A microfiber scaffold, organoid tissue patch, and methods of making and using the same

Abstract

The application discloses a kind of microfiber scaffolds, organoid tissue patches and preparation method and application thereof.The microfiber scaffold is prepared using melt electrospinning process, by introducing stepwise translation deposition path in the same deposition plane, so that the continuous deposition of multiple microfibers forms stable convergence node structure at predetermined position, thereby constructing microfiber network with regular pore and continuous support topology.The stepwise translation deposition includes at least one repeated deposition with preset in-plane offset after the completion of the first pass fiber deposition, to reduce the electrostatic interference between fibers and promote node formation.The scaffold structure is stable, open and thin, suitable as a tissue engineering support structure.The application also provides an organoid tissue patch composed of the scaffold loaded organoids and a construction method thereof, which can be used to cover tissue injury interface, such as post-hepatectomy section repair.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and regenerative medicine technology, specifically relating to a microfiber scaffold, an organoid tissue patch, its preparation method, and its applications. Specifically, this invention relates to a method for preparing a microfiber scaffold with a convergence node, achieved by controlling the stepwise translation of the fiber deposition path within the same deposition plane during molten electrowriting. The method involves stably converging multiple microfibers at predetermined positions to form a fiber convergence node. The microfiber scaffold can serve as a tissue engineering support structure, providing a physical support interface for three-dimensional cell constructs, and is suitable for tissue engineering applications requiring a balance between structural stability and pore connectivity. Background Technology

[0002] Tissue engineering and regenerative medicine aim to repair, replace, or reconstruct the function of damaged tissues or organs by constructing engineered scaffold systems with biocompatibility, structural stability, and functional support capabilities. As a crucial component of tissue engineering systems, scaffold materials not only need to provide the physical support required for cell adhesion and growth but also need to mimic the spatial structure and mechanical properties of the natural extracellular matrix to a certain extent, in order to maintain the morphological stability and functional state of cells. Among various scaffold types, microfiber scaffolds, due to their fiber scale closely resembling the natural extracellular matrix network, exhibit excellent adaptability in cell adhesion, migration, alignment, and three-dimensional tissue construction, and have been applied in various tissue engineering research fields, including liver, heart, bone, and soft tissue.

[0003] Currently, the fabrication of precision microfiber scaffolds mainly relies on molten electrospinning technology. This technology, by controlling the molten polymer jet and nozzle movement path, can achieve the directional deposition of micron-sized fibers, overcoming the shortcomings of electrospinning technology such as random fiber arrangement, insufficient structural consistency, and limited repeatability. However, in actual fabrication, when the fiber spacing decreases or the deposition density increases, the electrostatic interaction between fibers is significantly enhanced, easily leading to problems such as fiber misalignment, unstable deposition trajectories, or node position drift. This makes it difficult to form a stable and continuous connection structure in the fiber intersection area, affecting the consistency and repeatability of the overall scaffold topology.

[0004] To improve the overall stability of microfiber scaffolds, existing technologies typically employ methods such as increasing the number of fiber layers, introducing multilayer stacked structures, or reducing pore size to enhance the scaffold's load-bearing capacity and resistance to deformation. However, these methods often come at the cost of sacrificing pore connectivity or increasing overall thickness, which hinders the diffusion and exchange of nutrients, oxygen, and metabolic products, and also limits the practical deployment of scaffolds in space-constrained environments. Furthermore, multilayer stacked structures require high consistency in manufacturing processes, and alignment errors between layers can easily accumulate. During in vivo application, increased thickness may also affect the adhesion between the scaffold and host tissue. This is especially true in interfacial applications after tissue damage repair or resection, where scaffolds typically need to adhere to or cover tissue surfaces at relatively thin thicknesses while simultaneously withstanding multiple complex effects such as mechanical disturbances, fluid erosion, and localized stress changes. In these applications, the scaffold not only needs to maintain overall structural stability but also needs to maintain an open pore structure to support cell load and substance exchange. Scaffold structures that rely solely on increasing material usage or layer stacking to enhance stability often fail to meet interfacial adhesion and practical operational requirements due to increased thickness or limited pore size. Therefore, this type of interface application scenario poses new engineering constraints to microfiber scaffolds, namely, to achieve stable formation of fiber intersection nodes and maintain the overall structure reliably in complex environments without increasing the layered structure or reducing pore connectivity.

