Three-dimensional Hydrogels of Alginate/chitosan Semi-interpenetrating Polymer Networks and Nanocelluloses

The hydrogels are advanced materials used in biomedical applications during wound healing, controlled drug release and to prepare scaffolds. In this work are prepared hydrogels of alginate/chitosan (Alg/Ch) semi-interpenetrating polymer networks (semi-IPN’s) and nanocelluloses. The hydrogels after preparation by freeze drying are namely simply as gels. The cellulose nanocrystals (CNC’s) are obtained from acid hydrolysis of bleached Eucalyptus pulps and oxidized cellulose nanocrystals (CNCT’s) prepared by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical catalyzed reaction as known as TEMPO reaction. The cellulose nanobers (NFC’s) are obtained from mechanical shearing of cellulose pulps and oxidized NFC’s by TEMPO-mediated reaction (NFCT’s). The nanocellulose suspension and gels are characterized by FTIR at ATR mode, TGA, XRD, TEM, SEM, X-ray computed microtomography (micro-CT) and DMTA. The addition of CNC’s, NFC’s, CNCT’s or NFCT’s in the microstructure of gels increases their dimensional stabilities. The best results are obtained when CNCT’s and NFCT’s are added. The mechanical properties and dimensional stability of Alg/Ch semi-IPN’s increase after controlled thermal post-treatment. The heating during thermal post-treatment boosts the physicochemical interactions in the microstructures of semi-IPN’s. The biological assays show biocompatibility of broblast cells on the substrates, and differentiation and proliferation up seven days. The optimized mechanical properties, dimensional stability and biocompatibility of the gels studied in this work are important parameters for potential biomedical applications of these biomaterials. 50 tungsten X-ray A CCD camera of 1.3 megapixel resolution was attached to the lens scintillator with a 1:6 zoom range. Projections were recorded between 0 and 360° at an angular increment of 0.50°. It were studied samples of Alg/Ch and CNCT’s or NFCT’s (at 50 wt%). A cross-section of approximately 12 mm edge length was selected and analyses recorded at 40 kV and 800 µA. The pixel size of approximately 10 µm was reached. Image reconstruction was performed using the Feldkamp algorithm. The visualization and quantitative analysis of the volumes were carried out with Thermo software


Introduction
Synergistic effects arise when polymers are blended to prepare interpenetrating or semi-interpenetrating polymer networks also known as IPN's or semi-IPN's, respectively. These polymer networks enable preparation of materials and controlled and advanced properties (Naseri et al., 2016). The chitosan (Ch) is a polyelectrolyte obtained by physicochemical modi cation reaction of chitin and generally used to prepare biocompatible substrates (Li et al., 2005;Zhou, 2011). The alginate (Alg) is an anionic polyelectrolyte naturally biosynthesized by brown algaes. The Ch and Alg are some main polyelectrolytes used to prepare biocompatible materials. used for controlled release of sodium diclofenac. It was observed drug releasing up to 12 h in the intestine. Li et al. (2009) show the cytocompatibility and cell viability of chitosan/alginate scaffolds. The cell morphology, proliferation and bioadhesion on the material were studied in vitro. The analyzes of proteins extracted during cell growth showed the production of a speci c type of collagen. This behavior was not observed on the crosslinked Ch-based substrate itself. This study suggests that chitosan/alginate semi-IPN's enable cell proliferation, increase the expression of the HTB-94 chondrocyte phenotype and can be used as an alternative to prepare scaffolds. However, any mechanical measurement was described. Rani et al. (2011) carried out the synthesis of chitosan/alginate semi-IPN's to be used as scaffolds. Silver nanoparticles were added in the microstructure as antibacterial agent. It can be postulate based on their results the potential use of this kind of material in biomedical areas and the needs of complementary studies as mechanical and thermal properties, and porous microstructures of chitosan/alginate hydrogels.
The rst part of our research project was published in a recent paper (Siqueira et al., 2019) in which was studied alginate/nanocelluloses hydrogels. This article can be considered primary studies towards optimization of thermal and mechanical properties, experimental procedures and biological features. The alginate/nanocellulose hydrogels were submitted to cytotoxicity and biocompatibility assays. However, the use of sodium alginate presents some limitations as price, synthesis, availability and mechanical properties. Based on these assumptions additional studies to improve or to impart new properties into these materials are needed.
