Photosensitive agarose bio-ink for 3D printing, preparation method and application

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to photosensitive agarose bio-ink for 3D printing as well as a preparation method and application of the photosensitive agarose bio-ink. The bio-ink comprises 2%-6% (m / v) of itaconylated agarose (AIA), 0.1%-1.0% (m / v) of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphate and 0-0.1% (m / v) of a light absorber. According to the AIA, an itaconic acid group is introduced into an agarose molecular chain, so that the hydrophilicity of agarose is remarkably improved, good flowability of the agarose is maintained at normal temperature, rapid photo-crosslinking curing is achieved by means of a photoinitiator, and the AIA is suitable for digital light treatment and other printing technologies based on surface exposure. The modification strategy effectively overcomes the technical bottleneck that natural agarose is difficult to flow and form at normal temperature due to thermally induced gel characteristics. The agarose bio-ink disclosed by the invention shows excellent biocompatibility, biological activity and tissue adhesion, and the cell-loaded AIA artificial skin has excellent healing promoting capability on diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a photosensitive agarose bio-ink for 3D printing, its preparation method, and its application. Background Technology

[0002] 3D printing technology has made significant progress in recent years in fields such as tissue engineering and regenerative medicine. As a core material in 3D printing, the performance of bio-inks directly affects their printing quality and final application results. Natural polymer materials, due to their good biocompatibility, low immunogenicity, and modifiability, are gradually becoming an important choice for developing bio-inks. Agarose, as a natural polysaccharide, possesses good biocompatibility, low immunogenicity, and wide availability, and has been widely used in tissue engineering and cell carriers.

[0003] However, agarose faces significant technical challenges in bioprinting applications due to its unique thermotropic gelling properties. The inherent thermotropic gelling nature of agarose limits its ability to maintain a liquid state at room temperature, making high-precision 3D printing difficult. Agarose typically requires heating to high temperatures (approximately 90°C) to completely dissolve and gels before cooling to room temperature. This characteristic is particularly detrimental to photopolymerization printing technologies such as DLP (Digital Light Processing), where the initial high-temperature environment may damage the loaded bioactive substances (such as cells and growth factors), and the gelation that occurs during printing is difficult to control, leading to low printing accuracy and structural collapse. Therefore, unmodified agarose presents numerous difficulties in 3D printing, especially DLP printing, which requires room-temperature, high-precision operation.

[0004] To overcome this limitation, researchers have performed various chemical modifications on agarose to improve its flowability. For example, introducing photosensitive groups such as methacrylic anhydride (MA) can impart photocuring properties to agarose. However, such modifications often result in excessively high ink viscosity, and the flowability at room temperature remains unsatisfactory, ultimately affecting printing accuracy. Furthermore, modified agarose often needs to be maintained in a liquid state at relatively high temperatures for printing, which is detrimental to maintaining cell viability during cell-borne printing. On the other hand, while introducing hydrophilic functional groups such as phosphates and carboxyl groups can improve flowability, it often significantly weakens the mechanical strength of the cured gel, increasing the risk of collapse of the three-dimensional structure after printing. In addition, existing modification strategies primarily focus on adapting to extrusion printing or controlled-release applications, and research on their compatibility with surface-exposure-based DLP printing technology remains insufficient.

[0005] In the treatment of chronic diabetic wounds, wound healing is often slow due to the complex pathological microenvironment caused by hyperglycemia, low local pH, and persistent inflammation, and existing treatment methods are often ineffective. Agarose, as a natural polysaccharide, possesses excellent biostability and low immunogenicity, making hydrogels based on it a promising candidate to maintain long-term structural and functional stability in the complex microenvironment of diabetic wounds. This characteristic gives it an advantage over some easily degradable or inflammatory synthetic polymers. However, the thermotropic gelation properties of traditional agarose limit its application in bioprinting, preventing the fabrication of functional wound dressings with complex structures and cell-loading capabilities using 3D printing technology.

[0006] Therefore, developing an agarose-based bio-ink that maintains good flowability at room temperature, can form three-dimensional structures with sufficient mechanical strength through photopolymerization, and simultaneously retains excellent biocompatibility and tissue adhesion is of great significance for advancing the application of bioprinting technology in tissue engineering, diabetic wound repair, and drug delivery. The development of this novel bio-ink not only overcomes the shortcomings of traditional agarose in terms of flowability and mechanical strength, but also ensures the stability and functionality of the printed structure while maintaining cell viability, providing a natural polysaccharide-based photopolymerization material system with great clinical application potential for diabetic wound repair. Summary of the Invention

[0007] To address the technical bottleneck of traditional agarose's difficulty in flowing and molding at room temperature due to its thermogenic gel properties, as well as the problems of existing agarose modification strategies such as the need for high-temperature liquid maintenance and weakened mechanical strength, a novel photosensitive agarose bio-ink is proposed to achieve excellent biocompatibility while endowing the material with room-temperature flowability and excellent 3D printing processing performance.

[0008] The technical solution adopted by this invention to solve its technical problem is: to provide a photosensitive agarose bio-ink for 3D printing, its preparation method, and its application. This bio-ink contains the following components: 2%–6% (m / v) AIA, 0.1%–1.0% (m / v) LAP, and 0–0.1% (m / v) light absorber.

[0009] Preferably, the agarose is an agarose derivative containing photocrosslinkable double bonds obtained by modifying agarose with itaconic anhydride.

[0010] Furthermore, the degree of modification of the itaconylated agarose is 10% to 90%, more preferably 34% to 73%.

[0011] Optionally, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate, preferably at a concentration of 0.5% (m / v).

[0012] Optionally, the light absorber is a biocompatible dye, such as one or more of lemon yellow and brilliant blue.

[0013] Preferably, the bio-ink maintains liquid fluidity at 20–40°C.

