Tissue engineering airway mucosa: A ... - Wiley Online Library

The Laryngoscope C 2013 The American Laryngological, V

Rhinological and Otological Society, Inc.

Systematic Review

Tissue Engineering Airway Mucosa: A Systematic Review Nicholas Hamilton, BSc, MBChB; Anthony J. Bullock, BSc, PhD; Sheila MacNeil, BSc, PhD; Sam M. Janes, BSc, MBBS, MSc, PhD; Martin Birchall, MA, MB, BChir, MD Objectives/Hypothesis: Effective treatments for hollow organ stenosis, scarring, or agenesis are suboptimal or lacking. Tissue-engineered implants may provide a solution, but those performed to date are limited by poor mucosalization after transplantation. We aimed to perform a systematic review of the literature on tissue-engineered airway mucosa. Our objectives were to assess the success of this technology and its potential application to airway regenerative medicine and to determine the direction of future research to maximize its therapeutic and commercial potential. Data Sources and Review Methods: A systematic review of the literature was performed searching Medline (January 1996) and Embase (January 1980) using search terms “tissue engineering” or “tissue” and “engineering” or “tissue engineered” and “mucous membrane” or “mucous” and “membrane” or “mucosa.” Original studies utilizing tissue engineering to regenerate airway mucosa within the trachea or the main bronchi in animal models or human studies were included. Results: A total of 719 papers matched the search criteria, with 17 fulfilling the entry criteria. Of these 17, four investigated mucosal engineering in humans, with the remaining 13 studies investigating mucosal engineering in animal models. The review demonstrated how an intact mucosal layer protects against infection and suggests a role for fibroblasts in facilitating epithelial regeneration in vitro. A range of scaffold materials were used, but no single material was clearly superior to the others. Conclusion: The review highlights gaps in the literature and recommends key directions for future research such as epithelial tracking and the role of the extracellular environment. Key Words: Tissue engineering; airway mucosa; respiratory epithelium; regenerative medicine; tracheal surgery. Laryngoscope, 124:961–968, 2014

INTRODUCTION Airway mucosa forms a continuous lining extending from the oral and nasal cavities to the terminal bronchioles of the lung. It is composed of an epithelial layer overlying a connective tissue layer known as the lamina propria (Fig. 1). The epithelium is stratified squamous within the oral cavity, pharynx, laryngeal inlet, true vocal cord, and most of the epiglottis. The rest of the larynx and respiratory tract in humans are lined by respiratory epithelium consisting of pseudostratified ciliated columnar cells with interspersed mucus secreting goblet cells. Airway mucosa acts as a barrier against infection and has several key physiological functions such as the humidification of inspired air and the clearance of secre-

From the University College London Ear Institute (N.H., M.B.); and the Lungs for Living Research Centre (S.J.), University College London, United Kingdom; and the Department of Materials Science and Engineering (A.B., S.M.), Sheffield University, Sheffield, United Kingdom. Editor’s Note: This Manuscript was accepted for publication October 2, 2013. Review performed at University College London Ear Institute. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Nick Hamilton, University College London Ear Institute, University College London, London, U.K. E-mail: [email protected] DOI: 10.1002/lary.24469

Laryngoscope 124: April 2014

tions via the mucociliary escalator. Injuries involving small surface areas can repair through migration of cells from the wound edge, and injuries involving a distance greater than a few millimeters result in collagen deposition, fibrosis, and scarring.1 Chronic upper airway disease has many causes (Table I) and represents a substantial health burden. Worldwide, 650,000 people are diagnosed with head and neck cancer each year, with many requiring substantial resections of soft tissue, including airway mucosa.2 In addition, diseased segments of airway mucosa may scar causing disabilities, including stenosis and airway obstruction. Current methods aimed at restoring airway mucosa use either myocutaneous flaps or split skin grafts, resulting in a stratified squamous layer that lacks the ciliation and mucus-secreting glands of the columnarlined airway. This is of particular concern within the upper respiratory tract, where poor mucociliary clearance requires patients to nebulize daily and can result in life-threatening airway obstruction from mucus plugging. Grafting is associated with donor site morbidity and limited by the suitability and availability of donor tissue. These limitations make flaps and grafting suboptimal or unsuitable for portions of the airway such as the trachea and larynx. Transplantation has been proposed for large defects, but it is associated with serious Hamilton et al.: Tissue Engineering Airway Mucosa

