Bioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement

Bioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement Irene Wacker, Willi Wagner, Ronald Curticean, Endre Majorovits, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7619875/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The plant-derived biopolymer pectin has been explored as sealant in lung surgery for several years. Adhesion to the lung surface is thought to be mediated by the interpenetration of pectin’s heteropolysaccharide chains with the sugar-chains on glycoproteins and glycolipids of the mesothelial glycocalyx. To improve rational design of carbohydrate-based sealants, a mechanistical understanding of the adhesion process is necessary. Therefore, we first established a method to preserve pectin ultrastructure by limiting hydration during sample preparation for electron microscopy. This allows us to demonstrate distinct morphologies of pectins extracted from various plant sources. The network-like morphology of lemon pectin - closely resembling glycocalyx structure - provoked us to explore the interaction between the two by applying a pectin film to the surface of murine lung in a surgical setting. The ultrastructural visualisation of the sealant-tissue assembly shows a tight association between the carbohydrate fibres, likely driven by their polymer-typical chain entanglement. Biomedical Engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Usage of carbohydrate-based biopolymers such as cellulose, [ 1 ] chitosan, [ 2 ] or pectin [ 3 , 4 ] in a biomedical context has a longstanding tradition. Applications range from wound dressing to drug delivery, mediated by bioadhesion, [ 5 ] and more specifically by mucoadhesion in the latter case. [ 6 – 8 ] In particular the chemical properties of pectin, a complex, branched carbohydrate, and its bio-adhesiveness make it an interesting, biocompatible candidate as e. g. inert carrier for drug delivery. [ 9 ] Even more interestingly, the usage of pectin films as potential sealants in surgery has been explored in the work of Servais et al . [ 10 – 12 ] where interaction of a variety of pectin formulations with the mesothelium of internal organs was investigated. [ 10 ] In a murine surgical model setting it was found that pectin film exhibits a strong bioadhesion e.g. to lung pleura and could limit air leaks after pleural injury, [ 11 ] comparing favorably against commercially available surgical products. When adhesion of the sealant to the pleura was inhibited by desiccation or removal of the glycocalyx from the pleura by enzyme treatment, sealant failure was observed. [ 12 ] This indicates a potential interaction mechanism by an initial swelling of the dry film when applied to the pleura allowing entanglement of pectin’s carbohydrate chains with the sugar chains on the glycoproteins and glycolipids of the glycocalyx covering the mesothelial cells. The mechanism could therefore be based on a simple polymer-typical inter-chain entanglement, which – if verified – can be optimized further by future chemical modifications of the pectin material. To prove the hypothesis that adhesion is caused by entanglement of pectin carbohydrate chains with the extracellular glycocalyx on visceral organs the interaction of the different polysaccharides needs to be shown directly, preferably in an in vivo/in situ surgical setting. Separate visualization of the individual components of the proposed interaction complex – pectin on one side and the glycocalyx on top of the mesothelial cell layer on the other side – has been achieved previously in ex situ , non-surgical experiments in different ways: The nanostructure of high methoxyl sugar acid pectin gels analyzed by AFM exposed curvilinear strands with junctions and branch points. [ 13 ] TEM analysis of pure and mixed high methoxyl and low methoxyl pectin gels with different chemical modifications and varying concentrations of the gelling agents Ca 2+ , sucrose and citrate, embedded in resin and sectioned before imaging, revealed highly porous networks of thin filaments. [ 14 , 15 ] Similar networks were shown in a study comparing TEM data from resin embedded pectin gels with SAXS measurements on native pectin gels, indicating that structural information above 20 nm is largely consistent between the two methods. [ 16 ] Super-resolution light microscopy of the glycocalyx of cell lines and primary human tumor cells enabled 10–20 nm precision in localization of two components of cell surface glycans after subjecting the cells to metabolic labeling and bioorthogonal chemistry. [ 17 ] Ultrastructural TEM imaging of the glycocalyx on mesothelium of visceral organs after high pressure freezing and freeze substitution demonstrated a much thicker extracellular layer than detected with conventional chemical fixation methods. [ 18 ] Despite of these promising results regarding visualization of pectin and glycocalyx as separate entities, their combination in a surgical setting remains challenging for several reasons: 1. The pectin we are using is not an already stabilized gel as used before, [ 14 , 15 ] but a glass-phase film without any additives that will experience a limited hydration in contact with the organ it is placed on. Addition of an aqueous chemical fixative would invariably lead to dissolution of the film. 2. Even freeze substitution of pectin alone after high pressure freezing - which proved very successful for glycocalyx visualization [ 18 ] - did not work in previous experiments. Therefore, the alleged gold standard cryo electron microscopy cannot be easily applied here. In addition, the surgical hybrid model (a plant-based pectin patch applied onto a mammalian visceral organ) cannot be vitrified without many additional sample preparation steps such as the excision of the organ, sample punching for high pressure freezing, cryo FIB-milling and lamella extraction for cryoTEM. [ 19 – 21 ] Ultrastructural visualization of a pectin film in situ , i.e., adhering to a visceral surface thus requires a sample preparation that fulfills both, preservation of the pectin film itself in aqueous media and preservation of the tissue the film is interacting with. To achieve these combined goals, we sought to advance known protocols for biomaterial and tissue preparation facilitating an optimal stabilization of the different material components. To limit the number of animals needed we first established a more straightforward protocol for glass-phase pectin films using a chemical preparation method that allows to keep the films intact by limited hydration during processing for ultrastructural electron microscopy. The validity of this method was tested by analyzing different types of pectin from several plant sources. Finally, we analyzed the adhesion of pectin films to the lung surface at ultrastructural level and revealed entanglement between pectin carbohydrate polymer chains and the glycocalyx of the mesothelial cell layer of visceral organs. Results Stabilization of pectin films in vitro by reducing free water Since pectin films dissolve within a few minutes when brought into contact with aqueous solutions such as buffered fixatives, conventional embedding for ultrastructural analysis is impossible. To define conditions that would prevent dissolution of the pectin film, we systematically tested all solutions used during a typical chemical embedding procedure for their effect on pectin films (Table 1 ). We found that none of the components of the fixatives, applied individually or in different combinations together could prevent pectin dissolution. However, during the dehydration steps in organic solvents (15% − 100% ethanol, acetone) which are necessary before resin embedding, the films remained stable and even lost weight, presumably reducing their water content (above 70% ethanol). Therefore, we decided to add a low percentage of ethanol – the actual amount depending on the type of pectin being investigated (see below) - to the fixatives and all subsequent washing steps to keep pectin films intact. An exemplary workflow is illustrated in Fig. 1 . Starting with a dry film (Fig. 1 A) limited hydration is achieved by combining ethanol, ruthenium red, and conventional aldehyde fixatives in a Pipes buffer. Ruthenium red is known to intercalate into certain binding sites present in pectin, [ 22 ] stabilizing it to some extent but primarily introducing a heavy metal as indicated by the red color in Fig. 1 Bi and Bii. The Ru ions serve as seeds for another heavy metal, Os which is added as aqueous OsO 4 in the next step, leading to a blackening of the film (Fig. 1 C). Together, both metals generate contrast for subsequent imaging in an electron microscope. Dehydration of the film in an ascending ethanol series followed by acetone produces a still partially hydrated, blackened film (Fig. 1 D) which is then either air dried for direct analysis or embedded in epoxide resin for sectioning. For direct analysis in a scanning electron microscope (SEM), the dried film is broken into small pieces to expose a fracture face and mounted on an aluminum stub (Fig. 1 E). Alternatively, ultrathin sections are cut from the embedded sample (Fig. 1 G), placed on a piece of silicon wafer (Fig. 1 H), and also imaged in an SEM. Comparison of the two sample types (Fig. 1 F, I) shows a fibrous network for this particular pectin on both preparation routes. Table 1 Behavior of lemon pectin films after 15 min incubation in solvents used during a typical preparation protocol for EM imaging Pipes GA RR ethanol status pectin HF Water dissolved -/- 0.1 M Pipes x dissolved -/- 2.5% GA x dissolved -/- 25% ethanol x swollen film 4.0/3.4 50% ethanol x swollen film 1.74 70%ethanol x film 1 100% ethanol x shrunk film 0.4 acetone shrunk film 0.9 Combine 2 x 25% swollen film 4.1 Combine 2 x x dissolved -/- Combine 2 x 25% swollen film 6.2 Combine 3 x x x dissolved -/- Combine 3 x x 25% swollen film 6.6 Combine 4 x x x 20% swollen film 8.6 GA stands for glutaraldehyde, RR for Ruthenium Red HF stands for hydration factor, defined by weight(after incubation) / weight(initial) To determine the optimal hydration state for imaging a given type of pectin, here pectin from lemon, we varied the concentration of ethanol in the fixative. Pieces of pectin film were cut to a size sufficiently large to minimize relative measuring errors and weighed at different steps in the course of the embedding protocol. To illustrate the reliability of this gravimetric analysis, fig. S1 shows the dehydration kinetics of lemon pectin stabilized with 15% ethanol from two separate experiments with 10 min or 20 min incubation time for each dehydration step, respectively. All values were normalized to the initial weight of the dry film, plotted are averages ± SD from triplicates for each experiment. During embedding procedures, weights were only obtained after the initial fixation (“hydrated”), after dehydration in ethanol and acetone (“dehydrated”) and after air drying (Fig. 2 A, B). In the presence of 40% ethanol, the maximal hydration achieved was about 5x the weight of the original film, while in 15% ethanol this value rose to 13x. Samples prepared with 40% ethanol in the fixative showed a very dense, layered structure of the fracture face view in the SEM (Fig. 2 Ai). Embedded samples were rather difficult to cut into