On the bacteriostatic activity of hyaluronic acid composite films

Biofilm-related infections and contamination of biomaterials are major problems in the clinic. These contaminations are frequently caused by Staphylococcus aureus and are a pressing issue for implantable devices, catheters, contact lenses, prostheses, and wound dressings.

Carbohydrate Polymers 260 (2021) 117803 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol On the bacteriostatic activity of hyaluronic acid composite films Fernanda Zamboni a, b, Chinonso Okoroafor a, Michael P Ryan c, J Tony Pembroke c, Michal Strozyk a, Mario Culebras a, Maurice N Collins a, b, * a Bernal Institute, School of Engineering, University of Limerick, Ireland Health Research Institute, University of Limerick, Ireland c Department of Chemical Sciences, Bernal Institute, University of Limerick, Ireland b A R T I C L E I N F O A B S T R A C T Keywords: Hyaluronic acid Carbon nanofiber Bacteriostatic Staphylococcus aureus Biofilm-related infections and contamination of biomaterials are major problems in the clinic These contami­ nations are frequently caused by Staphylococcus aureus and are a pressing issue for implantable devices, catheters, contact lenses, prostheses, and wound dressings Strategies to decrease contamination and biofilm related in­ fections are vital for the success of implantable biomaterials In this context, hyaluronic acid (HA), a naturally derived carbohydrate polymer, known to be biocompatible, degradable, and immunomodulatory, has shown some antimicrobial activity effects Due to its poor structural stability, crosslinking strategies, and the incor­ poration of reinforcing fibres in HA gels is required to produce tailored gels for varying applications Whilst carbon-based reinforcing materials, such as carbon nanofibers (CNF), present some intrinsic antimicrobial ac­ tivity related to their high surface area, herein, a crosslinking strategy to enhance the mechanical properties and regulate the rate of degradation of HA is presented We utilise bis-(β-isocyanatoethyl) disulphide (BIED) as the crosslinker with the gel reinforced using 0.25 wt% CNF The effects of CNF and BIED on the structural, me­ chanical, thermal, and swelling behaviour are examined These new HA derivatives exhibit excellent mechanical properties and are capable of withstanding physiological stresses in vivo Antimicrobial activity of the HA de­ rivatives were tested against Staphylococcus aureus and the results reveal antibacterial effect These carbohydrate based materials have potential application on surfaces within clinical settings where staphylococcal contami­ nation is currently an issue Introduction cells and endothelial cells during wound healing remodelling promotes colonization of Gram-positive bacteria such as S aureus (Serra et al., 2015) They are also opportunistic pathogens that cause a variety of self-limiting to life-threatening diseases in humans S aureus as a bac­ terium is among the most common cause of staphylococcal infections and is responsible for various diseases including mild skin infections (impetigo and folliculitis), invasive diseases (wound infections and osteomyelitis, and toxin mediated diseases (such as food poisoning, scaled skin syndrome and toxic syndrome) (Bukowski, Wladyka, & Dubin, 2010; Mitchell & Howden, 2005) S aureus infection largely impacts orthopaedics, trauma, and cardiology with treatment consisting of complex and long term antibiotic courses (Ibberson et al., 2016) HA is a non-sulphated glycosaminoglycan (GAG) in the extracellular matrix (ECM) of numerous soft connective tissues, consisting of alter­ nating units of D-glucuronic acid and N-acetyl-D-glucosamine (Collins & Birkinshaw, 2013) HA is a carbohydrate polymer best known for its Staphylococcus aureus (S aureus) is a Gram-positive, non-motile, nonspore forming facultative anaerobic coccid bacterium The organism is approximately 0.5–1.5 μm in diameter and in physiological conditions grows by aerobic respiration at a temperature range from 15 ◦ C to 45 ◦ C (Jorgensen, Pfaller, & Carroll, 2015) They are normally found on the skin and mucous membranes of the nose in 30 % of the healthy human population, normally displaying no symptoms (Bhattacharya, Wozniak, Stoodley, & Hall-Stoodley, 2015) These bacteria are transmitted via contaminated surfaces, air-borne and direct contact to predisposed humans