Kit of parts for preparing biocompatible polymer hydrogels

Abstract

The present invention provides a kit-of-parts suitable for preparing a biocompatible polymer hydrogel, the kit-of-parts comprising at least two containers each containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein mixing of these components allows for the formation of a biocompatible polymer hydrogel, wherein the peroxidase and hydrogen peroxide are present in different containers; the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, and more preferably 55:45 to 45:55; the concentration of Dex-TA is in the range of 2 to 7 wt.% relative to the total aqueous composition of the kit-of-parts; and the concentration of HA-TA is in the range of 2 to 7 wt.% relative to the total aqueous composition of the kit-of-parts. A kit-of-parts, wherein A comprises repeating units of unsubstituted and TA-substituted dextran, and the amount of TA-substituted repeating units in DEX-TA (DSDEX) measured by proton NMR is 5 mol% to 20 mol% relative to the total number of unsubstituted and TA-substituted repeating units; HA-TA comprises repeating units of unsubstituted and TA-substituted hyaluronic acid, and the amount of TA-substituted repeating units in HA-TA (DSHA) measured by proton NMR is 5 mol% to 20 mol% relative to the total number of unsubstituted and TA-substituted repeating units; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; peroxidase is present at a concentration of 0.25 to 10 units / ml relative to the total aqueous composition of the kit-of-parts; and hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt%.

Description

[Technical Field]

[0001] The present invention relates to a kit of parts suitable for preparing a biocompatible polymer hydrogel, a method for producing a biocompatible polymer hydrogel, a mixed polymer composition, and a biocompatible polymer hydrogel.

[0002] The present invention further relates to the use of the biocompatible polymer hydrogels of the present invention to repair damaged cartilage in order to prevent or reduce pain in human or animal joints. [Background technology]

[0003] Cartilage is a connective tissue found in many parts of the body of higher animals, including humans, such as the joints between bones and the bronchi. Cartilage is made up of specialized cells called chondrocytes, which produce large amounts of an extracellular matrix composed of collagen fibers, proteoglycans, and elastin fibers. Cartilage is avascular and aneuric, meaning it lacks blood vessels and nerves, resulting in poor self-regeneration. Cartilage can be damaged in various ways, including injury, accidents, and age-related degeneration due to arthritis (particularly osteoarthritis). People with cartilage damage suffer from joint pain, stiffness, and reduced range of motion, which interfere with daily activities. Functional and physical limitations can also lead to anxiety and depression. In these cases, pain management and total or partial joint replacement with artificial joints are the most common treatments. However, pain management does not address the underlying cause and provides only temporary relief. Meanwhile, joint replacement is typically reserved for elderly patients in the late stages of the disease. Because the lifespan of artificial joints is limited, joint replacement surgery is not recommended for young patients. If cartilage damage caused by sports injuries or accidents is not treated early, young patients often develop osteoarthritis. For these reasons, alternative treatments to joint replacement surgery and early repair of cartilage damage are important to prevent the onset of joint diseases such as osteoarthritis. Therefore, preventive treatments for defective joints to avoid the onset of osteoarthritis in humans or animals are being studied.

[0004] Although there is no curative treatment for cartilage defects to date, tissue engineering is a promising method for regenerating degenerated or lost cartilage in humans or animals. Tissue engineering approaches generally involve the use of cells placed within a three-dimensional scaffold, which acts as a temporary artificial extracellular matrix (ECM). Hydrogels are water-insoluble, three-dimensional polymer chain networks capable of retaining large amounts of water. They are a type of biomaterial designed for use in the human body and are therefore used in biomedical applications. Biocompatible polymer hydrogels can serve as temporary scaffolds for inducing chondrocyte differentiation and differentiation of chondrocytes and / or their progenitor cells, resulting in the formation of de novo cartilage tissue. The use of biocompatible polymer hydrogels has several advantages. They can be prepared in situ from mixed polymer compositions applied locally via minimally invasive surgical procedures. Furthermore, they can fill irregularly shaped defects and can easily incorporate cells and bioactive molecules.

[0005] Therefore, biocompatible polymer hydrogels have attracted much attention in cartilage tissue engineering in recent years. Mixed polymer compositions are typically prepared to undergo chemical crosslinking reactions to form biocompatible polymer hydrogels through a gelation process.

[0006] Enzymatic cross-linking of dextran-tyramine conjugates (Dex-TA) in the presence of horseradish peroxidase (HRP) and hydrogen peroxide has been disclosed (Jin R. et al., Biomaterials. 2009(13):2544-51 and Jin R. et al., Biomaterials, 2007(18):2791-800).

[0007] WO 2011 / 059326 discloses a kit of parts suitable for preparing a biocompatible polymer hydrogel comprising a heparin-tyramine (Hep-TA) conjugate, a dextran-tyramine conjugate (Dex-TA), hydrogen peroxide, and peroxidase.

[0008] The gelation process and the formation of suitable polymer hydrogels are complex. During the polymerization (gelation) of a mixed polymer composition, numerous networks are formed. A key issue in this process is bond formation, which leads to cluster formation. As more clusters form, gelation ultimately occurs. Methods known in the art for preparing biocompatible polymer hydrogels have several drawbacks. A fast gelation time for a mixed polymer composition makes handling the mixed polymer composition difficult during application because the biocompatible polymer hydrogel may not form in situ but may form during injection. A very slow gelation process requires a long procedure time, making it not only very difficult to implement but also cost-ineffective. Polymer hydrogels exhibit relatively large volume changes in response to small changes in external stimuli (e.g., temperature) as they form a polymer hydrogel network and expand the polymer. If the polymer hydrogel swells too much, it may be pulled out of the application site by joint movement. Polymer hydrogels may have low crosslink network densities, resulting in a low storage modulus for the polymer hydrogel, which may degrade too quickly to regenerate adequate cartilage, and may be susceptible to mechanical erosion between two diarthrodial joints.

[0009] Conventional kits of parts suitable for preparing biocompatible polymer hydrogels and conventional biocompatible polymer hydrogels are far from optimal.

[0010] The object of the present invention is to overcome at least some of these drawbacks, and more specifically to develop an effective kit of parts suitable for the preparation of biocompatible polymer hydrogels, and effective biocompatible polymer hydrogels that can be advantageously used to repair damaged cartilage to prevent or reduce pain in human or animal joints.

[0011] As a result of extensive research, the present inventors have solved at least part of the above-mentioned problems and developed an effective kit of parts suitable for preparing biocompatible polymer hydrogels, which can be used to repair damaged cartilage. Summary of the Invention [Means for solving the problem]

[0012] The present invention provides a kit of parts suitable for preparing a biocompatible polymer hydrogel, comprising at least two containers each containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein the components are mixed to allow the formation of a biocompatible polymer hydrogel, Peroxidase and hydrogen peroxide are in separate containers. the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; The concentration of HA-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight of the total aqueous composition of the kit of parts; Regarding parts kits.

[0013] The kit of parts suitable for preparing the biocompatible polymer hydrogel according to the present invention can be used to repair damaged cartilage, allowing cartilage regeneration and preventing or reducing pain in human or animal joints. [Brief explanation of the drawings]

[0014] [Figure 1] This figure shows the gelation time (seconds) of biocompatible polymer hydrogels of the present invention prepared using 0.03 wt % (dark black) or 0.05 wt % (light black) hydrogen peroxide. These biocompatible polymer hydrogels were prepared using 3 Units / ml of peroxidase (HRP). [Figure 2] This figure shows the storage modulus (kPa) of biocompatible polymer hydrogels of the present invention prepared using 0.03 wt % (dark black) or 0.05 wt % (light black) hydrogen peroxide. These biocompatible polymer hydrogels were prepared using 3 Units / ml of peroxidase (HRP). [Figure 3] This figure shows the swelling behavior of biocompatible polymer hydrogels of the present invention prepared using 0.03 wt % (dark black) or 0.05 wt % (light black) hydrogen peroxide. These biocompatible polymer hydrogels were prepared using 3 Units / ml of peroxidase (HRP). [Figure 4] This figure shows the degradation over time of biocompatible polymer hydrogels of the present invention prepared using 0.03 wt % (dark black) or 0.05 wt % (light black) hydrogen peroxide. These biocompatible polymer hydrogels were prepared using 3 Units / ml of peroxidase (HRP). [Figure 5] 1 shows a template sheet used to evaluate the storage modulus and swelling behavior of the biocompatible polymer hydrogel of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a kit of parts suitable for preparing a biocompatible polymer hydrogel, comprising at least two containers each containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein the components are mixed to allow the formation of a biocompatible polymer hydrogel, Peroxidase and hydrogen peroxide are in separate containers. the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; The concentration of HA-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight of the total aqueous composition of the kit of parts; Regarding parts kits.

[0016] The above-mentioned tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide are mixed to obtain a mixed polymer composition, within which the gelation process is initiated to obtain a biocompatible polymer hydrogel.

