A method for orienting and locking a fibrin matrix and uses thereof

Abstract

This invention belongs to the field of biomedical polymer materials and tissue engineering technology, and relates to a method and application for locking the orientation of fibrin matrix, including: 1. preparing a fibrin precursor system; 2. loading it into a strain-constructing unit; 3. determining the intermediate gelation stage; 4. applying controllable local principal strain: applying controllable local principal strain to the fibrin network during the intermediate gelation stage, so that the local principal stretch ratio of the target region reaches 1.2 to 2.2, inducing the rearrangement of fibrin fibers and pore structures in the target region along the direction of the local principal strain; 5. locking the orientation structure: allowing the fibrin network to continue to complete gelation, fixing the fibrin orientation structure and pore orientation structure, and obtaining an oriented fibrin matrix; this invention can construct random, unidirectional, gradient, or partitioned oriented in vitro fibrin matrices, and is suitable for in vitro cell migration, tumor invasion, fibrosis microenvironment simulation, drug evaluation, and organ-on-a-chip model construction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and tissue engineering technology, specifically relating to a method and application for locking the orientation of fibrin matrix. Background Technology

[0002] The extracellular matrix (ECM) not only provides a supportive environment for cell adhesion but also regulates cell extension, migration, invasion, proliferation, differentiation, and drug response through fiber orientation, pore morphology, mechanical state, and spatial distribution. In muscle, tendons, nerves, blood vessels, myocardium, tumor stroma, and fibrotic tissues, the ECM is often not completely random but exhibits varying degrees of directional arrangement. Constructing orientation-controllable three-dimensional fibrous matrix is ​​of great significance for in vitro tissue models, tumor invasion models, organ-on-a-chip research, drug evaluation, and tissue engineering studies.

[0003] Existing methods for constructing oriented microenvironments include microgrooves, micropillar arrays, surface patterning, electrospun oriented fibers, micro / nano-printed oriented fibers, magnetic or electric field induction, extrusion printing shear orientation, microfluidic flow-induced orientation, and cell-induced self-organization. While these methods can induce cell or matrix orientation to some extent, they still have some limitations. Surface patterning methods are mostly applied to two-dimensional or quasi-two-dimensional interfaces, making it difficult to form stable orientations within soft three-dimensional matrices; electrospun or prefabricated fiber scaffolds mainly rely on exogenous solid fibers as contact guiding structures, making it difficult to completely simulate the three-dimensional fiber matrix inside natural hydrogels; external field induction methods such as magnetic and electric fields often require the introduction of responsive particles or special materials; extrusion printing and flow orientation methods are limited by nozzles, channels, and shear fields, restricting the spatial control of the orientation region and orientation direction; methods relying on cell-induced self-organization have long cycles, and the orientation results are greatly affected by cell type, cell density, and culture time.

[0004] Existing research confirms that mechanical strain can induce fiber rearrangement, pore elongation, or anisotropy in fibrous biopolymer gels such as collagen and fibrin. Furthermore, existing techniques utilize pre-deformed elastic cavities to release strain during collagen fiber formation, thereby inducing the orientation of collagen fibers within a three-dimensional gel. These studies demonstrate the feasibility of inducing fiber orientation during the formation or stress-induced process of fibrous hydrogels. However, current techniques primarily focus on collagen systems, deformation of mature gels under stress, continuous stretching culture, release from pre-deformed elastic cavities, or cell-induced remodeling. A method is lacking that utilizes fibrin as the primary fiber network, the mechanical state of fibrin during gelation as the criterion, local principal strain as the control target, and subsequent gelation to achieve orientation locking.

[0005] Fibrin is a typical natural fibrous hydrogel, formed by the gradual polymerization of fibrinogen under the action of thrombin to create a three-dimensional nanofiber network. It possesses excellent cell compatibility, degradability, and cell adhesion capabilities, and is now widely used in tissue engineering, angiogenesis, tumor models, wound repair, and cell migration research. The fibrin network undergoes a continuous transformation during its formation, from solution formation, fiber nucleation, initial connection, partial cross-linking, to complete gelation. During this process, the fibrin network can exhibit an intermediate gelation state suitable for strain-induced orientation and structural locking: at this stage, the network has formed a primary connecting framework that can effectively transfer stress, but the overall structure is not fully cross-linked and stabilized. The fibers still possess the kinetic conditions for slippage, rotation, pore stretching, and overall structural rearrangement, making it an ideal window for implementing strain-induced orientation and locking anisotropic structures. Current technologies cannot identify and utilize this specific intermediate gelation stage for directional control, making it difficult to stably prepare structurally controllable and biocompatible oriented fibrin three-dimensional matrices, thus hindering the further development of biomimetic in vitro models, organ-on-a-chip, and tissue engineering technologies. Summary of the Invention

[0006] This invention provides the following technical solution: a method for locking the orientation of fibrin matrix, comprising the following steps: Step S1: Preparation of fibrin precursor system: The fibrinogen solution and thrombin solution are mixed to obtain a precursor system in which fibrin polymerization can occur. Fibrin is the main fiber network that forms the oriented structure.

