Composite bio-ink for constructing tissue engineering skin through 3D printing as well as preparation method and application of composite bio-ink

Abstract

The invention relates to the technical field of material preparation, in particular to composite bio-ink for constructing tissue engineering skin through 3D printing and a preparation method and application of the composite bio-ink. The oxidized sodium alginate-gelatin bio-ink forms a Schiff base network through oxidized sodium alginate and gelatin to complete gelation. The preparation method comprises the following steps: performing in-situ reduction on a # imgabs0 # solution through polydopamine to obtain an AgNPs colloidal solution; the composite bio-ink provided by the invention has good printability and mechanical properties, and can be used for 3D bio-printing; the prepared scaffold has excellent biocompatibility, also has the functions of self-healing, resisting bacteria and promoting proliferation and differentiation of skin fibroblasts, can be used for preparing tissue engineering skin scaffolds and promoting wound healing and functional reconstruction of skin tissues, and has a relatively high application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of material preparation, and in particular to a composite bio-ink for 3D printing to construct tissue-engineered skin, and a preparation method and application thereof. Background Art

[0002] As the largest organ in the human body, the skin plays a vital role in maintaining a stable internal environment and protecting against external stimuli and pathogenic invasion. Medical research has shown that full-thickness skin defects larger than 4 cm in diameter cannot heal on their own and require skin transplantation for optimal recovery. For patients with skin defects caused by burns, soft tissue necrosis, tumor resection, and other injuries, the only clinical option has long been autologous skin transplantation due to the risks of immune rejection and genetic infection. This "rob Peter to pay Paul" approach not only limits the donor area but also poses secondary trauma and infection risks to the patient. Therefore, the demand for skin substitutes is extremely urgent.

[0003] The emergence of tissue engineering technology has provided new treatment options for patients with large-area skin defects, and the rise of 3D bioprinting has further spurred its development. 3D bioprinting offers significant advantages over traditional tissue engineering scaffold fabrication methods. It can fabricate tissue engineering scaffolds of any complex shape and structure from scratch, ignoring time and space constraints. Furthermore, precise assembly of various biomaterials can be achieved through effective control of scaffold porosity, fill rate, and pore size. 3D bioprinting requires the use of bioinks, which provide a growth and reproduction environment for cells and active substances during the construction of three-dimensional structures. For example, patent publication number CN119367606A discloses "A Double-Crosslinked Collagen DLP 3D Printing Bioink, Preparation Method, and Application." This invention utilizes a double-crosslinking mechanism to create a collagen bioink with excellent fluidity and photocuring properties. DLP 3D printing can produce hydrogels with excellent mechanical properties, fidelity, and degradation resistance, while addressing the cytotoxicity issues associated with photoinitiators in conventional photocuring printing. However, this bio-ink lacks antimicrobial properties and can easily breed bacteria and cause inflammation over extended use, making it unsuitable for wounds prone to infection and requiring cleanliness. Patent Publication No. CN117205367A discloses a "dialdehyde cellulose / gelatin composite bio-ink, its preparation method, and application." This invention accelerates gelation and improves reaction efficiency by adjusting the reaction mass ratio of gelatin and dialdehyde cellulose and adjusting the pH, while maintaining the mechanical properties of the prepared bio-ink. By using dialdehyde cellulose, a natural crosslinker, this invention avoids the toxic residues produced by chemical crosslinkers. However, the maximum tensile strength of this bio-ink is only approximately 10 kPa, which is insufficient for certain wound sites and has limited application. Patent Publication No. CN115887772A discloses a "gelatin / sodium alginate hydrogel-based 3D printing bio-ink and its application." This invention improves the mechanical properties of 3D printed scaffolds by separately modifying gelatin and sodium alginate and then combining photocatalytic redox crosslinking with metal ion-mediated ionic crosslinking to create a dual crosslinking system. However, this bio-ink does not enhance biological functions such as antibacterial and self-healing. At the same time, the chemical photoinitiators used will introduce certain cytotoxicity, which may have a negative impact on cell proliferation and differentiation.

