CN116531562A - Large-aperture injectable gel and application thereof in wound healing - Google Patents
Large-aperture injectable gel and application thereof in wound healing Download PDFInfo
- Publication number
- CN116531562A CN116531562A CN202310630010.2A CN202310630010A CN116531562A CN 116531562 A CN116531562 A CN 116531562A CN 202310630010 A CN202310630010 A CN 202310630010A CN 116531562 A CN116531562 A CN 116531562A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- rutin
- injectable gel
- sulfated hyaluronic
- pore size
- 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.)
- Granted
Links
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- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims abstract description 17
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Classifications
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0014—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- 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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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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Abstract
The invention discloses a large-aperture injectable gel and application thereof in wound healing, and belongs to the technical field of biological medicines. The invention takes S-HA-PEI as a base, and modifies succinylated rutin on the side chain of S-HA-PEI to crosslink with polyaldehyde compound to prepare novel hydrogel. The product has super-large pore diameter, swelling rate at least up to 1700%, and is suitable for absorbing seepage in chronic wound. Meanwhile, the hydrogel does not have hemolysis, has good biocompatibility, is excellent in promoting wound tissue repair as a cell scaffold, and has good application prospect in the field of tissue repair.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a large-aperture injectable gel and application thereof in wound healing.
Background
Chronic wounds, represented by diabetic ulcers, are a type of wound that is caused by a variety of etiologies, and is still unable to heal, or even has no tendency to heal, by standardized treatment. The chronic wound has the characteristics of complex occurrence mechanism, higher recurrence rate, high cost and high disability rate, and brings heavy burden to patients and families. How to promote the regeneration of skin tissues after severe injury of skin is an important subject in the current research of promoting chronic wound healing.
The use of an ideal dressing to protect a wound from secondary injury and infection is the primary step in wound care. The hydrogel is a three-dimensional cross-linked polymer network formed by hydrophilic high polymer materials, so that the wound is in a moist state, and a non-adhesive environment is provided. The compound has strong swelling capacity, can absorb and retain a large amount of water, helps the wound to continuously absorb exudates, and is an ideal chronic wound dressing. Along with the continuous improvement of clinical requirements on wound repair, the requirements of various functional hydrogels are gradually generated, and various antibacterial, antioxidant and charged hydrogels are generated. In the development of a variety of functional hydrogels, promoting healing has always been an important challenge in the design and construction of hydrogels.
Disclosure of Invention
In order to solve the problems, the invention provides a hydrogel based on sulfated hyaluronic acid, branched polyethylenimine, succinylated rutin and polyaldehyde compounds, wherein the sulfated hyaluronic acid and the branched polyethylenimine undergo amide condensation to form a nanoparticle structure, and the product and the succinylated rutin are crosslinked to reform a soluble polymer. The amino group on the surface branched polyethylenimine of the polymer and the aldehyde Cheng Xifu alkali structure in the polyaldehyde compound form a cross-linked network to prepare the hydrogel, and experiments prove that the hydrogel has excellent effects in promoting wound healing and resisting scars.
It is a first object of the present invention to provide a large-pore injectable gel comprising a crosslinked network structure formed of sulfated hyaluronic acid, branched polyethylenimine, succinylated rutin and polyaldehyde compounds therein.
Further, ions such as calcium ions and the like are also included in the crosslinked network structure.
Further, the polyaldehyde compound is selected from the group consisting of, but not limited to, an aldehyde polysaccharide, glutaraldehyde, trioxaldehyde, polyacrolein, and the like.
Further, polysaccharides include, but are not limited to, hyaluronic acid, alginate, dextran, chitosan, heparan sulfate, fucoidan, and the like.
The second object of the present invention is to provide a method for preparing the above-mentioned large-pore injectable gel, comprising the steps of:
s1, cross-linking sulfated hyaluronic acid and branched polyethylenimine to form a sulfated hyaluronic acid-branched polyethylenimine grafted polymer, and then cross-linking the sulfated hyaluronic acid-branched polyethylenimine grafted polymer with succinylated rutin to obtain a sulfated hyaluronic acid-branched polyethylenimine-rutin polymer;
s2, mixing the sulfated hyaluronic acid-branched polyethylenimine-rutin polymer with a polyaldehyde compound, and reacting to form the large-aperture injectable gel.
