Pre-vascularized artificial flaps and methods of making the same

Abstract

This application provides a prevascularized artificial flap and its preparation method, belonging to the field of tissue engineering materials. The preparation method includes: printing matrix bio-ink on a substrate to form a first matrix layer in a pre-cured state; printing fiber-reinforced ink on the pre-cured first matrix layer to form a fiber-reinforced layer; before the fiber-reinforced layer is fully cured, printing vascular bio-ink on the fiber-reinforced layer to form a vascular layer, and partially curing the vascular layer during or after printing; printing matrix bio-ink on the partially cured vascular layer to form a second matrix layer in a pre-cured state; and performing an overall curing treatment on the first matrix layer, fiber-reinforced layer, vascular layer, and second matrix layer to obtain a prevascularized artificial flap. This application can form a three-dimensional active tissue with a stable structure and a continuous vascular network, thereby reducing the risk of interlayer delamination.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering materials technology, specifically to a prevascularized artificial flap and its preparation method. Background Technology

[0002] Extensive full-thickness skin and soft tissue defects caused by severe burns, trauma, or tumor resection pose a significant challenge to clinical reconstructive surgery. While autologous flap transplantation is hailed as the "gold standard" in clinical treatment, providing viable tissue with complete blood supply and mechanical support, it suffers from inherent drawbacks such as significant donor site damage, limited availability, difficulty in achieving "ready-to-use" results, and a high risk of vascular anastomosis failure. Therefore, the development of tissue-engineered artificial flaps that can mimic the structure and function of natural tissue and can be transplanted immediately has significant clinical implications and promising application prospects.

[0003] Among the related technologies is a method for constructing three-dimensional vascularized myocutaneous flaps based on coaxial printing. This technology can print perfusion channels and load various cells, which has made progress in vascularization construction.

[0004] However, the multi-layered structure formed by this technology is achieved through post-implantation folding and physical adhesion using rat tail collagen. The functional layers are only physically bonded interfaces, which poses a risk of interlayer delamination due to weak interfacial bonding after long-term implantation. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present application provides a prevascularized artificial flap and its preparation method. The prevascularized artificial flap prepared by the prevascularized artificial flap preparation method can achieve bio-integration between functional layers, forming a three-dimensional active tissue with stable structure and interconnected vascular network, thereby reducing the risk of interlayer delamination.

[0006] To achieve the above objectives, in a first aspect, embodiments of this application provide a method for preparing a prevascularized artificial flap, comprising: We offer matrix bio-inks, vascular bio-inks, and fiber-reinforced inks. The matrix bio-ink is printed on the substrate to form a first matrix layer in a pre-cured state; Fiber-reinforced ink is printed onto a pre-cured first matrix layer to form a fiber-reinforced layer; Before the fiber reinforcement layer is fully cured, vascular bio-ink is printed on the fiber reinforcement layer to form a vascular layer, and the vascular layer is partially cured during or after the printing process. The matrix bio-ink is printed onto the partially cured blood vessel layer to form a second matrix layer in a pre-cured state. The first matrix layer, fiber reinforcement layer, vascular layer and second matrix layer are solidified as a whole to obtain a prevascularized artificial flap.

[0007] Furthermore, the curing process includes physical curing and / or chemical curing.

[0008] Furthermore, physical curing includes at least one of thermal curing and photocuring.

[0009] Furthermore, chemical curing includes at least one of chemical crosslinking curing, ionic crosslinking curing, and enzyme-catalyzed crosslinking curing.

[0010] Furthermore, matrix bioinks and vascular bioinks include photoinitiators.

[0011] Furthermore, the matrix bio-ink includes at least one of collagen, gelatin, hyaluronic acid, silk fibroin, sodium alginate, and chitosan.

[0012] Furthermore, the vascular bio-ink includes at least one of gelatin, sodium alginate, fibrinogen, matrix gelatin and methacrylamide gelatin, as well as vascular endothelial cells and / or angiogenesis factors.

[0013] Furthermore, the fiber-reinforced ink includes at least one of polycaprolactone, polylactic acid, and polyglycolic acid.

[0014] Secondly, the present invention also proposes a prevascularized artificial flap, which is prepared by the above-mentioned method for preparing a prevascularized artificial flap; wherein the vascular layer includes a biomimetic channel.

