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Bacterial cellulose sponges obtained with green cross-linkers for

tissue engineering

Adriana Nicoleta Frone, Denis Mihaela Panaitescu, Cristian Andi

Nicolae, Augusta Raluca Gabor, Roxana Trusca, Angela Casarica,
Paul Octavian Stanescu, Dora Domnica Baciu, Aurora Salageanu

PII: S0928-4931(19)32360-4
DOI: https://doi.org/10.1016/j.msec.2020.110740
Reference: MSC 110740

To appear in: Materials Science & Engineering C

Received date: 26 June 2019

Revised date: 20 December 2019
Accepted date: 9 February 2020

Please cite this article as: A.N. Frone, D.M. Panaitescu, C.A. Nicolae, et al., Bacterial
cellulose sponges obtained with green cross-linkers for tissue engineering, Materials
Science & Engineering C (2020), https://doi.org/10.1016/j.msec.2020.110740

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as the addition of a cover page and metadata, and formatting for readability, but it is
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© 2020 Published by Elsevier.

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Bacterial cellulose sponges obtained with green cross-linkers for tissue engineering

Adriana Nicoleta Frone1, Denis Mihaela Panaitescu1*, Cristian Andi Nicolae1, Augusta Raluca Gabor1,

Roxana Trusca2, Angela Casarica3, Paul Octavian Stanescu2, Dora Domnica Baciu4, Aurora Salageanu4
1
National Institute for Research & Development in Chemistry and Petrochemistry - ICECHIM, 202 Splaiul

Independentei, 060021 Bucharest, Romania;

2
University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania;
3
National Institute for Chemical Pharmaceutical Research and Development, 112 Calea Vitan, 031299, Bucharest,

Romania

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Cantacuzino National Medical-Military Institute for Research and Development, 103 Spl. Independentei, 050096

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Bucharest, Romania

Corresponding author e-mail address: panaitescu@icechim.ro; phone number: +4 021 312 30 68

Abstract:
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Three-dimensional (3D) porous structures with controlled pore size and interconnected pores, good mechanical
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properties and biocompatibility are of great interest for tissue engineering. In this work we propose a new strategy to

obtain highly porous 3D structures with improved properties using bacterial cellulose (BC) and eco-friendly additives
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and processes. Glucose, vanillin and citric acid were used as non-toxic and cheap cross-linkers and γ-

aminopropyltriethoxysilane was used to partially replace the surface OH groups of cellulose with amino groups. The
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efficiency of grafting and cross-linking reactions was confirmed by Fourier transform infrared spectroscopy and X-ray
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photoelectron spectroscopy. The morphological investigation of BC sponges revealed a multi-hierarchical organization

after functionalization and cross-linking. Micro-computed tomography analysis showed 80-90% open porosity in

modified BC sponges. The thermal and mechanical properties of the sponges were influenced by the cross-linker type

and concentration. The strength-to-weight ratio of BC sponges cross-linked with glucose and citric acid was 150% and

120% higher compared to that of unmodified BC sponge. In vitro assays revealed that the modified BC sponges are non-

cytotoxic and do not trigger an inflammatory response in macrophages. This study provides a simple and green method

to obtain highly porous cellulose sponges with hierarchical design, biocompatibility and good mechanical properties.

Keywords: bacterial cellulose, nanofibrous sponge, computed tomography, surface functionalization, cytotoxicity

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1. Introduction

Bacterial cellulose (BC) has attracted a lot of interest due to its extraordinary properties: high purity, water-uptake

capability, good biocompatibility and hemocompatibility, cell adhesion and proliferation, no allergenic reaction after

implantation, non-toxicity of itself and its degradation products and also good mechanical properties [1-5]. All these

recommend BC for biomedical applications. Nanofibrillated cellulose (NFC) and cellulose nanocrystals (CNC) isolated

from plants and wood were also studied for potential application in biomedicine [1,6-9]. NFC modified by tempo-

oxidation and carboxymethylation showed no toxicity in vitro and in vivo, however, both developed an early mild

inflammatory response [8]. It has been observed that NFC with a very high purity shows a low endotoxin level and may

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be applied in wound healing and scaffolding [7]. However, cotton CNC in higher concentration induced cell death and

changes in the gene expression of mammalian dermal fibroblast [9]. Contrarily, BC does not need extensive purification

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because it is not contaminated by hemicelluloses or lignin and it has better biocompatibility than plant or wood

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nanocellulose due to its biosynthesis [6]. Moreover, the cell growth and adhesion on BC was thoroughly evaluated and
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no inflammation or immune rejection was observed [10].

A key element in tissue engineering is the three-dimensional (3D) biomaterial scaffold which mimics the
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architecture of the extracellular matrix (ECM). ECM provides structural support for cell attachment, proliferation and

differentiation [11,12]. For this purpose, the 3D scaffolds should possess a high porosity (>80%) [13] and a network of
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interconnected pores that ensures cell migration, diffusion of nutrients, clearance of wastes at the same time promoting

cell adhesion and growth. In many tissues (heart, cartilages, bones), the ECM has a fiber-sponge complex structure [14],
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therefore a fibrous matrix with sponge-like architecture could successfully mimic ECM. The nanofibrillated network of
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BC is similar to that of the main component of the ECM, collagen, with respect to biocompatibility, fibers size and

assembling. In addition, BC is less immuno-stimulatory than collagen [15].

However, the pore size of the cellulose network in BC membranes is low, generally from 20 nm to 10 µm,

which limits cell penetration and migration [16,17]. Larger pore size is needed for effective transfer of nutrients and

vascularization [18,19]. The pore size and interconectivity are dictated by the size of the cells which will migrate in the

scaffold and the type of tissue to be reconstructed [18-20]. Pore sizes between 250 and 500 µm ensure chondrocytes

proliferation and ECM production, between 20 and 125 µm are recommended for regeneration of adult mammalian skin

and between 100 and 350 µm for regeneration of bone [19]. The shape and the size of the pores as well as porosity can

be adjusted by the method and conditions used to obtain cellulose scaffolds [19,20]. Salt crystals, agarose

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microparticles, paraffin, starch or gelatin microspheres were used to obtain cellulose scaffolds with different pore sizes

and properties [16,21,22]. Cellulose 3D scaffolds with the pore size of 200–500 µm, good biocompatibility and cell

adhesion were obtained by a salt leaching technique applied to cotton linters solutions in ionic liquid [21]. Cellulose

sponges for drug delivery were obtained by dissolving microcrystalline cellulose in NaOH/water solvent, non-solvent

extraction and freeze-drying [23]. Gao et al. obtained BC sponges showing good cells proliferation and growth by a

green method; crushed BC fabrics dispersed in water were freeze-dried then immersed in polyethylene glycol, soaked in

deionized water and freeze-dried again [17]. Microporous BC scaffolds for bone tissue regeneration were prepared by

incorporating paraffin wax spheres in the culture medium and removing the paraffin by cyclic washing using a

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surfactant [22]. Similarly, gelatin microspheres, 100 - 200 μm in diameters, were used as a porogen in a BC solution in

N-methylmorpholine - N-oxide monohydrate and led to 3D scaffolds with regular microporosity but high percentage of

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gelatin entrapped in the scaffold [5].

