WO2018000486A1 - 一种关节润滑材料及其制备方法 - Google Patents

一种关节润滑材料及其制备方法 Download PDF

Info

Publication number
WO2018000486A1
WO2018000486A1 PCT/CN2016/090772 CN2016090772W WO2018000486A1 WO 2018000486 A1 WO2018000486 A1 WO 2018000486A1 CN 2016090772 W CN2016090772 W CN 2016090772W WO 2018000486 A1 WO2018000486 A1 WO 2018000486A1
Authority
WO
WIPO (PCT)
Prior art keywords
chitin
carboxymethyl chitosan
carboxymethyl
reaction
cmcts
Prior art date
Application number
PCT/CN2016/090772
Other languages
English (en)
French (fr)
Inventor
吴奕光
杨帆
江长兵
吴灿光
薛清辉
Original Assignee
深圳市阳光之路生物材料科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市阳光之路生物材料科技有限公司 filed Critical 深圳市阳光之路生物材料科技有限公司
Publication of WO2018000486A1 publication Critical patent/WO2018000486A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/042—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the invention relates to the technical field of medical biomaterials, in particular to an O-carboxymethyl chitosan (O-CMCTS) hydrogel for joint lubrication.
  • O-CMCTS O-carboxymethyl chitosan
  • the artificial total joint replacement materials currently used for the treatment of osteoarthrosis are mostly hard materials such as metal, ceramic and ultra-high molecular polyethylene.
  • the mechanical properties of these prosthetic materials are quite different from those of natural joints, and it is difficult to form a lubricating film on the surface of artificial joints.
  • lubrication of artificial joints with artificial joint lubrication materials is an important measure to reduce friction and wear and improve service life.
  • an injectable bone joint lubricating material and a preparation method thereof discloses an injectable bone joint lubricating material and a preparation method thereof, and the injectable bone joint lubricating material comprises nanocrystalline cellulose and hyaluronic acid substances,
  • the hyaluronic acid substance is a water-soluble salt of hyaluronic acid or hyaluronic acid. Since hyaluronic acid (sodium) is easily degraded by hyaluronidase in the body, multiple injections are required, which prolongs the course of treatment and increases the cost of treatment; in addition, the injection of hyaluronic acid after gel formation in vitro is more difficult.
  • a temperature-sensitive chitosan hydrogel and a preparation method thereof discloses a temperature-sensitive injectable chitosan (CTS) hydrogel, which is cross-linked with a cross-linking agent ⁇ -glycerophosphate ion.
  • CTS temperature-sensitive injectable chitosan
  • a temperature-sensitive injectable hydrogel is prepared, and the prepared hydrogel can be used as a drug carrier and a tissue engineering scaffold.
  • CTS is only soluble in dilute aqueous acid solutions, and this hydrogel requires the use of a large amount of ionic cross-linking agent, so that its biocompatibility, especially in vivo, is affected.
  • the object of the present invention is to provide a covalently crosslinked O-CMCTS hydrogel which has better biocompatibility, biodegradability and bioadsorption than CTS hydrogel.
  • the invention provides a joint lubricating material comprising a covalently crosslinked O-CMCTS hydrogel having a gelation time of 0.5-10 min, a water content of >70% and a water retention rate of >65%.
  • the covalently crosslinked O-CMCTS hydrogel is obtained by gelation of glutaraldehyde (GA) and O-CMCTS, and the molar ratio of the aldehyde group in GA to the amino group in O-CMCTS is 1. : 4-16, the degree of deacetylation (DD) of O-CMCTS is > 25%.
  • the invention also provides a preparation method of the joint lubricating material as described above, comprising the following steps:
  • O-CMCH O-carboxymethyl chitin
  • O-CMCTS was gelled with GA to prepare a covalently crosslinked O-CMCTS hydrogel.
  • CMCTS carboxymethyl chitosan
  • the currently reported preparation of carboxymethyl chitosan (CMCTS) is mostly based on CTS.
  • the alkalized CTS is dispersed in isopropanol, and CTS is carboxylated with chloroacetic acid under heating or microwave irradiation.
  • Get CMCTS The preparation method is simple and easy to operate, but because of the two hydroxyl groups (C 6 -OH and C 3 -OH) and one amino group (C 2 -NH 2 ) in the molecular structure of CTS, carboxymethylation is in this 3
  • the reaction can occur at any position, so the product has three kinds of N, O-CMCTS, N-CMCTS and O-CMCTS, so the method has poor selectivity and the product structure is not simple.
  • chitin is used as a raw material in the existing reaction, and concentrated alkali and chloroacetic acid are added to combine deacetylation and carboxymethylation in one step.
  • This method has few steps, but the selectivity is also poor, and the prepared product has a single structure; in addition, the method cannot control the degree of deacetylation (DD) and carboxymethyl substitution (DS) of the product.
  • the invention prepares O-CMCTS with single product structure, controls products DD and DS, and uses chitin as raw material to prepare O- by carboxymethyl substitution reaction and deacetylation reaction respectively by using steps A1 and A2 as described above. CMCTS.
  • step A1 comprises the following steps:
  • chitin is chitin derived from shrimp shell or crab shell or carp cartilage, and its degree of deacetylation is ⁇ 10%; the molar ratio of chitin repeating unit to base is 1:6-11; It is one of NaOH or KOH.
  • the dispersing agent in step A12 is one of n-propanol, isopropanol and ethanol, and the volume thereof is added in a volume ratio of chitin to the dispersing agent of 1 g: 10-30 mL; chitin and chloroacetic acid are added.
  • the molar ratio is 1:0.8-2.5; the reaction temperature is 10-30 ° C; the reaction time is 2-6 h.
  • the post-treatment is: filtering the reaction liquid, dissolving the residue in water, adding acid to adjust the pH to 8; filtering, leaving the filtrate; ultrafiltration of the filtrate, and filtering the ultrafiltration Drying gives O-CMCH with a molecular weight greater than 10,000, with a DD of 10%-20% and a DS of 50%-100%.
  • the step A2 comprises the steps of: mixing the O-CMCH with the lye to carry out the reaction, adding the acid to adjust the pH to 8, after thoroughning, filtering, and the filtrate is precipitated with ethanol; re-dissolving, precipitating and filtering, and retaining The residue was filtered and dried to give O-CMCTS.
  • the mass percentage concentration of the lye in the step A2 is 30%-60%
  • the base is one of KOH or NaOH
  • the mass ratio of O-CMCH to alkali is 1:5-15
  • the reaction temperature is 60-
  • the reaction time is 2-6 h at 100 ° C
  • the O-CMCTS has a DD of 25% to 90% and a DS of 50% to 100%.
  • chitin is alkalized with a low concentration of lye at a lower temperature to obtain alkalized chitin, and then carboxymethylated with chloroacetic acid at a low temperature to prepare O-CMCH, the optimized The procedure allows its carboxymethyl substitution to occur only at C 6 -OH without C 2 -NH 2 and better control of the DS value of O-CMCH.
  • O-CMCH is deacetylated with a high concentration of lye at a high temperature to prepare O-CMCTS, which can better control the DD and DS of O-CMCTS.
  • the O-CMCH having a molecular weight of more than 10,000 can be better separated from the O-CMCH having a molecular weight of less than 10,000, and the O-CMCH having a large molecular weight is separated, and then obtained by the subsequent step A2.
  • Large molecular weight O-CMCTS Large molecular weight O-CMCTS.
  • reaction formula of the reaction of O-CMCTS with GA to form O-CMCTS hydrogel in step A3 is as follows:
  • the invention adopts the reaction of GA and O-CMCTS to prepare a hydrogel, and the electrostatic adsorption of the carboxylate anion at the C6 position on the O-CMCTS with the C-position amino quasi-cation reduces the reaction speed of GA and amino group, which is beneficial to GA and O.
  • - CMCTS is evenly mixed, and the gel formed is uniform and not easily broken.
  • step A3 comprises the steps of: dissolving O-CMCTS in water, formulating O-CMCTS solution, filtering and sterilizing the solution, adding GA solution, and gelling in a constant temperature water bath to obtain covalently crosslinked O-CMCTS Hydrogels.
