Physico-mechanical characterization of liquid versus solid

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Physico-mechanical characterization of liquid versus solid applications of visible light crosslinked tissue sealants Esmat Jalalvandi, Patrick Charron, and Rachael Ann Oldinski ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00785 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 24, 2019

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ACS Applied Bio Materials

Physico-mechanical characterization of liquid versus solid applications of visible light crosslinked tissue sealants

Esmat Jalalvandi,a Patrick Charron a and Rachael Ann Oldinski *a,b,c

a. Department of Mechanical Engineering, College of Engineering and Mathematical Sciences, University of Vermont, Burlington, VT, USA b. Department of Electrical and Biomedical Engineering, College of Engineering and Mathematical Sciences, University of Vermont, Burlington, VT, USA c. Materials Science Program, University of Vermont, Burlington, VT, USA * Corresponding Author [email protected]

Abstract The limitations of commercially available tissue sealants has resulted in the need for a new tissue adhesives with adequate adhesion, improved mechanical properties, and innocuous degradation products. To address current limitations, a visible light crosslinking method for the preparation of hydrogel tissue sealants, based on natural polymers (chitosan and/or alginate), is presented. Water-soluble chitosan was generated via modification with vinyl groups. To form hydrogels, alginate and chitosan were crosslinked by green light illumination, with or without the use of a bi-functional crosslinker. Evaluation of the mechanical properties through rheological characterization demonstrated an increased viscosity of polymer blends, and differences in shear moduli despite similar gelation points upon photo-crosslinking. A comparative study on the burst pressure properties of liquid versus solid material applications was performed to determine if the tissue sealants can perform under physiological lung pressures and beyond using different application methods. Higher burst pressure values were obtained for the sealants applied as a liquid compared to the solid application. The hydrogel tissue sealants revealed no cytotoxic effects towards primary human mesenchymal stem cells. This is the first report of a direct comparison between hydrogel tissue sealants of the same formulation applied in liquid versus solid form.

Keywords: alginate, chitosan, tissue sealant, burst pressure, green light crosslinking ACS Paragon Plus Environment

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1. Introduction The use of sutures and staples in surgery is often limited by their insufficient function to prevent fluid leaks from wounds and a lack of elasticity. Thus, there is a need to develop materials to replace sutures and staples in surgical procedures, or enhance the tissue sealing properties of current clinical methods.1-2 Amongst alternative materials, special attention has been paid to hydrogel-based sealants. Their unique and interesting mechanical and physiochemical properties are promising towards the application of a tissue sealant, which include: 1) protecting the injured tissue from infections, 2) creating and maintaining a moist environment encouraging various stages of wound healing, 3) ease of application and replacement, 4) ability to deliver healing agents at the wound site, and 5) oxygen permeability.3-4

Hydrogels are often non-reactive with biological tissue, non-irritating and, if biodegradable, result in innocuous degradation products.5-6 A hydrogel-based tissue sealant adheres to tissue through either molecular crosslinks or physical entanglements. In regard to the former, tissue adhesives bind to extracellular matrix (ECM) components through non-covalent forces such as charge-charge interactions, dipole-dipole interactions, van der Waals repulsion, and hydrogen bonding.7-8 The physical interaction between adhesives and ECM networks is a function of charge or ionization, pH of the media, and the entanglement/interlocking of the adhesive hydrogel with underlying tissue.9 Currently, Progel® (BARD) is the only surgical sealant approved specifically for pleural air leaks, comprised of albumin and a polyethylene glycol-based crosslinker, applied in conjugation with sutures. Limitations of the current clinical lung sealant include the high cost of human-based albumin and elevated risk of an immune response.10-11 Other commercially available sealants, such as cyanoacrylate-based materials (e.g., Super Glue) and fibrin tissue adhesives, are limited by inadequate mechanical strength, and the lack of degradability and/or biocompatibility; in addition these sealants were not designed for use with lungs.12-13 Therefore, the development of novel nontoxic surgical adhesives, with suitable adhesive strength and tunable mechanical properties, is desired.

