CN115192779A - Preparation method of Pluronic F127/hyaluronic acid composite hydrogel biological ink - Google Patents
Preparation method of Pluronic F127/hyaluronic acid composite hydrogel biological ink Download PDFInfo
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- CN115192779A CN115192779A CN202211033973.6A CN202211033973A CN115192779A CN 115192779 A CN115192779 A CN 115192779A CN 202211033973 A CN202211033973 A CN 202211033973A CN 115192779 A CN115192779 A CN 115192779A
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- pluronic
- hyaluronic acid
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- mixed solution
- methacrylic anhydride
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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
- 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
- A61L27/60—Materials for use in artificial skin
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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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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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
- 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
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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
- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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Abstract
The invention discloses a preparation method of Pluronic F127/hyaluronic acid composite hydrogel biological ink, which comprises the steps of adding a prepolymer solution of Pluronic F127 modified by mercapto capping into a prepolymer solution of methacrylic anhydride modified hyaluronic acid, carrying out a pre-crosslinking reaction, and then carrying out a light crosslinking reaction under the irradiation of an ultraviolet lamp. The thiol-terminated modified Pluronic F127 disclosed by the invention forms micelles by self-assembly in an aqueous medium, shows sol-gel phase change behavior along with temperature change, and is beneficial to fixing a printing structure through photo-crosslinking. The composite hydrogel biological ink prepared by the invention has good mechanical property and water retention, rapid gel behavior and biocompatibility, can be used for carrying stem cells for printing, and is beneficial to accelerating the process of healing skin wounds.
Description
Technical Field
The invention belongs to the technical field of medical biomaterials, and relates to a preparation method of Pluronic F127/hyaluronic acid composite hydrogel biological ink.
Background
The skin is an important soft tissue in human body, and has the functions of preventing external bacterial infection and protecting human. Severe skin wounds, except superficial wounds, generally lose regenerative repair capacity, and the original wound closure mechanism cannot achieve the repair of skin tissues. In this case, the skin wound repair can be carried out only by surgery, for example, by autologous skin transplantation, but this often results in unsatisfactory therapeutic effect due to donor deficiency or necrosis of the graft. The advent of tissue engineering has provided a means for treating skin wounds with potential advantages in repairing damaged tissue. Three-dimensional (3D) bioprinting is a promising technology for manufacturing skin substitutes and plays an important role in tissue engineering, however, it remains a challenge to develop bio-inks suitable for wound repair, 3D bioprinting, and engineered manufacturing. The ideal bio-ink should have sufficient mechanical properties, good printing properties, and support the survival of cells within the scaffold and maintain its biological function. Hydrogels have characteristics similar to the natural extracellular matrix, can maintain a moist environment at the wound interface, absorb body fluids, permeate oxygen, and also facilitate cell adhesion, proliferation, nutrient transport, and metabolic waste, and thus it is of great importance to develop hydrogel-based bio-inks for full-thickness skin defect treatment.
Hyaluronic Acid (HA) is a natural non-sulfated glycosaminoglycan, a natural polymer that is non-toxic, biodegradable and biocompatible, and HAs been widely used clinically as a dermal filler. The large number of carboxyl and hydroxyl groups available in the structure of hyaluronic acid, as a major component of the extracellular matrix, make it highly hydrophilic, playing a crucial role in hemostasis, modulating inflammation, promoting re-epithelialization, and HA-based hydrogels are the most attractive materials in 3D bioprinting due to their adjustable physicochemical and biological properties.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a preparation method of Pluronic F127/hyaluronic acid composite hydrogel bio-ink for repairing full-thickness skin defects.
