A personalized auricle reconstruction 3d bioprinting composite ink

Abstract

The present application relates to the field of tissue engineering and 3D bioprinting technology, and particularly relates to a kind of personalized auricle reconstruction 3D bioprinting composite ink.The composite ink includes gelatin methacryl hydrogel matrix, acellular cartilage matrix micro-fragment, biomimetic reinforcing phase, natural visible light crosslinking system, dynamic covalent crosslinking component, patient's own auricle cartilage cells and adipose-derived mesenchymal stem cells, and slow-release microspheres loaded with transforming growth factor-β3.The composite ink occurs shear thinning under the action of nozzle shear, is crosslinked by 405nm visible light after deposition to form an auricle scaffold with elasticity, self-repairing property and controllable degradability, and the internal nutrient transport is improved by biomimetic microchannels.The composite ink can adapt to the personalized model generated by scanning data of the healthy side auricle of the patient, improve the printing accuracy of complex auricle structure, cell survival rate and long-term morphological stability, promote cartilage matrix deposition and new cartilage formation.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and 3D bioprinting technology, specifically to a personalized auricle reconstruction 3D bioprinting composite ink. Background Technology

[0002] Congenital microtia is a relatively common congenital craniofacial defect. Patients typically have missing auricle contours, incomplete structural development, or bilateral asymmetry of the auricles. This not only affects appearance but can also have long-term impacts on the patient's psychological state and social interactions. Current auricle reconstruction techniques mainly use autologous rib cartilage sculpting or porous polyethylene artificial scaffold implantation. Autologous rib cartilage reconstruction requires harvesting multiple rib cartilages from the patient's thoracic region, which has drawbacks such as large donor site trauma, long operation time, high difficulty in cartilage sculpting, and significant influence of the surgeon's experience on the reconstructed auricle shape. While artificial scaffolds can reduce the amount of rib cartilage harvested, their biocompatibility and tissue fusion ability are limited. Postoperative problems may include scaffold exposure, infection, fibrous encapsulation, and long-term morphological instability, making it difficult to simultaneously meet the requirements of personalized morphological reconstruction, tissue regeneration, and long-term safety.

[0003] With the development of tissue engineering and 3D bioprinting technology, bio-inks are formed by combining active seed cells with biodegradable biomaterials. Based on the CT or MRI data of the patient's healthy auricle, a model of the affected auricle is created. Complex anatomical structures such as the helix, antihelix, concha, and triangular fossa can be replicated through layer-by-layer deposition. This provides a new technical approach to reduce the trauma of rib cartilage harvesting and improve the symmetry of bilateral auricles. However, the overall thickness of the auricle is usually only 1-5 mm, and some helix edges are less than 1 mm. The printing material needs to have good fluidity under the shearing action of the nozzle to continuously pass through the 18-22G micro-nozzle, and it also needs to quickly recover high viscosity and complete the shaping after extrusion to avoid filament breakage, nozzle blockage, edge diffusion, and structural collapse. Therefore, the rheological properties, cross-linking speed, and shape retention ability of the bio-ink directly determine the replication accuracy of the personalized auricle structure.

[0004] Auricular reconstruction bio-inks also need to meet requirements such as cell compatibility, cartilage phenotype maintenance, mechanical support, and controllable degradation. Natural auricular cartilage has a certain degree of bending stiffness and elastic recovery ability. The printed scaffold needs to withstand surgical operations and subcutaneous tissue pressure in the early stage of implantation and maintain the three-dimensional contour of the auricle in the long term. After the new cartilage is gradually deposited, the carrier material needs to degrade at a rate adapted to cartilage regeneration. If the scaffold strength is insufficient or the degradation is too fast, it is easy to cause thinning of the helix edge and collapse of the overall shape. If the material degrades too slowly or remains for a long time, it may induce an inflammatory response. At the same time, the cross-linking components, light conditions, and material residues used in the printing process may also damage the seed cells, reduce the survival rate of cells after printing, and thus affect the secretion of cartilage matrix and the quality of regenerated tissue.

[0005] Existing collagen-based bioinks exhibit good cell affinity, but their mechanical strength and structural retention are relatively weak, typically requiring an additional protective shell for shaping. Traditional gelatin methacrylamide bioinks can improve printing accuracy through photocrosslinking, but the chemical photoinitiators used may cause cytotoxicity, and the elasticity and crack resistance of a single crosslinked network are insufficient. Decellularized cartilage matrix can retain extracellular matrix components of cartilage such as glycosaminoglycans, type II collagen, and elastin, but its rheological properties are difficult to control stably; it easily clogs nozzles at high concentrations and struggles to maintain its shape at low concentrations. Although synthetic polymers such as polycaprolactone and polylactic-co-glycolic acid copolymers can improve scaffold strength, their cell adhesion and cartilage induction capabilities are limited. Therefore, current technologies struggle to simultaneously achieve multiple properties such as high-precision printing, low-toxicity crosslinking, elastic support, dynamic degradation, cell survival, and cartilage regeneration within the same bioink system.

[0006] The technical problem this invention aims to solve is that existing auricular reconstruction bio-inks cannot simultaneously meet the requirements of printability, biocompatibility, mechanical stability, and cartilage regeneration. This invention provides a personalized 3D bioprinting composite ink for auricular reconstruction. Through the synergistic effect of a hydrogel matrix, decellularized cartilage matrix microfragments, a natural visible light cross-linking system, dynamic covalent cross-linking components, composite seed cells, and cartilage-inducing sustained-release components, the composite ink exhibits shear-thinning and continuous extrusion properties within the nozzle. After deposition, it can rapidly cross-link while maintaining the fine structure of the auricle, while reducing damage to cell activity during the cross-linking process. This improves the elasticity, self-repair ability, and long-term deformation resistance of the scaffold. Furthermore, through a biomimetic cartilage microenvironment, sustained release of cartilage-inducing factors, and microchannel nutrient delivery, it promotes cell survival within the scaffold, maintains the cartilage phenotype, and facilitates the deposition of new cartilage matrix. This ensures that the degradation process of the carrier material matches the auricular cartilage regeneration process, thereby solving the problems of existing bio-inks such as easy clogging, easy collapse, low cell survival rate, scaffold brittleness, insufficient internal cell nutrition, and poor long-term morphological stability after implantation. Summary of the Invention

[0007] To address the challenges of existing auricular reconstruction bio-inks in simultaneously achieving high performance in micro-nozzle extrusion, precise shaping of complex auricular structures, seed cell survival rate, scaffold elasticity, and long-term morphological stability, this invention provides a personalized auricular reconstruction 3D bioprinting composite ink. Through the synergistic configuration of a hydrogel matrix, decellularized cartilage matrix micro-fragments, a natural visible light crosslinking system, dynamic covalent crosslinking components, a composite seed cell system, and cartilage-inducing sustained-release components, the composite ink exhibits shear-thinning and continuous extrusion properties within the printing nozzle. After deposition, it can rapidly restore its structural support capacity and achieve final shaping upon visible light irradiation. Simultaneously, it provides a growth environment for the loaded cells that closely resembles the extracellular matrix of natural auricular cartilage. This results in a printed scaffold that combines high-precision shaping, low cytotoxicity, elastic recovery, self-repair, controllable degradation, and cartilage-inducing properties.

[0008] This invention also aims to address the problems of insufficient mechanical strength of traditional collagen-based inks, poor elasticity of traditional gelatin methacrylamide inks and potential cell damage from photoinitiation systems, easy nozzle clogging or deposition collapse when using decellularized cartilage matrix alone, and insufficient cell affinity of synthetic polymer scaffolds. By using composite cell co-culture, continuous release of growth factors, and biomimetic microchannel nutrient delivery, it improves the situation of cell hypoxia, insufficient nutrition, and uneven cartilage matrix deposition inside large-volume auricular scaffolds, so that the degradation process of carrier materials is connected with the process of new cartilage formation, thereby improving the long-term morphological maintenance ability after implantation of reconstructed auricles.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] This invention provides a personalized 3D bioprinting composite ink for auricular reconstruction, comprising a hydrogel matrix, a biomimetic reinforcing phase, a natural visible light crosslinking system, a dynamic covalent crosslinking component, a seed cell system, and a cartilage-inducing sustained-release component. The hydrogel matrix includes at least gelatin methacrylamide, the biomimetic reinforcing phase includes decellularized cartilage matrix microfragments, the natural visible light crosslinking system includes at least one of proanthocyanidins and riboflavin, the seed cell system includes autologous auricular chondrocytes and adipose-derived mesenchymal stem cells, and the cartilage-inducing sustained-release component includes sustained-release microspheres loaded with transforming growth factor-β3. The composite ink exhibits reduced viscosity and continuous extrusion under shearing action applied by the printing nozzle. After leaving the nozzle and depositing on the printing platform, it recovers its structural support properties and subsequently forms an elastic, self-healing, and controllable degradable auricular cartilage biomimetic scaffold upon visible light irradiation.

[0011] Furthermore, the hydrogel matrix also includes oxidized hyaluronic acid, carboxymethyl chitosan, and zwitterionic monomer MPC. The oxidized hyaluronic acid and carboxymethyl chitosan form a reversible dynamic cross-linking network within the hydrogel matrix, enabling the composite ink to exhibit stress relaxation, reversible bond breaking, and network recombination capabilities after printing. The zwitterionic monomer MPC is dispersed within the dynamic cross-linking network, which enhances the hydrophilicity, cell compatibility, and ion conductivity of the composite ink, and improves the material exchange conditions of cells within a high-water-content network.

[0012] Furthermore, the biomimetic reinforcing phase also includes at least one of polyvinyl alcohol, polyethylene oxide, nano-silica, and polycaprolactone microfibers. The polyvinyl alcohol is used to form a load-bearing skeleton inside the printing holder, the polyethylene oxide is used to improve the flow continuity of the composite ink in the nozzle, and the nano-silica and polycaprolactone microfibers are used to improve the bending support, elastic recovery and long-term shape maintenance of the printing holder, so that the composite ink can be smoothly extruded when subjected to shearing action, and maintain the curved contour and thin-walled structure of the corresponding areas of the helix, antihelix, concha cavity and triangular fossa after deposition.

