CN115177790A - Preparation method of hyaluronic acid and lubricin protein synergistically modified collagen matrix - Google Patents
Preparation method of hyaluronic acid and lubricin protein synergistically modified collagen matrix Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (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-carboxylic acid Chemical compound 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 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 81
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- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 claims description 3
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
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- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
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- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- MBLBDJOUHNCFQT-UHFFFAOYSA-N n-(3,4,5,6-tetrahydroxy-1-oxohexan-2-yl)acetamide Chemical compound CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
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- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
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Abstract
The invention discloses a preparation method of a collagen matrix based on synergistic modification of hyaluronic acid and lubricin protein, which improves the anti-protein adsorption and lubrication promotion properties of the collagen matrix and belongs to the technical field of biological materials. According to the invention, the hyaluronic acid and the lubricin protein are directly connected to the surface of the collagen by utilizing the special molecular structures of the hyaluronic acid and the lubricin protein, so that the collagen matrix modified by the hyaluronic acid and the lubricin protein is obtained. The modified collagen surface prepared by the method has excellent lubricating property, protein adsorption resistance and biocompatibility, can be conveniently and automatically modified on the collagen surface, can be well combined with the damaged cartilage collagen surface, overcomes the defects of insufficient lubricating property and poor protein resistance of the unmodified collagen surface, is simple and convenient, and has good feasibility and practicability.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of a collagen matrix synergistically modified by hyaluronic acid and lubricin protein.
Background
Osteoarthritis is a disabling chronic joint disease, with up to 10% of men and 18% of women suffering from osteoarthritis in the world's population over the age of 60. Osteoarthritis causes damage and loss of cartilage matrix tissue, such as degradation and shedding of molecules like Collagen (COL), proteoglycans, aggrecan, etc. from the cartilage surface, which in turn leads to abnormal deposition and increased friction of cartilage matrix surface proteins. Healthy articular cartilage is a highly effective water-based tribological system with a very low coefficient of friction and its unique mechanical properties depend on the interaction of components covering the surface of the cartilage matrix. The cartilage matrix consists of up to 70-80% water and complex macromolecules, and the COL matrix accounts for about 60-80% of the dry weight of adult articular cartilage and is considered to be the major structural component of the cartilage matrix. The collagen structural surface of the diseased articular cartilage has early wear and high friction, and simultaneously has more abnormal protein deposition and chondrocyte apoptosis.
Hyaluronic Acid (HA) is composed of D-glucuronic acid and D-N-acetylglucosamine which are alternately linked by beta-1,4 glycosidic bonds and beta-1,3-glycosidic bonds, and is an important glycosaminoglycan of the cartilage extracellular matrix and a main component of synovial fluid. Studies have found that HA HAs excellent lubricating properties and good anti-inflammatory properties and plays an essential role in the treatment of osteoarthritis. Although hyaluronic acid alone can temporarily alleviate the progression of cartilage damage, it is not fundamentally regenerative for repair of cartilage tissue.
Disclosure of Invention
In order to overcome the defects of poor non-specific property and insufficient lubricating property of collagen surface protein and achieve the aim of better treating cartilage injury, the invention provides a preparation method of a collagen matrix modified by hyaluronic acid and lubricin protein in a synergistic manner.
Therefore, the invention adopts the following technical scheme:
a preparation method of a collagen matrix cooperatively modified by hyaluronic acid and lubricin protein comprises the following steps:
1) Preparing a collagen matrix:
preparing a phosphate buffer solution with the pH value of 7.3-7.5, dissolving collagen in a proper amount of the phosphate buffer solution to prepare a collagen solution with the concentration of 50 mu g/mL, then dropwise adding 100 mu L of the collagen solution to the surface of the substrate, incubating for 1-3h, and washing with the phosphate buffer solution to remove molecules which are not adsorbed on the substrate, so as to obtain a collagen substrate modified on the surface of the substrate;
2) Preparing a collagen matrix modified by hyaluronic acid and lubricin protein in a synergistic way:
dripping 100 mu L of mixed solution of hyaluronic acid and lubricin protein on the surface of the collagen matrix obtained in the step 1), incubating for 1-3h, then thoroughly washing the surface with the phosphate buffer solution and drying with nitrogen to prepare the collagen matrix modified by the hyaluronic acid and the lubricin protein in a synergistic way, wherein the concentrations of the hyaluronic acid and the lubricin protein in the mixed solution are 1mg/mL and 50 mu g/mL-200 mu g/mL respectively.