[0007] Step S2, Loading into the strain building unit: Place the fibrin precursor system into or into a building unit capable of forming a preset local principal strain field; the building unit is selected from one or more combinations of elastic cavities, flexible chip chambers, microfluidic culture chambers, deformable scaffolds, microfiber grids, elastic membranes, culture frames with anchored boundaries, releasable pre-deformable carriers, and external mechanical loading devices; the building unit is provided with geometric structures, anchoring structures, boundary constraint structures, elastic recovery structures, or deformation retention structures for defining the local principal strain field of the target region.

[0008] Step S3: Determine the intermediate gelation stage: Identify when the fibrin precursor system enters the intermediate gelation stage; the intermediate gelation stage is when the fibrin network has formed a preliminary connecting skeleton that can transmit stress, but it is not completely solidified and still has the ability to slip, rotate, stretch pores and rearrange the overall structure.

[0009] Step S4: Apply controllable local principal strain: Apply controllable local principal strain to the fibrin network during the intermediate gelation stage, so that the local principal stretch ratio of the target area reaches 1.2 to 2.2, and induce the fibrin fibers and pore structure in the target area to rearrange along the local principal strain direction.

[0010] Step S5, Orientation Structure Locking: The fibrin network is continuously gelled, and the fibrin orientation structure and pore orientation structure formed in step S4 are fixed to obtain an oriented fibrin matrix.

[0011] Preferably, in step S3, the intermediate gelation stage is determined by rheological parameters. The determination criteria are: the storage modulus G′ is greater than the loss modulus G″, and the storage modulus G′ is 5% to 90% of the storage modulus of the fibrin gel after final solidification; the storage modulus of the fibrin gel after final solidification is the storage modulus measured at the stable plateau stage or the preset final solidification time point under the same material composition and temperature conditions.

[0012] Preferably, in step S3, the parameters for determining the intermediate gelation stage are further selected from one or more combinations of the following: standing time after mixing, turbidity, fluorescence intensity, microscopic imaging image, system temperature, fibrinogen concentration, and thrombin concentration.

[0013] Preferably, in step S4, the local principal strain ratio of the target area is 1.3 to 2.0; the controllable local principal strain is applied by one or more combinations of uniaxial tension, biaxial tension, multiaxial tension, compression, release pre-deformation, bending, torsion, multi-point traction, local anchor point displacement, elastic membrane deformation, flexible chip deformation, and support grid deformation; the local principal strain ratio is determined by one or more methods of finite element simulation, digital image correlation, marker point displacement analysis, microscopic image registration, mesh tracking, and fluorescent microbead displacement analysis.

[0014] Preferably, the orientation structure locking method in step S5 includes any one of the following: a. Maintain a controllable local principal strain until the fibrin network is completely gelled; b. After applying a controllable local principal strain, release the external force and allow the fibrin network to continue gelling; c. Pre-deformation is applied to the building blocks in advance. During the intermediate gelation stage, the pre-deformation is released and the local principal strain is generated by the elastic recovery of the blocks. Then, the gelation is completed and the orientation is locked.

[0015] Preferably, the building blocks include deformation-retaining building blocks and elastic recovery-driven building blocks. Deformation-retaining building blocks can maintain or partially maintain the target deformation after external force is applied, reducing the damage to the orientation structure caused by unloading rebound. Elastic recovery-driven building blocks are in a pre-compressed, pre-stretched, bent, or torsional state before the introduction of the fibrin precursor system, releasing the pre-deformation during the intermediate gelation stage and applying local principal strain to the fibrin network through elastic recovery. The design control parameters of the building blocks include material modulus, structural stiffness, geometry, fiber diameter, thickness, anchor point position, boundary constraint method, elastic recovery capability, and deformation-retaining capability.

[0016] Preferably, the building unit is a microfiber lattice; the microfiber lattice unit structure is selected from one or more combinations of rhombus, parallelogram, hexagon, triangle, curve, gradient, and grid; the microfiber lattice preparation material is selected from one or more combinations of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, polyglycerol sebacic acid, polydimethylsiloxane, polyurethane, gelatin methacrylamide hydrogel, conductive polymer, piezoelectric polymer, shape memory polymer, and metal elastic component.

[0017] Preferably, the building block is a flexible organ-on-a-chip; the flexible organ-on-a-chip includes a culture chamber, a perfusion channel, a deformable loading unit, and a boundary anchoring structure; the culture chamber is used to accommodate the fibrin precursor system and cells; the deformable loading unit is used to apply controllable local principal strain during the gelation stage; the perfusion channel is used to input and output one or more of the following: culture medium, drugs, cytokines, immune cells, and detection probes.

[0018] Preferably, the fibrin precursor system further includes excipients selected from one or more of the following: cells, collagen, gelatin, hyaluronic acid, alginate, matrix gel, decellularized matrix, laminin, fibronectin, polypeptide hydrogel, growth factors, drugs, and fluorescent markers. The cells are selected from one or more combinations of tumor cells, fibroblasts, cancer-associated fibroblasts, endothelial cells, smooth muscle cells, immune cells, stem cells, nerve cells, and skeletal muscle cells.