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Fig. 1. Anatomical site of tracheal injury in trials reviewed. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

morbidity from immunosuppression and has ethical and practical problems. Tissue engineering uses engineering principles, biology, and material science to provide tissue. The aim is to produce new tissue that is biocompatible in terms of cell type, function, longevity, and immunogenicity. Tissue engineering has been applied clinically to replace damaged sections of the trachea and larynx3–8; however, observed regeneration of mucosa is slow and suboptimal, thereby significantly limiting functional recovery. The development of an effective engineered mucosal solution would greatly extend the reach of these and other hollow organ replacement technologies. Indeed, effective early mucosalization may tip cost benefit significantly toward commercial viability. Tissue engineering of mucosa has achieved good outcomes in various settings. Injury to the cornea can result in a depletion of the limbal stem cells that regulate corneal epithelial growth, leading to a loss of corneal translucency and vision. It is now possible to take a 1-mm section of limbal epithelium from a healthy donor eye, expand the stem cells ex vivo on amniotic membrane or fibrin, and then transplant the resulting sheet to replace damaged cornea. Modifications to this technique include the use of a mouse 3T3 fibroblast-feeder layer to promote expansion of epithelial cells and the use of explant or suspension culture methods. A systematic review of all 28 reported cases between 1997 and 2011 concluded that there is insufficient evidence to support one technique over another, although it did find an overall success rate of 76%.9 The use of buccal mucosa to treat urethral strictures in substitution urethroplasty is now accepted practice in strictures greater than 3 cm.10 However, such grafting in long segment strictures or revision cases results in significant donor site morbidity. Therefore, tissue-engineering products have been developed, the most successful of which utilizes autologous buccal epithelial cells and oral fibroblasts expanded ex vivo and Laryngoscope 124: April 2014

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then seeded onto decellularized cadaveric human dermis and matured at an air–liquid interface to produce a sheet of tissue that is structurally, functionally, and histologically similar to buccal mucosa.11 Five patients with long segment urethral strictures have been treated in this way, with the engineered mucosa remaining in place in three of the five patients.12 One patient experienced hyperproliferation and fibrosis of the proximal portion of the graft, requiring a partial excision and replacement with native buccal mucosa. Another had to have the entire graft excised and replaced due to significant penile shaft fibrosis. Although the recurrence of stricture is not uncommon in substitute urethroplasty, the lack of a rich capillary network in the engineered mucosa compared to native mucosa and the possibility of a host immune response have been suggested as possible causes for the significant fibrosis in two of the five patients.12 Although endoscopic resection of early stage esophageal adenocarcinoma has improved the morbidity and

TABLE I. Aetiology of Chronic Upper Airway Disease. Malignant Benign

squamous cell carcinoma Idiopathic

adenoid cystic carcinoma idiopathic subglottic stenosis

Iatrogenic

postintubation tracheal stenosis airway stenting tracheostomy radiotherapy

Traumatic Autoimmune

laryngeal fracture airway fire/burn Wegener’s granulomatosis sarcoidosis mucous membrane pemphigoid

Congenital

congenital subglottic stenosis laryngomalacia

Hamilton et al.: Tissue Engineering Airway Mucosa



963

2008

2008

Nomoto

2009

Go

Ni

2010

Kobayashi

2009

2010

Heikal

Nakamura

2010

Delaere

2009

2011

Jungebluth

Okano

2012

Year

Eliott

Author

Rat

Rabbit

Canine

Rabbit

Pigs

Rat

Sheep

Human

Human

Human

Type

27

12

18

24

20

7

12

1

1

1

No of Subjects

Tracheal fibroblasts

A cellular

Bone marrow aspirate or Mesenchymal stem cells or blood

Tracheal fibroblasts

Tracheal epithelium 1 MSC chondrocytes

Tracheal epithelial cells 1 gingival fibroblasts 1/adipose stem cells

Fibrin respiratory epithelial cell layer

Buccal mucosal graft (not expanded)

Bone marrow mononuclear cells

Mesenchymal stem cells and autologus epithelial cells

Cell Type

Collagen coated polyprolene mesh

Titanium mesh soaked in silk fibrin



Decellularized cadaveric tracheal segment


Fibrin-fibroblast layer 1titanium

8-cm tracheal allograft

Nanocomposite polymer

7cm decellularised trachea

Scaffold Type

1.5mm:3mm tracheal defects grafted

Semicircular tracheal defect 7th-12th rings implanted

5cm circumferential resection

5:9mm tracheal defect grafted

6cm tracheal segment transplanted


3:2cm tracheal mucosal defect grafted

Pre-implanted in forearm (4 months) and transplanted immuno supressed

6cm trachea 1 bilateral bronci transplanted

Tissue engineered tracheal transplant

Method

Cell expansion and differentiation

Cell differentiation and extent of stenosis

Incorporation of prosthesis, stenosis, exposure of mesh and cell differentiation

Bronchoscopic and histological examination at 7 & 14 days

Infection, stenosis, survial

Histological examination in vitro and in vivo

Macroscopic assessment of scarring, histology of mucosa and fluorescence analysis

