bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatibility and antimicrobial activity

bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatibility and antimicrobial activity

Journal Pre-proof Green synthesis of bacterial cellulose/bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatib...

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Journal Pre-proof Green synthesis of bacterial cellulose/bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatibility and antimicrobial activity

Mohamed Abdelraof, Mohamed S. Hasanin, Mohammad M. Farag, Hanaa Y. Ahmed PII:

S0141-8130(19)32944-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.07.144

Reference:

BIOMAC 12896

To appear in:

International Journal of Biological Macromolecules

Received date:

21 April 2019

Revised date:

14 July 2019

Accepted date:

24 July 2019

Please cite this article as: M. Abdelraof, M.S. Hasanin, M.M. Farag, et al., Green synthesis of bacterial cellulose/bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatibility and antimicrobial activity, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.07.144

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© 2018 Published by Elsevier.

Journal Pre-proof Green synthesis of bacterial cellulose/bioactive glass nanocomposites: Effect of glass nanoparticles on cellulose yield, biocompatibilityand antimicrobial activity Mohamed Abdelraofa, Mohamed S. Hasaninb*, Mohammad M. Faragc, Hanaa Y. Ahmedd a Microbial Chemistry Department, National Research Centre, 12622, Dokki, Cairo, Egypt. b Cellulose and Paper Department, National Research Centre, 12622, Dokki, Cairo, Egypt. c Glass Research Department, National Research Centre, 33 El-Bohooth St, Dokki, Giza 1262, Egypt. d The Regional Center of Mycology and Biotechnology- Al-Azhar University, Egypt.

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Abstract

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Despite the advantages of bacterial cellulose (BC) over traditional cellulose, its

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low yield and little bioactivity makes a limitation to be used in an industrial scale. This paper was mainly dual aimed to increase the BC yield using a nanobioactive glass (NBG),

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and in situsynthesize BC/NBG bioactive nanocomposites by a novel and simple green

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method.Accordingly, the composites were prepared via insitu fermentation approach by

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incorporation of NBG particles into BC producing culture medium. The effect of NBG addition on the production process of cellulose, biocompatibility, bioactivity and

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antimicrobial activity were investigated. The results showed that NBG was enhanced and increased the BC yield and this has been achieved by maintaining these NBG on the pH value of the culture medium during the fermentation period. Moreover, it was effectively improved biocompatibility and antimicrobial properities of BC. This study evidenced that BC/NBG composite can be expected to be widely applied in biomedical industries such as bone regeneration and wound healing with the unique of being not harmful to humans. Keywords Bacterial cellulose; green synthesis and nanobioactive glass

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Journal Pre-proof 1. Introduction Bacterial cellulose (BC) is a unique material made in surface culture by such rodshaped Gram-negative bacteria as Gluconacetobacter xylinum[1]. BC is characterized by excellent purity which makes it a good candidate in various biomedical applications, such as artificial skin for humans with extensive burns, artificial blood vessels for microsurgery [2], scaffolds for tissue engineering of cartilage [3], and wound-dressing [4]. In addition, a high hydrophlicity, good sorption of liquids, porosity, density, and high strength make

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BC-based materials suitable for such biomedical applications[5]. Moreover, BC has been

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proved to be a nontoxic, biocompatible, biodegradable and non-allergenic material, and

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can be safely sterilized without any change in its characteristics [6].However, a low yield

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of BC produced makes a limitation of its biomedical applications in a wide scale production. Therefore, there have been several works proposed to solve this problem. As

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reported before, glucose was considered as the most commonly carbon and energy

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sources for the biosynthesis of BC, but, this substrate under metabolic process was rapidly converted to gluconic acid. This by-product was subsequently accumulated in the culture

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medium and caused a decrease in the pH value, which in turn reduce the BC productivity. For that, some chemicals in the bacterial cellulose culture medium was suggested by some the authors in order to overcome this problem [7]. For instance, Li et al., 2012 [8] were studied the addition of some chemicals, such as ethanol and sodium citrate to the medium which decrease the impactof gluconic acidin the culture medium.In this respect, the incorporation of some green additives to the cultural medium which represented as a buffering capacity could be improved the BC productivity and developed of its characters. However, to

the best of our knowledge, few studies were focused on the green

modification of BC characteristics through in situ method. Recently, BC reinforcement can be take place using in situ approach during the fermentation process, which has a lot

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Journal Pre-proof of benefits such as easy operation, high efficiency, avoiding use of harmful materials, etc [9]. Hence, in situ modification by incorporating external substance into bacterial culture medium is a pormising method to improve the BC characteristics. On the other hand, BC in its natural state is not a bioactive material, which limits its application in different biomedical fields. Moreover, the physical stability and relatively low degradation rate of cellulose inside the human body could present challenges for BC to be utilized in certain biomedical applications. Accordingly, different bioactive materials, such as, hydroxyapatite (HAp) [10, 11] has been

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incorporated in BC culture medium and bioactive glass was hybrid with cellulose nanocrystals

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[12, 13] to improve its bioactivity and to make its degradation to be tunable. However, it has been reported that Hap showed a low degradation behavior compared to other calcium phosphate

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ceramics [14], and the stoichiometric composition causes a difficulty to increase HAp degradation

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by a composition change. In contrast, bioactive glasses have wide ranges of compositions, which

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makes a possibility to control their degradation. Bioactive glasses are the most interesting bioceramic materials for bone defects and soft tissue treatments during the last decades and they

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belong to the second and the third generations of biomaterials. That is because of their unique ability to convert to HAp in vivo, and their ability to bond with bone and soft tissues [15, 16]. The

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first bioactive glass which was discovered by Hench and coworkers in the late 1960’s and early 1970’s was prepared by the conventional melting method. It was encoded later as 45S5 or Bioglass® [17]. However, melting-derived bioactive glasses still have some limitation, such as inhomogeneity, difficulty to obtain nanoscale particles and low surface area. In 1991, the incorporation of sol-gel chemistry gave rise to a new generation of bioactive glasses with a great potential to develop better implants for biomedical applications [18, 19]. The incorporation of bioactive glass nanoparticles into biodegradable polymer, has given a new material combines between the glass bioactivity and desirable mechanical properties of the polymer which opened the field to be applied in more versatile biomedical applications, such as, orthopedic implants, bone grafting, and tissue engineering applications [20-22]. Moreover, it has been reported that 45S5 bioglass itself possessed an antimicrobial activity [23-26]. However, most of the previous

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Journal Pre-proof studies were proposed to add bioactive glasses to cellulose to improve its bioactivity did not prepare these composites in situ by incorporation of bioactive glasses in BC producing culture medium, and they simply hybride previously synthesized cellulose nanocrystal with such materials by physical method. In addition, there was no a specific study investigated the effect of glass nanoparticles on the BC culture media, and how itcan influence the growth rate, the yield and bioactivity of BC. Thus, in situ modification by adding exogenous substrate to the culture medium is a potential method to improve the

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properties of BC in order to increase its productivity, bioactivity and antimicrobial

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activity. In this work a promising clean method was carried out to prepare bioactive BC/NBG

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composite in situ. The growth processes of BC in the presence of bioactive glass were evaluated in

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detail. In vitro cytotoxicity of BC/NBG composite was examined by measuring the viability of Vero cells, and the antimicrobial effect was evaluated against the medically relevant bacterial and

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fungal strains. This is the first report on the preparation of modified BC based on bioactive glass

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via in situ procedure for biomedical applications.

