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Title:
POLYSACCHARIDE FOR USE IN REGENERATIVE MEDICINE AND/OR TISSUE ENGINEERING
Document Type and Number:
WIPO Patent Application WO/2020/075135
Kind Code:
A1
Abstract:
The present disclosure relates to the polysaccharide arabinoxylan and its use in medicine. More specifically, the present disclosure relates to arabinoxylan and its use as a biomaterial in regenerative medicine and/or tissue engineering purposes, in particular for use in peripheral nerve regeneration.

Inventors:
LÓPEZ CEBRAL RITA (PT)
ANTUNES CORREIA DE OLIVEIRA JOAQUIM MIGUEL (PT)
QUINTEROS LOPES HENRIQUEZ DA SILVA TIAGO JOSÉ (PT)
GONÇALVES DOS REIS RUI LUÍS (PT)
Application Number:
PCT/IB2019/058701
Publication Date:
April 16, 2020
Filing Date:
October 11, 2019
Export Citation:
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Assignee:
ASSOCIATION FOR THE ADVANCEMENT OF TISSUE ENGINEERING AND CELL BASED TECH & THERAPIES A4TEC ASSOCIAC (PT)
International Classes:
A61P25/00; A61K31/715; C08B37/00
Domestic Patent References:
WO2018042405A12018-03-08
Foreign References:
US20180235258A12018-08-23
Other References:
LINN BERGLUND ET AL: "Promoted hydrogel formation of lignin-containing arabinoxylan aerogel using cellulose nanofibers as a functional biomaterial", RSC ADVANCES, vol. 8, no. 67, 14 November 2018 (2018-11-14), pages 38219 - 38228, XP055661441, DOI: 10.1039/C8RA08166B
DONALD ADUBA ET AL: "Polysaccharide Fabrication Platforms and Biocompatibility Assessment as Candidate Wound Dressing Materials", BIOENGINEERING, vol. 4, no. 4, 18 January 2017 (2017-01-18), pages 1, XP055661371, DOI: 10.3390/bioengineering4010001
DONALD C. ADUBA ET AL: "Fabrication, characterization, and in vitro evaluation of silver-containing arabinoxylan foams as antimicrobial wound dressing : ARABINOXYLAN FOAMS AS ANTIMICROBIAL WOUND DRESSING", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A, vol. 104, no. 10, 3 June 2016 (2016-06-03), pages 2456 - 2465, XP055661425, ISSN: 1549-3296, DOI: 10.1002/jbm.a.35783
L. BACAKOVA ET AL: "Polysaccharides in Cell Carriers for Tissue Engineering: the Use of Cellulose in Vascular Wall Reconstruction", PHYSIOL. RES., 28 February 2014 (2014-02-28), pages S29 - S47, XP055665641, Retrieved from the Internet [retrieved on 20200205]
IZYDORCZYK M S ET AL: "Barley @b-glucans and arabinoxylans: Molecular structure, physicochemical properties, and uses in food products-a Review", FOOD RESEARCH INTERNATIONAL, ELSEVIER, AMSTERDAM, NL, vol. 41, no. 9, 1 November 2008 (2008-11-01), pages 850 - 868, XP025584169, ISSN: 0963-9969, [retrieved on 20080422], DOI: 10.1016/J.FOODRES.2008.04.001
KUEN YONG LEE ET AL: "Electrospinning of polysaccharides for regenerative medicine", ADVANCED DRUG DELIVERY REVIEWS, vol. 61, no. 12, 1 October 2009 (2009-10-01), pages 1020 - 1032, XP055009067, ISSN: 0169-409X, DOI: 10.1016/j.addr.2009.07.006
Attorney, Agent or Firm:
PATENTREE (PT)
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Claims:
C L A I M S

1. Composition comprising arabinoxylan for use in regenerative medicine and/or tissue engineering.

2. Composition according to the previous claim for use in peripheral nerve regeneration.

3. Composition according to any of the previous claims for use in treatment or regeneration of neurilemma cell.

4. Composition according to any of the previous claims for use in treatment or therapy of diseases that are improved or prevented by regeneration of neurilemma cell.

