MEANS FOR USE IN PREPARATION OF HYDROGEL BASED ON HYDROXYPHENYL DERIVATIVE OF HYALURONAN, METHOD OF HYDROGEL PREPARATION AND USE THEREOF

Abstract

Materials and methods for preparing a hydrogel are disclosed. Specifically, two separate solutions A and B, of which the solution A comprises enzyme horseradish peroxidase and the solution B comprises hydrogen peroxide is provided. At least one of solution A or B comprises calcium ions in the form of a pharmaceutically acceptable salt, and further the solution A and/or the solution B comprises hydroxyphenyl derivative of hyaluronan of a particular formula. A hydrogel based on hydroxyphenyl derivative of hyaluronan and a method of preparing and using the same is also disclosed.

Claims

1. Means for use in a preparation of a hydrogel, comprising two separate solutions A and B, of which the solution A comprises enzyme horseradish peroxidase and the solution B comprises hydrogen peroxide, at least one solution A or B comprising calcium ions in the form of a pharmaceutically acceptable salt and further the solution A and/or B comprises a hydroxyphenyl derivative of hyaluronan having a general formula I ##STR00005## wherein n is in the range of from 2 to 7500 and wherein R is OH or a substituent NHR.sub.2CONHR.sub.1ArOH having a general formula II, ##STR00006## wherein Ar is a phenylene group and R.sub.1 is an ethylene group, or Ar is an indolylene group and R.sub.1 is an ethylene group, or Ar is a hydroxyphenylene group and R.sub.1 is a carboxyethylene and wherein R.sub.2 is an alkylene of group having from 3 to 7 carbon atoms.

2. The means of claim 1, wherein the solution A comprises enzyme horseradish peroxidase of activity 0.5 to 2.25 U/mL, wherein at least one solution A or B comprises calcium ions in a concentration of from 0.05 to 0.55 mol/L, and wherein at least one solution A or B comprises the hydroxyphenyl derivative of hyaluronan of the general formula I of in a concentration of from 10 to 125 mg/mL.

3. The means of claim 1, wherein the hydroxyphenyl derivative of hyaluronan comprises a mass average molar mass of from 4×10.sup.4 to 3×10.sup.6 g/mol and a degree of substitution of from 1 to 10%.

4. The means of claim 1, wherein the solution A comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 4×10.sup.4 to 3×10.sup.6 g/mol and a degree of substitution of from 1 to 10%, at a concentration of from 10 to 125 mg/mL, the horseradish peroxidase, having an activity of from 0.5 to 2.25 U/mL, and calcium ions in a concentration of from 0.05 to 0.55 mol/L; and wherein the solution B comprises: the hydroxyphenyl derivative of hyaluronan of the general formula I having a mass average molar mass of from 4×10.sup.4 to 3×10.sup.6 g/mol and a degree of substitution of from 1 to 10%, at a concentration of from 10 to 50 mg/mL, and hydrogen peroxide in a concentration of from 2 to 10 mmol/L.

5. The means of claim 1, wherein the solution A comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 1×10.sup.5 to 1×10.sup.6 g/mol and a degree of substitution of from 2 to 5%, in a concentration of from 10 to 35 mg/mL, the horseradish peroxidase, having an activity of from 0.5 to 2.25 U/mL, and calcium ions in a concentration of from 0.05 to 0.55 mol/L; and and wherein the solution B comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from −1×10.sup.5 to 1×10.sup.6 g/mol and a degree of substitution of from 2 to 5%, in a concentration of from 26 mg/mL, and hydrogen peroxide in a concentration of from 3 to 7 mmol/L.

6. The means of claim 1, wherein the solution A comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 2×10.sup.5 g/mol to 4×10.sup.5 g/mol and a degree of substitution of from 2 to 5%, in a concentration of 20 mg/mL, the horseradish peroxidase, having an activity of from 1.0 to 1.3 U/mL, and calcium ions in a concentration of from 0.25 to 0.32 mol/L; and wherein the solution B comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 2×10.sup.5 g/mol to 4×10.sup.5 g/mol and a degree of substitution of from 2 to 5%, in a concentration of 26 mg/mL, and hydrogen peroxide in a concentration of from 4 to 5 mmol/L.

7. The means of claim 1, wherein the solution A comprises: the horseradish peroxidase, having an activity of from 1.8 to 2.2 U/mL, and calcium ions in a concentration of from 0.45 to 0.55 mol/L; and wherein the solution B comprises: the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 2×10.sup.4 to 4×10.sup.5 g/mol and a degree of substitution of from 2 to 4%, in a concentration of 26 mg/mL, and hydrogen peroxide in a concentration of from 4 to 5 mmol/L.

8. The means of claim 1, wherein the solution A comprises: the horseradish peroxidase, having an activity of from 0.8 to 2.2 U/mL, calcium ions in a concentration of from 0.02 to 0.5 mol/L, and the hydroxyphenyl derivative of hyaluronan, having a mass average molar mass of from 9×10.sup.4 to 4×10.sup.5 g/mol and a degree of substitution of from 1 to 10%, in a concentration of from 10 to 50 mg/mL; and wherein the solution B comprises: hydrogen peroxide in a concentration of from 4 to 5 mmol/L.

9. A method of preparing a hydrogel comprising a covalently crosslinked hydroxyphenyl derivative of hyaluronan, using the means of claim 1, the method comprising: providing the two separate solutions A and B; mixing the solution A with blood or with at least one transfusion preparation comprising fibrinogen, to give a precursor solution A; and mixing the precursor solution A with the solution B.

10. The method of claim 9, wherein 10 to 60% (v/v) of the blood or the transfusion preparation comprising fibrinogen is added into the solution A to give the precursor solution A.

11. The method of claim 9, wherein the precursor solution A is mixed with the solution B in volume-volume ratio of 1:1.

12. A hydrogel prepared by the method of claim 9, comprising enzyme horseradish peroxidase having an activity of from 0.1 to 2.5 U/mL, calcium ions in a concentration of from 0.01 to 0.11 mol/L, the covalently crosslinked hydroxyphenyl derivative of hyaluronan in a concentration of from 5 to 50 mg/mL, and the blood or the transfusion preparation comprising fibrinogen in a concentration of from 5 to 30% (v/v).

13. A cosmetics, medicine, or regenerative medicine preparation comprising the hydrogel of claim 12.

14. The preparation of claim 13, further defined as a wound dressing, a preparation for soft tissue augmentation, a preparations for viscosupplementation of synovial fluid, a matrix for controlled drug release, a fillings for tissue defects, or a scaffolds for tissue engineering.

15. The preparation of claim 14, further defined as a preparations comprising seeded and unseeded scaffolds for treatment of defects of joint cartilage and bone defects.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 provides a plot showing the effect of concentration of calcium ions on gelation rate of samples containing the transfusion preparation—30% (v/v) plasma, 13 mg/mL solution HATA, HRP 0.4 U/mL, H.sub.2O.sub.2 2.3 mM, various concentrations of CaCl.sub.2.2H.sub.2O in hydrogel.

[0024] FIG. 2 provides a plot showing the effect of concentration of Ca.sup.2+ (mol/L) on proliferation of 3T3 cells.

[0025] FIG. 3 provides a plot showing the cytotoxicity of hydrogel extracts containing various concentrations of calcium ions

[0026] FIG. 4 provides a plot showing the cytotoxicity of hydrogel extracts containing various ratios of blood/blood plasma. Concentration of calcium ions in the gel was equal to 0.1 mol/L. Given % are % (v/v).

[0027] FIG. 5 provides a plot showing the effect of concentration of Ca.sup.2+ ions in hydrogels containing 30% (v/v) ratio of blood plasma.

[0028] FIG. 6 provides an image showing the cell adhesion on the surface of hydrogels—staining LIVE/DEAD. Given % are % (v/v).

[0029] FIG. 7 provides an image showing a 3D culture of cells—staining LIVE/DEAD (arrows—adhered cells). Given % are % (v/v).

DETAILED DESCRIPTION

[0030] The following detailed description is merely exemplary in nature and is not intended to limit the disclosure or the application and uses of the subject matter as described herein. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description.

[0031] Means for use for the hydrogel preparation the principle thereof comprises two separate solutions A and B, where solution A contains enzyme horseradish peroxidase and solution B contains hydrogen peroxide, whereas at least one solution A or B contains calcium ions in the form of pharmaceutically acceptable salt and further solution A and/or B contains hydroxyphenyl derivative of hyaluronan of general formula I

##STR00003##

where n is in the range 2 to 7500 and R is OH or substituent NHR.sub.2CONHR.sub.1ArOH of general formula II,

##STR00004##

[0032] where Ar is phenylene and R.sub.1 ethylene or Ar is indolylene and R.sub.1 is ethylene or Ar is hydroxyphenyl and R.sub.1 is carboxyethylene and R.sub.2 is alkylene of 3 to 7 carbon atoms.

