CN110251681B - Functionalized chitosan porous microsphere for cartilage injury repair and preparation method thereof - Google Patents
Functionalized chitosan porous microsphere for cartilage injury repair and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a functionalized chitosan porous microsphere for repairing cartilage injury and a preparation method thereof. Adding a chitosan acetic acid solution into an organic solvent, stirring to form a W/O emulsion, dropwise adding the W/O emulsion into a precooled glass plate containing the same organic solvent, adding absolute ethyl alcohol for dehydration after emulsion droplets are solidified into balls, and finally adding a sodium hydroxide solution to neutralize the acetic acid remained in the microspheres. Adding glutaraldehyde into the microspheres washed to be neutral for further chemical crosslinking, removing free glutaraldehyde by using glycine, cleaning, and freeze-drying to obtain the chitosan porous microspheres. The micromolecular drug KGN is connected with the chitosan porous microsphere in a chemical bonding mode, so that the differentiation bioactivity of the bone marrow mesenchymal stem cells is endowed. The chitosan porous microsphere prepared by the invention has a three-dimensional network porous structure with a bionic structure, and provides space and conditions for the formation and growth of new cartilage cells. The preparation method is simple, has a controllable structure and good sphericity, and has a wide application prospect in the field of cartilage tissue regeneration.
Description
Technical Field
The invention belongs to the field of biomedical polymer materials, and relates to a functionalized chitosan porous microsphere for cartilage injury repair and a preparation method thereof.
Background
Cartilage damage is a disease that is quite common in the clinic, especially in Osteoarthritis (OA) patients due to aging of the population. Cartilage tissue is a terminally differentiated tissue, lacks blood vessels, and the supply of nutrients is primarily derived from synovial fluid, and thus is difficult to repair itself after injury. At present, various disadvantages exist in clinical drug therapy and surgical therapy. The development of tissue engineering brings a new opportunity for repairing cartilage damage. The traditional tissue engineering technology is to implant a biomaterial scaffold into a cartilage defect, but secondary operation cannot be avoided, so that great psychological and economic burden is undoubtedly added to a patient. In recent years, research shows that Mesenchymal Stem Cells (MSCs) implanted into cartilage injury parts by means of a tissue engineering method have a good effect of regeneration and repair, many researches on the repair of cartilage injuries by MSCs are concentrated on Bone Marrow Mesenchymal Stem Cells (BMSCs), and a large number of in vivo and in vitro researches show that the cartilage differentiation of BMSCs and the cartilage repair of autologous BMSCs have a good effect. The phenomenon of dedifferentiation of cartilage tissues formed by the differentiation of BMSCs under the stimulation of in vitro induction factors is easy to occur, thereby limiting the further clinical application.
Kartogenin (KGN) is a small molecule compound screened from various heterocyclic drug-like molecules by Johnson et al through a high-throughput screening technology (Johnson K, zhu S, treblay MS, et al science, 2012,336, 717-721.), and has a very strong ability to promote BMSCs to differentiate into chondrocytes. The mechanism is mainly that the runt related transcription factor gene (RUNX) is regulated, and mainly the RUNX1 gene promotes the differentiation of chondrocytes, the proliferation of the chondrocytes and the like. In addition, the small molecular compound can also obviously inhibit the reduction of extracellular matrix and proteoglycan caused by interleukin 1 beta (interleukin 1 beta, IL-1 beta) secretion. And can also promote the synergistic effect of chondrocyte secretion lubricin and Transforming Growth Factor beta 1 (Transforming Growth Factor beta 1, TGF-beta 1) and Bone Morphogenetic protein 7 (Bone Morphogenetic 7, BMP-7). The pure KGN has short in-vivo retention time and low drug utilization rate, a drug sustained-release material is needed to carry out effective loading on the drug, and meanwhile, a drug sustained-release stent which needs to be implanted into the body can provide a proper survival microenvironment for the differentiation of BMSCs into chondrocytes.
