ES2693919A1 - NOVEL FRYTIC ACID RETICULATING AGENTS (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Novel phytic acid crosslinking agents. The present invention describes novel, stable and biodegradable protein and/or polysaccharide membranes, particularly chitosan and chitosan/hyaluronic acid, useful as scaffolds for bone tissue engineering. These membranes were fabricated by mixtures of polysaccharides and proteins of interest and novel crosslinking agents consisting of phytic acid (PA) derivatives obtained by a condensation reaction between phytic acid (PA) with a hydroxy compound.

Description

NOVELFUL PHYSICAL ACID RETICULATING AGENTS

The invention relates to the synthesis of novel crosslinking agents based on phytic acid (PA) derivatives of PA condensates with hydroxy compounds and their procurement procedure

This type of natural crosslinking agent proved useful in the preparation of gels

10 stable biodegradable crosslinks of proteins and / or polysaccharides, useful in biotechnology and biomedicine.

STATE OF THE TECHNIQUE

15 Myoinositol hexaphosphate or phytic acid (PA) is a natural and ubiquitous antioxidant that constitutes 1-5% by weight of most cereals, nuts, legumes and oil seeds. PA has the ability to bind minerals, proteins and polysaccharides. However, despite the possible energy inherent in the six phosphoric ester bonds, phytic acid is very stable at neutral and alkaline pH. The

20% phosphorus release requires acid hydrolysis. The chelation potential of PA is shown in its high affinity with polyvalent cations. Zinc complexes are one of the most stable, followed by those with iron, calcium and magnesium, among others. As an antioxidant, PA is extraordinarily stable and its antioxidant activity is closely related to its high binding affinity for

25 iron Forming a unique iron chelate, suppresses oxidative reactions catalyzed by iron in biological materials. In addition to this, other clinical advantages of PA are its effects against cancer and its ability to reduce cholesterol and blood lipids.

30 Many areas of PA use include industrial and medical applications. In the biomedical field, PA has been used as a natural cross-linking agent for proteins and polysaccharides. The cations of the natural polymer are able to bind directly with PA anions by electrostatic charges. Polysaccharides can also bind to PA by forming hydrogen bonds. The advantages of PA

35 compared to other multivalent non-toxic polyanions, such as tripolyphosphate (TPP), are that the number of anions capable of reacting with cations is twice greater than TPP. Chitosan (Ch) has been crosslinked with PA by gelation

ionic for the encapsulation of insulin in a chitosan matrix for oral insulin administration. Hemoglobin / gelatin / fibrinogen nanofiber frames cross-linked with PA have been developed for ischemic myocardial regeneration.

5 The preparation of chitosan has been attempted with combinations of polyol or sugar phosphate salts (polyol salts bearing a single anionic head, such as glycerol phosphate salts (G), sorbitol, fructose or glucose) as a novel approach to produce injectable neutral solutions of Ch that produce gels in situ after

10 implantation in vivo. Particularly, the acid solutions of Ch can be neutralized with glycerol phosphate (GP) and reach a pH close to 7 without precipitation of the polymer. The resulting solution acquires heat-sensitive properties so that the chitosan / GP solutions remain in a liquid state at low temperature and form a gel at 37 ° C. The process of gelling Ch with GP can

15 involve different types of interactions: (1) electrostatic attraction between amino groups of Ch and phosphate groups of GP (ionic crosslinking); (2) hydrogen bond between the Ch chains as a result of reduced electrostatic repulsion after neutralization of the Ch solution with GP; and (3) at physiological temperature, the release of protons from the remaining protonated amino groups can

20 lead to the appearance of Ch-Ch attractive forces (i.e. van der Waals, hydrogen bonding and hydrophobic interactions) that reinforce the physical structure of the hydrogel.

Different articles have been devoted to studying sterilization, characterization and

25 cytotoxicity of Ch / GP heat sensitive systems. Spherical Ch / GP hydrogel particles prepared using two sequential gelation steps for the encapsulation of cells and bioactive agents were applied. Injectable thermo-gelling nanogels were prepared in a simple way by combining self-assembled nanocapsules of amphiphilically modified Ch with GP and G to

30 encapsulate an antiepileptic drug.

A Ch / GP formulation has been marketed by Piramal Life Sciences (Bio-Orthopedics Division) and used to stabilize the microfracture-based blood clot by dispersing the soluble Ch solution through whole blood and

35 by implanting the mixture through bone marrow access holes in a cartilage lesion.

On the other hand, heat sensitive Ch / GP systems have been prepared in the presence of proteins. Such is the case of Ch / GP / collagen hydrogel formulations prepared to encapsulate stem cells derived from adult human bone marrow, which demonstrate excellent cell compatibility, representing a new alternative

5 as a framework for bone tissue engineering. In other works, an injectable temperature sensitive Ch / GP / collagen hydrogel was prepared for the culture of adipose tissue derived stem cells that show great biocompatibility and possibilities of tissue engineering applications.

10 Uniting the properties of phytic acid (PA) and the benefits of glycerol phosphate (GP) mentioned above, this work has been dedicated to the synthesis of novel natural cross-linking agents based on phytic acid derivatives (PA) of PA condensates with hydroxylic compounds, preferably glycerol; and its use in the preparation of stable biodegradable crosslinked gels of proteins and / or

15 polysaccharides

DESCRIPTION OF THE INVENTION

The present invention describes novel stable and biodegradable membranes of

20 proteins and / or polysaccharides, particularly chitosan and chitosan / hyaluronic acid, useful as frameworks for bone tissue engineering.

These membranes were manufactured by mixtures of polysaccharides and proteins of interest and novel crosslinking agents consisting of phytic acid derivatives

25 (PA) obtained by a condensation reaction between phytic acid (PA) with a hydroxyl compound.

Phytic Acid (PA) 30 The advantages of using these novel crosslinkers with respect to phytic acid lie in

its lowest cytotoxicity and its highest compatibility with natural polymers. In addition, cross-linked formulations provide antioxidant properties and the ability to complex with polyvalent cations, of great importance in tissue regeneration processes.

Thus, a first aspect of the present invention relates to a compound obtained by a condensation reaction between phytic acid and with a hydroxyl compound in molar ratios ranging from 1:10 to 10: 1, and said compound having a molecular weight less than 5,000 g / mol.

The term "phytic acid" in the present invention refers to inositol hexaphosphate, and any isomer that includes the configurations cis, epi, alo, muco, neo, D-quiro, Lquiro and scyl.

