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einforcement of Esophageal Anastomoses Withn Extracellular Matrix Scaffold in a Canine Modellejandro Nieponice, MD, Thomas W. Gilbert, and Stephen F. Badylak, DVM, MD, PhD

epartments of Surgery and Bioengineering, and McGowan Institute for Regenerative Medicine, University of Pittsburgh,
ittsburgh, Pennsylvania
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Background. The gastric pull-up procedure, a standardntervention after radical esophagectomy, is associatedith high morbidity and mortality due to leaks and

tricture. A previous preclinical study showed that anxtracellular matrix (ECM) scaffold with autologous mus-le tissue could be used to repair a complete circumfer-ntial defect in the cervical esophagus. The aim of theresent study was to determine if healing of end-to-endnastomoses of the esophagus could be improved byeinforcement with an ECM scaffold.

Methods. Twelve female mongrel dogs underwent aomplete transection of either the cervical esophagusn � 6) or the gastroesophageal junction (n � 6). Aortion of the endomucosa at the anastomotic site wasesected and replaced with an ECM scaffold in contactith the subjacent muscle and the muscle was anasto-osed. The measured end points included macroscopic

nd microscopic evaluation and quantification of the
sophageal diameter at the anastomotic site.
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tive Medicine, 100 Technology Dr, Suite 200, University of Pittsburgh,ittsburgh, PA 15219; e-mail: [email protected].

2006 by The Society of Thoracic Surgeonsublished by Elsevier Inc

Results. No anastomotic leaks or systemic complica-ions were observed in the ECM-treated animals. Mor-hologic findings in both groups showed complete mu-osal covering of the surgery site. The remodeledsophageal tissue showed angiogenesis and completepithelialization. Intact, organized layers of muscle tis-ue were present between the native muscularis externand the submucosal layer and effectively bridged theransected ends.

Conclusions. The ECM scaffold altered the defaultechanism of esophageal repair. Scar tissue formationith associated stricture was virtually eliminated, and the

sophageal healing response was characterized by the re-lacement with structurally normal tissue layers. Thesendings suggest that the high morbidity rate associatedith esophagectomy procedures may be reduced by thisCM augmentation procedure at the anastomotic site.

(Ann Thorac Surg 2006;82:2050–8)
© 2006 by The Society of Thoracic Surgeons
egenerative medicine approaches using extracellularmatrix (ECM) scaffolds derived from the porcine

mall intestinal submucosa and urinary bladder (UBM)ave been shown to promote site-specific constructive

issue remodeling in preclinical [1–3] and clinical appli-ations [4–6], including musculoskeletal reconstruction,ower urinary tract repair, vascular replacement, andermatologic wound repair. A recent study showed thatn ECM scaffold in the presence of autologous muscleells induced constructive remodeling of esophageal tis-ue after creation of a full circumferential defect in aanine model [1]. This ECM scaffold approach could alsootentially benefit patients undergoing reconstructiveurgery of the esophageal tract by reinforcing surgicalnastomoses, a more conservative application that mayave immediate clinical translation.The most commonly accepted approach for restoring

ood transit after radical esophagectomy is a “gastricull-up”, which involves mobilizing the stomach through

he mediastinum after shaping it into a tube and anasto-osing it to the remaining cervical esophagus [7]. When

ccepted for publication June 12, 2006.

ddress correspondence to Dr Badylak, McGowan Institute for Regener-

he stomach is not available for anatomic or pathologiceasons, a colonic or small bowel interposition can besed [8]. Although these procedures are successful forome patients, they are associated with a high degree oforbidity, including anastomotic leakage, scarring, and

tricture [9, 10].Mobilization of the abdominal organs into the medias-

inal cavity compromises the blood supply to these tis-ues, which decreases the healing capacity of the tissuend contributes to complications at the anastomotic site11]. Anastomotic leakage is one of the most commonomplications and is considered an independent riskactor in the prognostic outcome [12]. Attempts to modifyhe surgical technique to reduce the incidence of leakageave had limited success [13, 14]. Another commonomplication is postoperative scarring and stricture ofhe anastomosis, which can require endoscopic dilationn up to 50% of patients [7].