In this study it was decided to add chitosan (cationic polyelectrolyte) and alginate (anionic polyeletrolyte) to prepare semi-interpenetrating polymer networks (semi-IPN's) and nanocelluloses to optimize the material, to impart new properties and to increase some others not presented when only alginate and nanocelluloses were used to prepare biomaterials. Some advantages of the blend between these polyelectrolytes are related to economical concerns (actually the price of alginate is 1.5 higher than chitosan), mechanical properties and biocompatibility effects (Ch is a polyelectrolyte derived from chitin which present impressive properties and interesting biocompatibility).
The material prepared by alginate/chitosan presents classical and rich theoretical and experimental information about physicochemistry domains and the drawbacks related to its preparation. This article putt other readers at close contact with very important theories (physycochemistry of solution, suspension, and polyelectrolytes; colloidal sciences; and surface and interface physicochemical interactions, etc.) to prepare advanced materials, and the experimental limitations related by the structures of these chemicals at molecular level.
CNC's and NFC's were added in the gels at different mixture order, pH, ionic strength and concentration in this work. Un-and modi ed nanocelluloses (TEMPO oxidized) were used to increase or to impart new properties. It is expected that the careful empirical studies and characterization techniques evidence the bene ts and synergistic effects between the components of the semi-IPN's.
The addition of nanocelluloses in Alg/Ch semi-IPN's, mainly oxidized cellulose nanocrystals and nano brils (CNCT's and NFCT's, respectively) presented the very promising results. These features are very important for structural applications, transport of biological uids and nutrients; and cell attachment, growth, differentiation and proliferation. To the best of our knowledge any study was still published describing all these parameters (biocompatibility, addition of un-and modi ed nanocelluloses, mechanical and thermal properties, dimensional stability, porous structures and physicochemical interactions). These materials also present remarkable ecological and economical concerns and advances towards sustainability and green chemistry.
The Ch is a polyelectrolyte slightly soluble in water and potential candidate for application in biomedical engineering. Its structure is formed by β-(1-4)-2-amino-2-deoxy-D-glucose units. The polymer chains of Ch are similar to cellulose and in acidi ed medium Ch is a polycation due to protonation of amine groups. The Alg is an anionic polyelectrolyte and its linear chain is soluble in aqueous media. The Alg is well known due to their healing and anti-tumoral properties. It consists of β-D-manuronic acid (M) and α-L-guluronic acid (G) units linked by glycosidic bonds. Alg is considered a polyanion at neutral or alkaline medium due to carboxyl groups in its structure.
The physicochemical interactions between Alg and Ch mainly through electrostatic interactions is a challenge. This blend can be an interesting candidate to prepare stable hydrogels and to impart improved structural homogeneity, dimensional stability and mechanical and biological properties when compared to other prepared from Alg or Ch themselves or by polymers derived from sources as petroleum (Li et al., 2005).
The hydrogels were prepared with Alg of high molecular weight supplied by Sigma-Aldrich (M w = 1 x 10 6 g.mol -1 , M/G ratio = 1.56 and viscosity of 250 cP at 25°C and 2 wt%). The Ch was supplied by Phytomare (M w < 100 kDa and average degree of deacetylation of 90%). Glacial acetic acid (C 2 H 4 O 2 at 99.8%) and calcium chloride (CaCl 2 .2H 2 O) were purchased of Vetec Química Fina Ltda (Brazil).
The phosphate buffer solution (PBS) was prepared by mixture of sodium chloride (NaCl), potassium chloride (KCl) and potassium phosphate (K 3 PO 4 ). All these chemicals were supplied by Synth. The sodium phosphate monohydrate (Na 3 PO 4 .H 2 O) supplied by Vetec Química Fina Ltda and orthophosphoric acid (H 3 PO 4 at 85%) supplied by Neon. All these chemicals were supplied at pure degree and used as received.

Extraction, oxidation reaction and characterization of nanocelluloses
The cellulose nano brils (NFC's) and nanocrystals (CNC's) were extracted and isolated, respectively, from bleached kraft cellulose pulp.
The NFC's were obtained from mechanical shearing. The bleached cellulose bers were suspended in water at 2 wt% and grounded in a Supermass Colloider mill (MKZA10-20J CE Masuko Sangyo, Japan).
