CN109513045B - Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof - Google Patents
Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof Download PDFInfo
- Publication number
- CN109513045B CN109513045B CN201811382935.5A CN201811382935A CN109513045B CN 109513045 B CN109513045 B CN 109513045B CN 201811382935 A CN201811382935 A CN 201811382935A CN 109513045 B CN109513045 B CN 109513045B
- Authority
- CN
- China
- Prior art keywords
- protein
- hydrogel
- platelet
- layer
- plasma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention discloses a protein-based hydrogel with double layers and different internal pore diameter structures and a preparation method thereof. The double-layer hydrogel takes platelet-rich lysate plasma as a main matrix, has unique double-layer different internal pore diameter structures and high porosity, is simple in preparation method, and is prepared under a low-temperature environment, so that the double-layer hydrogel is beneficial to preservation of bioactive molecules such as proteins in the platelet-rich lysate plasma. On the other hand, the small-pore gel layer of the double-layer hydrogel is stronger in compactness and hardness than the large-pore gel layer, so that the double-layer hydrogel has important application prospects in the fields of damage repair of soft and hard two layers of different structural tissues and the like.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a protein-based hydrogel with double layers and different internal pore diameter structures and a preparation method thereof.
Background
The hydrogel has a three-dimensional microporous structure inside, so that the hydrogel is beneficial to spreading, migration and proliferation of cells inside the gel, and is convenient for absorption and excretion of nutrients and metabolites, and has wide application prospects in the biomedical fields including cell culture, tissue regeneration and the like.
However, some tissue injuries (e.g. periodontitis) not only have superficial damage but also involve deep tissue, and the structures and densities of superficial and deep tissues are different [ Hajishengalis G. Nature Reviews Immunology,2015,15(1):30-44 ], which results in that the internal microporous structure and the density of the hydrogel itself are not well adapted to the structure of the tissue defect.
To solve this problem, bilayer hydrogels with different internal microporous structures have been the focus of research. The bilayer hydrogels that have been prepared so far are basically bonded together by non-covalent bonding means (e.g., hydrogen bonding) [ references Tavakoli J, Mirzaei S, Tang Y. polymers,2018,10(3):305 ], and the bilayer hydrogels prepared by these methods have weak bonding between the two gels and the two gels are easily separated by heat, shear force, and the like. Therefore, the double-layer hydrogel which takes the protein component as the matrix and is connected by covalent bonds is prepared, has different internal pore size structures, has important application value for the damage repair of tissues with a multi-layer structure, and has not been reported so far.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a preparation method of protein-based hydrogel with double layers with different internal pore sizes.
In order to achieve the above objects, a first aspect of the present invention provides a protein-based hydrogel having two layers with different internal pore sizes, wherein a water-soluble polymer containing carboxyl groups is used as a cross-linking agent, a first protein-based hydrogel layer having first pores is prepared at a low temperature by an amide reaction between amino groups in a protein and carboxyl groups in the cross-linking agent, and then a second protein-based hydrogel layer having second pores is prepared on the first protein-based hydrogel layer by the amide reaction, the two hydrogel layers are bonded together by chemical bonds, and the first pores and the second pores are arranged differently.
The further setting is that the mass percentage of the protein component exceeds 90 percent, the porosity is more than 50 percent, and the internal pore diameter of the two layers of hydrogel is 30-200 microns.
The invention also provides a preparation method of the protein-based hydrogel, which comprises the following steps:
(1) preparing a protein-based first hydrogel layer by taking platelet-rich lysate plasma freeze-dried powder, a cross-linking agent, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and phosphate buffer solution as raw materials and performing an amide reaction;
(2) and (2) adopting the same raw materials as those in the step (1), changing the mass ratio between the platelet-rich lysate plasma freeze-dried powder and the cross-linking agent, and preparing a protein-based second hydrogel layer on the surface of the protein-based first hydrogel layer through amide reaction superposition, wherein the pore diameters of the inner pores of the protein-based first hydrogel layer and the inner pores of the protein-based second hydrogel layer are unequal.