[0004] It can be seen that the current bio-ink has a relatively single function, and the tissue engineering skin scaffold prepared cannot match the complex patient environment, which ultimately affects the patient's healing effect. Summary of the Invention

[0005] In response to the problems raised in the above background technology that the current bio-ink is not comprehensive in function and has limited adaptability scenarios, the present invention provides a composite bio-ink for 3D printing to construct tissue-engineered skin, as well as its preparation method and application. The components of the bio-ink are non-toxic and have no side effects. It is used to prepare 3D printed hydrogel scaffolds, which have good biocompatibility and stability, and do not have immune rejection and cytotoxicity. It can be used as a skin repair and regeneration material in skin tissue engineering.

[0006] The present invention is specifically achieved through the following technical solutions.

[0007] The present invention provides a method for preparing a composite bio-ink for 3D printing to construct tissue-engineered skin, comprising the following steps: Sodium periodate is used as an oxidant and mixed with a sodium alginate aqueous solution to oxidize the sodium alginate, and then post-treated to obtain oxidized sodium alginate.

[0008] Gelatin is added to an aqueous solution of oxidized sodium alginate to obtain a mixture, and the mixture is allowed to stand under alkaline conditions, so that the amino groups of the gelatin react with the aldehyde groups of the oxidized sodium alginate to obtain a mixed solution of oxidized sodium alginate and gelatin.

[0009] Polydopamine solution and The solutions are mixed, and under alkaline conditions, polydopamine reduces silver ions into silver nanoparticles in situ to obtain polydopamine-nanosilver colloid.

[0010] A mixed solution of oxidized sodium alginate-gelatin and polydopamine-nanosilver colloid are mixed to obtain a composite bio-ink.

[0011] Preferably, the concentration of the sodium alginate aqueous solution is 2 wt% to 5 wt%, and the molar ratio of sodium periodate to sodium alginate is 1:1 to 1:1.5, preferably 1:1.2. The conditions for oxidizing the sodium alginate are: stirring at room temperature and in the dark. Post-treatment refers to precipitating the reaction solution with anhydrous ethanol after the reaction, followed by suction filtration, washing, and drying, and repeating the above process multiple times. The volume ratio of the reaction solution to anhydrous ethanol is 1:4.

[0012] Preferably, in the mixture, the concentration of gelatin is 5wt%~10wt%, and the mass ratio of oxidized sodium alginate to gelatin is 1:1~2.

[0013] Preferably, when the Schiff base reaction occurs, the alkaline condition means that the pH is adjusted to 8 to 9 using sodium hydroxide. The sodium hydroxide is added in the form of an aqueous solution with a concentration of 1 mol / L.

[0014] Preferably, in the step of preparing the polydopamine-nanosilver colloid, the alkaline condition refers to adjusting the pH to 8-10 with sodium hydroxide.

[0015] Preferably, The concentration of the solution is 1000mg / L~1500mg / L, the concentration of the polydopamine solution is 400mg / L~800mg / L, The ratio of solution to polydopamine solution is 0.5~2:1.

[0016] Preferably, the concentration of nanosilver in the composite bio-ink is 0.5 to 1 part per ten thousand.

[0017] The present invention provides a composite bio-ink for 3D printing to construct tissue-engineered skin, which is prepared by the above-mentioned preparation method.

[0018] The present invention provides an application of a composite bio-ink for 3D printing and constructing tissue-engineered skin. The 3D printing method comprises the following steps: Place the composite bio-ink into the printing syringe.

[0019] Adjust the printing process parameters of the bioprinter for printing.

[0020] After printing, the obtained three-dimensional scaffold is immersed in In aqueous solution.

[0021] Optimally, the printing process parameters are: needle diameter 0.41mm-0.84mm, print height 0.15mm-0.3mm, layer height 5-10 layers, line spacing 0.4mm-1mm, print speed 1mm / s-10mm / s, and extrusion speed 1mm / s-10mm / s. The temperature during 3D printing is 25°C, and the cooling plate is pre-cooled to 5-10°C.

[0022] The preparation method of PDA solution is as follows: disperse DA in deionized water at room temperature, then add 1 mol / L sodium hydroxide solution, maintain the pH at 9.0, and stir thoroughly for 30 minutes to ensure uniform mixing for later use.