  • the mass percentage concentration of the O-CMCTS solution in step A3 is 1% to 3%
  • the mass percentage concentration of GA is 2%
  • the molar ratio of GA to O-CMCTS is aldehyde group in GA and amino group in O-CMCTS.
  • the reaction was carried out at a ratio of 1:4-16, the reaction temperature was 37 ° C, and the gelation time was 0.5-10 min.
  • the obtained O-CMCTS hydrogel had a water content of >70% and a water retention rate of >65%.
  • the covalently crosslinked O-CMCTS hydrogel provided by the invention has the advantages that the gelation time of the covalently crosslinked O-CMCTS hydrogel is controllable, the water content is high and the water retention rate is high, and has good It is biocompatible and can maintain a certain capacity and elasticity of the joint for a certain period of time.
  • the O-CMCTS provided by the invention has the advantages of simple preparation principle, easy operation, low cost, single product structure, controllable by DD and DS, and further reaction with GA to obtain controllable gelation time, high water content and high water retention rate. Valence cross-linked O-CMCTS hydrogel.
  • the preferred embodiment of the present invention further includes the following beneficial effects: the O-CMCTS prepared according to the method, the DD and DS of the O-CMCTS can be controlled at 25%-90% and 50%-100%, respectively, the O-CMCTS Completely reacted with GA, without GA residue, the prepared O-CMCTS hydrogel has a uniform structure, high swelling ratio and good biocompatibility.
  • FIG. 1 is a reaction scheme for preparing an O-carboxymethyl chitosan hydrogel by gelation of O-carboxymethyl chitosan and glutaraldehyde according to an embodiment of the present invention.
  • FIG. 2 is a schematic view showing the change of viscosity of samples G1, G2, and G3 with time according to an embodiment of the present invention.
  • Figure 3 is a graph showing the degradation rates of samples G1, G2 and G3 in lysozyme/PBS solution according to an example of the present invention.
  • FIG. 4 is a cell diagram of samples G1, G2, G3 and a blank group cultured for 24 hours according to an embodiment of the present invention
  • FIG. 4a is a sample G1
  • FIG. 4b is a sample G2
  • FIG. 4c is a sample G3
  • FIG. 4d is a blank group.
  • Preparation of A2 and O-CMCTS Weigh 2.0g of O-CMCH prepared in step A1, and weigh 10g of NaOH to a concentration of 30% by weight of NaOH based on the ratio of O-CMCH to NaOH: 1:5. The solution, the O-CMCH was dispersed in the NaOH solution, stirred uniformly, and deacetylated at 60 ° C for 4 h. After completion of the reaction, the mixture was cooled, and an appropriate amount of hydrochloric acid was added to the viscous material to adjust the pH to about 8, and the mixture was thoroughly stirred until dissolved. Filtration with a 400 mesh filter cloth, distilling off most of the water in the filtrate, and then precipitating the product with absolute ethanol and filtering.
  • Preparation of covalently crosslinked O-CMCTS hydrogel 0.5 g of O-CMCTS prepared in the step A2 is weighed and dissolved in deionized water to prepare an O-CMCTS solution having a mass percentage of 1%. The core funnel was filtered, and the filtrate was placed in an autoclave for 30 min. A GA solution having a mass concentration of 2% was prepared in a fume hood, and the filter was sterilized by filtration. Add 5 mL of prepared sterile O-CMCTS solution to the sterile vial on a clean bench. Add the appropriate amount of GA solution according to the molar ratio of aldehyde group in GA to O-CMCTS of 1:4.
  • Preparation of A1, O-CMCH Weigh 5.0 g of chitin, and weigh 11.03 g of KOH into a 25% by weight KOH solution according to the ratio of chitin repeating unit to KOH molar ratio of 1:8.0.
  • the chitin powder was dispersed in a KOH solution, stirred uniformly, and vacuumed in a vacuum drying oven for 30 min. After the alkali permeation was completed, it was placed in a -20 ° C refrigerator and frozen overnight. The next day, the alkalized chitin was transferred into the reaction vessel, 75 mL of dispersing agent isopropanol was added, and the mixture was uniformly stirred at room temperature.
  • Preparation of A2 and O-CMCTS Weigh 2.0 g of O-CMCH obtained in the step A1, and weigh 16.0 g of KOH to a mass percentage of 45% by mass ratio of O-CMCH to KOH of 1:8.0.
  • O-CMCH was dispersed in a KOH solution, stirred uniformly, and deacetylated at 75 ° C for 5 h.
  • the mixture was cooled, and an appropriate amount of hydrochloric acid was added to the viscous material to adjust the pH to about 8, and the mixture was thoroughly stirred until dissolved. Filtration with a 400 mesh filter cloth, distilling off most of the water in the filtrate, and then precipitating the product with absolute ethanol and filtering.
  • Preparation of covalently crosslinked O-CMCTS hydrogel 0.5 g of O-CMCTS prepared in the step A2 is weighed and dissolved in deionized water to prepare an O-CMCTS solution having a mass concentration of 2%. The core funnel was filtered, and the filtrate was placed in an autoclave for 30 min. A GA solution having a mass concentration of 2% was prepared in a fume hood, and the filter was sterilized by filtration. Add 5 mL of the prepared sterile O-CMCTS solution to the sterile vial on the ultra-clean workbench.
  • Preparation of A1, O-CMCH Weigh 5.0g of chitin, according to the ratio of chitin repeating unit to NaOH molar ratio of 1:9.0, weigh 8.87g NaOH to form a 35% by weight NaOH solution, the carapace The powder was dispersed in a NaOH solution, stirred uniformly, and vacuumed in a vacuum oven for 30 minutes. After the alkali was completely infiltrated, it was placed in a refrigerator at -20 ° C overnight. On the next day, the alkalized chitin was transferred into the reaction vessel, 90 mL of dispersing agent isopropanol was added, and the mixture was stirred at room temperature.
  • Preparation of A2 and O-CMCTS Weigh 2.0 g of O-CMCH prepared in the step A1, and weigh 20 g of NaOH to a mass percentage of 50% by mass ratio of O-CMCH to NaOH of 1:10. NaOH solution, O-CMCH was dispersed in NaOH solution, stirred uniformly, and deacetylated at 80 ° C for 6 h. After completion of the reaction, the mixture was cooled, and an appropriate amount of hydrochloric acid was added to the viscous material to adjust the pH to about 8, and the mixture was thoroughly stirred until dissolved. Filtration with a 400 mesh filter cloth, distilling off most of the water in the filtrate, and then precipitating the product with absolute ethanol and filtering.
  • Preparation of covalently crosslinked O-CMCTS hydrogel 0.5 g of O-CMCTS prepared in the step A2 is weighed and dissolved in deionized water to prepare an O-CMCTS solution having a mass concentration of 3%. The core funnel was filtered, and the filtrate was placed in an autoclave for 30 min. A GA solution having a mass concentration of 2% was prepared in a fume hood, and the filter was sterilized by filtration. Add 5 mL of prepared sterile O-CMCTS solution to the sterile vial on a clean bench. Add the appropriate amount of GA solution according to the ratio of the amino group in the GA to the amino group in the O-CMCTS ratio of 1:8. Shake for 10 seconds to mix the two uniformly, and then observe the gelation process in a 37 ° C constant temperature water bath with a gelation time of 3.0 min. The water content was determined to be 84.3%, and the water retention rate was 85.6%.
  • Preparation of A1, O-CMCH Weigh 5.0g of chitin, according to the ratio of chitin repeating unit to NaOH molar ratio of 1:10.0, weigh 9.85g of NaOH to form a 30% by mass NaOH solution, chitin The powder was dispersed in a NaOH solution, stirred uniformly, and vacuumed in a vacuum drying oven for 30 minutes. After the alkali was completely infiltrated, it was placed in a -20 ° C refrigerator and frozen overnight. The next day, the alkalized chitin was transferred into the reaction vessel, 100 mL of the dispersing agent ethanol was added, and the mixture was uniformly stirred at room temperature.
