CN117487198A - Self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and preparation method thereof - Google Patents
Self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and preparation method thereof Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 173
- 108010035532 Collagen Proteins 0.000 title claims abstract description 173
- 229920001436 collagen Polymers 0.000 title claims abstract description 173
- 239000000017 hydrogel Substances 0.000 title claims abstract description 64
- 238000004132 cross linking Methods 0.000 title claims abstract description 61
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 title claims abstract description 54
- 229910052708 sodium Inorganic materials 0.000 title claims abstract description 54
- 239000011734 sodium Substances 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 229920002385 Sodium hyaluronate Polymers 0.000 claims abstract description 141
- 229940010747 sodium hyaluronate Drugs 0.000 claims abstract description 141
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims abstract description 141
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims abstract description 50
- 125000003172 aldehyde group Chemical group 0.000 claims abstract description 47
- 239000002904 solvent Substances 0.000 claims abstract description 46
- 125000003277 amino group Chemical group 0.000 claims abstract description 23
- 230000001590 oxidative effect Effects 0.000 claims abstract description 16
- 238000004108 freeze drying Methods 0.000 claims abstract description 15
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 111
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
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- 238000000034 method Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 12
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- 238000000502 dialysis Methods 0.000 description 12
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- 150000004753 Schiff bases Chemical group 0.000 description 6
- 238000011049 filling Methods 0.000 description 6
- 230000008093 supporting effect Effects 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
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- 150000003384 small molecules Chemical class 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
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- OQILSTRGJVCFAG-UHFFFAOYSA-N 1-(oxiran-2-ylmethoxy)butan-1-ol Chemical compound CCCC(O)OCC1CO1 OQILSTRGJVCFAG-UHFFFAOYSA-N 0.000 description 1
- CYCBPQPFMHUATH-UHFFFAOYSA-N 4-(oxiran-2-ylmethoxy)butan-1-ol Chemical compound OCCCCOCC1CO1 CYCBPQPFMHUATH-UHFFFAOYSA-N 0.000 description 1
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- 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
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- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/246—Intercrosslinking of at least two polymers
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- 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
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- C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
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Abstract
The application provides self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and a preparation method thereof. A self-crosslinking sodium hyaluronate-recombinant collagen hydrogel comprising: at least one sodium hyaluronate and at least one recombinant collagen comprising free amino groups, the remainder being a solvent. The molecular weight of the sodium hyaluronate is 10000-5000000. Sodium hyaluronate is oxidized by sodium periodate, and adjacent hydroxyl groups in sodium hyaluronate are oxidized into aldehyde groups. Recombinant collagen contains the free amino groups in an amount of 10-1000. The preparation method of the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel comprises the following steps: preparing sodium hyaluronate into sodium hyaluronate solution, oxidizing sodium hyaluronate with sodium periodate, dialyzing and freeze-drying to obtain sodium hyaluronate solution containing aldehyde group; adding the recombinant collagen into a solvent to prepare a recombinant collagen solution; and dropwise adding the recombinant collagen solution into the continuously stirred sodium hyaluronate solution to obtain the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel.
Description
Technical Field
The application relates to the field of hydrogels for tissue engineering, in particular to a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and a preparation method thereof.
Background
The recombinant collagen is a collagen raw material which can be produced by a bioengineering technology, and the basic structural units can be formed by artificial design, so that the recombinant collagen has the advantages of no risk of animal-derived collagen viruses, low rejection, good biocompatibility and high safety. For better application in the field of tissue engineering, recombinant collagen is usually designed as water-soluble collagen, however, collagen is very easily degraded in aqueous solution, and the shelf form of lyophilized powder avoids the problem of collagen hydrolysis, but temporary reconstitution before use increases the complexity of clinical operation and also has the risk of introducing pollution. Moreover, the pure collagen aqueous solution does not have any isolating, filling and supporting effects, so that the optimal effect can be realized by combining the recombinant collagen with other raw materials.