The platelet-rich lysate plasma freeze-dried powder is further prepared by the following steps:
a1. centrifuging the collected blood at the rotating speed of 1000-;
a2. rapidly freezing the yellowish liquid by liquid nitrogen, and then melting in a water bath at 37 ℃, and repeatedly operating for 3-5 times;
a3. centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 9000-;
a4. and (3) freeze-drying the platelet-rich lysate plasma to obtain the platelet-rich lysate plasma freeze-dried powder.
It is further provided that the cross-linking agent is a carboxyl-containing natural polysaccharide or a water-soluble synthetic polymer. Specifically, natural polysaccharide hyaluronic acid, sodium alginate, etc. or synthetic polymer such as polyacrylic acid, etc. can be selected.
The molar ratio of EDC, NHS and the cross-linking agent is further set to be 1 (1-1.2) to 3-4.
The mass ratio of the platelet-rich lysate plasma freeze-dried powder to the cross-linking agent in the steps (1) and (2) is (50-5): 1, the reaction temperature is-10 ℃ to-30 ℃, and the reaction time is 16-24 hours.
The properties of the protein-based bilayer hydrogels described above were characterized as follows:
and observing the sizes of the inner apertures of different layers of the prepared protein-based double-layer hydrogel in a dry state by adopting a scanning electron microscope, calculating the swelling rate and porosity of the double-layer hydrogel by using a weighing method, and testing the storage modulus of the double-layer hydrogel by using a rheometer.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention adopts a freezing gel method to prepare the protein-based hydrogel with double layers of different internal pore diameter structures. The method is simple and convenient, and the low-temperature environment is favorable for maintaining the biological activity of the protein.
(2) The protein-based hydrogel prepared by the invention has a double-layer structure with different internal pore sizes, and is beneficial to the damage repair of tissues with two layers of different structures.
(3) The protein-based hydrogel prepared by the invention also has high porosity, so that the regenerative cells can be conveniently spread and mutually cross-linked in the gel, and the tissue injury repair is promoted.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.
FIG. 1 is an optical photograph of a protein-based hydrogel (platelet-rich lysate plasma provides the protein component and sodium alginate is the cross-linking agent) with two layers of different internal pore sizes prepared in example 1;
FIG. 2 is a scanning electron micrograph of a protein-based hydrogel (platelet-rich lysate plasma provides the protein component and sodium alginate is the cross-linking agent) with two layers of different internal pore size structures prepared in example 1;
FIG. 3 shows the swelling ratio (left) and porosity (right) of the protein-based hydrogel with two layers of different internal pore size structures (platelet-rich lysate plasma provides the protein component and sodium alginate is the cross-linking agent) prepared in example 1;
figure 4 modulus characterization of protein-based hydrogels with bilayer different internal pore size structures (platelet rich lysate plasma provides the protein component and sodium alginate is the cross-linking agent) prepared in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are used for distinguishing two entities with the same name but different names or different parameters, and it should be noted that "first" and "second" are merely for convenience of description and should not be construed as limitations of the embodiments of the present invention, and they are not described in any more detail in the following embodiments.
EXAMPLE 1 preparation of a protein-based bilayer hydrogel with a Small pore size in the lower layer and a Large pore size in the upper layer (sodium alginate as a crosslinker)
a) Extraction of platelet-rich lysate plasma: blood was collected at 1 ml, centrifuged at 1700 rpm for 20 minutes, centrifuged at 3000 rpm for 15 minutes, and the upper pale yellow liquid was collected to remove the lower red blood cell solution. Quickly freezing the yellowish liquid with liquid nitrogen, thawing in 37 deg.C water bath, and repeating the operation for 3-5 times. And centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 10000 r/min for 5 minutes, and collecting supernatant to obtain the platelet-rich lysate plasma. The freeze-dried platelet-rich lysate plasma powder obtained by freeze drying is used as a reactant 1 and is stored in a refrigerator at the temperature of minus 80 ℃.
