Biomechanics of a Plant-Derived Sealant for Corneal Injuries

Abstract

Purpose: The corneal epithelium has a glycocalyx composed of membrane-associated glycoproteins, mucins, and galactin-3. Similar to the glycocalyx in visceral tissues, the corneal glycocalyx functions to limit fluid loss and minimize frictional forces. Recently, the plant-derived heteropolysaccharide pectin has been shown to physically entangle with the visceral organ glycocalyx. The ability of pectin to entangle with the corneal epithelium is unknown.

Methods: To explore the potential role of pectin as a corneal bioadhesive, we assessed the adhesive characteristics of pectin films in a bovine globe model.

Results: Pectin film was flexible, translucent, and low profile (80 µm thick). Molded in tape form, pectin films were significantly more adherent to the bovine cornea than control biopolymers of nanocellulose fibers, sodium hyaluronate, and carboxymethyl cellulose (P < 0.05). Adhesion strength was near maximal within seconds of contact. Compatible with wound closure under tension, the relative adhesion strength was greatest at a peel angle less than 45 degrees. The corneal incisions sealed with pectin film were resistant to anterior chamber pressure fluctuations ranging from negative 51.3 ± 8.9 mm Hg to positive 214 ± 68.6 mm Hg. Consistent with these findings, scanning electron microscopy demonstrated a low-profile film densely adherent to the bovine cornea. Finally, the adhesion of the pectin films facilitated the en face harvest of the corneal epithelium without physical dissection or enzymatic digestion.

Conclusions: We conclude that pectin films strongly adhere to the corneal glycocalyx.

Translational Relevance: The plant-derived pectin biopolymer provides potential utility for corneal wound healing as well as targeted drug delivery.

Introduction

Biopolymers, including polysaccharides and polypeptides, play an important role in tissue engineering, drug delivery, and wound healing. Composed of a repetitive sequence of monomers, biopolymers are chemically diverse, abundant, and biodegradable. The most abundant biopolymers in nature are polysaccharides. Polysaccharides are characterized by monosaccharide subunits linked by glycosidic bonds. Polysaccharide biopolymers used in biomedical applications include alginate,¹ cellulose,² chitin,³ and pectin.⁴ A particularly intriguing heteropolysaccharide is pectin. Chemically, pectin resembles the glycosaminoglycan molecules in the extracellular matrix of mammalian tissues.^5,6 Moreover, pectin demonstrates a branched-chain structure with notable biosimilarity to the glycocalyx of visceral organs.^7,8 

Pectin is a structural polymer in the middle lamella between plant cells.⁹ Pectin entangles with other pectin chains and cellulose microfibrils to function as the glue between plant cells. The capacity to entangle with other polysaccharide chains has led to the use of pectin as a bioadhesive.¹⁰ Pectin films have demonstrated significant adhesivity to visceral organs, including the lung, bowel, liver, and heart.^11–15 The potential adhesivity of pectin to the cornea is unknown. 

The corneal epithelium has a prominent glycocalyx composed of membrane-associated glycoproteins, mucins, and galactin-3.^16,17 Similar to the glycocalyceal functions in visceral tissues, the corneal glycocalyx appears to limit fluid loss and minimize frictional forces.¹⁸ The pectin bioadhesive applied to the corneal glycocalyx could contribute to patient care in multiple potential settings—after both routine corneal incisions and traumatic injury. In sutureless clear corneal incisions, a bioadhesive sealant “tape” could reduce the incidence of wound leak even in the presence of unpredictable variations in intraocular pressure.^19–21 In traumatic injuries, the potential to embed drugs in the pectin “free volume” provides an additional opportunity for targeted drug delivery.²² 

To explore the potential role of pectin as a corneal bioadhesive, we assessed the biomechanics of pectin bound to the corneal glycocalyx. Pectin's adhesivity provided insight into the functional entanglement of pectin chains with the corneal glycocalyx. An additional demonstration of pectin adhesion to the cornea, peel force applied to the pectin film provided a practical mechanism for the en face harvest of corneal epithelium. 

