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MORPHOLOGIC ASSESSMENT OF EXTRACELLULAR MATRIX SCAFFOLDS FOR PATCH

TRACHEOPLASTY IN A CANINE MODEL

RUNNING TITLE: PATCH TRACHEOPLASTY WITH ECM SCAFFOLDS

Thomas W. Gilbert, PhD 1, Sebastien Gilbert, MD 1, 2, 3, Matthew Madden 1,

Susan D. Reynolds, PhD 4, Stephen F. Badylak, DVM, MD, PhD 1

1 McGowan Institute for Regenerative Medicine, Department of Surgery,

University of Pittsburgh, Pittsburgh, PA 2 Heart, Lung, and Esophageal Institute, University of Pittsburgh Medical Center, Pittsburgh, PA

3Veterans’ Affairs Pittsburgh Health Care System, Pittsburgh, PA 4Department of Pediatrics, National Jewish Medical Research Center, Denver, CO

Presented at the Annual Meeting of the Society of Thoracic Surgeons, Fort Lauderdale, FL January 2008. Keywords: Word Count:

Corresponding author: Stephen F. Badylak McGowan Institute for Regenerative Medicine University of Pittsburgh 100 Technology Drive, Suite 200 Pittsburgh, PA 15219 P: (412) 235-5144 F: (412) 235-5110 badylaks@upmc.edu

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Abstract

BACKGROUND: The optimal management of benign tracheal stricture remains surgical resection.

Surgery is not always an option because of the challenges posed by anastomotic tension and a tenuous

blood supply. Regenerative medicine approaches, such as extracellular matrix (ECM) scaffold

technology, may alleviate some of the limitations to tracheal replacement. ECM scaffolds facilitate site-

specific tissue remodeling when used to reconstruct a variety of soft tissue structures.

METHODS: A 1cm wide x 2cm long defect was created in the ventral trachea of 15 dogs and repaired

with one of three acellular biologic scaffolds; urinary bladder matrix (UBM), UBM crosslinked with

carbodiimide (UBMC), and decellularized tracheal matrix (DTM). The grafts were evaluated via periodic

bronchoscopy and by macroscopic and microscopic morphologic examination at either 2 months or 6

months.

RESULTS: The UBM, UBMC and DTM groups showed no evidence of stenosis or tracheomalacia. The

UBM, UBMC, and DTM groups all showed deposition of organized collagenous tissue at the site of

scaffold placement and an intact epithelial layer. Scattered areas of mucociliary differentiation were

present at the edges of the graft site. There was no evidence cartilage observed within the remodeled

tissue at 6 months.

CONCLUSIONS: ECM scaffolds promotes healing of significant size tracheal defects without stricture

and with some, but not all, of the necessary structures required for tracheal reconstruction, including

complete coverage with ciliated epithelialium and dense organized collagenous tissue.

Word Count: 229

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Introduction

Tracheal stricture and tracheomalacia are associated with significant morbidity. Approximately

150,000 patients experience complications associated with endotracheal intubation and mechanical

ventilation each year in the US, including many patients with brain and/or spinal cord injuries which

require chronic intubation and ventilatory support [1, 2]. Primary or secondary tracheal malignancies,

although rare, often require resection of long segments of trachea, which can lead to stricture. Chronic

airway strictures pose a significant clinical challenge and are associated with high mortality rates (11-

24%) [3]. Despite advances in surgical techniques, resection of airway stricture with primary repair can

result in excessive anastomotic tension, tissue ischemia, and failure to heal. Although stents can be used

effectively as a palliative therapy, their use may be associated with a high complication rate in the

treatment of non-malignant airway strictures [4]. Stated differently, the surgical management of benign

and malignant tracheal pathology represents a significant and clinically relevant problem, and there is a

need for alternative approaches for tracheal reconstruction.

A variety of regenerative medicine approaches have been proposed for long (>5 cm), full

circumferential tracheal replacement, ranging from collagen scaffolds supported by silicone stents to

cartilaginous tubes created by in vitro culture methods [5-8]. However, these tracheal replacement

strategies have been inadequate as a result of incomplete epithelialization with associated stricture, or a

lack of mechanical integrity resulting in tracheomalacia [9].