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2. Materials and methods 2.1.Materials

The following chemicals were used to synthesize bioactive glass nanoparticles.; tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate, HNO3 and ethanol. All chemical, reagents and microbial culture medium used in this work were in analytical grade without any purification required before use. The BC-producing strain Gluconacetobacter xylinum ATCC 10245 was obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA. The important clinically strains werekindly supplied by Microbiology and Immunology Department, Faculty of Medicine, Al-Azhar University. 2.2.Methods 4

Journal Pre-proof 2.2.1. Synthesis of bioactive glass nanoparticles (NBG) Bioactive glass nanoparticles, based on 70 SiO2, 24 CaO and 6 P2O5 (in mole %), were prepared by sol gel method. Typically, TEOS was dissolved in 2 M HNO3 and ethanol. The mixture was allowed to stir for about 1 h to assure complete hydrolysis. During stirring, the following sequence of chemicals added; TEP and calcium nitrate tetrahydrate with time interval 30 minand the solution left to stir for 4 h. Concentrated ammonia solution was dropped into the solution while vigorously stirring. The sol was

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rapidly transformed intoa white gel which stirred mechanically to avoid the formation of

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bulk gel. The final gel dried at 80 ºC for 1 d in a drying oven. Finally, the dried sample

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was stabilized at 600 ºC in a bench top muffle furnace to remove residual organics and

nanocomposite.

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2.2.2. Preparation of pure BC

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nitrates. The prepared powder was ground and used thereafter for in situ preparation of

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The BC-producing strain G. xylinum ATCC 10245, was cultured in 100 mL of mannitol broth medium in a 500 ml flask with shaking at 180 rpm for 24 h at 30oC.G.

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xylinum was counted by plating on mannitol agar medium. Pure BC films were produced by cultivating bacteria G. xylinum in a liquid Hestrin Schramm (HS) culture medium Hestrin Schramm 1954 [27] composed of glucose, 2.0%; peptone, 0.5%; yeast extract, 0.5%; disodium phosphate, 0.27%; citric acid, 0.115%. The pH was adjusted to 6.0 and the liquid medium was placed in 500-mL Erlenmeyer flasks. After autoclaving at 121oC for 15 min, the media (100 mL each) were each inoculated with 8% of the 1-day-old broth from the mannitol broth medium. Then the fermentation was conducted under static conditions at 30oC for 7 d. To eliminate impurities such as bacteria and other metabolite substances, the harvested BC was purified by immersing in 0.1 M NaOH at 70oC for 30 min, followed by repeatedly washings with deionized water

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Journal Pre-proof until reaching neutral pH. The as-prepared BC films were stored in deionized water and placed in the refrigerator before use. 2.2.3. Production of BC/NBG composites NBG composite was first added to the HS culture medium with various concentrations which the pH value was notably increased with the increase of the NBG concentrations (0.05-0.75 g/ 100 ml). Subsequently, the culture medium was subjected to the sterilization process at 121oC in an autoclave for 20 min. After a similar fermentation

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process to that described above for the preparation of pure BC, BC/NBG gels were

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collected from the top of the culture medium and then purified in a manner similar to that

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of prepared pure BC. Considering the production efficiency, an optimum value of 0.150

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w/v% of bioactive glass (NBG) addition level was selected, and the composite prepared under this condition was used as the basic material for further studies. Consequently, Pure

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BC, BC/NBG and NBG-CaCl2 gels were dried at 70 °C for 24 h in order to evaluate the

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yield concentration in g L−1. The concentration of glucose was determined by using the DNS method and measured at 540 nm [28] the pH of the remaining medium was

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measured at the end of the fermentation process for both culture media. 2.3.Characterizationsof materials Transmission electron microscope (TEM), Model JEM2010, Japan, was used to investigate particle size and morphology of the synthesized sampels. The thermal gravimetric analysis (TGA) was carried out using TGA Q500 device. Investigation of the structural change of different samples was performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy (Spectrum Two IR Spectrometer PerkinElmer, Inc., Shelton, USA). All spectra were obtained by 32 scans and 4 cm−1 resolution in wave numbers ranging from 4000 to 450 cm−1. IR calculations which are including crystallity index (Cr.I.) [29] and main hydrogen bond strength (MHBS) [30]

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Journal Pre-proof were calculated. The crystal structure was determined using XRD (Modeldiffractometer, Schimadzu 7000, Japan.). The zeta potential of the prepared samples was measured, using

NicompTM 380 DLS size analyzer, USA. Laser light scattering is used at 18ᵒ. The surfaces of prepared samples were investigated by field emission scanning electron microscope (SEM) coupled with energy dispersive X-ray analysis; Model Quanta 250 FEG (Field Emission Gun) attached with EDX Unit (Energy Dispersive X-ray Analyses) for EDX and

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mapping, with accelerating voltage 30 KV.

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2.4.Biocompatibility studies

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2.4.1. In vitro bioactivity test

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Simulated body fluid (SBF), prepared according to Kokubo and Takadama [31], was used to study the in vitro bioactivity of the nanocomposites. The samples were

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immersed in the solution with 100 mg in 10 ml SBF in polyethylene tubes. At

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predetermined times (1, 3, 7, 14 and 21 days), 2 ml of the solution was collected and replaced by fresh one with the same volume. The concentrations of Ca2+ ions PO43- ions in

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the collected solutions were measured by using colorimetric kits (BIODIGNOSTIC, Egypt), as well as, the change of pH of SBF measured as a function of time. Moreover, the change of samples surfaces before and after immersion in SBF was investigated by SEM.

2.4.2. Cell viability test The selected samples were tested for cytotoxic effect against Vero cell line. When the vero cells reached confluence (75–90 %) (usually 24 h), the cell suspension was prepared in complete growth medium (DMEM) supplemented with 50 mg/mL gentamycin. The aliquots of 100 µL of cell suspension 1× 105 were added to each well on

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Journal Pre-proof a 96-well tissue culture plate. The blank wells contained complete DMEM medium in place of cell suspension. The cells were incubated for 24 h at 37°C in a humidified incubator with 5% CO2. After the formation of a complete monolayer cell sheet in each well of the plate, the glass samples were added. Serial two-fold dilutions of the compounds were added into a 96-well tissue culture plate using a multichannel pipette (eppendorff, Germany). After treatment (24 h), the culture supernatant was replaced by fresh medium. Then the cells in each well were incubated at 37°C with 100 mL of MTT

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solution (5 mg/mL) for 4 h. After the end of incubation the MTT solution was removed,

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then 100 mL of DMSO was added to each well. The absorbance was detected at 570 nm

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using a microplate ELISA reader (SunRise TECAN, USA).The absorbance of untreated

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cells was considered as 100%. The results were determined by three independent experiments [32, 33]. This experiment was performed as previously described in the

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procedure for cytotoxic activity. After the end of the treatment, the plates were inverted to

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remove the medium, the wells were washed three times with 100 µl of phosphate buffered saline (PH 7.2) and then the cells were fixed to the plate with 10% formalin for 15 min at

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room temperature. The fixed cells were then stained with 100 µl of 0.25% crystal violet for 20 min. The stain was removed and the plates were rinsed using deionised water to remove the excess of stain then allowed to dry. The cellular morphology was observed using an inverted microscope (CKX41; Olympus, Japan) equipped with the digital microscopy camera to capture the images representing the morphological changes compared to control cells.