[0013] Furthermore, the decellularized cartilage matrix microfragments are obtained from auricular cartilage through decellularization, pulverization, and enzymatic hydrolysis. The decellularization process removes immunogenic cell residues from the cartilage tissue and ensures that the resulting decellularized cartilage matrix retains no less than 90% of the natural glycosaminoglycans and total collagen components. The particle size of the decellularized cartilage matrix microfragments is 10-20 micrometers, and they are uniformly dispersed in the hydrogel matrix. The physical stacking between the microfragments enhances the support capacity of the composite ink in a static state, and the orientation and rearrangement of the microfragments under the shearing action of the nozzle reduces the extrusion resistance. Thus, while retaining the active components of the cartilage extracellular matrix, the composite ink is adapted to continuous printing with 18-22G micro-nozzles.

[0014] Furthermore, the natural visible light crosslinking system uses at least one of proanthocyanidins and riboflavin as a photosensitive crosslinking agent. Under 405nm visible light irradiation, it triggers the formation of a primary crosslinking network of gelatin methacrylamide, enabling the printed deposition layer to quickly solidify and reducing the diffusion, collapse, or interlayer misalignment of the auricular curved surface structure. The dynamic covalent crosslinking component forms a reversible bonded structure within the primary crosslinking network. When the printed scaffold is subjected to local compression during or after implantation, resulting in microcracks, the reversible bonded structure can reorganize and restore network continuity, thereby improving the problem of insufficient elasticity and brittle cracking under stress in single photocrosslinked hydrogel scaffolds.

[0015] Furthermore, the ratio of patient-derived autologous auricular chondrocytes to adipose-derived mesenchymal stem cells is 3:2, and the total cell density of both in the composite ink is 15-20 million per milliliter. The patient-derived autologous auricular chondrocytes are used to secrete type II collagen, glycosaminoglycans, and auricular cartilage extracellular matrix. The adipose-derived mesenchymal stem cells enhance the anti-apoptotic and proliferative capacity of the patient-derived autologous auricular chondrocytes through paracrine function and differentiate into chondrocytes under cartilage-inducing conditions to supplement the number of cells required for cartilage regeneration and reduce the need for large-scale material harvesting from the patient's residual auricular tissue.

[0016] Furthermore, the sustained-release microspheres loaded with transforming growth factor-β3 are uniformly dispersed between the hydrogel matrix and the seed cell system. The sustained-release microspheres are configured to continuously release transforming growth factor-β3 during the first 4 weeks after implantation of the printed scaffold. By continuously forming a cartilage-inducing microenvironment, the microspheres promote the secretion of type II collagen and glycosaminoglycans by the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells, accelerate the formation of new cartilage matrix, and match the deposition rate of new cartilage with the degradation rate of the composite ink carrier. This avoids premature degradation of the carrier causing auricular collapse or long-term carrier residue causing local inflammatory reactions.

[0017] Furthermore, after printing, the composite ink forms an interconnected biomimetic microchannel network with a porosity of 60%-70% and a pore diameter of 100-200 micrometers. The inner wall of the microchannel is modified with active polypeptides that can specifically bind to vascular endothelial cells, causing vascular endothelial cells in the surrounding tissues of the implantation site to adhere and migrate along the microchannels, guiding blood vessels to grow into the printed scaffold. This improves the delivery conditions of oxygen, nutrients, and metabolites within the large-volume auricular scaffold, reducing the probability of apoptosis or necrosis of cells in the central region of the scaffold due to hypoxia and insufficient nutrient supply.

[0018] Furthermore, the composite ink is configured to be extruded and printed in a sterile environment at 37°C through an 18-22G nozzle, and after deposition, it is irradiated with 405nm visible light for 30 seconds to complete rapid cross-linking and shaping. The printing resolution of the resulting auricular scaffold is no higher than 100 micrometers, and it can replicate the curved surface structure and thin-walled structure of the helix, antihelix, scaphoid fossa, concha, and triangular fossa. The flexural elastic modulus of the printed scaffold is 1.5-2.2MPa, so that the printed scaffold has the morphological support required in the early stage of implantation, the elastic recovery ability after being subjected to force, and the flexibility that is compatible with natural auricular cartilage.

[0019] Furthermore, the patient's autologous auricular cartilage cells are derived from the patient's residual auricular cartilage tissue. The composite ink is matched with the digital model of the affected auricle generated by mirroring the CT or MRI data of the patient's healthy auricle. By planning the printing path of the digital model of the affected auricle, the composite ink is deposited layer by layer according to the contour, thickness and curvature of different auricular anatomical regions. After the composite ink is mixed with cells, it is loaded into a sterile printing syringe. The printed auricular scaffold is placed in a biodegradable protective shell containing sterile nutrient solution and can be stably transported at 2-8℃ for no less than 72 hours. The survival rate of seed cells is no less than 80% within 7 days after printing.

[0020] In practical applications, autologous cartilage samples can be obtained from the patient's residual auricular cartilage tissue. The cartilage samples are enzymatically digested to separate primary chondrocytes, which are then expanded under culture conditions without exogenous animal-derived components. The expanded autologous auricular chondrocytes are mixed with adipose-derived mesenchymal stem cells in a set ratio, and then uniformly compounded with hydrogel matrix, biomimetic reinforcement phase, natural visible light crosslinking system, dynamic covalent crosslinking components, and cartilage-induced sustained-release components in a sterile environment to form a composite ink. At the same time, a digital model of the affected auricle is generated by mirroring the scanning data of the patient's healthy auricle. The printing path is planned according to the structural characteristics of the helix, antihelix, concha cavity, and triangular fossa, so that the composite ink is deposited layer by layer through micro-nozzles to form a personalized auricular scaffold.

[0021] This invention utilizes gelatin methacrylamide to form a high-water-content network suitable for cell embedding and cartilage matrix secretion, utilizes decellularized cartilage matrix microfragments to increase the static viscosity of the ink and provide signals from the natural cartilage extracellular matrix, and utilizes a biomimetic reinforcing phase to provide mechanical support for the initial implantation of the printed scaffold. The hydrogel matrix, decellularized cartilage matrix microfragments, and biomimetic reinforcing phase work together to make the composite ink flow easily under shear during the printing stage and maintain its shape rapidly after shearing stops, and to make the printed scaffold possess both cell affinity and structural stability.

[0022] The natural visible light crosslinking system enables the printed deposition layer to form a stable primary crosslinking network after being irradiated with visible light. The dynamic covalent crosslinking component further forms a reversible crosslinking network. The primary crosslinking network is used to maintain the spatial contour of the printed auricle, while the dynamic crosslinking network is used to absorb and release external stress and repair local microcracks. The two crosslinking networks interact with each other, enabling the printed support to maintain the curved surface and thin-walled structure of the auricle while possessing bending, resilience, and self-healing properties.

[0023] The patient's autologous auricular chondrocytes are responsible for the secretion of cartilage-specific matrix. Adipose-derived mesenchymal stem cells enhance the regenerative capacity of the cell system through paracrine and chondrogenic differentiation. Transforming growth factor-β3 sustained-release microspheres continuously provide cartilage induction signals in the early stages of implantation. Decellularized cartilage matrix provides the natural matrix environment required for cell adhesion and cartilage phenotype maintenance. The composite cell system, sustained-release induction system, and cartilage matrix environment work together to promote the deposition of type II collagen and glycosaminoglycans, thereby improving the rate of new cartilage formation and tissue uniformity.

[0024] The interconnected biomimetic microchannel network provides a pathway for the delivery of oxygen, nutrients, and metabolic products. The active peptides modified on the inner wall of the microchannels promote the adhesion of vascular endothelial cells and the ingrowth of blood vessels, allowing the nutrient supply from external tissues to gradually enter the scaffold. This improves the cell survival conditions in the central region of the large-volume auricular scaffold and enables the cartilage matrix to be uniformly deposited from the surface of the scaffold inward. As the new cartilage tissue gradually forms, the composite ink carrier degrades synchronously, and the load-bearing function of the scaffold is gradually transferred from the artificial cross-linked network to the new cartilage tissue.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Through the composite configuration of hydrogel matrix, decellularized cartilage matrix micro-fragments and biomimetic reinforcing phase, the composite ink has good shear thinning and continuous extrusion performance in 18-22G micro-nozzles, and quickly recovers its structural support capacity after leaving the nozzle. This can reduce the probability of nozzle blockage, filament breakage, deposition diffusion and local collapse, and improve the printing clarity of fine anatomical structures such as the helix edge, antihelix, concha cavity and triangular fossa.

[0026] 2. A natural visible light cross-linking system is constructed using at least one of proanthocyanidins and riboflavin, and rapid cross-linking and shaping are achieved using 405nm visible light. This reduces the damage caused to the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells by traditional chemical photoinitiation systems. At the same time, the dynamic covalent cross-linking network can bond and recombine after the scaffold develops microcracks, enabling the printed scaffold to have rapid shaping, elastic recovery, stress relaxation, and damage self-repair properties.

[0027] 3. A composite seed cell system is constructed using the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells. This system works in conjunction with transforming growth factor-β3 sustained-release microspheres and decellularized cartilage matrix to enable the patient's autologous auricular chondrocytes to continuously secrete cartilage-specific extracellular matrix. This allows the adipose-derived mesenchymal stem cells to replenish the number of regenerated cells through paracrine secretion and cartilage differentiation, thereby improving the cells' anti-apoptotic ability, cartilage phenotype stability, and the efficiency of new cartilage formation.