[0014] Furthermore, the present invention also provides a method for preparing the aforementioned photosensitive novel agarose bio-ink, comprising the following steps: (1) dissolving agarose in water, heating and stirring until completely dissolved, and controlling the reaction temperature at 45-50℃; then slowly adding itaconic anhydride, while maintaining the pH value of the reaction system between 8 and 9 with 6 M NaOH solution, and continuing the reaction for 7-8 hours; after the reaction is completed, dialysis purification of the product is performed for 3 days, and finally freeze-drying is performed to obtain itaconic anhydride-modified agarose solid containing crosslinkable double bonds.

[0015] (2) Dissolve the lyophilized AIA powder from step (1) in a suitable solvent (e.g., water or phosphate buffered saline (PBS) with pH 7.0-7.4) to prepare an AIA solution with a concentration of 2% to 6% (m / v); add 0.1% to 1.0% (m / v) of photoinitiator LAP and optionally 0% to 0.1% (m / v) of light absorber to the solution; then mix it evenly and remove air bubbles by vortexing or centrifugation to obtain a bio-ink that can be used for DLP printing.

[0016] Optionally, the photosensitive agarose bio-ink can be used to prepare tissue engineering scaffolds, wound dressings, or drug delivery carriers, and can be formed by surface exposure-based photopolymerization 3D printing technology.

[0017] Preferably, the tissue engineering scaffold is a tissue engineering scaffold for skin, blood vessels, cartilage, bone, or cornea.

[0018] Furthermore, the present invention also provides a dressing for repairing diabetic wounds, which is formed by 3D printing technology from the bio-ink (optionally loaded with active cells).

[0019] Furthermore, the present invention also provides the use of the dressing in the preparation of medical devices or products for the repair of diabetic wounds.

[0020] The present invention has the following beneficial effects:

[0021] (1) This invention, through an itaconic anhydride modification strategy, endows the material with the ability to maintain stable liquid flow at room temperature (20-40℃) while retaining the excellent biocompatibility of agarose. This characteristic fundamentally solves the technical problem that traditional agarose cannot be directly applied to high-precision DLP 3D printing due to its thermotropic gel properties.

[0022] (2) The degree of modification of AIA in this invention can be precisely controlled within a wide range (e.g., 10%-90%) by adjusting the reaction feed ratio. Experiments have confirmed that AIA bio-inks with different degrees of modification (e.g., 34%, 49%, and 73%) all exhibit excellent room temperature fluidity. By systematically controlling the degree of modification, the viscosity, light transmittance, gelation kinetics, and mechanical strength of the cured hydrogel of the bio-ink can be flexibly optimized, providing a customizable material basis for adapting to different printing resolution requirements and tissue engineering application scenarios.

[0023] (3) The AIA bio-ink of the present invention has suitable rheological properties and photosensitivity, and exhibits excellent processing adaptability in digital light processing (DLP) 3D printing. The ink can spread rapidly and respond to specific wavelength light (such as 405nm) to initiate cross-linking, stably constructing complex three-dimensional structures with micron-level resolution. The printed components have smooth surfaces, complete structures, and tight interlayer bonding, with no obvious collapse or deformation.

[0024] (4) Exhibiting excellent stability and healing-promoting activity in diabetic wound repair: The hydrogel scaffold constructed with the AIA bio-ink of this invention still retains the excellent chemical and structural stability of the natural agarose backbone, and can maintain its physical morphology and function for a long time in the complex microenvironment of high sugar, pH fluctuations and abnormal enzyme activity in diabetic wounds. This scaffold can not only serve as a physical barrier to cover the wound, but also as an excellent cell carrier to support the survival, proliferation and spread of loaded cells (such as fibroblasts and keratinocytes), thereby significantly promoting reepithelialization and collagen deposition in the wound and accelerating the tissue repair process.

[0025] (5) This invention provides a new strategy for the design of natural polysaccharide-based photocurable bio-inks, and specifically provides an active material system that can be processed by 3D printing technology, can load cells, and has a controllable structure, which has important application value in biomedical fields such as tissue engineering scaffold construction and diabetic wound repair. Attached Figure Description

[0026] Figure 1 Preparation and physicochemical property characterization of photosensitive agarose

[0027] Note: a) Schematic diagram of the reaction between agarose and four acid anhydrides; b) Flowability of agarose and acid anhydride-modified derivatives at room temperature (37℃); c) 1 d) H-NMR spectrum; e) FTIR spectrum; f) XRD spectrum analysis; g) UV-vis full-band transmittance curve analysis; h) Quantitative analysis of water contact angle on hydrogel surface; DSC thermal analysis curve.

[0028] Figure 2 AIA Modification Screening and Formulation Optimization

[0029] Note: AIA with different degrees of modification (a) 1 a) H-NMR spectrum; b) FTIR spectrum; c) Ink rheological viscosity test; d) SEM microstructure of cured gel; ef) Rheological modulus and strain scan of cured gel; g) Swelling performance test; h) Effect of LAP concentration on curing kinetics; i) LAP cytotoxicity test; j) Effect of light absorber concentration on curing time.

[0030] Figure 3 AIA Bio-Ink DLP Printing Performance Demonstration

[0031] Note: This exhibit showcases two-dimensional meshes, three-dimensional scaffolds, multi-layered stacked structures, and biomimetic organ models printed using AIA bio-ink.

[0032] Figure 4 Performance comparison of AIA and HAMA hydrogels

[0033] Note: a) Macroscopic demonstration of the compression recovery performance of the AIA-printed ear model; b) Adhesion demonstration of AIA hydrogel on different substrate surfaces; c) Photograph of AIA hydrogel adhesion under dynamic finger joint bending; d) Photograph of HAMA hydrogel peeling under dynamic finger joint bending; e) Comparison of ultimate compressive stress-strain curves of AIA and HAMA hydrogels; f) Stress-strain curve of AIA hydrogel after 10 cycles of compression; g) Stress-strain curve of HAMA hydrogel after 10 cycles of compression; h) Quantitative statistical graph of the adhesion strength of AIA and HAMA on pigskin surface; i) Comparison of actual photos of AIA and HAMA hydrogel adhesion on pigskin surface; j) Comparison of SEM microstructure of AIA and HAMA hydrogels.

