Journal of Surgical Research 147, 168–171 (2008)

Evaluation of Small Intestinal Submucosa as Scaffolds for IntestinalTissue Engineering

Min Lee, Ph.D.,* Paul C. Y. Chang, M.D.,† and James C. Y. Dunn, M.D., Ph.D.*,†,1

*Department of Bioengineering, †Department of Surgery, University of California, Los Angeles, California

Submitted for publication December 17, 2007

doi:10.1016/j.jss.2008.03.012

Background. Biodegradable scaffolds have beenused for regenerating the small intestine. The aim ofthis study was to evaluate the feasibility of small in-testinal submucosa (SIS) as scaffolds for intestinal re-generation in a rat model.

Materials and methods. A tubular one-ply or four-plySIS was interposed between isolated jejunal segmentsin rats. The scaffolds were harvested at 2, 4, and 8weeks after implantation, and the specimens were ex-amined grossly and histologically.

Results. Significant contractions were observed inSIS scaffolds after implantation. The one-ply SIS con-tracted to 44% of its initial length at 2 weeks andcontinued to contract to 6% of its initial length at 8weeks. The contraction of four-ply SIS scaffolds wasless than that of the one-ply SIS, reaching 29% of itsinitial length at 8 weeks. Minimal epithelial andsmooth muscular regeneration was observed in theSIS scaffolds after implantation.

Conclusions. A significant shrinkage was observedin the SIS scaffolds after implantation. Although thefour-ply SIS contracted less than the one-ply SIS, nei-ther scaffold supported significant amount of intesti-nal regeneration. © 2008 Elsevier Inc. All rights reserved.

Key Words: small intestinal submucosa; scaffold; con-traction; tissue engineering; intestine.

INTRODUCTION

Short bowel syndrome (SBS) is the malabsorptivestate that occurs after a massive loss of the smallintestine [1]. Current therapies for SBS including totalparenteral nutrition, surgical lengthening procedures,and small intestinal transplantation are associatedwith significant complications. The long-term use of

1 To whom correspondence and reprint requests should be addressedat UCLA Division of Pediatric Surgery, Box 709818, 10833 Le Conte

Avenue, Los Angeles, CA 90095. E-mail: jdunn@mednet.ucla.edu.

1680022-4804/08 $34.00© 2008 Elsevier Inc. All rights reserved.

parenteral nutrition is associated with liver failure [2];the efficacy of surgical lengthening procedures remainsto be defined [3, 4], and intestinal transplantation islimited by donor availability and complications fromimmunosuppression [5].

A potential therapy for patients with SBS is intesti-nal tissue engineering using absorbable biomaterials[6, 7]. In the selection of the biomaterials, the scaffoldsneed to provide the necessary support until the con-struct is remodeled. Small intestinal submucosa (SIS)is an acellular, collagen-based matrix that has beenused as a scaffold for tissue engineering in a variety ofsystems such as the bladder, blood vessels, and theabdominal wall [8–10]. One drawback in the use of SISis its poor mechanical properties. In previous inves-tigations, the regeneration of the intestinal mucosausing SIS was reported, but a significant contractionof the SIS graft was observed after implantation[11–15]. The reported intestinal regeneration mayresult from the contraction of SIS graft rather thantrue intestinal tissue growth. The purpose of thisstudy was to evaluate a multilayered SIS as a scaf-fold for the intestinal tissue regeneration to reducethe contraction of the graft.

MATERIALS AND METHODS

Animal Care

Female Lewis rats (Charles River Laboratories, Wilmington, MA)weighing 200 to 225 g were used as recipients. All animals weremaintained and handled in compliance with the institutional regu-lations established and approved by the Animal Research Committeeat the University of California, Los Angeles. Rats were housed incages under standard laboratory conditions and fed rat chow andwater ad libitum.

Preparation of Scaffold

One-ply and four-ply SIS were purchased from Cook Biotech

(West Lafayette, IN). A tubular SIS scaffold (5 mm in diameter, 20

169LEE, CHANG, AND DUNN: SMALL INTESTINAL SUBMUCOSA AS SCAFFOLDS

mm in length) was created by wrapping the SIS sheet around asilicon tube (10 French Red Robinson catheter; Bard, Covington,GA), and the edges of the sheet were sewn together using interrupted6-O Prolene sutures (Ethicon, Somerville, NJ). SIS scaffolds weresoaked with saline before implantation.