2.5.Antimicrobial activity tests The antimicrobial activity of the prepared BC/NBG composite was examined against five Gram-negative bacteria (Escherichia coli; Pseudomonas aeruginosa;

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Journal Pre-proof Salmonella typhimurium; Klebsiella pneumonia; and Proteus vulgaris), two Grampositive bacteria (Bacillus subtilis and , Staphylococcus aureus) and two fungi (Candida albicans and Aspergillus parasiticus). The microorganisms were cultured with different BC/NBG composites to evaluate their antimicrobial activity. Dried composites were sliced into small pieces and sterilized at 121oC for 15 min. The dilution series of each sample was prepared in appropriate broth in test tubes. The final concentrations tested from each of BC, NBG and BC/NBGwere 200, 150, 100, 50, 25, mg/mL of broth.

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Powders of each of sample were first mixed and vortexed with the broth cultur medium

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and then bacterial and fungal suspension were added to the mixture. Microbial cultures

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without any material served as controls. The viability of the bacterial and fungal

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suspensions incubated with different concentrations of each sample was assessed using solid agar plates. After 24 h cultivation in broth containing BC, NBG, and BC/NBG, 50

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µL samples from the suspensions were plated. The growth of bacteria and fungi was

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evaluated after cultivation on agar plates at 37oC for 48 h based on colony forming unit (CFU). Absence of growth on the plates was an indicator of bactericidal or fungicidal.

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The percentage inhibiting efficiency (% inhibit) was calculated as [(NC- NS)/NC] X 100, where NC is the number of microbial grown in the absence of each of BC, BG and BC/BGN and NS are the number of microbial grown in the presence of BC, NBG and BC/BGN composite, individually. The antimicrobial activities of the BC/NBG composite were also investigated through the agar well diffusion procedure in compared with BC and NBG, separatly. The test strains were incubated in nutrient broth medium at 37oC and 180 rpm for 24 h. All the microbial suspension was swapped over the medium. Dried samples of BC/NBG were then sterilized at 121oC for 15 min. and then suspended with sterilized deionized water. Wells of 3 mm diameter were made and loaded with 100µl of BC, NBG, and BC/NBG samples. The plates were incubated at 37oC for 24 h for bacterial

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Journal Pre-proof strains and 4 days for fungal strains and the inhibition zone was observed, each experiment was conducted in duplicate. Statistical analyses The experimental data represented in this work were expressed as the average ± standard deviation (SD) for n = 3 and were analyzed using standard analysis of Student’s t-test. The level of significance (p-value) was set at < 0.05.

3. Results and discussion

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3.1.Production of BC/NBG composite

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In this study, the BC/NBG composites were biosynthesized by incorporating NBG

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to the culture medium, using in situ production method which including the formation of

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BC pellicle structure with promising bioactivity, biocompatibility and bioavailability properties. Pure BC was also produced as a control. The conjugation between BC and

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NBG was takenplace, as illustratedin Figure 1a,forming in situ hydrogen bonds between

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the hydroxyl groups (C-OH)at the surfaces of cellulose polymer and the silanol groups (Si-OH) on the glass [34].

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In the static surface culture, cloud-like cellulose pellicles were first observed in the broth liquid. Subsequently, some cellulose tangles were found on the culture surface, which then closed together to form a thin cellulose film. The pellicle was initially sheer, but as it grew continuously, it became intensive and an opaque white. The growth process of BC in the presence of NBG or NBG/CaCl2 were similar, and there were no siginfiant differences between them. Generally,the observation of BC fragments with necked eye showed that they were distinctive on the surface of the culture after 2 d of culture with 0.1 % w/v of NBG particles, while, only a very small amount of BC fragments can be observed in the culture liquid without NBG particles. By increasing the glass percentage to 0.75 %, the culture medium became more turbid and the BC film did not appear on the

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Journal Pre-proof broth surface until day 6. After 7 d, the results showed that BC film prepared in 0.150 % NBG was significantly (p < 0.05) demonstrated the greatest thickness (about 6 mm), while in 0.5 % of NBG obtained a minimum thickness (about 2 mm). Precisely, the addition of NBG particles to the medium was significantly increased the BC yield to reach to its maximum value at glass amount of 0.15 %, which followed by aprogressive decrease as the glass amounts increased. (Figure 1b(i)). Where, BC yield without NBG after 4 d was 0.71 ± 0.05 g/l, which was significantly increased (p < 0.0002) to its

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maximum value at 0.15 % glass to become 1.64 ± 0.07 g/l, while, it reached to around

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0.07± 0.01 g/l at the ultimate glass percentage (0.75 %) after the same period of

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incubation. Moreover, the incubation time was affected significantly on the BC yield at

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glass percentage from 0 % to 0.35 %, where, there was a significant difference in the yield between day 4 and day 5 (p < 0.05), and between day 5 and day 6 (p < 0.05), while, the

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difference between day 6 and 7 was insignificant (p ~ 0.45). In contrast, at higher glass

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percentages, the incubation time was insignificant. To deeper understood these differences in results, the final pH and residual sugar content in the incubated media were measured.