  • O-CMCTS Weigh 2.0g of O-CMCH prepared in step A1, according to O-CMCH The ratio of mass ratio to NaOH is 1:12, 24.0 g of NaOH is weighed into a 55% by weight NaOH solution, O-CMCH is dispersed in NaOH solution, stirred uniformly, and deacetylated at 85 ° C for 3 h. . After completion of the reaction, the mixture was cooled, and an appropriate amount of hydrochloric acid was added to the viscous material to adjust the pH to about 8, and the mixture was thoroughly stirred until dissolved. Filtration with a 400 mesh filter cloth, distilling off most of the water in the filtrate, and then precipitating the product with absolute ethanol and filtering.
  • Preparation of covalently crosslinked O-CMCTS hydrogel 0.5 g of O-CMCTS prepared in the step A2 is weighed and dissolved in deionized water to prepare an O-CMCTS solution having a mass concentration of 2%. The core funnel was filtered, and the filtrate was placed in an autoclave for 30 min. A GA solution having a mass concentration of 2% was prepared in a fume hood, and the filter was sterilized by filtration. Add 5 mL of prepared sterile O-CMCTS solution to the sterile vial on a clean bench. Add the appropriate amount of GA solution according to the molar ratio of aldehyde group in GA to O-CMCTS in the ratio of 1:12. Shake for 10 seconds to mix the two evenly, and then observe the gelation process in a constant temperature water bath at 37 ° C. The gelation time was 4.5 min. The water content was determined to be 83.7%, and the water retention rate was 82.4%.
  • Preparation of A1 and O-CMCH Weigh 5.0 g of chitin, and weigh 15.17 g of KOH into a KOH solution with a concentration of 35% by mass according to the ratio of chitin repeating unit to KOH molar ratio of 1:11.0. The powder was dispersed in a KOH solution, stirred uniformly, and vacuumed in a vacuum oven for 30 minutes. After the alkali was completely infiltrated, it was placed in a refrigerator at -20 ° C overnight. The next day, the alkalized chitin was transferred into the reaction vessel, 150 mL of dispersing agent isopropanol was added, and the mixture was uniformly stirred at room temperature.
  • Preparation of A2 and O-CMCTS Weigh 2.0 g of O-CMCH obtained in the step A1, and weigh 30.0 g of KOH to a mass percentage of 60% according to a ratio of O-CMCH to KOH of 1:15.
  • the KOH solution was prepared by dispersing O-CMCH in a KOH solution, stirring uniformly, and deacetylating at a constant temperature of 100 ° C for 2 h. After completion of the reaction, the mixture was cooled, and an appropriate amount of hydrochloric acid was added to the viscous material to adjust the pH to about 8, and the mixture was thoroughly stirred until dissolved. Filter with a 400 mesh filter cloth, distill off most of the water in the filtrate and use absolute ethanol. The product was precipitated and filtered.
  • Preparation of covalently crosslinked O-CMCTS hydrogel 0.5 g of O-CMCTS prepared in the step A2 is weighed and dissolved in deionized water to prepare an O-CMCTS solution having a mass concentration of 3%. The core funnel was filtered, and the filtrate was placed in an autoclave for 30 min. A GA solution having a mass concentration of 2% was prepared in a fume hood, and the filter was sterilized by filtration. Add 5 mL of prepared sterile O-CMCTS solution to the sterile vial on a clean bench. Add the appropriate amount of GA solution according to the ratio of the molar ratio of aldehyde groups in the GA to the amino group in O-CMCTS of 1:16. Shake for 10 seconds to mix the two uniformly, and then observe the gelation process in a constant temperature water bath at 37 ° C. The gelation time was 8.2 min. The water content was determined to be 82.0%, and the water retention rate was 80.6%.
  • O-CMCTS hydrogels prepared in Examples 3, 4, and 5 were selected and labeled as samples G1, G2, and G3, respectively, for viscosity testing, degradation rate testing, and cytotoxicity testing.
  • Fig. 2 The process of measuring the viscosity of the sample by a rheometer is shown in Fig. 2.
  • the abscissa is time and the ordinate is viscosity.
  • Figure 3 shows the results of the degradation rate of samples G1, G2 and G3 in lysozyme/PBS solution. Due to the effect of the degree of crosslinking on the degradation rate of hydrogel, the sample G3 hydrogel initially showed a large swelling, and then gradually Degradation, after 60 days of degradation, the remaining mass is the lowest, about 65% of the mass before degradation. Therefore, the degree of cross-linking can be controlled by adjusting the ratio of GA to O-CMCTS, and the rate of hydrogel degradation can be adjusted to meet the needs of cell growth and tissue repair.
  • the three hydrogel samples were immersed in 75% alcohol for 2 hours at room temperature, then immersed in sterile distilled water, and the filter paper was blotted.
  • the test sample per 6 cm 2 surface area was 1 mL physiological. Brine extraction.
  • the material was placed in a leaching container, and the test sample was completely immersed in the liquid, the leaching temperature was 37 ⁇ 1 ° C, and the leaching time was 48 h.
  • the material extract was sterilized by filtration through a filter having a mesh size of 0.22 ⁇ m, and the cytotoxicity was measured by MTT assay.
  • the data of the test group and the blank control are shown in the following table:
  • the survival rate of the relative blanks was greater than 70%, indicating that the three hydrogels were not potentially cytotoxic.
  • the survival rates of the 50% and 100% extracts of the test samples were both high and no repeated tests were required.
  • FIGS. 4a-4d The fibroblasts cultured in the hydrogel extract are shown in Figures 4a-4d, and 4a, 4b, 4c and 4d are cell diagrams of G1, G2, G3 and 24 h cultures, respectively. It can be seen from the figure that the cell morphology is normal, the adherent growth is good, there is no rounding, suspension and death, the cell density is not significantly reduced, the cell growth is not inhibited, and the cells of each group show a good growth state. It is indicated that all three hydrogels are cytotoxic and meet the cytotoxicity requirements of national standards for medical materials.
  • the acute systemic toxicity test recommended in the GB/T 16886.11-2011/ISO 10993-11:2006 "Medical Device Biological Evaluation Part 11: Systemic Toxicity Test" standard requires hydrogel biocompatibility evaluation. Twenty mice were randomly divided into a sample group and a negative control group, 5 in each group. The sample group and the negative control group were injected with the test sample extract and the negative control solution from the abdominal cavity at an injection dose of 50 mL/kg, and then the animals were returned to their respective cages for feeding.
  • mice After the completion of the injection, the mice were observed immediately and at 4h, 24h, 48h and 72h, the general state, toxicity performance and number of dead animals of the animals in the test group and the control group were observed, and the body weights of the animals were recorded after 72 hours of observation.
  • the observation results of each animal are shown in the following table. During the 72h observation period, the animals in the test group did not react more than the negative control group, no animal death and toxicity occurred, and the body weight increased. There was no difference compared with the negative control. The hydrogel has no systemic acute toxicity.
  • O-CMCTS hydrogel has no obvious cytotoxicity, non-irritation and good histocompatibility in vitro. After two months of in vitro digestion, the residual rate is higher and remains above 65%. Therefore, the O-CMCTS hydrogel prepared by the invention has good biocompatibility and can maintain a certain capacity and elastic characteristics of the joint in a certain period of time, and can be used as a joint lubricant, and is suitable for local injection of joints and promoting cartilage tissue. repair.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Vascular Medicine (AREA)
  • Surgery (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)