Sodium hyaluronate is a natural biological molecule with extremely high moisture retention, viscoelasticity and lubricity, widely exists in skin and other tissues, has extremely good moisture retention, has the functions of isolation, filling and support after being implanted, and is widely applied to facial filling, postoperative anti-adhesion and the like. However, the crosslinking mode of sodium hyaluronate is usually chemical crosslinking, in which unremoved free small molecules such as 1,4 butanediol glycidyl ether (BDDE) are toxic, and the small molecules of the crosslinking agent are still gradually exposed during degradation after the crosslinked sodium hyaluronate is implanted into a human body, so that a certain safety risk is provided. The products currently in use on the market generally are gels formed by mechanical mixing of recombinant collagen and crosslinked sodium hyaluronate, in which the recombinant collagen and crosslinked sodium hyaluronate only exert their respective effects, while retaining their respective drawbacks.
The current recombinant collagen and sodium hyaluronate mainly have the following technical problems:
1. the aqueous solution of recombinant collagen is unstable and easily hydrolyzed because the molecular structure contains easily hydrolyzed chemical bonds, which are easily broken in water, resulting in the destabilization of recombinant collagen.
2. The recombinant collagen aqueous solution has no isolation, filling and supporting effects, and has limited application when used alone, and cannot exert the optimal effect of the recombinant collagen. The above effect is not shown because the linear structure of the recombinant collagen molecule limits its morphology and function in water.
3. The co-and/or separate crosslinking of recombinant collagen with sodium hyaluronate generally requires the use of chemical crosslinking agents. These cross-linking agents can react with recombinant collagen and sodium hyaluronate molecules to form cross-linked structures. However, these crosslinking agents present a certain safety risk.
4. Blending of recombinant collagen with crosslinked sodium hyaluronate does not improve the stability of recombinant collagen. This is because the structure after blending does not change the hydrolytic properties of the recombinant collagen, and there is still a risk of hydrolysis.
Therefore, the development of a sodium hyaluronate-recombinant collagen hydrogel that can spontaneously achieve crosslinking without the need for additional crosslinking agents is a need for a solution to the problem of those skilled in the art.
Disclosure of Invention
The embodiment of the application provides a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and a preparation method thereof, which are used for solving the problems existing in the related art, and the technical scheme is as follows:
in a first aspect, embodiments of the present application provide a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel comprising: at least one sodium hyaluronate and at least one recombinant collagen comprising free amino groups, the remainder being a solvent.
In one embodiment of the above technical solution, the molecular weight of the sodium hyaluronate is 10000-5000000.
In one embodiment of the above technical solution, the sodium hyaluronate is oxidized by sodium periodate, and adjacent hydroxyl groups in the sodium hyaluronate are oxidized into aldehyde groups.
In one embodiment of the above aspect, the recombinant collagen comprises a number of free amino groups ranging from 10 to 1000.
In one embodiment, the recombinant collagen comprises recombinant human collagen, or recombinant humanized collagen, or recombinant human-like collagen.
In one embodiment of the above method, the solvent is water for injection, or 0.9% physiological saline, or PBS phosphate buffer.
In one embodiment, the mass of the sodium hyaluronate is 0.2% -30% of the system.
In one embodiment, the mass of the recombinant collagen accounts for 0.05% -15% of the system.
In a second aspect, embodiments of the present application provide a method for preparing a self-crosslinked sodium hyaluronate-recombinant collagen hydrogel, for preparing a self-crosslinked sodium hyaluronate-recombinant collagen hydrogel according to any one of the above, comprising:
preparing sodium hyaluronate into sodium hyaluronate solution, oxidizing sodium hyaluronate with sodium periodate, dialyzing and freeze-drying to obtain sodium hyaluronate solution containing aldehyde group;
adding the recombinant collagen into a solvent to prepare a recombinant collagen solution;
and dropwise adding the recombinant collagen solution into the continuously stirred sodium hyaluronate solution to obtain the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel.
In one embodiment, the duration of stirring is 10-30min.