The invention idea is as follows: in the invention, methacrylic anhydride modified hyaluronic acid (HAMA) and sulfhydryl terminated modified Pluronic F127 (F127-SH) are mixed to prepare composite hydrogel with photosensitive and temperature-sensitive properties as biological ink. Firstly, disulfide bonds are formed between sulfydryl groups in F127-SH in the mixing process and self-associate to form micelles through hydrophobic interaction at 37 ℃, and then under ultraviolet irradiation, a stable structure is further formed through thiol-ene click reaction between the sulfydryl groups in F127-SH and olefins in HAMA. Pluronic F127 with temperature-sensitive property can adjust the viscosity of biological ink in the printing process, hyaluronic acid with excellent biological function can repair defected skin, and rapid click reaction based on thiol-ene is favorable for reducing damage to cells caused by long-time illumination.
In order to solve the technical problem, the invention discloses a preparation method of Pluronic F127/hyaluronic acid composite hydrogel biological ink, which comprises the steps of adding a pre-polymerization solution of sulfhydryl-terminated modified Pluronic F127 into a pre-polymerization solution of methacrylic anhydride modified hyaluronic acid, carrying out pre-crosslinking reaction, and then carrying out light crosslinking reaction under the irradiation of an ultraviolet lamp to obtain the Pluronic F127/hyaluronic acid composite hydrogel biological ink.
The preparation method of the sulfhydryl-terminated modified Pluronic F127 comprises the following steps:
(1) Reacting p-4-nitrophenyl chloroformate and triethylamine with Pluronic F127 to obtain activated Pluronic F127;
(2) And (2) reacting beta-mercaptoethylamine with the activated Pluronic F127 obtained in the step (1) to obtain the sulfhydryl-terminated Pluronic F127.
Specifically, the preparation method of the sulfhydryl-terminated modified Pluronic F127 comprises the following steps:
(1) Dissolving Pluronic F127 and triethylamine in an organic solvent to obtain a mixed solution A; dissolving p-chlorobenzoic acid-4-nitrophenyl ester in an organic solvent to obtain a mixed solution B; adding the mixed solution A into the mixed solution B for reaction to obtain activated Pluronic F127;
(2) Dissolving the activated Pluronic F127 obtained in the step (1) in an organic solvent to obtain a mixed solution C; dissolving beta-mercaptoethylamine in an organic solvent to obtain a mixed solution D; adding the mixed solution C into the mixed solution D for reaction to obtain mercapto-terminated Pluronic F127;
and (3) performing the step (1) and the step (2) under the protection of inert gas.
Specifically, in the step (1), the molar ratio of Pluronic F127 to triethylamine to 4-nitrophenyl p-chloroformate is 1:2 to 4:5 to 7; the organic solvent is dichloromethane, the amount of the dichloromethane is used for dissolving the solid in the mixed solution, and the viscosity of the mixed solution is moderate; the reaction is carried out at the temperature of 22-27 ℃ for 40-60 h.
Specifically, in the step (2), the molar ratio of the activated Pluronic F127 to the beta-mercaptoethylamine is 1:10 to 20; the organic solvent is dichloromethane, the amount of the dichloromethane is used for dissolving the solid in the mixed solution, and the viscosity of the mixed solution is moderate; the reaction is carried out at the temperature of 22-27 ℃ for 20-30 h.
The preparation method of the methacrylic anhydride modified hyaluronic acid comprises the following steps: reacting methacrylic anhydride with hyaluronic acid to obtain methacrylic anhydride modified hyaluronic acid, controlling the pH value of a reaction system to be between 8 and 10 within 14 hours before reaction, and keeping the pH value of the reaction system to be neutral at 7.35 to 7.45 after the total reaction time is 24 hours.
Specifically, the preparation method of the methacrylic anhydride modified hyaluronic acid comprises the following steps: dissolving hyaluronic acid in deionized water to obtain a mixed solution E, then adding methacrylic anhydride into the mixed solution E, carrying out a light-shielding reaction in an ice bath, controlling the pH value of a reaction system to be between 8 and 10 within 14 hours before the reaction, keeping the pH value of the reaction system to be neutral to be between 7.35 and 7.45 after the total reaction time is 24 hours, dialyzing the reaction liquid, and freeze-drying to obtain the hyaluronic acid.