[0034] Figure 5 In vitro cell compatibility assessment of AIA hydrogel

[0035] Note: a) Fluorescence staining of live and dead cells after cell encapsulation and culture; b) Cytoskeleton staining; c) Cell scratch pattern; d) Image J quantitative analysis of live cell count; e) Image J quantitative analysis of cytoskeleton fluorescence intensity; f) Image J quantitative analysis of scratch closure rate.

[0036] Figure 6 Cell Printing Performance and Skin Organoid Construction of AIA Bio-ink

[0037] Note: a) Schematic diagram of the structural design of skin organoids constructed with AIA bio-ink; b) Study on the printing performance of AIA bio-ink loaded with different types of cells (L929, HaCaT, HFF-1 and BMSCs).

[0038] Figure 7 In vivo evaluation of AIA artificial skin for repairing diabetic wounds

[0039] Note: a) Schematic diagram of establishing a diabetic rat model; b) Apparent image of wound healing; c) Heat map of wound area change; d) Image J quantitative analysis of wound closure.

[0040] Figure 8 Histopathological analysis of wound tissue

[0041] Note: a) H&E stained section of wound tissue on day 14; b) Masson trichrome stained section on day 14; c) Sirius red stained section on day 14; d) Image J quantitative analysis of epidermal thickness; e) Image J quantitative analysis of collagen volume percentage; f) Image J quantitative analysis of type I / III collagen ratio.

[0042] Figure 9 Assessment of inflammation reduction and angiogenesis in wound tissue

[0043] Note: a) Image J quantitative analysis of relative MPO expression; b) Image J quantitative analysis of relative IL-6 expression; c) Immunofluorescence staining image of MPO in wound tissue on day 7; d) Immunofluorescence staining image of IL-6 in wound tissue on day 7; e) Immunofluorescence staining image of α-SMA in wound tissue on day 7; f) Immunofluorescence staining image of CD31 in wound tissue on day 7; g) Image J quantitative analysis of neovascularization density; h) Image J quantitative analysis of neovascularization diameter.

[0044] Figure 10 Transcriptomic analysis of wound repair mechanisms

[0045] Note: a) Venn diagram of differentially expressed genes (DEGs); b) Volcano plot of differentially expressed genes; c) Gene expression clustering heatmap; d) GO functional enrichment analysis; e) KEGG pathway enrichment analysis of upregulated genes; f) KEGG pathway enrichment analysis of downregulated genes; g) RT-qPCR quantitative analysis results of key genes (TNF-α, IL-6, IL-10, Arg-1, PPARγ, Fabp4); h) Schematic diagram of the molecular mechanism of the PPAR signaling pathway. Detailed Implementation

[0046] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are further illustrations of the present invention, but are not intended to limit the scope of the invention. All other equivalent substitutions or obvious modifications that can be conceived by those skilled in the art without creative effort based on the technical solutions of the present invention should be considered to be included within the protection scope of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] The agarose raw material used in the embodiments of the present invention can be prepared by referring to patent CN202510136975.5, but the scope of protection of the present invention is not limited to agarose of this specific source or molecular weight.

[0050] For consistency, the material before printing will be referred to as "bio-ink" or "ink" in the following text, and the three-dimensional network structure formed after 3D printing and curing will be referred to as "hydrogel".

[0051] Example 1: Preparation and Characterization of Photosensitive Agarose

[0052] Itaconic acid anhydride was used to modify agarose to prepare itaconicated agarose (AIA). A certain amount of agarose powder was weighed and added to ultrapure water to prepare a 3% (m / v) suspension. The suspension was heated and stirred until completely dissolved. The solution was cooled to 45°C and kept at a constant temperature. Under this constant temperature condition, itaconic acid anhydride (IA) was slowly added dropwise to the solution. To evaluate the effects of different anhydride modifications, Acya (acrylic anhydride-modified agarose), AMA (methacrylic anhydride-modified agarose), and Amla (maleic anhydride-modified agarose) were prepared simultaneously with acrylic anhydride, methacrylic anhydride, and maleic anhydride modifications. During the reaction, the anhydride and agarose were controlled to be in an equal molar ratio, and the pH of the system was monitored in real time using a pH meter. The pH of the reaction system was strictly maintained between 8.0 and 9.0 by adding NaOH solution dropwise. The reaction was carried out under constant temperature of 45°C with continuous stirring for 7 hours. Figure 1 a) After the reaction was completed, the reaction solution was placed in a dialysis bag and purified by dialysis in deionized water for 3 days, with the dialysis solution being changed every 8 hours to completely remove unreacted small molecule anhydrides and byproduct salts. The dialysis solution was lyophilized to obtain white flocculent or sponge-like modified products, labeled Acya, AMA, AIA, and Amla, respectively.

[0053] 1H NMR spectrum ( 1H-NMR characterization: 5-10 mg of the lyophilized sample was dissolved in 1 mL of deuterated DMSO, and the results were as follows. Figure 1 c shows that, compared with unmodified agarose, all modified samples exhibited characteristic signals at chemical shifts of δ ≈ 5.8–6.2 ppm, attributable to double bond protons, proving that the unsaturated structure has been successfully introduced.

[0054] Fourier transform infrared (FTIR) characterization: Approximately 1-2 mg of lyophilized sample powder was mixed with approximately 100 mg of dried, spectroscopically pure potassium bromide (KBr) powder. The mixture was thoroughly ground into a uniform fine powder in an agate mortar. A transparent thin film was prepared using a pelleting method under 10 MPa pressure, and then a full-band scan was performed. The results are shown below. Figure 1 The data shows that the modified sample exhibits a distinct new absorption peak near 1720 cm⁻¹, which is attributed to the stretching vibration of the newly formed ester group C=O. Meanwhile, the intensity of the broad -OH peak near 3400 cm⁻¹ is relatively weakened, indicating that the hydroxyl groups on the agarose backbone have undergone ring-opening esterification with the acid anhydride.

