ECM-imitated ordered hydrogel material for tendon repair and preparation method of ECM-imitated ordered hydrogel material

Abstract

The invention belongs to the technical field of tendon repair scaffold materials, and particularly relates to an ECM-imitated ordered hydrogel material for tendon repair and a preparation method. The preparation method comprises the following steps: performing annealing treatment on PLLA electrospun yarns, and then freezing and slicing to obtain PLLA short rods; the annealing treatment is carried out twice at the temperature of 100-150 DEG C; mixing the PLLA short rods with a matrix raw material solution, adding the mixture into a mold, and carrying out primary cross-linking treatment to obtain primary hydrogel; performing twisting treatment on the primary hydrogel to obtain dehydrated hydrogel; and carrying out secondary cross-linking treatment on the dehydrated hydrogel and a cross-linking agent to obtain the ECM-imitated ordered hydrogel material for tendon repair.

Description

An ECM-like ordered hydrogel material for tendon repair and its preparation method Technical Field

[0001] This invention belongs to the field of tendon repair scaffold material technology, specifically relating to an ECM-like ordered hydrogel material for tendon repair and its preparation method. Background Technology

[0002] Because tendon tissue has low cell density and low metabolic rate, its natural healing ability is limited. After tendon injury, scar tissue with poor mechanical properties often forms, making it difficult to restore the original mechanical function. Tissue engineering has provided a new solution for tendon repair, the key of which lies in constructing a scaffold material that can mimic the natural tendon extracellular matrix (ECM).

[0003] Natural polymer hydrogels, represented by collagen, have become a research hotspot in tissue engineering due to their excellent biocompatibility, biodegradability, and ability to provide a water-containing three-dimensional microenvironment similar to natural ECM. Particularly in tendon repair, hydrogels can provide a favorable environment for the adhesion, growth, and migration of seed cells, promoting tissue regeneration. However, traditional hydrogels generally suffer from insufficient mechanical properties, such as low strength, low modulus, and poor toughness, resulting in overall mechanical performance far inferior to natural tendons. Furthermore, the microstructure of hydrogels is typically a disordered porous network, lacking the highly ordered collagen fiber bundle arrangement typical of tendons, making it difficult to achieve the anisotropic characteristics associated with tendon mechanical and biological functions.

[0004] Currently, structural regulation of hydrogels through physical or chemical means has become an important direction for improving their mechanical properties and biological functions. For example, methods such as directional freeze-drying, electrospinning, microstructure regulation, and densification are used to construct fiber-oriented structures or increase gel density to improve their mechanical properties and cell-guiding capabilities. Although these technologies have made some progress, they still have important limitations: some methods require complex instruments or introduce non-natural components, which may affect biocompatibility; some densification methods are difficult to precisely control fiber orientation or easily form overly dense structures, hindering cell migration and nutrient transport.

[0005] Furthermore, natural tendons possess significant piezoelectric properties, enabling them to generate bioelectric signals during mechanical loading, regulating cell behavior and ECM remodeling. This property plays a crucial role in tendon regeneration. Research has addressed this need by attempting to endow hydrogels with electroresponsiveness through the addition of piezoelectric materials such as BaTiO3, ZnO, and PVDF. However, these composite materials often suffer from poor piezoelectric particle dispersion, unstable interfacial bonding, insufficient biocompatibility, and discontinuous piezoelectric signals, making it difficult to simulate the bioelectric environment of natural tendons. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an ECM-like ordered hydrogel material for tendon repair and its preparation method.

[0007] The present invention aims to solve the following specific technical problems: solve the problem of incompatibility between "mechanical properties" and "bioactivity": provide a three-dimensional scaffold material that has both excellent mechanical properties (such as high modulus and high strength) and good biocompatibility and can support cells, and overcome the defects of traditional hydrogel materials in the repair of load-bearing tissues such as tendons due to insufficient mechanical properties, which are prone to deformation or damage.

[0008] Achieving a high degree of biomimicry in structure: providing a multi-level scaffold that can actively simulate the highly ordered collagen fiber orientation structure of the natural tendon extracellular matrix, overcoming the difficulty of controlling fiber orientation in existing compaction techniques (such as plastic compression), and the potential sacrifice of biocompatibility in existing orientation techniques (such as directional freezing) due to the introduction of complex equipment or non-natural components.

