NATURAL COMPOSITION COMPRISING ALGINATE AND CELLULOSE NANOFIBERS ORIGINATING FROM BROWN SEAWEED

Abstract

A natural composition for 3D printing comprising alginate from brown seaweed and cellulose nanofibers, wherein the cellulose nanofibers originate from cellulose from the same brown seaweed sample(s) as the alginate.

Claims

1. A natural composition for 3D printing comprising: alginate from brown seaweed, and cellulose nanofibers, wherein the cellulose nanofibers originate from cellulose from the same brown seaweed sample(s) as the alginate.

2. The natural composition of claim 1, wherein a solid content of the natural composition is 2-10 wt %.

3. A method for preparing a natural composition comprising alginate and cellulose nanofibers, wherein the method comprises the steps of: providing a material of brown seaweed, purifying the material to remove impurities from the brown seaweed comprising the alginate and cellulose, and nanofibrillating the cellulose of the purified material.

4. The method of claim 3, wherein the step of purifying the material comprises the use of one or more cellulose bleaching substances.

5. A method for preparing a shaped material comprising alginate and cellulose nanofibers, the method comprising the method of claim 3, and further steps of: forming a shaped material of the composition, and crosslinking the alginate.

6. The method of claim 5, wherein the step of crosslinking the alginate comprises the use of a bivalent or trivalent cation, a peroxide, a vinylsilane, UV light, EDC/NHS, gamma radiation or any combination thereof.

7. The method of claim 6, wherein the bivalent or trivalent cation is one or more of Ca.sup.2+, Ba.sup.2+, Mg.sup.2+, Sr.sup.2+, Al.sup.3+ or Fe.sup.3+.

8. A shaped material comprising the composition of claim 1, wherein the alginate is cross-linked.

9. The natural composition of claim 1, wherein the brown seaweed is selected from the group consisting of Laminaria digitata, Laminaria hyperborean, Macrocystis pyrifera, Ascophyllum nodosum, Sargassum spp., Laminaria japonica, Ecklonia maxima and Lessonia nigrescens.

10. The natural composition of claim 1, wherein the concentration of cellulose is 10-40 wt % and the concentration of alginate is 20-60 wt %.

11. (canceled)

12. (canceled)

13. The method of claim 3, wherein the brown seaweed is selected from the group consisting of Laminaria digitata, Laminaria hyperborean, Macrocystis pyrifera, Ascophyllum nodosum, Sargassum spp., Laminaria japonica, Ecklonia maxima and Lessonia nigrescens.

14. The shaped material of claim 8, wherein the brown seaweed is selected from the group consisting of Laminaria digitata, Laminaria hyperborean, Macrocystis pyrifera, Ascophyllum nodosum, Sargassum spp., Laminaria japonica, Ecklonia maxima and Lessonia nigrescens.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIGS. 1a and 1b show SEM images of the cell wall structure of the raw materials, stripe and blade, respectively (scale bar: 100 μm). In FIGS. 1c and 1d are photographs of the raw materials and in FIGS. 1e and 1f photographs of the bleached structures. In FIGS. 1g and 1h 0optical microscopy (OM) images (above) and polarized optical microscopy (POM) images (below) at different fibrillation processing time (scale bar: 200 μm) are shown. Measured size distribution of the obtained nanofibers (scale bar: 600 nm) are shown in FIGS. 1i and 1j.

[0034] FIG. 2 shows rheological data for the inks, S-A-CNF and B-A-CNF, respectively. In FIG. 2a is shown flow curves, in FIGS. 2b and 2c are photographs of the ink gels at 2 wt. %. In FIG. 2d is shown the storage modulus G′, and in FIG. 2e the loss modulus G″ measured over time where a CaCl.sub.2 solution was added 50 s after the measurement was started.

[0035] FIG. 3. shows compression evaluation of 3D printed S-A-CNF and B-A-CNF to determine their mechanical properties after crosslinking. In FIG. 3a compressive stress-strain curves up to 60% strain are shown. In FIG. 3b is shown photographs of the hydrogels after crosslinking. In FIG. 3c is shown the compressive stress and in FIG. 3d the compressive modulus at 30 and 60% strain.

DETAILED DESCRIPTION

[0036] In the following is described a method for producing a composition and matrix comprising alginate and nanofibrillated cellulose originating from alginate and cellulose from the same brown seaweed species Laminaria digitate sample(s). The thus produced composition and matrix are evaluated and compared to reference material comprising nanofibrillated cellulose from other cellulose sources. It is to be understood that the methods steps and chemicals presented below are mere examples and should not be construed as limiting for the methods and composition/matrix. It is shown that the composition/matrix obtained from Laminaria digitate has characteristics similar to alginate/CNF compositions known in the art.