ACS Paragon Plus Environment

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Polysaccharides, including chitosan, alginate, dextran and hyaluronic acid, are studied for use in hydrogel preparations for biomaterials due to their biocompatibility, biodegradability, and abundant natural resources.14-16 Chitosan, a β-1,4-linked polymer of glucosamine derived from arthropods, has beneficial biological properties; it can modulate the function of inflammatory cells in wound management, making this polysaccharide attractive for use in wound dressings and drug delivery systems.17-19 Alginate is an anionic biopolymer typically obtained from brown seaweed and has been extensively used for various biomedical applications.20-21 While alginate and chitosan exhibit different functional groups, both are biodegradable and biocompatible, and can potentially improve adhesion to tissues through van der Waals and charge-charge interactions, and/or physical entanglement.20,

22-23

Polysaccharide-based hydrogel networks may be formed by reacting a

component having nucleophilic groups (e.g., chitosan) with a component having electrophilic groups (e.g., aldehyde functionalized polysaccharides) to create a crosslinked (Schiff base) matrix. For example, a Schiff base reaction between oxidized dextran and chitosan showed five times the strength of commercially available fibrin glue; oxidized alginate and gelatin have also been investigated.24-26 However, the components of these hydrogels must be stored separately to avoid premature crosslinking, and the resulting hydrogels typically degrade quickly due to rapid hydrolysis of crosslinking sites and result in cytotoxic by-products.27-29 Therefore, tissue sealant crosslinking toxicity needs to be reduced, and storage and ease-of-use need to be increased.30

Photo-crosslinking is a rapid and convenient way to produce hydrogel polymer networks. Both ultraviolet (UV) and visible light sources are used to initiate crosslinking; however, the use of UV is associated with mild negative side effects in vivo.31 Indeed, some cellular activities are adversely affected by UV exposure.32-33 Visible light crosslinking has been employed as a safe alternative to UV photo-crosslinked hydrogels, without compromising material properties such as viscosity and stiffness, resulting in materials with comparable mechanical properties.34-35 Herein, we present a visible green light crosslinking method for the preparation of several tissue sealants based on chemically modified chitosan, alginate, and polymer blends of the biopolymers. It was



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hypothesized that the novel hydrogel formulations, as injectable liquid and solid patch applications, would prevent air leaks under physiological lung pressure through adhesion to a collagen substrate. In the current study, chitosan was modified to induce water solubility, and chitosan and alginate were both modified to introduce photo-responsive vinyl groups onto their respective backbones and were crosslinked by green light illumination with or without the presence of a bifunctional crosslinker. The various modified polymers and polymer blends were examined for potential future use as lung tissue sealants. A comparative study was performed on the burst pressure properties of liquid versus solid applications.

2. Materials and Methods 2.1 Materials Sodium alginate (Manugel, G/M:0.6; 190,000-240,000 g.mol-1) was donated by FMC Biopolymer. Succinic anhydride (99%, Acros Organic), sodium hydroxide (Acros Organic), glycidyl methacrylate (GM), chitosan (CS; 190,000-310,000 g.mol-1, lot# SLBF 5331V, PCode: 1001508341

and

product

of

Iceland),

N,Nˊ-methylenebisacrylamide

(MBAA,

99%),

dimethylformamide (DMF), dimethyl sulfoxide (DMSO), Eosin Y (EY), triethanolamine (TEA), 1vinyl-2-pyrrolidinone (VP), thiazolyl blue tetrazolium bromide (MTT), and trypsin-EDTA were purchased from Sigma-Aldrich and used without further purification. Cell culture media (minimum essential media, MEM) was acquired from GibcoTM. Primary human mesenchymal stem cells (MSCs, derived from bone marrow) were purchased from Rooster Bio. Human MSC-screened fetal bovine serum (FBS) was purchased from Atlanta Biologics. Penicillin-streptomycin was purchased from Corning Cellgro. Phosphate buffer saline tablets (PBS, without calcium and magnesium) was purchased from MP Biomedical, Inc., and used according to the manufacturer’s instructions.

2.2 Polymer Synthesis and Characterization Chitosan was modified with succinic anhydride to produce a water-soluble derivative of chitosan, namely N-succinyl chitosan, following the previously reported method.36 Briefly, chitosan (3 g) was dispersed in DMF (80 mL) along with succinic anhydride (3.5 g, 35 mmol) and reacted for 4 h at