Specifically, the molar ratio of hydroxyl in the hyaluronic acid to double bonds in the methacrylic anhydride is 1:15 to 20; the concentration of the hyaluronic acid in the mixed solution E is 0.5-1.5 g/mL.
Specifically, the concentration of the thiol-terminated modified Pluronic F127 (F127-SH) in the prepolymerization solution is 5 to 20% g/mL; the concentration of the methacrylic anhydride modified hyaluronic acid (HAMA) in the pre-polymerization solution is 1-3%g/mL; the volume ratio of the prepolymer solution of the thiol-terminated modified Pluronic F127 to the prepolymer solution of the methacrylic anhydride modified hyaluronic acid is 1:1.
specifically, the solvent in the pre-polymerization solution is a phosphate buffer solution containing 2-hydroxy-4 '- (2-hydroxyethoxy) -2-methylpropiophenone, and the concentration of the 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone is 0.1-0.3 percent of g/mL; the composite hydrogel bio-ink has a final concentration of thiol-terminated modified Pluronic F127 of 2.5-10 g/mL, preferably 7.5% by weight, a final concentration of methacrylic anhydride modified hyaluronic acid of 1.0% by weight, and a final concentration of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone of 0.1% by weight.
Wherein the phosphate buffer solution consists of sodium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value is 7.2-7.4.
Specifically, the pre-crosslinking reaction is carried out at the temperature of 36-40 ℃ for 1-3 min; the photocrosslinking reaction has the ultraviolet irradiation wavelength of 365nm and the ultraviolet irradiation power of 5mW/cm 2 The ultraviolet irradiation time is 1-2 min.
The Pluronic F127/hyaluronic acid composite hydrogel bio-ink prepared by the preparation method is also within the protection scope of the invention.
The Pluronic F127/hyaluronic acid composite hydrogel biological ink is applied to preparation of skin wound closure materials. The technology for preparing the skin wound closing material is preferably 3D biological printing.
Preferably, F127-SH/HAMA composite hydrogel (SM 7.5) having a final concentration of 7.5% g/mL F127-SH, 1%g/mL HAMA and 0.1% g/mL 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (I2959) is selected as the bio-ink printing stem cell-bearing scaffold.
Wherein, in the stem cell-carrying scaffold, P6 generation stem cells are adopted.
The process parameters of the scaffold prepared from the composite hydrogel biological ink are as follows: 1.3bar pressure, 20mm/s speed, 27G printing needle, adopting the suspension printing mode.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
1. the composite hydrogel has the advantages of quick gelling time, good mechanical property, water retention property and cell compatibility, and is beneficial to healing of skin wounds.
2. The free radical gel synthesis time is slow, but the thiol-terminated modified Pluronic F127 and methacrylic anhydride modified hyaluronic acid provided by the invention have the advantage that the gel synthesis time of the composite hydrogel is greatly shortened by adopting stepwise chain growth based on thiol-ene 'click' reaction.
3. Generally, too thick bio-ink increases the damage to cells by shear force, and too thin bio-ink causes deposition of cells which is not conducive to extrusion and clogging of the printing needle. According to the composite hydrogel, the thiol-terminated modified Pluronic F127 shows sol-gel phase change behavior along with temperature change, the viscosity of the biological ink can be adjusted, and photo-crosslinking is beneficial to fixing of a final printing structure and can improve the mechanical property of the composite hydrogel biological ink.
4. According to the invention, hyaluronic acid is selected as a base material, pluronic F127 is introduced, internal crosslinking sites of the composite hydrogel are increased along with the increase of the concentration of F127-SH, the crosslinking density is increased, a more compact internal structure is caused, and the composite hydrogel has good water retention and is beneficial to wound healing.