The preparation method of the phosphate buffer solution comprises the following steps: 3.63g of disodium hydrogen phosphate dodecahydrate, 0.27g of potassium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were dissolved in 1L of deionized water and adjusted to the desired pH.
Preferably, the hyaluronic acid has a molecular weight in the range of 9000-3000000.
Preferably, the concentration of lubricin protein in the mixture of step 2) is 100. Mu.g/mL.
The lubricin protein used in the present invention is a recombinant lubricin protein.
The substrate may be a joint, cartilage tissue or the surface of a culture dish.
The collagen matrix synergistically modified by the hyaluronic acid and the lubricin protein prepared by the method can be used in the field of biomedicine, and is particularly suitable for preparing cartilage repair materials, treating osteoarthritis and improving the protein adsorption resistance and lubricating performance of collagen on the surface of damaged cartilage.
According to the invention, through the special intermolecular interaction of natural hyaluronic acid and lubricin protein and collagen, the hyaluronic acid and lubricin protein are synergistically modified on the surface of the collagen, and the collagen matrix prepared by the method not only retains the migration and growth of the collagen in chondrocytes and the adhesion capability of the collagen in damaged cartilage tissues, but also has excellent non-specific adsorption resistance and lubrication promotion performance. Lubricin protein (LUB) is a highly glycosylated macromolecular protein secreted by chondrocytes and synoviocytes, is a major component of synovial fluid and cartilage surfaces, and synergistically reduces cartilage wear with HA.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1) The surface of the collagen matrix synergistically modified by the hyaluronic acid and the lubricin protein prepared by the method has good non-specific adsorption performance of anti-protein, and the non-specific adsorption quantity of lysozyme (with positive charges) and bovine serum albumin (with negative charges) is respectively reduced to 0ng/cm 2 、0ng/cm 2 The non-specific adsorption quantity of the collagen matrix is far lower than that of the unmodified collagen matrix, so that various side effects or infection problems caused by protein and bacteria adsorption can be greatly reduced.
2) The surface of the collagen matrix synergistically modified by the hyaluronic acid and the lubricin protein prepared by the method has excellent lubricating property, the friction coefficient in Phosphate Buffered Saline (PBS) simulating a physiological environment can be as low as about 0.07, and the defect of poor lubricating property of unmodified collagen is overcome.
3) The hyaluronic acid and lubricin protein synergistically modified collagen matrix prepared by the method has high surface abrasion resistance, and the abrasion resistance pressure in phosphate buffer solution (PBS, simulated physiological environment) can reach 10.83MPa and is far higher than that of unmodified collagen.
4) The preparation method is simple and convenient, has good biocompatibility and has good feasibility and practicability.
Drawings
FIG. 1 is a schematic diagram of the structure of a collagen matrix synergistically modified with hyaluronic acid and lubricin protein according to the present invention;
FIG. 2 is a graph showing the non-specific adsorption real-time curve change of Lysozyme (LYS) at 2mg/mL on the surface of collagen in example 3 modified in cooperation with hyaluronic acid and lubricin;
FIG. 3 is a graph showing the non-specific adsorption real-time curve of Bovine Serum Albumin (BSA) on the surface of collagen modified in cooperation with hyaluronic acid and lubricin protein in example 3;
FIG. 4 is a graph of the friction performance of collagen synergistically modified with hyaluronic acid and lubricin protein in example 1;
FIG. 5 is a graph of the friction performance of collagen synergistically modified with hyaluronic acid and lubricin protein in example 2;
fig. 6 is a graph showing the frictional properties of collagen synergistically modified with hyaluronic acid and lubricin protein in example 3.