ultrathin sections, displaying many cracks and tears (arrowheads) in the cross section (Fig. 2 Aii) and showing a very densely packed material at higher magnification (Fig. 2 Aiii). When reducing the ethanol content of the fixative to 15% much smoother sections were produced (Fig. 2 Bi) showing fibrous networks (Fig. 2 Bii). The density of filaments was higher at the edge of the sample (Fig. S2) with slightly different morphologies depending on the nature of the edge: Edges arising from cutting the small piece used for embedding are denoted as “cut” in the macroscopic image (Fig. S2A) and by brackets in the cross section (Fig. 2 Bi). The cut pectin film surface shows a denser packing of filaments, creating a skin effect (Fig. S2 D). The original surface of the film, i.e. the interface to air or plastic dish produced edges in the cross section without a clear skin (Fig. S2 C). In the center of the sample filaments were more loosely packed, leading to comparable densities in the two different radial profiles extending about 100 micrometers from the two types of edges (Fig. S2 E, F and movie_1). The fracture face of lemon pectin prepared with 15% ethanol exposed a more homogeneous aspect in overview (Fig. S3A) and again a fibrous network at higher magnification (Fig. S3Ai), consistent with the findings from the cross section (Fig. S3Aii). Ultrastructural signatures of various types of pectin Having established a reliable workflow for visualizing the ultrastructure of partially hydrated pectin films we investigated films, fabricated from pectin isolated from different plant sources. Optimal hydration had to be defined for each pectin type, as a state in which the film could be carried through the fixation steps without dissolving but at the same time giving an ultrastructure that was not just a compact block of material (as e.g. in Fig. 2Aiii). From all films tested potato pectin required the highest concentration of ethanol (40%) to remain stable, whereas the other pectin types could be stabilized with lower ethanol concentrations of 15% or 20%. A comparison of the fracture faces in Fig. 3 (top row) indicates differences in the structural composition: While orange pectin (Fig. 3 Ai) and soy pectin (Fig. 3 Bi) had a filamentous appearance - with higher porosity of the network for orange (Fig. 3 Ai), the fracture face of potato pectin (Fig. 3 Ci) exposed a more granular globular arrangement of coarser and more solid material. These findings are consistent with images of the corresponding cross sections (Fig. 3 bottom row), suggesting that the interconnected networks of orange and soy pectin were easier to stabilize than the agglomeration of globular particles in the case of potato pectin. Analysis of more films fabricated from pectin or mixtures of pectin with carboxymethylcellulose (CMC), another carbohydrate polymer, confirms these observations as shown in fig. S3 for lemon pectin (Fig. S3A), lemon pectin with CMC (Fig. S3B), sugar beet pectin with CMC (Fig. S3C), and CMC alone (Fig. S3D) as control. In all cases images of the fracture faces (left and central column) are consistent with images obtained from cross sections (right column). Lemon pectin shows a similar fibrillar organization as orange pectin, whereas the other materials appear as a mixture of globular and filamentous components. The globular components (arrowheads) are more obvious in the fracture face images, the filaments (arrows) in the cross sections. Pectin adhesion to lung surface in a surgical setting As shown previously, [ 7 ] pectin films can bind to visceral organs and serve as sealant and potential drug delivery device. So far, the basis for the structural adhesion on the ultrastructural level was not known since visualization of the interface between pectin and organ surfaces had not been achieved yet. It has been hypothesized that a hydration-mediated microstructural entanglement between the carbohydrates of the glycocalyx – an extensive network of carbohydrate filaments on the lung mesothelial cells [ 12 ] - and the lemon pectin chains would mediate the adhesion of the pectin film. With our new workflow for pectin imaging, we set out to test this hypothesis. Small pieces (3 x 3 mm) of lemon pectin film (arrowheads in Fig. 4 A) were placed on the surfaces of liver and lung of a mouse under a lethal dose of anesthetics. Pieces of tissue with adherent pectin patches were excised and immersion fixed in our optimized fixative with 15% ethanol and 0.15% Ruthenium Red added. To demonstrate that the inclusion of 15% ethanol in the fixative did not impair tissue preservation we imaged control tissue taken from the same animal. As shown in fig. S4 mesothelial cells and a few cell layers immediately below them appear darker stained than deeper tissue layers, almost displaying a negative contrast. This is presumably caused by limited diffusion of the highly charged Ruthenium red into the topmost tissue layers but not further as described before [14]. Pneumocytes, specifically type 2 alveolar epithelial cells, also exhibit dense cytoplasm with lamellar bodies (Fig. S5B, C). Secreted surfactant is visualized adjacent to these cells (Fig. S5D). Membrane-bound organelles such as Golgi stacks and endoplasmic reticulum (Fig. S6A), coated vesicles and coated pits (Fig. S6C) were visualized in a cell with less dense cytoplasm (Fig. S6B). Movie_2 shows the different regions of interest described in the context of the entire tissue piece. After embedding and trimming of a tissue sample with attached pectin film, the surface of the block was imaged in a light microscope under epi-illumination conditions (Fig. 4 B). The interface between the pectin patch and the tissue is clearly visible (arrows, see also fig. S7 C, D). Higher resolution images from the regions marked in green show well preserved tissue features of the lung surface with the pectin patch (P), the lungs glycocalyx, the mesothelial cell layer (M), the basement membrane and subpleural alveoli (A) from left to right (Fig. S7D-F) but no discernible details in the area of the pectin material (red rectangle). SEM imaging of the corresponding regions on ultrathin sections (Fig. 4 C-E) reveals one corner of the pectin patch only (Fig. 4 C), and pectin attached to the tissue surface (Fig. 4 D). The tightness of the interaction of pectin with the tissue is illustrated in Fig. 4 E at high magnification, where also the network character of pectin becomes obvious. Due to this very tight entanglement between pectin and tissue observed in these samples it was not possible to distinguish glycocalyx from pectin. As a next step we tried to achieve a less tight interaction by perfusion fixing the sacrificed animal for about 10 min to give the pectin film more time to hydrate before being excised with the underlying piece of tissue. Utilizing such a protocol we could identify regions on the lung surface where parts of the pectin patch had delaminated from the tissue surface (Fig. 5 A, star) but smaller pieces were still attached (Fig. 5 A orange box, B). In such regions at high magnification the glycocalyx could clearly be visualized with spherical microvilli cross sections and fine strands between them and also emanating from the cell surface (Fig. 5 C). To distinguish microvilli cross sections from “glycocalyx globules”, [ 12 ] presumably crossing points of filaments in a projection view, we did a 3D analysis by FIBSEM nanotomography (data in movie_3). 3D reconstruction of an image stack recorded with a voxel size of 5nm from such a region targeted in a 500 nm thick section by darkfield light microscopy (Fig. S8) shows the microvilli (yellow structures in Fig. 5 D) and between them and the cell surface a filamentous material (dark blue in Fig. 5 D). Peripheral filamentous structures, presumably pectin, are annotated in light blue (see also movies_4a-c for different angles of the 3D rendering and segmentation). From data generated by combining the signal from 9 consecutive single images (5 nm apart from each other in z-direction, the sum representing a section of 45 nm thickness) it is obvious that fine strands originating from the cell surface (Fig. 5 E) are physically entangled with pectin chains. Discussion In order to visualize on the nanoscale a possible entanglement of the heteropolysaccharide pectin with polysaccharides of the glycocalyx on lung mesothelial cells, it was paramount to combine the requirements for good ultrastructural tissue preservation with stabilization of pectin through the entire course of the electron microscopy sample preparation. This was achieved by limited hydration which helped to stabilize pectin during the chemical fixation and has not been employed before. The pectin films used here for application on the lung were pure pectin without any additives which would be hydrated by uptake of water from the organ surface. In pectin gels, stabilization for EM imaging was achieved by lowering the pH and adding high concentrations (30% − 50%) of sucrose, as well as complexing ions such as Ca 2+ to the fixatives. [ 14 , 15 ] Although low concentrations of Ca ions have been used successfully in fixatives for EM before, [ 22 ] pH values as low as 3.0 are not desirable for animal tissue fixation since average pH values in mammalian cytoplasm are usually between 7.0 and 7.4. [ 22 ] Furthermore, the addition of sucrose would increase osmolarity of the fixative to such an extent that the tissue would massively loose water, leading to shrinkage artifacts. Seeking another way to limit water uptake into the films we found that the presence of a certain amount of ethanol in the aqueous fixative is limiting hydration of the film which then allowed further processing for ultrastructural electron microscopy. Gravimetric analysis at different time points during embedding revealed that an initial hydration factor (after fixation) between 10 and 15 produced pectin morphologies with well separated fibrillar or globular arrangements – depending on the pectin type. Higher hydration factors led to dissolution of the films, while lower factors resulted in rather densely packed material where no fine structural features were discernible. Even in a hydration regime which just prevented dissolution of the film (15% ethanol for lemon and orange pectin) a frequent observation was that of a hydration gradient with a looser packing of filaments in the centre of the film (cf fig. S2). This skin effect has been observed before on pectin gels embedded for TEM [ 10 ] and was attributed to the presence of Ruthenium Red which we also used as a staining agent. Analysis of lung tissue subjected to this modified embedding procedure – optimized for retention of pectin films - demonstrated well preserved structural features (Fig.s S4-S6) with a high degree of ultrastructural information. As a next step we investigated the interaction of pectin with the glycocalyx on lung mesothelium. Although pectin had been used as surgical sealant before - either single [ 10 – 12 ] or in combination with other polymers [ 9 ] - the structural details of its adherence mechanism to the mesothelium have only been speculative. As for other wound closure systems or for mucoadhesion in general, entanglement models have been proposed, [ 6 – 8 ] assuming an interpenetration of the sealant polymer chains with the carbohydrate chains of