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Although benefic in tissue engineering, high porosity and big pores compromise the structural integrity of the
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scaffolds and their mechanical properties [24]. In several approaches, chemical modification of cellulose by cross-

linking was tried to improve the mechanical properties and stability in the body environment [23-25]. For example,
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epichlorhydrin and glutaraldehyde were used as cross-linkers in cellulose - NaOH solution [23] and in poly (vinyl

alcohol)/nanocellulose mixtures [25] in water. BC/gelatin composite sponges were obtained by dissolving BC in 1-
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butyl-3-methylimidazolium chloride and gelatin in dimethyl sulfoxide, mixing the solutions and adding glutaraldehyde

as a cross-linker [10]. However, epichlorhydrin and glutaraldehyde show acute toxicity [26] and extensive washing (e.g.
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four days, 12 times a day) is necessary for removing the solvent and unreacted aldehyde and to mitigate cellular toxicity
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[10].

Most of the methods proposed so far to obtain 3D cellulose structures for tissue engineering use toxic or

expensive solvents, are very complicated or do not assure the complete removal of the porogen or the desired properties

[5,10,22]. Simultaneous achievement of high porosity, good mechanical properties, biocompatibility and cell adhesion is

a difficult task. Therefore, this work proposes a new strategy to obtain biocompatible, lightweight 3D cellulose

structures with improved mechanical properties and high porosity starting from bacterial cellulose membranes obtained

from apples residues by fermentation. The new strategy consists in the mechanical defibrillation of BC membranes,

surface functionalization and cross-linking using green cross-linkers and, finally, freeze-drying. Functionalization and

cross-linking were used for balancing a high porosity with good mechanical properties. Glucose (GL), vanillin (V) and

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citric acid (CA) were selected as green cross-linkers. Glucose is a nontoxic and cheap alternative to the widely used

cross-linking agents, such as glutaraldehyde or epichlorohydrin, and it may act as a cryoprotector of cellulose during

freeze-drying. Glucose was used as a cross-linking agent of gelatin in gelatin/bacterial cellulose sponges for tissue

engineering [27]. Vanillin, a phenolic aldehyde, is obtained from a tropical orchid species and it is considered as a

―safe‖ additive being used as a flavor and for the preservation of food [28]. In addition, vanillin has bioactive properties,

being used as a constituent in cosmetic preparations and some medications. Due to the presence of aldehyde groups in

its structure, vanillin can also be used as a cross-linking agent [29]. Citric acid is a nontoxic cross-linker, widely used in

the food and pharmaceutical industry and a key intermediate in the metabolism of living organisms [30]. It was used for

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cross-linking cellulose with other modifiers, like poly(ethylene glycol) [31]. In our work, vanillin, glucose and citric

acid were used for the first time as cross-linkers of amino modified cellulose.

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The hypothesis underpinning this research is that the efficiency of these green cross-linkers may be increased

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by replacing some of the OH surface groups of cellulose by amino groups, which are more reactive. Therefore, surface

OH groups of cellulose were partially replaced with amino groups using γ-aminopropyltriethoxysilane (APS). APS is
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often used to modify cellulose for biomedical applications because it has low toxicity and high reactivity [32] and it may
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provide antibacterial activity to cellulose [33]. Then, glucose, vanillin and citric acid were covalently bonded to

cellulose by the reaction between the NH2 from the functionalized cellulose and the aldehyde or carboxyl groups of the
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cross-linkers. Compared to other methodologies this new one is more advantageous due to the lack of cellulose

solubilization by toxic or expensive solvents and removal of porogen or toxic cross-linking agents. The cross-linked BC-
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sponges were characterized to understand the influence of surface modification on the structure, thermal and mechanical
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properties as well as cell viability and inflammatory response. Moreover, the morphology of these cellulose sponges was

thoroughly investigated to highlight the hierarchical arrangement from nanometric to macroscopic lengths.

2. Experimental section

2.1. Materials

Glacial acetic acid (99.8%), absolute ethanol (99.5%) and chloroform were purchased from Chimreactiv (Bucharest,

Romania). Reagent grade γ-aminopropyltriethoxysilane (99%), glucose and citric acid with a purity higher than 99.5%

and high purity vanillin (Ph.Eur. 0.998%) were purchased from Sigma-Aldrich (Germany). All the reagents were used

without further purification.

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2.2. Synthesis of bacterial cellulose membranes

Gluconacetobacter Xylinus from Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures was

used as bacterial strain and fruit residues extract as carbon source. 50 mL of culture medium containing 7.5% glucose

equivalents from apples residues, 2% glycerin, 0.2% ammonium sulfate and 0.5% citric acid, was inoculated with 10%

(v/v) inoculum and kept in an autoclave at 121 °C for 15 min. The incubation was carried out under static conditions at

30 °C for a period of two weeks. The resulted membranes were washed thoroughly with distilled water and treated with

1 N NaOH at 30 °C for 2 days for cell lysis. The membranes were then treated with sodium azide water solution

(0.02%) to reduce microbial contamination and neutralized with 1% acetic acid. BC membranes (Fig. S1a) were washed

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with distilled water and kept at 4 °C until use.

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2.3. Surface modification of BC

Nanocellulose was obtained from bacterial cellulose membranes by mechanical defibrillation. The fresh membranes

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were first disintegrated with a blender for 15 min giving a gel, and then using a vertical colloid mill with recirculation
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for two hours. Finally, the suspension was homogenized with a Microfluidizer LM20 (Microfluidics, USA, ten passes).

Nanocellulose obtained by this method was denoted as BC.

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For the chemical grafting of BC, 1 g APS was pre-hydrolyzed in a 90/10 vol% ethanol/water mixture (100 mL)
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at room temperature for 2 h. Glacial acetic acid was added to adjust the pH of the silane solution to 4 and the solution

was stirred continuously. After the stabilization of the pH, an amount of 200 g BC water suspension (0.7 wt% BC) was
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added to the 100 mL silane solution (0.5 mol APS/mol anhydroglucose unit) and stirred at ambient temperature for 2 h.

Then, the mixture was heated under reflux for 2 h to perform the chemical reaction. After cooling, the mixture was
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frozen (-20 °C) for at least 24 h and then lyophilized (FreeZone Plus 2.5L Freeze Dry System, Labconco, USA) at -85

°C under vacuum (0.008 mbar) for 72 h, resulting amino modified BC sponge (sample denoted as BCA). The cross-

linking of BCA was performed using glucose, vanillin and citric acid. The schematic representation of the methods used

to obtain BCA and cross-linked BCA sponges is shown in Fig. 1 and the possible reactions on the surface of BC are

schematically presented in Fig. 2 (a-d).