They are also easily spread in hospitals, particularly in surgical wards (Forster et al., 2013) S aureus are usually detected in the top layer of wounds and are the most common bacterium isolated from chronic wounds (Gjodsbol et al., 2006) The interaction between keratinocytes, fibroblasts, Langerhans * Corresponding author at: Bernal Institute, School of Engineering, University of Limerick, Ireland E-mail address: Maurice.collins@ul.ie (M.N Collins) https://doi.org/10.1016/j.carbpol.2021.117803 Received 30 November 2020; Received in revised form 18 December 2020; Accepted 24 December 2020 Available online 14 February 2021 0144-8617/© 2021 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 intrinsic hydrating properties, which acts as a space filler, lubricant, and osmotic buffer that is found in the synovial fluid, in the vitreous fluid and in the extra cellular matrix (ECM) of many tissues in the body in addition to other important properties such as its immunomodulatory activity which is primarily dependent on its molecular size (F Zamboni, Vieira, Reis, Oliveira, & Collins, 2018) HA has already been widely used for applications in wound healing (Teh, Shen, Friedland, Atlas, & Marano, 2012), drug delivery (Burdick & Prestwich, 2011), and as scaffolds for tissue engineering (Souness, Zamboni, Walker, & Collins, 2018; F Zamboni et al., 2017) HA and its derivates offer long-term safety and proven ability to reduce bacterial `, De Vecchi, Bortolin, Morelli, adhesion and biofilm formation (Romano & Drago, 2017) HA is bacteriostatic, but not bactericidal, and exhibits dose-dependent effects on different microorganisms in the planktonic phase Concerning to possible orthopaedics applications, the analysis of different coatings on titanium surfaces has shown that HA significantly decreased S aureus adherence and its density on titanium surfaces (Harris & Richards, 2004) However, unmodified HA has poor residence time in vivo, but this can be tailored via crosslinking reactions Carbon nanofiber (CNF) composites have gained a lot of attention in the scientific community due to their interesting mechanical, electrical and thermal properties (Feng, Xie, & Zhong, 2014) CNFs also offer benefits for wound healing and are finding application in wound dres­ sing applications as they absorb exudates and regulate wound moisture This is attributed to their surface area, and porous structure (Eatemadi, Daraee, Zarghami, Melat Yar, & Akbarzadeh, 2016), while antimicrobial properties of CNFs depend on their structural characteristics (Song et al., 2015) In this study, we propose the combination of CNFs and HA in order to produce nanocomposite films with improved mechanical performance and better antimicrobial properties In addition, we provide a full comprehensive study of their Structure–property–function relationships in terms of swelling behaviour, cell viability and antimicrobial effect HA was heterogeneously crosslinked using a recently discovered cross­ linker by the current authors, bis-(β-isocyanatoethyl) disulfide (BIED), a di-isocyanate crosslinker that reacts with the hydroxyl groups of HA yielding the formation of urethane linkages between the HA polymeric chains (Fernanda Zamboni, Ryan, Culebras, & Collins, 2020) The pro­ duced films were then optimized in terms of their mechanical and antibacterial performance through the incorporation of carbon nano­ fibers These materials were targeted as potential therapeutic coatings on dressings for wound healing drop-wisely with vigorous stirring 100 mL of toluene was added into the reaction mixture The toluene layer containing the acyl azide (the re­ action intermediate) was separated The acyl azide was completely converted to isocyanate via curtis rearrangement upon heating Bis-(β-isocyanatoethyl) disulfide was obtained by evaporation using a rotary (yield 38 %) The schematics of the chemical reactions is shown in Fig 2.2 Heterogenous crosslinking of HA films For each experiment, 0.3 g (3 wt.%) of the chosen molecular weight of HA was dissolved in 10 mL of double-distilled water and placed in a water bath at 37 ◦ C overnight For the experiment with HA-CNF blend, 1.450 g (2.9 wt.%) of HA and 0.125 g (0.25 wt.