Crosslinked Bacterial Cellulose Hydrogels For Biomedical Applications

29 The skin, fundamental barrier that protects internal tissues, prevents pathogen invasion, and 30 maintains the body fluid equilibrium, may be compromised upon traumas, such as incisions and 31 burns. The healing process of such wounds is costly and usually hindered by the patient’s 32 physiological conditions, associated diseases, inflammation and external factors, namely bacterial 33 infections. Recently, increasing attention has been given to bacterial cellulose-based membranes to 34 be applied as dressings for healing purposes. Bacterial cellulose is an attractive biomaterial due to 35 its unique structural characteristics such as high porosity, high water retention capacity, high 36 mechanical strength, low density, and biodegradability. One drawback of bacterial cellulose 37 hydrogels is that, after the first dehydration, the water retention capacity is hindered. In this work 38 we produced, modified, and characterized hydrated and de-hydrated BC membranes. Two 39 crosslinking methods were adopted (using citric acid and epichlorohydrin as crosslinking agents), 40 and the results obtained from the characterizations such as water retention capacity, mechanical 41 properties or contact angle were compared to those of unmodified bacterial cellulose. We 42 demonstrate that the cross-linked bacterial cellulose membranes present physical properties suitable 43 to be used as surgical and burn wound dressings when hydrated, or as exuding wound dressings, 44 diapers dressing or sanitary pads when dehydrated. 45 46 47 49 50 51 52


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Cellulose is the most abundant natural polymer in the world and of great economic importance being 55 a linear homopolysaccharide composed by D-glucopyranose units linked by glycosidic bonds 56 (Fernandes et al. 2017). The largest source of this material is the cell wall of plants, but it is also 57 produced by fungi, protozoa and prokaryotes (Morgan et al. 2016). Much of the cellulose by-58 products, such as paper, textiles, and construction materials, are removed from cotton and wood.

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But issues of sustainability and preservation of the environment have led to the search for alternative 60 non-conventional materials, such as bacterial cellulose (BC). For many industrial applications, 61 vegetable cellulose is inconvenient, due to its association with other biopolymers such as properties, such as high tensile strength, elasticity, durability, and high-water retention capacity.

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This biopolymer can absorb over 200 times its own mass in water. BC is biocompatible, inert, non-85 toxic, and non-allergenic and thus its use as dressings results in better healing. It provides a moist 86 environment for wounds, presents adequate structure and mechanical robustness, in addition to being 87 selectively permeable. It is also promising for the incorporation of antimicrobial agents and can be 88 used in several fields of health sciences, in applications such as structures for bone regeneration, 89 drug delivery systems, new vascular grafts, or supports for tissue engineering (Mbituyimana et al. 90 2021). BC has already been used quite successfully in wound-healing applications, proving that it 91 could become a high-value product in the field of biotechnology (Fursatz et al. 2018

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In this work, chemical modifications were performed on bacterial cellulose, by crosslinking the 105 biopolymer with two different crosslinkers, namely Citric Acid (CA) and Epichlorohydrin (ECH),

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to increase its water retention capacity after the first dehydration. These modified membranes were 107 characterized, and their properties were compared with unmodified BC.

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Bacterial Cellulose production     Considering that the water retention capacity of the bacterial cellulose is reduced after the first 134 dehydration, meaning that after drying for the first time the BC will not be able to absorb the same 135 amount of water with respect to its own weight, two methods of BC crosslinking were used to 136 counteract this fact. By crosslinking the BC, a 3D structure is promoted that reduces the collapse of 137 the pore structure, and thus sustains a higher value of water retention capacity of the BC membranes.

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BC crosslinking is typically obtained by linking at least two hydroxyl groups of single cellulose 139 molecules or two or more hydroxyl groups of adjacent cellulose molecules, resulting in a stiffer 140 polymer and preserving its 3D structure (Qi et al. 2016).

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Two methods of crosslinking were applied, one using citric acid and the other epichlorohydrin as 142 the crosslinking agents as can be seen in Fig. 1.

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Citric acid crosslinking 150 Citric acid (CA) is a tricarboxylic acid based on propane-1,2,3-tricarboxylic acid with a hydroxy 151 substituent at position 2. Citric acid is a very suitable crosslinker for wound dressing, not only due 152 to its non-toxicity, but also due to the stable crosslinking bonds that it forms with cellulose.

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Water loss rate

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The water loss assays were conducted by placing BC samples in a ventilated oven at 35 ºC and by 183 weighting them at periodic time intervals.

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Water uptake rate and water retention capacity

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The hydrophilic properties of BC membranes were studied through the water absorption capacity as 187 a function of time. The water absorption capacity depends on the degree of interaction between water 188 and the cellulose molecules. Furthermore, the water absorption capacity is related to the structure of 189 the BC membrane, since the larger is the size of the pores in the membrane, the greater is the water 190 absorption capacity and, consequently, the greater is the degree of water retention.

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To test the water retention capacity (maximum water absorption) of the different membranes,

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In Fig. 2 A

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Water uptake rate

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To measure the water uptake rate, disks with a diameter of approximately 50 mm were used. After 282 dehydration, they were initially weighted and placed in Milli-Q water and left at room temperature.

