CN113999404B

CN113999404B - Preparation method of double-crosslinked stem cell sphere hydrogel for osteoarthritis

Info

Publication number: CN113999404B
Application number: CN202111177562.XA
Authority: CN
Inventors: 张磊; 司维; 隋悦; 李勇; 季维智
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2024-01-30
Anticipated expiration: 2041-10-09
Also published as: CN113999404A

Abstract

The invention discloses a preparation method of double-crosslinked stem cell sphere hydrogel for osteoarthritis, belonging to the field of biological material preparation. Can be used as adhesive substitute for synovial fluid after cartilage operation, and can be used for treating osteoarthritis, subchondral injury, osteoporosis, synovitis, tenosynovitis, tendinitis, tendinosis, and interstitial cystitis. The hydrogel is injectable double-crosslinked hydrogel, and is prepared by modifying hyaluronic acid and chondroitin sulfate which are derived from joint cavity matrix through hydroxyl groups and activating double crosslinking through EDC. The double-crosslinked stem cell pellet hydrogel obtained by the reaction has good mechanical property, controllable degradation rate, contribution to survival of 3D cell pellets, long-acting moisture preservation, lubrication and slow release of biological factors, high drug loading rate and light responsiveness, and potential application value in the fields of injectable drug slow release carriers, minimally invasive treatment and the like.

Description

Preparation method of double-crosslinked stem cell sphere hydrogel for osteoarthritis

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of double-crosslinked stem cell sphere hydrogel for osteoarthritis.

Technical Field

Osteoarthritis has 80% of the incidence in people over 55 years old. There are about 1.1 hundred million knee osteoarthritis patients in our country. The characteristics of high morbidity and high disability rate become a national heavy medical and economic burden. The regeneration and repair capacity of the articular cartilage is very limited. Current drug treatment can only relieve pain but cannot radically cure. Arthritis progresses to the end stage and only joint replacement surgery can be selected.

Sodium Hyaluronate (HA) and chondroitin sulfate (ChS) are dextran aldehyde acids which have no species specificity and widely exist in tissues such as placenta, amniotic fluid, articular cartilage and the like to play roles in lubrication and nourishing. In addition, it has effects of influencing osteoclast differentiation, promoting cartilage formation, and promoting dermal fibroblast generation. Injectable hydrogels, which are easily applied to minimally invasive surgery specific sites and to the convenience of patients, have attracted widespread attention for clinical medicine. However, one of the main limitations of using natural hydrogels to design injectable drug-carrying/cell systems is that they have weak mechanical properties, uncontrollable rheological properties and degradability, and greatly limited their slow release and therapeutic effects, so that the comprehensive improvement of the mechanical properties and degradability of injectable hydrogels has become a research hotspot in the biomedical hydrogels field.

However, conventional degradable natural polymer hydrogels have the following disadvantages: the hydrogel has poor mechanical properties, is difficult to adapt to various mechanical environments where joints are located, and limits the application of the hydrogel as an osteochondral replacement material; secondly, the thickness of the cartilage layer is limited, the cartilage injury can cause the injury of subchondral bone, the injury of subchondral bone can influence the metabolism of the cartilage layer, and the simple cartilage substitute material is not beneficial to the repair of the morphology and the structure function of the cartilage; the double-layer hydrogel is similar to the articular cartilage in structure, and is favorable for repairing the articular cartilage, but the current research shows that the interface bonding property of the upper layer and the lower layer of the double-layer hydrogel is poor, the gradient function repair can not be realized, and meanwhile, the problems of high short-term release, low long-term activity and the like of the upper layer of hydrogel are easily caused by swelling and soaking adsorption factors.

Based on the method, the grafting rate of the methacryloyl HA and ChS is controlled by the amount of methacrylic anhydride, and double-network activation crosslinking is carried out, so that the injectable composite gel material with controllable degradation rate and enhanced mechanical property is obtained, and the 3D stem cell sphere is combined, so that the free radical oxidation resistance is stronger, the lubrication and slow release efficiency is higher, and the treatment of osteoarthritis and the recovery of joint function are more facilitated.