5. The invention selects hyaluronic acid and Pluronic F127 as hydrogel raw materials, the two raw materials can generate crosslinking action through simple modification, the preparation is simple, and the raw materials are commercialized. Therefore, the selection, the establishment, the popularization and the promotion of the gel method have very important values in tissue engineering and regenerative medicine.
6. The composite hydrogel biological ink disclosed by the invention adopts a suspension printing mode, has the capability of printing a complex structure and ensures the survival rate of printed cells.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 (a) nuclear magnetic map of modified Pluronic F127; FIG. 1 (b) is a nuclear magnetic spectrum of modified hyaluronic acid;
FIG. 2 is a compressive stress-strain curve of a composite hydrogel;
FIG. 3 is a water retention image of a composite hydrogel;
FIG. 4 shows the growth of cells in the scaffold during printing of the composite hydrogel bio-ink SM7.5 stem cell-loaded cells;
fig. 5 is a complex structure image printed by composite hydrogel bio-ink SM 7.5;
FIG. 6 is a photograph and quantitative analysis of the dorsal wound of hydrogel scaffold treated mice;
figure 7 is a Masson staining picture of the wound of the hydrogel scaffold treated mouse and a collagen quantitative analysis.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1
(1) Weighing 10g of Pluronic F127, placing the Pluronic F127 into a two-neck flask, vacuumizing for 30min, introducing nitrogen for 5min, repeating the step twice, adding 70mL of dichloromethane and 300 mu L of triethylamine, and fully and uniformly stirring to obtain a mixed solution A. 0.96g of p-chlorobenzoic acid-4-nitrophenyl ester (p-NPC) is weighed, vacuumized for 30min, aerated with nitrogen for 5min, the step is repeated twice, 10mL of dichloromethane is added, and the mixture is fully and uniformly stirred to obtain a mixed solution B. And adding the mixed solution A into a constant-pressure dropping funnel of 50mL, dropwise adding into the mixed solution B, and finishing dropwise adding within 30 min. Reacting for 48h at 25 ℃, after the reaction is finished, preparing a saturated NaCl solution, carrying out salt washing, standing and separating by a separating funnel, taking a lower organic layer, drying overnight by anhydrous magnesium sulfate, carrying out suction filtration by a sand core funnel, carrying out rotary evaporation on an obtained organic phase, and rotating to a viscous state. Then adding cold ether for precipitation, centrifuging, drying in a vacuum drying oven overnight to obtain white powdered activated product (F127-NPC), and drying in a dryer for use.
(2) Weighing 1g of F127-NPC, putting the mixture into a two-neck flask, vacuumizing the two-neck flask for 30min, introducing nitrogen for 5min, repeating the step twice, adding 50mL of dichloromethane, and fully and uniformly stirring to obtain a mixed solution C. 0.086g of beta-mercaptoethylamine was weighed, evacuated for 30min and purged with nitrogen for 5min, the procedure was repeated twice, 10mL of dichloromethane was added, and the mixture was stirred well to obtain a mixed solution D. And adding the mixed solution C into the mixed solution D, reacting for 24h at 25 ℃, then performing salt washing, standing for liquid separation, drying, suction filtration, rotary evaporation, precipitation, centrifugation, drying and dialysis for three days, putting the dialyzed product into a freeze dryer for freeze drying to finally obtain a white product F127-SH, namely the sulfhydryl-terminated Pluronic F127, and storing in a dryer in a dark place.
(3) 10.00mg of F127, 10.00mg of F127-NPC and 10.00mg of 127-SH are respectively dissolved in CDCl 3 (600. Mu.L). Recording samples by NMR spectrometer 1 H NMR spectrum.
In the nuclear magnetic spectra of F127 and F127-SH (FIG. 1 (a)), it can be seen that F127 (F127-NPC) shows two new signal peaks around delta =7.37-7.39ppm (e-F) and 8.26-8.28ppm (g-h) after activation, which are the nuclear magnetic peaks of-CH-on the benzene ring after activation. The broad multiplet of δ =1.12ppm (a) is then the methyl group (-CH) of the PPO block in F127 3 ) δ =2.62-2.65ppm (i-j) and 1.41ppm (h) is the upper methylene (-CH) group of cysteamine 2 ) Nuclear magnetic peaks of the group and sulfhydryl hydrogen protons, which confirm the successful synthesis of F127-SH.
