Review

Airway tissue engineering: an update Jonathan M Fishman†, Katherine Wiles, Mark W Lowdell, Paolo De Coppi, Martin J Elliott, Anthony Atala & Martin A Birchall

1.

Introduction

2.

Airway tissue engineering

3.

Scaffolds

4.

Cells

5.

Bioreactors

6.

Tissue-engineered airway

†

skeletal muscle 7.

Transplantation of tissue-engineered airways

8.

Tissue engineering the larynx

9.

Tissue engineering lungs

10.

Conclusion

11.

Expert opinion

UCL Institute of Child Health, Department of Surgery, London, UK

Introduction: Prosthetic materials, autologous tissues, cryopreserved homografts and allogeneic tissues have thus far proven unsuccessful in providing long-term functional solutions to extensive upper airway disease and damage. Research is therefore focusing on the rapidly expanding fields of regenerative medicine and tissue engineering in order to provide stem cell-based constructs for airway reconstruction, substitution and/or regeneration. Areas covered: Advances in stem cell technology, biomaterials and growth factor interactions have been instrumental in guiding optimization of tissue-engineered airways, leading to several first-in-man studies investigating stem cell-based tissue-engineered tracheal transplants in patients. Here, we summarize current progress, outstanding research questions, as well as future directions within the field. Expert opinion: The complex immune interaction between the transplant and host in vivo is only beginning to be untangled. Recent progress in our understanding of stem cell biology, decellularization techniques, biomaterials and transplantation immunobiology offers the prospect of transplanting airways without the need for lifelong immunosuppression. In addition, progress in airway revascularization, reinnervation and ever-increasingly sophisticated bioreactor design is opening up new avenues for the construction of a tissue-engineered larynx. Finally, 3D printing is a novel technique with the potential to render microscopic control over how cells are incorporated and grown onto the tissue-engineered airway. Keywords: airway management, angiogenesis, artificial organs, biocompatible materials, bioreactors, larynx, regenerative medicine, stem cells, tissue engineering, tissue scaffolds, trachea, transplantation Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

Tissue engineering as a field is reaching maturity, growing at an estimated 22.5% per year and yielding promising results [1]. Within tissue engineering, airway reconstruction has been one of the first fields to witness clinical application. Macchiarini et al. performed the first tissue trachea transplant in 2008 [2]. The trachea is amenable to regeneration since it is a hollow luminal organ with a relatively simple function (i.e., it acts as a simple conduit for the passage of air into the lungs). However, the trachea is also exposed to the exterior environment, making it imperative that such constructs are biocompatible and highly functional. Since our last review in 2011, some of the areas for further study that we identified have been addressed. These include the optimization of available technology and the application to more nuanced aspects of reconstruction [3]. This review will focus on recent advances within the field of upper airway tissue engineering (i.e., trachea and larynx) but will conclude with some exciting recent developments with respect to tissue-engineering lower airways (i.e., lungs).

10.1517/14712598.2014.938631 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

1

J. M. Fishman et al.

Article highlights. .

.

.

.

.

Tissue-engineering large airways represents an attractive alternative to conventional airway reconstructive techniques such as the use of autologous tissues (e.g., pericardial patches, slide tracheoplasty), allogeneic tissues and tracheal homografts. Several patients to date have received tissue-engineered airways. In some cases up to 5 years of follow-up data have been reported with encouraging results. Studies have either utilized a decellularized or synthetic scaffold approach and either an in vitro, in vivo or in situ tissue-engineering approach. It is currently unclear which technique is best in relation to airway tissue engineering. The encouraging results obtained thus far in relation to tracheal tissue engineering have provided an impetus to engineer increasingly more complex organs and tissues (e.g., larynx and esophagus). Full-scale Phase I/II clinical trials are now required and are currently underway in the UK (RegenVOX study).

This box summarizes key points contained in the article.

In order to successfully tissue engineer a functional upper airway, an understanding of its normal anatomy and physiology is required. Anatomically, the trachea is a longitudinally flexible fibroelastic tube that forms a conduit between the larynx and the lungs. The anterior and lateral sides of the trachea are formed from C-shaped rings of hyaline cartilage, leaving a relatively deficient membranous posterior wall covered in the smooth muscle, trachealis. The trachea is lined internally with a pseudostratified columnar epithelium containing goblet cells. Mucous production by the latter, when wafted up the airway by the former, protects the lungs from infection. The trachea receives its blood supply from branches of the inferior thyroid and bronchial arteries. The vagus and recurrent laryngeal nerve innervate the trachea, detecting sensations of pain and triggering the secretomotor function of the goblet cells. Sympathetic supply from nerve roots T1 -- T4 innervates the blood vessels and the surrounding smooth muscles. Building an upper airway through tissue-engineering techniques requires a careful consideration of the various layers that make up the airway; not only the epithelium and cartilage layers but also the connective tissue layer, which not only serves as the compartment containing blood vessels and nerves but also as the compartment containing glands, are important sources of mucus and fluid. Tissue reconstruction of the trachea has, thus far, primarily relied on a donor scaffold seeded with mesenchymal stem cells (to regenerate cartilage) and epithelial cells (to regenerate the epithelium on the luminal, or inner, aspect). Neovascularization and reinnervation have been investigated but not clinically implemented. Airway tissue reconstruction is essential in patients who have advanced tracheal stenosis. For most airway reconstruction, end-to-end anastomoses can be performed. However, when less than half (~ 6 cm) of the adult tracheal length is still 2

viable (one-third of the pediatric length), replacement of the trachea is necessary to maintain a functional airway. This extent of damage is usually due to trauma, congenital disease or neoplastic change. Replacement of the airway can be performed using an allogeneic transplant, a synthetically engineered trachea coated with autologous cells, or an allogeneic tracheal scaffold coated in autologous cells. Allograft transplantation of the airway has been attempted in clinical practice following the first unsuccessful transplant of laryngeal tissue in 1969 [4]. Transplantation of the larynx, pharynx and/or trachea has been met with skepticism due to the ethical implications of subjecting a patient to life-long immunosuppression for a procedure that is not life-saving and that which carries considerable morbidity and mortality [5]. This is especially true given the potential life span of pediatric patients with congenital malformations. Further, as with any transplant, there are major risks of reperfusion injury, rejection and infection [6]. The advantages of transplantation are primarily concerned with quality of life and have been undertaken on compassionate grounds. The first successful case of laryngeal transplantation was performed on a 40-year-old male with a stenotic larynx and pharynx. By 40 months post-transplant, the patient had regained voice, inflection, range and ability to swallow without signs of rejection [7]. A subsequent patient who underwent larynx, trachea, thyroid, pharynx and parathyroid gland transplant had robust revascularization, neuromuscular innervation and sensation of taste and smell [8]. Although these results are promising, the greatest drawback of transplantation is utility. Eligible patients include the rare few with benign resectable disease or a traumatic injury. By contrast, there are 1000 laryngectomies performed in the UK for head and neck cancer each year [5]. Giving immune suppression to this population would be at best risky and at worse fatal due to the high risk of tumor recurrence [9]. Given the advances in tissue engineering over the past decade, it is considerably more likely that improved functionality post-laryngectomy will come from a tissueengineering approach rather than from allotransplantation. An alternative to airway transplantation is the use of aortic allografts [10]. In a study of 15 patients who had undergone transcervical partial laryngectomy with aortic allograft reconstruction, all 15 patients had their tracheostomy tubes removed and resumed laryngeal phonation without major surgical complications [11]. The advantage of this technique would be in the emergency setting as current tissue-engineering methods take minimum 17 days [12]. Nevertheless, aortic allografts are not without their drawbacks. An earlier study identified the formation of fistulas between the esophagus and the graft in three of the four transplanted patients [13]. Animal studies in the pig noted that, although the aortic allograft formed an effective conduit, the major tracheal components remained immature and did not reproduce the function or structure of the native trachea [14]. In a similar study in sheep, 6 of 10 sheep required euthanasia post-transplant due to severe tracheitis, pneumonia, graft necrosis or airway

Expert Opin. Biol. Ther. (2014) 14(10)

