Figure 1.
Hematoxylin and eosin histology of neo-natal intestine.
At 3–5 days post-partem, normal rat intestine (A) was harvested and stripped of muscle (B). The resulting muscle strip (C) was cultured along with ISMCs isolated from the muscle strip enzymatically. Both circular (C) and longitudinal (L) smooth muscle was visible in the whole intestine and isolated MS. 400x magnification, 200-µm scale bar.
Figure 2.
Phase contrast images of ISMCs and MS grown in culture for 3, 7, and 14 days after harvest from neonates.
Smooth muscle cells (top row) were evenly dispersed after seeding and reached confluency at about 14 days in culture. Adherent MS (bottom row) were about 200-µm in diameter with cells emanating outward on the culture surface until reaching confluency after approximately 14 days in culture. 100x magnification, 200-µm scale bar.
Figure 3.
Immunofluorescence of cultured cells after 14 days.
Smooth muscle cells and muscle strips were grown in culture for 14 days and stained for markers of smooth muscle, glial, and neural lineage. SMA and DES immunofluorescence confirmed smooth muscle lineage in both cultures. Both samples lacked MHC, a marker of smooth muscle maturity. Cultured MS but not cultured ISMCs contained S100 and BTUB, markers for enteric glial and neural cells. 100x magnification, 200-µm scale bar.
Figure 4.
Co-immunofluorescence of S100 and BTUB in MS at Day 14.
Overlapping networks of S100 (red) and BTUB (green) in cultured MS was observed. Arrows point to BTUB-positive extensions from the MS, arrowheads point to MS. 100x magnification, 200-µm scale bar.
Figure 5.
mRNA expression of cultured ISMC and MS 14 for after neonatal harvest.
There was no significant difference in mRNA expression for smooth muscle markers between cultured ISMC and MS, including SMA, DES, and MHC. There was a significant increase in mRNA expression of BTUB in cultured MS (p<0.05, *; n = 6). Samples were normalized to smooth muscle from the harvest with GAPDH as a control gene.
Figure 6.
Contraction of cultured MS 14 days after neonatal harvest.
A roughly 10% change in projected area was observed on a MS cultured for 14 days, showing the retained contractile function of cultured MS (S1 Video). White arrow points to observed MS, 100x magnification, 200-µm scale bar.
Figure 7.
Calcium indicator intensity in native smooth muscle stripped from day 5 rat pup.
An increase in calcium indicator intensity is in native smooth muscle just prior to depolarization (A) and 0.7 seconds later (B), after depolarization. Regions of black indicate depolarization, while regions of white are decreases in calcium intensity as an artifact of the smooth muscle movement during contraction. 100x magnification, 200-µm scale bar. A plot of calcium indicator intensity (C) within the native smooth muscle show the intense, periodic waves of depolarization.
Figure 8.
Calcium indicator intensity in cultured MS after 14 days.
An increase in calcium indicator intensity is observed between images of the MS just prior to depolarization (A) and 0.7 seconds later (B), after depolarization (S2 Video). Muscle strips and the surrounding cells that formed a “ganglia” are labeled. Regions of black indicate depolarization. 100x magnification, 200-µm scale bar. A plot of calcium indicator intensity (C) within MS and the “ganglia” show the periodic depolarization waves in the MS culture.
Figure 9.
Calcium indicator intensity in cultured ISMC after 14 days.
The changes in calcium indicator of ISMC were less intense than in MS in an image just prior to (A) and 0.7 seconds after depolarization (B). Calcium intensity fluctuations (C) were not periodic (S3 Video). 100x magnification, 200-µm scale bar.
Figure 10.
Demonstration of pharmacological response of MS to cholinergic stimulation.
At day 14, the cholinergic agonist carbachol was added to cultured MS to induce MS depolarization after incubating in calcium indicator (S4 Video). Calcium intensity increased in MS between images taken just before (A) and immediately after (B) adding carbachol. A plot of calcium intensity (C) in the observed muscle strip indicates a pharmacological sensitivity to cholinergic agonists. MS were also incubated with the cholinergic antagonist atropine along with calcium indicator to reduce MS cholinergic sensitivity (S5 Video). MS with atropine were observed before (D) and after addition of carbachol (E). A plot of calcium indicator intensity shows reduced intensity after incubation in atropine compared to MS without atropine, while the addition of carbachol (arrow) did not increase intensity of the calcium indicator. An arrow points to observed MS, an arrowhead indicates addition of cholinergic stimulation on intensity profiles.100x magnification, 200-µm scale bar.
Figure 11.
Demonstration of pharmacological response of ISMC to cholinergic stimulation.
At day 14, the cholinergic agonist carbachol was added to cultured ISMC to induce depolarization after incubating in calcium indicator. Calcium intensity increased in ISMC between images taken just before (A) and immediately after (B) adding carbachol. A plot of calcium intensity (C) in the observed ISMCs indicates a pharmacological sensitivity to cholinergic agonists. An arrow points to observed MS, an arrowhead indicates addition of cholinergic stimulation on intensity profiles.100x magnification, 200-µm scale bar.
Figure 12.
Histological analysis of implant sections.
Scaffolds with either GFP-expressing ISMC or MS were retrieved after two weeks in vivo and were visualized in cross-section with H&E staining (A, B). A representative image of an ISMC-seeded explant (A, 40x, 500-µm scale) shows laser-cut pores (arrows) to improve cellular and vascular infiltration through dense PCL scaffold (PCL) from the “serosal” to luminal side of the implant. GFP-expressing MS (B, 200x, 100-µm scale) on the “serosal” surface of a PCL implant is outlined with a dotted line. Scaffolds with GFP-expressing MS were immunofluorescently labeled with antibodies to GFP or SMA (C, D). GFP-expressing and MS (C) survived the 2 week implantation and migrated out from the MS. Non-GFP-expressing cells from the host also permeated the PCL implant and had SMA immunofluorescence (D). 40x magnification, 500-µm scale bar.
Figure 13.
Immunofluorescent imaging of ISMC- or MS-seeded implant.
After two weeks in vivo, ISMC and MS implants on scaffolds were identified with anti-GFP immunofluorescence. Both ISMC and MS expressed SMA, but ISMC had less immunofluorescence for DES and MHC compared to MS. ISMC also showed reduced S100 immunofluorescence compared to MS. Merged images of SMA (green) and s100 (red) show no co-localization of immunofluorescence in either ISMC or MS. BTUB immunofluorescence was decreased in ISMC implants compared to MS implants. Nuclei were indicated with DAPI. 200x magnification, 100-µm scale bars.