[0023] The present invention provides a composite bio-ink prepared by the above-mentioned preparation method for 3D printing and constructing tissue-engineered skin. The bio-ink provided by the present invention can exhibit different mechanical properties by adjusting the component ratio of OSA and Gel, thereby meeting the needs of wound repair in different skin areas. At the same time, the bio-ink has good biocompatibility and is non-cytotoxic. The 3D bio-hydrogel scaffold prepared using this bio-ink is completely non-toxic, does not have immune rejection reactions, and can promote the proliferation and differentiation of skin fibroblasts. Therefore, the 3D composite bio-ink provided by the present invention can be used as a skin repair material in the field of skin tissue engineering.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The invention uses sodium periodate as an oxidant to oxidize sodium alginate to obtain oxidized sodium alginate; gelatin is added to the aqueous solution of oxidized sodium alginate, and the solution is allowed to stand under alkaline conditions, so that the amino groups of gelatin react with the aldehyde groups of oxidized sodium alginate to obtain a mixed solution of oxidized sodium alginate and gelatin; polydopamine solution and The solutions are mixed. Under alkaline conditions, polydopamine reduces silver ions to silver nanoparticles in situ, resulting in a polydopamine-nanosilver colloid. A mixed solution of sodium alginate-gelatin and polydopamine-nanosilver colloid are then mixed to create a composite bio-ink. This bio-ink combines the advantages of biomaterials and nanoparticles, exhibiting not only excellent biocompatibility and mechanical strength but also inhibiting bacterial growth, promoting skin cell proliferation, adhesion, differentiation, and maturation, and accelerating wound recovery. It meets clinical mechanical and biological requirements, is highly compatible with the complex patient environment, and helps promote wound healing.

[0025] The present invention provides a 3D-printed hydrogel bioscaffold prepared using the method described above, effectively addressing the problem of wound repair in different skin locations. By adjusting the ratio of the hydrogel bioscaffold matrix material, tissue-engineered skin scaffolds with varying mechanical properties can be prepared to meet the needs of wound repair in different skin locations.

[0026] The present invention introduces 3D printing technology and Schiff base cross-linking network to give the skin scaffold a tight pore structure, realizing controllable production of different pore sizes and arrangements, which is beneficial to simulating vascular function, helping cells transport nutrients and eliminate metabolism, and promoting wound healing.

[0027] The present invention reduces the The AgNPs were obtained from the solution, avoiding the agglomeration problem caused by directly adding AgNPs. The prepared tissue-engineered skin scaffold had an antibacterial effect of more than 99% against Gram-positive and Gram-negative bacteria, meeting the needs of clinical treatment of skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the bio-ink preparation process provided by the present invention.

[0029] Figure 2 Schematic diagram of the Schiff base reaction.

[0030] Figure 3 This is a comparison chart of the mechanical tensile strength of different samples in Test Example 1.

[0031] Figure 4 Internal microscopic morphology of different samples in Test Example 2.

[0032] Figure 5This is a diagram showing the proliferation / differentiation of cells in different samples in Test Example 3.

[0033] Figure 6 This is a comparison chart of the antibacterial effects of sample C and sample D in test example 4 at different nanosilver concentrations.

[0034] Figure 7 Schematic diagram of the locations of the sample and control groups implanted into the skin wounds of New Zealand rabbits in Test Example 5; A1, A2, and A3 are sample groups, B1 is a blank control group, and B2 and B3 are autologous skin transplantation controls.

[0035] Figure 8 This is the postoperative observation picture of the New Zealand rabbit skin wound in Test Example 5.

[0036] Figure 9 This is a tissue section image after the sample in Test Example 6 was implanted into the skin wound of a New Zealand rabbit.

[0037] Figure 10 These are actual pictures of sample A, sample B, sample C and sample D. DETAILED DESCRIPTION

[0038] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention is further described below with reference to specific examples and accompanying drawings. However, the examples are not intended to limit the present invention. The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0039] In order to enable bio-ink to be used for repairing skin defects in different parts of the body, the effects of different matrix material ratios on the physical and chemical properties of bio-ink scaffolds were studied, so that it can be directly applied to wounds in different skin parts by adjusting the material ratio.

[0040] The present invention provides a method for preparing a 3D printing composite bio-ink for tissue engineering skin construction, such as Figure 1 As shown, the following steps are included: Sodium periodate is used as an oxidant and mixed with a sodium alginate aqueous solution to oxidize the sodium alginate, and then post-treated to obtain oxidized sodium alginate.

[0041] Gelatin is added to an aqueous solution of oxidized sodium alginate to obtain a mixture, and the mixture is allowed to stand under alkaline conditions, so that the amino groups of the gelatin react with the aldehyde groups of the oxidized sodium alginate to obtain a mixed solution of oxidized sodium alginate and gelatin.