Example 2
1) 1g of hyaluronic acid (HA, mw: 200-400 kDa) is dissolved in deionized water, when the solution is fully dissolved to form a mixed solution E with the hyaluronic acid concentration of 1g/mL, the mixed solution E is reacted in ice bath at 4 ℃ in a dark place, 8mL of Methacrylic Anhydride (MA) is added, 5M NaOH is prepared to adjust the pH value of the reaction, the pH value of the reaction system is controlled between 8 and 10 within 14h before the reaction, after the reaction is fully performed for 24h, the pH value of the reaction system is detected and kept between 7.35 and 7.45, the reaction solution is placed in a dialysis bag (8000-14000 Da) for dialysis for 3 days, water is changed every 4h for the first two days, and water is changed every three times every day for the next several days. And (4) putting the dialyzed product into a freeze dryer for freeze drying to finally obtain a white spongy sample, and storing the white spongy sample in the dryer in a dark place.
2) 10.00mg of HA and 10.00mg of HAMA were dissolved in D, respectively 2 O (600. Mu.L), using NMR spectrometerRecording of samples 1 H NMR spectrum.
3) In the nuclear magnetic spectra of Methacrylic Anhydride (MA) and HAMA (fig. 1 (b)), it can be seen that HA grafted by MA HAs two new signal peaks around δ =6.10ppm (a) and 5.71ppm (b), and a bifurcated peak pattern around δ =1.86ppm (c), which are the nuclear magnetic peak of double bond hydrogen and the nuclear magnetic peak of methyl hydrogen on methacrylate group, respectively, and the nuclear magnetic peak at δ =1.96ppm (d) is the peak of methyl hydrogen on hyaluronic acid side chain, which proves that hyaluronic acid HAs been successfully methacrylated.
Example 3
(1) Dissolving HAMA prepared in example 2 in 0.1% g/mL of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone phosphate buffered water to give a pre-polymerization solution containing HAMA at a concentration of 1%g/mL, placing the pre-polymerization solution in a glass sample bottle, pre-crosslinking at 37 deg.C for 2min, and placing at a wavelength of 365nm and a power of 5mW/cm 2 Irradiating the solution for 1min by ultraviolet light to perform photocrosslinking to obtain HAMA hydrogel.
(2) The F127-SH prepared in example 1 was dissolved in 0.1% g/mL of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone phosphate buffered water to prepare prepolymerization solutions containing F127-SH at different concentrations, wherein the concentrations of F127-SH in the prepolymerization solutions were 5%g/mL, 10% g/mL, 15% g/mL and 20% g/mL, respectively; dissolving HAMA prepared in example 2 in 0.1 g/mL of phosphate buffered water of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone to prepare a pre-polymerization solution containing HAMA, wherein the concentration of HAMA in the pre-polymerization solution is 2%g/mL; at the temperature of 4 ℃, two parts of prepolymerization liquid are mixed according to the volume ratio of 1:1 mixing so that the final concentration of HAMA in the system is 1%g/mL and the final concentration of F127-SH in each group is 2.5% g/mL, 5%g/mL, 7.5% g/mL, 10% g/mL, carrying out a pre-crosslinking reaction at 37 ℃ for 2min, followed by 5mW/cm at 37 ℃ 2 And irradiating for 1min by ultraviolet (365 nm) to perform photocrosslinking to obtain the composite hydrogel, and respectively marking the composite hydrogel as SM2.5, SM5, SM7.5 and SM10.