Methods

Animals

Bovine globes were harvested from a local abattoir and transported in sealed containers at room temperature. Ischemic times were routinely less than 4 hours. After euthanasia, globes were harvested from adult (300–400 g) Wistar rats (Charles River, Wilmington, MA, USA). The care and nurturing of the animals was consistent with guidelines of the American Association for Accreditation of Laboratory Animal Care (Bethesda, MD, USA) and the Association for Research in Vision and Ophthalmology Animal Statement. In addition, the animal protocol and care was approved by the Brigham & Women's Institutional Animal Care and Use Committee. 

Pectin Films

The citrus pectin powder used in this report was obtained from a commercial source (Cargill, Minneapolis, MN, USA). The characterization of the high methoxyl citrus pectin has been detailed elsewhere.²³ Briefly, the proportion of galacturonic acid residues in the methyl ester form determined the degree of methoxylation. The high-methoxyl pectins used in this study demonstrated a greater than 50% degree of methoxylation. The pectin powder was stored in low humidity at 25°C. To create a pectin film, the powder was dissolved at room temperature with a stepwise hydration protocol that gradually increased water content to avoid undissolved powder.²⁴ The complete dissolution of the pectin was achieved by a high-shear 10,000-rpm rotor-stator mixer (L5M-A; Silverson, East Longmeadow, MA, USA). Plateau viscosity was monitored using a digital tachometer and ammeter (DataLogger; Silverson). The dissolved pectin, typically at 3% (w/w), was poured into custom molds. 

Control Biopolymers

Nanocellulose fiber (NCF) powder, obtained from the University of Maine (Process Development Center, Orono, ME, USA), and carboxymethylcellulose (CMC), obtained from a commercial source (Cargill, Minneapolis, MN, USA), were dissolved at 25°C by a controlled increase in water similar to previous reports.¹⁰ Both NCF and CMC dissolution was achieved with progressive hydration followed by mixing with a high-shear 10,000-rpm rotor-stator mixer (L5M-A; Silverson). The dissolved NCF and CMC was poured into standardized molds and cured for further studies. The sodium hyaluronate/CMC polymer was obtained from commercial sources (SepraFilm, Genzyme, Cambridge, MA, USA). 

Corneal Incision

The corneal incision was a created with a No. 15 scalpel blade with a Bard-Parker No. 3 handle oriented orthogonal to the corneal surface and penetrating through the cornea. The surgical incision was 8 × 0.4 mm. The transcorneal penetration was confirmed with fluid egress from the anterior chamber. 

Spectrophotometry Measurements

Color measurements of the pectin film were obtained with a Minolta spectrophotometer (CM-508d; Minolta, Ramsey, NJ, USA) with a pulsed xenon arc light source.²⁵ The spectrophotometer had a 400- to 700-nm wavelength range at a 20-nm pitch. Light transmittance was calibrated with a white standard; multicolor cellophane reference standards (Outus, Chicago, Canada) were used for comparison. 

Amsler Grid

Visual distortion from the pectin film was assessed by projecting pectin film over half of a black and white Amsler grid. Images were obtained with a 12-megapixel color camera at a variety of focal lengths without magnification. 

Adhesion Testing

Pectin–cornea adhesion experiments were performed with a custom fixture designed for the TA-XT plus with a 5-kg load cell (Stable Micro Systems, Godalming, Surrey, UK). The fixture was composed of a 30-mm diameter flat-ended stainless steel cylindrical probe with pectin mounted to the surface with double-sided proprietary adhesive (Research Division, 3M, St. Paul, MN, USA). The cornea was mounted to a hemispherical foam mount (21 mm diameter) with cyanoacrylate (Vetbond; 3M). The cylindrical probe descended onto the cornea at a velocity of 0.5 mm/s to a compression force of 5 N. The compression force was maintained for variable development times followed by probe withdrawal at 0.5 mm/s. Force and distance recordings were obtained at 500 points per second (pps). 