2.4. Preparation of cross-linked BC structures

A 10% GL solution was added to the BCA suspension (1 wt% BCA) to obtain a mass ratio of 1:1 BC:GL. The

suspension was homogenized by magnetic stirring for 15 minutes, and then it was placed in an air-circulation oven at

100 °C for 90 minutes to initiate the condensation reaction between the amino group on BCA and the GL. After cooling,

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the sample was frozen (-20 °C) for at least 24 h and then lyophilized at -85 °C under vacuum (0.008 mbar) for 72 h,

resulting BCA-GL. The reaction of aminated cellulose with glucose is similar to the Maillard reaction and is initiated by

the condensation of the carbonyl group of the reducing glucose with the amino group of APS-functionalized BC (Fig.

2b). Further, Amadori products may form cross-linked structures with other amino groups [34].

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Fig. 1 Schematic representation of the methods used to obtain BCA and cross-linked BCA sponges
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An amount of vanillin was added to ethyl alcohol to obtain a 2.5% solution and left at room temperature under
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magnetic stirring until complete dissolution. The vanillin solution was added to the water suspension containing 1 wt%
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BCA to obtain a proportion of 5% and 50% of vanillin:cellulose (mass ratio). After magnetic stirring for 3 h at 50 °C

and sonication for 1 h using an ultrasonic bath (Elmasonic S40h), the mixtures were frozen for at least 24 h (-20 °C) and

lyophilized at -85 °C under vacuum (0.008 mbar) for 72 h. The resulted 3D structures were denoted as BCA-5V and

BCA-50V (Fig. 1). The possible reactions in the case of BCA cross-linking with vanillin are shown in Fig. 2c. Both

Schiff base formation and acetalization were proposed as mechanisms for the cross-linking of chitosan with vanillin [29].

An aqueous solution of CA (2.5%), prepared by magnetic stirring at room temperature, was added over the

BCA water suspension (1 wt%) in the amounts corresponding to the concentrations of 5 wt% and 50 wt% of CA related

to the amount of cellulose. The mixtures were subjected to cross-linking under reflux at 80 °C for 24 h. Subsequently,

the mixtures were sonicated for 1 h, then frozen for at least 24 hours (-20 °C) and lyophilized.

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Fig. 2 (a) Grafting of aminosilane moieties at the surface of BC (BCA); (b) Cross-linking of BCA by glucose (BCA-

GL); (c) Possible reactions in the case of vanillin cross-linked BCA; (d) Cross-linking of BCA with citric acid

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The 3D structures were denoted as BCA-5CA and BCA-50CA (Fig. 1). One possible reaction between the

amino groups on functionalized cellulose and citric acid is shown in Fig. 2d. Esterification of hydroxyl groups of

cellulose by citric acid may also take place. BC was lyophilized in the same conditions with the cross-linked samples

and served as a reference besides BCA.

2.5 Characterization

2.5.1 Fourier transform infrared (FTIR) spectroscopy

The FTIR - Attenuated Total Reflectance (ATR) analysis was carried out in duplicate on a Tensor 37 spectrophotometer

from Bruker (USA), with an ATR setup. Data were collected at room temperature from 4000 to 400 cm-1 with 16 scans

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per spectrum at a resolution of 4 cm-1.

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2.5.2 Scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX)

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Cross-section morphology of sponges was assessed by scanning electron microscopy. The sponge samples were cracked
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in liquid nitrogen and the cross-sections were sputter-coated with gold and characterized by SEM-EDX. For this

analysis, a Quanta Inspect F scanning electron microscope (FEI-Philips, USA), equipped with a field emission gun was
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used. This worked at an accelerating voltage of 30 kV with a resolution of 1.2 nm. The composition was analyzed with

an energy dispersive X-ray (EDX) spectrometer coupled to SEM, with a resolution of 133 eV at MnKα.
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2.5.3 X-ray Photoelectron Spectroscopy (XPS)

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XPS measurements were done using a K-Alpha spectrometer (Thermo Scientific, USA) equipped with a

monochromated Al Kα source (1486.6 eV) under ultrahigh vacuum conditions (2 × 10 −9 mbar). Charging effects were
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compensated by a flood gun. The XPS spectra were recorded as survey spectra (step of 1 eV) at a pass energy of 200 eV

2.3.4 Thermogravimetric analysis (TGA)

TGA was performed using a TGA Q5000 (TA Instruments, USA). Samples of about 5 mg were placed in aluminum

pans and heated from 25 ºC to 700 ºC, at a heating rate of 10 ºC/min under nitrogen flow (50 mL/min).

2.5.5 Dynamic mechanical analysis (DMA)

The BC sponges were analyzed using a DMA Q800 (TA Instruments, USA) operating in compression mode. The

specimens for the compression test were cylinders with a height of 5-6 mm and a diameter of 14.5 mm. The sponges

were tested at 30 °C using a force ramp of 0.1 N/min from 0.001 N to 10.000 N and a compression clamp. The density

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of the sponges was calculated by dividing the mass of the sponge by its volume. Measurements were carried out in

triplicates.

2.5.6 Micro-computed tomography (CT)

Micro-computed tomography analysis was carried out using a high-resolution Bruker μ-CT SkyScan 1272 (Germany)

equipment. The samples were scanned without filter, using a source of 50 kV, 200 µA, with an exposure time of 250

ms/frame. Projections were acquired over a range of 180°, with a rotation step of 0.2°. The image pixel size

corresponded to 6 µm. The tomograms were reconstructed using the NRecon software (Bruker, Germany) and analysed

with the CTAn software (Bruker, Germany). After thresholding, the binary images consisted of only black and white

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associated to the pores and sample itself. The images were processed by 3D analysis for the quantification of total and

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open porosity, structure separation and structure thickness. ―Wall thickness‖ (structure thickness) describes the width of

the samples walls as a function of the product between the number of white pixels and the scanning resolution.