Experimental Section

[0037] Materials. Brown seaweed, (Laminaria digitata) was provided by The Northern Company Co. (Træna, Norway) and used as a raw material. The fast-growing seaweed was cultivated in the North Atlantic Ocean on the Norwegian west coast and harvested in May 2017. Laminaria digitata consists of a holdfast (root-like), stipe (stem-like) and blade (leaf-like) structure (Misurcova et al., 2012). Its carbohydrate composition vary with season, geographic location, and age (Manns et al., 2017), as well as between the different parts of the seaweed (stipe and blade) (Black et al., 1950). Fresh samples were stored in wet condition in closed bags in a freezer before use. The stipe and blade of the seaweed were prepared in separate batches for comparison and utilization of the entire structure. Both materials, stipe and blade, were purified and nanofibrillated using equivalent processing conditions.

[0038] The chemicals used in the purification process, sodium hydroxide (NaOH), sodium chlorite (NaClO.sub.2), acetic acid (CH.sub.3COOH)), chemical composition (sodium bromide (NaBr)), and ionic crosslinking (calcium chloride (CaCl.sub.2.2H.sub.2O)) of laboratory grade were purchased from Sigma-Aldrich (Stockholm, Sweden) and were used as received. Deionized water was used for all experiments.

[0039] Preparation. The stipe and blade of the seaweed were left in room temperature for about 24 h in order to defrost and thereafter cut into smaller pieces, here about 1-3 cm.sup.2, prior to purification using bleaching with NaClO.sub.2 (1.7%) in an acetate buffer (pH 4.5) 80° C. for 2 h. In the purification process all colour was removed and the material was thereafter washed until a neutral pH was reached. The solid recovery was calculated as yields according to the following equation:

Yield (%)=W.sub.1/W.sub.0×100  (1),

where W.sub.1 indicates the dry weight of the sample after the bleaching and W.sub.0 indicates the initial dry weight of the seaweed. The presented yield is based on the average of three different batches.

[0040] The materials were nanofibrillated using an MKZA6-3 Supermasscolloider ultrafine friction grinder (Masuko Sangyo Co. Japan) with coarse silica carbide (SiC) grinding stones, and at a concentration of 2 wt. %. The nanofibrillation was operated in contact mode with a gap of the two disks set to −90 μm, at 1500 rpm. The total processing was 40 min and 30 min for the stipe and blade material, respectively. The prepared inks were denoted S-A-CNF (stipe) and B-A-CNF (blade).

[0041] The energy consumption for the fibrillation process was established by direct measurement of power using a power meter, Carlo Gavazzi, EM24 DIN (Italy) and the processing time. The energy demand was calculated from the product of power and time and the energy consumption for the fibrillation process is expressed as kWh per dry weight kg of the nanofibers. Samples were collected at regular intervals to assess the degree of fibrillation. The process was finalized when a plateau in viscosity was reached and no larger structures could be observed by microscope. The prepared inks were kept in a refrigerator at 6° C. prior to 3D printing of the hydrogels.

[0042] 3D printing of biomimetic hydrogels. Cylindrical disks of S-A-CNF and B-A-CNF were 3D printed using the INKREDIBLE 3D bioprinter, CELLINK AB (Gothenburg, Sweden); a pneumatic-based extrusion bioprinter. The solid discs (10 mm diameter, 4 mm high, 6 layers) were designed in the CAD software 123D Design (Autodesk) and the created STL files were subsequently converted into g-code using Repetier-Host (Repetier Server) software. A nozzle diameter of 0.5 mm was used at a pressure of 5 kPa and dosing distance of 0.05 mm. The two ink formulations were 3D printed directly onto a glass petri dish and crosslinked thereafter in a bath of a 90 mM aqueous solution of CaCl.sub.2 for 30 min directly on the petri dish and finally washed with deionized water. The printability was evaluated with concern to printer parameters and shape fidelity.

[0043] Chemical composition. The composition of the bleached stipe and the blade were assessed in terms of alginate and cellulose content; starting with a dry weight of 10 g. For the isolation of alginate, the procedure of Zubia et al., 2008 was followed using a formaldehyde alkali treatment method. The precipitate was washed with absolute ethanol followed by acetone, prior to drying for 24 h at 40° C. The alginate fraction was expressed as a percentage of dry weight.