(3) The compression performance of the hydrogel was tested using a 20mm upper clamp in the rheometer, 500 μ L of hydrogel was injected into a 2mL glass bottle, temperature, light-gelled and added to the parallel plates. The sample was compressed at 10.0 μm/s to reach 100% strain at 37 ℃ to obtain a stress-strain curve. FIG. 2 is a compressive stress-strain curve of a hydrogel, which reflects the mechanical properties of the hydrogel; among them, SM7.5 and SM10 showed higher breaking stress compared to hydrogel HAMA, probably because the introduction of F127-SH increased the crosslinking density of the hydrogel, thereby improving the mechanical properties of the hydrogel. The maximum breaking stress of the SM10 hydrogel reaches 0.097MPa, and the fact that the internal crosslinking sites of the hydrogel network are increased along with the increase of the concentration of F127-SH and the mechanical strength of the hydrogel is further enhanced can be obtained.
Example 4
(1) The hydrogels SM2.5, SM5, SM7.5, SM10, HAMA prepared in example 3 were applied to this example.
(2) By using
Preparing a round hydrogel sample by using a mold, soaking the round hydrogel sample in a PBS solution, recording the mass Wt after the round hydrogel sample is swelled and balanced, placing the hydrogel in a constant-temperature shaking table at 37 ℃, taking out the hydrogel every 10 hours to measure the mass Wd, and calculating the formula: weight (%) = Wd/Wt × 100%.
The change of the water retention of the F127-SH/HAMA composite hydrogel with time in the range of 0-60 h is shown in figure 3. After 24 hours, the water retention rate of HAMA is less than 20%, and the water retention rate of the composite hydrogel group is between 40% and 60%. With the increase of the concentration of F127-SH, the water retention rate of the composite hydrogel gradually increases. The high water retention rate is due to the excellent water retention property and high crosslinking density of hyaluronic acid, so that water molecules are not easy to exude.
Example 5
(1) The hydrogel SM7.5 prepared in example 3 was applied to this example.
(2) Stem cells cultured to the P6 generation are digested and centrifuged, SM7.5 (prepared in example 3) bio-ink is prepared and cells are resuspended at 1.6 × 106 cells/mL at 4 ℃, the mixed bio-ink is placed in a sterile printing cartridge, a cell-carrying scaffold structure (7.5 mm in diameter and 1.5mm in height) is printed at a speed of 20mm/s and a printing needle of 27G under a pressure of 1.3bar, a large hydrogel mixed by sterilized bio-ink material and stem cells is used as a control group and is compared with the stem cell-carrying printing scaffold, after 1, 3 and 7 days of culture, the proliferation activity of the cells in the scaffold is detected by using a CCK-8 kit, the program of a microplate reader is set at 450nm to test the OD value of each group in the well plate, blank wells are used as background plates for deducting the OD value generated by the well as the relative calculation formula of the cells: cell viability (%) = [ (ODs-ODb)/(ODc-ODb) ]. Times.100%
In order to observe the living/dead condition of the cells in the scaffold more intuitively, the cells are stained by adopting a Calcein-AM/PI kit. Add 5. Mu.L Calcein (Calcein-AM) and 5. Mu.L Propidium Iodide (PI) to 10mL serum-free medium, add 100. Mu.L per well, incubate for 40min, wash twice with PBS after staining to remove excess staining agent. The samples were observed in an inverted fluorescence microscope. As shown in fig. 4, cells grew well on the scaffold compared to the bulk unprinted hydrogel, and the cell-loaded scaffold group had more viable cells and exhibited good proliferative viability than the unprinted hydrogel group.
Example 6
(1) The hydrogel SM7.5 prepared in example 3 was applied to this example.
(2) Different shapes are set in the printing system for printing, the printed plane structure grid boundary is clear and a complex three-dimensional structure is printed in fig. 5, and the result proves that the biological ink material (SM 7.5) has the capability of printing the complex structure.
Example 7
(1) The hydrogel SM7.5 prepared in example 3 was applied to this example.