Biologic scaffolds composed of naturally-occurring extracellular matrix (ECM) promote site-

specific tissue remodeling in both pre-clinical studies and for numerous clinical applications [10]. ECM

scaffolds are degraded rapidly with concomitant site-specific deposition of host tissues. Degradation

appears to be critical to the constructive remodeling response as matricryptic peptides formed as a result

of parent molecule breakdown exhibit inherent bioactivity, including bacteriostasis [11, 12] and

chemotaxis for differentiated and progenitor cells [13]. For repair of tracheal defects, one form of ECM

scaffold, small intestinal submucosa (SIS-ECM) has shown moderate success for small defects (less than

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½ the circumference) and defects with adequate structural support, but did not fully restore functional

tracheal tissue [14-16].

The purpose of the present study was to determine whether an ECM scaffold with specific

features, such as a basement membrane ECM or cartilaginous ECM, might enhance the remodeling

response for a complex tissue like the trachea. Three different forms of ECM scaffold were used to repair

a partial (i.e., non-circumferential) defect in the ventral trachea in a canine model. In one group, the

defect was repaired with an 8 layer multilaminate ECM scaffold referred to as urinary bladder matrix

(UBM-ECM), which retains a basement membrane structure [17]. The second group was repair UBM-

ECM patch, an 8 layer UBM-ECM patch that was crosslinked with 10mM carbodiimide (UBMC) to

provide additional mechanical support to the device. The third group was repaired with a decellularized

full thickness patch of porcine tracheal matrix (DTM) that contained both a basement membrane and the

remnants of tracheal cartilaginous rings.

Materials and Methods

Study Design

Fifteen mongrel dogs weighing 19.5 kg ± 0.3 kg were each subjected to surgical resection of a 1

cm wide × 2 cm long defect (~ 30% of circumference and 3 rings long) of the ventral cervical trachea.

The dogs were divided into 3 groups of five, with tracheoplasty performed with one of three ECM

patches; multilaminate UBM-ECM, multilaminate UBMC, or DTM. Three of the five animals in each

group were sacrificed at 2 months after surgery, and the other two animals were sacrificed at 6 months

after surgery. The remodeling ECM patches were evaluated by bronchoscopic assessment at 1, 2, and 6

months, and by gross morphology and qualitative and semi-quantitative histologic assessment at the time

of sacrifice. All animal procedures were performed in compliance with the 1996 “Guide for The Care and

Use of Laboratory Animals” and approved by the Institutional Animal Care and Use Committee at the

University of Pittsburgh.

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Harvest of Porcine Tissues

Urinary bladders and tracheas were harvested from market weight pigs (approximately 110-130

kg) immediately after sacrifice at an abattoir and transported to the lab on ice. Residual external

connective tissues were then removed. The urinary bladders were further cleaned by repeated rinses to

remove all residual urine. The urothelial layer was removed by soaking of the material in 1 N saline for

15 minutes. All tissues were frozen at -80°C until time for continued processing, usually no more than 2

months.

UBM-ECM Device Preparation

Urinary bladders were thawed and the tunica serosa, tunica muscularis externa, tunica

submucosa, and most of the muscularis mucosa were mechanically delaminated from the bladder tissue.

The remaining basement membrane of the tunica epithelialis mucosa and the subjacent tunica propria,

collectively termed urinary bladder matrix (UBM), were then decellularized and disinfected by immersion

in 0.1% (v/v) peracetic acid, 4% (v/v) ethanol, and 96% (v/v) deionized water for 2 h. The UBM-ECM

material was then washed twice for 15 min with phosphate buffered saline (PBS) (pH = 7.4) and twice for

15 min with deionized water.

Multilayer tubes were created by wrapping hydrated sheets of UBM around a 22 mm perforated

tube/mandrel that was covered with umbilical tape for a total of eight complete revolutions (i.e., a layer

tube) [18]. The constructs were subjected to a vacuum of 710 to 740 mm Hg (Leybold, Export, PA,

Model D4B) for 10 to 12 h to remove the water and form a tightly coupled multilaminate device. Each

tubular device was cut to approximately 2 cm wide x 3 cm long pieces that were terminally sterilized with

ethylene oxide.