[0015] Furthermore, the biomimetic channel is at least one of a spiral network and a tree-like fractal network; and / or, The biomimetic channel has multiple branch channels, with a center-to-center distance of 2mm-20mm between adjacent branch channels; and / or, The biomimetic channel has a porous structure with a radially gradient, and a porosity of 30%-80%; and / or, The walls of the biomimetic channel include an endothelial cell layer; and / or, The fiber reinforcement layer is arranged in a mesh pattern; and / or, The porosity of the first and second matrix layers is 70%-95%.

[0016] The beneficial effects of this application are as follows: In the technical solution of this application, the first matrix layer is printed with fiber-reinforced ink while in a pre-cured state. At this time, the matrix material has not yet formed a dense cross-linked network, and the fiber-reinforced ink can partially penetrate to the surface of the first matrix layer, forming a mechanical interlock. The fiber-reinforced layer is printed with vascular bio-ink while not fully cured, causing molecular chain entanglement and chemical bonding between the vascular layer and the fiber-reinforced layer. After partial curing, the vascular layer helps maintain its morphological and structural stability, avoiding structural collapse or deformation, which would affect the use of the subsequent vascular layer. The surface still retains active reactive groups, which can undergo cross-linking reactions with the subsequently printed second matrix layer. Finally, through overall curing, covalent bonds and interpenetrating networks are formed in the interfacial regions between the layers, solving the interlayer delamination problem caused by weak interfacial bonding in traditional multilayer structures, and improving the structural integrity and long-term stability of the artificial flap.

[0017] In summary, by controlling the curing sequence of each functional layer, an interpenetrating network structure is formed at the interface between adjacent layers. Utilizing the fluidity and wettability of the material during its incomplete curing phase, an "anchored interlocking" structure is formed between the fiber reinforcement layer and the matrix layer, and a "seamless embedding" structure is formed between the vascular network and the matrix layer. Finally, through unified cross-linking, functional layers with different physicochemical properties permeate, interweave, and fuse at the interface, fundamentally eliminating the macroscopic interface and delamination risks present in traditional lamination processes. This allows the artificial flap to maintain long-term structural integrity and stability even when subjected to complex mechanical environments after implantation.

[0018] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0020] Figure 1 This application provides a schematic diagram of the structure of an embodiment of a prevascularized artificial flap; Figure 2 for Figure 1 Mid-section view.

[0021] Explanation of reference numerals in the attached figures: 100. Prevascularized artificial flap; 1. First matrix layer; 2. Fiber reinforcement layer; 3. Vascular layer; 31. Bionic channel; 4. Second matrix layer. Detailed Implementation

[0022] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0024] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0025] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0026] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0027] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0028] In the description of the embodiments of this application, the technical terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0029] In the description of the embodiments of this application, unless otherwise expressly specified and limited, technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. For those skilled in the art, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0030] Extensive full-thickness skin and soft tissue defects caused by severe burns, trauma, or tumor resection pose a significant challenge to clinical reconstructive surgery. While autologous flap transplantation is hailed as the "gold standard" in clinical treatment, providing viable tissue with complete blood supply and mechanical support, it suffers from inherent drawbacks such as significant donor site damage, limited donor availability, difficulty in achieving "ready-to-use" results, and a high risk of vascular anastomosis failure. Therefore, the development of tissue-engineered artificial flaps that can mimic the structure and function of natural tissues and can be transplanted immediately has significant clinical implications and promising application prospects.

[0031] One related technology provides a method for constructing a three-dimensional vascularized myocutaneous flap based on coaxial printing. This technology can print perfusion channels and load various cells, which represents an advancement in vascularization construction.

[0032] However, the multi-layered structure formed by this technology is achieved through post-implantation folding and physical adhesion using rat tail collagen. The functional layers are only physically bonded interfaces, which poses a risk of interlayer delamination due to weak interfacial bonding after long-term implantation.