[0044] The cellulose content was extracted following the method described by Siddhanta et al., 2009. In brief, the samples were defatted repeatedly with MeOH, followed by 600 ml NaOH (0.5M) solution at 60° C. overnight, washed and dried in room temperature. For removal of any remaining minerals, the dried material was re-suspended in a 200 ml solution of hydrochloric acid (5% v/v), washed and dried for 24 h at 40° C. The cellulose fraction was denoted as a percentage on a dry weight basis.

[0045] Polarized Optical Microscopy (POM). A polarizing microscope, Nikon Eclipse LV100N POL (Japan) and the imaging software NIS-Elements D 4.30 was used to assess the nanofibrillation process. Reference images without polarization filter were also captured. Viscosity. Viscosity measurements were also performed during the nanofibrillation using a Vibro Viscometer SV-10, (A&D Company, Ltd, Japan), at a constant shear rate. The velocity (shear rate) of the sensor plates keeps periodically circulating from zero to peak because sine-wave vibration is utilized, at a frequency of 30 Hz. The viscosity measurements were repeated once the temperature of the samples had been stabilized to 22.3±1.0° C. to confirm that a plateau in viscosity had been reached during fibrillation. The presented values are an average of three measurements for each sample.

[0046] Atomic Force Microscopy (AFM). The morphology was studied after the nanofibrillation using an Atomic Force Microscopy (AFM). The fibrillated sample suspension (0.01 wt-%) was dispersed and deposited by spin coating onto a clean mica for imaging. The measurements were performed on a Veeco Multimode Scanning Probe, USA in tapping mode, with a tip model TESPA (antimony (n) doped Si), Bruker, USA. The nanofiber size (width) was measured from the height images using the Nanoscope V software and the average values and deviations presented are based on 50 different measurements. All measurements were conducted in air at room temperature.

[0047] Scanning Electron Microscopy (SEM). The cross-sections of the stipe and blade were observed using a using a SEM JCM-6000 NeoScope (JEOL, Tokyo, Japan) at an acceleration voltage of 15 kV to study their cell wall structures. In addition, the cross-section of the nanofilms were observed. All samples were coated using a coating system machine (Leica EM ACE200, Austria) with a platinum target. The coating was performed within a vacuum of approximately 6×10.sup.−5 mbar, under a current of 100 mA, for 20 s to obtain a coating thickness of 25 nm.

[0048] Rheology. The rheological behaviour of the hybrid-inks, S-A-CNF and B-A-CNF were analysed using the Discovery HR-2 rheometer (TA Instruments, UK) at 25° C. A cone-plate (20 mm) was used and the shear viscosity was measured at shear rates from 0.01-1000 s.sup.−1. Furthermore, the change in moduli while cross-linking the ink was measured with a plate-plate configuration (8 mm, gap 500 μm). The oscillation frequency measurements were conducted at 0.1% strain, based on oscillation amplitude sweeps to establish the LVR, and at a frequency of 1 Hz for 10 min. 50 s after the measuring was started, a 1 mL drop of 90 mM CaCl.sub.2 solution was added around the inks causing gelling while simultaneously measuring the storage and loss modulus.

[0049] Compression properties. Uniaxial unconfined compression tests of the 3D printed and cross-linked hydrogels were carried out using a dynamic mechanical analyser DMA Q800 (TA Instruments, New Castle, USA) at 25° C. The hydrogels were preloaded using a load of 0.05 N, and subsequently compressed up to a strain of 100%, and at a strain rate of 10% min.sup.−1. The materials were compared by the stress and tangent modulus at 30% and 60% compressive strain level, respectively. The disks with dimensions of 10 mm in diameter and a height of 4 mm were tested 6 times for each material; the average results are reported.

Results and Discussion

[0050] Purification and characterization of raw material. The yield and chemical composition after the pretreatment of the raw materials is presented in Table 1.

TABLE-US-00001 TABLE 1 Yield calculation, and cellulose and alginate content after purification Initial Weight Total Raw weight after bleaching yield Cellulose Alginate Materials [g] [g] [%] [wt. %] [wt. %] Stipe 70 49.7 .sup. 71 ± 8 33 ± 6 45 ± 13 Blade 70 51.8 74.2 ± 7 23 ± 3 46 ± 11
The objective of the purification of the seaweed was to remove the colour pigments and other impurities, while maintaining as much of the inherently high alginate content found in brown seaweed, together with the cellulose content. Indeed, the yield of the stipe and blade were as high as 71% and 74%, respectively after the bleaching procedure (Table 1). These values can be compared to that of wood after direct bleaching, namely about 70%, yet mainly composed of hollocellulose.