For the carbodiimide crosslinked group, the 2 cm x 3 cm UBM devices were immersed in 10 mM

1-ethyl-3-(dimethylaminopropyl)-carbodiimide hydrochloride (Sigma-Aldrich, St. Louis, MO) for 24

hours at room temperature. Each device was rinsed three times with sterile deionized water for 15

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minutes to remove any residual crosslinking agent. The devices were vacuum-pressed around the same

mandrel as previously described to dry specimens and terminally sterilized with ethylene oxide.

Tracheal Tissue Matrix Scaffold Preparation

The tracheas were subjected to the following chemical treatment to decellularize the tissue: 1

hour in 0.25%Trypsin/0.05%EDTA at 37°C, 48 hours in deionized water at 4°C, and 48 hours in 3%

Triton X-100 at 4°C. The tissue was rinsed heavily to remove residual detergent, and then disinfected

with peracetic acid as described for UBM-ECM. The DTM material was frozen at -20°C and lyophilized.

Devices were then cut (2 cm x 3 cm) from the ventral portion of the trachea, (i.e., from the portion that

contained cartilaginous tissue) and terminally sterilized with ethylene oxide.

Surgical Procedure

All animals were sedated with acepromazine (0.1 mg/kg IM), followed by intravenous

administration of thiopental (12-25 mg/kg IV), intubation and isoflurane (1.5-3%) maintenance of

surgical place anesthesia. Cefazolin (15 mg/kg IV) was administered prior to surgical preparation and

skin incision. Using aseptic technique, the proximal cervical trachea was exposed through a midline neck

incision. A 1 cm wide × 2 cm long portion of the ventral tracheal wall was surgically removed. The

defect was then repaired with one of the ECM scaffold devices with at least 5 mm of overlap around the

edges of the defect (Figure 1A). All grafts were secured using absorbable 4-0 polydiaxonone suture (PDS

Ethicon, Summerville, NJ). Non-resorbable polypropylene sutures (Prolene Ethicon, Summerville, NJ)

were used to mark the corners of the repair site. The scaffold placement site was tested for air leaks by

submerging in saline while applying a Valsalva maneuver. The repair was also examined by

bronchoscopy to verify graft placement and airway patency.

Postoperative Care

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The dogs were recovered from anesthesia, extubated, and monitored until resting comfortably in a

sternal position. The dogs were cage housed overnight and returned to their larger run housing (3.05 m ×

4.27 m) on postoperative day 1. Oral prophylactic antibiotics were administered (cephalothin/ cephalexin

(35 mg/kg) twice daily) for 7 to 9 days. The dogs received intravenous acepromazine (0.1 mg/kg) and

butorphanol (0.05 mg/kg) for 2 days, followed by subcutaneous or intramuscular buprenorphine (0.01 to

0.02 mg/kg) every 12 hours thereafter for analgesia as needed.

Clinical and Bronchoscopic Assessment

Each animal had a predetermined follow-up period of either 2 or 6 months at which time the

subject was sacrificed. Bronchoscopic examinations were conducted at predetermined intervals (1, 2, and

6 months) after surgery to evaluate scaffold remodeling. Airway stenoses were evaluated using a flexible

bronchoscope, and visually quantified as a percent decrease in the ventrodorsal diameter of the trachea.

Strictures were classified as mild (< 25%), moderate (25-50%) or severe (> 50%). Documented

bronchoscopic data included: appearance of the graft surface and its relationship to the native trachea,

signs of inflammation (e.g.: exudate, granulation tissue), presence or absence of tracheomalacia, and

stricture formation.

Morphologic Assessment

Animals were euthanized by sedation with acepromazine (0.01 mg/kg SC) and butorphanol (0.05

mg/kg), induced anesthesia with 5% Isoflurane for 5 minutes, and administration of Pentobarbital Sodium

IV (390 mg / 4.5 kg BW). Immediately after euthanasia, the scaffold placement site, including native

tracheal tissue surrounding the graft site was harvested. The excised sample was exposed by a

longitudinal incision in the distal-to-proximal direction along the membranous portion of the trachea. The

exposed mucosal surface was examined and photographed. The tissue was immersed in 10% neutral

buffered formalin for histologic preparation. Tissue was trimmed longitudinally, sectioned, and stained by

conventional histologic staining with Masson’s Trichrome and Periodic Acid Schiff (PAS), and with

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immunohistochemical staining for p63. The p63 staining was performed after antigen retrieval with citric

acid buffer (pH = 6.0). The primary antibody was a mouse anti-human p63 (Dako, Inc., Carpinteria, CA;

Cat # M7247) at a 1/25 dilution for 1 hour at room temperature. The secondary antibody used was anti-

mouse IgG at 1/500 followed by detection with HRP-strepavidin also at 1/500. DAB was the substrate

and the slides were counterstained with Harris' hematoxylin. The areas examined included the native

tissue, the proximal and distal interfaces between the remodeled and native tissue, and the middle region

of the remodeled area.