[0019] The beneficial effects of this invention are: 1. This invention uses fibrin as the main fiber network for forming the orientation structure, and utilizes the intermediate gelation stage in the fibrin gelation process to achieve internal matrix orientation, avoiding complete reliance on prefabricated orientation scaffolds, strong external fields, or shear flow.

[0020] 2. This invention determines the intermediate gelation stage by using rheological parameters, time, optical signals, microstructure signals, or a combination thereof, so that loading occurs at a stage where the fibrin network can transmit stress but has not yet fully solidified. This can reduce ineffective rearrangement caused by premature loading and elastic rebound caused by late loading, thereby improving the stability of the oriented structure.

[0021] 3. By designing the local principal stretch ratio and local principal strain direction, the matrix orientation is no longer determined solely by the overall stretch amount, but by the local principal strain field of the target area, which is beneficial for constructing large-area, partitioned, gradient, or multi-directional orientation structures.

[0022] 4. This invention does not require the single material modulus of the support or carrier to be a necessary limitation. Instead, it allows material modulus, structural stiffness, geometry, fiber diameter, thickness, boundary constraints, elastic recovery, and deformation retention capacity to be optional controllable factors, thereby making it compatible with both deformation retention-driven and elastic recovery-driven construction paths.

[0023] 5. This invention enables the switchable construction of random / oriented matrices by adjusting the loading method, loading direction, and loading amplitude under the same fibrous material system and similar culture conditions, which is beneficial for studying the influence of matrix orientation on cell behavior. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process flow of a fibrin matrix orientation locking method and its application according to the present invention; Figure 2 This is a schematic diagram illustrating the relationship between the fibrin gelation state and the orientation locking effect of the present invention; Figure 3 This is a graph showing the relationship between the fibrin gelation state and the orientation locking effect of the present invention. Figure 4 These are schematic diagrams and results of the oriented fibrous matrix structure of the present invention; Figure 5 This is a schematic diagram of the random / orientation-controllable in vitro tumor microenvironment chip of the present invention; Figure 6 This is a schematic diagram of the partitioned oriented fibrous matrix of the present invention; Figure 7 This is a comparison diagram of the tensile loading state and results of the fibrin gel of the present invention; Figure 8 This is a schematic diagram of the method steps of the present invention. Detailed Implementation

[0025] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] like Figures 1-8 As shown, this embodiment presents a fibrin matrix orientation locking method. It is used to construct a stable oriented fiber network during fibrin gel formation, overcoming the problems of uncontrollable fiber arrangement within natural fibrous hydrogels and the tendency for the oriented structure to weaken or disappear after the release of external force. This invention features a simple process, good cell compatibility of the material system, and the ability to construct random, unidirectional, gradient, or partitioned oriented fibrin matrices in vitro.

[0027] This embodiment uses fibrin as the main fiber network. Controlled deformation is applied at the stage where the fibrin network is not yet fully solidified but has already formed a preliminary interconnected structure. This causes the fibrin fibers and pore structure to rearrange along a predetermined direction, and the oriented structure is then fixed through a subsequent gelation process. This yields a fibrin matrix that is orientationally stable, cell-compatible, and oriented tunable, suitable for in vitro cell migration, tumor invasion, fibrosis microenvironment simulation, drug evaluation, and organ-on-a-chip model construction.

[0028] The fibrin matrix orientation locking method of this embodiment includes the following steps: S1, Preparation of the fibrin precursor system. A fibrinogen solution is mixed with a thrombin solution to form a precursor system capable of fibrin polymerization; wherein fibrin forms the main fibrous network that shapes the oriented structure.

[0029] S2, a building block into which a fibrin precursor system can be placed or contacted to form a pre-defined local principal strain field. The building block includes an elastic cavity, a flexible chip chamber, a microfluidic culture chamber, a deformable scaffold, a microfiber lattice, an elastic membrane, a culture frame with anchored boundaries, a releasable pre-deformable carrier, an external mechanical loading device, or a combination thereof. The building block has a geometry, anchoring structure, boundary constraint structure, elastic recovery structure, or deformation-retaining structure for defining the local principal strain field of the target region.

[0030] S3 indicates that the fibrin precursor system is in the intermediate gelation stage. The intermediate gelation stage is when the fibrin network has formed a preliminary connection structure that can transmit stress, but it has not yet fully solidified and still has the ability to rearrange its structure.

[0031] In a preferred embodiment, the intermediate gelation stage is determined by the storage modulus G′ and the loss modulus G″, wherein G′ is greater than G″ and G′ is lower than the storage modulus of the final solidified fibrin gel. Further, in the intermediate gelation stage, G′ is 5%–90% of the storage modulus of the final solidified fibrin gel; preferably 10%–80%; more preferably 20%–70%. The storage modulus of the final solidified fibrin gel refers to the storage modulus measured when the fibrin gel reaches a stable plateau stage under the same material composition and temperature conditions, or the storage modulus measured at a preset final solidification time point.

[0032] In another embodiment, the intermediate gelation stage is determined by post-mixing time, rheological parameters, turbidity, fluorescence intensity, microscopic imaging, temperature, fibrinogen concentration, thrombin concentration, or a combination thereof.