b) Activation of sodium alginate: a2% sodium alginate solution was prepared by dissolving 2 g of sodium alginate (viscosity average molecular weight ≥ 2000cps) in 100 ml of phosphate buffered saline (PBS, pH 7.2). To 240 microliters of sodium alginate solution, 15 mg of EDC was added, and mixed well with shaking. After 30 min reaction on a shaker, 9 mg of NHS was added and mixed well.
c) Preparation of a protein-based small-pore gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (c) sequentially adding 60 microliters of 40% platelet-rich lysate plasma solution and 90 microliters of sodium alginate solution activated according to the step (b) into a 200-microliter centrifuge tube, uniformly mixing, quickly transferring into a polytetrafluoroethylene die with the diameter of 8 millimeters and the depth of 6 millimeters, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
d) Preparing a protein-based macroporous gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (c) sequentially adding 60 microliters of 40% platelet-rich lysate plasma solution, 30 microliters of phosphate buffered saline (PBS, PH 7.2) and 60 microliters of sodium alginate solution reactivated according to the step b into a 200 microliter centrifuge tube, uniformly mixing, quickly transferring to the surface of the protein-based small-pore gel layer in the mold in the step c, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃. And observing the sizes of the inner apertures of different layers of the double-layer hydrogel in a dry state by adopting a scanning electron microscope after the reaction is finished.
The product obtained in this example is a protein-based hydrogel with two layers of different internal pore sizes (platelet-rich lysate plasma provides the protein component, and sodium alginate is the cross-linking agent), as shown in fig. 1. Scanning electron microscopy shows that random micropores are formed inside the double-layer hydrogel, the pore size of the small-pore gel layer is 40-50 micrometers, and the pore size of the large-pore gel layer is 100-150 micrometers (as shown in figure 2).
EXAMPLE 2 preparation of a protein-based bilayer hydrogel with a Small pore size in the lower layer and a Large pore size in the upper layer (hyaluronic acid as a crosslinker)
a) Extraction of platelet-rich lysate plasma: blood was collected at 1 ml, centrifuged at 1700 rpm for 20 minutes, centrifuged at 3000 rpm for 15 minutes, and the upper pale yellow liquid was collected to remove the lower red blood cell solution. Quickly freezing the yellowish liquid with liquid nitrogen, thawing in 37 deg.C water bath, and repeating the operation for 3-5 times. And centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 10000 r/min for 5 minutes, and collecting supernatant to obtain the platelet-rich lysate plasma. The freeze-dried platelet-rich lysate plasma powder obtained by freeze drying is used as a reactant 1 and is stored in a refrigerator at the temperature of minus 80 ℃.
b) Activation of hyaluronic acid: a1% hyaluronic acid solution was prepared by dissolving 1 g hyaluronic acid (number average molecular weight 100000-200000) in 100 ml phosphate buffered saline (PBS, pH 7.2). 5 mg of EDC were added to 250. mu.l of hyaluronic acid solution and mixed well with shaking. After 30 min reaction on a shaker, 4 mg of NHS was added and mixed well.
c) Preparation of a protein-based small-pore gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (c) sequentially adding 30 microliters of 40% platelet-rich lysate plasma solution, 25 microliters of phosphate buffered saline (PBS, pH 7.2) and 95 microliters of the hyaluronic acid solution activated according to the step b into a 200 microliter centrifuge tube, uniformly mixing, quickly transferring into a polytetrafluoroethylene mold with the diameter of 8 millimeters and the depth of 6 millimeters, and reacting in a refrigerator at the temperature of-20 ℃ for 24 hours.
d) Preparing a protein-based macroporous gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (3) sequentially adding 30 microliters of 40% platelet-rich lysate plasma solution, 45 microliters of phosphate buffered saline (PBS, PH 7.2) and 75 microliters of hyaluronic acid solution reactivated according to the step b into a 200-microliter centrifuge tube, uniformly mixing, quickly transferring to the surface of the protein-based small-pore gel layer in the mould in the step c, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
The product obtained in this embodiment is a protein-based hydrogel (platelet-rich lysate plasma provides protein components, hyaluronic acid is a cross-linking agent) with two layers of different internal pore structures, wherein the pore size of the small-pore gel layer is 50-80 microns, and the pore size of the large-pore gel layer is 120-180 microns.