the glycocalyx in our case. However, a successful visualization of a postulated interaction complex for mucoadhesion at the ultrastructural level had not been achieved even using cryofixation and freeze substitution of a model system consisting of rat intestinal mucus and a mucoadhesive polymer. [ 23 ] Here we visualize for the first time the postulated interaction complex in both, LM and EM. We show that a pectin film can firmly adhere to the mesothelial surface and survive the lengthy embedding procedure for EM analysis. In darkfield epi-illumination LM, the partially hydrated film appears as milky substance tightly attached to the lung surface. Ultrastructurally, this film shows the typical fibrillar network also observed for isolated lemon pectin. In samples with a short hydration time the attachment is so close that it is not possible to distinguish between the two components of the complex, animal glycocalyx and plant-derived pectin. Extended hydration times produced samples with pectin patches adhering less strongly. Here individual filaments originating from the surface of mesothelial cells and from microvilli could be detected (cf Fig. 5 C, E) intermingling with a fibrillar network that extends several micrometers away from the tissue surface. Although we cannot know the origin of a specific fibril within the putative interaction zone – plant or animal – the morphological convergence is remarkable, especially for the lemon pectin that we chose. Since it is known that after chemical fixation the width of the observable glycocalyx is about 10 times smaller than the 13 µm observed after cryopreparation [ 18 ] we cannot give a reliable measure for the thickness of the transition zone. Analysis by 3D stimulated Raman spectral imaging of the transition zone between pectin and porcine intestinal serosa in fully hydrated, non-fixed state found a thickness of about 15 µm [ 24 ] but could not provide ultrastructural detail. In view of the structural similarity of the glycocalyx with pectin’s fibrous structure described here, the question arises whether pectin as a natural sealant can be considered more than just biocompatible: The carbohydrate-based material might rather be regarded as a bifunctional sealant, which engages in perfect polymer-chain entanglement of its branched carbohydrates with the glycocalyx surface of the target organ, while at the same time presenting a glycocalyx-like surface to other internal organs in the body. Based on emerging findings on the role of the glycocalyx in vascular endothelial and immune functions [ 25 – 27 ] it is tempting to speculate whether the structural homology of the plant-derived material pectin to glycans produced by the body itself will extend to a functional potential regarding signaling or mechanical properties. This will certainly need more research in the future. To conclude: Using the concept of limited hydration by adding a low amount of ethanol during fixation of pure high-methoxy pectin films we found a way to stabilize hydrated films and visualize their ultrastructure. SEM images of pectin films fabricated from several plant sources revealed different ultrastructural signatures for the various films with consistent morphology for each pectin type when comparing fracture faces with cross sections. Putative entanglement of lemon pectin’s carbohydrate chains with the glycocalyx on lung mesothelium was analyzed by single section high resolution imaging and FIBSEM nanotomography of pectin patches adhering to the surface of mouse lung. A dense material strongly attached to the mesothelial cells was visualized by darkfield light microscopy of embedded samples. At the nanoscale, a meshwork of filaments arising from cell surfaces and microvilli was found, extending up to several hundred micrometers above organ surface. These results support the initial hypothesis, that certain types of pectin – here lemon pectin – consist of fibrous material facilitating an entanglement with complementary polymer chains in the glycocalyx of mammalian visceral organs. This entanglement is in fact expected for such homologous glycan-glycan polymeric structures and gives a direct mechanical explanation for the bioadhesion of pectin patches in a surgical setting. This finding might also open new routes for optimization of adhesion and subsequent resorption by chemical modifications focused on improving entanglement and kinetics of swelling and disintegration after wound healing. It will be interesting to test whether our approach of limited hydration is also applicable to rationally designed composite bio-derived hydrogels with advanced stability and toughness. [ 28 – 30 ] Methods Materials and reagents Ruthenium Red (“for microscopy”) and tannic acid (ACS reagent) were purchased from Sigma-Aldrich; PIPES (buffer grade) from AppliCHem PanReac, paraformaldehyde (16% EM grade), glutaraldehyde (25%, EM grade), OsO 4 (crystals for making 5% stock in ethanol), Uranyl acetate, and lead citrate (3% ready to use solution) from Science Services. All components for the Epon mix (42.4 g glycid ether 100, 29.6 g dodecenylsuccinic acid anhydride, 18.4 g methyl-5-norbornene-2,3-dicarboxylic anhydride and 2.4 g benzyldimethylamine as initiator) were bought from Serva. All other materials were reagent grade. Fabrication of pectin films The pectins used in this study were obtained from two commercial sources (Cargill, Minneapolis, MN, USA; Megazyme, Bray, Ireland). They included lemon pectin, orange pectin, potato pectin (Pectic Galactan), soy pectin (Rhamnogalacturonan), and sugar beet pectin (Arabinan). Pectin powder was stored in low humidity at 25°C. Carboxymethylcellulose (CMC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Preparation of the pectin films has been described in detail elsewhere. [ 31 ] Briefly, the pectin powder was dissolved to 3% (w/w) at 25°C by a step-wise addition of deionized water to first induce swelling and softening of the particles before fluidization and full dissolution. To complete dissolution a high-shear 10,000 rpm rotor-stator mixer (L5M-A; Silverson, East Longmeadow, MA) was used, monitoring the viscosity via the instrument’s data logger software. The dissolved pectin was poured into custom molds and cured to glass-phase films. Animals All experiments were conducted in agreement with national and international guidelines. Three twelve weeks old male and female mice, wild type C57BL/6 were anesthetized prior to euthanasia. The care of the animals was consistent with guidelines of the German Animal Welfare Law. Studies were approved by the animal care and use committees of University Hospital Heidelberg under the registration number T-30/23. The animals were euthanized with intraperitoneal injection of 100 mg/kg ketamine hydrochloride with 10 mg/kg xylazine hydrochloride. Mice were placed in supine position, the thoracic and abdominal cavity were opened and the right ventricle cannulated with a 20G catheter, an incision was made to the inferior caval vene to later allow drainage of blood and fixative. The trachea was cannulated using a blunt 20-G cannulas the lung air inflated to 25 cm H 2 O pressure. Small pectin patches were carefully applied to the wet surfaces of visceral organs. The animal was then vascular perfusion fixed via the right ventricle using the primary fixative (1.25% glutaraldehyde, 2% paraformaldehyde, 0.15% Ruthenium red, and 15% ethanol in 0.1M Pipes buffer). Small organ samples containing pectin patches were harvested after 10 min of perfusion fixation and submerged in the same fixative. Gravimetric analysis Pectin films (40-100µm thick) were cut in 4 x 10 mm pieces and weighed. After treatment with the fixatives (see below), i.e. after about 2 h in aqueous solution, the hydrated films were weighed again. Since observation in the SEM samples requires dry samples, the hydrated films were slowly dehydrated through a graded ethanol series and acetone, weighed again and then air dried. For some experiments additional weight measurements were taken after each dehydration step. The weight values were normalized to the initial weight and the obtained “hydration factor” plotted in Excel. Sample preparation for microscopy Pectin films were cut in 2 x 5 mm pieces, sometimes also in a triangular shape, and incubated for 30 min in the primary fixative, consisting of 2.5% glutaraldehyde, 0.15% Ruthenium red, and 15% to 40% (depending on the type of pectin) ethanol in 0.1M Pipes buffer pH 6.8. After washing for 30 min with 20% ethanol in 0.1M Pipes (2–3 solution changes), the secondary fixation was for 1h in 1% OsO 4 , 0.15% Ruthenium red, 20% ethanol in 0.1M Pipes. After washing as above the samples were dehydrated in a graded ethanol series (50%, 70%, 90%, 100%) followed by ethanol/acetone (1:1) and dried acetone twice, each step for 10 min. Then samples were either air-dried or infiltrated with 30% Epon in dried acetone for 1h, 70% Epon in dried acetone for 1h, and 100% Epon overnight, followed by fresh 100% Epon for 4 h. For polymerization (2 d at 65°C) the samples were transferred to silicon molds filled with 100% Epon. For visualization of pectin patches on mouse lung, the protocol was slightly adjusted: The primary fixative was 1.25% glutaraldehyde, 2% paraformaldehyde, 0.15% Ruthenium red, and 15% ethanol in 0.1M Pipes buffer. After the secondary fixative (prepared as above) an additional step was introduced to increase tissue contrast: Overnight incubation with 2% Uranyl acetate in 25% ethanol. Dehydration steps were increased to 15min/step and the initial infiltration steps to 2h/step. Air-dried pieces of pectin films were cut into smaller pieces and mounted with silver paint (Acheson, Plano, Wetzlar, Germany) onto aluminum stubs. Resin-embedded samples were processed further by ultramicrotomy: Sections (70–100 nm for 2D imaging, 500 nm for nanotomography) were produced using a Powertome PC ultramicrotome (RMC Boeckeler, Tucson, USA). For thin sections an Ultra 35° diamond knife was used, for thick sections a HistoJumbo 45° (both Diatome, Biel, Switzerland). Sections were collected on pieces of Si wafer before post-staining with 3% aqueous uranyl acetate followed by 3% aqueous lead citrate. Light and electron microscopy An upright microscope (AxioImager 2, Carl Zeiss Microscopy, Oberkochen, Germany) with epi-illumination and long-distance objectives was used in brightfield or darkfield mode for mapping of embedded sample through the trimmed blockface or of thick sections on pieces of silicon wafer. The software package ZENconnect, (Carl Zeiss Microscopy, Oberkochen, Germany) allowed a seamless transfer of these datasets to the instruments used for ultrastructural analysis, see below. SEM hierarchical imaging at 1.5 keV landing energy with a 20 µm aperture using SE and ESB detectors was performed with the Atlas 5 AT software package on a Ultra55 SEM (Carl Zeiss Microscopy). FIBSEM nanotomography was carried out on a Crossbeam 540 (Carl Zeiss Microscopy) at 2 keV landing energy, 1.0 nA beam current with an isotropic voxel size of 5 nm in x, y, and z. FIB milling was performed at 30 kV with an ion beam current of 700 pA. The ZENconnect LM dataset was imported into the Atlas 3D software package for targeting of relevant pre-selected regions. InLens and ESB (1000 V grid) detectors were used simultaneously for image recording. Image