[0028] 4. By using an interconnected microchannel network with a porosity of 60%-70% and a pore diameter of 100-200 micrometers, the material transport conditions inside the scaffold are improved. The active peptides on the inner wall of the microchannels promote the adhesion of vascular endothelial cells and the ingrowth of blood vessels, so that the cells inside the auricular scaffold can obtain a more continuous supply of oxygen and nutrients, reduce the risk of hypoxia and necrosis of cells in the central area of ​​the large-volume cartilage scaffold, and improve the uniformity of the distribution of newly formed cartilage tissue inside the scaffold. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments. Those skilled in the art can reproduce the technical solution of the present invention and achieve its claimed technical effects based on the content disclosed in this specification. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial improvements and adjustments made based on the core concept of the present invention should fall within the scope of protection of the present invention.

[0030] Example 1: Preparation and application of personalized auricle reconstruction 3D bioprinting composite ink 1. Construction of the raw material system The personalized auricle reconstruction 3D bioprinting composite ink of this embodiment includes a hydrogel matrix, a biomimetic reinforcing phase, a natural visible light crosslinking system, a dynamic covalent crosslinking component, a seed cell system, and a cartilage-induced sustained-release component. The hydrogel matrix is ​​mainly composed of gelatin methacryloyl and compounded with oxidized hyaluronic acid, carboxymethyl chitosan, and zwitterionic monomer MPC. The biomimetic reinforcing phase includes decellularized cartilage matrix microfragments, and one or more of polyvinyl alcohol, polyethylene oxide, nano-silica, and polycaprolactone microfibers are selected according to the target mechanical properties of the printed scaffold. The natural visible light crosslinking system includes at least one of proanthocyanidins and riboflavin. The seed cell system includes autologous auricle chondrocytes and adipose-derived mesenchymal stem cells. The cartilage-induced sustained-release component includes sustained-release microspheres loaded with transforming growth factor-β3.

[0031] Gelatin methacrylamide is used to construct the main hydrogel network capable of embedding active cells, and forms a primary cross-linked structure that maintains the three-dimensional contour of the auricle after visible light irradiation; oxidized hyaluronic acid and carboxymethyl chitosan are used to form a dynamic covalent cross-linked network with reversible bonding ability, which allows the printing scaffold to release local stress when subjected to bending, compression or local contact, and restores the network continuity through dynamic bonding after microcracks are formed; zwitterionic monomer MPC is used to improve the hydrophilicity and ion exchange conditions of the composite ink, so as to maintain a microenvironment with high water content and suitable for nutrient delivery around the embedded cells.

[0032] Polyvinyl alcohol is used to form an auxiliary load-bearing skeleton, improving the overall support of the printed auricle in the early stages of implantation; polyethylene oxide is used to regulate the flow continuity of the hydrogel matrix in the nozzle, reducing the probability of filament breakage and uneven extrusion during printing; nano-silica, as an inorganic reinforcing filler dispersed in the hydrogel network, improves the bending stiffness and deformation resistance of the printed scaffold; polycaprolactone microfibers, as a biodegradable fiber reinforcing component, are used to limit excessive swelling or contour diffusion of the hydrogel network during long-term cultivation and early implantation.

[0033] All the above materials undergo biosafety and aseptic quality control before being used for cell loading. The hydrogel component should be free of harmful residues that could affect cell survival, the decellularized cartilage matrix should be free of immunogenic cell residues, the amount of natural visible light crosslinking agent added should be such that rapid shaping can be achieved under 405nm visible light conditions without reducing the activity of seed cells, and the amount of nano-silica and polycaprolactone microfiber added should be such that the mechanical properties of the scaffold can be improved without causing nozzle clogging.

[0034] 2. Preparation of decellularized cartilage matrix microfragments Auricular cartilage tissue suitable for preparing cartilage extracellular matrix was selected. The cartilage tissue was cleaned and non-cartilage appendages were removed to reduce the influence of fat, connective tissue and surface impurities on subsequent decellularization. Subsequently, the cartilage tissue was decellularized to remove cells and immunogenic cellular components. At the same time, the treatment intensity was controlled to avoid excessive loss of natural glycosaminoglycans, total collagen, type II collagen and elastin.

[0035] After the decellularization process is completed, the resulting cartilage matrix is ​​thoroughly washed to remove residual treatment solution and exfoliated cell components from the decellularization process. The washed cartilage matrix is ​​then dried at low temperature or freeze-dried to form a dry matrix that is easy to pulverize. The dried decellularized cartilage matrix is ​​mechanically pulverized and particle size classified, and cartilage matrix micro-fragments with a particle size of 10-20 micrometers are selected as the biomimetic reinforcing phase of the composite ink.

[0036] The decellularized cartilage matrix microfragments after particle size classification are subjected to moderate enzymatic hydrolysis to expose more active sites on the surface of the microfragments that can bind to the hydrogel matrix, while avoiding the complete degradation of the microfragments into soluble components that lose their physical thickening effect. The treated decellularized cartilage matrix microfragments should be able to be uniformly dispersed in the hydrogel matrix. Under static conditions, the physical stacking between particles improves the support capacity of the composite ink, and under nozzle shearing action, the extrusion resistance is reduced through particle orientation and rearrangement.

[0037] The residual cellular components, glycosaminoglycans, and total collagen in decellularized cartilage matrix microfragments were tested. The decellularization process should remove immunogenic cell residues and ensure that the retention of natural glycosaminoglycans and total collagen is not less than 90% of the corresponding components in the original cartilage tissue. If the test results do not meet this requirement, the intensity of the decellularization process should be adjusted or the processing time should be shortened to avoid excessive loss of cartilage active components due to the pursuit of cell removal.

[0038] While micro-fragments with a particle size of less than 10 micrometers can easily pass through the printing nozzle, their particle stacking and rheological regulation are weak, making it difficult to fully improve the structural support capacity after ink deposition. Although micro-fragments with a particle size of more than 20 micrometers can improve static viscosity, they are prone to local aggregation and nozzle clogging when using 20G or finer nozzles. Therefore, in this embodiment, the particle size of decellularized cartilage matrix micro-fragments is controlled at 10-20 micrometers to balance rheological regulation, nozzle passability, and cartilage microenvironment construction.

[0039] 3. Preparation of hydrogel matrix and dynamic cross-linked network Under aseptic conditions, a gelatin methacrylamide matrix solution is prepared to fully hydrate the gelatin methacrylamide and form a uniform, transparent or translucent hydrogel precursor. Oxidized hyaluronic acid is added while maintaining the flowability of the matrix solution, so that the oxidized hyaluronic acid is uniformly dispersed in the gelatin methacrylamide matrix solution. Then, carboxymethyl chitosan is added to gradually form a dynamic covalent bond structure between the oxidized hyaluronic acid and carboxymethyl chitosan.

[0040] The order of adding oxidized hyaluronic acid and carboxymethyl chitosan should avoid excessive local concentrations that could lead to premature gelation. During the mixing process, the shear strength should be controlled to ensure that the formed dynamic cross-linked network is evenly distributed in the gelatin methacryloyl matrix. The degree of dynamic cross-linking is determined by the ability of the composite ink to maintain its deposition morphology when stationary, to flow when subjected to nozzle shearing, to undergo stress relaxation after being subjected to external force, and to restore network continuity after the external force is removed.

[0041] During the formation of the dynamic cross-linked network, the zwitterionic monomer MPC is added, which is uniformly dispersed in the composite network formed by gelatin methacryloyl, oxidized hyaluronic acid and carboxymethyl chitosan. MPC does not bear the main mechanical support of the auricle scaffold. Its main purpose is to improve the hydrophilicity, water retention and ion exchange conditions of the hydrogel network, and reduce the decrease in cell activity caused by local material transfer restriction after high-density cell embedding.

[0042] The prepared hydrogel matrix should exhibit a significant shear-thinning trend, meaning it maintains high viscosity under low shear or static conditions, decreases viscosity after shearing intensifies, and recovers a certain structural strength after shearing is relieved. When the hydrogel matrix still exhibits significant flow after standing, its static support capacity can be improved by increasing the degree of dynamic cross-linking or increasing the dispersion of decellularized cartilage matrix microfragments. When the extrusion resistance of the hydrogel matrix in the printing nozzle is too high, the degree of dynamic cross-linking can be appropriately reduced or the extrusion continuity can be adjusted using polyethylene oxide.

[0043] 4. Combination of biomimetic reinforcing phases After forming the hydrogel matrix, polyvinyl alcohol is added according to the size of the target auricle model, the thickness of the helix edge, and the support performance required in the early stage of implantation. The polyvinyl alcohol should be evenly distributed in the hydrogel matrix and form a load-bearing structure that is interpenetrating or intertwined with the gelatin methacrylamide network, so that the printed scaffold can disperse local stress when it is bent, and reduce permanent deformation of the helix edge and the antihelix bulge.

[0044] Polyethylene oxide is added to adjust the continuous extrusion performance of the composite ink. The amount of polyethylene oxide added is not primarily aimed at increasing the final strength of the print holder, but rather at ensuring that the composite ink can stably pass through the 18-22G nozzle, and that the extruded filament is continuous and without significant fluctuations in thickness. When the composite ink is printed through a 20G nozzle, the amount of polyethylene oxide is preferably determined through rheological testing, so that the high shear force within the nozzle can reduce the ink viscosity, and the ink can quickly recover its support state after extrusion.

[0045] Before addition, nano-silica is uniformly dispersed to avoid the formation of large agglomerates. The dispersed nano-silica is then slowly added to the hydrogel matrix, allowing it to be uniformly distributed in the gelatin methacrylamide network and the dynamic covalent cross-linked network. The interfacial interaction between the inorganic particles and the polymer network improves the bending stiffness and compressive resilience of the printed scaffold, and makes the mechanical properties of the scaffold close to those of natural auricular cartilage.

[0046] Polycaprolactone is added in the form of microfibers. The size of the microfibers should be suitable for passing through the selected printing nozzle and to avoid the formation of tangles between the fibers. The polycaprolactone microfibers form a dispersed fiber-reinforced structure in the hydrogel matrix, which limits the excessive swelling and morphological diffusion of the hydrogel in the early stage of scaffold implantation. As the newly formed cartilage matrix is ​​gradually deposited, the polycaprolactone microfibers and other biodegradable carriers gradually degrade, so that the load-bearing function of the scaffold is transferred to the newly formed cartilage tissue.