[0033] To address the aforementioned technical problems, in a first aspect, embodiments of this application provide a method for preparing a prevascularized artificial flap 100, comprising: We offer matrix bio-inks, vascular bio-inks, and fiber-reinforced inks. The matrix bio-ink is printed on the substrate to form the first matrix layer 1 in a pre-cured state; Fiber-reinforced ink is printed onto the pre-cured first matrix layer 1 to form fiber-reinforced layer 2; Before the fiber reinforcement layer 2 is fully cured, vascular bio-ink is printed on the fiber reinforcement layer 2 to form vascular layer 3, and vascular layer 3 is partially cured during or after the printing process. The matrix bio-ink is printed onto the partially cured vascular layer 3 to form a second matrix layer 4 in a pre-cured state; The first matrix layer 1, the fiber reinforcement layer 2, the vascular layer 3, and the second matrix layer 4 are integrally solidified to obtain a prevascularized artificial flap 100.

[0034] like Figure 1 and Figure 2 As shown, in the technical solution of this application, the first matrix layer 1 is printed with fiber-reinforced ink while in a pre-cured state. At this time, the matrix material has not yet formed a dense cross-linked network, and the fiber-reinforced ink can partially penetrate to the surface of the first matrix layer 1, forming a mechanical interlock. The fiber-reinforced layer 2 is printed with vascular bio-ink while in a partially cured state, causing molecular chain entanglement and chemical bonding between the vascular layer 3 and the fiber-reinforced layer 2. After partial curing, the vascular layer 3 helps maintain its own morphological and structural stability, avoiding structural collapse or deformation, which would affect the subsequent use of the vascular layer 3. The surface still retains active reactive groups, which can undergo cross-linking reactions with the subsequently printed second matrix layer 4. Finally, through overall curing treatment, covalent bonds and interpenetrating network structures are formed in the interface regions between the layers, solving the interlayer delamination problem caused by weak interfacial bonding in traditional multilayer structures, and improving the structural integrity and long-term stability of the artificial flap.

[0035] In summary, by controlling the curing sequence of each functional layer, an interpenetrating network structure is formed at the interface of adjacent layers. Utilizing the fluidity and wettability of the material during its incomplete curing, an "anchored interlocking" structure is formed between the fiber-reinforced layer 2 and the matrix layer, and a "seamless embedding" structure is formed between the vascular network and the matrix layer. Finally, through unified cross-linking, functional layers with different physicochemical properties permeate, interweave, and fuse at the interface, fundamentally eliminating the macroscopic interface and delamination risks present in traditional lamination processes. This allows the artificial flap to maintain long-term structural integrity and stability even when subjected to complex mechanical environments after implantation.

[0036] Furthermore, addressing the challenge of "difficult clinical operation," this application achieves immediate surgical operability. Through an internal fiber network reinforcement layer, the artificial flap possesses excellent tensile and tear resistance, enabling it to withstand surgical suturing, clamping, and other procedures without tearing or deformation. Simultaneously, the entire artificial flap is digitally customized, allowing for personalized adjustments to its shape, thickness, vascular network topology, and fiber reinforcement pattern based on the patient's three-dimensional wound imaging data. It can also flexibly integrate various cells and growth factors to construct a truly "personalized, functional, and ready-to-use" active artificial flap.

[0037] Understandably, the specific physical configurations of each layer, including thickness, pore distribution, fiber orientation, and the topology of the vascular network, can be adapted to the anatomical characteristics and mechanical requirements of the target repair site. For example, for weight-bearing areas such as the soles of the feet or joints, the thickness and fiber density of the fiber-reinforced layer 2 can be appropriately increased, and the distribution density of the vascular network can be optimized to match local metabolic needs; while for areas with high aesthetic requirements, such as the face, the overall thickness can be reduced and a more refined vascular branching pattern can be adopted to achieve better tissue integration and aesthetic restoration.

[0038] Understandably, the specific printing steps can be implemented in various ways, such as using a multi-nozzle collaborative printing system. In this system, the first nozzle carries matrix bio-ink, the second carries fiber-reinforced ink, and the third carries vascular bio-ink. Each nozzle sequentially ejects ink according to a pre-set 3D model path, thus forming different functional layers. During printing, the curing status of each layer can be monitored in real time, and printing parameters, such as nozzle movement speed, extrusion pressure, curing light intensity, or temperature field distribution, can be dynamically adjusted to meet the needs of different artificial skin flaps.

[0039] In some embodiments, the curing process includes physical curing and / or chemical curing.

[0040] In this embodiment, the curing rate and degree of crosslinking can be accurately controlled by using physical curing and chemical curing in synergy or alone. Specifically, physical curing has a rapid response and is suitable for scenarios requiring immediate shaping or precise local control; chemical curing can form a more stable and durable covalent bond network, making the overall structure more stable. When the two are used in combination, the advantages of both can be combined to produce artificial flaps with excellent mechanical properties and biocompatibility.