analysis For registration of nanotomography stacks the TrakEM module of the open-source software Fiji was used. [ 32 ] Registered substacks were annotated manually in Fiji [ 33 ] and visualized in Chimera. [ 34 ] All other operations on images (merging of slices by the command z-projection (from stack operations) using “sum slices”), filtering etc. were also done in Fiji. Declarations Contributions S.J.M. and W.W. posed the initial question how to visualize the ultrastructure of a hydrated pectin film. The concept of limited hydration was explored and established by I.W. Pectin preparation J.M.P., mouse handling and surgical experiments W.W., embedding and sectioning of samples R.C., LM and SEM imaging R.C. & I.W., FIBSEM nanotomography E.M. Visualization in 3D and movies by I.W. & R.R.S. The project was administered by S.J.M. and R.R.S., I.W. and R.R.S. drafted the manuscript and all authors reviewed and edited text and figures. Ethics declarations The authors declare no competing interests. Acknowledgements I.W., R.C., and R.R.S. acknowledge key funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy for the Excellence Cluster ‘3D Matter Made to Order’ (EXC-2082/1–390761711). I.W., R.C. and R.R.S. also acknowledge funding by the German Ministry for Research and Education under the Project ‘NanoPatho’, FKZ 13N14476, as well as support by the University Computing Centre of the University Heidelberg regarding Research Data Management/ELN and the data storage service SDS@hd supported by the Ministry of Science, Research and the Arts Baden-Württemberg (MWK) and the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. Data Availability All data will be made available via the data repository heiDATA under a DOI which will be assigned after acceptance of the paper. 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Supplementary Files WackerSI20250731.docx Bioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement WackerMovie1.mp4 Hydration gradient of lemon pectin – fly-in: overview to detail Wackermovie2.mp4 Wackermovie3.mp4 FIBSEM nanotomography data - fly-through Wackermovie4a.mp4 Annotated FIBSEM data – 3D visualization from different angles Wackermovie4b.mp4 Annotated FIBSEM data – 3D visualization from different angles Wackermovie4c.mp4 Annotated FIBSEM data – 3D visualization from different angles Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7619875","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515273217,"identity":"9d4a303c-92fb-47ca-98fa-e4d045438844","order_by":0,"name":"Irene Wacker","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-2993-5665","institution":"BioQuant, Universität Heidelberg, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany","correspondingAuthor":true,"prefix":"","firstName":"Irene","middleName":"","lastName":"Wacker","suffix":""},{"id":515273218,"identity":"4c9d841a-763b-4c50-9f3d-5e1ccb4feddb","order_by":1,"name":"Willi Wagner","email":"","orcid":"","institution":"Diagnostic Radiology and Neuroradiology, Greifswald University Hospital, Ferdinand-Sauerbruch-Strasse 1, Greifswald, Germany","correspondingAuthor":false,"prefix":"","firstName":"Willi","middleName":"","lastName":"Wagner","suffix":""},{"id":515273219,"identity":"1385c597-df24-4fff-b8c9-6629b43630e0","order_by":2,"name":"Ronald Curticean","email":"","orcid":"","institution":"BioQuant, Universität Heidelberg, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Curticean","suffix":""},{"id":515273220,"identity":"75c42eb1-ae7d-4953-9b4d-02495ea73439","order_by":3,"name":"Endre Majorovits","email":"","orcid":"","institution":"Carl Zeiss Microscopy GmbH, Carl-Zeiss Str. 22, D-73447 Oberkochen, Germany","correspondingAuthor":false,"prefix":"","firstName":"Endre","middleName":"","lastName":"Majorovits","suffix":""},{"id":515273221,"identity":"e4cb48de-7cc6-4d10-a1b3-b9fb09f6ea08","order_by":4,"name":"Jennifer M. Pan","email":"","orcid":"","institution":"Laboratory of Adaptive and Regenerative Biology, Brigham \u0026 Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"M.","lastName":"Pan","suffix":""},{"id":515273222,"identity":"faf3f271-eb72-4064-bda1-2b50c8ab9626","order_by":5,"name":"Steven J. Mentzer","email":"","orcid":"https://orcid.org/0000-0003-1496-6044","institution":"Laboratory of Adaptive and Regenerative Biology, Brigham \u0026 Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"J.","lastName":"Mentzer","suffix":""},{"id":515273223,"identity":"3a7d99a7-6e6f-4730-9dd5-aa95acd3f35b","order_by":6,"name":"Rasmus R. 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To introduce contrast and conductivity for SEM imaging, OsO\u003csub\u003e4\u003c/sub\u003e is used (C). After dehydration by an ascending ethanol series followed by acetone (D) samples are then either air-dried (E) or embedded in epoxy resin (G). Fracture faces of air dried samples mounted on aluminium stubs (E) are directly imaged in an SEM (F), while resin-embedded samples are cut into ultrathin sections and placed on pieces of silicon wafer (H) before SEM imaging (I).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/a901884e9cdb2f999c092237.jpeg"},{"id":91515655,"identity":"b1dec6fd-1ac7-424e-8129-b17fb1b069c3","added_by":"auto","created_at":"2025-09-17 09:15:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":544593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetermination of optimal hydration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePectin films (lemon) were either hydrated in the presence of 40% (top row) or 15% (bottom row) ethanol. The block diagrams show the degree of hydration (weight normalized to the original dry weight) at different steps during preparation for SEM imaging. Maximal hydration occurs after fixation (first bar), decreases during dehydration in organic solvents (middle bar) and reaches the original value after air drying (right bar). Films hydrated in the presence of high ethanol concentration show a layered appearance, both as fracture faces (Ai) and in cross sections (Aii) with very dense material packing visualized at high magnification (Aiii). Note the high number of cracks (arrowheads) in the cross section. At low ethanol concentration the material is more isotropically packed (Bi), presenting a filamentous network (Bii). Sample edges inside brackets are cut faces, outside brackets are original interfaces (to air/dish respectively). For more details see Figures S2, S3 and movie_1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/1dc47623392372257ac20206.jpeg"},{"id":91515654,"identity":"bf99a08f-2b12-4c0f-9ac9-4664f05a54ad","added_by":"auto","created_at":"2025-09-17 09:15:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":821204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePectins from different plants show different morphologies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFracture faces from air-dried pectin films stained with ruthenium red and OsO\u003csub\u003e4\u003c/sub\u003e (top row) and ultrathin cross sections from films embedded in epoxide resin after staining (bottom row) of pectin isolated from orange (A), soy (B), or potato (C). Scale bars are 1 mm (top) or 400 nm (bottom).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/823aadb14b34b0373d4328d2.jpeg"},{"id":91516111,"identity":"ca582de4-5c02-4aa3-812c-4cb6b90d0330","added_by":"auto","created_at":"2025-09-17 09:23:01","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":589311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePectin attachment to visceral organ surface\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Pectin patches (arrowheads) placed on murine lung and liver. (B) After embedding and trimming of the block, the interface (arrows) between pectin patch (P) and lung tissue (L) is clearly visible on the blockface (epi-illumination light microscopy) in bright field mode of entire blockface combined with darkfield mode for selected regions of lung tissue (green rectangles, overlays in software ZEN connect, see also Figure S7). C) to E) SEM images of sections showing edge of pectin patch (C), pectin–lung interface at intermediate (D) and high (E) magnification. P – Pectin, M – Mesothelial cell layer, A – Pulmonary alveoli\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/8bdaa958f2f1882980e31b36.jpeg"},{"id":91515660,"identity":"ea712488-86c4-4918-96ac-b2e6fcfd0663","added_by":"auto","created_at":"2025-09-17 09:15:01","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":499579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEntanglement of pectin with glycocalyx\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCross section of pectin patches (P) at lung (L) surface. (A) overview, star indicates delaminated pectin patch, (B) detail with tightly attached pectin patch, (C) high resolution image showing glycocalyx filaments emanating from cell surface. (D) 3D rendering of a FIBSEM nanotomography stack (recorded with 5 nm voxel size, see also movie_3) from a 500 nm thick section. Lung surface represented in orange, microvilli in yellow, fibrils close to surface in blue, peripheral fibrils in light blue. See also movies _4a-c for better impression of 3D data. (E) Sum over 9 slices (45 nm in z-direction) showing microvilli cross sections (arrowheads) and thin filaments arising from the lung surface.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/298097ddf6ef73e6f693330c.jpeg"},{"id":91517203,"identity":"26f409d8-9b84-42ed-9c78-0b08e0feb860","added_by":"auto","created_at":"2025-09-17 09:31:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3487647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/2d3f2623-2b7b-4322-a975-c57d1a343c02.pdf"},{"id":91515670,"identity":"e88a242d-2e04-417e-896e-3d3b6179a252","added_by":"auto","created_at":"2025-09-17 09:15:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6914168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"WackerSI20250731.docx","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/b39ca8f19031ebe2911c04b5.docx"},{"id":91515674,"identity":"ab2885c1-8409-4fb4-927c-e97baea06cc8","added_by":"auto","created_at":"2025-09-17 09:15:01","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29356526,"visible":true,"origin":"","legend":"\u003cp\u003eHydration gradient of lemon pectin – fly-in: overview to detail\u003c/p\u003e","description":"","filename":"WackerMovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/7487a4d128b7347ea305a868.mp4"},{"id":91515675,"identity":"ba059013-0431-428a-a529-29905a4c452b","added_by":"auto","created_at":"2025-09-17 09:15:02","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27581002,"visible":true,"origin":"","legend":"","description":"","filename":"Wackermovie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/7a27b456b043fa9014c8d622.mp4"},{"id":91516112,"identity":"66ba2451-9e96-4314-983a-a52a64f307a2","added_by":"auto","created_at":"2025-09-17 09:23:01","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4703645,"visible":true,"origin":"","legend":"\u003cp\u003eFIBSEM nanotomography data - fly-through\u003c/p\u003e","description":"","filename":"Wackermovie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/4fc84e5ceb07e0933e7c9659.mp4"},{"id":91516116,"identity":"8b59a99f-fd18-44ca-a37a-064858e95f99","added_by":"auto","created_at":"2025-09-17 09:23:01","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3535789,"visible":true,"origin":"","legend":"\u003cp\u003eAnnotated FIBSEM data \u0026nbsp;– 3D visualization from different angles\u003c/p\u003e","description":"","filename":"Wackermovie4a.