Scaffold Implantation

All procedures were performed under isoflurane inhalational an-esthesia. A midline abdominal incision was made, and a well-vascularized 3-cm jejunal segment 10 cm distal to the ligament ofTreitz was separated from continuity. Intestinal continuity was re-stored by anastomosing the intestine in an end-to-end fashion usinginterrupted 6-O Prolene sutures. The isolated segment of the jeju-num was divided in the middle, and a tubular SIS was interposedbetween the two ends of the divided jejunal segment, and the anas-tomoses between the tubular scaffold and the intestinal segmentwere completed in an end-to-end fashion with interrupted 6-O Pro-lene sutures (Fig. 1). The anastomotic sites were additionallymarked with silk sutures for later identification. A silicone tube (8French Red Robinson catheter; Bard) was inserted as a stent acrossthe anastomoses. Both ends of the intestinal segment were broughtto the skin level as jejunostomies as described by Wang et al. [11].The scaffold was wrapped with omentum and was secured in placewith 6-O Prolene sutures. The abdominal wall and skin was closed intwo layers with a running 3-O Vicryl (Ethicon) and 3-O silk sutures.The isolated segment of the jejunum was flushed with saline throughthe jejunostomies twice a week.

Histological Evaluation

Animals were sacrificed at 2, 4, and 8 weeks after implantation (n �2 per time point). Tissue specimens were harvested, and the lengthof the scaffolds was measured between the silk sutures that wereused to mark the anastomoses between the jejunum and the tubularscaffold. Tissue specimens were fixed in 10% neutral buffered forma-lin and were embedded in paraffin. Tissue sections were cut at 5 �mand were stained with hematoxylin and eosin.

Statistical Analysis

Student’s t-tests were performed for statistical analysis. The sig-

FIG. 1. Photograph of the interposed SIS graft. A tubularizedSIS scaffold was interposed in the middle of an isolated jejunalsegment and was anastomosed to the native intestine in an end-to-end fashion. (Color version of figure is available online.)

nificance level was defined as P � 0.05.

RESULTS

Macroscopically, the anastomotic sites were incorpo-rated into the adjacent native intestine without leak-age or material disruption. The longitudinal shrinkageof the tubular scaffold was observed in all cases (Table1). A significant contraction was observed 2 weeks afterthe implantation of the tubularized one-ply SIS (Fig.2A and B). There was an approximately 56% decreasein the length of the implanted one-ply SIS at 2 weeks,and this tubular scaffold further contracted to 19% ofthe initial length at 4 weeks (Fig. 2C and D). At 8weeks, two suture lines of the tubular scaffold with thejejunum nearly came together (94% contraction) (Fig.2E and F). Histologically, at 2 weeks after implanta-tion, epithelial regeneration was not observed on theSIS graft (Fig. 2B). At 4 weeks, some mucosal growthwas observed on both sites of the anastomoses (Fig.2D). The luminal surface of the SIS graft at 8 week wasmostly lined with mucosal epithelium except for itscentral region (Fig. 2F).

Contraction of the tubularized four-ply SIS was lessthan that of the one-ply SIS (P � 0.05, Fig. 3). Therewas an approximately 30% decrease in the length ofthe implanted four-ply SIS tube at 2 weeks (Fig. 3Aand B), and 60 and 70% contraction at 4 and 8 weeks,respectively (Fig. 3C-F). Histologically, by 4 weeks,only some mucosal growth was observed near the anas-tomotic sites of the SIS graft with the native intestine(Fig. 3D). The luminal surface of the SIS graft at 8weeks was covered with mucosal epithelium except forbare areas in the central region (Fig. 3F).

DISCUSSION

SIS, a naturally derived biodegradable biomaterial,has been extensively studied as a scaffold for the re-generation of a variety of tissues [8–10]. Previous stud-ies demonstrated that SIS was able to induce the re-generation of the small intestine, but a significantcontraction of the SIS was observed [11–15]. A 20 to40% shrinkage in the length of regenerated small in-testine has been reported using SIS in a similar rat

TABLE 1

Extent of Longitudinal Shrinkage of ImplantedTubular Scaffold

One-ply SIS Four-ply SIS

2 weeks 56 � 10% 32 � 5%*4 weeks 81 � 6% 60 � 4%*8 weeks 94 � 2% 71 � 6%*

* Significant difference compared with one-ply SIS. P � 0.05.