The advantages or beneficial effects in the technical scheme at least comprise:
the application adopts an innovative method to solve the problems of instability and easy hydrolysis of the recombinant collagen aqueous solution. Hydroxyl groups in sodium hyaluronate are oxidized into aldehyde groups by utilizing sodium periodate, and the aldehyde groups are self-assembled with amino groups in recombinant collagen to form Schiff base groups. The content of aldehyde groups in sodium hyaluronate and the content of amino groups in recombinant collagen can be controlled by adjusting process parameters and genetic design, so that the proportion and the content between the sodium hyaluronate and the recombinant collagen are adjusted to influence the crosslinking degree.
The cross-linked structure of the recombinant collagen in the hydrogel is realized through spontaneous reaction between aldehyde groups and amino groups, molecular entanglement of different molecular weights and formation of hydrogen bonds without the assistance of any other cross-linking agent. The crosslinked structure can improve the stability of the recombinant collagen and avoid the safety risk of using toxic small molecule crosslinking agents.
By this self-crosslinking method, the present application successfully prepares a complex of sodium hyaluronate and recombinant collagen. The composite not only has good stability, but also realizes the functions of isolation, filling and supporting. Therefore, the problem that the use of recombinant collagen alone is limited is solved, so that the recombinant collagen can be better applied to the field of tissue engineering.
The method can not only improve the stability of the recombinant collagen, but also avoid the use of toxic small molecule cross-linking agents, thereby improving the safety of the product. Meanwhile, the property of the compound can be controlled by adjusting the crosslinking degree, and hydrogels with different viscosities can be prepared, so that the hydrogel has wider application potential.
The foregoing summary is for the purpose of the specification only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will become apparent by reference to the drawings and the following detailed description.
Drawings
In the drawings, the same reference numerals refer to the same or similar parts or elements throughout the several views unless otherwise specified. The figures are not necessarily drawn to scale. It is appreciated that these drawings depict only some embodiments according to the disclosure and are not therefore to be considered limiting of its scope.
FIG. 1 is a schematic diagram of self-crosslinking sodium hyaluronate-recombinant collagen hydrogels prepared in example 1 and example 6.
FIG. 2 is an infrared spectrum of the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel of example 1.
FIG. 3 is a graph showing the supporting effect of injection of the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel of example 6.
FIG. 4 is a graph showing the unsupported effect of the aqueous recombinant collagen solution of comparative example 1 after injection.
Detailed Description
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
In a first aspect, embodiments of the present application provide a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel comprising: at least one sodium hyaluronate and at least one recombinant collagen comprising free amino groups, the remainder being a solvent.
In one embodiment, the sodium hyaluronate has a molecular weight of 10000-5000000.
In one embodiment, the sodium hyaluronate is oxidized by sodium periodate, and adjacent hydroxyl groups in the sodium hyaluronate are oxidized to aldehyde groups.
The following is a reaction formula for oxidizing sodium hyaluronate with sodium periodate. The preparation scheme of sodium hyaluronate containing aldehyde groups in the reaction formula can be adopted, and adjacent hydroxyl groups in sodium hyaluronate are oxidized into aldehyde groups by utilizing sodium periodate.
In one embodiment, the recombinant collagen comprises a number of free amino groups ranging from 10 to 1000.
In one embodiment, the recombinant collagen comprises recombinant human collagen, or recombinant humanized collagen, or recombinant human-like collagen.
In one embodiment, the solvent is water for injection, or 0.9% physiological saline, or PBS phosphate buffer.
In one embodiment, the sodium hyaluronate comprises 0.2% -30% by mass of the system.
In one embodiment, the recombinant collagen comprises 0.05% to 15% by mass of the system.
In a second aspect, embodiments of the present application provide a method for preparing a self-crosslinked sodium hyaluronate-recombinant collagen hydrogel, for preparing a self-crosslinked sodium hyaluronate-recombinant collagen hydrogel according to any one of the above, comprising:
preparing sodium hyaluronate into sodium hyaluronate solution, oxidizing sodium hyaluronate with sodium periodate, dialyzing and freeze-drying to obtain sodium hyaluronate solution containing aldehyde group;
adding the recombinant collagen into a solvent to prepare a recombinant collagen solution;
and dropwise adding the recombinant collagen solution into the continuously stirred sodium hyaluronate solution to obtain the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel.