[0033] In the means according to one embodiment of the present invention, solution A contains horseradish peroxidase of activity 0.5 to 2.25 U/mL, preferably 1.8 to 2.2 U/mL, more preferably 1.0 to 1.3 U/mL and solution B contains hydrogen peroxide at 2 to 10 mmol/L, preferably 3 to 7 mmol/L, more preferably 4 to 5 mmol/L, whereas at least one solution A or B contains calcium ions at concentration 0.05 to 0.55 mol/L, preferably 0.25 to 0.55 mol/L, more preferably 0.25 to 0.45 mol/L, the most preferably 0.25 to 0.32 mol/L and hydroxyphenyl derivative of hyaluronan of the general formula I at the concentration 10 to 125 mg/mL, preferably 10 to 35 mg/mL, more preferably 20 to 26 mg/mL.

[0034] Means according to the present invention comprises two aqueous solutions A and B, one of which contains horseradish peroxidase (solution A) and the other hydrogen peroxide (solution B), whereas at least one of the solutions contains hydroxyphenyl derivative of hyaluronan of general formula I, and at the same time at least one of the solutions contains calcium ions in the form of pharmaceutically acceptable salts and in water well soluble salts. Pharmaceutically acceptable calcium salts are chosen from the group consisting of calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium hydrogen carbonate or monocalcium phosphate, preferably calcium chloride, calcium hydrogen carbonate, calcium lactate, calcium acetate and monocalcium phosphate.

[0035] Hydroxyphenyl derivative of hyaluronan of general formula I may be prepared following the protocols in CZ303879 that is included by this reference.

[0036] Polysaccharides including hyaluronan and derivatives prepared thereof belong among polymers made of mixtures of macromolecules of various sizes and form ununiform (polydisperse) systems. Molar mass of such polymers may be expressed as number average molar mass (Mn) or mass average molar mass (Mw). The ratio of these two types of average molar masses of polymer chains (Mw/Mn) expresses the measure of ununiformity (polydispersity) of polymer sample and is referred to as polydispersity index (PI). According to another embodiment of the present invention is Mw of hydroxyphenyl derivative of hyaluronan of general formula I in the range 4×10.sup.4 to 3×10.sup.6 g/mol, preferably 1×10.sup.5 to 1×10.sup.6 g/mol, more preferably 2×10.sup.4 to 4×10.sup.5 g/mol. PI is in the range 1 to 3.

[0037] According to another embodiment of the present invention is the degree of substitution (DS) of hydroxyphenyl derivative of hyaluronan of general formula I in the range 1 to 10%, preferably 2 to 5%, more preferably 2 to 4%.

[0038] Solutions A, B or the precursor solution A are aqueous solutions, preferably 0.9% aqueous solution of sodium chloride, i.e. isotonic solutions, i.e. with the same osmolarity (308 mOsmol) as blood plasma.

[0039] Solution A of the described means is used in a preparation of the precursor solution of hydrogel, where solution A of the means is mixed with blood or the transfusion preparation with a content of fibrinogen resulting in the formation of pA. Mixing solutions pA and B according to the present invention in volume ratio 1:1 results in formation of gel-forming mixture that after crosslinking of hydroxyphenyl derivative of hyaluronan gives hydrogel containing blood or the transfusion preparation with content of fibrin. The means and the method of preparation of hydrogels described in this document enables the preparation of hydrogels, in which blood or the transfusion preparation with the content of fibrin makes 1 to 30 vol. % of the total volume of the final hydrogel.

[0040] According to another embodiment of the present invention the means comprises solutions A and B, where the solution A comprises: [0041] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 4×10.sup.4 g/mol to 3×10.sup.6 g/mol (and with the polydispersity index 1-3), in which the DS is in the range 1 to 10%, whereas it is in the concentration 0 to 125 mg/mL, preferably 10 to 125 ml/mL [0042] horseradish peroxidase of activity 0.5 to 2.25 U/mL [0043] calcium ions in the concentration 0.05 to 0.55 mol/L and solution B comprises: [0044] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 4×10.sup.4 g/mol to 3×10.sup.6 g/mol (and with polydispersity index 1-3), in which the DS is in the range 1 to 10%, whereas the concentration is 10 to 50 mg/mL hydrogen peroxide in the concentration range 2 to 10 mmol/L.

[0045] According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises: [0046] hydroxyphenyl derivative of hyaluronan of the general formula I of mass average molar mass in the range 1×10.sup.5 g/mol to 1×10.sup.6 g/mol (and with polydispersity index 1-3), in which DS is in the range 2 to 5%, whereas it is in the concentration 0 to 35 mg/mL, preferably 10 to 35 mg/mL, [0047] horseradish peroxidase of activity 0.5 to 2.25 U/mL [0048] calcium ions at the concentration 0.05 to 0.55 mol/L

[0049] And the solution B comprises: [0050] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass 1×10.sup.5 g/mol to 1×10.sup.6 g/mol (and with polydispersity index 1-3), in which DS is in the range 2 to 5%, whereas it is at the concentration 26 mg/mL [0051] hydrogen peroxide in the range of concentration 3 to 7 mmol/L.

[0052] According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises: [0053] horseradish peroxidase of activity 1.8 to 2.2 U/mL [0054] calcium ions at the concentration 0.45 to 0.55 mol/L

[0055] and the solution B comprises: [0056] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which the DS is in the range 2 to 4%, whereas it is at the concentration 26 mg/mL [0057] hydrogen peroxide in the concentration range 4 to 5 mmol/L.

[0058] According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises: [0059] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which the DS is in the range 2 to 4%, whereas it is at the concentration 20 mg/mL [0060] horseradish peroxidase of activity 1.0 to 1.3 U/mL [0061] calcium ions of concentration 0.25 to 0.32 mol/L

[0062] and the solution B comprises: [0063] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which the DS is in the range 2 to 4%, whereas it is at the concentration 26 mg/mL [0064] hydrogen peroxide in the concentration range 4 to 5 mmol/L.

[0065] According to yet another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises: [0066] horseradish peroxidase of activity 0.8 to 2.2 U/mL [0067] calcium ions at the concentration 0.02 to 0.5 mol/L [0068] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 9×10.sup.4 g/mol to 4×10.sup.5 g/mol, with polydispersity index 1 to 10% and at the concentration in the range 10 to 50 mg/mL, and the solution B comprises: [0069] hydrogen peroxide in the concentration range 4 to 5 mmol/L.

[0070] Another embodiment of the present invention is the means of the preparation of the hydrogel comprising the covalently crosslinked hydroxyphenyl derivative of hyaluronan that is made by preparation of two separate solutions A and B described above, and then the solution A is mixed with blood or with at least one transfusion preparation with content of fibrinogen resulting in formation of the precursor solution A, that is mixed with the solution B. Solutions A and B of the means according to the present invention are preferably used in the method according to the present invention.

[0071] 10 to 60% (v/v) of blood or the transfusion preparation with content of fibrinogen is preferably added to the solution A, more preferably 30 to 60% (v/v). The precursor solution A with the solution B is preferably mixed in volume ratio 1:1.

[0072] According to a preferred embodiment of the method of the present invention is at first the solution A of the means mixed with blood or the transfusion preparation with fibrinogen content resulting in the precursor solution of hydrogel pA that comprises: [0073] hydroxyphenyl derivative of hyaluronan of general formula I of mass average molar mass in the range 4×10.sup.4 g/mol to 3×10.sup.6 g/mol (and with polydispersity index 1-3) with substitution degree 1 to 10%, at concentration 0 to 50 mg/mL, preferably 10 to 50 mg/mL, [0074] horseradish peroxidase of activity 0.2 to 0.9 U/mL, [0075] calcium ions at concentration 0.02 to 0.22 mol/L, [0076] 10 to 60% (v/v) of blood or transfusion preparation with the content of fibrinogen, that is further mixed in volume ratio 1:1 with the solution B of above means that comprises: [0077] hydroxyphenyl derivative of hyaluronan of general formula I of mass average molar mass in the range 4×10.sup.4 g/mol (and with polydispersity index 1-3), in which is DS in the range 1 to 10%, whereas it is at the concentration 10 to 50 mg/mL, [0078] hydrogen peroxide in the range of concentration 2 to 10 mmol/L.