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As itcould be seen from Figure 1b(ii) the final pH of the medium without NBG particles was decreased remarkably from 5.01 ± 0.15 (at day 4) to 3.70± 0.2 (at day 7), whereas, glucose concentration of the fermentation broth (Figure 2b(iii)) was slightly changed in the first 3 days. In this period, glucose was mainly used for the growth of G .xylinum. Thus, very little cellulose could be observed. During the next 3 days, the glucose concentration decreased rapidly and almost linearly with incubation time, meanwhile, a large amount of cellulose was formed. With the prolonging time, the pH of culture medium was reached to ~ 3.70, far below the pH that suitable for the growth of the bacteria and formation of BC. However, the pH values were increased progressively as the amounts of NBG particles increased, and there were no significant differences (p >

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Journal Pre-proof 0.05) of pH values at different incubation times. The amount of the residual sugar content in the medium became neglected. The same result was achieved in the presence of NBG particles and CaCl2, in which the final pH remains unchanged and the bacterial cellulose yield was increased. Sugar consumption and pH decrease were directly proportion to the BC productivity. Where, a pH decrease for HS medium was caused by the rapid enzymatic conversion of glucose into gluconic acid by four enzymatic steps. These steps were the

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phosphorylation of glucose by glucokinase, the isomerization of glucose-6-phosphate to

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glucose-1-phosphate by phosphoglucomutase, the synthesis of UDP glucose by UDPG-

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pyrophosphorylase and finally the cellulose synthase reaction [35]. Interestingly, the

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significant enhancement of BC production in the presence of NBG particles was directly related to the buffering capacity of these particles which maintained BC production in the

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optimal pH range during the static cultivation period. On the other hand, as a result ofhigh

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surface activity, NBG particles possessed a more ability to penetrate the cell membrane of bacterial cells during the fermentation process, which reduced the transfer resistance of

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oxygen and nutrients. Furthermore, the increase in pH was likely because of the leaching of Ca2+ ions from the glass surface and produced a neutralization of the surrounding medium pH leading to the leakage of glucans action. In other words, when the culture medium was modified with excessive NBG amount (more than 0.5% w/v) a total BC/NBG weight was redused to 43 %. This can be explained byahigh concentration of NBG was increased the viscosity, and so, decreased the molecular diffusivity of oxygen in the aqueous phase. This effect was describedby the fact that the bacterial cells could not move freely at the intitial stage, because of the spatial obstacle of NBG resulting in a dropofthe oxygen transfer rate and cell movement, resulting in a significant decrease in cellulose yield [36]. Subsequently, the band-like

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Journal Pre-proof cellulose could be induced by a low mobility of cells in the high viscous culture medium due to physical constraints. The physical constraints on cell movement, such as viscous medium and the spatial obstacle, were supposed to be very important for the changes of cellulose production and structure [36]. Hirai, et al. [37] proposed that the specific movements of the cells may be hindered, and then the performance of enzyme systems in a synthesizing terminal complex subunit (TCs) may be depressed. The high amount of NBG in biological fluids resulted in an increase of osmotic pressure and pH due to the

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leaching of alkali ions from the glass surface, thus making the surrounding environment

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hostile to microbial growth [38].

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Thereby, The addition of NBG nanoparticles with a percentage of 0.15% was

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yielded a very dense network of BC/NBG composite (2.20 g/L) after 7 d of incubation, as illustrated in Figure 1c, that was 1.9-fold greater than the control (1.16 g/L), whereas, it

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became lower than the latter one when the percentage of NBG particles was more than

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0.50 %. As a result, 0.15 % was selected as an optimal NBG addition level with or

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without calcium chloride for further studies.

3.2.Composite Characterization 3.2.1. NBG distribution in thecompoistes The homogeneity of biocactive composite materials important to obtain isotropic properties of them.Accordingly, TEM and elemental mapping by EDX were performed. Firstly, the morphology and particle size of NBG particles were investigated by TEM analysis, as shown in Figure 2A(a). It can be noted from the figure that the synthesized NBG particles were presented in the nano-scale, and their particle sizes in the range between 14 and 30 nm with the almost spherical shape. BC appeared as slides-like shape with fiber behavior in deep composition (Figure 2A(b)). Moreover, the figure showed

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Journal Pre-proof that the NBG particles were well-distributed in BC polymer matrix (Figure 2A (c)) which indicated to a good homogeneity between NBG particles and BC. However, addition of CaCl2 to the bacterial medium was caused formation of larger aggregates of NBG particles throughout the BC matrix (Figure 2A(d)). This can be explained by the attraction of negatively charged glass particles (as it will be discussed later) to the positively charged Ca2+ ions.Moreover, elemental analyses of C, Si, Ca and P by EDX were performed to investigate the degree of distribution of NBG particles in the polymer

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matrix, and mapping photos for this elements are represented in Figure 2B. As shown in

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the figure, all elements were distributed homogeneously in BC polymer for BC/NBG and

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BC/NBG/CaCl2 composites, which indicated to a high homogeneity degree between glass

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and polymer phases.

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3.2.2. XRD

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The XRD analysis gives useful information about interaction and crystals morphology of the material. Figure 3a illustrated the XRD of BC, BC/NBG and

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BC/NBG-CaCl2 patterns. As reported previously in our work the BC produced from HS media gives crystallinity around 77.5%. However, the incorporation of bioactive glass nanoparticles into BC made the intermolecular structure more amorphous as shown in the diffraction patterns of the composites,BC/NBG and BC/NBG-CaCl2. Where the intensities of peaks that appeared at 14.5o and 22.9o stood for the crystal plane (1-10), and (200), respectively [39]. These results can likely be attributed to the in situ production condition of BC, where, NBG particles participated in the polymerization process of BC and they defragmented and deformed the BC chains, and so, NBG particles were added their amorphous character on the final formed composites. 3.2.3. FT-IR 14

Journal Pre-proof FT-IR is a useful tool to confirmthe behaviour of cellulose function groups toward BC/NBG and BC/NBG-CaCl2. Figure 3b illustrats the FT-IR from 400 to 4000 cm-1 of BC, BC/NBG and BC/NBG-CaCl2. BC spectrum showed the characteristic peaks of cellulose type including 3388, 2941, 2839, 1493, 1209 and 860 cm-1 which attributed to hydroxyl group stretching vibration,hydroxyl groups stretching vibration of methylene (– CH2–) band, symmetric stretching vibration of methyl (–CH3–), asymmetric deformation vibration of methyl as well as methylene, asymmetric deformation vibration of methyl

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and methylene stretching vibration of C–O–C in the sugar ring, respectively. Composition

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of BC with BC/NBG and BC/NBG-CaCl2 showed no shift in these all characteristic

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groups of BC, but, the OH stretching group appearedto be more broad this result in

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tabbing of ions into cellulose 3D-network. New peaks were appeared in the composite materials corresponded to NBG. Where, the peak observed at 458 cm-1 in sample

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BC/NBG was attributed to Si–O–Si bending vibration modes, and it was shifted to a

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higher vibration (477 cm-1) for sample BC/NBG-CaCl2. Moreover, the band noticed at 789 cm−1 was assigned to the O-Si-O bending mode of orthosilicateSiO44− [40].

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Asymmetric stretching vibration of Si-O-Si was noticed at 1060 cm−1. A small shoulder observed at 1216 cm−1 was also assigned to Si-O-Si bending mode [41]. Additinoally, the FT-IR calculations were cleared that the Cr.I. of theamples BC, BC/NBG and BC/NBGCaCl2 were 1.3, 1.0 and 1.1 respectivily. Morever, MHBS of the tested samples were areoud 1.1 without significant difference this may be due to the interaction between NBG and NBG-CaCl2 were inter molecules with BC and hydrogen bonds still connected together via BNG particles . Hence, the FT-IR calculatiosdata wereamphazized the XRD results, as well as, cleared theinteration between NBG and BC in two composites. 3.2.4. TGA

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Journal Pre-proof The thermal behaviour of BC and its composites was investigated by TGA. Figure 3c illustrats the TGA curves of native BC and BC/NBG, as well as, BC/NBG-CaCl2. From the figure it can be noted that the NBG and NBG-CaCl2 were improved the BC thermal stability. All tested samples showed water evaporation around100 oC. There were obvious differences among the samples during the decomposition stage. Where, BC showed low thermal stability in comparison toBC/NBG and BC/NBG-CaCl2. Moreover, BC/NBG-CaCl2 was showed close thermal decomposition stage to BC/NBG. However,

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BC/NBG-CaCl2 was showed higher thermal stability than BC/NBG with low remaining

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weight as a result of the presence chlorine ion as illustrated in Table 1.