[0005] On the other hand, in organoid applications, organoids, as three-dimensional cell constructs with self-organizing capabilities and organ-specific functions, are considered important candidates for cell replacement therapy and tissue repair. However, existing organoid culture systems typically rely on animal-derived matrix materials to maintain their spatial structure and functional phenotype during in vitro expansion and in vivo application. These matrix materials are complex in composition, exhibit significant batch-to-batch variations, making standardized preparation difficult, and pose potential safety and compliance issues during clinical translation. Furthermore, organoids lacking stable physical support are prone to displacement, morphological collapse, or structural rearrangement in interfacial applications, further increasing the requirements for scaffold structural stability and microscopic topological control.

[0006] In summary, existing technologies generally face the following technical bottlenecks: On the one hand, in the fabrication process of microfiber scaffolds, there is a lack of a control method that can stably achieve the formation of fiber intersection nodes in a single deposition plane from the perspective of manufacturing path and forming mechanism; on the other hand, while meeting the requirements of thin structure and high porosity connectivity, existing scaffold systems are difficult to take into account the engineering requirements for structural stability in complex interface application scenarios.

[0007] Therefore, it is still necessary to provide a new method for preparing microfiber scaffolds to solve the problems of unstable fiber intersection and uncontrollable structure, and to provide a reliable structural basis for the subsequent construction of implantable organoid tissue patches. Summary of the Invention

[0008] The purpose of this invention is to address the technical problems of unstable fiber junction nodes and insufficient structural consistency in the fabrication process of existing microfiber scaffolds, as well as the difficulty in balancing structural stability under conditions of thinness and high porosity connectivity. Starting from the level of manufacturing path and forming mechanism, this invention proposes a microfiber scaffold, organoid tissue patch, its preparation method and application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a microfiber scaffold based on a melt electrowriting process, comprising the following steps: The polymer material is heated to a molten state, extruded through a nozzle to form a jet, and then fibers are deposited on the substrate. During fiber deposition, a stepwise translational deposition strategy within the same deposition plane is employed. This strategy includes: firstly, completing a first pass of fiber deposition with a predetermined in-plane fiber spacing; then, while maintaining the deposition plane unchanged, performing at least one subsequent fiber deposition operation along a direction parallel to the previous deposition path, with each subsequent deposition applying a predetermined in-plane lateral translational displacement relative to the previous deposition path; wherein the predetermined in-plane fiber spacing is 100-200 μm, and the predetermined in-plane lateral translational displacement is 30-70 μm. The stepwise translational deposition strategy enables multiple microfibers to converge and fuse at predetermined locations, forming a convergent node structure.

[0010] Furthermore, the in-plane fiber spacing is 150 μm, the in-plane lateral translational displacement is 50 μm, and at least two subsequent fiber deposition operations are performed along the parallel direction.

[0011] Furthermore, by changing the fiber deposition orientation, multi-directional fiber arrangement is formed within the same deposition plane, thereby obtaining a microfiber network with a mesh-like structure; the multi-directional orientation includes 0°, 45°, 90° and / or 135° orientation.

[0012] Furthermore, the polymer material is a biodegradable polymer; preferably poly(ε-caprolactone) (PCL).

[0013] Secondly, the present invention provides a microfiber scaffold prepared by the above method; The microfiber scaffold is a non-layered two-dimensional network structure, comprising multiple continuous microfibers and converging nodes formed by the fusion connection of multiple microfibers in the same deposition plane. The converging nodes are interconnected by continuous fibers to form a continuous in-plane mechanical transmission path; the microfibers and converging nodes together enclose an open porous structure.

[0014] Furthermore, the diameter of the microfiber is in the micrometer range, preferably 5.32±0.70μm; the characteristic pore size of the open pore structure is 30-60μm.

[0015] Thirdly, the present invention provides an organoid tissue patch, comprising: A supporting substrate, wherein the supporting substrate is the aforementioned microfiber scaffold; Organoids loaded on the surface or in the interfiber spaces of the microfiber scaffold; the organoids are attached to the microfiber scaffold via the extracellular matrix or directly to form a bioactive composite system.