[0033] S4, during the intermediate gelation stage, a controllable local principal strain is applied to the fibrin network so that the local principal stretch ratio of the target region reaches 1.2 to 2.2, preferably 1.3 to 2.0, more preferably 1.4 to 1.8, and more preferably 1.5 to 1.7, thereby causing the fibrin fibers and pore structure in the target region to rearrange along the direction of the local principal strain.

[0034] Controllable local principal strain can be achieved through uniaxial tension, biaxial tension, multiaxial tension, compression, release pre-deformation, bending, torsion, multi-point traction, local anchor point displacement, elastic membrane deformation, flexible chip deformation, support grid deformation, or combinations thereof. The local principal strain ratio can be determined through finite element simulation, digital image correlation, marker point displacement analysis, microscopic image registration, mesh tracing, fluorescent microbead displacement analysis, or combinations thereof.

[0035] S5, the fibrin network continues to gel, thereby locking the fibrin orientation structure and pore orientation structure formed in step S4, and obtaining the oriented fibrin matrix.

[0036] In one embodiment, the fibrin network continues to gel while maintaining a controlled local principal strain. In another embodiment, the external force is released after the controlled local principal strain is applied, and the fibrin network continues to gel. In yet another embodiment, the fibrin network continues to gel after recovery deformation is generated by releasing pre-deformation through building blocks.

[0037] In this invention, the material modulus, structural stiffness, geometry, fiber diameter, thickness, anchor point location, boundary constraint method, elastic recovery capability, and deformation retention capability of the building unit collectively determine its strain transfer, elastic recovery, and deformation retention effects on the fibrin precursor system. This invention does not require a fixed material modulus as a necessary condition, but rather uses the ability of the target region to obtain a preset local principal strain and lock the orientation structure during subsequent gelation as the design basis.

[0038] The building blocks can be either deformation-retaining or elastic recovery-driven. Deformation-retaining building blocks maintain or partially maintain the target deformation after external force is applied, reducing the damage to the oriented structure caused by unloading rebound. Elastic recovery-driven building blocks are in a pre-compressed, pre-stretched, bent, or torsional state before being introduced into the fibrin precursor system, and release the pre-deformation during the intermediate gelation stage, applying local principal strain to the fibrin network through elastic recovery.

[0039] In one embodiment, the building block is a microfiber lattice. The microfiber lattice may have rhomboid units, parallelogram units, hexagonal units, triangular units, curved units, gradient units, mesh units, or combinations thereof. The microfiber lattice may be made of polycaprolactone, polylactic acid, polylactic-co-hydroxyacetic acid copolymer, polyglycerol sebacic acid, polydimethylsiloxane, polyurethane, gelatin methacrylamide hydrogel, conductive polymers, piezoelectric polymers, shape memory polymers, metallic elastic components, or combinations thereof.

[0040] In another embodiment, the building block is a flexible organ-on-a-chip. The flexible organ-on-a-chip includes a culture chamber, a perfusion channel, a deformable loading unit, and a boundary anchoring structure. The culture chamber is used to contain the fibrin precursor system and cells; the deformable loading unit is used to apply controllable local principal strain to the culture chamber during the fibrin gelation stage; the perfusion channel is used for the input and output of culture medium, drugs, cytokines, immune cells, detection probes, or combinations thereof.

[0041] The fibrin precursor system may also include cells, collagen, gelatin, hyaluronic acid, alginate, matrix gel, decellularized matrix, laminin, fibronectin, polypeptide hydrogel, growth factors, drugs, fluorescent markers, or combinations thereof. Cells include tumor cells, fibroblasts, cancer-associated fibroblasts, endothelial cells, smooth muscle cells, immune cells, stem cells, nerve cells, skeletal muscle cells, or combinations thereof.

[0042] The present invention also provides an oriented fibrin matrix, which is prepared by the above method. The oriented fibrin matrix includes an oriented fibrin fiber network and an oriented pore structure formed by the fibrin fiber network. Further, the long axis of the fibrin fiber network, the pores, and / or the long axis of the embedded cells are aligned along a predetermined direction.

[0043] The present invention also provides an in vitro model or organ-on-a-chip, comprising the above-described oriented fibrin matrix. The in vitro model or organ-on-a-chip may include a chip substrate, a culture chamber, a perfusion channel, a deformable loading unit, and an oriented fibrin matrix. The culture chamber may include a tumor cell culture area, a fibrin stroma area, a vascular-like channel area, an immune cell channel area, or a combination thereof.

[0044] This invention also provides the application of the above-mentioned oriented fibrous matrix, in vitro model, or organ-on-a-chip in the preparation of products for in vitro cell migration evaluation, in vitro tumor invasion evaluation, in vitro tumor stroma remodeling evaluation, in vitro angiogenesis evaluation, in vitro immune cell infiltration evaluation, in vitro drug penetration evaluation, in vitro drug sensitivity evaluation, in vitro fibrosis microenvironment simulation, or in vitro tissue engineering model construction. The above applications are for in vitro research or in vitro drug evaluation purposes and do not involve methods for the diagnosis and treatment of diseases in humans or animals.