Further, in step S1, the structure of the sulfated hyaluronic acid-branched polyethyleneimine graft polymer is as follows:
wherein R, R' is independently selected from SO 3 - Or H, m is an integer between 10 and 10000, and n is an integer between 1 and 100000.
Further, in step S1, the structure of the sulfated hyaluronic acid-branched polyethylenimine-rutin polymer is as follows:
wherein R, R' is independently selected from SO 3 - Or H, R3 is selected from succinylated rutin or H, m is 10-10000, n is an integer between 1 and 100000.
Further, the succinylated rutin structure is shown as follows:
further, the sulfated hyaluronic acid has a molecular weight of 1000-1000000.
Further, the molecular weight of the branched polyethyleneimine is 50-1000000.
Further, in step S1, the mass ratio of the sulfated hyaluronic acid, the branched polyethyleneimine and the succinylated rutin is 1-100:1-100:1-100.
Further, in step S2, the molar ratio of the sulfated hyaluronic acid-branched polyethylenimine-rutin polymer to the polyaldehyde compound is 1-100:1-100.
Further, in step S2, after the reaction with the polyaldehyde compound, a step of incubating the reaction product with an ionic solution is further included.
A third object of the present invention is to provide the use of the above-described large pore size injectable gel for preparing tissue engineering materials.
A fourth object of the present invention is to provide the use of the above-described large pore injectable gel in the preparation of wound healing products.
It is a fifth object of the present invention to provide the use of the large pore injectable gel described above for the preparation of anti-inflammatory products.
The invention has the beneficial effects that:
according to the invention, the succinylated RUTIN is connected to S-HA-PEI to synthesize an S-HA-PEI-RUTIN structure, the compound presents typical amphoteric high molecular structural characteristics, and the compound is incubated with the hydroformylation polysaccharide, so that the excessive amino on the PEI surface can be crosslinked with aldehyde groups to form imino dynamic bonds, and the ultra-macroporous injectable hydrogel is obtained. The material shows higher biocompatibility in vitro. Meanwhile, the material has strong water absorption, and the water absorption rate is at least up to 1700%. The wound healing experimental result shows that the hydrogel can promote tissue regeneration at the initial stage of wound healing, remarkably accelerate the healing speed of chronic wounds and improve inflammatory cell infiltration. In vivo degradation and safety experiments show that the hydrogel has no influence on liver and kidney functions of mice, and the hydrogel has good safety and biocompatibility. In conclusion, the hydrogel prepared by the invention can provide a scaffold for cell growth, promote chronic wound healing and play a role in anti-inflammatory.
Drawings
FIG. 1 is an infrared spectrum of a hydrogel;
FIG. 2 is an SEM image of a hydrogel, (a) S-HA-P-R-A-HA; (b) S-HA-P-R-a-SA;
(c)S-HA-P-R-A-SA’；(d)S-HA-P-R-A-SA-Ca 2+ ；(e)S-HA-P-R-A-SA’-Ca 2+ ；
FIG. 3 is a frequency sweep dynamic rheological profile of a hydrogel, (a) S-HA-P-R-A-HA; (b) S-HA-P-R-a-SA; (c) S-HA-P-R-a-SA'; (d) S-HA-P-R-A-SA-Ca 2+ ；(e)S-HA-P-R-A-SA’-Ca 2+ ；
FIG. 4 is the swelling ratio of hydrogels;
FIG. 5 shows the haemolysis rates of different hydrogels;
FIG. 6 shows proliferation of NIH-3T3 cells;
FIG. 7 is a graph of the effect of complete excision wound healing in diabetic mice, (a, b) day 0, 3, 5, 8, 10, 16 diabetic wounds and wound area; (c) H & E staining of wound tissue on days 3, 5, 8, 10; (d) Masson trichrome representative staining patterns of wound tissue at days 3, 5, 8 (< 0.05 for p, <0.01 for p, <0.001 for p);
FIG. 8 shows measurement of serum ALT, AST, TP, CREA-S as an index of liver and kidney function, NC as a normal control group;
FIG. 9 shows in vivo degradation of mice after 12 days of gel implantation subcutaneously, (a) NC; (b) S-HA-P-R-A-HA; (c) S-HA-P-R-A-SA; (d) S-HA-P-R-A-SA'.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1 preparation of hydrogels
(1) Preparation of sulfated hyaluronic acid
(1) Pretreatment of resin
Soaking the resin in hot water at 50-60 deg.c for about 15min for 1 time until no brown color and little foam are produced, adding 5% concentration hydrochloric acid solution, soaking, wet packing for 4 hr, penetrating deionized water in 2 times the volume of the column layer through the resin layer, and flushing with deionized water to pH 7.0.