[0016] Fourthly, the present invention provides a method for constructing the above-mentioned organoid tissue patch, comprising the following steps: The microfiber scaffolds were surface-treated and then incubated with a cell adhesion-promoting agent and sterilized. The organoids obtained from in vitro culture are resuspended in the culture medium and dropped onto the surface of the scaffold, allowing the organoids to enter the interfibrillary spaces and attach under the action of gravity or surface tension. After culture, the organoid tissue patch is obtained.

[0017] Furthermore, the surface treatment includes immersing the scaffold in an alkaline solution to alter the surface state of the fibers; preferably, the alkaline solution is a 0.1 mol / L sodium hydroxide solution.

[0018] Furthermore, the organoid is a bile-derived liver organoid.

[0019] Fifthly, the present invention provides the application of the above-mentioned organoid tissue patch in the preparation of medical devices for tissue damage repair.

[0020] Specifically, the above-mentioned organoid tissue patch is used in the preparation of implants for covering the cut surface or soft tissue defect interface after liver resection.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The method of the present invention controls the deposition path of microfibers by performing stepwise translational deposition in the same deposition plane, so that the microfibers deposited multiple times will converge in a controlled manner in a predetermined intersection area, thereby forming a regular convergence node structure. This avoids the problems of node position shift, node missing or inconsistent node shape caused by electrostatic interaction between fibers under high-density deposition conditions, and improves the repeatability and consistency of microfiber network structure.

[0022] (2) The microfiber scaffold prepared by the above method is a non-layered two-dimensional network structure, and the nodes are formed in the same deposition plane. The formed converging nodes are formed by melting and connecting multiple microfibers in the same deposition plane. The nodes are interconnected by continuous fibers, so that the entire microfiber network forms a continuous in-plane mechanical transmission path. The structural continuity is achieved without increasing the number of fiber layers or the amount of material used, avoiding the increase in thickness and manufacturing complexity caused by the layered structure. Through the regular distribution of the converging nodes, the pore boundaries in the microfiber network are supported by the node structure, so that the pores are not prone to collapse or morphological instability while maintaining the connectivity. This is beneficial for use in application scenarios that require both spatial openness and structural integrity.

[0023] (5) The microfiber scaffold obtained based on the above manufacturing control method is thin and has a continuous structure, making it suitable for attachment or deployment in the interface area formed by tissue damage or resection. Therefore, it can be used to load organoids and form tissue patches. Its regular node network structure provides physical support and spatial constraints for organoids, restricts the displacement of the scaffold in the local area, provides a structural basis for the positioning and spreading of organoids on the scaffold surface, and is conducive to the overall preservation of the patch during storage and operation. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation of the microfiber scaffold in Example 1.

[0025] Figure 2 The diagram shows the structure of the microfiber scaffold obtained in Example 1, where A is a structural observation diagram and B is an electron microscope observation diagram.

[0026] Figure 3 This is a diagram of the organoid tissue patch culture process, with the scale bar at 100 μm.

[0027] Figure 4 The results show the structural stability of the microfiber scaffold.

[0028] Figure 5The results are shown in Figure 1. Tensile test results of the microfiber scaffold; A shows the tensile test results along the 0° and 45° directions, demonstrating the influence of fiber orientation on stress-strain behavior; B is a schematic diagram of the tensile test of the PCL scaffold; C is a representative stress-strain curve of the tensile test of the PCL scaffold.

[0029] Figure 6 The results show the cryopreservation and resuscitation activity of microfiber scaffolds loaded with organoids.

[0030] Figure 7 The images show in vivo and ex vivo imaging results of organoid tissue patches. A is a surgical image of a mouse; B is a Kaplan-Meier curve showing the survival rate of mice in different treatment groups during postoperative follow-up (*P < 0.05); C is an in vivo fluorescence imaging image of mice; D is an ex vivo fluorescence imaging image of the major organs (heart, liver, spleen, lung, and kidney) of mice after surgery, showing the distribution of fluorescence signals in each organ; E is a quantitative statistical analysis of the fluorescence signal intensity in each ex vivo major organ (****P < 0.0001, NS: no statistical significance). Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Example 1: Method for preparing microfiber scaffolds This embodiment provides a method for preparing a microfiber scaffold. The microfiber scaffold is prepared using a melt electrowriting (MEW) process. The equipment used is a MEW system (BP6601, Yongqinquan Intelligent Equipment, China), and the printing material is medical-grade poly(ε-caprolactone) (PCL, EFL-PCL-80K).