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125 °C. The product was filtered and air dried. Dried product was dissolved in sodium hydroxide solution (5% w/v, 100 mL), and stirred at 65 °C for 18 h under N2 protection. The mixture was filtered to remove undissolved particles and then dialyzed (Spectra/Por dialysis tubing, MWCO 6000-8000) against distilled water, followed by lyophilization to acquire N-succinyl chitosan (3.1 g). N-succinyl chitosan was further modified to introduce photo-reactive species. N-succinyl chitosan (3 g, 13.4 mmol) was dissolved in distilled water (150 mL) and pH was set to 11 using sodium hydroxide solution. The reaction vessel was purged with N2 for 10 min. GM (19 g, 130 mmol) was added in excess to the chitosan solution and reacted for 18 h under inert atmosphere at 60 °C. The final mixture was precipitated in cold ethanol to obtain methacrylated N-succinyl chitosan (GM-CS), which was re-dissolved in water and dialyzed against water for 2 days followed by lyophilization (2.4 g). Methacrylated alginate was synthesized following the same method outlined above.37 Sodium alginate (3.0 g, 13.8 mmol) was dissolved in distilled water (200 mL) and pH was set to 11 using sodium hydroxide solution. The solution was flushed with N2 for 10 min followed by the addition of GM (3.0 g, 19.7 mmol). The mixture was heated to 60 °C for 18 h under inert atmosphere. After cooling to room temperature, the mixture was poured into a large amount of cold ethanol, and the precipitate was collected, dissolved in water, and dialyzed against distilled water for 2 days to remove ethanol and GM trace. Lyophilization resulted in methacrylate alginate (GM-Alg, 2.3 g).

The lyophilized polymers were characterized using Fourier transform infrared (FTIR) spectroscopy at room temperature, while polymers in the liquid state were characterized using hydrogen (i.e., proton) nuclear magnetic resonance (1H-NMR) spectroscopy techniques. FTIR spectra (8 scans) were recorded on an IRAffinity-1S SHIMADZU spectrometer with a diamond attenuated total reflectance (ATR) top-plate. 1H-NMR spectra were recorded on a Bruker AVANCEIII spectrometer at 500 MHz at 30 °C (16 scans). 1H-NMR spectra of chitosan and N-succinyl chitosan were collected on polymers dissolved in D2O/DCl. 1H-NMR spectra of GM-CS, alginate and GM-Alg were collected on polymers dissolved in D2O.



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2.3 Polymer Blend Solutions and Patch Preparation Table 1 lists the components and weight/volume ratios for each hydrogel group prepared and tested. Polymer concentrations were selected based on viscosity values, to maintain injectable formulations. The polymer and blend concentrations were chosen based on our earlier work; in addition, our focus was to investigate the differences between alginate, chitosan, and the effect of blending the two polymers.38-40 Some of the hydrogel groups were prepared with or without the short-chain crosslinker MBAA (6% w/w). The concentration of MBAA was kept constant in all group to determine whether it can have a significant effect on the properties of the hydrogels.41-43 Photosensitive initiator solution was prepared in PBS, pH 7.4, under red light to prevent premature photocrosslinking. The concentration of each of the following components were chosen based on an absorption assay conducted on solutions containing Eosin Y (EY), EY supplemented with triethanolamine

(TEOA),

EY

supplemented

with

1-vinyl-2-pyrrolidinone

(VP),

and

EY

supplemented with TEOA and VP.44 The solution with the highest absorbance at 530 nm was chosen as the photoinitiator system for subsequent tests. According to previously published studies, the photo-sensitive initiator solution was prepared as follows: EY (1 mmol.L-1, photosensitizer), TEOA (125 mmol.L-1, initiator), VP (20 mmol.L-1, catalyst).34,

38, 40, 45

The hydrogel

precursor solutions (PBS, pH 7.4) were prepared with the photo-sensitive initiator solution, which were then used for the liquid-based tissue sealants. To fabricate tissue sealant hydrogel patches, the polymer precursor solutions were prepared with the photo-sensitive initiator solution and injected between two sheets of polytetrafluoroethylene (Teflon®) with a 1-mm spacer using a syringe and 20-gauge needle, taking care to avoid the formation of air bubbles. The molds were rapidly frozen using liquid N2 then lyophilized. Once dry, the molds were disassembled, and the dry polymer films were cut into individual patches using circular biopsy punches (8-mm diameter). It is important to note that the patch fabrication process was carried out under red light to avoid pre-crosslinking the patches.