[0055] Room temperature flowability test: Each sample was prepared into a 3% (m / v) aqueous solution and placed in a 37℃ constant temperature incubator. The flowability was observed by inverting the sample. Results are as follows: Figure 1 As shown in b, unmodified agarose (Aga) and its modified products, such as acrylic anhydride (Acya) and methacrylic anhydride (AMA), readily form gels at this temperature, exhibiting gelation characteristics. In contrast, samples modified with itaconic anhydride (AIA) and maleic anhydride (Amla) maintain good flowability and remain as transparent liquids under the same conditions. Further observation revealed that the Amla sample exhibits a self-assembly tendency and partially gels after a period of time, demonstrating instability in its flow state. AIA, on the other hand, demonstrates superior flowability and system stability.

[0056] X-ray diffraction (XRD) was used to perform XRD tests on the lyophilized samples, and the results are as follows: Figure 1 As shown in e, unmodified agarose (Aga) exhibits a clear crystallization diffraction peak at 19°. After modification with acid anhydride, the diffraction peak intensities of Acya and AMA samples are significantly reduced; while the diffraction peaks of Amla and AIA samples are broader and weaker, indicating that the intermolecular hydrogen bond network is weakened after modification, and the system tends to be more amorphous.

[0057] Optical performance testing: Each sample was prepared into a 3% (m / v) aqueous solution, and its transmittance in the 200–800 nm wavelength range was measured using a UV-Vis spectrophotometer. The results are shown in 1f. AIA exhibits excellent transmittance in the visible light band, especially at a wavelength of 405 nm, where its transmittance is significantly higher than that of unmodified agarose and AMA.

[0058] Each sample was prepared into a hydrogel, and the water contact angle on its surface was measured using a contact angle meter. The results are as follows: Figure 1 As shown in g, the water contact angles of the unmodified agarose, Acya, and AMA gel surfaces are relatively large; while the water contact angles of the AIA and Amla gel surfaces are significantly reduced, with the AIA gel having the smallest contact angle, indicating that it has the strongest hydrophilicity.

[0059] Differential scanning calorimetry (DSC) was used to test the samples: the scanning temperature range was set to 20–100℃, and the results are as follows. Figure 1 Unmodified agarose exhibited a significant exothermic peak at approximately 35 °C, corresponding to its thermo-induced gelation transition; Acya and AMA samples showed similar phase transition behavior; Amla samples showed a broad endothermic peak at approximately 57 °C; while AIA did not show significant endothermic or exothermic peaks throughout the entire test range, indicating that it eliminated the thermo-induced gelation properties of natural agarose.

[0060] Example 2: Formulation optimization and performance testing of AIA bio-ink

[0061] After confirming that itaconic anhydride-modified agarose (AIA) has room temperature flowability, in order to screen the best bio-ink formulation suitable for DLP printing, this embodiment selected three AIA samples with different modification degrees (34%, 49% and 73%) for systematic physicochemical property characterization.

[0062] use 1 ¹H-NMR was used to quantitatively analyze the chemical structure and degree of substitution of the sample. The spectrum shows ( Figure 2 a) By calculating the ratio of the double bond proton integral area to the framework proton integral area and combining it with proton number normalization, the actual substitution degrees of the three samples were quantitatively determined to be 34%, 49% and 73%, respectively.

[0063] Characterization was performed using FTIR, and the results are as follows: Figure 2 As shown in b, the intensity of the stretching vibration peak of the ester carbonyl (C=O) group at approximately 1720 cm⁻¹ increases significantly with increasing modification degree, confirming that both the degree of esterification and the double bond content of AIA increase with increasing modification degree.

[0064] The rheological properties and mechanical properties after curing of three types of bio-inks were tested. The rheological test results at 25℃ showed ( Figure 2 c) The steady-state shear viscosity of AIA bio-ink increases significantly with increasing modification degree, indicating that as the modification degree increases, the number of carboxyl groups on the side chains of the molecular chain increases, promoting the entanglement between molecular chains and significantly increasing the viscosity of the solution.

[0065] Frequency scanning experiments show that ( Figure 2e) As the degree of IA modification increases, the storage modulus (G') of the gel decreases significantly, indicating that although increasing the degree of modification increases the photopolymerization active sites, the introduction of side chain groups also destroys the original physical crosslinking network of agarose. Under high degree of modification, the structural destruction of the physical crosslinking network dominates, exceeding the reinforcing effect of photocrosslinking, resulting in a decrease in the mechanical strength of the final hydrogel.

[0066] Strain scanning experiments also showed ( Figure 2 f), the highly modified sample underwent gel structure destruction at a low strain, indicating that its mechanical strength decreased with increasing modification degree.

[0067] The microstructure of the gel was observed using scanning electron microscopy (SEM). Figure 2 d) The results showed that as the degree of modification increased, the average pore size of the gel increased. Among them, 49% AIA gel exhibited a regular and uniform three-dimensional porous network structure, while the pore structure of 73% AIA gel appeared loose, which was consistent with its poor mechanical properties.

[0068] In addition, swelling behavior test ( Figure 2 g) also shows that the 73% AIA gel has a higher swelling rate and exhibits macroscopic morphological instability after prolonged soaking. In contrast, the 49% AIA gel maintains good structural integrity after swelling and exhibits a more stable network structure.

[0069] Real-time monitoring of the in-situ photocuring process of inks at different LAP photoinitiator concentrations ( Figure 2 (h) It was found that as the LAP concentration increased from 0.1% to 1.0%, the rate of increase of the storage modulus (G') accelerated, and the curing efficiency was significantly improved.