[0042] Polydopamine solution and The solutions are mixed, and under alkaline conditions, polydopamine reduces silver ions into silver nanoparticles in situ to obtain polydopamine-nanosilver colloid.

[0043] A mixed solution of oxidized sodium alginate-gelatin and polydopamine-nanosilver colloid are mixed to obtain a composite bio-ink.

[0044] The present invention provides a 3D printing composite bio-ink for tissue engineering skin construction, comprising oxidized sodium alginate (OSA), gelatin (Gel), polydopamine (PDA), silver nanoparticles (AgNPs) and deionized water; oxidized sodium alginate (OSA) in the bio-ink is a product obtained by oxidative modification of sodium alginate (SA), and has excellent biocompatibility, bioactivity and biodegradability, and is rich in aldehyde groups; gelatin (Gel) has thermosensitive properties and excellent biocompatibility, and its molecular chain is rich in amino groups. Figure 2 As shown, the amino groups of Gel and the aldehyde groups of OSA can undergo a Schiff base reaction in an alkaline environment. This not only enhances the crosslinking mechanical properties, but the Schiff base's self-healing properties also impart self-healing properties to the hydrogel. Damaged skin wounds, exposed to air, are prone to infection, which can delay wound healing. Hydrogels loaded with antimicrobial materials offer strong resistance to bacterial infection and are a promising clinical solution for addressing bacterial infections. Currently, AgNPs have been shown to be one of the most effective antimicrobial materials, but they tend to aggregate during use, reducing their antimicrobial efficacy. To address this, the present invention leverages the self-polymerization of dopamine (DA) in an alkaline environment to form polydopamine (PDA). PDA is used to in situ reduce silver ions, thus avoiding AgNP aggregation. Furthermore, the introduction of PDA provides more cell adhesion sites, enhancing cell activity and promoting wound healing. Therefore, tissue-engineered skin scaffolds prepared using this composite bio-ink possess promising mechanical properties and flexibility, promote cell proliferation and differentiation, and accelerate wound healing, making it a promising option for skin regeneration and reconstruction.

[0045] The present invention is described below by way of specific examples, but is not intended to limit the scope of the present invention. Experimental methods not specifically described herein were performed according to the corresponding commercial specifications, and the instruments, reagents, and consumables used in the examples were all commercially available unless otherwise specified.

[0046] Example 1 (1) SA was dissolved in deionized water and stirred at 40°C until completely dissolved to obtain a 5 wt% SA solution. Sodium periodate was added to the SA solution at a molar ratio of sodium periodate to SA of 1:1.2. The mixture was stirred at room temperature in the dark for 2 hours to obtain a reaction solution containing OSA. The reaction solution containing OSA was precipitated with anhydrous ethanol at a volume ratio of the reaction solution to anhydrous ethanol of 1:4. The mixture was then filtered, washed, and dried, and the process was repeated twice to obtain the product OSA.

[0047] (2) OSA was weighed and dissolved in deionized water. The mixture was stirred thoroughly at 40°C for two hours. Gel was then added and stirred for 1 hour to ensure complete mixing with OSA to prepare a mixture. The Gel content in the mixture was 5 wt % and the mass ratio of OSA to Gel was 1:1.5. 1 mol / L sodium hydroxide solution was added dropwise to the mixture until the pH reached 9. After standing for 30 minutes, the mixture was centrifuged at 3000 rpm for 3 minutes to remove bubbles for subsequent use to obtain an OSA-Gel mixed solution.

[0048] (3) Mix 600 mg / L PDA solution with 1400 mg / L The solution was mixed evenly at a volume ratio of 1:1, 1 mol / L NaOH solution was added, the pH was adjusted to 9, and magnetic stirring was continued for 30 min until the mixture was completely mixed to obtain PDA-AgNPs colloid.

[0049] (4) PDA-AgNPs colloid was added to the mixed solution of OSA-Gel and the AgNPs concentration was adjusted to 1 / 10,000 to obtain a composite bio-ink.

[0050] The composite bio-ink obtained by the above preparation method was placed in a printing syringe and 3D printed through a printing nozzle with a diameter of 0.6 mm to obtain a 3D printed scaffold; wherein the process parameters of the 3D printing process are: printing layer height 0.3 mm, layer height 5 layers, line spacing 1 mm, printing speed 10 mm / s, extrusion speed 10 mm / s; the temperature during the printing process was 25 ° C, and the cooling plate was pre-cooled to 5 ° C. The 3D printed scaffold was immersed in 2 wt% concentration of Cross-linking was performed in the solution to obtain a 3D printed hydrogel scaffold, which was recorded as sample A.

[0051] Example 2 (Gel content is 8wt%) The preparation steps of Example 2 were the same as those of Example 1, except that the content of Gel added in step (2) was 8 wt %. The obtained 3D printed hydrogel scaffold was recorded as Sample B.

[0052] Example 3 (Gel content: 10 wt%) The preparation steps of Example 3 were the same as those of Example 1, except that the content of Gel added in step (2) was 10 wt %. The obtained 3D printed hydrogel scaffold was recorded as Sample C.

[0053] Example 4 (Gel content is 10wt%, AgNPs concentration is 0.5 parts per ten thousand) The preparation steps of Example 4 were the same as those of Example 3, except that the PDA-AgNPs colloid was added in step (4) and the AgNPs concentration was adjusted to 0.5 parts per ten thousand. The resulting 3D printed hydrogel scaffold was designated as Sample D.

[0054] The specific steps are: (1) SA was dissolved in deionized water and stirred at 40°C until completely dissolved to obtain a 5 wt% SA solution. Sodium periodate was added to the SA solution at a molar ratio of sodium periodate to SA of 1:1.2. The mixture was stirred at room temperature in the dark for 2 hours to obtain a reaction solution containing OSA. The reaction solution containing OSA was precipitated with anhydrous ethanol at a volume ratio of the reaction solution to anhydrous ethanol of 1:4. The mixture was then filtered, washed, and dried, and the process was repeated twice to obtain the product OSA.

[0055] (2) OSA was weighed and dissolved in deionized water. The mixture was stirred thoroughly at 40°C for two hours. Gel was then added and stirred for 1 hour to ensure complete mixing with OSA to prepare a mixture. The Gel content in the mixture was 10 wt % and the mass ratio of OSA to Gel was 1:1.5. 1 mol / L sodium hydroxide solution was added dropwise to the mixture until the pH reached 9. After standing for 30 minutes, the mixture was centrifuged at 3000 rpm for 3 minutes to remove bubbles for subsequent use to obtain an OSA-Gel mixed solution.

[0056] (3) Prepare 600 mg / L PDA solution and mix it with 1400 mg / L The solution was mixed evenly at a volume ratio of 1:1, 1 mol / L NaOH solution was added, the pH was adjusted to 9, and magnetic stirring was continued for 30 min until the mixture was completely mixed to obtain PDA-AgNPs colloid.

[0057] (4) PDA-AgNPs colloid was added to the mixed solution of OSA-Gel and the AgNPs concentration was adjusted to 0.5 parts per ten thousand to obtain a composite bio-ink.

[0058] The composite bio-ink obtained by the above preparation method was placed in a printing syringe and 3D printed through a printing nozzle with a diameter of 0.6 mm to obtain a 3D printed scaffold; wherein the process parameters of the 3D printing process are: printing layer height 0.3 mm, layer height 5 layers, line spacing 1 mm, printing speed 10 mm / s, extrusion speed 10 mm / s; the temperature during the printing process was 25 ° C, and the cooling plate was pre-cooled to 5 ° C. The 3D printed scaffold was immersed in 2 wt% concentration of Cross-linking was performed in the solution to obtain a 3D printed hydrogel scaffold, which was recorded as sample D.

[0059] Test Example 1 The tensile strength of samples A, B, and C was tested using a CTM2500 universal material testing machine at a tensile speed of 5 mm / s. The termination condition for the experiment was sample fracture. Figure 3The following graphs show the tensile strength results for Samples A, B, and C. After testing, the tensile strengths of Samples A, B, and C were 11 kPa, 21 kPa, and 15 kPa, respectively. It can be seen that with increasing Gel content, the mechanical tensile properties of the scaffold initially increase and then decrease. This demonstrates that by adjusting the material ratio of the bio-ink, the mechanical properties of the skin scaffold can be modified to suit the needs of wounds in different skin locations.