Disclosure of Invention

Aiming at the defects in the research field, the invention provides the photopolymerisable injectable double-crosslinked stem cell sphere hydrogel for osteoarthritis and the preparation method thereof by utilizing the activated self-assembled double-crosslinked system, and the obtained double-crosslinked gelatin hydrogel has the characteristics of convenient use (injection-in-vivo solidification), excellent mechanical property, controllable degradation and good biocompatibility, contribution to survival of 3D stem cell spheres, long-acting repair of worn articular cartilage and batch preparation. In order to achieve the above purpose, the following technical scheme is adopted:

the invention prepares a double-crosslinked stem cell sphere hydrogel for osteoarthritis, which comprises the following steps:

(1) Adding sodium carbonate into deionized water, diluting and regulating the pH value of a sodium carbonate aqueous solution to be 7-10, adding hyaluronic acid according to the proportion of 5-20g/L, stirring and dissolving at the temperature of 30-55 ℃ to obtain a hyaluronic acid solution, adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the hyaluronic acid is 1-5:1, adding N, N-dimethylformamide according to the proportion of 1-5 ml/L, adding triethylamine according to the proportion of 0.2-0.6 g/L, reacting for 18-40 hours at normal temperature, dialyzing with deionized water at normal temperature, and freeze-drying to obtain the methacryloylated hyaluronic acid, wherein the reaction chemical formula is as follows:

(2) Dissolving chondroitin sulfate in deionized water according to the proportion of 1-20 g/L to prepare a chondroitin sulfate solution, adding methacrylic anhydride with the mass of 0.1-0.5 times of that of the chondroitin sulfate, and then stirring and reacting for 18-40h at the temperature of 4-10 ℃; dialyzing with deionized water at normal temperature, and freeze-drying to obtain methacryloyl chondroitin sulfate, wherein the reaction chemical formula is as follows:

(3) Dissolving dodecyl trimethyl ammonium chloride and sodium chloride in deionized water, adding sodium methylacrylamide, adding a mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adjusting pH to be acidic after complete dissolution, and reacting for 0.2-1.5h at room temperature, wherein in the mixed solution, the adding amount of dodecyl trimethyl ammonium chloride is 1-10 g/L, the adding amount of sodium chloride is 10-30 g/L, the adding amount of sodium methylacrylamide is 10-30 g/L, and the adding amount of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 5-20 g/L; filtering after the reaction is finished, regulating the pH of the filtrate to 7.0-7.4, adding 90-100wt% ethanol to obtain precipitate, washing and drying the precipitate to obtain the methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate, wherein the reaction chemical formula is as follows:

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) with a DMEM culture medium according to the proportion of the addition amount of 2.5-5 g/L under the condition of light shielding, adding 0.5-3% of methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate with the mass percentage concentration after dissolution, completely dissolving, filtering by a 0.22um-0.45um filter, and adding mesenchymal stem cell balls for mixing; the mixed solution is dripped into a culture dish by a pipetting gun, the irradiation time of a blue light lamp is 20-40s, the wavelength of the blue light lamp is 405-465nm, and the mixed solution is added into a DMEM culture medium for culture, so that the double-crosslinked stem cell pellet hydrogel for osteoarthritis is obtained.

Preferably, the conditions for dialysis in steps (1) to (2) of the present invention are: dialyzing at normal temperature for 3-7d, changing water every 6-18h, and keeping the molecular weight cut-off of the dialysis bag at 8kDa-14kDa.

The invention has the beneficial effects that: the grafting rate of the methacryloyl HA and ChS is controlled by the amount of methacrylic anhydride, and double-network activation crosslinking is carried out, so that the injectable composite gel material with controllable degradation rate and enhanced mechanical property is obtained, and the 3D stem cell sphere is combined, so that the free radical oxidation resistance is stronger, the lubrication and slow release efficiency is higher, and the treatment of osteoarthritis and the recovery of joint function are more facilitated.