The term "hydroxy compound" in the present invention refers to an alkyl substituted with at least one hydroxyl group. In preferred embodiments, the hydroxyl compound has more than two hydroxyl groups, and more preferably at least one of the hydroxyl groups is a Į-OH.

The term "alkyl" in the present invention refers to the radical of saturated aliphatic groups, which includes straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups . In preferred embodiments, a straight chain or branched chain alkyl has 2 to 10 in its skeleton, and more

25 preferably from 3 to 6.

In a preferred embodiment, the hydroxy compound is an aliphatic polyalcohol having three to six carbon atoms, and two to six hydroxyl groups, such as aliphatic glycols, glycerol, sorbitol, glucidol, and the like. More preferably, the

The hydroxy compound is glycerol.

In another preferred embodiment, the compound is obtained by a condensation reaction between phytic acid (PA) and glycerol (G) in molar ratios ranging from 1:10 to 10: 1. More preferably, the molar ratios are selected from

35 between a list consisting of 1: 7, 4: 1, 5: 1 and 6: 1. Even more preferably, the molar ratio is PA: G is 1: 7.

A further embodiment of the present invention relates to the use of the compound of the invention as defined above as a crosslinking agent.

A third embodiment of the present invention provides a process of obtaining 5 of the compound of the invention, wherein the process comprises at least the following stages:

(a) Reacting phytic acid (PA) with the corresponding hydroxyl compound in molar ratios ranging from 1:10 to 10: 1 at a temperature ranging from 100 ° C to 140 ° C;

10 (b) Dissolve the reaction product obtained from (a) in water;

(C)

Precipitate the solution of (b) in an organic alcohol solvent selected from 2-propanol, iso-butanol and n-butanol; Y

(d)

Lyophilization of the precipitate obtained from (c).

In a preferred embodiment, step (a) is carried out at a temperature ranging from 110 ° C to 120 ° C. Even more preferably, the reaction is carried out at 120 ° C.

In another preferred embodiment, the hydroxy compound in step (a) is a polyalcohol

20 aliphatic having three to six carbon atoms, and two to six hydroxyl groups, such as aliphatic glycols, glycerol, sorbitol, glucidol, and the like. More preferably, the hydroxy compound is glycerol.

In another preferred embodiment, the compound is obtained by a reaction of

Condensation between phytic acid (PA) and glycerol (G) in molar ratios ranging from 1:10 to 10: 1. More preferably, the molar ratios are selected from a list consisting of 1: 7, 4: 1, 5: 1 and 6: 1. Even more preferably, the molar ratio is PA: G is 1: 7.

In an even more preferred embodiment, the synthesis of the compound of the invention was carried out at 120 ° C by reacting phytic acid with glycerol, in a PA: G molar ratio of 1: 7.

Another aspect of the invention relates to the compound obtained from the process referenced above.

A further embodiment of the invention relates to a polymer membrane that

it comprises a mixture of the compound of the invention and at least one protein, a polysaccharide, or a combination thereof, wherein said membranes contain an amount of the crosslinking agent of the invention ranging from 2.5 to 50% by weight. .

In a preferred embodiment, the protein is selected from a list consisting of albumin, collagen, collagen / gelatin, fibrin, fibrinogen and fibroin. More preferably, the protein is collagen / gelatin.

In another preferred embodiment, the polysaccharide is selected from a list consisting of chitosan, hyaluronic acid, chondroitin sulfate and heparin. More preferably, the polysaccharide is selected from chitosan and hyaluronic acid. Even more preferably, the membrane comprises chitosan and hyaluronic acid.

In another preferred embodiment, the polymer membrane comprises a compound obtained by a condensation reaction between phytic acid and glycerol in the PA: G 1: 7 molar ratio, and chitosan.

In another preferred embodiment, the polymer membrane comprises a crosslinking agent obtained by a condensation reaction between phytic acid and glycerol in the PA: G 1: 7 molar ratio, and chitosan and hyaluronic acid.

The present invention further provides a process for obtaining the membrane

25 of the polymer of the invention, wherein the process comprises at least the following steps:

(a) Form a film of an aqueous solution containing at least one protein and / or polysaccharide in an acid solution,

(b) Wash the product of step (a) with a basic solution and water for neutralization of the residual acid solvent;

(C)

React the film of step (c) with an aqueous solution of the compound of the invention in an amount ranging from 2.5-50% by weight; Y

(d)

Wash and dry the polymer membrane resulting from (c).

The term "acid solution" in the present invention refers to an acid solution, usually one that has a pH of less than 7 and is capable of reacting with a basic solution. Preferably, the acid in the acid solution is selected

among, but not limited to, the group consisting of hydrochloric acid, sulfuric acid, formic acid, lactic acid, malic acid, succinic acid, acetic acid, methanesulfonic acid, benzenesulfonic acid, phosphoric acid, and the like, and suitable combinations of two or more of them. More preferably, the acid in the solution

5 of acid is acetic acid.

The term "basic solution" in the present invention refers to, but is not limited to, various solutions of a base (for example, sodium hydroxide, calcium hydroxide, ammonia, sodium carbonate, calcium carbonate) dissolved in water. The concentration of

A basic solution is preferably 0.001 N or higher, more preferably 0.01 N or higher, and still more preferably 0.1 N to 3.0 N.

In a preferred embodiment, the protein in step (a) is selected from a list consisting of albumin, collagen, collagen / gelatin, fibrin, fibrinogen and

15 fibroin More preferably, the protein is collagen / gelatin.

In another preferred embodiment, the polysaccharide in step (a) is selected from a list consisting of chitosan, hyaluronic acid, chondroitin sulfate and heparin. More preferably, the polysaccharide is selected from chitosan and acid

20 hyaluronic. Even more preferably, the membrane comprises chitosan and hyaluronic acid.

In another preferred embodiment, the crosslinking agent in step (c) is obtained by a condensation reaction between phytic acid and glycerol in the PA: G molar ratio.

25 1: 7.

In another preferred embodiment, the polysaccharides in step (a) is chitosan and hyaluronic acid and the cross-linking agent in step (c) is a cross-linking agent obtained by a condensation reaction between phytic acid and glycerol in relation

30 molar PA: G 1: 7.