[0079] Further is also described a preferred embodiment of the means of the preparation of the hydrogel according to the present invention, where the solution A of the means is at first mixed with blood or the transfusion preparation with content of fibrinogen giving the precursor solution of hydrogel pA that further comprises: [0080] hydroxyphenyl derivative of hyaluronan of the general formula I of mass average molar mass in the range 1×10.sup.5 g/mol to 1×10.sup.6 g/mol (and with polydispersity index 1-3) in which is DS 2 to 5%, whereas it is at the concentration 0 to 14 mg/mL, preferably 10 to 14 mg/mL, [0081] horseradish peroxidase of activity 0.2 to 0.9 U/mL [0082] calcium ions at the concentration 0.02 to 0.22 mol/L [0083] 10 to 60% (v/v) of blood or the transfusion preparation with fibrinogen content that is further mixed in volume ratio 1:1 with the solution B of above described means, that comprises: [0084] hydroxyfenyl derivative of hyaluronan of general formula I of mass average molar mass in the range 1×10.sup.5 g/mol to 1×10.sup.6 g/mol (and with polydispersity index 1-3), in which is DS 2 to 5%, whereas it is at concentration 26 mg/mL [0085] hydrogen peroxide in the range of concentration 3 to 7 mmol/L.

[0086] Further is described another preferred embodiment of the means of the preparation of the hydrogel according to the present invention, where the solution A is at first mixed with the transfusion preparation with content of fibrinogen resulting in the precursor solution of the hydrogel pA that comprises: [0087] horseradish peroxidase of activity 0.7 to 0.9 U/mL, [0088] calcium ions at concentration 0.18 to 0.22 mol/L, [0089] 60% (v/v) of blood plasma or the fraction of blood plasma reaches in blood platelets,

[0090] that is further mixed in the volume ratio 1:1 with the solution B of the above described means, that comprises [0091] hydroxyphenyl derivative of hyaluronan of the general formula I at the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which is DS in the range 2 to 4%, whereas it is at the concentration 26 mg/mL [0092] hydrogen peroxide in the concentration range 4 to 5 mmol/L.

[0093] Further is described yet another preferred embodiment of the preparation of the hydrogel according to the present invention, where the solution A is at first mixed with blood giving the precursor solution of the hydrogel pA that comprises: [0094] hydroxyphenyl derivative of hyaluronan of the general formula I of the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which DS is in the range 2 to 4%, whereas it is at the concentration 14 mg/mL [0095] horseradish peroxidase of activity 0.7 to 0.9 U/mL [0096] calcium ions at concentration 0.18 to 0.22 mol/L [0097] 30% (v/v) of blood,

[0098] That is further mixed in the volume ratio 1:1 with the solution B of the above described means, that comprises: [0099] hydroxyphenyl derivative of hyaluronan of the general formula (I) of the mass average molar mass in the range 2×10.sup.5 g/mol to 4×10.sup.5 g/mol (and with polydispersity index 1-3), in which DS is in the range 2 to 4%, whereas it is at the concentration 26 mg/mL [0100] hydrogen peroxide in the concentration range 4 to 5 mmol/L.

[0101] According to another embodiment of the present invention, hydrogels prepared by the means, as is described above, comprise enzyme horseradish peroxidase of activity 0.1 to 2.5 U/mL, preferably 0.25 to 0.45 U/mL, more preferably 0.35 to 0.45 U/mL, calcium ions at the concentration 0.01 to 0.11 mol/L, preferably 0.09 to 0.11 mol/L, the covalently crosslinked hydroxyphenyl derivative of hyaluronan at the concentration 5 to 50 mg/mL, more preferably 13 to 20 mg/mL and blood or the transfusion preparation with fibrinogen content at the concentration in the range 5 to 30% (v/v) preferably 15 to 30% (v/v). Hydrogels elasticity (G′) according to the present invention may fall in the range from 1 Pa to 3 kPa, preferably 1 Pa to 2 kPa.

[0102] According to another embodiment of the present invention, the hydrogel described above will be used for manufacturing of preparations in cosmetics, medicine or regenerative medicine.

[0103] Preferably, such preparation is chosen from a group of preparations comprising wound dressing, preparation for soft tissues augmentation, preparations for viscosupplementation of synovial fluid, matrix for controlled drug release, fillings of tissue defects, scaffolds for tissue engineering.

[0104] As stated above, the preparation according to the present invention enables the preparation of gel-forming mixture (hydrogel) that forms following mixing of hydrogel precursor solution A and solution B. Gel-forming mixture (hydrogel) contains hydroxyphenyl derivative of hyaluronan of the general formula I, blood or the transfusion preparation containing fibrinogen, horseradish peroxidase, hydrogen peroxide and calcium ions in the form of water soluble pharmaceutically acceptable salt. This gel-forming mixture enables forming of hydrogel based on the covalently crosslinked tyramine derivative of hyaluronan with content of blood or the transfusion preparation containing fibrin.

[0105] To the hydrogel crosslinking contributes the reaction catalyzed with horseradish peroxidase that leads to oxidation of hydroxyphenyl cores of tyramine resulting in dimer, or oligomer formation. Hydrogen peroxide is source of hydroxyl radicals for reaction catalyzed with horseradish peroxidase and serves as the crosslinking reaction initiator.

[0106] Surprisingly, it was found that the addition of sufficient amount of calcium ions into at least one solution A or B, as is stated above, makes the process of gelation more effective and increases the elasticity module of forming hydrogels. This phenomenon has not been observed in analogous hydrogels without content of blood or the transfusion preparation.

[0107] It was further found that the presence of calcium ions in the solution of hydroxyphenyl derivative or its mixture with blood or the transfusion preparation with content of fibrinogen itself does not lead, without the initiation of enzymatic crosslinking reaction of hydroxyphenyl derivative of hylauronan, to the formation of hydrogels. It is not only a manifestation of the blood coagulation that may result from the increased calcium concentration or the result from calcium ions interaction with the polyanionic chain of the hydroxyphenyl derivative of hyaluronan. It can't be excluded that all mentioned processes take place simultaneously in the gel-forming mixture of the hydrogel. It is likely, that the formation of the hydrogel occurs by interaction among individual forming polymer networks and their combined size exceeds critical limit and hydrogel forming occurs. Contrary to so far published systems, thus described composition of the means enables the preparation of the hydrogel based on the hydroxyphenyl derivative of hyaluronan of the general formula I also in the presence of blood.

[0108] In the case of blood use, above described gel-forming mixtures enable forming of the hydrogel at hydrogen peroxide concentrations that don't lead to damage of biological structures and at the same time enable use of horseradish peroxidase of activity that wouldn't lead itself to effective hydrogel forming in the presence of blood or the transfusion preparation with fibrinogen content. This significantly increases the possibility to use the means in the field of tissue engineering, regenerative medicine and further medicinal fields.

[0109] Similar phenomenon may be observed also in the case of hydrogels with the content of plasma. Hydrogen peroxide is more stable in plasma and thus the presence of calcium ions is not in this case essential for the hydrogel forming. Nevertheless, in this case too, the presence of calcium ions leads to acceleration of the process of gelation and increases the elastic modules of the prepared hydrogels. This phenomenon may be also explained by the parallel forming of the fibrin network and the network based on the hydroxyphenyl derivative of hyaluronan.

[0110] Higher concentration of calcium ions leads to inhibition of proliferation of cultured cells, what would limit the use of the means as material for medicinal purposes. In the means according to the present invention, the amount of calcium ions is optimized so that it is sufficient for support of forming hydrogels of the invention but at the same time enables use of prepared hydrogels of the invention as matrix for 2D and 3D cell culture. The possibility to use a combination of blood or transfusion preparations containing fibrinogen for the preparation of hydrogels based on hydroxyphenyl derivatives of hyaluronan was not so far published.

[0111] The term “blood” means blood drawn from a donor, human or animal. It refers to a heterologous donor or preferably autologous donor. It is a material for the preparation of transfusion preparations. The term “transfusion preparation with fibrinogen content” means the transfusion preparation that is made from blood by usual protocols and comprises fibrinogen. The transfusion preparation is selected from a group containing blood plasma, plasma fraction reaches in blood platelets (PRP). It does not refer to blood derivatives, that are industrially manufactured preparations that comprise especially albumin, coagulating factors and immunoglobulins of human origin.

EXAMPLES OF THE EMBODIMENTS OF THE INVENTION

[0112] DS=degree of substitution=100%*molar amount of modified disaccharide units of hyaluronan/molar amount of all disaccharide units of the derivative of hyaluronan. Degree of substitution was determined with .sup.1H NMR spectroscopy.

[0113] Mass average molar mass (Mw) and polydispersity index (PI) were determined using SEC-MALLS.