EXAMPLE 3 preparation of a protein-based bilayer hydrogel with a Small pore size lower layer and a Large pore size upper layer (polyacrylic acid as a crosslinker)
a) Extraction of platelet-rich lysate plasma: blood was collected at 1 ml, centrifuged at 1700 rpm for 20 minutes, centrifuged at 3000 rpm for 15 minutes, and the upper pale yellow liquid was collected to remove the lower red blood cell solution. Quickly freezing the yellowish liquid with liquid nitrogen, thawing in 37 deg.C water bath, and repeating the operation for 3-5 times. And centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 10000 r/min for 5 minutes, and collecting supernatant to obtain the platelet-rich lysate plasma. The freeze-dried platelet-rich lysate plasma powder obtained by freeze drying is used as a reactant 1 and is stored in a refrigerator at the temperature of minus 80 ℃.
b) Activation of polyacrylic acid: a3% polyacrylic acid solution was prepared by dissolving 3 g of polyacrylic acid (number average molecular weight 100000) in 100 ml of phosphate buffered saline (PBS, pH 7.2). 5 mg of EDC were added to 250. mu.l of polyacrylic acid solution and mixed well with shaking. After 30 min reaction on a shaker, 4 mg of NHS was added and mixed well.
c) Preparation of a protein-based small-pore gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (3) adding 81 microliters of 40% platelet-rich lysate plasma solution and 69 microliters of polyacrylic acid solution activated according to the step (b) into a 200-microliter centrifuge tube in sequence, mixing uniformly, quickly transferring into a polytetrafluoroethylene mold with the diameter of 8 millimeters and the depth of 6 millimeters, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
d) Preparing a protein-based macroporous gel layer: platelet-rich lysate plasma lyophilized powder was dissolved in phosphate buffered saline (PBS, PH 7.2) at a concentration of 40%. And (3) adding 81 microliters of 40% platelet-rich lysate plasma solution, 15 microliters of phosphate buffered saline (PBS, PH 7.2) and 54 microliters of polyacrylic acid solution reactivated according to the step b into a 200 microliter centrifuge tube in sequence, mixing uniformly, quickly transferring to the surface of the protein-based small-pore gel layer in the mould in the step c, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
The product obtained in this embodiment is a protein-based hydrogel (platelet-rich lysate plasma provides protein components, polyacrylic acid is a cross-linking agent) with two layers of different internal pore-size structures, wherein the pore size of the small-pore gel layer is 30-60 micrometers, and the pore size of the large-pore gel layer is 80-150 micrometers.
EXAMPLE 4 preparation of protein-based bilayer hydrogel with a lower layer of small pore size and an upper layer of large pore size (MC-3T3-E1 cell lysate provides the protein component)
a) Preparation of MC-3T3-E1 cell lysate: after the MC-3T3-E1 cells grow fully adherent to the wall in a culture flask, the cells are digested by pancreatin, then the culture medium is added for suspension, and the cells are centrifuged for 5 minutes at the rotating speed of 1000 rpm. The supernatant was aspirated, suspended in phosphate buffered saline (PBS, pH 7.2) and centrifuged at 1000 rpm for 5 minutes, and then aspirated to remove residual medium. The cells were suspended by adding 1 ml of phosphate buffered saline (PBS, pH 7.2), the cell suspension was rapidly frozen by liquid nitrogen, and then thawed in a water bath at 37 ℃ by repeating the operation 3 to 5 times. And centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 400 rpm for 10 minutes, and collecting the supernatant, namely the MC-3T3-E1 cell lysate. Freeze-drying the MC-3T3-E1 cell lysate freeze-dried powder as a reactant 1, and storing in a refrigerator at-80 ℃.
b) Activation of hyaluronic acid: a1% hyaluronic acid solution was prepared by dissolving 1 g hyaluronic acid (number average molecular weight 100000-200000) in 100 ml phosphate buffered saline (PBS, pH 7.2). 5 mg of EDC were added to 250. mu.l of hyaluronic acid solution and mixed well with shaking. After 30 min reaction on a shaker, 4 mg of NHS was added and mixed well.