[0045] Example Example 1: Construction of a unidirectional fibroin matrix based on the intermediate gelation stage.

[0046] Prepare fibrinogen and thrombin solutions. Mix the fibrinogen and thrombin solutions to achieve a final fibrinogen concentration of 0.5–50 mg / mL and a final thrombin concentration of 0.01–100 U / mL. In one specific embodiment, the final fibrinogen concentration is 3 mg / mL and the final thrombin concentration is 2 U / mL. Inject the mixed fibrin precursor system into the pores of a rhomboid microfiber framework. The rhomboid microfiber framework is made of polycaprolactone and has a structure that can be stretched along the diagonal direction.

[0047] After fibrinogen and thrombin are mixed, the fibrin network formation process is monitored. Fluorescently labeled fibrinogen microscopy reveals the gradual nucleation and connection of the fibrin network. Rheological time-varying measurements show the changes in storage modulus G′ and loss modulus G″ over gelation time. When the fibrin network enters the intermediate gelation stage (G′ is greater than G″ but still lower than the final coagulation G′), uniaxial stretching is applied to the microfibril framework.

[0048] In one specific embodiment, approximately 60% stretching is applied to the rhomboid microfiber lattice about 90 seconds after mixing, bringing the local principal stretch ratio in the target region close to 1.6. At this point, the fibrin network has formed a preliminary interconnected structure capable of transmitting stress, but it is not yet fully solidified and can undergo fiber rotation, pore elongation, and network rearrangement under external strain. Continued culturing after stretching allows the fibrin network to complete gelation. After gelation, the external force is released, and the resulting fibrin matrix retains the fiber orientation and pore long axis orientation along the stretching direction.

[0049] Example 2: The effect of loading at different gelation stages on orientation locking effect.

[0050] Using the same material system and microfiber lattice structure as in Example 1, uniaxial tension of the same amplitude was applied at different gelation stages. The first group was loaded in the early stage after mixing, when the fibrin network was insufficiently connected, external strain was difficult to transfer cooperatively within the network, fiber rearrangement was weak, and orientation retention was limited after unloading. The second group was loaded in the intermediate gelation stage, when the network had stress transfer capability but still retained rearrangement capability. Tension induced significant rearrangement of fibrin fibers and pore structures along the local principal strain direction, which was then fixed during subsequent gelation. The third group was loaded near or after complete gelation. At this stage, the network connection was strong, and instantaneous orientation could occur during stretching, but after unloading, due to elastic rebound, the orientation was significantly weakened or disappeared.

[0051] This embodiment illustrates that the intermediate gelation stage is a crucial stage for achieving fibrin matrix orientation locking. Loading too early prevents the network from effectively transferring stress, while loading too late results in significant elastic recovery of the network, both of which are detrimental to the formation of a stable orientation structure.

[0052] Example 3: Determining the intermediate gelation stage using rheological parameters.

[0053] The fibrin gelation process was monitored over time using a rotational rheometer. A mixture of fibrinogen and thrombin solutions was rapidly loaded onto the rheometer platform, and small-amplitude oscillation tests were performed at 37°C. The changes in G′ and G″ over time were recorded. The time interval for the transition of the fibrin network from a liquid-like state to an elastic support network was determined based on the rheological curves.

[0054] When G′ is less than or close to G″, the system remains predominantly liquid or weakly connected, with insufficient network stress transfer capacity. When G′ is greater than G″ but lower than the final solidification plateau value, the system enters the intermediate gelation stage, possessing both strain transfer and network rearrangement conditions. When G′ approaches the final solidification plateau value, the network tends to be fully solidified. Although it can undergo elastic deformation under loading, it is more prone to springback after unloading and is difficult to stably maintain its orientation.

[0055] In this embodiment, G′ is greater than G″ and G′ is 5%–90% of G′ after final coagulation as the criterion for the intermediate gelation stage; preferably 10%–80%; more preferably 20%–70%. This criterion can be adjusted according to fibrinogen concentration, thrombin concentration, temperature, ionic strength, and sample size.

[0056] Example 4: Effect of local principal stretch ratio on fibrin matrix orientation.

[0057] Using the same fibrin precursor system and building blocks as in Example 1, uniaxial stretching of different amplitudes was applied during the intermediate gelation stage. The local principal stretch ratio of the target region was determined by finite element analysis or image marker displacement analysis, and the orientation of fibrin fibers, pore long axis orientation, and embedded cell orientation were analyzed by fluorescence microscopy.

[0058] When the local principal stretch ratio is low, the fibrin network remains dominated by random pores and random fibers. When the local principal stretch ratio increases above 1.3, the fibrin pores gradually elongate, and the fiber orientation deflects along the local principal strain direction. When the local principal stretch ratio reaches 1.5–1.7, the long axes of the fibrin fibers and pores exhibit significant orientation, which is maintained after subsequent gelation. Further increasing the stretching amplitude leads to a plateau in orientation enhancement, while excessive deformation may increase the risk of carrier damage, interfacial deadhesion, or cellular stress. Therefore, controlling the local principal stretch ratio of the target region within 1.3–2.0, preferably 1.5–1.7, is beneficial for achieving a stable, reproducible, and cell-compatible oriented fibrin matrix.