The purpose of the present study was to evaluate these of an ECM scaffold in the remodeling of the anasto-otic site in two locations in a canine model: the cervical

sophagus and the gastroesophageal junction. We hy-othesized that the reinforcement of an end-to-end anas-

omosis with a UBM-ECM scaffold would minimize scarissue with stricture and promote a constructive, site
ppropriate remodeling response.
0003-4975/06/$32.00doi:10.1016/j.athoracsur.2006.06.036

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2051Ann Thorac Surg NIEPONICE ET AL2006;82:2050–8 REINFORCEMENT OF ESOPHAGEAL ANASTOMOSES

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aterial and Methods

verview of Experimental Designwelve healthy adult female mongrel dogs, weighingetween 17 and 24 kg, were divided into two equalroups of 6 dogs. In group 1, the cervical esophagus ofach dog was completely transected and anastomosed inn end-to-end fashion. In group 2, the gastroesophagealunction was completely transected and anastomosed inn end-to-end fashion. The anastomotic sites in bothroups were reinforced with a UBM-ECM scaffold thatas placed on the mucosal surface of the anastomosis.ight additional dogs were used as controls in which noBM-ECM scaffold was placed. All animal proceduresere performed in compliance with the Guide for The Care

nd Use of Laboratory Animals (National Institutes ofealth, 1996).All dogs survived until elective euthanasia was per-

ormed at prescribed time points (range, 30 to 180 days)r until stricture formed that prevented swallowing. Theeasured end points included esophageal function andorphologic characteristics of the anastomotic site.

roup 1: Cervical Esophageal Anastomosishe 6 dogs in this group underwent a complete transec-

ion of the esophagus in the mid-cervical region, withurgical resection of 1.5 cm of the full circumferentialndomucosa from both ends of the transection site. Thendomucosa was defined as the epithelium, basal lamina

ig 1. Schematic of the surgical procedures. Dogs were divided in twoervical region, and the endomucosal layer was resected on both proxold was telescoped inside the esophagus (center) and an end-to-ennderwent complete esophageal resection at the gastroesophageal jnd (left). A UBM funnel-shaped scaffold was telescoped inside (ceice (right).

basement membrane), and lamina propria, plus the w

unica submucosa, leaving only the muscularis externa.tubularized form of UBM-ECM scaffold was sutured to

he ends of the remaining endomucosa, and the ends ofhe abluminal muscularis externa were anastomosedFig 1A). Two control dogs underwent the same surgicalrocedure without the UBM-ECM scaffold placement

Table 1).

roup 2: Gastroesophageal Junction Anastomosishe 6 dogs in this group underwent a complete transec-

ion of the gastroesophageal junction to assess the re-odeling of the UBM-ECM in a relatively harsh environ-ent (ie, the environment to which the anastomosis is

ubjected after a gastric pull-up procedure). The endo-ucosa for a 1.5-cm length on the esophageal side of the

ransection was resected and a funnel-shaped UBM-CM scaffold was sutured proximally to the free end of

he endomucosa and then sutured distally through thetomach wall (Fig 1B). Three control dogs underwent aomplete transection of the gastroesophageal junctionhat was repaired without endomucosal resection orBM-ECM scaffold placement. Another 3 control dogsnderwent transection of the gastroesophageal junction,

ollowed by endomucosal resection of the esophageal seg-ent without UBM-ECM scaffold placement (Table 1).