The HAMA-ChSMA binary gel system prepared by the invention has the composition and structure of highly bionic extracellular matrix (ECM), and has good stability, water retention rate and mechanical property.

The invention takes hyaluronic acid and chondroitin sulfate as raw materials, uses methacrylic anhydride double bond to carry out acylation modification reaction to introduce photocrosslinking, and uses active reaction to introduce and obtain double-crosslinked hydrogel. The invention adopts photocrosslinking to generate an activated double network for the natural polymer hydrogel, so that the natural polymer hydrogel has excellent mechanical property and biocompatibility, provides a new thought and method for preparing the high-strength stem cell hydrogel, and is beneficial to the development and utilization of injectable functional hydrogel materials.

Drawings

FIG. 1 is a gel-forming diagram of a composite material photo stimulus (405 nm wavelength);

FIG. 2 is a double cross-linked stem cell pellet hydrogel construction;

FIG. 3 shows the proliferation assay of chondrocytes with injectable double crosslinked stem cell pellet hydrogels.

Detailed Description

The invention will be described in further detail with reference to specific embodiments, but the scope of the invention is not limited to the description.

Example 1

The preparation method of the double-crosslinked stem cell sphere hydrogel for osteoarthritis specifically comprises the following steps:

(1) Adding sodium carbonate into deionized water, diluting and regulating the pH value of an aqueous solution of sodium carbonate to be 9, adding hyaluronic acid according to the proportion of 20g/L, stirring and dissolving at 50 ℃ to obtain a hyaluronic acid solution, adding N, N-dimethylformamide according to the proportion of 3ml/L into the hyaluronic acid solution, adding triethylamine according to the proportion of 0.2g/L, adding sodium carbonate according to the proportion of 1.0mmol/L every 12h, reacting for 24h at normal temperature, dialyzing for 5d at normal temperature by using deionized water, changing water every 12h, retaining molecular weight of a dialysis bag to 8000-14000Da, and freeze-drying after dialysis is finished to obtain the methacryloylated hyaluronic acid.

(2) Dissolving chondroitin sulfate in deionized water at a ratio of 10g/L to obtain a chondroitin sulfate solution, adding methacrylic anhydride with the mass 0.3 times of that of the chondroitin sulfate, and stirring at 4 ℃ for reaction for 40 hours; dialyzing with deionized water at normal temperature for 5d, changing water every 12h, and freeze drying to obtain methacryloyl chondroitin sulfate with molecular weight cut-off of 8000-14000 Da.

(3) Dissolving dodecyl trimethyl ammonium chloride and sodium chloride in deionized water, adding sodium methylacrylamide, adding a mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adjusting pH to be acidic after complete dissolution, and reacting for 1h at room temperature, wherein in the mixed solution, the adding amount of dodecyl trimethyl ammonium chloride is 5g/L, the adding amount of sodium chloride is 15g/L, the adding amount of sodium methylacrylamide is 120g/L, and the adding amount of the mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 10g/L; filtering after the reaction is finished, regulating the pH of the filtrate to 7.2, adding 95wt% ethanol to obtain a precipitate, washing the precipitate, and drying the precipitate to obtain the methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) with a DMEM culture medium according to the proportion of 3g/L under the light-shielding condition, adding 2% of methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate with the mass percentage concentration after dissolution, completely dissolving, filtering by a 0.22um filter, and adding mesenchymal stem cell spheres for mixing; the mixed solution is dripped into a culture dish by a pipetting gun, the irradiation time of a blue light lamp is 30s, the wavelength of the blue light lamp is 405nm, and the mixed solution is added into a DMEM culture medium for culture, so that the injectable double-crosslinked stem cell pellet hydrogel for osteoarthritis is obtained.

