CN111228579B

CN111228579B - Injectable hydrogel, preparation method and application thereof, and joint lubricant

Info

Publication number: CN111228579B
Application number: CN202010073764.9A
Authority: CN
Inventors: 姜晓蕾; 彭显博; 陈文静; 王斯姝; 孔建; 薛会群
Original assignee: Saikesaisi Biotechnology Co ltd
Current assignee: Saikesaisi Biotechnology Co ltd
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2021-12-03
Anticipated expiration: 2040-01-21
Also published as: CN111228579A

Abstract

The invention provides an injectable hydrogel, a preparation method and application thereof, and a joint lubricant, and relates to the technical field of osteoarthritis treatment, wherein the injectable hydrogel comprises the following raw materials: macromolecular biological crosslinking agent, micromolecular crosslinking agent and carboxymethyl chitosan; the macromolecular biological cross-linking agent comprises at least one of oxidized sodium hyaluronate, oxidized sodium alginate, oxidized sodium carboxymethyl cellulose, oxidized starch and oxidized dextran; the small molecule cross-linking agent comprises an aldehyde cross-linking agent with a molecular weight of less than 200. The injectable hydrogel has a good effect on cartilage lubrication protection in osteoarthritis treatment, and can play a role in prolonging the treatment effect and reducing the administration frequency.

Description

Injectable hydrogel, preparation method and application thereof, and joint lubricant

Technical Field

The invention relates to the technical field of osteoarthritis treatment, in particular to an injectable hydrogel, a preparation method and application thereof and a joint lubricant.

Background

Osteoarthritis is a common chronic disease of middle-aged and elderly people, and is clinically manifested by arthralgia, swelling and stiffness, and deformity and dysfunction of joints in severe patients, and is also accompanied by secondary synovitis. The main pathological features of the medicine are progressive degeneration and destruction of articular cartilage, progressive loss of cell nutrients in cartilage tissue and secondary hyperosteogeny, reduction of cartilage cells and formation of osteophyte in middle and late stages. The prevalence rate of the traditional Chinese medicine is high in middle-aged and elderly people, 50% of people over 60 years old and 80% of people over 75 years old, the disability rate of the disease can reach 53%, and the traditional Chinese medicine becomes one of main diseases damaging the physical and mental health of the old.

Viscoelastant supplementation therapy is an important breakthrough in the field of osteoarthritis treatment in recent years, and is also a novel way to treat osteoarthritis and reduce the chance of joint morbidity. The viscoelastic supplementary products sold on the market at present mainly comprise two types of hyaluronic acid and carboxymethyl chitin. Hyaluronic acid degrades rapidly in the human body and needs to be injected every week. The carboxymethyl chitin can be stored in a human body for a longer time than that of hyaluronic acid, but the injection is still required to be performed once every 2 weeks.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an injectable hydrogel which has a good effect on cartilage lubrication protection in osteoarthritis treatment, and can achieve the effects of prolonging the treatment effect and reducing the administration times.

The invention provides an injectable hydrogel which comprises the following raw materials:

macromolecular biological crosslinking agent, micromolecular crosslinking agent and carboxymethyl chitosan;

the macromolecular biological cross-linking agent comprises at least one of oxidized sodium hyaluronate, oxidized sodium alginate, oxidized sodium carboxymethyl cellulose, oxidized starch and oxidized dextran;

the small molecule cross-linking agent comprises an aldehyde cross-linking agent with a molecular weight of less than 200.

Further, the molar ratio of aldehyde groups in the macromolecular biological cross-linking agent to the aldehyde groups in the micromolecular cross-linking agent is 0.05-20: 1;

preferably, the ratio of the total mole number of aldehyde groups in the macromolecular biological cross-linking agent and the small molecular cross-linking agent to the mole number of sugar units in the carboxymethyl chitosan is 0.05-1: 1;

preferably, the small molecule cross-linking agent comprises at least one of glutaraldehyde, glyoxal, malondialdehyde, and succindialdehyde.

Further, the oxidation degree of the macromolecular biological crosslinking agent is 10-80%.

Further, the carboxymethyl chitosan comprises at least one of N, O-CMCTS, O-CMCTS and N-CMCTS;

preferably, the carboxymethyl chitosan has an average degree of substitution of 50 to 200%.