BM-ECM Device Preparationorcine urinary bladders were harvested from market

ps. (A) Group 1 underwent complete esophageal section at the mid-and distal ends (left). A urinary bladder matrix (UBM) tubular scaf-astomosis was completed on top of the device (right). (B) Group 2on, and the endomucosa was resected on the proximal esophagealand an end-to-end anastomosis was completed on top of the de-

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2052 NIEPONICE ET AL Ann Thorac SurgREINFORCEMENT OF ESOPHAGEAL ANASTOMOSES 2006;82:2050–8

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fter sacrifice. Residual external connective tissues, in-luding adipose tissue, were trimmed and all residualrine was removed by repeated washes with tap water.he urothelial layer was removed by soaking the material

n 1 N saline. The tunica serosa, tunica muscularis ex-erna, tunica submucosa, and most of the muscularis

ucosa were mechanically delaminated from the blad-er tissue. The remaining basement membrane of the

unica epithelialis mucosa and the subjacent tunica pro-ria, collectively termed UBM, were then decellularizednd disinfected by immersion in 0.1% (v/v) peracetic acid�), 4% (v/v) ethanol, and 96% (v/v) deionized water forhours. The UBM-ECM material was then washed twice

or 15 minutes with phosphate-buffered saline (pH � 7.4)nd twice for 15 minutes with deionized water.The scaffolds were fabricated with two different shapes

o specifically match the anatomy of either the cervicalsophagus or the gastroesophageal junction. For group 1,ubular shaped scaffolds were fabricated to match thenatomy of the cervical esophagus. Briefly, multilayerubes were created by wrapping hydrated sheets of UBMround a 22-mm perforated tube/mandrel that was cov-red with umbilical tape for four complete revolutionsie, a four-layer tube) [1].

For group 2, funnel shaped scaffolds were fabricated toatch the anatomy of the gastroesophageal junction. A

ustom mold was constructed in the shape of a funnel,ith a perforated straight tube (25-mm diameter) con-ected to a bowl (70-mm radius of curvature). Before theCM was placed on the mold, the tube was wrapped withmbilical tape and the bowl was wrapped with cheese-loth to facilitate removal of the UBM after vacuumressing. Hydrated sheets of UBM-ECM were wrappedround the mold until 4 to 6 layers were present. Bothhe esophageal and the gastroesophageal constructsere then placed into plastic pouches and attached tovacuum pump (Model D4B, Leybold, Export, PA)

ith a condensate trap inline. The constructs were sub-ected to a vacuum of 710 to 735 mm Hg for 10 to 12 hourso remove the water and form a tightly coupled multi-aminate construct. After removal of each device from the

andrel, it was terminally sterilized with ethylene oxide.

urgical Proceduresach animal was anesthetized by intravenous adminis-

able 1. Overview of Experimental Design

xperimental Group Site of End-to-End A

E (group 1, n � 6) CEontrol for group 1 (n � 2) CEEJ anastomosis (group 2, n � 6) GEJontrol for group 2 (n � 3) GEJontrol for group 2 (n � 3) GEJ

E � cervical anastomosis; ECM � extracellular matrix; GEJ � g

ration of sodium thiopental and a surgical plane of a

nesthesia was maintained by intubation and inhalationf isoflurane in oxygen. The surgical area was shaved andrepared with standard draping for aseptic surgery.

roup 1midline cervical incision was made and tissue layers

ere dissected to isolate the esophagus. A completeransection of the esophagus was made, and 1.5 cm of thendomucosa was resected from cut ends of both theroximal and distal segments. A tubular UBM-ECMcaffold was telescoped within the remaining muscularisucosa and sutured to the proximal mucosal end with

wo short running sutures of either Prolene 5-0 (Ethicon,omerville, NJ) or PDS 5-0 (Ethicon), and to the distal endith four separate stitches equally spaced about the

ircumference. Finally an end-to-end anastomosis of theuscularis mucosa was performed with separate stitches

f PDS 3-0. The cervical wound was closed by layers withicryl 3-0 (Ethicon). The same procedure was repeated

or the control dogs without implanting the scaffold.