(blue light irradiation time was 25s, blue light wavelength was 405 nm) as shown in FIGS. 1 to 2. Effect of injectable double-crosslinked hydrogel loaded with 3D cell spheres on chondrocyte proliferation; as can be seen from fig. 3, the chondrocytes grow well under the stimulation of the hydrogel, and have remarkable proliferation behavior, which indicates that the double-crosslinked hydrogel has good biocompatibility and the effect of promoting the development of the chondrocytes.

Example 2

(1) Adding sodium carbonate into deionized water, diluting and regulating the pH value of an aqueous solution of sodium carbonate to be 10, adding hyaluronic acid according to the proportion of 5g/L, stirring and dissolving at 30 ℃ to obtain a hyaluronic acid solution, adding N, N-dimethylformamide according to the proportion of 5ml/L into the hyaluronic acid solution, adding triethylamine according to the proportion of 0.6g/L, adding sodium carbonate according to the proportion of 0.8mmol/L every 12h, reacting for 40h at normal temperature, dialyzing for 5d at normal temperature by using deionized water, changing water every 12h, retaining molecular weight of a dialysis bag to 8000-14000Da, and freeze-drying after dialysis is finished to obtain the methacryloylated hyaluronic acid.

(2) Dissolving chondroitin sulfate in deionized water according to the proportion of 2g/L to prepare a chondroitin sulfate solution, adding methacrylic anhydride with the mass of 0.5 times of that of the chondroitin sulfate, and then stirring and reacting for 30 hours at 8 ℃; dialyzing with deionized water at normal temperature for 5d, changing water every 12h, and freeze drying to obtain methacryloyl chondroitin sulfate with molecular weight cut-off of 8000-14000 Da.

(3) Dissolving dodecyl trimethyl ammonium chloride and sodium chloride in deionized water, adding sodium methylacrylamide, adding a mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adjusting pH to be acidic after complete dissolution, and reacting for 1.5 hours at room temperature, wherein in the mixed solution, the adding amount of dodecyl trimethyl ammonium chloride is 10g/L, the adding amount of sodium chloride is 30g/L, the adding amount of sodium methylacrylamide is 30g/L, and the adding amount of the mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 20g/L; filtering after the reaction is finished, regulating the pH of the filtrate to 7.0, adding 95wt% ethanol to obtain a precipitate, washing the precipitate, and drying the precipitate to obtain the methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) with a DMEM culture medium according to the proportion of 5g/L under the light-shielding condition, dissolving, adding 0.5 mass percent of methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate, completely dissolving, filtering by a 0.3um filter, and adding mesenchymal stem cell spheres for mixing; the mixed solution is dripped into a culture dish by a pipetting gun, the irradiation time of a blue light lamp is 20s, the wavelength of the blue light lamp is 405nm, and the mixed solution is added into a DMEM culture medium for culture, so that the injectable double-crosslinked stem cell sphere hydrogel for osteoarthritis is obtained, the mechanical property is strong, the degradation rate is slow, and the double-crosslinked hydrogel has good biocompatibility and the effect of promoting the development of chondrocytes.

Example 3

(1) Adding sodium carbonate into deionized water, diluting and regulating the pH value of an aqueous solution of sodium carbonate to be 7, adding hyaluronic acid according to the proportion of 10g/L, stirring and dissolving at 55 ℃ to obtain a hyaluronic acid solution, adding N, N-dimethylformamide according to the proportion of 1ml/L into the hyaluronic acid solution, adding triethylamine according to the proportion of 0.2g/L, adding sodium carbonate according to the proportion of 0.2mmol/L every 12h, reacting for 18h at normal temperature, dialyzing for 5d at normal temperature by using deionized water, changing water every 12h, retaining molecular weight of a dialysis bag to 8000-14000Da, and freeze-drying after dialysis is finished to obtain the methacryloylated hyaluronic acid.