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3.2.5. DLS

In the biomedical applications, zeta potential measurements play an important

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role in the explanation of the behavior of materials inside the biological system. Zeta

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potential measurments were used to evaluate the BC, BC/NBG and BC/NBG-CaCl2 as illustrated in Table 2. Zeta potential measurements were gave useful data towered the

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tested samples, where, the cell current of BC was decreased from 1.3mA to 1.15 mA in BC/NBG and to 0.6 mA in BC/NBG-CaCl2. These results were conferred that the NBG and NBG-CaCl2 involved in the BC network, however, the Ca2+ions in sample BC/NBGCaCl2 was madeacurrent movement through the cell more slowly. Hence, the Av. phase shift, rad/sec and Av. mobility, M.U were decrease with added NBG and BG-CaCl2these results were emphazised the above -obtained data [42].

3.3.Biological studies 3.3.1. In vitro bioactivity and degradation assessment

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Journal Pre-proof The method which still documented in the in vitro bioactivity investigation is immersing the material in SBF, and the criterion is that the material is bioactive or bioinert is its ability to form a bone-like apatite layer on their surface after soaking in SBF. Figure 4 shows the SEM and EDX analysis of the surface of the BC, BC/NBG and NBG-CaCl2 samples after immersion in SBF for 21 days. Despite there were no spherical aggregates corresponding to the formation of Ca phosphate species noticed on BC sample surface, EDX analysis detected Ca and P atoms on the surface. These can be explained by

of

penetration of Ca2+ and PO43-ions, contained in SBF, into the grooves or pores of BC

ro

sample without forming mature Ca-phosphate compound. Alternatively, these ions might

-p

be formed very minute Ca phosphate crystals inside the sample cavities which cannot be

re

observed by SEM. On the contrary, these aggregates were obviously observed on BC/NBG and BC/NBG-CaCl2 surfaces, and the glass particles distributed in the cellulose

lP

matrix were noticed. The EDX analysis of BC/NBG and BC/NBG-CaCl2 sample surfaces

na

wasdemonstrated that the amount of Ca on BC/NBG-CaCl2 sample was much higher than that on BC/NBG surface. This can be attributed to the surplusamount of CaCl2 added to

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the nutrient medium of bacteria.As a result, the addition of NBG particles in the bacteria nutrient medium to in situ synthesize nanocomposites was enhanced and developed the formation of bone-like apatite layers on their surfaces. The pH changes of SBF incubated BC, BC/NBG and BC/NBG-CaCl2 were measured at predetermined times. Figure 4ii(a) demonstrates the change of pH with time after soaking in SBF solution up to 21 days. It can be observed from the figure that the pH changes for all samples were almost similar, they abruptly increased in the first day of incubation from 7.4 to 8.48, 8.41 and 8.24 for BC, BC/NBG and BC/NBG-CaCl2, respectively, and they remained constant during the rest of soaking time. This abrupt increase can be explained by a slight degradation of cellulose to low molecular weight

17

Journal Pre-proof carbohydrates (glucose, arabinose anddesoxy saccharide) which was likely increased the pH values. The weight loss wasprobably due to dissolved substances from the degraded product of BC which aresoluble in SBF solution, such low molecular weight carbohydratesasglucose, arabinose, desoxy saccharide, glucoronic and gluconic acid. The change of concentrations of Ca2+and PO43-ions in SBF was also monitored as an indication of bon-like apatitelayer formation. Figure 4ii(b) and (c) present the concentration of both ions in SBF solution incubated BC, BC/NBG and BC/NBG-CaCl2

of

samples. It can be noted from the figure that all Ca2+concentrationswereslightly decreased

ro

with the time for all samples. Where, it decreased from 2.28 ± 0.25 mM, 2.23 ± 0.05 mM

-p

and 2.22 ± 0.09 m Mafter 1 d of immersion to 2.09 ± 0.05 mM, 2.10 ± 0.17 mM and 2.13

re

± 0.07 at the end of incubation time for BC, BC/NBG and BC/NBG-CaCl2, respectively. This was likely due to a formation of Ca phosphate crystals. On contrary, PO43-ion

lP

concentration was fluctuated in the solution contained BC sample, that was probably due

na

to repeated adsorption and desorption of PO43- ions in SBF. While, for BC/NBG-CaCl2 sample, the PO43-concentration was progressively increased during 3 - 14 d to reach 1.25

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± 0.03mM, and then, it was slightly decreased again to become 1.14 ± 0.23mM at the end of soaking time. The change of solution concentration contained BC/NBG sample showed similar behavior, but, the concentration was almost constant between 3 d and 14 d with a concentration around 8.4 mM, and it followed by increase again to be 1.20 ± 0.07 mM. Accordingly, PO43- ions concentration in SBF incubated BC/NBG-CaCl2was almost higher than that incubated BC/NBG sample, which can be assigned to, as mentioned before, an initial addition of CaCl2 to bacterial nutrient medium, which increased the concentration of Ca2+ ions in the cellulose polymer. These Ca2+ ions resulted in the formation of more Ca phosphate crystals which released more amounts of PO43-ions into the surrounding medium.

18

Journal Pre-proof 3.3.2. Cell viability test Figure 5 cleared the tested samples activity against Verocells. Native BC showed the low cell compatibilityas shown in Figure 5b incomparison with control in Figure 5a this effect did not refer to its toxicity but referred to the purity of BC which native from any ions so, BC changed the ionic culture media balance and effect the cells grow up. On the other hand Figure 5c and d illustrated the effect of NBG and NBG-CaCl2 toward the Vero cells and emphazised that the cells appearancewas normal like control without

of

neither necrotic effect nor shrinking changes (normal fibroblast likecells). In addition, the

ro

NBG-CaCl2was gave a slightmore viability toward normal cells than NBG as shown in

lP

3.4.Antimicrobial activity

re

inbilogical system regulation [43].