Need for airway prosthesis, lung function tests

Extent of epithelialization, stenosis, clinical outcome

Endoscopy, cytology, ventilation perfusion scan

Primary Outcome Measure

Fibroblast group showed regeneration of airway mucosa faster than controls

No stneosis, fully differentiated columnar epithelium at 12 weeks

Stenosis in five dogs, incomplete mucosal layer in all, respiratory mucosa found only at anastomotic ends

Yes

Yes

No

Yes

Yes

Combination of MSC externally and Resp epithelial cells on lumen resulted in reduced infection and stenosis Columnar ciliated epithelium found compared to stratified squamous on control

Yes

Yes

Yes

No

Yes

Complete Mucosa

Enhanced epithelialization with GFBs 1 ASCs compared to GFBs alone

Less fibrosis than controls, improved mucosal regeneration with fibroblasts

Airway prosthesis free and adequate lung function tests at one year

Improved clinical parameters, partial epithelialization at two months

Complete epithelialization on endoscopy (15 months), ciliated epithelia on cytology, normal perfusion scan

Outcome

TABLE II. Experimental Trials of Tissue Engineered Airway Mucosa Applied to the Upper Airway

Fibroblast promote respiratory mucosal regeneration

Only one control, poor growth at 4 weeks

Poor epithelialization, no preimplantation in omentum

Circumferential injury not examined

The mucosal to lining acts as a barrier to infection

Small defect examined

Fibroblast promote respiratory mucosal regeneration

Eventual implant 4.5cm

No histological analysis, long term mucosal integrity not assessed

Required dilatation up to 18 months post procedure

Comment

29

33

22

32

27

28

37

21

6

20

Ref


964


2005

Omori

2000

2006

Nomoto

Suh

2007

Yamashita

2004

2008

Macchiarini

Kim

2008

Year

Tada

Author

Canine

Canine

Human

Rat

Canine

Human

Rat

Type

17

10

1

9

5

1

28

No of Subjects

Buccal mucosal graft (not expanded)

Epithelial cells from abdominal skin

A cellular

Tracheal epithelial cells

Peripheral blood

Respiratory epithelial cells

A cellular

Cell Type

Gelatin coated polyprolene mesh

Gelatin coated polyprolene mesh

Collagenous gel on collagenous sponge

Collagenous gel on collagenous sponge


Decellularized cadaveric tracheal segment

Polyprolene mesh coated with Vitrigel and collagen

Scaffold Type

Transplanted to 5cm tracheal defect following preimplantation in omentum

Transplanted to 5cm tracheal defect following preimplantation in omentum

Right half of three rings resected and grafted

1.5:3mm tracheal defect grafted

1.5cm 2cm tracheal defect grafted

Left main bronchus segment transplanted


Method

TABLE II. (Continued)

Formation and histology of mucosa, stenosis, exposure of mesh.

Formation of mucosa, stenosis, exposure of mesh.

Epithelialization observed on endoscopy

Histo analysis at 3, 7, 14, 30 days

Cell phenotype, extent of macroscopic epithelialization, stenosis over one year

Macroscopic assessment, laser doppler of vasculature, assessment of cell type

Histology of vitrigel and regular sponge compared

Primary Outcome Measure

Yes

Yes

Complete incorporation by 2 months with buccal mucosal cells at 2 months differentiating to columnar at 6 months

Yes

Yes

Yes

Yes

Yes

Complete Mucosa

Almost complete incorporation of graft with host at two months

Good mucosal lining at 5 months

Ciliated columnar epithelium by 14 days

No stenosis, good epithelialization, partial differentiation

Good epithelialization with identical cell phenotype, good vascularisation

Ciliated columnar cells formed on vitrigel but not collagen sponge at 28days

Outcome

Respiratory mucosa found first at wound edges

Nil histological exam and short follow up

Non-circumferential injury

Small injury, no control

No control group

No long term follow up of cell

Small defect, short follow up

Comment

36

35

38

31

34

8

30

Ref

mortality associated with this condition, esophagectomy remains the preferred management when this involves greater than 5 cm of length; endoscopic resection results in stenosis in over 50% of cases.13,14 In 2011, extracellular matrix (ECM) derived from porcine small intestine was employed as a biological dressing following endoscopic circumferential resection of long segments in five patients with early adenocarcinoma.15 All five experienced the return of normal squamous epithelium throughout with no reported stenosis. This important case series showed that the application of ECM as a biological dressing may significantly improve mucosal healing and reduce adverse scarring. This outcome is believed to be due to the ECM, providing important proteomic cues to enhance repair through epithelial integrin-binding pathways.15 The use of ECM could provide a potential means of aiding reepithelization within the airway. However, as demonstrated by human tracheal transplant cases, this healing process is involved with a period of granulation, and sloughing that can lead to serious complications.8 For this reason, the delivery of a fully functioning intact airway mucosal layer at the point of implantation should be seen as the leading aim in long segment repairs, with the use of ECM perhaps reserved for shorter segments. Engineered mucosa has also been used as part of wholly tissue-engineered three-dimensional organ transplants. To address congenital bladder deficiency, autologous urothelial cells were expanded ex vivo and seeded onto a bladder-shaped matrix composed of natural, synthetic, or composite biomaterials. These products successfully treated 20 reported patients.16 They demonstrate the importance of epithelial culture conditions and the choice of biomaterial in restoring a functioning mucosal layer. Although these early successes demonstrate the potential utility of tissue-engineered mucosa, the therapeutic potential of such products for the treatment of airway disorders remains unclear. We aimed to perform a systematic review of the literature on tissueengineered airway mucosa. Our objectives were to assess the success of this technology to date and its potential application to airway regenerative medicine—and to determine the direction of future research to maximize its therapeutic and commercial potential.