(2) Synthesis of TBA-HA
1g of hyaluronic acid is dissolved in 100mL of deionized water, the HA aqueous solution is transferred to a resin column, the flow rate is adjusted to be small (the liquid is not stranded in a line at a speed), and the solution is dripped into the TBA aqueous solution, and the pH value of the final solution is not lower than 9. The HA-TBA was transferred to a dialysis bag of 8kDa for dialysis and lyophilization. And (3) after the sample is completely freeze-dried into flocculent, obtaining the sample, and placing the flocculent sample in a dryer for storage for later use.
(3) Synthesis of S-HA
Accurately weighing 1g TBA-HA sample, adding 200mL anhydrous DMF for complete dissolution for 2-3h, ice bath and N 2 40mL of anhydrous DMF solution containing 3g of pyridine sulfur trioxide was added under the condition, and after 1 hour of reaction, 10mL of deionized water was added to quench the reaction. The solution was adjusted to pH 8.5-9 with 1M NaOH, dialyzed, and lyophilized to give a sample designated S-HA.
(5) Reaction of sulfated hyaluronic acid with PEI (S-HA-PEI)
80mg PEI solution was dissolved in Hepes buffer to a concentration of 1mg/mL. 160mg of S-HA sample was precisely weighed, and appropriate amounts of EDC and NHS were added thereto and reacted in Hepes buffer for 0.5h. And adding 2mg/mL of S-HA reaction solution into 1mg/mL of PEI system, dialyzing, and freeze-drying to obtain a sample for later use and named S-HA-PEI.
(2) Synthesis of S-HA-PEI-RUTIN
(1) Synthesis of succinylated RUTIN (RUTIN)
2g of rutin and 3g of succinic anhydride are precisely weighed, dissolved in 80mL of anhydrous pyridine, and refluxed at 70 ℃. After the reaction, the anhydrous pyridine was removed by rotary evaporation at 60 ℃. A small amount of absolute ethyl alcohol was added to the product to dissolve it sufficiently, and absolute ethyl ether (1:20) was added to the solution under ice bath conditions to precipitate it. Then centrifugating at a temperature of 4 ℃ at a speed of 8000rpm for 10min, removing supernatant, adding anhydrous diethyl ether for washing for 2-3 times, and pumping the precipitate in vacuum drying for later use.
②S-HA-PEI-RUTIN
80mg of succinylated rutin, EDC and NHS are taken and reacted in PBS buffer solution for 2 hours under the condition that the concentration is 1mg/mL until the reaction solution becomes transparent green. 200mg of S-HA-PEI is taken and put into PBS buffer solution to make the concentration be 2mg/mL; 1mg/mL of succinylated rutin solution was added dropwise thereto, and dialyzed. The dialyzed sample was spin-distilled to 3mL at 60℃for further use.
R3=run or H.
(3) Synthesis of oxidized hyaluronic acid and oxidized sodium alginate
(1) Oxidized hyaluronic acid preparation: 1g of sodium hyaluronate was dissolved in 100mL of distilled water, and an aqueous solution of sodium periodate (0.5M, 5 mL) was added dropwise, followed by a reaction at room temperature in the absence of light for 2 hours. Then adding 1mL of ethylene glycol to quench the reaction, stirring for 1h at room temperature, dialyzing, and freeze-drying to obtain oxidized hyaluronic acid which is named as A-HA.
(2) Preparation of oxidized sodium alginate: 1g of sodium alginate is taken to prepare an aqueous solution with the mass fraction of 1 percent. 1mL of a 0.25M, 0.025M sodium periodate solution was added and oxidized in the dark for 24h. The oxidation reaction was stopped by adding 0.2mL of ethylene glycol for 15min. Dialysis, lyophilization and designated as A-SA (aldehyde substitution degree 0.5), A-SA' (aldehyde substitution degree 0.05).