Further, still include: an osmotic pressure regulator and/or a buffer;

preferably, the osmolality adjusting agent comprises sodium chloride;

preferably, the buffer comprises sodium dihydrogen phosphate and disodium hydrogen phosphate.

A method of preparing an injectable hydrogel as hereinbefore described comprising:

mixing a macromolecular biological crosslinking agent, a micromolecular crosslinking agent and carboxymethyl chitosan, and reacting to obtain the injectable hydrogel.

Further, comprising:

dissolving a macromolecular biological crosslinking agent and a micromolecular crosslinking agent in a first solvent to obtain a first solution;

dissolving carboxymethyl chitosan in a second solvent to obtain a second solution;

mixing the first solution and the second solution, and performing covalent crosslinking to obtain the injectable hydrogel;

preferably, the first solvent and the second solvent each independently comprise a mixture of a buffered saline solution and an osmotic pressure regulator, and/or a physiological saline.

Further, the covalent cross-linking has a cross-linking ratio of 0.05 to 1: 1;

preferably, the mass concentration of the second solution is 1-10% (w/v);

preferably, the volumes of the first solution and the second solution are the same when the first solution and the second solution are mixed;

preferably, the first solution and the second solution are mixed after filtration and sterilization.

Use of an injectable hydrogel as hereinbefore described for joint lubrication.

A joint lubricant comprising the injectable hydrogel as hereinbefore described.

Compared with the prior art, the invention can at least obtain the following beneficial effects:

in the raw materials of the injectable hydrogel, a macromolecular biological crosslinking agent and a micromolecular crosslinking agent react with amino of carboxymethyl chitosan to form the injectable hydrogel with viscoelasticity, and the obtained injectable hydrogel has a good effect on cartilage lubrication protection in osteoarthritis treatment and can play a role in prolonging the treatment effect and reducing the administration times.

In addition, the macromolecular biological cross-linking agent and the micromolecular cross-linking agent are aldehyde cross-linking agents and react with amino, so a certain competitive relationship exists, the macromolecular cross-linking agent reacts slowly due to steric hindrance, and the micromolecular cross-linking agent reacts quickly, so the gel forming time is controlled by adjusting the proportion of the macromolecular biological cross-linking agent and the micromolecular cross-linking agent, the gel forming time of the obtained injectable hydrogel can be 0.5-15 minutes, the gel forming time is not limited by the substitution sites of the substituent groups in the carboxymethyl chitosan, the raw material source is wide, and the cost is low.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the relative proliferation rates of cells when the different extracts of Experimental example 1 were used for culturing L929 fibroblasts.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In one aspect of the present invention, the present invention provides an injectable hydrogel comprising the following raw materials:

the small molecule cross-linking agent comprises an aldehyde cross-linking agent with a molecular weight of less than 200 (for example, 100, 86, 72, 58 and the like can be realized).

The molecular weight of the small-molecule crosslinking agent refers to the actual relative molecular weight of the small-molecule crosslinking agent.

In addition, the macromolecular biological cross-linking agent and the micromolecular cross-linking agent are aldehyde cross-linking agents and react with amino, so a certain competitive relationship exists, the macromolecular cross-linking agent reacts slowly due to steric hindrance, and the micromolecular cross-linking agent reacts quickly, so the time for forming gel is controlled by adjusting the proportion of the macromolecular biological cross-linking agent and the micromolecular cross-linking agent, the gel forming time of the obtained injectable hydrogel can be 0.5-15 minutes, the raw material source is wide, and the cost is low.

In some embodiments of the invention, the small molecule cross-linking agent comprises at least one of glutaraldehyde, glyoxal, malondialdehyde, and succindialdehyde.

In some embodiments of the invention, the molar ratio of aldehyde groups in the macromolecular biological crosslinker to the small molecule crosslinker is from 0.05 to 20:1 (e.g., can be 0.05: 1, 1:1, 3:1, 5:1, 7:1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, or 20:1, etc.). Relative to the above molar ratio range, when the molar ratio of aldehyde groups in the macromolecular biological cross-linking agent and the small molecular cross-linking agent is less than 0.05: 1, the gel forming time is less than 0.5min, which is not beneficial to fully mixing and carrying out subsequent operation; when the molar ratio of aldehyde groups in the macromolecular biological cross-linking agent to the aldehyde groups in the micromolecular cross-linking agent is more than 20:1 hour, the gelling time is too long, the exposure time is increased, and the clinical use is not facilitated.