Detailed Description
The preparation method of the present invention is described in detail below with reference to the accompanying drawings and examples.
Hyaluronic Acid (HA) and lubricin protein (LUB) have the following structures, respectively:
in the following examples, the preparation of phosphate buffer was as follows:
3.63g of disodium hydrogen phosphate dodecahydrate, 0.27g of potassium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were dissolved in 1L of deionized water to prepare a 10mM phosphate buffer solution of pH = 7.4.
In the following examples, the lubricin protein was a recombinant lubricin protein, purchased from Lubris Biopharma (Weston, MA).
The hyaluronic acid in the invention is food grade.
The collagen used in the invention is nontoxic, pyrogen-free and has a purity of more than 99.9%. Collagen used in the following examples was purchased from joint kinetic Anda (Tianjin) Biotechnology, inc.
The structure of the collagen matrix synergistically modified by hyaluronic acid and lubricin protein according to the present invention is schematically shown in FIG. 1.
For the convenience of preparation and performance testing, the following examples all use a chip with gold film used in mica or dissipative quartz crystal microbalance system as a substrate, which only serves as a support and has no effect on the function of the collagen matrix prepared. It is understood that in practical applications, joints, cartilage tissue or the surface of a culture dish, etc. may be used as a substrate.
Example 1
A preparation method of a collagen matrix cooperatively modified by hyaluronic acid and lubricin protein comprises the following steps:
1) Preparing a collagen matrix:
collagen (COL) was dissolved in phosphate buffered saline (pH = 7.4) (PBS) to prepare a COL solution of 50 μ g/mL, and then 100 μ L of the COL solution was dropped on the surface of the substrate (chip or mica), incubated for 1h, and washed with PBS (pH = 7.4) to remove molecules not adsorbed on the substrate, resulting in a collagen matrix modified on the surface of the substrate.
2) Synergistic modification of hyaluronic acid and lubricin protein on the surface of collagen matrix:
dripping 100 mu L of mixed solution of Hyaluronic Acid (HA) and lubricin protein (LUB) on the surface of the COL matrix obtained in the step 1), incubating for about 1h, then thoroughly washing the surface with PBS and drying with nitrogen, and finally obtaining the collagen matrix (COL/HA/LUB-1 h) synergistically modified by the hyaluronic acid and the lubricin protein. Wherein the concentrations of HA and LUB in the mixed solution are respectively 1mg/mL HA and 100 mug/mL.
Determination of nonspecific adsorption resistance:
the nonspecific adsorption resistance of the surface of the collagen synergistically modified by the hyaluronic acid and the lubricin protein is tested by the following specific method:
the obtained dissipative quartz crystal microbalance chip (COL/HA/LUB) with the collagen matrix synergistically modified by the hyaluronic acid and the lubricin protein is attached to a flow cell of a dissipative quartz crystal microbalance system. Phosphate buffer at pH 7.4 was used as the mobile phase at a flow rate of 50. Mu.L/min. After the baseline is stable, 2mg/mL bovine serum albumin or lysozyme is introduced, the protein solution is pushed to the surface of the chip by the mobile phase, a real-time change curve is determined by a dissipative quartz crystal microbalance system, the frequency change value is read, and the nonspecific adsorption quantity is calculated, which is shown in Table 1.
As is clear from the results shown in Table 1, the nonspecific adsorption amounts of bovine serum albumin and lysozyme to the surface of the collagen matrix modified with hyaluronic acid and lubricin prepared in example 1 were 3.87ng/cm, respectively 2 And 5.87ng/cm 2 Far lower than the collagen surface adsorption amount of the control group.
Friction coefficient measurement and lubrication performance evaluation:
the surface force instrument is adopted to measure the friction coefficient of the prepared hyaluronic acid and lubricin protein synergistically modified collagen surface, and the lubricating property of the hyaluronic acid and lubricin protein is evaluated, and the specific method comprises the following steps:
the mica sheet with the collagen matrix modified by the hyaluronic acid and the lubricin protein in a synergic mode is fixed in a surface dynamometer test system to measure the friction coefficient of the mica sheet, the measurement is repeated for 3 times, and the result is summarized as shown in figure 4.