[0070] Combined with cytotoxicity test results ( Figure 2 i), and finally selected a LAP concentration of 0.5% (m / v) as the optimal condition for balancing curing efficiency and biosafety. Simultaneously, to improve the accuracy of DLP printing, the effect of the amount of light absorber (such as lemon yellow) on the curing rate was investigated. The results showed that when the light absorber concentration was adjusted to 0.05% (m / v) ( Figure 2 j) The ink can ensure sufficient curing depth to achieve interlayer bonding, and effectively suppress curing of non-target areas caused by light scattering, thereby achieving rapid and high-precision interlayer molding.

[0071] Based on the above test results, AIA with a modification degree of 49% was determined to be the best optimized bio-ink formulation suitable for DLP 3D printing.

[0072] Example 3: 3D Printing Performance Study of AIA Bio-ink

[0073] AIA bio-ink at a concentration of 3.5% (m / v) was added to an optimized concentration as described in Example 2 (e.g., 0.5% (m / v) LAP and 0.05% (m / v) light absorber) and then directly used for DLP printing at room temperature. AIA ink maintains good liquid flowability without heating, allowing it to spread rapidly in the printer cartridge to form a uniform and stable liquid film—a characteristic not found in traditional agarose materials.

[0074] Under fixed exposure parameters and layer thickness conditions, various typical structures were printed to demonstrate its forming capabilities, including two-dimensional patterns, three-dimensional mesh frames, multi-layer stacked structures, and tissue / organ models. Figure 3 The printing results showed that AIA bio-ink exhibited excellent molding precision. The line widths, pore edges, and fine features of the printed structures were clearly distinguishable, with sharp boundaries and no obvious droplet diffusion or shape distortion. Furthermore, the layers of the printed structures were tightly bonded, and no interlayer separation or structural collapse was observed. Further printing of complex three-dimensional structures, such as auricular models and organoid structures with internal cavities, showed that these complex models could be stably molded, exhibiting good shape fidelity and structural retention. In addition, tests revealed that the hydrogel structures printed with AIA maintained their complete macroscopic morphology and dimensional stability even after prolonged immersion in PBS solution, indicating the stability of its cross-linked network structure, which provides a foundation for its application in biological environments.

[0075] The above results demonstrate that AIA bio-ink can achieve high-precision, low-defect microscale three-dimensional structure fabrication at room temperature using DLP technology without high-temperature pretreatment. This material system exhibits excellent printability and molding reliability, and has significant application value in biomanufacturing fields such as tissue engineering scaffolds, microfluidic chips, and organoid construction.

[0076] Example 4: Performance Study of AIA and HAMA

[0077] To objectively evaluate the overall performance of AIA hydrogel, this embodiment systematically compares it with methacryloyl hyaluronic acid (HAMA), which is commonly used in the field of bioprinting, in terms of shape recovery, interfacial adhesion, mechanical strength, and microstructure.

[0078] The physical flexibility and surface adhesion of the material were evaluated through macroscopic deformation and adhesion experiments. In the shape recovery test ( Figure 4 a) When a compressive force is applied to the printed AIA auricle model, the hydrogel can almost instantly recover to its original shape after the pressure is removed, indicating that its internal cross-linked network has excellent elastic recovery ability.

[0079] The adhesion properties of AIA materials were tested. Figure 4 (b) The results showed that the AIA hydrogel could adhere firmly to different substrate surfaces, including plastic (PP), glass, metal and various biological tissues, demonstrating good interfacial adaptability.

[0080] To simulate the adhesion stability of dynamic skin surfaces, a finger bending experiment was conducted. Figure 4 As shown in c, during continuous bending, the AIA hydrogel remained tightly adhered to the skin surface of the finger joint without detachment or displacement; in contrast, the HAMA gel exhibited significant edge lifting and peeling under the same bending conditions. Figure 4 d), indicating that it is difficult for it to adapt to dynamic deformation of the skin surface.

[0081] Ultimate compression and cyclic compression tests were conducted on the mechanical properties of the two materials. Ultimate compression stress-strain curves ( Figure 4 (e) shows that HAMA fractured at approximately 45% strain, while AIA withstood up to 85% ultimate strain, exhibiting superior toughness. Further fatigue tests demonstrated that the AIA hydrogel survived 10 cycles of constant strain compression (…). Figure 4 f), whose loading-unloading curves highly overlap, have a very small hysteresis loop and no significant stress decay; while HAMA's cyclic curve ( Figure 4 g) With increasing fatigue cycles, significant stress softening and energy dissipation are observed, indicating poor fatigue resistance and difficulty in maintaining the long-term stability of complex three-dimensional structures.

[0082] In the quantitative assessment of tissue adhesion, two hydrogels were applied to the surface of fresh pig skin tissue for testing. Physical observation was conducted. Figure 4 i) and quantitative data on adhesion strength ( Figure 4 (h) Consistently, HAMA gel showed weak adhesion to tissue and was prone to slippage; while AIA hydrogel, due to its rich content of free carboxyl groups and other polar groups in its molecular chain, could form a strong interfacial interaction with the tissue surface, and its peel strength was significantly higher than that of HAMA, maintaining stable adhesion even in moist skin environments. Scanning electron microscopy (SEM) observation results ( Figure 4 (j) The microscopic mechanism underlying the performance difference between the two was revealed: HAMA gel has a larger but unevenly distributed pore size and poor structural continuity; while AIA gel exhibits a regular, dense, and uniform three-dimensional porous network structure. This regular microstructure not only endows the material with more stable mechanical properties but also facilitates uniform cell distribution and material transport. In summary, AIA is significantly superior to HAMA in terms of shape memory, fatigue resistance, tissue adhesion, and microstructural uniformity, making it more suitable for high-precision bioprinting and wound repair.

[0083] Example 5: Biocompatibility Study of AIA

[0084] The biocompatibility of the AIA hydrogel prepared in this invention was evaluated using fibroblast L929 cells. Figure 5 L929 cells were encapsulated in AIA and HAMA hydrogels, respectively, with commercially available HAMA and Blank medium (without materials) serving as controls. Cells in the Blank group were directly seeded onto culture plates. Live / dead cell staining was performed after 1, 3, and 5 days of culture. The results are shown in Table 1. Cells in the Blank group showed uneven distribution; while cells in both AIA and HAMA hydrogels showed continuous and uniform green fluorescence signals, with very few dead cells (red fluorescence), indicating that cells in both hydrogels maintained high viability, with no significant difference between the two groups. Quantitative analysis of cell numbers in the three groups showed that cell proliferation levels in the AIA and HAMA groups were similar, and significantly higher than in the Blank group, indicating that AIA hydrogel provides good support for cell proliferation. Phalloidin staining revealed that the Blank group had a smaller spreading area and sparse actin fiber structure; while cells in both HAMA and AIA groups showed good spreading morphology with clear, extended actin stress fibers. Quantitative analysis of fluorescence intensity showed that the cytoskeleton development of the two hydrogel groups was comparable, and both were significantly better than that of the Blank group. Cell migration ability was assessed by a cell scratch assay. Twenty-four hours after scratch formation and addition to the corresponding culture systems, the Blank group showed the lowest scratch closure rate; while the scratch closure areas of both the AIA and HAMA groups were significantly larger. Quantitative analysis of migration area showed that the cell migration abilities of the AIA and HAMA groups were similar, and both were significantly higher than those of the Blank group. These results indicate that AIA hydrogel exhibits good cell compatibility in supporting cell survival and proliferation, promoting cell spreading, and migrating. Its cell compatibility is comparable to that of commercially available HAMA hydrogel and significantly better than that of the Blank group without material support. This suggests that AIA hydrogel is suitable as a carrier for cell encapsulation and delivery, and has important application value in fields such as 3D bioprinting and tissue engineering.

[0085] Example 6: Cell-loaded printing performance of AIA bio-ink and skin organoid construction

[0086] This embodiment uses DLP 3D printing technology to further systematically evaluate the biomimetic tissue construction capability and cell-carrying printing performance of AIA bio-ink. Figure 6 This embodiment designs a skin organoid model with a double-layer structure. Figure 6a) This model is based on the layered anatomical features of natural skin and employs a biomimetic design: the upper layer of bio-ink is loaded with human immortalized keratinocytes (HaCaT) to simulate the dense epidermis, while the lower layer of bio-ink is loaded with human foreskin fibroblasts (HFF-1) to simulate the matrix-rich dermis. Encapsulation results demonstrate that AIA bio-ink can successfully construct this anisotropic bilayer structure with tight interlayer bonding and clear boundaries, achieving precise spatial distribution and in-situ encapsulation of different cells.

[0087] Based on this design, to verify the broad applicability and printing accuracy of AIA bio-ink to the above-mentioned and other cell types, this study further selected four representative cell types for load printing tests, including L929 mouse fibroblasts, HaCaT, HFF-1, and bone marrow mesenchymal stem cells (BMSCs). Figure 6 As shown in b, the AIA bio-ink successfully loaded the aforementioned four cell types and printed complex 3D models with micron-level precision. Subsequent live-dead cell staining results showed that after one day of culture, the vast majority of the printed constructs in the field of view were live cells emitting green fluorescence, with very few red dead cells, indicating that the cell viability remained at an extremely high level. This result strongly confirms that AIA bio-ink not only possesses excellent structural processability, but also that the gentle photocuring process of DLP printing causes minimal damage to various cell types, including epithelial cells and stem cells, demonstrating broad application prospects in the construction of complex tissue engineering scaffolds.

[0088] Example 7: AIA skin organoids for diabetic wound repair

[0089] To evaluate the in vivo biocompatibility of AIA bio-ink and the bioactivity of artificial skin constructed after cell loading under complex pathological conditions, this embodiment established a full-thickness skin defect model on the back of diabetic SD rats for systematic study. In the experimental design, a circular wound with a diameter of 10 mm was prepared on the back of rats that had been successfully induced to have diabetes, and the rats were randomly divided into five groups for comparison: the HAMA porous gel group was used as the reference material control, the AIA porous gel group and the AIA artificial skin loaded with cells (AIA-cell-laden) group were used as the experimental groups, the untreated diabetic group (Control) was used as the negative control, and a wound group from non-diabetic healthy rats (Wild) was introduced as a reference for the physiological healing of acute wounds.

[0090] Macroscopic observation results of wound healing ( Figure 7(b) shows that during the 14-day observation period, the groups exhibited distinctly different healing trajectories. The Wild group, serving as a physiological repair control, showed normal acute wound healing characteristics, with the wound essentially closed by day 14, achieving a healing rate of nearly 80%. In contrast, the Control group, affected by the high glucose and chronic inflammatory microenvironment, showed significantly slower healing, with curled wound edges and a persistently large unhealed area. Compared to the Control group, the wounds in all three groups receiving material treatment (HAMA, AIA, and AIA-cell-laden) showed a clear progressive shrinkage trend over time, indicating that the material coverage effectively improved the local microenvironment.

[0091] To more intuitively illustrate the spatial distribution characteristics of wound contraction, this study generated a wound area heatmap. Figure 7 c), the color gradient changes in the graph clearly reflect the evolution of the wound area gradually shrinking over time in each group. Further analysis using a line graph of wound closure rate ( Figure 7 d) Quantitative kinetic analysis showed that rapid wound contraction was initiated in all treatment groups from days 4 to 7. However, the differences between groups widened further after entering the critical period of proliferation and remodeling from days 7 to 14. The AIA-cell-laden group exhibited the best healing-promoting performance, with the steepest curve slope, representing the fastest wound closure rate. The residual wound area at the same time point was significantly smaller than that of the HAMA group and the AIA-only group. Particularly noteworthy is that the final healing rate of the AIA-cell-laden group was significantly higher than that of the unloaded AIA group. This result confirms that AIA hydrogel can not only act as a physical scaffold to guide tissue regeneration but also as an excellent cell carrier to maintain the activity of loaded cells. Through the synergistic effect of the material and cells, it effectively breaks the healing stagnation of diabetic wounds, significantly accelerating wound closure and epithelialization. These in vivo experimental results strongly demonstrate that AIA bio-ink and the artificial skin scaffold constructed from it have good in vivo biocompatibility and bioactivity, and have great application potential in the repair of chronic diabetic wounds.