[0047] Decellularized cartilage matrix microfragments with a particle size of 10-20 micrometers were added to the above-mentioned composite hydrogel and mixed evenly. The decellularized cartilage matrix microfragments not only participated in the formation of static support structure as rheology regulating particles, but also provided cartilage extracellular matrix signals related to glycosaminoglycans, collagen and elastin to the embedded cells as biomimetic functional components, thereby avoiding the reduction of cell affinity by simply increasing the content of synthetic polymers to obtain mechanical properties.

[0048] 5. Construction of natural visible light crosslinking systems After completing the compounding of the hydrogel matrix and the biomimetic reinforcing phase, proanthocyanidins, riboflavin, or a combination of both are added to uniformly disperse the natural photosensitizing components in the composite ink matrix. The amount of proanthocyanidins and riboflavin added is controlled based on the fact that the composite ink can form a stable primary cross-linking network after being irradiated with 405nm visible light for 30 seconds, and does not cause a significant decrease in the survival rate of seed cells after printing.

[0049] This embodiment does not add traditional high-concentration chemical photoinitiators. Instead, it uses natural photosensitive components to trigger gelatin methacryl crosslinking under visible light irradiation, reducing the damage to seed cells caused by ultraviolet light and traditional chemical photoinitiators. Visible light irradiation is performed immediately after printing to fix the newly deposited helix, antihelix, and concha cavity structures before significant flow or collapse occurs.

[0050] The primary cross-linking network formed by visible light plays a role in immediate shaping after printing, while the dynamic covalent cross-linking network formed by oxidized hyaluronic acid and carboxymethyl chitosan plays a role in stress relaxation and damage repair. The two cross-linking networks work together to give the printed scaffold both spatial stability to maintain the anatomical contour of the auricle and flexibility and resilience close to that of natural auricular cartilage.

[0051] The cross-linked samples were subjected to bending and recovery tests. When the sample could not recover its original shape after bending, the integrity of the dynamic cross-linking network should be improved or the composition of the biomimetic reinforcing phase should be adjusted. When the sample had high strength but brittle fracture occurred, the strength should not be increased by simply increasing the degree of photocrosslinking, but the stress dissipation capacity should be improved by using a dynamic covalent cross-linking network. When the sample was too soft and difficult to maintain its three-dimensional shape, the reinforcing effect formed by polyvinyl alcohol, nano-silica, or polycaprolactone microfibers could be improved.

[0052] 6. Addition of TGF-β3 sustained-release microspheres Prepare sustained-release microspheres loaded with transforming growth factor-β3, so that transforming growth factor-β3 is embedded or bound inside the biodegradable microspheres and can be released gradually as the microspheres degrade; the sustained-release microspheres should have a particle size suitable for dispersion in the composite hydrogel, should not cause significant aggregation in the printing nozzle, and should not reduce the survival rate of seed cells due to local high concentration.

[0053] TGF-β3 sustained-release microspheres were added to the composite hydrogel that had been compounded but not yet added to the seed cells, so that the sustained-release microspheres were evenly distributed between the hydrogel matrix, decellularized cartilage matrix micro-fragments and the biomimetic reinforcing phase. During the mixing process, excessive shearing was avoided on the sustained-release microspheres to reduce the probability of microsphere rupture and premature release of growth factors.

[0054] TGF-β3 sustained-release microspheres are configured to continuously release transforming growth factor-β3 during the first 4 weeks after scaffold implantation, maintaining a continuous cartilage-inducing environment inside the printed scaffold during the early stages of cartilage regeneration. The continuous release method prevents the rapid diffusion or inactivation of free TGF-β3 in a short period of time, and enables the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells to continuously secrete type II collagen and glycosaminoglycans.

[0055] The TGF-β3 sustained-release process works in conjunction with the degradation process of the carrier material. In the early stages of implantation, the cross-linked network formed by the composite ink and the biomimetic reinforcement phase provide morphological support. In the first four weeks, the continuous release of growth factors promotes the formation of cartilage matrix. Subsequently, as the newly formed cartilage gradually matures, the carrier material gradually degrades, causing the scaffold's load-bearing structure to gradually transform from an artificial hydrogel network into newly formed cartilage tissue.

[0056] 7. Acquisition and expansion of seed cells Approximately 0.5 grams of autologous cartilage sample was taken from the patient's residual auricular cartilage tissue. The cartilage sample was washed and enzymatically digested to obtain primary auricular chondrocytes. The sampling process was based on the principle of obtaining a sufficient amount of primary cells while avoiding additional significant trauma to the patient. The separated primary auricular chondrocytes were cultured in serum-free chondrocyte expansion medium.

[0057] Primary auricular chondrocytes were expanded under sterile conditions for 2-3 weeks to increase the number of primary cells to meet the requirements for printing auricular scaffolds. During the expansion process, exogenous animal-derived components that may cause immune responses were avoided. Cell morphology, proliferation status, contamination and cartilage phenotype were tested, and cells with good activity and no contamination were selected for the preparation of composite ink.

[0058] Adipose-derived mesenchymal stem cells were obtained and expanded. Cell activity and differentiation potential of the expanded adipose-derived mesenchymal stem cells were tested. In this embodiment, adipose-derived mesenchymal stem cells were used to enhance the anti-apoptotic ability of auricular chondrocytes through paracrine function and differentiate into chondrocytes in a chondrogenic microenvironment composed of TGF-β3 and decellularized chondrocyte matrix.

[0059] The patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells were mixed at a cell ratio of 3:2. This allowed the autologous auricular chondrocytes to undertake the main function of cartilage-specific matrix secretion, while the adipose-derived mesenchymal stem cells were responsible for paracrine support and replenishing the number of chondrocytes. Compared with using only autologous auricular chondrocytes, this co-culture system can reduce the amount of residual auricular cartilage tissue obtained and improve the expansion capacity and anti-apoptotic capacity of the seed cell system.

[0060] 8. Mixing of cells with composite hydrogel matrix The composite hydrogel matrix, after material compounding and sterility testing, was placed at a temperature suitable for maintaining fluidity. Before adding seed cells, it was confirmed that the composite hydrogel did not have obvious particle aggregation, premature gelation, or bubble aggregation. The patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells, mixed in a 3:2 ratio, were slowly added to the composite hydrogel matrix to ensure that the cells were evenly dispersed in the composite ink.

[0061] The final cell density in the composite ink is controlled at 15-20 million per milliliter to ensure that there are enough cartilage regeneration cells in the printed scaffold, while avoiding excessively high cell density that could lead to increased local viscosity of the ink, uneven cell distribution, and intensified competition for oxygen and nutrients. When used for smaller-sized local repair structures of the auricle, the cell density can be adjusted within the range of 10-20 million per milliliter according to the printing volume.

[0062] The cell mixing process was carried out using a low-shear method to avoid cell membrane damage, TGF-β3 sustained-release microsphere rupture, or agglomeration of decellularized cartilage matrix microfragments caused by prolonged and vigorous stirring. After mixing, a small amount of composite ink was used to check the cell distribution to confirm that the patient's autologous auricular cartilage cells and adipose-derived mesenchymal stem cells were evenly distributed in different sampling areas.

[0063] The mixed composite ink is loaded into a sterile printing syringe, minimizing air bubbles during loading. After loading, the syringe is kept in conditions suitable for cell survival and ink flow, and printing is completed within a specified time. If material stratification, cell sedimentation, or particle aggregation occurs after the composite ink is placed in the syringe, the static viscosity and dynamic cross-linking degree of the hydrogel matrix should be readjusted.

[0064] 9. Establishment of personalized digital auricle models Obtain CT, MRI, or 3D scan data of the patient's healthy auricle, perform contour segmentation and 3D reconstruction on the healthy auricle data to form a digital model of the healthy auricle including the helix, antihelix, scaphoid fossa, concha, triangular fossa, and surrounding structures of the tragus; mirror the healthy auricle model according to the relationship between the patient's head midline and the auricle position to obtain a personalized digital auricle model that matches the affected side.

[0065] The mirror model is adapted to the patient's individual anatomical conditions by considering the subcutaneous space behind the affected ear, the local skin coverage, and the size of the healthy ear. The overall size of the model, the curvature of the outer edge of the helix, the height of the antihelix, and the depth of the concha are matched with the patient's individual anatomical conditions. The overall thickness of the auricle model is controlled within the range of 1-5 mm according to different anatomical regions, and the edge of the helix can be set as a thin-walled structure of less than 1 mm.

[0066] Printing path planning is performed on personalized digital auricle models, dividing the auricle framework into an outer contour preservation area, a local load-bearing area, a cartilage regeneration area, and a microchannel nutrient delivery area. The continuity of the path is improved in areas prone to deformation, such as the helix and antihelix, and the support path is optimized in concave areas such as the concha and triangular fossa to avoid structural collapse caused by local suspension or excessive interlayer spacing during the printing process.

[0067] Based on the requirements of biomimetic microchannels, interconnected channel structures are set up inside the model, and the porosity of the scaffold is controlled at 60%-70%, and the microchannel pore size is controlled at 100-200 micrometers. This allows the channels to provide pathways for the delivery of oxygen, nutrients and metabolites while maintaining overall mechanical support. For local scaffolds that focus on cell migration and proliferation, the porosity can be further increased while ensuring structural stability.

[0068] The digital model with the completed path planning is simulated and printed for inspection. The focus is on checking for path interruptions, material accumulation, or local thinning in areas such as the helix edge (less than 1 mm), the junction of the helix crus and the concha, the bifurcation of the upper and lower crus of the antihelix, and the triangular fossa. Corrections are made before the actual printing.

[0069] 10. Functionalization of the Inner Wall of Bionic Microchannels The hydrogel pathway used to construct microchannels is pre-introduced with active peptides that can specifically bind to vascular endothelial cells, allowing the active peptides to be deposited on the inner wall of the microchannel along with the composite ink. The amount of active peptides added is determined to promote the adhesion and migration of vascular endothelial cells on the inner wall of the microchannel, without altering the main rheological and crosslinking properties of the composite ink.