Peel Force Testing

Pectin–cornea peel adhesion experiments were performed with a custom fixture designed for the TA-XT plus with 5-kg load cell (Stable Micro Systems). The cornea was mounted to a hemispherical foam mount (21 mm diameter) with cyanoacrylate (Vetbond). The pectin–corneal adhesion was established with a 5- to 10-second development time. The adherent pectin film was withdrawn at 0.5 mm/s with real-time video recording. The simultaneous withdrawal force and peel angle recordings were obtained at 500 pps. 

Transcorneal Pressure Testing

Similar to commercial pressure decay leak testing,²⁶ the anterior chamber of the eye was cannulated with a 14-g catheter (Angiocath; BD Insyte, Sandy, UT, USA) and sealed with cyanoacrylate (Vetbond). A clear corneal incision was created remote to the catheter. The anterior chamber was exposed to stepped plateau pressures at 5-mm Hg increments. The plateau pressure was monitored for 20 seconds. A stable plateau pressure within 2 mm Hg was required for an additional 5-mm Hg increase. After establishing the baseline control, the pectin film was applied, and the sequence was repeated until a loss of the pressure plateau was identified. 

Scanning Electron Microscopy

After coating with 20- to 25-A gold in an argon atmosphere, the pectin films were imaged using a Philips XL30 ESEM scanning electron microscope (Philips, Eindhoven, Netherlands) at 15 Kev and 21 µA. Distance calibration was integrated into standardized automation. 

In Situ Silver Staining

Freshly euthanized rat or procured bovine globes were gently rinsed with phosphate-buffered saline (PBS) and incubated in 5% D-glucose (Gibco Laboratories, Grand Island, NY, USA) for 3 minutes. The corneal surface was treated with 0.4% Silver Nitrate (Sigma-Aldrich, Saint Louis, MO, USA) for 30 seconds and submerged briefly in 5% D-glucose solution before exposure to 254 nm UV light (Thermo Scientific, Waltham, MA, USA) for 60 seconds. 

En Face Isolation of Corneal Epithelium

The en face isolation is similar to the approach used on the lung.²⁷ Briefly, the bovine globe or euthanized rat globe was subjected to en face corneal isolation. Pectin films were gently applied to the corneal surface. After a 5-second development time, the corneal epithelium was peeled off at an angle of 120 degrees with a steady rate of 2 mm per second. A thin film of PBS was maintained on the specimen to prevent dehydration. The technique produced large continuous sheets of corneal epithelium. Skillful application of the technique produced mostly viable cells assessed morphologically and is consistent with previous findings.²⁷ The slide was then submerged in PBS for 60 minutes on a shaker to allow pectin to dissolve. The slides were then washed three times, fixed with −20°C acetone, and mounted with DAPI-containing medium (Vector Laboratories, Burlingame, CA, USA). 

Corneal Epithelial Cell Image Acquisition

The corneal epithelial cells were imaged using a Nikon Eclipse TE2000 inverted epifluorescence microscope system as previously described.²⁷ Briefly, image acquisition was controlled using MetaMorph 7.10 software (Molecular Devices, Downingtown, PA, USA). The 14-bit fluorescent images were digitally recorded with an electron multiplier CCD (EMCCD) camera (C9100-02; Hamamatsu, Shizuoka, Japan). Epithelial image stacks, systematically imaging the epithelial sample, were reconstructed into an integrated montage. The corneal epithelial cells were segmented using a machine learning algorithm previously described.²⁷ 

Statistical Analysis

The statistical analysis was based on measurements in at least three different samples. The unpaired Student's t-test for samples of unequal variances was used to calculate statistical significance. The data were expressed as mean ± 1 SD. The significance level for the sample distribution was defined as P < 0.05. 

Results

Physical Properties of Pectin Film

The pectin film in these studies was cured as a translucent and flexible 80-µm-thick rectangular strip (Fig. 1A). Potentially relevant for corneal application, the pectin film demonstrated slight yellow coloration and minimal optical distortion (Figs. 1B, 1C). On the surface of the cornea, the pectin film was strongly adherent to the corneal surface within seconds of application (Fig. 1D). 

