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. 2018 Feb;24(3-4):199-206.
doi: 10.1089/ten.TEA.2017.0042. Epub 2017 Jun 30.

Structural Heteropolysaccharide Adhesion to the Glycocalyx of Visceral Mesothelium

Andrew B Servais  1 , Arne Kienzle  1 , Cristian D Valenzuela  1 , Alexandra B Ysasi  1 , Willi L Wagner  1   2 , Akira Tsuda  3 , Maximilian Ackermann  2 , Steven J Mentzer  1
Affiliations

Structural Heteropolysaccharide Adhesion to the Glycocalyx of Visceral Mesothelium

Andrew B Servais et al. Tissue Eng Part A. 2018 Feb.

Abstract

Bioadhesives are biopolymers with potential applications in wound healing, drug delivery, and tissue engineering. Pectin, a plant-based heteropolysaccharide, has recently demonstrated potential as a mucoadhesive in the gut. Since mucoadhesion is a process likely involving the interpenetration of the pectin polymer with mucin chains, we hypothesized that pectin may also be effective at targeting the glycocalyx of the visceral mesothelium. To explore the potential role of pectin as a mesothelial bioadhesive, we studied the interaction of various pectin formulations with the mesothelium of the lung, liver, bowel, and heart. Tensile strength, peel strength, and shear resistance of the bioadhesive-mesothelial interaction were measured by load/displacement measurements. In both high-methoxyl pectins (HMP) and low-methoxyl pectins, bioadhesion was greatest with an equal weight % formulation with carboxymethylcellulose (CMC). The tensile strength of the high-methoxyl pectin was consistently greater than low-methoxyl or amidated low-methoxyl formulations (p < 0.05). Consistent with a mechanism of polymer-glycocalyx interpenetration, the HMP adhesion to tissue mesothelium was reversed with hydration and limited by enzyme treatment (hyaluronidase, pronase, and neuraminidase). Peel and shear forces applied to the lung/pectin adhesion resulted in a near-interface structural failure and the efficient isolation of intact en face pleural mesothelium. These data indicate that HMP, in an equal weight % mixture with CMC, is a promising mesothelial bioadhesive for use in experimental and therapeutic applications.

Keywords: adhesion; glycocalyx; mesothelium; pectin.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Murine visceral mesothelium and glycocalyx. (A) Scanning electron micrograph of the murine lung visceral pleura. The “flagstone” appearance of the mesothelium was demonstrated above the cut surface of the lung (arrows). (B) TEM of the visceral pleural mesothelium demonstrated microvilli (bracket) and the underlying mesothelial basement membrane (black arrows). The intervillar glycocalyx was not seen in TEM. (C) Staining visceral mesothelium with the green fluorescent LEL (Vector Laboratories) demonstrated the glycocalyx (arrows) in the lung (C1), liver (C2), bowel (C3), and heart (C4). The blue nuclei reflected Hoechst 33342 (Sigma) counterstain. Scale bars = 60 μm. LEL, Lycopersicon esculentum lectin; TEM, transmission electron microscopy.
<b>FIG. 2.</b>
FIG. 2.
Load/displacement measurements. The adhesion of mesothelium to the pectin-based bioadhesive was assessed by three factors: (A) tensile strength, (B) peel strength, and (C) shear resistance. The tissue was applied—with ∼0.1 N force and 3–5 min development time—to the firm pectin-based substratum that was composed of 50% pectin and 50% CMC. Loads were applied at a controlled rate to a suture passed through the tissue within 2 mm of the adhesive interface. The lung demonstrated tensile strength (A) greater than peel strength (B) or shear resistance (C). The adhesion of lung to equal weight % pectin and CMC is shown. Notably, peel and shear forces applied to the lung demonstrated near-interface parenchymal separation (yield point) that facilitated the isolation of the pleural mesothelium. The results represent median values of N = 5 replicates. CMC, carboxymethylcellulose.
<b>FIG. 3.</b>
FIG. 3.
Tensile strength of visceral mesothelium adhesion to varied mixtures of pectin and CMC. (A–C) The adhesion strength of CMC and different weight ratios of HMP, amidated low-methoxyl (LMA), and nonamidated low-methoxyl (LMC) pectin were tested for relative tensile strength; liver adhesion is shown. The area of the bubble reflects relative adhesion strength of the different mixtures scaled to 100. The greatest adhesion strength was demonstrated in equal weight (%) ratio of all three pectins and CMC. (D) Comparison of adhesion strength of HMP, LMA, and LMC and equal weight (%) ratio mixtures of CMC tested against all 4 mesothelial tissues. HMP demonstrated consistently greater adhesion than comparable LMA and LMC pectin (asterisk, p < 0.05). (E) Comparison of equal weight ratio of HMP and CMC (50%) and CMC with no pectin (0%) for all four mesothelial tissues. The 50% mixture was significantly greater than the 0% mixture for all four tissues (asterisk, p < 0.05). Error bars = 1 SD of triplicate samples. LMA, amidated low-methoxyl; HMP, high-methoxyl pectin; SD, standard deviation.
<b>FIG. 4.</b>
FIG. 4.
Bioadhesion of visceral mesothelium after pretreatment with glycocalyx-directed enzymes. The lung (A), liver (B), bowel (C), and heart (D) tissue was treated with neuraminidase (Neur), pronase (Pro), or hyaluronidase (Hyal) at established concentrations before adhesion on a 50% HMP and CMC substratum. The 50% HMP (Max) and 0% pectin (Min) provided control comparisons for the enzyme effects on tensile strength. Tensile strength was diminished by all three enzymes; however, a significant quantitative variation in enzyme inhibition was noted. Box plots indicate median values and 25th and 75th percentile; whiskers represent variability outside the upper and lower quartiles. Data represent replicate samples of N = 3 mice.
<b>FIG. 5.</b>
FIG. 5.
Shear and peel force isolation of en face pleural mesothelium. A combination of shear force and peel force applied to the lung-pectin adhesion resulted in the separation of the mesothelium from the subjacent lung. (A) SEM of the resulting mesothelial layer showed the typical “flagstone” appearance of the free surface of the mesothelium (ellipse) and alveolar remnants on the deep surface of the layer (arrow). (B) Fluorescent nuclear staining demonstrated an intact monolayer (scale bar = 100 μm). (C) Silver staining demonstrated intact tight junctions (scale bar = 50 μm). SEM, scanning electron microscopy.
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