[0070] Active peptides can bind to functional groups in the hydrogel matrix, or remain on the channel surface through affinity interactions with decellularized cartilage matrix microfragments or microchannel wall materials. After scaffold implantation, vascular endothelial cells in the surrounding tissue can migrate into the scaffold along the functionalized microchannels, allowing peripheral blood vessels to gradually grow into the scaffold.

[0071] The microchannels are not used to enable the auricular cartilage to form a large number of mature blood vessels, but to improve the nutrient delivery conditions inside the scaffold in the early stages of implantation and to promote the establishment of a material exchange interface between the surrounding tissue and the scaffold. As new cartilage gradually forms, the microchannels can continuously provide oxygen and nutrients to the cells inside the scaffold and remove metabolic products, reducing the probability of apoptosis or necrosis of cells in the central region due to hypoxia and insufficient nutrition.

[0072] 11. 3D Bioprinting A sterile printing syringe containing composite ink is installed in a custom auricle bio-3D printer. The forming chamber of the printing equipment is kept sterile and the ambient temperature is controlled at 37°C to maintain the extrudable state of the composite ink and the activity of seed cells. A 18-22G nozzle is selected according to the local structure of the digital model, with a 20G nozzle being preferred to complete the printing of the auricle support while balancing printing accuracy and continuous extrusion performance.

[0073] Before formal printing, conduct short-distance trial extrusion to observe whether the extruded filament is continuous, whether the diameter is uniform, and whether the material can maintain its shape after extrusion stops. When the extrusion pressure continues to rise or the filament is interrupted, check whether the decellularized cartilage matrix microfragments, nano-silica, and polycaprolactone microfibers aggregate. When the material flattens rapidly after extrusion, check whether the static viscosity and dynamic cross-linking degree of the hydrogel meet the requirements.

[0074] Following the printing path of a personalized digital auricle model, composite ink is deposited layer by layer. First, the bottom contour and basic load-bearing layer of the auricle are formed, and then the concha cavity, antihelix, triangular fossa and helix structure are gradually formed. This ensures continuous contact between adjacent printed layers and avoids the formation of interlayer separation interfaces that affect the overall mechanical properties. During the printing process, a biomimetic microchannel network with a porosity of 60%-70% and a pore size of 100-200 micrometers is formed simultaneously.

[0075] The printing time for the entire personalized auricular support is controlled at 8-10 minutes, and the printing resolution is no higher than 100 micrometers. Through the shear-thinning properties of the composite ink, its viscosity is reduced in the nozzle and it is continuously extruded. Through the particle stacking of decellularized cartilage matrix micro-fragments, dynamic cross-linking network and biomimetic reinforcing phase, it quickly recovers its support capacity after leaving the nozzle, thereby reducing the diffusion of the helix edge, the filling of the concha cavity and the collapse of the antihelix bulge.

[0076] 12. Visible light crosslinking and shaping Immediately after printing, the auricular support is irradiated with 405nm visible light for 30 seconds, so that the gelatin methacrylamide forms a primary cross-linked network under the action of at least one natural photosensitive component among proanthocyanidins and riboflavin, and fixes the spatial contours of the helix, antihelix, concha, and triangular fossa.

[0077] The irradiation process should cover different surfaces and concave areas of the auricular scaffold to avoid insufficient cross-linking at the bottom of the concha or below the antihelix; the irradiation intensity should be such that the structure can be fixed within 30 seconds without causing a significant increase in scaffold temperature or a decrease in cell activity, and the scaffold strength should not be increased by extending the irradiation time alone.

[0078] After visible light crosslinking, the dynamic covalent crosslinking network formed by oxidized hyaluronic acid and carboxymethyl chitosan continues to exist in the primary crosslinking network. When the scaffold is subjected to external force, the dynamic bonds can undergo reversible breakage to dissipate stress. After the external force is removed, the dynamic bonds reform, restoring the network continuity of the scaffold. Thus, the auricular scaffold possesses both initial morphological stability, bending flexibility, and micro-damage repair capabilities.

[0079] 13. Post-printing quality inspection The appearance and size of the cross-linked auricular framework were inspected. The integrity of the helix edge, antihelix crus, concha cavity and triangular fossa were checked. The surface of the framework was checked for obvious discontinuities, material accumulation, closure of channels or local collapse. The printed framework was compared with the personalized digital model in three dimensions to confirm that the main anatomical structures could be replicated and that the overall shape matched the mirror image model of the patient's healthy ear.

[0080] The porosity and microchannel size of the stent were tested to confirm that the porosity of the preferred embodiment was 60%-70%, the microchannel pore size was 100-200 micrometers, the channels remained connected and did not experience large-area closure due to cross-linking shrinkage or material flow; the distribution of active peptides on the inner wall of the channel was tested to confirm that the active peptides were located on the channel surface that could contact vascular endothelial cells.

[0081] The flexural modulus of the printed scaffold was tested and controlled within the range of 1.5-2.2 MPa, preferably close to 1.8 MPa. When the flexural modulus was lower than the target range, the supporting effect of the biomimetic reinforcing phase or the degree of primary crosslinking was increased. When the flexural modulus was higher than the target range and the scaffold exhibited obvious brittleness, excessive crosslinking was reduced and the stress dissipation effect of the dynamic crosslinking network was improved.

[0082] The stent was subjected to repeated bending and local compression tests. After the external force was removed, it was observed whether the stent could restore the original auricular contour. When the stent developed microcracks, the crack edges were kept in contact and the network continuity was observed under conditions suitable for dynamic bond exchange to evaluate the self-healing performance of the dynamic covalent crosslinked network.

[0083] Cell viability was sampled from the printed scaffolds to observe the cell distribution and survival in the scaffold surface, central region, and around the microchannels. The survival rate of seed cells should be no less than 80% within 7 days after printing, preferably achieved by using low-toxicity visible light crosslinking and a dynamic hydrogel microenvironment to achieve a cell survival rate of over 90%.

[0084] 14. Cold chain preservation and transportation The qualified personalized auricular support is placed in a biodegradable protective shell containing sterile nutrient solution. The internal shape of the protective shell matches the printed auricular support to limit displacement, compression or partial folding of the support during transportation, while allowing the sterile nutrient solution to contact the surface of the support.

[0085] The protective shell containing the auricular support is placed in a cold chain transport box at 2-8℃ to ensure that the printed support maintains structural integrity and cell activity during a storage and transport period of no less than 72 hours. During the transport process, the support is prevented from freezing directly, and the effects of continuous vibration and local compression on the helix edge and the helix ridge structure are reduced.

[0086] After transport, the stent is re-inspected to check whether the auricle contour has been significantly deformed, whether the nutrient solution inside the protective shell is contaminated or abnormally turbid, and to perform a bioactivity test on the surface and internal cells of the stent. After passing the re-inspection, the stent can be used for subsequent in vitro culture, animal implantation verification, or clinical application research that meets medical management requirements.

[0087] 15. Implantation and Postoperative Observation During implantation verification, a subcutaneous receiving space matching the personalized auricular framework is formed behind the recipient's ear according to the size of the framework. The printed auricular framework is removed from the biodegradable protective shell and implanted into the receiving space, so that the direction of the helix, concha, and earlobe is consistent with the personalized design direction. After adjusting the position, fixation and incision are completed.

[0088] In the early stages of implantation, the three-dimensional shape of the auricle is maintained by a combination of gelatin methacrylamide primary cross-linking network, dynamic covalent cross-linking network, polyvinyl alcohol load-bearing structure, nano-silica and polycaprolactone microfibers. TGF-β3 sustained-release microspheres continuously release chondrogenic factors in the first 4 weeks, and decellularized cartilage matrix provides natural cartilage extracellular matrix signals to seed cells. Adipose-derived mesenchymal stem cells support the survival and proliferation of autologous auricular chondrocytes through paracrine and chondrogenic differentiation.

[0089] After implantation, the biomimetic microchannel network establishes a material exchange pathway between the surrounding tissues and the scaffold. The active peptides on the inner wall of the channel promote the adhesion and migration of vascular endothelial cells, enabling nutrients to be delivered into the scaffold. As type II collagen and glycosaminoglycans are gradually deposited, new cartilage tissue is formed inside the scaffold and undertakes more and more mechanical support.

[0090] Subsequent three-dimensional contour and imaging follow-up can be conducted at 1 month, 3 months, 6 months and 12 months to evaluate the maintenance of the auricular framework morphology, distribution of new cartilage, degree of material degradation and local inflammatory response; when conditions permit in animal experiments or clinical research, histological staining can be used to evaluate the formation of type II collagen, glycosaminoglycans and cartilage lacunae.

[0091] Example 2: PVA, GelMA and Nano-Silica Reinforced Composite Ink In this embodiment, polyvinyl alcohol, gelatin methacrylamide, and nano-silica constitute the main material system. Polyvinyl alcohol forms the load-bearing framework, gelatin methacrylamide forms the cell embedding and visible light cross-linking network, and nano-silica is dispersed in the composite network formed by the two to improve the biomechanical properties of the scaffold. Decellularized cartilage matrix microfragments with a particle size of 10-20 micrometers are added to improve the biomimetic properties and shear thinning properties of cartilage.

[0092] The compatibility between polyvinyl alcohol, gelatin methacrylamide, and nano silica was adjusted based on the digital model of the auricle and the nozzle specifications. When printing thinner auricular edges, the main control objectives were to improve the shape retention of ink after deposition and the interlayer bonding. When printing the bottom of the concha cavity and the main body of the support, the main control objectives were to improve the overall bending support and the stability of the internal channels.

[0093] The composite ink is loaded into the printing syringe and printed using an 18G nozzle for multi-scale porous structure printing. When the printing scaffold is mainly aimed at cell migration and proliferation, the porosity can reach more than 80%. When printing a complete auricular scaffold while taking into account morphological support and internal nutrient delivery, it is preferable to control the porosity at 60%-70% to avoid the reduction of the load-bearing capacity of the helix and antihelix structure due to excessive porosity.