Figure 1.

 

Physical and optical properties of pectin film. (A) Pectin film is flexible and semi-transparent. (B) The pectin film causes some discoloration but no distortion of an Amsler grid. (C) The slight yellow color of the pectin was not associated with a significant impact on light absorbance in the visual range (black line, circle). Blue, red, yellow, and orange transparent optical standards are shown for comparison. (D) Pectin film is shown sealing a corneal incision in the bovine cornea (white arrow).

Pectin–Corneal Tensile Adhesion Strength

To investigate pectin adhesion to the bovine cornea, pectin film and the bovine cornea were mounted on custom fixtures designed for an TA-XT Plus material analyzer (Stable Micro Systems) (Fig. 2A). The pectin film engaged the cornea with a compression force of 5 N. Although near-maximal adhesion was obtained within seconds of contact, a conventional development time of 60 seconds was used (Fig. 2A). The force required for probe withdrawal was recorded at 500 pps. The pectin–corneal adhesion was uniformly greater than 3 N with evidence of a distinct debonding curve (Fig. 2B, arrow). In contrast, control biopolymers including NCF, sodium hyaluronate, and CMC demonstrated significantly lower adhesion strength (Fig. 2C, P < 0.05) and work of cohesion (Fig. 2D, P < 0.05). 

Figure 2.

 

Adhesive strength of pectin film. (A) Schematic demonstrating the custom experimental design. Pectin film was mounted on a cylindrical probe (yellow). The bovine cornea (white) was fixed to a foam hemispherical mount. The pectin film and cornea were compressed at 5 N for 60 seconds followed by probe withdrawal at 0.5 mm/s. The force required for probe withdrawal was measured at 500 pps. (B) The adhesion curves demonstrated a peak force of greater than 3 N and evidence of a prominent debonding curve (arrow). (C) The peak force of the pectin film was significantly greater than three other biopolymers, including NCF, hyaluronate, and CMC (P < 0.05). (D) The comparison of pectin's work of adhesion, reflecting the area under the adhesion curve, was also significant (P < 0.05). The boxes span the interquartile range with the median marked with an X and the whiskers defining the data range. Minimum of six biologic replicates per data point.

Peel Strength of Pectin–Corneal Adhesion

Wound films require low-angle peel strength to facilitate reinforcement across linear wounds. To simulate tension across the corneal incision, we measured real-time interface angles and peel force at peel rates of 0.5 mm/s (Figs. 3A–C). Consistent with optimal performance in wound closure, the pectin films demonstrated maximal peel resistance at peel angles less than 45 degrees (Fig. 3D). 

Figure 3.

 

Peel strength of pectin film. (A) Schematic demonstrating the custom experimental design. The bovine cornea (white) was fixed to a foam hemispherical mounting fixture. The pectin film and cornea were compressed at 5 N for 60 seconds followed by probe withdrawal at 0.5 mm/s. The force required for probe withdrawal was measured at 500 pps. (B) Simultaneous video recordings (12 megapixels) were obtained at 30 fps with time-based correction. Peel angles were measured after processing with standard MetaMorph filters (Molecular Devices). Instantaneous measurements of peel angle (red arrows) and adhesive force (blue arrow) were made. (C) The adhesion curves demonstrated a greater peel force at peel angles less than 45 degrees. (D) The comparison of peel adhesion strength was significantly greater at 30 degrees than 60 or 120 degrees (P < 0.05). Because of the variable surface area of the film, the adhesive strength was expressed as relative adhesive force. The boxes span the interquartile range with the median marked with an X and the whiskers defining the data range. Minimum of four biologic replicates per data point.

