffolds for vocal fold regeneration have been primarily based on chemical modification of hyaluronic acid (HA), with reactive chemical groups that can rapidly cross-link in situ after injection. Hyaluronic acid is a common scaffold material owing to its biocompatibility and properties similar to native SLP. Most hydrogels are designed to improve the SLP because it is the area most important to phonation and is the most common site of injury. However, most vocal fold defects usually involve more than 1 layer. Kutty and Webb3 developed a 2-layer hydrogel system consisting 1724

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of methacrylated HA and acrylated polyethylene glycol derivatives to replace a multilayered structure. Gel formulations were developed that approximated the rheologic (HA) and tensile (polyethylene glycol) properties of the SLP and the vocal ligament, respectively. The hydrogel materials were found to support fibroblast spreading, proliferation, and collagen/glycosaminoglycan synthesis. The hydrogels could be varied in duration from weeks to months independent of the mechanical properties.4 Studies also suggest that injectable HA-based hydrogels may accelerate tissue remodeling through amplification of biochemical responses during the acute healing phase.5,6

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Cells Type

Source

Stem cells differentiated

Autologous heterologous

Bioactive factors enable cell proliferation and direct differentiation

Scaffolds provide structural support and aid in cellular differentiation

Bioengineered Tissue Bioactive factors

Scaffold Biologic Trachea Dermis Intestine etc.

vs

Scaffolds combined with bioactive factors provide form and function to the developing construct

Synthetic Ceramics Composites Metals Polymers

Cytokines Growth factors Hormones Morphogenetic proteins etc.

FIGURE 2. Tissue engineering fundamentals. Tissue engineering requires the synergistic combination of cells, scaffolds, and bioactive factors. Cells develop into the desired tissue, scaffolds provide structure and support, and bioactive factors aid in differentiation and cell nutrition.

The use of acellular biological scaffolds is an alternative to hydrogels. These scaffolds are decellularized to remove immunogenic foreign epitopes, followed by recellularization. There are many benefits to this type of scaffold. The tissue has a similar composition, mechanical property, and architecture as that of native tissue. They release growth factors and peptides that stimulate angiogenesis, recruit endogenous marrow-derived cells, and govern tissue remodeling. They also ease attachment, migration, and infiltration of host cells; attract biofactors; and can degrade within a few months.7-10 Decellularized matrices have been widely studied in animal models and humans. Xu et al11 used a saline-based osmotic approach to create a decellularized bovine lamina propria and epithelium. They observed early infiltration of host fibroblasts and inflammatory cells after Mayo Clin Proc. n December 2014;89(12):1722-1733 www.mayoclinicproceedings.org

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implantation in freshly injured rat vocal folds. Human vocal fold fibroblasts attached, proliferated, and synthesized ECM containing collagen, HA, decorin, and fibronectin. Histologic results showed that the scaffolds were degraded after 3 months, with no fibrotic tissue formation or calcification.12 Repopulated ECM scaffolds were shown to exhibit elastic shear modulus and dynamic viscosity comparable with human vocal mucosa at physiologic frequencies (100-250 Hz). Cell Transplantation. Cells are vital to tissue engineering. They are involved in the initial construct development, maintain function after implantation, and improve functioning of the other tissue components. Many cell types have been studied for vocal fold regeneration, but fibroblasts and stem cells seem the most promising to date.