[0060] Test Example 2 Samples A, B, and C were freeze-dried in a -80°C freeze-drying oven for 72 h to obtain freeze-dried scaffold samples. The sample surfaces were then sprayed with gold using an ion sputtering apparatus, and the internal microstructure of the freeze-dried scaffolds was observed using a scanning electron microscope (MAIA3LMH, TESCAN, Czech Republic). Figure 4 The microstructure of the freeze-dried scaffolds is shown in Figure 2. Testing revealed that the micromorphology of each scaffold exhibited a densely packed pore structure, demonstrating successful crosslinking of the gel and OSA. The pore diameters of each scaffold ranged from 20 to 50 microns. This dense pore structure helps retain moisture on the wound surface, maintaining a moist environment, promoting wound healing and reducing inflammation. It also aids in the transport of nutrients and waste, mimicking the function of blood vessels, helping cells transport nutrients and eliminate metabolic waste, and, to a certain extent, enhancing cell proliferation and migration.

[0061] Test Example 3 The live-dead staining assay was used to examine the effects of Samples A, B, and C on the proliferation and differentiation of skin fibroblasts. A blank control group, containing only culture medium and cells without sample addition, was also established. Skin fibroblasts cultured in six-well plates were stained with Calcein-AM / PI reagent and observed using a fluorescence microscope. Calcein-AM stains live cells, producing green fluorescence, while propidium iodide (PI) stains dead cells, producing red fluorescence. After three days, the live-to-dead cell ratio was assessed to assess the proliferation and differentiation of the skin fibroblasts within the hydrogel scaffolds.

[0062] As a 3D printed biological scaffold, the most important thing is that the cells in the scaffold can maintain a high survival rate. Figure 5 The results show the proliferation and differentiation of fibroblasts in samples A, B, and C. The test results show that the cell survival rate in sample A reached 84%, the cell survival rate in sample B was 91%, and the cell survival rate in sample C was 93%, while the blank control group was 79%. Furthermore, compared with the blank control group, the cells in each sample showed a significant deformation trend, further confirming that the samples promote the differentiation and maturation of fibroblasts.

[0063] Test Example 4 The antibacterial properties of the composite hydrogel against Gram-positive representative bacteria Staphylococcus aureus (S. aureus) and Gram-negative representative bacteria Escherichia coli (E. coli) were tested. 100ul of bacterial suspension was drawn from a 20ml sterile centrifuge tube and then diluted to a concentration of Cfu / ml was kept aside for future use. Samples C and D were sterilized by irradiation with ultraviolet light for 30 min, and then the concentration was 2ul of the bacterial suspension with a cfu / ml content was inoculated onto sample C and sample D, and then transferred to a 37°C constant temperature incubator for incubation for 3 hours. After the incubation period, 1ml of PBS solution was added to the sample using a pipette, and the bacteria on the surface of the sample were rinsed into the PBS solution. Subsequently, 100ul of PBS solution was aspirated and transferred to a culture dish containing LB solid culture medium. 5-6 flat-plate coating beads were poured into the culture dish and shaken for 2 minutes to spread the liquid evenly in the culture dish. Finally, the culture dish was placed in a 37-degree constant temperature incubator for incubation for 12 hours. After 12 hours, the number of colonies in the culture dishes of sample C and sample D was compared to detect the antibacterial properties of the bioscaffolds with different concentrations of AgNPs.

[0064] Figure 6 The following are the antibacterial test results. It can be seen that in the blank control group without AgNPs, bacteria practically covered the entire culture dish. Sample D, containing 0.5 parts per million of AgNPs, achieved an antibacterial efficacy exceeding 90% against Staphylococcus aureus and Escherichia coli, though some colonies still remained. Sample C, containing 1 part per million of AgNPs, had zero colonies, indicating that sample C achieved an antibacterial efficacy exceeding 99%. These results demonstrate the excellent antibacterial properties of the composite hydrogel and further confirm that tissue-engineered skin scaffolds prepared using this bioink can inhibit bacterial growth in wounds, reduce inflammation, and accelerate wound healing in clinical treatment.