c) Preparation of a protein-based small-pore gel layer: the lyophilized MC-3T3-E1 cell lysate powder was dissolved in phosphate buffered saline (PBS, pH 7.2) at a concentration of 40%. And (3) adding 48 microliters of 40% MC-3T3-E1 cell lysate solution and 102 microliters of the hyaluronic acid solution activated according to the step b into a 200 microliter centrifuge tube in sequence, mixing uniformly, quickly transferring into a polytetrafluoroethylene mold with the diameter of 8 millimeters and the depth of 6 millimeters, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
d) Preparing a protein-based macroporous gel layer: the lyophilized MC-3T3-E1 cell lysate powder was dissolved in phosphate buffered saline (PBS, pH 7.2) at a concentration of 40%. And (c) sequentially adding 60 microliters of 40% MC-3T3-E1 cell lysate solution and 90 microliters of the hyaluronic acid solution reactivated according to the step b into a 200 microliter centrifuge tube, uniformly mixing, quickly transferring to the surface of the protein-based small-pore gel layer in the mould in the step c, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
The product obtained in this embodiment is a protein-based hydrogel (MC-3T3-E1 cell lysate provides protein components, hyaluronic acid is a cross-linking agent) with two layers of different internal pore sizes, wherein the pore size of the small-pore gel layer is 40-70 microns, and the pore size of the large-pore gel layer is 80-120 microns.
EXAMPLE 5 preparation of protein-based bilayer hydrogel with lower non-oriented wells and upper oriented wells (bovine serum albumin provides the protein component)
a) Preparation of Bovine Serum Albumin (BSA) solution: a5% solution of Bovine Serum Albumin (BSA) was prepared by dissolving 5 g of BSA (66 kDa molecular weight) in 100 ml of phosphate buffered saline (PBS, pH 7.2).
b) Activation of sodium alginate: a2% sodium alginate solution was prepared by dissolving 2 g of sodium alginate (viscosity average molecular weight ≥ 2000cps) in 100 ml of phosphate buffered saline (PBS, pH 7.2). To 240 microliters of sodium alginate solution, 15 mg of EDC was added, and mixed well with shaking. After 30 min reaction on a shaker, 9 mg of NHS was added and mixed well.
c) Preparation of protein-based non-oriented pore gel layer: and (c) sequentially adding 70 microliters of 5% Bovine Serum Albumin (BSA) solution, 30 microliters of phosphate buffer saline (PBS, PH 7.2) and 50 microliters of sodium alginate solution activated according to the step (b) into a 200-microliter centrifuge tube, uniformly mixing, quickly transferring into a polytetrafluoroethylene mold with the diameter of 8 millimeters and the depth of 6 millimeters, and reacting for 24 hours in a refrigerator at the temperature of-20 ℃.
d) Preparation of protein-based oriented pore gel layer: firstly, standing a copper column in a container filled with a proper amount of liquid nitrogen to pre-freeze the copper column, then sequentially adding 70 microliters of 5% Bovine Serum Albumin (BSA) solution, 30 microliters of phosphate buffer saline (PBS, PH 7.2) and 50 microliters of sodium alginate solution reactivated according to the step b into a 200-microliter centrifuge tube, rapidly transferring the mixture to the surface of the protein-based non-directional pore gel layer in the mold in the step c after uniformly mixing, then placing the mixture on the pre-frozen copper column in advance, and transferring the pre-frozen copper column into a refrigerator at the temperature of-20 ℃ to react for 24 hours after the hydrogel is completely frozen.
The product obtained in this example is a protein-based hydrogel with two layers of different internal pore-size structures (bovine serum albumin provides a protein component, and sodium alginate is a cross-linking agent), wherein the first layer is a non-directional pore gel layer, and the second layer is a directional pore gel layer.