[0092] Example 8: Histopathological analysis of wound tissue

[0093] To thoroughly evaluate the effects of AIA hydrogel on the microstructure and immune microenvironment of diabetic wound repair, this study systematically analyzed the histopathology and immunofluorescence of wound tissues from each group (Control, HAMA, AIA, and AIA-cell-laden) on days 7 and 14. First, H&E staining was performed... Figure 8 a) and Masson staining ( Figure 8(b) Morphological changes and collagen deposition in the wound tissue were observed on day 14. Results showed that, compared to the Control group, both the AIA and HAMA groups effectively accelerated wound re-epithelialization and promoted denser and more orderly collagen fiber deposition in the dermis, thus significantly reducing the residual wound area. The AIA-cell-laden group exhibited the best repair effect on day 14, with intact new epidermal structure, the highest maturity of the dermal collagen matrix, and the most observed skin appendage regeneration. Further analysis using Sirius red staining (…) Figure 8 c) The collagen types were distinguished. Under a polarized light microscope, type I collagen showed strong yellow birefringence, while type III collagen showed weak green birefringence.

[0094] To quantify the above histological observations, this study statistically analyzed the epidermal thickness of each group ( Figure 8 d) Total collagen content ( Figure 8 e) and the ratio of type I to type III collagen ( Figure 8 f). The results showed that the epidermal thickness of the AIA-cell-laden group was closest to that of normal skin, and its total collagen content was significantly higher than that of other groups, indicating that AIA material can effectively promote collagen remodeling.

[0095] Example 9: Assessment of inflammation resolution and angiogenesis in wound tissue

[0096] Regarding the regulation of the immune microenvironment, this embodiment assessed the inflammatory status of wound tissue on day 7 through quantitative analysis and immunofluorescence staining. Quantitative analysis of the relative expression levels of the neutrophil marker myeloperoxidase (MPO) and the pro-inflammatory factor interleukin-6 (IL-6) was conducted. Figure 9 (a, 9b) shows that the levels of inflammatory factors in the AIA-cell-laden group were significantly lower than those in the Control group. This trend was visually confirmed by immunofluorescence staining. Figure 9 In the AIA-cell-laden group (c, 9d), the positive fluorescence signal intensity of MPO and IL-6 was significantly reduced. This indicates that AIA hydrogel, especially after cell loading, can effectively inhibit neutrophil infiltration and the secretion of inflammatory factors, thereby accelerating inflammation resolution and creating a favorable low-inflammatory microenvironment for tissue repair.

[0097] By detecting smooth muscle actin (α-SMA) in wound tissue on day 7, Figure 9 e) Platelet endothelial cell adhesion molecule (CD31, Figure 9The expression of α-SMA was used to assess angiogenesis. α-SMA labeled pericytes and myofibroblasts, while CD31 labeled vascular endothelial cells. Immunofluorescence results showed an increase in the number of new blood vessels in both the AIA and HAMA groups, but the AIA-cell-laden group had the highest angiogenesis density and more mature and complete vascular morphology. Further quantitative statistical analysis confirmed that the AIA-cell-laden group had the highest angiogenesis density (f) Figure 9 g) and vessel diameter ( Figure 9 h) were significantly superior to other groups. This confirms that the cell-loaded AIA hydrogel has a better effect on promoting vascular endothelial cell proliferation, migration and vascular maturation, and can provide sufficient blood supply and nutrition for newly formed granulation tissue.

[0098] Example 10: Transcriptomic and Molecular Biological Analysis of Wound Repair Mechanisms

[0099] To further explore the molecular mechanism by which AIA-cell-laden promotes wound repair in diabetic patients, this study performed transcriptome sequencing on wound tissues from the AIA-cell-laden and Control groups, and used qPCR to verify and examine systemic changes in gene expression. Venn diagrams were used to analyze these changes. Figure 10 a) and volcano map ( Figure 10 b) Quantitative screening of differentially expressed genes (DEGs) in the two groups was performed. Compared with the Control group, the AIA-cell-laden group identified 132 significantly differentially expressed genes, of which 70 genes were significantly upregulated and 62 genes were significantly downregulated. Following this, a hierarchical clustering heatmap was generated. Figure 10 c) shows that the two groups of samples exhibit distinctly different clustering patterns, indicating that treatment induced a systematic remodeling of the gene expression profile in wound tissue.

[0100] To clarify the biological functions of these differentially expressed genes, GO enrichment analysis was performed. Figure 10 d) The results showed that differentially expressed genes were significantly enriched in lipid metabolism, responses to oxidative stress, and phylogenetic processes, suggesting that treatment initiated metabolic repair and antioxidant defense programs. KEGG pathway enrichment analysis further revealed specific signaling networks: upregulated genes ( Figure 10 e) The genes were mainly enriched in key metabolic regulatory pathways such as the PPAR signaling pathway and fatty acid metabolism, indicating that the treatment activated the metabolic regulatory network centered on PPAR; while downregulated genes ( Figure 10 f) Significantly enriched in infection and immune response-related pathways, confirming that treatment promotes the transformation of the wound microenvironment from a "pro-inflammatory" state to a "repair" state.