5. Composition according to any of the previous claims for use in treatment or therapy of diseases that respond positively to the regeneration of neurilemma cell.

6. Composition according to any of the previous claims for use in treatment or therapy of Charcot-Marie-Tooth disease, Guillain-Barre syndrome, schwannomatosis, chronic inflammatory demyelinating polyneuropathy, or leprosy.

7. Composition for use according to any of the previous claims further comprising a polymer selected from the following list: natural polymer, synthetic polymer or semi synthetic polymer, in particular chitosan, collagen or chondroitin sulfate.

8. Composition for use according to any of the previous claims, wherein the composition comprises 0.1-50% (warabinoxyian/vComposition) of arabinoxylan.

9. Composition for use according to the previous claim, wherein the composition comprises 0.5-30% (warabinoxyian/vComposition) of arabinoxylan.

10. Composition for use according to the previous claims 8-9, wherein the composition comprises 0.5-10% (wa rabinoxyian/vComposition) of arabinoxylan.

11. Composition for use according to any of the previous claims, wherein arabinoxylan has a viscosity between 1- 100 cSt, preferably 5-50 cSt, more preferably 10.2-31.4 cSt, at 30 °C.

12. Composition for use according to any of the previous claims, wherein arabinoxylan has a molecular weight between 5-900 KDa, preferably 50-400 kDa, more preferably 56.7-323 kDa.

13. Composition for use according to any of the previous claims comprising, carboxymethyl-arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, for use in medicine, preferably to arabinoxylan, carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan.

14. Composition comprising arabinoxylan for use according to any of the previous claims, further comprising an excipient selected from: filler, binder, disintegrant, coating, sorbent, anti-adherent, lubricant, glidant, preservative, antioxidant, coloring agent, solvent and co-solvent, buffering agent, chelating agent, viscosity imparting agent, surface active agent, humectant, or mixtures thereof.

15. Composition comprising arabinoxylan for use according to any of the previous claims, further comprising a polysaccharide selected from: glycosaminoglycans, cellulose, alginate, fucoidan, dextrin, carrageenan, gellam gum, guar gum, or mixtures thereof.

16. Composition comprising arabinoxylan for use according to any of the previous claims, further comprising a protein selected from: collagen, laminin, albumin, keratin or fibronectin, or mixtures thereof.

17. Composition comprising arabinoxylan for use according to any of the previous claims, further comprising cell culture media or other buffered media.

18. Composition comprising arabinoxylan for use according to any of the previous claims, wherein the composition is in the form of a hydrogel.

19. Composition comprising arabinoxylan for use according to any of the previous claims, wherein the composition is administered by topical route, enteral route or parenteral route.

Description:
POLYSACCHARIDE FOR USE IN REGENERATIVE MEDICINE AND/OR TISSUE

ENGINEERING

Technical field

[0001] The present disclosure relates to the polysaccharide arabinoxylan and its use in medicine. More specifically, the present disclosure relates to arabinoxylan and its use as a biomaterial in regenerative medicine and/or tissue engineering purposes, in particular for use in peripheral nerve regeneration.

Background Art

[0002] Peripheral nerve (PN) injuries are worldwide health problem.

[0003] The limited natural regenerative capacity of the peripheral nervous system makes the recovery from these lesions frequently unsuccessful.

[0004] The socio-economic and healthcare implications of inefficient peripheral nerve recovery are numerous.

[0005] Artificial 3D systems for nerve guidance have shown fair success in promoting PN regeneration (PNR) of short gap lesions. But the results were suboptimal in cases of long nerve gap injuries. Accordingly, novel 3D platforms should be developed that are able to better guide the regeneration of the injured axons. Natural biomaterials are preferred over synthetic or semi-synthetic ones for this purpose.

[0006] Polysaccharides as chitosan and gellan gum have been utilized by different groups to prepare these 3D systems, leading to very interesting results, but sub-optimal.

[0007] The search for new polymers is an important step in obtaining these 3D systems. In this sense, the application of novel biomaterials to the PNR area is expected to compensate for the deficient results observed to date.

[0008] Arabinoxylans (ABXs) are a major hemicellulosic component of many cereals.