%) of CNF were dissolved in 48.4 mL of distilled water yielding a 50 g aqueous solution which was homogenized using ultrasound-assisted dispersion at an amplitude of 10 microns for 10 The CNFs used in this project are 98 % carbonbased, measuring 100 nm in diameter and 20− 200 μm in length (Sup­ plementary Fig 1) The HA and HA-CNF films were prepared via casting their respective aqueous solution onto a clean petri dish, followed by drying at 25 ◦ C for 120 h The resulting films were then cut in pieces of 1cm2 and subse­ quently placed in the vacuum oven at 40 ◦ C (1000 psi) for h Heterogeneous crosslinking was performed on the vacuum dried HA and HA-CNF films The films (1 cm2) were placed in 10 mL of acetonewater (80:20 by volume respectively) solution containing 200 μL of BIED, pH was adjusted using 2.0 M hydrochloric acid (pH of 3.0) The crosslinking reaction was allowed to proceed at 25 ◦ C for 72 h After the reaction, HA films were washed with distilled water and dried at room temperature, until further use 2.3 Chemical structural analysis Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy was used to evaluate the chemical structure of the hydrogel films At room temperature, the IR spectra were recorded using a Perkin Elmer spectrum 2000 FTIR spectrophotometer between 4000–650 cm− with 8.0 cm− resolution The spectra obtained resulted from an average of 20 scans 2.4 Swelling studies HA films were immersed in Phosphate Buffer Saline (PBS, pH 7.4) and their swelling behaviour assessed by measuring their swelling ratio as a function of immersion time The vacuum dried films were weighed as Wd and then swollen in PBS at 37 ◦ C in a water bath for two weeks The films were removed at regular intervals, dried with filter paper to remove surface/excess PBS and weighed as Ws The percentage swelling, was calculated, as follows: Materials and methods HA with an average MW of 0.14 and 1.2 MDa was supplied by Shanghai Easier Industrial Development Co LTD (Shanghai, China) as dry powder CNFs, methanol, hydrazine monohydrate, 3,3′ -dithiopro­ pionic acid, sodium nitrite, concentrated sulfuric acid, hydrochloric acid, acetone, toluene, phosphate buffer saline (PBS) and all other consumables were supplied by Sigma-Aldrich (St Louis, MO, USA) The strain of Staphylococcus aureus used was ATCC 29,213 and obtained from the American Type Culture Collection SR = Ws Wd (1) 2.5 Crosslink density 2.1 Synthesis of Bis-(β-isocyanatoethyl) disulfide (BIED) crosslinker The crosslinked HA films were dried in the vacuum oven at 40 ◦ C (1000 psi) for h and subsequently swollen in PBS until they reached their equilibrium water content The crosslink densities were assessed by volumetric swelling and applying a simplified version of Flory-Rehner equation as described by Collins and Birkinshaw (2007) ⎡ Mc = ̃ ρρ ρs (QM − 1)] ∗ V1 (1 − / ⎣1 + / BIED was prepared following the experimental procedure previously reported in (Fernanda Zamboni et al., 2020) Briefly, dithiopropionic acid (10 g) was mixed with 100 mL of methanol in the presence of sulfuric acid (6 mL) as the catalyst and refluxed for h to yield a solution containing diethyl dithiodipropionate (yield 90 %) The solution con­ taining diethyl dithiodipropionate was mixed with 98 % hydrazine monohydrate (10 mL) and refluxed for h to obtain dithiodipropionyl dihydrazide, which is a white precipitate after recrystallization in an ice bath (yield 97 %) Dithiodipropionyl dihydrazide was then dissolved in a 100 mL hydrochloric acid M 10 mL of sodium nitrite 11 M was added χ )ῡ (2) where Mc is the average molecular weight between crosslinks, ρρ is the F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 Fig Schematics of the chemical reactions involved in the synthesis of BIED in (Fernanda Zamboni et al., 2020) density of the dry polymer (1.229 gcm− 3) and ρs is the density of PBS (1.0 gcm− 3) QM is the swelling ratio determined experimentally by comparing the mass of the material before and after immersion, ῡ is the specific volume of the dry polymer (0.814 gcm− 3), V1 is the molar vol­ ume of the solvent (18 cm3/mol for PBS), and χ is the Flory polymer solvent interaction parameter (0.473) The effective crosslink density, ve, was then calculated as follows: ve = ρρ with the reference (an empty) pan placed on the right of the thermal analyser The experiment was carried out at 20 cc/mm of nitrogen gas flow with an increasing temperature from ◦ C to 200 ◦ C at a rate of 10 ◦ C/min Triplicate samples were tested to ensure reproducibility 2.7 Tensile testing The non-crosslinked and crosslinked HA and HA-CNF