Another aspect of the invention relates to the membranes obtained from the aforementioned process.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning that is commonly understood by a person skilled in the art to which the present invention belongs. They can

methods and materials similar or equivalent to those described herein be used in the practice of the present invention. Throughout the description and claims, the word "understand" and its variations are not intended to exclude other technical characteristics, additives, components or stages. Objects, advantages and

Additional features of the invention will be apparent to those skilled in the art upon examination of the description or may be learned by the practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

10 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. 1H NMR spectra of phytic acid, glycerol, GPhy1: 7 and GPhy5: 1 in D2O at 25 ° C. FIG. 2. 13C NMR spectra of glycerol, phytic acid, GPh1: 7 and GPhy5: 1 in D2O a

15-25 ° C. FIG. 3. 31P NMR spectra of initial PA and GPhy1: 7 in D2O at 25 ° C. FIG. 4. Cellular viability of the response of human fibroblasts to different concentrations of GPhy1: 7, GPhy4: 1, GPhy5: 1 and GPhy6: 1. FIG. 5. ATR-FTIR spectra of ChGPhy1: 7 and initial Ch membranes.

20 FIG. 6. Water withdrawal of ChGPhy1: 7 membranes in PBS (pH 7.4, 37 ° C). The results are given as mean ± of (n = 5). Statistical analysis (ANOVA) of each sample was performed at times 2 and 7 days with respect to 1 day (* p <0.05). FIG. 7. ATR-FTIR spectra of Ch / HAGPhy1: 7 membranes.

25 FIG. 8. SEM images of Ch / HAGPhy1: 7 membranes of different conc. of crosslinker: a) Ch / HA2.5% (1: 7), b) Ch / HA5% (1: 7), c) Ch / HA10% (1: 7).

FIG. 9. Water uptake of Ch / HAGPhy1: 7 membranes in PBS (pH 7.4, 37 ° C). The results are given as mean ± of (n = 5). Statistical analysis 30 (ANOVA) of each sample was performed at times 7 and 10 days with respect to

at 1 day (* p <0.05) and with respect to 2 days (#p <0.05). FIG. 10. SEM photographs of the Ch10% membrane (1: 7) at 4 days (a, b), 7 days (c, d) and 14 days (e, f) immersion in SBF1,5. FIG. 11. Results of cell viability of human fibroblasts in the presence of

35 extracts of ChGPh1: 7 membranes. The results are shown as mean ± of (n = 8). Significant differences (* p <0.05 and *** p <0.001) of membrane results with respect to control are indicated in the graph.

FIG. 12. Alamar blue results for ChGPh1: 7 membranes. All results are shown as mean ± of (n = 8). The asterisk represents a significant difference between the samples with respect to the control (** p <0.005 and *** p <0.001).

5 FIG. 13. SEM images of ChGPh1 membranes: 7 to 24 h (ad), 4 (eh), 7 (il) and 14 (mp) days after seeding of human fibroblasts for Ch (ad), Ch2 membranes, 5% (1: 7) (eh), Ch5% (1: 7) (il), Ch10% (1: 7) (mp) respectively.

FIG. 14. Optical microscopy images of Crystal-stained cell attached to 10 ChGPhy membranes (1: 7) after 14 days of seeding human fibroblasts. a) Ch2.5% (1: 7), b) Ch5% (1: 7), c) Ch10% (1: 7).

FIG. 15. Results of the cell viability of human fibroblasts in the presence of ChGPh4: 1, ChGPh5: 1 and ChGPh6: 1 membrane extracts. The results are shown as mean ± of (n = 8).

15 FIG. 16. Alamar blue results for ChGPh membranes (5: 1), (6: 1), (4: 1). All results are shown as mean ± of (n = 8). The asterisk (*) represents a significant difference between the samples with respect to the control (* p <0.05, ** p <0.005, *** p <0.001).

20 EXAMPLES

EXAMPLE 1. Synthesis of crosslinking agents (GPhy)

GPhy synthesis was carried out at 120 ° C in bulk by reacting phytic acid (PA) and glycerol (G), in molar ratios of PA: G in the range 1:10 to 10: 1. Left

25 that the condensation reaction proceeds for 12 h with mechanical agitation. Then, the reaction product was dissolved in water and precipitated in 2-propanol.

The isolated product was dissolved in a minimum amount of water, and lyophilized. GPhy crosslinkers (GPhy1: 7, GPhy4: 1, GPhy5: 1 and GPhy6: 1) were stored at

30 room temperature

EXAMPLE 2. Characterization of GPhy crosslinkers Purified crosslinkers were characterized by ATR-FTIR spectroscopy along with their precursors, G and PA. The ATR-FTIR spectra of the different

35 crosslinkers were quite similar and showed the main representative bands present in the spectra of both precursors. Particularly for GPhy1: 7, a broadband at 3248 cm-1 due to voltage vibrations of

links associated with O-H and N-H; bands at 2943 and 2888 cm -1 due to asymmetric and symmetric vibrations of C-H bonds of the inositol ring. At a lower wave number, a band at 1460 cm-1 due to G C-H bonds of the inositol ring and G residues, the band that in particular increased and was displaced with respect to PA (1399 cm

5 1); a band at 1176 cm-1 attributed to X P = O, also displaced with respect to PA (1186 cm-1); an intense peak at 1035 cm-1 (secondary peak at 1004 cm-1) attributed to X CO of alcohols, glycosidic bonds X P-O and P-O-C and X C-O; finally, peaks at 918, 815 (weak) and 715 (weak) cm-1 due to X P-O and P-O-C links, peaks that also changed with respect to PA.

10 The crosslinkers were subsequently characterized by NMR spectroscopy. Fig. 1 shows 1H NMR spectra of two crosslinkers and precursors. The glycerol spectrum showed a multiplet between 3.5 and 3.8 ppm that can be assigned to 1CH2, 2CH2 and CH protons. The spectrum of GPhy1: 7 showed modified signals in the range of

15 of the initial glycerol and new signals in the range 3.82-3.98 ppm, attributed to CH2-OP, and in the range 3.9-4.10 ppm, due to CH-OP in the reacted glycerol residues, respectively. Finally, between 4.1 and 4.6 ppm, three signals of very low intensity for the GPhy1: 7 crosslinker, assigned to protons, protons H1-H5 in unmodified PA. The resonance of the proton H6 in the residue of

20 PA The spectra of the crosslinkers GPhy4: 1, GPhy5: 1 and GPhy6: 1 were quite similar to each other, but differ somewhat from GPh1: 7, as shown in Fig. 1 in which the spectrum of GPhy5: 1 is shown for comparison purposes. The main difference is that the new multiplet in the 3.82-3.98 ppm range disappears and new signals increase in the 3.98-4.10 ppm range.