[0009] Providing stable and controllable bioelectric activity: This invention provides a hydrogel scaffold that obtains a piezoelectric response by introducing a functional material with piezoelectric properties, enabling the material to generate bioelectric signals similar to those of natural tendons under stress conditions, thereby promoting directed cell growth and tissue regeneration; at the same time, it overcomes the problems of easy aggregation of piezoelectric particles, unstable interfacial bonding, insufficient biosafety, and poor signal stability in the prior art.

[0010] Optimize the structural morphology and application adaptability of materials: Provide a novel three-dimensional structure that is more suitable for tendon repair than existing film-type or injection-type piezoelectric materials. While possessing good cell biological properties, it maintains stable piezoelectric response and predictable mechanical properties by reasonably dispersing and fixing piezoelectric components, thereby solving the problems of uncontrollable strength after injection molding of materials or difficulty in adhering to complex shapes of film-type materials.

[0011] To achieve the above objectives, the technical solution of the present invention is as follows.

[0012] The first aspect of this invention provides a method for preparing an ECM-like ordered hydrogel material for tendon repair, comprising the following steps: annealing PLLA electrospinning, followed by cryo-slicing to obtain PLLA short rods; the annealing treatment is performed twice at 100℃ to 150℃; mixing the PLLA short rods with a matrix raw material solution, adding the mixture to a mold, and performing a first crosslinking treatment to obtain a primary hydrogel; torturing the primary hydrogel to obtain a dehydrated hydrogel; and performing a second crosslinking treatment on the dehydrated hydrogel and a crosslinking agent to obtain an ECM-like ordered hydrogel material for tendon repair.

[0013] Invention Concept: This invention utilizes a series of ordered process steps to progressively control the structural morphology of materials, thereby obtaining functional materials that possess both dense structure, orientation characteristics, and stable performance. Specifically, firstly, a two-step annealing process is used to thermally regulate the material system, promoting the rearrangement and ordering of the internal structure in stages, laying the foundation for subsequent morphology formation. Subsequently, cryo-slicing is performed at low temperatures to maintain the structural state formed after annealing and obtain an initial structure with controllable morphology and size. Based on this, a first crosslinking step is used to fix the initially formed three-dimensional network structure, ensuring structural stability during subsequent processing. Next, a torsion process is used to mechanically dehydrate the material, further densifying the internal structure and introducing a certain degree of structural orientation during dehydration. Finally, a second crosslinking step further fixes the structure after densification and orientation control, thereby stabilizing the final morphology and properties of the material. Through the synergistic effect of these steps, a material morphology with a specific degree of densification and orientation characteristics is formed.

[0014] In this invention, the purpose of annealing is to promote the rearrangement and ordering of the internal structure of the material, thereby providing the basic conditions for the formation and enhancement of the piezoelectric properties of the material. By performing annealing treatments at two different temperatures, the structural state of the material can be controlled in stages, which is beneficial to improving the piezoelectric properties.

[0015] The purpose of cryosectioning is to shape the material under low temperature conditions to maintain the structural characteristics formed after annealing, avoid structural collapse of the material under room temperature conditions, and provide samples with consistent morphology and structure for subsequent crosslinking treatment.

[0016] The purpose of the first cross-linking is to fix the initial structure formed after annealing and freeze-slicing, so that the material maintains a stable three-dimensional network structure during subsequent processing and prevents the structure from becoming loose or deformed.

[0017] The purpose of dehydration by twisting is to promote the expulsion of moisture from the material through mechanical means, thereby further densifying the material structure. At the same time, a certain degree of structural orientation is introduced during the dehydration process, which is beneficial to the improvement of the material's mechanical properties.

[0018] The role of the second crosslinking is to further fix the densified and oriented structure formed after torsion dehydration, thereby stabilizing the final morphology and properties of the material and preventing the structure from springing back or loosening during subsequent use.

[0019] Unlike the single-step annealing process used in existing technologies, this invention employs a two-step annealing process. By controlling the rearrangement process of the internal structure of the material in stages, it is beneficial to the formation and enhancement of piezoelectric properties while ensuring structural stability. The above effects are difficult to achieve with a single annealing process.