films were dried in the vacuum oven at 40 ◦ C (1000 psi) for h and their me­ chanical properties analysed using dynamic mechanical analysis (DMA) Q800 1535 (TA instrument), operating in the DMA Controlled Force mode with a gauge length of 12.74 mm The stress/strain test was per­ formed on samples maintained at an isothermal temperature of 25 ◦ C, and a force ramp rate of 3.0 N/min to an upper force limit of 18.0 N (3) Mc 2.5.1 Mesh size The mesh sizes of the swollen HA (C) and HA-CNF (C) were deter­ mined using the following equation obtained from Collins and Birkin­ shaw (2007) ⎡ √̅̅̅̅̅̅ ρρ ξ = 0.1748 M c ⎣1 + (4) (QM − 1)] ρs 2.8 In vitro cell proliferation study / NIH/3T3 fibroblast cell line obtained from the American Type Cul­ ture Collection was cultured in high glucose Dulbecco Modified Medium supplemented with glutamine, antibiotics (penicillin + streptomicyn) and 10 % foetal bovine serum (FBS) Cells were maintained in the incubator at 37 ◦ C in a humidified atmosphere with 5% CO2 until 80 % confluent Cells were trypsinized and seeded into the test conditions, where medium contained CNF at concentrations of 0.25 %, 0.1 %, 0.05 %, 0.01 % and 0.005 % (w/v) for 1, and days Presto blue cell 2.6 Differential scanning calorimetry (DSC) The non-crosslinked and crosslinked HA and HA-CNF films were dried in the vacuum oven at 40 ◦ C (1000 psi) for h and analysed using a differential scanning calorimeter (Perkin Elmer DSC 6) The samples were enclosed in the DSC pans to avoid oxidation and placed on the left F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 viability assay was performed as described by the manufacturer In summary, 10 % Presto Blue reagent was added to the samples and incubated for 20 After the incubation period, the fluorescence originated from the reduction of resazurin by the cells was read in a microplate reader at the excitation and emission wavelengths of 535 and 615 nm, respectively NIH/3T3 fibroblasts were cultured as described above and seeded onto the films containing 0.1 or 1.2 MDa HA and CNF Prior from the cell seeding, all films were sterilized using UV radiation for h each side within an aseptic environment using laminar flow Alamar blue cell viability assay was performed as described by the manufacturer In summary, 10 % Alamar Blue reagent was added to the samples and incubated for 1, and days continuously At each time point, 10 μL of the medium from each sample was transferred to a 96-well plate A microplate reader at the excitation and emission wavelengths of 540 and 590 nm, respectively, was used to read the fluorescence of the samples 2.9 In vitro antimicrobial study S aureus was cultured in Luria Bertani (LB) Broth to monitor the growth kinetics by spectrophotometry 10 g of LB broth was dissolved in a bottle containing 500 mL distilled water (according to manufacturer’s specification) and sterilized via autoclaving Then mL of the sterilized LB Broth was poured into a set of sterile tubes and inoculated with inocula from sub-cultured strains of S aureus The vacuum dried noncrosslinked and crosslinked HA and HA-CNF films were sterilized via UV radiation for h prior to addition into the inoculated tubes and placed in the incubator at 37 ◦ C to allow the microorganisms to grow Aliquots of 200 μL of the cultured media from each sample was added into cuvettes pre-filled with 800 μL of fresh medium, and the optical density (OD600) of each sample was recorded every hour for h Mean growth rate (MGR) was obtained as the slope of the linear region after plotting the ln OD x time for each sample, and the mean relative growth index (MRGI) was calculated from the equation below: ( ) MGRsample MRGI = x100 (5) MGR control Where the MGR of S aureus only is the control 2.10 Statistical analysis Data are presented as mean ± standard deviation (s.d.) and analysed using one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s HSD test P-values < 0.05 (*) were considered significant Fig Representative HA films HA films are transparent (A), while CNFreinforced HA films show characteristic black colour (B) Schematic reaction of BIED crosslinking HA via urethane linkages (C) FTIR Spectrum of BIED in black, HA (N) in red, HA (C) in green, HA-CNF (N) in blue and HA-CNF (C) in turquoise (D) Results and discussion HA and CNF-reinforced HA films are shown in Fig (A and B, respectively) These films are then heterogeneously