25 attributed to the resonance of CH-O-P. In addition, the signals due to the resonance of unmodified phytic protons increased. Proton NMR analysis of different crosslinkers seems to indicate that the reduction in the G content in the condensation reaction favors the reactivity of the ȕ-hydroxy groups while the increase in G enhances the reactivity of the Į-hydroxy groups.

30 13C NMR spectra of GPhy1: 7 and GPhy5: 1 crosslinkers and precursors are shown in Fig. 2. The 13C NMR spectrum of PA showed four peaks at 75.3, 78.0, 78.5 and 79, 3 ppm that were assigned to C4 and C6; C2; C1 and C3; and C5. The initial 13C NMR spectrum of G showed a peak at 62.5 ppm that was assigned to CH2 carbons and a peak at

35 72.0 ppm assigned to CH carbons. The 13C NMR spectrum of the GPhy1: 7 crosslinker showed peaks at 62.1, 62.5, 65.8, 70.8, 71.1, 72.0, 72.2, 72.4 and 74.3, of which, the last four signals can be provisionally attributed to carbons of

CH-O-PO3H. The 13C NMR spectrum of the GPhy5: 1 crosslinker, on the other hand, was quite complex and should be analyzed in depth using new experiments. However, it indicates that the modification of phosphate groups is carried out for the PA: G molar ratio of 5: 1. Therefore, the corresponding spectra of GPhy6: 1 and

5 GPhy4: 1 resembled those of GPhy5: 1.

The 31P NMR spectrum of PA is widely discussed in the literature and most authors reported resonance of four peaks. The 31P NMR spectrum of commercial PA analyzed in this work (Fig. 3) showed peaks in the range -2 to 2 ppm

10 that were assigned to P5 (G 0.4), P1 and P3 (G 0.04), P4 and P6 (G 0.53) and P2 (G 0.94). The 31 P NMR spectrum of GPhy1: 7 (Fig. 3) showed peaks in the range -3 to 3 ppm, which indicates that the chemical environment of the P nuclei changed after the condensation reaction.

15 The molecular weights of the GPh1: 7 and GPhy5: 1 crosslinkers were determined by high resolution mass spectrometry (HR-MS) and the number average molecular weight (Mn), weight average molecular weight (Mw) was calculated. and polydispersity (PDI). For GPhy5: 1, the values of Mw and PDI were 869 g / mol, 1226 g / mol and 1.41 respectively, and for GPhy1: 7 it was 841 g / mol, 1262 g / mol and

20 1.50 respectively.

The thermal properties of GPhy crosslinkers were analyzed by DSC and TGA techniques. The GPhy1: 7 DSC thermogram showed a glass transition with a glass transition temperature (Tg) of 47 ° C indicating an amorphous character of this

Crosslinker as a consequence of the presence of glycerol residues in its structure. However, GPhy4: 1, GPhy5: 1 and GPhy6: 1 did not show vitreous transitions that were attributed to higher PA contents in these crosslinkers.

Thermogravimetric (TGA) and differential thermogravimetric (DTGA) data of

30 GPhy crosslinkers and precursors are shown in Table 1. Weight loss of GPhy1: 7 was experienced in three stages. The first with a Tmax of approximately 125 ° C can be attributed to the loss of water contained in the sample. The second stage of degradation with maximum speed at 215 ° C would imply the degradation of the rest of G (Tmax = 228 ° C) and part of the phytate residues (Tmax = 215 ° C) and the last stage with

Maximum speed at 337 ° C would correspond to the degradation of the rest of phytate. The residue of GPhy1: 7 was 52%. This value was particularly reduced with respect to that of PA, which indicates the content of G, which gives no residue at all. From these

residue values of both GPh1: 7 and PA, the present inventors estimated the amount of G incorporated in this crosslinker after the condensation reaction, which was 3 mol%. In the case of GPhy4: 1, GPhy5: 1 and GPhy6: 1, the present inventors estimated that this value was 2 mol%.

Table 1. Results of thermal degradation of GPhy1: 7 and precursors under a nitrogen atmosphere.

Sample

Tmax (ºC) Residue (%)

1st stage

2nd stage 3rd stage 4th stage

G

228 0

PA

117 215 332 394 75

GPhy1: 7

125 215 337 - 52

GPhy4: 1

141 223 346 466 69

GPhy5: 1

137 242 356 451 69

GPhy6: 1

144 244 354 70

EXAMPLE 3. Cytotoxicity of GPhy crosslinkers

Cellular toxicity was evaluated using human embryonic skin fibroblast (HFB,

10 Innoprot). The cells were cultured in Dulbecco-modified Eagle medium (DMEM) supplemented with HEPES (for HFB cells) and 10% fetal bovine serum (FBS), 100 ml units of penicillin, 100 μg ml of streptomycin and 200 mM L-glutamine. A humidified atmosphere at 37 ° C with 5% CO2 and 95% air was used for cell culture growth. For cell experiments, they were collected

15 cells using 0.25% trypsin and 1 mM EDTA in Hanks buffer.

The cytotoxicity evaluation of PA, GPhy and commercial ȕ-GP was determined by MTT assay. A stock solution of the corresponding sample was prepared in DMEM and successive dilutions were tested. The cells were seeded at a density

20 of 9 x 105 cells / ml in complete medium and incubated at confluence. After 24 h of incubation, the medium was replaced with the corresponding dilution and incubated at 37 ° C in humidified air with 5% CO2 for 24 h. A solution of MTT in hot PBS (0.5 mg / ml) was prepared and the plates were incubated at 37 ° C for 3 h. Excess medium and MTT were removed and 100 µl of DMSO was added to all

25 wells This was mixed for 10 min and absorbance was measured with a Biotek SYNERGY HT plate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. The percentage of relative cell viability (CV) was calculated from Ec (2): CV (%) = 100 x (DOS - DOB) / (DOC - DOB) Ec (2)

where DOS, DOB and DOC are the optical density of formazan production for the sample, blank and control, respectively. A dose-response curve of relative cell viability was plotted to delineate the concentrations of the sample tested that reduced the production of MTT-formazan by 50% (IC50 value).