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/7406c41d6880502b21d7eb77.mp4"},{"id":91515663,"identity":"a7894a4f-7301-419a-91c6-70789e125f43","added_by":"auto","created_at":"2025-09-17 09:15:01","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3472507,"visible":true,"origin":"","legend":"\u003cp\u003eAnnotated FIBSEM data \u0026nbsp;– 3D visualization from different angles\u003c/p\u003e","description":"","filename":"Wackermovie4b.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/7ce07ce97de3a52a9b943f1c.mp4"},{"id":91516113,"identity":"4f95afc8-49b8-4288-b3e5-8919cdc235e0","added_by":"auto","created_at":"2025-09-17 09:23:01","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1737413,"visible":true,"origin":"","legend":"\u003cp\u003eAnnotated FIBSEM data \u0026nbsp;– 3D visualization from different angles\u003c/p\u003e","description":"","filename":"Wackermovie4c.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7619875/v1/0a02a15dde694c949d6ff240.mp4"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUsage of carbohydrate-based biopolymers such as cellulose,\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e chitosan,\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e or pectin\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e in a biomedical context has a longstanding tradition. Applications range from wound dressing to drug delivery, mediated by bioadhesion,\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e and more specifically by mucoadhesion in the latter case.\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn particular the chemical properties of pectin, a complex, branched carbohydrate, and its bio-adhesiveness make it an interesting, biocompatible candidate as e. g. inert carrier for drug delivery.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e Even more interestingly, the usage of pectin films as potential sealants in surgery has been explored in the work of Servais \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e where interaction of a variety of pectin formulations with the mesothelium of internal organs was investigated.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e In a murine surgical model setting it was found that pectin film exhibits a strong bioadhesion e.g. to lung pleura and could limit air leaks after pleural injury,\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e comparing favorably against commercially available surgical products.\u003c/p\u003e\u003cp\u003eWhen adhesion of the sealant to the pleura was inhibited by desiccation or removal of the glycocalyx from the pleura by enzyme treatment, sealant failure was observed.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e This indicates a potential interaction mechanism by an initial swelling of the dry film when applied to the pleura allowing entanglement of pectin\u0026rsquo;s carbohydrate chains with the sugar chains on the glycoproteins and glycolipids of the glycocalyx covering the mesothelial cells. The mechanism could therefore be based on a simple polymer-typical inter-chain entanglement, which \u0026ndash; if verified \u0026ndash; can be optimized further by future chemical modifications of the pectin material.\u003c/p\u003e\u003cp\u003eTo prove the hypothesis that adhesion is caused by entanglement of pectin carbohydrate chains with the extracellular glycocalyx on visceral organs the interaction of the different polysaccharides needs to be shown directly, preferably in an \u003cem\u003ein vivo/in situ\u003c/em\u003e surgical setting.\u003c/p\u003e\u003cp\u003eSeparate visualization of the individual components of the proposed interaction complex \u0026ndash; pectin on one side and the glycocalyx on top of the mesothelial cell layer on the other side \u0026ndash; has been achieved previously in \u003cem\u003eex situ\u003c/em\u003e, non-surgical experiments in different ways: The nanostructure of high methoxyl sugar acid pectin gels analyzed by AFM exposed curvilinear strands with junctions and branch points.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e TEM analysis of pure and mixed high methoxyl and low methoxyl pectin gels with different chemical modifications and varying concentrations of the gelling agents Ca\u003csup\u003e2+\u003c/sup\u003e, sucrose and citrate, embedded in resin and sectioned before imaging, revealed highly porous networks of thin filaments.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Similar networks were shown in a study comparing TEM data from resin embedded pectin gels with SAXS measurements on native pectin gels, indicating that structural information above 20 nm is largely consistent between the two methods.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e Super-resolution light microscopy of the glycocalyx of cell lines and primary human tumor cells enabled 10\u0026ndash;20 nm precision in localization of two components of cell surface glycans after subjecting the cells to metabolic labeling and bioorthogonal chemistry.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Ultrastructural TEM imaging of the glycocalyx on mesothelium of visceral organs after high pressure freezing and freeze substitution demonstrated a much thicker extracellular layer than detected with conventional chemical fixation methods.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDespite of these promising results regarding visualization of pectin and glycocalyx as separate entities, their combination in a surgical setting remains challenging for several reasons: 1. The pectin we are using is not an already stabilized gel as used before,\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e but a glass-phase film without any additives that will experience a limited hydration in contact with the organ it is placed on. Addition of an aqueous chemical fixative would invariably lead to dissolution of the film. 2. Even freeze substitution of pectin alone after high pressure freezing - which proved very successful for glycocalyx visualization\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e - did not work in previous experiments. Therefore, the alleged gold standard cryo electron microscopy cannot be easily applied here. In addition, the surgical hybrid model (a plant-based pectin patch applied onto a mammalian visceral organ) cannot be vitrified without many additional sample preparation steps such as the excision of the organ, sample punching for high pressure freezing, cryo FIB-milling and lamella extraction for cryoTEM.\u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eUltrastructural visualization of a pectin film \u003cem\u003ein situ\u003c/em\u003e, i.e., adhering to a visceral surface thus requires a sample preparation that fulfills both, preservation of the pectin film itself in aqueous media and preservation of the tissue the film is interacting with. To achieve these combined goals, we sought to advance known protocols for biomaterial and tissue preparation facilitating an optimal stabilization of the different material components. To limit the number of animals needed we first established a more straightforward protocol for glass-phase pectin films using a chemical preparation method that allows to keep the films intact by limited hydration during processing for ultrastructural electron microscopy. The validity of this method was tested by analyzing different types of pectin from several plant sources. Finally, we analyzed the adhesion of pectin films to the lung surface at ultrastructural level and revealed entanglement between pectin carbohydrate polymer chains and the glycocalyx of the mesothelial cell layer of visceral organs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eStabilization of pectin films in vitro by reducing free water\u003c/p\u003e\n\u003cp\u003eSince pectin films dissolve within a few minutes when brought into contact with aqueous solutions such as buffered fixatives, conventional embedding for ultrastructural analysis is impossible. To define conditions that would prevent dissolution of the pectin film, we systematically tested all solutions used during a typical chemical embedding procedure for their effect on pectin films (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that none of the components of the fixatives, applied individually or in different combinations together could prevent pectin dissolution. However, during the dehydration steps in organic solvents (15% \u0026minus;\u0026thinsp;100% ethanol, acetone) which are necessary before resin embedding, the films remained stable and even lost weight, presumably reducing their water content (above 70% ethanol). Therefore, we decided to add a low percentage of ethanol \u0026ndash; the actual amount depending on the type of pectin being investigated (see below) - to the fixatives and all subsequent washing steps to keep pectin films intact. An exemplary workflow is illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Starting with a dry film (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) limited hydration is achieved by combining ethanol, ruthenium red, and conventional aldehyde fixatives in a Pipes buffer. Ruthenium red is known to intercalate into certain binding sites present in pectin,\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e stabilizing it to some extent but primarily introducing a heavy metal as indicated by the red color in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eBi and Bii. The Ru ions serve as seeds for another heavy metal, Os which is added as aqueous OsO\u003csub\u003e4\u003c/sub\u003e in the next step, leading to a blackening of the film (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Together, both metals generate contrast for subsequent imaging in an electron microscope. Dehydration of the film in an ascending ethanol series followed by acetone produces a still partially hydrated, blackened film (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD) which is then either air dried for direct analysis or embedded in epoxide resin for sectioning. For direct analysis in a scanning electron microscope (SEM), the dried film is broken into small pieces to expose a fracture face and mounted on an aluminum stub (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Alternatively, ultrathin sections are cut from the embedded sample (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG), placed on a piece of silicon wafer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH), and also imaged in an SEM. Comparison of the two sample types (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF, I) shows a fibrous network for this particular pectin on both preparation routes.