When sodium hyaluronate is prepared as a sodium hyaluronate solution, water for injection (or purified water) is actually used. The water for injection (or purified water, sterile water, deionized water) used in this portion can be separately and additionally formulated, which does not affect the final result due to the conventional technical means; the amount of water for injection can also be calculated into the whole solvent amount, the change of the solvent addition amount has little influence on the final result, and the solvent used in the part can limit the range as follows: sodium hyaluronate is added into 15% -85% solvent to prepare sodium hyaluronate solution.
When preparing the solution of sodium hyaluronate containing aldehyde groups, the solvent is used.
The specific reaction principle of the steps is as follows: preparing sodium hyaluronate into sodium hyaluronate solution, adding sodium periodate into the sodium hyaluronate solution, oxidizing sodium hyaluronate by the sodium periodate, washing out the sodium periodate by dialysis after the oxidation reaction is finished, changing the concentration of the sodium hyaluronate solution in the dialysis process, freeze-drying the oxidized sodium hyaluronate solution (after washing out the sodium periodate by dialysis) to obtain freeze-dried fibers (the form of the product obtained after freeze-drying is unimportant in the application, and the fibers are the appearance of a sample obtained in the real process), and adding the freeze-dried fibers into a part of solvent to prepare the sodium hyaluronate solution containing aldehyde groups; adding the recombinant collagen into a solvent to prepare a recombinant collagen solution; and dropwise adding the recombinant collagen solution into the continuously stirred sodium hyaluronate solution to obtain the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel. More specifically, the gel forming process of the present application is; the recombinant collagen solution is dripped into the sodium hyaluronate solution containing aldehyde groups, and the two solutions form a compact cross-linked structure. Before the crosslinking reaction reaches a certain critical value, the crosslinking degree is insufficient to separate out the gel from the system, but the appearance, viscosity and other properties of the system are improved, such as the gel shown in fig. 1 (b). When the cross-linking reaction breaks through the critical value, the gel gradually separates out from the solution after reaching a certain degree, and the gel can be fished out from the aqueous solution, as shown in the gel form of fig. 1 (a).
The following is a cross-linking reaction formula of recombinant collagen and a sodium hyaluronate solution containing aldehyde groups. The free amino groups in the recombinant collagen are self-crosslinked with aldehyde groups in the oxidized sodium hyaluronate to form Schiff base groups. The sodium hyaluronate and the recombinant collagen form a cross-linked structure in an aqueous solution through molecular entanglement, schiff base groups, hydrogen bonds and the like with different molecular weights, and finally the hydrogel is formed.
In one embodiment, the duration of stirring is from 10 to 30 minutes.
The sodium hyaluronate and the recombinant collagen form Schiff base groups, molecular entanglement of different molecular weights, hydrogen bond formation and the like in the aqueous solution through crosslinking, so that a firm hydrogel network is obtained, and the stability of the recombinant collagen is improved.
The present application crosslinks sodium hyaluronate and recombinant collagen to form a hydrogel by introducing sodium hyaluronate. The composite not only has good stability, but also realizes the functions of isolation, filling and supporting. Therefore, the problem that the use of recombinant collagen alone is limited is solved, so that the recombinant collagen can be better applied to the field of tissue engineering.
The cross-linked structure of the recombinant collagen in the hydrogel is realized through spontaneous reaction between aldehyde groups and amino groups, molecular entanglement of different molecular weights and formation of hydrogen bonds without the assistance of any other cross-linking agent. The crosslinked structure can improve the stability of the recombinant collagen and avoid the safety risk of using toxic small molecule crosslinking agents.