roup 2midabdominal incision was made, the triangular liga-ent of the liver was sectioned, and the diaphragm

iatus was dissected to expose the gastroesophagealunction. A complete transverse section of the gastro-sophageal junction was made, and 1.5 cm of the esoph-geal endomucosa and a 5-mm ring of gastric mucosaere resected. The funnel shaped UBM-ECM device waslaced within the luminal position at the point of thenastomosis between the structures. The tubular seg-ent of the device was anastomosed to the esophagealucosa with two short running sutures of PDS 5-0, and

he flared portion was fixed to the gastric wall with foureparate stitches of PDS 4-0 (Fig 2). The gastroesophagealnd-to-end anastomosis was completed by restoring theuscularis externa with separate 3-0 PDS sutures.The abdominal cavity was closed by layers with Vicryl

-0, Prolene 1-0, and PDS 3.0 respectively. A chest tubeas placed on the left side of the thorax because theleural cavity was opened during hiatus dissection.The same procedure was repeated for control dogs,ith the exception that no device was implanted, and in

hree animals, the esophageal mucosa was not removed

mosis Endomucosal Resection ECM Scaffold

Yes Tubular shapeYes No scaffoldYes Funnel shapeYes No scaffoldNo No scaffold

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ostsurgical Carehe dogs were recovered from anesthesia, extubated,nd monitored in the recovery room until they wereesting comfortably in a sternal position. The dogs wereept in a cage overnight and returned to their larger runousing on postoperative day 1. They were given oralrophylactic antibiotics consisting of cephalothin/ephalexin (35 mg/kg) twice daily for 7 to 9 days. Theogs also received intravenous acepromazine (0.1 mg/kg)nd butorphanol (0.05 mg/kg) for 2 days, followed byubcutaneous or intramuscular buprenorphine (0.01 to.02 mg/kg) every 12 hours thereafter for analgesia aseeded. The group 2 dogs (including controls) were given0 mg of omeprazole daily.Dogs were fed from an elevated/raised platform. The

og’s daily nutrition was calculated and divided into twoo three daily feedings. Gruel/soft food was provided for

week postoperatively. The animals were reintroducedo solid food over a 2-week period, with oral intaketarting 36 hour after surgery. The dogs were weighedeekly and housed in a run measuring approximately

0 � 14 ft to allow freedom to ambulate. Endoscopicxaminations were conducted at approximately monthlyntervals to evaluate esophageal structure.

orphologic Examinationmmediately after euthanasia, the scaffold placement site,ncluding native esophageal and gastric tissue both prox-mal and distal to the graft site, was harvested. Thexcised sample was opened by making a longitudinalncision in the distal-to-proximal direction. The exposed

ucosal surface was examined, and the tissue was thenmmersed in 10% neutral buffered formalin.

The luminal circumference of the esophagus was mea-ured 4 to 5 cm proximal to the anastomotic level and athe anastomotic site to determine the degree of stricture.esults were expressed as percent reduction of the cir-umference between the anastomotic site and the proxi-al normal tissue. Tissue was trimmed longitudinally

cross the anastomosis, sectioned, and stained with bothematoxylin and eosin and Masson’s trichrome stains.

ig 2. Macroscopic photograph of the urinary bladder matrix funnel-geal anastomosis. The flare part of the device was mounted first on the esophagus, and finally, the end-to-end anastomosis between the e

he areas examined included the native tissue, the prox- s

mal and distal anastomoses of the scaffold, and theid-scaffold region.

esults

linical Outcomesll dogs in both groups (including controls) recoveredell from the surgical procedure and had a favorable

linical outcome immediately after surgery. After oralntake was reestablished, the dogs in group 1 had noigns of leak or infection in the cervical wound, exceptne of the control dogs had an untreatable leak at thenastomosis level that required euthanasia. In group 2,here were no signs of abdominal distress, except in onef the control dogs had an episode of fever and lethargy8 hours after the surgery that was successfully treatedith antibiotics and conservative care. One dog in grouphad an untreatable stricture of the proximal anastomo-

is that required euthanasia at day 39. In group 2, one ofhe control dogs had an untreatable stricture at day 14hat required euthanasia. All the other dogs had anneventful recovery, showing normal appetite, normalydration, normal weight, and normal activity.