crosslinked using BIED as shown in Fig 2-C The FTIR spectrum of BIED (shown in Fig 2d) clearly depicts characteristic spectral bands at 2270 cm− which is characteristic of the isocyanate functional group (C-N-O), 1731 cm-1 – O) stretching, 1516.2 cm-1 which indicates an ester carbonyl group (C– – C bond), 1260.3 (OH– bending) and 1030 cm-1 (C–N group) (C– (Fernanda Zamboni et al., 2020) The FTIR spectra of HA (N) and HA-CNF (N) clearly show bands at 3349 cm− which is characteristic of O–H, 2921 cm− which indicates the asymmetric vibration of alkanes (C–H stretching), 1638 cm− (Amide-Carbonyl functional group), 1420 cm− (COO- stretching) and 1023 cm− (CO–H linkage stretching) The FTIR spectra of the cross­ linked HA (C) and HA-CNF (C) look similar and show additional peaks at the spectral bands of 1731 cm− which indicates an ester carbonyl group – O) stretching, 1606 cm− (CN group), 1528 cm–− (CC bending), (C– – − (COC–– stretching) of the urethane linkage, in accor­ 1228 cm– dance with the literature (Fernanda Zamboni et al., 2020) HA-CNF (C) generally reached higher swelling ratios and took longer times to reach swelling maximas than the HA (C) films this is possibly attributed to the high surface-to-volume ratio of CNFs creating free volume for PBS diffusion, see Fig This result is in general agreement with other reports which show increased swelling behaviour of nano­ fibers incorporated into hydrogel systems (Kai et al., 2012; Llor­ ´mez & Serrano-Aroca, 2018; Ornaghi, Bianchi, Ornaghi, & ens-Ga Jacobi, 2019) Heterogeneously crosslinked HA films show varied matrix parame­ ters dependent on HA MW and CNF-reinforcement (Table 1) The higher water uptake is associated with lower molecular weights with higher volumes of end groups, which is in accordance with the hydrogel mesh size The decreased effective crosslink density and increased mesh size observed in the 0.1 MDa HA-CNF (C) film can be attributed to CNF F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 NIH/3T3 cells with HA crosslinked films (F Zamboni et al., 2017) CNF-reinforced HA crosslinked films were unable to be photographed due to its opacity under the optical microscope The assessment of the cell proliferation after exposure to different concentrations of CNF is shown in Fig 7a All testing conditions show increased proliferation over time Interestingly, the group with higher CNF concentration (0.25 % w/v) showed a similar proliferation profile to the control group The proliferation of NIH/3T3 fibroblasts on different HA films is shown in Fig 7b Overall, all samples increased proliferation over time The reinforcement of HA films by the addition of CNF does not show negative effects on cell proliferation, which is sup­ ported by other publications (Bhattacharyya, Guillot, Dabboue, Tranchant, & Sal­ vetat, 2008; Steel, Azar, & Sundararaghavan, 2020) However, it is observed that crosslinked films displayed a smaller increase in prolif­ eration than their non-crosslinked counterparts CNF and carbon nano­ tubes (CNT) have been shown to increase adhesion of different cell types ´mez, & Serrano-Aroca, to a variety of polymeric films (Salesa, Llorens-Ga 2020) We have previously shown that BIED-crosslinked HA films modulate the immune system (Fernanda Zamboni et al., 2020) Moreover, CNT are also shown to decrease the production of nitric oxide and Fig The swelling behaviour of low MW (0.1 MDa) and high MW (1.2 MDa) HA films incorporation CNFs possess a small portion of hydroxyl groups on their surface (associated with the carbon vapour grown deposition process) (Bubert et al., 2002), of which can react with the isocyanate group of BIED, competing with hydroxyl groups of HA, thus, decreasing the HA crosslinking efficiency The thermal behaviour of different HA films is shown in Fig HA (N) shows the presence of a broad endothermic peak around 110 ◦ C, which is associated with the loss of moisture remaining after the initial drying procedure and an exothermic peak representing degradation around 230 ◦ C, in agreement with the literature (Collins & Birkinshaw, 2007) On the other hand, the thermal behaviour of HA-CNF (N) revealed the presence of two narrow exothermic peaks at around 230 ◦ C and 260 ◦ C, which indicates that the degradation of HA is disrupted by the interaction with CNF This is not surprising considering that the BIED is likely linking the CNF to the HA The thermal