MATERIALS AND METHODS A systematic review of literature on mucosal tissue engineering was performed with specific reference to airway mucosa. Databases searched were Medline (1996–December 2012) and Embase (1980–December 2012) using search terms “tissue engineering” or “tissue” and “engineering” or “tissue engineered” and “mucous membrane” or “mucous” and “membrane” or “mucosa.” Reference lists were scanned for further relevant articles. Identified international experts in otolaryngology, cardiothoracic surgery, and tissue-engineering sciences were approached for their views on the most important published and unpublished studies in this field. Inclusion criteria for articles in this review were those reporting original studies in which tissue engineer-


ing had been used to repair or to replace airway mucosa within the trachea or bronchi in animal models or human studies.

RESULTS A total of 719 articles were found matching the search criteria, of which 17 fulfilled the entry criteria (Table II). Of the 17 tracheal and main bronchi studies, nine examined reconstructing mucosa in noncircumferential tracheal injuries, with one of these being in a human. Four studies examined repairing circumferential injuries in animal models; four examined circumferential injuries in humans (Fig. 1). In all of the studies reporting on the use of engineered mucosa following an anterior tracheal injury, complete mucosalization was achieved over time. In the studies examining circumferential injuries, one of the four animal studies and one of the four human studies reported incomplete mucosalization. The time at which complete mucosalization was reported varied from 7 days to 15 months.

DISCUSSION The Role of Scaffolds in Tissue-Engineered Airway Mucosa Epithelial cell survival in vivo requires support from a three-dimensional ECM. The ECM is believed to bind to cell surface receptors known as integrins in order to activate intracellular signaling pathways that control gene expression, cytoskeletal organization, and cell morphology.17 Therefore, it is highly likely that epithelial cells used to generate tissue-engineered products need to be delivered on a three-dimensional scaffold that effectively mimics ECM while also providing appropriate biomechanical support. Matrices used to date include synthetic polymers, decellularized tissues, collagen, and amniotic membrane. The relative merits of these materials as applied to regenerative medicine have been discussed in other reviews.18,19

Tissue Engineering to Repair Circumferential Mucosal Defects in Humans In 2008, the first stem cell-based, tissue-engineered organ transplant was performed using a decellularized human trachea seeded with respiratory epithelial cells and mesenchymal stem cell (MSC)-derived chondrocytes within a bioreactor.6 This construct was transplanted to replace a 5-cm stenosed segment of left main bronchus and trachea in a 30-year-old woman. Endoscopic examination at 4 days showed that the graft was almost indistinguishable from the adjacent bronchial mucosa, and by 1 month no macroscopic difference between the engineered and existing mucosa could be found, although mucociliary clearance remained limited for a further 6 months with some buildup of mucus. The lung function tests normalized following the intervention and the patient is working and free from stents, tracheostomy, or further intervention 5 years later. This study demonstrated proof of principle that tissue engineering could be successfully used in combination with stem cells to Hamilton et al.: Tissue Engineering Airway Mucosa