As can be seen from fig. 4, the collagen matrix modified based on hyaluronic acid and lubricin protein (COL/HA/LUB-1 h) prepared in example 1 had a surface friction coefficient of 0.07, which is much lower than that of a single collagen surface (0.48), and thus had good lubricating properties.
Example 2
A preparation method of a collagen matrix cooperatively modified by hyaluronic acid and lubricin protein comprises the following steps:
1) Preparing a collagen matrix:
COL was dissolved in PBS (pH = 7.4) to prepare a solution of 50 μ g/mL, and then 100 μ L of the COL solution was dropped onto the surface of a substrate (chip or mica), incubated for 2h, and washed with PBS to remove molecules that were not adsorbed to the substrate, resulting in a collagen matrix modified on the surface of the substrate.
2) Synergistic modification of hyaluronic acid and lubricin protein on the surface of collagen matrix:
dripping 100 mu L of mixed solution of HA and LUB on the surface of the COL matrix obtained in the step 1), incubating for 2h, then thoroughly washing the surface with PBS and drying with nitrogen, and finally obtaining the collagen matrix (COL/HA/LUB-2 h) modified by the cooperation of hyaluronic acid and lubricin protein. The concentrations of HA and LUB in the mixed solution are respectively 1mg/mL solution and 100 mug/mL.
Determination of nonspecific adsorption resistance:
the test method was the same as in example 1. The test results are shown in Table 1.
As is clear from the results shown in Table 1, the non-specific adsorption amounts of bovine serum albumin and lysozyme to the surfaces of the collagen matrices modified with hyaluronic acid and lubricin prepared in example 2 were 1.22ng/cm 2 And 2.31ng/cm 2 Far lower than the collagen surface adsorption amount of the control group.
Evaluation of Friction Properties:
the wear rate of the prepared hyaluronic acid and lubricin protein modified collagen surface is measured by a surface force instrument, and the lubricating property is evaluated by the following specific method:
the mica sheet with the collagen matrix modified by the hyaluronic acid and the lubricin protein in a synergic mode is fixed in a surface dynamometer test system to measure the abrasion speed of the mica sheet, the measurement is repeated for 3 times, and the result is summarized as shown in figure 5.
As can be seen from FIG. 5, the surface (COL/HA/LUB-2 h) of the collagen matrix modified with hyaluronic acid and lubricin-based protein prepared in example 2 had a wear rate of 19 μm/s, which was much higher than that of a single collagen surface (3 μm/s), indicating that it had high wear resistance and excellent lubricating properties.
Example 3
A preparation method of a collagen matrix cooperatively modified by hyaluronic acid and lubricin protein comprises the following steps:
1) Preparing a collagen matrix:
COL was dissolved in PBS (pH = 7.4) to prepare a solution of 50 μ g/mL, and then 100 μ L of the COL solution was dropped on the surface of the substrate (chip or mica), incubated for 3h, and washed with PBS to remove molecules not adsorbed on the substrate, and finally a collagen matrix modified on the surface of the substrate was obtained.
2) Synergistic modification of hyaluronic acid and lubricin protein on the surface of collagen:
dripping 100 mu L of mixed solution of HA and LUB (the concentration is 1mg/mL and 100 mu g/m respectively) on the surface of the COL matrix obtained in the step 1), incubating for about 3h, then thoroughly washing the surface with PBS and drying with nitrogen, and finally obtaining the collagen matrix (COL/HA/LUB-3 h) synergistically modified by hyaluronic acid and lubricin.
Determination of nonspecific adsorption resistance:
the test method was the same as in example 1. The test results are shown in table 1, fig. 2 and fig. 3.