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Autologous fibroblasts derived from buccal mucosa were first investigated by Chhetri et al.13 Fibroblasts were injected into a unilateral full-thickness lamina propria injury in a canine model. Each animal received 3 injections of approximately 25 million cells at weekly intervals beginning 6 to 8 weeks after injury. Vibratory and acoustic performance returned to near baseline levels by 29 weeks. The fibroblast-treated vocal folds stained positive for ECM components. Histologic analysis demonstrated increased fibroblasts, collagen, and reticulin; decreased elastin; and equal HA relative to the uninjured vocal folds. Stem cells are a popular option for vocal fold regeneration. They come in many forms but essentially break down into embryonic and adult. Embryonic stem cells have demonstrated some benefit in vocal fold regeneration, but the technical and ethical hurdles currently preclude widespread investigation. Autologous adultderived cells are easier to study and have shown some benefit. Bone marrowederived mesenchymal stem cells (BMSCs) injected before or during injury have been shown to prevent the gross appearance of scar in dogs14 and to improve rheologic properties in rabbits.15 Adipose-derived mesenchymal stem cells (ASCs) are a particularly promising cell type for the treatment of scarred vocal folds. In vitro trials have shown that ASCs secrete several growth factors that balance collagen and HA in the ECM. Hepatocyte growth factor (HGF) is believed to be a major component of these and is discussed later in the article. Briefly, studies have shown that ASCs secrete HGFs that attenuate collagen production and fibroblast proliferation in culture. The ASCs can also produce elastic fibers, which typically does not occur in a scar environment.16 The ASCs have been used to tissue engineer a multilayered structure for vocal fold replacement.16 In this model, ASCs differentiated into epithelial and mesenchymal lineages by physical and biochemical stimulation. Bioactive Factors. Growth factors are peptide molecules that function to regulate cell proliferation and differentiation. They have been studied extensively and have shown clinical success in human clinical studies. Basic fibroblast growth factor (bFGF) and HGF warrant specific mention. Hirano et al17 first described using bFGF in a human vocal fold in 2008. Injection into an 1726

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atrophic vocal fold demonstrated improvement of aerodynamic and acoustic parameters 1 week after injection and lasted up to 3 months. Other studies by the same group showed that bFGF significantly increased fibroblast production of HA and decreased collagen deposition in aged rat vocal folds.18 The bFGF has also been found to upregulate HA synthase and matrix metalloproteinase-2 production,19 downregulate expression of procollagen I, and increase expression of fibronectin and HGF.20 Hepatocyte growth factor is a pleiotropic cytokine with a favorable profile for vocal fold healing. It has strong antifibrotic potency,21 increases HA and elastin synthesis, decreases collagen synthesis, induces cell growth and migration, and is highly angiogenic. Hepatocyte growth factor and its receptor c-Met have been detected in epithelial and gland cells of normal vocal folds and in the lamina propria after injury.22 Hirano et al23 performed a one-time injection of HGF into rabbit vocal folds after unilateral mucosal stripping. Six months after injury, HGF-injected vocal folds exhibited significantly decreased collagen accumulation in response to injury and no significant changes in collagen, elastin, or HA levels relative to the uninjured contralateral vocal fold. Mechanotransduction. Vocal fold maturation seems to depend on phonation-derived mechanical stimulation and hormonal signaling. Neonatal vocal folds are homogenous tissues lacking the layered microstructure observed in the adult.24 Interestingly, a study by Sato et al25 demonstrated that vocal fold vibration induces differentiation of the vocal fold lamina propria. In their study, vocal folds unphonated (no vibration) for 11 years and 2 months were investigated by light and electron microscopy. They found atrophy of the lamina propria, with no differentiation and few ECMs. This is similar to the configuration of newborn vocal folds. Studies using high-frequency vibration bioreactors indicate that vibration significantly affects matrix regulation in the SLP.26,27 Vibration was found to significantly increase messenger RNA expression levels of fibronectin, matrix metalloproteinase-1, HA synthase 2, CD44, fibromodulin, and decorin compared with samples without vibration. Collagen and elastin expression was relatively unchanged.

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Laryngeal Superstructure Promising examples of bioengineered laryngeal structures have been reported. A porcinederived xenogeneic ECM has been successfully used for reconstruction of the larynx in adult dogs.28 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. Whole human larynges have also been successfully decellularized.10 Minimal major histocompatibility complex immunostaining was observed, and only a few chondrocytes were detectable. Both bFGF and vascular endothelial growth factor were maintained. These are important for angiogenesis and neovascularization of the graft. The mechanical response of the cartilaginous laryngeal structures was similar to native tissues and reflected their different composition (elastic cartilage in the epiglottis and hyaline cartilage in cricoid and thyroid cartilages). These studies represent a significant step toward successful laryngeal superstructure bioengineering. Once these techniques are refined, they might provide a functional partial laryngectomy reconstruction option or create a scaffold for total laryngeal regeneration. Although vocal fold motion has not yet been achieved, proper positioning and architecture might enable improved voicing and airway protection. APPLICATIONS IN CRANIOFACIAL RECONSTRUCTION AND PLASTIC SURGERY Regenerative applications in reconstructive surgery are rapidly expanding. Generally, efforts are either replicative or restorative and involve bone, cartilage, soft tissue, or a composite of these tissue types. This discussion focuses on bone and cartilage. Despite a long history of autograft, homograft, and xenograft use to shoulder the reconstructive burden, emerging trends in tissue engineering technology, such as biocompatible polymer construct, concomitant with the increasing safety and prevalence of stem cell therapies ushers forth a new era of facial restoration with less obligatory donor site morbidity, decreased graft rejection, decreased graft involution, and increased patient satisfaction. The challenge to researchers is how to integrate these in a safe, predictable, and efficacious manner. Mayo Clin Proc. n December 2014;89(12):1722-1733 www.mayoclinicproceedings.org