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eBehavior of lemon pectin films after 15 min incubation in solvents used during a typical preparation protocol for EM imaging\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eethanol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003estatus pectin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHF\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edissolved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1 M Pipes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edissolved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.5% GA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edissolved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e25% ethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0/3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e50% ethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%ethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100% ethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eshrunk film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eacetone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eshrunk film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edissolved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edissolved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCombine 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswollen film\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eGA stands for glutaraldehyde, RR for Ruthenium Red\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eHF stands for hydration factor, defined by weight(after incubation) / weight(initial)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the optimal hydration state for imaging a given type of pectin, here pectin from lemon, we varied the concentration of ethanol in the fixative. Pieces of pectin film were cut to a size sufficiently large to minimize relative measuring errors and weighed at different steps in the course of the embedding protocol. To illustrate the reliability of this gravimetric analysis, fig. S1 shows the dehydration kinetics of lemon pectin stabilized with 15% ethanol from two separate experiments with 10 min or 20 min incubation time for each dehydration step, respectively. All values were normalized to the initial weight of the dry film, plotted are averages\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from triplicates for each experiment.\u003c/p\u003e\n\u003cp\u003eDuring embedding procedures, weights were only obtained after the initial fixation (\u0026ldquo;hydrated\u0026rdquo;), after dehydration in ethanol and acetone (\u0026ldquo;dehydrated\u0026rdquo;) and after air drying (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). In the presence of 40% ethanol, the maximal hydration achieved was about 5x the weight of the original film, while in 15% ethanol this value rose to 13x. Samples prepared with 40% ethanol in the fixative showed a very dense, layered structure of the fracture face view in the SEM (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eAi). Embedded samples were rather difficult to cut into ultrathin sections, displaying many cracks and tears (arrowheads) in the cross section (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eAii) and showing a very densely packed material at higher magnification (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eAiii). When reducing the ethanol content of the fixative to 15% much smoother sections were produced (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eBi) showing fibrous networks (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eBii). The density of filaments was higher at the edge of the sample (Fig. S2) with slightly different morphologies depending on the nature of the edge: Edges arising from cutting the small piece used for embedding are denoted as \u0026ldquo;cut\u0026rdquo; in the macroscopic image (Fig. S2A) and by brackets in the cross section (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e Bi). The cut pectin film surface shows a denser packing of filaments, creating a skin effect (Fig. S2 D). The original surface of the film, i.e. the interface to air or plastic dish produced edges in the cross section without a clear skin (Fig. S2 C). In the center of the sample filaments were more loosely packed, leading to comparable densities in the two different radial profiles extending about 100 micrometers from the two types of edges (Fig. S2 E, F and movie_1). The fracture face of lemon pectin prepared with 15% ethanol exposed a more homogeneous aspect in overview (Fig. S3A) and again a fibrous network at higher magnification (Fig. S3Ai), consistent with the findings from the cross section (Fig. S3Aii).\u003c/p\u003e\n\u003cp\u003eUltrastructural signatures of various types of pectin\u003c/p\u003e\n\u003cp\u003eHaving established a reliable workflow for visualizing the ultrastructure of partially hydrated pectin films we investigated films, fabricated from pectin isolated from different plant sources. Optimal hydration had to be defined for each pectin type, as a state in which the film could be carried through the fixation steps without dissolving but at the same time giving an ultrastructure that was not just a compact block of material (as e.g. in Fig.\u0026nbsp;2Aiii). From all films tested potato pectin required the highest concentration of ethanol (40%) to remain stable, whereas the other pectin types could be stabilized with lower ethanol concentrations of 15% or 20%.\u003c/p\u003e\n\u003cp\u003eA comparison of the fracture faces in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (top row) indicates differences in the structural composition: While orange pectin (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e Ai) and soy pectin (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e Bi) had a filamentous appearance - with higher porosity of the network for orange (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eAi), the fracture face of potato pectin (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e Ci) exposed a more granular globular arrangement of coarser and more solid material.\u003c/p\u003e\n\u003cp\u003eThese findings are consistent with images of the corresponding cross sections (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e bottom row), suggesting that the interconnected networks of orange and soy pectin were easier to stabilize than the agglomeration of globular particles in the case of potato pectin. Analysis of more films fabricated from pectin or mixtures of pectin with carboxymethylcellulose (CMC), another carbohydrate polymer, confirms these observations as shown in fig. S3 for lemon pectin (Fig. S3A), lemon pectin with CMC (Fig. S3B), sugar beet pectin with CMC (Fig. S3C), and CMC alone (Fig. S3D) as control. In all cases images of the fracture faces (left and central column) are consistent with images obtained from cross sections (right column). Lemon pectin shows a similar fibrillar organization as orange pectin, whereas the other materials appear as a mixture of globular and filamentous components. The globular components (arrowheads) are more obvious in the fracture face images, the filaments (arrows) in the cross sections.\u003c/p\u003e\n\u003cp\u003ePectin adhesion to lung surface in a surgical setting\u003c/p\u003e\n\u003cp\u003eAs shown previously,\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e pectin films can bind to visceral organs and serve as sealant and potential drug delivery device. So far, the basis for the structural adhesion on the ultrastructural level was not known since visualization of the interface between pectin and organ surfaces had not been achieved yet. It has been hypothesized that a hydration-mediated microstructural entanglement between the carbohydrates of the glycocalyx \u0026ndash; an extensive network of carbohydrate filaments on the lung mesothelial cells\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e - and the lemon pectin chains would mediate the adhesion of the pectin film.\u003c/p\u003e\n\u003cp\u003eWith our new workflow for pectin imaging, we set out to test this hypothesis. Small pieces (3 x 3 mm) of lemon pectin film (arrowheads in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) were placed on the surfaces of liver and lung of a mouse under a lethal dose of anesthetics. Pieces of tissue with adherent pectin patches were excised and immersion fixed in our optimized fixative with 15% ethanol and 0.15% Ruthenium Red added. To demonstrate that the inclusion of 15% ethanol in the fixative did not impair tissue preservation we imaged control tissue taken from the same animal. As shown in fig. S4 mesothelial cells and a few cell layers immediately below them appear darker stained than deeper tissue layers, almost displaying a negative contrast. This is presumably caused by limited diffusion of the highly charged Ruthenium red into the topmost tissue layers but not further as described before [14]. Pneumocytes, specifically type 2 alveolar epithelial cells, also exhibit dense cytoplasm with lamellar bodies (Fig. S5B, C). Secreted surfactant is visualized adjacent to these cells (Fig. S5D). Membrane-bound organelles such as Golgi stacks and endoplasmic reticulum (Fig. S6A), coated vesicles and coated pits (Fig. S6C) were visualized in a cell with less dense cytoplasm (Fig. S6B). Movie_2 shows the different regions of interest described in the context of the entire tissue piece.\u003c/p\u003e\n\u003cp\u003eAfter embedding and trimming of a tissue sample with attached pectin film, the surface of the block was imaged in a light microscope under epi-illumination conditions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The interface between the pectin patch and the tissue is clearly visible (arrows, see also fig. S7 C, D). Higher resolution images from the regions marked in green show well preserved tissue features of the lung surface with the pectin patch (P), the lungs glycocalyx, the mesothelial cell layer (M), the basement membrane and subpleural alveoli (A) from left to right (Fig. S7D-F) but no discernible details in the area of the pectin material (red rectangle). SEM imaging of the corresponding regions on ultrathin sections (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-E) reveals one corner of the pectin patch only (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), and pectin attached to the tissue surface (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The tightness of the interaction of pectin with the tissue is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE at high magnification, where also the network character of pectin becomes obvious. Due to this very tight entanglement between pectin and tissue observed in these samples it was not possible to distinguish glycocalyx from pectin.\u003c/p\u003e\n\u003cp\u003eAs a next step we tried to achieve a less tight interaction by perfusion fixing the sacrificed animal for about 10 min to give the pectin film more time to hydrate before being excised with the underlying piece of tissue. Utilizing such a protocol we could identify regions on the lung surface where parts of the pectin patch had delaminated from the tissue surface (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, star) but smaller pieces were still attached (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA orange box, B). In such regions at high magnification the glycocalyx could clearly be visualized with spherical microvilli cross sections and fine strands between them and also emanating from the cell surface (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). To distinguish microvilli cross sections from \u0026ldquo;glycocalyx globules\u0026rdquo;,\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e presumably crossing points of filaments in a projection view, we did a 3D analysis by FIBSEM nanotomography (data in movie_3). 