[0051] An alginate content of 25-30 wt. % and cellulose content of 10-15 wt. % have previously been reported for the raw seaweed, Laminaria Digitata harvested in Scotland during May (Schiener et al., 2015). From Table 1, after bleaching, the alginate and cellulose contents were higher, yet their relative percentage to each other was maintained. The stipe measured a higher cellulose content, though the significance is questionable considering the standard deviations, which might reflect the heterogeneity of the raw material even within a specie (Manns et al., 2014). There are only a limited number of studies that have measured the compositional content of the different parts of brown seaweed, and for Laminaria Digitata, a cellulose content of 6-8 wt. % and 3-5 wt. % have been reported for the stipe and the blade, respectively (Black et al., 1950). However, the cellulose content is highly dependent on several factors such as: measuring methods, geographical, seasonal, and age to mention a few (Schiener et al., 2015).

[0052] Nanofibrillation process and characterization of inks. The nanofibrillation of the purified materials was carried out using viscosity measurements and POM/OM to assess the degree of fibrillation throughout the process. The route from the raw materials to nanoscale is shown in FIG. 1.

[0053] The viscosity may be used as an indication of the degree of fibrillation, where the viscosity plateau has signified a strong network formation of separated nanofibers with a maintained length (Berglund et al., 2016).

[0054] The increased viscosity and plateau of both S-A-CNF and B-A-CNF were clearly observed from the samples measured in room temperature, namely 3289, and 2102 mPas, respectively. When comparing these viscosity values to that of wood pulp, the viscosity plateau at 1565 mPa s was significantly lower and reached first after 90 min of fibrillation.

[0055] In FIGS. 1c and 1d photographs of the different parts of brown seaweed, stipe and blade, are shown. From the cross-sectional views, FIGS. 1a and 1b, differences of the cell wall structures of the different parts of brown seaweed, stipe and blade, are apparent. A more organized structure was observed for the stipe (FIGS. 1a, 1c), compared to the more layer-like structure of the blade (FIGS. 1b, 1d), displaying a wide range of pore-sizes. Completely white structures were obtained after the bleaching process (FIGS. 1e, 1f). In FIGS. 1g and 1h optical microscopy (OM) images (above) and polarized optical microscopy (POM) images (below) at different fibrillation processing time (scale bar: 200 μm) are shown. The nanofibrillation of the stipereached a maximum viscosity at an energy demand of 1.5 kWh/kg. In comparison, the blade had a slightly lower energy demand throughout the process, and the maximum viscosity was reached at an energy demand of 1.0 kWh/kg. The slightly higher energy demand of the stipe could be explained by its higher cellulose content (Table 1), which might acquire more energy to be separated. In addition, the arrangement of cellulose and alginate in the stipe appear to be more consolidated in thicker cell walls as seen in FIG. 1a). The nanofibers of S-A-CNF and B-A-CNF were in average 7±3 and 6±3 nm, respectively. Measured size distribution of the obtained nanofibers (scale bar: 600 nm) are shown in FIGS. 1i and 1j.

[0056] The measured energy consumption was, remarkably low for the nanofibrillation of both seaweed structures, in comparison to that of commercially bleached wood karft pulp, that reached a maximum viscosity at 8.4 kWh/kg under the similar processing conditions (Berglund et al., 2017). The importance of hemicellulose present for the process efficiency of nanofibrillation of wood pulp have previously been reported using ultrafine grinding (Iwamoto et al., 2008). The low energy demand suggest that the presence of alginate during nanofibrillation may act beneficial for the separation of nanofibers.

[0057] 3D printability and characterization of biomimetic hydrogels. The rheological behaviour of the inks were studied to evaluate their suitability for 3D printing. In FIG. 2a a shear-thinning behaviour is observed for both S-A-CNF and B-A-CNF inks, similar to viscosity curves previously reported for commercial alginate mixed with CNF (Abouzeid et al., 2018), as well as pure CNF (Markstedt et al., 2015). For S-A-CNF, the initial viscosity was 1224 Pa s and it decreased to 0.3 Pa s upon increasing the shear rate to 1000 1/s, in comparison to B-A-CNF which initially was lower at 578 Pa s, and dropped to 0.2 Pa s at a shear rate of 1000 1/s. Also, the higher viscosity of S-A-CNF compared to B-A-CNF can be visually seen in FIG. 2b and FIG. 2c. The high viscosity at low shear rates and the shear thinning behaviour with increasing shear rates provide shape fidelity during printing. To maintain the structural integrity after printing, crosslinking of the alginate is required, however. Hence, the gelling behaviour of the inks was studied by measuring the loss—(G′) and storage (G″) modulus as a function of time while crosslinking with CaCl.sub.2 (see FIGS. 2d and 2e). Both the storage modulus, FIG. 2d, and loss modulus, FIG. 2e, displayed an instant increase upon addition of CaCl.sub.2 solution at 50 s, and become gradually linear after additionally 50 s. The time was measured for additionally 5 min to confirm this plateau. The higher storage modulus of S-A-CNF reflects a higher degree of cross-linking, in turn resulting in a higher strength or mechanical rigidity.