[0020] When annealing is performed only once or the annealing temperature is below 100 °C, the thermal energy is insufficient to drive the effective rearrangement of the internal structure of the material, making it difficult to form a structural state that is conducive to improving piezoelectric properties.

[0021] The choice of annealing temperature is closely related to the thermal stability of the material system and the energy required for the rearrangement of the internal structure of the material. The annealing temperature range selected in this invention can effectively promote the structural ordering while ensuring the stability of the material structure.

[0022] Preferably, the specific operation of the annealing process is as follows: the first annealing process is carried out at 100℃~110℃; and the second annealing process is carried out at 140℃~150℃.

[0023] Preferably, the time for both the first and second annealing processes is 8 to 10 hours.

[0024] Preferably, the specific operation of cryo-slicing is as follows: the annealed electrospun fibers are divided into small pieces, placed in water for freezing, and then the frozen ice blocks containing electrospun fibers are sliced ​​along the direction perpendicular to the spinning to obtain PLLA short rods.

[0025] Preferably, the matrix raw material solution is obtained by mixing collagen, PBS buffer, NaOH and water; the concentration of collagen is 5 mg / mL.

[0026] Preferably, the conditions for the first crosslinking treatment are: temperature of 35℃~37℃ and time of 1h~2h.

[0027] Preferably, the specific operation of the torsion treatment is as follows: fix both ends of the primary hydrogel, apply torque to the fixed primary hydrogel, wherein the torsion angle is 300° / mm, and obtain a dehydrated hydrogel.

[0028] In this invention, when the torsion angle is too low, the mechanical action generated by the material during the torsion process is insufficient, making it difficult to effectively remove internal moisture, resulting in low dehydration efficiency and difficulty in achieving ideal structural densification. When the torsion angle is too high, such as 600° / mm, it is prone to breakage. Therefore, the preferred torsion angle is 300° / mm. Through torsion treatment, the material is mechanically dehydrated, further densifying the internal structure and introducing a certain degree of structural orientation during the dehydration process.

[0029] Preferably, the crosslinking agent is a 0.1 mM genipin solution; the specific operation of the second crosslinking treatment is as follows: the dehydrated hydrogel is immersed in the crosslinking agent and soaked at 37°C for 16-24 hours to obtain an ECM-like ordered hydrogel material for tendon repair.

[0030] The second aspect of the present invention provides an ECM-like ordered hydrogel material for tendon repair, which is prepared by the method described in the first aspect for preparing an ECM-like ordered hydrogel material for tendon repair.

[0031] The beneficial effects of the present invention are as follows: 1. By regulating the orientation and compaction of the internal structure of the hydrogel, the present invention improves the mechanical strength and elastic modulus of the hydrogel scaffold while maintaining the material's good biocompatibility and three-dimensional porous structure, so that it can meet the initial mechanical support performance requirements for the repair of load-bearing tissues such as tendons.

[0032] 2. This invention constructs an ordered structure arranged in a specific direction inside the hydrogel, which is closer to the orientation characteristics of collagen fibers in the extracellular matrix of natural tendons in terms of structural morphology and mechanical response. This is beneficial for simulating the anisotropic structure of tendon tissue and provides a structural basis for the orderly regeneration of tissue.

[0033] 3. This invention introduces a functional material with piezoelectric properties during the hydrogel preparation process, enabling the resulting scaffold to generate a stable piezoelectric response during stress deformation, thereby endowing the material with bioelectric function and making up for the shortcomings of traditional hydrogel materials in bioelectric stimulation.

[0034] 4. The hydrogel scaffold obtained by the present invention has a good balance in terms of structural biomimicry, mechanical properties and functional integration. Compared with existing membrane or simple injection materials, it has better application potential in adapting to complex stress environments and irregular damage sites. Attached Figure Description

[0035] Figure 1 is a schematic diagram of the structure of the customized mold used in an embodiment of the present invention. Wherein, 1 is the mold body; 2 is the groove; and 3 is the partition.