Example 1

Synthesis of Tyramine Derivative of HA (HA-TA)

Synthesis of 6-amino-N-[2(4hydroxyphenyl)ethyl]hexanamide

[0114] 6-[(terc. butoxycarbonyl)amino]hexane acid (1.00 g, 4.3 mmol) was dissolved in 50 mL tetrahydrofuran (THF). 1,1′ carbodiimidazole (0.70 g, 4.3 mmol) was added to the acid solution. The reaction mixture was heated at 50° C. for sixty minutes. The container was then washed with inert gaz. Tyramine (0.59 g, 4.3 mmol) was added to the reaction mixture. The mixture was further heated for another 2 hours. THF was then removed by distillation under reduced pressure. The residue was dissolved in 50 mL ethylacetate. The solution was washed with 150 mL purified water (divided into three parts). Organic layer was dried over molecular sieve. Ethylacetate was removed by distillation under reduced pressure. The residue was heated for 6 hours at reflux. The solvent was removed by distillation under reduced pressure. The residue was dissolved in 50 mL ethylacetate. The solution was washed with 150 mL of purified water (divided into three parts). Organic layer was dried over molecular sieve. Ethylacetate was removed by distillation under reduced pressure.

[0115] m=0.75 g (70% theory)

[0116] .sup.1H NMR (D.sub.2O, ppm) δ: 1.17 (m, 2H, γ-CH.sub.2-hexane acid); 1.48 (m, 2H, (3-CH.sub.2-hexane acid); 1.58 (m, 2H, 6-CH.sub.2-hexane acid); 2.17 (t, 2H, —CH.sub.2—CO—); 2.73 (m, 2H, —CH.sub.2-Ph); 2.91 (m, 2H, —CH.sub.2—NH.sub.2); 3.42 (m, 2H, —CH.sub.2—NH—CO—); 6.83 (d, 2H, arom); 7.13 (d, 2H, arom).

[0117] .sup.13C NMR (D.sub.2O, ppm) δ: 24 (γ-C-hexane acid); 26 (δ-C-hexane acid); 33 (β-C-hexane acid); 35 (—C—CO—); 39 (—C—NH.sub.2); 40 (C-Ph); 63 (—C—NH—CO—); 115 (C3 arom); 126 (C1 arom); 130 (C2 arom.); 153 (C4 arom); 176 (—CO—).

Preparation of Aldehyde Derivative (HA-CHO)

[0118] Hyaluronan (10.00 g, Mw=2×10.sup.6 g.Math.mol.sup.−1) was dissolved in 750 mL 2.5% (w/w) solution Na.sub.2HPO.sub.4.12H.sub.2O. The solution was cooled to 5° C. 2.60 g NaBr and 0.05 g 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl was added to resulting solution. After thorough homogenization of the solution, 3 mL of solution NaClO (10-15% available C12) were added to reaction mixture. Reaction proceeded under continuous stirring for 15 min. Reaction was quenched by addition of 100 mL 40% solution of propan-2-ol. Product was purified by ultrafiltration and isolated by precipitation with propan-2-ol.

[0119] IR (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0120] .sup.1H NMR (D.sub.2O) δ: 2.01 (s, 3H, CH.sub.3—), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., —O—CH(OH)—), 5.27 (geminal glykol —CH—(OH).sub.2).

[0121] a) Preparation of Tyramine Derivative of HA with C.sub.6 spacer (Mw≈5×10.sup.4 g.Math.mol.sup.−1, DS≈6%)

[0122] Aldehyde derivative of HA (≈6×10.sup.4 g.Math.mol.sup.−1, DS=9%) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted with acetic acid to 3. 6-amino-N-[2-(4-hydroxyphenyl)ethyl]hexanamide (intermediate (I)) (1.25 g, 5 mmol) was added to solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Complex picoline-borate (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. Product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Humidity and residual propan-2-ol were removed from precipitate by drying in hot-air drier (40° C., 3 days).

[0123] IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0124] .sup.1H NMR (D.sub.2O) δ: 1.25 (t, 2H, γ-CH.sub.2-aminohexane acid), 1.48 (m, 2H, δ-CH.sub.2-aminohexane acid) 1.51 (m, 2H, β-CH.sub.2-aminohexane acid), 2.01 (s, 3H, CH.sub.3—), 2.65 (m, 2H, Ph-CH.sub.2—), 2.73 (m, 2H, ε-CH.sub.2-aminohexane acid), 3.37-3.93 (m, skelet of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., —O—CH(OH)—), 6.59 (d, 2H, arom.), 7.01 (d, 2H. arom).

[0125] SEC MALLS: Mw=5.4×10.sup.4 g.Math.mol.sup.−1; PI=1.61

[0126] DS (.sup.1H NMR): 6.2%

[0127] b) Preparation of Tyramine Derivative of HA with C.sub.6 spacer (Mw≈3×10.sup.5 g.Math.mol.sup.−1, DS≈2%)

[0128] Aldehyde derivative of HA (Mw 3×10.sup.5 g.Math.mol.sup.−1, DS 9%) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted with acetic acid to 3. 6-amino-N-[2-(4-hydroxyphenyl)ethyl]hexanamid (intermediate (I)) (0.625 g, 2.5 mmol) was added to the solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Complex picoline-borate (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Humidity and residual propan-2-ol were removed from precipitate by drying in hot-air drier (40° C., 3 days).

[0129] IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0130] .sup.1H NMR (D.sub.2O) δ: 1.25 (t, 2H, γ-CH.sub.2-aminohexane acid), 1.48 (m, 2H, δ-CH.sub.2-aminohexane acid) 1.51 (m, 2H, β-CH.sub.2-aminohexane acid), 2.01 (s, 3H, CH.sub.3—), 2.65 (m, 2H, Ph-CH.sub.2—), 2.73 (m, 2H, ε-CH.sub.2-aminohexane acid), 3.37-3.93 (m, skelet of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., —O—CH(OH)—), 6.59 (d, 2H, arom.), 7.01 (d, 2H. arom).

[0131] SEC MALLS: Mw=2.81×10.sup.5 g.Math.mol.sup.−1; PI=1.49

[0132] DS (.sup.1H NMR): 2.2%

[0133] c) Preparation of Tyramine Derivative of HA with C.sub.6 spacerem (Mw≈1.5×10.sup.6 g.Math.mol.sup.−1, DS=1%)

[0134] Aldehyde derivative of HA (≈1.5×10.sup.6 g.Math.mol.sup.−1, DS=4%) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted with acetic acid to 3. 6-amino-N-[2-(4-hydroxyfenyl)ethyl]hexanamid (meziprodukt (I)) (0.625 g, 2.5 mmol) was added to solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Picoline-borate complex (0.270 g, 2.5 mmol) was then added to reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Humidity and residual propan-2-ol were removed from precipitate by drying in hot-air drier (40° C., 3 days).

[0135] IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0136] .sup.1H NMR (D.sub.2O) δ: 1.25 (t, 2H, γ-CH.sub.2-aminohexane acid), 1.48 (m, 2H, 6-CH.sub.2-aminohexane acid) 1.51 (m, 2H, β-CH.sub.2-aminohexane acid), 2.01 (s, 3H, CH.sub.3—), 2.65 (m, 2H, Ph-CH.sub.2—), 2.73 (m, 2H, ε-CH.sub.2-aminohexane acid), 3.37-3.93 (m, skelet of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., —O—CH(OH)—), 6.59 (d, 2H, arom.), 7.01 (d, 2H. arom).

[0137] SEC MALLS: Mw=1.45×10.sup.6 g.Math.mol.sup.−1; PI=1.65

[0138] DS (.sup.1H NMR): 1.1%

[0139] d) Preparation of Tyramine Derivative of HA with C6 spacerem (Mw≈1.0×10.sup.5 g.Math.mol.sup.−1, DS=8.5%)

[0140] Aldehyde derivative of HA (≈1.0×10.sup.5 g.Math.mol.sup.−1, DS=10%) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted with acetic acid to 3. 6-amino-N-[2-(4-hydroxyfenyl)ethyl]hexanamid (intermediate (I)) (0.625 g, 2.5 mmol) was added to solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Complex picoline-borate (0.270 g, 2.5 mmol) was then added to the mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Humidity and residual propan-2-ol were removed from precipitate by drying in hot-air drier (40° C., 3 days).