Abstract

一种关节润滑材料及其制备方法，该关节润滑材料为共价交联型O-羧甲基壳聚糖水凝胶，其凝胶化时间为0.5-10min，含水量高于70％，保水率高于65％，由脱乙酰度＞25％的O-羧甲基壳聚糖与戊二醛进行凝胶化反应制得。该共价交联型O-羧甲基壳聚糖水凝胶具有生物相容性，可作为关节润滑剂，适用于关节局部注射，能在一定时间内使关节保持一定的容量及弹性特征，促进软骨组织修复。

Description

一种关节润滑材料及其制备方法 技术领域
本发明涉及医用生物材料技术领域，特别是涉及到一种用于关节润滑的O-羧甲基壳聚糖(O-CMCTS)水凝胶。
背景技术
随着生活水平的提高，人类的平均寿命逐渐延长，人口老龄化成为当今很重要的社会问题，预计到2050年左右，全国的老年人口将达到全国总人口的1/3，骨关节退行性病变是中老年人常见的疾病，且65岁以上老年人的患病率达50％。另外，中青年人长期过度运动导致关节软骨磨损也是临床常见疾病，且有持续上升趋势，严重影响生活质量。因此，开发有效的人工关节润滑材料直接注射进软骨磨损的关节腔内以治疗此类关节疾病十分必要。
此外，目前治疗骨关节病所采用的人工全关节置换材料多为金属、陶瓷和超高分子聚乙烯等硬质材料。这些假体材料的机械性能与天然关节相差甚远，且难以在人工关节的表面形成润滑膜。为实现人工关节优异的摩擦学性能，用人工关节润滑材料对人工关节进行润滑是降低摩擦磨损、提高使用寿命的重要措施。
国内专利《一种可注射骨关节润滑材料及其制备方法》公开了一种可注射骨关节润滑材料及其制备方法，该可注射骨关节润滑材料包括纳米晶体纤维素与透明质酸类物质，所述透明质酸类物质为透明质酸或透明质酸的水溶性盐。由于透明质酸(钠)易被体内透明质酸酶降解，因此需多次注射，既延长了疗程，又增加了治疗成本；另外，透明质酸体外形成凝胶后的注射难度加大。
国内专利《一种温度敏感的壳聚糖水凝胶及其制备方法》公开了一种温敏型可注射壳聚糖(CTS)水凝胶，采用交联剂β-甘油磷酸钠离子交联CTS制备得到一种温敏型可注射水凝胶，制备的水凝胶可作为药物载体及组织工程支架使用。但CTS仅溶于稀酸水溶液中，而且这种水凝胶需要使用大量的离子交联剂，使得其生物相容性，特别是体内组织相容性受到影响。
发明内容
本发明的目的是提供一种共价交联型O-CMCTS水凝胶，该水凝胶具有较CTS水凝胶更好的生物相容性、生物可降解性、生物吸附性等优异性，可作为关 节润滑剂，适用于关节局部注射，且能在一定时间内使关节保持一定的容量及弹性特征，促进软骨组织修复。
本发明提供一种关节润滑材料，该材料包含共价交联型O-CMCTS水凝胶，其凝胶化时间为0.5-10min，含水量＞70％，保水率＞65％。
优选地，该共价交联型O-CMCTS水凝胶由戊二醛(GA)与O-CMCTS进行凝胶化反应制得，GA中的醛基与O-CMCTS中氨基的摩尔比为1∶4-16，O-CMCTS的脱乙酰度(DD)＞25％。
本发明还提供一种如上所述关节润滑材料的制备方法，包括如下步骤：
A1.以甲壳素为原料，与氯乙酸反应制备O-羧甲基甲壳素(O-CMCH)；
A2.O-CMCH与碱反应制备O-CMCTS；
A3.O-CMCTS与GA进行凝胶化反应制备共价交联型O-CMCTS水凝胶。
目前报道的羧甲基壳聚糖(CMCTS)的制备大都以CTS为原料，将碱化的CTS分散在异丙醇中，在加热的条件或微波照射条件下，用氯乙酸羧化CTS，从而得到CMCTS。该方法制备原理简单，操作易行，但因CTS分子结构中有2个羟基(C6-OH和C3-OH)和1个氨基(C2-NH2)，羧甲基化在这3个位置均可发生反应，因此产物有N，O-CMCTS、N-CMCTS和O-CMCTS三种，故此方法选择性差，制备的产物结构不单一。
为降低生产成本，现有反应中用甲壳素为原料，加入浓碱和氯乙酸，将脱乙酰化和羧甲基化结合在一起，一步到位。这种方法步骤少，但同样选择性差，制备的产物结构不单一；另外，此方法无法控制产物的脱乙酰度(DD)及羧甲基取代度(DS)。
本发明为制备产物结构单一的O-CMCTS，控制产物DD和DS，以甲壳素为原料，采用如上所述的A1和A2步骤，分别进行羧甲基取代反应和脱乙酰基反应来制备O-CMCTS。
优选地，步骤A1包括如下步骤：
A11.将甲壳素加入质量百分比浓度为20％-35％的碱液中，待碱渗透完全后进行冷冻，冷冻完全后解冻得碱化甲壳素；
A12.将碱化甲壳素加入分散剂，并加入氯乙酸进行反应；
A13.反应结束后，进行后处理得到O-CMCH。
进一步地优选，步骤A11中甲壳素为来源于虾壳或蟹壳或鱿鱼软骨中的甲壳素，其脱乙酰度≤10％；甲壳素重复单元与碱的摩尔比为1∶6-11；碱为NaOH或KOH中的一种。
进一步地优选，步骤A12中分散剂为正丙醇、异丙醇和乙醇中的一种，其体积按甲壳素的质量与分散剂的体积比为1g∶10-30mL加入；甲壳素与氯乙酸的摩尔比为1∶0.8-2.5；反应温度为10-30℃；反应时间为2-6h。
进一步地优选，步骤A13中，所述的后处理为：将反应液过滤，滤渣溶于水，加酸调节pH至8；过滤，留取滤液；将滤液进行超滤，超滤后的滤渣进行干燥，得分子量大于10000的O-CMCH，其DD为10％-20％，DS为50％-100％。
优选地，步骤A2包括如下步骤：将O-CMCH与碱液混合进行反应，反应结束后加酸调节pH至8，充分搅拌后过滤，滤液用乙醇进行沉淀；再次溶解、沉淀并过滤，留取滤渣，干燥得O-CMCTS。
进一步地优选，步骤A2中碱液的质量百分比浓度为30％-60％，碱为KOH或NaOH中的一种；O-CMCH与碱的质量比为1∶5-15；反应温度为60-100℃，反应时间为2-6h，所制得的O-CMCTS的DD为25％-90％，DS为50％-100％。
通过上述优化的步骤A1，甲壳素在较低温条件下与低浓度的碱液进行碱化得到碱化甲壳素，再在低温下与氯乙酸进行羧甲基化反应制备O-CMCH，该优化的步骤使其羧甲基取代只发生在C6-OH而不发生在C2-NH2，且能更好地控制O-CMCH的DS值。通过上述优化的步骤A2，O-CMCH与高浓度的碱液在高温下进行脱乙酰基反应制备O-CMCTS，能更好地控制O-CMCTS的DD和DS。
另通过如上所述A13的后处理步骤，能更好地将分子量大于10000的O-CMCH与分子量小于10000的O-CMCH相分离，分离得到大分子量的O-CMCH，进而通过后续的步骤A2得到大分子量的O-CMCTS。
步骤A3中O-CMCTS与GA反应生成O-CMCTS水凝胶的反应式如下所示：
Figure PCTCN2016090772-appb-000001
本发明采用GA与O-CMCTS反应制备水凝胶，因O-CMCTS上C6位的羧酸根阴离子与C2位氨基准阳离子的静电相吸作用，降低GA与氨基的反应速度，有利于GA与O-CMCTS混合均匀，形成的凝胶均匀，不易破碎。