Chitosan (CS) is a natural high molecular polysaccharide extracted from marine crustacean, and has excellent biocompatibility and biodegradability. In addition, the chitosan surface is rich in active functional group amino, which is beneficial to graft modification. The chitosan microsphere has more holes and high specific surface area, can improve the drug loading rate and provide a certain space for the survival of the BMSCS. In previous researches, a pore-forming agent is used in the preparation process of the chitosan porous microspheres, and the addition of the pore-forming agent makes subsequent removal difficult, the process is complex, and the environmental burden is heavy (CN 1927892A). Patent CN 103804712A discloses a preparation method of porous and pore-size-controllable chitosan microspheres, the invention uses electrostatic spraying and vacuum freeze-drying technology to prepare the chitosan porous microspheres with adjustable pore size and specific surface area, but the particle size and pore size of the chitosan porous microspheres prepared by the method are smaller, and the function of cell microcarrier can not be satisfied. Therefore, the development of the functionalized chitosan porous microsphere which has simple preparation process and low cost and can meet the requirements of drug slow release and cell microcarrier has important application prospect.
Disclosure of Invention
In view of the above, the invention provides a functionalized chitosan porous microsphere for cartilage injury repair and a preparation method thereof, the chitosan porous microsphere obtained by the preparation method is bonded with KGN, has good biodegradability, can promote cells to grow and proliferate on the surface and in pores of the microsphere, can further promote BMSCs to differentiate into chondrocytes, and has good application prospect in the treatment of OA.
The invention provides a functionalized chitosan porous microsphere for repairing cartilage injury, wherein the diameter of the chitosan porous microsphere is 100-500 mu m, the pore size is 10-30 mu m, micropores on the surface and in the chitosan porous microsphere are connected with a granular micromolecular drug KGN through covalent bonds, and the micromolecular drug KGN is uniformly distributed on the surface and in the pores of the chitosan porous microsphere.
Preferably, the diameter of the chitosan porous microsphere is 150-250 μm, the pore size is 15-25 μm, the surface of the chitosan porous microsphere and the internal micropores are communicated with each other, and the small molecule drug KGN is uniformly distributed on the surface of the chitosan porous microsphere and in the internal micropores.
The second aspect of the invention provides a preparation method of a functionalized chitosan porous microsphere for cartilage injury repair, which comprises the following steps:
s1, adding chitosan into an acetic acid solution with the mass fraction of 1-3%, stirring, heating and dissolving, cooling and defoaming after the solution is clarified to obtain a chitosan acetic acid solution serving as an aqueous phase (W) for emulsification;
s2, selecting a water-insoluble organic solvent as an oil phase (O), dropwise adding the chitosan acetic acid solution obtained in the step S1 into the pre-emulsified oil phase, and stirring to form a W/O emulsion;
s3, dropwise adding the W/O emulsion obtained in the step S2 into the oil phase obtained in the pre-cooling step S2, removing the oil phase after standing at a low temperature of-15 to-25 ℃, adding absolute ethyl alcohol, standing at a low temperature of-15 to-25 ℃, adding alkali liquor after removing the absolute ethyl alcohol, standing at a low temperature of-15 to-25 ℃, removing the alkali liquor, and washing to be neutral;
and S4, adding glutaraldehyde into the neutral solution obtained in the step S3 for chemical crosslinking reaction, removing glutaraldehyde after the reaction is finished to obtain a microsphere solution, adding a glycine solution into the microsphere solution, washing with deionized water, and then freeze-drying to obtain the chitosan porous microsphere.
Preferably, in step S1, the molecular weight of the chitosan is 110 to 150kDa, and more preferably 110kDa; the degree of deacetylation is 80% or more, more preferably 95%.
Preferably, in the step S1, the heating and dissolving temperature is 35 to 80 ℃, and more preferably 50 ℃; the mass fraction of chitosan in the formed chitosan acetic acid solution is 1 to 4%, and more preferably 2.5%.
Preferably, in step S2, the volume ratio of the chitosan acetic acid solution to the oil phase is 1: (4 to 8), more preferably 1:6. the oil phase is one or more of petroleum ether, n-octane or n-heptane.
More preferably, in step S2, an emulsifier is added to the oil phase for pre-emulsification, wherein the addition amount of the emulsifier is 2 to 6% by mass of the oil phase, and more preferably 4%; the emulsifier is Span surfactant or a mixture of Span surfactant and Tween surfactant, and further preferably Span80.