[0009] Arabinoxylans present a highly branched structure characterized by a b-1,4- xylopyranose backbone, branched by very short side chains of a-1,2- and a-1,3- linked arabinofuranose. Glucuronic acid and its 4-0- methyl ether are also present in terminal non reducing positions on the side chains.

[0010] Cereal arabinoxylans also contain ferulic acid, attached by an ester linkage to arabinoxyl units.

[0011] The known antioxidant capacity of these polymers is influenced by the content of this ferulic acid.

[0012] Around 95% of the biomass produced around the world is a lignocellulosic material not edible by humans. This makes its utilization as a raw material for the obtaining of chemicals and biofuels an attractive eco-friendly and socio-economic strategy. Indeed, most of lignocellulosic biomass is mainly formed by three interesting components: cellulose (40- 50%), hemicelluloses (20-30%) and lignin (10-25%).

[0013] Accordingly, the utilization of arabinoxylans from biomass constitutes the opportunity to give a second live to wastes that otherwise would suppose an environmental concern.

[0014] ABXs are known to form permanent gels after being in contact with oxidizing agents at room temperature. More specifically, oxidative coupling reactions occur that cross-link polysaccharide chains forming covalent hydrogels. This property of ABX has been widely exploited by the food industry. However, it has never been applied to the area of PNR or any other regenerative medicine and/or tissue engineering application.

[0015] These facts are disclosed in order to illustrate the environmental and socio-economic problems addressed by the present disclosure.

General Description

[0016] The present disclosure relates to the polysaccharide arabinoxylan and its use in medicine, preferably it relates to arabinoxylan and its use as a biomaterial in regenerative medicine and/or tissue engineering purposes.

[0017] Two ABXs were studied: Arabinoxylan with low viscosity (ABX LV) and arabinoxylan with medium viscosity (ABX MV). The viscosity and molecular weight of ABX LV were 10.2 cSt (1% w/v; Ostwald C-type viscometer, 30 °C) and 56.7 Kd (MAALS), respectively. The viscosity and molecular weight of ABX MV were 31.4 cSt (1% w/v; Ostwald C-type viscometer, 30 °C) and 323 Kd (MAALS), respectively. These data were given by the supplier. [0018] The present disclosure relates to arabinoxylan, carboxymethyl-arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, for use in medicine, preferably to arabinoxylan, carboxymethyl-arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, for use in regenerative medicine and/or tissue engineering, more preferably to a composition comprising arabinoxylan, carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, for use in regenerative medicine and/or tissue engineering, in particular it relates to a composition comprising arabinoxylan, carboxymethyl-arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, for use in the treatment or prevention of a tissue disease or a tissue defect, both human and veterinarian.

[0019] In an embodiment, said composition comprising arabinoxylan, carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may be for use in both human and veterinarian.

[0020] In an embodiment, the composition of the present disclosure may be used in treatment or regeneration of neurilemma cell.

[0021] In an embodiment, the composition of the present disclosure may be used in treatment or therapy of diseases that are improved or prevented by regeneration of neurilemma cell.

[0022] In an embodiment, the composition of the present disclosure may be used in treatment or therapy of diseases that respond positively to the regeneration of neurilemma cell.

[0023] In an embodiment, the composition of the present disclosure may be used in treatment or therapy of Charcot-Marie-Tooth disease, Guillain-Barre syndrome, schwannomatosis, chronic inflammatory demyelinating polyneuropathy, or leprosy.

[0024] In an embodiment, said composition comprising arabinoxylan, carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may be for use in peripheral nerve regeneration, in particular, Schwann cells cultured in culture medium supplemented with arabinoxylan showed with good viability and positive metabolic activity profiles.

[0025] In an embodiment, said composition may further comprise a polymer selected from the following list: natural polymer, synthetic polymer or semi-synthetic polymer, in particular chitosan, collagen or chondroitin sulfate.