(4) Preparation of hydrogels
Taking 1mL of S-HA-PEI-RUTIN concentrated solution, respectively and rapidly mixing with 1mL of A-HA, A-SA and A-SA', and forming hydrogel in a period of time. The prepared hydrogels are named S-HA-P-R-A-HA, S-HA-P-R-A-SA and S-HA-P-R-A-SA', respectively. Then soaking the hydrogel S-HA-P-R-A-SA and S-HA-P-R-A-SA' in CaCl 2 In aqueous solution (2 mL, 0.05%) overnight at 4℃to form double crosslinked hydrogels, which were designated S-HA-P-R-A-SA-Ca, respectively 2+ 、S-HA-P-R-A-SA’-Ca 2+ . Experiments show that the aldehyde group substitution degrees of different polysaccharides can form hydrogels.
Example 2 characterization of hydrogels
(1) Fourier transform infrared spectroscopy
As shown in the infrared spectra of the hydrogels of fig. 1, the main peaks of the five sets of hydrogels did not show significant differences. S-HA-P-R is crosslinked with an aldehyde compound through an amide bond to form a hydrogel. Wherein at 1650cm -1 Characteristic absorption of the stretching vibration of the c=o bond was observed at 1260cm -1 Deformation vibration of c=n was observed.
(2) Scanning electron microscope
S-HA-P-R-A-HA, S-HA-P-R-A-SA', S-HA-P-R-A-SA-Ca 2+ 、S-HA-P-R-A-SA’-Ca 2+ Five sets of hydrogels were made into cylinders 25mm in diameter and 3mm in thickness and the microstructure of the gels was observed using a scanning electron microscope. The hydrogels were placed in liquid nitrogen for 10min,freeze-drying after brittle failure. The freeze-dried solid is adhered on conductive gel, sprayed with metal in vacuum, and observed with an accelerating voltage of 5 kV.
The morphology of five sets of hydrogels after lyophilization is shown in FIG. 2, wherein (a) S-HA-P-R-A-HA; (b) S-HA-P-R-a-SA; (c) S-HA-P-R-a-SA'; (d) S-HA-P-R-A-SA-Ca 2+ ；(e)S-HA-P-R-A-SA’-Ca 2+ . By SEM images of the respective sets of hydrogels, five sets of hydrogels were clearly observed to exhibit similar porous structures. There was a difference in average pore size between the different sets of hydrogels. The pore diameter of S-HA-P-R-A-HA is about 300 μm, and the pore diameters of S-HA-P-R-A-SA and S-HA-P-R-A-SA' are about 400 μm; and pass through Ca 2+ The treated hydrogel S-HA-P-R-A-SA-Ca 2+ 、S-HA-P-R-A-SA’-Ca 2+ The aperture is about 1 mm. Since the pore diameter of the hydrogel is related to the water absorption capacity, the larger the pore diameter is, the higher the water absorption speed is, the stronger the water absorption capacity is, and the capacity of absorbing liquid seepage in a wound is enhanced.
(3) Rheology test
S-HA-P-R-A-HA, S-HA-P-R-A-SA', S-HA-P-R-A-SA-Ca 2+ 、S-HA-P-R-A-SA’-Ca 2+ Five sets of hydrogels were made into cylinders of 40mm diameter and 2mm thickness, and the hydrogels were frequency scanned (0.1-10 Hz) using a rheometer with a 40mm diameter sample stage in the linear viscoelastic region of 0.5-100 rad/s. All samples were tested at room temperature.
As shown in fig. 3, wherein (a) S-HA-P-R-a-HA; (b) S-HA-P-R-a-SA; (c) S-HA-P-R-a-SA'; (d) S-HA-P-R-A-SA-Ca 2+ ；(e)S-HA-P-R-A-SA’-Ca 2+ The frequency sweep dynamic rheogram of five sets of hydrogels was continuously examined. The results showed that the storage modulus (G') and loss modulus (G ") of the five sets of hydrogels were independent of frequency, indicating that the hydrogels had stable structures. All samples showed viscoelastic gel characteristics (G 'over the frequency range tested'>>G "). In addition, storage modulus determines the mechanical properties of the biomaterial and is a key property affecting hydrogels as bioscaffold. The G ' value of A is 50, the G ' values of the hydrogels of groups B and C are in the range of 150-180, and the G ' values of D and E are in the range of 300-400.