优选地，步骤A3包括如下步骤：O-CMCTS溶于水，配制成O-CMCTS溶液，溶液过滤并灭菌，加入GA溶液，在恒温水浴中进行凝胶化得共价交联型O-CMCTS水凝胶。
进一步地优选，步骤A3中O-CMCTS溶液的质量百分比浓度为1％-3％，GA的质量百分比浓度为2％，GA与O-CMCTS按GA中的醛基与O-CMCTS中氨基的摩尔比为1∶4-16进行反应，反应温度为37℃，凝胶化时间为0.5-10min，所制得的O-CMCTS水凝胶含水量＞70％，保水率＞65％。
本发明提供的共价交联型O-CMCTS水凝胶，具有的优势在于：该共价交联型O-CMCTS水凝胶凝胶化时间可控，含水量高且保水率高，具有良好的生物相容性且能在一定时间内使关节保持一定的容量及弹性。本发明提供的O-CMCTS制备原理简单，操作易行，成本低廉，产物结构单一，DD及DS均可控，进而与GA反应得到凝胶化时间可控，含水量高并保水率高的共价交联型O-CMCTS水凝胶。
本发明优选地实施例还包括如下的有益效果：按照该方法制备的O-CMCTS，其O-CMCTS的DD和DS能分别控制在25％-90％和50％-100％，该O-CMCTS与GA完全反应，无GA残留，制备得到的O-CMCTS水凝胶结构均匀，溶胀率高，生物相容性好。
附图说明
图1为本发明实施例由O-羧甲基壳聚糖与戊二醛进行凝胶化反应制备O-羧甲基壳聚糖水凝胶的反应式。
图2为本发明实施例样品G1、G2和G3的粘度随时间变化过程示意图。
图3为本发明实施例样品G1、G2和G3在溶菌酶/PBS溶液中的降解率。
图4为本发明实施例样品G1、G2、G3和空白组培养24h的细胞图；图4a为样品G1，图4b为样品G2，图4c为样品G3，图4d为空白组。
具体实施方式
实施例1
A1、O-CMCH的制备：称取5.0g甲壳素，按甲壳素重复单元与NaOH摩尔比为1∶6的比例，称取5.9gNaOH配成质量百分比浓度为20％的NaOH溶液，将甲壳素粉末分散于NaOH溶液中，搅拌均匀，于真空干燥箱中抽真空30min，待碱渗透完全后放入-20℃冰箱冷冻过夜。次日解冻，将碱化甲壳素转入反应釜内，加入分散剂正丙醇50mL，室温下搅拌均匀，分四次加入1.86g氯乙酸(1h内加完)，于10℃恒温反应6h。反应结束后抽滤，将滤渣溶于水后加盐酸调节pH至8左右，再用400目滤布过滤，滤液超滤5次，每次体积压缩比为1/5，超滤膜截留分子量大于10,000，超滤后的浓溶液置于60℃烘箱内鼓风干燥，得到O-CMCH，其DD＝10.22％，DS＝50.21％。
A2、O-CMCTS的制备：称取2.0g步骤A1中制得的O-CMCH，按O-CMCH与NaOH质量比为1∶5的比例，称取10gNaOH配成质量百分比浓度为30％的NaOH溶液，将O-CMCH分散于NaOH溶液中，搅拌均匀，于60℃恒温条件下脱乙酰反应4h。反应结束后冷却，向粘状物中加入适量盐酸调节pH至8左右，充分搅拌至溶解。用400目滤布过滤，蒸去滤液中大部分水分后用无水乙醇沉淀出产物并过滤，滤渣溶于水后再用无水乙醇沉淀，经3次沉淀加过滤后的滤渣置于60℃烘箱内鼓风干燥，得到O-CMCTS，其DD＝25.42％，DS＝50.06％。
A3、共价交联型O-CMCTS水凝胶的制备：秤取0.5g步骤A2中制得的O-CMCTS，溶于去离子水中配制成质量百分比浓度为1％的O-CMCTS溶液，用砂芯漏斗过滤，滤液置于高压灭菌锅中湿热灭菌30min。在通风橱中配制质量百分比浓度为2％的GA溶液，滤膜过滤除菌。在超净工作台上量取5mL配制好的无菌O-CMCTS溶液加入无菌西林瓶中，按照GA中醛基与O-CMCTS中氨基摩尔比为1∶4的比例，加入适量的GA溶液，摇晃10秒使两者混合均匀，然后置于37℃恒温水浴中观察其凝胶化过程，其凝胶化时间为9.8min。测定其含水量为71.5％，保水率为68.20％。
实施例2
A1、O-CMCH的制备：称取5.0g甲壳素，按甲壳素重复单元与KOH摩尔比为1∶8.0的比例，称取11.03g KOH配成质量百分比浓度为25％的KOH溶液， 将甲壳素粉末分散于KOH溶液中，搅拌均匀，于真空干燥箱中抽真空30min，待碱渗透完全后放入-20℃冰箱冷冻过夜。次日解冻，将碱化甲壳素转入反应釜内，加入分散剂异丙醇75mL，室温下搅拌均匀，分四次加入2.33g氯乙酸(1h内加完)，于15℃恒温反应5h。反应结束后抽滤，将滤渣溶于水后加盐酸调节pH至8左右，再用400目滤布过滤，滤液超滤5次，每次体积压缩比为1/5，超滤膜截留分子量大于10,000，超滤后的浓溶液置于60℃烘箱内鼓风干燥，得到O-CMCH，其DD＝14.79％，DS＝81.35％。
A2、O-CMCTS的制备：称取2.0g步骤A1中制得的O-CMCH，按O-CMCH与KOH质量比为1∶8.0比例，称取16.0g KOH配成质量百分比浓度为45％的KOH溶液，将O-CMCH分散于KOH溶液中，搅拌均匀，于75℃恒温条件下脱乙酰反应5h。反应结束后冷却，向粘状物中加入适量盐酸调节pH至8左右，充分搅拌至溶解。用400目滤布过滤，蒸去滤液中大部分水分后用无水乙醇沉淀出产物并过滤，滤渣溶于水后再用无水乙醇沉淀，经3次沉淀加过滤后的滤渣置于60℃烘箱内鼓风干燥，得到O-CMCTS，其DD＝50.55％，DS＝81.20％。
A3、共价交联型O-CMCTS水凝胶的制备：秤取0.5g步骤A2中制得的O-CMCTS，溶于去离子水中配制成质量百分比浓度为2％的O-CMCTS溶液，用砂芯漏斗过滤，滤液置于高压灭菌锅中湿热灭菌30min。在通风橱中配制质量百分比浓度为2％的GA溶液，滤膜过滤除菌。在超净工作台上量取5mL配制好的无菌O-CMCTS溶液加入无菌西林瓶中，按照GA中醛基与O-CMCTS中氨基摩尔比为1∶6的比例，加入适量的GA溶液，摇晃10秒使两者混合均匀，然后置于37℃恒温水浴中观察其凝胶化过程，其凝胶化时间为0.5min。测定其含水量为89.5％，保水率为88.5％。
实施例3
A1、O-CMCH的制备：称取5.0g甲壳素，按甲壳素重复单元与NaOH摩尔比为1∶9.0的比例，称取8.87g NaOH配成质量百分比浓度为35％的NaOH溶液，将甲壳素粉末分散于NaOH溶液中，搅拌均匀，于真空干燥箱中抽真空30min，待碱渗透完全后放入-20℃冰箱冷冻过夜。次日解冻，将碱化甲壳素转入反应釜内，加入分散剂异丙醇90mL，室温下搅拌均匀，分四次加入3.49g氯乙酸(1h内加完)，于20℃恒温反应3.5h。反应结束后抽滤，将滤渣溶于水后加 盐酸调节pH至8左右，再用400目滤布过滤，滤液超滤5次，每次体积压缩比为1/5，超滤膜截留分子量大于10,000，超滤后的浓溶液置于60℃烘箱内鼓风干燥，得到O-CMCH，其DD＝15.29％，DS＝72.62％。