B [0026] In an embodiment, said composition may comprise 0.1-50% (w ar abmoxyian /v C om P osition) of arabinoxylan, preferably 0.5-30% (w a rabinoxyian/v C omposition), more preferably 0.5-10% (Warabinoxylan/Vcomposition) O G it may Comprise 0.1-50% (Wcarboxymethyl-arabinoxylan/Vcomposition) Of carboxymethyl-arabinoxylan, preferably 0.5-30% (w C arboxymethyi-arabinoxyian/v C omposition), more preferably 0.5-10% (Wcarboxymethyl-arabinoxylan /Vcomposition) OG it may Comprise 0.1-50% (Wmethacrylated- arabinoxyian/vcomposition) of methacrylated-arabinoxylan, preferably 0.5-30% (w me thacryiated- arabinoxylan/Vcomposition), more preferably 0.5-10% (Wmethacrylated -arabinoxylan/Vcomposition) OG it may comprise 0.1-50% (w a rabinoxyian-suifate/v C omposition) of arabinoxylan-sulfate, preferably 0.5-30% (Warabinoxylan-sulfate/Vcomposition) / more preferably 0.5-10% (Warabinoxylan-sulfate /Vcomposition) OG it ay comprise 0.1-50% (w a zo-arabinoxyian/v C omposition) of azo-arabinoxylan, preferably 0.5-30% (w azo - arabinoxylan/Vcomposition) / more preferably 0.5-10% (Wazo-arabinoxyla n/Vcomposition).

[0027] In an embodiment, said composition may comprise arabinoxylan, carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, wherein said arabinoxylan, carboxymethyl-arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may have a viscosity between 1- 100 cSt at 30 °C, preferably 5-50 cSt at 30 °C, more preferably 10.2-31.4 cSt at 30 °C. The viscosity is determined by preparing a 1% solution and using an Ostwald C-type viscometer set at 30°C against distilled water.

[0028] In an embodiment, arabinoxylan, carboxymethyl-arabinoxylan, methacrylated- arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may have a molecular weight between 5-900 KDa, preferably 50-400 kDa, more preferably 56.7-323 kDa.

[0029] In an embodiment, the composition now disclosed may further comprise an excipient selected from: filler, binder, disintegrant, coating, sorbent, antiadherent, lubricant, glidant, preservative, antioxidant, coloring agent, solvent and co-solvent, buffering agent, chelating agent, viscosity imparting agent, surface active agent, humectant, or mixtures thereof.

[0030] In an embodiment, the composition now disclosed may further comprise a polysaccharide, preferably wherein the polysaccharide is selected from: glycosaminoglycans, cellulose, alginate, fucoidan, dextrin, carrageenan, gellam gum, guar gum, or mixtures thereof.

[0031] In an embodiment, the composition now disclosed may further comprise a protein, preferably wherein the protein is selected from: collagen, laminin, albumin, keratin or fibronectin, or mixtures thereof. [0032] In an embodiment, the composition now disclosed may further comprise a cell culture media or other buffered media.

[0033] In an embodiment, the composition now disclosed may be in the form of a hydrogel.

[0034] In an embodiment, the composition now disclosed may be administered by topical route, enteral route or parenteral route. Topical routes include application into the skin and mucous. Parenteral administration routes include intra-arterial, intra-articular, intracavitary, intracranial, epidural, intradermal, intralympathic, intramuscular, intraocular, intrasynovial, intravenous, or subcutaneous. Enteral routes include oral and gastro-intestinal.

[0035] In a preferred embodiment, dosage of the composition can be adapted to the administration route, as well as to the patient profile, including age, gender, condition, disease progression, or any other phenotypic or environmental parameters.

[0036] In an embodiment, ABX has diverse ratios of arabinose:xylose and the mono- to di- substituted Xylp residues, in particular the ratio is 0.59.

[0037] In an embodiment, ABX has different proportions of unsubstituted, mono- and di- substituted Xylp residues, such as ratio of mono- (M) to di-substituted (D) Xylp residues are between 0.83 - 1.01.

[0038] In an embodiment, arabinoxylan or its derivatives, for example carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may be used in the treatment or prevention of bone, cartilage, osteochondral, muscle, musculoskeletal, ocular, skin, vascular, liver, kidney, pancreatic, peripheral nerve, spinal cord or brain diseases, human or veterinarian.