3D reconstruction of an image stack recorded with a voxel size of 5nm from such a region targeted in a 500 nm thick section by darkfield light microscopy (Fig. S8) shows the microvilli (yellow structures in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) and between them and the cell surface a filamentous material (dark blue in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Peripheral filamentous structures, presumably pectin, are annotated in light blue (see also movies_4a-c for different angles of the 3D rendering and segmentation). From data generated by combining the signal from 9 consecutive single images (5 nm apart from each other in z-direction, the sum representing a section of 45 nm thickness) it is obvious that fine strands originating from the cell surface (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE) are physically entangled with pectin chains.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn order to visualize on the nanoscale a possible entanglement of the heteropolysaccharide pectin with polysaccharides of the glycocalyx on lung mesothelial cells, it was paramount to combine the requirements for good ultrastructural tissue preservation with stabilization of pectin through the entire course of the electron microscopy sample preparation. This was achieved by limited hydration which helped to stabilize pectin during the chemical fixation and has not been employed before.\u003c/p\u003e\u003cp\u003eThe pectin films used here for application on the lung were pure pectin without any additives which would be hydrated by uptake of water from the organ surface. In pectin gels, stabilization for EM imaging was achieved by lowering the pH and adding high concentrations (30% \u0026minus;\u0026thinsp;50%) of sucrose, as well as complexing ions such as Ca\u003csup\u003e2+\u003c/sup\u003e to the fixatives.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Although low concentrations of Ca ions have been used successfully in fixatives for EM before,\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e pH values as low as 3.0 are not desirable for animal tissue fixation since average pH values in mammalian cytoplasm are usually between 7.0 and 7.4. \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e Furthermore, the addition of sucrose would increase osmolarity of the fixative to such an extent that the tissue would massively loose water, leading to shrinkage artifacts. Seeking another way to limit water uptake into the films we found that the presence of a certain amount of ethanol in the aqueous fixative is limiting hydration of the film which then allowed further processing for ultrastructural electron microscopy. Gravimetric analysis at different time points during embedding revealed that an initial hydration factor (after fixation) between 10 and 15 produced pectin morphologies with well separated fibrillar or globular arrangements \u0026ndash; depending on the pectin type. Higher hydration factors led to dissolution of the films, while lower factors resulted in rather densely packed material where no fine structural features were discernible. Even in a hydration regime which just prevented dissolution of the film (15% ethanol for lemon and orange pectin) a frequent observation was that of a hydration gradient with a looser packing of filaments in the centre of the film (cf fig. S2). This skin effect has been observed before on pectin gels embedded for TEM\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e and was attributed to the presence of Ruthenium Red which we also used as a staining agent.\u003c/p\u003e\u003cp\u003eAnalysis of lung tissue subjected to this modified embedding procedure \u0026ndash; optimized for retention of pectin films - demonstrated well preserved structural features (Fig.s S4-S6) with a high degree of ultrastructural information.\u003c/p\u003e\u003cp\u003eAs a next step we investigated the interaction of pectin with the glycocalyx on lung mesothelium. Although pectin had been used as surgical sealant before - either single\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e or in combination with other polymers \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e - the structural details of its adherence mechanism to the mesothelium have only been speculative. As for other wound closure systems or for mucoadhesion in general, entanglement models have been proposed,\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e assuming an interpenetration of the sealant polymer chains with the carbohydrate chains of the glycocalyx in our case. However, a successful visualization of a postulated interaction complex for mucoadhesion at the ultrastructural level had not been achieved even using cryofixation and freeze substitution of a model system consisting of rat intestinal mucus and a mucoadhesive polymer.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e Here we visualize for the first time the postulated interaction complex in both, LM and EM. We show that a pectin film can firmly adhere to the mesothelial surface and survive the lengthy embedding procedure for EM analysis. In darkfield epi-illumination LM, the partially hydrated film appears as milky substance tightly attached to the lung surface. Ultrastructurally, this film shows the typical fibrillar network also observed for isolated lemon pectin. In samples with a short hydration time the attachment is so close that it is not possible to distinguish between the two components of the complex, animal glycocalyx and plant-derived pectin. Extended hydration times produced samples with pectin patches adhering less strongly. Here individual filaments originating from the surface of mesothelial cells and from microvilli could be detected (cf Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, E) intermingling with a fibrillar network that extends several micrometers away from the tissue surface.\u003c/p\u003e\u003cp\u003eAlthough we cannot know the origin of a specific fibril within the putative interaction zone \u0026ndash; plant or animal \u0026ndash; the morphological convergence is remarkable, especially for the lemon pectin that we chose. Since it is known that after chemical fixation the width of the observable glycocalyx is about 10 times smaller than the 13 \u0026micro;m observed after cryopreparation \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e we cannot give a reliable measure for the thickness of the transition zone. Analysis by 3D stimulated Raman spectral imaging of the transition zone between pectin and porcine intestinal serosa in fully hydrated, non-fixed state found a thickness of about 15 \u0026micro;m \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e but could not provide ultrastructural detail.\u003c/p\u003e\u003cp\u003eIn view of the structural similarity of the glycocalyx with pectin\u0026rsquo;s fibrous structure described here, the question arises whether pectin as a natural sealant can be considered more than just biocompatible: The carbohydrate-based material might rather be regarded as a bifunctional sealant, which engages in perfect polymer-chain entanglement of its branched carbohydrates with the glycocalyx surface of the target organ, while at the same time presenting a glycocalyx-like surface to other internal organs in the body. Based on emerging findings on the role of the glycocalyx in vascular endothelial and immune functions \u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e it is tempting to speculate whether the structural homology of the plant-derived material pectin to glycans produced by the body itself will extend to a functional potential regarding signaling or mechanical properties. This will certainly need more research in the future.\u003c/p\u003e\u003cp\u003eTo conclude: Using the concept of limited hydration by adding a low amount of ethanol during fixation of pure high-methoxy pectin films we found a way to stabilize hydrated films and visualize their ultrastructure. SEM images of pectin films fabricated from several plant sources revealed different ultrastructural signatures for the various films with consistent morphology for each pectin type when comparing fracture faces with cross sections. Putative entanglement of lemon pectin\u0026rsquo;s carbohydrate chains with the glycocalyx on lung mesothelium was analyzed by single section high resolution imaging and FIBSEM nanotomography of pectin patches adhering to the surface of mouse lung. A dense material strongly attached to the mesothelial cells was visualized by darkfield light microscopy of embedded samples. At the nanoscale, a meshwork of filaments arising from cell surfaces and microvilli was found, extending up to several hundred micrometers above organ surface.\u003c/p\u003e\u003cp\u003eThese results support the initial hypothesis, that certain types of pectin \u0026ndash; here lemon pectin \u0026ndash; consist of fibrous material facilitating an entanglement with complementary polymer chains in the glycocalyx of mammalian visceral organs. This entanglement is in fact expected for such homologous glycan-glycan polymeric structures and gives a direct mechanical explanation for the bioadhesion of pectin patches in a surgical setting. This finding might also open new routes for optimization of adhesion and subsequent resorption by chemical modifications focused on improving entanglement and kinetics of swelling and disintegration after wound healing. It will be interesting to test whether our approach of limited hydration is also applicable to rationally designed composite bio-derived hydrogels with advanced stability and toughness.\u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMaterials and reagents\u003c/p\u003e\u003cp\u003eRuthenium Red (\u0026ldquo;for microscopy\u0026rdquo;) and tannic acid (ACS reagent) were purchased from Sigma-Aldrich; PIPES (buffer grade) from AppliCHem PanReac, paraformaldehyde (16% EM grade), glutaraldehyde (25%, EM grade), OsO\u003csub\u003e4\u003c/sub\u003e (crystals for making 5% stock in ethanol), Uranyl acetate, and lead citrate (3% ready to use solution) from Science Services. All components for the Epon mix (42.4 g glycid ether 100, 29.6 g dodecenylsuccinic acid anhydride, 18.4 g methyl-5-norbornene-2,3-dicarboxylic anhydride and 2.4 g benzyldimethylamine as initiator) were bought from Serva. All other materials were reagent grade.\u003c/p\u003e\u003cp\u003eFabrication of pectin films\u003c/p\u003e\u003cp\u003eThe pectins used in this study were obtained from two commercial sources (Cargill, Minneapolis, MN, USA; Megazyme, Bray, Ireland). They included lemon pectin, orange pectin, potato pectin (Pectic Galactan), soy pectin (Rhamnogalacturonan), and sugar beet pectin (Arabinan). Pectin powder was stored in low humidity at 25\u0026deg;C. Carboxymethylcellulose (CMC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Preparation of the pectin films has been described in detail elsewhere.\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e Briefly, the pectin powder was dissolved to 3% (w/w) at 25\u0026deg;C by a step-wise addition of deionized water to first induce swelling and softening of the particles before fluidization and full dissolution. To complete dissolution a high-shear 10,000 rpm rotor-stator mixer (L5M-A; Silverson, East Longmeadow, MA) was used, monitoring the viscosity via the instrument\u0026rsquo;s data logger software. The dissolved pectin was poured into custom molds and cured to glass-phase films.