Cell counts were performed for secretory cells identified by positive PAS staining and basal cells

identified by p63 positive staining within longitudinal sections of the remodeled tissue in each specimen

using 200X images in the MetaVue™ Software package (Molecular Devices, Sunnyvale, CA). The

length of the scaffold was also measured so that the number of cells could be related to the distance from

the edge of the remodeled graft as defined by the presence of host cartilage. For qualitative comparison,

the cell counts were interpolated for cell counts over every 1000 µm and the average number of cells in

that region was averaged for each type of scaffold at each time point and presented from the proximal

edge of the defect to the point of interest.

Semi-quantitative assessment of the presence of ciliated cells was conducted. A score from 0-3

was assigned to each 200X field (Table 1). An interpolated score was then calculated for each 1000 µm

region across the remodeled area.

Results

Clinical Outcomes

All dogs in each group recovered without complications from the surgical procedure, and had an

uneventful early post-operative course. Three animals, two from the UBM group and one from the

UBMC group, had to be euthanized between 1 and 3 weeks post-surgery due to the development of

subcutaneous emphysema that resulted from small air leaks formed at the site of suture placement. These

three animals were replaced in the study. One other dog had a small amount of subcutaneous emphysema

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which resolved promptly following evacuation of the neck incision. All other animals had an uneventful

post-surgical course until elective sacrifice at either 2 or 6 months after surgery.

Bronchoscopic Examination

Bronchoscopic evaluation of the remodeled tracheal defect sites repaired with UBM, UBMC, and

DTM groups all showed a smooth, shiny surface consistent with the presence of an epithelial layer and

neovascularization at 1 month. In the UBMC group, there was evidence a whitish exudate throughout the

first 2 months. One dog in the UBMC group and one dog in the DTM group showed mild asymptomatic

tracheal stenosis at one month, but in both cases the stenosis was completely resolved at 2 months. One

dog in the UBM group developed a small asymptomatic nodule at the distal aspect of the graft site which

may have been related to the sutures. At 6 months, the remodeled patch appeared similar to that

observed at two months, with the exception that the vascular response was less pronounced and the newly

formed tissue had more of a whitish appearance (Figure 1B).

Macroscopic Observations

For all three groups, gross observations showed remodeling of the scaffold with dense, fibrous

connective tissue with a shiny epithelial layer covering the entire surface (Figure 1C). New blood vessels

were evident in the remodeled tissue and these vessels were slightly more visible at 2 months as opposed

to 6 months. There was evidence of inflammation localized around the sutures at two months, but this

also diminished by 6 months as the sutures had resorbed. The remodeled scaffold material in all groups

was pliable without visible evidence of mechanical support from cartilage formation.

Microscopic Observations

Histologic examination of the UBM, UBMC, and DTM grafts showed complete degradation and

replacement of the device with host tissue by two months after surgery (Figure 2A, 3A, & 4A

respectively). The site of remodeling for all three devices showed dense, organized collagenous tissue

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with complete epithelialization. In all three groups, there was histologic evidence of neovascularization at

2 months that diminished slightly by 6 months. The UBMC group showed slightly increased

mononuclear cell numbers in the dense collagenous tissue subjacent to the epithelium compared to either

the UBM or DTM groups. There was no evidence of cartilage formation within any of the remodeled

scaffold materials.

UBM

All of the remodeled ECM scaffolds showed an intact epithelial cell layer, but the degree of

maturity of the epithelial layer varied with the type of scaffold used and the time point at which it was

evaluated. At 2 months, the remodeled UBM graft showed ciliated columnar epithelium over the

majority of the graft (Figure 2A & 2B). There was a uniform distribution of secretory cells (Figure 2C)

and basal cells (Figure 2D) present in the remodeled UBM graft, but the frequency of each cell type was

less than that found in the normal trachea. At 6 months, one animal in the UBM group showed complete

coverage of the remodeled tissue with a ciliated columnar epithelium, while the other animal showed a

lack of ciliated cells (Figure 2B). The number of secretory cells was unchanged at 6 months compared

with the 2 month time point, with the exception that there were generally fewer secretory cells in the

center of the remodeled UBM (Figure 2C). The number of basal cells present at 6 months increased

compared to the 2 month time point (Figure 2D).