The stability of the recombinant collagen can be improved by the reticular structure formed by crosslinking the recombinant collagen and the sodium hyaluronate, and the reticular structure can prevent breakage and diffusion of molecular chains.
Example 1
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. each 50g of sodium hyaluronate having a molecular weight of 300,000 and 1,500,000 was weighed and prepared as a solution, and in this example, sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen containing about 10 amino groups on a molecular chain into the rest of solvent to prepare solution, wherein the solvent is phosphate buffer solution. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 15% in the solution system and the sodium hyaluronate containing aldehyde groups was 5% in the solution system. Stirring was continued for 30min to finally obtain a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, see fig. 1 (b), which was prepared in example 1.
Among them, a process of oxidizing sodium hyaluronate with sodium periodate is a prior art, and thus the process is not described.
Example 2
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. each of 500,000 and 1,000,000 molecular weight sodium hyaluronate was weighed 50g and prepared as a solution, and in this example, sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 70 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is phosphate buffer solution. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 13% in the solution system and the sodium hyaluronate containing aldehyde groups was 8% in the solution system. Stirring is continued for 30min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 3
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. sodium hyaluronate of molecular weight 200,000 and 1,500,000 was weighed 75g and 25g, respectively, and prepared as solutions, in this example sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 70 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is phosphate buffer solution. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 13% in the solution system and the sodium hyaluronate containing aldehyde groups was 8% in the solution system. Stirring is continued for 30min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 4
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. sodium hyaluronate of molecular weight of 10,000, 800,000 and 5,000,000 was weighed 45g, 35g and 20g, respectively, and prepared as solutions, in this example sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 70 amino groups on a molecular chain into the rest of solvent to prepare solution, wherein the solvent is physiological saline. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 5% in the solution system and the sodium hyaluronate containing aldehyde groups was 0.2% in the solution system. Stirring is continued for 30min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 5
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. 100g of sodium hyaluronate having a molecular weight of 200,000 was weighed and prepared as a solution, and in this example, sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 50 amino groups on a molecular chain into the rest of solvent to prepare solution, wherein the solvent is physiological saline. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was present at a ratio of 0.05% in the solution system and the sodium hyaluronate containing aldehyde groups was present at a ratio of 0.2% in the solution system. Stirring is continued for 10min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 6
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. sodium hyaluronate of 400,000 and 1,500,000 molecular weight was weighed 50g each and prepared as a solution, in this example sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 700 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is water for injection. The recombinant collagen solution was then added drop-wise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was present in a 10% ratio in the solution system and the sodium hyaluronate containing aldehyde groups was present in a 25% ratio in the solution system. Stirring was continued for 10min to finally obtain a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, see fig. 1 (a), which was prepared in example 6.
Example 7
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. 100g of sodium hyaluronate having a molecular weight of 800,000 was weighed and prepared as a solution, and in this example, sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 700 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is water for injection. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 12% in the solution system and the sodium hyaluronate containing aldehyde groups was 30% in the solution system. Stirring is continued for 10min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 8
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. 100g of sodium hyaluronate having a molecular weight of 5,000,000 was weighed out and prepared as a solution, and in this example, sodium hyaluronate solution was prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 1000 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is water for injection. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 15% in the solution system and the sodium hyaluronate containing aldehyde groups was 12% in the solution system. Stirring is continued for 10min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Example 9
The embodiment provides a preparation method of self-crosslinking sodium hyaluronate-recombinant collagen hydrogel, which comprises the following steps:
1. sodium hyaluronate of molecular weight 800,000 and 1,500,000 were weighed 50g each and prepared as solutions, in this example sodium hyaluronate solutions were prepared using water for injection. And (3) oxidizing the sodium hyaluronate by sodium periodate, and then completing dialysis and freeze-drying to obtain the sodium hyaluronate freeze-dried fiber containing aldehyde groups.
