Patent Application
20220378976
The present invention relates to a means for use in a preparation of a hydrogel based on a hydroxyphenyl derivative of hyaluronan (HATA) with content of blood or transfusion preparations with content of fibrinogen, that may serve as biomaterials usable in the field of tissue engineering, regenerative medicine and further medicinal fields. The present invention further relates to the hydrogel based on the hydroxyphenyl derivative of hyaluronan and the method of preparation and the use thereof.
Polymer hydrogels are an important group of materials that are used in the development of medicinal means in the field of tissue engineering and regenerative medicine. Polymer hydrogels are in these areas used for example as matrix for controlled release of biologically active agents and cell cultures, may be used for soft tissues augmentation, as temporal substitution of intercellular matrix, etc.
From a physicochemical point of view, polymer hydrogel is a disperse system composed of disperse medium—water and disperse portion that is formed of a three-dimensional macromolecular network. This network forms a continuous structure that permeates through the whole disperse environment. Continuous is thus not only the disperse environment but also the disperse portion. Hydrogels are materials with elastic properties that result from the presence of this three-dimensional macromolecular network.
According to the nature of interactions participating in the forming of polymer network, the gels can be divided into physically crosslinked and chemically crosslinked (covalent gels). The polymer network of covalent hydrogels may be formed by e.g. polymerization of monomers present in a solution or by crosslinking of originally linear chains of precursor polymers. This may be accomplished e.g. by mutual reaction of suitable functional groups of polymer chains or by using crosslinking agents containing two or more functional groups able to react with an initial polymer. During the crosslinking, the polymer network dimension gradually grows until the whole volume of the disperse system is permeated with the polymer network. The forming of the infinite network is called a gelation point. Growing of the number of knot points and number of involved polymer chains need not, in fact, stop at this point. The mass of the gel portion may continue growing at the expense of disperse environment mass. Further progress of crosslinking reaction then leads to an increase in network density of the hydrogel that may influence e.g. stability, mechanical properties and diffusion parameters of the hydrogel environment.
It is preferred, in a number of medicinal applications that the polymer network formation and hydrogel forming occurs directly in the spot determined for the material application, or in situ. Hydrogel formation may be induced by specific conditions (temperature, pH) or by acting of external stimuli (physical—UV irradiation, chemical—reaction initiator, addition of crosslinking agent, etc.).
Suitable method usable for gel preparation in situ must meet a number of parameters:
Hyaluronan is a polysaccharide from the group of glycosaminoglycans that consists of units consisting of D-glucuronic acid and N-acetylglucosamine. It is the polysaccharide that is well soluble in aqueous environment, where it forms, depending on molecular mass and concentration, viscous solutions to viscoelastic hydrogels. Hyaluronan is a natural component of intercellular matrix of tissues. By binding to specific topical cellular receptor, the molecule of hyaluronan is able to interact with cells in its environment and regulate their metabolic processes. Hydrogels based on hyaluronan undergo in the organism natural degradation by acting of specific enzymes (hyaluronases), or by acting of reactive oxygen species, resulting in their gradual absorption following their implantation into the organism. For achieving mechanically more resistant materials and due to slowing of their biodegradation a number of hydrogel kinds was developed based on covalently crosslinked hyaluronan. Such hydrogels are often used as materials for viscosupplementation of synovial fluid, soft tissue augmentation, scaffold structures for cultivation and implantation of cells, etc.
In the past, various kinds of hyaluronan derivatives were developed that are able to undergo sol-gel transition under physiological conditions in situ. For example, phenolic derivatives of hyaluronan may be used for this purpose. Calabro et al. describe in the documents EP1587945B1 and EP1773943B1 the process of preparation of phenolic hyaluronan derivatives with the reaction of carboxylates present in the structure of D-glucuronic acid of hyaluronan, with aminoalkyl derivatives of phenol e.g. tyramine. Products of this reaction are hyaluronan amides.
Crosslinking of phenolic hyaluronan derivatives may be initiated by an addition of peroxidase (e.g. horseradish peroxidase) and a diluted solution of hydrogen peroxide. Horseradish peroxidase (Horseradish peroxidase, HRP, E.C. 1.11.1.7) is currently widely used as catalyst of organic and biotransformation reactions. Hydrogels based on hydroxyphenyl derivatives of hyaluronan may be used as injectable matrix for controlled release of agents or as materials suitable for cultivation and implantation of cells. Wolf et al. describe, in the document CZ303879, a hyaluronan conjugate with tyramine containing aliphatic linker inserted between the chains of polymer and tyramine. The presence of aliphatic linker enables improved efficiency of the crosslinking reaction and gives the network higher elasticity.