[0041] In some embodiments, physical curing includes at least one of thermal curing and photocuring.

[0042] In this embodiment, thermosetting utilizes increased temperature to promote polymer chain movement and accelerate cross-linking reactions, making it suitable for heat-sensitive biomaterial systems. Gradient curing can be achieved through precise temperature control. Photocuring, on the other hand, relies on photoinitiators to generate free radicals or cationic active species under specific wavelengths of light, initiating rapid polymerization of monomers or prepolymers. Photocuring offers advantages such as fast curing rates and small heat-affected zones. Both physical curing methods can be flexibly selected or combined based on the physicochemical properties of the ink material itself.

[0043] In some embodiments, chemical curing includes at least one of chemical crosslinking curing, ionic crosslinking curing, and enzyme-catalyzed crosslinking curing.

[0044] In this embodiment, chemical crosslinking curing involves the reaction of the bifunctional or multifunctional crosslinking agent in the crosslinking agent with the active groups on the polymer chain to form a stable covalent bond network. The crosslinking density and mechanical properties can be precisely controlled by adjusting the crosslinking agent concentration and reaction conditions. Ionic crosslinking curing involves the formation of coordination bonds between polyvalent metal ions and anionic groups on the polymer chain. Enzymatic crosslinking curing involves the crosslinking reaction of the substrate catalyzed by natural enzymes. Natural enzymes are characterized by mild conditions and excellent biocompatibility, and can retain the function of bioactive molecules to the greatest extent. These three chemical curing methods can be flexibly selected or combined according to the material properties and biological requirements of the vascular layer 3, fiber reinforcement layer 2, and matrix layer.

[0045] In some embodiments, matrix bioinks and vascular bioinks include photoinitiators.

[0046] In this embodiment, by introducing a photoinitiator into the matrix bio-ink and the vascular bio-ink, the printed matrix layer and vascular layer 3 can rapidly initiate a free radical polymerization reaction under light of a specific wavelength, which is beneficial for precisely controlling the curing range and degree.

[0047] For example, the photoinitiator may be one or more combinations of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), 2-hydroxy-2-methyl-1-phenylpropanone (Irgacure 1173), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone (Irgacure 2959), 1-hydroxycyclohexylphenyl ketone (Irgacure 184), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (Irgacure 819), 2-isopropylthioxanthone (ITX), ethyl 4-dimethylaminobenzoate (EDB), 4-p-toluene-mercaptobenzophenone (MTA), riboflavin, or eosin Y. The aforementioned photoinitiators can efficiently generate free radicals under ultraviolet or visible light irradiation, initiating polymerization reactions of acrylate, methacrylate, or vinyl monomers, and exhibit good biocompatibility.

[0048] In some embodiments, the matrix bio-ink includes at least one of collagen, gelatin, hyaluronic acid, silk fibroin, sodium alginate, and chitosan.

[0049] In this embodiment, by selecting natural polymer materials such as collagen, gelatin, hyaluronic acid, and silk fibroin as components of the matrix bioink, better biocompatibility is achieved. Specifically, collagen, as a major structural protein of the extracellular matrix, has good cell adhesion and biodegradability, providing a natural adhesion microenvironment for cells. Gelatin contains an arginine-glycine-aspartic acid sequence, which can promote cell adhesion and proliferation. Hyaluronic acid has excellent water retention and lubrication properties, and can regulate the water content and rheological properties of the matrix. Silk fibroin has excellent mechanical strength and toughness. The above materials can be used alone or compounded in specific proportions. By adjusting the molecular weight, concentration, and cross-linking method of each component, precise control of the porosity, degradation rate, and mechanical properties of the matrix layer can be achieved, thereby matching the histological needs of different repair sites.

[0050] In some embodiments, the vascular bio-ink includes at least one of gelatin, sodium alginate, fibrinogen, matrix gelatin, and methacrylamide gelatin, as well as vascular endothelial cells and / or angiogenesis factors.