[0094] After printing, the scaffold was irradiated with 405nm visible light for 30 seconds to complete cross-linking. Cell adhesion, cell proliferation and mechanical properties were tested on the resulting scaffold. The composite ink formed by PVA, GelMA and nano silica can make the mechanical properties of the scaffold closer to those of human ear cartilage. Its tensile modulus matching degree can be increased by more than 40%, and the cell adhesion rate and proliferation rate can be increased by 30%-50% compared with traditional scaffolds.

[0095] This embodiment is suitable for auricular scaffolds that require high mechanical support in the early stages of implantation. It improves the support performance by using polyvinyl alcohol and nano-silica, and maintains cell compatibility by using gelatin methacrylamide and decellularized cartilage matrix, so that the material enhancement effect and the construction of the cartilage regeneration microenvironment can be achieved simultaneously.

[0096] Example 3: OHA, CMCS and MPC dynamic network composite inks In this embodiment, a hydrogel matrix is ​​formed using gelatin methacrylamide, oxidized hyaluronic acid, carboxymethyl chitosan, and zwitterionic monomer MPC. Gelatin methacrylamide forms a visible light-curable network, oxidized hyaluronic acid and carboxymethyl chitosan form a dynamic covalent cross-linked network, and MPC is used to improve hydrophilicity, cell-friendly properties, and ion transport conditions.

[0097] In the preparation process, a uniform gelatin methacrylamide matrix is ​​first formed, then oxidized hyaluronic acid and carboxymethyl chitosan are added to form a dynamic cross-linked structure. Subsequently, MPC, decellularized cartilage matrix microfragments, at least one of proanthocyanidins and riboflavin, TGF-β3 sustained-release microspheres, and seed cell system are added.

[0098] The composite ink in this embodiment can decrease in viscosity under nozzle shearing action. After shearing stops, it can restore its structural support capacity through dynamic bond reforming and decellularized cartilage micro-fragments re-stacking. The primary network formed after visible light crosslinking is used to fix the auricle contour, and the dynamic network is used to dissipate stress when the support is bent or compressed.

[0099] When simulating cuts or local cracks in the stent, the two sides of the crack are brought into contact again to observe whether the dynamic cross-linking network can restore continuity. Compared with a stent that only has an irreversible optical cross-linking network, the stent in this embodiment can reduce the probability of local cracks continuing to expand and improve the resilience of the helix edge after being compressed.

[0100] Example 4: Composite ink of GelMA, PEO, PCL and decellularized cartilage matrix In this embodiment, gelatin methacrylamide is used as the cell loading and photocrosslinking matrix, polyethylene oxide is used to improve the extrusion continuity during the printing process, polycaprolactone microfibers are used to improve the initial mechanical stability of the scaffold, and decellularized cartilage matrix microfragments are used to provide natural cartilage extracellular matrix components and regulate static viscosity.

[0101] During preparation, polyethylene oxide is uniformly added to the gelatin methacrylamide matrix to form a stable and continuous extruded filament in the ink within an 18-22G nozzle. Then, polycaprolactone microfibers that can pass through the nozzle are added to disperse the microfibers within the hydrogel network. Finally, decellularized cartilage matrix microfragments with a particle size of 10-20 micrometers are added.

[0102] This embodiment utilizes polyethylene oxide to reduce the risk of filament breakage during continuous printing, uses polycaprolactone microfibers to limit swelling and deformation of the scaffold in the early stages of implantation, and uses decellularized cartilage matrix to induce seed cells to maintain the natural cartilage phenotype, thereby avoiding insufficient cell adhesion due to relying solely on synthetic materials such as polycaprolactone to obtain support performance.

[0103] This composite ink is suitable for printing areas such as the outer edge of the helix, the main trunk of the antihelix, and the edge of the concha cavity, which require both thin-wall molding and long-term shape maintenance. When planning the printing path, the fiber reinforcement path and the hydrogel deposition path can be adjusted according to the load-bearing requirements of different areas.

[0104] Example 5: Co-cultured cells and TGF-β3 sustained-release system In this embodiment, the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells are used to form a seed cell system. The two are mixed at a cell ratio of 3:2, and the mixed cells are added to a composite hydrogel matrix to achieve a final cell density of 15-20 million cells per milliliter.

[0105] Autologous auricular chondrocytes are used to maintain the specificity of auricular cartilage and secrete type II collagen and glycosaminoglycans. Adipose-derived mesenchymal stem cells enhance the anti-apoptotic ability of chondrocytes through paracrine function and differentiate into chondrocytes under continuous induction by TGF-β3 to supplement the number of cells required for cartilage regeneration.

[0106] Low concentrations of TGF-β3 sustained-release microspheres were added to the composite ink to enable the growth factor to be continuously released during the first 4 weeks after implantation. During the culture or implantation verification process, the formation of type II collagen and glycosaminoglycans at different time points were detected to evaluate the synergistic effect between the co-cultured cell system and the sustained-release induction system.

[0107] After co-culturing autologous chondrocytes and adipose-derived mesenchymal stem cells and adding TGF-β3 sustained-release microspheres, the elastic modulus of the newly formed cartilage can reach 90% of that of natural cartilage after 8 weeks of implantation, and its regeneration rate is faster than that of the system using only a single chondrocyte.

[0108] Example 6: Bionic Microchannel Auricular Support In this embodiment, interconnected biomimetic microchannels are constructed in a personalized digital auricle model. The porosity is set to 60%-70%, the microchannel pore size is set to 100-200 micrometers, and the microchannels extend from the surface of the support to the interior to form a material transport network covering the helix, antihelix, concha cavity, and central region of the support.

[0109] An active polypeptide that can specifically bind to vascular endothelial cells is added to the composite ink used to form the inner wall of the microchannel, so that the active polypeptide is retained on the channel surface after printing and cross-linking; the scaffold is placed in a culture environment containing vascular endothelial cells or implanted for verification, and the adhesion and migration of vascular endothelial cells on the inner wall of the channel are observed.

[0110] A dense scaffold without biomimetic microchannels was set up as a control to compare cell survival in the surface and central regions of the scaffold. In the dense scaffold without microchannels, oxygen and nutrients mainly diffuse from the surface of the scaffold to the interior, and cell activity is prone to decline in the central region of the scaffold. In contrast, the scaffold with interconnected microchannels can improve the internal nutrient delivery conditions.

[0111] After implantation of a traditional non-porous scaffold, only cells within a surface area of ​​about 1 mm can survive. However, after setting up biomimetic microchannels, the cell survival rate inside the scaffold can be maintained at over 85% for a long time. This indicates that a porosity of 60%-70%, a channel pore size of 100-200 micrometers, and functionalization of the inner wall can jointly improve the survival conditions of cells inside a large-volume auricular scaffold.

[0112] Example 7: Personalized ear printing and cold chain transportation We acquire 3D scan data of the patient's healthy auricle, generate a model of the affected auricle through mirroring, and perform surface smoothing, local thickness correction, and printing path planning on the model to preserve anatomical structures such as the helix, antihelix, concha, and triangular fossa, and to match the size of the printed scaffold to the implantation space behind the patient's affected ear.

[0113] The composite ink was printed in a sterile chamber at 37°C using a 20G nozzle. The printing process was controlled to take 8-10 minutes. After printing, the auricle support was irradiated with 405nm visible light for 30 seconds to quickly set the shape. The printed support was then 3D scanned, and the scanned model was superimposed on the original digital model for comparison to evaluate the contour similarity and the accuracy of local structure replication.

[0114] When the rheological properties of the composite ink, the printing path, and the nozzle condition meet the requirements, the printing resolution can reach below 100 micrometers; after rheological property optimization, the printing resolution can reach up to 50 micrometers, the accuracy of replicating the fine structure of the auricle can exceed 95%, and the similarity between the overall shape and the mirror model of the patient's healthy ear can reach more than 98%.

[0115] The qualified printed scaffolds were placed in a biodegradable protective shell containing sterile nutrient solution and stored and transported at 2-8℃ for 72 hours. The scaffold morphology, bending elasticity and cell activity were tested before and after transport. The protective shell was used to limit the folding of the helix edge or the deformation of the antihelix caused by transport vibration, and the sterile nutrient solution was used to maintain the survival environment of the cells on the surface and inside the scaffold.

[0116] Example 8: In vitro performance verification 1. Rheological property testing Rheological properties of cell-free composite inks were tested to detect viscosity changes under different shear conditions and viscosity recovery after the shear conditions were removed. Qualified composite inks should have the ability to maintain the deposition morphology when at rest, exhibit a significant decrease in viscosity under simulated nozzle extrusion shear, and recover some structural strength after the shear conditions are removed.

[0117] Extrusion tests were conducted using 18G, 20G, and 22G nozzles to observe the extrusion continuity, filament uniformity, and clogging under different nozzle conditions. When the 18G nozzle could extrude continuously but the 22G nozzle was prone to clogging, nozzle compatibility could be improved by reducing the aggregation of large-particle-size reinforcing components or increasing the regulating effect of polyethylene oxide.

[0118] 2. Print conformity test Test models containing straight lines, arcs, cross layers, thin walls, and recessed structures were printed to observe the diffusion of the printed filaments after deposition, the bonding between adjacent layers, and the retention of the recessed structures. Further models of the helix edge, antihelix bifurcation, and concha cavity were printed to test whether 0.5 mm-level structures could be stably replicated.

[0119] After printing, the material was irradiated with 405nm visible light for 30 seconds. The structural dimensions before and after irradiation were measured to evaluate the effect of visible light crosslinking on the rapid shaping of the deposited layer. When blurred edges appeared after printing, the static viscosity, the dispersion state of decellularized cartilage matrix microfragments, and the light coverage were checked.

[0120] 3. Cell viability test Cell viability was tested using composite ink containing seed cells before printing, immediately after printing, and on days 1, 3, and 7 of culture. Samples were taken from the surface of the scaffold, the central region, and around the microchannels. The basic requirement was met when the cell viability was not less than 80% on day 7 after printing.