[0065] Test Example 5 An acute full-thickness skin defect model was made using New Zealand rabbits. Sample B was used as an example to verify the effectiveness and biocompatibility of the tissue engineering scaffold prepared by the bio-ink of the present invention in repairing the defect. A blank control group and an autologous skin transplantation control group were also set up. Three full-thickness skin defect wounds with a diameter of 2.5 cm were made on both sides of the back of the New Zealand rabbit 3 cm away from the spine. The front and back defect wounds on the same side were 3 cm apart, and there were 6 defect wounds for each rabbit. After cleaning the wound, sample B was trimmed to the same diameter and applied tightly to the skin defect. The autologous skin was well sutured with non-absorbable sutures (autologous skin has contractility). The placement of each sample and control group is shown in the figure. Figure 7 The effect of sample B on skin defect repair was observed one, two, and three weeks after surgery. A1, A2, and A3 were sample groups, B1 was a blank control group, and B2 and B3 were autologous skin transplantation control groups.

[0066] The healing of the New Zealand rabbits was assessed by taking photos. Figure 8 As shown. One week after treatment, the wounds in sample groups A1, A2, and A3 all shrank significantly, reaching a diameter of approximately 1.5 cm. The wounds in the blank control group B1 shrank significantly, reaching a diameter of approximately 0.8 cm to 1 cm. In the autologous skin transplantation control groups B2 and B3, the skin healed well, with the grafts intact and rosy in color. Some dorsal hair had grown, and most sutures were intact. Two weeks after treatment, the scars in sample groups A1, A2, and A3 shrank to approximately 1 to 1.5 cm in diameter, with varying degrees of scab formation. In the blank control group B1, the scars shrank significantly, reaching a diameter of approximately 0.8 cm, with a darker color and an uneven healing surface. In the autologous skin transplantation control groups B2 and B3, the grafts showed little shrinkage, were fully intact, and had a rosy, whiteish color. Some stye marks were still visible. After three weeks, the scars in sample groups A1, A2, and A3 shrank to approximately 0.8 cm, but the scars were irregularly long and had some dorsal hair growing out. The blank control group, B1, showed significant scar contraction, an uneven healing surface, and a tense appearance relative to the surrounding normal skin. The autologous skin graft control groups, B2 and B3, showed the skin grafts were indistinguishable from normal skin, with the dorsal hair having largely grown out, and the overall skin elasticity around the grafts was moderate. Comparisons of the experimental sample groups with the blank control group and the autologous skin graft control group revealed that the newly formed tissue in the sample groups was similar to normal tissue, similar to the recovery capacity of the autologous skin graft control group, confirming that the tissue engineering scaffolds prepared with this bio-ink are suitable for animal studies.

[0067] Test Example 6 In order to evaluate the effect of tissue engineering scaffolds on the repair of full-thickness skin defects, wounds were subjected to histological analysis. The tissues were fixed with 4% formaldehyde solution, embedded in paraffin, and sliced into 5 μm sections. H&E staining was used to evaluate the histological structure of the skin at different times of wounds in New Zealand rabbits, the sample residue, and the difference from normal skin. Figure 9 shown.

[0068] One week after treatment, the healing sites in sample groups A1, A2, and A3 were filled with granulation tissue and had an uneven surface. The healing tissue lacked sweat glands, hair follicles, sebaceous glands, and other skin accessory structures. The epidermis had begun to form locally, with dense new capillaries at the junction with normal tissue. In the blank control group, B1, the healing sites were essentially the same as those in the sample groups, but the healing surface was significantly wider than in the sample groups, and epidermal formation was not evident. In the autologous skin transplantation control groups, B2 and B3, the connective tissue in the dermis showed no significant difference from that in normal tissue, with a vague boundary. A small number of sweat glands and hair follicles were present in the dermis, and the epidermis was fully formed. After two weeks of treatment, the healing sites in groups A1, A2, and A3 were filled with granulation tissue, though the surface remained partially uneven (see A1). Sweat glands, hair follicles, and small blood vessels were partially visible within the healing tissue (see A2). In the blank control group B1, the healing sites were filled with granulation tissue, lacking skin appendages. The healing surface was wider than in the sample group, but the epidermis was largely formed. In the autologous skin transplantation control groups B2 and B3, the epidermis and subcutaneous connective tissue showed no significant differences from normal tissue. The number of sweat glands, hair follicles, sebaceous glands, small blood vessels, and hair shafts in the dermis was significantly reduced compared to normal tissue. After three weeks of treatment, the healing surfaces in groups A1, A2, and A3 were significantly shortened at all locations. The junction between the dermis and normal tissue was more pronounced in A1 and A2, and the epidermis was well formed. The dermis in A3 showed a higher number of sweat glands, hair follicles, sebaceous glands, and small blood vessels, closely resembling normal tissue. In the blank control group (B1), the healing surface was significantly shortened, with local cavities in the dermis and no obvious skin appendages. The junction with normal tissue was more obvious, but the epidermis was well formed. In the autologous skin transplantation control groups (B2 and B3), the epidermis, dermis, and subcutaneous connective tissue showed no significant differences from normal tissue, and all normal skin structures were present.