[0141] IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0142] .sup.1H NMR (D.sub.2O) δ: 1.25 (t, 2H, γ-CH.sub.2-aminohexane acid), 1.48 (m, 2H, δ-CH.sub.2-aminohexane acid) 1.51 (m, 2H, β-CH.sub.2-aminohexane acid), 2.01 (s, 3H, CH.sub.3—), 2.65 (m, 2H, Ph-CH.sub.2—), 2.73 (m, 2H, ε-CH.sub.2-aminohexane acid), 3.37-3.93 (m, skelet of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., —O—CH(OH)—), 6.59 (d, 2H, arom.), 7.01 (d, 2H. arom).

[0143] SEC MALLS: Mw=9.5×10.sup.4 g.Math.mol.sup.−1; PI=1.55

[0144] DS (.sup.1H NMR): 8.6%

[0145] e) Preparation of Tyramine Derivative of HA with C6 spacerem (Mw≈2.5×10.sup.6 g.Math.mol.sup.−1, DS=1%)

[0146] Aldehyde derivative of HA (≈2.5×10.sup.6 g.Math.mol.sup.−1, DS=4%) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted with acetic acid to 3. 6-amino-N-[2-(4-hydroxyfenyl)ethyl]hexanamide (intermediate (I)) (0.625 g, 2.5 mmol) was added to solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Complex picoline-borate (0.270 g, 2.5 mmol) was then added to the mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Humidity and residual propan-2-ol were removed from precipitate by drying in hot-air drier (40° C., 3 days).

[0147] IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm.sup.−1.

[0148] .sup.1H NMR (D.sub.2O) δ: 1.25 (t, 2H, γ-CH.sub.2-aminohexane acid), 1.48 (m, 2H, δ-CH.sub.2-aminohexane acid) 1.51 (m, 2H, β-CH.sub.2-aminohexane acid) 2.01 (s, 3H, CH.sub.3—), 2.65 (m, 2H, Ph-CH.sub.2—), 2.73 (m, 2H, ε-CH.sub.2-aminohexane acid), 3.37-3.93 (m, skelet of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer, —O—CH(OH)—), 6.59 (d, 2H, arom.), 7.01 (d, 2H. arom).

[0149] SEC MALLS: Mw=2.5×10.sup.6 g.Math.mol.sup.−1; PI=1.65

[0150] DS (.sup.1H NMR): 1%

Example 2

The Influence of the Presence of Calcium Ions on the Formation of the Hydrogel in the Presence of Blood

[0151] The rate of gelation was measured with rotational rheometer AR-G2 (TA Instrument) using geometry board-board (40 mm) and space size 400 μm. 525 μL sample was applied on bottom static board. Pre-shear 2000 L/s per sec was chosen for sample homogenization. Time sweep mode was chosen for determination of gelation rate at frequency 1 Hz and displacement 0.001 rad at 37° C. Elastic and viscose module was determined from the dependency of elastic G′ on viscose G″ module on time t. Tyramine derivative of hyaluronan synthetized following procedure according to example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for sample preparation.

[0152] Elastic G′ and viscose G″ module of individual samples read at 300 s are presented in Table 1.

TABLE-US-00001 TABLE 1 Composition of tested gel-forming mixtures Proportion Concentration Concentration Activity Concentration of blood in HA-TA CaC1.sub.2•2H.sub.2O HRP H.sub.2O.sub.2 sample G′ G″ (mg/mL) (mol/L) (U/mL) (mmol/L) (%) (Pa) (Pa) 1 20 — — — 15 0.17 1.23 2 20 0.1 — — 15 0.34 1.06 3 20 0.1 0.4 — 15 0.20 1.15 4 20 — 0.4 — 15 0.24 1.27 5 20 — — 2.3 15 0.07 0.92 6 20 0.1 0.4 2.3 15 21.80 11.50 7 20 0.4 2.3 15 0.52 1.99

[0153] From given data it is apparent that gelation occurs only at the combination of hydroxyphenyl derivative of hyaluronan with horseradish peroxidase, hydrogen peroxide and calcium ions with blood (sample 6)—elastic module G′ is higher than viscose module G″. In the other samples (samples 1 to 5 and 7) is elastic module lower than the viscose module and the gelation of given mixtures does not occur even after 24 hours.

Example 3

Influence of Calcium Ions on the Efficiency of the Gelation in the Presence of Blood and Transfusion Preparations

[0154] Viscoelastic properties of hydrogels were determined with rotational rheometer AR-G2 (TA Instruments) using method strain sweep at constant value of frequency 1 Hz and displacement in the range 10.sup.−3 to 2 radians. Geometry with rougher surface (cross-hatched) was used in order to avoid slipping of prepared hydrogels during measurement. Hydrogels of diameter 17.5 mm were used for the measurement. Hydrogels were scored after 60 minutes of preparation. Tyramine derivative of hyaluronan synthetized following procedure according to Example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the solution preparation.

[0155] Viscoelastic properties (elastic G′ and viscose G″ module) of hydrogels prepared without transfusion preparations are shown in Table 2. The table also demonstrates the difference in the elastic module in hydrogels prepared in the absence and the presence of calcium ions.

[0156] No increase in the elastic module depending on the calcium ions presence was observed in hydrogels prepared in the absence of blood or transfusion preparations. On the contrary, hydrogels with the content of blood plasma or blood plasma reaches in blood platelets (PRP) prepared in the presence of calcium ions showed elevated values of the elastic module in comparison with analogous gels prepared in the absence of calcium. Precursor solutions with blood content but without calcium were not able form hydrogel at all.

TABLE-US-00002 TABLE 2 Composition of tested gel-forming mixtures and properties of hydrogels formed thereof Conc. Conc. Activity Conc. HA-TA CaC1.sub.2•2H.sub.2O HRP H.sub.2O.sub.2 G′ G″ (mg/mL) (mol/L) (U/mL) (mmol/L) (v/v) (Pa) SD (Pa) SD 1 13 0.1 0.4 2.3 mM Without 1028.9 48.8 2.08 0.47 0 blood 1052.6 34.4 4.17 1.53 2 0.1 30% 578.3 27.2 2.35 0.18 0 plasma 433.7 8.5 2.07 0.05 3 0.1 30% 548.2 65.5 2.46 0.27 0 PRP 360.8 11.7 2.94 0.15 4 20 0.1 Without 1764.0 151.8 1.82 1.06 0 blood 1664.8 91.6 1.86 1.01 5 0.1 15% 59.0 1.9 20.73 1.85 0 Blood No gelling 6 30 0.1 30% 1.7 0.3 0.41 0.06 0 Blood No gelling

Example 4

Influence of Calcium Ions on the Rate of the Hydrogel Forming in the Presence of Blood Plasma

[0157] Gelation rate was determined using method described in Example 2.

[0158] Influence of calcium ions on the rate of gelation of samples containing the transfusion preparation— 30% (v/v) plasma is shown in FIG. 1. The time of gelation decreases with increasing calcium ions concentration.

Example 5

Influence of Calcium Ions on Cell Proliferation

[0159] FIG. 2 shows the viability of cells (fibroblast cell line 3T3) cultured in the presence of calcium ions (0.1-0.001 mol/L). The cells were seeded on 24-well culture panel at a density of 15 000 cell/well. The medium was removed the next day; and fresh medium contained calcium ions at tested concentrations. Cell proliferation was determined at intervals 24, 48 and 72 h with assay for determination of ATP using CellTiter-Glo assay. DEMI water in 10% volume of medium was used as the control.

[0160] Norm ISO 10993-5-2009 states the limit for cytotoxicity as reduction of viability of influenced cell by more than 30% in comparison with control. Such reduction in cell viability below this limit occurs at concentrations 0.1 mol/L to 0.05 mol/L Ca.sup.2+. The presence of calcium ions in concentration ≤0.01/L does not negatively influence cell viability.

Example 6

[0161] Cytotoxicity of Extracts from Hydrogels Containing Various Calcium Ions Concentration

[0162] Hydrogels were prepared from gel-forming mixture that came from mixing precursor solution pA and solution B in the volume ratio 1:1 (for composition of solutions and forming hydrogels see Table 3). Calcium chloride was used as source of calcium ions. Tyramine derivative of hyaluronan synthetized according to procedure of Example 1 (Mw=2.8×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions.

[0163] Sterile prepared hydrogels were transferred into a plastic test tube and covered with a culture medium in ratio 9:1 (9 mL medium/1 g gel). Extraction was performed for 72 h at 37° C. Cells 3T3 were seeded into 24-well culture panel at a density of 15 000 cells/well. The medium was replaced the next day with test extract. Cell proliferation was recorded at the intervals 24, 48 and 72 h by means of ATP determination with CellTiter-Glo assay. Culture medium was used as control. FIG. 3 describes cell viability in the presence of extracts of the hydrogel containing various calcium ion concentrations. The limit value of calcium ions concentration in the hydrogel for cell viability was 0.1 mol/L. Exceeding this limit resulted in hydrogel extracts cytotoxicity.