(2) Dissolving chondroitin sulfate in deionized water according to the proportion of 20g/L to prepare a chondroitin sulfate solution, adding methacrylic anhydride with the mass 0.1 times of that of the chondroitin sulfate, and then stirring and reacting for 18 hours at the temperature of 4 ℃; dialyzing with deionized water at normal temperature, and lyophilizing to obtain methacryloyl chondroitin sulfate.

(3) Dissolving dodecyl trimethyl ammonium chloride and sodium chloride in deionized water, adding sodium methylacrylamide, adding a mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adjusting pH to be acidic after complete dissolution, and reacting for 0.2h at room temperature, wherein in the mixed solution, the adding amount of dodecyl trimethyl ammonium chloride is 2g/L, the adding amount of sodium chloride is 10g/L, the adding amount of sodium methylacrylamide is 10g/L, and the adding amount of the mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 5g/L; filtering after the reaction is finished, regulating the pH of the filtrate to 7.4, adding 90wt% ethanol to obtain a precipitate, washing the precipitate, and drying the precipitate to obtain the methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) with a DMEM culture medium according to the proportion of 2.5g/L under the light-shielding condition, dissolving, adding 0.5 mass percent of methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate, completely dissolving, filtering by a 0.45um filter, and adding mesenchymal stem cell spheres for mixing; the mixed solution is dripped into a culture dish by a pipetting gun, the irradiation time of a blue light lamp is 40s, the wavelength of blue light is 465nm, and the mixed solution is added into a DMEM culture medium for culture, so that the injectable double-crosslinked stem cell pellet hydrogel for osteoarthritis is obtained, the mechanical property is low, the degradation rate is high, and the double-crosslinked hydrogel has good biocompatibility and the effect of promoting the development of chondrocytes.

Claims

1. The preparation method of the double-crosslinked stem cell sphere hydrogel for osteoarthritis is characterized by comprising the following steps of:

(1) Adding sodium carbonate into deionized water, diluting and regulating the pH value of an aqueous solution of sodium carbonate to be 7-10, adding hyaluronic acid according to the proportion of 5-20g/L, stirring and dissolving at the temperature of 30-55 ℃ to obtain a hyaluronic acid solution, adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the hyaluronic acid is 1-5:1, adding N, N-dimethylformamide according to the proportion of 1-5 ml/L, adding triethylamine according to the proportion of 0.2-0.6 g/L, reacting for 18-40 hours at normal temperature, dialyzing with deionized water at normal temperature, and freeze-drying to obtain the methacryloylated hyaluronic acid;

(2) Dissolving chondroitin sulfate in deionized water according to the proportion of 1-20 g/L to prepare a chondroitin sulfate solution, adding methacrylic anhydride with the mass of 0.1-0.5 times of that of the chondroitin sulfate, and then stirring and reacting for 18-40h at the temperature of 4-10 ℃; dialyzing with deionized water at normal temperature, and freeze-drying to obtain methacryloyl chondroitin sulfate;

(3) Dissolving dodecyl trimethyl ammonium chloride and sodium chloride in deionized water, adding sodium methylacrylamide, adding a mixed solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, adjusting pH to be acidic after complete dissolution, and reacting for 0.2-1.5h at room temperature, wherein in the mixed solution, the adding amount of dodecyl trimethyl ammonium chloride is 1-10 g/L, the adding amount of sodium chloride is 10-30 g/L, the adding amount of sodium methylacrylamide is 10-30 g/L, and the adding amount of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 5-20 g/L; filtering after the reaction is finished, regulating the pH of the filtrate to 7.0-7.4, adding 90-100wt% ethanol to obtain precipitate, washing the precipitate, and drying to obtain the methacryloyl hyaluronic acid-methacryloyl chondroitin sulfate;

2. The method for preparing the double-crosslinked stem cell pellet hydrogel for osteoarthritis according to claim 1, wherein: the conditions for dialysis in steps (1) to (2) are: dialyzing at normal temperature for 3-7d, changing water every 6-18h, and keeping the molecular weight cut-off of the dialysis bag at 8kDa-14kDa.