2. Firstly, adding sodium hyaluronate freeze-dried fiber containing aldehyde groups into part of solvent to prepare solution, adding recombinant collagen with about 800 amino groups on a molecular chain into the rest solvent to prepare solution, wherein the solvent is water for injection. The recombinant collagen solution was then added dropwise to a continuously stirred sodium hyaluronate solution, wherein the recombinant collagen was 15% in the solution system and the sodium hyaluronate containing aldehyde groups was 20% in the solution system. Stirring is continued for 20min, and finally the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel is obtained.
Comparative example 1
The recombinant collagen comprising about 800 amino groups on the molecular chain is prepared into a solution, and the solvent is water for injection. Wherein the proportion of the recombinant collagen in the solution system is 15 percent.
The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel prepared in example 9 was immersed in an aqueous solution at 5 ℃ to prepare 5 groups of parallel samples in total, the batch mass was recorded, sampling was performed at time nodes of 2 weeks, 5 weeks, 8 weeks, and 10 weeks, and freeze-dried, the weight of the freeze-dried samples was tested, and the sample weight loss rate was calculated according to formula (1).
Wherein W is Original source For the sample feeding quality, W Terminal (A) To the final weight of the lyophilized sample.
The recombinant collagen solution prepared in comparative example 1 was placed in an environment of 5 ℃ and the molecular weight of the recombinant collagen was tested by electrophoresis after 1 week to determine degradation thereof. The results are shown in the following table.
The above degradation data demonstrate that the stability of the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel of example 9 is better than the recombinant collagen aqueous solution of comparative example 1.
Referring further to FIG. 2, an infrared spectrum of the self-crosslinked sodium hyaluronate-recombinant collagen hydrogel of example 1 is shown, which shows 1653cm -1 At the schiff base c=n peak, evidence of the presence of schiff base, i.e. the crosslinking of recombinant collagen with sodium hyaluronate.
Referring to FIG. 3, the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel of example 6 was used for injection, and immediately after the hydrogel was injected subcutaneously, a clear hydrogel sample was observed
Referring to fig. 4, the injection of the aqueous recombinant collagen solution of comparative example 1 was used, and it was apparent that there was no supporting property, and the sample was dispersed subcutaneously after injection, and the sample was unable to maintain its basic form and remained subcutaneously. The sample was not visible after moisture was absorbed.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The foregoing is merely specific embodiments of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art can easily think of various changes or substitutions within the technical scope of the present application, and these should be covered in the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. A self-crosslinking sodium hyaluronate-recombinant collagen hydrogel comprising: at least one sodium hyaluronate and at least one recombinant collagen comprising free amino groups, the remainder being a solvent.
2. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 1, wherein the sodium hyaluronate has a molecular weight of 10000-5000000.
3. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 2, wherein the sodium hyaluronate is oxidized by sodium periodate and adjacent hydroxyl groups in the sodium hyaluronate are oxidized to aldehyde groups.
4. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 1, wherein the recombinant collagen comprises a number of free amino groups ranging from 10 to 1000.
5. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel of claim 4, wherein the recombinant collagen comprises recombinant human collagen, or recombinant humanized collagen, or recombinant human-like collagen.
6. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 1, wherein the solvent is water for injection, or 0.9% physiological saline, or PBS phosphate buffer.
7. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to any one of claims 1-6, wherein the sodium hyaluronate is present in an amount of 0.2% -30% by mass of the system.
8. The self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 7, wherein the mass of the recombinant collagen is 0.05% -15% of the system.
9. A method for preparing a self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to any one of claims 1 to 8, comprising:
preparing sodium hyaluronate into sodium hyaluronate solution, oxidizing sodium hyaluronate with sodium periodate, dialyzing and freeze-drying to obtain sodium hyaluronate solution containing aldehyde group;
adding the recombinant collagen into a solvent to prepare a recombinant collagen solution;
and dropwise adding the recombinant collagen solution into the continuously stirred sodium hyaluronate solution to obtain the self-crosslinking sodium hyaluronate-recombinant collagen hydrogel.
10. The method for preparing self-crosslinking sodium hyaluronate-recombinant collagen hydrogel according to claim 9, wherein the continuous stirring time is 10-30min.
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