Airway tissue engineering

obstruction when the stents were removed [15]. The mechanisms of tracheal regeneration seen with aortic allografts remain unclear, although recent evidence has implicated recipient-derived bone marrow-derived mesenchymal stem cell migration into the grafts as playing a pivotal role in the process [16]. More research into aortic graft viability in these human and animal studies is warranted. The potential for the use of tracheal allografts without immunosuppression has also been investigated. Delaere et al. reported an allograft transplant that was revascularized in situ in the host forearm for 9 months before being transplanted into the trachea. The patient was given 7.5 months of immune suppression pre- and post-operatively [17]. Delaere et al. followed up these initial findings with further tracheal allografts in four more patients (three with long segment tracheal stenosis and one with low-grade chondrosarcoma). They made several changes to their protocol in order to promote epithelialization and revascularization. In particular, immune suppression was withdrawn at 4 months postoperatively in the second patient and 1 month postoperatively in the third and fourth patients. Although the donor tracheas in these patients were fully revascularized in the forearm after 2 months, the repopulation of the mucosa was not complete. Further, immune rejection in patients 2 -- 4 resulted in a partial loss of the allograft due to mucosal necrosis postoperatively. For the fifth patient, the potential for resurgence of a chondrosarcoma, a low-grade malignancy, was deemed minimal during the 3-month immune suppression period. In order to prevent mucosal failure, the recipient’s buccal mucosa was grafted onto the trachea and revascularized. Incisions were also created through the intercartilaginous ligaments in order to improve the vascularization of the mucosa. At 6 months postoperatively, patient 5 had patent anastomoses [18]. These results suggest that, although allografts may potentially be used without immune suppression, there is still a strong possibility for immunogenic tissue necrosis. Additionally, with the reported positive outcomes of tracheal tissue engineering, the need for immune suppression in allografts undermines their therapeutic potential. 2.

Airway tissue engineering

Langer and Vacanti historically proposed three theoretical types of tissue engineering: cells-only, tissue-inducing or cells grown on a matrix [19]. Methods using only cells face the limitation that the infused cells may not function adequately and could be subject to immune rejection. In addition, they lack a template for adequate tissue formation. A tissueinducing substance requires the production of large numbers of the molecules but could theoretically be advantageous once harnessed as it would facilitate targeted regeneration. The use of cells in conjunction with a matrix could permit implantation of a construct into a human target. The latter approach has been the technique of choice due to the need for replacement of long-segment diseased trachea. Advances

have been made in the three areas that we identified in our previous review: the choice of cells for seeding, the scaffold on which to seed the cells and the bioreactor in which to harvest the construct (Figure 1). 3.

Scaffolds

One of the most active sources of debate within the field of tissue engineering is the choice of scaffold [20]. An ideal scaffold should be bioactive, biocompatible (failure to elicit an adverse host response, immunologically driven or otherwise), nontoxic and noncarcinogenic. It should also mimic the internal environment and maintain the mechanical properties of the trachea. In particular, the scaffold should maintain air and liquid tight seals as well as structural support (lateral rigidity and longitudinal flexibility) in order to protect the airway and permit epithelialization. Scaffold types can broadly be divided into synthetic and allogeneic decellularized tracheas. Synthetic scaffolds Two types of synthetic tracheal scaffolds have seen clinical application, one made by a propylene mesh tube covered with collagen sponge and the other made using a polymer, polyhedral oligomeric silsesquioxane (POSS)-poly(carbonateurea)urethane (PCU) (POSS nanocages covalently bonded to PCU polymer chains) [21]. The former developed cartilage and underwent epithelialization (nuclei, capillaries and regenerated epithelium) by day 14 in vitro. However, the new cartilage did not function as a framework and did not develop the properties of an endogenous trachea [22]. The construct was seeded with autologous venous blood and was transplanted into four patients affected by stenosis or cancer of the trachea. At 3 years postoperatively, good epithelialization was still observed. No rejection was found. An air leakage occurred in one of the grafts but the other three were functional [23]. How closely this structure mimics the endogenous trachea is unknown. A bioartificial POSS-PCU trachea-bronchial nanocomposite was seeded with bone marrow mononuclear cells and was transplanted into a patient with extensive cancer of the trachea. The patient was given G-CSF and epoetin-b over 14 days in order to promote cartilage formation and neovascularization, respectively. Initial investigations revealed necrotic connective tissue secondary to a fungal infection, but the patient was asymptomatic and tumor-free at 5 months. The scaffold had patent anastomoses, was lined with a vascularized mucosa and was partly covered with epithelium. Cell markers suggested the presence of cell-mediated wound repair, extracellular matrix (ECM) remodeling and vascularization of the graft [24]. The advantages of this polymer are that it can be tailor-made to fit the patient’s size, can be designed to have certain biomechanical properties and is ethically sound. It should be noted, however, that epithelialization and neovascularization were difficult to achieve on this bioartificial composite [25]. Theoretically, a smart polymer that could incorporate signaling 3.1

Expert Opin. Biol. Ther. (2014) 14(10)

3

J. M. Fishman et al.

1. Cells only

2. Tissue-modifying factor

Synthetic scaffold or

3. Cells

Donor trachea

Bioreactor in vitro culture

Figure 1. Different ways to tissue-engineer a trachea are shown. Figures 1) and 2) demonstrate an ‘in vivo’ tissue-engineering approach whereby cells are seeded onto scaffolds at the time of implantation, with or without the use of exogenous bioactive molecules (growth factors); the latter may assist in the maintenance, proliferation and differentiation of cells. Figure 3) demonstrates an ‘in vitro’ tissue-engineering approach whereby both epithelial cells and chondrocytes are seeded onto scaffolds within an ex vivo bioreactor prior to implantation. The bioreactor maintains conditions similar to that seen in the in vivo microenvironment.

molecules to promote cell--scaffold interaction and eventually dissolve leaving a native tissue could meet all of the criteria of a tissue-engineered airway. More recently, alternative polymers (such as electrospun polyethylene terephthalate and polyurethane scaffolds) have been designed that more closely mimic the 3D structure of the native ECM [26]. Future studies are warranted directly comparing such scaffolds with other synthetic scaffolds, such as those composed of POSS-PCU, for airway tissue engineering. Decellularization of human allografts Human allografts and porcine xenografts have also been extensively investigated in vitro as possible scaffold sources [27]. The decellularized human tracheal allograft is the current scaffold of choice as it is the least immunogenic, although use of porcine tissues in the future may offer an unlimited source of organs for transplantation. With respect to the latter, the a-gal epitope, previously felt to be an insurmountable barrier to xenotransplantation by causing hyperacute rejection, has not been shown to pose a significant problem when porcine tissues are decellularized and stripped of surface epitopes [27]. Decellularization is, nevertheless, a stringent process because even a small adverse host immune response could lead to graft rejection, which in the case of airways would be disastrous. An accepted definition of decellularization is the presence of < 50 ng dsDNA/mg ECM 3.2

4

by dry weight, < 200 base pair DNA fragment or lack of visible nuclear material when stained with 4¢,6-diamidino-2-phenylindole or hematoxylin and eosin [20,28,29]. The detergentenzymatic method (DEM) is employed to decellularize donor tissue as it is better than formalin and acetone at decreasing antigenicity without altering the basement membrane complex [30]. In the DEM, the trachea is subjected up to 25 cycles of decellularization in order to fully remove DNA and human leukocyte antigen classes I and II, while preserving basement membrane structure, hierarchical structure and angiogenic response. Even at its fastest airway implementation (17 cycles, 5 weeks), this process is still inappropriate for prospective manufacture in emergency situations [31]. While the DEM method does not affect cell attachment and vascularization, it contributes to mechanical instability of the trachea by loss of glycosaminoglycans (GAGs) [32]. However, non-decellularized tissue scaffolds lose GAGs in vivo without losing tensile properties, suggesting that there are other contributors to mechanical stability [33]. Using the DEM in pig allografts and mice xenografts, Jungebluth et al. determined that maximum force and rupture force were lower in decellularized tissue than in native tissue [34]. Tissue deformity percentage was also higher in the decellularized tissue compared to control tracheal tissue. It has been suggested that this increase in airway compliance could contribute to

Expert Opin. Biol. Ther. (2014) 14(10)

Airway tissue engineering

chondrocyte loss and subsequent postoperative stenosis [35]. Therefore, it is important to find the balance between decellularized tissues that are sufficiently non-immunogenic without losing biomechanical integrity. An innovative decellularization process has recently been proposed and implemented. Gillies et al. demonstrated improved removal of DNA and intracellular proteins and retention of GAGs from the dense tissue tibialis anterior using latrunculin B, hyper and hypotonic solutions and DNase [36]. However, the mechanical properties of the trachea were not significantly improved in this new method as compared to the DEM. Less-aggressive treatments such as freeze thaw, lyophilization and chemical dehydration may be more suitable for the maintenance of chondrocytes [20]. These types of decellularization have not been successfully implemented within a clinical setting. Ultimately, preservation of airway biomechanical properties through decellularization would optimize scaffold integrity and longevity post-transplant. 4.