5 For the GPhy1: 7 crosslinker, cell viability was approximately 70% in a concentration range of 5-80 mg / ml, indicating the absence of cytotoxicity according to the evaluation of ISO 10993-5: 2009. However, cell viability showed a dose-response behavior for the other crosslinkers (Fig. 4). TO

From the linear portions of the corresponding curves, IC50 was determined as the concentration that reduced the production of MTT-formazan by 50%. IC50 values of GPhy4: 1, GPhy5: 1 and GPhy6: 1 were 17.5 mg / ml, 25 mg / ml and 19 mg / ml, respectively. For comparison purposes, the cytotoxicity of commercial PA and ȕGP was analyzed under the same experimental conditions and the IC50 values were

15 8 and 9 mg / ml, respectively. The results of the present inventors demonstrated that the biocompatibility of GPhy crosslinkers increased with respect to those of PA and GP crosslinkers currently used in the literature and commercial formulations.

EXAMPLE 4. Preparation of chitosan membranes (Ch) Ch membranes were prepared before crosslinking as follows: Ch (2% by weight) was dissolved in acetic acid solution (1% v / v), placed in A glass mold was transferred to an oven at 37 ° C and held there until complete evaporation of the solvent. Then, the Ch membrane obtained was washed

25 with 1 N NaOH solution and water until neutral pH. The purified Ch membrane was immersed in 40 ml of an aqueous solution of GPhy (2.5, 5, 10 or 50% by weight in different experiments) and kept overnight at 37 ° C. Then, the corresponding crosslinked membrane was washed with distilled water and dried.

30 Membranes were obtained by crosslinking Ch with GPhy1: 7 in different proportions (hereinafter, ChGPhy1: 7 membranes). The names and composition of Ch membranes prepared with this GPhy crosslinker are reported in Table 2. The most likely mechanism of the crosslinking reaction would be due to ionic interactions between phosphate anions of the crosslinker and NH3 + groups.

35 of Ch. In addition, hydrogen bond interactions between the GPhy1: 7 crosslinker and the polysaccharide can contribute to membrane stability.

Table 2. Names and composition of ChGPhy1: 7 membranes.

Sample Name

GPhy1: 7 (% by weight with respect to Ch)

Ch2.5% (1: 7)

2.5

Ch5% (1: 7)

5

Ch10% (1: 7)

10

EXAMPLE 5. Characterization of GPhy crosslinkers of ChGPhy1: 7 membranes ChGPhy1: 7 membranes were analyzed by ATR-FTIR spectroscopy. The

5 main absorption bands in the spectra of the ChGPhy1: 7 membranes and the changes with respect to the Ch spectrum are shown in Fig. 5 and can be summarized as follows: the band between 3600 and 3000 cm-1 assigned to X OH and associated NHs became wider, which may indicate the presence of interactions between amino groups of Ch and phosphate groups of GPhy1: 7, but also between hydroxyl groups

10 of Ch and G molecules of GPh1: 7; a new peak appears at 2972 cm-1 and increases with the content of crosslinker GPhy1: 7; the bands in the range 1700 -1500 cm-1 became wider, the band to 1647 cm-1 because the amide I was reduced, the band to 1582 cm-1 because G NH shifted to wave number lower with respect to Ch (1588 cm-1) indicating ionic interactions between amino groups and groups

15 GPhy phosphate and a secondary peak appears at 1540 cm-1 and increases with the crosslinker content; in the 1100 and 1000 cm-1 range the peak at 1066 cm-1 increases and the peak at 992 cm-1 decreases and moved to 998 cm-1 with the content of GPhy1: 7 in the membrane. The FTIR results of the present inventors are in agreement with those reported for Ch and proteins crosslinked with PA.

20 SEM and EDX analyzes were applied to evaluate surface morphology and elementary membrane composition. The surface morphology of the membranes was predominantly smooth regardless of the content of GPhy1: 7. The EDX spectra of the ChGPhy1: 7 membranes showed the peaks of the

25 main elements C, O, N at the characteristic energy levels of 0.27, 0.52, 0.39 keV, respectively. In addition, a peak was observed at the energy level of P (2.01 keV) belonging to the polyanions of GPhy1: 7, confirming the crosslinking reaction. EDX data of membranes are given in Table 5. Table 5. EDX results of ChGPhy1: 7 membranes.

Membrane

C / N CO C / P P / N

Ch2.5% (1: 7)

4.6 1.1 361.5 78.5

Ch5% (1: 7)

4.2 1.1 126.7 30.0

Ch10% (1: 7)

4.0 3.5 104.8 25.8

The C / O ratio increased slightly with the concentration of crosslinker, which indicates the presence of a higher O content from GPhy1: 7, while the C / P and N / P ratios in particular decreased. Results were reported

5 similar for electro-spun protein nanofibers crosslinked with PA.

The thermal degradation of ChGPhy1: 7 membranes by TGA under air atmosphere was analyzed and the results are shown in Table 6. The degradation of membranes occurred in two main stages, the first with a maximum velocity at about 10 300 ° C and the second at about 600 ° C. The thermal degradation of ChGPhy1: 7 membranes may be mainly associated with the dehydration of saccharide rings, depolymerization and decomposition of molecular units in Ch chains. All samples of ChGPhy1: 7 membranes presented residues that show the presence of phosphates in the

15 samples that come from the GPhy1: 7 crosslinker.

Table 6. Results of TGA and DTGA of ChGPhy1: 7 membranes under air atmosphere.

Sample

Tmax Residue (%)

1st stage

2nd stage

Ch

306 582 0

Ch2.5% (1: 7)

296 599 2.0

Ch5% (1: 7)

290 598 1.4

Ch10% (1: 7)

287 582 1.55

20 The swelling behavior of crosslinked membranes in PBS pH 7.4 at 37 ° C was studied. The evolution of swelling over time is presented in Fig.

6. Water collection was very fast, reaching a maximum in 1 day for all samples. At this time, the membrane crosslinked with 5% GPhy1: 7 reached a value of approximately 100%, while the membranes crosslinked with 25 2.5 and 10% GPhy1: 7 approximately 85%, indicating that the 5% can be an optimum crosslinker concentration. At 2 days, water withdrawal decreased slightly, although not significantly. This result is normally associated with the elastic behavior of polymers in which the relaxation of macromolecules occurs as a result of immersion in a medium of

30 hydration At 7 days, water uptake for the Ch5% (1: 7) and Ch10% (1: 7) membranes was stabilized, while it decreased significantly for those

manufactured with 2.5% crosslinker. This decrease in swelling may be due to the breakage of some ionic crosslinks that is more evident in less crosslinked samples. Swelling of uncrosslinked Ch samples also decreased significantly, which can be attributed to disintegration in the

5 medium. So, the 2.5% concentration of GPhy1: 7 seems to be insufficient to give membrane stability in the short term. On the contrary, the present inventors can say that concentrations of both 5 and 10% of GPhy1: 7 provided membranes with adequate swelling behavior and stability for use in biomedical applications.