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Bony Craniofacial Components Induced Bone Restoration. With significant overlap in orthopedics, otolaryngology, oral and maxillofacial surgery, and neurosurgery, osteogenesis is perhaps the most widely explored regenerative field in facial plastic and reconstructive surgery. Effective replication of bone requires coordinated manipulation of cells, bioactive signaling molecules, and biomimetic, biodegradable scaffolds.29 A representative disease process for which restorative regenerative therapies are being explored is osteoradionecrosis of the mandible, a devastating phenomenon encountered in 5% to 15% of patients after head and neck irradiation, where the bone becomes gradually replaced by fibrous tissue.30,31 As the disease progresses, treatment initially calls for rigorous oral hygiene, followed by hyperbaric oxygen therapy or bone sequestrectomy, and then even osseous or osteocutaneous free tissue transfer.32 In swine models, the use of BMSCs with hydroxyapatite/tricalcium phosphate as a carrier vehicle has shown promising reparative results with respect to damaged bone.33 Likewise, initial results in limited human trials have shown equally impressive bone restoration with the use of platelet-poor plasma derivative fortified with mineral scaffold and autologous BMSCs, some studies even denoting inferior alveolar nerve recovery and resolution of fistulae.34 Bone Replacement. Even more challenging are cases where bone is actually missing or needs to be removed, as in the instance of trauma, tumor, or congenital deformity. Examples of this include segmental mandibulectomy defects in the lower third of the face, maxillary and alveolar defects in the middle third, and calvarial defects in the upper third. These defects have to be replaced with a graft or implant, the success of which is measured in terms of osteoinduction, osteoconduction, and osseointegration. Osteoinduction is the ability of a graft to recruit immature cells to develop into preosteoblasts and, ultimately, new bone. Osteoconduction is bone growth on the surface of an implant or graft. Osseointegration is the stable anchorage achieved at the bone-implant interface.35 To date, the standard of care when reconstructing segmental mandibulectomy defects is free tissue transfer. The reasons for this are multifactorial, but the overwhelming motivation is the 91% to 99% success rate of most free tissue

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transfer endeavors, regardless of the flap used.36 Nonvascularized bone grafting from the iliac crest, fibula, radial forearm, scapula, and rib has been used for oromandibular reconstruction, with success rates ranging from 70% to 88%. The success of nonvascularized bone grafting dramatically declines for defects that are 5 to 6 cm in size, cross the midline, have had previous irradiation, and are subject to wound contamination.37,38 Although a variety of flaps are used for oromandibular reconstruction, the fibula free flap is the most frequently used because of low complications, ease of harvest, optional skin paddle, and an overwhelming 25 cm of dense cortical bone hospitable to dental implantation.39 Despite the high success rate of free tissue transfer, the challenges faced by both surgeon and patient include increased operative time, donor site defect, comorbidities influencing ease of harvest (ie, vascular disease, limited anastomotic vessels secondary to previous surgery), increased length of hospitalization, and a re-exploration rate of 5% to 25%.36 It is for these reasons that a regenerative solution would be ideal. Advances in orthopedic polymer and ceramic technology along with improving safety and efficacy of stem cell therapies have brought forth new technologies for addressing critically sized defects, defined as “a large disruption in bony material that cannot spontaneously heal.”40 There has been an influx of in vivo animal research using prefabricated scaffolds impregnated with growth factors such as bone morphogenetic protein (BMP), some of these scaffolds being implanted after in vitro seeding with BMSCs. For instance, Terheyden et al41-43 published 3 experiments using a minipig model for scaffold prefabrication and subsequent mandibular implantation. The investigators manufactured a scaffold composed of natural xenogeneic deproteinized bone soaked in BMP-7 and subsequently implanted this in the latissimus dorsi muscle for incubation. Microvascular transplant of this implant to a mandibular defect was then performed with success. Insights such as the need for a perforated scaffold to aid in vasculogenesis, the correlation between increased BMP-7 concentration and bone growth, and the optimal site and time for prefabricated flap incubation were gleaned from these animal studies.41-43 Similar mandibular studies have been performed in dogs using calcium phosphate ceramics and collagen membrane with delayed bone marrow 1728