[0036] Figure 2 shows the stress-strain curves of crosslinked hydrogel samples with different contents of PLLA provided in Examples 1 to 2 and Comparative Example 1 of the present invention.

[0037] Figure 3 shows the biocompatibility characterization of cross-linked hydrogel tissue cells provided in Example 4 of the present invention. Among them, (a) is a schematic diagram of the survival and morphology of cells after staining after 1 day of culture; (b) is a schematic diagram of the survival and morphology of cells after staining after 3 days of culture; and (c) is a schematic diagram of the survival and morphology of cells after staining after 7 days of culture.

[0038] Figure 4 is a stress-strain curve of a hydrogel that has undergone self-assembly crosslinking without torsion and further crosslinking, as provided in an embodiment of the present invention.

[0039] Figure 5 is a stress-strain curve of a hydrogel with a unit length torsion angle of 300° and no further cross-linking provided by an embodiment of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] To overcome the limitations of hydrogels, current research has developed a variety of advanced hydrogel modification techniques.

[0043] Directional structural enhancement technologies, such as directional freeze-drying and bioelectrospinning, can significantly improve the mechanical properties of hydrogels by altering their microstructure to achieve directional fiber alignment, resulting in moduli ranging from tens to hundreds of megapascals. However, these methods also have many limitations, such as poor biocompatibility during preparation (potentially using toxic cross-linking agents or organic solvents), the need for specialized and complex equipment, or the introduction of additional non-natural material components, thus restricting their clinical translation prospects.

[0044] Densification techniques, such as plastic compression and gel extraction-extrusion, physically squeeze out some of the water from the hydrogel, promoting densification and improving its mechanical properties to some extent. However, these methods either struggle to precisely control the structural orientation of the fibers within the hydrogel, making it unable to effectively mimic the anisotropy of tendons; or the preparation process lacks controllability, resulting in limited mechanical reinforcement and poor batch-to-batch repeatability.

[0045] To endow hydrogels with bioelectric activity, current research often employs piezoelectric enhancement techniques by incorporating piezoelectric materials, such as PVDF, BaTiO3, or ZnO nanoparticles, piezoelectric crystals, or piezoelectric nanofibers, into the hydrogel network to generate electrical signals under mechanical stimulation. However, these methods generally suffer from the following problems: piezoelectric particles tend to aggregate within the hydrogel, leading to localized stress concentration or uneven electrical response; the interface between piezoelectric particles and the hydrogel matrix is ​​weak, making them prone to detachment or migration during deformation or long-term use; the interface between exogenous piezoelectric particles and the colloidal layer may pose risks to biocompatibility or stability; and the uneven distribution of piezoelectric components makes it difficult to match the overall piezoelectric properties with the continuous signal of natural tendons.

[0046] Specifically, the fundamental structural defect lies in the fact that, from a mechanical perspective, the existing three-dimensional network structure of injectable hydrogels typically has high water content and low cross-linking density, making them inherently soft and weak, with an elastic modulus far lower than that of real tendon tissue, thus failing to provide effective mechanical support in vivo. Furthermore, methods of reinforcement through physical compression or doping with rigid particles can easily lead to an overly dense structure or the formation of stress concentration points, damaging its porous structure and hindering cell migration and nutrient delivery.

[0047] From the perspective of biomimetic structures, existing technologies (such as plastic compression) produce fibers with disordered or random arrangements, which cannot simulate the highly ordered collagen fiber bundle structure of natural tendons. On the other hand, technologies that can achieve oriented structures (such as oriented freezing) have poor compatibility with bioactive components (such as living cells) during the preparation process, making it difficult to load cells while constructing the structure, and the process is complex and costly.

[0048] From a functional perspective, existing techniques for imparting piezoelectricity to hydrogels through exogenous piezoelectric particles generally suffer from problems such as uneven particle dispersion, easy aggregation, and poor interfacial bonding. This leads to unstable piezoelectric responses and may pose risks such as particle detachment, biocompatibility issues, and long-term mechanical property degradation. Furthermore, the electrical signals under this "particle doping" mechanism often lack continuity and directionality, making it difficult to simulate the natural piezoelectric behavior of tendons.