Cells

Choice of cells Anatomically, the two main cell types that are required for seeding airway scaffolds are chondrocytes, to regenerate the tracheal cartilage, and epithelial cells, to line the airway. The ‘ideal’ cell type must be stable and functional, abundant, fail to elicit an adverse host immune response and noninvasive for the purpose of harvesting (Figure 2). Epithelial cells are readily available in the form of nasal epithelial cells and grow with relative ease. Unfortunately, in vivo, they have not adequately recreated the pseudo-stratified columnar epithelium characteristic of the trachea, leading to difficulties in mucociliary clearance and post-transplant infection [37]. Whether epithelial cells are derived from primary cell cultures, differentiated from autologous fibroblasts (induced pluripotent stem cells [iPSCs]), or derived from elsewhere, optimization of epithelial cell cultures and differentiation protocols is required in order to generate large numbers of fully functional cells for tissue engineering airways. Chondrocytes have been more challenging to harvest and grow effectively, and a wide variety of cell types have been tested. These range from embryonic stem cells (ESCs) with the highest expansion capability to differentiated adult cells which have only very limited expansion capability [38]. These cell types may also be divided by source: autologous, allogeneic or xenogeneic. A xenograft model of human bone marrowderived mesenchymal stem cells (BM-MSCs) injected into scarred rabbit vocal folds demonstrated that the cells did not seem to survive at 3 months even though immune suppression was administered every second day. The tendency to thick epithelium and fibrosis strongly suggests that xenogeneic cells do not differentiate and expand appropriately when transplanted [39]. One of the most studied allogeneic cell sources is human ESCs (hESCs). When injected into the scarred vocal folds of 4.1

rabbits, hESCs yielded human cartilage, muscle, and epithelial tissue that mixed with native rabbit tissue. At 1 month follow up, no anaplasia was found [40]. However, the burden of proof of no anaplasia with hESCs is very high, and it is unlikely that they will be used in a non-life-saving procedure. Further, hESCs present ethical concerns of access and usage that render them a highly regulated and potentially inconsistent source of cells. Another cell group that has been investigated in conjunction with airway tissue engineering is stem and progenitor cells from the fetus, umbilicus and amnion. Amniotic fluid stem (AFMSCs) and progenitor cells are generally preferred over fetal and umbilical placental cells as the former demonstrate good expansion but lower risk of malignancy than the latter. AF-MSCs are more effective at chondrogenesis on pediatric scaffolds rather than adult ones. For the 1/100,000 pediatric patients who have laryngotracheal agenesis, the choice of cell may well prove to be those from the amniotic fluid as they are accessible and can be transplanted at birth [41]. Interestingly, AF-MSCs exhibit a proangiogenic effect. Although the mechanism of this effect is unknown, it could be mediated by recruiting host repair cells [42,43]. Research into how this angiogenic response occurs could inform adult tissue engineering. The first preclinical regulatory validation of a diaphragmatic tendon engineered using AF-MSCs has been brought [44]. Thus, the application of AF-MSCs in the pediatric population may be on the horizon. Autologous cells would be preferable to allogeneic or xenograft cells as ethical issues are assuaged and immune matching would minimize local inflammation and graft failure. However, autologous cells may not be easily acquired from the patient due to procedural difficulty or underlying malignancy. Attempts to use a readily available, well-differentiated cell types such as adipose tissue have failed in animal models due to scaffold breakdown and lack of appropriate cell differentiation and structure [45]. Autologous iPSCs are an attractive solution due to their expansion capability and noninvasive harvesting from skin fibroblasts. The iPSCs are cells that were initially well differentiated but have been reprogrammed using a cocktail of factors into a progenitor or stem cell [46]. However, iPSCs have been found to have ‘memory’ of their former cell types and do not fully function as their reprogrammed cell type [47]. Another concern is that epigenetic and genetic changes may occur during the reprogramming, especially tumorigenic mutations. Realistically, however, this caveat is true to all cell-based therapies that undergo manipulation. Recent advancements in iPSCs have been promising, as the field has made some leaps toward generating unlimited quantities of cells [48]. In particular, iPSCs have been made to differentiate into functional airway and lung epithelium [49-51]. One of the greatest concerns regarding iPSCs to date is the costly introduction of external growth factors and proteins. Methodologies for generating iPSCs cheaply using physical stress or chemical strain have been proposed, but a successful

Expert Opin. Biol. Ther. (2014) 14(10)

5

J. M. Fishman et al.

1. ESCs

3. Fibroblast iPSCs

2. AFS cells

4. MSCs

Bioreactor in vitro culture

Figure 2. Stem cells may be derived from a multiplicity of different cell sources for airway tissue engineering, including ESCs (1), AFS cells (2), iPSCs (3) and MSCs (4). Cells derived from primary cell cultures are not shown in the figure, which are generally post-mitotic and therefore yields and proliferation rates tend to be lower. AFS cells: Amniotic fluid stem cells; ESCs: Embryonic stem cells; iPSCs: Induced pluripotent stem cells; MSC: Mesenchymal stem cell.

implementation remains elusive. As the techniques for generating and then reprogramming iPSCs continue to improve, the resulting cells may turn out to be a cost-effective and functionally appropriate choice. There are some stem cells in the adult humans that can be harvested. At the moment, autologous BM-MSCs are the most commonly implemented cell type in clinical applications of tracheal engineering. These are accessible (albeit not as easily as iPSCs) with no potential for immunogenicity, a safe track record in clinical practice, and high yield [52]. Interestingly, BM-MSCs are comparable in vitro to human vocal fold fibroblasts when assessed by cell viability and cell surface markers [53]. Human vocal fold fibroblasts are not available commercially and the primary cells are difficult to acquire; therefore, BM-MSCs may well find a second use in laryngeal tissue engineering. Which cell factors to use? Determining which factors to use to promote chondrocyte growth and differentiation has presented some obstacles in tissue engineering [54,55]. Growth factors have the potential for malignancy and yet can promote cell differentiation. Known growth factors involved in chondrocyte growth include 4.2

6

TGF-b, IGF, fibroblast growth factor, bone morphogenetic protein, hedgehog and wingless (human homolog, Wnt). From a cost perspective, animal products would be considerably cheaper than human growth factors. However, animal products introduce the potential for a human immune response. More research is needed to determine which growth factors should be introduced, and if synthetic nonimmunogenic factors (such as human recombinant erythropoietin [EPO]) can be generated. Growth factors have, nevertheless, been investigated in conjunction with BM-MSCs [56]. The paracrine effects during remodeling processes are clearly important in cell-based therapy and its importance should not be underestimated. TGF-b has been most extensively studied. Cals et al. demonstrated that TGF-b3 is a more effective subtype in promoting BMMSC chondrogenesis than TGF-b1 [57]. Another animal study found increased BM-MSC expansion following the use of human recombinant EPO and G-CSF both intraoperatively and postoperatively. Further, the BM-MSCs had increased expression of EPO receptors and anti-apoptotic genes. A contraindication for the use of G-CSF is that this factor expands myeloid cells -- mainly neutrophils -- which may then weaken cell anchoring [24,54].

Expert Opin. Biol. Ther. (2014) 14(10)

Airway tissue engineering

Accurately imitating the in vivo environment and its complex interaction of growth factors may be impossible. However, the clinical application of growth factors could be their seeding directly onto the scaffold intraoperatively and then complemented by further delivery postoperatively. As a gold standard, in a scenario in which growth factors are better understood, a cocktail of growth factors delivered during cell seeding in vitro could help guide cell expansion and differentiation. 5.