The CNC's were also obtained from bleached kraft pulp in the Laboratory of Physicochemistry of UFMG (Brazil). The hydrolysis reaction was carried out in sulfuric acid solution at 65% (v/v), 50 o C and constant mechanical stirring for 50 min. Three dilutions were performed with milli-Q water to stop the reaction. The suspension was submitted to centrifugation steps to remove the excess of acid. The supernatant was eliminated and the precipitate solubilized, and added in dialysis membranes. Changes of water of dialysis bath were carried out up to neutral pH. The suspension of CNC's was submitted to ultrasound treatment for 2 min (Unique Sonicator, 40 kHz) and ltered through membranes of acetate cellulose (Sartorius).
The NFC's and CNC's were oxidized by TEMPO catalyzed reaction. The oxidation of hydroxyl groups in nanocelluloses was performed based on the method described by Saito and Isogai (2004).
Cellulose pulp at 1.5 wt% was used to prepare TEMPO-oxidized cellulose nano bers (NFCT's). It was added 0.016 g of TEMPO radical in the aqueous reaction medium, 0.100 g of NaBr and 5.35 mL of NaClO at 14 wt%. The pH of the medium was kept at 10 by addition of sodium hydroxide solution. The NFCT's were washed by distilled water and centrifugation steps up to neutral pH. At the end suspensions at 2 wt% of NFCT's were obtained.
Suspension of TEMPO-oxidized cellulose nanocrystals (CNCT's) was prepared based on the experimental procedures described by Saito et al. (2007). The 0.100 mmol TEMPO radical and 1.00 mmol NaBr were solubilized in water per gram of cellulose. The suspension of CNC's at 2 wt% were added into a three bottom neck ask. The pH of the suspension was kept at 10 by the addition of sodium hydroxide solution. The oxidation reaction was started by addition of sodium hypochlorite (NaClO). The CNCT's were washed by centrifugation steps and putted in dialysis membranes (6-8 kDa) up to neutral pH.
Conductometric titrations were performed as described by Saito and Isogai (2004) to determine the degree of oxidation (DO) of the nanocelluloses. Approximately 50.0 mg of TEMPO oxydized nanocelluloses were suspended in 0.0500 mol.L -1 hydrochloric acid solution and the pH adjusted up to 2.7 to protonate acid groups present on the nanocelluloses. The titration was carried out by dropwise of 0.0100 mol.L -1 sodium hydroxide solution.

Alg/Ch semi-IPN's
To prepare Alg/Ch semi-IPN's a three-dimensional network is desirable between carboxylic groups in Alg and amine groups in Ch structure. Some physicochemical interactions, as for example between divalent metal cations (M 2+ ), as calcium ions, and negatively charged groups as carboxylate contribute to the formation of the microstructure. Alg/Ch/nanocellulose gels were prepared in aqueous media. Both polyelectrolytes present ionic functional groups in their chains at speci c experimental conditions. This is possible at the pH close to the pKa values of the functional groups (Azzam et al., 2016;Li et al., 2009;Siqueira et al., 2019;Saito et al., 2007;Saito and Isogai, 2004;Senel et al., 2000). The Alg shows pKa value in the range of 3.38 and 3.65 for sequences of M and G, respectively, and Ch pKa value close to 6.3. In this work the optimized pH to prepare PEC's and semi-IPN's is in the range between 3.4 and 6.3. It was decided to use an optimized pH of 5.3 after trials.
The Algand Ch-based gels were also crosslinked by calcium ions (Ca 2+ ) to enable comparative data (blank samples). The Ch solution was prepared at pH 5.3 and 2.0 wt% and Alg solution at pH 3.8 and 2.0 wt%. The Alg and Ch solutions were slowly added under mild mechanical stirring (340 rpm) at 60 o C for 60 min. The PEC's were cooled in liquid nitrogen and freeze dried for 48 h. The gels were added in a bath of 2.0 wt% of calcium chloride solution for 15 min. At this time the interactions between negatively charged groups (carboxylate groups) and Ca 2+ can takes place and the microstructure of Alg/Ch semi-IPN's be formed. It is worth pointed out that trials to determine an optimized contact time of Alg/Ch semi-IPN's in calcium chloride solution bath were previously performed (between 5 min and 24 h). The Alg/Ch semi-IPN's crosslinked by calcium ions were washed in distilled water to remove excess, cooled in liquid nitrogen and freeze dried for 48 h.