ndoscopic Examination ResultsROUP 1. The endoscopic examination of the dogs in thisroup showed the formation of a normal appearingucosal surface in the area where the device was im-

lanted. No signs of stricture were found at the level ofhe anastomosis through endoscopic examination. Theurface transition from the distal segment of the device tohe normal native esophagus showed no difference in

orphology, and the anastomotic lines were difficult toetect and required identification of the sutures. The

ransition from the proximal native esophagus to theroximal end of the device had a mild scarring that didot prevent passing the 6F endoscope and did not affect

he clinical outcome in 5 of 6 dogs. There was no evidenceor active inflammation, erosion, scarring, or necrosis inhe rest of the remodeled UBM-ECM scaffold.

The endoscopic examination of the control dogs

d scaffold (inset) designed to match the anatomy of the gastroesoph-stric end (left), the tubular part (arrow) was telescoped later insidegus and the stomach was completed on top (right).

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nd lack of epithelialization, with a severe inflammatoryeaction at the anastomosis level in the dog that had theeak at day 7.ROUP 2. The endoscopic examination of the dogs in thisroup showed a complete epithelialization of the anasto-otic site with a normal appearing transition from

sophageal mucosa to the gastric folds at approximatelymonth. Mild redness was evidenced in some spots,

ikely due to increased reflux of gastric contents afterurgical manipulation of the gastroesophageal junction.he anastomotic site was not detectable, and there wereo signs of stricture at that level. The proximal anasto-osis of the UBM-ECM scaffold also had an increased

nflammatory reaction and mild scarring that did notffect the clinical outcome in any of the dogs. Thiscarring was more pronounced in 1 of the 6 dogs and wasilated endoscopically to prevent further complications.The control dogs that did not undergo endomucosal

esection had an increased inflammatory response in therea of the anastomosis, and clear scar tissue formationas visible between the esophageal and the gastric

egments. The control dogs that underwent endomucosaesection had an irregular mucosal covering with scat-ered foci of glistening, shiny surface mixed with aeddened, eroded surface at the level of the anastomosis.everal mild adhesions were present at the anastomoticite that were easily disrupted with the scope and werelinically asymptomatic, except for one dog that had anntreatable stricture at day 14 and the scope could notass through the stenosis.

acroscopic AppearanceROUP 1. The anastomotic area showed complete remod-ling of the endomucosal and muscular layers withinimal scarring. In several dogs, the site where the

omplete section had been made was difficult to find andas detected only by identifying the sutures (Fig 3).carring was evident on the abluminal surface between

he two edges of the muscular layer. The remodeledevice could be identified by the lack of folds thatharacterize a mature esophageal lining. A completely

ig 3. Macroscopic photograph of cervical anastomosis reinforced withe native anastomosis was difficult to distinguish by eye (arrow). Thnce of the Prolene sutures. In the inner surface (right), complete epitolds in the remodeled area compared with native tissue.

pithelialized luminal layer was evident, however, and �

he transition with the native tissue was very smooth.here were no macroscopic differences between dogs inroup 1 and the control dogs that had an uneventfulecovery. In the control dog that had a leak, a hole of 3/8f the circumference was evident at the anastomosis

evel. In this group, there were no noticeable differencesn circumference reduction, although it was slightly moreevere in the control dogs (25.6% � 10.9% for UBM-ECMreated versus 30.1% � 0.2% for controls; Fig 4).