[0069] Comparing the experimental group samples with the blank control group and the autologous skin transplantation control group, it was found that the epidermis of the sample group was well formed, and the dermis contained sweat glands, hair follicles, sebaceous glands, small blood vessels and other structures, which were similar to normal tissues. The healing effect was similar to that of the autologous skin transplantation control group. It can be verified that the tissue-engineered skin printed by the bio-ink of the present invention has certain practicality, and its functionality can replace animal autologous skin for skin transplantation operations.

[0070] The physical and chemical performance data of each sample are shown in Table 1, and the actual pictures of each sample are shown in Figure 10 shown.

[0071] Table 1 Physical and chemical properties of tissue engineering skin scaffold samples with different ratios Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications of the present invention fall within the scope of the claims and their equivalents, such changes and modifications are intended to be included.

Claims

1. A method for preparing a composite bio-ink for 3D printing of tissue-engineered skin, characterized in that: The following steps are involved: Sodium periodate is used as an oxidant, mixed with a sodium alginate aqueous solution, and the sodium alginate is oxidized to prepare oxidized sodium alginate; adding gelatin to an aqueous solution of oxidized sodium alginate to obtain a mixture, and allowing the mixture to stand under alkaline conditions so that the amino groups of the gelatin react with the aldehyde groups of the oxidized sodium alginate to form a Schiff base reaction, thereby obtaining a mixed solution of oxidized sodium alginate and gelatin; Polydopamine solution and The solutions are mixed, and under alkaline conditions, polydopamine reduces silver ions into silver nanoparticles in situ to obtain polydopamine-nanosilver colloid; A mixed solution of oxidized sodium alginate-gelatin and polydopamine-nanosilver colloid are mixed to obtain a composite bio-ink.

2. The preparation method according to claim 1, characterized in that The concentration of the sodium alginate aqueous solution is 2wt%~5wt%, the molar ratio of sodium periodate to sodium alginate is 1:1~1:1.5, and the conditions for oxidizing the sodium alginate are: stirring at room temperature and in the dark.

3. The preparation method according to claim 1, characterized in that In the mixture, the concentration of gelatin is 5wt%~10wt%, and the mass ratio of oxidized sodium alginate to gelatin is 1:1~2.

4. The preparation method according to claim 1, characterized in that When the Schiff base reaction occurs, the alkaline condition means that the pH is adjusted to 8-9 with sodium hydroxide.

5. The preparation method according to claim 1, characterized in that In the step of preparing the polydopamine-nanosilver colloid, the alkaline condition refers to adjusting the pH to 8-10 with sodium hydroxide.

6. The preparation method according to claim 1, characterized in that The concentration of the solution is 1000mg / L~1500mg / L, the concentration of the polydopamine solution is 400mg / L~800mg / L, The volume ratio of the solution and the polydopamine solution is 0.5~2:

1.

7. The preparation method according to claim 1, characterized in that The concentration of nanosilver in the composite bio-ink is 0.5 to 1 part per ten thousand.

8. A composite bio-ink for 3D printing of tissue-engineered skin, characterized in that: The method is described in any one of claims 1 to 7.

9. The use of the composite bio-ink for 3D printing and constructing tissue-engineered skin according to claim 8, characterized in that: The 3D printing method includes the following steps: placing the composite bio-ink of claim 8 into a printing syringe; Adjusting the printing process parameters of the bioprinter for printing; After printing, the obtained three-dimensional scaffold is immersed in in solution.

10. The use according to claim 9, characterized in that The printing parameters are: needle diameter 0.41mm~0.84mm, printing height 0.15mm~0.3mm, layer height 5~10 layers, line spacing 0.4mm~1mm, printing speed 1mm / s~10mm / s, extrusion speed 1mm / s~10mm / s.

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