Test example 6 Performance test of protein-based hydrogel having two layers of different inner pore size structures (platelet-rich lysate plasma provides the protein component and sodium alginate is the cross-linking agent) prepared in example 1
The bilayer hydrogel prepared in example 1 was freeze-dried and weighed as W1And then soaking the mixture in deionized water overnight and weighing the mixture as W2Then, the water in the soaked hydrogel was sucked off with filter paper and weighed as W3。
According to the formula: swelling ratio W2/W1Porosity ═ W2-W3)/W2X 100%, the swelling ratio and porosity of the bilayer hydrogel were calculated, respectively, and the results are shown in fig. 3. The protein-based bilayer hydrogel prepared in example 1 had a swelling ratio of about 13.8 and a porosity of about 51.9%.
The storage modulus and loss modulus of the protein-based hydrogel having two layers of different internal pore size structures prepared in example 1 were measured by a rheometer, and the results are shown in fig. 4. The storage modulus of the protein-based bilayer hydrogel is about 31000 pascal, and the loss modulus is about 3600 pascal, which shows that the protein-based bilayer hydrogel prepared by the method has good mechanical properties.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
While the invention has been described with reference to several particular embodiments, it is to be understood that the invention is not limited to the specific embodiments disclosed. The invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
Claims (6)
1. A protein-based hydrogel with double layers of different internal pore size structures is characterized in that the preparation method comprises the following steps:
(1) preparing a protein-based first hydrogel layer, wherein a platelet-rich lysate plasma freeze-dried powder, a cross-linking agent, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and phosphate buffer solution are used as raw materials, and the protein-based first hydrogel layer with first pores is prepared at a low temperature through an amide reaction between amino groups in protein and carboxyl groups of the cross-linking agent;
(2) and (2) adopting the same raw materials as those in the step (1), changing the mass ratio between the platelet lysate plasma freeze-dried powder and the cross-linking agent, preparing a protein-based second hydrogel layer on the surface of the protein-based first hydrogel layer through amide reaction superposition, bonding the two hydrogel layers together through chemical bonds, and setting the pore diameters of the inner pores of the protein-based first hydrogel layer and the inner pores of the protein-based second hydrogel layer to be unequal.
2. The protein-based hydrogel according to claim 1, wherein the bilayer of the protein-based hydrogel has a different internal pore size structure, and wherein: the mass percentage of the protein component is more than 90 percent, the porosity is more than 50 percent, and the inner pore diameter of the two hydrogel layers is 30-200 microns.
3. The protein-based hydrogel according to claim 1, wherein the bilayer of the protein-based hydrogel has a different internal pore size structure, and wherein: the platelet-rich lysate plasma freeze-dried powder is prepared by the following steps:
a1. centrifuging the collected blood at the rotating speed of 1000-;
a2. rapidly freezing the yellowish liquid by liquid nitrogen, and then melting in a water bath at 37 ℃, and repeatedly operating for 3-5 times;
a3. centrifuging the liquid subjected to repeated freeze thawing at the rotating speed of 9000-;
a4. and (3) freeze-drying the platelet-rich lysate plasma to obtain the platelet-rich lysate plasma freeze-dried powder.
4. The protein-based hydrogel according to claim 1, wherein the bilayer of the protein-based hydrogel has a different internal pore size structure, and wherein: the cross-linking agent is carboxyl-containing natural polysaccharide or water-soluble synthetic polymer.
5. The protein-based hydrogel according to claim 1, wherein the bilayer of the protein-based hydrogel has a different internal pore size structure, and wherein: the molar ratio of EDC, NHS and the cross-linking agent is 1 (1-1.2) to 3-4.