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grafting,44 in rabbits using hydroxyapatite/polyamide with or without BMP-7etransduced mesenchymal stem cells,45,46 and even in macaque monkeys using seeded hydroxyapatitecoated poly (E-caprolactone)47 with varying levels of successful osteoinduction, osteoconduction, and osseointegration. Maxillary defects are traditionally treated with obturation, local pedicled flaps (such as temporoparietal flaps), and nonosseous or osseous free tissue transfer, with fibula and scapula being the most common.48 Cranioplasty for calvarial defects depends on autologous, nonvascularized bone grafting for small- to mediumsized cranial defects and on titanium mesh implants or polymethylmethacrylate for larger defects.49 Tissue engineering animal studies for these sites have shown promising results. Bioengineered bone composed of ASCs incubated in osteogenic medium on a scaffold of hydroxyapatite/b-tricalcium phosphate was successfully used to repair maxillary-alveolar defects in dogs.50 Regarding cranioplasty, 15  15-mm cranial defects were created in New Zealand white rabbits and then were treated with ASCs osteoinduced with BMP-2 on an acellular collagen

FIGURE 3. Human model for a bioengineered mandibular osseous flap. Computed tomographic scan demonstrating the final position of a titanium mesh and hydroxyapatite scaffold after being seeded with stem cells, implanted into the latissimus dorsi muscle, and transferred via microvascular technique to a segmental mandibulectomy critical defect. Black arrow shows area with adequate bone formation, white arrow shows island with minor growth. Bone densities were measured outside [1] and inside [2] blocks. From Biomaterials,53 with permission.

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sponge. This study showed 96.9% ossification in the treatment group.51,52 Successful animal models have given way to human clinical trials. In 2006, Warnke et al53 fashioned a titanium mesh and hydroxyapatite scaffold coated with BMP-7 and seeded with BMSCs and then implanted it into the latissimus muscle to induce ectopic bone formation. This implant was then successfully transplanted using the microvascular technique into a critical mandibular defect in a former patient with floor of the mouth cancer (Figure 3).53 Similarly, other prefabricated mandibular and maxillary implants have been placed into critical defects using either local pedicled flaps54 or the microvascular technique.55,56 In efforts to generate a single surgery and a single operative site technique, in situ bone formation has also been explored. In 2013, Finnish researchers published on a 10-cm anterior segmental mandibulectomy defect treated with a tissue-engineered construct of b-tricalcium phosphate granules, recombinant human BMP-2, and Good Manufacturing Practiceelevel ASCs. During dental implantation 10 months later, bone cores were collected. Histologic and in vitro analysis demonstrated osteogenesis.57 Since that time, at least a dozen other cases of in situ bone formation have been applied to the calvarium, mandible, and even nasal septum with variable success. Many of these cases have used a similar b-tricalcium phosphate, BMP-2, and Good Manufacturing Practiceelevel ASC construct.58,59 Cartilaginous Craniofacial Components A generous supply of autologous cartilage is paramount to the reconstructive surgeon to facilitate successful nasal and auricular reconstruction. Autologous cartilage is the gold standard. Allogenic materials have a risk of rejection, disease transmission, and resorption. Synthetic materials have a risk of immunogenicity, extrusion, and infection.60 Typically, local autologous cartilage from the surgical site may be reshaped or reoriented to fit the needs of the patient. However, in the setting of previous surgery for septal defects or severe (grade III/IV) microtia, there may be a paucity of cartilage from the primary operative site, and an alternative harvest site (ie, costal cartilage) must be used, with added morbidity to the patient. Bioengineered cartilage has the advantage of minimal donor site morbidity, large tissue Mayo Clin Proc. n December 2014;89(12):1722-1733 www.mayoclinicproceedings.org