TABLE-US-00003 TABLE 3 Concentration of individual components in the precursor solution pA and solution B and the gel-forming mixture produced by mixing the solution pA and the solution B Precursor solution pA Solution pB Gel-forming mixture (hydrogel) HRP Ca.sup.2+ HA-TA H.sub.2O.sub.2 HRP Ca.sup.2+ HA-TA H.sub.2O.sub.2 (U/mL) (mol/L) (mg/mL) (mmol/L) (U/mL) (mol/L) (mg/mL) (mmol/L) 0.8 0.1 26 4.6 0.4 0.05 13 2.3 0.14 0.07 0.2 0.1 1 0.5

Example 7

Cytotoxicity of Extracts of Hydrogel Containing Blood or Blood Plasma

[0164] Preceding the preparation of the hydrogel, solution A was mixed with appropriate blood or blood plasma amount producing precursor solution pA. Hydrogels were produced from gel-forming mixture that resulted from mixing precursor solution pA with addition of blood or blood plasma and solution B in volume ratio 1:1 (for composition of solutions and produced hydrogels see table 4). Calcium chloride was used as source of calcium ions. Tyramine derivative of hyaluronan synthetized following procedure according to Example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for solution preparation. Sterile prepared hydrogels were transferred into the plastic test tube and covered with the culture medium in ratio 9:1 (9 mL medium/1 g gel). Extraction proceeded for 72 h at 37° C. 3T3 cells were seeded into 24-well culture panel at a density of 15 000 cells/well. The medium was replaced with tested extract the next day. Cell proliferation was monitored at intervals 24, 48 and 72 h by means of determination of ATP using CellTiter-Glo assay. Culture medium was used as control. The results presented in FIG. 4 show that extracts of hydrogels with 15-30% (v/v) proportion of blood or blood plasma and with 0.1 mol/L content of calcium ions do not have negative influence on cell viability.

TABLE-US-00004 TABLE 4 The influence of blood plasma and blood on the cytotoxicity of extracts of hydrogels-Concentration of single components in the precursor solution pA and the solution B and the gel-forming mixture produced by mixing the solution pA and the solution B. Precursor solution pA Solution B Gel-forming mixture (hydrogel) HRP Ca.sup.2+ HA-TA HA-TA HRP Ca.sup.2+ HA-TA (U/mL) (mol/L) (mg/mL) (v/v) (mg/mL) (mmol/L) (U/mL) (mol/L) (mg/mL) (mmol/L) (v/v) 0.8 0.2 0   26 4.6 0.4 0.1 13 2.3 0 Plasma 26 4.6 13 2.3 Plasma 60% 30% 14 Plasma 26 4.6 20 2.3 Plasma 30% 15% 14 Blood 26 4.6 20 2.3 Blood 30% 15% indicates data missing or illegible when filed

Example 8

Cytotoxicity of Extracts of the Hydrogel Containing the Portion of Blood Plasma and Various Concentration of Calcium Ions

[0165] Further, the influence of calcium ions concentration present in hydrogel with the content of blood plasma on the cell viability was studied. For the evaluation of this parameter, the extracts of hydrogels were again prepared. The solution A was mixed with the appropriate amount of blood or blood plasma giving the precursor solution pA before the hydrogel preparation. Hydrogels were prepared from the gel-forming mixture that was produced by the mixing precursor solution pA and the solution B in the volume ratio 1:1 (for the composition of solutions and produced hydrogels see Table 5). Calcium chloride was used as source of calcium ions. Tyramine derivative of hyaluronan synthetized following the procedure according to Example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for solutions preparation. Sterile prepared hydrogels were transferred into a plastic test tube and covered with the culture medium in ratio 9:1 (9 mL medium/1 g gel). Extraction proceeded for 72 h at 37° C. 3T3 cells were seeded into 24-well culture panel at a density of 15 000 cells/well. The medium was replaced for the tested extract the next day. Cell proliferation was monitored at intervals 24, 48 and 72 h by means of determination of ATP using CellTiter-Glo assay. Culture medium was used as control. The results presented in FIG. 5 show that extracts prepared from hydrogels containing 30% (v/v) portion of cell plasma and calcium ions in concentration range 0 to 0.1 mol/L do not induce cytotoxicity.

TABLE-US-00005 TABLE 5 Influence of calcium ions concentration on the cytotoxicity of extracts of hydrogels containing cell plasma-Concentrations of individual components in the precursor solution pA and the solution B and in the gel-forming mixture produced by the mixing solution pA and the solution B Precursor solution pA Solution B Gel-forming mixture (hydrogel) HRP Ca.sup.2+ HA-TA HA-TA H.sub.2O.sub.2 HRP Ca.sup.2+ HA-TA (U/mL) (mol/L) (mg/mL) (v/v) (mg/mL) (mmol/L) (U/mL) (mol/L) (mg/mL) (mmol/L) (v/v) 0.8 0 0 Plasma 26 4.6 0.4 0 13 2.3 Plasma 60% 30% 0.1 0 Plasma 26 4.6 0.05 13 2.3 Plasma 60% 30% 0.2 0 Plasma 26 4.6 0.1 13 2.3 Plasma 60% 30% indicates data missing or illegible when filed

Example 9

Test of Cell Adhesion to Hydrogel Layers

[0166] The solution A was mixed with appropriate amount of blood, blood plasma or PRP giving the precursor solution pA. Hydrogels were produced from the gel-forming mixture that was produced by the mixing precursor solution pA with addition of blood or the transfusion preparation and the solution B in the volume ratio 1:1 (for the composition of solutions and produced hydrogels see Table 6). Calcium chloride was used as a source of calcium ions. Tyramine derivative of hyaluronan synthetized following the procedure according to Example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of the solutions. Tested hydrogels were layered upon 24-well culture panels. Stem cells were then seeded upon the surface of hydrogel layer at a density of 13 000 cells/well. Cell growth was monitored for 4 days using LIVE/DEAD staining.

[0167] Stem cell adhesion on the surface of the material manifests itself by typical changes in cell morphology where the cells adopt elongated shape. FIG. 6 documents that the cells are not able to adhere to the hydrogel without the addition of blood or the transfusion preparation. In this case the cells keep, even after 4 days of culture, spherical shape and do not manifest marks of interaction with the surface of the gel. Likewise, their proliferation is suppressed. Hydrogels with the content of blood or transfusion preparations with the fibrinogen content (blood plasma, PRP), on the contrary, support adhesion and proliferation of the cells. Cells adopt on their surface typical elongated shape and their count increases with the time of culture. The experiment also showed that the presence of calcium in structure of hydrogel is not an obstacle for culture upon the hydrogel surface.

TABLE-US-00006 TABLE 6 2D adhesion of stem cells to the hydrogel surface-Concentrations of individual components in the precursor solution pA and the solution B and the gel- forming mixture produced by mixing the solution pA and the solution B Precursor solution pA Solution B Gel-forming mixture (hydrogel) HRP Ca.sup.2+ HA-TA HA-TA H.sub.2O.sub.2 HRP Ca.sup.2+ HA-TA (U/mL) (mol/L) (mg/mL) (v/v) (mg/mL) (mmol/L) (U/mL) (mol/L) (mg/mL) (mmol/L) (v/v) 0.8 0.1 0 0 26 4.6 0.4 0.05 13 2.3 0 0 Plasma 26 4.6 13 2.3 Plasma 60% 30% 14 Plasma 26 4.6 20 2.3 Plasma 30% 15% 0 PRP 26 4.6 13 2.3 PRP 60% 30% 14 PRP 26 4.6 20 2.3 PRP 30% 15% indicates data missing or illegible when filed

Example 10

3D Cell Culture

[0168] Cells of 3T3 mouse fibroblast cell line were used for encapsulation. Cell pellet was suspended in the solution A before the hydrogel preparation. The solution A was then mixed with the appropriate amount of blood, blood plasma or PRP producing the precursor solution pA. Hydrogels were then prepared by the mixing precursor solution A and the solution B in volume ratio 1:1 (for composition of solutions and produced hydrogels see Table 7). Calcium chloride was used as a source of calcium ions. Tyramine derivative of hyaluronan synthetized following the procedure according to Example 1 (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of the solutions. Sterile prepared hydrogels were transferred into the culture panel and covered with the culture medium where they were incubated for 4 days at 37° C., 5% CO.sub.2. Cell growth was monitored for 4 days using LIVE/DEAD staining. The results showed in FIG. 7 document that the system enables encapsulation of viable cells into hydrogels and their subsequent 3D culture. Cells in hydrogels containing transfusion preparation are able of adhesion.