Preferably, in step S2, magnetic stirring is adopted for the stirring, the stirring time is 0.5 to 2 hours, and the magnetic stirring rotation speed is 300 to 600rpm, and more preferably 500rpm.
Preferably, in step S3, the low temperature of the low-temperature standing is preferably-20 ℃. The low-temperature standing time is 12-48 h.
Preferably, in step S3, the alkali liquor is 0.5-2.5 mol/L sodium hydroxide solution.
Preferably, in step S3, the W/O emulsion is dropped into a glass plate containing the precooled oil phase, and then the operations such as low-temperature standing and removal are performed. The diameter of the glass plate is 10 to 18cm, and more preferably 18 cm.
Preferably, in step S4, the addition amount of the glutaraldehyde is 5-15% of the mass of the neutral solution, and the chemical crosslinking reaction lasts 2-12 hours. The mass fraction of the glycine in the glycine solution is 1-6%. Preferably, the method further comprises the step of S5 KGN modification of the chitosan porous microspheres: and (5) adding the chitosan porous microspheres obtained in the step (S4) into the activated KGN solution of 10-50 mg/mL, oscillating for 12-24 h, dialyzing for 24-72 h by using a dialysis membrane with the molecular weight cutoff of 3.5-20 kDa, and finally freezing and drying to obtain the functionalized chitosan porous microspheres.
More preferably, the KGN is activated for 0.5 to 2h by EDC/NHS, and the molar ratio of EDC to NHS in EDC/NHS activation is (4 to 1): 1.
the third aspect of the invention provides the application of the functionalized chitosan porous microspheres for cartilage injury repair in cartilage injury treatment medicines and BMSCs three-dimensional culture. Specifically, cells can adhere to the surface of the functionalized chitosan porous microsphere and grow in the inner hole of the microsphere.
Compared with the prior art, the invention has the beneficial effects that:
the functionalized chitosan porous microsphere provided by the invention has good sphericity, certain strength and toughness, large specific surface area, network-like continuous porous structure, pore size favorable for the growth of cells, space and conditions for the formation and growth of new chondrocytes, and favorable for the formation of three-dimensional culture of cells-microspheres.
The invention further covalently connects KGN to the surface and the internal structure of the chitosan porous microsphere, thereby greatly improving the drug-loading rate of the drug, and compared with a physical drug-loading mode, the drug release of KGN is slower because KGN carries the drug by a covalent grafting mode.
The functionalized chitosan porous microsphere provided by the invention has good biodegradability, and can promote the proliferation and differentiation of adhered BMSCs to chondrocytes.
The preparation method adopted by the invention is simple, the structure is controllable, the sphericity is good, and the preparation method has wide application prospect in the field of cartilage tissue regeneration.
Drawings
FIGS. 1a to 1c are surface field emission scanning electron micrographs of chitosan porous microspheres prepared in example 1 of the present invention; wherein, fig. 1a is a single microsphere surface field emission scanning electron microscope image, and fig. 1b and fig. 1c are partial enlarged views of the microsphere surface.
FIGS. 2 a-2 c are surface field emission scanning electron micrographs of KGN-modified chitosan porous microspheres prepared in example 2 of the present invention; wherein, FIG. 2a is a surface field emission scanning electron microscope image of a single microsphere, and FIGS. 2b and 2c are partial enlarged views of the microsphere surface.
FIGS. 3 a-3 c are laser confocal microscope photographs of BMSCs and KGN modified chitosan porous microspheres co-cultured for 7 days. Wherein, FIG. 3a is DAPI staining diagram, FIG. 3b is FITC labeled phalloidin staining diagram, and FIG. 3c is two dyes composite staining diagram.
Detailed Description
In order to facilitate understanding of the present invention, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following specific examples.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
The invention provides a functionalized chitosan porous microsphere for cartilage injury repair, wherein the diameter of the chitosan porous microsphere is 100-500 mu m, the pore size is 10-30 mu m, the surface and the interior of the chitosan porous microsphere are connected with a granular micromolecular drug KGN through covalent bonds, and the micromolecular drug KGN is uniformly distributed on the surface and in the pores of the chitosan porous microsphere.