6. The protein-based hydrogel according to claim 1, wherein the bilayer of the protein-based hydrogel has a different internal pore size structure, and wherein: the mass ratio of the platelet-rich lysate plasma freeze-dried powder to the cross-linking agent in the steps (1) and (2) is (50-5): 1, the reaction temperature is-10 ℃ to-30 ℃, and the reaction time is 16-24 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811382935.5A CN109513045B (en) | 2018-11-20 | 2018-11-20 | Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811382935.5A CN109513045B (en) | 2018-11-20 | 2018-11-20 | Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109513045A CN109513045A (en) | 2019-03-26 |
| CN109513045B true CN109513045B (en) | 2021-01-15 |
Family
ID=65776468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811382935.5A Active CN109513045B (en) | 2018-11-20 | 2018-11-20 | Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109513045B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228214A (en) * | 2020-02-25 | 2020-06-05 | 中国科学院大学温州研究院(温州生物材料与工程研究所) | Erythrocyte membrane-based hydrogel contact lens and preparation method and application thereof |
| CN111558081B (en) * | 2020-05-19 | 2021-01-29 | 西北大学 | Method for preparing tannin modified double-layer hydrogel |
| CN111704802A (en) * | 2020-07-09 | 2020-09-25 | 中国科学院大学温州研究院(温州生物材料与工程研究所) | Probiotic metabolite-based hydrogel and preparation method thereof |
| CN114931583B (en) * | 2022-05-27 | 2023-09-22 | 四川大学 | Preparation method of core-shell near-infrared light-controlled sequential release hydrogel |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1679972A (en) * | 2005-02-02 | 2005-10-12 | 江汉大学 | Burn dressing of chitin gel with gradient structure and its preparation |
| WO2007103775A2 (en) * | 2006-03-03 | 2007-09-13 | Washington University In St. Louis | Biomaterials having nanoscale layers and coatings |
| WO2009023276A1 (en) * | 2007-08-15 | 2009-02-19 | The Board Of Trustees Of The Leland Stanford Junior University | Sequential coupling of biomolecule layers to polymers |
| CN102573943A (en) * | 2009-10-23 | 2012-07-11 | 世元世龙技术株式会社 | Composition for inducing tissue regeneration by activating platelet-rich plasma (PRP), and method for manufacturing same |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
| WO2014116717A1 (en) * | 2013-01-22 | 2014-07-31 | Prolynx Llc | Sealants having controlled degradation |
| CN105749342A (en) * | 2016-04-29 | 2016-07-13 | 华南理工大学 | Bi-phase bone cartilage repairing support and preparing method thereof |
| CN106267357A (en) * | 2016-08-09 | 2017-01-04 | 上海交通大学 | A kind of repair the two-layer compound hydrogel of osteochondral tissue, preparation method and application |
| CN106822183A (en) * | 2016-12-26 | 2017-06-13 | 上海斯能得医疗科技有限公司 | A kind of photosensitive platelet rich plasma gel and its production and use |
| CN107384856A (en) * | 2017-07-28 | 2017-11-24 | 重庆赛纳思生物科技有限公司 | A kind of method for preparing platelet lysates liquid |
| CN107513165A (en) * | 2017-09-15 | 2017-12-26 | 长春工业大学 | A kind of high intensity adhesive double hydrogel and preparation method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7166133B2 (en) * | 2002-06-13 | 2007-01-23 | Kensey Nash Corporation | Devices and methods for treating defects in the tissue of a living being |
| US8497023B2 (en) * | 2008-08-05 | 2013-07-30 | Biomimedica, Inc. | Polyurethane-grafted hydrogels |
-
2018
- 2018-11-20 CN CN201811382935.5A patent/CN109513045B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1679972A (en) * | 2005-02-02 | 2005-10-12 | 江汉大学 | Burn dressing of chitin gel with gradient structure and its preparation |
| WO2007103775A2 (en) * | 2006-03-03 | 2007-09-13 | Washington University In St. Louis | Biomaterials having nanoscale layers and coatings |
| WO2009023276A1 (en) * | 2007-08-15 | 2009-02-19 | The Board Of Trustees Of The Leland Stanford Junior University | Sequential coupling of biomolecule layers to polymers |
| CN102573943A (en) * | 2009-10-23 | 2012-07-11 | 世元世龙技术株式会社 | Composition for inducing tissue regeneration by activating platelet-rich plasma (PRP), and method for manufacturing same |
| WO2014116717A1 (en) * | 2013-01-22 | 2014-07-31 | Prolynx Llc | Sealants having controlled degradation |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