[0049] From the perspective of application structure, existing materials are mostly pre-formed rigid films or injection-molded materials with uncontrollable mechanical properties. The former is difficult to fit complex damaged areas, while the latter cannot guarantee the structure and strength after in-situ molding in the body. There is a lack of an ideal form that can adapt to the shape and have a predetermined reinforcing structure.

[0050] In summary, there is still a lack of a comprehensive functional material that simultaneously possesses good mechanical properties, an ordered ECM-like structure, the ability to support cells, and stable piezoelectric response. This has become a key bottleneck restricting the development of tendon tissue engineering. Therefore, there is an urgent need to develop a scaffold material that can uniformly and stably load piezoelectric components into a three-dimensional biomimetic structure and generate stable piezoelectric signals under mechanical stress.

[0051] The present invention aims to provide a tendon repair material that combines a biomimetic ECM structure, suitable mechanical properties, and intrinsic piezoelectric activity. Specifically, the present invention aims to: construct a biomimetic ECM and a mechanical reinforcement structure: provide a novel hydrogel scaffold with high density and ordered fiber orientation, so that the material can achieve high modulus, high strength, and high toughness without relying on large-scale external reinforcement methods, approaching the mechanical level of natural tendons.

[0052] Achieving efficient cell loading and cell guidance: Forming a three-dimensional scaffold with an ECM-like oriented fiber structure that supports cell adhesion, proliferation and migration, and guides cells to align in the direction of the fibers.

[0053] Achieving stable and controllable piezoelectric properties: By precisely controlling the type, dispersion method and fixation site of piezoelectric components, the scaffold can generate a stable piezoelectric response in the physiological mechanical environment to simulate the electromechanical behavior of tendon tissue and promote tissue regeneration.

[0054] Optimize the application adaptability of materials: The shape and structure of the material can be shaped or processed according to the needs of the defect area, combining initial mechanical support with good biological properties, making it more suitable for tendon repair applications than existing membrane or injection materials.

[0055] As shown in Figure 1, the customized PDMS mold includes a mold body 1, which has a groove 2, and a partition 3 is embedded in the groove 2. The length of the mold body 1 is 36mm, the width is 20mm, and the depth is 30mm. The Chinese name for PDMS is polydimethylsiloxane.

[0056] Groove 2 has a middle section and enlarged areas at both ends of the middle section. The size of the enlarged areas gradually increases away from the middle section, forming a fan-shaped structure. Specifically, the total length of groove 2 is 26mm, and it is located in the middle of mold body 1. The length of the middle section of groove 2 is 12mm, and the maximum width of the enlarged areas is 10mm. Groove 2 is mainly used to fill collagen solution, and the filling depth of groove 2 is 25mm.

[0057] The partition 3 is fixed in the middle of the groove 2 and arranged along the length of the mold body 1. The partition 3 is 20mm long and 2mm wide. The partition 3 is required to be embedded 3mm into the bottom of the mold body 1 and 5mm above the top plane of the mold body 1. It can be freely disassembled for easy demolding.

[0058] The technical solution of the present invention will be further described below through specific embodiments. Unless otherwise specified, the methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0059] Poly-L-lactide oligomer, abbreviated as PLLA, is a hydroxyl-terminated form of poly-L-lactide oligomer. Its main chain is composed of repeating L-lactide units, and the molecular chain ends with chemically active hydroxyl groups (usually double-terminated hydroxyl groups).

[0060] Example 1: A method for preparing an ECM-like ordered hydrogel material for tendon repair, comprising the following steps: Step 1, preparing PLLA short rods: Dissolve 400 mg of PLLAOH (PLLAOH is the hydroxyl-terminated form of poly-L-lactic acid with a molecular weight of 1 million) in hexafluoroisopropanol to obtain a PLLA solution with a concentration of 40 mg / mL. Then, electrospin the PLLA solution with a concentration of 40 mg / mL. Anneal the obtained electrospun fibers at 100°C for 10 h. After naturally cooling to room temperature, anneal at 150°C for 10 h. Finally, after cooling to room temperature, complete the high-temperature annealing to obtain the annealed electrospun fibers.