[0051] In this embodiment, a bioactive vascularized structure is constructed by combining natural or modified polymeric materials with active cells and growth factors. Specifically, gelatin and its derivatives (such as methacrylamide gelatin) have good cell adhesion and thermo- / photosensitive crosslinking properties, allowing for rapid shaping after printing; sodium alginate and its derivatives can form a stable gel through ionic crosslinking, making them suitable as a support material for vascular channels; fibrinogen can form a fibrin gel under the action of thrombin, mimicking the temporary matrix in the natural coagulation process; the matrix gel is rich in basement membrane components such as laminin and collagen IV, which can promote the formation of tubular structures by endothelial cells. The above materials can be used alone or in specific proportions. By loading vascular endothelial cells (such as human umbilical vein endothelial cells and human microvascular endothelial cells) and / or vascular growth factors (such as vascular endothelial growth factor VEGF and basic fibroblast growth factor bFGF), they can promote the proliferation, migration, and self-assembly of endothelial cells after printing, ultimately forming a biomimetic vascular network with physiological functions, thus realizing the pre-vascularized construction of artificial flaps.

[0052] In some embodiments, the fiber-reinforced ink includes at least one of polycaprolactone, polylactic acid, and polyglycolic acid.

[0053] In this embodiment, by selecting biodegradable synthetic polymers such as polycaprolactone, polylactic acid, and polyglycolic acid as components of the fiber-reinforced ink, excellent mechanical support properties can be provided for the artificial flap. Specifically, polycaprolactone has good flexibility and a slow degradation rate, enabling it to maintain structural stability long-term after implantation. Polylactic acid has high tensile strength and modulus, which can improve the tensile load-bearing capacity of the artificial flap. Polyglycolic acid has a fast degradation rate and excellent mechanical strength. The above materials can be used alone or compounded in specific proportions to suit the needs of different artificial flaps.

[0054] Secondly, the present invention also proposes a prevascularized artificial flap 100, which is prepared by the above-mentioned method for preparing the prevascularized artificial flap 100; wherein, the vascular layer 3 includes a biomimetic channel 31.

[0055] Understandably, the biomimetic channel 31 can simulate the branching structure of the natural vascular system, enabling the perfusion and flow of blood or culture medium, providing a continuous supply of nutrients and a channel for the removal of metabolic waste for the artificial flap. The geometry of the biomimetic channel 31 can be designed according to the vascular anatomy of the target tissue.

[0056] Understandably, the bionic channel 31 has at least two ports for anastomosis with the host blood vessels, forming a complete physiological circulation path from one port into the artificial flap, through which sufficient material exchange occurs, and out through the other port. This is used to simulate the microcirculation function of natural tissues and fundamentally solve the risk of ischemic necrosis in the graft center.

[0057] In some embodiments, the biomimetic channel 31 is at least one of a spiral network and a tree-like fractal network.

[0058] In this embodiment, the spiral network, by mimicking the tortuous course of arteries in tissue, reduces blood flow resistance and enhances the shear stress adaptability of the vessel wall. The tree-like fractal network, through its progressively decreasing diameter design, achieves efficient blood distribution and collection, closely matching the fractal structure of the natural vascular system, which is beneficial for the uniform transport of oxygen and nutrients. Both topologies can be constructed individually or designed in combination, depending on the blood supply characteristics of the target repair site.

[0059] In some embodiments, the biomimetic channel 31 has multiple branch channels, and the center distance between two adjacent branch channels is 2mm-20mm.

[0060] In this embodiment, by limiting the center distance between two adjacent branch channels to the range of 2mm-20mm, it can be ensured that the diffusion distance of nutrients does not exceed the critical threshold of tissue necrosis.

[0061] In some embodiments, the wall of the biomimetic channel 31 has a porous structure with a radial gradient and a porosity of 30%-80%.

[0062] In this embodiment, the smaller pore size of the porous structure can reduce blood leakage in the early stage of perfusion, while the larger pore size of the porous structure is conducive to the ingrowth of host tissue cells and angiogenesis, promoting deep integration of the graft and the host tissue.

[0063] In some embodiments, the wall of the biomimetic channel 31 includes an endothelial cell layer.

[0064] In this embodiment, the endothelial cell layer can mimic the endothelial lining structure of natural blood vessels, giving the channel good blood compatibility and antithrombotic ability.

[0065] In some embodiments, the fiber reinforcement layer 2 is arranged in a mesh pattern.