TABLE-US-00007 TABLE 7 3D culture of cells-Concentration of individual components in the precursor solution A and the solution B and in the hydrogel after mixing of solutions pA and B. Precursor solution pA Solution B Gel-forming mixture (hydrogel) HRP Ca.sup.2+ HA-TA HA-TA H.sub.2O.sub.2 HRP Ca.sup.2+ HA-TA (U/mL) (mol/L) (mg/mL) (v/v) (mg/mL) (mmol/L) (U/mL) (mol/L) (mg/mL) (mmol/L) (v/v) 0.8 0.2 0   26 4.6 0.4 0.1 13 2.3 0 Plasma 26 4.6 13 2.3 Plasma 60% 30% 0 PRP 26 4.6 13 2.3 PRP 60 30% 60% 14 Blood 26 4.6 20 2.3 Blood 30% 15% indicates data missing or illegible when filed

Example 11

[0169] Preparation of Hydrogels with Blood Content (5% v/v)

[0170] Derivative HA-TA (Mw=5.4×10.sup.4 g.Math.mol.sup.−1, DS 6.2%) was used for preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.8 ml) of the means for hydrogel preparation having composition: [0171] CaCl.sub.2.2H.sub.2O . . . 0.223 mol/L [0172] HA-TA . . . 55.556 mg/mL,

[0173] by addition of 0.2 ml of blood. Further, 2 ml of solution B of the means were used for hydrogel preparation. Table 8 shows composition of precursor solution pA and solution B.

TABLE-US-00008 TABLE 8 The composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood 5% (v/v). Solution pA Solution B HRP 0.9 U/mL H.sub.2O.sub.2 9.2 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  50 mg/mL HA-TA  50 mg/mL Blood 10% (v/v)

[0174] Hydrogel with the content 5% (v/v) of blood was prepared by mixing the solution pA and the solution B in volume ratio 1:1.

[0175] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinking hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0176] HRP . . . 0.45 U/mL [0177] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0178] crossHA-TA . . . 50 mg/mL [0179] Blood . . . 5% (v/v)

Example 12

[0180] Preparation of Hydrogels with Blood Content (5% v/v)

[0181] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.8 mL) of the means for hydrogel preparation of composition: [0182] HRP . . . 0.889 U/ml [0183] CaCl.sub.2.2H.sub.2O . . . 0.223 mol/L [0184] HA-TA . . . 15.556 mg/mL,
by addition of 0.2 ml of blood. Further, 2 ml of solution B of the means were used for hydrogel preparation. Table 9 shows composition of precursor solution pA and solution B.

TABLE-US-00009 TABLE 9 The composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood 5% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL HA-TA  14 mg/mL Blood 10% (v/v)

[0185] Hydrogel with content 5% (v/v) of blood was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0186] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0187] HRP . . . 0.4 U/mL [0188] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0189] crossHA-TA . . . 20 mg/mL [0190] Blood . . . 5% (v/v)

Example 13

[0191] Preparation of Hydrogels with Blood Content (5%)

[0192] Derivative HA-TA (Mw=1.45×10.sup.6 g.Math.mol.sup.−1, DS 1.1%) was used for preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.8 ml) of the means for hydrogel preparation of composition: [0193] HRP . . . 0.556 U/ml [0194] CaCl.sub.2.2H.sub.2O . . . 0.223 mol/L [0195] HA-TA . . . 11.112 mg/mL,
by addition of 0.2 ml of blood. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 10 shows composition of the precursor solution pA and the solution B.

TABLE-US-00010 TABLE 10 The composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood 5% (v/v). Solution pA Solution B HRP 0.5 U/mL H.sub.2O.sub.2 2.3 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  10 mg/mL HA-TA  10 mg/mL Blood 10% (v/v)

[0196] The hydrogel with the content 5% (v/v) of blood was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0197] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0198] HRP . . . 0.25 U/mL [0199] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0200] crossHA-TA . . . 10 mg/mL [0201] Blood . . . 5% (v/v)

Example 14

[0202] Preparation of the Hydrogel with the Blood Content (15% v/v)

[0203] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (1.4 ml) of the means for hydrogel preparation of composition: [0204] HRP . . . 1.143 U/ml [0205] CaCl.sub.2.2H.sub.2O . . . 0.286 mol/L [0206] HA-TA . . . 20 mg/mL,
by addition of 0.6 ml of blood. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 11 shows composition of the precursor solution pA and the solution B.

TABLE-US-00011 TABLE 11 Composition of the precursor solution pA and the solution B for the preparation of hydrogel with the content of blood 15% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL HA-TA  14 mg/mL Blood 30% (v/v)

[0207] The hydrogel with the content 15% (v/v) of blood was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0208] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0209] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0210] crossHA-TA . . . 20 mg/mL [0211] Blood . . . 15% (v/v)

Example 15

[0212] Preparation of Hydrogel with Blood Content (30% v/v)

[0213] Derivative HA-TA (Mw=5.4×10.sup.4 g.Math.mol.sup.−1, DS 6.2%) was used for the preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (0.8 ml) of the means for hydrogel preparation of composition: [0214] HRP . . . 25 U/ml [0215] CaCl.sub.2.2H.sub.2O . . . 0.5 mol/L [0216] HA-TA . . . 0.125 mg/mL,
by addition of 1.2 ml of blood. Further, 2 ml of solution B of the means were used for the hydrogel preparation. Table 12 shows composition of the precursor solution pA and the solution B.

TABLE-US-00012 TABLE 12 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood 15% (v/v). Solution pA Solution B HRP 0.9 U/mL H.sub.2O.sub.2 9.3 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  50 mg/mL HA-TA  50 mg/mL Blood 60% (v/v)

[0217] Hydrogel with the content 30% (v/v) of blood was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0218] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0219] HRP . . . 0.45 U/mL [0220] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0221] crossHA-TA . . . 50 mg/mL [0222] Blood . . . 30% (v/v)

Example 16

[0223] Preparation of the Hydrogel with the Blood Content (30% v/v)

[0224] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (0.8 ml) of the means for hydrogel preparation of composition: [0225] HRP . . . 2 U/ml [0226] CaCl.sub.2.2H.sub.2O . . . 0.5 mol/L [0227] HA-TA . . . 0.35 mg/mL
by addition of 1.2 ml of blood prepared the precursor hydrogel solution pA. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 13 shows the composition of the precursor solution pA and the solution B.

TABLE-US-00013 TABLE 13 Composition of the precursor solution pA and the solution B for the preparation of hydrogel with the content of blood 30% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL HA-TA  14 mg/mL Blood 60% (v/v)

[0228] Hydrogel with the content 30% (v/v) of blood was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0229] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0230] HRP . . . 0.4 U/mL [0231] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0232] crossHA-TA . . . 20 mg/mL [0233] Blood . . . 30% (v/v)

Example 17

[0234] Preparation of the Hydrogel with the Blood Plasma Content (5% v/v)

[0235] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.8 ml) of the means for hydrogel preparation of composition: [0236] HRP . . . 0.889 U/ml [0237] Ca(HCO.sub.3).sub.2 0.223 mol/L
was by the addition of 0.2 ml of the blood. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 14 shows composition of the precursor solution pA and the solution B.

TABLE-US-00014 TABLE 14 Composition of the precursor solution pA and the solution B for the preparation of hydrogel with the content of blood 5% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L Ca(HCO.sub.3).sub.2 0.2 mol/L HA-TA  26 mg/mL Blood plasma 10% (v/v)

[0238] Hydrogel with the content 5% (v/v) of blood plasma was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0239] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations: [0240] HRP . . . 0.4 U/mL [0241] Ca(HCO.sub.3).sub.2 . . . 0.1 mol/L [0242] crossHA-TA . . . 13 mg/mL [0243] Blood plasma . . . 5% (v/v)

Example 18

[0244] Preparation of Hydrogels with the Blood Plasma Content (15% v/v)

[0245] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.4 ml) of the means for hydrogel preparation of composition: [0246] HRP . . . 1.143 U/ml [0247] CaCl.sub.2.2H.sub.2O . . . 0.286 mol/L
by addition of 0.6 ml of blood. As the precursor solution pB were used 2 ml of the solution B of the means for hydrogel preparation. Table 15 shows composition of the precursor solution pA and solution B.