Bioreactors

Integral to the optimization of cell seeding on airway tissue scaffolds is the function of the bioreactor. Initially, bioreactors were static, 2D in vitro culture systems. A number of improvements have been made to bioreactors to facilitate appropriate cell growth as well as to closely mimic conditions in vivo [58]. Chang et al. developed a sterilizable, double-chamber, rotating construct bioreactor [59] in order to seed both chondrocytes and epithelial cells in their differing environments [60]. The rotation between liquid and gaseous phases also generates the shear stress necessary to promote cell growth and differentiation. It is worth noting that the structure of the bioreactor leads to blood flow within and around the graft as well as bidirectionally, forming a different microenvironment to that of the human body [24]. What, if any, implications this has for cell growth and differentiation have yet to be determined. Improvements in the mechanical stimulation of tracheal constructs have yielded promising results. Dynamic deformational loading and sheer of chondrocytes embedded in a 3D scaffold led to increased GAG synthesis and an improved mechanical profile [61]. Translational and rotational strain of human BM-MSCs in collagen gel improved both cell alignment and fiber formation [62]. Niklason et al. demonstrated that pulsatile radial stress of tubular scaffolds seeded with smooth muscle cells improves organization of blood vessels [63]. An interesting improvement that could be applied to laryngeal tissue engineering is the use of a bioreactor that is capable of vibrating two cell-seeded substrates in a sinusoidal frequency such that the substrates contact each other [53]. As bioreactors develop mechanical properties similar to the host environment, cell growth and differentiation may be optimized. Although mechanical similarity to the in vivo environment is being addressed, there are a number of other areas for improvement to bioreactors [38]. One of the more novel bioreactors is made of eggshell, facilitating gas exchange. Interestingly, similar to cartilage, the eggshell-based gas exchange increases in volume during agitation [64]. The incorporation of disposable or sterilizable components into the bioreactor is a functional but vital aspect of bioreactor design. The further improvement of component parts would permit optimization of fluid handling and, consequently, of delivery of nutrients to cells. In particular, a gold-standard bioreactor would have a variety of measurement capabilities within the construct such as detection of pressure and concentration of nutrients and cytokines. The capability for adjustment in

reaction to the information generated by the bioreactor would permit adaptability of the bioreactor (and its operator) to the internal environment of the scaffold. 6.

Tissue-engineered airway skeletal muscle

One of the areas of the greatest improvement has been in the development of the skeletal muscle. Growth of skeletal muscle would be invaluable to patients who have lost motor function in the airway vital to the actions of swallowing, phonation and coughing. An accessible source of skeletal muscle is of paramount importance as satellite cells cannot regenerate damaged muscle when the loss is > 20%. In theory, tissue-engineered skeletal muscle cells should meet the following criteria: be from an accessible source with high yield, be able to grow homogenously for a long time, be susceptible to in vitro transduction with vectors encoding therapeutic genes and be able to reach sites of muscle regeneration through a systemic delivery route [65]. Regarding the first criterion, the urinary bladder [66] and small intestine submucosa in pigs [67] have aided in the de novo formation of large amounts of skeletal muscle. How applicable this technique would be in humans has yet to be determined. Stem cells have also been investigated as inducers of skeletal muscle. Mesangioblasts have been tested in vitro and in vivo in mouse [68] and dog [69] muscular dystrophy. Kanemaru et al. induced skeletal muscle lineage cells from human, rat and canine BM-MSCs with 89% efficiency and transplanted the cells into injured posterior cricoarytenoid muscles. The muscle choice is significant as the posterior cricoarytenoid is the only abductor muscle in the human larynx. The authors found that autologous induced muscle cells effectively restored vocal fold moment, whereas control and allograft transplants did not [70]. The use of stem cells has been furthered by the first reported transplantation of iPSCs into a mouse muscular dystrophy model [71]. Not only did the iPSCs resolve the dystrophic phenotype, but they also replenished the depleted progenitor supply. We have recently successfully regenerated skeletal muscle using a decellularized scaffold of the rabbit cricoarytenoid muscle. Decellularization achieved DNA clearance and decreased levels of MHC classes I and II. Further, the scaffold’s structural integrity was preserved. Following xenotransplantation in rats, six rabbit grafts showed minimal signs of rejection at 2 weeks. However, there were many remaining questions that we feel are worth addressing. Which cells infiltrated the muscle scaffold -- host or donor? What happens to the grafts if they are left in situ for longer periods of time? How functional is this muscle [72]?

Transplantation of tissue-engineered airways

7.

The application of tissue-engineered transplants to clinical practice has expanded extensively in the past 6 years.

Expert Opin. Biol. Ther. (2014) 14(10)

7

J. M. Fishman et al.

Table 1. Survival of tissue-engineered tracheae between 2008 and 2014 according to published* outcomes.

Biologic adults Synthetic adults Biologic children Synthetic children

Alive

Dead

Survival %

1 0 1 0

1 2* 1* 2*

50 (50) 0 (0) 50 (50) 0 (0)

*Including ‘published’ in the popular press only where indicated.

Tissue-engineered transplants have been investigated in a wide range of organs and tissues, including the small bowel, trachea, larynx and lung [25,73-78]. In particular, the urethra is another tubular structure that has been clinically transplanted using a biodegradable poly(glycolic acid) scaffold with autologous urethral muscle, epithelial cells and lymphatics in five patients needing reconstruction. At 6 years postoperatively, the grafts showed no signs of immunogenicity [79]. Tracheal and urethral constructs have been the vanguard of clinical application because of the tractability of the tissue type: a hollow tube that does not require extra consideration in order to generate specialized functionality. Within the context of upper airway tissue engineering, several adult and pediatric patients to date have received either decellularized or synthetic-based scaffolds seeded with autologous stem cells, as summarized in Table 1.

Adult tissue-engineered tracheal transplants Macchiarini et al. transplanted a tissue-engineered left main bronchus in a 30-year-old female with advanced bronchomalacia. The team used a detergent-enzyme decellularized trachea allograft seeded with nasal epithelial cells and BM--MSC-derived chondrocytes in a rotating construct bioreactor [2]. The decellularization process lasted 6 weeks with 25 rounds of enzyme-detergent washing. No immunosuppressant drugs were given postoperatively. Encouragingly, the graft was completely covered with mucosa at 1 month. At 18 months postoperatively, there were no signs of chronic rejection. Further, laser Doppler indicated a healthy adjacent microvascular bed from postoperative day 4 and active mucosal bleeding from day 30. The nasal epithelial cells grew too fast and may have apoptosed, suggesting that a different epithelial cell type should be explored. We called for longerterm follow up on tissue-engineered transplants prior to undertaking a large-scale trial [3]. A 5-year follow up has since been published indicating no clinical immune response and no serological signs of rejection in the patient [80]. These results are promising, as they demonstrate that the protocol is both safe and effective. However, the patient had a recurrent stenosis at the native trachea near the site of the anastomoses. This stenosis developed where a tracheal resection was performed 8 years prior, suggesting the 7.1