(4) Swelling Performance test
S-HA-P-R-A-HA, S-HA-P-R-A-SA', S-HA-P-R-A-SA-Ca, respectively 2+ 、S-HA-P-R-A-SA’-Ca 2+ Four sets of hydrogels were lyophilized, immersed in 2mL PBS buffer (pH 7.4), left to stand and weighed (W0). The gel was taken out and water on the surface of the gel was wiped off with paper at predetermined time intervals, and mass changes (Wt) thereof with time were recorded, respectively, and the swelling ratio was calculated according to the following formula.
Swelling ratio (%) =w t /W 0 ×100％。
The swelling ratios of the five hydrogels are plotted as a function of time as shown in FIG. 4. The freeze-dried hydrogel shows strong water absorption capacity. The water absorption degree reaches equilibrium within 100 min. Wherein the swelling ratio of S-HA-P-R-A-HA is about 1700%, and the swelling ratios of S-HA-P-R-A-SA and S-HA-O-R-A-SA-Ca are about 2+ HAs a swelling ratio of about 1700% similar to that of S-HA-P-R-A-HA, and S-HA-P-R-A-SA' -Ca 2+ The swelling ratio of (2) was about 2000%. Therefore, the five groups of hydrogels have rapid swelling properties and can be used as wound dressings to absorb tissue exudates.
(5) Calculation of the Haemolysis Rate (HR)
Firstly, taking fresh blood from a mouse body, taking blood from eyeballs, adding the blood into a centrifuge tube containing an anticoagulant, and preparing whole blood of the mouse; 5mL of whole blood of the mouse is taken, and 5mL of physiological saline is added for dilution for later use. Test tubes of the experimental group (different hydrogels, 10mL of physiological saline), the negative control group (10 mL of physiological saline) and the positive control group (10 mL of distilled water) were put into a 37 ℃ water bath for incubation for 30min, taken out, diluted mouse whole blood (0.2 mL) was added, put into a 37 ℃ water bath again for continuous heat preservation for 1h, then the solutions of the test tubes were centrifuged at 2500rpm for 5min, and the absorbance value of the supernatant at 545nm was measured. The haemolysis rate was calculated according to equation 4.1:
where Dt is the absorbance of the experimental group, dnc is the absorbance of the negative control group, dpc is the absorbance of the positive control group (n=3).
The percent hemolysis = [ (Dt-Dnc)/(Dpc-Dnc) ]x100%.
The basic performance requirement of biological materials is good compatibility. The hemolysis rate lower than 5% can prove that the biological material has good blood compatibility, and can be applied to biological medical materials. As shown in fig. 5, which shows the hemolysis rates of different hydrogels, it can be seen that the hemolysis rates of all the hydrogels are lower than 5%, which indicates that the prepared hydrogels show good blood compatibility and are expected to become candidate materials for novel wound dressing.
Example 3 cytotoxicity assay
(1) Grouping of experimental cells
Blank group: adding only PBS;
control group: culturing the cells in a medium;
drug administration group: the medium containing the samples cultures cells, grouped according to sample concentration (0, 50, 100, 200, 400, 800 μg/mL).
(2) Cytotoxicity test
NIH-3T3 cells were grown at 6X 10 3 The culture was inoculated into 96-well plates (100. Mu.g/well), DMEM medium was added thereto, and the culture was continued for 24 hours, followed by pipetting the supernatant. Media containing samples were added and 5 duplicate wells were placed in each group. After incubation at 37℃for 24h, 10. Mu.L of CCK-8 was added, and incubated at 37℃in the dark for 40min, and absorbance was measured at a wavelength of 450nm using an enzyme-labeled instrument. Cell viability (Cell viability) was calculated according to the formula:
wherein, C450 and Csample, ccontrol are the absorbance at 450nm of the blank group, the administration group and the control group, respectively.
As a result, NIH-3T3 cells were cultured with sample media containing 0, 50, 100, 200, 400, 800. Mu.g/mL, respectively, as shown in FIG. 6. After 24h incubation, the proliferation efficiency of the cells was comparable to that of the control group, and the difference in concentration caused only slight absorption fluctuations, but no significant effect. The prepared sample has good biocompatibility with cells.