[0059] Example 5: Deformation-retaining building blocks for constructing oriented fibrous matrix.

[0060] Polycaprolactone microfiber frameworks with different fiber diameters or unit structures were prepared. A fibrin precursor system was injected into each framework, and the same nominal stretching was applied during the intermediate gelation stage. The orientation retention effect of each fibrin matrix was compared using microscopic imaging and image analysis.

[0061] When the lattice fibers are too thin, their mechanical strength is insufficient, making them prone to breakage or structural instability under significant tension, and they are unable to stably transfer strain. When the lattice fibers are too thick or the structural stiffness is too high, the lattice stores more elastic energy after tension, which may cause significant rebound of the fibrin matrix upon unloading, resulting in a decrease in orientation retention. Selecting microfiber lattices with appropriate fiber diameter, structural stiffness, and unit geometry can achieve a balance between mechanical stability and low rebound, allowing the fibrin matrix to maintain a high degree of orientation after unloading.

[0062] This embodiment illustrates that the building blocks are not merely containers, but crucial structural units used to regulate the local principal strain field and maintain the structural state after unloading. The design of the building blocks should not be based solely on the intrinsic modulus of the material, but should comprehensively consider material modulus, structural stiffness, fiber diameter, geometry, boundary constraints, elastic energy storage, and deformation retention capacity.

[0063] Example 6: Construction of oriented fibrous matrix using elastic recovery-driven building blocks.

[0064] Elastic recovery-driven building blocks are fabricated. These building blocks can be pre-stretched elastic cavities, pre-compressed elastic cavities, flexural elastic membranes, torsional elastic scaffolds, shape memory scaffolds, or releasable pre-deformable chips. First, the building blocks are placed in a pre-compressed, pre-stretched, bent, or torsional state, and a fibrin precursor system is introduced into them. After the fibrin network enters the intermediate gelation stage, the pre-deformation of the building blocks is released, allowing them to elastically recover. During the elastic recovery process, a preset local principal strain is applied to the fibrin network, causing the fibrin fibers and pore structure to rearrange their orientation. Subsequently, the fibrin network continues to gel, locking the orientation structure.

[0065] This embodiment illustrates that elastic recovery can be one of the ways to drive fibrin network rearrangement. Unlike deformation-preserving pathways, this pathway does not require low resilience of building blocks, but rather requires that their deformation recovery occur during the intermediate gelation stage of the fibrin and that a predetermined local principal strain field be generated in the target region.

[0066] Example 7: Construction of a random / orientation-switched fibrin matrix.

[0067] Two groups of fibrin matrices were prepared using the same fibrinogen concentration, thrombin concentration, and culture conditions. In the randomized group, the fibrin precursor system was injected into the culture chamber without external strain, allowing it to gel naturally to obtain a randomly oriented fibrin matrix. In the oriented group, after injecting building blocks that could form a preset local principal strain field into the fibrin precursor system, controllable local principal strain was applied during the intermediate gelation stage to achieve a local principal stretch ratio of 1.3–2.0 in the target region, followed by continued gelation to obtain an oriented fibrin matrix.

[0068] Since the randomized and oriented groups use the same material system and are controlled solely by strain loading, this method can be used to study the effect of matrix orientation, a single variable, on cell behavior. It can be used to compare differences in cell migration direction, migration speed, cell morphology, invasion depth, matrix degradation, and drug response in randomized and oriented matrices.

[0069] Example 8: Construction of in vitro tumor stroma microenvironment chip.

[0070] A flexible tumor-stromal microenvironment chip was fabricated. The chip includes a tumor cell culture region, a fibrin-stromal region, perfusion channels, and deformable loading units. Tumor cells were seeded in the tumor cell culture region, and a precursor system containing fibrinogen, thrombin, and cancer-associated fibroblasts was injected into the fibrin-stromal region. Depending on experimental needs, endothelial cells could also be seeded in the perfusion channels to form a vascular-like interface, or immune cells could be added to the perfusion fluid to simulate the immune cell infiltration process.

[0071] When the fibrin network in the fibrin stroma region enters the intermediate gelation stage, a preset local principal strain is applied to the fibrin stroma region through traction at both ends of the chip, local anchor point traction, elastic membrane deformation, or release pre-deformation. By adjusting the loading direction and anchor point position, the fibrin fibers can be aligned along the direction of tumor cell invasion, perpendicular to the direction of tumor cell invasion, or form a gradient orientation structure. After further gelation, an in vitro tumor microenvironment chip containing oriented fibrin stroma is obtained.

[0072] This chip can be used to observe in vitro the behaviors of tumor cells, including migration along the matrix, invasion, mass migration, single-cell migration, matrix degradation, vascular chemotaxis, immune cell infiltration, and drug response. This application is for in vitro research and in vitro drug evaluation purposes and does not involve the diagnosis or treatment of diseases in humans or animals.

[0073] Example 9: Construction of partitioned oriented fibroin matrix.