The second aspect of the invention provides a preparation method of functionalized chitosan porous microspheres for cartilage injury repair, which comprises the following steps:
s1, adding chitosan into an acetic acid solution with the mass fraction of 1-3%, stirring, heating and dissolving, cooling and defoaming after the solution is clarified to obtain a chitosan acetic acid solution serving as an aqueous phase (W) for emulsification;
s2, selecting a water-insoluble organic solvent as an oil phase (O), dropwise adding the chitosan acetic acid solution obtained in the step S1 into the pre-emulsified oil phase, and stirring to form a W/O emulsion;
s3, slowly dripping the W/O emulsion obtained in the step S2 into the oil phase obtained in the pre-cooling step S2, standing at a low temperature of between 15 ℃ below zero and 25 ℃ below zero, sucking out a n-heptane solution to remove an oil phase after chitosan emulsion drops are solidified into balls, standing at a low temperature of between 15 ℃ below zero and 25 ℃ below zero to dewater after replacing with absolute ethyl alcohol, standing at a low temperature of between 15 ℃ below zero and 25 ℃ below zero after removing the absolute ethyl alcohol, removing residual acetic acid in the microspheres, and washing to be neutral after removing the alkali liquor;
and S4, adding glutaraldehyde into the neutral solution obtained in the step S3 for chemical crosslinking reaction, removing glutaraldehyde after the reaction is finished to obtain a microsphere solution, adding a glycine solution into the microsphere solution to neutralize free glutaraldehyde in the microspheres, washing with deionized water, then freeze-drying to obtain chitosan porous microspheres, and modifying the chitosan porous microspheres with KGN.
The invention adopts a method of combining emulsification and thermal phase separation to obtain the porous chitosan microspheres, the organic solvent is not solidified (still in a liquid state) at a lower temperature of (-15 to-25 ℃), the spherical chitosan emulsion drops contain water, and the emulsion drops are frozen and solidified into spheres at a low temperature. Water forms small ice crystals in the chitosan matrix at low temperatures. Separating the solidified microspheres, further dehydrating the microspheres by using ethanol, and removing ice crystals to form the porous chitosan microspheres. The emulsification process is controllable, simple and feasible, and has higher drug loading rate compared with a double emulsification mode. Furthermore, the chitosan is covalently linked with KGN drugs with carboxyl groups through EDC and NHS activation by utilizing the amino functional groups of the chitosan. The covalent linkage mode can be slowly released in the slow degradation process of the chitosan, and is an erosion release mechanism.
The third aspect of the invention provides the application of the functionalized chitosan porous microspheres for cartilage injury repair in cartilage injury treatment medicines and BMSCs three-dimensional culture.
The following will describe the functionalized chitosan porous microspheres for cartilage damage repair and the preparation method thereof in detail with reference to 3 specific examples.
Example 1
The embodiment provides a preparation method of chitosan porous microspheres for cartilage injury repair, which comprises the following steps:
1. weighing a certain amount of chitosan (molecular weight is 110kDa, degree of deacetylation is 95%) and adding into 1wt.% acetic acid solution, heating to 50 ℃, magnetically stirring for dissolving, cooling and defoaming after the solution is clarified for later use. The mass fraction of chitosan was 2.5wt.%, as aqueous phase (W) for emulsification;
2. preparing 72mL of n-heptane solution (O) containing 4wt.% Span80, slowly dropwise adding 12mL of the chitosan solution into the pre-emulsified n-heptane solution, and magnetically stirring at 500rpm for 1h to form W/O emulsion;
3. the glass plate (18 cm) containing n-heptane was pre-cooled to-20 deg.C, and the W/O emulsion was slowly and uniformly added dropwise to the glass plate and cooled at-20 deg.C for 24h. After the chitosan emulsion drops are solidified into spheres, sucking out the n-heptane solution, replacing the n-heptane solution with absolute ethyl alcohol and dehydrating for 24 hours at the same temperature. After dehydration, replacing the solution with 1mol/L sodium hydroxide solution, standing the solution at the temperature of minus 20 ℃ for 24 hours, and removing the residual acetic acid in the microspheres;
4. the sodium hydroxide solution was removed from the glass plate and washed repeatedly with deionized water until neutral. Chemical crosslinking was performed for 6h with the addition of 8wt.% glutaraldehyde. And after removing glutaraldehyde, adding a glycine solution with the mass fraction of 4wt.% to neutralize free glutaraldehyde in the microspheres, and finally, repeatedly washing with deionized water and freeze-drying for later use.