behaviour of both crosslinked HA and HA-CNF films show a sharp endothermic peak at around 195 ◦ C and 185 ◦ C, respec­ tively which is attributed to the scission of the disulphate bond For the tensile test results the films were subjected to the same test conditions and representative stress-strain plots of each film are shown in Fig The Young’s moduli for all films were calculated and results are shown in Fig 5c As expected, HA MW strongly influences the me­ chanical properties of the films High MW HA films (1.2 MDa) show decreased Young’s modulus, in comparison to low MW HA films (0.1 MDa) Although, for all samples, crosslinking the films with BIED, drastically increased the mechanical strength of the films The low MW HA-CNF (C) films have the highest modulus, due to the reinforcing effect of CNF combined with BIED crosslinking to HA Optical micrographs of the cell attachment to different substrates are shown in Fig Interestingly, cell attachment does not seem to differ when CNF content in the cell culture medium is increased For HA crosslinked films, it is observed that cells attach to the surface of the films independently of HA MW This could be explained due to cellmediated interactions via CD44 and RHAMM cell surface receptors on Fig Representative DSC thermograms of different HA films Table Matrix characteristics from heterogeneously crosslinked HA films HA MW Reinforcement Maximum water uptake Mesh size (nm) Crosslinking density (mol/cm3) 0.1 MDa – CNF – CNF 26.68 (±1.32) 31.56 (±1.46) 14.2 (±1.8) 12.75 (±2.32) 258.9 (±15.4) 316 (±17.4) 120.3 (±18.8) 106.1 (±23.7) 5.6*10− 4.2*10− 1.8*10− 2.2*10− 1.2 MDa (±4.9*10-7) (±3.6*10-7) (±4.2*10-6) (±7.5*10-6) MW between crosslinks (g/mol) 2.2*105 (±1.8*104) 2.9*105 (±2.3*104) 7.2*104 (±1.6 *104) 6.1*104 (±2*104) F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 Fig Representative stress-strain plots of various HA films A) 1.2 MDa, B) 0.1 M Da and C) Young’s modulus of 0.1 and 1.2 MDa HA films pro-inflammatory cytokines by macrophages (Khang, 2015) This augers well for the films produced in this study and their ability to potentially mitigate inflammatory reactions, especially when targeting wound healing and skin derivate applications The non-crosslinked and crosslinked HA and HA-CNF films show antimicrobial activity against S aureus The growth rate of S aureus is shown in Fig When comparing the MGR between the control (SA) group and the HA samples from both MWs tested, it shows that all crosslinked samples significantly decrease MGR (p < 0.05), thus having a higher bacterio­ static activity The decrease of MGR for crosslinked samples is not associated with the crosslinker itself The MGR of S aureus cultured in LB broth containing only BIED at the same concentration used to crosslink HA films is 0.0202 (p > 0.05) Both 1.2 MDa and 0.1 MDa HA (N) films not show bacteriostatic activity (p > 0.05) The bacteriostatic effect between low and high MW HA groups, shows that 1.2 MDa HA samples [HA (C) and HA-CNF (N)] present significant higher bacteriostatic effects than their low MW counterparts (p < 0.05) Pointing out the highest decrease in MGR is related to crosslinked 1.2 MDa HA films (0.016 ± 0.00055) S aureus produces hyaluronidase (a.k.a hyaluronate lyases) encoded in the hysA gene, which cleaves the β-1,4 glycosidic bond of HA by β-elimination in a processive manner This results in unsaturated disaccharides as the final product of complete digestion It has been hypothesize that the bacte­ riostatic effect of HA is due to the saturation of the bacterial hyaluron­ idase by the excess of HA, which prevents the bacteria from proliferating, because they have elevated levels of ECM component to digest, which affects tissue permeability (Carlson et al., 2004) The increased bacteriostatic effect observed in the crosslinked HMW HA shows the dependency of the hyaluronate lyase to degrade the HMW HA network into small fragments, which is less evident for LMW HA The addition of CNF to the HMW HA crosslinked network can decrease the number of interactions between HA and the bacteria, which compro­ mises the digestion of HA through bacterial hyaluronate lyases, there­ fore reducing the bacteriostatic effect In Table 2, the comparison between the mean growth indexes and F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 Fig Cell attachment after days incubation on different substrates