1Department of Surgery, Brigham and Women's Hospital, and 2Department of Surgery and 3Division of Endocrinology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115
Submitted 30 August 2002 ; accepted in final form 2 August 2003
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ABSTRACT |
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tissue engineering; neomucosa; glucagon-like peptide-2; sodium-glucose cotransporter 1
Although the clinical promise of tissue engineering has already begun to be realized, its greatest impact, at least in the near future, may lie in the creation of models for studying physiological processes, disease pathogenesis, and molecular therapeutics (14). A prototype tissue-engineered neointestine that mimics many structural and functional features of the native small intestine has been developed (19, 32). In an approach first reported by Choi and Vacanti (9), intestinal cell clusters are implanted onto biodegradable polymer scaffolds to generate viable neointestinal tissues. When implanted into rats, these tissues develop into cystlike structures that can be anastomosed to native intestine and contain neomucosa with morphological and transporter properties that are strikingly similar to those of normal small intestine (34). We have also reported that the immunocytes of this neomucosa exhibit similar topographic distributions and population densities to those of native jejunum (26).
A fundamental question exists: does tissue-engineered neointestine have the capacity to respond to regulatory signals that regulate physiological processes in the gastrointestinal tract? In this study, we tested the ability of neointestinal epithelium to respond to glucagon-like peptide-2 (GLP-2). GLP-2 is an endogenous regulatory peptide that has potent trophic activity that is specific for the intestinal mucosa (5, 11, 23, 36, 38). GLP-2 also has been reported to modulate intestinal transporter activity and expression (7). Our findings suggest that GLP-2 enhances epithelial proliferation and increases Na+-glucose cotransporter 1 (SGLT1) expression in the neointestine. Tissue-engineered neointestine represents a potential novel approach for treating patients suffering from intestinal insufficiency.
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MATERIALS AND METHODS |
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Animals. Six-day-old neonatal Lewis rats were used as donors for intestinal epithelial organoid unit isolation. Adult male Lewis rats weighing 150-200 g served as syngeneic recipients. Animals were purchased from Charles River Laboratories (Wilmington, MA) and were housed in accordance with National Institutes of Health guidelines for the care of laboratory animals. Rats were maintained under a 12-h photoperiod and were allowed free access to rat chow and water until the beginning of the study protocol.
Polymer fabrication. Biodegradable polymer tubes, 10 mm in length, 5 mm in outer diameter, and 2 mm in internal diameter, were created from sheets of a nonwoven mesh of polyglycolic acid fibers (Smith and Nephew, Heslington, UK) and sprayed on the outer surface with 5% poly-L-lactic acid (24). The polymer was coated with 200 µl of 0.1% collagen solution (Vitrogen 100; Collagen, Palo Alto, CA).
Epithelial organoid unit isolation and polymer seeding. Intestinal epithelial organoid units were isolated by a process modified from that described by Choi et al. (8). These organoid units are clusters of intestinal cells surrounding a mesenchymal core. In brief, total small bowel was harvested without mesentery from 6-day-old neonatal Lewis rats, luminally lavaged with HBSS, longitudinally incised along the antimesenteric border, and sharply dissected into full-thickness 2 x 2-mm sections. After three washings in 4°C HBSS, they were transferred into an enzyme solution containing 800 U/ml collagenase I (Sigma, St. Louis, MO) and 0.25 mg/ml dispase (Boehringer-Mannheim, Indianapolis, IN) and placed on an orbital shaker for 25 min at 37°C. The digestion was immediately stopped with three 4°C washes with a solution of high-glucose DMEM (GIBCO-BRL, Gaithersburg, MD) with 4% heat-inactivated FBS (iFBS) and 4% sorbitol. The resulting organoid units were centrifuged between washes at 150 g for 5 min, and the supernatant was removed. Organoid units were reconstituted in high-glucose DMEM with 10% iFBS, counted by hemocytometer, and loaded at 1.0 x 105 units/polymer. Polymer tubes were internally loaded with organoid units by micropipette. They were allowed to attach for 1.5 h at 4°C before implantation into animals.