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achieve organ reconstruction in a human. An important part of the process used to generate the implant was the use of a dedicated bioreactor. This was a rotating device that enabled seeding on both sides of the graft and provided an air–liquid interface to promote differentiation of ciliated respiratory epithelium. A similar technique was applied to replace a 7-cm tracheal segment in a 12-year-old boy with congenital tracheal stenosis and a pulmonary sling.20 A cadaveric decellularized trachea was treated with autologous MSCs on the outside, and patches of tracheal epithelium from the excised trachea were used to regenerate the mucosal lining. The patient received granulocyte colony stimulating factor (G-CSF) prior to the surgery to mobilize hematopoietic stem cells and endothelial progenitors and to induce MSC proliferation. The graft was also soaked in human recombinant erythropoietin and GCSF, and transforming growth factor (TGF)-b was injected into the tracheal rings to increase angiogenesis, improve autologous MSC recruitment, and induce chondrocyte differentiation. The graft was found to revascularize within 1 week of surgery, although definite cytological evidence of a restored epithelial layer was not found until 1 year postprocedure. Furthermore, although the graft suffered from a lack of biomechanical strength up to 18 months that manifested as tracheomalacia, the patient has not required any medical intervention since then and at 2 years had a fully functioning airway and had returned to school. In 2010, Delaere et al. implanted an 8-cm tracheal allograft into the forearm of a 55-year-old women with treatment-resistant traumatic long-segment tracheal stenosis.21 The graft was wrapped in autologous fascia and subcutaneous tissue from the recipient site, and the patient started on immunosuppressive therapy. At 34 days, donor mucosa was lost from the graft possibly due to rejection or ischemia, and autologous buccal mucosa used to partially replace this. At 4 months, mucosal lining was complete with vascularization supplied by the overlying radial forearm skin. The resultant free composite graft was transplanted to replace a 4.5-cm segment of the stenosed trachea. Immunosuppression was withdrawn successfully 6 months following transplantation, and the patient was well at 1 year. This study showed the potential for autologous buccal mucosa as an airway mucosal substitute. In 2011, Jungebluth et al. performed the first airway transplant to use a recellularized synthetic scaffold to replace 6 centimeters of trachea, the right main bronchus, and part of the left main bronchus in a 36-year-old male.6 A synthesized nanocomposite polymer was seeded with autologous mononuclear cells and incubated in a bioreactor. During implantation, the scaffold was reseeded with mononuclear cells and wrapped in greater omentum. Bronchoscopic examination at 1 week showed a patent airway, although mucosal analysis found necrotic connective tissue and fungal contamination. At 2 months, the infection had cleared, but large areas of granulation were found with signs of only partial epithelialization. To date, long-term outcome data has not been published and it is unclear whether such a scaffold is able to sustain regenerating respiratory epithelium. Laryngoscope 124: April 2014

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Repair of Circumferential Tracheal Mucosal Injuries in Animal Models Three studies used a tubular polyprolene mesh coated with either collagen or gelatin to repair an experimental 5-cm tracheal resection in dogs.22–24 Kim et al. seeded the scaffold with abdominal epithelial cells and found a complete mucosal lining at 2 months, although analysis of the epithelial phenotype was not reported. 23 Suh et al. lined the scaffold with buccal mucosa and found squamous epithelial cells at the center of the graft and respiratory epithelial cells along the edges at 2 months with a complete lining of respiratory epithelium at 6 months.24 Nakamura et al. seeded the same scaffold with a combination of bone marrow aspirate, mesenchymal stem cells (MSC), and peripheral blood, and found an increase in nonciliated cuboidal epithelial cells with increasing distance from the graft edge.31 Both of these studies suggest that native epithelium grows inward from the wound edges to eventually replace the transplanted mucosa, an observation that fits well with observed reendothelization of tissueengineered vascular grafts.25 Epithelialization in the Nakamura study was incomplete in all experimental groups. Unlike the other two studies, the graft was not preimplanted in omentum for 1 week prior to transplant. This technique is believed to improve vascular supply to the mucosal layer and in doing so to improve survival of the regenerated epithelial lining. Although an improved vascular supply to the mucosal layer has been shown to be important in epithelial survival in vivo,26 a well designed research study is still required to demonstrate the efficacy and mechanism of omental wraps in tissue engineering hollow organs and to confirm their hypothetical revascularization benefits. In pigs, an experimental 6-cm tracheal resection was reconstructed using a decellularized tracheal matrix seeded with either MSC-derived chondrocytes on the outer layer, respiratory epithelial cells on the inner layer, or a combination of the two.27 An acellular matrix was also implanted. Severe bacterial and fungal infections occurred on the inner surface of both the acellular and the chondrocyte-only groups, confirming the important role of the mucosal lining in acting as a barrier against infection. The grafts containing only respiratory epithelial cells suffered from severe stenosis. The grafts combining both chondrocytes and respiratory epithelial cells, however, avoided both infection and significant stenosis, alluding to a possible interplay between the mesenchymal layer and the regenerating epithelial cells.

Repair of Noncircumferential Tracheal Mucosal Injuries Tissue-engineering techniques have been used to repair noncircumferential tracheal mucosal defects in rat,28–31 rabbit,32,33canine,34–36 sheep,37 and in one human38 (Table II). A range of scaffolds and cells were used, including acellular matrices. In all cases, complete epithelialization was achieved over time. Although these studies indicate that tissue engineering can successfully reconstruct noncircumferential tracheal mucosal Hamilton et al.: Tissue Engineering Airway Mucosa

injuries, their clinical application is questionable; clinical observations show that such injuries tend to reepitheliaze well or can be managed effectively with existing reconstructive techniques.