1 m

170 JOURNAL OF SURGICAL RESEARCH: VOL. 147, NO. 2, JUNE 15, 2008

model [11]. Chen and Badylak also reported 80% de-crease in the area of the SIS patch at 1 year afterimplantation [15].

In this study, we investigated whether a multilay-ered SIS graft will undergo less contraction as com-pared to the one-ply SIS in a rat model. A significantdegree of shrinkage of the one-ply SIS tube was ob-

FIG. 2. Macroscopic findings of the luminal side of the SIS graft aSIS. The location of the graft is confirmed by the suture mark (arrows(D), and 8 weeks (F) after implantation of one-ply SIS (Scale bar �

FIG. 3. Macroscopic findings of the luminal side of the SIS graft aSIS. The location of the graft is confirmed by the suture mark (arrows

(D), and 8 weeks (F) after implantation of four-ply SIS (Scale bar � 1 m

served after implantation. This observation may bedue to the poor mechanical properties and the highdegradation rate of SIS. It has been reported that40–60% of implanted SIS was degraded within 4weeks, and most of material was removed from the siteof remodeling in 8 to 12 weeks [16, 17]. The contractionof the four-ply SIS tube was less than that of the

weeks (A), 4 weeks (C), and 8 weeks (E) after implantation of one-plyistological findings of the regenerated intestine 2 weeks (B), 4 weeksm). (Color version of figure is available online.)

weeks (A), 4 weeks (C), and 8 weeks (E) after implantation of four-plyistological findings of the regenerated intestine 2 weeks (B), 4 weeks

t 2). H

t 2). H

m). (Color version of figure is available online.)

171LEE, CHANG, AND DUNN: SMALL INTESTINAL SUBMUCOSA AS SCAFFOLDS

one-ply SIS tube probably due to the greater initialmechanical integrity to resist contraction. The contrac-tion of the SIS in this study was greater than thatreported by Wang and colleagues [11]. There was nomajor difference in the techniques used in the animalmodel [11], but the SIS used in this study was derivedfrom the porcine small intestine, whereas Wang’sgroup made their SIS from rodent intestines. This mayaccount for the observed differences in contractionrates. Another significant difference in this study wasthe lack of mucosal regeneration. While luminal con-tents from the native intestine will have stimulatoryeffects on the mucosal growth, we used the isolatedintestinal segment because Wang’s group had reportedmucosal regeneration in such models. The minimalepithelial and smooth muscular regeneration observedin the SIS scaffolds in this study suggests that previ-ously reported regeneration on SIS may result from thecontraction of SIS rather than true intestinal tissueregeneration. An ideal scaffold should maintain theproper mechanical support until complete tissue in-growth occurs. The use of synthetic biomaterials givesgreater advantages compared to natural materials inthat they can be processed to give a wide range ofphysical properties and degradation rate [18].

The observed minimal regeneration also suggeststhat the use of SIS alone is not sufficient, and the useof appropriate progenitor cells may be necessary for theregeneration of the components of the small intestine.Vacanti’s group has regenerated neomucosa on thepolyglycolic acid by seeding intestinal organoids [7].Hori et al. used autologous mesenchymal stem cells toregenerate an intestinal muscle layer with a collagensponge as a scaffold [19]. Nakase et al. reported the useof autologous smooth muscle cells isolated from stom-ach wall to regenerate the smooth muscle layer [20]. Itis likely that a cellular component of the small intes-tine is needed to initiate intestinal tissue regeneration.

This study provides a preliminary evaluation of theuse of SIS for intestinal tissue engineering. Althoughprevious reports have demonstrated the regenerationof small intestine using SIS [15, 21, 22], our findingshere suggest that the observed intestinal tissue forma-tion may have resulted from the contraction of thedefect covered by SIS rather than true tissue regener-ation. Further studies are needed to find the optimalscaffold materials providing the proper mechanicalsupport and facilitating intestinal tissue regeneration.

ACKNOWLEDGMENTS

This work was supported by the Fubon Foundation and the Amer-ican Surgical Association Foundation.

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