UBMC

For the UBMC group, ciliated epithelial cells were observed at the edges of the remodeled

scaffold, but no ciliated cells were present in the center of the patch (Figure 3A). The number of ciliated

cells present increased from 2 months to 6 months (Figure 3B). At both 2 and 6 months, the number and

distribution of secretory cells was less than observed in native trachea, but was similar to that observed

for the UBM group at 2 months (Figure 3C). There was no change in the number or distribution of

secretory cells between the 2 and 6 month time points. The remodeled UBMC showed no basal cells over

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the majority of the remodeled area at 2 months, but by 6 months, there was a uniform distribution of basal

cells similar to that observed for the UBM group (Figure 3D).

DTM

The DTM group showed large regions with no ciliated epithelial cells in the central portion of the

remodeled graft at 2 months, and this deficiency was more pronounced after 6 months (Figure 4A & 4B).

The DTM group showed the fewest number of secretory cells of the three groups at both time points with

a complete lack of secretory cells in the middle portion of the remodeled graft at 6 months (Figure 4C) .

At both time points, the middle portion of the remodeled grafts showed little or no presence of basal cells

(Figure 4D).

Comment

The UBM, UBMC and DTM forms of ECM facilitated closure of a critical size tracheal defect

with no evidence of clinically significant stenosis, tracheomalacia, or inflammation at either 2 months or

6 months. UBM, UBMC, and DTM were replaced with organized collagenous connective tissue and an

intact epithelial layer with areas of mucocilliary differentiation located primarily at the edges of the

remodeled tissue. However, secretory cells, basal cells, glandular structures and to some extent ciliated

cells were not fully restored. Importantly, there was no evidence of cartilage formation, which means

none of these scaffolds in their current form restored the mechanical support required for a full

circumferential tracheal replacement.

The results of the present study are similar to those observed when SIS-ECM was used for patch

tracheoplasty in both a rat and rabbit model [14, 15, 19]. The remodeled SIS-ECM formed a fibrous

connective tissue that showed ciliated epithelialization. No cartilage formation was found even when

thyroid or auricular cartilage was co-localized at the site of remodeling [15, 19]. In fact, the presence of

the cartilaginous tissue contributed to an increased rate of complications. The only study in which

neochondrogenesis has been reported associated with SIS-ECM repair of the trachea was in a porcine

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model in which SIS-ECM was used to coat a self-expanding metallic stent [16]. However, the presence

of the metallic stent led to significant luminal narrowing.

The use of carbodiimide to chemically crosslink UBM in the present study was intended to slow

enzymatic degradation and increase the strength of the scaffold [20]. It was hypothesized that the

crosslinked scaffold would remain at the site of implantation longer and provide mechanical support.

Although a temporal and quantitative evaluation of scaffold degradation was not conducted in the present

study, there was no histologic evidence to suggest delayed degradation of the UBMC scaffolds.

It was hypothesized that the DTM graft would provide the mature epithelial development and

chondrogenesis due to the organ specific composition and organization of the ECM. In fact,

epithelialization of the DTM scaffold was qualitatively worse than either UBM or UBMC with less

coverage with ciliated epithelium, fewer secretory cells, and fewer basal cells. The decreased population

of basal cells may have contributed to the lack of ciliated and secretory cells, as basal cells serve as a

progenitor cell for the tracheal epithelium [21, 22].

In a recent study, it was shown that lyophilization of UBM caused irreversible changes to the

ultrastructure of UBM, and slowed cell growth on and penetration into the scaffold in vitro [23]. A

similar phenomenon may explain the suboptimal results observed for the lyophilized DTM in vivo.