Kurisawa et al. describe, in the document U.S. Pat. No. 8,853,162, a method of a preparation of a hydrogel containing interpenetrating networks based on a hydroxyphenyl derivative of hyaluronan and fibrin. The method uses a reaction catalyzed with horseradish peroxidase for the crosslinking of hydroxyphenyl derivative of hyaluronan. Fibrin network forms from fibrinogen by acting of thrombin. This approach provides hydrogels that are more resistant to biodegradation processes in comparison with hydrogel based on fibrin alone. Hydrogels formed by combination of fibrin network and covalently crosslinked hyaluronan derivatives are used as matrices for cell culture and in future may be used as a part of healing preparations for tissue engineering. A disadvantage of this solution is the use of blood derivatives—thrombin and fibrinogen—obtained by homolog blood processing. This process is costly and at the same time does not eliminate the danger of infectious diseases transmission.
Hydrogel crosslinking is mediated by reaction catalyzed with horseradish peroxidase that results in oxidation of hydroxyphenyl cores of tyramine resulting in dimers, or oligomers thereof. Hydrogen peroxide is a source of hydroxyl radicals for reactions catalyzed by horseradish peroxidase and serves thus as crosslinking reaction initiator. In the environment of blood plasma or blood, the hydrogen peroxide undergoes degradation through interactions with other enzymes (e.g. catalase, myeloperoxidase), or with hematin present in hemoglobin. These are reactions that compete horseradish peroxidase mediated crosslinking reaction, which leads to a decrease in efficiency of hydrogel crosslinking. This phenomenon manifests itself in the case of blood plasma use as longer time of gelation and lower elastic module of formed hydrogel. In the case of hydrogel preparation with the use of full blood may this phenomenon result in the failure of the process of hydrogel formation.
Blood and transfusion preparations made of blood are in the field of regenerative medicine used as source of a number of biologically active agents that participate in tissue defects healing, regulation of inflammation reaction, differentiation of progenitor cells etc.
The possibility of using the combination of in situ crosslinking hydrogel and blood as material for support of tissue defects healing is from clinical point of view advantageous because it does not demand either preparation of transfusion preparation or blood derivative production. The possibility of using autologous blood in the same time eliminates the risk of infectious diseases transmission. Combination of blood and in situ gelling hydrogel for support of tissue defects healing is described in e.g. EP1294414 that uses a mixture of blood and chitosan-glycerol phosphate for fillings of tissue defects. The solutions of chitosan-glycerol phosphate are known for their ability to undergo the transition of sol-gel by increasing the solution temperature to 37° C. The use of this system leads to an accelerated production of blood clot and to acceleration of defect closure. Although this process is faster than the natural forming of blood clot, in clinical practice it demands temporal interruption of the intervention, that lasts typically about 15 minutes, and that is necessary for the gel to reach suitable mechanical properties.
Lee at al. describe the use of a hydroxyphenyl derivative of gelatin for a preparation of hydrogels with content of blood plasma fraction reaches in blood platelets that serves as a scaffold for culture of rabbit chondrocytes. Implantation of this scaffold into the model osteochondral defects lead to acceleration of healing of the damaged cartilage. The described system, however, was not used for preparation of hydrogels with blood content.
The goal of the present invention is to provide a means (a set or a kit, e.g. a kit-of-parts) that enables the effective preparation of the hydrogel.
In particular, means for use in a preparation of a hydrogel is provided. The means, e.g. a kit-of-parts, comprises two separate solutions: a solution A and a solution B. The solution A comprises enzyme horseradish peroxidase, and the solution B comprises hydrogen peroxide. At least one of the solutions A and B comprises calcium ions in the form of a pharmaceutically acceptable salt. At least one of the solutions A and B comprises a hydroxyphenyl derivative of hyaluronan having general formula I:
wherein n is in the range of from 2 to 7500, and wherein R is OH or a substituent NHR2CONHR1ArOH having a general formula II,
wherein Ar is a phenylene group and R1 is an ethylene group, or Ar is an indolylene group and R1 is an ethylene group, or Ar is a hydroxyphenylene group and R1 is a carboxyethylene, and wherein R2 is an alkylene group having from 3 to 7 carbon atoms.