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At different pre-established times, the discs were removed using tweezers, carefully placed on

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The data presented in Fig. 3

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Water loss rate

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The data presented in Fig. 4 A was calculated by dividing the time dependent values of BC+H20 300 mass by its initial value for normalization. There was no measurable difference in the water loss rate 301 between samples from unmodified BC and crosslinked BC (with AC and ECH). This is in 302 accordance with the fact that the assay started with the BC membranes fully hydrated (Fig. 4 B), 303 with the pores strained to their maximum dimensions, so that the access of the water molecules to 304 the atmosphere was approximately the same for all samples. Close to the dried state (Fig. 4 C), the 305 water loss rate was expected to be different from sample to sample and slightly higher in the 306 crosslinked samples due to their structure, but the weight values obtained at this stage of the assays 307 were so small (» 0.5 mg), that eventual differences were not observable.

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In the three spectra, the several peaks in the region between 1000-1200 cm -1 are characteristic of 333 C=O groups of the primary hydroxyl strong stretching vibration, that may be attributed to the 12 cellulose structure (Madivoli et al. 2016). The peak at 1641 cm -1 corresponds to the -CH2 bending 335 vibration, being more intense in the CA crosslinked sample. The peaks associated to C-OH 336 stretching and C-O-C bending vibrations that occur at 1031 cm -1 and 1107 cm -1 were also observed 337 for all the membranes produced (Brandes et al. 2016). The spectral signatures of BC and of the 338 modified BC with 2% ECH form were highly overlapped (Udoetok, Wilson, and Headley 2018).

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The peak at 1735 cm -1 , typical of ester carbonyl groups, was present in the BC modified by citric

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The BC mechanical properties have already been determined for single isolated fibers, evidencing 347 the extraordinary specific tensile strength (normalized by specific density). The specific tensile 348 strength of the BC fibers can achieve 598 MPa.g −1 .cm 3 , which is considerably higher than the novel

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In this work, the mechanical properties were determined not for isolated fibers but for the dehydrated 351 membranes, using uniaxial tensile testing. In Fig. 6, plots of the typical stress-strain behavior 352 obtained for each membrane are shown. The differences in the mechanical behavior between the 353 three studied membranes were obvious. The more pronounced differences were the higher tensile

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In Table 1, the average values of the mechanical properties obtained for the different BC membranes 360 are displayed and were extracted from the tensile tests.

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In Fig. 6, the crosslinking of the BC membranes induced a stiffer and more fragile behavior, when 365 compared with the unmodified membrane. The unmodified BC samples attained a high value of 366 maximum strain (39%), while for the crosslinked samples, this value decreased to 7.2% or 3.6% for 367 the crosslinking with 2% ECH and 50% CA, respectively. This stiffer and more fragile behavior was 368 not only evident in the strain but also in the ultimate tensile strength (UTS) and the Young's modulus.

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The unmodified BC samples presented an UTS of 33.9 MPa and a Young's modulus of 1.62 GPa,

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In the dehydrated state, the unmodified BC sample was much more ductile than the crosslinked ones,

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which were more brittle, although for the proposed application (wound dressings) all samples 383 presented suitable properties, being mechanically robust and resilient.

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Mechanical properties of the hydrated membranes

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The viscoelastic properties of BC membranes can be observed in Fig. 7

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Knowing that the higher the G' value, the more pronounced are the elastic properties, and the higher  contact angle when compared with the unmodified BC (Fig. 8 A). The static water contact angles of 428 BC crosslinked with 2% ECH at 0 s, 20 s and 120 s, were 65.1º ± 3º, 56.3º ± 3º and 50.5º ± 3º, 429 respectively and of BC crosslinked with 50% CA at 0 s, 20 s and 120 s, were 44.1º ± 2º, 40.5º ± 2º 430 and 40.6º ± 2º. As observed in Fig. 8 A, all the contact angles obtained for the 3 samples were lower 431 than 90º, which is characteristic of hydrophilic surfaces (Rbihi et al. 2020). It is evident that 432 membranes produced using both methods of crosslinking described in this paper, when compared to 433 the unmodified BC, present an improvement in the wettability capacity (Fig. 8 B). The differences

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which allows its application on heavily exudating wounds.

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Chemically, BC is equivalent to vegetal cellulose, being free of undesirable by-products, such as 466 hemicellulose, pectin, or lignin. BC is not only biodegradable and non-toxic, but also presents a 467 notable advantage in regard to vegetal cellulose, which is its high grade of intrinsic purity, that 468 enables its use with minimal post-processing.

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By crosslinking the BC membranes, the decrease in the water retention capacity after the first

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The two crosslinking methods, with CA and with ECH, resulted in differences in terms of 474 mechanical properties and water uptake capacity, although these were not very significative. The

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CA crosslinking showed advantages in the water uptake capacity and in the fact that CA is a harmless 476 compound, entitling it as better suited for biomedical applications.

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This works demonstrates that these BC membranes present physical properties suitable to be used