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Table 1. Hydrogel tissue sealant components and their respective weight/volume concentrations used in the preparation of polymer precursor solutions. All of the precursor solutions included 2.5 µL of EY/VP and 25 µL of TEA per 1 mL of polymer solution, prepared in PBS, pH 7.4

Hydrogel Group

Polymer Component Solution Concentrations (% w/v)

A B C D

E

F

GM-Alg

3

3

-

-

1.5 1.5

GM-CS

-

-

3

3

1.5 1.5

MBAA

-

6

-

6

-

6

2.4 Rheological Characterization Polymer precursor solution viscosities and gelation kinetics under visible green light exposure were quantified through rheological measurements using a TA rheometer, AR2000. The rheometer was equipped with a steel cone geometry (20 mm diameter, 1° cone angle) and a Peltier plate maintained at 37 °C. The viscosity of each precursor solution (without EY/VP, TEA) was measured at shear rates ranging from 1 to 100 (1/s), over a 60 second period; experimental groups included hydrogels outlined in Table 1, while control solutions comprised of sodium alginate (3%, w/v), Nsuccinyl chitosan (3% w/v), and a 1:1 blend of sodium alginate and N-succinyl chitosan (3% w/v). Polymer solutions were prepared in PBS, pH 7.4. Quantitative viscosity measurements were further used to qualitatively assess possible polymer degradation post chemical modification; viscosity was calculated using analytical software (TA Data Analysis).

Gelation kinetics for the various hydrogel groups were studied by performing oscillatory time sweeps at 10% radial strain and 1 Hz on polymer precursor solutions. Solutions of GM-Alg and GM-CS alone or a 1:1 blend of the two polymers were made up in PBS, pH 7.4, (3% w/v) with or without the short-chain crosslinker MBAA (6% w/w, Table 1). The photo-sensitive initiator solution was prepared under red light and added to the polymer solutions. The hydrogel precursor solutions



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were placed on the rheometer, data was recorded under ambient light for 30 seconds, and then the solutions were exposed to visible green light [525 nm, custom 9.84 cm diameter light-emitting diode (LED) array, NFLS-G30×3-WHT, SuperBright LEDs] for 10 min. Storage (Gʹ), loss (Gʹʹ) moduli, and the ratio of G″/G′ (tan δ) were calculated using analytical software (TA Data Analysis).

2.5 Burst Pressure Testing Quantitative burst pressure testing was performed on hydrogels, from liquid-based and solid-based sealants (n=3), according to standard ASTM F2392-04. A custom-built burst pressure testing device was employed as previously described, and set up in an incubator at 37 °C.38, 40 All burst pressure tests were conducted a 37 °C. Collagen-rich substrates (Collagen Casings, The Sausage Maker Inc.) were used as an in vitro test membrane and were hydrated for 30 min prior to testing. Substrates were tested intact on the burst pressure device, to ensure the absence of any defects. For dynamic testing, the collagen substrate was clamped down in the burst pressure device and pressurized with air (via a syringe pump) to a baseline of 12 inches of water (in.H2O), at an infusion rate of 75 mL/h, and held briefly. Once substrate integrity was confirmed, it was removed from the device, and punctured with a biopsy punch to create a controlled defect, which was five times smaller than the area covered by the hydrogel tissue sealants.

For liquid sealant applications, the punctured collagen substrate was clamped between a glass slide and a Teflon® mold, with a 1.5-cm diameter hole centered over the collagen puncture. Hydrogel precursor solutions were prepared in PBS buffer, pH 7.4, according to Table 1 with the photo-sensitive initiator (see above). The precursor solution (0.5 mL) was injected into the mold over the compromised collagen substrate, and photo-crosslinked for 5 min under visible green light. The collagen substrate with the hydrogel sealant was removed from the mold and loaded onto the burst pressure device. For the solid sealants (8 mm diameter), dry polymer patches were centered over the collagen puncture with no clamps and hydrated via the hydrated collagen substrate. The solid substrate was then photo-crosslinked for 5 min under green light. Air volume, via the syringe pump, was increased at a constant, controlled flow rate (75 mL/h) within the burst


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pressure chamber, until the seal failed. Failure was defined as a loss in pressure due to an air leak.38

2.6 Cytotoxicity An in vitro cell viability assay was performed to assess the cytotoxic effects the hydrogel groups. MSCs were thawed and seeded in T75 tissue culture flasks at a density of 0.3x106 cells per flask, in 10 mL of standard MSC growth media (MEM supplemented with 10% (v/v) FBS, 1% penicillinstreptomycin, and cultured at 37 °C, 5% CO2 to reach a confluent layer. MSCs were expanded to create a stock solution of 3×105 cells/mL (passage 5). Cytotoxicity assays were carried out in 24well tissue culture polystyrene plates, at a seeding density of 0.5×105 cells/mL. The precursor solutions for each hydrogel group were prepared in MEM (0.5 mL) and placed inside culture inserts (8 µm pore size, BD Falcon, USA) then exposed to green light for 5 min to form hydrogels. Once crosslinked, the hydrogels were rinsed with PBS (200 µL), the culture inserts were transferred to the pre-seeded well plates, and the plates were incubated at 37 °C, 5% CO2. Mitochondrial activity was determined after 48 h incubation for each hydrogel group and were compared to non-treated MSC controls using an MTT assay. After 48 h, the culture inserts and media were removed and MTT solution (5 mg/mL, 250 µL) was added to each well and incubated for 3 h. MTT was metabolically reduced by viable cells to a blue-violet formazan which was then dissolved in DMSO (250 µL). The absorbance of each well was measured at 570 nm to determine the percentage of viable cells using a microplate reader (H1Synergy, BioTek). MSC mitochondrial activity was compared to normalized non-treated controls.14