[0066] In this embodiment, the fiber reinforcement layer 2, arranged in a mesh pattern, can form an interwoven continuous skeleton in three-dimensional space, dispersing external stress and improving the overall tear resistance of the artificial flap. The mesh size and fiber diameter of the mesh structure can be adjusted according to the mechanical requirements of the target repair site.

[0067] In some embodiments, the porosity of the fiber reinforcement layer 2 is 60%-95%.

[0068] This design provides mechanical support without hindering cell migration and material exchange between the upper and lower matrix layers.

[0069] In some embodiments, the porosity of the first matrix layer 1 and the second matrix layer 4 is 70%-95%.

[0070] In this embodiment, by limiting the porosity of the first matrix layer 1 and the second matrix layer 4 to the range of 70%-95%, sufficient three-dimensional space can be provided for cell proliferation, migration and nutrient diffusion, while maintaining the structural integrity of the matrix layer.

[0071] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0072] I. Preparation Method Materials preparation: Matrix-based bio-ink: 3% (w / v) type I collagen and 1% (w / v) sodium hyaluronate were dissolved in a 0.1 mol / L acetic acid solution. Type I collagen, as a major component of the extracellular matrix, provides adhesion sites for cells; sodium hyaluronate is used to adjust the rheological properties and water retention of the ink; acetic acid is used to dissolve the collagen. The pH was then adjusted to 7.4 with sodium hydroxide solution and stored at 4°C for later use, yielding the matrix-based bio-ink.

[0073] Vascular bio-ink: Human umbilical vein endothelial cells are infused at a concentration of 1×10⁻⁶. 7 Cells / mL density was resuspended in an 8% (w / v) solution of methacrylamide gelatin (GelMA) containing 0.25% (w / v) photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) and kept at 22°C for printing to obtain vascular bio-ink. GelMA serves as a photocrosslinkable scaffold material, while LAP initiates the crosslinking reaction upon light irradiation.

[0074] Fiber-reinforced ink: Medical-grade polycaprolactone (PCL) particles with a molecular weight of 80,000 g / mol are directly selected as fiber-reinforced ink.

[0075] Example 1 This embodiment constructs a prevascularized artificial flap by step-by-step printing and precise control of the curing sequence.

[0076] First, the printing platform is preheated to 37°C. Using a printhead equipped with a 22G flat-tipped nozzle (approximately 0.41mm inner diameter), the bio-based matrix ink is printed according to the preset 3D model path under an extrusion pressure of 60kPa, forming a first matrix layer with a thickness of approximately 0.3mm in a pre-cured state. Here, "pre-cured state" refers to the matrix ink undergoing thermal gelation at 37°C to initially form its initial shape, but before a dense chemical cross-linked network has been formed.

[0077] The system then switches to a melt extrusion printhead, setting the printhead temperature to 85°C, the nozzle diameter to 50 μm, and the extrusion pressure to 400 kPa. Before the first matrix layer has fully solidified, fiber-reinforced ink (PCL) is immediately printed onto it using a pre-defined orthogonal grid path (grid spacing of 250 μm), forming a fiber reinforcement layer. The thickness of the fiber layer printed in a single pass is approximately 80 μm. The high-temperature PCL melt partially melts and embeds into the soft surface of the underlying first matrix layer, forming a mechanically interlocked structure.

[0078] Next, the process was switched to a temperature-controlled precision needle, with the needle inner diameter set to 200 micrometers, the printing temperature to 22°C, and the extrusion pressure to 30 kPa. Before the fiber-reinforced layer (PCL) had completely cooled and solidified, vascular bio-ink was immediately printed onto the grid of the PCL, forming a vascular layer with a biomimetic dendritic fractal network structure. During the printing of the vascular layer, a wavelength of 405 nm and a light intensity of 5 mW / cm² were used. 2 A blue light source is used to simultaneously scan and expose the vascular bio-ink along the printing path, rapidly pre-crosslinking the methacrylamide gelatin (GelMA) in the vascular bio-ink for approximately 1-2 seconds, thus partially curing the vascular layer. The purpose of this step is to maintain the tubular structure of the vascular layer, preventing collapse, while retaining unreacted active groups on its surface.

[0079] Then, the printer was switched back to a printhead equipped with a 22G flat-tipped needle. Using matrix bio-ink, under the same printing parameters as before (60Kpa extrusion pressure), the printhead was filled and covered above and around the partially cured vascular layer until the total thickness of the flap reached 2.5mm, completely embedding the entire vascular network in the matrix material to form a second matrix layer in a pre-cured state.