EXAMPLE 6. Preparation of GPhy crosslinked polysaccharide membranes containing more than one polysaccharide Crosslinked membranes were made containing more than one polysaccharide with mixtures of chitosan (Ch) and hyaluronic acid (HA) in a mass ratio Ch: HA of

15 60:40 using GPhy1: 7 as a crosslinker (hereinafter, Ch / HAGPhy1: 7 membranes), using the same protocol described for Ch membranes (Example 4).

Briefly, Ch (2% by weight) was dissolved in acetic acid solution (1% v / v) and, by

20 apart, HA (2% by weight) was dissolved in aqueous solution. Aliquots of both solutions were mixed with stirring and slightly acidified (1 N HCl) to obtain the complete dissolution of both polysaccharides, which was placed in a glass mold, transferred to an oven at 37 ° C and kept there until evaporation. Complete solvent. The purified membrane was crosslinked by immersion in solution

GPhy 25 (2.5, 5 or 10% by weight with respect to Ch, in different experiments) for 12 h at 37 ° C. The corresponding hydrogel membrane was removed from the mold, washed with 1 N NaOH and distilled water until neutral pH. The membrane was then dried at 37 ° C.

30 As with ChGPhy1: 7 membranes, the crosslinking reaction will be due to ionic interactions between GPhy1: 7 phosphate anions and protonated amino groups of Ch. However, in this case, the resulting material will be a network of semi-interpenetrated polymer (semi-IPN) formed by a network of crosslinked Ch in which linear HA macromolecules are entangled with those of Ch. In addition,

Most likely, intermolecular interactions such as hydrogen bonding, which intervene in the carboxylic, hydroxyl and amino groups belonging to different polysaccharides, contribute to stability.

dimensional of the semi-IPN hydrogel structure.

Names and composition of the membranes as they were produced are given in the

Table 7.

Table 7. Names and composition of Ch / HAGPhy1: 7 membranes.

Sample Name

Ch / HA (p / p) GPhy1: 7 (% by weight with respect to Ch)

Ch / HA2.5% (1: 7)

60:40 2.5

Ch / HA5% (1: 7)

60:40 5

Ch / HA10% (1: 7)

60:40 10

EXAMPLE 6. Characterization of Ch / HA membranes crosslinked with GPhy1: 7 The ATR-FTIR spectra of Ch / HAGPhy1: 7 membranes showed the characteristic bands of both polysaccharides and GPhy1: 7 crosslinker (Fig. 7).

10 The main bands appeared: between 3600 and 3200 cm-1 (XO-H and N-H associated); at 2924 and 2854 cm -1 (X C-H); at 1720 cm -1 (X C = O in carboxylic groups); at 1643/1634 cm -1 (X C = O of amide, amide I); at 1579 cm -1 (G N-H); at 1420 cm -1 (X COO- and G C-H); at 1373 cm -1 (symmetric G-CH3); at 1333 cm -1 (X C-N, amide III); to

12 1258/1262 cm-1 (X P = O); at 1150 cm-1 (X C-O-C asymmetric); at 1066, 1029, 995 and 984 cm -1 (X C-O of alcohols, glycosidic bonds X P-O and P-O-C, X C-O and pyranose structure vibration); at 893 and 721 cm-1 (X P-O and P-O-C).

The main differences between the spectra of the membranes of Ch / HAGPhy1: 7 and

Those of the Ch / HA membrane are the following: disappearance of a secondary peak with peaks at 2909-2862 cm-1, which are more pronounced in the uncrosslinked Ch / HA membrane; in the range 1700-1500 cm-1, an increase and displacement in the amide band I towards a lower wave number (1643 to 1634 cm-1) with respect to uncrosslinked membrane (1646 cm-1); the band at 1579 cm -1 moved with respect to the membrane

Ch / HA (1586 cm-1) as a result of ionic interactions between the NH3 + groups of the Ch / HAGPhy1: 7 membrane and the phosphate groups of GPh1: 7; a change in the intensities of the bands in the range 1150 and 950 cm-1.

Superficial morphology and elementary membrane composition were examined

30 of Ch / HAGPhy1: 7 by SEM and EDX, respectively. Fig. 6 shows the SEM images of Ch / HAGPhy1: 7 membranes. A highly structured, highly porous morphology was observed in all samples. Porosity tends to be reduced with crosslinker content, providing a network of reduced mesh size.

This superficial morphology seems to be very suitable for colonization and proliferation of cells in tissue engineering applications.

The EDX spectra of Ch / HAGPhy1: 7 membranes showed the peaks of the main elements C, O, N and P. The results of EDX are shown in Table 8. A decrease in the C / ratio can be clearly observed. P with the content of GPhy1: 7 in the membrane, in addition to a decrease in the N / P ratio, which confirms the crosslinking reaction.

Table 8. EDX results of Ch / HAGPhy1: 7 membranes.

Membrane

C / N CO C / P P / N

Ch / HA2.5% (1: 7)

- - - -

Ch / HA5% (1: 7)

4.8 1.1 188 39

Ch / HA10% (1: 7)

4.6 1.1 90 twenty

10 Thermal degradation of Ch / HAGPhy1: 7 membranes was analyzed by thermogravimetry. TGA and DTGA curves are shown in Table 9. The degradation patterns of all Ch / HAGPhy1: 7 membranes are quite similar, but showed differences with respect to the uncrosslinked Ch / HA membrane.

15 Regardless of the concentration of GPhy1: 7, degradation of Ch / HAGPhy1: 7 membranes took place in two main degradation stages, while that of Ch / HA membranes in three stages. This difference makes it clear that both polysaccharides are basically mixed in the uncrosslinked membranes, while they are well matted forming semi-IPN networks in the membranes of

20 Ch / HAGPhy1: 7. In addition, the Tmax values of the semi-IPN samples are higher compared to the Ch / HA sample, manifesting the cross-linked reaction and entanglement formation between both polysaccharides which, in addition, can be stabilized with hydrophilic interactions. Considering the concentrations of GPhy1: 7, 5% provided membranes that have the highest stability,

25 while 10% gave rise to membranes with the lowest thermal stability. Table 9. Results of thermal degradation of Ch / HAGPhy1: 7 membranes under air atmosphere.

