Enterogenesis in a clinically feasible model of mechanical small-bowel lengthening

Abstract

Background: Recent work indicates that mechanical force induces small-bowel growth, although methods reported do not have direct clinical application. We report a clinically feasible technique of enterogenesis and describe intestinal function in this model.

Methods: Using a pig model (n = 11), we stretched isolated small intestinal segments mechanically for 7 days in vivo with an intraluminal device. Control segments were not stretched. Morphology, histology, and epithelial proliferation were assessed. Absorption and epithelial barrier function were examined in an Ussing chamber.

Results: Stretch segments were significantly longer than Control segments and had nearly 2-fold greater surface area (P < .001). Mucosal thickness was much greater in Stretch than Control segments (772 +/- 134 vs. 647 +/- 75 microm, P = .02). Although villus height was reduced in Stretch and Control segments (353 +/- 76 vs. 324 +/- 76 microm, P = .6) versus native jejunum (522 +/- 87, P < .0005), crypt depth was increased dramatically in Stretch (450 +/- 95 microm) versus Control segments (341 +/- 64, P = .005). This observation was accompanied by a 2-fold increase in cellular proliferation (26.3 +/- 3.8 vs 12.1 +/- 6.6 % bromodeoxyuridine+, P < .05). Barrier function was intact ([3H]-mannitol permeation, 0.16 +/- 0.08%, vs native jejunum, 0.17 +/- 0.08%, P = .81). Glucose-mediated sodium transport was similar in Stretch versus native jejunum segments (60.0 +/- 23.5 vs 82.3 +/- 47.3 microA/cm2, P = .31), as was carbachol-induced chloride transport (82.4 +/- 72.2 vs 57.2 +/- 33.4 microA/cm2, P = .54) and alanine absorption (16.46 +/- 12.94 vs 23.53 +/- 21.31 microA/cm2, P = .53).

Conclusions: Mechanical stretching induces small intestinal growth, while maintaining function. Epithelial architecture does change, such that a decrease in villus height is offset by a marked increase in crypt depth and a 2-fold increase in epithelial proliferation. Epithelial barrier and absorptive functions remain intact. The device described may have direct clinical applicability.

Fig 1

Schematic representation of the hydraulic lengthening device. Top, Device before extension. Note the radio-opaque markers within both ends embedded in silicon, allowing for visualization with abdominal radiographs. Bottom, Device in a nearly extended state. Note that the larger (3-mL) syringe is filled, and fluid has now entered through the channel in the 3-mL plunger, allowing the 1-mL syringe to start filling.

Fig 2

Surgical creation of isolated intestinal segments. A, Mid-jejunum was utilized. B, A segment of intestine was taken out of continuity, while preserving its mesenteric pedicle. C, The device for bowel elongation is inserted into the lumen with the tubing to activate the device exiting the bowel (arrow). Note: For simplicity, only 1 segment is shown. For experimental purposes, 2 such intestinal segments were created in each animal to provide an in situ Control segment as well as an elongated segment. D, The isolated segments of bowel also were drained via a soft catheter (arrow) to prevent accumulation of mucus. Radio-opaque contrast injection demonstrates normal diameter and morphology of the isolated segment in vivo.

Fig 3

Epithelial cell proliferation was detected by measuring BrdU incorporation into actively dividing cells. Proliferation is expressed as the mean percent of BrdU-positive cells per crypt (±SD). Stretch segment proliferation was more than 2-fold greater than that of Control segment (*P < .01) and approached significance, compared with native jejunum (P = .05).

Fig 4

Representative histologic figures from nonoperated Jejunum, Stretch, and Control segments of pig intestine. All slides were stained for BrdU as a measure of proliferation. Note the marked increase in BrdU-positive crypt cells (brown) in the Stretch segment.

Fig 5

Cumulative transmucosal permeation of ³H-mannitol (mean ± SD%) during 90 minutes incubation in Ussing chambers. Time 0 represents the start of the incubation with ³H-mannitol after a 20-minute equilibration period. There was a steady linear permeation increment in transmucosal passage of ³H-mannitol over 90 minutes. Stretch segment of intestine did not differ from native jejunum, demonstrating intact mucosal barrier function.

Fig 6

Epithelial barrier function as measured by trans-epithelial resistance (TER, ω/cm²) is shown. TER was virtually identical between Stretch segments of intestine, compared with native jejunum, indicating normal mucosal barrier function.

Fig 7

Averaged baseline short circuit current (Isc, μA/ cm²) during the first 20 minutes after mounting intestinal samples in the Ussing chamber. Baseline Isc was significantly lower in Stretch intestinal segments than in native jejunum (P < .0001). However, stimulated ion transport in response to various agonists did not differ between Stretch and native jejunum (see text).