Surgical procedures. Twelve adult male Lewis rats were used as recipients of the organoid unit-polymer constructs. Under intraperitoneal pentobarbital anesthesia, an upper midline abdominal incision was made. The unit-polymer constructs were wrapped with omentum, secured with 6-0 prolene sutures (Ethicon, Somerville, NJ), and placed into the upper abdominal cavity. By 3 wk after the initial operation, the unit-polymer constructs had formed cystlike structures. The lumens of the cysts were opened longitudinally and anastomosed to the native jejunum 15 cm distal to the ligament of Treitz in a side-to-side fashion by using interrupted 6-0 silk sutures. Animals were allowed to recover from surgery for 1 wk before entering the study protocol described below. During this period, they were allowed free access to rat chow and water, with the rats consuming approximately equal amounts after postoperative day 1. Health assessment was performed daily.
GLP-2 administration. The adult rats bearing the neointestinal implants were allocated to one of two groups (n = 6 per group). Group I rats were administered subcutaneous injections of GLP-2 analog (1 µg/g body wt) reconstituted in 0.5 ml PBS (Invitrogen Life Technologies, Carlsbad, CA) twice daily for 10 days. Group II rats (controls) were administered subcutaneous injections of vehicle alone on an identical schedule. Animals were pair fed, and weights were measured daily throughout the study period.
Tissue harvesting. After 10 days of the GLP-2 treatment, animals received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; 50 mg/kg) 1 h before death. All rats were killed at the same time of day. Under Nembutal (sodium pentobarbital) anesthesia (50 mg/kg ip), a midline laparotomy was performed and the neointestinal cysts were excised. Cysts were measured, rinsed free of luminal debris, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA) and immersed in cold isopentane. The tissues were kept at -80°C for later studies. Adjacent 10-µm sections were cut in a cryostat for morphometric analysis (hematoxylin and eosin), determination of proliferative index (BrdU staining), apoptotic index [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining], GLP-2 receptor (GLP-2R) expression, in situ hybridization, and immunofluorescence.
Histological measurements. An observer unaware of the treatment status of animals from which the tissues were harvested measured 15 villi and 20 crypts per slide under a microscope (x10 magnification) using a commercially available computer-based morphometry program (Image-ProPlus, v. 4.0 for Windows 98; Media Cybernetic).
Immunofluorescence. Frozen sections were fixed in 4% paraformaldehyde for 15 min, blocked with 2% normal goat serum in PBS, and then incubated overnight at 4°C in rabbit anti-rat SGLT1 antiserum (1:500 in presence of 1% normal goat serum and 0.1% Triton X-100 in 0.2 mM PBS). The anti-SGLT1 antibody was provided by Professor E. Wright (Department of Physiology, University of California-Los Angeles School of Medicine, Los Angeles, CA). Sections were incubated at room temperature with a Cy3-conjugated goat anti-rabbit secondary antibody (1:200 for 1 h; Jackson Immunoresearch, West Grove, PA). Controls were incubated with the secondary antibody alone under identical conditions. Photographs were taken with a digital spot camera attached to a Nikon E600 microscope and were imported into Adobe Photoshop (Adobe Systems, Seattle, WA). Exposure settings and sharpness enhancement were identical for each picture.
Cellular proliferation: BrdU. Proliferating intestinal epithelial cells were detected by immunostaining for an S-phase marker, BrdU. Sections were treated with 10% normal horse serum in PBS for 30 min. The primary antibody was applied for 60 min by using a 1:50 dilution of mouse anti-BrdU (DAKO) in PBS containing 1% normal horse serum. To generate negative controls, mouse IgG (1 mg/ml; Vector Laboratories) was used as the primary antibody. Sections were then rinsed with 0.01 M PBS containing 0.05% Tween 20 and treated with 1:200 biotinylated horse anti-mouse IgG (Vector Laboratories) in PBS containing 1% normal horse serum for 30 min. Detection was performed with avidin-biotin-peroxidase diaminobenzidine chromagen substrate kits (Vector Laboratories) according to the kit instructions. All slides were viewed under a microscope (x40 magnification) by a blinded observer, and the number of brown BrdU-stained nuclei was calculated as a fraction of the total number of cells per crypt and expressed as a percentage. In total, 20 crypts per slide were selected by using previously described criteria (35).