-p

Figure 5e this may be tookeplace as resultof Ca2+ ions which play an effective role

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The efficacy of antimicrobial activity of BC, NBG particles and BC/NBG composite with different concentrations (200, 150, 100, 50 and 25 mg/mL) on clinically

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important aerobic bacteria and fungi was initially studied by CFU method as described previously. In this regared, BC did not show any antimicrobial effect, while NBG was had antimicrobial effect at 150 mg/ml for all bacteria except for Salmonella typhemerium and Pseudomonas aerginosa which were inhibited at 50 and 200 mg/ml, respectively (Table 3). Moreover, Aspergillus parasiticus could resist the effect of most NBG concentrations, but, at 200 mg/mL, NBG wasinhibited it with 91%. Even though the antimicrobial activity of BC/NBG compositewere found similar to NBG behavior, but BC/NBG composite was inhibited the microbial growth at low concentrations. Since, all microbial growth were completly hindered at 50 mg/mLexcept for Aspergillus parasiticus which inhibited at concentration of 100 mg/ml of BC/NBG composite. Otherwise, antimicrobial activity test

19

Journal Pre-proof was also demonestrated using agar diffusion method (Figure 6). Our findings were close to that reported previously [44, 45]. They discussed the inhibitory effect of different bioactive glass types on various clinically aerobic and anaerobic microorganisms was likely due to the dissolution process of the glass network in aqueous solution rabidly. The solution became saturated with ions such as sodium, calcium, phosphate, and silicate and their release resulted in a rise in pH and in osmotic pressure of its vicinity [46]. The optimal pH of all the microbial tested was close to neutral. Thus, the increase in pH and

of

osmotic pressure could partly explain the growth inhibition. In addition, the high

ro

concentrations of calcium released from the NBG can beresulted in perturbations of the

-p

membrane potential of bacteria[45]. Another possibility of the antimicrobial mechanism

re

of NBG was reported formerly in terms of reactive oxygen species (ROS) mechanism [47], where, the strength of the NBG composite was governed by the oxidative stress

lP

theory. This might be based on its nanosilica which considered as a main component of

na

bioactive glass synthesis. Under this nanosilica stress, microbial cells were created reactive oxygen species (ROS) i.e. oxygen free radical. Notably, this free radical reacted

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with the cellular proteins, DNA, membrane, etc., which may further led to causealeakage of essential metabolites and physically disrupting other key cell functions [47]. However, the BC/NBG composite was had antimicrobial susceptibility at low concentration when compared to NBG only. This phenomenon was probably due to a characterization of bacterial cellulosebya porous network structure in its architecture, which is beneficial for potential regulation and transfer of antibiotics or other medicines into the wound, meanwhile serving as an efficient physical barrier against any external infection [48].Moreover, BC has been proved to be commonly exhibited compatibility with biological tissue, as well as, significant bioavailability and biodegradability [6, 49]. Hence, it was clear that BC played an important role in the regulation and transfer of

20

Journal Pre-proof NBG and this related to the high bioavailability of BC which resulting in increase of antimicrobial activity.

Conclusion

Incorporation of NBG in BC via in situ fermentation can be modified the properties of the cellulose produced, increasing the antimicrobial susceptibility and biocompatibility. The inclusion of NBG in the culture medium was promoted a

of

remarkable yield improve, and the yield was 1.8 fold as high in the case of the samples

ro

obtained by adding of 0.150% BG to the homogenized BC. BC/NBG nanocomposite was

-p

displayed a significant antimicrobial activity at low amount, comparing to NBG and

re

BC.Thiscan be ascribed to the synergistic action of NBG and BC, in which, NBG caused hostile of the microbial growth while, BC make regulation of the NBG transfer and this

lP

due to its high bioavailability. Finally, the described approach proved to be successful for

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Acknowledgement

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the development of novel nanocomposite materials based on BC and NBG.

We would like to thank the National Research Centre, Egyptand The Regional Center of Mycology and Biotechnology- Al-Azhar University, Egyptfor a possibility to use their facilities.

Conflict of interest The authors declare that they have no conflict of interest.

References [1] Y.-C. Hsieh, H. Yano, M. Nogi, S. Eichhorn, An estimation of the Young’s modulus of bacterial cellulose filaments, Cellulose 15(4) (2008) 507-513. 21

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[2] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Bacterial synthesized cellulose—artificial blood vessels for microsurgery, Progress in polymer science 26(9) (2001) 1561-1603. [3] A. Svensson, E. Nicklasson, T. Harrah, B. Panilaitis, D. Kaplan, M. Brittberg, P. Gatenholm, Bacterial cellulose as a potential scaffold for tissue engineering of cartilage, Biomaterials 26(4) (2005) 419-431. [4] O.M. Alvarez, M. Patel, J. Booker, L. Markowitz, Effectiveness of a biocellulose wound dressing for the treatment of chronic venous leg ulcers: results of a single center randomized study involving 24 patients, Wounds-a Compendium of Clinical Research and Practice 16(7) (2004) 224-233. [5] W.K. Czaja, D.J. Young, M. Kawecki, R.M. Brown, The future prospects of microbial cellulose in biomedical applications, biomacromolecules 8(1) (2007) 1-12. [6] N. Lin, A. Dufresne, Nanocellulose in biomedicine: Current status and future prospect, European Polymer Journal 59 (2014) 302-325. [7] M. Abdelraof, M.S. Hasanin, H. El-Saied, Ecofriendly green conversion of potato peel wastes to high productivity bacterial cellulose, Carbohydrate polymers 211 (2019) 75-83. [8] Y. Li, C. Tian, H. Tian, J. Zhang, X. He, W. Ping, H. Lei, Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved, 2012. [9] B. Wang, G.-x. Qi, C. Huang, X.-Y. Yang, H.-R. Zhang, J. Luo, X.-F. Chen, L. Xiong, X.-D. Chen, Preparation of bacterial cellulose/inorganic gel of bentonite composite by in situ modification, Indian journal of microbiology 56(1) (2016) 72-79. [10] B. Fang, Y.-Z. Wan, T.-T. Tang, C. Gao, K.-R. Dai, Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds, Tissue Engineering Part A 15(5) (2009) 1091-1098. [11] K.A. Zimmermann, J.M. LeBlanc, K.T. Sheets, R.W. Fox, P. Gatenholm, Biomimetic design of a bacterial cellulose/hydroxyapatite nanocomposite for bone healing applications, Materials Science and Engineering: C 31(1) (2011) 43-49. . Chen, . .r. arcia, . Muno , . re de arra a, N. Garmendia, Q. Yao, A.R. Boccaccini, Cellulose Nanocrystals Bioactive Glass Hybrid Coating as Bone Substitutes by Electrophoretic Co-deposition: In Situ Control of Mineralization of Bioactive Glass and Enhancement of Osteoblastic Performance, ACS applied materials & interfaces 7(44) (2015) 24715-24725. [13] X. Wang, F. Cheng, J. Liu, J.-H. Smått, D. Gepperth, M. Lastusaari, C. Xu, L. Hupa, Biocomposites of copper-containing mesoporous bioactive glass and nanofibrillated cellulose: Biocompatibility and angiogenic promotion in chronic wound healing application, Acta biomaterialia 46 (2016) 286-298. [14] S. Schaefer, R. Detsch, F. Uhl, U. Deisinger, G. Ziegler, How Degradation of Calcium Phosphate Bone Substitute Materials is influenced by Phase Composition and Porosity, Advanced Engineering Materials 13(4) (2011) 342-350. [15] L.L. Hench, Bioactive materials: the potential for tissue regeneration, Journal of biomedical materials research 41(4) (1998) 511-518. [16] M.N. Rahaman, D.E. Day, B.S. Bal, Q. Fu, S.B. Jung, L.F. Bonewald, A.P. Tomsia, Bioactive glass in tissue engineering, Acta biomaterialia 7(6) (2011) 2355-2373. [17] L.L. Hench, R.J. Splinter, W. Allen, T. Greenlee, Bonding mechanisms at the interface of ceramic prosthetic materials, Journal of Biomedical Materials Research 5(6) (1971) 117-141. 8 M. Vallet‐ egí, C. agel, A. . Salinas, lasses with medical applications, European ournal of Inorganic Chemistry 2003(6) (2003) 1029-1042. 9 . i, A. Clark, . Hench, An investigation of bioactive glass powders b sol‐gel processing, Journal of Applied Biomaterials 2(4) (1991) 231-239. [20] M. Sato, T.J. Webster, Nanobiotechnology: implications for the future of nanotechnology in orthopedic applications, Expert review of medical devices 1(1) (2004) 105-114.