[0039] In an embodiment, arabinoxylan or its derivatives, for example carboxymethyl- arabinoxylan, methacrylated-arabinoxylan, arabinoxylan-sulfate or azo-arabinoxylan, may be used in the preparation of hydrogels or other scaffolds for tissue engineering and regenerative medicine, human or veterinarian.

[0040] In an embodiment, arabinoxylan or its derivatives may be used in the preparation of hydrogels, nanoparticles or other vehicles for human or veterinarian drug/cell delivery.

[0041] In an embodiment, high molecular weight chitosan or its derivatives may be used as human or veterinarian viscosupplements.

[0042] In an embodiment, the composition may further comprise a hydrogel or a plurality of hydrogels. Preferably, wherein the hydrogel may be selected from a list consisting of carbopol, matrigel, hyaluronic acid, dextran, alginate, collagen, gellan gum, or mixtures thereof. The composition further comprising a hydrogel, or a plurality of hydrogels has the advantage of acting as a matrix for improved cellular migration and proliferation.

[0043] In an embodiment, the composition may further comprise an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, or mixtures thereof.

[0044] In an embodiment, the compositions may be combined with other excipients or active substances used in the context of veterinarian and human medicine.

[0045] The present disclosure provides arabinoxylan or its derivatives to use in regenerative medicine or tissue engineering (human or veterinarian). Moreover, the present disclosure provides the use of arabinoxylan or its derivatives in the manufacture of a medicament for regenerative medicine or tissue engineering (human or veterinarian).

[0046] The disclosure provides the composition described above to use in human or veterinarian therapy. Further, the present disclosure provides the use of the composition described above in the manufacture of a medicament to use in human or veterinarian regenerative medicine or tissue engineering.

[0047] The disclosure provides the composition described above to use in human or veterinarian drug delivery.

[0048] The disclosure provides the composition described above to use in human or veterinarian cell delivery.

[0049] The disclosure provides the composition described above to use in human or veterinarian diagnosis.

[0050] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objectives, advantages and features of the solution will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the solution.

Brief Description of the Drawings

[0051] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the present disclosure. [0052] Figure 1: Representative example of the FTIR spectrum obtained for the studied arabinoxylans, correspondent to ABX LV. The characteristic bands related to the ABX chemical structure are indicated.

[0053] Figure 2: 1 H NMR spectra obtained for the studied arabinoxylans. A) ABX LV and B) ABX MV. The spectra show the integration of the peaks correspondent to the Araf HI regions (d 5.4, Integral I, and d 5.3 to 5.22, Integral II) and the H2 region correspondent to the un substituted Xylp (d 3.32, Integral III).

[0054] Figure 3: XRD spectra correspondent to the studied arabinoxylans. A predominant amorphous character can be appreciated.

[0055] Figure 4: Thermograms correspondent to the DSC analysis of the studied arabinoxylans. A) ABX LV and B) ABX MV. Both endothermic and exothermic peaks are observed.

[0056] Figure 5: Representative image of the syringeability and injectability of both ABXs. The image corresponds to an ABX MV solution.

[0057] Figure 6: Inverted microscope evaluation of the viability of Schwann cells cultured in cell culture medium supplemented with the studied arabinoxylans.

[0058] Figure 7: Analysis of the viability of Schwann cells cultured in cell culture medium supplemented with the studied arabinoxylans. Calcein AM stained live cells green and propidium iodide stained dead cells red.

[0059] Figure 8: Analysis of the metabolic activity of Schwann cells cultured in cell culture medium supplemented with the studied arabinoxylans. Alamar Blue reagent was used for this evaluation. A) ABX LV and B) ABX MV.

[0060] Figure 9: Peripheral nerve regeneration scheme. A) Ongoing nerve regeneration after the implantation of an ABX scaffold. The scaffold is also a vehicle of pro-regenerative substances. B) Successfully repaired peripheral nerve and scaffold biodegradation.