Decellularized allogenic canine tracheas in a hydrated form have been evaluated for repair of full

circumferential resections of the intrathoracic trachea with considerable success [24, 25]. The detergent

treatment was similar that used in the present study. A key aspect of this approach was the viability of the

cartilage within the allograft. The presence of chondrocyte material within the cartilage was highly

predictive for successful patency of the tracheal allograft [24]. It is not clear whether the chondrocytes

were viable and therefore participated in the remodeling response, or if the preserved mechanical integrity

of the cartilage simply allowed the graft to maintain enough strength to resist the negative pressures of

respiration. If the integrity of the cartilage was the critical factor determining success, then the lack of

viable cartilage due to lyophilization may partially explain the lack of cartilage formation for the DTM in

the current study. Lyophilization of the DTM likely disrupted glycosaminoglycans in the cartilage and

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compromised the ability of the scaffold to provide mechanical support during the remodeling process. It

is unknown whether a dog would reject cellular material remaining in viable porcine cartilage, but in the

canine allograft experiments, the chondrocytes did not express major histocompatibility complex [25].

The current study showed that the presence of a vacuum pressed UBM graft or a lyophilized form

of decellularized tracheal matrix is insufficient to promote the complete regeneration of functional trachea

tissue. However, the results shed light on modifications that may be incorporated to an ECM-based

airway replacement graft to increase its regenerative potential. For instance, the use of a hydrated form of

ECM with preserved cartilage integrity may be necessary in order to realize functional tracheal

replacement.

Acknowledgements

The authors would like to recognize funding from the Commonwealth of Pennsylvania and the

Department of Veterans’ Affairs Competitive Pilot Project Fund (CP-048). The authors would also like

to acknowledge the advice of Jennifer DeBarr for histologic preparation of the specimens.

The authors had full control of the study design, methods used, outcome parameters, analysis of data, and

production of the written manuscript.

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References

1. Stauffer JL, Olson DE, Petty TL. Complications and consequences of endotracheal intubation and tracheotomy. A prospective study of 150 critically ill adult patients. Am J Med. 1981;70(1):65-76.

2. Wijkstra PJ, Avendano MA, Goldstein RS. Inpatient chronic assisted ventilatory care: a 15-year experience. Chest. 2003;124(3):850-856.

3. Cotton R. Management of subglottic stenosis in infancy and childhood. Review of a consecutive series of cases managed by surgical reconstruction. Ann Otol Rhinol Laryngol. 1978;87(5 Pt 1):649-657.

4. Gaissert HA, Grillo HC, Wright CD, Donahue DM, Wain JC, Mathisen DJ. Complication of benign tracheobronchial strictures by self-expanding metal stents. J Thorac Cardiovasc Surg. 2003;126(3):744-747.

5. Kanzaki M, Yamato M, Hatakeyama H, et al. Tissue engineered epithelial cell sheets for the creation of a bioartificial trachea. Tissue Eng. 2006;12(5):1275-1283.

6. Kojima K, Bonassar LJ, Roy AK, Vacanti CA, Cortiella J. Autologous tissue-engineered trachea with sheep nasal chondrocytes. J Thorac Cardiovasc Surg. 2002;123(6):1177-1184.

7. Teramachi M, Nakamura T, Yamamoto Y, Kiyotani T, Takimoto Y, Shimizu Y. Porous-type tracheal prosthesis sealed with collagen sponge. Ann Thorac Surg. 1997;64(4):965-969.

8. Yamashita M, Kanemaru S, Hirano S, et al. Tracheal regeneration after partial resection: a tissue engineering approach. Laryngoscope. 2007;117(3):497-502.

9. Grillo HC. Tracheal replacement: a critical review. Ann Thorac Surg. 2002;73(6):1995-2004.

10. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials. 2007;28(25):3587-3593.

11. Brennan EP, Reing J, Chew D, Myers-Irvin JM, Young EJ, Badylak SF. Antibacterial activity within degradation products of biological scaffolds composed of extracellular matrix. Tissue Eng. 2006.

12. Sarikaya A, Record R, Wu CC, Tullius B, Badylak SF, Ladisch M. Antimicrobial activity associated with extracellular matrices. Tissue Eng. 2002;8(1):63-71.

13. Li F, Li W, Johnson S, Ingram D, Yoder M, Badylak SF. Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells. Endothelium. 2004;11(3-4):199-206.

14. De Ugarte DA, Puapong D, Roostaeian J, et al. Surgisis patch tracheoplasty in a rodent model for tracheal stenosis. J Surg Res. 2003;112(1):65-69.