Nanocellulose suspensions at 1.14 wt% were rst dispersed in sodium Alg solution (2.0 wt%) for synthesis of Alg/Ch/nanocellulose semi-IPN's. The weight ratio between nanocelluloses, Alg and Ch in these studies are shown in Table 1. These mixtures were also kept under heating and stirring for 60 min, cooled and freeze dried for 48 h. The gels were added in 2.0 wt% calcium chloride bath for 15 min. The crosslinked materials were washed by distilled water to remove excess of calcium ions and freeze dried for 48 h.
Tab. 1: Weight ratio of nanocelluloses (wt%) for synthesis of Alg/Ch semi-IPN's. The infrared spectra of nanocelluloses, polyelectrolyes and semi-IPN's were recorded in a spectrometer (Perkin-Elmer Spectrum) at room temperature. The parameters were wavelength range between 4000 and 500 cm -1 , resolution of 2 cm -1 and 20 accumulation scans.
Thermogravimetric Analysis (TGA) Thermogravimetric analyzes of nanocelluloses and hydrogels were performed in a TGA-DTG-60 (Shimadzu) and in alumina crucibles. The analyses were performed at heating rate of 10°C.min 1 and in the temperature range between 25 and 600°C. A nitrogen ow of 200 mL.min -1 was used during the scans.

X-ray diffraction (XRD)
The nanocelluloses and gels were analyzed by X-ray diffraction (diffractometer Shimadzu model XRD-6000). The parameters used were Cu Kα radiation (λ = 0.155428nm), voltage of 30 kV and current of 30 mA. The spectra were collected in the scanning mode of 2º.min -1 at Bragg angle (2θ) range between 5 and 50º.

Dynamic Mechanical Thermal Analyses (DMTA)
The mechanical properties of the gels were studied by DMTA (Netzch model 242). The analyses were carried out in the temperature range between 20 and 80ºC, heating rate of 3ºC.min -1 , 1 Hz frequency and initial load of 5 N. Two trials were carried out to study the thermomechanical properties of the samples: i) the samples were submitted to heating-cooling/heating-cooling cycles in the furnace of the apparatus (25-80ºC/25-80ºC); and ii) the samples were thermally post-treated in an oven at 80ºC for 4 h and after analyzed by DMTA. The storage and loss moduli (E' and E'', respectively) were obtained from the viscoelastic behavior of the samples.

Scanning Electron Microscopy (SEM)
The morphological characterization of gels was performed in a double-beam scanning electron microscope (FEI Quanta FEG 3D). The samples were cooled in liquid nitrogen to avoid deformation of the gels during fracture. These samples were xed on supports and coated by carbon lms of approximately 15 nm of thickness. The images were recorded at secondary electron mode and acceleration voltage of 20 kV. The micrographs obtained by SEM were also used to determine the average size of pores in the gels with the Image J software. Fifteen measurements were taken in each sample.

X-ray computed microtomography (micro-CT)
Analyses of the three-dimensional morphology of the samples were carried out after cryo-facture in liquid nitrogen in a microtomograph (SkyScan 1174 Bruker, Germany). The parameters used were 50 keV and 40 W of tungsten X-ray source. A CCD camera of 1.3 megapixel resolution was attached to the lens scintillator with a 1:6 zoom range. Projections were recorded between 0 and 360° at an angular increment of 0.50°. It were studied samples of Alg/Ch and CNCT's or NFCT's (at 50 wt%). A cross-section of approximately 12 mm edge length was selected and analyses recorded at 40 kV and 800 µA. The pixel size of approximately 10 µm was reached. Image reconstruction was performed using the Feldkamp algorithm. The visualization and quantitative analysis of the volumes were carried out with Thermo Scienti c Avizo software (Thermo Fisher Scienti c, Oregon -USA).

Biological Assays
The biocompatibility and cytotoxicity of L929 broblast cells on Alg/Ch semi-IPN's will be published as soon as possible. This article presents indirect evidences of the potential use of these materials through analyses of bioadhesion and cell differentiation obtained from SEM images. The broblast cells were cultured on the surfaces of semi-IPN's. Samples of (5x5x1) mm 3 were submitted to sterilization by ultraviolet radiation for 30 min. The gels were immersed in fetal bovine serum (FBS) for 1 h and added into cell suspension (5x10 6 cells.mL -1 ). The cell cultures were incubated at 37ºC for 7 days in CO 2 atmosphere. The gels were washed with phosphate buffer solution (PBS). The samples were immersed in ethanol and dried in desiccator with vacuum, coated by thin lms of gold and observed in a SEM (Quanta FIB EGF 3D with FEI).