[0017] Detailed description of features The term "polymer hydrogel" as used herein refers to a three-dimensional hydrophilic polymer network. The polymer hydrogel of the present invention has a high water content, providing an environment similar to that of natural cartilage. Furthermore, the polymer hydrogel of the present invention allows sufficient transport of nutrients and waste products essential for cell growth. Preferably, the polymer hydrogel of the present invention is obtained in situ upon injection of the above-described mixed polymer composition.

[0018] The kit of parts of the present invention and the mixed polymer composition of the present invention comprise a polymer or functionalized polymer that can be selected from natural polymers, polymers present in cartilage tissue, modified natural polymers, or synthetic polymers such as polymers based on polyethylene glycol.

[0019] The polymers used in the present invention can be gelatin (amide type), collagen, polysaccharides (such as dextran), and glycosaminoglycans (GAGs) (including hyaluronic acid, chondroitin sulfate, keratin sulfate, heparin sulfate, and chitosan).

[0020] The polymers used in the present invention are preferably dextran and hyaluronic acid based polymers, which are biocompatible and easy to modify, making them ideal template materials for further functionalization.

[0021] As used herein, the term "biocompatible polymer hydrogel" refers to a polymer hydrogel that has the ability to come into contact with biological systems without causing serious adverse effects. Biocompatibility in this context means that the material does not cause a serious pathological reaction in the body to the biological material or that the material is not harmful to the patient. ISO standard tests can be used to assess the biocompatibility of polymer hydrogels, which take into account, among other things, issues of cytotoxicity, irritation, sensitization, acute, subacute, or subchronic systemic toxicity, pyrogenicity, and genotoxicity.

[0022] A kit of parts suitable for preparing the biocompatible polymer hydrogel of the present invention comprises at least two containers.

[0023] The term "container" as used herein refers to an object for holding or transporting an aqueous composition. Any type of container suitable for holding or transporting a substance can be used. Preferably, the container is suitable for holding or transporting a pharmaceutical substance or a substance such as tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide. Preferably, the container used in the kit of parts of the present invention is assembled into an applicator device.

[0024] Each container contains an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, while the peroxidase and hydrogen peroxide are contained in separate containers, thereby avoiding the gelation process and formation of a polymer hydrogel within the applicator device.

[0025] Tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA) are prepared by modifying dextran and hyaluronic acid by coupling reaction with tyramine (TA) according to procedures known in the art (Dex-TA reported by R Jin et al. in Journal of Controlled Release 2008;132:e24-e6, and Dex-TA conjugates and HA-TA conjugates disclosed in WO 2011 / 059326 and WO 2019 / 143247), both of which are incorporated herein by reference.

[0026] Although tyrosine (TA) was selected as the enzymatically crosslinkable reactive side chain, other reactive side groups, including hydroxyphenyl groups such as those found in tyrosine or dihydroxyphenyl groups such as those found in dopamine, can also be used. Tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA) were crosslinked in situ from biocompatible polymer hydrogels by forming tyramine-tyramine bonds in the presence of a catalyst such as horseradish peroxidase (HRP) and an oxidizing agent such as hydrogen peroxide (H2O2). Hyaluronic acid was advantageously selected because it is naturally present in articular tissue and synovial fluid and functions as a lubricant and shock absorber in joints. Furthermore, hyaluronic acid was advantageously selected because it is readily degraded by hyaluronidase, a hydrolytic enzyme present in the human body, and the degradation profile of the hydrogel may affect cell morphogenesis and tissue regeneration after implantation. Thus, the tyramine-functionalized hyaluronic acid (HA-TA) used in the present invention mimics the natural environment of the joint and also provides control over the degradation profile of the hydrogel.

[0027] As used herein, the term "tyramine-functionalized dextran (Dex-TA)" refers to a dextran molecule conjugated to a tyramine molecule linked by a urethane bond or by any bond that allows for conjugation of the dextran molecule with tyramine.

[0028] As used herein, the term "tyramine-functionalized hyaluronic acid (HA-TA)" refers to a dextran molecule conjugated to a tyramine molecule linked by an amide bond or any bond that allows for conjugation of the hyaluronic acid molecule with the tyramine.

[0029] The functional properties of the biocompatible polymer hydrogels of the present invention are influenced by many factors, with important properties being the shear storage modulus, swelling or shrinkage (related to the volume change after the biocompatible polymer hydrogel is formed in situ), glucose permeability, gelation time (the time it takes for the crosslinked polymer to change from a free-flowing solution to a gel or semi-gel state), viscosity, and degradation of the biocompatible polymer hydrogel.

[0030] As used herein, the term "shear storage modulus" refers to a parameter calculated from strain and stress measurements obtained when a biocompatible polymer hydrogel is subjected to an experiment in a shear plate-plate configuration. Storage modulus refers to the ability of a biocompatible polymer hydrogel to elastically store energy (energy stored in the elastic structure of the biocompatible polymer hydrogel). A high storage modulus is preferred so that the biocompatible polymer hydrogel is a rigid gel that can withstand any force that may be encountered in a joint. Preferably, the storage modulus is not too high to allow cell penetration and proliferation within the biocompatible polymer hydrogel.

[0031] As used herein, the term "swelling" refers to the increase in volume of a biocompatible polymer hydrogel after it is formed in situ. Swelling of a biocompatible polymer hydrogel, which is a porous, fluid-saturated material, can be triggered by various stimuli. Swelling induces deformation of the biocompatible polymer hydrogel, which can cause the hydrogel to be pushed out of the injection site (e.g., a joint) during movement and potentially remove the hydrogel from the defect site.

[0032] As used herein, the term "shrinkage" refers to the volume reduction of the mixed polymer composition upon conversion to a biocompatible polymer hydrogel. Preferably, the biocompatible polymer hydrogel shrinks slightly to prevent the biocompatible polymer hydrogel from swelling from the injection site and removing the hydrogel from the defect site during operation.

[0033] As used herein, the term "gel time" refers to the time it takes for a crosslinked polymer to change from a free-flowing solution to a gel or semi-gel state.

[0034] As used herein, the term "degradation" refers to the mass loss of a biocompatible polymer hydrogel sample after exposure to a 5 U / mL hyaluronidase solution at 37°C. Degradation is preferably not too rapid to allow sufficient time for chondrocytes to migrate into the biocompatible polymer hydrogel and generate extracellular matrix. In a preferred embodiment, the degradation rate is less than 50% by weight within the first 30 days after hydrogel formation.

[0035] The inventors sought a hydrogel with a composition that balanced a number of unique properties, including mechanical stability of the hydrogel against intra-articular forces, an optimal degradation time to promote healing of intra-articular defects, and an excellent gelation time that allows for injection without requiring a long wait for the gel to solidify in the joint. The kit of parts described in claim 1 defines multiple essential features that must be possessed together to obtain a biocompatible polymer hydrogel that can be used to treat cartilage defects. Each essential feature will be described in detail below, and a preferred range for each feature will be presented.

[0036] The primary factor affecting the functional properties of the biocompatible polymer hydrogel of the present invention is the weight ratio of Dex-TA to HA-TA.

[0037] The present inventors have found that preparing a biocompatible polymer hydrogel using only HA-TA results in a biocompatible polymer hydrogel with insufficient mechanical properties. When only Dex-TA is used, in-situ degradation does not occur or degrades very slowly. Instead, the present inventors have found that acceptable functional properties of the biocompatible polymer hydrogel of the present invention can be obtained when the total weight ratio of Dex-TA to HA-TA in the kit of parts of the present invention is 75:25 to 25:75, preferably 60:40 to 40:60, and most preferably 55:45 to 45:55.

[0038] The inventors of the present invention have found that the storage modulus and swelling / shrinkage ratio are improved when the weight ratio of Dex-TA / HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, and more preferably 55:45 to 45:55. The resulting biocompatible polymer hydrogel can withstand the force at the injection site (e.g., a joint) and provide an environment suitable for cell migration and proliferation. The inventors have found that if the weight ratio of Dex-TA / HA-TA is outside the above range, the biocompatible polymer hydrogel becomes too soft to withstand the force or too stiff to allow cells to migrate into the hydrogel.

[0039] Regarding the effect of the weight ratio of Dex-TA / HA-TA on the degradation of the biocompatible polymer hydrogel of the present invention, the inventors have found that degradation is slowed when the weight ratio of Dex-TA / HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, and more preferably 55:45 to 45:55. Such slow degradation provides sufficient time for cells from the surrounding tissue to migrate into the hydrogel, proliferate, and generate new extracellular matrix while healing the defect.

[0040] Regarding the effect of the weight ratio of Dex-TA / HA-TA on the gelation of the biocompatible polymer hydrogel of the present invention, the present inventors have found that when the weight ratio of Dex-TA / HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, and more preferably 55:45 to 45:55, the surgeon can have enough time to inject the hydrogel into the defect without having to wait a long time for the gel to solidify.