Apoptosis. Specimens were immerse-fixed in buffered formalin (4% vol/vol) for 12 h. These were embedded in paraffin, and 10-µm sections were taken. Specimens were exposed for 1 h at room temperature in a humidified chamber to a TUNEL mixture. This contained equilibration buffer (98 µl), biotinylated nucleotide mix (1 µl), and terminal deoxynucleotidyl transferase (1 µl). Terminal deoxynucleotidyl transferase was omitted from the TUNEL labeling mixture in the TUNEL-negative controls. Positive controls were obtained by pretreating with DNase before incubation with the TUNEL labeling mixture. Endogenous peroxidases were blocked by immersing the slides in 0.3% hydrogen peroxide for 3 min at room temperature. Sections were then incubated with streptavidin horseradish peroxidase at 1:500 in PBS for 30 min at room temperature and were visualized with diaminobenzidine. For TUNEL-stained sections, counterstaining was omitted. A cell with both TUNEL immunoreactivity and morphological features of apoptosis (e.g., pyknosis) was regarded as apoptotic. Levels of TUNEL staining were quantified by a blinded observer. The numbers of TUNEL-positive cells were counted in at least five fields (x40 magnification). The mean number of TUNEL-reactive cells was taken as representative of TUNEL staining for each specimen. The ratio of apoptotic to nonapoptotic cells (apoptotic index) in each villus was obtained and expressed as a percentage.
Northern blot analysis. RNA was extracted from tissue samples as described previously (33). Equal amounts of RNA were electrophoresed on 1% formaldehyde-agarose gels. Loading efficiency and RNA integrity were confirmed by ethidium bromide staining. RNA was transferred to Hybond-N (Amersham Biosciences, Piscataway, NJ) and hybridized to 32P-labeled cDNA probes. The probes used were rat SGLT1, glucose transporter GLUT2, sucrase (21), mouse villin (IMAGE Clone 373217; American Type Culture Collection, Manassas, VA), and GAPDH (12) cDNA. Blots were washed twice in 2x SSC (1x SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) and 0.1% SDS for 10 min at room temperature, once in 1x SSC and 0.1% SDS for 15 min at 65°C, and twice in 0.1x SSC and 0.1% SDS for 20 min at 65°C before exposure to Kodak XAR-5 film at -80°C. Densitometric analysis was performed by using NIH Image software. To compare the SGLT1, GLUT2, and sucrase expression levels among samples consisting of different proportions of intestinal mucosa, their hybridization signals were normalized to the corresponding villin signal before comparison.
In situ hybridization. Nonradioactive in situ hybridization was performed as described (3) by using a digoxigenin-labeled cRNA probe that contains 2.5 kb of the SGLT1 sequence. Frozen sections (10 µm) were cut in a cryostat and captured onto Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were then fixed, acetylated, and hybridized at 68°C over three nights to the probe (probe concentration 100 ng/ml). Hybridized probe was visualized by using alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche, Indianapolis, IN) and 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD). Sections were rinsed several times in 100 mM Tris, 150 mM NaCl, and 20 mM EDTA, pH 9.5, and were coverslipped with glycerol gelatin (Sigma). Control sections were prepared by substituting the sense probe transcript. Pictures were taken with a digital spot camera attached to a Nikon E600 microscope and were imported into Adobe Photoshop. Exposure settings and sharpness enhancement were identical for each picture.
In situ hybridization for GLP-2R expression. A 1.3-kb ribo-probe specific for GLP-2R was generated from mouse IMAGE clone 5363416 (GenBank accession no. BI734956
[GenBank]
; American Type Culture Collection, Manassas, VA). The IMAGE clone contained an insert of 4.4 kb, most of which was sequence from mouse chromosome 11 adjacent to the GLP-2R gene. The extraneous sequence was removed from the pCMV-SPORT6 vector by digestion with HindIII and religation. The distal 1.1 kb of the remaining insert showed >90% homology with the rat coding sequence, whereas the proximal 228 nt contained a 104-nt segment with 96% homology to the rat 5'-untranslated region described by Lovshin et al. (22). Sense and antisense transcripts were transcribed from the SP6 and T7 RNA polymerase sites, respectively, present in the vector. Nonradioactive in situ hybridization was performed as described above.
Statistical analysis. Comparison of continuous variables among groups was performed by using ANOVA, Tukey with honestly significant difference. All data are expressed as means ± SE. Significance was accepted at the 95% confidence level (P < 0.05). Computations were performed by using a statistical package (Statistica, v. 4.3; StatSoft, Tulsa, OK).
