CN115282326A

CN115282326A - Method for 3D printing of functional hydrogel wound dressing

Info

Publication number: CN115282326A
Application number: CN202210969585.2A
Authority: CN
Inventors: 程凤; 徐磊; 栗洪彬; 贺金梅; 黄玉东
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2022-11-04

Abstract

The invention discloses a method for 3D printing of a functional hydrogel wound dressing, which comprises the following steps: (1) Preparing printing ink containing TO-CNF, gelMA and photoinitiator; (2) Screening printing parameters of the ink, and determining the concentration and the printing parameters of the printable ink; (3) adding GelMA-DA to the printable ink; (4) adding PDA @ rGO into the composite ink; (5) Transferring the composite ink into an injector, centrifuging and defoaming, and precooling in a refrigerator; (6) Putting the syringe into the extruder3D printing is carried out in the 3D printer; (7) UV crosslinking and Ca crosslinking of printed hydrogels ²⁺ Complexing and crosslinking; and (8) performing photothermal antibiosis on the crosslinked wound dressing. The hydrogel prepared by the method has good photo-thermal antibacterial performance, conductivity, hemostatic property and biodegradability, and can keep the wound surface moist and effectively promote the wound surface healing.

Description

Method for 3D printing of functional hydrogel wound dressing

Technical Field

The invention belongs to the field of biological materials, relates to a preparation method of a wound dressing material, and particularly relates to a method for 3D printing of a functional hydrogel wound dressing.

Background

The skin is the largest organ of the human body and has the functions of protecting the body from damage and microbial attack and maintaining body fluid, electrolytes and nutritional ingredients. Once the skin has serious defects, the formed wounds can seriously affect the life and health of people. Wounds, especially extensive full-thickness wounds, require a long time to repair, and acute and chronic wounds remain a major clinical problem. Thus, there is an urgent need for a wound dressing that ideally meets the requirements of rapid wound healing, promotion of wound healing and reduction of scarring. Several wound dressings have been developed, including foams, electrospun nanofibers, films, hydrogels, and the like. The hydrogel dressing has the functions of keeping a wound surface moist environment, absorbing tissue exudates, permeating oxygen, cooling the wound surface and relieving pain of a patient, and is widely applied to the field of wound dressings.

Most wound dressing products currently on the market are not customizable to specific conditions, and 3D printing systems have the potential to provide customized innovative solutions in this area that can be rapidly prototyped or customized using 3D printing to produce designs using Computer Aided Design (CAD) via digitally controlled layer-by-layer deposition techniques.

Methacrylated gelatin (GelMA) hydrogels have been widely used in various biomedical applications due to their good biological and tunable physical properties. GelMA hydrogels closely resemble some of the basic properties of the native extracellular matrix (ECM) because of the presence of cell attachment and matrix metalloproteinase-responsive peptide motifs, which allow cells to proliferate and diffuse in GelMA-based scaffolds. From the processing point of view, gelMA can be crosslinked by ultraviolet light to form hydrogel with adjustable mechanical properties, but GelMA hydrogel has poor mechanical strength and cannot meet the requirement of being used as a wound dressing. The Cellulose Nanofiber (CNF) has wide sources, good biocompatibility and excellent mechanical strength, and has huge application prospects in the field of biomaterials. Carboxyl can be introduced on CNF by adopting a Tempo oxidation method, so that the hemostatic performance of the wound dressing is improved.

Research shows that when the temperature is higher than 50 ℃, the bacteria can be effectively killed, and normal cells of a human body cannot be damaged when the temperature is lower than 55 ℃. Therefore, photothermal antibiosis is an antibiosis strategy with good antibacterial performance and avoiding abuse of antibiotics, and has great clinical application potential in treatment of skin wound infection. Graphene is a widely used photo-thermal agent, and the graphene-doped wound dressing can be rapidly heated under a near-infrared laser of 808 nm. Besides, the doping of the graphene endows the dressing with excellent photo-thermal antibacterial performance, and the dressing also has excellent conductivity, so that the wound infection can be effectively cured, and the wound healing is promoted.

The current hydrogel wound dressings have the following problems: poor antibacterial performance and antibiotic dependence; poor adhesion, difficulty in adhering to the wound surface; the device cannot be customized according to different conditions of individual wounds; the air permeability is poor; the mechanical property is poor.

Disclosure of Invention

In order to solve the problems of the wound dressing, the invention provides a method for 3D printing of a functional hydrogel wound dressing. The hydrogel wound dressing with good conductivity, adhesiveness, photothermal antibacterial property and hemostatic property is prepared by adopting a 3D printing hydrogel forming process, and the problems that the traditional wound dressing is poor in antibacterial performance (mostly adopting antibiotics) and wet adhesiveness, and cannot meet the personalized requirements of users according to the complexity of wound surfaces are solved, so that the application of the wound dressing in various wound surfaces is limited.

The purpose of the invention is realized by the following technical scheme:

a method of 3D printing a functional hydrogel wound dressing, comprising the steps of:

step (1) of preparing a printing ink containing 0 to 3% of TO-CNF, 5 to 30% of GelMA and 0.5% of a photoinitiator by volume mass%, wherein:

the photoinitiator is 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) or phenyl-2,4,6-trimethyllithium benzoylphosphonate (LAP);

and (2) screening printing parameters of the ink obtained in the step (1) to determine the concentration of the printable ink and printer parameters, wherein:

the printable ink concentrations are: 0% of TO-CNF, 15% of GelMA and 0.5% of photoinitiator; 0.5 percent of TO-CNF, 10 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1 percent of TO-CNF, 5 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1.5 percent of TO-CNF, 5 TO 10 percent of GelMA and 0.5 percent of photoinitiator;

the printer parameters are as follows: the extrusion distance of the spray head is 0.05mm, and the moving speed of the spray head is 1.5-3.17 mm/s; the extrusion distance of the spray head is 0.2mm, and the moving speed of the spray head is 3.17mm/s; the extrusion distance of the spray head is 0.35mm, and the moving speed of the spray head is 4.83mm/s;

step (3) of adding GelMA-DA to the printable ink of step (2) to prepare a composite ink containing GelMA-DA in an amount of 5 to 20% by volume/mass;

step (4) adding PDA @ rGO into the composite ink prepared in the step (3), and controlling the content of the PDA @ rGO to be 0-8 mg/ml;

step (5) moving the composite ink obtained in the step (4) into an injector, performing centrifugal defoaming, and precooling in a refrigerator;

step (6), placing the syringe filled with the precooled composite ink in the step (5) into an extrusion type 3D printer, and performing 3D printing on the composite hydrogel by adopting the printable ink concentration, the printing speed and the extrusion pressure determined in the step (2);

step (7) UV crosslinking and Ca treatment of the hydrogel printed in step (6) ²⁺ The method comprises the following steps:

firstly, carrying out ultraviolet irradiation treatment on hydrogel at a distance of 5-15 cm from an ultraviolet lamp for 200-300 s, and then taking down the photo-crosslinked hydrogel and soaking the hydrogel in the volume1-3% of CaCl by mass fraction ₂ Soaking in the solution for 2-10 min and taking out; then soaking the mixture in PBS buffer solution for 10-15 min and taking out;

step (8) performing photothermal antibiosis on the wound dressing crosslinked in the step (7), and specifically comprising the following steps:

adopting a near infrared laser with the wavelength of 808nm and the wavelength of 0.6 to 1.2w/cm ² The power density of (2) is used for irradiating the wound dressing for 5-15 min.

Compared with the prior art, the invention has the following advantages:

1. compared with the traditional wound dressing, the 3D printing hydrogel wound dressing is adopted, and the method is easy to operate, has customizability, safe materials and low toxicity; the wound dressing prepared by the method has excellent application prospect.

2. According to the invention, by using the 3D printing forming method, the wound dressing with the shape customized according to different conditions is prepared, the requirements of different patients can be met, and the mesh-structure dressing prepared by 3D printing has good air permeability and can accelerate the healing of wounds.

3. According to the invention, the dopamine is grafted on GelMA, so that the adhesive force of the dressing is improved, and the defect that hydrogel is used as a wound dressing is overcome. The addition of PDA @ rGO endows the dressing with photo-thermal antibacterial performance, can generate a temperature higher than 50 ℃ under a near-infrared laser of 808nm, can effectively resist bacteria, overcomes the problems of poor antibacterial performance, antibiotics abuse and the like of the traditional wound dressing, and can effectively absorb active oxygen, reduce inflammation and accelerate wound healing due to the introduction of polydopamine.

Drawings

FIG. 1 is a flow chart for the preparation of a functional hydrogel wound dressing according to the present invention;

FIG. 2 is a NMR chart of gelatin, gelMA and GelMA-DA in the present invention;

FIG. 3 is a scanning electron micrograph of TO-CNF according TO the present invention;

FIG. 4 is a diagram showing an infrared absorption spectrum of TO-CNF in the present invention;

FIG. 5 is a diagram illustrating the printable parameters of different ink compositions;

FIG. 6 is a chart illustrating printable parameters of printing speed and pressure in accordance with the present invention;

FIG. 7 is a graph of the ultraviolet absorption spectrum of GelMA-DA in accordance with the present invention;

FIG. 8 is a graph of the ultraviolet absorption spectrum of PDA @ rGO in the present invention;

FIG. 9 is a graph of the infrared absorption spectrum of PDA @ rGO in the present invention;

fig. 10 is a rheological test chart of the printed ink.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.

The invention provides a method for 3D printing of a functional hydrogel wound dressing, which comprises the following steps as shown in figure 1:

step (1) preparation of GelMA: 10g of gelatin was dissolved in 100ml of PBS (pH =7.2 to 7.4) buffer (until no distinct particles were visible to the naked eye) sufficiently at 50 ℃,4 to 8ml of methacrylic anhydride was slowly added dropwise at a rate of one drop per second, and after completion of addition, the reaction was carried out at 50 ℃ for 2 hours at a stirring rate of 240 rpm. After the reaction, 100ml of PBS buffer was added to dilute the reaction solution, and the mixture was stirred well for 10min. And filling the product into a 10000-molecular cut-off dialysis bag after the reaction is finished, dialyzing for 5-7 d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished.

Step (2) preparation of GelMA/TO-CNF composite ink: firstly, a photoinitiator 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) or phenyl-2,4,6-trimethylbenzoyllithium phosphonate (LAP) (the volume mass percentage is 0.5%) is dissolved in deionized water at 80 ℃, and then TO-CNF and GelMA are respectively taken and added into the deionized water with the photoinitiator TO prepare GelMA/TO-CNF composite ink, wherein the volume mass fraction of the TO-CNF in the composite ink is 0-3%, and the volume mass fraction of the GelMA in the composite ink is 5-30%.

Screening printable parameters: firstly, placing the composite ink prepared in the step (2) into a centrifuge tube, centrifuging for 3min at the rotating speed of 3000rpm by using a centrifuge, then taking out the centrifuge tube, transferring the ink into an injector, precooling for 5min by using a refrigerator, taking out, placing into an extrusion type 3D printer, and printing a mesh square with the pattern of 10mm multiplied by 10 mm. And screening printing parameters of the prepared composite ink, and determining the concentration of the printable GelMA/TO-CNF composite ink and the printable extrusion speed and pressure. The screening method of the printing parameters comprises the following steps: and (3) screening the printing parameters by adopting a 21G needle head according TO the TO-CNF/GelMA concentration, the extrusion speed and the pressure respectively, and taking the structural integrity of the printed product as the available printing parameters. The specific printing parameters are as follows:

the printable ink concentrations were: 0% of TO-CNF, 15% of GelMA and 0.5% of photoinitiator; 0.5 percent of TO-CNF, 10 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1 percent of TO-CNF, 5 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1.5 percent of TO-CNF, 5 TO 10 percent of GelMA and 0.5 percent of photoinitiator;

the printer parameters are: the extrusion distance of the spray head is 0.05mm, and the moving speed of the spray head is 1.5-3.17 mm/s; the extrusion distance of the spray head is 0.2mm, and the moving speed of the spray head is 3.17mm/s; the extrusion distance of the spray head is 0.35mm, and the moving speed of the spray head is 4.83mm/s;

step (4) preparation of GelMA-DA: dissolving 0.84g of GelMA in 20ml of PBS buffer solution, adjusting the pH value to between 5 and 6, and heating to 50 ℃ for full dissolution; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.4-0.8 g dopamine hydrochloride, reacting for 12-24 h at 37 ℃, and N in the whole process ₂ And the dopamine hydrochloride is protected and prevented from being oxidized. After the reaction, dialyzed for 3 days under acidic condition, and stored by freeze drying.

Step (5) preparation of PDA @ rGO: adding 10-40 mg of graphene oxide and 20mg of dopamine hydrochloride into 200ml of Tris (pH = 8.5) buffer solution, and carrying out ultrasonic treatment for 30min; then heating the solution in water bath at 60 ℃, and violently stirring for reacting for 18-24 h; after the reaction is finished, the reaction is stopped by respectively washing the reaction kettle with absolute ethyl alcohol and deionized water for a plurality of times, and the product is washed and fully dried.

Step (6) preparation of GelMA/TO-CNF/GelMA-DA/PDA @ rGO composite ink: adding 5-20% of GelMA-DA and 0-8 mg/ml of PDA @ rGO on the basis of the GelMA/TO-CNF composite ink in the step (2); after adding, heating to 50 ℃ and fully stirring, then carrying out ultrasonic treatment for 30min, and repeating stirring after finishing ultrasonic treatment to fully disperse PDA @ rGO.

And (7) firstly putting the composite ink in the step 6 into a centrifuge at 3000rpm, centrifuging and defoaming for 3min, then transferring into a 10ml syringe, and putting into a refrigerator for precooling for 10min.

And (8) performing 3D printing of the composite ink: loading the syringe filled with the precooled composite ink into a 3D printer; and (4) then, performing 3D printing on the composite ink by adopting the two sets of parameters of the ink proportioning parameter, the printing speed and the extrusion pressure which are researched in the step (3).

Step (9) crosslinking of the hydrogel wound dressing: firstly, carrying out ultraviolet irradiation treatment on the extruded and printed hydrogel at a distance of 10cm from an ultraviolet lamp for 240s, then taking down the photo-crosslinked hydrogel and soaking the hydrogel in CaCl with the volume mass fraction of 1-3% ₂ Soaking in the solution for 2-10 min and taking out; then soaking in PBS buffer solution for 10-15 min and taking out.

Step (10), photo-thermal antibacterial of the wound dressing: adopting a near-infrared laser with the wavelength of 808nm, and respectively using the wavelength of 0.6-1.2 w/cm ² The power density of (a) was used to irradiate the dressing for 10min, the temperature change was recorded, and the photothermal antimicrobial effect was evaluated by plate counting.

The method has the following advantages:

1. the GelMA/TO-CNF composite hydrogel is printed by using the extrusion type 3D printing system, so that the injectability of the hydrogel is fully utilized, and the hydrogel dressing with excellent performance is successfully printed. 3D printed hydrogel scaffolds for wound healing have many advantages: i) The formulation or dosage can be customized according to individual needs; ii) dimensional characteristics of the wound dressing, such as topography, structure, are customizable; iii) A simple material loading process; iv) a wide range of materials can be used; v) the mesh design allows efficient oxygen permeation.

2. By adding the TO-CNF, the mechanical strength of the GelMA hydrogel is effectively improved, and the stability of the composite ink during extrusion is improved. And carboxyl is introduced into CNF oxidized by TEMPO, the hemostatic performance of the wound dressing is improved, and the CNF can be subjected to complexing crosslinking with metal ions and crosslinking with ultraviolet light of GelMA to form an interpenetrating crosslinked network.

3. The prepared hydrogel has the characteristics of strong adhesion, soft texture, capability of being attached to skin and the like, has good photothermal antibacterial performance, conductivity, hemostatic property and biodegradability, can keep the wound surface moist and effectively promote the wound surface to heal, can meet the requirements of customizing different conditions of personal wounds, and can solve the problems of poor air permeability, poor mechanical property and the like of the traditional dressing.

4. Is suitable for preparing complex wound dressing materials.

Example 1

Step (1) preparation of GelMA: 10g of gelatin was dissolved in 100ml of PBS buffer (until no particles were visible) at 50 ℃ and 4ml of methacrylic anhydride was slowly added dropwise at a rate of one drop per second, after which the mixture was reacted at 50 ℃ for 2 hours with stirring at 240 rpm. After the reaction, 100ml of PBS buffer was added to dilute the reaction solution, and the mixture was stirred well for 10min. And filling the product into a 10000-molecular-weight cut-off dialysis bag after the reaction is finished, dialyzing for 7d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished. The NMR spectra of gelatin and GelMA are shown in FIG. 2.

Step (2) preparation of GelMA/TO-CNF composite ink: firstly, a photoinitiator 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) (the volume mass percentage is 0.5%) is dissolved in deionized water at 80 ℃, and then a scanning electron microscope picture and an infrared picture of TO-CNF (shown in figures 3 and 4) and GelMA are respectively taken TO be added into 1ml of deionized water of the photoinitiator TO prepare GelMA/TO-CNF composite ink, wherein the volume mass fraction of the TO-CNF in the composite ink is 0-1.5%, and the volume mass fraction of the GelMA in the composite ink is 5-15%.

Screening printable parameters: firstly, placing the composite ink prepared in the step (2) into a centrifuge tube, centrifuging for 3min at the rotating speed of 3000rpm by using a centrifuge, then taking out the centrifuge tube, transferring the ink into an injector, precooling for 5min by using a refrigerator, then taking out the ink, and placing the ink into an extrusion type 3D printer. The printed pattern was 10mm by 10mm mesh squares. The prepared composite ink was subjected to screening of printing parameters, and the printable ink concentration and printable speed and pressure were determined, and the printable parameters were as shown in fig. 5 and 6.

Step (4) preparation of GelMA-DA: dissolving 0.84g GelMA in 20ml PBS buffer solution, adjusting pH to 5.5, heating to 50 deg.C for dissolving completely; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.4g dopamine hydrochloride, reacting for 24h at 37 ℃, N ₂ And the dopamine hydrochloride is protected and prevented from being oxidized. Dialyzing for 3d under acidic condition after the reaction is finished, and freeze-drying and storing. The nuclear magnetic resonance hydrogen spectrum of GelMA-DA is shown in FIG. 2, and the ultraviolet-visible absorption spectrum is shown in FIG. 7.

Step (5) preparation of PDA @ rGO: adding 20mg of graphene oxide and 20mg of dopamine hydrochloride into 200ml of Tris buffer solution, and carrying out ultrasonic treatment for 30min; then heating the solution in water bath at 60 ℃, and violently stirring for reacting for 24 hours; after the reaction is finished, the reaction is stopped by respectively washing the reaction kettle with absolute ethyl alcohol and deionized water for a plurality of times, and the product is washed and fully dried. The ultraviolet-visible absorption spectrum and infrared absorption spectrum of PDA @ rGO are shown in FIGS. 8 and 9.

Step (6) preparation of GelMA/TO-CNF/GelMA-DA/PDA @ rGO composite ink: adding GelMA-DA (10%) and PDA @ rGO (4 mg/ml) on the basis of the GelMA/TO-CNF composite ink in the step (2); heating to 50 ℃ after adding, fully stirring, then carrying out ultrasonic treatment for 30min, and repeatedly stirring after finishing ultrasonic treatment to ensure that the PDA @ rGO is fully dispersed.

And (7) firstly putting the composite ink in the step (6) into a centrifuge at 3000rpm for centrifuging and defoaming for 3min, then transferring into a 10ml syringe, and putting into a refrigerator for precooling for 10min.

And (8) performing 3D printing of the composite ink: loading the syringe filled with the precooled composite ink into a 3D printer; and (4) then, performing 3D printing on the composite ink by adopting the two groups of parameters of the ink proportioning parameter, the printing speed and the extrusion pressure which are researched in the step (3). The rheological test chart of the composite ink is shown in fig. 10.

Step (9), crosslinking of the hydrogel wound auxiliary material: firstly, toCarrying out ultraviolet irradiation treatment on the extruded and printed hydrogel at a distance of 10cm from an ultraviolet lamp for 240s, and then taking down the photo-crosslinked hydrogel and soaking the hydrogel in CaCl with the volume mass fraction of 3% ₂ Soaking in the solution for 5min, and taking out; then, the sample was soaked in PBS buffer for 15min and then taken out.

Step (10), photo-thermal antibacterial of the wound dressing: using a near infrared laser of 808nm at a wavelength of 0.7w/cm ² The power density of (a) was used to irradiate the dressing for 10min, the temperature change was recorded, and the photothermal antimicrobial effect was evaluated by plate counting.

Example 2

Step (1) preparation of GelMA: 10g of gelatin was sufficiently dissolved in 100ml of PBS buffer (until no particles were visible to the naked eye) at 50 ℃, then 8ml of methacrylic anhydride was slowly added dropwise at a rate of one drop per second, and after completion of the addition, the reaction was carried out at a temperature of 50 ℃ for 2 hours with a stirring speed of 240 rpm. After the reaction, 100ml of PBS buffer was added to dilute the reaction solution, and the mixture was sufficiently stirred for 10min. And (3) filling the product into a 10000 dialysis bag with the molecular weight cutoff after the reaction is finished, dialyzing for 5d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished.

Step (2) preparation of GelMA/TO-CNF composite ink: firstly, dissolving a photoinitiator 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) (with the volume mass percent of 0.05%) in deionized water at 80 ℃, and then respectively taking TO-CNF and GelMA in 1ml of deionized water added with the photoinitiator TO prepare GelMA/TO-CNF composite ink, wherein the volume mass fraction of the TO-CNF in the composite ink is 1-3%, and the volume mass fraction of the GelMA in the composite ink is 10-30%.

Screening printable parameters: firstly, placing the composite ink prepared in the step 2 into a centrifuge tube, centrifuging for 3min at the rotating speed of 3000rpm by using a centrifuge, then taking out the centrifuge tube, transferring the ink into an injector, precooling for 5min by using a refrigerator, then taking out, and placing into an extrusion type 3D printer. The printed pattern was 10mm by 10mm mesh squares. And screening printing parameters of the prepared composite ink, and determining the printable ink concentration, the printable speed and the printable pressure.

Step (4) preparation of GelMA-DA: dissolving 0.84g GelMA in 20ml PBS buffer solution, adjusting pH to 5.5, heating to 50 deg.C for dissolving completely; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.4g dopamine hydrochloride, reacting at 37 deg.C for 24h, and reacting for N ₂ And (4) protecting the dopamine hydrochloride from being oxidized. Dialyzing for 3d under acidic condition after the reaction is finished, and freeze-drying and storing.

Step (5) preparation of PDA @ rGO: adding 20mg of graphene oxide and 20mg of dopamine hydrochloride into 200ml of Tris buffer solution, and carrying out ultrasonic treatment for 30min; then heating the solution in water bath at 60 ℃, and violently stirring for reacting for 24 hours; after the reaction is finished, the reaction is respectively washed by absolute ethyl alcohol and deionized water for a plurality of times to stop the reaction, and the product is washed and fully dried.

Step (9), crosslinking of the hydrogel wound auxiliary material: firstly, carrying out ultraviolet irradiation treatment on the extruded and printed hydrogel at a distance of 10cm from an ultraviolet lamp for 240s, then taking down the photo-crosslinked hydrogel and soaking the hydrogel in CaCl with the volume mass fraction of 3% ₂ Soaking in the solution for 5min, and taking out; then, the sample was soaked in PBS buffer for 15min and then taken out.

This example differs from example 1 in that: in the embodiment, gelMA with higher substitution degree is prepared, and GelMA and TO-CNF with higher density are adopted as printing parameters for printing.

Example 3

Step (1) GelMA preparation: 10g of gelatin was sufficiently dissolved in 100ml of PBS buffer (until no particles were visible to the naked eye) at 50 ℃, 4ml of methacrylic anhydride was slowly added dropwise at a rate of one drop per second, and after completion of the addition, the reaction was carried out at a temperature of 50 ℃ for 2 hours with a stirring speed of 240 rpm. After the reaction, 100ml of PBS buffer was added to dilute the reaction solution, and the mixture was stirred well for 10min. And (3) filling the product into a 10000 dialysis bag with the molecular weight cutoff after the reaction is finished, dialyzing for 5d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished.

Step (2) preparation of GelMA/TO-CNF composite ink: firstly, a photoinitiator 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) (with the volume mass fraction of 0.5%) is dissolved in deionized water at 80 ℃, and then TO-CNF and GelMA are respectively taken and added into 1ml of deionized water with the photoinitiator TO prepare GelMA/TO-CNF composite ink, wherein the volume mass fraction of the TO-CNF in the composite ink is 0-1.5%, and the volume mass fraction of the GelMA is 5-15%.

Step (4) preparation of GelMA-DA: dissolving 0.84g GelMA in 20ml PBS buffer solution, adjusting pH to 5.5, heating to 50 deg.C for dissolving completely; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.8g dopamine hydrochloride, reacting at 37 deg.C for 24h, and reacting for N ₂ And the dopamine hydrochloride is protected and prevented from being oxidized. Dialyzing for 3d under acidic condition after the reaction is finished, and freeze-drying and storing.

Step (5) preparation of PDA @ rGO: adding 20mg of graphene oxide and 20mg of dopamine hydrochloride into 200ml of Tris buffer solution, and carrying out ultrasonic treatment for 30min; then heating the solution in water bath at 60 ℃, and violently stirring for reacting for 24 hours; after the reaction is finished, the reaction is stopped by respectively washing the reaction kettle with absolute ethyl alcohol and deionized water for a plurality of times, and the product is washed and fully dried.

Step (6) preparation of GelMA/TO-CNF/GelMA-DA/PDA @ rGO composite ink: adding GelMA-DA (20%) and PDA @ rGO (4 mg/ml) on the basis of the GelMA/TO-CNF composite ink in the step (2); after adding, heating to 50 ℃ and fully stirring, then carrying out ultrasonic treatment for 30min, and repeating stirring after finishing ultrasonic treatment to fully disperse PDA @ rGO.

And (8) performing 3D printing of the composite ink: loading the syringe filled with the precooled composite ink into a 3D printer; and (4) then, performing 3D printing on the composite ink by adopting the two groups of parameters of the ink proportioning parameter, the printing speed and the extrusion pressure which are researched in the step (3).

And (9) crosslinking of the hydrogel wound auxiliary material: firstly, carrying out ultraviolet irradiation treatment on the extruded and printed hydrogel at a distance of 10cm from an ultraviolet lamp for 240s, then taking down the photo-crosslinked hydrogel and soaking the hydrogel in CaCl with the volume mass fraction of 3% ₂ Soaking in the solution for 5min, and taking out; then, the sample was soaked in PBS buffer for 15min and then taken out.

Step (10), photo-thermal antibacterial of the wound dressing: using a near infrared laser of 808nm at a wavelength of 0.7w/cm ² The dressing was irradiated for 10min at power density, the temperature change was recorded, and the photothermal antimicrobial effect was evaluated by plate counting.

This example differs from example 1 in that: this example produced GelMA-DA having a higher degree of substitution and its concentration in the composite ink increased from 10% to 20%, providing a higher adhesion to the material.

Example 4

Step (1) preparation of GelMA: 10g of gelatin was dissolved in 100ml of PBS buffer (until no visible particles were observed) at 50 ℃, 4ml of methacrylic anhydride was slowly added dropwise at a rate of one drop per second, and after completion of the addition, the reaction was carried out at 50 ℃ for 2 hours with a stirring speed of 240 rpm. After the reaction, 100ml of PBS buffer was added to dilute the reaction solution, and the mixture was stirred well for 10min. And filling the product into a 10000-molecular-weight cut-off dialysis bag after the reaction is finished, dialyzing for 5d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished.

Step (2) preparation of GelMA/TO-CNF composite ink: firstly, a photoinitiator 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone (Irgacure 2959) (the volume mass percent is 0.5%) is dissolved in deionized water at 80 ℃, then TO-CNF and GelMA are respectively taken TO be added into 1ml of deionized water with the photoinitiator TO prepare GelMA/TO-CNF composite ink, the volume mass fraction of the TO-CNF in the composite ink is 0-1.5%, and the volume mass fraction of the GelMA is 5-15%.

Step (3) screening printable parameters: firstly, placing the composite ink prepared in the step 2 into a centrifuge tube, centrifuging for 3min at the rotating speed of 3000rpm by using a centrifuge, then taking out the centrifuge tube, transferring the ink into an injector, precooling for 5min by using a refrigerator, then taking out, and placing into an extrusion type 3D printer. The printed pattern was 10mm by 10mm mesh squares. And screening printing parameters of the prepared composite ink, and determining the printable ink concentration, the printable speed and the printable pressure.

Step (4) preparation of GelMA-DA: dissolving 0.84g GelMA in 20ml PBS buffer solution, adjusting pH to 5.5, heating to 50 deg.C for dissolving completely; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.4g dopamine hydrochloride, reacting at 37 deg.C for 24h, and reacting for N ₂ And the dopamine hydrochloride is protected and prevented from being oxidized. Dialyzing for 3d under acidic condition after the reaction is finished, and freeze-drying and storing.

Step (6) preparation of GelMA/TO-CNF/GelMA-DA/PDA @ rGO composite ink: adding GelMA-DA (10%) and PDA @ rGO (8 mg/ml) on the basis of the GelMA/TO-CNF composite ink in the step (2); heating to 50 ℃ after adding, fully stirring, then carrying out ultrasonic treatment for 30min, and repeatedly stirring after finishing ultrasonic treatment to ensure that the PDA @ rGO is fully dispersed.

Step (10), photo-thermal antibacterial of the wound dressing: using a near infrared laser at 808nm, respectively at 1.2w/cm ² The power density of (a) was used to irradiate the dressing for 10min, the temperature change was recorded, and the photothermal antimicrobial effect was evaluated by plate counting.

This example differs from example 1 in that: in the embodiment, the concentration of PDA @ rGO is increased to 8mg/ml, and the power density of a 808nm laser is increased to 1.2w/cm ² 。

Claims

1. A method of 3D printing a functional hydrogel wound dressing, characterized in that the method comprises the steps of:

step (1) preparing a printing ink containing 0 to 3% of TO-CNF, 5 to 30% of GelMA and 0.5% of photoinitiator by volume mass percentage;

step (2) screening printing parameters of the ink obtained in the step (1) to determine the concentration and the printing parameters of the printable ink;

step (7) UV crosslinking and Ca treatment of the hydrogel printed in step (6) ²⁺ Complexing and crosslinking;

and (8) performing photothermal antibiosis on the wound dressing crosslinked in the step (7).

2. The method of 3D printing a functional hydrogel wound dressing according to claim 1, characterized in that the method of screening the printing parameters is: and (3) screening the printing parameters by adopting a 21G needle head according TO the TO-CNF/GelMA concentration, the extrusion speed and the pressure respectively, and taking the structural integrity of the printed product as the available printing parameters.

3. The method of 3D printing a functional hydrogel wound dressing according to claim 1, wherein the printable ink concentrations are: 0% of TO-CNF, 15% of GelMA and 0.5% of photoinitiator; 0.5 percent of TO-CNF, 10 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1 percent of TO-CNF, 5 TO 15 percent of GelMA and 0.5 percent of photoinitiator, 1.5 percent of TO-CNF, 5 TO 10 percent of GelMA and 0.5 percent of photoinitiator.

4. The method of 3D printing a functional hydrogel wound dressing of claim 1, wherein the printer parameters are: the extrusion distance of the spray head is 0.05mm, and the moving speed of the spray head is 1.5-3.17 mm/s; the extrusion distance of the spray head is 0.2mm, and the moving speed of the spray head is 3.17mm/s; the extrusion distance of the spray head is 0.35mm, and the moving speed of the spray head is 4.83mm/s.

5. The method of 3D printing of a functional hydrogel wound dressing according to claim 1 or 3, characterized in that the photoinitiator is 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone or phenyl-2,4,6-trimethylbenzoyllithium phosphonate.

6. The method of 3D printing a functional hydrogel wound dressing according to claim 1, characterized in that the GelMA-DA is prepared as follows: dissolving 0.84g of GelMA in 20ml of PBS buffer solution, adjusting the pH value to be between 5 and 6, and heating to 50 ℃ for full dissolution; then adding 0.2g EDC and 0.12g NHS, reacting for 30min, adding 0.4-0.8 g dopamine hydrochloride, reacting for 12-24 h at 37 ℃, and N in the whole process ₂ Protection; after the reaction, dialyzed for 3 days under acidic condition, and stored by freeze drying.

7. The method of 3D printing a functional hydrogel wound dressing according to claim 6, wherein the GelMA is prepared as follows: fully dissolving 10g of gelatin in 100ml of PBS buffer solution at 50 ℃, slowly dripping 4-8 ml of methacrylic anhydride at a speed of one drop per second, and reacting at the temperature of 50 ℃ for 2 hours at a stirring speed of 240 rpm; after the reaction is finished, adding 100ml of PBS buffer solution for dilution, and fully stirring for 10min; and filling the product into a 10000-molecular cut-off dialysis bag after the reaction is finished, dialyzing for 5-7 d at 40 ℃, changing water twice every day, and freeze-drying and storing after the dialysis is finished.

8. The method of 3D printing a functional hydrogel wound dressing according to claim 1, characterized in that the pda @ rgo is prepared as follows: adding 10-40 mg of graphene oxide and 20mg of dopamine hydrochloride into 200ml of Tris buffer solution, and carrying out ultrasonic treatment for 30min; then heating the solution in water bath at 60 ℃, and violently stirring for reacting for 18-24 h; after the reaction is finished, the reaction is respectively washed by absolute ethyl alcohol and deionized water for a plurality of times to stop the reaction, and the product is washed and fully dried.

9. The method of 3-D printing a functional hydrogel wound dressing according to claim 1, characterized in that the UV cross-linking and Ca ²⁺ The specific steps of complex crosslinking are as follows: firstly, carrying out ultraviolet irradiation treatment on hydrogel at a distance of 5-15 cm from an ultraviolet lamp for 200-300 s, then taking down the photo-crosslinked hydrogel and soaking the hydrogel in CaCl with the volume mass fraction of 1-3% ₂ Soaking in the solution for 2-10 min and taking out; then soaking in PBS buffer solution for 10-15 min, and taking out.

10. The method of 3D printing a functional hydrogel wound dressing according to claim 1, characterized in that the specific steps of the photothermal antimicrobial are as follows: adopting a near infrared laser with the wavelength of 808nm and the wavelength of 0.6 to 1.2w/cm ² The power density of (2) is used for irradiating the wound dressing for 5-15 min.