8

possibility of an underlying predilection for scarring at surgical sites. It is worth noting that airway complications in the left main bronchus postoperatively have been commented on before in lung transplant recipients [81]. One suggestion is that, postoperatively, reduced blood flow and impaired mucosa may have contributed to this stenosis. Addressing neovascularization is, therefore, paramount to transplant viability. Since the first tissue-engineered transplant, another patient, a 76-year-old male with severe tracheal stenosis received a tissue-engineered transplant. The trachea underwent a 17cycle DEM decellularization protocol in the bioreactor of Macchiarini et al. for 3 weeks [2,37]. Epithelial cells were cultured from the inferior turbinate and transplanted along with MSC-derived chondrocytes. Unfortunately, the patient died from a cardiac arrest secondary to severe stenosis of two coronary arteries. At autopsy, the tracheal anastomoses were intact, the submucosa had reformed, intact chondrocytes were present and one multilayer of squamous epithelium was found. The lumina of capillaries and red platelets were seen, suggesting that some neovascularization had occurred. However, there were no new arterioles or venules since the blood vessels seen contained no tunica media or adventitial layers. The autopsy, importantly, revealed that there was no sign of immune rejection, suggesting that a 17-cycle DEM is safe to use in human transplants [37]. In both patients, chondrogenesis was effective, but epithelialization was problematic. Nasal epithelial cells seem to be inadequate both in terms of the numbers of cells obtained, survival and formation of the appropriate histological subtype. Although squamous metaplasia is not uncommon in the airways of smokers, the formation of a pseudostratified columnar epithelium with goblet cells and the resultant mucociliary clearance would minimize the infectious risk in patients postoperatively. Pediatric tracheal transplants Congenital tracheal stability and bronchopulmonary malformations affect 2% of live births [82]. There are many potential uses for tissue engineering in the pediatric population, including those born with bronchopulmonary and genitourinary malformations, extracardiac cavopulmonary connections, various conditions affecting heart valves, and myelomeningoceles. In pediatrics, the time to the second operation must be long in concordance with the patient’s expected life span. Vascularization, epithelialization, absence of shortening and mechanical stability must all be optimized [83]. Further, the human trachea increases by several centimeters in adolescence. Therefore, adaptive growth is an extra criterion for pediatric tissue engineering. To our knowledge, only one pediatric stem-cell based tissue-engineered transplant has been reported in the medical literature. A 12-year-old boy with congenital tracheal stenosis received a transplant from a decellularized (17 cycles, 3 weeks) trachea seeded with G-CSF, BM-MSCs and autologous tracheal epithelium intraoperatively. As the operation was an 7.2

Expert Opin. Biol. Ther. (2014) 14(10)

Airway tissue engineering

emergency, no in vitro epithelial or chondrogenic growth prior to transplantation was performed. The graft was coated with human recombinant EPO and TGF-b intraoperatively to promote angiogenesis and chondrogenesis, respectively. G-CSF was also given for 3 days preoperatively, and intravenous recombinant EPO was given postoperatively. The graft revascularized after 1 week, but there was a strong neutrophil response at 8 weeks, generating extracellular traps of luminal neutrophils. Further, the stent used to keep the airway patent was quick to dissolve. The resultant loss of vertical stability may have contributed to mild collapse of the proximal graft and the need for two further stents. Complete epithelialization and return of biomechanical strength were established at 15 months postoperatively [84]. The introduction of G-CSF may have increased BM-MSC mobilization but may also have contributed to the resulting DNA neutrophil extracellular traps seen, leading to the length of time taken for mucosal clearance and epithelialization and increased potential for infections. TGF-b is also a promoter of scar tissue formation and an inhibitor of epithelialization, suggesting that it may have played a role in the delayed epithelialization [85]. Further, intraoperative seeding of the cells may have contributed to their delayed growth. In the first case of adult tissue engineering, mucosal coverage was achieved at 1 month and mucociliary clearance was achieved at 6 months [2]. This pediatric study, therefore, confirms that care should be taken with the application of growth factors as they may cause immunogenicity or unwanted knock-on effects, which could harm mucosal regeneration. It also suggests that, in their current form, tissue-engineered airways require improvement for application to the emergency situation where an ‘in vivo’ tissue-engineering approach is required. 8.

Tissue engineering the larynx

Given the large number of laryngectomies performed to treat head and neck cancer, a tissue-engineered larynx and trachea could provide a solution for patients who have undergone failed radiotherapy for cancer. Half of the larynx can be removed without total loss of function [22]. Thus, in theory a bioengineered construct would not need to fully restore neuromuscular activity in order to achieve functional breathing, a patent airway, and to some extent swallowing and voice. Baiguera et al. have developed a human laryngeal scaffold using 25 cycles of detergent-enzymatic decellularization and retaining the structures of the epiglottis, false vocal cords, true vocal cords, cricoid cartilage and thyroid cartilage. No nuclear material remained, biomechanical properties were intact and residual angiogenic factors suggested a strong potential angiogenic response [75]. Although the scaffold may be a tractable problem to untangle, the difficulties lie in the neovascularization, nervous innervation and muscle regeneration of a potential laryngeal transplant following total laryngectomy.

9.

Tissue engineering lungs

Although reconstructing the larynx or trachea is not life-saving, tissue-engineered lungs could have myriad uses for the thousands of patients who currently await a lung transplant. In vitro methods have been promising. Rat and sheep lung acellular scaffolds have been generated using four cycles of a DEM that decreases the time of production and better preserves architecture and ECM components [76]. The use of fewer cycles of DEM is not unprecedented. A decellularized small bowel scaffold only underwent one cycle of DEM as, at four cycles, the crypt and villous structure was lost [78]. Using four cycles of decellularization may be warranted in the clinical application as, in the lung, it is essential that architecture is preserved, and the need for immune suppression is already established. However, this would render airway tissue engineering no more effective than lung transplantation in addressing a surgical solution to lung cancer. Interestingly, another study in rats used a phosphate buffer with low pressure for 2 -- 3 h and maintained lung architecture while removing 99% of DNA and MHC I and II. However, although collagen was preserved, elastin and GAGs were removed. The decellularized lungs exhibited altered compliance, increased opening pressure and decreased levels of surfactant as compared to controls. Encouragingly, overall stress and tensile stress were similar to non-tissue-engineered lungs. Cells were prepared by seeding of neonatal rat lung epithelial cells and microvascular lung endothelial cells for 8 days. The choice of cells is particularly important as, in another study using carcinomatous human alveolar basal epithelial cells, overgrowth of the airways led to edema and obliteration even though the transplant anastomoses were patent [86]. When implanted in rats for 45 -- 120 min, the lungs participated in gas exchange [77]. Although tissue-engineered lung transplantation is still in its earliest stages, developments are being made. Current research is assessing how bioreactor cues such as stress, pressure and oxygen concentration affect scaffold seeding along with the best choice of cell to generate functional lung tissue. Lungs present a unique problem in that the precision of lung engineering is such that loss of structural integrity is unacceptable. The advances in tissue engineering airway could continue to inform lung tissue engineering. 10.

Conclusion

Tissue engineering using synthetic or human cadaveric decellularized scaffolds is, for the moment, the theoretically most viable alternative to allotransplantation. Tissue engineering presents the possibility for a biocompatible, nontumorigenic, adaptable mode for reconstruction of a failing airway [19]. The trachea has been a prime locale for studying organ replacement using tissue-engineering techniques for several reasons. The trachea is a hollow tube with a relatively simple anatomy.

Expert Opin. Biol. Ther. (2014) 14(10)

9

J. M. Fishman et al.

Moreover, the trachea is connected to two structures that would prove invaluable if they were also tissue engineered -- the lungs and the larynx. Finally, tracheal tissue engineering has already provided an option for patients with advanced tracheal stenosis leading to significantly improved quality of life. Such engineering could potentially provide a surgical treatment for patients with radiotherapy-resistant tumors of the airways resulting in loss of the trachea, larynx or pharynx. Even though there has been considerable progress in the field of airway tissue engineering in the past 3 years, outstanding research areas abound. We are only beginning to understand the long-term clinical results of trachea tissue transplants. Learning what the seeded cells give rise to in vivo and what cellular growth is host response will give insight into what concoction of cells is necessary for optimal transplant function. We have learned from the first two case studies that nasal epithelial cells do not function analogously to endogenous tracheal epithelium in airway constructs. The highlighted importance of epithelialization for graft viability suggests an important role not only for endogenous fibroblast activation but also for angiogenesis. How angiogenesis can be appropriately promoted is an intriguing and critical area for future research. Finally and perhaps most importantly, ‘success’ needs to be clearly defined outlining the primary outcome measures in each case. Future studies should focus on gathering long-term functional (e.g., mucociliary clearance), as well as survival and morphological (regeneration) data, with all three end points being utilized as determinants of success. With the advances currently being made in the development of tissue-engineered skeletal muscle, the potential implementation of laryngeal tissue transplantation is approaching. In order to progress in this particular application of airway tissue engineering, adequate reinnervation of tissue-engineered skeletal muscle must be achieved. Further, if tissue engineering is to be used in patients with neoplasms, proving that tissue-engineered airways are, in the long-term, both functional and biocompatible is of paramount importance. The recent pediatric tracheal transplantation is a step forward in assessing the former. The very recently published follow-up study of Macchiarini et al.’s initial transplant is encouraging for the latter. As the number and longevity of case studies increases, long-term follow up will determine how viable these tissue-engineered transplants are. In addition, full-scale Phase I/II clinical trials are currently underway in the UK (RegenVOX study) and results are eagerly awaited.