[0041] The second factor is the concentration of Dex-TA and HA-TA relative to the total aqueous composition of the kit-of-parts. The inventors have found that acceptable functional properties of the biocompatible polymer hydrogels of the present invention can be obtained when the concentration of Dex-TA is in the range of 2-7 wt %, preferably 3-6 wt %, and more preferably 3-5 wt %, relative to the total aqueous composition of the kit-of-parts, and when the concentration of HA-TA is in the range of 2-7 wt %, preferably 3-6 wt %, and more preferably 3-5 wt %, relative to the total aqueous composition of the kit-of-parts. The inventors have investigated the storage modulus and swelling / shrinkage of the biocompatible polymer hydrogels. Furthermore, the degradation of the biocompatible polymer hydrogels over the first 30 days after hydrogel formation, as well as the gelation time and viscosity of the biocompatible polymer hydrogels, were also investigated.

[0042] Table 1 below summarizes the data and the effect on the functional properties of the biocompatible polymer hydrogels observed for both Dex-TA and HA-TA concentrations.

[0043] [Table 1]

[0044] The inventors of the present invention observed that when the concentration of Dex-TA and HA-TA was less than 2 wt%, the storage modulus of the biocompatible polymer hydrogel was immeasurably reduced due to gel collapse, and in fact, the gel was too soft to withstand the forces at the injection site (e.g., joint).

[0045] When the concentrations of both Dex-TA and HA-TA were greater than 2 wt% or less than 7 wt%, the storage modulus was measurable, and the biocompatible polymer hydrogel was able to withstand a certain amount of force at the injection site (e.g., a joint) and provide a suitable environment for cell migration and proliferation.

[0046] If the concentration of Dex-TA and HA-TA exceeds 7 wt%, the hydrogel network will not be properly formed, and the viscosity of the polymer solution will increase, which will inhibit cross-linking of the hydrogel.

[0047] When the concentrations of Dex-TA and HA-TA are 3–5 wt %, the storage modulus is optimal, the biocompatible polymer hydrogel can withstand the forces at the injection site (e.g., joint), and provide an excellent environment for cell migration and proliferation.

[0048] The second important factor is the shrinkage of the hydrogel during use. The inventors of the present invention observed that when the concentration of Dex-TA and HA-TA was less than 2 wt%, the hydrogel contracted so much that the measuring gel disc was damaged by the hydrogel, causing the hydrogel to deform and peel off from the injection site, making it impossible to measure its value.

[0049] When the concentrations of both Dex-TA and HA-TA are above 2 wt % or below 7 wt %, swelling / shrinkage is measurable and the deformation of the biocompatible polymer hydrogel is within an acceptable range.

[0050] If the concentration of Dex-TA and HA-TA exceeds 7% by weight, the swelling of the hydrogel becomes too great, causing the hydrogel to deform during operation and protrude from the injection site.

[0051] When the concentration of Dex-TA and HA-TA is 3 to 5 wt%, the hydrogel does not swell excessively, protrude excessively, or shrink excessively, and the biocompatible polymer hydrogel does not become dislodged or peel off from the injection site.

[0052] Another important factor is the degradation of the hydrogel during use to regenerate healthy cartilage tissue within the joint. The inventors of the present invention predict that if the concentration of Dex-TA and HA-TA is less than 2 wt%, the degradation of the biocompatible polymer hydrogel will be so rapid in the first 30 days after hydrogel formation that the gel may degrade before cartilage is regenerated. This product is considered ineffective for cartilage repair.

[0053] When the concentrations of both Dex-TA and HA-TA are greater than 2% by weight or less than 7% by weight, degradation is within acceptable limits, allowing sufficient time for chondrocytes to migrate into the biocompatible polymer hydrogel and generate extracellular matrix, making the product effective for cartilage repair.

[0054] When the concentration of Dex-TA and HA-TA exceeds 7% by weight, the degradation is very slow, resulting in prolonged healing of the defect due to the slow degradation of the polymer network, and therefore the product is not very effective for cartilage repair.

[0055] At concentrations of 3-5% by weight, Dex-TA and HA-TA degrade slowly, providing sufficient time for cells to migrate from the surrounding tissue into the hydrogel, proliferate, and produce new extracellular matrix while healing the defect. This product is highly effective for cartilage repair.

[0056] Another important factor is the gelation of the liquid composition into a hydrogel after injection into the joint. The inventors of the present invention have found that when the concentration of Dex-TA and HA-TA is less than 2% by weight, the gelation time is 25 seconds, and the transition from liquid to solid occurs very rapidly, which causes problems during application of the hydrogel by the surgeon.

[0057] Ideally, the hydrogel should gel not too quickly to allow application by the surgeon, but at the same time not too slowly to allow treatment to occur within a reasonable time frame. This is observed when the concentrations of both Dex-TA and HA-TA are greater than 2 wt% or less than 7 wt%, especially when the Dex-TA and HA-TA concentrations are between 3 and 5 wt%, allowing the surgeon sufficient time to inject the gel into the defect without having to wait long for the gel to solidify.

[0058] The inventors of the present invention observed that when the concentration of Dex-TA and HA-TA was less than 2% by weight, the viscosity of the hydrogel was so low that the hydrogel flowed out from the injection site.

[0059] When the concentrations of both Dex-TA and HA-TA are greater than 2 wt % or less than 7 wt %, the viscosity of the hydrogel is within an acceptable range and the hydrogel does not flow out of the injection site.

[0060] If the concentration of Dex-TA and HA-TA exceeds 7% by weight, the viscosity becomes too high, inhibiting gel formation and causing mixing problems.

[0061] When the concentrations of Dex-TA and HA-TA were 3-5 wt%, no mixing problems were observed, and the viscosity was such that the gel could be easily extruded from the syringe as a liquid without excessive force. This facilitated the surgeon's application of the hydrogel by injection. Furthermore, the gel was shown to be fixed in the defect without the risk of flowing out of the injection site.

[0062] When DEX-TA and / or HA-TA are present in the container, the concentration of each of DEX-TA and HA-TA in the container is preferably in the range of 2 to 10% by weight.

[0063] The third factor is the degree of substitution (DS) of tyramine in dextran and hyaluronic acid. The term "degree of substitution (DS)" in relation to dextran and hyaluronic acid refers to the (average) number of substituents (TA) attached per repeat unit, taking into account that the repeat unit of dextran is a monosaccharide, while the repeat unit of hyaluronic acid is a disaccharide.

[0064] The present inventors have found that DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and that DEX-TA (DS DEX It has been found that acceptable functional properties of the biocompatible polymer hydrogel of the present invention can be obtained when the amount of TA-substituted repeating units of (a) is 5 mol % to 20 mol %, preferably 6% to 17%, more preferably 7% to 15%, even more preferably 7.5% to 12%, and even more preferably 7.5% to 10.5%, based on the total number of unsubstituted and TA-substituted repeating units.

[0065] DEX-TA(DS DEX When the hydroxyl group is between 5 mol% and 20 mol%, the storage modulus is such that the hydrogel can withstand the forces at the injection site (e.g., a joint) and provide a suitable environment for cells to migrate and proliferate within.

[0066] The present inventors have investigated the effects of DEX-TA (DS DEX If the DS is outside the above range, the biocompatible polymer hydrogel is too soft to withstand the force and is extruded from the injection site, or is too hard for cells to migrate into the hydrogel. Dex It is predicted that water-insoluble products may be formed when the temperature is high.

[0067] DS DEX It has been observed that the degradation of biocompatible polymer hydrogels is slightly slower when DS is less than 5 mol%, while DS > 12 mol% DEXThe formation of biocompatible polymer hydrogels is very slow. Ideally, degradation should not be too rapid, allowing sufficient time for cells from the surrounding tissue to migrate into the hydrogel, proliferate, and produce new extracellular matrix while healing the defect.

[0068] The present inventors have found that HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and that the HA-TA (DS HA It has been found that acceptable functional properties can be obtained when the amount of TA-substituted repeat units of (a) is 5 mol % to 20 mol %, preferably 6% to 17%, more preferably 7% to 15%, and even more preferably 10% to 14.5%, based on the total number of unsubstituted and TA-substituted repeat units.

[0069] The fourth factor is the weight-average molecular weight (Mw) of DEX-TA and HA-TA. The present inventors have found that acceptable functional properties of the ecological synthetic polymer hydrogel of the present invention can be obtained when the weight-average molecular weight (Mw) of DEX-TA is 15 to 650 kDa, preferably 20 to 125, and more preferably 30 to 60, and when the weight-average molecular weight (Mw) of HA-TA is 15 to 550 kDa, preferably 15 to 300, and more preferably 15 to 60.

[0070] The preparation process of biocompatible polymer hydrogels is also influenced by many factors, especially the viscosity, injectability, and gelation time of the mixed polymer composition that must be considered to obtain an effective process.

[0071] As used herein, the term "injectable" means that the mixed polymer composition can be introduced into the body using an injection applicator device, which may have a needle.

[0072] The first factor to consider in preparing the biocompatible polymer hydrogel of the present invention is the amount of peroxidase. The present inventors have found that an effective method for obtaining the biocompatible polymer hydrogel of the present invention is achieved when peroxidase is used at a concentration of 0.25 to 10 units / ml relative to the total aqueous composition of the kit-of-parts. It is preferred that peroxidase be used at a concentration of 1 to 5 units / ml, more preferably 2 to 4 units / ml, and even more preferably 2 to 3.5 units / ml relative to the total aqueous composition of the kit-of-parts.