(3) Diabetes induction model
S-HA-P-R-A-SA' compared with S-HA-P-R-A-SA, only sea is producedDifferences in the extent of modification of alginic acid aldehyde groups. However, the degree of hydroformylation of alginic acid is different, and no obvious physical and chemical property difference is caused. Therefore, only S-HA-P-R-A-SA and its combination with Ca are selected during the biological activity assay phase 2+ The crosslinked product was further determined. All animal protocols were approved by the university of Jiangnan laboratory animal administration and animal welfare ethics committee, numbered JN.NO20220615c0600830. After 64 male mice were adaptively bred for 7 days, streptozotocin (STZ, 70 mg/kg) was intraperitoneally injected for 4 consecutive days to prepare a diabetes model. Normal diet feeding was performed for the next 10 days. On day 14 of model construction, mice with blood glucose levels exceeding 13.5mmol/L were selected. Randomly divided into 4 groups, respectively model group (NC), S-HA-P-R-A-HA group, S-HA-P-R-A-SA-Ca 2+ A group. C57BL/6 mice were first anesthetized with Avertin anesthetic (240 mg/kg). The anesthetized mice were fixed with 3M tape with the back facing upward, dehaired, sterilized with ethanol, and a round wound 0.9cm x 0.9cm in size was cut in the middle of the back. The different sets of gels were cut into small cylinders of 0.9cm diameter and 0.3cm height, implanted subcutaneously and the skin was sutured. And (5) after the mice wake up, placing the mice back to a clean area for normal feeding. All mice were removed each time at 3, 5, 8, 10, 16 days post-surgery to measure the size of their wounds and photographed. To further determine the dynamic regeneration process of the tissue, 3 mice were removed at a time during the above period, sacrificed and wound tissue was removed and fixed with 4% paraformaldehyde. Then H is carried out&E staining, and performing a Maron trichromatic staining analysis to evaluate wound tissue regeneration.
A completely resected wound was created in STZ-induced diabetic mice, different sets of hydrogels were implanted on the wound, and the wound healing process was observed within 0, 3, 5, 8, 10, 16 days. As shown in fig. 7 (a, b), it can be seen from the pictures of the wounds of mice that NC group mice have too slow wound healing rate; whereas the hydrogel groups all promoted wound healing to varying degrees. Wherein, the S-HA-P-R-A-HA group HAs obvious wound diminishing condition in the first 5 days, and the wound area is 0.6cm 2 Becomes 0.3cm 2 The method comprises the steps of carrying out a first treatment on the surface of the By day 10, the wound area was 0.1cm 2 Up to day 16The wound area is only 0.06cm 2 The method comprises the steps of carrying out a first treatment on the surface of the At the same time, on the healed skin, signs of hair follicle growth were observed. Compared with NC group, S-HA-P-R-A-SA and S-HA-P-R-A-SA-Ca 2+ Both groups also promoted wound healing to some extent. The healing rate is always slower than that of S-HA-P-R-A-HA.
In addition, H is applied to the skin of the wound&E staining analysis. The results as shown in fig. 7 (c) show: the NC group wound did not begin to shrink slightly until day 8, and the S-HA-P-R-A-HA group began to shrink at day 3, S-HA-P-R-A-SA and S-HA-P-R-A-SA-Ca 2+ The mean started to appear as wound contraction at day 5. Although all three sets of hydrogels promoted wound healing to some extent, they exhibited different effects throughout the wound healing process. Wherein the S-HA-P-R-A-HA group HAs no obvious inflammatory cell infiltration throughout, and the cambium of the neutrophils is less, which indicates that the repair type macrophages are increased, and the phagocytosis of the neutrophils is enhanced. However, S-HA-P-R-A-SA and S-HA-P-R-A-SA-Ca 2+ Both groups had a degree of inflammatory cell infiltration throughout the wound healing process. Secondly, in the process of wound healing in the NC group, the healing tissue of the NC group presents a disordered state; whereas the S-HA-P-R-A-HA group HAs a morphology similar to that of normal tissue in the collagenous layer of the wound from the beginning to the end of wound healing, the hydrogel group may promote wound healing without scar generation. Therefore, the hydrogel S-HA-P-R-A-HA can be used as a cell scaffold for early wound healing, thereby promoting tissue regeneration.
(4) In vivo safety of hydrogels
After normal rearing of the mice after the operation of (3) for 12 days, 3 mice were subjected to sudden death and whole blood was taken from each group. Whole blood was left to settle naturally for 4h at 4 ℃. Centrifuge at 4℃and 4000rpm for 10min. The supernatant was carefully placed in a new ep tube and the ALT, AST, CREA-S, TP value was determined immediately.