Pressure Testing of Pectin–Corneal Adhesion

To test outflow resistance of the pectin film after a corneal incision, the anterior chamber of the bovine globes was cannulated and progressively pressurized until a wound leak was detected (Fig. 4A). The pressurization pattern was an incremental 5-mm Hg step with an intervening 20-second plateau; the system was sensitive to ±2 mm Hg. The simulated clear corneal incision in the bovine cornea without pectin film failed to generate a pressure greater than 50 mm Hg (Fig. 4B). In contrast, the incision sealed with the pectin film resulted in a pressure range consistently greater than 200 mm Hg (214 ± 68.6 mm Hg) (Fig. 4B). To test inflow resistance of the pectin film, the anterior chamber of the bovine cornea was mounted in a custom pressure chamber. The depressurization pattern was an incremental 5-mm Hg decrease in pressure with an intervening 20-second plateau. In this simulacrum, the pectin tape reliably sealed transcorneal pressure gradients greater than 51.3 ± 8.9 mm Hg (Fig. 4). 

Figure 4.

 

Pressure resistance of pectin film. (A) Schematic demonstrating the custom experimental design. Separate custom fixtures were used for testing elevated and reduced pressures across the bovine cornea. In both assays, a progressive transcorneal pressure gradient was created; the yield point was identified by a statistically significant drop in the pressure gradient. (B) In the increased pressure condition, the yield point was identified by a significant decrease in transcorneal pressure rise (arrow). (C) Similarly, in the decreased pressure condition, the yield point was identified by an increase in the negative transcorneal pressure gradient (arrow). Sudden or complete sealant failure was not observed. The peak pressure gradient in the increased (D) and decreased (E) pressure conditions was highly significant relative to the no-film control (P < 0.001). CCI, clear corneal incision; +PT, plus pectin film. The boxes span the interquartile range with the median marked with an X and the whiskers defining the data range; minimum of four biologic replicates per data point.

Scanning Electron Microscopy of Pectin–Corneal Adhesion

To demonstrate the physical interaction of pectin tape with the corneal surface, pectin tape was applied to the bovine cornea and imaged with scanning electron microscopy (SEM). SEM demonstrated intimate adhesion of the pectin to the corneal epithelium (Fig. 5). The SEM images demonstrated intimate adhesion without detectable interfacial gaps or separation (Fig. 5, arrows). 

Figure 5.

 

Scanning electron microscopy of the bovine cornea and pectin film. The pectin film (arrows) is adherent to the bovine cornea (c). (A) Overview of the bovine cornea (c) with adherent pectin film. (B–D) Pectin–corneal interface is shown (arrows).

En Face Isolation of Corneal Epithelium

The adhesivity of pectin to the glycocalyx has been used to isolate pleural epithelium.²⁷ To explore the utility of pectin to isolate corneal epithelium, we applied pectin films to both bovine globe corneas and recently euthanized rat corneas (Fig. 6). With in situ labeling with silver stain, subsequent stripping of the bovine (Figs. 6A–C) and rat (Figs. 6D–E) corneas produced sheets of corneal epithelium. Morphometric analysis of the epithelium demonstrated comparable cell area (bovine, 2314 ± 2273 µm²; rat, 2273 ± 436 µm²) and cell perimeters (bovine, 202 ± 193 µm; rat ,193 ± 16 µm), P > 0.39. 

Figure 6.

 

En face harvest of the corneal epithelium. Silver staining of the corneal epithelium in bovine (A–C) and rat (D–F) models. The corneal epithelium was stained in situ, then harvested by peel force applied at a 120° angle at 2 mm/s. Dark lines reflect superficial silver staining, whereas lighter staining (yellow arrows) suggests deeper layers. (G) Fluorescein staining of the harvested region of the bovine cornea (white outline). (H) Fluorescence microscopy of the red silver stain and the blue nuclear (DAPI) stain imaged with a 610-nm-long pass filter. (I) Morphometric comparison of the bovine and rat corneal cell area (mean ± 1 SD). The boxes span the interquartile range with the median marked with an X and the whiskers defining the data range.

Discussion

In this report, we demonstrated the physical and mechanical properties of pectin adhesion to the bovine corneal epithelium and glycocalyx. The low-profile pectin film was previously shown to be translucent, flexible, fracture resistant, and bioabsorbable.^{10,15,23,28,29} Here, we showed that the pectin film was also rapidly and strongly adherent to the corneal surface―effectively sealing corneal incisions. The strongly adherent pectin was also used for en face harvest of the corneal epithelium. We conclude that pectin film is a potentially useful adjunct in the treatment of corneal injury and a potentially valuable tool in corneal research. 