A method of preparing a hydrogel using the kit-of-parts, i.e., the solutions A and B, is also provided, along with a preparation (e.g. a cosmetic, medicine, or regenerative medicine preparation) comprising or prepared from the same.
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.
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
where n is in the range 2 to 7500 and R is OH or substituent NHR2CONHR1ArOH of general formula II,
where Ar is phenylene and R1 ethylene or Ar is indolylene and R1 is ethylene or Ar is hydroxyphenyl and R1 is carboxyethylene and R2 is alkylene of 3 to 7 carbon atoms.
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.
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.
Hydroxyphenyl derivative of hyaluronan of general formula I may be prepared following the protocols in CZ303879 that is included by this reference.
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 4×104 to 3×106 g/mol, preferably 1×105 to 1×106 g/mol, more preferably 2×104 to 4×105 g/mol. PI is in the range 1 to 3.
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%.
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.
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.
According to another embodiment of the present invention the means comprises solutions A and B, where the solution A comprises:
According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises:
And the solution B comprises:
According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises:
and the solution B comprises:
According to another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises:
and the solution B comprises:
According to yet another preferred embodiment of the present invention the means comprises solutions A and B, where the solution A comprises:
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.
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.
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:
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:
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:
that is further mixed in the volume ratio 1:1 with the solution B of the above described means, that comprises
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:
That is further mixed in the volume ratio 1:1 with the solution B of the above described means, that comprises:
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 1H NMR spectroscopy.
Mass average molar mass (Mw) and polydispersity index (PI) were determined using SEC-MALLS.
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.
m=0.75 g (70% theory)
1H NMR (D2O, ppm) δ: 1.17 (m, 2H, γ-CH2-hexane acid); 1.48 (m, 2H, (3-CH2-hexane acid); 1.58 (m, 2H, 6-CH2-hexane acid); 2.17 (t, 2H, —CH2—CO—); 2.73 (m, 2H, —CH2-Ph); 2.91 (m, 2H, —CH2—NH2); 3.42 (m, 2H, —CH2—NH—CO—); 6.83 (d, 2H, arom); 7.13 (d, 2H, arom).
13C NMR (D2O, ppm) δ: 24 (γ-C-hexane acid); 26 (δ-C-hexane acid); 33 (β-C-hexane acid); 35 (—C—CO—); 39 (—C—NH2); 40 (C-Ph); 63 (—C—NH—CO—); 115 (C3 arom); 126 (C1 arom); 130 (C2 arom.); 153 (C4 arom); 176 (—CO—).
Hyaluronan (10.00 g, Mw=2×106 g·mol−1) was dissolved in 750 mL 2.5% (w/w) solution Na2HPO4.12H2O. 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.
IR (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 2.01 (s, 3H, CH3—), 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)2).
a) Preparation of Tyramine Derivative of HA with C6 spacer (Mw≈5×104 g·mol−1, DS≈6%)
Aldehyde derivative of HA (≈6×104 g·mol−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).
IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2-aminohexane acid), 1.48 (m, 2H, δ-CH2-aminohexane acid) 1.51 (m, 2H, β-CH2-aminohexane acid), 2.01 (s, 3H, CH3—), 2.65 (m, 2H, Ph-CH2—), 2.73 (m, 2H, ε-CH2-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).
SEC MALLS: Mw=5.4×104 g·mol−1; PI=1.61
DS (1H NMR): 6.2%
b) Preparation of Tyramine Derivative of HA with C6 spacer (Mw≈3×105 g·mol−1, DS≈2%)
Aldehyde derivative of HA (Mw 3×105 g·mol−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).
IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2-aminohexane acid), 1.48 (m, 2H, δ-CH2-aminohexane acid) 1.51 (m, 2H, β-CH2-aminohexane acid), 2.01 (s, 3H, CH3—), 2.65 (m, 2H, Ph-CH2—), 2.73 (m, 2H, ε-CH2-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).
SEC MALLS: Mw=2.81×105 g·mol−1; PI=1.49
DS (1H NMR): 2.2%
c) Preparation of Tyramine Derivative of HA with C6 spacerem (Mw≈1.5×106 g·mol−1, DS=1%)
Aldehyde derivative of HA (≈1.5×106 g·mol−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).
IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2-aminohexane acid), 1.48 (m, 2H, 6-CH2-aminohexane acid) 1.51 (m, 2H, β-CH2-aminohexane acid), 2.01 (s, 3H, CH3—), 2.65 (m, 2H, Ph-CH2—), 2.73 (m, 2H, ε-CH2-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).