[0080] After printing, the entire construct, comprising the first matrix layer, fiber reinforcement layer, vascular layer, and second matrix layer, was placed under light at a wavelength of 405 nm and an intensity of 20 mW / cm². 2The entire flap is exposed to blue light for 90 seconds for overall curing. This step further thermally gels the collagen in the first and second matrix layers, while simultaneously allowing unreacted GelMA in each layer to undergo full cross-linking. Ultimately, the functional layers are tightly connected at the interface through covalent bonds and molecular chain entanglement, forming an integrated three-dimensional active tissue, thus obtaining the prevascularized artificial flap of Example 1.

[0081] Example 2 The difference from Example 1 is that the vascular bio-ink is composed of: DMEM medium containing 2% (w / v) sodium alginate and 5% (w / v) fibrinogen, in which 1×10 7 HUVECs with cells / mL were printed. After printing the vascular layer, instead of photocuring, 20 μL of 50 mM CaCl2 solution was immediately sprayed to perform ionic cross-linking and partial curing, so that the sodium alginate was initially gelled. The remaining steps were roughly the same as in Example 1, resulting in the prevascularized artificial flap of Example 2.

[0082] Example 3 The difference from Example 1 is that the fiber-reinforced ink used is polylactic acid-glycolic acid copolymer (PLGA, 85 / 15), the printing temperature is adjusted to 120°C, the nozzle diameter is adjusted to 80 μm, and the fiber mesh spacing is adjusted to 1.0 mm. The remaining steps are roughly the same as in Example 1, resulting in the prevascularized artificial flap of Example 3.

[0083] Example 4 The difference from Example 1 is that the overall curing process adopts a dual curing method: first, light curing (20 mW / cm² 405nm blue light, 60 seconds) is performed, and then the construct is immersed in a PBS solution (pH 7.4) containing 5 mM genipin and chemically crosslinked and cured at 37°C for 24 hours. The remaining steps are roughly the same as in Example 1, resulting in the prevascularized artificial flap of Example 4.

[0084] Example 5 The difference from Example 1 is that the curing time of the vascular layer is after printing. That is, the entire vascular network is printed first, and then the entire vascular network area is scanned and exposed for 60 seconds using a wide beam of 405nm blue light (5 mW / cm²) to partially cure it. The remaining steps are roughly the same as in Example 1, resulting in the prevascularized artificial flap of Example 5.

[0085] Example 6 The difference from Example 1 lies in the printing process of the fiber-reinforced layer and the vascular layer. This example uses a coaxial printing nozzle, with the inner nozzle (150 μm diameter) introducing vascular bio-ink and the outer nozzle (300 μm diameter) introducing fiber-reinforced ink (PCL). During printing, both inks are extruded simultaneously, forming a composite fiber with PCL as the outer shell and cell-loaded GelMA as the core. This composite fiber is deposited on the first matrix layer before complete curing, directly forming a composite layer that provides both mechanical support and vascular access. Subsequent steps are largely the same as in Example 1, resulting in the prevascularized artificial flap of Example 6.

[0086] Comparative Example 1 The difference from Example 1 is that no fiber reinforcement layer is printed, but the remaining steps are largely the same as in Example 1, resulting in the prevascularized artificial flap of Comparative Example 1.

[0087] Comparative Example 2 The difference from Example 1 is that each layer was allowed to cure completely before printing the next layer. The remaining steps are roughly the same as in Example 1, resulting in the prevascularized artificial flap of Comparative Example 2.

[0088] Comparative Example 3 The difference from Example 1 is that the second matrix layer is printed directly after the vascular layer is printed (i.e., no partial curing is performed), and the remaining steps are roughly the same as in Example 1, resulting in the prevascularized artificial flap of Comparative Example 3.

[0089] II. Testing Methods 1. Suture retention strength test: The edges of the prevascularized artificial flap were sutured to a standard clamp using 4-0 polypropylene sutures. A universal testing machine was used to stretch the flap at a speed of 10 mm / min until the sutures tore or the flap ruptured. The maximum force value (unit: N) was recorded. Five samples were tested in each group, and the average value was taken.