Future Work One of the unknown questions in attempts to engineer airway mucosa is whether the epithelial layer survives following transplantation or is replaced by recipient epithelium migrating in from the wound edge, as is the case for endothelialization of tissue-engineered vessels. Immunohistochemistry of murine tracheal allografts showed the infiltration of recipient basal cells followed by complete regeneration of the mucosal layer with recipient epithelium. Studies in this review also suggest epithelium from the wound edge migrates inward.29,37,39 However, no report exists documenting a real-time assessment of tissue-engineered airway epithelial cell fate in vivo. The use of lentiviral vectors to deliver fluorescent proteins into target cells, and the use of fluorescent, targeted quantum dot nanoparticles have provided real-time imaging of cell fate in nonrespiratory animal models.40–42 Such methods could provide a specific marker for the tissue-engineered airway epithelial cells that can be monitored over time in vivo with endoscopy or by immunofluorescence staining of biopsies/ brushings. This could determine the fate of the engineered epithelium and provide an experimental model for the development of techniques promoting transplanted epithelial survival in vivo. Epithelial-mesenchymal interactions are essential for epithelial homeostasis and repair and should be considered when engineering airway mucosa.43,44 Fibroblasts in the submucosa secrete growth factors, including keratinocyte growth factor, which promote expansion and differentiation of respiratory mucosa ex vivo.45 Fibroblasts integrated into an acellular scaffold improve the density and speed of regenerating epithelial cells in animal studies,29,32,37 but their effect on engineered respiratory epithelium in vivo is unclear. This requires clarification as their inclusion in some putative production processes adds complexity and cost and may limit commercial viability. Alternative approaches to harness epithelialmesenchymal interactions use isolated growth factors. Topically applied basic fibroblast growth factor and vascular endothelial growth factor (VEGF) improved epithelization in experimental tracheal transplants, and TGF b-3 improved epithelial regeneration following laryngeal injury.46,47 Such factors are rapidly degraded in vivo; therefore, their direct effect is short-lived. Incorporating growth factors into slow-release nanoparticles may increase longevity of effect, for example, as applied to the fabrication of VEGF-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles embedded in thermosensitive hydrogel in porcine bladder acellular matrix.48 Attempts at tissue-engineering stratified squamous mucosa have proved achievable, as demonstrated by the MacNeil group,11,12 but attempts at engineering a columnar ciliated epithelium with good mucociliary function Laryngoscope 124: April 2014

following transplantation has proved more challenging. In the absence of such function, potentially lifethreatening mucostasis and secondary infection may ensue. Recent improvements in the understanding of the cellular basis of respiratory epithelial repair include an appreciation of the central role of basal cells, the identification of likely stem cell niches, and the role of Wnt catenin signaling pathways.49 Certain ECM core protein and signaling peptides also appear important for attachment and differentiation of epithelial cells.50 Some these processes might be good targets for novel therapeutics that lead to the regeneration of a robust, functioning airway epithelium. Three-dimensional bioprinting uses inkjet technology whereby living cells with or without growth factors suspended in a collagen gel are ejected from an inkjet nozzle; therefore, a programmable, cellularized and complex three-dimensional structure could be generated for respiratory epithelial regeneration. However, at this time bioprinters can only print constructs composed of one or two cell types, and they probably are not sufficiently sophisticated to truly replicate the epithelial three-dimensional environment.51 Viral gene therapy to ciliate respiratory epithelium has been extensively studied as a potential treatment for cystic fibrosis and primary ciliary dyskinesia. Viral vectors have induced and enhanced ciliation in respiratory epithelium ex vivo and in some animal models, although the clinical translation of this technology has been slow due to problems with the permeability and survivability of viral vectors within the respiratory tract.52,53 An alternative could be nonviral gene therapy, which has the advantage of avoiding an adverse immune response, is cheaper, and avoids the safety concerns associated with the use of viruses. Nonviral vectors involve a cationic lipid or polymer that joins with negatively charged nucleic acid. Although these complexes can transfect cells in vitro, the in vivo use is limited by a lack of specificity, a strong interaction with blood constituents, concerns over toxicity, and uptake by the reticuloendothelial system. A combination nonviral vectors with other nanotechnologies such as silica nanotubes produced multifunctional hybrid nonviral vectors that overcame many of these limitations.54 To our knowledge, these techniques have not been applied to respiratory epithelial regeneration. Mucosal engineering offers a range of potential new therapeutics for unmet clinical needs, but ultimate implementation for wider health benefit depends on successful commercialization. Cost is likely to play a critical role in this; current tissue-engineering treatments require fabrication within a facility that follows good manufacturing practice standards, which is prohibitively expensive for widespread clinical use. However, progress is now being made to reduce the cost of goods through innovative manufacturing changes and streamlining the bioprocessing. This will increase the cost-to-benefit ratio, enabling a wider group of patients to potentially be treated, and thus enabling greater economies of scale that further reduce the cost of goods. Simpler competitor techniques, such as the use of extracellular membrane or ECM to Hamilton et al.: Tissue Engineering Airway Mucosa

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provide “biological dressings”15 should also be considered in comparative efficacy and cost benefit studies.