TABLE 1 amount of nonspecific adsorption on different surfaces
As is clear from the results shown in Table 1, the nonspecific adsorption amounts of bovine serum albumin and lysozyme by synergistically modifying the collagen matrix surface (COL/HA/LUB-3 h) based on hyaluronic acid and lubricin prepared in example 3 were 0ng/cm, respectively 2 And 0ng/cm 2 。
Evaluation of Friction Properties:
the surface force instrument is adopted to measure the friction coefficient of the prepared hyaluronic acid and lubricin protein synergistically modified collagen surface, and the lubricating property of the hyaluronic acid and lubricin protein is evaluated, and the specific method comprises the following steps:
the mica sheet with hyaluronic acid and lubricin protein synergistically modified collagen surface is fixed in a surface dynamometer test system to measure the abrasion pressure, the measurement is repeated for 3 times, and the result is summarized as shown in figure 6.
As can be seen from FIG. 6, the wearing pressure of the collagen surface modified based on hyaluronic acid and lubricin protein (COL/HA/LUB-3 h) prepared in example 3 was 10.83MPa, which is much higher than the wearing rate of a single collagen surface (1.87 MPa), indicating that it HAs high wear resistance and excellent lubricating properties.
Claims (6)
1. A preparation method of a collagen matrix cooperatively modified by hyaluronic acid and lubricin protein comprises the following steps:
1) Preparing a collagen matrix:
preparing a phosphate buffer solution with the pH value of 7.3-7.5, dissolving collagen in a proper amount of the phosphate buffer solution to prepare a collagen solution with the concentration of 50 mu g/mL, then dropwise adding 100 mu L of the collagen solution to the surface of the substrate, incubating for 1-3h, washing with the phosphate buffer solution, and removing molecules which are not adsorbed to the substrate to obtain a collagen substrate modified on the surface of the substrate;
2) Preparing a collagen matrix modified by hyaluronic acid and lubricin protein in a synergistic way:
dripping 100 mu L of mixed solution of hyaluronic acid and lubricin protein on the surface of the collagen matrix obtained in the step 1), incubating for 1-3h, then thoroughly flushing the surface with the phosphate buffer solution and blow-drying with nitrogen to prepare the collagen matrix synergistically modified by hyaluronic acid and lubricin protein, wherein the concentrations of hyaluronic acid and lubricin protein in the mixed solution are 1mg/mL and 50 mu g/mL-200 mu g/mL respectively.
2. The method according to claim 1, wherein the phosphate buffer is prepared by: 3.63g of disodium hydrogen phosphate dodecahydrate, 0.27g of potassium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were dissolved in 1L of deionized water and adjusted to the desired pH.
3. The method of claim 1, wherein: the molecular weight range of the hyaluronic acid is 9000-3000000.
4. The production method according to claim 1, characterized in that: in the mixed solution in the step 2), the concentration of the lubricin protein is 100 mug/mL.
5. The method of manufacturing according to claim 4, characterized in that: the lubricin protein is a recombinant lubricin protein.
6. The production method according to any one of claims 1 to 5, characterized in that: the substrate is the surface of a joint, cartilage tissue or a culture dish.
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| US20140369975A1 (en) * | 2012-01-19 | 2014-12-18 | The Johns Hopkins University | Biomaterials comprising hyaluronic acid binding peptides and bifunctional biopolymer molecules for hyaluronic acid retention and tissue engineering applications |
| CN105899527A (en) * | 2013-10-22 | 2016-08-24 | 卢布里斯有限责任公司 | Production of recombinant lubricin |
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| US20140369975A1 (en) * | 2012-01-19 | 2014-12-18 | The Johns Hopkins University | Biomaterials comprising hyaluronic acid binding peptides and bifunctional biopolymer molecules for hyaluronic acid retention and tissue engineering applications |
| CN105899527A (en) * | 2013-10-22 | 2016-08-24 | 卢布里斯有限责任公司 | Production of recombinant lubricin |
| CN109054030A (en) * | 2018-06-28 | 2018-12-21 | 华南理工大学 | A kind of amphoteric ion polymer brush and the preparation method and application thereof based on hyaluronic acid |
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