\u003c/p\u003e\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003e All experiments were conducted in agreement with national and international guidelines. Three twelve weeks old male and female mice, wild type C57BL/6 were anesthetized prior to euthanasia. The care of the animals was consistent with guidelines of the German Animal Welfare Law. Studies were approved by the animal care and use committees of University Hospital Heidelberg under the registration number T-30/23. The animals were euthanized with intraperitoneal injection of 100 mg/kg ketamine hydrochloride with 10 mg/kg xylazine hydrochloride. Mice were placed in supine position, the thoracic and abdominal cavity were opened and the right ventricle cannulated with a 20G catheter, an incision was made to the inferior caval vene to later allow drainage of blood and fixative. The trachea was cannulated using a blunt 20-G cannulas the lung air inflated to 25 cm H\u003csub\u003e2\u003c/sub\u003eO pressure. Small pectin patches were carefully applied to the wet surfaces of visceral organs. The animal was then vascular perfusion fixed via the right ventricle using the primary fixative (1.25% glutaraldehyde, 2% paraformaldehyde, 0.15% Ruthenium red, and 15% ethanol in 0.1M Pipes buffer). Small organ samples containing pectin patches were harvested after 10 min of perfusion fixation and submerged in the same fixative.\u003c/p\u003e\u003cp\u003eGravimetric analysis\u003c/p\u003e\u003cp\u003ePectin films (40-100\u0026micro;m thick) were cut in 4 x 10 mm pieces and weighed. After treatment with the fixatives (see below), i.e. after about 2 h in aqueous solution, the hydrated films were weighed again. Since observation in the SEM samples requires dry samples, the hydrated films were slowly dehydrated through a graded ethanol series and acetone, weighed again and then air dried. For some experiments additional weight measurements were taken after each dehydration step. The weight values were normalized to the initial weight and the obtained \u0026ldquo;hydration factor\u0026rdquo; plotted in Excel.\u003c/p\u003e\u003cp\u003eSample preparation for microscopy\u003c/p\u003e\u003cp\u003ePectin films were cut in 2 x 5 mm pieces, sometimes also in a triangular shape, and incubated for 30 min in the primary fixative, consisting of 2.5% glutaraldehyde, 0.15% Ruthenium red, and 15% to 40% (depending on the type of pectin) ethanol in 0.1M Pipes buffer pH 6.8. After washing for 30 min with 20% ethanol in 0.1M Pipes (2\u0026ndash;3 solution changes), the secondary fixation was for 1h in 1% OsO\u003csub\u003e4\u003c/sub\u003e, 0.15% Ruthenium red, 20% ethanol in 0.1M Pipes. After washing as above the samples were dehydrated in a graded ethanol series (50%, 70%, 90%, 100%) followed by ethanol/acetone (1:1) and dried acetone twice, each step for 10 min. Then samples were either air-dried or infiltrated with 30% Epon in dried acetone for 1h, 70% Epon in dried acetone for 1h, and 100% Epon overnight, followed by fresh 100% Epon for 4 h. For polymerization (2 d at 65\u0026deg;C) the samples were transferred to silicon molds filled with 100% Epon.\u003c/p\u003e\u003cp\u003eFor visualization of pectin patches on mouse lung, the protocol was slightly adjusted: The primary fixative was 1.25% glutaraldehyde, 2% paraformaldehyde, 0.15% Ruthenium red, and 15% ethanol in 0.1M Pipes buffer. After the secondary fixative (prepared as above) an additional step was introduced to increase tissue contrast: Overnight incubation with 2% Uranyl acetate in 25% ethanol. Dehydration steps were increased to 15min/step and the initial infiltration steps to 2h/step.\u003c/p\u003e\u003cp\u003eAir-dried pieces of pectin films were cut into smaller pieces and mounted with silver paint (Acheson, Plano, Wetzlar, Germany) onto aluminum stubs.\u003c/p\u003e\u003cp\u003eResin-embedded samples were processed further by ultramicrotomy: Sections (70\u0026ndash;100 nm for 2D imaging, 500 nm for nanotomography) were produced using a Powertome PC ultramicrotome (RMC Boeckeler, Tucson, USA). For thin sections an Ultra 35\u0026deg; diamond knife was used, for thick sections a HistoJumbo 45\u0026deg; (both Diatome, Biel, Switzerland). Sections were collected on pieces of Si wafer before post-staining with 3% aqueous uranyl acetate followed by 3% aqueous lead citrate.\u003c/p\u003e\u003cp\u003eLight and electron microscopy\u003c/p\u003e\u003cp\u003eAn upright microscope (AxioImager 2, Carl Zeiss Microscopy, Oberkochen, Germany) with epi-illumination and long-distance objectives was used in brightfield or darkfield mode for mapping of embedded sample through the trimmed blockface or of thick sections on pieces of silicon wafer. The software package ZENconnect, (Carl Zeiss Microscopy, Oberkochen, Germany) allowed a seamless transfer of these datasets to the instruments used for ultrastructural analysis, see below.\u003c/p\u003e\u003cp\u003eSEM hierarchical imaging at 1.5 keV landing energy with a 20 \u0026micro;m aperture using SE and ESB detectors was performed with the Atlas 5 AT software package on a Ultra55 SEM (Carl Zeiss Microscopy).\u003c/p\u003e\u003cp\u003eFIBSEM nanotomography was carried out on a Crossbeam 540 (Carl Zeiss Microscopy) at 2 keV landing energy, 1.0 nA beam current with an isotropic voxel size of 5 nm in x, y, and z. FIB milling was performed at 30 kV with an ion beam current of 700 pA.\u003c/p\u003e\u003cp\u003eThe ZENconnect LM dataset was imported into the Atlas 3D software package for targeting of relevant pre-selected regions. InLens and ESB (1000 V grid) detectors were used simultaneously for image recording.\u003c/p\u003e\u003cp\u003eImage analysis\u003c/p\u003e\u003cp\u003eFor registration of nanotomography stacks the TrakEM module of the open-source software Fiji was used.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e Registered substacks were annotated manually in Fiji \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e and visualized in Chimera.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e All other operations on images (merging of slices by the command z-projection (from stack operations) using \u0026ldquo;sum slices\u0026rdquo;), filtering etc. were also done in Fiji.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eContributions\u003c/h2\u003e\n\u003cp\u003eS.J.M. and W.W. posed the initial question how to visualize the ultrastructure of a hydrated pectin film. The concept of limited hydration was explored and established by I.W. Pectin preparation J.M.P., mouse handling and surgical experiments W.W., embedding and sectioning of samples R.C., LM and SEM imaging R.C. \u0026amp; I.W., FIBSEM nanotomography E.M. Visualization in 3D and movies by I.W. \u0026amp; R.R.S. The project was administered by S.J.M. and R.R.S., I.W. and R.R.S. drafted the manuscript and all authors reviewed and edited text and figures.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eEthics declarations\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eI.W., R.C., and R.R.S. acknowledge key funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany\u0026rsquo;s Excellence Strategy for the Excellence Cluster \u0026lsquo;3D Matter Made to Order\u0026rsquo; (EXC-2082/1\u0026ndash;390761711). I.W., R.C. and R.R.S. also acknowledge funding by the German Ministry for Research and Education under the Project \u0026lsquo;NanoPatho\u0026rsquo;, FKZ 13N14476, as well as support by the University Computing Centre of the University Heidelberg regarding Research Data Management/ELN and the data storage service SDS@hd supported by the Ministry of Science, Research and the Arts Baden-W\u0026uuml;rttemberg (MWK) and the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG.\u003c/p\u003e\n\u003ch3\u003eData Availability\u003c/h3\u003e\n\u003cp\u003eAll data will be made available via the data repository heiDATA under a DOI which will be assigned after acceptance of the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSeddiqi H, Oliaei E, Honarkar H, Jin J, Geonzon LC, Bacabac RG, Klein-Nulend J (2021) Cellulose and its derivatives: towards biomedical applications. 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J Biol Mat Res Part A 108:246\u0026ndash;253\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M, Tomancak P, Hartenstein V, Douglas RJ (2012) TrakEM2 software for neural circuit reconstruction. PLoS ONE 7:e38011\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P (2012) Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676\u0026ndash;682\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera\u0026mdash;a visualization system for exploratory research and analysis. J Comput Chem 25:1605\u0026ndash;1612\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"d8b4e237-84bc-4c73-b812-0995d491f276","identifier":"10.13039/501100001659","name":"Deutsche Forschungsgemeinschaft","awardNumber":"3D Matter Made to Order’ (EXC-2082/1 – 390761711)","order_by":0},{"identity":"b6c94843-207d-477b-8c17-92159b8dc4cf","identifier":"10.13039/501100002347","name":"Bundesministerium für Bildung und Forschung","awardNumber":"NanoPatho’, FKZ 13N14476","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"BioQuant, Universität Heidelberg","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7619875/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7619875/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe plant-derived biopolymer pectin has been explored as sealant in lung surgery for several years. Adhesion to the lung surface is thought to be mediated by the interpenetration of pectin\u0026rsquo;s heteropolysaccharide chains with the sugar-chains on glycoproteins and glycolipids of the mesothelial glycocalyx. To improve rational design of carbohydrate-based sealants, a mechanistical understanding of the adhesion process is necessary. Therefore, we first established a method to preserve pectin ultrastructure by limiting hydration during sample preparation for electron microscopy. This allows us to demonstrate distinct morphologies of pectins extracted from various plant sources. The network-like morphology of lemon pectin - closely resembling glycocalyx structure - provoked us to explore the interaction between the two by applying a pectin film to the surface of murine lung in a surgical setting. The ultrastructural visualisation of the sealant-tissue assembly shows a tight association between the carbohydrate fibres, likely driven by their polymer-typical chain entanglement.\u003c/p\u003e","manuscriptTitle":"Bioadhesion of plant heteropolysaccharides to animal glycocalyx by polymer-typical chain entanglement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 09:14:56","doi":"10.21203/rs.3.rs-7619875/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f205963d-d804-478d-8bc1-2c2005caeea8","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54733817,"name":"Biomedical Engineering"}],"tags":[],"updatedAt":"2025-09-17T09:14:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 09:14:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7619875","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7619875","identity":"rs-7619875","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}