A2、O-CMCTS的制备：称取2.0g步骤A1中制得的O-CMCH，按O-CMCH与NaOH质量比为1∶10的比例，称取20g NaOH配成质量百分比浓度为50％的NaOH溶液，将O-CMCH分散于NaOH溶液中，搅拌均匀，于80℃恒温条件下脱乙酰反应6h。反应结束后冷却，向粘状物中加入适量盐酸调节pH至8左右，充分搅拌至溶解。用400目滤布过滤，蒸去滤液中大部分水分后用无水乙醇沉淀出产物并过滤，滤渣溶于水后再用无水乙醇沉淀，经3次沉淀加过滤后的滤渣置于60℃烘箱内鼓风干燥，得到O-CMCTS，其DD＝75.89％，DS＝72.70％。
A3、共价交联型O-CMCTS水凝胶的制备：秤取0.5g步骤A2中制得的O-CMCTS，溶于去离子水中配制成质量百分比浓度为3％的O-CMCTS溶液，用砂芯漏斗过滤，滤液置于高压灭菌锅中湿热灭菌30min。在通风橱中配制质量百分比浓度为2％的GA溶液，滤膜过滤除菌。在超净工作台上量取5mL配制好的无菌O-CMCTS溶液加入无菌西林瓶中，按照GA中醛基与O-CMCTS中氨基摩尔比为1∶8的比例，加入适量的GA溶液，摇晃10秒使两者混合均匀，然后置于37℃恒温水浴中观察其凝胶化过程，其凝胶化时间为3.0min。测定其含水量为84.3％，保水率为85.6％。
实施例4
A1、O-CMCH的制备：称取5.0g甲壳素，按甲壳素重复单元与NaOH摩尔比为1∶10.0的比例，称取9.85gNaOH配成质量百分比浓度为30％的NaOH溶液，将甲壳素粉末分散于NaOH溶液中，搅拌均匀，于真空干燥箱中抽真空30min，待碱渗透完全后放入-20℃冰箱冷冻过夜。次日解冻，将碱化甲壳素转入反应釜内，加入分散剂乙醇100mL，室温下搅拌均匀，分四次加入4.66g氯乙酸(1h内加完)，于25℃恒温反应3h。反应结束后抽滤，将滤渣溶于水后加盐酸调节pH至8左右，再用400目滤布过滤，滤液超滤5次，每次体积压缩比为1/5，超滤膜截留分子量大于10,000，超滤后的浓溶液置于60℃烘箱内鼓风干燥，得到O-CMCH，其DD＝16.61％，DS＝98.74％。
A2、O-CMCTS的制备：称取2.0g步骤A1中制得的O-CMCH，按O-CMCH 与NaOH质量比为1∶12的比例，称取24.0gNaOH配成质量百分比浓度为55％的NaOH溶液，将O-CMCH分散于NaOH溶液中，搅拌均匀，于85℃恒温条件下脱乙酰反应3h。反应结束后冷却，向粘状物中加入适量盐酸调节pH至8左右，充分搅拌至溶解。用400目滤布过滤，蒸去滤液中大部分水分后用无水乙醇沉淀出产物并过滤，滤渣溶于水后再用无水乙醇沉淀，经3次沉淀加过滤后的滤渣置于60℃烘箱内鼓风干燥，得到O-CMCTS，其DD＝80.24％，DS＝98.70％。
A3、共价交联型O-CMCTS水凝胶的制备：秤取0.5g步骤A2中制得的O-CMCTS，溶于去离子水中配制成质量百分比浓度为2％的O-CMCTS溶液，用砂芯漏斗过滤，滤液置于高压灭菌锅中湿热灭菌30min。在通风橱中配制质量百分比浓度为2％的GA溶液，滤膜过滤除菌。在超净工作台上量取5mL配制好的无菌O-CMCTS溶液加入无菌西林瓶中，按照GA中醛基与O-CMCTS中氨基摩尔比为1∶12的比例，加入适量的GA溶液，摇晃10秒使两者混合均匀，然后置于37℃恒温水浴中观察其凝胶化过程，其凝胶化时间为4.5min。测定其含水量为83.7％，保水率为82.4％。
实施例5
A1、O-CMCH的制备：称取5.0g甲壳素，按甲壳素重复单元与KOH摩尔比为1∶11.0的比例，称取15.17g KOH配成质量百分比浓度为35％的KOH溶液，将甲壳素粉末分散于KOH溶液中，搅拌均匀，于真空干燥箱中抽真空30min，待碱渗透完全后放入-20℃冰箱冷冻过夜。次日解冻，将碱化甲壳素转入反应釜内，加入分散剂异丙醇150mL，室温下搅拌均匀，分四次加入5.82g氯乙酸(1h内加完)，于30℃恒温反应2h。反应结束后抽滤，将滤渣溶于水后加盐酸调节pH至8左右，再用400目滤布过滤，滤液超滤5次，每次体积压缩比为1/5，超滤膜截留分子量大于10,000，超滤后的浓溶液置于60℃烘箱内鼓风干燥，得到O-CMCH，其DD＝19.37％，DS＝89.68％。
A2、O-CMCTS的制备：称取2.0g步骤A1中制得的O-CMCH，按O-CMCH与KOH质量比为1∶15的比例，称取30.0g KOH配成质量百分比浓度为60％的KOH溶液，将O-CMCH分散于KOH溶液中，搅拌均匀，于100℃恒温条件下脱乙酰反应2h。反应结束后冷却，向粘状物中加入适量盐酸调节pH至8左右，充分搅拌至溶解。用400目滤布过滤，蒸去滤液中大部分水分后用无水乙醇 沉淀出产物并过滤，滤渣溶于水后再用无水乙醇沉淀，经3次沉淀加过滤后的滤渣置于60℃烘箱内鼓风干燥，得到O-CMCTS，其DD＝90.00％，DS＝89.62％。
A3、共价交联型O-CMCTS水凝胶的制备：秤取0.5g步骤A2中制得的O-CMCTS，溶于去离子水中配制成质量百分比浓度为3％的O-CMCTS溶液，用砂芯漏斗过滤，滤液置于高压灭菌锅中湿热灭菌30min。在通风橱中配制质量百分比浓度为2％的GA溶液，滤膜过滤除菌。在超净工作台上量取5mL配制好的无菌O-CMCTS溶液加入无菌西林瓶中，按照GA中醛基与O-CMCTS中氨基摩尔比为1∶16的比例，加入适量的GA溶液，摇晃10秒使两者混合均匀，然后置于37℃恒温水浴中观察其凝胶化过程，其凝胶化时间为8.2min。测定其含水量为82.0％，保水率为80.6％。
如上所述实施例1-5中步骤A3由O-CMCTS与GA进行凝胶化反应制备O-CMCTS水凝胶的反应式如图1所示。
相关测试
选取实施例3、4、5中制备的O-CMCTS水凝胶，分别标记为样品G1、G2和G3，对其进行粘度测试、降解率测试以及细胞毒性测试。
粘度测试
通过流变仪测定各个O-CMCTS水凝胶形成过程中粘度随时间的变化过程，并目测凝胶化时间。
通过流变仪测定样品粘度的变化过程如图2所示，横坐标为时间，纵坐标为粘度。
从图2中可以看出G1、G2和G3的粘度随时间的变化趋势基本一致，一开始粘度都是随着时间的增加而增大，在某个时刻达到最大值，然后再减小。当水凝胶形成后，样品发生了相变，由液态变为固态。
因只有流动的流体才具有粘度，当水凝胶形成后，样品内的流体逐渐变少，则粘度会变小。因此，我们可以判定图中出现的最大粘度对应的时刻即为凝胶化时间。
根据图2所得凝胶化时间和目测的凝胶化时间如下表所示：
Figure PCTCN2016090772-appb-000002
目测的凝胶化时间和流变仪测定的凝胶化时间虽有差异，但差异不大，且趋势一致。
降解率测试