[0074] A deformable culture framework with multiple anchor points and multiple culture zones was prepared. Each culture zone could be injected with the fibrin precursor system, but different culture zones exhibited different local principal strain directions due to differences in boundary anchoring methods and local traction directions. During the intermediate gelation stage, loading was performed simultaneously or in stages, causing the first culture zone to form a fibrin matrix aligned horizontally, the second culture zone to form a fibrin matrix aligned vertically, and the third culture zone to form an obliquely or gradient-oriented matrix.

[0075] This embodiment can be used to construct spatially heterogeneous extracellular matrix models, such as to simulate different fiber orientation environments in tumor margin regions, fibrotic interfaces, perivascular matrix, tissue damage boundaries, or cell migration channels.

[0076] Example 10: Construction of a cell-oriented fibrous matrix.

[0077] One or more of the following cell types are incorporated into a fibrin precursor system: tumor cells, fibroblasts, cancer-associated fibroblasts, endothelial cells, immune cells, stem cells, nerve cells, skeletal muscle cells, smooth muscle cells, or other in vitro cultured cells. The cell-containing precursor system is injected into the building blocks, and a controlled local principal strain is applied during the intermediate gelation stage, causing the fibrin fibers and pore structure to rearrange their orientation along the local principal strain direction. After gelation, the embedded cells are guided by the oriented fibrin matrix, exhibiting cell extension, protrusion extension, or migration bias along the matrix orientation direction.

[0078] This embodiment illustrates that the present invention can be used not only for the construction of cell-free fibrin matrix, but also for the construction of cellular three-dimensional microenvironments. By controlling matrix orientation, cell-matrix interactions can be studied in vitro.

[0079] Example 11: Evaluation method for orientation structure.

[0080] Oriented fibrin matrix can be evaluated using the following methods: observing fibrin fiber orientation using fluorescently labeled fibrinogen or immunostaining; calculating fiber orientation index, angular distribution, orientation half-width, or anisotropy index using image analysis; calculating pore long axis direction, pore aspect ratio, and pore orientation index through pore segmentation; for cell-containing systems, cell orientation can be analyzed through the long axis of the cell nucleus, cytoskeleton, cell processes, or cell migration trajectory; and evaluating the effect of the loading process on cell viability through live / dead staining, metabolic activity detection, or cell function detection.

[0081] Comparative Example 1: Naturally gelled fibrous matrix without applied strain.

[0082] After injecting the fibrin precursor system into the same carrier, gelation was directly achieved without applying strain. The resulting fibrin matrix exhibited a random distribution of fiber orientation and pore long axis. Compared to Example 1, this comparative example could not form a stable unidirectional fibrin orientation structure.

[0083] Comparative Example 2: Strain applied after complete gelation.

[0084] The fibrin precursor system was injected into the same carrier and allowed to fully gel before applying strain of the same magnitude as in Example 1. Although the fibrin network may exhibit transient deformation and local orientation under loading, the orientation structure was significantly weakened or disappeared after unloading due to the elastic rebound of the mature network. This comparative example illustrates that applying strain after complete gelation is insufficient to achieve stable orientation locking.

[0085] Comparative Example 3: Applying strain too early.

[0086] When strain is applied immediately after fibrinogen and thrombin are mixed, the fibrin network has not yet formed an effective interconnected structure, making it difficult for external strain to be co-transmitted within the network. After gelation, the resulting fibrin matrix exhibits a low degree of orientation. This comparative example demonstrates that applying strain too early is detrimental to the co-orientation rearrangement of the fibrin network.

[0087] In summary, this invention applies controllable local principal strain during the intermediate gelation stage of the fibrin network. Leveraging the network's ability to transmit stress and undergo rearrangement at this stage, it induces the orientation rearrangement of fibrin fibers and pore structures along the strain direction, locking the orientation structure during subsequent gelation. This solves the problem in existing technologies where orientation is difficult to maintain stably after unloading due to elastic rebound. The method of this invention is controllable; a stable oriented fibrin matrix can be obtained simply by adjusting the loading timing and strain amplitude. It allows for single-variable control of random / orientation processes and can construct spatially heterogeneous matrix structures such as partitioned orientation and gradient orientation. It is suitable for constructing three-dimensional in vitro microenvironment models containing cells and can be used in various scenarios such as cell-matrix interaction studies, in vitro tumor microenvironment construction, and drug evaluation, exhibiting good biocompatibility and application flexibility.

[0088] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for locking the orientation of fibrin matrix, characterized in that, Includes the following steps: Step S1: Preparation of fibrin precursor system: The fibrinogen solution and thrombin solution are mixed to obtain a precursor system in which fibrin polymerization can occur. Fibrin is the main fiber network that forms the oriented structure. Step S2, Loading into the strain construction unit: The fibrin precursor system is placed into or brought into contact with a construction unit capable of forming a preset local principal strain field; the construction unit is selected from one or more combinations of elastic cavities, flexible chip chambers, microfluidic culture chambers, deformable scaffolds, microfiber grids, elastic membranes, culture frames with anchored boundaries, releasable pre-deformable carriers, and external mechanical loading devices; the construction unit is provided with geometric structures, anchoring structures, boundary constraint structures, elastic recovery structures, or deformation-retaining structures for defining the local principal strain field of the target region; Step S3: Determine the intermediate gelation stage: Identify that the fibrin precursor system has entered the intermediate gelation stage; the intermediate gelation stage is when the fibrin network has formed a preliminary connecting skeleton that can transmit stress, but it is not completely solidified and still has the ability to slip, rotate, stretch pores and rearrange the overall structure. Step S4: Apply controllable local principal strain: Apply controllable local principal strain to the fibrin network during the intermediate gelation stage, so that the local principal stretch ratio of the target area reaches 1.2 to 2.2, and induce the fibrin fibers and pore structure in the target area to rearrange along the local principal strain direction; Step S5, Orientation Structure Locking: The fibrin network is continuously gelled, and the fibrin orientation structure and pore orientation structure formed in step S4 are fixed to obtain an oriented fibrin matrix.