2.7 Statistical Analysis Quantitative results were statistically analysed by one-way analysis of variance (ANOVA) with Bonferroni post-test, for burst pressure data analysis and cytotoxicity assay results. Data are reported as mean ± standard deviation. For all analyses, the statistical significance was set at p ≤ 0.05.



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3. Results and Discussion 3.1 Synthesis and Characterization In its native state, chitosan is not water soluble at neutral pH; however, the polymer needs to dissolve in an aqueous solution prior to the methacrylation reaction. The N-succinyl-substitution method was used to improve chitosan aqueous solubility in preparation for methacrylation and visible light crosslinking. The chemical structure, 1H-NMR and FTIR spectra of N-succinyl chitosan are shown in Figure 1A. The chemical modification resulted in the disruption of strong intermolecular interactions of chitosan (e.g., hydrogen bonding) caused by the free amine groups on the chitosan backbone. 1H-NMR was used to characterize the polymer modification in the aqueous state, while FTIR was used to characterize the polymer in a dehydrated state. When Nsuccinylated chitosan was compared to the 1H-NMR spectrum of non-modified chitosan in D2O/DCl solvent, the degree of deacetylation was calculated to be 78 mol%.46 The 1H-NMR peak centered at 1.9 ppm corresponds to the hydrogens of the acetyl group of chitosan (h), whilst that at 2.5 ppm corresponds to the methylene groups in succinylated units (g). Signals between 3.3 and 3.9 ppm are attributed to protons of the saccharide rings (b-e). The content of free amine groups, succinylated units, were calculated to be 28 mol% and 56 mol% after the solubilization of chitosan. The FTIR spectrum of N-succinyl chitosan showed stretching vibrations of –NH2 and –OH at 3430 cm-1, the weak band of CH2 stretching at 2925 cm-1, the C=O stretching of amide I band at 1630 cm-1 and the amide II band at 1560 cm-1. The peak at 1415 cm-1 represents –COOH symmetric stretching vibration. The peak observed at 1070 and 1050 cm-1 belong to the secondary hydroxyl group and the primary hydroxyl group present in cyclic and primary alcohol, respectively.47

Methacrylation of N-succinyl chitosan was carried out in water, as excess GM reacted with nucleophile moieties existing on the polymer backbone, in this case either free amine or hydroxyl groups. The chemical structure, and 1H-NMR and FTIR spectra, respectively, of methacrylated-Nsuccinyl chitosan (GM-CS), are shown in Figure 1A. The degree of methacrylation was calculated to be 50 mol% using 1H-NMR spectrum. The integral of the signal corresponding to the three protons of methyl group (6.1 ppm, 13) was compared to the integral of the protons of the chitosan


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backbone (7-10) to calculate the degree of modification by GM. Also, new peaks appeared at 1.9 and 5.6 ppm, representing the two protons of vinyl groups (1, 2), which were compared to the integral of the signal related to the chitosan backbone (7-10) to further confirm the degree of modification. The FTIR spectrum of GM-CS showed the characteristic absorption peak of C=C around 1680 cm-1. Prominent absorption bands at 3300 cm-1 (for O–H, N-H stretching vibrations) and 2930 cm-1 (for C–H stretching vibrations) were observed for GM-CS.

Alginate was modified via methacrylation in aqueous solution. GM-Alg was synthesized by reacting alginate hydroxyl groups with GM (Figure 1B). In the 1H-NMR spectrum of GM-Alg, the presence of two signals around 6.1 and 5.6 ppm corresponds to the two protons of vinyl moieties (aʹ, bʹ), while the peak at 1.86 ppm represents the three protons of CH3 groups (kʹ). A 50 mol% degree of methacrylation was observed from the 1H-NMR spectrum of GM-Alg. The degree of methacrylation was calculated through a direct comparison of the integral of the signal related to the three protons of methyl group (kʹ) with the integral of the signal corresponding to the protons on alginate backbone (gʹ-jʹ). The characteristic absorption peak of C=C was observed around 1680 cm-1 in the FTIR spectrum of GM-Alg. Stretching vibration of –OH at 3455 cm-1 and the weak band of CH2 stretching at 2930 cm-1 were also observed.