[0073] The term "Units / ml" (also referred to as "pyrogallol units / ml") with respect to peroxidase refers to the concentration of peroxidase expressed in "activity units" related to the enzymatic activity controlling the formation of a biocompatible polymer hydrogel. Specifically, it refers to the amount of enzyme that forms 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 and 20°C, as determined using spectrophotometric analysis.

[0074] Regarding the effect of the amount of peroxidase on the gelation time of the mixed polymer composition of the present invention, the present inventors monitored the gelation of the mixed polymer composition and observed that decreasing the amount of peroxidase increased the gelation time, improving the handling and usability of the kit-of-parts of the present invention. As shown in Figure 1, an optimal gelation time ranging from 35 to 60 seconds was obtained when a peroxidase concentration of 3 Units / ml was used relative to the total aqueous composition of the kit-of-parts. This gelation time improves the handling and usability of the kit-of-parts of the present invention.

[0075] The amount of peroxidase also affects the storage modulus of the biocompatible polymer hydrogel of the present invention, as shown in Figure 2, and the swelling / shrinkage of the biocompatible polymer hydrogel of the present invention, as shown in Figure 3. The optimal storage modulus is in the range of 30 to 60 kPa, which is obtained when peroxidase is used at a concentration of 3 Units / ml relative to the total aqueous composition of the kit of parts. Furthermore, when peroxidase is used at a concentration of 3 Units / ml relative to the total aqueous composition of the kit of parts, swelling is also avoided and an optimal shrinkage rate is obtained in the range of -10% to 10%.

[0076] Any peroxidase known to those skilled in the art and suitable for preparing biocompatible polymer hydrogels can be used. Preferably, the peroxidase used in the present invention is selected from horseradish peroxidase (HRP), soybean peroxidase, myeloperoxidase, lactoperoxidase, Nitrospira recombinant peroxidase, and Lyngbya recombinant lysyl oxidase. The preferred peroxidase used in the present invention is horseradish peroxidase (HRP).

[0077] The second factor is the amount of hydrogen peroxide. The inventors have found that an effective method for obtaining the biocompatible polymer hydrogel of the present invention is achieved by using hydrogen peroxide at a concentration of 0.01 to 0.10 wt.% based on the total aqueous composition of the kit-of-parts. The preferred concentration is 0.02 to 0.07 wt.%, more preferably 0.025 to 0.06 wt.%, and even more preferably 0.03 to 0.055 wt.% based on the total aqueous composition of the kit-of-parts.

[0078] Regarding the effect of hydrogen peroxide content on the gel time of the mixed polymer composition, the inventors found that using a hydrogen peroxide concentration of 0.17 wt% based on the total aqueous components of the kit-of-parts resulted in a gel of the mixed polymer composition in 13 minutes. Lowering the hydrogen peroxide concentration to 0.1 wt% based on the total aqueous components of the kit-of-parts resulted in a gel of 197 seconds. Further lowering the hydrogen peroxide concentration to 0.01 wt% based on the total aqueous components of the kit-of-parts resulted in a gel of 14 seconds. The optimal gel time for improving the handling and usability of the kit-of-parts of the present invention was found to be in the range of 35 to 60 seconds, which was achieved using a hydrogen peroxide concentration of 0.05 wt% based on the total aqueous components of the kit-of-parts (as shown in Figure 1).

[0079] Furthermore, when hydrogen peroxide was used at 0.05 wt.%, optimal storage modulus and shrinkage / swelling of the biocompatible polymer hydrogel of the present invention were obtained, as shown in Figures 2 and 3. The optimal storage modulus was in the range of 30 to 60 kPa, which is the range in which the biocompatible polymer hydrogel can withstand the forces of the injection site (e.g., a joint) while remaining soft enough for cells to migrate and proliferate within the biodegradable polymer hydrogel. Furthermore, when hydrogen peroxide was used at 0.05 wt.% of the total aqueous composition of the kit of parts, swelling was also avoided, and optimal shrinkage rates were obtained in the range of -10% to 10%.

[0080] Therefore, when hydrogen peroxide was used at 0.05 wt%, the biocompatible polymer hydrogel had the appropriate rigidity, making it less susceptible to mechanical erosion between two moving joints, and the optimal density of the biocompatible polymer hydrogel was achieved, allowing chondrocytes to migrate and grow into the biocompatible polymer hydrogel, thereby enabling cartilage regeneration.

[0081] Finally, when hydrogen peroxide was used at 0.05 wt%, optimal biocompatible polymer hydrogels were consistently obtained, also characterized by gradual degradation, as shown in Figure 4, which allowed time for chondrocytes to migrate and grow into the biocompatible polymer hydrogel.

[0082] Based on the above, we found that the amount of both peroxidase and hydrogen peroxide affected the gelation time of the mixed polymer composition, the storage modulus, and the shrinkage / swelling (measured as gap size) of the biocompatible polymer hydrogel. Therefore, we evaluated their effects by considering various combinations of peroxidase (HRP) and hydrogen peroxide concentrations shown in Table 2. The gelation measurements were performed at temperatures between 18 and 22°C, and the storage modulus and gap size measurements were performed at 20 ± 0.1°C, with a fixed polymer concentration of 8.9 wt% (4.45 wt% Dex-TA + 4.45 wt% HA-TA).

[0083] [Table 2]

[0084] [Table 3]

[0085] Table 2 shows that the combination of a high hydrogen peroxide concentration (maximum) and a low peroxidase (HRP) concentration (minimum) results in very slow gelation, requiring long treatment times that are not only difficult to implement but also cost-ineffective. Conversely, the combination of a low hydrogen peroxide concentration (minimum) and a high peroxidase (HRP) concentration (maximum) results in very fast gelation, making handling difficult and inconvenient to use the kit of parts of the present invention.

[0086] Furthermore, low hydrogen peroxide concentrations (minimum) result in the formation of a network with low cross-link density, which is reflected in both a low storage modulus and a high degree of swelling, and movement can cause the gel to be pulled out of the injection site (e.g., joint).

[0087] Additionally, the resulting biocompatible polymer hydrogels of the present invention have been shown to have a relatively low storage modulus, a lack of swelling, and excellent structural support compared to conventional biomaterials and biocompatible polymer hydrogels. Furthermore, the slow degradation time of the polymer hydrogels allows for sufficient structural support at the defect site, thereby providing more time for chondrocyte migration and growth and enabling the regeneration of new cartilage. Therefore, in the kit-of-parts of the present invention, advantageous biocompatible hydrogel / cell constructs can be obtained when peroxidase is present at a concentration of 0.25 to 10 units / ml and hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt% relative to the total aqueous composition of the kit-of-parts. It is preferable that the peroxidase is present at a concentration of 1 to 5 units / ml and the hydrogen peroxide is present at a concentration of 0.02 to 0.07% by weight, more preferably 2 to 4 units / ml and 0.025 to 0.06% by weight, and even more preferably 2 to 3.5 units / ml and 0.03 to 0.055% by weight, relative to the total weight of the aqueous composition of the parts kit.

[0088] One preferred embodiment of the present invention is a kit of parts suitable for preparing a biocompatible polymer hydrogel, comprising at least two containers each containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein mixing of these components allows for the formation of a biocompatible polymer hydrogel, Peroxidase and hydrogen peroxide are in separate containers. The total weight ratio of Dex-TA to HA-TA is 55:45 to 45:55, The concentration of Dex-TA is in the range of 3 to 5% by weight based on the total aqueous composition of the kit of parts; The concentration of HA-TA is in the range of 3 to 5% by weight based on the total aqueous composition of the kit of parts; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeat units is 7.5 mol % to 10.5 mol % based on the total number of unsubstituted and TA-substituted repeat units; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeat units is 10 mol % to 14.5 mol % based on the total number of unsubstituted and TA-substituted repeat units; DEX-TA has a weight average molecular weight (Mw) of 30 to 60 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 60 kDa, The peroxidase is present at a concentration of 2 to 3.5 Units / ml and the hydrogen peroxide is present at a concentration of 0.03 to 0.055% by weight of the total aqueous composition of the kit of parts; Regarding parts kits.

[0089] The inventors of the present invention have observed that the above-described kit of parts provides an ideal biocompatible polymer hydrogel with a storage modulus of approximately 30-60 kPa, which can withstand the forces of an injection site (e.g., a joint) and provides an ideal environment for cells to migrate and proliferate within. The swelling / shrinkage rate is approximately -10% to 10%, preventing excessive swelling and shrinkage and preventing the hydrogel from being dislodged or peeled off from the injection site during joint movement. The degradation of the biocompatible polymer hydrogel within the first 30 days after hydrogel formation is also optimal. The degradation is not too rapid, providing sufficient time for cells to migrate into the hydrogel from surrounding tissue and proliferate and produce new extracellular matrix while healing the defect. The gelation time, which is the time it takes for the crosslinked polymer to transform from a free-flowing solution into a gel or semi-gel state, is 40-50 seconds, allowing the surgeon sufficient time to inject the hydrogel into the defect without having to wait for the hydrogel to solidify. Finally, because the viscosity is approximately 18–20 mPa·s, the hydrogel can be extruded as a liquid from a syringe without mixing issues or the application of excessive force. Furthermore, the hydrogel was shown to be firmly fixed in the defect without the risk of spillage.