| CN105749342A (en) * | 2016-04-29 | 2016-07-13 | 华南理工大学 | Bi-phase bone cartilage repairing support and preparing method thereof |
| CN106267357A (en) * | 2016-08-09 | 2017-01-04 | 上海交通大学 | A kind of repair the two-layer compound hydrogel of osteochondral tissue, preparation method and application |
| CN106822183A (en) * | 2016-12-26 | 2017-06-13 | 上海斯能得医疗科技有限公司 | A kind of photosensitive platelet rich plasma gel and its production and use |
| CN107384856A (en) * | 2017-07-28 | 2017-11-24 | 重庆赛纳思生物科技有限公司 | A kind of method for preparing platelet lysates liquid |
| CN107513165A (en) * | 2017-09-15 | 2017-12-26 | 长春工业大学 | A kind of high intensity adhesive double hydrogel and preparation method |
Non-Patent Citations (3)
| Title |
|---|
| Cost-Effective Double-Layer Hydrogel Composites for Wound Dressing Applications;Javad Tavakoli et al;《polymers》;20180312;第1-15页 * |
| Hyaluronic acid-human blood hydrogels for stem cell transplantation;Connie Y. Chang et al;《Biomaterials》;20120813;第8026-8033页 * |
| The fabrication of biomimetic biphasic CAN-PAC hydrogel with a seamless interfacial layer applied in osteochondral defect repair;Jinfeng Liao et al;《Bone Research》;20170704;第1-15页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109513045A (en) | 2019-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109513045B (en) | Protein-based hydrogel with double layers of different internal pore diameter structures and preparation method thereof | |
| Kang et al. | Fabrication of porous gelatin scaffolds for tissue engineering | |
| Hwang et al. | Poly (ethylene glycol) cryogels as potential cell scaffolds: effect of polymerization conditions on cryogel microstructure and properties | |
| KR101114712B1 (en) | A Platelet rich plasma using regeneration constituent manufacturing method thereof | |
| JP4762785B2 (en) | Gelatin sponge | |
| JPH10501173A (en) | Polymer microbeads and method for producing the same | |
| CN108409988B (en) | Preparation method of spongy macroporous polyvinyl alcohol hydrogel | |
| Hakam et al. | Evaluation of fibrin-gelatin hydrogel as biopaper for application in skin bioprinting: An in-vitro study | |
| CN110172163B (en) | Method for preparing silk fibroin-tyramine modified hyaluronic acid hydrogel material based on physical-chemical double crosslinking | |
| KR20200034663A (en) | Hydrogel and biomedical applications for cell culture | |
| Wang et al. | Using a mild hydrogelation process to confer stable hybrid hydrogels for enzyme immobilization | |
| Whittaker et al. | Engineering DN hydrogels from regenerated silk fibroin and poly (N-vinylcaprolactam) | |
| CN103893824B (en) | Three-dimensional bone tissue engineering scaffold material with different stiffness and preparation method of material | |
| CN105713106A (en) | Double-crosslinked sodium alginate hydrogel and preparation method and application thereof | |
| CN109111580B (en) | Platelet-rich lysate plasma-based hydrogel with micron porous structure | |
| CN108785752A (en) | Fibroin albumen based porous materials and preparation method thereof | |
| Bonina et al. | pH-sensitive hydrogels composed of chitosan and polyacrylamide–preparation and properties | |
| CN102198291B (en) | Polysaccharide-based nerve repair stent material with continuous gradient property and preparation method thereof | |
| CN110698719A (en) | Preparation of polyvinyl alcohol-based hydrogel | |
| CN113663127A (en) | Nano composite hydrogel and preparation method and application thereof | |
| CN105920679A (en) | Preparation method of skin stent material having three-dimensional gradient pore structure | |
| EP2793963A2 (en) | Continuous matrix with osteoconductive particles dispersed therein, method of forming thereof, and method of regenerating bone therewith | |
| CN114854053B (en) | Polyethylene glycol-chitosan double-network hydrogel and preparation method and application thereof | |
| CN114642628B (en) | M1 type macrophage lysate-based hydrogel and preparation method and application thereof | |
| Liu et al. | Porous poly (DL-lactic acid) modified chitosan-gelatin scaffolds for tissue engineering |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |