CN109731130B - Method for preparing hydrogel wound dressing by low-temperature biological 3D printing technology - Google Patents
Method for preparing hydrogel wound dressing by low-temperature biological 3D printing technology Download PDFInfo
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Abstract
The invention provides a method for preparing a hydrogel wound dressing by adopting a low-temperature biological 3D printing technology, which comprises the following steps of: (1) preparing a sodium alginate/gelatin composite hydrogel matrix material; (2) loading vascular endothelial growth factor VEGF and antibacterial peptide LL-37 into matrix materials respectively; (3) preparing the wound dressing with the three-dimensional structure by using a multi-nozzle low-temperature biological 3D printing technology; (4) the wound dressing with a stable three-dimensional structure is obtained by a solidification process and a freeze drying technology. The invention has the following advantages that (1) the invention has good functions of promoting granulation tissue regeneration and antibiosis. (2) The controllable space and concentration distribution of the biological factors are realized, the whole dressing preparation process is mild, and the activity of the biological factors is ensured. (3) The final product is in a dry stable state and is easy to store and transport for a long time.
Description
Technical Field
The invention relates to the field of medical products, in particular to a preparation method of a low-temperature biological 3D printing hydrogel wound dressing.
Background
Extensive and severe skin soft tissue trauma due to trauma, burns, disease, etc., is a formidable clinical problem. Skin flap technology is currently the main treatment in the clinic. However, the application of flap technology is limited due to the lack of donor areas, high price, additional surgical risk, and surgical pain. In recent years, it has become a promising wound treatment strategy to use artificial wound dressing to assist and even replace skin flap.
At present, some 3D printing dressing products exist, for example, a 3D bioprinted medical dressing disclosed in patent No. CN105031713A and a preparation method thereof are used to prepare a personalized medical dressing with good air permeability and water absorbability for patients, but the bone repair scaffold prepared by 3D printing at present has a single function and no antibacterial property, and is often easily infected by bacteria in application to cause a series of inflammations and complications.
Also for example, patent No. CN106729988A discloses a 3D printed bone repair scaffold with antibacterial property and a preparation method thereof, the scaffold is a multilayer columnar structure with a good three-dimensional pore structure, and is composed of polycaprolactone, polydopamine and antibacterial peptide LL 37. The bone repair scaffold has good biocompatibility and antibacterial capacity, good osteogenesis capacity and bone conduction capacity, and has the effect of promoting the growth of new bone tissues on bone defect parts. The repair bracket mainly aims at bone repair, has poor regeneration effect on exposed wound granulation tissues, has high dispersion speed of effective components, has short interval time for replacing the bracket, increases treatment cost and aggravates nursing tasks.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of a novel hydrogel wound dressing which is based on a multi-nozzle low-temperature biological 3D printing technology, takes a hydrogel material as a base material, loads bioactive factors and has a controllable macro-micro topological structure.
In order to realize the purpose of the invention, the technical scheme is as follows:
a preparation method of a low-temperature biological 3D printing hydrogel wound dressing comprises the following steps: the preparation method comprises the following steps of (1) preparing a sodium alginate/gelatin composite hydrogel matrix material: dissolving sodium alginate with the mass fraction of 2-15% in deionized water, swelling for 6 hours at normal temperature by water absorption, and fully and mechanically stirring for 30 minutes; dissolving gelatin with the mass fraction of 2% -15% in deionized water, and electromagnetically stirring for 30 minutes at 40 ℃; and finally, blending the sodium alginate solution and the gelatin solution according to the mass ratio of 1: 5-5: 1, and fully stirring at 40 ℃ until a uniform blended solution is obtained.
(2) Compounding the biological factor with the hydrogel matrix material: VEGF and LL-37 solutions with appropriate concentrations are respectively added into the sodium alginate/gelatin composite hydrogel matrix material, and stirred for 5-10 minutes at 37 ℃, and finally, hydrogel solutions loaded with 1-20 mu g/mL VEGF and 50-300 mu g/mL LL-37 are respectively obtained.
(3) Preparing the wound dressing based on a low-temperature biological 3D printing technology: the VEGF-and LL-37-loaded matrix materials are respectively loaded into different 3D printing material cavities, and the wound dressing with controllable macro-micro porous topological structure and biological factor space customized distribution is designed and printed by multi-nozzle biological 3D printing equipment. Preferably, the printing needle head is 20-27G, the temperature of the material cavity is 25-37 ℃, the ambient temperature is 15-30 ℃, the temperature of the low-temperature forming platform is-10 ℃ to-50 ℃, the relative humidity is 35-65%, the air pressure of compressed air is 10-150 kPa, the movement speed of the three-dimensional platform is 3-20 mm/s, and the height of the printing layer is 100-500 mu m.
(4) The post-treatment process of the wound dressing comprises the following steps: freezing the printed wound dressing on a horse in a refrigerator at the temperature of minus 20-minus 25 ℃ for 1-3 hours, and drying for at least 10 hours by a freeze drying technology; then, soaking the dried hydrogel dressing in a solution containing 0.2-2% by mass of carbodiimide (EDC), 0.2-2% by mass of N-hydroxysuccinimide (NHS) and 0.5-5% by mass of calcium chloride (CaCl)2) The curing solution is used for 10 to 20 minutes; and (3) cleaning the solidified dressing with distilled water, and freeze-drying for 24 hours by using a freeze-drying technology to obtain the final dried dressing.
The prepared hydrogel wound dressing comprises a plurality of functional layers, wherein each functional layer comprises a VEGF-loaded hydrogel layer or an LL-37-loaded hydrogel layer.
Preferably, the uppermost functional layer and the lowermost functional layer are each a hydrogel layer loaded with LL-37.
Preferably, the resulting dried dressing is soaked in physiological saline or a balanced salt solution for 3 minutes to return the gel from the freeze-dried state to the hydrogel state, and the use state is switched as needed.
The hydrogel is a three-dimensional network structure material formed by macromolecule crosslinking, and is an ideal material in the field of wound regeneration and repair because the hydrogel contains a large amount of water, is soft and similar to biological tissues, and has the advantages of excellent biocompatibility, degradability, absorption and the like. However, it is difficult to satisfy the complicated requirements of the regenerative medicine field simultaneously with a single hydrogel material. Therefore, the composite hydrogel material with excellent mechanical and biological properties is an ideal wound dressing base material.
The sodium alginate is a byproduct obtained after extracting iodine and mannitol from brown algae such as kelp or gulfweed, is a natural polysaccharide, and has good moisture retention and biomechanical properties; the gelatin, as a deformation product of collagen, has excellent biocompatibility, and has the advantages of hemostasis function, promotion of wound regeneration rate and the like. The sodium alginate and gelatin composite hydrogel can meet the comprehensive requirements of the wound dressing base material on excellent mechanical property and biological property.
VEGF is a mitogen of specific artery and vein and lymphatic endothelial cells, can promote angiogenesis in vitro, and makes the fused vascular endothelial cells invade human collagen matrix to form a capillary-like structure, which has strong angiogenesis capacity and can promote regeneration of granulation tissue; LL-37 is the only known human Cathelicidin antibacterial peptide, has multiple biological properties such as antibiosis, promoting vascularization and the like, can inhibit the formation of bacterial biomembranes by using the micro-gram concentration of LL-37, has little drug resistance generated by LL-37, and is an ideal substitute product and synergist of antibacterial drugs. VEGF and LL-37 are loaded into the sodium alginate/gelatin hydrogel, and the hydrogel material can be used as an excellent carrier of biological factors, so that the biological factors are protected and slowly released.
The macro-micro topology of the material can influence the cell behavior and is an extremely important factor in the field of regenerative medicine. However, due to the limitation of the traditional preparation technology, the hydrogel wound dressing products on the market at present mainly take a film shape, a sheet shape and a paste shape, and have a single structure, which is an important factor of lacking a topological structure. The biological 3D printing technology can print various bioactive materials (including cells and biological factors), particularly the multi-nozzle technology can realize simultaneous printing of various materials, and a biological functional device with complex components and structures is constructed in a customized manner. The wound dressing is prepared by a multi-nozzle biological 3D printing technology, and the concentration and space accurate distribution of functional components such as VEGF, LL-37 and the like can be realized. Moreover, the controllable macro-micro porous structure is beneficial to the migration and proliferation of cells and can accelerate the regeneration rate of the wound surface.
According to the scheme, the obtained bioactive hydrogel wound dressing can be in a dry and stable final state by designing a proper post-treatment process (a curing process and a freeze-drying process), and long-term storage and transportation are facilitated. Meanwhile, in the initial stage of wound surface regeneration and repair, the macro-micro topological structure of dressing design can be maintained.
Compared with the prior art, the 3D printing hydrogel wound dressing provided by the invention has the following advantages:
(1) the wound dressing is loaded with biological factors VEGF and antibacterial peptide LL-37, and has good functions of promoting granulation tissue regeneration and resisting bacteria.
(2) Through the multi-nozzle low-temperature biological 3D printing technology, the wound dressing with the macro-micro controllable topological structure is printed, the controllable space and concentration distribution of biological factors are realized, the whole dressing preparation process is mild, the activity of the biological factors is guaranteed, the designed porous structure can promote the migration and proliferation of cells, and the speed of wound regeneration and repair is accelerated.
(3) The final product is in a dry stable state through the post-treatment processes of solidification and freeze drying, and is easy to store and transport for a long time.
(4) The gel is convenient to use, can be cut at will according to the shape of a wound, and can be recovered to the hydrogel state from the freeze-dried state after being soaked in normal saline or balanced salt solution for 3 minutes.
(5) The requirement for antibiotics during treatment is low, and infection can be effectively prevented even if no antibiotics are used, and the gel can maintain macro-micro topological structure during the treatment, and gradually degrade and release carried functional components along with the growth of granulation tissues.
Drawings
Figure 1 SEM image of hydrogel wound dressing prepared in example one.
Fig. 2 shows the results of the wound healing experiment of the hydrogel wound dressing prepared in the first embodiment.
Fig. 3 shows the cell experiment test results of the hydrogel wound dressing prepared in the first embodiment.
Figure 4 animal test results of hydrogel wound dressing prepared in example one.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
The first embodiment is as follows:
a preparation method of a low-temperature biological 3D printing hydrogel wound dressing comprises the following steps: dissolving 8% of Sodium Alginate (SA) in mass fraction in deionized water, swelling for 6 hours at normal temperature by water absorption, and fully and mechanically stirring for 30 minutes; dissolving Gelatin (GA) with the mass fraction of 8% in deionized water, and electromagnetically stirring for 30 minutes at 40 ℃; and finally, blending the sodium alginate solution and the gelatin solution according to the mass ratio of 1:1, and fully stirring at 40 ℃ until a uniform blended solution containing 4% of SA and 4% of GA is obtained.
(2) Compounding the biological factor with the hydrogel matrix material: adding 1mL of 20 mu g/mL VEGF solution into 9mL of 4% SA/4% GA blending solution, and stirring at 37 ℃ for 5 minutes to finally obtain a 2 mu g/mL VEGF loaded hydrogel matrix material; 1mL of a 2mg/mlL LL-37 solution was added to 9mL of a 4% SA/4% GA blend solution and stirred at 37 ℃ for 5 minutes to finally obtain a 200. mu.g/mL LL-37 loaded hydrogel matrix material.
(3) The VEGF-and LL-37-loaded hydrogel matrix material is respectively filled into two different 3D printing material cavities, preferably a printing needle head 23G, the temperature of the material cavity is 30 ℃, the ambient temperature is 20 ℃, the temperature of a low-temperature forming platform is-20 ℃, the relative humidity is 50%, the air pressure of compressed air is 40kPa, and the movement speed of a three-dimensional platform is 15 mm/s. The wound dressing with controllable macro-micro porous topological structure and biological factor space customized distribution is printed by multi-nozzle biological 3D printing equipment. The dressing has the overall dimension of 15mm multiplied by 1.6mm, and comprises 6 layers, wherein the uppermost layer and the lowermost layer are hydrogel matrix materials loaded with LL-37, the middle layer is hydrogel matrix materials loaded with VEGF, hydrogel filaments between the two adjacent layers are vertically arranged, the height of a printing layer is 400 mu m, and the filament spacing is 600 mu m.
(4) Freezing the printed wound dressing in a refrigerator at-20 ℃ for 3 hours, and drying for 12 hours by a freeze-drying technology; subsequently, the dried hydrogel dressing was soaked in a solution containing 1% by mass of carbodiimide (EDC), 0.25% by mass of N-hydroxysuccinimide (NHS) and 1% by mass of calcium chloride (CaCl) at 4 deg.C2) For 10 minutes; and (3) cleaning the solidified dressing with distilled water, and freeze-drying for 24 hours by using a freeze-drying technology to obtain the final dried dressing.
Example two:
a preparation method of a low-temperature biological 3D printing hydrogel wound dressing comprises the following steps: (1) dissolving Sodium Alginate (SA) with the mass fraction of 2% in deionized water, swelling for 6 hours at normal temperature by water absorption, and fully and mechanically stirring for 30 minutes; dissolving Gelatin (GA) with the mass fraction of 15% in deionized water, and electromagnetically stirring for 30 minutes at 40 ℃; and finally, blending the sodium alginate solution and the gelatin solution according to the mass ratio of 5:1, and fully stirring at 40 ℃ until a uniform blended solution containing 4% of SA and 4% of GA is obtained.
(2) Compounding the biological factor with the hydrogel matrix material: adding 1mL of 20 mu g/mL VEGF solution into 9mL of 4% SA/4% GA blending solution, and stirring at 37 ℃ for 5 minutes to finally obtain a 2 mu g/mL VEGF loaded hydrogel matrix material; 1mL of a 2mg/mL LL-37 solution was added to 9mL of a 4% SA/4% GA blend solution and stirred at 37 ℃ for 5 minutes to finally obtain a 200. mu.g/mL LL-37 loaded hydrogel matrix material.
(3) The VEGF-and LL-37-loaded hydrogel matrix material is respectively filled into two different 3D printing material cavities, preferably a printing needle head of 20G, the temperature of the material cavity is 37 ℃, the ambient temperature is 15 ℃, the temperature of a low-temperature forming platform is-10 ℃, the relative humidity is 35%, the air pressure of compressed air is 100kPa, and the movement speed of a three-dimensional platform is 20 mm/s. The wound dressing with controllable macro-micro porous topological structure and biological factor space customized distribution is printed by multi-nozzle biological 3D printing equipment. The dressing has the overall dimension of 15mm multiplied by 3.2mm, and comprises 8 layers, wherein the uppermost layer and the lowermost layer are hydrogel matrix materials loaded with LL-37, the middle four layers are hydrogel matrix materials loaded with VEGF, hydrogel filaments between the two adjacent layers are vertically arranged, the height of a printing layer is 500 mu m, and the filament spacing is 600 mu m.
(4) Freezing the printed wound dressing in a refrigerator at-25 ℃ for 1 hour, and drying for 10 hours by a freeze-drying technology; subsequently, the dried hydrogel dressing was soaked in a solution containing 0.2% by mass of carbodiimide (EDC), 2% by mass of N-hydroxysuccinimide (NHS) and 5% by mass of calcium chloride (CaCl) at 4 deg.C2) For 20 minutes; and (3) cleaning the solidified dressing with distilled water, and freeze-drying for 24 hours by using a freeze-drying technology to obtain the final dried dressing.
Example three:
a preparation method of a low-temperature biological 3D printing hydrogel wound dressing comprises the following steps: (1) dissolving Sodium Alginate (SA) with the mass fraction of 2% in deionized water, swelling for 6 hours at normal temperature by water absorption, and fully and mechanically stirring for 30 minutes; dissolving Gelatin (GA) with the mass fraction of 15% in deionized water, and electromagnetically stirring for 30 minutes at 40 ℃; and finally, blending the sodium alginate solution and the gelatin solution according to the mass ratio of 5:1, and fully stirring at 40 ℃ until a uniform blended solution containing 4% of SA and 4% of GA is obtained.
(2) Compounding the biological factor with the hydrogel matrix material: adding 1mL of 20 mu g/mL VEGF solution into 9mL of 4% SA/4% GA blending solution, and stirring at 37 ℃ for 5 minutes to finally obtain a 2 mu g/mL VEGF loaded hydrogel matrix material; 1mL of a 2mg/mL LL-37 solution was added to 9mL of a 4% SA/4% GA blend solution and stirred at 37 ℃ for 5 minutes to finally obtain a 200. mu.g/mL LL-37 loaded hydrogel matrix material.
(3) The VEGF-and LL-37-loaded hydrogel matrix material is respectively filled into two different 3D printing material cavities, preferably a printing needle 27G, the temperature of the material cavity is 25 ℃, the ambient temperature is 30 ℃, the temperature of a low-temperature forming platform is-10 ℃, the relative humidity is 65%, the air pressure of compressed air is 100kPa, and the movement speed of the three-dimensional platform is 3 mm/s. The wound dressing with controllable macro-micro porous topological structure and biological factor space customized distribution is printed by multi-nozzle biological 3D printing equipment. The dressing has the overall dimension of 15mm multiplied by 3.2mm, and comprises 8 layers, wherein the uppermost layer and the lowermost layer are hydrogel matrix materials loaded with LL-37, the middle four layers are hydrogel matrix materials loaded with VEGF, hydrogel filaments between the two adjacent layers are vertically arranged, the height of a printing layer is 500 mu m, and the filament spacing is 600 mu m.
(4) Freezing the printed wound dressing in a refrigerator at-20 ℃ for 2 hours, and drying for 12 hours by a freeze-drying technology; subsequently, the dried hydrogel dressing was soaked in a solution containing 2% by mass of carbodiimide (EDC), 0.2% by mass of N-hydroxysuccinimide (NHS) and 0.5% by mass of calcium chloride (CaCl) at 4 deg.C2) For 20 minutes; and (3) cleaning the solidified dressing with distilled water, and freeze-drying for 24 hours by using a freeze-drying technology to obtain the final dried dressing.
SEM image of hydrogel wound dressing prepared in example one: (a) and (3) a top view of the dressing, wherein hydrogel forming filaments are vertically arranged between two adjacent layers, and the distance between the adjacent filaments in each layer is 600 microns. (b) The side view of the dressing, the top and bottom two layers are LL-37 loaded hydrogel matrix material, the middle four layers are VEGF loaded hydrogel matrix material, eight layers, and the height of the printing layer is set to be 400 μm. The final overall dimensions of the dressing are 15mm by 3.2 mm.
Wound healing tests were performed on the dressing obtained in example one, and the test methods and results were as follows:
1. anaesthetizing rabbits: the rabbits were anesthetized with seralazine hydrochloride (0.1-0.2 mL/Kg).
2. Shaving hairs: hair was shaved off from the rabbit cranium using electric hair clippers, followed by epilation treatment with 4% (w: v) H2S.
3. And (3) disinfection: disinfecting with 5% iodophor from inside to outside for three times.
4. Constructing the exposed wound surface of the bone: the skin wound surface with the size of 1.0cm x 1.0cm is cut by adopting an ophthalmologic operation scissors, and a sharp blade cuts open periosteum and gradually separates to form the exposed wound surface of the skull bone of the rabbit.
5. Gel dressing change: covering the prepared 3D printed active hydrogel dressing on the wound surface, covering the wound with sterile vaseline gauze slightly larger than the wound surface, suturing with skin to form fixation, covering and fixing the wound with common gauze blocks by the same method on the outer layer, and replacing the vaseline gauze with the replacement liquid in the blank group as a control.
6. And (3) observation of curative effect: dressing change is carried out on the wound every three days, the wound healing condition is observed, and the wound healing rate is calculated. The results are shown in FIG. 2.
The hydrogel wound dressings prepared in example one were subjected to cell culture tests, all using self-extracted rat skin fibroblasts (NSFs). The results are shown in FIG. 3: (a) 24h DAPI cell adhesion staining diagram, and the result proves that the prepared hydrogel wound dressing has good adhesion effect on NSFs; (b) after 3 days of culture, the result proves that the NSFs can migrate into the hydrogel dressing through the designed macroscopic holes, so that the cell proliferation speed is accelerated; (c-d) SEM pictures after culturing for seven days, and the result proves that the NSFs have a very good spreading form on the surface of the wound dressing. All cell test results prove that the hydrogel wound dressing prepared in the first example has excellent biological performance.
The test results of the hydrogel wound dressing prepared in the first example are shown in the attached figure 4: (a) the hydrogel wound dressing is in a dry state before use, can be directly pasted on an injured wound after being soaked in normal saline for five minutes after being unpacked, and is convenient to use. (b) A full-thickness skin defect model (with the size of 15mm multiplied by 15 mm) is established at the top of the head of a rabbit (skull periosteum is scraped), and comparison shows that the regeneration and repair speed of the wound surface of the gel dressing group is obviously higher than that of a control group, so that the hydrogel wound dressing prepared in the first embodiment has a good effect of promoting the regeneration and repair of the skin wound surface.
Claims (7)
1. The application of the hydrogel wound dressing prepared by the low-temperature biological 3D printing technology in preparation of a material for repairing exposed wound of a bone is characterized in that the hydrogel wound dressing comprises six functional layers, the uppermost layer and the lowermost layer are hydrogel matrix materials loaded with LL-37, the middle layer is a hydrogel matrix material loaded with VEGF, and hydrogel filaments between the two adjacent layers are vertically arranged; the preparation method of the hydrogel wound dressing comprises the following steps:
(1) the preparation of the sodium alginate/gelatin composite hydrogel matrix material comprises the following steps: dissolving sodium alginate in deionized water, and mechanically stirring; dissolving gelatin in deionized water; finally, blending the sodium alginate solution and the gelatin solution until a uniform blended solution is obtained, so as to obtain a hydrogel matrix material;
(2) compounding the biological factor with the hydrogel matrix material: respectively adding VEGF and LL-37 solutions into a hydrogel matrix material, and stirring at 37 ℃ for 5-10 minutes to respectively obtain 1-20 mu g/mL VEGF loaded hydrogel solutions and 50-300 mu g/mL LL-37 loaded hydrogel solutions;
(3) preparing the wound dressing based on a low-temperature biological 3D printing technology: respectively loading the VEGF-and LL-37-loaded matrix materials into different 3D printing material cavities, and printing wound dressing through multi-nozzle biological 3D printing equipment;
(4) the post-treatment process of the wound dressing comprises the following steps: freezing the printed wound dressing on a horse in a refrigerator at the temperature of minus 20-minus 25 ℃ for 1-3 hours, and drying for at least 10 hours by a freeze drying technology; then, soaking the dried wound dressing in a curing solution for 10-20 minutes; and (3) cleaning the cured wound dressing with distilled water, and freeze-drying for 24 hours by using a freeze-drying technology to obtain the final dried dressing.
2. The application of claim 1, wherein the sodium alginate solution and the gelatin solution are blended according to a mass ratio of 1: 5-5: 1.
3. The application of claim 1, wherein the mass fraction of sodium alginate is 2% -15%.
4. The use according to claim 1, wherein the mass fraction of gelatin is 2% to 15%.
5. The use of claim 1, wherein the sodium alginate is dissolved in deionized water, swollen with water at room temperature for 6 hours, and then stirred.
6. The application of the printing needle head as claimed in claim 1, wherein the printing needle head of the multi-nozzle biological 3D printing equipment is 20G-27G, the temperature of a material cavity is 25-37 ℃, the ambient temperature is 15-30 ℃, the temperature of a low-temperature forming platform is-10 ℃ to-50 ℃, the relative humidity is 35-65%, the air pressure of compressed air is 10-150 kPa, the movement speed of a three-dimensional platform is 3-20 mm/s, and the height of a printing layer is 100-500 μm.
7. The use according to claim 1, wherein the curing solution comprises 0.2 to 2 mass% of carbodiimide, 0.2 to 2 mass% of N-hydroxysuccinimide and 0.5 to 5 mass% of calcium chloride.
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