FIG. 1 shows a field emission scanning electron microscope photograph of the chitosan porous microsphere prepared in this example, from which it can be seen that the chitosan porous microsphere has good sphericity, is continuous inside and outside, has a continuous porous structure, and has a pore size in the range of 10-50 μm.
Example 2
The embodiment provides a preparation method of a functionalized chitosan porous microsphere for cartilage injury repair, which comprises the following steps:
1. weighing a certain amount of chitosan (molecular weight is 110kDa, degree of deacetylation is 95%) and adding into 2wt.% acetic acid solution, heating to 40 ℃, magnetically stirring for dissolving, cooling and defoaming after the solution is clarified for later use. The mass fraction of chitosan was 3wt.%, as aqueous phase (W) for emulsification;
2. preparing 65mL of n-heptane solution (O) containing 3wt.% of Span80, slowly dropwise adding 13mL of the chitosan solution into the pre-emulsified n-heptane solution, and magnetically stirring at 600rpm for 0.5h to form W/O emulsion;
3. a glass plate (15 cm) containing n-heptane was pre-cooled to-23 deg.C, and the W/O emulsion was slowly and uniformly added dropwise to the glass plate and cooled at-23 deg.C for 12h. After the chitosan emulsion drops are solidified into spheres, sucking out the n-heptane solution, replacing the n-heptane solution with absolute ethyl alcohol and dehydrating the n-heptane solution for 12 hours at the same temperature. After the dehydration is finished, replacing 1.5 mol/L sodium hydroxide solution to stand for 12h at the temperature of minus 23 ℃, and removing the residual acetic acid in the microspheres;
4. the sodium hydroxide solution in the glass plate was removed and washed repeatedly with deionized water until neutral. 12wt.% glutaraldehyde was added for chemical crosslinking for 6h. After removing glutaraldehyde, adding a glycine solution with the mass fraction of 4wt.% to neutralize free glutaraldehyde in the microspheres, finally repeatedly washing with deionized water, and freeze-drying for later use.
5. Activating 30mg/mL KGN by EDC/NHS (molar ratio is 2. The drug loading of the drug-loaded microspheres was 6.8% by HPLC.
The KGN-modified chitosan porous microspheres are characterized by means of a field emission scanning electron microscope, and the results are shown in figure 2. The result shows that the chitosan porous microspheres loaded with KGN have a three-dimensional network structure (fig. 2a, fig. 2b, and fig. 2 c), and pores are interconnected, and it is clearly seen in fig. 2c that KGN is distributed in the chitosan porous microspheres (indicated by arrows).
Example 3
The embodiment provides a preparation method of a functionalized chitosan porous microsphere for cartilage injury repair, which comprises the following steps:
weighing a certain amount of chitosan (with the molecular weight of 150kDa and the deacetylation degree of 90%) and adding the chitosan into 0.5wt.% acetic acid solution, heating to 60 ℃, magnetically stirring for dissolution, cooling and defoaming after the solution is clarified for later use. The mass fraction of chitosan was 1.5wt.%, as aqueous phase (W) for emulsification;
preparing 84mL of n-heptane solution (O) containing 4wt.% of Span80, slowly dropwise adding 12mL of the chitosan solution into the pre-emulsified n-heptane solution, and magnetically stirring at 400rpm for 2h to form W/O emulsion;
the glass plate (10 cm) containing n-heptane was pre-cooled to-15 deg.C, and the W/O emulsion was slowly and uniformly added dropwise to the glass plate and cooled at-15 deg.C for 24h. After the chitosan emulsion drops are solidified into spheres, sucking out the n-heptane solution, replacing the n-heptane solution with absolute ethyl alcohol and dehydrating for 24 hours at the same temperature. After the dehydration is finished, replacing the sodium hydroxide solution with 2mol/L, standing the solution for 24 hours at the temperature of minus 15 ℃, and removing the residual acetic acid in the microspheres;
the sodium hydroxide solution was removed from the glass plate and washed repeatedly with deionized water until neutral. Chemical crosslinking was carried out for 8h with the addition of 10wt.% glutaraldehyde. And after removing glutaraldehyde, adding a glycine solution with the mass fraction of 5wt.% to neutralize free glutaraldehyde in the microspheres, and finally, repeatedly washing with deionized water and freeze-drying for later use.