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Figure 1. (A) The chemical structure of chitosan, the intermediate polymer, N-succinyl chitosan, and GM-CS are shown with protons labelled, which correspond to the 1H-NMR spectra. The FTIR spectra of chitosan, N-succinyl chitosan and GM-CS are shown at the right of the figure. (B) The chemical structure of alginate and GM-Alg are shown with the corresponding protons labelled, which correspond to the 1H-NMR spectra. The FTIR spectra of alginate and GM-Alg are shown at the right of the figure. 1H-NMR spectra of chitosan and N-succinyl chitosan were collected on polymers dissolved in D2O/DCl at 30 °C. 1H-NMR spectra of GM-CS, alginate and GM-Alg were collected on polymers dissolved in D2O at 30 °C. FTIR spectra of chitosan, N-succinyl chitosan, GM-CS, alginate and GM-Alg were collected on lyophilized polymers at room temperature.

3.2 Rheological Measurements The viscoelastic properties of the hydrogel precursor solutions, chitosan intermediate (N-succinyl chitosan), and the control (sodium alginate) were quantified via rheological analysis. Viscosity values versus increasing shear rates for eight different polymer solutions are presented in Figure 2. As shown, the viscosities of the polymer solutions containing chitosan (GM-CS, and GMCS+MBAA) were slightly lower compared to the GM-Alg and GM-Alg+MBAA solutions, which may


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be due to the higher electrolyte nature and chain mobility of alginate, even in the modified state (Figure 1). It is also shown that the addition of the low molecular weight linker, MBA, decreased the viscosity of the GM-CS solution; however, the effect of MBAA on the viscosity of GM-Alg was not as apparent. One limitation of polymer chemical modification is a reduction of the molar mass, which is reflected in the data as a decrease in the solution viscosity.38, 48-49 However, the decrease in GM-CS viscosity was partially recovered through blending two polymers, GM-Alg and GM-CS. For an injectable tissue sealant application, a higher viscosity is advantageous to enhance retention of the hydrogel polymer precursor solution in situ while crosslinking takes place. Thus, the GM-Alg and GM-Alg:GM-CS solutions may lead to higher retention and hydrogels with more entanglements with underlying tissues. Indeed, these hypotheses were verified after burst pressure testing of the various materials.

Figure 2. The viscosity values for eight different hydrogel precursor solutions prepared in PBS pH 7.4, at 37 °C were collected over a 60 second period. A chemical intermediate (N-succinyl chitosan) and a sodium alginate control were also analysed. Alginate solutions are identified by square markers (■), chitosan-based solutions are identified by diamond markers (♦), and blends of alginate and chitosan-based solutions are identified by circles (●).

The gelation kinetics for sets of hydrogels (Table 1, groups A-F) was analysed by an oscillating time sweep during the photo-crosslinking process. The storage moduli, G′, and loss moduli, G″,



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were measured after a 30 second delay followed by exposure to green light for 10 min at 37 °C (Figure 3A-F). In all sample groups, at the very beginning with Gʹ lower than G″, the systems behaved like viscous ﬂuids. The crossover point between G′ and G″ is defined as the gelation point. The gelation point indicates a liquid to gel transition where a viscous liquid phase turns to an elastic solid phase, which can be observed for all hydrogel sets less than 1 min after exposure to green light. Comparing the chemically modified alginate and chitosan, there appeared to be no differences in the gelation points of the two materials. Both moduli rose quickly as crosslinking continued, where the build-up rate of Gʹ was much higher than that of G″ due to the crosslinking between the radical species and the formation of a solid network.

Figure 3. The material properties of hydrogels were recorded during an oscillatory time sweep at 37 °C, 1 Hz and 10% shear strain. (A) GM-Alg, (B) GM-Alg+MBAA, (C) GM-CS, (D) GM-CS+MBAA, (E) GM-Alg:GM-CS, and (F) GM-Alg:GM-CS+MBAA are presented. The gelation point is defined as the crossover point between G′ (storage modulus, gray line) and G″ (loss modulus, black line). (G) G′, (H) G″, and (I) tan δ values for all the hydrogel groups represent values at 550 seconds. Data are reported as mean ± standard deviation (n = 3).