11.

Expert opinion

Immunogenicity Immunogenicity is one of the most challenging obstacles to overcome in airway tissue engineering. The case reports of tissue-engineered transplants have reported fungal infections immediately post-transplant, suggesting that some local immune suppression or modulation may be occurring [2,37,84]. Further, Berg et al. reported in their histopathological findings that the tissue-engineered trachea had a lymphoplasmacytic infiltrate in the submucosa close to the cartilage [37]. There are many variables that can affect the host response: manufacturing, rate of scaffold degeneration and presence of cross-species antigens. Animal studies have shown that naturally occurring allogeneic scaffolds can lose up to 60% of their mass within the first 4 weeks of transplantation [87]. However, one scaffold type generated an acute cell response when transplanted but was then replaced by muscle and connective tissue after 3 months. Therefore, the immune response may be part of the remodeling process. By contrast, another scaffold type did not have a strong host immune response but was nonfunctional, completely replaced by fibrotic tissue [27]. It remains to be understood how scaffolds interact immunologically with their hosts in both animal and human studies. In our recent study of skeletal muscle scaffold xenografts, it was found that the scaffold was immunomodulatory, demonstrating anti-inflammatory effects [88]. A decrease in T-cell proliferation activity and a switching of macrophage response to an M2 type supported the qualitative lack of a host immune response. There was also a shift from a TH1 phenotype, usually associated with graft rejection, toward a TH2 phenotype. This particular shift is notable because a TH2 type response is associated with transplant/graft acceptance [27]. Although it is known that decellularized allograft scaffolds are hypoimmunogenic, it is interesting to consider that our xenograft scaffold delayed immune rejection by up to 4 weeks in vivo [88]. Possibilities for the mechanism of immune modulation include the unmasking of peptides during the decellularization process, stimulation of cytokines and the induction of unrelated T cells or the activation of nonspecific bystanders. This effect may also include a contribution from regulatory (suppressor) T cells, which may also play a role in the effects seen, as described in this study and elsewhere [88,89]. The ramifications of scaffold-mediated immune modulation for a patient with an underlying cancer are currently unknown. 11.1

Reinnervation Reinnervation of laryngeal muscle to generate a functional tissue-engineered larynx is also on the horizon. In the past 3 years, two studies have emerged that have shed some light onto this problem by using electrodes to stimulate the muscle. Cheetham et al. used functional electrical stimulation of the posterior cricoarytenoid muscle in the horse in order to 11.2

As tracheal tissue engineering is implemented clinically, research will continue to address the more nuanced aspects of functionality. In particular, understanding the transplant immune response may facilitate application of this technique to cancer patients. Promoting angiogenesis and reinnervation 10

of skeletal muscle would improve and broaden the clinical applications of airway tissue engineering.

Expert Opin. Biol. Ther. (2014) 14(10)

Airway tissue engineering

enable vocal cord adduction. The horse is a particularly appropriate model animal as degeneration of vocal muscles in horses is unilateral and slow (as in the case of humans) and the larger size of the equine vocal muscles reduces some of the technical challenges of electrode implantation in smaller mammals. The prosthesis was a modified cochlear implant stimulator with an output of 12 electrodes. The patency of the airway was maintained during strenuous exercise even under recurrent laryngeal nerve block [90]. In the second study, F€orster et al. used functional electrical stimulation in mini-pigs in order to innervate the posterior cricoarytenoid muscle. Their study incorporated seven cases, each with bilateral electrodes stabilized by the cricoid cartilage. Maximal glottal opening was reached before the adductors were co-activated. Although the electrode was successfully implanted, the surgery was invasive. An endoscopic approach would be more clinically applicable. Further, although the electrode was functional, it is essential to demonstrate that the electrode is also mechanically stable in the long term [91]. Angiogenesis Angiogenesis is vital to prevent graft necrosis, generate thicker tissue, assist innervation and improve graft function [92]. In particular, angiogenesis is of paramount importance for the growth of a viable mucosa with adequate mucociliary clearance [93]. Although some tissue-engineered constructs, such as skin, do not require angiogenic support, neovascularization is essential in any tissue thicker than 400 µm [94]. Like epithelial cells and BM-MSCs, a cell type with angiogenic potential must be stable and functional, abundant, nonimmunogenic and noninvasive to harvest. As endothelial cells are generally highly immunogenic, an autologous source is preferable. Although endothelial cells from the saphenous vein yield large numbers, the procedure to harvest them is highly invasive. By contrast, cells harvested from skin sites have a poor yield. Alternatives include endothelial progenitor cells and iPSCs [95]. Currently, there is no adequate cell source for revascularization. Encouragingly, studies have shown that acellular scaffolds are capable of revascularization when only growth factors are incorporated. One possibility is the use of factors implicated in the healing of burns. This includes the administration of factors CXCL12/CXCR4 for activation of endogenous haematopoietic and endothelial stem cells [96]. One concern 11.3

with dispensing growth factors is that they may not be reaching their targets [97]. Suggestions for improved usage include slow release devices or targeted encapsulation mimicking targeted drug delivery [98]. Alternatively, angiogenic cells could be seeded along with the other cells and growth factors in vitro in order to fully participate in the complex regenerative process. There is still a danger of an immune response being launched against allogeneic human or xenogeneic growth factors and so synthetic cues are preferable. 3D printing We have addressed some of the alternatives to allogeneic scaffold-based tissue engineering including transplantation and use of synthetic scaffolds. We believe that radical improvement to tissue engineering could come from 3D printing. One of the primary concerns with synthetic scaffolds is that they fail to adequately mimic in vivo microarchitecture. Advances in 3D printing to create effective macrostructures can be combined with other technologies such as near-field electrode spinning and projection stereolithography [99,100]. Gauvin et al. used stereolithography to cover a 3D printed scaffold with human endothelial cells. The endothelial cells not only maintained their cell type but also generated a dense, confluent covering [100]. 3D-bioprinting has already been successfully utilized in a clinical setting to create a customized, bioresorbable airway splint which was subsequently implanted into a child with tracheobronchomalacia [101]. Improvement of the cell seeding process on a microscopic level could lead to better functionality of tracheal constructs and insight with which to address tissue engineering the larynx or lung. 11.4

Acknowledgment JM Fishman and Katherine Wiles have contributed equally to this work.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents, received or pending, or royalties.

Expert Opin. Biol. Ther. (2014) 14(10)

11

J. M. Fishman et al.

Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

2.

..

3.

..

4.

Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet 1999;354:S32-S4 Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372:2023-30 First-in-human stem cell-based tissueengineered organ transplant worldwide demonstrating ‘proof-of-concept’. In this case, an in vitro tissue-engineering approach was utilized. Fishman JM, De Coppi P, Elliott MJ, et al. Airway tissue engineering. Expert Opin Biol Ther 2011;11:1623-35 Previous expert opinion review of the field. Kluyskens P, Ringoir S. Follow up of a human larynx transplantation. Laryngoscope 1970;80:1244-50

5.

Birchall M, Macchiarini P. Airway transplantation: a debate worth having? Transplantation 2008;85:1075-80

6.

Birchall M. Human laryngeal allograft: shift of emphasis in transplantation. Lancet 1998;351:539-40

7.

Strome M, Stein J, Esclamado R, et al. Laryngeal transplantation and 40-month follow-up. N Engl J Med 2001;344:1676-9 First case of laryngeal allotransplantation worldwide.

.

8.

.

Farwell DG, Birchall MA, Macchiarini P, et al. Laryngotracheal transplantation. Laryngoscope 2013;123:2502-8 Second case of laryngeal allotransplantation worldwide.

9.

Birchall M. Tongue transplantation. Lancet 2004;363:1663

10.

Seguin A, Radu D, Holder-Espinasse M, et al. Tracheal replacement with cryopreserved, decellularized, or glutaraldehyde-treated aortic allografts. Ann Thorac Surg 2009;87:861-7

11.

12

Zeitels SM, Wain JC, Barbu AM, et al. Aortic homograft reconstruction of partial laryngectomy defects: a new technique. Ann Otol Rhinol Laryngol 2012;121:301-6

12.