2. The fibrin matrix orientation locking method according to claim 1, characterized in that, In step S3, the intermediate gelation stage is determined by rheological parameters. The determination criteria are: the storage modulus G′ is greater than the loss modulus G″, and the storage modulus G′ is 5% to 90% of the storage modulus of the fibrin gel after final solidification. The storage modulus of the fibrin gel after final solidification is the storage modulus measured at the stable plateau stage or the preset final solidification time point under the same material composition and temperature conditions.

3. The fibrin matrix orientation locking method according to claim 1, characterized in that, In step S3, the parameters for determining the intermediate gelation stage are also selected from one or more combinations of the following: standing time after mixing, turbidity, fluorescence intensity, microscopic imaging image, system temperature, fibrinogen concentration, and thrombin concentration.

4. The fibrin matrix orientation locking method according to claim 1, characterized in that, In step S4, the local principal stretching ratio of the target area is 1.3 to 2.0; The controllable local principal strain is applied in one or more combinations of uniaxial tension, biaxial tension, multiaxial tension, compression, release pre-deformation, bending, torsion, multi-point traction, local anchor point displacement, elastic membrane deformation, flexible chip deformation, and support grid deformation. The local principal stretch ratio is determined by one or more of the following methods: finite element simulation, digital image correlation, marker displacement analysis, microscopic image registration, mesh tracking, and fluorescent microbead displacement analysis.

5. The fibrin matrix orientation locking method according to claim 1, characterized in that, The orientation structure locking method in step S5 includes any of the following: a. Maintain a controllable local principal strain until the fibrin network is completely gelled; b. After applying a controllable local principal strain, release the external force and allow the fibrin network to continue gelling; c. Pre-deformation is applied to the building blocks in advance. During the intermediate gelation stage, the pre-deformation is released and the local principal strain is generated by the elastic recovery of the blocks. Then, the gelation is completed and the orientation is locked.

6. The fibrin matrix orientation locking method according to claim 1, characterized in that, The building blocks include deformation-retaining building blocks and elastic recovery-driven building blocks. The deformation-retaining building blocks can maintain or partially maintain the target deformation after external force is applied, reducing the damage to the orientation structure caused by unloading and rebound. The elastic recovery-driven building blocks are in a pre-compressed, pre-stretched, bent or torsional state before the introduction of the fibrin precursor system. During the intermediate gelation stage, the pre-deformation is released, and local principal strain is applied to the fibrin network by relying on elastic recovery. The design control parameters of the building unit include material modulus, structural stiffness, geometry, fiber diameter, thickness, anchor point position, boundary constraint method, elastic recovery capability, and deformation retention capability.

7. The fibrin matrix orientation locking method according to claim 1, characterized in that, The building unit is a microfiber lattice; the structure of the microfiber lattice unit is selected from one or more combinations of rhombus, parallelogram, hexagon, triangle, curve, gradient, and grid. The microfiber lattice material is selected from one or more combinations of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, polyglycerol sebacate, polydimethylsiloxane, polyurethane, gelatin methacrylamide hydrogel, conductive polymer, piezoelectric polymer, shape memory polymer, and metal elastic components.

8. The fibrin matrix orientation locking method according to claim 1, characterized in that, The building block is a flexible organ-on-a-chip; the flexible organ-on-a-chip includes a culture chamber, a perfusion channel, a deformable loading unit, and a boundary anchoring structure. The culture chamber is used to accommodate the fibrin precursor system and cells; The deformable loading unit is used to apply controllable local principal strain during the gelation stage; The perfusion channel is used for the input and output of one or more of the following: culture medium, drugs, cytokines, immune cells, and detection probes.

9. The fibrin matrix orientation locking method according to claim 1, characterized in that, The fibrin precursor system also contains excipients selected from one or more of the following: cells, collagen, gelatin, hyaluronic acid, alginate, matrix gel, decellularized matrix, laminin, fibronectin, polypeptide hydrogel, growth factors, drugs, and fluorescent markers. The cells are selected from one or more combinations of tumor cells, fibroblasts, cancer-associated fibroblasts, endothelial cells, smooth muscle cells, immune cells, stem cells, nerve cells, and skeletal muscle cells.

10. The application of the fibrin matrix orientation locking method according to any one of claims 1 to 9 in the preparation of oriented fibrin matrix, in vitro model or organ-on-a-chip.

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