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To further evaluate the results from rheological studies in a more precise way, a time point was selected (550 second, after completion of crosslinking process) and the Gʹ, G″ and tan δ values were compared between all hydrogel groups. In Figure 3G, the Gʹʹ results indicate that the GMAlg was significantly stiffer compared to the other hydrogels. Interestingly, GM-CS was less stiff compared to GM-Alg; however, the addition of the MBAA crosslinking increased slightly the stiffness of GM-CS and blending the GM-CS with GM-Alg further increased the stiffness. The differences in Gʹ between GM-Alg and GM-CS may be due to the relatively more mobile alginate chain, due to fewer bulky side groups (see Figure 1), and the ability for GM-Alg to form more chemical crosslinks and chain entanglements, increasing the hydrogel stiffness. The GM-CS repeat unit contains more side chains compared to GM-Alg due to the succinylation process which creates a steric hindrance and could affect the availability of crosslinking sites. More importantly, the effect of the MBAA was not mimicked in the alginate-based hydrogels, where the addition of the MBAA decreased hydrogel stiffness; these results may indicate that the GM-Alg hydrogels reached maximum crosslink density without MBAA and the addition of the short chain MBA interfered with the GM-Alg network. In Figure 3H, the G″ values follow similar trends compared to G′. GM-Alg achieved the highest G″ values. However, the addition of the MBAA crosslinker did help to recover some of the stiffness in the GM-Alg:GM-CS hydrogels (groups E versus F). When tan δ was calculated for each hydrogel group, the material response and energy dissipation properties of the materials were revealed. While the GM-Alg hydrogel group exhibited the highest G′ and relatively high tan δ vales, the GM-Alg+MBAA, exhibited a low G″ values with the highest tan δ. Thus, the GM-Alg hydrogels are more elastic (i.e., a high ratio of solid network versus molecular mobility) compared to the GM-Alg+MBAA group. As expected, the short chain MBAA is likely not as integrated with the solid network. As further discussed in the burst pressure results (see below), the rheological results indicate that the MBAA crosslinker was not integrated into the solid network of the GM-Alg hydrogel, contributing to high energy dissipation with no significant difference in the burst pressures.



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3.3 Burst Pressure Analysis Burst pressure testing was carried out by pressurizing sealed collagen substrates with air until the system began to leak, at which point the burst pressure was recorded. Burst pressure measurements were used to quantify the ability of chemically modified alginate and chitosanbased hydrogels as potential lung tissue sealants. To perform effectively, a tissue sealant must adhere to under-lying tissue and exhibit material strength exceeding physiological loading conditions. Specifically, physiological lung pressure is approximately 12 in.H2O and thus any material developed to serve as a lung tissue sealant must exhibit and sustain a burst pressure property greater than 12 in.H2O.40

Liquid-Based Sealant The burst pressure results for all liquid-based sealants are presented in Figure 4A. The liquid precursors were deposited on a defected collagen membrane and crosslinked via green light for 5 min. The results indicate that the highest burst pressure values were exhibited by groups A and B, GM-Alg and GM-Alg+MBAA, with 74.0 ± 9.5 and 81.6 ± 3.5 in.H2O, respectively. Chitosan-based hydrogels, groups C and D, exhibited values of 17.8 ± 3.7 and 17.6 ± 1.5 in.H2O, respectively. The lower values for the chitosan-based sealants can be explained by the chain rigidity and possible decreased degree of crosslinking, as discussed in the rheological results. These limitations will also reduce the ability of the materials to dissipate energy resulting in network damage and hydrogel failure during loading (Figure 3I). GM-CS synthesis was obtained after a two-step chemical process, which potentially lowered the molar mass of final product as discussed in the viscosity results. The use of GM-Alg mixed with GM-CS (groups E and F) increased the burst pressure values compared to groups C and D. This is due to the presence of GM-Alg, providing more entanglements with the collagen substrate during the study, and the ability of the alginate-based hydrogels to dissipate more energy during loading, thereby reducing physical damage to the material while maintaining a seal with the underlying substrate.


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Figure 4. Burst pressure values for (A) liquid-based sealants and (B) solid-based sealants at 37 °C (n=3). A-F on the x-axis in both graphs represent the groups prepared according to Table 1. (C) Green light crosslinking of liquid-based sealants over the collagen puncture using a Teflon mold. (D) Fabrication of solid-based sealants using an 8 mm biopsy punch.