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availability, and the ability to be shaped to the individual needs of the patient. Cartilage cells can be harvested from an area such as the septum or auricle, expanded as a monolayer, seeded onto a scaffold to assume a 3-dimensional construct, and implanted. Growth and differentiation can be influenced by the introduction of growth factors, such as insulin-like growth factor 1, fibroblast growth factor (FGF), plateletderived growth factor, transforming growth factor (TGF), epidermal growth factor, and members of the BMP family. Exogenous stress can also influence growth.61 In addition to the harvest of mature chondrocytes, the use of ASCs and BMSCs has also been explored.62 Bioengineered constructs for nasal reconstruction have been investigated in human and animal studies. Yanaga et al63 injected gelatinous chondroid matrix with chondrocytes derived from auricular cartilage into the nasal dorsa of 75 patients and monitored them for 6 years. This matrix turned from a soft gel to a hard neocartilage in 2 to 3 weeks and yielded satisfactory and long-lasting results.63 Up to this point, however, animal studies have dominated the literature, with cell sources ranging from human nasal septal cartilage to rabbit BMSCs and scaffolding ranging from polyvinyl alcohol to poly(lactic-co-glycolic acid) (PLGA).62 For example, PLGA scaffolding with rabbit BMSCs has been used to generate cartilage with precise nasal alar morphologic properties when implanted into nude mice.64 Alginate hydrogels with human nasal cartilage chondrocytes have also been injected into nude mice to develop injectable cartilage for rhinoplasty.65 When it comes to auricular cartilage, Vacanti’s “auriculosaurus” was perhaps the most dramatic introduction the public was given to potential human application of regenerative medicine. Polyglycolic acid scaffolds were fashioned into the 3-dimensional construct of a human ear, seeded with bovine chondrocytes, and implanted into pockets on the dorsa of 10 athymic mice.66 Although successful with the retention of this complex structure and growth of new cartilage, most notable was the public reaction to the images of this “earmouse.” Using this as a jumping point, much research was conducted using synthetic polymers, particularly aliphatic polyesters, such as polyglycolic acid and PLGA, because they are Food and Drug Administration approved. Quite a few other

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animal studies have tested the utility of other scaffolds, including other synthetics, such as polylactic acid, poly-L-lactide acid, poly-ε-caprolactone, poly-4-hydroxybutyrate, and even fibrous collagen with coiled titanium wire. Hydrogels such as sodium alginate, pluronics, and fibrin gel polymer have also been trialed. Growth factors, including bFGF, FGF-2, TGF-b, and insulin-like growth factor 1, have shown a positive effect on cartilage growth and the potential for clinical application. Some of the greatest challenges faced by these constructs were shrinkage and distortion of this complex 3-dimensional shape.67 Recently, successful attempts have even been made to oxidize commercially available, alloplastic, porous, high-density polyethylene implants (Medpor; Stryker) and subsequently spray these with a fibrin hydrogel seeded with expanded auricular cartilage cells. These implants showed no evidence of skin necrosis, implant exposure, or extrusion compared with nonseeded controls after implantation into the dorsum of athymic mice.68 One human study used injection implantation of cultured chondrocytes into the subcutaneous pocket on the fascia of the lower abdomen. Six months later, the neocartilage block was surgically harvested and carved into an auricle for implantation. The 4 patients who had this done had stable implants with no evidence of rejection at follow-up ranging from 2 to 5 years.69 APPLICATIONS IN OTOLOGY AND HEARING Regenerative medicine applications for otology span the distance from the pinna, as discussed previously herein, to the auditory nerve. Although a comprehensive review of all the basic, translational, and human research for every application is too broad for the scope of this article, several representative areas are discussed. Tympanic Membrane The current standard for closing tympanic membrane perforations is tympanoplasty using connective tissue, such as temporalis fascia or tragal perichondrium. Adipose tissue can sometimes be used if the perforation is of smaller size. In the hopes of encountering a regenerative solution, Hakuba et al70 divided 24 guinea pigs into 3 groups of 8. Total tympanic membrane perforations were created in all the animals, with 8 acting as controls, 8 getting saline-gelatin hydrogel implants, and 8 getting bFGF-gelatin hydrogel implants. All the animals in the lattermost group 1730