Sample

Tmax (ºC)

1st stage

2nd stage 3rd stage

Ch / HA

271 497 560

Ch / HA2.5% (1: 7)

292 586

Ch / HA5% (1: 7)

286 602

The swelling behavior of crosslinked membranes in PBS pH 7.4 at 37 ° C was studied. The evolution of swelling over time is presented in Fig.

9. Ch / HAGph1: 7 membranes showed swelling profiles quite

5 similar to those observed for ChGPhy1: 7 membranes. Water collection was very fast, reaching a maximum between 1 and 2 days for all samples and giving values ranging between 90 and 115%. The highest swelling was observed for the crosslinked membrane with 5% GPhy1: 7, and the lowest for that with 10% crosslinker, which again indicates that 5% of GPhy1: 7 could be a

10 optimal concentration. Then, the water uptake decreased significantly to values of approximately 80-90% for the membranes of Ch / HA5% (1: 7) and Ch / HA10% (1: 7) respectively, and approximately 70% for Ch / HA2.5% (1: 7). The decrease in swelling can be attributed to some release of uncrosslinked HA chains, but also to some breakage of ionic bonds, more

15 pronounced for samples manufactured with the lowest crosslinker content.

From the results of swelling experiments, the present inventors can say that concentrations of both 5 and 10% of GPhy1: 7 provide membranes with adequate and good swelling behavior

20 stability for biomedical applications.

EXAMPLE 7. Formation of apatite-like layer on Ch membranes crosslinked with GPhy.

Crosslinked Ch membranes with 10% GPhy1: 7 crosslinked were used

25 so-called Ch10% membranes (1: 7) for mineralization tests. The formation of a layer similar to apatite on the surface of the membranes was studied over time by impregnating the sample in SBF1.5 at 37 ° C. The growth of a layer similar to apatite was confirmed by experimental techniques such as SEM, EDX and ATR-FTIR.

30 Fig. 10 shows the SEM photographs of the surface of Ch10% membranes

(1: 7) after 4, 7 and 14 days of impregnation in SBF1,5. At 4 days, the surface of the membranes showed precipitation of aggregates that were concentrated in localized areas, indicative of Ca-P deposits. At 7 days, the precipitates appeared more scattered on the surface and at 14 days, the aggregates practically

35 colonized the entire surface. At higher increase (2000x), it could be seen that the precipitates were formed by spherical particles that presented the

Typical cauliflower morphology attributed to the apatite-like layer. The EDX spectra of the precipitates obtained at different periods of time showed the typical bands of Ca and P giving Ca / P ratios of 1.92, 1.45 and 1.66 within 4, 7 and 14 days, respectively, values that approximate those of the Ca / P ratio of the biological apatite (1.66). These results suggest that Ch membranes prepared with GPhy crosslinkers may have application in tissue regeneration processes that involve bone, such as subchondral cartilage repair.

EXAMPLE 8. Biological behavior of crosslinked polysaccharide membranes

In this example, the biological behavior of Ch and Ch / HA membranes manufactured with different GPhy crosslinkers was evaluated by respective MTT assays.

The corresponding sample was placed in DMEM free of FBS. Then, extracts of the medium were extracted in different periods of time (1, 2, 7 and 14 days) and replaced with fresh medium. The extracts were filtered and used for cytotoxicity assays. Thermanox® (TMX) discs were used as a negative control. Separately, cells were seeded at a density of 9 x 104 cells / ml in complete medium, and incubated until confluence. After 24 h of incubation, the medium was replaced with the corresponding extract and incubated at 37 ° C in humidified air with 5% CO2 for 24 h. The rest of the test was carried out as explained above.

The analysis of variance (ANOVA) of the results for materials tested with respect to the control at the level of significance of * p <0.05 was performed.

The names and composition of the membranes studied are summarized in Table 10.

Table 10. Names and composition of Ch and Ch / HA membranes prepared with GPhy crosslinkers

Name of the membrane family

Sample Name Ch / HA (p / p) GPhy content (% by weight with respect to Ch) Type of crosslinker

ChGPhy1: 7

Ch2.5% (1: 7) 100: 0 2.5 GPhy1: 7

ChGPhy1: 7

Ch5% (1: 7) 100: 0 5 GPhy1: 7

ChGPhy1: 7

Ch10% (1: 7) 100: 0 10 GPhy1: 7

Ch / HAGPhy1: 7

Ch / HA2.5% (1: 7) 60:40 2.5 GPhy1: 7

Ch / HAGPhy1: 7

Ch / HA5% (1: 7) 60:40 5 GPhy1: 7

Ch / HAGPhy1: 7

Ch / HA10% (1: 7) 60:40 10 GPhy1: 7

ChGPhy4: 1

Ch50% (4: 1) 100: 1 fifty GPhy4: 1

ChGPhy5: 1

Ch50% (5: 1) 100: 1 fifty GPhy5: 1

ChGPhy6: 1

Ch50% (6: 1) 100: 1 fifty GPhy6: 1

With respect to membranes manufactured with only Ch, the cytotoxicity results of ChGPhy1: 7 membranes on human fibroblasts are shown in Fig. 11. It can be seen that cell viability in the presence of extracts taken between 4

5 h and 7 d was approximately 100% or higher, indicating the absence of cytotoxicity. Interestingly, cell viability increases statistically in the presence of extracts from the Ch10% sample (1: 7) taken after 14 d.

Cell proliferation in ChGPhy1 membranes was analyzed quantitatively: 7

10 after different times after sowing and the results are presented in Fig. 12. For Ch10% membranes (1: 7), cell proliferation increased in particular with respect to Ch after 14 days after sowing , which indicates that the higher presence of the phytate derivative in this composition makes it more evident that this compound may have a stimulation activity on the

15 fibroblast proliferation.

Cell morphology

The materials were placed in a 24-well plate (in duplicate), seeded with cells at a density of 5 x 104 cells / ml and incubated at 37 ° C.