SEC MALLS: Mw=1.45×106 g·mol−1; PI=1.65
DS (1H NMR): 1.1%
d) Preparation of Tyramine Derivative of HA with C6 spacerem (Mw≈1.0×105 g·mol−1, DS=8.5%)
Aldehyde derivative of HA (≈1.0×105 g·mol−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).
IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2-aminohexane acid), 1.48 (m, 2H, δ-CH2-aminohexane acid) 1.51 (m, 2H, β-CH2-aminohexane acid), 2.01 (s, 3H, CH3—), 2.65 (m, 2H, Ph-CH2—), 2.73 (m, 2H, ε-CH2-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).
SEC MALLS: Mw=9.5×104 g·mol−1; PI=1.55
DS (1H NMR): 8.6%
e) Preparation of Tyramine Derivative of HA with C6 spacerem (Mw≈2.5×106 g·mol−1, DS=1%)
Aldehyde derivative of HA (≈2.5×106 g·mol−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).
IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm−1.
1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2-aminohexane acid), 1.48 (m, 2H, δ-CH2-aminohexane acid) 1.51 (m, 2H, β-CH2-aminohexane acid) 2.01 (s, 3H, CH3—), 2.65 (m, 2H, Ph-CH2—), 2.73 (m, 2H, ε-CH2-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).
SEC MALLS: Mw=2.5×106 g·mol−1; PI=1.65
DS (1H NMR): 1%
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=2.81×105 g·mol−1, DS 2.2%) was used for sample preparation.
Elastic G′ and viscose G″ module of individual samples read at 300 s are presented in Table 1.
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.
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 10−3 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=2.81×105 g·mol−1, DS 2.2%) was used for the solution preparation.
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.
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.
Gelation rate was determined using method described in Example 2.
Influence of calcium ions on the rate of gelation of samples containing the transfusion preparation— 30% (v/v) plasma is shown in
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 Ca2+. The presence of calcium ions in concentration ≤0.01/L does not negatively influence cell viability.
Cytotoxicity of Extracts from Hydrogels Containing Various Calcium Ions Concentration
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=2.8×105 g·mol−1, DS 2.2%) was used for the preparation of solutions.
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.
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=2.81×105 g·mol−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
indicates data missing or illegible when filed
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=2.81×105 g·mol−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
indicates data missing or illegible when filed
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=2.81×105 g·mol−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.
Stem cell adhesion on the surface of the material manifests itself by typical changes in cell morphology where the cells adopt elongated shape.
indicates data missing or illegible when filed
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=2.81×105 g·mol−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% CO2. Cell growth was monitored for 4 days using LIVE/DEAD staining. The results showed in
indicates data missing or illegible when filed
Preparation of Hydrogels with Blood Content (5% v/v)
Derivative HA-TA (Mw=5.4×104 g·mol−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:
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.
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinking hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of Hydrogels with Blood Content (5% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of Hydrogels with Blood Content (5%)
Derivative HA-TA (Mw=1.45×106 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of the Hydrogel with the Blood Content (15% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of Hydrogel with Blood Content (30% v/v)
Derivative HA-TA (Mw=5.4×104 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of the Hydrogel with the Blood Content (30% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
Preparation of the Hydrogel with the Blood Plasma Content (5% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations:
Preparation of Hydrogels with the Blood Plasma Content (15% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations:
Preparation of the Hydrogel with the Blood Plasma Content (30% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood plasma in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 5% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 5% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 15% v/v)
Derivative HA-TA (Mw=1.45×106 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30%)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30)
Derivative HA-TA (Mw=1.45×106 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Content of Blood Plasma Fraction Reaches in Blood Platelets (PRP, 30% v/v)
Derivative HA-TA (Mw=9.5×104 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and PRP in following concentrations:
Preparation of Hydrogels with the Blood Content (5% v/v)
Derivative HA-TA (Mw=2.81×105 g·mol−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:
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.
Thus prepared hydrogel contains enzyme horseradish peroxidase, calcium ions, covalently crosslinked hydroxyphenyl derivative of hyaluronan (crossHA-TA) and blood in following concentrations:
| Number | Date | Country | Kind |
|---|---|---|---|
| PV2019-360 | Jun 2019 | CZ | national |
This application is the National Stage of International Application No. PCT/CZ2020/050043, filed on 9 Jun. 2020, which claims priority to and all advantages of CZ Application No. PV2019-360, filed on 10 Jun. 2019, the contents of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CZ2020/050043 | 6/9/2020 | WO |