[0090] In the kit of parts of the present invention described above, the aqueous composition in each container contains 80 to 99.9 wt %, preferably 83 to 99.8 wt %, more preferably 86 to 99.8 wt %, and most preferably 89 to 99.8 wt % of water, saline, phosphate buffer, or phosphate buffered saline.

[0091] As used herein, the term "water saline solution" refers to saline solution (also called saline), a mixture of sodium chloride (salt) and water, which is commonly used in medicine.

[0092] As used herein, the term "phosphate buffered saline" (also known as PBS) refers to a buffer solution having a pH of about 7.4.

[0093] As used herein, the term "phosphate buffered saline" refers to an aqueous salt solution containing the salt as disodium hydrogen phosphate, sodium chloride, potassium chloride, or potassium dihydrogen phosphate, with a pH of about 7.4. This buffer is useful for maintaining a constant pH, and the osmolality and ionic concentration of the solution are consistent with those of the human body (isotonicity).

[0094] Water, saline, phosphate buffer, or phosphate buffered saline are commonly used in medicine.

[0095] Typically, Dex-TA and HA-TA can be added to water, saline, phosphate buffer, or phosphate buffered saline (preferably phosphate buffered saline having a pH of about 7.4) to obtain a polymer solution. Peroxidase can also be dissolved in water, saline, phosphate buffer, or phosphate buffered saline to obtain a desired peroxidase concentration. To obtain a hydrogen peroxide solution of a desired concentration, a hydrogen peroxide solution can be prepared, preferably freshly prepared, in water, saline, phosphate buffer, or phosphate buffered saline.

[0096] A preferred embodiment of the present invention comprises: a. Tyramine-functionalized dextran (Dex-TA), b. Tyramine-functionalized hyaluronic acid (HA-TA), C. peroxidase, a first container containing an aqueous composition A comprising: d. hydrogen peroxide, and e. optionally, at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably both Dex-TA and HA-TA; a second container containing an aqueous composition B comprising: A kit of parts comprising: the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; The concentration of HA-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight of the total aqueous composition of the kit of parts; Regarding parts kits.

[0097] The detailed description of features relating to the present invention set forth immediately under the heading "Detailed Description of Features" applies equally to this preferred embodiment unless otherwise stated in the following text.

[0098] In this specific embodiment, the kit of parts of the invention comprises two containers: a first container containing aqueous composition A as defined above, and a second container containing aqueous composition B as defined above.

[0099] The volume to volume ratio of composition A to composition B is in the range of 0.1:1 to 10:1, preferably in the range of 0.5:1 to 7:1, and more preferably in the range of 0.8:1 to 5:1.

[0100] Components A and B of the kit-of-parts described above are both fluid in the temperature range of 0°C to 37°C and have a viscosity of 1 mPa·s to 50 Pa·s, preferably 1 to 100 mPa·s, more preferably less than 50 mPa·s, and even more preferably less than 35 mPa·s, measured at 25±0.1°C and a shear rate of 10 Hz with a rheometer in double-gap configuration (for low viscosity) or cone-and-plate configuration (for high viscosity) as appropriate.

[0101] The term "fluid" as used herein refers to a substance, e.g., a liquid, that can flow, does not have a fixed shape, conforms to the shape of its container, and offers little resistance to external stresses, and whose molecules move freely relative to one another.

[0102] In the kit of parts of this particular embodiment of the present invention described above, composition A or composition B each contains 80 to 99.9 wt %, preferably 83 to 99.8 wt %, more preferably 86 to 99.8 wt %, and most preferably 89 to 99.8 wt % of water, saline, phosphate buffer, or phosphate buffered saline.

[0103] The terms "saline," "phosphate buffer," and "phosphate-buffered saline" have the same definitions as described above. The preparation of solutions of Dex-TA, HA-TA, hydrogen peroxide, and peroxidase (e.g., HRP) using water, saline, phosphate buffer, or phosphate-buffered saline is the same as described above.

[0104] In certain embodiments, composition A is prepared by mixing Dex-TA, HA-TA, and a peroxidase solution in phosphate buffered saline in one container, while composition B is prepared in a second container by mixing either hydrogen peroxide alone in phosphate buffered saline or a hydrogen peroxide solution in phosphate buffered saline with a Dex-TA, HA-TA solution in phosphate buffered saline.

[0105] Any type of assembled kit known to those skilled in the art that includes at least two containers suitable for preparing biocompatible polymer hydrogels can be used for the present invention. Preferably, the outlets of the containers are connected to a mixing element, such as a static mixer, to effectively mix the contents of the different containers.

[0106] Preferably, the kit of parts of the present invention is an injection kit. Commonly known commercially available injection kits such as the Nordson Medical Applicator Assembly device or the Sulzer Medmix K-system can be used.

[0107] Preferably, the kit has separate outlets for each container to reduce the risk of cross-contamination during the application process. Preferably, the contents of each container are injected simultaneously and preferably mixed before leaving the kit. In a preferred embodiment, the kit of parts suitable for preparing a biocompatible polymer hydrogel according to the present invention is a syringe. The kit of parts suitable for preparing a biocompatible polymer hydrogel according to the present invention can also be sold as a pre-filled syringe.

[0108] The kit of parts of the present invention described above can be used at the surgical or incision site to repair damaged cartilage in order to prevent or reduce pain in the joints of humans or animals.

[0109] Administration by injection allows for the in situ formation of microgels and macrogels at the injection site, such as a joint cavity. Surgery into a site such as a joint cavity can be performed by incision under open vision or arthroscopic surgery. The incision is made depending on the size and location of the defect. A retractor is often used to ensure visualization of the defect. Rough and loose defective cartilage is removed, and the size and depth of the defect are measured. The defect is carefully debridemented to remove cracks and degenerated cartilage. The defect is then positioned horizontally and confirmed to be dry. The biocompatible polymer hydrogel is then carefully injected to fill the defect to the edges. After several minutes, the stability of the biocompatible polymer hydrogel is confirmed by gently moving the application site, for example, by bending the knee if the biocompatible polymer hydrogel is applied to the knee. The incision is then closed according to standard procedures.

[0110] The present invention provides a. mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition; b. subjecting the mixed polymer composition to a temperature of 15 to 38°C for 10 to 200 seconds to form a biocompatible polymer hydrogel; A method for producing a biocompatible polymer hydrogel comprising: the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7 wt% of the mixed polymer; The concentration of HA-TA is in the range of 2 to 7 wt% of the mixed polymer; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 Units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt% of the mixed polymer composition. It also relates to methods.

[0111] The detailed description of features relating to the present invention set forth immediately under the heading "Detailed Description of Features" applies equally to this embodiment unless otherwise stated in the following text.

[0112] A method for producing a biocompatible polymer hydrogel includes mixing an aqueous composition containing tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition. Mixing can be performed by procedures known in the art. Preferably, mixing is performed using a cannula equipped with a blending connector or a static mixer connectable to an applicator device.

[0113] The inventors have determined the concentrations of Dex-TA and Ha-TA shown above based on a specific weight ratio of Dex-TA and HA-TA, a specific DS DEX and DS HAIt has been found that by using specific weight-average molecular weights of DEX-TA and HA-TA, and specific concentrations of peroxidase and hydrogen peroxide as shown above, a biocompatible polymer hydrogel can be formed at a temperature of 15 to 38° C. for a time period of 10 to 200 seconds, preferably 20 to 100 seconds, and more preferably 30 to 60 seconds. Specifically, the temperature is 35 to 38° C. when the method is performed in vivo, and 18 to 22° C., preferably 17 to 20° C. when the method is performed in vitro.

[0114] In a preferred embodiment, the present invention comprises: a. mixing aqueous composition A and aqueous composition B to obtain a mixed polymer composition; b. subjecting the mixed polymer composition to a temperature of 15 to 38°C for 10 to 200 seconds to form a biocompatible polymer hydrogel; A method for producing a biocompatible polymer hydrogel comprising: aqueous composition A comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), and peroxidase; aqueous composition B comprises hydrogen peroxide, and optionally also comprises at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably both Dex-TA and HA-TA; the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7% by weight based on the total of Compositions A and B; The concentration of HA-TA is in the range of 2 to 7% by weight based on the total of compositions A and B; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DSHA ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight based on the total weight of the aqueous compositions A and B. Regarding the method.

[0115] The detailed description of features relating to the present invention set forth immediately under the heading "Detailed Description of Features" applies equally to this preferred embodiment unless otherwise stated in the following text.

[0116] The present invention provides a mixed polymer composition obtained by mixing an aqueous composition containing tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7 wt% of the mixed polymer composition; The concentration of HA-TA is in the range of 2 to 7 wt% of the mixed polymer composition; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa, HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa, The peroxidase is present at a concentration of 0.25 to 10 Units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt% of the mixed polymer composition. It also relates to mixed polymer compositions.