Davidson MB, Mustafa K, Girdwood RW. Tracheal replacement with an aortic homograft. Ann Thorac Surg 2009;88:1006-8

13.

Wurtz A, Porte H, Conti M, et al. Surgical technique and results of tracheal and carinal replacement with aortic allografts for salivary gland--type carcinoma. J Thorac Cardiovasc Surg 2010;140:387-93.e2

23.

Omori K, Tada Y, Suzuki T, et al. Clinical application of in situ tissue engineering using a scaffolding technique for reconstruction of the larynx and trachea. Ann Otol Rhinol Laryngol 2008;117:673-8

24.

Jungebluth P, Alici E, Baiguera S, et al. Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Lancet 2011;378:1997-2004 First demonstration of airway tissue engineering using a synthetic scaffold (polyhedral oligomeric silsesquioxanepoly(carbonate-urea)urethane) approach.

14.

Jaillard S, Holder-Espinasse M, Hubert T, et al. Tracheal replacement by allogenic aorta in the pig. Chest 2006;130:1397-404

15.

Tsukada H, Ernst A, Gangadharan S, et al. Tracheal replacement with a silicone-stented, fresh aortic allograft in sheep. Ann Thorac Surg 2010;89:253-8

25.

Seguin A, Baccari S, Holder-Espinasse M, et al. Tracheal regeneration: evidence of bone marrow mesenchymal stem cell involvement. J Thorac Cardiovasc Surg 2013;145:1297-304.e2

Totonelli G, Maghsoudlou P, Fishman JM, et al. Esophageal tissue engineering: a new approach for esophageal replacement. World J Gastroenterol 2012;18:6900

26.

Delaere P, Vranckx J, Verleden G, et al. Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med 2010;362:138-45

Jungebluth P, Haag JC, Lim ML, et al. Verification of cell viability in bioengineered tissues and organs before clinical transplantation. Biomaterials 2013;34:4057-67

27.

Badylak SF, Gilbert TW. Immune response to biologic scaffold materials. Semin Immunol 2008;20:109-16

28.

Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials 2006;27:3675-83

29.

Reing JE, Brown BN, Daly KA, et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials 2010;31:8626-33

30.

Conconi MT, Coppi PD, Liddo RD, et al. Tracheal matrices, obtained by a detergent -- enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells. Transpl Int 2005;18:727-34

31.

Baiguera S, Jungebluth P, Burns A, et al. Tissue engineered human tracheas for in vivo implantation. Biomaterials 2010;31:8931-8

32.

Haykal S, Soleas JP, Salna M, et al. Evaluation of the structural integrity and extracellular matrix components of tracheal allografts following cyclical decellularization techniques: comparison of three protocols. Tissue Eng Part C Method 2012;18:614-23

33.

Partington L, Mordan N, Mason C, et al. Biochemical changes caused by

16.

17.

18.

.

Delaere P, Vranckx J, Den Hondt M, Leuven Tracheal Transplant Group. Tracheal allograft after withdrawal of immunosuppressive therapy. N Engl J Med 2014;370(16):1568-70 Case series of tracheal allotransplantation using a heterotopic vascularization approach, followed by withdrawal of immunosuppressive therapy and orthotopic transplantation.

19.

Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920-6

20.

Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011;32:3233-43

21.

22.

Gaudio C, Baiguera S, Ajalloueian F, et al. Are synthetic scaffolds suitable for the development of clinical tissueengineered tubular organs? J Biomed Mater Res A 2014;102(7):2427-47 Nomoto Y, Okano W, Imaizumi M, et al. Bioengineered prosthesis with allogenic heterotopic fibroblasts for cricoid regeneration. Laryngoscope 2012;122:805-9

Expert Opin. Biol. Ther. (2014) 14(10)

..

Airway tissue engineering

decellularization may compromise mechanical integrity of tracheal scaffolds. Acta Biomater 2013;9:5251-61 34.

35.

36.

37.

38.

39.

Jungebluth P, Go T, Asnaghi A, et al. Structural and morphologic evaluation of a novel detergent--enzymatic tissue-engineered tracheal tubular matrix. J Thorac Cardiovasc Surg 2009;138:586-93 Ma R, Li M, Luo J, et al. Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage. Biomaterials 2013;34:1790-8 Gillies AR, Smith LR, Lieber RL, et al. Method for decellularizing skeletal muscle without detergents or proteolytic enzymes. Tissue Eng Part C Methods 2010;17:383-9 Berg M, Ejnell H, Kova´cs A, et al. Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report. Tissue Eng Part A 2013;20:389-97 Jungebluth P, Moll G, Baiguera S, et al. Tissue-engineered airway: a regenerative solution. Clin Pharmacol Ther 2012;91:81-93 Cedervall J, A¨hrlund Richter L, Svensson B, et al. Injection of embryonic stem cells into scarred rabbit vocal folds enhances healing and improves viscoelasticity: short -- term results. Laryngoscope 2007;117:2075-81

40.

41.

42.

43.

Svensson B, Nagubothu RS, Cedervall J, et al. Injection of human mesenchymal stem cells improves healing of scarred vocal folds: analysis using a xenograft model. Laryngoscope 2010;120:1370-5 Lange P, Fishman JM, Elliott MJ, et al. What can regenerative medicine offer for infants with laryngotracheal agenesis? Otolaryngol Head Neck Surg 2011;145:544-50 Mirabella T, Cilli M, Carlone S, et al. Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model. Biomaterials 2011;32:3689-99 Mirabella T, Poggi A, Scaranari M, et al. Recruitment of host’s progenitor cells to sites of human amniotic fluid stem cells

implantation. Biomaterials 2011;32:4218-27

and adipose tissue. Tissue Eng Part B Rev 2010;16:435-44

44.

Turner CG, Klein JD, Steigman SA, et al. Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells. J Pediatr Surg 2011;46:57-61

56.

Ronziere M, Perrier E, Mallein-Gerin F, et al. Chondrogenic potential of bone marrow-and adipose tissue-derived adult human mesenchymal stem cells. Biomed Mater Eng 2010;20:145-58

45.

Wood MW, Murphy SV, Feng X, et al. Tracheal reconstruction in a canine model. Otolaryngol Head Neck Surg 2014;150(3):428-33

57.

46.

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76

Cals F, Hellingman C, Koevoet W, et al. Effects of transforming growth factor-beta subtypes on in vitro cartilage production and mineralization of human bone marrow stromal -- derived mesenchymal stem cells. J Tissue Eng Regen Med 2012;6:68-76

58.

Plunkett N, O’Brien FJ. Bioreactors in tissue engineering. Technol Health Care 2011;19:55-69

59.

Chang CH, Lin FH, Lin CC, et al. Cartilage tissue engineering on the surface of a novel gelatin--calcium -phosphate biphasic scaffold in a double -- chamber bioreactor. J Biomed Mater Res B Appl Biomater 2004;71:313-21

60.

Asnaghi M, Jungebluth P, Raimondi MT, et al. A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: from concept to clinical trial. Biomaterials 2009;30:5260-9

61.

Mauck R, Byers B, Yuan X, et al. Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomech Model Mechanobiol 2007;6:113-25

62.

Li YJ, Batra NN, You L, et al. Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation. J Orthop Res 2004;22:1283-9

63.

Niklason L, Gao J, Abbott W, et al. Functional arteries grown in vitro. Science 1999;284:489-93

47.

Kim K, Doi A, Wen B, et al. Epigenetic memory in induced pluripotent stem cells. Nature 2010;467:285-90

48.

Tabar V, Studer L. Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nat Rev Genet 2014;15:82-92

49.

Ghaedi M, Mendez JJ, Bove PF, et al. Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor. Biomaterials 2014;35:699-710

50.

Mou H, Zhao R, Sherwood R, et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell 2012;10:385-97

51.

52.

53.

54.

55.

Wong AP, Bear CE, Chin S, et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 2012;30:876-82 Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol 2007;211:27-35

64.

Gaston J, Quinchia Rios B, Bartlett R, et al. The response of vocal fold fibroblasts and mesenchymal stromal cells to vibration. PLoS One 2012;7:e30965

Lee H-P, Chen H-C, Lai C-W, et al. Eggshell as a novel biomimetic reactor for in vitro tissue culture. J Chin Inst Chem Eng 2005;36:321-30

65.