Detailed Description

[0061] The present disclosure refers to the physicochemical characterization of the polysaccharide arabinoxylan, as well as its biological performance once in contact with the most important cells interplaying for peripheral nerve regeneration, the Schwann cells. [0062] The present disclosure refers to the application of this polysaccharide in peripheral nerve regeneration, as well as any other regenerative medicine/tissue engineering application.

[0063] In an embodiment, the ability of ABX to form 3D structures widely utilized with food industry purposes, are used as regenerative medicine/tissue engineering scaffolds.

[0064] In an embodiment, different 3D structures can benefit from the incorporation of arabinoxylan for their improved performance as regenerative medicine/tissue engineering scaffolds.

[0065] In an embodiment, the mixture of arabinoxylan and other natural, synthetic or semi synthetic polymers is of potential interest for regenerative medicine/tissue engineering applications.

[0066] In an embodiment, the chemical structure of the studied arabinoxylans (ABX) was characterized by Fourier Transform Infrared spectroscopy (FTIR) spectroscopy.

[0067] In an embodiment, the FTIR spectrum was obtained using Shimadzu IRPrestige 21 spectromer (Shimadzu, Europe).

[0068] In an embodiment, the FTIR samples were prepared as potassium bromide tablets at room temperature. The spectrum was collected by averaging 32 scans with a resolution of 4 cm 1 , corresponding to the 4000-400 cm 1 spectrum region.

[0069] In an embodiment, the FTIR spectra obtained for both ABX LV and ABX MV were very similar. In this way, the spectrum of ABX LV was selected as representative (see Figure 1). The peaks at 3344 cm 1 are related to the stretching vibrations of the hydroxyl (O-FI) groups present in the polysaccharides. Meanwhile, the stretching vibrations of C-H are represented in the peaks at 2873 and 2920 cm 1 . The peak at 1643 is due to H-O-H stretching, consequence of the hydration water present in polysaccharides. This peak may also be related to the presence of carboxylate groups (C = O stretching vibration), for instance those present in ferulic acid. Deformation modes of -CH2 are represented in the region from 1315 to 1433 cm 1 . Absorptions in the region between 875 and 1190 are due to C-C, C-OFI and C-O-C stretching. Finally, the peak at 896 cm 1 represents the glycosidic linkages existent between the sugar units. Accordingly, the FTIR spectra confirmed the chemical features of arabinoxylan.

[0070] In an embodiment, the composition of the studied ABXs was further studied by Nuclear Magnetic Resonance (NMR), confirming that the polymer now disclosed is ABX, and permitted to determine the arabinose:xylose ratio, the mono- to di-substituted Xylp residues ratio and the proportion of unsubstituted, mono- and di-substituted Xylp residues.

[0071] In an embodiment, for the obtaining of the 1 H-NM R spectra of arabinoxylan 700 mI of a 4.28 mg/ml D2O solution were placed in 5 mm NMR tubes.

[0072] In an embodiment, the spectra were recorded under the Burker Avance III spectral conditions: resonance frequency of 400,13 M Hz, with Is pulse and 3,98 ms acquisition time. MestReNova Software 9.0 (Mestre-lab Research) was used for spectral processing. Chemical shifts are reported in ppm (d). Coincidences with the descriptions by other authors confirmed that the obtained NMR spectra (displayed in Figure 2 (A and B)) correspond to that of arabinoxylan. Briefly, the signals correspondent to the anomeric protons of the arabinofuranosyl units (Araf) arise in the 5.2-5.4 ppm region. Meanwhile, those correspondent to the xylopyranosyl (Xylp) appear at d 4.4- 4.7 ppm. Single Araf units linked to the 3-0- of a Xylp residue (d 5.4) are distinguishable from those attached to 3-0- and 2-0- of the same Xylp residue (d 5.3 and 5.22, respectively). A weak doublet present at d 4.66 corresponds to the HI of the di-substituted Xylp residues. Meanwhile, at d 3.32 the H2 signal of unsubstituted Xylp can be seen. This signal is at the edge of the carbohydrate envelope and is not overlapped by any other signals. Taking into account the previous assignments, it is possible to calculate the arabinose:xylose ratio (A/X), as well as the ratio of mono- (M) to di- substituted (D) Xylp residues (M/D). The following signals were integrated for this purpose: the two Araf HI regions separately (d 5.4, Integral I, and the region from d 5.3 to 5.22, including major and minor signals, Integral II) and signal relative to the H2 of un-substituted Xylp (d 3.32, Integral III). The following equations led to the results:

I + II

{A /X) =

III + (I + 0.5 x II)

I

(M /D) =

0.5 x II

[0073] Also, if U represents the proportion of unsubstituted Xylp residues the ratio of substituted to unsubstituted Xylp residues is given by:

I + 0.5 x III

(M + D)/U

ΪΪΪ

[0074] Then, knowing that (M + D + U) = 1, the last two equations may be used to determine the proportions of M, D and U. [0075] The results show differences in the substitution degree between the two studied arabinoxylans (see Table ).

[0076] Table 1 : Differences in the substitution degree between the two studied arabinoxylans

[0077] Table indicating the quantitative NMR results for the determination of the arabinose:xylose ratio (A/X) and the mono- (M) to di-substituted (D) Xylp residues ratio (M/D), as well as the proportion of unsubstituted (U), mono- (M) and di-substituted (D) Xylp residues.

[0078] In an embodiment, the analyses of crystalline phases and amorphous content within the studied materials was obtained by X-Ray Diffraction (XRD) and permitted to determine the crystalline phases and amorphous content of the studied ABXs.

[0079] In an embodiment, a conventional Bragg-Brentano diffractometer (D8 Advance DaVinci, Bruker, Germany) was used, equipped with CuKa radiation produced at 40 kV and 40 mA. Data sets were collected in the 2q range of 5-60 9 , with a step size of 0.02 9 and Is for each step. The analyses were performed at RT. The absence of narrow consecutive peaks in the XRD spectra, and the presence of a well-defined peak instead, indicates that the polymer only presents a certain crystallinity (see Figure 3). In addition, the broad range of this peak, from 10 to 30 9 (2 Theta), points to the predominance of amorphous structure in both the analysed ABX. A difference between the two samples is however observed. This difference is related to the larger area of the ABX MV peak with respect to that of ABX LV, which indicates larger crystallinity and a smaller crystalline size in ABX MV.

[0080] In an embodiment, the thermodynamical properties of the studied arabinoxylans were evaluated by Differential Scanning Calorimetry (DSC).

[0081] In an embodiment, the DSC analysis was performed on a DSC Q100 apparatus (TA Instruments Inc., USA). [0082] In an embodiment, the DSC samples were packed in a TA aluminium pan (3.5-4 mg sample weight), which was covered with a suitable aluminium cover. An empty pan was used as a reference. Both temperature and heat flux were previously calibrated with Indium. All samples equilibrated at 0 °C, an isothermal step of 2 min and a progressive heating from 0 °C to 350 °C, at a rate of 10 °C/min. The measurements were performed under dry nitrogen atmosphere, at a flow rate of 50 ml/min. The obtained thermogram for ABX LV shows two different peaks (see Figure 4A). The first one (from 20 to 130 °C) is an endothermic peak correspondent to the absorbance of heat that leads to the evaporation of water absorbed within the ABX (Tg = 70.35 °C). The second peak is due to ABX thermal degradation. High amount of energy is needed for the rupture of glycosidic bonds, which is then liberated by the small ABX products. At this point the water bound to the ABX may act as plasticiser, lowering the Tm (Tm = 301.90 °C) and leading to the melting of ABX. However, in the thermogram obtained for ABX MV three peaks can be distinguished (see Figure 4B). The first one (from 20 to 130 °C) is also an endothermic peak correspondent to the evaporation of the water absorbed within the ABX. In this case the value for Tg is 71.59 °C. The most remarkable difference is the double melting peak of the thermal decomposition curve. It has been described in the literature that the majority of the bimodal melting profiles are due to melting and recrystallization (Tm = 281.92 °C), followed by a final melting at higher temperatures (Tm = 292.32 °C). This behaviour was reported to be consequence of the crystal size distribution. Also, in this case the water bound to the ABX may act as plasticiser.