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RESULTS |
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Macroscopic appearance of neointestinal cysts. All anastomoses between the neointestinal cysts and the native intestine were patent. In no case was the lumen of the native intestine obstructed at the site of the anastomosis. The lumen of the anastomosed cysts contained intestinal luminal material that did not differ grossly from that found in the lumen of the native intestine. The gross appearance of a representative neointestinal cyst anastomosed to native jejunum is shown in Fig. 1. The mean cyst diameter was 1.6 ± 0.2 cm in the GLP-2-treated animals and 1.5 ± 0.1 cm in the control animals (P > 0.05).
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Effects of GLP-2 on histology, cellular proliferation, and apoptosis. Robust-appearing crypt-villus architecture was observed for neointestine harvested from both GLP-2-treated and control rats (Fig. 2). Mean villus height and mean crypt depth were significantly greater for neointestine harvested from rats that had received GLP-2 than for neointestine harvested from control animals (420 ± 19 vs. 240 ± 10 µm and 135 ± 12 vs. 82 ± 4 µm, respectively; P < 0.05; Fig. 3). Similarly, the percentage of crypt cells stained by using antibody directed against BrdU was greater in neointestine harvested from GLP-2-treated rats than neointestine harvested from control animals (66.9 ± 1.8 vs. 57.4 ± 2.5%; P < 0.05; Fig. 4A). These proliferation rates are within the range (60.5 ± 4.7%) previously reported for neointestinal tissue (34). The apoptotic index in tissue harvested from treated animals was 0.12 ± 0.15 vs. 0.82 ± 1.2 in that from control animals (P < 0.05; Fig. 4B). The apoptotic index in tissue from control animals is similar to that previously reported (34) for neointestine that has been anastomosed to native jejunum (0.77 ± 0.18%). Apoptotic cells were most numerous at the tips of the neointestinal villi (Fig. 4B, inset).
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Mean villus height and mean crypt depth in native jejunum harvested from rats that had received GLP-2 were greater than those in native jejunum harvested from control animals (750 ± 91 vs. 507 ± 28 µm and 154 ± 14 vs. 105 ± 12 µm, respectively; P < 0.05; Fig. 3). Similarly, mean crypt depth in colon harvested from a site 10 cm distal to the ileocecal junction in GLP-2-treated rats was greater than that in colon harvested from the corresponding location in control rats (33.2 ± 6.2 vs. 22.4 ± 6.2 µm; P < 0.05).
Effects of GLP-2 on SGLT1, sucrase, and GLUT2 mRNA expression. We analyzed mRNA extracted from neointestinal mucosa for SGLT1 expression by Northern blotting. We normalized the signals obtained for SGLT1, sucrase, and GLUT2 to that for villin, as a proxy for the quantity of epithelial cells in the sample. In separate studies conducted in rats, intestinal villin expression was unaffected by GLP-2 administration, as assessed by Northern blot analysis (data not shown). The expression level of SGLT1 mRNA, which ran as a single prominent band at 2.8 kb, was 2.4-fold greater in neointestine harvested from GLP-2-treated rats than that in neointestine harvested from control animals (P < 0.01; Fig. 5). Hybridization for sucrase with our probe detects two transcripts: the full-length mRNA at 6.2 kb as well as a second transcript running just below the 28S rRNA. Sucrase mRNA expression was 2.3-fold greater (P < 0.05) in neointestine from GLP-2-treated animals than in neointestine from control animals (Fig. 5A). No significant alterations in neointestinal GLUT2 mRNA expression, shown here as the typical 2.6-kb transcript (Fig. 5A), with respect to GLP-2 treatment status were evident. SGLT1, sucrase, and GLUT2 mRNA expression in native jejunum were unaffected by GLP-2 treatment status.
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To evaluate the topographical distribution of SGLT1 mRNA expression in the tissue-engineered intestine, we performed in situ hybridization. Hybridization with antisense probe resulted in labeling of the enterocytes in the villi, whereas no labeling was seen with the sense probe. Representative results are shown in Fig. 6, A-C, top. SGLT1 mRNA expression was found to be limited to the villi, with greatest expression evenly distributed in the lower two-thirds and with lesser expression in the tips. Little or no signal was detected in the crypt cells or underlying substratum. This expression pattern is reminiscent of that reported for native small intestine (6, 17). Labeling intensity was greater for neointestine harvested from GLP-2-treated rats than neointestine harvested from control animals, a finding that parallels those observed on Northern blotting. Similarly, the labeling intensity was greater in native jejunum harvested from GLP-2-treated rats than in native jejunum from control animals (Fig. 6, A-E, top).