Jungebluth P, Bader A, Baiguera S, et al. The concept of in vivo airway tissue engineering. Biomaterials 2012;33:4319-26

Fishman JM, Tyraskis A, Maghsoudlou P, et al. Skeletal muscle tissue engineering: which cell to use? Tissue Eng Part B Rev 2013;19:503-15

66.

Brown BN, Valentin JE, Stewart-Akers AM, et al. Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component. Biomaterials 2009;30:1482-91

Puetzer JL, Petitte JN, Loboa EG. Comparative review of growth factors for induction of three-dimensional in vitro chondrogenesis in human mesenchymal stem cells isolated from bone marrow Expert Opin. Biol. Ther. (2014) 14(10)

13

J. M. Fishman et al.

67.

Valentin JE, Turner NJ, Gilbert TW, et al. Functional skeletal muscle formation with a biologic scaffold. Biomaterials 2010;31:7475-84

68.

Galvez BG, Sampaolesi M, Brunelli S, et al. Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. J Cell Biol 2006;174:231-43

69.

70.

71.

72.

73.

.

74.

.

Sampaolesi M, Blot S, D’antona G, et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 2006;444:574-9 Kanemaru SI, Kitani Y, Ohno S, et al. Functional regeneration of laryngeal muscle using bone marrow--derived stromal cells. Laryngoscope 2013;123:2728-34 Tedesco FS, Gerli MF, Perani L, et al. Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy. Sci Transl Med 2012;4:140ra89-9 Fishman JM, Ansari T, Sibbons P, et al. Decellularized rabbit cricoarytenoid dorsalis muscle for laryngeal regeneration. Ann Otol Rhinol Laryngol 2012;121:129-38 Badylak SF, Weiss DJ, Caplan A, et al. Engineered whole organs and complex tissues. Lancet 2012;379:943-52 Recent review of the field. Atala A, Kasper FK, Mikos AG. Engineering complex tissues. Sci Transl Med 2012;4:160rv12-12 Recent review of the field.

75.

Baiguera S, Gonfiotti A, Jaus M, et al. Development of bioengineered human larynx. Biomaterials 2011;32:4433-42

76.

Maghsoudlou P, Georgiades F, Tyraskis A, et al. Preservation of microarchitecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment. Biomaterials 2013;34:6638-48

77.

78.

14

Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010;329:538-41 Totonelli G, Maghsoudlou P, Garriboli M, et al. A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration. Biomaterials 2012;33:3401-10

79.

Raya-Rivera A, Esquiliano DR, Yoo JJ, et al. Tissue-engineered autologous urethras for patients who need reconstruction: an observational study. Lancet 2011;377:1175-82

80.

Gonfiotti A, Jaus MO, Barale D, et al. The first tissue-engineered airway transplantation: 5-year follow-up results. Lancet 2014;383:238-44 Five year follow up of the first patient to receive a tissue-engineered airway.

..

89.

Haykal S, Zhou Y, Marcus P, et al. The effect of decellularization of tracheal allografts on leukocyte infiltration and of recellularization on regulatory T cell recruitment. Biomaterials 2013;34:5821-32

90.

Cheetham J, Regner A, Jarvis JC, et al. Functional electrical stimulation of intrinsic laryngeal muscles under varying loads in exercising horses. PLoS One 2011;6:e24258

91.

F€orster G, Arnold D, Bischoff SJ, et al. Laryngeal pacing in minipigs: in vivo test of a new minimal invasive transcricoidal electrode insertion method for functional electrical stimulation of the PCA. Eur Arch Otorhinolaryngol 2013;270:225-31

81.

Takao M, Namikawa S, Yuasa H, et al. Airway complications after lung transplantation: is there a left-sided predilection? J Thorac Cardiovasc Surg 1995;109:815-17

82.

Boogaard R, Huijsmans SH, Pijnenburg MW, et al. Tracheomalacia and bronchomalacia in children: incidence and patient characteristics. Chest 2005;128:3391-7

92.

Ott HC. Engineering tissues for children: building grafts that grow. Lancet 2012;380:957-8

Rouwkema J, Rivron NC, van Blitterswijk CA. Vascularization in tissue engineering. Trends Biotechnol 2008;26:434-41

93.

Hamilton N, Bullock AJ, MacNeil S, et al. Tissue engineering airway mucosa: a systematic review. Laryngoscope 2014;124(4):961-8

94.

Auger FA, Gibot L, Lacroix D. The pivotal role of vascularization in tissue engineering. Annu Rev Biomed Eng 2013;15:177-200

95.

Kim S, Von Recum H. Endothelial stem cells and precursors for tissue engineering: cell source, differentiation, selection, and application. Tissue Eng Part B Rev 2008;14:133-47

96.

Avniel S, Arik Z, Maly A, et al. Involvement of the CXCL12/ CXCR4 pathway in the recovery of skin following burns. J Invest Dermatol 2006;126:468-76

97.

Yla¨-Herttuala S, Rissanen TT, Vajanto I, et al. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 2007;49:1015-26

98.

Levenberg S, Rouwkema J, Macdonald M, et al. Engineering vascularized skeletal muscle tissue. Nat Biotechnol 2005;23:879-84

99.

Derby B. Printing and prototyping of tissues and scaffolds. Science 2012;338:921-6

83.

84.

..

85.

Elliott MJ, De Coppi P, Speggiorin S, et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet 2012;380:994-1000 Two year follow up of the first tissueengineered tracheal replacement in a pediatric patient. In this case, an in vivo tissue-engineering approach was utilized. Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelialmesenchymal cell transition. Mol Biol Cell 2005;16:1987-2002

86.

Ott HC, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 2010;16:927-33

87.

Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, et al. Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair. J BoneJoint Surg 2007;89:621-30

88.

.

Fishman JM, Lowdell MW, Urbani L, et al. Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model. Proc Natl Acad Sci 2013;110:14360-5 Demonstration of the immunomodulatory effects of decellularized scaffolds. Expert Opin. Biol. Ther. (2014) 14(10)

100. Gauvin R, Chen Y-C, Lee JW, et al. Microfabrication of complex porous tissue engineering scaffolds using 3D

Airway tissue engineering

projection stereolithography. Biomaterials 2012;33:3824-34 101.

Zopf DA, Hollister SJ, Nelson ME, et al. Bioresorbable airway splint created with a three-dimensional printer. N Engl J Med 2013;368(21):2043-5

Affiliation

Jonathan M Fishman†1 BM BCh (Oxon) MA (Hons, Cantab) BA (Hons) MRCS (Eng) DOHNS (RCS Eng) PhD, Katherine Wiles2 BA (Hons), Mark W Lowdell3 PhD, FRCPath, Paolo De Coppi4 MD PhD, Martin J Elliott5 MD FRCS, Anthony Atala6 MD & Martin A Birchall7 MA (Hons, Cantab) MB BChir MD (Cantab) FRCS (Eng, Gen) FRCS (Eng, Otol) FRCS (ORL) FMedSci † Author for correspondence 1 Clinical Lecturer, UCL Institute of Child Health, Department of Surgery, 30 Guilford Street, London WC1N 1EH, UK Tel: +44 07989 331573; E-mail: [email protected] 2 Academic Centre, School of Biomedical Sciences, 1st Floor, Henriette Raphael Building, Guy’s Campus, London, SE1 1UL, UK 3 University College London, Department of Haematology, Royal Free Campus, London NW3 2QG, UK 4 Reader and Consultant Paediatric Surgeon, Great Ormond Street Hospital for Children and UCL Institute of Child Health, Department of Paediatric Surgery, 30 Guilford Street, London WC1N 1EH, UK 5 Professor of Cardiothoracic Surgery, Great Ormond Street Hospital for Children, Great Ormond Street, London WC1N 3JH, UK 6 Professor of Urology and Director, Wake Forest Institute of Regenerative Medicine, Richard H. Dean Biomedical Building, 391 Technology Way, Winston-Salem, NC 27101, USA 7 Professor of Laryngology, UCL Ear Institute, Nose and Ear Hospital, The Royal National Throat, Nose and Ear Hospital, 330-332 Gray’s Inn Road, London WC1X 8EE, UK

Expert Opin. Biol. Ther. (2014) 14(10)

15