[0061] The annealed electrospun yarn was cut into 3mm×3mm square pieces. Water was added to an ice box, and the square pieces were laid flat on the water and frozen overnight. The next day, more water and pieces were added to freeze the ice, so that each piece had two pieces of electrospun yarn, resulting in ice blocks. The ice blocks were then sliced ​​on a freeze cutter along the direction perpendicular to the spinning to obtain PLLA short rods.

[0062] Step 2, Preparation of primary hydrogel: Take 8 mg of PLLA short rods, 800 μL of 10× (10-fold concentrated) PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water. Mix them evenly to obtain a collagen solution with a final concentration of 5 mg / mL. Then, add the neutralized collagen solution dropwise into the groove of a custom PDMS mold, as shown in Figure 1, and place it at 37℃ for crosslinking for 1 hour to obtain the primary hydrogel, denoted as TC300-1PL.

[0063] Step 3, fixing the hydrogel and achieving its dehydration and further cross-linking: The primary hydrogel is removed from the groove of the PDMS mold. The two ends of the annular primary hydrogel are fixed using paperclips, with the distance between the fixed ends matching the inner ring length of the primary hydrogel to avoid stretching it before dehydration. Subsequently, a torque is applied to the primary hydrogel using a torsion device to achieve rapid dehydration. The torsion speed is (π / 4) / s, and the torsion angle per unit length of the primary hydrogel is θ = 300° / mm.

[0064] The dehydrated hydrogel was placed in a 0.1 mM genipin solution for further cross-linking for 24 hours to obtain a cross-linked hydrogel sample, which is an ECM-like ordered hydrogel material for tendon repair.

[0065] Example 2: A method for preparing an ECM-like ordered hydrogel material for tendon repair, differing from Example 1 in that the amount of PLLA short rods added to the hydrogel is different; the specific preparation method includes the following steps: Step 1, preparing PLLA short rods: Take 400 mg of PLLAOH (molecular weight of 1 million) and dissolve it in hexafluoroisopropanol to obtain a PLLA solution with a concentration of 40 mg / mL. Then, electrospin the PLLA solution with a concentration of 40 mg / mL. Anneal the obtained electrospun fibers at 100°C for 10 h, wait for them to cool naturally to room temperature, and then anneal them at 150°C for 10 h. Finally, after cooling to room temperature, complete the high-temperature annealing to obtain the annealed electrospun fibers.

[0066] The annealed electrospun yarn was cut into 3mm×3mm square pieces. Water was added to an ice box, and the square pieces were laid flat on the water and frozen overnight. The next day, more water and pieces were added to freeze the ice, so that each piece had two pieces of electrospun yarn, resulting in ice blocks. The ice blocks were then sliced ​​on a freeze cutter along the direction perpendicular to the spinning to obtain PLLA short rods.

[0067] Step 2, Preparation of primary hydrogel: Take 16 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water. Mix them evenly to obtain a collagen solution with a final concentration of 5 mg / mL. Then, drop the neutralized collagen solution into the groove of a custom PDMS mold, as shown in Figure 1, and place it at 37℃ for crosslinking for 1 hour to obtain the primary hydrogel, denoted as TC300-2PL.

[0068] Step 3, fixing the hydrogel and achieving its dehydration and further cross-linking: The primary hydrogel is removed from the groove of the PDMS mold. The two ends of the annular primary hydrogel are fixed using paperclips, with the distance between the fixed ends matching the inner ring length of the primary hydrogel to avoid stretching it before dehydration. Subsequently, a torque is applied to the primary hydrogel using a torsion device to achieve rapid dehydration. The torsion speed is (π / 4) / s, and the torsion angle per unit length of the primary hydrogel is θ = 300° / mm.

[0069] The dehydrated hydrogel was placed in a 0.1 mM genipin solution for further cross-linking for 24 hours to obtain a cross-linked hydrogel sample, which is an ECM-like ordered hydrogel material for tendon repair.

[0070] Example 3: A method for preparing an ECM-like ordered hydrogel material for tendon repair, comprising the following steps: Step 1, preparing PLLA short rods: carried out according to the method of Example 2.