[0101] To further verify the reliability of the transcriptome sequencing results, inflammation-related genes (TNF-α, IL-6, IL-10, Arg-1) and key genes of the PPAR pathway (PPAR, Fabp4) were selected for RT-qPCR detection. The results showed ( Figure 10 Compared to the Control group, the expression of pro-inflammatory cytokines TNF-α and IL-6 was significantly downregulated in the AIA-cell-laden group, while the expression of anti-inflammatory cytokines IL-10 and the M2 macrophage marker Arg-1 was significantly upregulated. Furthermore, the expression levels of the key metabolic regulator PPAR and its downstream target gene Fabb4 were significantly increased. This is highly consistent with the transcriptomic results, and can be further confirmed by the schematic diagram of the PPAR signaling pathway molecular mechanism (…). Figure 10 (h) This strongly confirms that AIA-cell-laden accelerates the healing of diabetic chronic wounds by specifically activating the PPAR signaling pathway, driving a dual mechanism of anti-inflammatory and metabolic repair.

[0102] In summary, this invention provides the first preparation of a novel photosensitive agarose (AIA). Compared to natural agarose, AIA increases molecular polarity and improves hydrophilicity by introducing itaconic acid groups, enabling it to dissolve at room temperature (20-40℃) and maintain good liquid flowability, thus overcoming the technical limitation of natural agarose requiring high-temperature dissolution.

[0103] This invention provides a photosensitive agarose bio-ink suitable for DLP 3D printing, comprising: 2%–6% (m / v) AIA, 0.1%–1.0% (m / v) LAP, and 0–0.1% (m / v) light absorber. This AIA bio-ink retains the inherent biocompatibility of agarose while effectively supporting cell survival, proliferation, and migration. Compared to common agarose, the AIA bio-ink exhibits higher light transmittance, lower contact angle, and more suitable rheological properties, ensuring uniform film deposition and high-precision molding during DLP printing. The AIA bio-ink possesses good room-temperature flowability and uniformity, and by optimizing the modification degree, hydrogels with stable mechanical properties and suitable network structures can be obtained. This ink is suitable for DLP 3D printing technology, enabling the fabrication of complex three-dimensional structures with micron-level precision.

[0104] AIA bio-inks exhibit excellent cell compatibility, supporting the survival, proliferation, and spread of various cell types, including L929 fibroblasts, bone marrow mesenchymal stem cells (BMSCs), and human immortalized keratinocytes (HaCaT). Even when loaded with high-density cells for DLP printing, it maintains micron-level printing precision and high cell viability, demonstrating significant application value in the field of biomanufacturing.

[0105] The AIA porous gel prepared from this bio-ink exhibited good in vivo biocompatibility and bioactivity in a diabetic wound model. Experimental results showed that the cell-loaded artificial skin (AIA-cell-laden) was superior to both HAMA porous gel and unloaded AIA porous gel in promoting wound closure, epithelial regeneration, and collagen deposition, effectively promoting tissue repair in diabetic wounds. This invention provides a new solution for developing high-performance photocurable bio-inks and diabetic wound repair materials based on natural polysaccharides.

Claims

1. A photosensitive agarose bio-ink for 3D printing, its preparation method and application, characterized in that, The bio-ink contains the following components: 2%~6% (m / v) itaconylated agarose, 0.1%~1.0% (m / v) photoinitiator, and 0~0.1% (m / v) light absorber.

2. The photosensitive agarose bio-ink according to claim 1, wherein the agarose is an agarose derivative containing photocrosslinkable double bonds obtained by modifying agarose with itaconic anhydride.

3. The photosensitive agarose bio-ink according to claim 1 or 2, characterized in that, The degree of modification of the itaconylated agarose is 10% to 90%.

4. The photosensitive agarose bio-ink according to claim 3, characterized in that, The degree of modification of the itaconylated agarose is 34% to 73%.

5. The photosensitive agarose bio-ink according to claim 1, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

6. The photosensitive agarose bio-ink according to claim 6, characterized in that, The concentration of the photoinitiator is 0.5% (m / v).

7. The photosensitive agarose bio-ink according to claim 1, characterized in that, The light absorber is selected from one or more of lemon yellow and brilliant blue.

8. The photosensitive agarose bio-ink according to claim 8, characterized in that, The concentration of the light absorber is 0.05% (m / v).

9. The photosensitive agarose bio-ink according to claim 1, characterized in that, The bio-ink maintains liquid fluidity at 20–40°C.

10. A method for preparing the photosensitive agarose bio-ink according to any one of claims 1-10, characterized in that, Includes the following steps: (1) Preparation of itaconylated agarose: Dissolve agarose in water, heat and stir until completely dissolved, and control the reaction temperature at 45-60℃; slowly add itacony anhydride to the solution, while maintaining the pH of the reaction system between 8 and 9 with an alkaline solution, and continue the reaction for 7-8 hours; after the reaction is completed, the product is purified by dialysis and freeze-dried to obtain itaconylated agarose. (2) Preparation of bio-ink: Dissolve the itaconylated agarose obtained in step (1) in a solvent to prepare a solution with a concentration of 2% to 6% (m / v); add 0.1% to 1.0% (m / v) of photoinitiator and 0% to 0.1% (m / v) of optional light absorber to the solution; mix evenly and remove bubbles to obtain the photosensitive agarose bio-ink.

11. The preparation method according to claim 11, characterized in that, The alkaline solution mentioned in step (1) is a 6M sodium hydroxide solution, and the solvent mentioned in step (2) is water or phosphate buffer (PBS).

12. The use of the photosensitive agarose bioink according to any one of claims 1-10 in the preparation of tissue engineering scaffolds, wound dressings, or drug delivery carriers, wherein, The bio-ink is formed using a surface-exposure-based photopolymerization 3D printing technology.

13. A wound dressing, characterized in that, The wound dressing is formed by 3D printing technology using the photosensitive agarose bio-ink as described in any one of claims 1-10.

14. The wound dressing according to claim 13, characterized in that, The bio-ink is loaded with active cells, which are selected from one or more of fibroblasts, keratinocytes, and bone marrow mesenchymal stem cells.

Images