[0121] By adopting a natural visible light crosslinking system and a dynamic hydrogel network, the damage to seed cells during the printing process is reduced. The optimized crosslinking system can increase the cell survival rate from about 70% in the traditional system to more than 92%. When the cell activity is lower than the target value, the illumination time, the concentration of natural photosensitizing components, the nozzle shearing action, and the cell mixing process are checked.

[0122] 4. Mechanical property testing The printed composite ink is used to make standard mechanical specimens or specimens are cut from different regions of the auricle support to test the flexural modulus, compression recovery performance and shape retention after repeated bending; the flexural modulus is controlled in the range of 1.5-2.2 MPa, preferably about 1.8 MPa.

[0123] Apply local compression to the sample and then remove the external force to observe whether the sample can rebound quickly; pre-inflate microcracks in the sample and re-contact the crack interfaces to observe whether the dynamic covalent cross-linked network can restore the interfacial continuity, in order to evaluate the self-healing performance and fatigue resistance of the scaffold.

[0124] 5. Cartilage matrix formation test The printed scaffolds were cultured to induce cartilage formation, and the formation of type II collagen, glycosaminoglycans and other extracellular matrix of cartilage was detected. The differences between the autologous auricular chondrocyte culture group and the autologous auricular chondrocyte and adipose-derived mesenchymal stem cell co-culture group were compared.

[0125] The release cycle of TGF-β3 sustained-release microspheres was tested to confirm that they could continuously release transforming growth factor-β3 in the first 4 weeks. The results of type II collagen and glycosaminoglycan detection at different time points were combined to determine whether the growth factor release process matched the cartilage matrix formation process.

[0126] 6. Degradation Matching Test The printed scaffold is placed in a simulated in vivo environment or implanted in animals for verification. Changes in scaffold quality, volume, mechanical properties, and cartilage matrix content are monitored regularly. During the gradual degradation of the carrier material, the overall mechanical properties of the scaffold should not suddenly decrease, but should be gradually supported by the newly formed cartilage matrix.

[0127] When the scaffold material degrades significantly before the formation of new cartilage, it indicates that the degradation rate is too fast, and the stability of the cross-linked network should be improved or the reinforcing phase should be adjusted. When new cartilage has already formed but a large amount of material remains, it indicates that the degradation rate is too slow, and the degree of irreversible cross-linking should be reduced or the degradable reinforcing component should be adjusted.

[0128] Example 9: Animal Verification A rabbit ear cartilage defect model was selected for verification. The porous bionic auricular cartilage scaffold prepared according to Example 1 was implanted into the cartilage defect site. A control group without composite ink scaffold or a control group with traditional scaffold was set up. During the implantation process, the scaffold was kept in stable contact with the defect edge so that the loaded cells and cartilage induction components could play a role in the defect area.

[0129] After implantation, observe local inflammation, scaffold exposure, morphological deformation, and wound healing, and conduct imaging and histological evaluations at set times. The histological evaluation focuses on observing the distribution of newly formed cartilage tissue, cartilage lacunae formation, type II collagen and glycosaminoglycan deposition, and scaffold material degradation.

[0130] After using a porous biomimetic scaffold printed with PVA, GelMA and nano-silica composite ink, the cartilage regeneration effect in the rabbit ear cartilage defect model was better than that in the control group. Histological staining showed that the structure of the newly formed cartilage was highly similar to that of the natural ear cartilage, indicating that there is a synergistic effect between the mechanical support of the composite material, the biomimetic induction of the decellularized cartilage matrix, the co-cultured cell system and the microchannel nutrient delivery.

[0131] The elastic modulus of newly formed cartilage was measured at 8 weeks after implantation. The elastic modulus of the newly formed cartilage was about 90% of that of natural cartilage when autologous chondrocytes and adipose-derived mesenchymal stem cells were co-cultured at a ratio of 3:2 and scaffolded with TGF-β3 sustained-release microspheres were added. This indicates that the sustained-release induction and co-culture cell system can accelerate the maturation of newly formed cartilage.

[0132] Comparative Example 1: Single GelMA Bio-ink This comparative example uses only gelatin methacrylamide as the main hydrogel matrix, without adding decellularized cartilage matrix microfragments, dynamic covalent crosslinking components, polyvinyl alcohol, nano silica and polycaprolactone microfibers, and uses the same auricle model for printing and crosslinking.

[0133] Due to the lack of particle stacking effect of decellularized cartilage matrix micro-fragments, the ink is difficult to maintain a high static support capacity while improving fluidity. The helix edge and antihelix ridge are prone to diffusion before cross-linking. Due to the lack of biomimetic reinforcing phase and dynamic covalent cross-linking network, the cross-linked scaffold has insufficient elasticity and crack resistance, and is prone to local cracks when squeezed.

[0134] This comparative example shows that although single GelMA can achieve shaping through photocrosslinking, it cannot simultaneously meet the requirements of printing and maintaining the shape of complex auricular structures, long-term mechanical maintenance, micro-damage repair, and the construction of natural cartilage microenvironment.

[0135] Comparative Example 2: Composite ink without decellularized cartilage matrix microfragments This comparative example retains gelatin methacryloyl, oxidized hyaluronic acid, carboxymethyl chitosan, biomimetic reinforcing phase, natural visible light crosslinking system and seed cell system, but does not add decellularized cartilage matrix micro-fragments with a particle size of 10-20 micrometers.

[0136] Although the composite ink can maintain a certain viscosity through the polymer network, it lacks the physical stacking effect of microparticles in a static state, resulting in reduced shear thinning and rapid recovery performance after extrusion. At the same time, the seed cells lose the extracellular matrix signal of cartilage formed by natural glycosaminoglycans, collagen and elastin, leading to a decrease in the maintenance of cartilage phenotype and the ability to secrete cartilage-specific matrix.

[0137] This comparative example shows that decellularized cartilage matrix microfragments are not only bioactive fillers, but also play a role in regulating rheological properties. Their combination with the hydrogel matrix can simultaneously improve printing performance and cartilage regeneration performance.

[0138] Comparative Example 3: Single chondrocytes without TGF-β3 sustained-release microspheres This comparative example uses only the patient's own auricular chondrocytes as seed cells, without adding adipose-derived mesenchymal stem cells and TGF-β3 sustained-release microspheres. The remaining material composition and printing conditions are the same as in Example 1.

[0139] Due to the lack of paracrine support from adipose-derived mesenchymal stem cells and the lack of chondrocyte replenishment, the anti-apoptotic and proliferative capacity of autologous auricular chondrocytes in the high-density three-dimensional scaffold is relatively insufficient; and because TGF-β3 is not continuously released through sustained-release microspheres, a stable chondrogenic environment cannot be maintained inside the scaffold in the first 4 weeks.

[0140] This comparative study demonstrates that autologous auricular chondrocytes, adipose-derived mesenchymal stem cells, and TGF-β3 sustained-release microspheres form a synergistic relationship. The co-cultured cell system increases cell number and anti-apoptotic ability, while the sustained-release system continuously induces the formation of type II collagen and glycosaminoglycans.

[0141] Comparative Example 4: Composite ink without dynamic covalent cross-linked network This comparative example retains gelatin methacrylamide, biomimetic reinforcing phase, natural visible light crosslinking system, seed cell system, and TGF-β3 sustained-release microspheres, but does not include oxidized hyaluronic acid and carboxymethyl chitosan used to form a dynamic covalent crosslinking network.

[0142] When a printed scaffold is exposed to visible light, it can form a primary cross-linked network and maintain its shape. However, under repeated bending or local compression conditions, the primary cross-linked network mainly bears stress through irreversible structure. When the local stress exceeds the network's tolerance, cracks are easily generated. Once cracks form, they are difficult to recover through network recombination.

[0143] This comparative example shows that the dynamic covalent crosslinking network does not simply improve the initial strength, but rather enhances the flexibility, self-healing properties, and long-term deformation resistance of the scaffold through reversible bond breakage, stress release, and bond recombination.

[0144] Comparative Example 5: Dense Auricular Support Without Bionic Microchannels This comparative example uses the same composite ink as Example 1, but the printed model does not have interconnected microchannels with a porosity of 60%-70% and a pore size of 100-200 micrometers, nor does it have vascular endothelial cell-binding active peptides on the inner wall of the channels.

[0145] After dense scaffold implantation or long-term culture, cells on the surface of the scaffold can obtain nutrients through the surrounding culture medium or tissue, while oxygen and nutrients in the central region of the scaffold mainly rely on long-distance diffusion, which can easily lead to reduced cell activity and uneven cartilage matrix deposition.

[0146] This comparative example shows that the cell compatibility of composite inks alone cannot solve the problem of nutrient delivery inside large-volume auricular scaffolds. It is necessary to improve the long-term survival conditions of cells inside the scaffolds by connecting microchannels and functionalizing the inner walls of the channels.

[0147] Batch quality control implementation methods Each batch of decellularized cartilage matrix microfragments was tested for residual cellular components, glycosaminoglycan retention, total collagen retention, particle size distribution, sterility, and endotoxins. The glycosaminoglycan and total collagen retention should both be no less than 90%, the main microfragment particle size should be in the range of 10-20 micrometers, and it was confirmed that there were no large agglomerates that could clog the 18-22G nozzle.

[0148] Each batch of cell-free composite ink was tested for appearance, uniformity, rheological properties, nozzle passability, visible light crosslinking time, flexural modulus, and dynamic repair performance. Continuous extrusion tests were conducted using 18G, 20G, and 22G nozzles to confirm that the ink did not have obvious filament breakage, continuous pressure increase, or nozzle clogging, and to confirm that it could be set within 30 seconds of 405nm visible light irradiation after printing.

[0149] Each batch of cell-containing composite ink was tested for cell origin, cell ratio, cell density, cell viability, sterility, and uniformity of distribution. The ratio of autologous auricular chondrocytes to adipose-derived mesenchymal stem cells should be 3:2, and the final cell density is preferably 15-20 million per milliliter.