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―Porosity‖ (structure separation) describes the width of the samples pores as a function of the product between the
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number of black pixels and the scanning resolution.
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2.5.7 In vitro cytotoxicity

The potential cytotoxicity of surface modified, cross-linked BC sponges and of their hydrolytic degradation products
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was evaluated against L929 murine fibroblasts. L929 cells, purchased from European Collection of Authenticated Cell

Cultures (ECACC, collection of Public Health England), were cultivated in DMEM (Dulbecco's Modified Eagle
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Medium, Lonza, Belgium) supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Germany) and antibiotics,
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100 U/ml penicillin - 100 μg/ml streptomycin (Lonza, Belgium). About 5 mg of BC-based formulations were incubated

overnight at room temperature in 1 mL serum-free culture medium under sterile conditions. L929 cells were seeded in a

96-well plate at a density of 5×104 cells/well and allowed to adhere overnight at 37 ºC in humidified atmosphere with

5% CO2. After culture medium removal, cells were exposed for 24 h to undiluted and a dilution series (50.00%, 25.00%,

12.50%, 6.25%, and 3.13%) of the sample extracts. Each eluate and the control were tested in triplicate wells. Cell

viability was estimated by MTT assay, a method based on the reduction of soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl tetrazolium bromide (MTT, Sigma-Aldrich, USA) to blue insoluble formazan by cellular enzymes in

metabolically active cells. Briefly, at the end of incubation period, culture media was removed from the cells and fresh

culture medium with 5% MTT was added to each well. The plates were incubated for additional 3 hours. Formazan

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crystals were dissolved overnight at 37 ºC with lysis buffer containing 20% sodium dodecyl sulfate (Sigma-Aldrich,

USA), 50% N,N-dimethylformamide, 0.4% acetic acid and 0.04 hydrochloric acid (Merck, USA). Sample absorbance

which directly correlates with the number of metabolically active cells was measured at 570 nm using a microplate

reading spectrophotometer (Thermo Scientific, USA). Cell viability was calculated as follows:

Cell viability (%) = [absorbance (sample)/ absorbance (control)] × 100. (1)

2.5.8 Evaluation of pro-inflammatory effect

RAW 264.7 murine macrophages, purchased from ECACC, collection of Public Health England, were cultivated in the

same conditions with L929 murine fibroblasts. Experiments were run in triplicate. For stimulation experiments, RAW

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264.7 cells were seeded in a 96-well plate at a density of 5×104 cells/well and cultured overnight to allow adherence and

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to reach the exponential growth phase. After culture medium removal, cells were exposed for 24 h to serial dilutions

(50.00%, 25.00%, 6.25%, and 3.13%) of the sample extracts. Lipopolysaccharide (LPS, Invivogen, USA) was used as

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positive control (final concentration 2 μg/mL). Supernatants were harvested after 3 h for tumor necrosis factor-α (TNF-
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α) measurement and after 24 h for nitric oxide (NO) determination. The amount of NO was measured by the
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accumulation of nitrite in the culture supernatants, collected after 24 h of stimulation, using a colorimetric reaction with

the Griess reagent (0.1% (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride and 1% (w/v) sulfanilamide containing
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5% (w/v) H3PO4). 80 μL of cell culture supernatants were mixed with equal volume of the Griess reagent for 10 min in

the dark. The standard curve was created by using known concentrations of sodium nitrite, and the absorbance was
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measured at 540 nm using a microplate reader spectrophotometer (Thermo Scientific, USA). TNF-α levels were

measured on cell supernatants collected after 3 h of stimulation using an ELISA kit (DuoSet, R&D Systems Inc., USA).
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Data were normalized to cell number determined by MTT assay using unstimulated cells as reference.

2.5.9 Water uptake ability

The ability of BC cross-linked scaffolds to take up water was evaluated by weighing the scaffolds (w0) and immersing in

deionized water for 24 h. The weights after immersion and the removal of the excess water using a filter paper (wi) were

measured and the swelling degree (S) was calculated by the formula:

( ) (2)
The experiments were repeated three times and the results were presented as mean ± standard deviation. The

significant (p < 0.05) and very significant (p < 0.01) differences were evaluated for mechanical, porosity, swelling and

biological tests using the Student t-test.

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3. Results and discussion

3.1 Structural analysis by FTIR, EDX and XPS

Fig. 3a shows the FTIR spectra of pristine BC and APS treated BC (BCA). A narrower infrared band, between 3500 and

3200 cm-1, was observed in BCA compared to BC. This band is assigned to hydrogen-bonded hydroxyl groups of

cellulose, showing weaker intermolecular interactions and reduced hydrogen bonding in BCA due to the grafting

reaction with APS [2,35].

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Fig. 3 FTIR spectra of BC and BCA sponges (a); FTIR spectra of BCA cross-linked with vanillin (5% and 50% V),

citric acid (5% and 50% CA) and glucose (BCA-GL) (b); FTIR spectra of BCA-V in the range from 1800 to 1200 cm-1

(c); FTIR spectra of BCA-CA in the range 1850 - 700 cm-1 (d)

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New shoulders were observed at 2929 and 2973 cm-1 in BCA. They are characteristic to the C-H stretching

vibration in the propyl moiety of APS, the asymmetric vibration in CH 2 at 2929 cm-1 and that in CH3 at 2973 cm-1 [36].

The small peak at 1724 cm-1 in BCA, may be due to the carbonyl groups, showing a slight oxidation of cellulose

because of the chemical and thermal treatments. The effectiveness of the chemical grafting of cellulose with APS was

also supported by the new bands at 1625 cm-1 and 1505 cm-1, which are characteristic to the asymmetric and symmetric

deformations of the NH2 moiety [37,38]. Other new peaks appeared at 466 cm-1 and 796 cm-1. They may be assigned to

Si-O-C asymmetric bending [39] and Si-C/Si-O bonds [40], respectively, also proving the grafting of the aminosilane on

cellulose. The shoulders at 960/930 cm-1 show the presence of free silanol groups not involved in grafting or cross-

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linking reactions [38,41].

The addition of the cross-linkers (V, CA and GL) induced further changes in the FTIR spectra of BCA (Fig.

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3b). V is an aromatic aldehyde and the benzene ring may have different effects on the cross-linking reaction depending

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on the environment. The band characteristic to the bending vibration of phenolic OH, which appears at about 1266 cm-1

in pristine V [42] was shifted at 1293 cm-1 in the cross-linked BCA (Fig. 3c); however the band at 1514 cm-1 which may
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be associated to the stretching of the benzene ring did not change its position compared to pristine V. The band at 1595 cm-
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is mostly assigned to the amino group of silanes [43] and its intensity decreased in the case of BCA-50V, showing the

involvement of the amino group in the cross-linking reaction. The shoulder at 1639 cm-1 may be ascribed to the stretching
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vibration of C=N group [43,44] proving the reaction between the carbonyl group of vanillin and the amino group of BCA and

the formation of a Schiff base (Fig. 2c).

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FTIR spectra of CA cross-linked cellulose (Fig. 3b) show a broad band between 2500 and 2700 cm-1 which
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may be associated to the OH stretching vibrations of carboxylic acids (CA in this case). It is remarkable that the band

observed at 1505 cm-1 in BCA was missing in the cross-linked samples showing the involvement of NH2 group in the

cross-linking reactions (Fig. 3d). The peak characteristic to C=O vibrations was shifted from about 1730 cm-1 in CA to

1710-1720 cm-1, which is specific to the ester bonding in BCA-CA [45]. This shows that the cross-linking reaction

consumed CA and led to ester linkages with cellulose. This overlapped with the absorption band of C=O (amide group)

at about 1650 cm-1, which results from the reaction of the amino group of BCA with CA (Fig. 2d). The peak at 1398 cm-
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can be assigned to the symmetric stretching of the carboxylic group in unreacted CA [46].