After incubation periods of 24 h, 4 and 8 days, the cells were fixed with 2.5% glutaraldehyde buffered in 0.1 M PBS. The dried samples were examined under scanning electron microscopy (SEM) using a Philips XL 30 device at an acceleration voltage of 25 keV.

25 The morphology of bound cells was also analyzed by optical microscopy. Samples were placed in a 24-well plate (in duplicate), seeded with cells at a density of 2x104 cells / ml and incubated for 14 days at 37 ° C. Then, the medium was removed, the samples were washed with PBS, the cells were left with a 2.5% solution of glutaraldehyde in distilled water for 15 min at

room temperature and finally washed twice with distilled water. The cells were stained with a solution of Crystal Violet (1 mg / ml in PBS) and incubated for 15 min at room temperature. Then, the samples were washed with distilled water and dried at room temperature. All samples were examined

5 by optical microscopy using a NIKON ECLIPSE TE2000-S microscope.

The morphology of cells adhered to Ch ChGPh1: 7 membranes was examined by SEM at different periods of time after sowing (Fig. 13).

10 At 24 h, the cells adopt spherical morphology in each membrane. However, after 4 days, more elongated cells were observed in ChGPhy1: 7 membranes compared to those of simple Ch, and, after 7 and 14 days, cell colonization was very good for ChGPhy1: 7 membranes, and qualitatively higher compared to that of Ch, forming the typical monolayer,

Particularly more evident for the composition of Ch10% (1: 7).

Additionally, ChGPhy1: 7 membrane-bound cell morphology was analyzed by optical microscopy and the results are shown in Fig. 14. After 14 days of seeding, epifluorescence microscopy images revealed morphology.

20 elongated cells cultured in all membranes, but a denser mesh was observed in the membranes of Ch10% composition (1: 7).

The cytotoxicity results of Ch50% membranes prepared with GPhy4: 1, GPhy5: 1 and GPhy6: 1 named as the membranes of ChGPhy4: 1, ChGPhy5: 1 and

25 ChGPhy6: 1 are shown in Fig. 15. For this family of membranes, the absence of cytotoxicity in human fibroblasts was measured again for extracts taken within 9 days, which shows cell viability values of approximately 100% in all cases and not statistically different with respect to the control of Ch.

30 Cell proliferation was determined using the Alamar blue test and the results are shown in Fig. 15. It was evident that within 48 h from cell proliferation it was considerably and statistically higher in ChGPhy4: 1 membranes, ChGPhy5: 1 and ChGPhy6: 1 compared to those of Ch,

35 again confirming the stimulation activity with respect to fibroblast growth due to the GPhy compound, which in these crosslinked membranes is present in a higher concentration (50%). Cell decline

quantified after 4, 7 and 14 days in the crosslinked samples may be directly related to the highest cell density obtained in the first 48 h, which interrupted the cells continue to proliferate due to lack of space.

5 With regard to samples made with both Ch and HA and GPhy1: 7, called Ch / HAGPy1: 7 membranes, this family of membranes was also biocompatible in the MTT test. The results of Fig. 17 show that cell viability in the presence of extracts of all membrane compositions

10 taken up to 14 days was approximately 100%.

Claims (15)

1. Compound obtained by a condensation reaction between phytic acid and with a hydroxyl compound in molar ratios ranging from 1:10 to 10: 1, and said compound having a molecular weight of less than 5,000 g / mol. 2. A compound according to claim 1, wherein the hydroxy compound is a C2-C10 alkyl substituted with at least one hydroxyl group. 3. A compound according to any one of claims 1 or 2, wherein the hydroxyl compound is an aliphatic polyalcohol having three to six carbon atoms, and two to six hydroxyl groups. 4. Compound according to any of claims 1 to 3, wherein the compound Hydroxyl is selected from the list consisting of aliphatic glycols, glycerol, sorbitol and glucidol. 5. Compound according to any of claims 1 to 4, wherein the compound Hydroxyl is glycerol. twenty 6. A compound according to claim 5, wherein the molar ratios are selected from a list consisting of 1: 7, 4: 1, 5: 1 and 6: 1. 7. Use of the compound according to any one of claims 1 to 6 as a crosslinking agent. 8. A process for obtaining the compound according to any one of claims 1 to 6, wherein the process comprises at least the following steps: (a) React phytic acid (PA) with the hydroxyl compound Corresponding in molar ratios ranging from 1:10 to 10: 1 at a temperature ranging from 100 ° C to 140 ° C;

(b)

Dissolve the reaction product obtained from (a) in water;

(C)

Precipitate the solution of (b) in an organic alcohol solvent

selected from 2-propanol, iso-butanol and n-butanol; and 35 (d) Lyophilization of the precipitate obtained from (c). 9. The process according to claim 8, wherein step a) is carried out at a temperature ranging from 110 ° C to 120 ° C, preferably at 120 ° C. 10. A polymer membrane comprising a mixture of the compound according to any one of claims 1 to 6 and at least one protein, a polysaccharide, or 5 a combination thereof, wherein said membrane contains an amount of the crosslinking agent ranging from 2.5 to 50% by weight. 11. Polymer membrane according to claim 10, wherein the polysaccharide is selected from a list consisting of chitosan, hyaluronic acid, chondroitin sulfate and heparin. 12. Polymer membrane according to claim 11, wherein the polysaccharide is selected from chitosan and hyaluronic acid. 13. A polymer membrane according to any of claims 10 to 12, comprising chitosan and hyaluronic acid. 14. Polymer membrane according to any of claims 10 to 12, which it comprises a compound obtained by a condensation reaction between phytic acid and glycerol in the molar ratio PA: G 1: 7, and chitosan. 15. Polymer membrane according to any of claims 10 to 13, comprising a compound obtained by a condensation reaction between phytic acid and glycerol in the PA: G 1: 7 molar ratio, and chitosan and hyaluronic acid. 16. A process for obtaining the polymer membrane according to any of claims 10 to 15, wherein the process comprises at least the following steps: (a) Form a film of an aqueous solution containing at least one protein and / or polysaccharide in an acid solution,

(b)

Wash the product of step (a) with a basic solution and water for neutralization of the acid residual solvent;

(C)

React the film of step (c) with an aqueous solution of the

compound according to any one of claims 1 to 6 in an amount ranging from 2.5-50% by weight; Y (d) Wash and dry the polymer membrane resulting from (c). 17. Use of the polymer membrane according to any of claims 9 to 15 as frameworks for bone tissue engineering.