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[21] R. Murugan, S. Ramakrishna, Development of nanocomposites for bone grafting, Composites Science and Technology 65(15) (2005) 2385-2406. [22] M. Peter, N. Binulal, S. Soumya, S. Nair, T. Furuike, H. Tamura, R. Jayakumar, Nanocomposite scaffolds of bioactive glass ceramic nanoparticles disseminated chitosan matrix for tissue engineering applications, Carbohydrate Polymers 79(2) (2010) 284-289. [23] I. Allan, M. Wilson, H. Newman, Particulate Bioglass® reduces the viability of bacterial biofilms formed on its surface in an in vitro model, Clinical oral implants research 13(1) (2002) 53-58. [24] S. Begum, W.E. Johnson, T. Worthington, R.A. Martin, The influence of pH and fluid dynamics on the antibacterial efficacy of 45S5 Bioglass, Biomedical Materials 11(1) (2016) 015006. [25] J. Pratten, S.N. Nazhat, J.J. Blaker, A.R. Boccaccini, In vitro attachment of Staphylococcus epidermidis to surgical sutures with and without Ag-containing bioactive glass coating, Journal of biomaterials applications 19(1) (2004) 47-57. [26] M.M. Farag, W.M. Abd-Allah, H.Y.A. Ahmed, Study of the dual effect of gamma irradiation and strontium substitution on bioactivity, cytotoxicity, and antimicrobial properties of 45S5 bioglass, Journal of Biomedical Materials Research Part A 105(6) (2017) 1646-1655. [27] S. Hestrin, M. Schramm, Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose, Biochemical Journal 58(2) (1954) 345. [28] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Analytical chemistry 31(3) (1959) 426-428. [29] M.L. Nelson, R.T. O'Connor, Relation of certain infrared bands to cellulose crystallinity and crystal latticed type. Part I. Spectra of lattice types I, II, III and of amorphous cellulose, Journal of applied polymer science 8(3) (1964) 1311-1324. [30] I. Levdik, M. Inshakov, E. Misyurova, V. Nikitin, Study of pulp structure by infrared spectroscopy, Tr. Vses Nauch. Issled. Irst. Tsellyul Bum. Prom 52 (1967) 109-111. [31] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity?, Biomaterials 27(15) (2006) 2907-2915. [32] Y.-L. Cheng, W.-L. Chang, S.-C. Lee, Y.-G. Liu, H.-C. Lin, C.-J. Chen, C.-Y. Yen, D.-S. Yu, S.-Z. Lin, H.-J. Harn, Acetone extract of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity, Life sciences 73(18) (2003) 2383-2394. [33] A.H. Basta, H. El-Saied, M.M. El-Deftar, A.A. El-Henawy, H.H. El-Sheikh, E.H. Abdel-Shakour, M.S. Hasanin, Properties of modified carboxymethyl cellulose and its use as bioactive compound, Carbohydrate polymers 153 (2016) 641-651. [34] Y. Chen, X. Zhou, X. Yin, Q. Lin, M. Zhu, A novel route to modify the interface of glass fiberreinforced epoxy resin composite via bacterial cellulose, International Journal of Polymeric Materials and Polymeric Biomaterials 63(4) (2014) 221-227. [35] P. Ross, H. Weinhouse, Y. Aloni, D. Michaeli, P. Weinberger-Ohana, R. Mayer, S. Braun, E. De Vroom, G. Van der Marel, J. Van Boom, Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid, Nature 325(6101) (1987) 279. [36] Z. Yan, S. Chen, H. Wang, B. Wang, C. Wang, J. Jiang, Cellulose synthesized by Acetobacter xylinum in the presence of multi-walled carbon nanotubes, Carbohydrate Research 343(1) (2008) 73-80. [37] A. Hirai, M. Tsuji, H. Yamamoto, F. Horii, In situ crystallization of bacterial cellulose III. Influences of different polymeric additives on the formation of microfibrils as revealed by transmission electron microscopy, Cellulose 5(3) (1998) 201-213. [38] L. Drago, M. Toscano, M. Bottagisio, Recent evidence on bioactive glass antimicrobial and antibiofilm activity: A mini-review, Materials 11(2) (2018) 326.

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[39] H.-J. Son, H.-G. Kim, K.-K. Kim, H.-S. Kim, Y.-G. Kim, S.-J. Lee, Increased production of bacterial cellulose by Acetobacter sp. V6 in synthetic media under shaking culture conditions, Bioresource Technology 86(3) (2003) 215-219. [40] E. Zhang, C. Zou, G. Yu, Surface microstructure and cell biocompatibility of siliconsubstituted hydroxyapatite coating on titanium substrate prepared by a biomimetic process, Materials Science and Engineering: C 29(1) (2009) 298-305. [41] S. Hesaraki, M. Gholami, S. Vazehrad, S. Shahrabi, The effect of Sr concentration on bioactivity and biocompatibility of sol–gel derived glasses based on CaO–SrO–SiO2–P2O5 quaternary system, Materials Science and Engineering: C 30(3) (2010) 383-390. [42] S. Ibrahim, H. El Saied, M. Hasanin, Active paper packaging material based on antimicrobial conjugated nano-polymer/amino acid as edible coating, Journal of King Saud University-Science (2018). [43] R.P. Rubin, G.B. Weiss, W. James Jr, Calcium in biological systems, Springer Science & Business Media2013. [44] O. Leppäranta, M. Vaahtio, T. Peltola, D. Zhang, L. Hupa, M. Hupa, H. Ylänen, J.I. Salonen, M.K. Viljanen, E. Eerola, Antibacterial effect of bioactive glasses on clinically important anaerobic bacteria in vitro, Journal of Materials Science: Materials in Medicine 19(2) (2008) 547-551. [45] E. Munukka, O. Leppäranta, M. Korkeamäki, M. Vaahtio, T. Peltola, D. Zhang, L. Hupa, H. Ylänen, J.I. Salonen, M.K. Viljanen, Bactericidal effects of bioactive glasses on clinically important aerobic bacteria, Journal of Materials Science: Materials in Medicine 19(1) (2008) 27-32. [46] L. Hench, J. Wilson, Introduction to Bioceramics, World Sci, Publ. Co., New Jersey, EUA (1993). [47] S. Janardan, P. Suman, G. Ragul, U. Anjaneyulu, R. Shivendu, N. Dasgupta, C. Ramalingam, S. Swamiappan, K. Vijayakrishna, A. Sivaramakrishna, Assessment on the antibacterial activity of nanosized silica derived from hypercoordinated silicon (iv) precursors, RSC Advances 6(71) (2016) 66394-66406. [48] M. Andresen, P. Stenstad, T. Møretrø, S. Langsrud, K. Syverud, L.-S. Johansson, P. Stenius, Nonleaching antimicrobial films prepared from surface-modified microfibrillated cellulose, Biomacromolecules 8(7) (2007) 2149-2155. [49] J. Luan, J. Wu, Y. Zheng, W. Song, G. Wang, J. Guo, X. Ding, Impregnation of silver sulfadiazine into bacterial cellulose for antimicrobial and biocompatible wound dressing, Biomedical Materials 7(6) (2012) 065006.