The cross-linking of amino-modified cellulose by GL is observed in the FTIR spectra by the broadening of the

OH band and the appearance of a new peak at 3301 cm-1 (Fig. 3b); these changes are due to the new hydrogen bonds

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induced by the addition of GL in equal proportion with BCA. The new peak observed at 1737 cm-1 shows that GL has

participated in the cross-linking reaction forming an Amadori product [47] (Fig. 2b). The absence of the band at 1505

cm-1, characteristic to amino group [37], shows the involvement of NH2 group in the cross-linking reactions.

The grafting of the silane on BC forming Si-O-C, Si-O-Si and Si-OH groups cannot be clearly detected by

FTIR because of overlapping with the vibrations of cellulose. Therefore, EDX analysis was performed to highlight the

elemental composition in the cross-section of pristine BC and BCA sponges (Fig. S2). EDX results show the presence of

Si and N elements in the cross-section of BCA sponge and none of these elements in BC. The high atomic percentage of

N (3.8%) may be due to the low accuracy of EDX when analyzing light elements [48] or to the presence of N in low

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concentration in the neat BC membranes because of the residual proteins from biosynthesis [2].

To clarify these issues, the BC and uncross-linked and cross-linked BCA sponges were analyzed by XPS and

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the survey spectra are shown in Fig. 4. Besides carbon and oxygen, centered at 285 and 532 eV, the XPS spectrum of

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neat BC also contains nitrogen, at 399 eV, showing the presence of N traces in BC from the cellulose producing bacteria
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[2,49]. The XPS elemental composition on the surface of functionalized and cross-linked BC structures is shown in

Table 1. The presence of Si was observed in all BCA sponges and no silicon was found on the surface of neat BC, which
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demonstrates the presence of the grafted silane after functionalization and cross-linking. The atomic concentration of Si

in BCA (1.43%) corresponds to a grafting ratio of about 1/6 (1 APS at every 6 anhydroglucose units), which was
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estimated considering the theoretical structure of BC (11) and grafted APS (7) [50].
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Fig. 4 XPS survey spectra of BC and BCA sponges cross-linked with CA, V and GL

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A degree of surface substitution (DSS, the number of silyl groups/ anhydroglucose unit) close to 0.2 was

determined for BCA using the atomic concentration of Si determined by XPS [2]. This is a relatively small grafting ratio

[50,51], which was preferred for avoiding any cytotoxic effect of APS and for a better control of the porous structure

and mechanical properties. However, the slight toxicity of amino groups in APS, if any, was eliminated after cross-

linking as also reported for APS-functionalized hydroxyapatite [52] studied for in vivo biomedical applications.

Table 1. Atomic concentration of elements from XPS analysis on the surface of BC scaffolds

O1s C1s N1s Si2p

Samples O/C
(%) (%) (%) (%)

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BC 37.48 61.47 1.05 - 0.61
BCA 35.76 60.65 2.16 1.43 0.59

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BCA-50V 35.51 62.25 1.24 1.00 0.57
BCA-50CA 36.08 61.81 1.11 1.00 0.58
BCA-GL 36.51 61.58 0.93 0.98 0.59

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The slight variation in the concentration of C and O after cross-linking (Table 1) also confirms the APS
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grafting and the cross-linking reactions: (i) the lower O content in BCA and cross-linked BCA compared to BC due to
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the lower concentration of O in APS compared to BC; (ii) higher C concentration in BCA-50V due to the higher C

content in vanillin. Finally, ATR-FTIR, EDX and XPS confirmed the grafting of amino groups on BC surface and the
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cross-linking reactions involving the cross-linkers and the amino groups of BCA.

3.2 Thermal analysis

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The thermal degradation profiles of functionalized and cross-linked BC are shown in Fig. 5. A major degradation step
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was observed for all the samples, at 367 ºC for BC, 364 ºC for BCA, 361 ºC for BCA-50V, 347 ºC for BCA-50CA and

358 ºC for BCA-GL. This is due to the dehydration and depolymerization of cellulose and to the decomposition of the

cyclic structures which are formed at high temperature [53]. Slightly lower decomposition temperature, considered as

the temperature of the maximum degradation rate (Td) in the derivative curves, was noticed for the cross-linked samples

because of the chemical reactions. The largest difference, of 20 ºC compared to BC, was observed for BCA-50CA. This

may be due to the unbound CA (also observed by FTIR) which begins to decompose at a much lower temperature, 212

ºC [54] vs. 320 ºC for BC. Indeed, Widsten et al determined a proportion of at least 10% unbound/bound CA in the case

of linerboard cellulose cross-linked by CA [55]. Moreover, CA has anti-inflammatory effect [56] and a small amount of

residual CA in the sponges may have benefic effect for biomedical application.

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Fig. 5 TGA and DTG curves of cross-linked BCA sponges; only the samples modified by the highest concentration of
the cross-linking agent (V or CA) are presented

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The residue at 700 ºC was 4% for BC and higher after functionalization and cross-linking: 12.5% for BCA,

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14% for BCA-50V, 7.5% for BCA-50CA and 13% for BCA-GL. It is worth mention that a broad degradation peak was
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observed in the case of BCA-GL, between 150 and 250 ºC; this is due to the degradation of glucose which takes place in

this temperature range [57]. Therefore, the differences observed in the thermal degradation of BC after cross-linking
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highlight the chemical modifications induced by the cross-linking agents and the effect of unreacted products. It is worth

mention that the natural cross-linkers used in this work led to minor changes in the thermal stability of BCA considering
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the dramatic decrease of Td (> 50 °C) in the case of glutaraldehide cross-linked biopolymers [58].
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3.3 Morphology of BC sponges by SEM

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The cross-section morphology of BC sponges was investigated by SEM. An increasingly larger magnification and a

small increasing step were used to capture any morphological changes. This detailed analysis revealed a multi-

hierarchical organization in the BC sponges after functionalization and cross-linking (Fig. 6, 7, S3). All the images

highlight the presence of large pores and channels and a high degree of interconnectivity, occurring as a result of freeze-

drying, which is an eco-friendly and easy to scale-up method. Indeed, ice crystals are formed during freezing of BC

aqueous suspensions and BC nanofibers are concentrated in the interstitial regions between the ice crystals and on their

surface [59-61]. Therefore, all the samples contain 2D sheet-like structures formed by the aggregation of nanofibers due

to their high concentration between the ice crystals [61-63]. Although identical BC/BCA concentration was used in all the

samples, the different cross-linking agent and reaction conditions influenced the porous architecture as revealed by the

SEM images taken at several magnifications (Fig. 6, 7, S3).