Activating 40mg/mL KGN by EDC/NHS (molar ratio is 1.
After the chitosan porous microspheres modified with KGN and BMSCs are co-cultured for 7 days, the cells are observed by a laser confocal microscope, as shown in FIG. 3, wherein cell nuclei are stained by DAPI (blue), cytoskeleton is stained by FITC-labeled phalloidin (green), cells are uniformly distributed in the chitosan porous microspheres, and the cells can be seen to grow in the pores of the microspheres.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. A preparation method of functionalized chitosan porous microspheres for cartilage injury repair comprises the following steps:
s1, adding chitosan into an acetic acid solution with the mass fraction of 1-3%, stirring, heating and dissolving, cooling and defoaming after the solution is clarified to obtain a chitosan acetic acid solution as an emulsifying water phase, wherein the molecular weight of the chitosan is 110-150 kDa, and the deacetylation degree is more than 80%;
s2, selecting a water-insoluble organic solvent as an oil phase, dropwise adding the chitosan acetic acid solution obtained in the step S1 into the pre-emulsified oil phase, and stirring to form a W/O emulsion;
s3, dropwise adding the W/O emulsion obtained in the step S2 into the oil phase obtained in the pre-cooling step S2, removing the oil phase after standing at a low temperature of-15 to-25 ℃, adding absolute ethyl alcohol, standing at a low temperature of-15 to-25 ℃, adding alkali liquor after removing the absolute ethyl alcohol, standing at a low temperature of-15 to-25 ℃, removing the alkali liquor, and washing to be neutral;
s4, adding glutaraldehyde into the neutral solution obtained in the step S3 for chemical crosslinking reaction, removing glutaraldehyde after the reaction is finished to obtain a microsphere solution, adding a glycine solution into the microsphere solution, washing with deionized water, and then freeze-drying to obtain the chitosan porous microspheres;
and S5, adding the chitosan porous microspheres obtained in the step S4 into the activated KGN solution of 10-50 mg/mL, oscillating for 12-24 h, dialyzing for 24-72 h by using a dialysis membrane with the molecular weight cutoff of 3.5-20 kDa, and finally freeze-drying to obtain the functionalized chitosan porous microspheres.
2. The method for preparing the functionalized chitosan porous microspheres for cartilage damage repair according to claim 1, wherein: in the step S1, the heating and dissolving temperature is 35-80 ℃, and the mass percent of chitosan in the formed chitosan acetic acid solution is 1-4%.
3. The method for preparing the functionalized chitosan porous microspheres for cartilage damage repair according to claim 1, wherein: in step S2, the volume ratio of the chitosan acetic acid solution to the oil phase is 1: (4-8); the oil phase is one or more of petroleum ether, n-octane or n-heptane.
4. The method for preparing the functionalized chitosan porous microspheres for cartilage damage repair according to claim 3, wherein: in the step S2, adding an emulsifier into the oil phase for pre-emulsification, wherein the addition amount of the emulsifier is 2-6% of the mass of the oil phase; the emulsifier is Span surfactant or a mixture of Span surfactant and Tween surfactant.
5. The method for preparing the functionalized chitosan porous microspheres for cartilage damage repair according to claim 1, wherein: s4, adding the glutaraldehyde in an amount which is 5-15% of the mass of the neutral solution, wherein the chemical crosslinking reaction lasts for 2-12 h; the mass fraction of the glycine in the glycine solution is 1-6%.
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| WO2018067545A1 (en) * | 2016-10-04 | 2018-04-12 | University Of Florida Research Foundation, Inc. | Chondroprotective nanoparticles for the treatment of osteoarthritis |
| CN106729987A (en) * | 2017-01-26 | 2017-05-31 | 福州大学 | A kind of collagen/chitosan/Sodium Hyaluronate compound rest of load KGN |
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