[0090] 2. Interlayer bonding strength test: Prevascularized artificial flaps were prepared into dumbbell-shaped standard specimens and subjected to tensile tests. The maximum tensile strength (unit: MPa) was recorded when a fracture surface appeared. Five samples were tested in each group, and the average value was taken.

[0091] III. Analysis of Test Results for Each Embodiment and Comparative Example Table 1. Suture retention strength (N) of Examples 1-6 and Comparative Examples 1-3 Table 2. Interlayer bonding strength (MPa) of Examples 1-6 and Comparative Examples 1-3 III. Conclusion Analysis: As shown in Table 1, the prevascularized artificial flaps prepared in Examples 1-6 of this application all exhibited excellent suture retention strength (average value > 4.8 N), far exceeding that of the comparative examples. Among them, Example 6 achieved the integrated construction of fibers and blood vessels through coaxial printing, resulting in the highest strength (6.26 N). Comparative Example 1 showed a significant decrease in strength due to the lack of a fiber reinforcement layer. Comparative Example 2 had the lowest strength because the layers were only physically bonded, making the interfaces extremely prone to separation under stress. Comparative Example 3 had an incomplete structure due to the incomplete curing of the vascular layer; although its strength was higher than that of Comparative Example 2, it was still far lower than that of the other examples.

[0092] As shown in Table 2, the interlayer bonding strength (tensile strength) of Examples 1-6 is relatively high (average value > 0.38 MPa), and the fractures mostly occur within the matrix layer or fiber layer, rather than at the interlayer interface. This proves that the "interfacial interpenetrating network structure" of this application successfully achieves the integration of each functional layer, and the interlayer bonding strength even exceeds the cohesive strength of the material itself. In contrast, Comparative Example 1 fractured at the blood vessel / matrix interface, Comparative Example 2 fractured at the physical bonding interface, and Comparative Example 3 fractured at the collapse point of the structurally weak blood vessel layer, and their bonding strengths were all far lower than those of the Examples.

[0093] Therefore, by precisely controlling the curing sequence of each functional layer, this application eliminates the macroscopic interface in traditional multilayer structures, improves the structural integrity, interlayer bonding force and mechanical properties of artificial flaps, and can meet the stringent requirements for flap suturing operations in clinical transplantation.

[0094] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for preparing a pre-vascularized artificial skin flap, characterized by, include: We offer matrix bio-inks, vascular bio-inks, and fiber-reinforced inks. The matrix bio-ink is printed on a substrate to form a first matrix layer in a pre-cured state; The fiber-reinforced ink is printed onto the pre-cured first matrix layer through a melt extrusion printhead to form a fiber-reinforced layer; Before the fiber reinforcement layer is fully cured, the vascular bio-ink is printed on the fiber reinforcement layer to form a vascular layer, and the vascular layer is partially cured during or after the printing process. The matrix bio-ink is printed onto the partially cured blood vessel layer to form a second matrix layer in a pre-cured state; The first matrix layer, the fiber reinforcement layer, the vascular layer and the second matrix layer are integrally solidified to obtain a prevascularized artificial flap; The matrix bio-ink includes at least one of collagen, gelatin, hyaluronic acid, silk fibroin, sodium alginate, and chitosan; The vascular bio-ink comprises 8% w / v methacrylamide gelatin, as well as vascular endothelial cells and / or angiogenesis factors. The fiber-reinforced ink includes at least one of polycaprolactone, polylactic acid, and polyglycolic acid.

2. The method for preparing a prevascularized artificial flap according to claim 1, characterized in that, The curing process includes physical curing and / or chemical curing.

3. A prevascularized artificial flap, characterized in that, The prevascularized artificial flap is prepared by the method described in claim 1 or 2; wherein, The vascular layer includes biomimetic channels.

4. The prevascularized artificial flap according to claim 3, characterized in that, The biomimetic channel is at least one of a spiral network and a tree-like fractal network; and / or, The biomimetic channel has multiple branch channels, and the center distance between two adjacent branch channels is 2mm-20mm; and / or, The biomimetic channel has a porous structure with a radially gradient, and a porosity of 30%-80%; and / or, The wall of the biomimetic channel includes an endothelial cell layer; and / or, The fiber reinforcement layer is arranged in a mesh pattern; and / or, The porosity of the first matrix layer and the second matrix layer is 70%-95%.

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