[0117] The detailed description of features relating to the present invention set forth immediately under the heading "Detailed Description of Features" applies equally to this preferred embodiment unless otherwise stated in the following text.

[0118] The inventors have found that if the mixed polymer composition gels too quickly, it cannot be easily applied to a surgical or incision site. The inventors have found that the optimal gelation time of the mixed polymer composition of the present invention is achieved by adjusting the concentrations of Dex-TA and HA-TA shown above to a specific weight ratio of Dex-TA to HA-TA, a specific DS DEX and DS HA It has been found that when the above-mentioned weight-average molecular weights of DEX-TA and HA-TA are combined with the above-mentioned specific concentrations of peroxidase and hydrogen peroxide, gelation of the mixed polymer composition, and thus formation of a biocompatible polymer hydrogel, can be achieved at a temperature of 15 to 38°C for 10 to 200 seconds, preferably 20 to 100 seconds, and more preferably 30 to 60 seconds.

[0119] The optimum gel time found by the inventors allows the mixed polymer composition to be a "pourable composition."

[0120] The term "injectable composition" refers to a solution capable of forming a polymer hydrogel after injection. Thus, the mixed polymer composition is sufficiently fluid to allow injection of the mixed polymer composition. The injectable composition offers the advantages of forming microgels and macrogels in situ at the injection site, such as a joint cavity, having excellent conformability to irregularly shaped defects, and allowing easy migration and growth of chondrocytes into the biocompatible polymer hydrogel, thereby enabling cartilage regeneration. Alternatively, the mixed polymer composition may first form a microgel, which is then injected directly into the joint cavity.

[0121] Injection of the mixed polymer composition into the surgical or incision site takes only a few seconds. After injection, a waiting period of several minutes is required for the formation and settling of the biocompatible polymer hydrogel. Furthermore, the stability of the biocompatible polymer hydrogel is confirmed by gently moving the application site, for example, bending the knee if the biocompatible polymer hydrogel is applied to the knee. The incision is then closed. This minimally invasive and simple injection procedure is a highly advantageous technique compared to traditional implantation surgery, which is commonly used in cases of severe cartilage damage.

[0122] Injection of the mixed polymer composition into the surgical or incision site can be accomplished by methods known in the art. Preferably, a needle or cannula is used. If a needle is used, the needle preferably has a gauge of 12G to 23G, more preferably 16G to 21G.

[0123] To allow for easy application of the mixed polymer composition of the present invention, the mixed polymer composition must have an appropriate viscosity.

[0124] The inventors have found that the initial viscosity of the mixed polymer composition at a fixed polymer concentration of 8.9 wt % (4.45 wt % Dex-TA + 4.45 wt % HA-TA) at TO is 15 to 25 mPa·s, preferably 18 to 20 mPa·s, when measured with a rheometer in double-gap or cone-plate configuration as appropriate, at 25±0.1°C and a shear rate of 10 Hz.

[0125] The term "TO" refers to the first time that the tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide come into contact with each other before the gelation process begins. Therefore, the viscosity measurement at TO is performed in the absence of hydrogen peroxide, which prevents the initiation of the gelation process and, therefore, the formation of the biocompatible polymer hydrogel of the present invention. Once the gelation process begins, the viscosity of the mixed polymer composition increases until the formation of the biocompatible polymer hydrogel.

[0126] The present invention provides a biocompatible polymer hydrogel obtained by reacting tyramine-functionalized dextran (Dex-TA) with tyramine-functionalized hyaluronic acid (HA-TA) in the presence of peroxidase and hydrogen peroxide, the total weight ratio of Dex-TA to HA-TA is 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS DEX ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the proton NMR analysis of HA-TA (DS HA ) the amount of TA-substituted repeating units is 5 mol % to 20 mol %; The biocompatible polymer hydrogel has a shear storage modulus of 10 to 100 kPa as measured by an oscillatory rheology device and exhibits a shrinkage rate α of -20% to 20%, wherein α is represented by the formula (I):

number

[0127] The detailed description of features relating to the present invention set forth immediately under the heading "Detailed Description of Features" applies equally to this preferred embodiment unless otherwise stated in the following text.

[0128] The shear storage modulus of the biocompatible polymer hydrogel of the present invention was evaluated using an oscillatory rheology instrument. Samples for oscillatory rheology measurements were prepared in a mold 9 mm wide and 1.6 mm high to obtain a cylindrical sample. After preparation, the cylindrical biocompatible polymer hydrogel sample (gel disk) was incubated overnight in phosphate-buffered saline until equilibrium swelling was reached. The gel disk was then removed from the PBS, gently tapped with tissue paper to remove excess PBS, and placed in an oscillatory rheology instrument equipped with an 8 mm diameter spindle. The spindle was lowered onto the aligned gel disk until a normal force of 0.05 ± 0.005 N was obtained. Measurements were then performed in oscillatory mode at 20 ± 0.1 °C, a strain amplitude of 0.01%, and a frequency of 1 Hz.

[0129] The degradation of the biocompatible polymer hydrogels of the present invention was determined by incubating the hydrogels in hyaluronidase enzyme at 37°C for a set period of time. The biocompatible polymer hydrogels were prepared in molds measuring 9 mm wide and 1.6 mm high to obtain cylindrical samples. The biocompatible polymer hydrogel cylindrical samples (gel disks) were then incubated overnight in phosphate-buffered saline to reach equilibrium swelling. The biocompatible polymer hydrogels were weighed after overnight equilibrium swelling. The phosphate-buffered saline was then removed, and the biocompatible polymer hydrogels were incubated in 5 U / mL hyaluronidase solution at 37°C. The degradation of the biocompatible polymer hydrogels was then measured by weighing them at different time points and calculating the degradation as the percentage of the biocompatible polymer hydrogel remaining by weight. The remaining weight percentage of the biocompatible polymer hydrogel can be calculated as wt / wi*100%, where wt is the weight of the biocompatible polymer hydrogel at the time point of interest and wi is the initial weight of the biocompatible polymer hydrogel after equilibrium swelling.

[0130] The present inventors have surprisingly found that the biocompatible polymer hydrogel of the present invention obtained by the reaction shown above is not susceptible to mechanical erosion between two moving joints.

[0131] The biocompatible polymer hydrogel of the present invention does not swell or swells very little, and has excellent density, which allows chondrocytes to migrate and proliferate in the biocompatible polymer hydrogel of the present invention.

[0132] The shear storage modulus characterizing the biocompatible polymer hydrogel of the present invention is 10 to 100 kPa, preferably 15 to 90 kPa, more preferably 20 to 70 kPa, and most preferably 30 to 60 kPa, when measured using an oscillatory rheology device, while the shrinkage ratio α is -20% to 20%, preferably -15% to 15%, and more preferably -10% to 10%, when measured using the above formula (I).

[0133] Furthermore, the decomposition time of the polymer hydrogel of the present invention is slow enough to provide sufficient support at the defect site, which allows more time for chondrocytes to migrate and grow, enabling cartilage regeneration.

[0134] The polymer hydrogel of the present invention is characterized by being degraded in vitro by 10% to 50%, preferably 10% to 40%, more preferably 10% to 30%, and even more preferably 10% to 20% after 30 days in hyaluronidase having a concentration of 5 units / ml.

[0135] The term "Units / ml" with respect to hyaluronidase refers to the concentration of hyaluronidase expressed in "activity units" of enzyme activity that produces the same reduction in turbidity as 1.0 unit of the international standard.

[0136] The present invention also relates to the use of the biocompatible polymer hydrogel of the present invention as a pharmaceutical for preventing or suppressing pain symptoms in the joints of a human or animal, wherein the pain is selected from the group consisting of inflammatory pain, arthritis pain, osteoarthritis pain, osteochondritis dissecans pain, and traumatic defects due to trauma or accident or slip or fall.

[0137] As used herein, the term "use as a pharmaceutical for the treatment or prevention of joint pain symptoms" refers to a treatment aimed at preventing or minimizing joint pain caused by damaged or defective tissue, and the provision of the polymer hydrogel of the present invention aims to improve the regeneration of damaged or defective tissue and reduce pain. Preferably, the damaged or defective tissue is cartilage tissue.

[0138] This painful condition may result from diseases such as inflammation, arthritis, osteoarthritis, or osteochondritis dissecans, or may result from traumatic injury due to an injury or accident or trip or fall.

[0139] The polymer hydrogel may further comprise a drug or agent that can be released according to a slow or sustained release mechanism, and thus the biocompatible polymer hydrogel can be used as a sole therapy, as an adjunct, or in combination with other known therapies.

[0140] The present invention also relates to a method for treating or preventing joint pain in a human or animal, comprising administering a therapeutically effective amount of the biocompatible polymer hydrogel of the present invention.

[0141] A therapeutically effective amount of the biocompatible polymer hydrogel of the present invention can be administered to a surgical or incision site by injecting a therapeutically effective amount of the mixed polymer composition of the present invention over a period of time typically between 2 and 10 seconds.