CONCLUSION Tissue-engineering techniques have been applied successfully to regenerate airway mucosa for large airways in the laboratory, animals, and in humans. We did not find evidence that one scaffold material or cell type was superior at regenerating airway mucosa; comparative studies are needed. An established epithelial layer within an engineered airway implant may protect against infection as well as improve mucociliary function. Few studies assessed the regenerated airway mucosa in detail. A multidisciplinary scientific approach to tissue-engineering airway mucosa, incorporating a strong element of learning from discovery science, as well as considering practical aspects of clinical application, is most likely to yield effective novel therapies for patients with advanced mucosal disorders.

BIBLIOGRAPHY 1. Nauta A, Gurtner GC, Longaker MT. Wound healing and regenerative strategies. Oral Dis 2011;17:541–549. 2. Parkin, F Bray, J Ferlay, P Pisani. Global cancer statistics. CA Cancer J Clin 2005;55:74–108. 3. Zeitels SM, Wain JC, Barbu AM, Bryson PC, Burns JA. Aortic homograft reconstruction of partial laryngectomy defects: a new technique. Ann Otol Rhinol Laryngol 2012;121:301–306. 4. Wurtz A, Porte H, Conti M, et al. Surgical technique and results of tracheal and carinal replacement with aortic allografts for salivary glandtype carcinoma. J Thorac Cardiovasc Surg 2010;140:387–393. 5. Wurtz A, Porte H, Conti M, et al. Tracheal replacement with aortic allografts. N Engl J Med 2006;355:1938–1940. 6. Jungebluth P, Alici E, Baiguera S, et al. Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Lancet 2011;10:378:1997–2004. 7. 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. doi: 10.1016/S0140–6736(12)60737-5. Epub 2012. 8. Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372:2023–2030. 9. Baylis O, Figueiredo F, Henein C, Lako M, Ahmad S. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem 2011;112:993–1002. 10. Bhargava S, Chapple CR. Buccal mucosal urethroplasty: is it the new gold standard? BJU Int 2004;93:1191–1193. 11. Bhargava S, Chapple CR, Bullock AJ, Layton C, MacNeil S. Tissue-engineered buccal mucosa for substitution urethroplasty. BJU Int 2004;93: 807–811. 12. Bhargava S, Patterson JM, Inman RD, MacNeil S, Chapple CR. Tissueengineered buccal mucosa urethroplasty-clinical outcomes. Urol 2008;53: 1263–1269. 13. Chennat J, Konda VJ, Ross AS, et al. Complete Barrett’s eradication endoscopic mucosal resection: an effective treatment modality for high-grade dysplasia and intramucosal carcinoma–an American single-center experience. Am J Gastroenterol 2009;104:2684–2692. 14. Pouw RE, Seewald S, Gondrie JJ, et al. Stepwise radical endoscopic resection for eradication of Barrett’s oesophagus with early neoplasia in a cohort of 169 patients. Gut 2010;59:1169–1677. 15. Badylak SF. Esophageal preservation in five male patients after endoscopic inner-layer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold. Tissue Eng Part A 2011;17:1643–1650. 16. Atala A. Tissue engineering of human bladder. Br Med Bull 2011;97:81–104. 17. Larjava H, Koivisto L, Hakkinen L, Heino J. Epithelial integrins with special reference to oral epithelia. J Dent Res 2011;90:1367–1376. 18. Garg T, Singh O, Arora S, Murthy R. Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst 2012;29:1–63. 19. Rustad KC, Sorkin M, Levi B, Longaker MT, Gurtner GC. Strategies for organ level tissue engineering. Organogenesis 2010;6:151–157. 20. 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;15:380:994–1000. 21. Delaere P, Vranckx J, Verleden G, De Leyn P, Van Raemdonck D. Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med 2010;14:362:138–145.