TABLE-US-00015 TABLE 15 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood plasma 15% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL Blood plasma 30% (v/v)

[0248] Hydrogel with the content 15% (v/v) of blood plasma was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0249] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations: [0250] HRP . . . 0.4 U/mL [0251] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0252] crossHA-TA . . . 13 mg/mL [0253] Blood plasma . . . 15% (v/v)

Example 19

[0254] Preparation of the Hydrogel with the Blood Plasma Content (30% v/v)

[0255] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (0.8 ml) of the means for hydrogel preparation of composition: [0256] HRP . . . 2 U/ml [0257] CaCl.sub.2.2H.sub.2O . . . 0.5 mol/L
by addition of 1.2 ml of blood. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 16 shows composition of the precursor solution pA and the solution B.

TABLE-US-00016 TABLE 16 Composition of the precursor solution pA and the solution B for preparation of hydrogel with content of blood 30% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL Blood plasma 60% (v/v)

[0258] Hydrogel with the content 30% (v/v) of blood was prepared by mixing the solution pA and the solution B in volume ratio 1:1.

[0259] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations: [0260] HRP . . . 0.4 U/mL [0261] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0262] crossHA-TA . . . 13 mg/mL [0263] Blood plasma . . . 30% (v/v)

Example 20

[0264] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 5% v/v)

[0265] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (0.8 ml) of the means for hydrogel preparation of composition: [0266] HRP . . . 0.889 U/ml [0267] Calcium acetate . . . 0.223 mol/L
by addition of 0.2 ml PRP. 2 ml of the solution B of the means were used for the hydrogel preparation. Table 17 shows composition of the precursor solution pA and the solution B.

TABLE-US-00017 TABLE 17 Composition of the precursor solution pA and thesolution B for the preparation of hydrogel with the content of PRP 5% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L Calcium acetate 0.2 mol/L HA-TA  26 mg/mL PRP 10% (v/v)

[0268] Hydrogel with the content 5% (v/v) of PRP was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0269] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0270] HRP . . . 0.4 U/mL [0271] Calcium acetate . . . 0.1 mol/L [0272] crossHA-TA . . . 0.13 mg/mL [0273] PRP . . . 5% (v/v)

Example 21

[0274] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 5% v/v)

[0275] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (1.8 ml) of the means for the hydrogel preparation of composition: [0276] HRP . . . 0.889 U/ml [0277] Calcium lactate . . . 0.023 mol/L
by addition of 0.2 ml of PRP. 2 ml of the solution B of the means were used for the hydrogel preparation. Table 18 shows composition of the precursor solution pA and the solution B.

TABLE-US-00018 TABLE 18 Composition of the precursor solution pA and the solution B for the preparation of hydrogel with the content of PRP 5% (v/v). Solution pA Solution B HRP  0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L Calcium lactate 0.02 mol/L HA-TA  26 mg/mL PRP 10% (v/v)

[0278] The hydrogel with the content 5% (v/v) of PRP was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0279] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0280] HRP . . . 0.4 U/mL [0281] Calcium lactate . . . 0.1 mol/L [0282] crossHA-TA . . . 13 mg/mL [0283] PRP . . . 5% (v/v)

Example 22

[0284] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 15% v/v)

[0285] Derivative HA-TA (Mw=1.45×10.sup.6 g.Math.mol.sup.−1, DS 1.1%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from solution A (1.4 ml) of the means for the hydrogel preparation of composition: [0286] HRP . . . 1.143 U/mL [0287] CaCl.sub.2.2H.sub.2O . . . 0.286 mol/L
by addition of 0.6 ml of PRP. 2 ml of the solution B of the means were used for the hydrogel preparation. Table 19 shows composition of the precursor solution pA and the solution B.

TABLE-US-00019 TABLE 19 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of PRP 15% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL PRP 30% (v/v)

[0288] The hydrogel with the content 15% (v/v) of PRP was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0289] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0290] HRP . . . 0.4 U/mL [0291] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0292] crossHA-TA . . . 13 mg/mL [0293] PRP . . . 15% (v/v)

Example 23

[0294] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30%)

[0295] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (0.8 ml) of the means for the hydrogel preparation of composition: [0296] HRP . . . 2 U/mL [0297] CaCl.sub.2.2H.sub.2O . . . 0.5 mol/L
by addition of 1.2 ml of PRP. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 20 shows composition of the precursor solution pA and the solution B.

TABLE-US-00020 TABLE 20 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of PRP 30% (v/v). Solution pA Solution B HRP 0.8 U/mL H2O2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  26 mg/mL PRP 60% (v/v)

[0298] The hydrogel with the content 30% (v/v) of PRP was prepared by mixing the solution pA and the solution B in volume ratio 1:1.

[0299] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0300] HRP . . . 0.4 U/mL [0301] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0302] crossHA-TA . . . 13 mg/mL [0303] PRP . . . 30% (v/v)

Example 24

[0304] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30)

[0305] Derivative HA-TA (Mw=1.45×10.sup.6 g.Math.mol.sup.−1, DS 1.1%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (0.8 ml) of the means for the hydrogel preparation of composition: [0306] HRP . . . 2 U/mL [0307] Ca(H.sub.2PO.sub.4).sub.2 . . . 0.05 mol/L
by addition of 1.2 ml of PRP prepared the precursor hydrogel solution pA. Further, 2 ml of the solution B of the means were used for the hydrogel preparation. Table 21 shows composition of the precursor solution pA and the solution B.

TABLE-US-00021 TABLE 21 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of PRP 30% (v/v). Solution pA Solution B HRP  0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L Ca(H.sub.2PO.sub.4).sub.2 0.02 mol/L HA-TA  26 mg/mL PRP 60% (v/v)

[0308] The hydrogel with the content 30% (v/v) of PRP was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0309] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0310] HRP . . . 0.0.4 U/mL [0311] CaCl.sub.2.2H.sub.2O . . . 0.01 mol/L [0312] crossHA-TA . . . 13 mg/mL [0313] PRP . . . 0.30% (v/v)

Example 25

[0314] Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30% v/v)

[0315] Derivative HA-TA (Mw=9.5×10.sup.4 g.Math.mol.sup.−1, DS 8.6%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (0.8 ml) of the means for the hydrogel preparation of composition: [0316] HRP . . . 2 U/ml [0317] Ca(H.sub.2PO.sub.4).sub.2 . . . 0.05 mol/L [0318] HA-TA . . . 100 mg/mL
by addition of 1.2 ml of PRP. 2 ml of the solution B of the means were used for the hydrogel preparation. Table 22 shows composition of the precursor solution pA and the solution B.

TABLE-US-00022 TABLE 22 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of PRP 30% (v/v). Solution pA Solution B HRP  0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L Ca(H.sub.2PO.sub.4).sub.2 0.02 mol/L HA-TA   40 mg/mL Blood plasma 60% (v/v)

[0319] The hydrogel with the content 30% (v/v) of PRP was prepared by mixing the solution pA and the solution B in the volume ratio 1:1.

[0320] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations: [0321] HRP . . . 0.4 U/mL [0322] Ca(H.sub.2PO.sub.4).sub.2 . . . 0.01 mol/L [0323] crossHA-TA . . . 20 mg/mL [0324] Blood plasma . . . 30% (v/v)

Example 26

[0325] Preparation of Hydrogels with the Blood Content (5% v/v)

[0326] Derivative HA-TA (Mw=2.81×10.sup.5 g.Math.mol.sup.−1, DS 2.2%) was used for the preparation of solutions of the means for the hydrogel preparation. The precursor hydrogel solution pA was prepared from the solution A (1.8 ml) of the means for the hydrogel preparation of composition: [0327] HRP . . . 0.889 U/mL [0328] CaCl.sub.2.2H.sub.2O . . . 0.223 mol/L [0329] HA-TA . . . 15.556 mg/mL,
by addition of 0.2 mL of blood. 2 mL of solution B of the means were used for the hydrogel preparation. Table 23 shows composition of the precursor solution pA and the solution B.

TABLE-US-00023 TABLE 23 Composition of the precursor solution pA and the solution B for the preparation of the hydrogel with the content of blood 5% (v/v). Solution pA Solution B HRP 0.8 U/mL H.sub.2O.sub.2 4.6 mmol/L CaCl.sub.2•2H.sub.2O 0.2 mol/L HA-TA  14 mg/mL Blood 10% (v/v)

[0330] The hydrogel with the content 5% (v/v) of PRP was prepared by mixing the solution pA and the solution B in volume ratio 1:1.

[0331] Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations: [0332] HRP . . . 0.4 U/mL [0333] CaCl.sub.2.2H.sub.2O . . . 0.1 mol/L [0334] crossHA-TA . . . 7 mg/mL [0335] blood . . . 5% (v/v)