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developed a fibrous layer in addition to an epithelial and mucosal layer, with complete closure of the perforation.70 This study is one of many to use bFGF as a bioactive molecule to aid in the closure of tympanic membrane perforations.71-73 In addition to gelatin hydrogels, absorbable gelatin compressed sponge (Gelfoam, Pfizer), HA, decellularized dermis, chitosan, alginate, and even rice paper have been explored as potential scaffolds, with the bulk of the literature exploring epidermal growth factor and FGF families as bioactive molecules. Platelet-derived growth factor and TGF-b have also been used. Although many implants have been explored with timed release of bioactive molecules, relatively few have been implanted with seeded cells. It is theorized that progenitor cells in the remnant tympanic membrane are sufficient to populate the scaffold successfully.74 Ossicles Although the small shape and relative tissue uniformity of the ossicles is enticing, the tenuous blood supply, hidden location, and availability of comparable prostheses have led to few studies on the topic. Nonetheless, several researchers have looked at various biocompatible and biodegradable polymer constructs. One such construct is a combined poly(propylene fumarate)/poly (propylene fumarate)-diacrylate scaffold fashioned into partial ossicular replacement prostheses. The porosity of these prostheses has been evaluated using microecomputed tomography, and their capability to support human mesenchymal stem cell growth has been quantitatively and qualitatively evaluated in vitro.75 In addition, these tissue-engineered scaffolds have been compared with cadaveric ossicles in terms of immunopositivity, distribution of ECM molecules, and osteochondrogenic markers and have been found to be remarkably similar histologically.75 Application of this technology to translational studies and human trials is forthcoming. Hair Cells Stem cell therapy as a treatment for deafness has been thoroughly explored for the past decade, with hair cell and auditory neuron regeneration receiving the bulk of the attention.76 During this period, attempts to convert stem cells into inner ear mechanosensitive hair cells and sensory neurons have encountered difficulty in phenotypic conversion and aggregation. In efforts to

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circumvent this issue, protocols that call for the stepwise differentiation of embryonic stem cells into hairlike cells have arisen. In a 2013 Nature article, Koehler et al77 describes such a protocol in which mouse embryonic stem cells are differentiated in 3-dimensional culture. Precise temporal control of BMP, TGF-b, and FGF resulted in prosensory cells emerging from otic placodes to generate hair cells bearing stereocilia bundles and kinocilium. These hairlike cells also manifested mechanosensitive properties, with synapses to sensory neurons also derived in the culture. This in vitro model of inner ear differentiation has exciting implications for future application.77 CONCLUSION Tissue regeneration in Oto-HNS is complicated by multiple tissue types and various specialized functions. However, significant advancements have been achieved in this field that promise to benefit millions of patients. Functional reconstruction of head and neck defects through bone and cartilage regeneration will decrease operative time and remove the need for a donor site. In turn, this will dramatically improve healing and patient suffering. Regeneration of auditory system components has the potential to restore hearing in those who have become deaf and may develop hearing in patients with congenital deafness. Engineering of laryngeal structures can allow patients to regain their identity through self-expression, to eat and breathe without difficulty, and to survive cancers that would have been otherwise terminal. Regenerative medicine is an exciting new field that has the potential to revolutionize disease management. Continued research and discovery are extending the boundaries of our capabilities. The possibilities for tissue regeneration are theoretically endless, limited only by our knowledge and imagination. Abbreviations and Acronyms: ASC = adipose-derived mesenchymal stem cell; bFGF = basic fibroblast growth factor; BMP = bone morphogenetic protein; BMSC = bone marrowederived mesenchymal stem cell; ECM = extracellular matrix; FGF = fibroblast growth factor; HA = hyaluronic acid; HGF = hepatocyte growth factor; Oto-HNS = otorhinolaryngologyehead and neck surgery; PLGA = poly (lactic-co-glycolic acid); SLP = superficial lamina propria; TGF = transforming growth factor Correspondence: Address to David G. Lott, MD, Mayo Clinic Arizona, 5777 E Mayo Blvd, Phoenix, AZ 85054 Mayo Clin Proc. n December 2014;89(12):1722-1733 www.mayoclinicproceedings.org

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([email protected]). Individual reprints of this article and a bound reprint of the entire Symposium on Regenerative Medicine will be available for purchase from our website www.mayoclinicproceedings.org. The Symposium on Regenerative Medicine will continue in an upcoming issue.

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