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Fig. 6 SEM images of BC, BCA and cross-linked BCA sponges

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Fig. 7 SEM images showing the structure of the ―walls‖ in BCA sponges

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A lower degree of organization was observed in the SEM images of pristine BC (Figs. 6, S3). Pores of 300 µm

in size or larger and a multitude of pores of 40 - 200 µm were noticed. A more organized structure, with 500 µm pores

in the foreground and pores of 50-150 µm in the background (Fig. 6- BCA), was observed after APS functionalization.

This self-assembled structure is obvious in the low magnification image of BCA (Fig. 6-bottom-right corner) and in Fig.

S3 (BCA) where a ―cylinder‖ was investigated in depth. This structure has some similarities with Venus’ flower basket

(Euplectella aspergillum), a tube-shaped sea sponge with exceptional flexibility and toughness [64]. A fine ―lacy‖

structure with a multitude of small pores, 20 - 60 µm in size, was observed in Fig. 6 for BCA-5V. At a higher

concentration of V (Fig. 6, BCA-50V), sheets of BC nanofibers and a large distribution of pore sizes were visible. A

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―lacy‖ structure with micrometric pores and channels, having sizes ranging from a couple of microns to more than 150

µm, were visible in BCA-CA samples (Figs. 6). A more clear organization, close to an ―egg-box‖ structure, was noticed

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for BCA-50CA (S3). This ―egg-box‖ structure appears in cross-linked alginate and is characterized by special

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mechanical properties and high water diffusion [65]. A labyrinth of chambers and channels with a narrower distribution
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of sizes, generally from 100 to 150 µm and a high degree of organization was revealed by SEM in the case of BCA-GL

(Figs. 6 and S3). This self-assembled structure resulting from GL cross-linking may be related to better mechanical
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properties.

In summary, almost all the 3D structures resulting from BCA cross-linking show a network of chambers and
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channels, 100 – 500 µm in size, interconnected with a finer network of micrometric pores, less than 100 µm in size.

Moreover, the walls of the pores and channels in cross-linked BCA scaffolds are formed by a fine network of cellulose
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nanofibers (Fig. 7), with the thickness of 15 – 50 nm, which gives better mechanical strength and flexibility besides
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better diffusion of water and nutrients. The small differences observed between the sponges at this hierarchical level

may come from the different cross-linking agent and reaction conditions. The denser network in the case of BCA-GL is,

probably, determined by the high amount of GL which partially covered the pores. Contrarily, freeze-dried

undefibrillated BC membranes show mostly submicronic pores (Fig. S1b and c), which does not allow the penetration of

cells, the effective transfer of nutrients and vascularization.

3.4 Investigation of BC sponges by computed tomography

Micro-computed tomography (micro-CT) allows the 3D visualization of the porous structures resulted from BC

functionalization and cross-linking, giving information on open porosity, thickness of the walls and connectivity (Fig.

8). For comparison, the micro-CT image of pristine BC sponge is shown in Fig. 8. No orientation or arrangement is

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obvious in this image; however, a lot of large pores, more than 1 mm in size, may be observed, some of them being

encircled in Fig. 8. This emphasizes a poor distribution of pores in the section of the BC sponge.

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Fig. 8 Micro-computed tomography images of BC sponge and differently cross-linked BCA sponges

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It is remarkable that a special arrangement was observed in the case of BCA (Fig. 8), the figure showing a

cross-sectional image of the oriented cylinders highlighted by SEM. A finer porous structure was observed in BCA-

50CA and BCA-50V, in good agreement with the ―lacy‖ structure observed by SEM. A higher degree of organization

was observed in the case of BCA-50CA and BCA-GL, and a denser structure in the last case, similar to SEM

observations. The porosity (P) is high in all the samples, cross-linked or not, ranging from 79% in BCA-50CA to 92% in

BC, which is critically important for tissue engineering. Although the differences are small, a slight reduction in P was

observed after functionalization and cross-linking (Table 2). The difference is statistically significant for BCA-50CA (p

< 0.05), which also showed a lower mean pore size (p < 0.01). Smaller porosity was also reported for Aloe Vera-based

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sponges after the treatment with gellan gum [66].

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Table 2 Porosity (P); wall thicknesses (WT) distribution - proportion of WT up to 24 µm (WT 24) and 36 µm (WT36);

mean pore size (from the Gaussian distribution fitting)

Samples P -p
WT24 WT36 Mean pore size
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(%) (%) (%) (µm)
BC 92.1±5.6 98.6±7.7 99.9±1.1 97.8±6.7
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BCA 86.1±0.4 86.3±1.1 99.4±0.2 86.9±1.5

BCA-50V 83.5±5.0 75.4±6.5* 98.4±1.7 85.8±4.3
BCA-50CA 79.1±4.8* 69.7±7.0** 96.8±2.3 69.9±6.5**
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BCA-GL 84.2±2.5 92.9±1.7 99.6±0.1 78.0±4.0*

Significantly (*) and very significantly (**) different from BC: * p < 0.05; ** p < 0.01
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Micro-CT analysis showed that the wall thickness did not exceed 36 µm, with a high proportion below 24 µm
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(Table 2). For example, the wall thickness was below 24 µm in great proportion (98.6%) in the BC sponge. The pore

size distribution was Gaussian in all the cases (Fig. 9), however several differences were noticed.

Fig. 9 Pore size distribution of BCA sponges obtained from micro-computed tomography analysis

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A lower pore size and narrower distribution were obtained for BCA-50CA, where more than 78% of the pore

sizes range from 50 to 100 µm. The widest pore size distribution was noticed for BCA-50V, where 76% of the pore size

was in the range from 50 to 150 µm. Although BCA-GL showed a wide distribution of pore sizes, more than 60% were

from 50 to 100 µm. Pores larger than 300 µm were noticed in the BC sponge. The quantitative information obtained

from micro-tomography analysis shows the influence of the different cross-linking methods on the 3D architecture of

BCA sponges. At this micrometric resolution, X-ray CT has some limitations in the detection of very small pores [67],

however the pore size distribution in the range of larger pores, from 50 to 200 µm in our case, was well represented.

Moreover, the presence of a network of large micrometric pores was also confirmed by the SEM analysis.