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22. Nakamura, et al. In situ tissue engineering for tracheal reconstruction using a luminar remodeling type of artificial trachea. J Thorac Cardiovasc Surg 2009;138:811–819. 23. Kim J, et al. Replacement of a tracheal defect with a tissue-engineered prosthesis: early results from animal experiments. J Thorac Cardiovasc Surg 2004;128:124–129. 24. Suh SW, Kim J, Baek CH, Han J, Kim H. Replacement of a tracheal defect with autogenous mucosa lined tracheal prosthesis made from polypropylene mesh. ASAIO J 2001;47:496–500. 25. Nelson GN, et al. Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo. FASEB J 2008;22:3888–3895. 26. Kaully T, Kaufman-Francis K, Lesman A, Levenberg S. Vascularization— the conduit to viable engineered tissues. Tissue Eng Part B Rev 2009;15: 159–169. 27. Go T, et al. Both epithelial cells and mesenchymal stem cell-derived chondrocytes contribute to the survival of tissue-engineered airway transplants in pigs. J Thorac Cardiovasc Surg 2010;139:437–443. 28. Kobayashi K, et al. A tissue-engineered trachea derived from a framed collagen scaffold, gingival fibroblasts and adipose-derived stem cells. Biomaterials 2010;31:4855–4863. 29. Kobayashi K, et al. Effect of fibroblasts on epithelial regeneration on the surface of a bioengineered trachea. Ann Otol Rhinol Laryngol 2008;117:59–64. 30. Tada Y, et al. 30 Regeneration of tracheal epithelium utilizing a novel bipotential collagen scaffold. Ann Otol Rhinol Laryngol 2008;117:359–365. 31. Nomoto Y, Suzuki T, Tada Y, et al. Tissue engineering for regeneration of the tracheal epithelium. Ann Otol Rhinol Laryngol 2006;115:501–506. 32. Okano W, et al. Bioengineered trachea with fibroblasts in a rabbit model. Ann Otol Rhinol Laryngol 2009;118:796–804. 33. Ni Y, et al. Radiologic and histologic characterization of silk fibroin as scaffold coating for rabbit tracheal defect repair. Otolaryngol Head Neck Surg 2008;139:256–261. 34. Yamashita M, et al. Tracheal regeneration after partial resection: a tissue engineering approach. Laryngoscope 2007;117:497–502. 35. Kim J, et al. Replacement of a tracheal defect with a tissue-engineered prosthesis: early results from animal experiments. Thorac Cardiovasc Surg 2004;128:124–129. 36. Suh SW, Kim J, Baek CH, Han J, Kim H. Replacement of a tracheal defect with autogenous mucosa lined tracheal prosthesis made from polypropylene mesh. ASAIO J 2001;47:496–500. 37. Heikal MY, et al. Autologous implantation of bilayered tissue-engineered respiratory epithelium for tracheal mucosal regenesis in a sheep model. Cells Tissues Organs 2010;192:292–302. 38. Omori K, et al. Regenerative medicine of the trachea: the first human case. Ann Otol Rhinol Laryngol 2005;114:429–433. 39. Genden EM, Iskander AJ, Bromberg JS, Mayer L. tracheal allografts undergo reepithelialization with recipient-derived epithelium. Arch Otolaryngol Head Neck Surg 2003;129:118–23. 40. Deroose CM, et al. Noninvasive monitoring of long-term lentiviral vectormediated gene expression in rodent brain with bioluminescence imaging. Mol Ther 2006;14:423–431. 41. Pfeifer A, et al. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol Ther 2001;3:319–322. 42. Merian J, Gravier J, Navarro F, Texier I. Fluorescent nanoprobes dedicated to in vivo imaging: from preclinical validations to clinical translation. Molecules 2012;17:5564–5591. 43. Mackenzie I, Rittman G, Bohnert A, Breitkreutz D, Fusenig NE. Influence of connective tissues on the in vitro growth and differentiation of murine epidermis. Epithelial Cell Biol 1993;2:107–119. 44. El Ghalbzouri A, Ponec M. Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components. Wound Repair Regen 2004;12:359–367. 45. Kobayashi K, Nomoto Y, Suzuki T, et al. Effect of fibroblasts on tracheal epithelial regeneration in vitro. Tissue Eng 2006;12:2619–2628. 46. Govindaraj S, Gordon R, Genden EM. Effect of fibrin matrix and vascular endothelial growth factor on reepithelialization of orthotopic murine tracheal transplants. Ann Otol Rhinol Laryngol 2004;113:797–804. 47. Sung SW, Won T. Effects of basic fibroblast growth factor on early revascularization and epithelial regeneration in rabbit tracheal orthotopic transplantation. Eur J Cardiothorac Surg 2001;19:14–18. 48. Geng H, Song H, Qi J, Cui D. Sustained release of VEGF from PLGA nanoparticles embedded thermo-sensitive hydrogel in full-thickness porcine bladder acellular matrix. Nanoscale Res Lett 2011;6:312. 49. Butler C, Birchall M, Giangreco A. Interventional and intrinsic airway homeostasis and repair. Physiology (Bethesda) 2012;27:140–147. 50. Badylak, SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater 2009;5:1–13. 51. Chang CC, Boland ED, Williams SK, Hoying JB. Direct-write bioprinting three-dimensional biohybrid systems for future regenerative therapies. J Biomed Mater Res B Appl Biomater 2011;98:160–170. 52. McIntyre JC, et al. Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model. Nat Med 2012;18:1423–1428. 53. Chhin B, et al. Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy. PLoS Genet 2009;5: e1000422. 54. Guo X, Huang L. Recent advances in nonviral vectors for gene delivery. Acc Chem Res 2012;45:971–979.