nary bladder matrix scaffold. In the external view (left), the site ofn of the scaffold to native tissue can be determined from the pres-

ization was observed with no signs of stricture. There was a lack of

ig 4. Percentage reduction in the circumference of the esophagust the anastomotic levels compared with the normal esophagus (4 tocm proximal to the implant). No differences were observed between

he urinary bladder matrix (UBM) treated group and the controln the cervical esophagus. At the level of the gastroesophageal junc-ion, the control group without endomucosal resection had an in-reased reduction of the circumference that approached significancep � 0.057) compared with the treated group. The control groupith endomucosal resection (experimental control) showed a greater

eduction of the circumference than both the clinical control and thereated group, although there was considerable variability. Data rep-esent means � SD. e � UBM treated; � experimental control;

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ROUP 2. As in group 1, the anastomotic area was com-letely remodeled with a continuous mucosal layer andlmost no signs of scarring (Fig 5). In contrast, the contrologs in which the endomucosa was left intact and noevice was implanted had a clear endomucosal gap at thenastomotic level. The control dogs in which the endo-ucosa was resected had a stricture at the anastomotic

evel, and the resected area was covered by a thinpithelial layer that was clearly distinguished from thedjacent normal mucosa (Fig 5). The control dogs had aoticeable reduction of the circumference at the anasto-osis level, with a reduction of 47.4% � 25.9% with

ndomucosal resection and 34.5% � 9.5% without endo-ucosal resection. The reduction in the circumference

ended to be less for the UBM-treated group (15.9% �3.7%), although significance was not detected. The com-arison of the UBM-treated group with the control groupith endomucosal resection yielded a p � 0.159 due to

he substantial variability observed in the control group.he UBM-treated group approached statistical signifi-

ig 5. Macroscopic photograph of gastroesophageal anastomosis. Thence at the transition between esophageal and gastric mucosa (arrow)ucosal resection. The control group in which there was no endomuc

arrow). The control group with endomucosal resection but no scaffoldomucosa was resected was only covered by a thin epithelial layer an

ig 6. Histologic appearance of cervical anastomosis. The urinary blasubmucosal layer with organized collagen, with no inflammatory re

pithelialized, and a new muscle tissue layer was regenerated between
tain; original magnification �4 [left]) and �10 [right]).
ance compared with the control group without endomu-osal resection (p � 0.057; Fig 4).

icroscopic ExaminationROUP 1. Histologic examination showed a continuous

ntact mucosal layer from the proximal to the distalative esophagus across the anastomotic site where thecaffold was placed. The mucosa was characterized by atratified squamous epithelium upon an intact basementembrane. Rete pegs of the epithelium were not as

ronounced as those in the adjacent native esophagealucosa (Fig 6). The muscularis externa at the anastomo-

is site showed bands of organized collagen with numer-us islands of skeletal muscle cells that appeared to be

ndependent from the muscle fascicles of the adjacentative esophagus. The tissues from the control dog thatad an uneventful recovery also had a continuous intactucosal layer but also had a subjacent accumulation of

olymorphonuclear and mononuclear inflammatoryells, characteristic of chronic active inflammation. The

p treated with urinary bladder matrix (left) had a normal appear-out gaps, and was completely epithelialized in the area of the endo-esection (center) showed a clear gap at the anastomosis levelt) had no gaps at the anastomotic level, but the area where the en-

re was a stricture ring at the anastomotic level (arrow).

matrix-extracellular matrix device was completely incorporated andor fibrosis, was observed. The remodeled scaffold was completely

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og that had a leak at day 7 showed a clear lack ofpithelialization with a pronounced inflammatoryesponse.ROUP 2. Histopathologic examination of dogs that re-eived the UBM-ECM device showed a normal transitionrom esophageal to gastric epithelium, with no signs ofctive or chronic inflammation in the subjacent tissueFig 7). The repair site between the transected edges ofhe muscle tissue was very narrow and filled with par-ially organized collagen. The submucosal tissue con-isted of organized connective tissue with an abundantapillary supply. There was an absence of inflammatoryells in any of the remodeled scaffold materials that werexamined beyond 4 weeks after surgery. The tissues fromhe control dogs in which the endomucosa was resectedut no UBM-ECM devices were placed showed signs ofhronic and active inflammation beneath a transitionaleoepithelium that had a hyperplastic response withccumulated debris on the luminal surface (Fig 7). In theog that had the untreatable stricture, the epithelializa-