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3.5 Dynamic mechanical properties

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The compression properties of BC sponges obtained with green cross-linking agents were investigated by DMA. The

compression strength values at 50% strain (σ50) are given in Fig. 10a. BC and BCA showed small σ50 values, 2 and 3

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kPa, similar to other reports [68]. CA led to the doubling of the compression strength compared to BCA when it was
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used in high concentration. Smaller variation of σ50 was observed after vanillin cross-linking, regardless the

concentration. The most important increase in strength was noticed for BCA-GL with a four times increase of the σ50.
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The compression strength of BCA-50CA and BCA-GL sponges, 6.7 and 11.4 kPa, is in the range recommended for soft
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tissues, for example for cartilage repair [68,69]. Similar compression strength values, between 5 and 8 kPa, were

obtained in the case of highly porous bacterial cellulose/chitosan scaffolds for cartilage tissue engineering [68] or for
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cross-linked nanocellulose sponges designed as implants for bone defects [70]. When comparing the compression

strength values at 70% strain or maximum compression strength (Fig. 10c), BCA-50CA and BCA-GL sponges showed
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16 and 36 kPa, which are higher or similar with previous reports [68,70,71]. Moreover, these values are similar to that

obtained for crosslinked BC - gelatin - hydroxyapatite composites (about 20 kPa) designed for bone tissue engineering [71].

Several factors might contribute to this remarkable increase of strength observed for BCA-GL, such as a higher

density, better pore size distribution, a more organized structure and cross-linking [68,71]. Cross-linking may improve

the compression strength by the intermolecular cross-links between cellulose chains due to the condensation reaction of

the carbonyl group of glucose with the amino group of BCA (Fig. 2b). This further contributed to a more efficient load

transfer, a uniform stress distribution and no stress concentration [14].

Nevertheless, the compression strength strongly depends on the density of the sponges [72] and the specific

strength may be more appropriate for comparing the properties of cellulose sponges. The specific compression strength

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was calculated for 50% strain considering the density of the sponges (Table S1). When the density was taken into account

(Fig. 10b), an increasing tendency of the compression strength compared to that of BCA was observed in the case of

BCA-5V and BCA-50V, depending on vanillin concentration.

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Fig. 10 The compression strength (a) and specific compression strength of BC sponges (b); stress-strain curves (c);

water up-take ability of BC scaffolds after 24 h of incubation (** for p<0.01; * for p<0.05)

Remarkably, all the cross-linked samples showed better compression strength that unmodified BC or BCA, the

differences being, statistically, very significant (p<0.01), except for BCA-5V (p<0.05). This confirms once more the

efficiency of the ―green‖ cross-linkers used in this approach. The strength-to-weight ratio of BCA-GL was higher with

150% and that of BCA-50CA with 120% compared with that of unmodified BC sponge. Interestingly, both BCA-GL

and BCA-50CA sponges showed lower mean pore size (Table 1) than uncross-linked BCA, with 13% and 26%, which

may be correlated with the better compression strength. The highest mean pore size was noticed for the BC sponge

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which showed the poorest mechanical properties. Moreover, the ―egg-box‖ structure observed in the SEM image of

BCA-50CA and the high degree of organization revealed by the SEM images of BCA-GL coupled with the denser

network of the cell walls may be related to the higher mechanical strength of the sponges obtained by these methods.

Although cross-linking led to a slight decrease of the mean pore size and open porosity, it also determined the

doubling or tripling of the mechanical strength and allowed a better control of the porous structure. Thus, cross-linking

BC with ―green‖ additives is a promising way to simultaneously obtain good porosity and mechanical strength, which is

important for tissue engineering.

3.6 Water uptake ability

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High water uptake ability is required for tissue engineered scaffolds to allow the transport of nutrients and the growth of

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cells [19,27]. The water uptake of BC scaffolds was measured after 24 h of incubation for complete swelling (Fig. 10d).

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Both APS functionalization and cross-linking led to a decrease of the swelling ability of BC due to the reduced number

of OH groups available for water adsorption; S decreased from 1552% for BC to 1341% for BCA, 1136% for BCA-
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50V, 935% for BCA-50CA and 306% for BCA-GL. Similar S value (1600%) was reported for BC sponges [10].
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However, only the difference between BC-GL and all the other scaffolds was statistically very important and the

difference between BCA and BCA-50CA was statistically important (Fig. 10d). The different swelling behavior of
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BCA-GL may be due to a higher cross-linking degree which generally leads to lower water uptake [27] or to a denser

network with GL partially covering the pores, as observed by SEM. It is worth mention that uncross-linked BC and,
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partially, BCA sponges were not stable in the water environment and the measurements were altered by the nanofibers

dislocated from the scaffolds. In contrast, all the cross-linked BCA scaffolds were stable in water and the water uptake
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of BCA-50V and BCA-50CA was about 12.4 and 10.4 times of their dry weight, which is in the range obtained for other

BC sponges for tissue engineering [27].

3.7 In vitro cytotoxicity

Although bacterial cellulose has been proved a safe biocompatible material [73,74], it is important to examine the

possible cytotoxic effects of newly synthesized BC-derived materials and their degradation products. L929 cell viability

was evaluated for undiluted and serial sample dilutions by MTT assay (Fig. 11a).

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Fig. 11 Cell viability measured by MTT test for BC, BCA, BCA-50CA, BCA-50V, and BCA-GL sponges; L929 cell

culture without BC samples served as a control (a); TNF-α level in BC-derived materials supernatants (50.00% dilution)

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incubated with RAW 264.7 macrophages; untreated cells acted as negative control and LPS-treated cells as positive
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control (b)
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In vitro assays revealed that BC sponges are non-cytotoxic for L929 fibroblast cells, regardless the chemical

treatment; both surface functionalization and cross-linking led to negligible toxicity. There was no significant difference
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(p > 0.05) in cell viability compared to the control. This is very important for tissue engineering. Previous study reported

different results depending on the treatment. In particular, Alexandrescu et al. [75] showed that microfibrillated cellulose
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from wood pulp was not cytotoxic either before or after treatments by TEMPO oxidation or polyethyleneimine cross-

linking, but modification with cetyl trimethylammonium bromide led to high toxicity. The lack of cytotoxic effect may
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result from the green nature of the cross-linking agents used in our tests in opposition to the cytotoxic effect of

glutaraldehyde or other aldehydes with biocidal activity [76].

3.7 Evaluation of pro-inflammatory effect

NO and TNF-α (a prototype of proinflammatory cytokines) were measured in RAW 264.7 macrophage cell line to

assess the influence of surface chemical modification of BC on activation of macrophage secretory function (Fig. 11b

and Table 3). Previous studies have shown the importance of cellulose surface chemistry in the modulation of the

biological response [7,77]. Lopes et al. [77] studied the in vitro biological responses to nanofibrillated cellulose prepared

by enzymatic treatment of bleached sulfite pulp. They observed an inflammatory response for NFC in contrast to

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microcrystalline cellulose (MCC) and functionalized NFC, which showed lower TNF-α level. Different results were

reported by Vartiainen et al. [78] which observed no effect on TNF-α level in case of NFC obtained from bleached wood

pulp by grinding while MCC had an inflammatory response.