图3为样品G1、G2和G3在溶菌酶/PBS溶液中降解率测试结果，由于交联程度对水凝胶降解速率的影响，样品G3水凝胶在最初呈现出较大的溶胀，而后逐渐降解，降解60天后剩余质量最低，约为降解前质量的65％。因此，可以通过调节GA与O-CMCTS的比例来控制交联程度，调节水凝胶降解速度，以满足细胞生长和组织修复的需求。
细胞毒性测试
室温下将三种水凝胶供试品于75％酒精中浸泡2h消毒，再用无菌蒸馏水浸泡，滤纸吸干，按照GB/T 16886.12的要求，每6cm2表面积的供试品采用1mL生理盐水浸提。将材料置入浸提容器，供试品全部浸没于液体内，浸提温度为37±1℃，浸提时间为48h。将材料浸提液用筛目为0.22μm的过滤器过滤除菌后，进行MTT法测试细胞毒性。试验组、空白对照的数据见如下表：
Figure PCTCN2016090772-appb-000003
相对空白的存活率均大于70％，表明三种水凝胶均不具有潜在的细胞毒性。试验样品50％与100％浸提液存活率均较高，不需重复试验。
水凝胶浸提液培养的成纤维细胞见图4a-4d，图中4a、4b、4c和4d分别是加样G1、G2、G3和空白组培养24h的细胞图。从图中可以看出细胞形态正常，贴壁生长良好，基本无圆缩、悬浮和死亡，细胞密度无明显减少，细胞生长也无受到抑制，各组细胞均表现出良好的生长状态。说明三种水凝胶均无细胞毒性，符合国标对医用材料的细胞毒性要求。
根据GB/T 16886.11-2011/ISO 10993-11：2006《医疗器械生物学评价第11部分：全身毒性试验》标准中推荐的急性全身毒性试验要求开展水凝胶生物相容性评价。将20只小鼠，随机分成样品组和阴性对照组，每组5只。样品组和阴性对照组按50mL/kg的注射剂量自腹腔分别注入被测样品浸提液和阴性对照液，之后将动物返回各自的笼子里饲养。注射完毕后，观察小鼠即刻以及4h、24h、48h和72h观察试验组和对照组中动物的一般状态、毒性表现和死亡动物数，并在72h观察后记录动物体重。
每只动物的观察结果如下表，在72h观察期内，试验组动物反应不大于阴性对照组，没有出现动物死亡和毒性反应，体重均有增长，与阴性对照相比无任何差异，说明三种水凝胶无全身急性毒性反应。
Figure PCTCN2016090772-appb-000004
Figure PCTCN2016090772-appb-000005
由此可见，O-CMCTS水凝胶在体外无明显的细胞毒性、无刺激性、组织相容性良好，体外酶解两个月后，其残留率较高，保持在65％以上。因此，本发明制备的O-CMCTS水凝胶具有良好的生物相容性且能在一定时间内使关节保持一定的容量及弹性特征，可作为关节润滑剂，适用于关节局部注射，促进软骨组织修复。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的技术人员来说，在不脱离本发明构思的前提下，还可以做出若干等同替代或明显变型，而且性能或用途相同，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种关节润滑材料，其特征在于，该材料包含共价交联型O-羧甲基壳聚糖水凝胶，其凝胶化时间为0.5-10min，含水量＞70％，保水率＞65％。
  2. 如权利要求1所述的关节润滑材料，其特征在于，所述共价交联型O-羧甲基壳聚糖水凝胶由戊二醛与O-羧甲基壳聚糖按戊二醛中醛基与O-羧甲基壳聚糖中氨基的摩尔比为1∶4-16进行凝胶化反应制得，O-羧甲基壳聚糖的脱乙酰度＞25％。
  3. 如权利要求1所述的关节润滑材料的制备方法，其特征在于，包括如下步骤：
    A1.以甲壳素为原料，与氯乙酸反应制备O-羧甲基甲壳素；
    A2.O-羧甲基甲壳素与碱反应制备O-羧甲基壳聚糖；
    A3.O-羧甲基壳聚糖与戊二醛进行凝胶化反应制备共价交联型O-羧甲基壳聚糖水凝胶。
  4. 如权利要求3所述的制备方法，其特征在于，步骤A1包括如下步骤：
    A11.将甲壳素加入质量百分比浓度为20％-35％的碱液中，待碱渗透完全后进行冷冻，冷冻完全后解冻得碱化甲壳素；
    A12.将碱化甲壳素加入分散剂，并加入氯乙酸进行反应；
    A13.反应结束后，进行后处理得到O-羧甲基甲壳素。
  5. 如权利要求4所述的制备方法，其特征在于，步骤A11中甲壳素为来源于虾壳或蟹壳或鱿鱼软骨中的甲壳素，其脱乙酰度≤10％；甲壳素重复单元与碱的摩尔比为1∶6-11；碱为NaOH或KOH中的一种；
    步骤A12中分散剂为正丙醇、异丙醇和乙醇中的一种，其体积按甲壳素的质量与分散剂的体积比为1g∶10-30mL加入；甲壳素与氯乙酸的摩尔比为1∶0.8-2.5；反应温度为10-30℃；反应时间为2-6h；
    步骤A13中，所述的后处理为：将反应液过滤，滤渣溶于水，加酸调节pH至8；过滤，留取滤液；将滤液进行超滤，超滤后的滤渣进行干燥，得分子量大于10000的O-羧甲基甲壳素，其脱乙酰度为10％-20％，羧甲基取代度为50％-100％。
  6. 如权利要求3所述的制备方法，其特征在于，步骤A2包括如下步骤：将O-羧甲基甲壳素与碱液混合进行反应，反应结束后加酸调节pH至8，充分搅拌后过滤，滤液用乙醇进行沉淀并过滤；再次溶解、沉淀和过滤，留取滤渣， 干燥得O-羧甲基壳聚糖。
  7. 如权利要求6所述的制备方法，其特征在于，步骤A2中碱液的质量百分比浓度为30％-60％，碱为KOH或NaOH中的一种；O-羧甲基甲壳素与碱的质量比为1∶5-15；反应温度为60-100℃，反应时间为2-6 h，所制得的O-羧甲基壳聚糖脱乙酰度为25％-90％，羧甲基取代度为50％-100％。
  8. 如权利要求3所述的制备方法，其特征在于，步骤A3包括如下步骤：O-羧甲基壳聚糖溶于水，配制成O-羧甲基壳聚糖溶液，过滤并灭菌，加入戊二醛溶液，在恒温水浴中进行凝胶化得共价交联型O-羧甲基壳聚糖水凝胶。
  9. 如权利要求8所述的制备方法，其特征在于，步骤A3中O-羧甲基壳聚糖溶液的百分比浓度为1％-3％，戊二醛的质量百分比浓度为2％，戊二醛与O-羧甲基壳聚糖按戊二醛中的醛基与O-羧甲基壳聚糖中氨基的摩尔比为1∶4-16进行反应，反应温度为37℃，凝胶化时间为0.5-10min，所制得的O-羧甲基壳聚糖水凝胶含水量＞70％，保水率＞65％。
  10. 如权利要求1-9任一所述的共价交联型O-羧甲基壳聚糖水凝胶，在制备可注射骨关节润滑材料中的应用。
PCT/CN2016/090772 2016-06-28 2016-07-20 一种关节润滑材料及其制备方法 WO2018000486A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610492982.X 2016-06-28
CN201610492982.XA CN106046397A (zh) 2016-06-28 2016-06-28 一种关节润滑材料及其制备方法