[0033] In the preparation process, PCL is heated to a molten state and extruded through a nozzle to form a continuous polymer jet, which is then deposited onto a substrate to deposit fibers. The fiber diameter is controlled within the micrometer scale using process parameters. During fiber deposition, a stepwise translational deposition strategy within the same deposition plane is employed. Taking a 0° orientation as an example, the first fiber deposition is completed with an in-plane fiber spacing of 150 μm. Subsequently, while keeping the deposition plane unchanged, at least two subsequent fiber deposition operations are performed in a direction parallel to the previous deposition path, each time applying an in-plane lateral translational displacement of approximately 50 μm relative to the previous deposition path. Through this stepwise deposition method, multiple fibers converge at predetermined positions and form stable connections. By changing the fiber deposition orientation to 45°, 90°, and 135°, multi-directional fiber arrangements can be formed within the same deposition plane, thereby obtaining a microfiber network scaffold with a convergent node structure (see...). Figure 1 ).

[0034] Structural observation of the obtained scaffold ( Figure 2 A in the text). Electron microscopy observations show ( Figure 2 (B) The scaffold is composed of multiple continuous microfibers with a fiber diameter of approximately 5.32 ± 0.70 μm. The fibers form a converging node structure at their intersection regions, where multiple fibers are stably connected. The scaffold, together with the fibers and the converging nodes, encloses an open porous structure. Examples are provided for 0° and 45° orientations: at 0° orientation, the characteristic pore size is approximately 50.01 ± 0.53 μm; at 45° orientation, the characteristic pore size is approximately 34.08 ± 2.50 μm.

[0035] Example 2: Method for constructing organoid tissue patches Using the microfiber scaffold described in Example 1 as a supporting substrate, organoid tissue patches were constructed. After the scaffold was prepared, it underwent surface treatment and sterilization. The surface treatment consisted of immersing the scaffold in a 0.1 mol / L sodium hydroxide solution for 5 minutes to alter the fiber surface state, followed by washing with sterile phosphate-buffered saline (PBS). To promote cell adhesion, the treated scaffold was incubated in a cell adhesion promoter for 1 hour, sterilized with 75% ethanol, and then rinsed with sterile PBS.

[0036] Bile-derived liver organoids obtained through in vitro culture were collected, resuspended in culture medium, and uniformly dropped onto the scaffold surface at a predetermined volume. After standing at a constant temperature for a certain period, the organoids were allowed to enter the interfibrous spaces and complete initial attachment under surface tension. When seeded onto the scaffold, the cells adhered tightly to the fibrous network and maintained a spherical morphology. Continued culture until day 3 confirmed that they maintained stable attachment and high viability. Figure 3 ).

[0037] Experimental Example 1: Structural Stability Testing of Microfiber Scaffolds The microfiber scaffold prepared in Example 1 was placed in phosphate-buffered saline (PBS) and incubated at 37°C with shaking at 200 rpm. The scaffold structure was observed on days 0, 3, and 7. Microscopic observation showed that under the above incubation conditions, the overall scaffold structure remained continuous, and no obvious breakage, node detachment, or overall structural disintegration was observed. Figure 4 ).

[0038] Experimental Example 2: Mechanical Property Testing of Microfiber Scaffolds Tensile tests were conducted on the microfiber scaffold prepared in Example 1 using a universal testing machine. During the tests, the load-displacement relationship of the scaffold under different tensile directions was recorded, and the corresponding mechanical response curves were obtained. The test results showed that within the tested strain range, the scaffold did not fracture or become unstable, exhibiting a continuous mechanical response. Figure 5 ).