(2) 40 ICR male mice (20 g) were anesthetized by intraperitoneal injection of chloral hydrate (0.1 mL/10 g), and then dorsal hairs were removed to make 27 mm circular defect wounds on the dorsal surface. After being sterilized by medical alcohol, 3 groups of mice randomly divided into 5 groups were selected and placed with the stem cell-loaded printing scaffold, the stem cell-unloaded printing scaffold and the stem cell-loaded hydrogel-unprinted. Fixing the support structure at the wound part with transparent medical adhesive tape, and respectively adopting a Tegaderm for the rest two groups TM 3M TM Commercial product served as positive control and saline treated group as negative control. Wound size was observed at 3, 7 and 14 days after treatment and photographed and recorded using the following formula,% wound shrinkage = [ original wound area-wound area on day of treatment = [% ]]Original wound area x 100%.
As shown in fig. 6, the wound area was observed to decrease in all treatment groups, and the wound quantified closure rates of the control group, the commercial group, the cell-free printed scaffold group, and the cell-free hydrogel group were about 14.95%, 27.45%, 42.00%, and 49.95% respectively after 3 days of treatment, while the cell-free printed scaffold group was 67.00% and was statistically different from the other groups (p < 0.01). On day 7 of treatment, the closure rate of the cell-loaded printed scaffold group was 88.82% compared to the control and commercial groups (49.18%, 69.87%), and the cell-loaded printed scaffold group exhibited a faster wound healing rate than the other groups throughout the treatment, achieving substantially full coverage of hair at 14 days, indicating that it had a promoting effect on wound healing.
(3) Masson staining (fig. 7) was used to observe collagen deposition upon wound healing, and at day 3, more collagen deposition began to occur in the scaffold group compared to the control, with little observation in the commercial group. After 7 days of treatment, different amounts of collagen deposition occurred in each group, and the amount of collagen deposition was further increased in the printed group. After 14 days of treatment, collagen deposition from the cell-loaded printing scaffold group appeared more dense and similar to healthy skin. Quantitative data analysis showed that the wounds treated with the cell-loaded printing scaffold group had the highest collagen density of about 74.09%, while the cell-loaded non-printing hydrogel group, the non-cell-loaded printing scaffold group, the commercial group, and the control group were 63.54%, 61.75%, 49.89%, and 47.55%, respectively. The superior wound repair effect exhibited by the cell-free printed scaffold group compared to the commercial group and the control group may be due to the role of hyaluronic acid in wound healing. Whereas the cell-loaded printing scaffold group showed the best wound healing, probably the role of hyaluronic acid with stem cells.
The invention provides a method and a method for preparing Pluronic F127/hyaluronic acid composite hydrogel bio-ink, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the invention, and the modifications and embellishments should be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (13)
- The preparation method of the Pluronic F127/hyaluronic acid composite hydrogel biological ink is characterized in that a prepolymer solution of sulfhydryl-terminated modified Pluronic F127 is added into a prepolymer solution of methacrylic anhydride modified hyaluronic acid to carry out a pre-crosslinking reaction, and then a light crosslinking reaction is carried out under the irradiation of an ultraviolet lamp, so that the Pluronic F127/hyaluronic acid composite hydrogel biological ink is obtained.
- 2. The method of claim 1, wherein the thiol-terminated modified Pluronic F127 is prepared by the steps of:(1) Reacting p-4-nitrophenyl chloroformate, triethylamine and Pluronic F127 to obtain activated Pluronic F127;(2) And (3) reacting beta-mercaptoethylamine with the activated Pluronic F127 obtained in the step (1) to obtain the sulfhydryl terminated Pluronic F127.
- 3. The method of claim 2, wherein the thiol-terminated modified Pluronic F127 is prepared by the steps of:(1) Dissolving Pluronic F127 and triethylamine in an organic solvent to obtain a mixed solution A; dissolving p-chlorobenzoic acid-4-nitrophenyl ester in an organic solvent to obtain a mixed solution B; adding the mixed solution A into the mixed solution B for reaction to obtain activated Pluronic F127;(2) Dissolving the activated Pluronic F127 obtained in the step (1) in an organic solvent to obtain a mixed solution C; dissolving beta-mercaptoethylamine in an organic solvent to obtain a mixed solution D; adding the mixed solution C into the mixed solution D for reaction to obtain mercapto-terminated Pluronic F127;and (3) performing the step (1) and the step (2) under the protection of inert gas.
- 4. The method according to claim 3, wherein in step (1), the molar ratio of Pluronic F127, triethylamine and 4-nitrophenyl p-chloroformate is from 1:2 to 4:5 to 7; the organic solvent is dichloromethane; the reaction is carried out at the temperature of 22-27 ℃ for 40-60 h.
- 5. The method according to claim 3, wherein in step (2), the molar ratio of activated Pluronic F127 to β -mercaptoethylamine is 1:10 to 20; the organic solvent is dichloromethane; the reaction is carried out at the temperature of 22-27 ℃ for 20-30 h.
- 6. The method according to claim 1, wherein the methacrylic anhydride-modified hyaluronic acid is prepared by: reacting methacrylic anhydride with hyaluronic acid to obtain methacrylic anhydride modified hyaluronic acid, controlling the pH value of a reaction system to be between 8 and 10 within 14 hours before reaction, and keeping the pH value of the reaction system to be neutral at 7.35 to 7.45 after the total reaction time is 24 hours.
- 7. The method according to claim 6, wherein the methacrylic anhydride-modified hyaluronic acid is prepared by: dissolving hyaluronic acid in deionized water to obtain a mixed solution E, then adding methacrylic anhydride into the mixed solution E, carrying out light-shielding reaction in ice bath, controlling the pH value of the reaction system to be between 8 and 10 within 14 hours before the reaction, keeping the pH value of the reaction system to be neutral 7.35 to 7.45 after the total reaction time is 24 hours, dialyzing the reaction solution, and freeze-drying to obtain the hyaluronic acid.
- 8. The method according to claim 7, wherein the molar ratio of hydroxyl groups in the hyaluronic acid to double bonds in the methacrylic anhydride is 1:15 to 20; the concentration of the hyaluronic acid in the mixed solution E is 0.5-1.5 g/mL.
- 9. The method of claim 1, wherein the concentration of said mercapto-terminated modified Pluronic F127 in the prepolymerization solution is 5-20% g/mL; the concentration of the methacrylic anhydride modified hyaluronic acid in the pre-polymerization solution is 1-3%g/mL; the volume ratio of the prepolymer solution of the thiol-terminated modified Pluronic F127 to the prepolymer solution of the methacrylic anhydride modified hyaluronic acid is 1:1.
- 10. the process according to claim 1, wherein the solvent in the solution for preliminary polymerization is a phosphate buffer solution containing 2-hydroxy-4 '- (2-hydroxyethoxy) -2-methylpropiophenone, and the concentration of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone is 0.1 to 0.3% g/mL; the composite hydrogel bio-ink, the final concentration of the thiol-terminated modified Pluronic F127 was 2.5-10% g/mL, the final concentration of the methacrylic anhydride modified hyaluronic acid was 1.0% g/mL, the final concentration of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone was 0.1% g/mL.
- 11. The preparation method of claim 1, wherein the pre-crosslinking reaction is carried out at a temperature of 36-40 ℃ for 1-3 min; the photocrosslinking reaction has the ultraviolet irradiation wavelength of 365nm and the ultraviolet irradiation power of 5mW/cm 2 The ultraviolet irradiation time is 1-2 min.
- 12. The preparation method of any one of claims 1 to 11 is used for preparing Pluronic F127/hyaluronic acid composite hydrogel bio-ink.
- 13. Use of the Pluronic F127/hyaluronic acid composite hydrogel bio-ink according to claim 12 for the preparation of wound closure material for skin.
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