ion was incomplete, and a variable amount of dense,isorganized, fibrous connective tissue (scarring) wasresent.The tissues from the control dogs with no endomucosal

esection showed a clear tissue retraction, with scarringt the level of the anastomosis and a considerablemount of fibrous connective tissue that formed a largenastomotic gap (Fig 7). A typical foreign body reactionas always evident and limited to the tissues immedi-

tely surrounding the sutures in both groups.No evidence of the ECM material could be identified in

ny of the dogs in both groups, consistent with completeegradation of the scaffold [15].

omment

urgical procedures involving the esophagus are associ-ted with increased morbidity and mortality rates [7, 9,0, 16]. The present study showed that the xenogeneicCM bioscaffold derived from porcine urinary bladderltered the normal healing response to site-specific con-tructive remodeling of esophageal tissue when used as aeinforcement of native esophageal anastomosis. The

ig 7. Histologic appearance of gastroesophageal anastomosis. The treubmucosal layer and continuity with the underlying muscular layer.ar matrix scaffold (center) had a distinct invagination of the mucosaue. The control group with endomucosal resection and with no scaffocollection of dense collagenous connective tissue with an abundance

yperplastic response (trichrome stain; original magnification �4 [lef

onstructive remodeling response observed in this study m

eplaced the usual scarring process observed in surgicalnastomoses of the digestive tract in a clinical setting.It was interesting to note that the control dogs in which

he endomucosa was resected had a better healing re-ponse than the controls where the endomucosa was leftntact, despite an increased inflammatory infiltrate. Theogs that had the UBM scaffold as reinforcement showednear normal macroscopic and microscopic appearance,ith absence of inflammation or detrimental scar tissue.his difference between the control dogs and the UBM-CM treated dogs was more noticeable at the level of theastroesophageal junction, suggesting that ECM scaf-olds promote constructive remodeling despite the harshnvironment caused by gastric reflux [17, 18].We have previously shown that when an ECM scaffold

s placed in direct contact with autologous muscle tissue,ven relatively small amounts (ie, 30% circumference),he reconstitution of the esophagus is ad integrum [1].urthermore, that study also showed that the mechanicalehavior of the UBM scaffold changed during the remod-ling process from a very stiff, relatively noncompliantaterial to a tissue that had characteristics similar to

ative esophageal tissue.Similar remodeling responses using ECM bioscaffolds

ave been seen in other body locations [2, 3]. Numerouseports of lower urinary tract reconstruction including therinary bladder, urethra, and ureter show similar replace-ent of the ECM scaffold by an epithelial cell population,

ubmucosa, and smooth muscle cell layer. In these loca-ions, the presence of a muscle cell population in contactith an ECM scaffold enhanced but was not essential for

emodeling without scar tissue formation [19].Replacement of muscular body wall structures with

heets of skeletal muscle mixed with collagenous connec-ive tissue and adipose tissue have been reported with anCM scaffold derived from porcine small intestinal sub-ucosa [3, 6]. ECM scaffolds have now been used in more

han 500,000 patients worldwide for numerous clinicalpplications.Although the mechanisms of esophageal scarring and

tricture formation are largely unknown, they are likelyelated to the presence of a harsh environment (ie, distalsophagus with gastric reflux), proteolytic enzymes, and

group (left) showed a normal epithelial transition with an organizedcontrol group with no endomucosal resection and with no extracellu-

level of suture placement (arrow) and an accumulation of scar tis-plantation (right) showed no invagination of the mucosa, but ratherflammatory cells subjacent to the neoepithelium that indicated ater], and �10 [right].

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last proliferation and contractility [20–23]. These mech-nisms are likely aggravated when a leak occurs due ton even more aggressive inflammatory response andnfection.