The hydrogel is implanted subcutaneously and then normally fed for 12 days, and serum is taken to detect liver and kidney functions. As shown in FIG. 8, ALT, AST, TP, CREA-S showed no significant difference from the normal control group, and no abnormality in liver and kidney function was observed after gel implantation in the dermis of the mouse, and no disturbance was observed. It was therefore concluded that this type of hydrogel has a high degree of safety and biocompatibility.
(5) In vivo degradation of hydrogels
After the 24 male mice were adaptively bred for 7 days, they were randomly divided into 4 groups according to mass, which were model group (NC), S-HA-P-R-A-HA group, S-HA-P-R-A-SA-Ca, respectively 2+ A group. Surgery was identical to (3) sudden death of mice on day 10 post-surgery. Taking out incised skin tissue, fixing with 4% paraformaldehyde solution, and further performing H&E staining. The sections were analyzed under an optical microscope to observe the material degradation under the skin.
The hydrogel was implanted into the dermis of the mice and in vivo degradation of the hydrogel was observed. As shown in FIG. 9, the S-HA-P-R-A-HA hydrogel was completely degraded in vivo (FIG. 9 b), and the skin tissue morphology of mice was not significantly different from that of the normal group. Whereas S-HA-P-R-A-SA and S-HA-P-R-A-SA' did not degrade completely within 14 days, but allowed cells to enter the interior of the residual cell scaffold; experiments show that the hydrogels have stronger cell compatibility, can be used as scaffolds for tissue regeneration, and promote the growth of cells.
Comparative example 1
The sulfated hyaluronic acid-branched polyethylenimine and the oxidized hyaluronic acid are directly incubated, and the sulfated hyaluronic acid and the branched polyethylenimine are subjected to amide condensation to form nanoparticles, but the nanoparticles are not in a water-soluble structure, so that hydrogel cannot be directly formed with oxidized polysaccharide. Therefore, the modification of the succinylated rutin promotes the dissolution of the sulfated hyaluronic acid-branched polyethylenimine and promotes the cross-linking of the sulfated hyaluronic acid-branched polyethylenimine and the aldehyde polysaccharide to form a macromolecular network, thereby preparing the hydrogel structure.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A large pore size injectable gel characterized by: the large-aperture injectable gel comprises a cross-linked network structure formed by sulfated hyaluronic acid, branched polyethylenimine, succinylated rutin and polyaldehyde group compound.
2. The large pore size injectable gel of claim 1, wherein: the cross-linked network structure also comprises ions.
3. The large pore size injectable gel of claim 1, wherein: the polyaldehyde compound comprises one or more of aldehyde polysaccharide, glutaraldehyde, trioxaldehyde and polyaldehyde.
4. A method of preparing a large pore size injectable gel according to any one of claims 1 to 3, comprising the steps of:
s1, cross-linking sulfated hyaluronic acid and branched polyethylenimine to form a sulfated hyaluronic acid-branched polyethylenimine grafted polymer, and then cross-linking the sulfated hyaluronic acid-branched polyethylenimine grafted polymer with succinylated rutin to obtain a sulfated hyaluronic acid-branched polyethylenimine-rutin polymer;
s2, mixing the sulfated hyaluronic acid-branched polyethylenimine-rutin polymer with a polyaldehyde compound, and reacting to form the large-aperture injectable gel.
5. The method according to claim 4, wherein in step S1, the structure of the sulfated hyaluronic acid-branched polyethyleneimine graft polymer is as follows:
wherein R, R' is independently selected from SO 3 - Or H, m is an integer between 10 and 10000, and n is an integer between 1 and 100000.
6. The method according to claim 4, wherein in step S1, the structure of the sulfated hyaluronic acid-branched polyethylenimine-rutin polymer is as follows:
wherein R, R' is independently selected from SO 3 - Or H, R3 is selected from succinylated rutin or H, m is an integer between 10 and 10000, and n is an integer between 1 and 100000.
7. The method of manufacturing according to claim 4, wherein: in step S2, after the reaction with the polyaldehyde compound, a step of incubating the reaction product with an ionic solution is further included.
8. Use of a large pore size injectable gel according to any one of claims 1 to 3 for the preparation of tissue engineering materials.
9. Use of a large pore size injectable gel according to any one of claims 1 to 3 for the preparation of a wound healing product.
10. Use of a large pore size injectable gel according to any of claims 1-3 for the preparation of an anti-inflammatory product.
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