Results And Discussion
It is described in the literature that negatively charged carboxylate groups of Alg and positively charged amine groups of Ch interact in optimized physicochemical conditions. These polyelectrolytes give rise formation of polyelectrolyte macroion complexes (PMC's) also known as polyelectrolyte complexes (PEC's) (Berger et al., 2004;Isogai et al., 2011). It was used in this work Alg and Ch as polyelectrolytes and calcium chloride (CaCl 2 ) as crosslinking agent to prepare biocompatible materials due to economic and ecological concerns and towards a green chemistry.
Figure 1 depicts ionic and/or secondary interactions in the microstructure of Alg/Ch and Alg/Ch/nanocellulose semi-IPN's. It is expected at least some types of interactions as: ionic or electrostatic interactions of carboxylate groups in Alg structure and calcium ions; interactions of carboxylate groups in TEMPO-oxidized nanocelluloses, carboxylate groups of Alg chains and calcium ions; secondary interactions (hydrogen bonds, van der Waals, induced and permanent dipole) of functional groups in the structure of Alg/Ch/nanocelluloses; ionic/electrostatic interactions of Alg and Ch; and ionic/electrostatic interactions of carboxylate groups of CNCT's and NFCT's and amine groups of Ch.
As stated and well explained and justi ed by French (2017) the repeating unit of cellulose is often considered to be cellobiose instead of glucose. This review presents some arguments regarding the repeating unit in cellulose molecules and crystals based on biosynthesis, shape, crystallographic symmetry, and linkage position. The statement that cellobiose could be the repeating unit of cellulose instead of glucose needs take some care when regarding the chemical bonds in the structure, reactivity and properties (Nishiyama et al. 2002;Kouwijzer et al. 1995). There is almost universal agreement that cellulose is a polymer of β-(1-4)-linked D-glucopyranosyl units and glucose is repeatedly added during biosynthesis of cellulose chains. One common argument used by some authors to consider cellobiose as the repeating unit of celullose is due to the fact that cellobiose is obtained by hydrolysis of cellulose. However, cellobiose is one of the products of acid hydrolysis and prolonged hydrolysis results at glucose. One report (JCBN, 1982) states that ''polysaccharides composed of only one kind of monosaccharide are described as homopolysaccharides'' and another report (JCBN, 1983) adds a statement that ''The repeating unit in a homopolysaccharide is a sugar residue". This review support the glucose residues as the repeating unit of cellulose and also in agreement with International Union of Pure and Apllied Chemistry (IUPAC) and International Union of Biochemistry and Molecular Biology (IUBMB).
It's worth pointed out that Figure 1 is just an attempt to describe the complex structure of Alg/Ch/nanocellulose semi-IPN's, and possible physicochemical interactions between their components. Any picture could describe completely the microstructure formed. In this paper it was considered glucose as the repeating unit of cellulose to prepare these pictures even if some between then present more than one glucose unit. The pictures are based on articles that describe the egg-box model as one possibility to explain these interactions. One possibility is the formation of dimers around cation ions added in polyelectrolyte solutions to prepare hydrogels (Li et al., 2007;Donati et al., 2005). The materials synthesized can be considered physical gels due to reversible feature of the interactions in their microstructures (non-covalent bonds). Figure 2 presents images of the morphology and dimensional stability of the Alg/Ch/nanocellulose semi-IPN's after optimization of physicochemical parameters as mixing order, concentration, pH, ionic strength and temperature. The Support Information (SI 1 ) shows the photographs of Alg/Ch semi-IPN's without addition of nanocelluloses. The freeze-dried and thermally post-treated gels present good dimensional stability. However, their surface areas show some insights. The surface of gel at contact with the mold and air during preparation present smooth and rough surfaces, respectively. The microstructure shows slow shrinkage after thermal post-treatment at 80 o C for 4 h, but the materials still kept their dimensional stabilities. It is worth pointed out that porous structures and rough surface areas are very interesting for nutrient ux and cell attachment, respectively. The shrinkage of some samples is probably due to water elimination, reconformation of polyelectrolytes (extended-coil interconversion), and increase of ionic crosslinking density or secondary interactions in the microstructure of the gels. All these phenomena can contribute to decrease the distance between components in the microstructure and to increase physicochemical interactions between them (ionic, electrostatic, van der Waals, permanent and induced dipole). The microstructure is kept intact when compared with the gels without addition of nanocelluloses. After some optimization studies it were added 36 and 50 wt% of nanocelluloses to prepare dimentionatly stable Alg/Ch/nanocellulose semi-IPN's. Figure 3 presents FTIR at ATR mode analyses of Alg and Ch polyelectrolytes (Figure 3.A) and ionic crosslinked Alg/Ch/nanocellulose gels (Figure 3.B). The Support Information (SI 2 ) shows FTIR spectra for un-and oxidized nanocelluloses. The high degree of desacetylacetion of Ch used in this work (ca. 93%) to prepare the gels enables electrostatic interactions between Alg and Ch, and gives rise the formation of semi-IPN's whose present good dimensional stability in water and phosphate buffer solution (PBS). The blend Alg/Ch/oxidized nanocellulose semi-IPN's show better results. The addition of nanocelluloses at 36 and 50 wt% were bene cial to keep the dimensional stability. The ionic crosslinking due to calcium ions was also important for interactions in the Alg and Alg/nanocellulose phases (calcium ions/carboxylate groups). The interactions between the crosslinked Alg or Alg/nanocelullose phases and Ch phase allow formation of a complex and pore network in the gel microstructures. As can be observed in the FTIR spectra all the main absorption bands of Alg, Ch and nanocelluloses are also observed in the spectra of each constituent itself even if some bands show overlapping or small displacement of wavelength absorption. These spectra show evidences of ionic or secondary interactions and formation of physical gels. Any new absorption band was observed when the components and the blends are compared which is an evidence that covalent bonds were not formed (chemical gels). The nanocellullose spectra (SI 2 ) show some main absorption bands at 3300 cm -1 attributed to stretching vibrations of -OH groups; at 2900 cm -1 attributed to stretching vibrations of -CH groups; at 1610 and 1411 cm -1 attributed to symmetric and asymmetric stretching vibrations of carboxylate groups, respectively; and at 1430 cm -1 attributed to stretching of methyl groups. The absorption bands in the range 1200 to 920 cm -1 are attributed to stretching vibrations of the polysaccharide structures as also observed in Alg and Ch spectra (Azzam et al., 2016).
There are some evidences that the interactions between Alg, Ch and nanocelluloses are secondary interactions based on FTIR analyses. In this work the Alg/Ch/nanocellulose gels show broadening and overlapping of the absorption band at 1600 cm -1 attributed to stretching vibrations of carboxyl and amine groups. Other evidence is observed at 1400 cm -1 also attributed to carboxyl and amine groups. The absorption band in the range 3200 to 3600 cm -1 shows a broadening in Alg/Ch/nanocellulose gels. This band is attributed to stretching vibrations of -OH and -NH groups, and hydrogen bonds. The displacement and overlapping of characteristic absorption bands of Alg, Ch and nanocelluloses when compared to the absorption bands of the gels are evidences of secondary interactions between functional groups (mainly carboxylate and amine). Any new absorption band was observed in the gels spectra as postulated before. The high charge density of these polyelectrolytes give rise mainly to formation of physicochemical interactions between them as also observed by Lawrie et al. (2007). Figure 4.A presents X-ray diffractogram patterns of Alg and Ch, and Figure 4.B crosslinked Alg/Ch and Alg/Ch/nanocellulose gels at 50 wt% concentration. The diffractograms show characteristic patterns of polymer materials (when compared as for example with the ne and well de ned patterns for metallic or ceramic materials), and more speci cally polysaccharides structures (French, 2014).
XRD patterns can show small displacements of 2θ values, intensity and/or crystalline/non-crystalline ratio when articles are compared due to experimental procedures, physicochemical parameters for synthesis of polymers or cellulose source for extraction of CNC's and preparation of CNF's.
Some tools as the software and model used during data interpretation and the experimental parameters for sample preparation and parameters for XRD analyses can also affect the results. As one example and stated by French (2014) the patterns calculated by Mercury ® program are isotropic but there is no way to input crystallite shape information that can affect the relative peak heights and widths.
In this work X-ray analyses are used for comparison purposes, and to take some additional qualitative information about the microstructural organization of the complex blends between un-and crosslinked Alg/Ch/nanocelluloses to prepare hydrogels and their biocompatibility. Attempts to take quantitative information on XRD analyses will be issues of future works as well as cell viability and other correlations between microstructure and biological features. As observed crosslinked Alg/Ch gels and mainly Alg diffractogram pattern without addition of nanocelluloses have broaden diffraction peaks suggesting defective crystallization and probably low crystallinity. Smitha et al. (2005) stated based on their studies that the mixture of Alg and Ch decreases the crystallinity when compared to the crystallinity of this polymers. The crosslinked Alg/Ch gels show lower cristallinity than pure Ch and Alg probably due to fast physicochemical interactions with calcium ions and the gelation process. Li et al. (2009) observed that typical peaks of Ch disappeared after mixture with Alg and the PEC's showed an amorphous morphology similar to Alg. It was stated that the introduction of Alg into Ch disrupted the crystalline structure of Ch. Li et al. (2007) also observed by XRD analyses formation of junction zones with different crystallinity in Ca-alginate gels. This was con rmed by the measurements of Ca-alginate gel beads prepared at different pH and slow crosslinking rate, which leads to a higher crystallinity and more perfect ordering. Reversible aggregation of junction zones and their impacts on XRD patterns were also observed during dehydration and rehydration (or swelling). Nagahama et al. (2009) observed based on XRD results that there was good compatibility and interaction between gelatin and chitosan molecules in the membranes synthesized. The peak intensity ratio of chitosan membrane was reduced when gelatin was added. A little decrease in the crystallinity of chitosan/gelatin membranes was attributed to formation of hydrogen bonds.
After addition of CNC's, CNCT's, CNF's and CNFT's in Alg/Ch gels at 50 wt% is observed a more perfect ordering, characteristic peaks of these nanostructures and diffractogram patterns more representative of high crystallinity. These results corroborate with a more uniform and organized microstructure and probably formation of hydrogen bonds and ionic interactions between un-and oxidized nanostructures and Alg/Ch-based gels.
The thermal stability of Alg, Ch and Alg/Ch/nanocellulose semi-IPN's were analyzed from TGA-DTG curves. The Figures in SI 3 present the curves of TGA obtained in nitrogen atmosphere and their rst derivative (DTG). It was observed three main events of weight loss. The rst one takes place between 20 and 175ºC and approximately 15% of weight loss attributed to water elimination and small fragments of glycoside structures. The second one in the range of 175 to 385ºC and ca. 40% weight loss can be considered the main event. It is attributed to degradation reactions of glycoside structure of Alg and Ch polyelectrolytes and nanocelluloses. The third one at approximately 385ºC is attributed to degradation of the crystalline regions of these components whose are more thermally stable. It is worth pointed out that in synthetic air or oxidant atmosphere the degradation of glycoside-based structures is generally displaced towards lower temperatures than nitrogen atmosphere. The residues (ash content) at approximately 600ºC are probably due to calcium inorganic salts as also described by Han et al. (2010).
The thermal degradation of glycoside-based structures was also studied by other authors. The presence of hemicelluloses in the structure of NFC's can displace the temperature of thermal degradations towards lowest values mainly in oxidant atmosphere (Yang et al., 2007). Isogai et al. (2011) describe the formation of sodium carboxylates in the structure of TEMPO oxidized nanocelluloses. These functional groups decrease the thermal stability of the nanocelluloses and displace the degradation temperature towards lowest values. In our work an increase of thermal stability and approximately 30 up to 40% of ash content at 600ºC were observed for Alg/Ch semi-IPN's prepared by addition of TEMPO-oxidized nanocelluloses (CNCT's or NFCT's). This can be partially explained by the ionic crosslinking of the carboxylate groups/calcium ions. These ionic or electrostatic interactions can increase the thermal stability of gels and displace the degradation temperatures of the microstructure towards highest values.
The two concentrations of nanocelluloses used in this work (36 and 50 wt%) show similar results when studied by FTIR-ATR, DRX and TGA-DTG. However, the better dimensional stability and mechanical properties were observed when 50 wt% of nanocelluloses were added to prepare gels. Based on these results were carried out on Alg/Ch/oxidized nanocellulose gels at 50 wt% analyses of DMTA, SEM and Xray micro CT images and cell growth assays. Table 2 shows storage moduli obtained by dynamic mechanical thermal analyses (DMTA) of the gels.
The samples were thermally post-treated in the furnace of the apparatus during a consecutive heatingcooling/heating-cooling cycle between 25 up to 80ºC. The results were compared at 35, 37 and 42ºC whose are temperatures for some biomedical applications. The biomaterials prepared by natural polymers or their derivatives and biopolymers generally present favorable microstructure for cell bioadhesion and proliferation. The poor mechanical properties and dimensional stability are generally drawbacks for their applications. The storage moduli (E') show low values during rst heating/cooling cycle as observed by DMTA analyses. This can be explained by the polyelectrolyte structures and fast ionic crosslinking in the calcium chloride bath. In these conditions polymer conformation and full ionic crosslinking can be limited before an ultimate state. When thermal post-treatment is carried out the polyelectrolyte structures are submitted to relaxations close to glass transitions values and the polymer chains can approach each other and to interact more effectively.
Some additional degree of ionic crosslinking of calcium ions and carboxylate groups of Alg and CNCT's and NFCT's is expected. These behaviors (polymer relaxation and conformation and additional ionic crosslinking) can explain the remarkable increase of E' after the rst heating cycle up to 80ºC and during the second heating-cooling cycle (Siqueira and Botaro, 2013).
The increase of mechanical properties of semi-IPN's was also observed and described by authors when nanocelluloses are added in some polyelectrolytes (De France et al., 2017;Kumar et al. 2017).  The gels prepared by addition of nanocelluloses (CNC, CNCT, NFC and NFCT) presented bigger pore sizes and rough walls than gels without them. These features can be partially explained by the impressive properties of nanocelluloses (high surface area, reactivity, stiffness and mechanical properties) (Siqueira et al., 2019;Lin and Duffresne, 2014). The synergistic effects of the addition of nanocelluloses, mainly TEMPO-oxidized, calcium crosslinking and thermal post-treatment improve the mechanical properties and increases the surface available for cell attachment. the insets represent a magni cation of 1000x). Figure 6 shows images obtained by X-ray micro CT of Alg/Ch/nanocellulose semi-IPN's. This powerful technique enables to take qualitative and quantitative information of volume, size, shape, distribution and interconnectivity of pores (Isaac et al., 2015). The results corroborate with SEM images as a complementary tool to take some additional information about the internal microstructures of the gels. In this work the results of X-ray micro CT will be used for comparative purposes.
The Figure 6.A shows an oriented structure of pores for Alg/Ch/CNCT semi-IPN's and cracks probably due to the stiffness of CNCT's (rigid rod-like structures) added in the gels. The Figure 6.B presents Alg/Ch/NFCT semi-IPN's. It can be observed higher pore sizes, more uniform distribution and ribbed and roughness surfaces for these gels than CNCT-based gels.
It was used the software Image J to take some quantitative information when compared Alg/Ch/nanocellulose IPN's for comparative purposes and based on the work of Isaac et al. (2015). The CNCT's and NFCT's-based gels present 50 and 80% of pores in their microstructures, respectively. Both oxidized nanocelluloses-based gels showed thickness of pore walls of 16 up to 20 µm but NFCT's-based gels presented the highest homogeneity.  The L929 broblast cells are attached in the gel microstructure as observed in the micrographs. The rough surface of the gels, the polyelectrolyte charges, the nanocellulose and the pore surface are essential parameters for attachment. The morphology of cells is kept approximately spherical or extended which are some evidences of proliferation, differenciation and/or biocompatibility on the substrate (Dan et al., 2016;Domingues et al., 2014).
It was clearly possible to observe cell attachment of L929 broblast cells on Alg/Ch/NFCT IPN's mainly due to surface area of the NFCT's and functional groups as carboxylate and amino (Figure 7.E and F).
These groups in uence the cell adhesion, proliferation and differentiation due to physicochemical interactions of proteins of L929 broblast cells (Rashad et al., 2017). It can be also observed surprisingly the formation of thin and long laments derived from broblast cells that improve the attachment (SI 4 ).
These laments show high degree of adherence and biocompatibility of the cells on these gels.

Conclusions
The Alg/Ch/nanocellulose semi-IPN's present good dimensional stability and rough surface area, and enable cell attachment, growth, proliferation and differentiation for time interval as long as 30 days. The physicochemical parameters (pH, ionic strength, charge density, order of mixture, oxidation reaction and nanocellulose concentration), ionic crosslinking and thermal post-treatment in uences directly the microstructure of the gels as observed by SEM and X-ray micro CT images. The roughness of the surface and complex structure of pores of these gels when nanocelluloses are added collaborate to cell attachment and growth. The rst draft of the manuscript was written by Eder Siqueira and all authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript.

Declarations
Ethics approval The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
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