[0150] The dispersion uniformity and release cycle of TGF-β3 sustained-release microspheres were tested to confirm that the sustained-release microspheres did not rupture or aggregate significantly and could continuously release transforming growth factor-β3 in the first 4 weeks. The porosity, channel pore size and channel connectivity of the biomimetic microchannel scaffold were tested, and the optimal porosity was 60%-70% and the channel pore size was 100-200 micrometers.

[0151] The personalized auricular scaffolds that have been printed and cross-linked are subjected to three-dimensional contour matching, mechanical properties, cell viability and cold chain stability tests. The printing resolution should not be higher than 100 micrometers, the flexural modulus should be 1.5-2.2 MPa, the cell viability should not be less than 80% within 7 days after printing, and there should be no obvious structural deformation or abnormal decrease in cell viability after storage or transportation at 2-8℃ for 72 hours.

[0152] Scale-up Implementation During batch preparation, decellularized cartilage matrix microfragments, hydrogel matrix, biomimetic reinforcing phase, natural visible light crosslinking components, and TGF-β3 sustained-release microspheres are prepared separately and subjected to individual quality testing. Only after each component passes the test is the entire batch aseptically combined to avoid the entire batch of cell-containing composite ink being scrapped due to the failure of a single component.

[0153] The hydrogel matrix and biomimetic reinforcing phase can be prepared in advance in batches, but seed cells and TGF-β3 sustained-release microspheres should be added close to the printing time. The material preparation area, cell expansion area, composite ink mixing area and bioprinting area are set up in separate zones to reduce the risk of cross-contamination and to allow material performance testing and cell quality testing to be performed separately.

[0154] For personalized auricle printing tasks for different patients, the same basic material system and quality control standards can be used. The printing path, material usage, and local enhancement methods can be adjusted according to the size, thickness, and curvature of the patient's healthy auricle model, without changing the 3:2 ratio of the patient's autologous auricle chondrocytes to fat-derived mesenchymal stem cells, the TGF-β3 sustained release requirement for the first 4 weeks, and the basic mechanical performance requirements of the scaffold.

[0155] When printing multiple auricular scaffolds continuously, a short-distance trial extrusion is performed after each change of the cell-containing printing syringe, and the nozzle is regularly checked for agglutinated cartilage matrix microfragments, polycaprolactone microfibers, or nano-silica aggregates. The printing chamber temperature is maintained at 37°C, and 405nm visible light crosslinking is performed immediately after printing to avoid inconsistencies in the contour accuracy and cell activity of different batches of scaffolds due to differences in waiting time.

[0156] Long-term stability observation implementation method The printed auricular scaffolds were cultured in vitro and then implanted for observation. During the in vitro culture process, the scaffold size, helix edge thickness, antihelix elevation height, concha cavity depth, flexural modulus, material degradation degree, and cell activity were recorded regularly to evaluate whether the scaffolds experienced continuous swelling, edge thinning, or local collapse.

[0157] Three-dimensional scans were performed at 1 month, 3 months, 6 months and 12 months during the implantation observation process. The auricle contour at each time point was compared with the digital model before implantation to evaluate the overall morphological retention rate and local structural changes. At the same time, the matching relationship between material degradation and new cartilage formation was evaluated by imaging or histological methods.

[0158] When the scaffold material degrades before the newly formed cartilage has formed a stable structure, the helix or antihelix region is prone to morphological decline, indicating that TGF-β3 release, cell activity, or the rate of material degradation needs to be adjusted. When the scaffold maintains a high level of material residue for a long time and local inflammation occurs, it indicates that the carrier degradation rate is too slow, and the proportion of long-term stable components should be reduced or the dynamic cross-linking network should be adjusted.

[0159] The optimized auricle reconstruction system maintained good shape and elasticity during follow-up. The auricle shape satisfaction rate was over 92% one year postoperatively. The complication rate was reduced by 40% compared to traditional autologous rib cartilage reconstruction, and no serious scaffold exposure or deformation events occurred. These results indicate that personalized structural replication, low-toxicity cross-linking, cartilage induction, microchannel nutrient delivery, and degradation matching jointly affect the long-term stability of the reconstructed auricle.

[0160] The above embodiments are used to illustrate the technical concept and implementation process of the present invention. Without departing from the synergistic relationship of hydrogel matrix, decellularized cartilage matrix reinforcement, natural visible light crosslinking, dynamic covalent crosslinking, composite seed cells, TGF-β3 sustained release, and biomimetic microchannels, the amount of material, nozzle model, printing path, and local enhancement method can be adjusted according to the patient's auricle size, printing equipment specifications, and implantation site conditions. All such adjustments fall within the scope of implementation of the technical solution of the present invention.

Claims

1. A personalized auricle reconstruction 3D bioprinting composite ink, characterized in that, Including hydrogel matrix, biomimetic reinforcing phase, natural visible light crosslinking system, dynamic covalent crosslinking component, seed cell system and cartilage-induced sustained-release component; The hydrogel matrix includes at least gelatin methacrylamide, the biomimetic reinforcing phase includes decellularized cartilage matrix microfragments, the natural visible light crosslinking system includes proanthocyanidins and / or riboflavin, the seed cell system includes patient autologous auricular cartilage cells and adipose-derived mesenchymal stem cells, and the cartilage-induced sustained-release component includes sustained-release microspheres loaded with transforming growth factor-β3. The composite ink undergoes shear thinning under extrusion shearing action and forms an auricular cartilage biomimetic scaffold with elasticity, self-healing and controllable degradation after being irradiated with visible light.

2. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The hydrogel matrix also includes oxidized hyaluronic acid, carboxymethyl chitosan, and zwitterionic monomer MPC. The oxidized hyaluronic acid and carboxymethyl chitosan participate in the construction of a reversible dynamic cross-linking network to impart stress relaxation, network recombination after damage, and adjustable degradation properties to the printing scaffold. The zwitterionic monomer MPC is uniformly dispersed in the dynamic cross-linking network to improve the hydrophilicity, cell-friendly properties, and ion conduction properties of the composite ink.

3. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The biomimetic reinforcing phase also includes at least one of polyvinyl alcohol, polyethylene oxide, nano-silica, and polycaprolactone microfibers. Polyvinyl alcohol forms a load-bearing skeleton, polyethylene oxide improves extrusion continuity, and nano-silica and polycaprolactone microfibers improve the bending support, elastic recovery, and long-term shape maintenance of the printing holder, so that the composite ink has both smooth flow in the nozzle and rapid shape retention after deposition.

4. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The decellularized cartilage matrix microfragments are obtained from auricular cartilage through decellularization, pulverization, and enzymatic hydrolysis. The decellularization process removes immunogenic cell residues and ensures that the resulting matrix retains no less than 90% of the natural glycosaminoglycans and total collagen components. The microfragments have a particle size of 10-20 micrometers and are dispersed in a hydrogel matrix. While retaining the active components of the cartilage, the static support and shear thinning properties of the composite ink are adjusted to make it suitable for continuous extrusion through 18-22G micro-nozzles.

5. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The natural visible light crosslinking system uses proanthocyanidins and / or riboflavin as photosensitive crosslinking aids, and triggers the formation of a primary crosslinking network by gelatin methacrylamide under 405nm visible light irradiation. The dynamic covalent crosslinking component forms a reversible bond structure in the primary crosslinking network that can be broken and reconnected, so that when the printed auricle is squeezed and microcracks are generated, the continuous network can be restored, and the toxic effects of additional chemical photoinitiators on the loaded cells are reduced.

6. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The ratio of the patient's autologous auricular chondrocytes to adipose-derived mesenchymal stem cells is 3:2, and the total cell density of the two in the composite ink is 15 million to 20 million per milliliter. The adipose-derived mesenchymal stem cells enhance the anti-apoptotic ability of chondrocytes through paracrine active factors and differentiate into chondrocytes under chondrocyte induction environment to supplement the number of seed cells, promote cell proliferation, and maintain the cartilage phenotype of the printed cells.

7. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The sustained-release microspheres loaded with transforming growth factor-β3 are uniformly dispersed between the hydrogel matrix and the seed cell system, and are configured to continuously release transforming growth factor-β3 for the first 4 weeks after implantation. This induces the patient's autologous auricular chondrocytes and adipose-derived mesenchymal stem cells to secrete type II collagen and glycosaminoglycans, so that the rate of new cartilage deposition matches the degradation rate of the composite ink carrier, reducing the risk of premature scaffold degradation and collapse or long-term material residue causing inflammation.

8. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The composite ink, after printing, forms an interconnected biomimetic microchannel network. The porosity of the biomimetic microchannel network is 60%-70%, the pore diameter is 100-200 micrometers, and the inner wall of the microchannel is modified with active polypeptides that can specifically bind to vascular endothelial cells to guide the blood vessels around the implantation site to grow into the scaffold, improve the delivery of oxygen and nutrients to the auricular scaffold, and maintain the survival of seed cells and cartilage matrix secretion inside the large-volume auricular scaffold.

9. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The composite ink is configured to be extruded and printed in a sterile environment at 37°C through an 18-22G nozzle, and then irradiated with 405nm visible light for 30 seconds after extrusion to complete the shaping. The printed scaffold can replicate the curved surface and thin-walled structure of the helix, antihelix, concha, and triangular fossa, with a printing resolution of no more than 100 micrometers. The flexural modulus of the scaffold is 1.5-2.2MPa to match the support performance, flexibility, and resilience of natural auricular cartilage.

10. The personalized auricle reconstruction 3D bioprinting composite ink according to claim 1, characterized in that, The patient's autologous auricular cartilage cells are derived from the patient's residual auricular cartilage tissue. The composite ink is matched with the digital model of the affected auricle generated based on the CT or MRI data of the patient's healthy auricle and is loaded into a sterile printing syringe for personalized printing. The printed auricular scaffold is placed in a biodegradable protective shell containing sterile nutrient solution and can be stably transported for no less than 72 hours under 2-8℃ conditions. The survival rate of seed cells is no less than 80% within 7 days after printing.