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Journal Pre-proof Figure 1: (a) conjugation between BC and NBG through hydrogen bond. (b) BC yield (g/L) (i), pH (ii) and residual sugar (g/L) (iii) after 4, 5, 6 and 7 d. (c) cloud-like cellulose pellicles of BC/NBG

Figure 2. (A) TEM of NBG particles, BC (with magnified part in the circule), BC/NBG and BC/NBG-CaCl2; a, b, c and d, respectively. (B) Elemental mapping by EDX analysis of BC/NBG and BC/NBG-CaCl2. Figure 3. XRD (a), FT-IR (b) and TGA (c) of BC, BC/NBG and BC/NBG-CaCl2

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Figure 4. (i) SEM micrographs and EDX analysis of BC, BC/NBG and BC/NBG-CaCl2 (a, b and c, respectively) after immersion in SBF for 21 days. (ii) pH (a) and concentration of Ca and P ions (b and c, respectively) in SBF incubated BC, BC/NBG and BC/NBG-CaCl2 samples for 21 d.

na

lP

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Figure 5: Morphological characteristics of Vero cells treated with and untraded sample observed after 24 h treatment under an inverted microscope. (a) Control Vero cells, (b) Vero cells treated with (BC 125µg mL-1), (c) Vero cells treated with (BC/NBG 125µg mL-1) and (d) Vero cells treated with (BC/NBG-CaCl2 12.5µg mL-1). Magnification: ×40.The cytotoxicity effect of BC, BC/NBG and BC/NBG-CaCl2.

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Figure 6: The antimicrobial activity of BC (1), NBG (2) and BC/NBG (3) by agar plat diffusion method:.P. vulgaris (a), E.coli(b), S. typhimurium (c) , P. aeruginosa (d), K. pneumonia (e), B. subtilis(f), S. aureus (g), C. albicans (h) and A. parasiticus(i).

25

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Table 1. Weight loss % of BC, BC/NBG and BC/NBG-CaCl2. Samples

Evaporation

Wt.

Temp.

Main decomposition

Residual

Decomposition

Remaining

Weight

content

temp.

Weight %

loss %

Wt.%

T0

T

W0

W

85

93

145 241

86

BC/NBG

90

128

174 249

89

BC/NBG

94

119

185 257

-p

90

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na

lP

re

-CaCl2

84

52

14

85

69

43

ro

BC

of

%

26

86

37 58

Journal Pre-proof Table 2. Zeta potential measurements of BC, BC/NBG and BC/NBG-CaCl2. zeta potential measurements Cell current,

Av. Phase

Av. Mobility, M.U.

mA

shift, rad/sec

BC

1.30

-8.79

-0.28

-4.0

BC/NBG

1.15

-10.07

-0.31

-4.43

BC/NBG -CaCl2

0.60

-11.66

-0.41

-5.93

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potential, mV

-p re lP na Jo ur 27

Av. Zeta

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Table 3. Determination of the antimicrobial susceptibility of each of NBG and BC/NBG nanocomposite with different concentrations by using CFU method 25 (mg/ml) NBG

Tested Strains

50 (mg/ml)

BC/NBG

NBG

100 (mg/ml)

BC/NBG

NBG

150 (mg/ml)

BC/NBG

NBG

200 (mg/ml)

BC/NBG

NBG

BC/NBG

Av.

S.D.

Av.

S.D.

Av.

S.D.

Av.

S.D.

Av.

S.D.

Av.

S.D.

Av.

S.D.

Av.

Av.

S.D.

Av.

S.D.

1.5

62.3

2.5

65.0

3.0

90.7

4.7

70.8

3.2

95.1

2.7

90.8

3.2

95.0

2.6

94.9

1.5

95.7

1.5

-

-

30.0

2.0

22.7

2.5

76.3

4.0

59.7

2.9

93.3

2.5

Pr. vulgaris

10.0

2.0

59.3

1.5

32.7

2.5

91.3

2.1

82.2

2.7

94.0

2.0

f o

S.D.

19.3

Klebsiella pneumonia

-

-

69.7

2.5

29.3

2.5

92.7

2.5

88.3

2.5

S. typhimurium

25.0

3.0

86.7

2.3

78.0

2.0

95.3

2.1

80.2

3.0

B. subtilis

6.7

1.3

40.7

3.1

32.7

3.1

86.3

4.5

83.9

2.4

S. aureus

-

-

53.0

2.0

27.3

2.5

95.0

2.0

79.7

3.2

C. albicans

-

-

32.3

3.5

19.3

1.5

84.0

3.5

67.9

A. parasiticus

-

-

-

-

-

-

34.3

3.2

27.0

E. coli Ps. Aeruginosa

l a

o J

28

3.9

95.8

1.4

95.0

2.0

94.5

1.5

90.7

3.1

97.1

2.0

93.7

1.5

96.2

2.6

92.5

2.2

95.4

2.5

95.5

2.5

96.4

1.9

93.9

2.0

93.2

3.0

93.9

1.0

95.1

1.9

93.3

1.2

92.2

2.6

93.1

3.1

96.8

2.5

95.0

1.3

96.0

1.0

96.5

1.3

93.1

3.8

93.9

2.6

95.0

2.6

94.3

2.9

97.4

0.5

2.5

91.2

3.9

95.3

2.4

95.4

1.4

96.4

1.9

97.3

1.2

2.6

77.3

4.5

67.8

5.5

76.7

3.5

94.0

1.0

94.4

1.9

r P

n r u

Av. = average; S.D. = standard deviation

p e

ro 87.4

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6