In some embodiments of the invention, the ratio of the total number of moles of aldehyde groups in the macromolecular biological crosslinker and the small molecule crosslinker to the number of moles of saccharide units in the carboxymethyl chitosan is 0.05 to 1:1 (e.g., may be 0.05: 1, 0.1:1, 0.3:1, 0.5:1, 0.7:1, 0.9: 1, or 1:1, etc.). Relative to the above molar ratio range, when the ratio of the total number of moles of aldehyde groups in the macromolecular biological crosslinking agent and the small molecular crosslinking agent to the number of moles of sugar units in the carboxymethyl chitosan is less than 0.05: 1, the content of the cross-linking agent is low, the gelling speed is slow, and even the gelling cannot be realized; when the ratio of the total mole number of aldehyde groups in the macromolecular biological crosslinking agent and the small molecular crosslinking agent to the mole number of sugar units in the carboxymethyl chitosan is more than 1:1, too high a crosslinking agent content, high gel strength, poor flexibility, and unsuitability for intra-articular injection.

In some embodiments of the invention, the degree of oxidation of the macromolecular bioconjugate ranges from 10% to 80% (e.g., can be 10%, 20%, 40%, 60%, or 80%, etc.). Compared with the oxidation degree range, when the oxidation degree of the macromolecular biological cross-linking agent is less than 10%, a large amount of macromolecular biological cross-linking agent is needed for providing aldehyde groups in a certain proportion, and the macromolecular biological cross-linking agent with low oxidation degree has large molecular weight, so that the macromolecular biological cross-linking agent has high viscosity when dissolved in a certain amount of solvent, even can not be completely dissolved, is not beneficial to full mixing reaction, and if the amount of the solvent is increased, the macromolecular biological cross-linking agent and the small molecular cross-linking agent are diluted, so that the reaction speed is reduced; when the oxidation degree of the macromolecular biological cross-linking agent is more than 80%, the macromolecular biological cross-linking agent is oxidized more thoroughly in the preparation process, the chain breaking is serious, the molecular weight of the product is low, the aldehyde group content is high, the performance is closer to that of a micromolecular cross-linking agent, and the regulation of the gelling time by the macromolecular steric hindrance effect is not facilitated.

The oxidation degree of the macromolecular biological cross-linking agent is the ratio of the number of the sugar units containing the dialdehyde groups to the total number of the sugar units.

In some embodiments of the invention, the carboxymethyl chitosan (CMCTS) comprises at least one of N, O-CMCTS, O-CMCTS, and N-CMCTS. Therefore, the material has wide sources and low price, and the gel forming time is not limited by the substitution sites of the substituent groups in the carboxymethyl chitosan.

In some embodiments of the invention, the carboxymethyl chitosan has an average degree of substitution of 50-200% (e.g., may be 50%, 80%, 100%, 120%, 150%, 180%, or 200%, etc.). Relative to the above substitution range, when the average substitution degree of carboxymethyl chitosan is less than 50%, biocompatibility is poor, and it is not suitable as a medical material; when the average degree of substitution of carboxymethyl chitosan is higher than 200%, most of amino groups are substituted, the content of free amino groups is low, the number of crosslinking sites is small, and effective crosslinking cannot be achieved.

In some embodiments of the invention, the injectable hydrogel further comprises: an osmotic pressure regulator and/or a buffer; preferably, the osmolality adjusting agent comprises sodium chloride; preferably, the buffer comprises sodium dihydrogen phosphate and disodium hydrogen phosphate. Thus, the injectable hydrogel obtained has better properties.

In another aspect of the present invention, there is provided a method for preparing the injectable hydrogel as described above, comprising:

mixing a macromolecular biological crosslinking agent, a micromolecular crosslinking agent and carboxymethyl chitosan, and reacting to obtain the injectable hydrogel. Therefore, the operation is simple and convenient, and the realization is easy.

It should be noted that the macromolecular biological crosslinking agent, the small molecular crosslinking agent and the carboxymethyl chitosan are consistent with the foregoing description, and will not be described in detail herein.

In some embodiments of the present invention, the above preparation method comprises:

and mixing the first solution and the second solution, and performing covalent crosslinking to obtain the injectable hydrogel.

In some embodiments of the invention, the first solvent and the second solvent each independently comprise a mixture of a buffered saline solution and an osmotic pressure regulator, and/or a physiological saline.

In some embodiments of the invention, the covalent crosslinks have a crosslinking ratio of 0.05 to 1:1 (e.g., can be 0.05: 1, 0.1:1, 0.3:1, 0.5:1, 0.7:1, 0.9: 1, or 1:1, etc.).

The crosslinking ratio is a ratio of the total number of moles of aldehyde groups in the macromolecular biological crosslinking agent and the small molecular crosslinking agent to the number of moles of sugar units in the CMCTS.

In some embodiments of the invention, the second solution has a mass concentration of 1-10% (w/v); the volumes of the first solution and the second solution are the same when the first solution and the second solution are mixed. Thus, injectable hydrogels with appropriate gel times can be obtained.

In some embodiments of the invention, the first solution and the second solution are filtered and sterilized prior to mixing.

In some embodiments of the invention, the injectable hydrogel is injected into the body after being fully mixed in vitro in joint cavity viscoelastic substance supplementation treatment, or the first solution and the second solution are respectively injected by using a duplex syringe and are mixed into the gel in situ.

In some embodiments of the present invention, the above preparation method comprises the steps of:

a. sodium periodate reacts with sodium Hyaluronate (HA) to prepare oxidized sodium hyaluronate (O-HA) containing dialdehyde;

b. dissolving O-HA and Glutaraldehyde (GA) in normal saline according to a certain proportion to obtain a first solution, wherein the aldehyde group content ratio of O-HA to GA is 0.05-20:1, and filtering to remove bacteria for later use;

c. dissolving carboxymethyl chitosan (CMCTS) in normal saline to obtain a second solution with the concentration of 1-10% (w/v), and filtering to remove bacteria for later use;

d. and c, uniformly mixing and stirring the solutions in the b and the c, and carrying out covalent crosslinking to obtain the hydrogel, wherein the crosslinking ratio (the number of aldehyde groups: the number of CMCTS sugar units) is 0.05-1: 1.

In another aspect of the invention, the invention provides the use of an injectable hydrogel as hereinbefore described for joint lubrication.

In another aspect of the invention, the invention provides a joint lubricant comprising the injectable hydrogel as described above.

Some embodiments of the present invention will be described in detail below with reference to specific examples. The embodiments described below and the features of the embodiments can be combined with each other without conflict.

Examples

In the following examples and comparative examples, unless otherwise specified, the details of CMCTS-1, CMCTS-2, CMCTS-3, CMCTS-4 and CMCTS-5 are as follows:

CMCTS-1: commercially available carboxymethyl Chitosan (carboxymethyl Chitosan manufactured by Heppe Medical Chitosan, Germany) contains N, O-CMCTS and O-CMCTS with the substitution degree of 129.8 percent;

CMCTS-2: commercially available carboxymethyl chitosan (carboxymethyl chitosan produced by Delaware sea-Li-an), with N, O-CMCTS, O-CMCTS, substitution degree of 85.5%;

CMCTS-3: the self-made carboxymethyl chitosan with the components of N, O-CMCTS and O-CMCTS with the substitution degree of 178.2 percent is prepared by the following specific steps:

(1) putting 10.0g of powdery chitosan into a four-neck flask, adding 500ml of isopropanol, and stirring at 30 ℃ for 1 h; (2) dissolving 45g of NaOH in 50ml of water, cooling to room temperature by using cold water, adding the solution into a four-neck flask, and stirring for 2 hours at 30 ℃ to obtain alkalized chitosan; (3) slowly adding 90.0g of chloroacetic acid into the alkalized chitosan for several times under the stirring state, then adjusting the temperature to 60 ℃, and reacting for 4 hours to obtain a carboxymethyl chitosan crude product; (4) washing the crude product with 400ml of 75% ethanol solution by mass fraction for 3 times, dissolving in 1000ml of purified water, adjusting pH to be neutral with 1mol/L hydrochloric acid, precipitating with absolute ethanol, filtering, and drying at 60 ℃ for 7h to obtain white powdery carboxymethyl chitosan;

CMCTS-4: carboxymethyl chitosan was prepared according to the following method:

suspending 10.0g of chitosan in 500ml of distilled water, standing overnight at room temperature, adding 6.0g of glyoxylic acid, stirring for reaction for 45min, then adding 2mol/L of NaOH solution to adjust the pH of the reaction solution to 12, and slowly dropping 100ml of aqueous solution containing 0.4g of NaBH₄The reaction mixture was stirred for 50 min. Pouring the reaction product into a beaker containing 2000ml of 80% ethanol, and fully stirringStirring for alcohol precipitation, vacuum-filtering with Buchner funnel until the solution is neutral, washing with 95% ethanol twice, dehydrating with anhydrous ethanol, and oven-drying the product at 60 deg.C to obtain white powdery carboxymethyl chitosan with substitution degree of 66.4% and N-CMCTS as component;

CMCTS-5: the preparation method of the carboxymethyl chitosan comprises the following steps:

1. preparation of O-CMCH: weighing 5.0g of chitin, and mixing the chitin with KOH according to the molar ratio of the chitin repeating unit to the KOH of 1: and 8.0, weighing 11.03g of KOH to prepare a KOH solution with the mass percentage concentration of 25%, dispersing chitin powder in the KOH solution, uniformly stirring, vacuumizing in a vacuum drying oven for 30min, and freezing in a refrigerator at the temperature of-20 ℃ for overnight after complete alkali penetration. The next day, the alkalized chitin is transferred into a reaction kettle, 75mL of isopropanol serving as a dispersing agent is added, the mixture is stirred uniformly at room temperature, 2.33g of chloroacetic acid is added in four times (the addition is finished within 1 hour), and the mixture is reacted for 5 hours at the constant temperature of 15 ℃. After the reaction is finished, carrying out suction filtration, dissolving filter residues in water, adding hydrochloric acid to adjust the pH value to about 8, filtering with 400-mesh filter cloth, carrying out ultrafiltration on the filtrate for 5 times, wherein the volume compression ratio of each time is 1/5, the molecular weight cut off by the ultrafiltration membrane is more than 10,000, and carrying out forced air drying on the ultrafiltered concentrated solution in a 60-DEG C drying oven to obtain O-CMCH;

2. preparation of O-CMCTS: weighing 2.0g of the O-CMCH prepared in the step 1, wherein the mass ratio of the O-CMCH to KOH is 1: weighing 16.0g of KOH according to the proportion of 8.0 to prepare a KOH solution with the mass percentage concentration of 45%, dispersing O-CMCH in the KOH solution, stirring uniformly, and performing deacetylation reaction for 5 hours at the constant temperature of 75 ℃. After the reaction is finished, cooling, adding a proper amount of hydrochloric acid into the sticky matter to adjust the pH value to about 8, and fully stirring until the hydrochloric acid is dissolved. Filtering with 400 mesh filter cloth, evaporating most water from the filtrate, precipitating with anhydrous ethanol to obtain product, filtering, dissolving the residue in water, precipitating with anhydrous ethanol, precipitating for 3 times, filtering, and drying in 60 deg.C oven by blowing to obtain O-CMCTS with substitution degree of 82.4%.

Examples 1 to 7, comparative example 1

The injectable hydrogel is prepared as follows:

dissolving 1.0g of commercially available carboxymethyl chitosan CMCTS-1 in 20ml of normal saline to prepare a solution with the concentration of 5%, and filtering and sterilizing the solution for later use; dissolving O-HA and GA in 20ml of normal saline to prepare a solution, wherein the molar ratio of aldehyde groups in the O-HA to the GA is 1:1, and filtering and sterilizing the solution for later use; mixing the two solutions (the crosslinking ratio is 0.3:1), stirring uniformly, pouring the beaker every 15s, and measuring the gelling time of different hydrogels;

wherein the oxidation degrees of O-HA in examples 1-7 are 5%, 10%, 20%, 40%, 60%, 80% and 90%, respectively, the oxidation degree of O-HA in comparative example 1 is 0, and the gelling times of the injectable hydrogels in examples 1-7 and comparative example 1 are shown in Table 1 below:

TABLE 1

Examples 8 to 14

The injectable hydrogel is prepared as follows:

dissolving 1.0g of carboxymethyl chitosan CMCTS-1 in 20ml of normal saline to prepare a solution with the concentration of 5%, and filtering and sterilizing the solution for later use; dissolving O-HA (with oxidation degree of 40%) and GA in 20ml of normal saline to prepare a solution, and filtering and sterilizing the solution for later use; mixing the two solutions (the crosslinking ratio is 0.5:1), stirring uniformly, pouring the beaker every 15s, and measuring the gelling time of different hydrogels;

wherein the ratio of the number of moles of aldehyde groups in O-HA to the total number of moles of aldehyde groups in O-HA and GA in examples 8-14 is shown in Table 2 below, and the gel formation time of the injectable hydrogels in examples 8-14 is shown in Table 2 below:

TABLE 2

Examples 15 to 19

The injectable hydrogel is prepared as follows:

dissolving 1.0g of commercially available carboxymethyl chitosan CMCTS-1 in 20ml of normal saline to prepare a solution with the concentration of 5%, and filtering and sterilizing the solution for later use; dissolving O-HA and GA in 20ml of normal saline, preparing a solution with the molar ratio of aldehyde groups in the O-HA to GA being 1:1, and filtering and sterilizing the solution for later use; mixing the two solutions, stirring uniformly, pouring the beakers at intervals of 15s, and measuring the gelling time of different hydrogels;

wherein the cross-linking ratio of the two solutions of examples 15-19 is shown in Table 3 below, and the gel formation time of the injectable hydrogel is shown in Table 3 below:

TABLE 3

Examples 20 to 24

The injectable hydrogel is prepared as follows:

dissolving 1.0g of CMCTS in 20ml of normal saline to prepare a solution with the concentration of 5%, and filtering and sterilizing the solution for later use; dissolving O-HA (oxidation degree of 40%) and GA in 20ml of normal saline, preparing a solution with the molar ratio of aldehyde groups in O-HA to GA being 1:1, and filtering and sterilizing the solution for later use; mixing the two solutions (the crosslinking ratio is 0.3:1), stirring uniformly, pouring the beaker every 15s, and measuring the gelling time of different hydrogels;

where the CMCTS in examples 20-25 are CMCTS-1, CMCTS-2, CMCTS-3, CMCTS-4, CMCTS-5, and CMCTS with a degree of substitution of 0, respectively, the gel-forming times for the injectable hydrogels of examples 20-24 are shown in Table 4 below:

TABLE 4

Example 25

The injectable hydrogel is prepared as follows:

dissolving 0.5g of CMCTS-1 in 10ml of normal saline to prepare a solution with the concentration of 5%, and filtering and sterilizing the solution for later use; dissolving O-HA (oxidation degree of 50%) and GA in 10ml of normal saline, preparing a solution with the molar ratio of aldehyde groups of O-HA to GA being 1:1, and filtering and sterilizing the solution for later use; mixing the above two solutions, stirring well (crosslinking ratio of 0.2:1), sucking 0.5ml with a syringe before hydrogel forming, and pushing out to obtain injectable hydrogel.

Example 26

The injectable hydrogel is prepared as follows:

dissolving 0.1g of CMCTS-2 in 2ml of normal saline to prepare a solution A with the concentration of 5%, and filtering and sterilizing for later use; dissolving O-HA (oxidation degree of 50%) and GA in 2ml of normal saline, preparing solution B with the molar ratio of aldehyde groups in O-HA and GA being 0.1:1, and filtering and sterilizing for later use; and respectively sucking the solution A and the solution B into two syringes of a double syringe, and mixing and pushing out the solution A and the solution B through the double syringe to form the injectable hydrogel, wherein the crosslinking ratio of the solution A and the solution B is 0.5:1 when the solution A and the solution B are mixed.

Example 27

The injectable hydrogel is prepared as follows:

dissolving 0.5g CMCTS-3 in 10ml normal saline to prepare a solution with the concentration of 5 percent, and filtering and sterilizing; dissolving O-HA (oxidation degree of 40%) and GA in 10ml of normal saline, preparing a solution with the molar ratio of aldehyde groups in O-HA to GA being 2:1, and filtering and sterilizing; mixing the above two solutions (crosslinking ratio of 0.1:1), stirring, sucking 0.5ml with syringe before hydrogel forming, and pushing out to obtain injectable hydrogel.

Example 28

The injectable hydrogel is prepared as follows:

dissolving 0.1g of CMCTS-5 in 2ml of normal saline to prepare a solution A with the concentration of 5%, and filtering and sterilizing; dissolving O-HA (oxidation degree of 50%) and GA in 2ml of normal saline, preparing solution B with the molar ratio of aldehyde groups in O-HA and GA being 0.5:1, and filtering for sterilization; and respectively sucking the solution A and the solution B into two syringes of a double syringe, and mixing and pushing out the solution A and the solution B through the double syringe to form the injectable hydrogel, wherein the crosslinking ratio of the solution A and the solution B is 0.7:1 when the solution A and the solution B are mixed.

Test example 1

Injectable hydrogel cytotoxicity test

The injectable hydrogels of examples 25 to 28 were each extracted with DMEM medium at a ratio of 0.1g/ml at 37 ℃ for 48 hours, and the extract was used for culturing L929 fibroblasts, and the relative proliferation rate of 24 hours of cells was measured by MTT method, as shown in FIG. 1, wherein the blank control group was cells cultured with DMEM medium.

The results show that the relative proliferation rate of the L929 fibroblasts is more than 70%, each injectable hydrogel has no obvious cytotoxicity, and in addition, the relative proliferation rate of the cells is gradually increased along with the improvement of the substitution degree of CMCTS, so that the biocompatibility is improved.

Example 29

The injectable hydrogel is prepared as follows:

dissolving 1g of CMCTS-4 in 20ml of normal saline to prepare a solution A with the concentration of 6%, and filtering and sterilizing; dissolving oxidized sodium alginate (O-SA) with oxidation degree of 50% and GA in 20ml of normal saline, wherein the molar ratio of aldehyde groups in O-SA and GA is 0.6:1, preparing a solution B, and filtering and sterilizing; and mixing the two solutions, and uniformly stirring, wherein the crosslinking ratio of the solution A to the solution B is 0.8: 1. The beaker was poured every 15s and the gelling time was measured to be 115 s.

Example 30

The injectable hydrogel is prepared as follows:

dissolving 1g of CMCTS-1 in 20ml of normal saline to prepare a solution A with the concentration of 3%, and filtering and sterilizing; dissolving oxidized carboxymethyl cellulose (O-CMC) with the oxidation degree of 60% and GA in 20ml of normal saline, preparing solution B with the molar ratio of aldehyde groups in the O-CMC and the GA being 0.3:1, and filtering and sterilizing; and mixing the two solutions, and uniformly stirring, wherein the crosslinking ratio of the solution A to the solution B is 0.4: 1. The beaker was poured every 15s and the gelling time was found to be 185 s.

Example 31

The injectable hydrogel is prepared as follows:

dissolving 1g of CMCTS-2 in 20ml of normal saline to prepare a solution A with the concentration of 8%, and filtering and sterilizing; dissolving oxidized dextran (O-Gn) with oxidation degree of 60% and GA in 20ml of normal saline, wherein the molar ratio of aldehyde groups in O-Gn and GA is 0.3:1, preparing a solution B, and filtering and sterilizing; and mixing the two solutions, and uniformly stirring, wherein the crosslinking ratio of the solution A to the solution B is 0.4: 1. The beaker was poured every 15s and the gelling time was found to be 165 s.

Example 32

The injectable hydrogel is prepared as follows:

dissolving 1g of CMCTS-2 in 20ml of buffer salt solution to prepare a solution A with the concentration of 6%, and filtering and sterilizing; dissolving oxidized sodium alginate (O-SA) with the oxidation degree of 60% and succinaldehyde in 20ml of buffer salt solution, preparing solution B with the molar ratio of aldehyde groups in the O-SA and the succinaldehyde of 0.5:1, and filtering for sterilization; and mixing the two solutions, and uniformly stirring, wherein the crosslinking ratio of the solution A and the solution B is 0.6:1, the buffer salt solution contains 0.5mg/ml of disodium hydrogen phosphate, 0.15mg/ml of sodium dihydrogen phosphate and 8mg/ml of sodium chloride. The beaker was poured every 15s and the gelling time was measured to be 105 s.

Comparative example 2

Injectable hydrogel was prepared as in example 28, except that the injectable hydrogel was prepared without O-HA.

Comparative example 3

An injectable hydrogel was prepared in the same manner as in example 28, except that the injectable hydrogel was prepared without GA.

Test example 2

Application of injectable hydrogel in lubricating joints of experimental rabbits

32 experimental animals of New Zealand rabbits were subjected to osteoarthritis modeling by removing joint capsule and synovial membrane model, and the model was divided into 8 groups, i.e., experiment group 1, experiment group 2, experiment group 3, experiment group 4, experiment group 5, experiment group 6, positive control group, and negative control group.

After molding 2w, each experimental group 1-6 was administered 0.5ml of the injectable hydrogel of example 25, example 26, example 27, example 28, comparative example 2 and comparative example 3, respectively, into the knee joint cavity; the positive control group is administered with medical chitosan (Shanghai Bisheng) for intra-articular injection, and is administered once every 2w for 3 times; the negative control group was given physiological saline every 2w for 3 times. The control group was sacrificed 2w after the last administration and the cartilage surface destruction was scored according to the criteria shown in table 5 below, and the scoring results are shown in table 6 below:

TABLE 5

TABLE 6

Statistical analysis on each group shows that the scores of 1-4 of each experimental group are lower than those of a negative control group, and significant difference exists; the scores of the experimental groups 1-4 are not significantly different from those of the positive control group. The scores of the experimental groups 5 and 6 are slightly lower than those of the negative control group, but the statistical difference is not significant, and the scores are significantly different than those of the positive control group. The injectable hydrogel prepared by the invention has good effect on cartilage lubrication protection in osteoarthritis treatment, and plays a role in prolonging treatment effect and reducing administration times through crosslinking.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An injectable hydrogel, comprising the following raw materials:

the small molecular cross-linking agent comprises an aldehyde cross-linking agent with the molecular weight of less than 200;

the molar ratio of aldehyde groups in the macromolecular biological cross-linking agent to aldehyde groups in the micromolecular cross-linking agent is 0.05-20: 1;

the ratio of the total mole number of aldehyde groups in the macromolecular biological cross-linking agent and the micromolecular cross-linking agent to the mole number of sugar units in the carboxymethyl chitosan is 0.05-1: 1;

the oxidation degree of the macromolecular biological cross-linking agent is 10-80%;

the average degree of substitution of the carboxymethyl chitosan is 50-200%.

2. The injectable hydrogel of claim 1, wherein the small molecule cross-linking agent comprises at least one of glutaraldehyde, glyoxal, malondialdehyde, and succindialdehyde.

3. The injectable hydrogel of claim 1 or 2, wherein said carboxymethyl chitosan comprises at least one of N, O-CMCTS, and N-CMCTS.

4. The injectable hydrogel of claim 1, further comprising: an osmotic pressure regulator and/or a buffer.

5. The injectable hydrogel of claim 4 wherein said tonicity modifier comprises sodium chloride.

6. The injectable hydrogel of claim 4, wherein said buffer comprises monobasic sodium phosphate and dibasic sodium phosphate.

7. A method of preparing an injectable hydrogel of any one of claims 1 to 6 comprising:

8. The method of claim 7, comprising:

9. The method of claim 8, wherein the first and second solvents each independently comprise a mixture of a buffered saline solution and an osmotic pressure regulator, and/or a physiological saline.

10. The method of claim 8, wherein the covalent crosslinking has a crosslinking ratio of 0.05 to 1: 1;

the crosslinking ratio refers to the ratio of the total number of moles of aldehyde groups in the macromolecular biological crosslinker and the small molecular crosslinker to the number of moles of sugar units in the CMCTS.

11. The method of claim 8, wherein the second solution has a mass concentration of 1-10% w/v.

12. The method of claim 8, wherein the first solution and the second solution are mixed in the same volume.

13. The method of claim 8, wherein the first solution and the second solution are filtered and sterilized prior to mixing.

14. Use of an injectable hydrogel according to any one of claims 1 to 6 for the preparation of a joint lubricating product.

15. A joint lubricant comprising the injectable hydrogel of any one of claims 1 to 6.