There are a number of reasons that UBM-ECM scaffolday enhance the normal esophageal wound-healing re-

ponse. Naturally occurring ECM scaffolds have shownesistance to infection after deliberate contaminationith bacteria or gastrointestinal contents [24–26]. This

ntimicrobial property exhibited in vivo is due to theresence of small peptides (5 to 16 kDa) that are formeduring the degradation of an ECM scaffold. These andimilar peptides have shown resistance to gram-positivend gram-negative bacteria in in vitro [27], and have alsoxhibited chemoattractant properties [28]. It is thoughthat the chemoattractant properties of the degradationroducts promotes the recruitment of bone marrow de-ived cells to the site of ECM remodeling that, in turn,espond to local mechanical and biochemical signals thatirect these cells down a tissue-specific differentiation

ineage [29, 30]. UBM-ECM has also been shown to retainn intact basement membrane that supports the rapidonfluent growth of epithelial cells [31]. Such cells areonsidered to be important for the successful develop-ent of a luminal structure [32].Attempts to improve the outcome of esophagogastric

nastomoses have included modification of the surgicalechnique from hand sewing to mechanical staples. How-ver, the nonanatomic position of the gastric tube in theastric pull-up, the lack of adequate blood supply, andhe acidic environment of the stomach drastically ham-er the healing potential, often leading to catastrophicomplications [9]. The use of UBM-ECM scaffolds to alterhe normal healing response might decrease the overall

orbidity and mortality associated with esophagealrocedures.The ability to withstand the harsh esophageal environ-ent can also be useful to attempt a less-invasive repair

f esophageal fistulae or perforations where total resec-ion is usually required. A recent report using ECMcaffolds as a reinforcement of mechanical sutures inejunal anastomoses in pigs showed increased leak pres-ure, suggesting the potential of this approach to preventndesired outcomes [33]. Furthermore, current mini-ally invasive therapies for Barrett disease, such as

ndomucosal resection or photodynamic therapy, coulde improved by the use of UBM-ECM scaffolds to pre-ent the scar healing and stricture associated with thoserocedures [34].One limitation of the present study is that the gastric

ull-up was not mimicked and the healing response wasot assessed under the tension at the anastomotic site,or was the compromised blood supply that exists in thelinical setting. However, ECM scaffolds are commonlysed in load-bearing applications with site-appropriateemodeling [2, 3]. Furthermore, previous studies suggesthat ECM scaffolds induce an aggressive angiogenesis inhe remodeling tissue [28, 35].

Another limitation is that the low number of control
ogs prevented a powerful statistical analysis. This deci-
ion was from the results of our previous study, in whichll control animals (complete circumferential resectionithout repair with UBM-ECM scaffold) experienced a

evere, untreatable stricture with convincing results [1].herefore, repeating a large number of controls for thistudy was not in accordance of the Animal Welfareegulations as suggested by our local Internal Animalare Use Committee.In summary, the findings of the present study suggest

hat it is possible to reinforce the surgical anastomoses ofative esophagogastric tissue and decrease postoperativeorbidity using a regenerative medicine approach with a

iodegradable ECM scaffold. The ECM scaffold promotesemodeling to site-specific esophageal tissue rather thanhe scar tissue that is usually associated with woundealing in mammals. The experimental configurationas specifically intended to address surgical anastomo-

es that are created during the gastric pull-up procedure,ut a similar reinforcement approach may also be appli-able to partial resection of the esophagus (ie, mucosec-omy in Barrett disease), which also tends to result in scarissue formation and esophageal stenosis [36, 37]. Thisonservative approach could rapidly be translated to thelinical setting and serve as the basis for future esopha-eal replacement or reconstruction using a regenerativeedicine approach.

e would like to acknowledge funding from the Common-ealth of Pennsylvania. We would also like to thank the staff at

he McGowan Animal Facility for their support during the studys well as Dr Alan Spievack and Dr Adolfo Badaloni for theircientific input.

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