Comparing the burst pressure values, the addition of the crosslinker, MBAA, did not have a significant effect on burst pressure data for both GM-Alg and GM-CS-based sealants. However, when MBAA was used in the GM-Alg:GM-CS (1:1) blend, it resulted in a significant increase in the burst pressure from 27.3 ± 9.4 in.H2O to 59.0 ± 3.6 in.H2O, respectively (Figure 4A). This significant increase was likely related to the high tan δ values for these hydrogels, and the addition of the crosslinker, MBAA. The MBAA crosslinker may act as a bridge and connect two different polymeric chains, GM-Alg and GM-CS, which were not initially able to show high burst pressure property (Figure 4A, groups E and F).

The liquid-based sealant values for the above studies formulations are higher than previously reported studies.38,

40

Annabi et al. synthesized a surgical sealant based on methacrylated

tropoelastin.30 The highest value of 47.82 ± 6.02 in.H2O was reported for a highly methacrylated



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tropoelastin solution, at a 20% (w/v) concentration. In addition, our sealants achieve burst pressures much higher than physiological pressures and burst pressures of sealants reported in the literature (fibrin glue = 16.5 in.H2O, Progel = 40.4 in.H2O).11 Previously, Fenn et al., investigated the effect of precursor concentration on the burst pressure properties. Different concentrations of methacrylated alginate were prepared and tested via burst pressure analysis. The highest burst pressure values were attributed to the precursors with high concentrations and subsequently, resulted in better sealing properties.40 The scope of the current study focused on the chemical modification and blends of two materials, not the concentration of the polymer solutions; however, it is expected that higher concentrations would result in higher burst pressures, as shown in the literature.

Solid-Based Sealant To compare the performance of liquid-based and solid-based tissue sealants, burst pressure properties were evaluated. Components of each solid-based sealant were prepared and mixed according to Table 1. The mixtures containing polymer precursors (3%, w/v) and photoinitiator solution were injected into a Teflon mold and thin films were obtained after lyophilization. These films were cut into circular shapes using 8 mm biopsy punches and were used to cover the defect area of the collagen substrate for burst pressure testing (Figure 4D). The patches were hydrated slightly on the collagen substrate prior to testing. The values obtained from burst pressure testing of the solid-based sealants indicated lower burst pressure values compared to liquid-based sealants (Figure 4B). This may be due to improper hydration which may have resulted in incomplete crosslinking, which may lower the effectiveness of sealing the membrane defect properly. However, the burst pressure properties of the solid-based sealants follow a similar trend for liquid-based materials with GM-Alg+MBAA showing the highest value of burst pressure followed by the GM-Alg group. While the burst pressure properties of the liquid-based sealants correlate with the tan δ values, the solid-based sealants do not, which may be due to the inconsistent crosslinking of the solid material applications.

3.4 In Vitro Cytotoxicity


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The mitochondrial activity of MSCs was assessed in the presence of six different groups of hydrogels compared to a non-modified cell control using the MTT assay.16 Absorbance of viable cells was detected at 570 nm and the corresponding results are shown in Figure 5. MSCs showed a greater than 80 % viability after 48 h of incubation in the presence of the hydrogels. No significant differences were observed between non-modified cells and the chemically-modified hydrogels.

Figure 5. MTT viability assay results for MSCs cultured in the presence of hydrogels for 48 h. There was no significant difference between non-modified MSC controls and the hydrogels. A-F on the x-axis represent the groups prepared according to Table 1.

4. Conclusions Chemically modified alginate- and chitosan-based hydrogels were successfully designed, fabricated, and studied as potential lung tissue sealant materials. Utilizing a visible light crosslinking system, hydrogel precursor solutions were prepared and characterized to determine rheological material properties, burst pressure strength, and MSC compatibility. Importantly, the novel tissue sealants remained adhered to the underlying substrate under dynamic conditions, mimicking lung inflation; the resulting hydrogels demonstrated efficacy as lung tissue sealants based on high burst pressures, specifically for the liquid-based alginate materials. Indeed, liquidbased tissue sealants demonstrated higher burst pressures compared to patch-based sealants. The highest burst pressure values for both liquid- and solid-based sealants was observed for GMAlg+MBAA hydrogels. The hydrogels showed no significant toxic effects towards MSCs. The results of the solid-based sealants limit their application as surgical sealants as most of the groups showed a burst pressure below the physiological pressure of lungs (12 in.H2O). Thus, the use of



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solid-based sealants needs to be amended to improve the performance of solid-based tissue sealants.

Conflicts of Interest There are no conflicts to declare.

Acknowledgements This work was funded in part by NIH Grant R01 EB020964 (Oldinski) and the University of Vermont College of Engineering and Mathematical Sciences.

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Physico-mechanical characterization of liquid versus solid

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