[0039] Experimental Example 3: Detection of cryopreservation and thawing activity of organoid tissue patches The organoid tissue patches from Example 2 were placed in commercial serum-free cryopreservation solution (CS10) and cryopreserved at -196 °C under liquid nitrogen. Cell viability was assessed at three preset time points: 1 month, 3 months, and 6 months. At each time point, the patches were rapidly thawed in a 37 °C water bath and then transferred to a cell culture incubator for 2 hours to achieve stable cell viability. The results showed that ( Figure 6 Compared to the control group (not cryopreserved) and the group 1 hour after cell seeding onto the scaffold, the organoid tissue patch after cryopreservation and thawing still maintained a high cell viability, suggesting that this type of tissue patch has good operational stability and application feasibility in the cryopreservation and thawing process.

[0040] Experimental Example 4: In vivo localization observation of organoid tissue patches In a 70% partial hepatectomy model in FRG mice, the PCL microfiber patch of this invention, loaded with differentiated liver organoids, was applied to the liver resection margin. Figure 7 (A) Compared with the untreated model group and the scaffold-only treatment group, the organoid tissue patch treatment group showed a trend of significantly improved survival during postoperative follow-up ( Figure 7 (B in the original text). In further localization observation, organoid tissue patches labeled with fluorescent dyes were placed on the surface of the residual liver in a partially hepatectomized mouse model, and in vivo fluorescence imaging and ex vivo imaging analysis of major organs were performed at set time points after surgery. The results showed that the fluorescence signal was mainly distributed in the liver region, and only low levels of signal or no obvious enrichment were detected in other major organs such as the heart, spleen, lungs, and kidneys. Figure 7 (CE in the text).

Claims

1. A method for preparing a microfiber scaffold based on a fused electrowriting process, characterized in that, The preparation method includes the following steps: The polymer material is heated to a molten state, extruded through a nozzle to form a jet, and then fibers are deposited on the substrate. During fiber deposition, a stepwise translational deposition strategy within the same deposition plane is employed. This strategy includes: firstly, completing a first pass of fiber deposition with a predetermined in-plane fiber spacing; then, while maintaining the deposition plane unchanged, performing at least one subsequent fiber deposition operation along a direction parallel to the previous deposition path, with each subsequent deposition applying a predetermined in-plane lateral translational displacement relative to the previous deposition path; wherein the predetermined in-plane fiber spacing is 100-200 μm, and the predetermined in-plane lateral translational displacement is 30-70 μm. The stepwise translational deposition strategy enables multiple microfibers to converge and fuse at predetermined locations, forming a convergent node structure.

2. The preparation method according to claim 1, characterized in that, The in-plane fiber spacing is 150 μm, the in-plane lateral translational displacement is 50 μm, and at least two subsequent fiber deposition operations are performed along the parallel direction.

3. The preparation method according to claim 1, characterized in that, By changing the fiber deposition orientation, a multi-directional fiber arrangement is formed within the same deposition plane, thereby obtaining a microfiber network with a mesh-like structure; the multi-directional orientations include 0°, 45°, 90° and / or 135° orientations.

4. The preparation method according to claim 1, characterized in that, The polymer material is a biodegradable polymer.

5. A microfiber scaffold, characterized in that, It is prepared by the method described in any one of claims 1-4.

6. An organoid tissue patch, characterized in that, include: A supporting substrate, wherein the supporting substrate is a microfiber scaffold as described in claim 5; Organoids loaded on the surface of the microfiber scaffold or in the interfiber spaces; The organoids are attached to the microfiber scaffold via the extracellular matrix or directly, forming a bioactive composite system.

7. A method for constructing an organoid tissue patch as described in claim 6, characterized in that, The construction method includes the following steps: The microfiber scaffolds were surface-treated and then incubated with a cell adhesion-promoting agent and sterilized. The organoids obtained from in vitro culture are resuspended in the culture medium and dropped onto the surface of the scaffold, allowing the organoids to enter the interfibrillary spaces and attach under the action of gravity or surface tension. After culture, the organoid tissue patch is obtained.

8. The construction method according to claim 7, characterized in that, The surface treatment specifically involves immersing the scaffold in an alkaline solution to alter the surface state of the fibers.

9. The construction method according to claim 7, characterized in that, The organoids described are bile-derived liver organoids.

10. The use of the organoid tissue patch as described in claim 6 in the preparation of a medical device for tissue damage repair.

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