15. Gubbels SP, Richardson M, Trune D, Bascom DA, Wax MK. Tracheal reconstruction with porcine small intestine submucosa in a rabbit model. Otolaryngol Head Neck Surg. 2006;134(6):1028-1035.

16. Park JW, Pavcnik D, Uchida BT, et al. Small intestinal submucosa covered expandable Z stents for treatment of tracheal injury: an experimental pilot study in swine. J Vasc Interv Radiol. 2000;11(10):1325-1330.

17. Brown B, Lindberg K, Reing J, Stolz DB, Badylak SF. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue Eng. 2006;12(3):519-526.

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18. Badylak SF, Vorp DA, Spievack AR, et al. Esophageal reconstruction with ECM and muscle tissue in a dog model. J Surg Res. 2005;128(1):87-97.

19. Zhang L, Liu Z, Cui P, Zhao D, Chen W. SIS with tissue-cultured allogenic cartilages patch tracheoplasty in a rabbit model for tracheal defect. Acta Otolaryngol. 2007;127(6):631-636.

20. Billiar K, Murray J, Laude D, Abraham G, Bachrach N. Effects of carbodiimide crosslinking conditions on the physical properties of laminated intestinal submucosa. J Biomed Mater Res. 2001;56(1):101-108.

21. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am J Respir Crit Care Med. 1998;157(6 Pt 1):2000-2006.

22. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol. 2004;164(2):577-588.

23. Freytes DO, Tullius RS, Valentin JE, Stewart-Akers AM, Badylak SF. Hydrated versus lyophilized forms of porcine extracellular matrix derived from the urinary bladder. J Biomed Mater Res A. 2008.

24. Liu Y, Nakamura T, Sekine T, et al. New type of tracheal bioartificial organ treated with detergent: maintaining cartilage viability is necessary for successful immunosuppressant free allotransplantation. Asaio J. 2002;48(1):21-25.

25. Liu Y, Nakamura T, Yamamoto Y, et al. A new tracheal bioartificial organ: evaluation of a tracheal allograft with minimal antigenicity after treatment by detergent. Asaio J. 2000;46(5):536-539.

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Table 1. Criteria for assigning ciliated cell scores.

Cilated Cell Score Criteria for Scoring

3 Complete coverage of epithelium with ciliated cells

2 Greater than 75% coverage of the epithelium with ciliated cells

1 Scattered evidence of ciliated cell coverage of the epithelium. (Greater

than 0%, but less than 75%)

0 No evidence of ciliated cell coverage of epithelium

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Figure Legends

Figure 1. A) Photograph of surgical placement of DTM (left) for patch tracheoplasty. B) Representative

bronchoscopic image of remodeled ECM at 6 months after surgical placement for patch

tracheoplasty. The remodeled area is the whitish area in on the top surface of the trachea. C)

Representative gross morphologic image of remodeled ECM at 6 months after surgical placement

for patch tracheoplasty. The remodeled area (arrow) consists of dense collagenous tissue covered

with a shiny epithelial layer. There is no evidence of cartilage formation in the remodeled area.

Figure 2. A) Histologic image of remodeled UBM stained with PAS showing columnar ciliated

epithelium with scattered secretory cells (200X). B) Average score of ciliated cells as a function

of longitudinal distance for all remodeled UBM specimens. C) Average secretory cell count as a

function of longitudinal distance from proximal to distal edge for the UBM specimens. D)

Average basal cell count as a function of longitudinal distance from proximal to distal edge for

the UBM specimens.

Figure 3. A) Histologic image of remodeled UBMC stained with PAS showing columnar ciliated

epithelium with scattered secretory cells (200X). B) Average score of ciliated cells as a function

of longitudinal distance for all remodeled UBMC specimens. C) Average secretory cell count as

a function of longitudinal distance from proximal to distal edge for the UBMC specimens. D)

Average basal cell count as a function of longitudinal distance from proximal to distal edge for

the UBMC specimens.

Figure 4. A) Histologic image of remodeled DTM stained with PAS showing columnar ciliated

epithelium with scattered secretory cells (200X). B) Average score of ciliated cells as a function

of longitudinal distance for all remodeled DTM specimens. C) Average secretory cell count as a

function of longitudinal distance from proximal to distal edge for the DTM specimens. D)

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Average basal cell count as a function of longitudinal distance from proximal to distal edge for

the DTM specimens.