Table 3 Concentration of nitric oxide (NO) in µmol/mL in RAW 264.7 murine macrophages culture supernatants

cultivated for 24 h. Untreated cells were used as negative control and LPS-treated cells as positive control.

Dilutions BC BCA BCA-50CA BCA-50V BCA-GL LPS Control

50 0 0 0 0 4.96±0.09** 26.76±3.26 0
25 0 0 0 0 0.46±0.1** 0

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6.25 0 0 0 0 0 0
3.125 0 0 0 0 0 0

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** p < 0.01 (very significantly different)

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In the case of functionalized and cross-linked BC sponges, TNF-α concentration was very low, similar to that

of the negative control for BC, BCA-50V and BCA-50CA (Fig. 11b, Table S2). This demonstrates that BC had no
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adverse immunological effects before and after cross-linking with V and CA. An increase of TNF-α concentration,
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representing only 4% of the positive control (LPS-stimulated cells), was obtained for BCA, but the effect disappeared

after cross-linking with V and CA, because of the involvement of the amino group, in agreement with previous work
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[52]. Nevertheless, a noticeable increase in TNF-α level was observed for BCA cross-linked with GL vs. control, which

was statistically very significant for 50% dilution (p < 0.01). Correlating with NO results, which showed significantly
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higher NO levels only for BCA-GL vs. control (Table 3) (p < 0.01), it may be supposed that GL influenced the

inflammatory response of BC. Indeed, Gonzalez et al. showed that high concentration of glucose triggers the secretion
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of TNF-α by macrophages [79]. The unreacted GL is, probably, responsible for this effect. Therefore, a careful adjusting

of BCA:GL ratio is necessary, which will be subject of future work. In conclusion, no obvious difference was observed

between the cytotoxicity and pro-inflammatory effect of unmodified and modified BC, except for BCA-GL, which

means that the good biocompatibility of BC was maintained after cross-linking.

4. Conclusions

A new strategy to obtain highly porous 3D cellulose structures by using simple and ―green‖ methods was

developed in this study. The 3D cellulose structures that combine lightweight and stiffness were prepared by freeze-

drying the water suspensions of bacterial cellulose nanofibers functionalized with aminosilane and cross-linked with

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―green‖ cross-linkers. Vanillin, glucose and citric acid were used as substitutes of common cross-linking agents, which

are toxic. The cross-section morphology of BC sponges, investigated by SEM, revealed a multi-hierarchical

organization and at least three different organization levels; (i) a network of chambers and channels, 100 – 500 µm in

size, interconnected with (ii) a finer pore network and (iii) the nanofibrous bacterial cellulose ―walls‖ which imparts

good strength and flexibility to the pores and channels. All the cross-linked BC sponges showed better compression

strength than unmodified BC sponge. The strength-to-weight ratio of BCA-GL and BCA-50CA was higher than that of

BC, with 150% and 120%, respectively. In vitro assays revealed that functionalized and cross-linked BC sponges eluates

are non-cytotoxic and do not trigger an inflammatory response in macrophages with the exception of BCA-GL, which

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requires further investigation. The doubling and tripling of the compression strength confirms the effectiveness of these

―green‖ cross-linkers in improving the mechanical properties while maintaining a high open porosity (≥80%) with the

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pore size between 100 and 500 microns and a good biocompatibility. Therefore, this study provides a new

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environmentally-friendly method to obtain biocompatible cellulose scaffolds suitable for soft tissue engineering.
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Acknowledgements
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This study was financially supported by a grant of Ministry of Research and Innovation–UEFISCDI, project number

PN-III-P4-ID-PCE-2016-0431, Contract no. 148/2017 (CELL-3D) within PNCDI III and by European Regional
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Development Fund through Competitiveness Operational Program 2014-2020, Priority axis 1, Project No. P_36_611,

MySMIS code 107066-INOVABIOMED. Authors thank Iuliana Biru, Politehnica University of Bucharest, for micro-
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computed tomography analysis.

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Author contribution statement

Bacterial cellulose sponges obtained with green cross-linkers for tissue engineering

Adriana Nicoleta Frone1, Denis Mihaela Panaitescu1*, Cristian Andi Nicolae1, Augusta Raluca Gabor1,

Roxana Trusca2, Angela Casarica3, Paul Octavian Stanescu2, Dora Domnica Baciu4, Aurora Salageanu4
1
National Institute for Research & Development in Chemistry and Petrochemistry - ICECHIM, 202 Splaiul

Independentei, 060021 Bucharest, Romania;

2
University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania;
3
National Institute for Chemical Pharmaceutical Research and Development, 112 Calea Vitan, 031299, Bucharest,

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Romania

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4
Cantacuzino National Medical-Military Institute for Research and Development, 103 Spl. Independentei, 050096

Bucharest, Romania

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Corresponding author e-mail address: panaitescu@icechim.ro; phone number: +4 021 312 30 68
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Adriana Nicoleta Frone – Methodology, Writing- Original draft preparation

Denis Mihaela Panaitescu - Conceptualization, Supervision, Writing, Reviewing and Editing

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Cristian Andi Nicolae – Investigation – TGA, Validation

Augusta Raluca Gabor – Investigation – DMA, Validation

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Roxana Trusca – Investigation – SEM analysis

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Angela Casarica - Methodology and Investigation – Bacterial cellulose biosynthesis

Paul Octavian Stanescu - Validation – Computed tomography

Dora Domnica Baciu – Investigation - In vitro tests

Aurora Salageanu – Validation and Writing - In vitro tests

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Conflict of Interest

Bacterial cellulose sponges obtained with green cross-linkers for tissue engineering

Adriana Nicoleta Frone1, Denis Mihaela Panaitescu1*, Cristian Andi Nicolae1, Augusta Raluca Gabor1,

Roxana Trusca2, Angela Casarica3, Paul Octavian Stanescu2, Dora Domnica Baciu4, Aurora Salageanu4
1
National Institute for Research & Development in Chemistry and Petrochemistry - ICECHIM, 202 Splaiul

Independentei, 060021 Bucharest, Romania;

2
University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania;
3
National Institute for Chemical Pharmaceutical Research and Development, 112 Calea Vitan, 031299, Bucharest,

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Romania

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4
Cantacuzino National Medical-Military Institute for Research and Development, 103 Spl. Independentei, 050096

Bucharest, Romania

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Corresponding author e-mail address: panaitescu@icechim.ro; phone number: +4 021 312 30 68
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The authors declare no conflict of interest.

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Highlights

 Nanocellulose was obtained from bacterial cellulose (BC) membranes by defibrillation

 Green cross-linkers (vanillin, glucose, citric acid) were used to obtain BC sponges
 Improved mechanical properties obtained with citric acid and glucose-G crosslinkers
 Crosslinked BC sponges are non-cytotoxic and have no inflammatory response except for G

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