A) Control; B) 0.005 % CNF suspended in cell culture medium; C) 0.01 % CNF suspended in cell culture medium; D) 0.05 % CNF suspended in cell culture medium; E) 0.1 % CNF suspended in cell culture medium; F) 0.25 % CNF suspended in cell culture me­ dium; G) 0.1 MDa HA crosslinked films; and H) 1.2 MDa HA crosslinked films Arrows show: Cell attached to the well (#), cell attached to the surface of the HA film (★), and CNF pre­ cipitate (*) mean doubling time are shown The antibacterial findings of this research agree with the earlier study of Pirnazar et al who reported that the three different MW formulations of recombinant HA used in their experiment exhibited varied bacteriostatic effects on S aureus depend­ ing on the MW and concentration of HA (Pirnazar et al., 1999) Conclusion Crosslinking HA films with BIED produced a gel-like biomaterial whose mechanical properties and functionality makes it suitable for various biomedical applications More so, experimental tests revealed that the addition of CNFs (0.25 wt.%) to HA films significantly improved the film mechanical property, and demonstrated no harmful cytotoxic effects Furthermore, the study of the antimicrobial activity of these F Zamboni et al Carbohydrate Polymers 260 (2021) 117803 Fig Mean growth rate of S aureus for each HA hydrogel formulation SA is the control group containing only S aureus in LB broth Table Bacteriostatic effects of various HA hydrogels HA MW 0.1 MDa 1.2 MDa Groups Mean Relative Growth Index Mean Doubling Time (min) Control HA (N) HA (C) HA-CNF (N) HA-CNF (C) HA (N) HA (C) HA-CNF (N) HA-CNF (C) 100 % 97.6 % 90.5 % 32.4 33.8 36.5 90.9 % 36.2 87.2 % 37.9 99.1 % 78.1 % 33.3 42.3 82.4 % 40.1 85.7 % 38.5 Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Maurice N Collins: Formal analysis, Funding acquisition, Investigation, Methodology, Project administra­ tion, Resources, Software, Supervision, Writing - original draft, Writing review & editing Fig a) Cell proliferation of NIH/3T3 fibroblasts exposed to different con­ centrations of CNF suspended in cell culture medium using Presto Blue assay b) Continuous cell proliferation assessment of NIH/3T3 fibroblasts on different HA films with or without CNF reinforcement using Alamar Blue assay Not cross­ linked (NC); Crosslinked (C) Acknowledgements Irish Research Council Postgraduate Scholarship (GOIPG/2015/ 3577) films revealed that they possess bacteriostatic properties against S aureus, where crosslinked HA-CNF films exhibited the highest anti­ bacterial effect without any bactericidal effect The combined benefits of mechanical properties, together with fibroblast proliferation and bacteriostatic activity, imply that these films can be further exploited for the development of wound healing dressings Appendix A Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2021.117803 References CRediT authorship contribution statement Bhattacharya, M., Wozniak, D J., Stoodley, P., & Hall-Stoodley, L (2015) Prevention and treatment of Staphylococcus aureus biofilms Expert Review of Anti-infective Therapy, 13(12), 1499–1516 Bhattacharyya, S., Guillot, S., Dabboue, H., Tranchant, J.-F., & Salvetat, J.-P (2008) Carbon nanotubes as structural nanofibers for hyaluronic acid hydrogel scaffolds Biomacromolecules, 9(2), 505–509 Bubert, H., Ai, X., Haiber, S., Heintze, M., Brüser, V., Pasch, E., … Marginean, G (2002) Basic analytical investigation of 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and associated proinflammatory cytokine release on transparent carbon nanotube/polymer composite nano-film Nanotechnology, 26(32), Article 325101 ´ (2018) Low-cost advanced hydrogels of calcium Llorens-G´ amez, M., & Serrano-Aroca, A Alginate/Carbon nanofibers with enhanced water diffusion and compression properties Polymers, 10(4), 405 ... to the scission of the disulphate bond For the tensile test results the films were subjected to the same test conditions and representative stress-strain plots of each film are shown in Fig The. .. disaccharides as the final product of complete digestion It has been hypothesize that the bacte­ riostatic effect of HA is due to the saturation of the bacterial hyaluron­ idase by the excess of HA, which... proliferation profile to the control group The proliferation of NIH/3T3 fibroblasts on different HA films is shown in Fig 7b Overall, all samples increased proliferation over time The reinforcement of