[0142] It is understood that the required therapeutically effective amount of the biocompatible polymer hydrogel or mixed polymer composition according to the present invention is determined, for example, by the volume of the area to be repaired or replaced, but also by the swelling behavior and degradation characteristics of the polymer hydrogel, and whether the polymer hydrogel is used as a monotherapy or in a combination therapy. If the therapy is also used to release a drug, the amount will also depend on the amount of drug incorporated into the polymer hydrogel, the pharmacokinetics of the drug, and the progression of the disease symptoms.

[0143] To establish a therapeutically effective amount and treatment regimen (e.g., dosage and frequency of administration) of the biocompatible polymer hydrogel or mixed polymer composition according to the present invention, known procedures (e.g., in vivo experiments, clinical trials, etc.), such as those conventionally employed in the pharmaceutical industry, can be used.

[0144] The invention will now be illustrated in a non-limiting manner by the following examples. [Example]

[0145] Abbreviation Dex (dextran), PNC (4-nitrophenyl chloroformate), LiCl (lithium chloride), TA (tyramine), DMF (N,N-dimethylformamide), TA·HCl (tyramine hydrochloride), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), NaCl (sodium chloride), HA (hyaluronic acid), PBS (phosphate buffered saline or buffer solution of approximately pH 7.4), H2O2 (hydrogen peroxide), NMR (nuclear magnetic resonance), wt% (weight percent), Eq. (molar equivalent).

[0146] Example 1 Synthesis of Dex-TA Synthesis of dextran-p-nitrophenyl carbonate (dextran-PNC) LiCl (4.0 g, dried at 115°C) and dextran (5.00 g, 30.8 mmol of repeating units (ru)) were weighed into a 500 mL three-neck round-bottom flask equipped with a stir bar. The flask was evacuated and refilled with nitrogen gas three times, then left under vacuum at 95°C for 1.5 hours. After sufficient drying, the flask was filled with nitrogen and 200 mL of anhydrous DMF was added via cannula while stirring. A thermometer was then attached to the flask, and the mixture was heated to 95°C while stirring. After the dextran was completely dissolved, the solution was cooled to 0°C and anhydrous pyridine (2.0 mL, 25.8 mmol) was added. Freshly sublimed para-nitrophenyl chloroformate (2.5 g, 12.4 mmol) was then added in portions, maintaining the temperature below 2°C. After 1 hour, the reaction mixture was poured into 1 L of ice-cold ethanol. The precipitate was filtered off (Por4) and washed with cold ethanol (3 × 100 mL) followed by diethyl ether (3 × 100 mL). After drying under vacuum, the product was obtained as a white powder (6.00 g, 30.7 mmol ru, 99% yield, DS 20%). 1 H-NMR (400 MHz): δ (ppm) = 3.0-4.0 (sugar ring protons, m, 6H); 4.2-5.8 (anomeric and hydroxyl protons, m, 4H); 7.58 (Ar o-CH, d, 2H); 8.34 (Ar m-CH, d, 2H).

[0147] Synthesis of dextran-tyramine Dextran-PNC (6.00 g, 30.7 mmol ru, 6.15 mmol p-nitrophenyl carbonate) was weighed into a 250 mL three-neck round-bottom flask equipped with a stir bar. The flask was evacuated and refilled with nitrogen gas three times. Then, while filling the flask with nitrogen gas and stirring, 100 mL of anhydrous DMF was added via cannula. After the dextran was completely dissolved, tyramine (1.69 g, 12.3 mmol) was added. After 1 h, the reaction mixture was poured into 1 L of ice-cold ethanol. The precipitate was filtered off (Por4) and washed with cold ethanol (3 × 100 mL) followed by diethyl ether (3 × 100 mL). After drying under vacuum, the crude product was obtained as a white powder. The crude product was dissolved in 75 mL of Milli-Q water and dialyzed (MWCO 3500 Da) against Milli-Q water for 3 days, followed by filter sterilization and lyophilization to give the product as a white foam (5.04 g, 28.0 mmol, 92% yield, 10% DS). 1H-NMR (400 MHz): δ (ppm) = 3.0-4.0 (sugar ring protons, m, 6H); 4.8-5.2 (anomeric and hydroxyl protons, m, 4H); 6.7-7.1 (Ar m-CH, d, 2H); 6.9-7.3 (Ar o-CH, d, 2H).

[0148] The DS of dextran-TA and dextran-PNC was calculated by comparing the integrals between 4.8 and 5.2 ppm (corresponding to the four anomeric protons from dextran) with the integrals of the aromatic protons of tyramine (6.7–7.1 and 6.9–7.3 ppm) or para-nitrophenyl (7.3–7.7 and 8.10–8.50 ppm). The DS of dextran is expressed as the percentage of modified sugar units in the dextran.

[0149] Example 2 Synthesis of HA-TA Sodium hyaluronate (5.00 g, 12.5 mmol) was dissolved in 500 mL of Milli-Q water (relative volume 100) in a 1 L round-bottom flask equipped with a stir bar. While stirring at room temperature, DMTMM (3.46 g, 12.5 mmol, 1 molar equivalent) and TA·HCl (2.17 g, 12.5 mmol, 1 molar equivalent) were added sequentially. The addition of DMTMM and TA·HCl was repeated after 24 and 48 hours. After 72 hours, 40 mL of NaCl (relative volume 8, saturated) was added to the reaction mixture, and the reaction mixture was poured into 2.5 L (relative volume 500) of cold ethanol. The crude product was isolated by centrifugation at 5000 rpm and then vacuum dried. The crude product was dissolved in 75 mL of Milli-Q water and dialyzed (MWCO 1000 Da) against Milli-Q water for 3 days. Filter sterilization and lyophilization gave the HA-TA product as a white foam (5.10 g, 12.4 mmol, 99% yield, 10% DS). 1 HNMR was used to confirm the successful synthesis of HA-TA: Baseline correction: spline method The peaks at 1.7–2.1 ppm, 6.7–7.1 ppm, and 6.9–7.3 ppm were integrated. The peak area of ​​the peak between 1.7 and 2.1 ppm was set to 3000. DS: Add up the peak areas of 6.7-7.1 ppm and 6.9-7.3 ppm and divide by 40. 1 H-NMR(400MHz,D2O):δ(ppm)=1.98(acetyl-CH3,s,3H);2.75(2-CH2,s,2H);2.90(1-CH2,s,2H);3.2-4.2(sugar ring,m,10H);4.34(s,1H);4.43(d,1H);6.84(Ar m-CH,d,2H);7.16(Ar o-CH,d,2H).

[0150] Example 3 A hydrogel parts kit suitable for preparing biocompatible polymer hydrogel formulations by premixing in a 4:1 ratio Add equal amounts of Dex-TA and HA-TA to PBS (phosphate-buffered saline, or a buffer solution with a pH of approximately 7.4) to obtain polymer solutions with concentrations ranging from 11.0 to 13.0 wt%. Dissolve HRP (horseradish peroxidase) in PBS to obtain a 30 U / mL HRP stock solution. Dilute 30 wt% HO with PBS to obtain 0.25 to 0.5 wt% HO.

[0151] The Dex-TA / HA-TA polymer solution and the HRP solution are premixed in a 4:1 v / v ratio. The Dex-TA / HA-TA polymer solution and the H2O2 solution are also premixed in a 4:1 v / v ratio. Both premixed solutions are then mixed in a 1:1 v / v ratio.

[0152] Example 4 A hydrogel parts kit suitable for preparing biocompatible polymer hydrogel formulations by premixing in a 7:1 ratio Add equal amounts of Dex-TA and HA-TA to PBS (phosphate-buffered saline, or a buffer solution with a pH of approximately 7.4) to obtain a polymer solution with a concentration ranging from 11.0 to 13.0 wt%. Dissolve HRP (horseradish peroxidase) in PBS to obtain a 30 U / mL HRP stock solution. Dilute 30 wt% H2O2 with PBS to obtain a 0.25 to 0.5 wt% H2O2. Premix the Dex-TA / HA-TA polymer solution and HRP solution in a 7:1 ratio. Mix this premix with H2O2 in a 4:1 ratio.

[0153] method Several methods were used to investigate the properties, structure, processability, and performance of the mixed polymer compositions and biocompatible polymer hydrogels of the present invention.

[0154] Viscosity measurement Viscosity measurements were performed on a TA Instruments Discovery HR20 in double-gap geometry. During viscosity measurements, the temperature was held constant at 25°C for 205 seconds at a shear rate of 10 s-1. This was repeated twice. Viscosity values ​​were calculated using software (Trios software, TA Instruments). The viscosity measured during the last 105 seconds of each measurement was averaged. The two average values ​​of the two repeated measurements were averaged to obtain the final viscosity. In this way, the average dynamic (shear) viscosity of the biocompatible polymer hydrogel was determined.