[0083] In an embodiment, the syringeability and injectability of both ABXs was confirmed by using a lab syringe with a needle, in particular a 1 ml syringe with a 27G x 1/2" (0.4 x 12 mm) needle. The behaviour of 5 mg/ml ABX solutions in mQ water was evaluated. See the evidence in Figure 5.

[0084] In an embodiment, the biological interaction with the main cells responsible for peripheral nerve regeneration, Schwann cells, was evaluated. In particular, the cytotoxicity of ABX was evaluated using a Schwann cell line. For this purpose, immortalized Schwann cells on passage 18 were cultured with High Glucose DMEM medium, including 10% FBS, 1% penicillin/streptomycin and 1% Sodium Pyruvate. The medium was supplemented with arabinoxylan: 33.33%, 50% and 66.66%. Cell medium without ABX acted as control. The cells were kept at 37 °C in 5% CO2, and analysed after 1, 3 and 7 days of culture.

[0085] In an embodiment, the viability of Schwann cells was preliminarily studied using an inverted microscope (Primovert, Zeiss, Gottingen, Germany). Figure 6 shows the results obtained in the case of the maximum ABX proportion studied, together with the controls. The results for the other 2 proportions studied were similar. After 1 day in culture the cells displayed the characteristic phenotype of SCs. With time the number of cells in each well visibly increased, and these cells showed in all cases their characteristic phenotype. This indicates the affinity of these cells for ABX.

[0086] In an embodiment, the viability of Schwann cells cultured with cell medium supplemented with ABX (33.33%, 50% and 66.66%) was confirmed after 1, 3 and 7 days. Calcein AM and propidium iodide staining were utilized for this purpose. Calcein stains live cells in green and dead cells in red. Accordingly, the culture wells were washed with PBS and 1 ml of culture medium supplemented with 1 pg Calcein AM and 2 pg propidium iodide was added to each well. After 20 minutes of incubation, the samples were washed with PBS and analyzed using a transmitted and reflected light microscope (Axio Imager Zlm, Zeiss, Jena, Germany). Again, the results were very similar between the 3 ABX concentrations studied and the controls. Figure 7 shows the results obtained in the case of the maximum ABX proportion studied, together with the controls. Spread green cells can be seen at all the studied time- points, and their number increases with time both in the samples and the controls. Dead red cells are scarce. These results confirm the suitability of ABX as a biomaterial for peripheral nerve regeneration purposes.

[0087] In an embodiment, the metabolic activity of Schwann cells was evaluated using Alamar Blue (AB), which yields a fluorescent signal and a colorimetric change when incubated with metabolically active cells. The medium in the different culture wells was substituted by cell culture medium containing 10% AB. Incubation occurred during 3 h, after which 100 pi from each well were transferred to white opaque 96-well plates. Three replicates were analysed for each well. Fluorescence was monitored at 590 nm emission wavelength (excitation wavelength 530 nm), using a microplate reader (FL 600, Bio-Tek Instruments). After each AB determination, PBS was utilized to remove AB remains, after which fresh medium supplemented with ABX was added. The metabolic activity values were calculated by normalization with the mean fluorescence value obtained for the controls. The results (Figure 8 A and B) indicated similar metabolic activity in all the analysed samples. In all cases the values of metabolic activity increased along the study (time-points 1 to 7 days), further confirming the suitability of ABX for peripheral nerve regeneration purposes.

[0088] In an embodiment, the ABX is exemplified as a medical device utilized in the promotion of peripheral nerve regeneration. Figure 9A represents the events occurring at the site of a nerve transection injury, after an ABX hydrogel was implanted. In addition to act as a supporting scaffold for the growing axons, the hydrogel is acting as a vehicle of regenerative cues, such as growth factors and supportive cells. Meanwhile, Figure B represents the successfully repaired nerve and the subsequent biodegradation of the engineered ABX scaffold.

[0089] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0090] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term "a polysaccharide" or "the polysaccharide" also includes the plural forms "polysaccharides" or "the polysaccharides," and vice versa. In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0091] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0092] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

IB [0093] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0094] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0095] The above described embodiments are combinable.

[0096] The following claims further set out particular embodiments of the disclosure.