[0058] 3D-printability and crosslinking enables the use of inks in a wide range of applications that for example requires specific shapes for wound dressing (Leppiniemi et al., 2017), or even 3D-printing of living tissues and organs (Markstedt et al., 2015). The printability and stability of 3D discs from S-A-CNF and B-A-CNF inks, as prepared at 2 wt. % solid content, were studied and the printing parameters were tuned through a trial-and-error method. Both inks could be printed without collapse of the structure, yet S-A-CNF displayed a better shape fidelity likely attributed to the higher viscosity.

[0059] A minor shrinkage of the diameter and some swelling in the centre, appearing as a slightly convex surface were observed after crosslinking of the discs. These tendencies of shape deformation after CaCl.sub.2 crosslinking have previously been reported for 3D printed alginate/CNF hydrogels (Markstedt et al., 2015; Leppiniemi et al., 2017). The behaviour might reflect inadequate homogeneity of the diffusion based CaCl.sub.2 crosslinking approach.

[0060] The ionic crosslinking of alginate using CaCl.sub.2 has been widely studied and by varying parameters such as crosslinking ratio (Freeman et al., 2017), and crosslinking time (Giuseppe et al., 2018) the mechanical properties of printed hydrogels can be tuned. However, other factor such as: molecular weight and M/G ratio, originating from the raw material and its alginate extraction process have a high influence both on crosslinking behaviour and fundamental mechanical behaviour.

[0061] The 3D printed S-A-CNF and B-A-CNF hydrogels were evaluated in compression to determine their mechanical properties after crosslinking, as presented in FIG. 3.

[0062] Since the compressive stress and strain curves revealed a viscoelastic non-linear stress-strain behaviour, the compressive modulus and stress at 30 and 60% strain were used for mechanical characterization (FIG. 3a) of the 3D printed hydrogels (see FIG. 3b).

[0063] In FIG. 3c and FIG. 3d, it is shown that S-A-CNF has an overall higher compressive property in comparison to B-A-CNF. This is in good agreement with the rheological behaviour and could be explained by a higher amount of CNF, reinforcing the structure.

[0064] However, the stiffness of alginate hydrogels is directly related to its crosslinking, and still the S-A-CNF with a lower amount of alginate displays a higher stiffness as seen in FIG. 3d.

[0065] In Laminara digitata, a higher amount of alginate rich in guluronic acid (G) were shown for the stipe when compared to the blade of the seaweed (Peteiro et al. 2018), thus equivalent with a lower M/G ratio in the stipe. Alginates with lower M/G ratio are known to display a higher affinity towards crosslinking (mechanical rigidity), and the gel strength of alginate is mainly dependent on content and length of the guluronic acid. A lower M/G ratio of the alginate in the S-A-CNF hydrogel, compared to that of B-A-CNF may further contribute to the higher compressive properties.

[0066] Notable is also that the maximum compressive stress could be measured at around 80% strain for the B-A-CNF hydrogel (175.2 kPa±3). At this strain the B-A-CNF hydrogel fractured, while the S-A-CNF hydrogel was compressed without any visual fractures. The combination of the alginate of S-A-CNF ink with its CNF content appear to assemble into a biomimetic hydrogel with high compressive stiffness and strength, yet highly flexible.

[0067] The above described composition may be used in bioprinting with living cells for example as bioinks in 3D bioprinting of soft-tissue.

[0068] Crucial for obtaining the properties of the composition discussed above, i.e. the rheological behaviour and in turn the printability of the composition, is the extraction process of both alginate and cellulose nanofibers. For example, alginate extraction-purification from brown seaweed using three different routes was shown by Gomez et al (2009) to result in significant differences in rheological and gelation behaviour. Another example by Hiasa et al (2016) demonstrated the difference between pectin-containing cellulose nanofibers (based on the natural raw material structure) opposed to the addition of commercial pectin to cellulose nanofibers. The commercial pectin that was added did not interact with the purified cellulose nanofibers, thus significantly limiting the dispersion properties (and, hence, printability) compared to the natural pectin-containing nanofibers. Hence, to obtain the printable composition described above, the alginate and cellulose nanofibers should originate from the same brown seaweed sample(s) and, hence, have a natural composition of alginate and cellulose.

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