Publications (1)

Publication Number Publication Date
WO2018000486A1 true WO2018000486A1 (zh) 2018-01-04

Family

ID=57166066

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/090772 WO2018000486A1 (zh) 2016-06-28 2016-07-20 一种关节润滑材料及其制备方法

Country Status (2)

Country Link
CN (1) CN106046397A (zh)
WO (1) WO2018000486A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975072A (zh) * 2023-02-11 2023-04-18 武汉轻工大学 一种可再分散纳米甲壳素及其制备方法

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106860871A (zh) * 2017-01-09 2017-06-20 安徽大学 一种羧甲基壳聚糖pH敏感性水凝胶药物载体的制备方法
CN107056986B (zh) * 2017-03-30 2020-10-09 池州学院 一种壳聚糖基高吸水树脂清洁生产工艺
CN107349477A (zh) * 2017-06-08 2017-11-17 西安交通大学 表面织构填充氧化石墨烯缓释润滑凝胶及其制备方法和用途
CN107349476B (zh) * 2017-06-13 2020-09-08 爱美客技术发展股份有限公司 仿生关节滑液及其制备方法
CN110522953B (zh) * 2018-05-24 2022-02-15 中国科学院兰州化学物理研究所 一种仿生关节润滑剂及其制备方法
CN109821023A (zh) * 2019-02-22 2019-05-31 江西光至金辉医疗制品有限公司 一种pH敏感型智能药物控制释放载体及其制备方法
CN110772376A (zh) * 2019-11-20 2020-02-11 韩熠 一种用于婴幼儿及儿童的医用眼贴及其制备方法
CN111228579B (zh) * 2020-01-21 2021-12-03 赛克赛斯生物科技股份有限公司 可注射水凝胶及其制备方法和应用以及关节润滑剂

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888988A (en) * 1995-05-08 1999-03-30 Chitogenics, Inc. Covalently linked N,O-carboxymethylchitosan and uses thereof
CN1962729A (zh) * 2006-11-29 2007-05-16 武汉理工大学 pH敏感型羧甲基壳聚糖水凝胶的制备方法
WO2008072230A1 (en) * 2006-12-11 2008-06-19 Chit2Gel Ltd. Novel injectable chitosan mixtures forming hydrogels
CN103937009A (zh) * 2013-01-18 2014-07-23 林潘海 一种几丁聚糖水凝胶的制备方法
CN104231112A (zh) * 2014-04-29 2014-12-24 深圳大学 一种6-o-羧甲基壳聚糖硫酸酯化产物的合成方法
US20150147397A1 (en) * 2012-05-17 2015-05-28 Cartiheal (2009) Ltd. Biomatrix Hydrogels and Methods of Use Thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888988A (en) * 1995-05-08 1999-03-30 Chitogenics, Inc. Covalently linked N,O-carboxymethylchitosan and uses thereof
CN1962729A (zh) * 2006-11-29 2007-05-16 武汉理工大学 pH敏感型羧甲基壳聚糖水凝胶的制备方法
WO2008072230A1 (en) * 2006-12-11 2008-06-19 Chit2Gel Ltd. Novel injectable chitosan mixtures forming hydrogels
US20150147397A1 (en) * 2012-05-17 2015-05-28 Cartiheal (2009) Ltd. Biomatrix Hydrogels and Methods of Use Thereof
CN103937009A (zh) * 2013-01-18 2014-07-23 林潘海 一种几丁聚糖水凝胶的制备方法
CN104231112A (zh) * 2014-04-29 2014-12-24 深圳大学 一种6-o-羧甲基壳聚糖硫酸酯化产物的合成方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG, GUANYUN ET AL.: "Preparation and the Swelling Characteristics of the O-carboxymethyl Chitosan Hydrogel", THE FOOD INDUSTRY, vol. 34, no. 8, 31 August 2013 (2013-08-31), pages 129 and 130, ISSN: 1004-471X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975072A (zh) * 2023-02-11 2023-04-18 武汉轻工大学 一种可再分散纳米甲壳素及其制备方法
CN115975072B (zh) * 2023-02-11 2024-04-30 武汉轻工大学 一种可再分散纳米甲壳素及其制备方法

Also Published As

Publication number Publication date
CN106046397A (zh) 2016-10-26

Similar Documents

Publication Publication Date Title
WO2018000486A1 (zh) 一种关节润滑材料及其制备方法
Amirian et al. In-situ crosslinked hydrogel based on amidated pectin/oxidized chitosan as potential wound dressing for skin repairing
Yu et al. A self-healing and injectable oxidized quaternized guar gum/carboxymethyl chitosan hydrogel with efficient hemostatic and antibacterial properties for wound dressing
EP2310448B1 (en) Three-dimensional nanocomposite materials consisting of a polysaccharidic matrix and metallic nanoparticles, preparation and use thereof
Zhang et al. Preparation and characterization of carboxymethyl chitosan/collagen peptide/oxidized konjac composite hydrogel
Weng et al. Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran
CA2740136C (en) Injectable in-situ crosslinked hydrogel and methods of making and using thereof
JP5357015B2 (ja) 組織工学用で活性化合物のキャリアとしての、多糖類混合物のハイドロゲル
Adali et al. The chondrocyte cell proliferation of a chitosan/silk fibroin/egg shell membrane hydrogels
KR20220113832A (ko) 젤란 검 하이드로겔(gellan gum hydrogels), 제조, 방법 및 그 용도
HUE026830T2 (en) Viscoelastic gels as new fillers
CN110790885B (zh) 一种聚乙烯醇/壳聚糖季铵盐抗菌自愈水凝胶及其制备方法和应用
WO2009101518A2 (en) Gellan gum based hydrogels for regenerative medicine and tissue engineering applications, its system, and processing devices
US6281341B1 (en) Hetero-polysaccharide conjugate and methods of making and using the same
CN111388755A (zh) 一种可注射型透明质酸/壳聚糖水凝胶及其制备方法
CN112587726B (zh) 复合水凝胶支架及其制备方法和应用
WO2017028625A1 (zh) 一种胸膜/脑膜补片及其制备方法
Amiryaghoubi et al. In situ forming alginate/gelatin hydrogel scaffold through Schiff base reaction embedded with curcumin-loaded chitosan microspheres for bone tissue regeneration
CN115429935B (zh) 一种可注射性的交联硫酸软骨素水凝胶及其制备方法
CN111499889A (zh) 一种硫酸软骨素镁透明质酸水凝胶及其制备方法与应用、一种凝胶产品
CN115624647B (zh) 一种复合创口愈合药物与膜精华液的生物膜医用敷料及其制备方法和应用
Das et al. Evaluation of Opuntia-carrageenan superporous hydrogel (OPM-CRG SPH) as an effective biomaterial for drug release and tissue scaffold
CN113041405B (zh) 一种含透明质酸和四氢嘧啶的人体润滑液及其制备方法和应用
CN114213676A (zh) 一种天然深共晶紫苏叶提取物水凝胶的制备方法
JPWO2018079812A1 (ja) ゲル組成物及びその製造方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16906884

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 16906884

Country of ref document: EP

Kind code of ref document: A1