[0155] A. Viscosity Measurement Geometry Double-gap configuration Cup inner diameter: 40.0 mm Bob inner diameter: 40.79mm Bob outer diameter: 43.88mm Cup outer diameter: 44.76mm Inner cylinder height: 55.0 mm Immersion height: 53mm Operating gap: 2mm Bob material: Aluminum Peltier heating

[0156] B. Measurement Settings Four stages of measurements were performed. 1. Shear sweep (not used for data processing, only for additional checks) T=25.0±0.1℃ Shear sweep 0.01~100Hz 10 points / decade 5 seconds measurement per point 2. Time sweep (used for data processing) T=25.0±0.1℃ 205-second measurement Shear rate = 10Hz Sampling interval = 5 seconds 3. Shear sweep (not used for data processing, only for additional checks) T=25.0±0.1℃ Shear sweep 0.01~100Hz 10 points / decade 5 seconds measurement per point 4. Time sweep (used for data processing) T=25.0±0.1℃ 205-second measurement Shear rate = 10Hz Sampling interval = 5 seconds

[0157] C. Viscosity Data Processing Only the data obtained in the second and fourth stages were used for data processing to obtain the viscosity of the solution. The viscosity data obtained in the latter half of the second stage (the last 105 seconds) was averaged. The viscosity data obtained in the latter half of the fourth stage (the last 105 seconds) was averaged. The viscosity of the solution was obtained by averaging the average values ​​of the last 105 seconds of the second and fourth stages.

[0158] Rheological measurements 1. Preparation of Hydrogel Discs Using Rheological Molds Use the pre-assembled mold shown in Figure 5 to prepare hydrogel discs. The mold sheet is placed in the mold block, and a glass slide is placed on top and secured with a binder clamp. The mixed components of the present invention are injected through the 1.5 mm hole on the left side of the mold and allowed to gel within the mold. The mold strip is then removed from the mold block, and the gel disc is extruded from the mold strip. The gel disc is then incubated in PBS at 2-8°C for 12-168 hours before measurement.

[0159] 2. Details of rheological measurements A. Rheometry Measurement Geometry Parallel Plate Configuration φ8mm spindle stainless steel Peltier heating B. Rheometry measurement setup The spindle is lowered onto the gel disc until a normal force (axial force) of 0.05±0.005 N is reached. The gap size is recorded when said normal force is reached.

[0160] Measurements were performed in oscillatory mode using the following settings: Strain amplitude = 0.01% Frequency = 1Hz Measurement time = 120 seconds Sampling interval = 5 seconds ·Temperature=20±0.1℃

[0161] C. Rheometry Data Processing Average the storage modulus data for the second half of the measurement (60–120 s).

[0162] Swelling / Shrinkage To investigate the swelling / shrinking behavior of biocompatible polymer hydrogels, an oscillatory rheology apparatus (TA Instruments-Discovery HR20) was used. Gel disks were prepared in 1.6 mm thick molds, placed in PBS at 2–7°C for 12–168 h, dried, and placed in an oscillatory rheometry apparatus equipped with an 8 mm diameter spindle. The spindle was lowered onto the aligned gel disc until a normal force of 0.05±0.005 N was recorded. The shrinkage rate α is calculated by the formula (I):

number

Claims

1. 1. A kit of parts suitable for preparing a biocompatible polymer hydrogel, comprising at least two containers each containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein the components are mixed to allow the formation of a biocompatible polymer hydrogel; Peroxidase and hydrogen peroxide are in separate containers. a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; the concentration of Dex-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; the concentration of HA-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol % based on the total number of unsubstituted and TA-substituted repeat units; the DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; the HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; the peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight based on the total aqueous composition of the kit of parts; Parts kit.

2. a. Tyramine-functionalized dextran (Dex-TA), b. Tyramine-functionalized hyaluronic acid (HA-TA); c. peroxidase, a first container containing an aqueous composition A comprising: d. hydrogen peroxide, and e. optionally, at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably both Dex-TA and HA-TA; a second container containing an aqueous composition B comprising:

10. The kit of parts of claim 1, comprising: a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; the concentration of Dex-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; the concentration of HA-TA is in the range of 2 to 7% by weight based on the total aqueous composition of the kit of parts; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; the DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; the HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; the peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight based on the total aqueous composition of the kit of parts; Parts kit.

3. 3. The kit of parts according to claim 1 or 2, wherein the peroxidase is selected from horseradish peroxidase (HRP), soybean peroxidase, myeloperoxidase, lactoperoxidase, Nitrospira recombinant peroxidase, and Lyngbya recombinant lysyl oxidase.

4. 4. The kit of parts according to claim 2 or 3, wherein the volume to volume ratio of composition A to composition B is in the range of 0.1:1 to 10:

1.

5. 5. The kit of parts according to any one of claims 2 to 4, wherein components A and B are both fluid in the temperature range of 0°C to 37°C and have a viscosity of from 1 mPa s to 50 Pa s, preferably from 1 mPa s to 100 mPa s, more preferably less than 50 mPa s, and even more preferably less than 35 mPa s, measured at 25±0.1°C and a shear rate of 10 Hz with a rheometer in double gap or cone and plate configuration as appropriate.

6. 10. The kit of parts of claim 1, wherein the aqueous composition in each container comprises 80 to 99.9% by weight of water, saline, phosphate buffer, or phosphate buffered saline.

7. The kit of parts according to any one of claims 2 to 5, wherein composition A or composition B each contains 80 to 99.9% by weight of water, saline, phosphate buffer, or phosphate buffered saline.

8. a. mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition; b. subjecting the mixed polymer composition to a temperature of 15-38°C for 10-200 seconds to form a biocompatible polymer hydrogel; A method for producing a biocompatible polymer hydrogel comprising: a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; the concentration of Dex-TA is in the range of 2 to 7% by weight of the mixed polymer composition; The concentration of HA-TA is in the range of 2 to 7 wt% of the mixed polymer composition; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; the DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; the HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt % of the mixed polymer composition. method.

9. a. mixing aqueous composition A and aqueous composition B to obtain a mixed polymer composition; b. subjecting the mixed polymer composition to a temperature of 15-38°C for 10-200 seconds to form a biocompatible polymer hydrogel; A method for producing a biocompatible polymer hydrogel comprising: the aqueous composition A comprises tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), and peroxidase; said aqueous composition B comprising hydrogen peroxide, and optionally also comprising at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably both Dex-TA and HA-TA; a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The concentration of Dex-TA is in the range of 2 to 7% by weight based on the total of Compositions A and B; The concentration of HA-TA is in the range of 2 to 7% by weight based on the total of Compositions A and B; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; the DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; the HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; the peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10% by weight based on the total weight of the aqueous compositions A and B; method.

10. A mixed polymer composition obtained by mixing an aqueous composition containing tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; the concentration of Dex-TA is in the range of 2 to 7% by weight of the mixed polymer composition; The concentration of HA-TA is in the range of 2 to 7 wt% of the mixed polymer composition; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; the DEX-TA has a weight average molecular weight (Mw) of 15 to 650 kDa; the HA-TA has a weight average molecular weight (Mw) of 15 to 550 kDa; The peroxidase is present at a concentration of 0.25 to 10 units / ml and the hydrogen peroxide is present at a concentration of 0.01 to 0.10 wt % of the mixed polymer composition. Mixed polymer compositions.

11. 11. The mixed polymer composition of claim 10, having a gel time of 10 to 200 seconds at a temperature of 17 to 38°C.

12. 12. The mixed polymer composition according to claim 10 or 11, wherein the mixed polymer composition has a viscosity at TO of 15 to 25 mPa s when measured with a rheometer at 25±0.1°C and a shear rate of 10 Hz in double gap or cone-plate configuration as appropriate.

13. A biocompatible polymer hydrogel obtained by reacting tyramine-functionalized dextran (Dex-TA) with tyramine-functionalized hyaluronic acid (HA-TA) in the presence of peroxidase and hydrogen peroxide, a total weight ratio of Dex-TA to HA-TA of 75:25 to 25:75, preferably 60:40 to 40:60, more preferably 55:45 to 45:55; The DEX-TA contains repeating units of unsubstituted and TA-substituted dextran, and the DEX-TA (DS ＤＥＸ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The HA-TA contains repeating units of unsubstituted and TA-substituted hyaluronic acid, and the HA-TA (DS ＨＡ ) the amount of TA-substituted repeat units is 5 mol % to 20 mol %; The biocompatible polymer hydrogel has a shear storage modulus of 10 to 100 kPa as measured by an oscillatory rheology device, and exhibits a shrinkage rate α of −20% to 20%, wherein α is represented by the formula (I): [Equation 1] is determined in accordance with α is the shrinkage rate (%) of the gel disc; h ｍｏｌｄ is the mold height, 1.6 mm; h ｄｉｓｃ is the height (mm) of the gel disc measured under a normal force of 0.05 N in the oscillatory rheology device; Biocompatible polymer hydrogel.

14. The biocompatible polymer hydrogel according to claim 13, which degrades in vitro by 10% to 50% after 30 days in hyaluronidase having a concentration of 5 Units / ml.

15. 15. The biocompatible polymer hydrogel of claim 13 or 14 for use as a medicament for preventing or suppressing pain symptoms in the joints of a human or animal, wherein the pain symptoms are selected from the group consisting of inflammatory pain, arthritis pain, osteoarthritis pain, osteochondritis dissecans pain, and traumatic defects due to trauma or accident or slip or fall.

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