Pectin is a structural heteropolysaccharide that comprises approximately 30% of the primary cell walls of plants.³⁰ One of the most complex polymers on earth, pectin has unique chemical and structural features. Chemically, pectin consists mainly of esterified D-galacturonic acid residues in (1→4) chains.^31,32 Pectin is bioabsorbable and widely recognized as a harmless food additive in North America and Europe. In the United States, pectin is affirmed GRAS (Generally Recognized as Safe), as defined in the Code of Federal Regulations.³³ Notably, pectin is a common vehicle for oral “gel caps” and has been used as a lung sealant in animal¹³ and human¹² models. 

Structurally, pectin is composed of branched polysaccharide chains. In the middle lamella between plant cells, these branched chains entangle with other pectin chains and cellulose microfibrils.^30,34 The physical entanglement between polymer chains provides an explanation for the bioadhesive function of pectin.¹⁰ In mammals, pectin has been shown to entangle with the surface glycocalyx of visceral organs, including the lung, heart, liver, and bowel.¹¹ Although there are limited structural data on the corneal epithelial glycocalyx, lectin staining and the available morphologic studies suggest that the corneal glycocalyx has a similar structure to the glycocalyx of visceral organs.^16,35,36 The data presented here―demonstrating strong pectin bioadhesion to the cornea―are consistent with the cornea and visceral organs sharing a similar glycocalyceal structure. 

Although there is a potential therapeutic role for pectin films in a variety of corneal injuries, an obvious application is the prevention of wound leakage after corneal surgery. The pectin film appears to be functionally capable of limiting wound leakage despite wide ranges in intraocular pressure. In addition to limiting wound leaks, the convenience of pectin film may also increase surgical flexibility. With an effective sealant, corneal surgery would not be restricted to complex incisional architecture.^37,38 The availability of pectin film may permit larger or secondary incisions when necessary without the increased risk of perioperative morbidity. Although pectin's biodurability on the cornea and compatibility with tears is unknown, previous studies of the pleural surface of the murine lung suggest a pectin half-life of 7 days.¹³ 

Importantly, pectin has an enviable safety profile. Pectin was evaluated and cleared toxicologically by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1981.³⁹ In the United States, pectin is affirmed GRAS, as defined in the Code of Federal Regulations 184.1588.³³ In post–World War II United States, pectin was used as an intravenous volume expander because of the need for blood products. To quote one study, “altogether, in patients who had received 4,000cc of pectin solution [intravenously], no changes were encountered which possibly could be attributed to the administration of pectin.”⁴⁰ In addition, pectin-related allergies are extremely rare.⁴¹ 

The multilayers of the corneal epithelium, although similar to skin, are distinct from the monolayer of visceral organ epithelium.^7,42 The anterior layer of the nonkeratinized, stratified corneal epithelium is composed of polygonal squamoid cells. In addition to tight junctions that produce a tear barrier and provide tissue integrity, the anterior layer also expresses a glycocalyx that is available for pectin bioadhesion.⁴³ Here, the pectin film was applied as a bioadhesive to entangle with the corneal glycocalyx and facilitate isolation of the corneal epithelium. Optical sectioning indicated that two or three layers of the flat polygonal cells were isolated as a single entity—perhaps providing a functional distinction from the deeper suprabasal (wing) cell layers.^42,44 Future work will be necessary to characterize the constituents of this layer. 

Acknowledgments

The authors thank Eric Donnenfeld for his helpful comments. 

Supported by NIH Grants HL134229 and HL007734, the German Research Foundation (SFB1066), and Thoracic Surgery Foundation. 

Disclosure: B.S. Liu, None; M. Liao, None; W.L. Wagner, None; H.A. Khalil, None; Z. Chen, None; M. Ackermann, None; S.J. Mentzer, None