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SGLT1 protein expression. To evaluate the topographical distribution of SGLT1 protein expression, we performed indirect immunofluorescence microscopy using anti-SGLT1 antiserum. For neointestine harvested from control rats, SGLT1 protein expression was localized to the apical membrane of the enterocytes in the middle and upper villi (Fig. 6B, bottom). For neointestine harvested from GLP-2-treated rats, SGLT1 protein expression was localized to the apical membrane of enterocytes along the entire length of the villi (Fig. 6C, bottom). Similar topographical distributions were observed in native jejunum; staining intensity appeared greater in tissue harvested from GLP-2-treated rats than in tissue from control animals (Fig. 6, D-E, bottom).
GLP-2R expression. In situ hybridization localized the expression of the putative GLP-2R to the lamina propria of the subepithelial layer of the neointestine (Fig. 7). GLP-2 treatment status had no apparent effect on GLP-2R expression location or density.
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DISCUSSION |
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To begin characterizing the physiology of the prototype tissue-engineered intestine, we tested its ability to respond to GLP-2, an endogenous regulatory peptide with potent trophic activity specific for the intestinal epithelium (8, 11, 16). Our findings clearly demonstrate that GLP-2 administration is associated with increases in villus height, crypt depth, and crypt cell proliferative index and a reduction in apoptosis in the epithelium of the neointestine. These results mirror the effects of GLP-2 on native small intestine in our study and in those previously reported (5, 11, 29, 36, 37).
A principal physiological action of the small intestinal epithelium is nutrient absorption. Because GLP-2 has been reported to enhance intestinal glucose uptake, we examined the effects of GLP-2 on SGLT1 expression in the tissue-engineered neointestine. Northern blot analysis clearly shows that GLP-2 administration is associated with an increase in the expression of SGLT1 mRNA in the epithelium of the neointestine. In situ hybridization and immunofluorescence of neointestine harvested from control animals suggest that the topographical distribution of SGLT1 mRNA and protein expression, respectively, approximate those observed for native small intestine. In addition, SGLT1 immunofluorescence suggests that GLP-2 administration leads to expanded distribution of SGLT1 protein expression to include the entire length of the villi. Previous studies have shown that anastomosis to the native intestine is a critical step that allows the neomucosa of the engineered intestinal cysts to acquire structural and functional features, including SGLT1 expression, that recapitulate those of native mucosa (26, 34). This finding suggests that exposure to luminal contents enhances the development of a more mature neomucosa. SGLT1 expression levels are also regulated by dietary (13) and circadian variables (10, 28). In our study, animals were pair fed, weight matched, and killed at the same time of day to minimize the potential confounding effects of these factors.
The effect of GLP-2 administration on the expression of two other GLP-2-responsive genes in the neointestinal mucosa was examined in our study. The expression of sucrase, but not of GLUT2, was observed to increase with GLP-2 treatment. This finding suggests differential regulation of sucrase and GLUT2 expression within the neointestinal mucosa, but the exact mechanisms will obviously require further study.
Where GLP-2R is located in the intestine has been controversial. Various authors have suggested that the GLP-2R is located on enterocytes (18), enteroendocrine cells (38), or neural elements of the intestine (4). Our finding that the putative GLP-2R is located in the subepithelial layer of the neointestine, and not in the epithelium itself, is consistent with the reported subepithelial localization of this receptor in the mouse intestine (4).
Our findings are important for several reasons. First, they demonstrate a previously undescribed feature of neointestine generated by using our tissue-engineering approach: that it has the capacity to respond to external regulatory signals in a manner similar to that of native intestine. Second, our findings suggest a strategy for expanding neointestinal absorptive surface area and, more importantly, nutrient absorptive capacity. Such strategies will have great utility in optimizing neointestine for clinical application in patients suffering from intestinal insufficiency. The feasibility of localized and controlled polymeric delivery of biologically active macromolecules in a dose-and time-dependent manner to tissue-engineered structures makes our findings on the activity of GLP-2 particularly promising (25, 27, 30). Finally, our study paves the way for future investigations designed to dissect the mechanisms of the action of GLP-2 and other regulatory agents on the complex physiological processes in the intestinal epithelium. The feasibility of deliberately modifying selected components of engineered tissues as they are assembled makes this latter possibility especially exciting.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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This work was presented at the 2002 Digestive Disease Week, May 19-22, 2002, San Francisco, CA.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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