[0071] Step 2, Preparation of TC300-1PL hydrogel embedded with cells: Take 10× culture medium (with a cell concentration of 5*10) 5 800 μL of collagen (cells / mL), 8 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water were mixed thoroughly to obtain a collagen solution with a final concentration of 5 mg / mL. The collagen solution with cells embedded was then dropped into the groove of a sterilized PDMS mold and incubated at 37°C for 1 hour to allow the collagen to crosslink into a hydrogel, thus obtaining a primary hydrogel tissue.

[0072] Step 3: Fix the hydrogel tissue and achieve its dehydration and further cross-linking: Dehydrate the primary hydrogel tissue according to the method in Step 3 of Example 2, demonstrating that the cells can survive and spread. Maintain a sterile environment, and then continue to incubate in an incubator for 1 to 7 days. Stain the tissue to observe cell survival and morphological changes, ultimately obtaining the desired hydrogel tissue. The specific method is as follows:

[0073] The primary hydrogel tissue was removed from the groove of the PDMS mold, and the two ends of the annular primary hydrogel tissue were fixed with paperclips. The distance between the fixed ends was consistent with the inner ring length of the primary hydrogel tissue to avoid stretching the primary hydrogel tissue before dehydration. Then, a torque was applied to the primary hydrogel tissue using a torsion device to achieve rapid dehydration of the primary hydrogel tissue. The torsion speed was (π / 4) / s, and the torsion angle per unit length of the primary hydrogel tissue was θ = 300° / mm.

[0074] The dehydrated hydrogel tissue was placed in a 0.1 mM genipin solution for further cross-linking for 24 hours to obtain a cross-linked hydrogel tissue sample, which is the ECM-like ordered hydrogel material for tendon repair.

[0075] Example 4: A method for preparing an ECM-like ordered hydrogel material for tendon repair, comprising the following steps: Step 1, preparing PLLA short rods: carried out according to the method of Example 2.

[0076] Step 2, Preparation of TC300-2PL hydrogel embedded with cells: Take 10× culture medium (with a cell concentration of 5*10) 5800 μL of collagen (cells / mL), 16 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water were mixed thoroughly to obtain a collagen solution with a final concentration of 5 mg / mL. The collagen solution with cells embedded was then dropped into the groove of a sterilized PDMS mold and incubated at 37°C for 1 hour to allow the collagen to crosslink into a hydrogel, thus obtaining a primary hydrogel tissue.

[0077] Step 3: Fix the hydrogel tissue and achieve its dehydration and further cross-linking: Dehydrate the primary hydrogel tissue according to the method in Step 3 of Example 2, demonstrating that the cells can survive and spread. Maintain a sterile environment, and then continue to incubate in an incubator for 1 to 7 days. Stain the tissue to observe cell survival and morphological changes, ultimately obtaining the desired hydrogel tissue. The specific method is as follows:

[0078] The primary hydrogel tissue was removed from the groove of the PDMS mold, and the two ends of the annular primary hydrogel tissue were fixed with paperclips. The distance between the fixed ends was consistent with the inner ring length of the primary hydrogel tissue to avoid stretching the primary hydrogel tissue before dehydration. Then, a torque was applied to the primary hydrogel tissue using a torsion device to achieve rapid dehydration of the primary hydrogel tissue. The torsion speed was (π / 4) / s, and the torsion angle per unit length of the primary hydrogel tissue was θ = 300° / mm.

[0079] The dehydrated hydrogel tissue was placed in a 0.1 mM genipin solution for further cross-linking for 24 hours to obtain a cross-linked hydrogel tissue sample, which is the ECM-like ordered hydrogel material for tendon repair.

[0080] Comparative Example 1: A method for preparing an ECM-like ordered hydrogel material for tendon repair, differing from Example 1 in that PLLA short rods were not added to the hydrogel. The specific preparation method includes the following steps: Step 1: Take 0 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water, mix them evenly to obtain a collagen solution with a final concentration of 5 mg / mL, then drop the neutralized collagen solution into the groove of a customized PDMS mold, and place it at 37°C for 1 hour for crosslinking to obtain a primary hydrogel, denoted as TC300.

[0081] Step 2, fixing the hydrogel and achieving its dehydration and further cross-linking: The primary hydrogel is removed from the groove of the PDMS mold. The two ends of the annular primary hydrogel are fixed using paperclips, with the distance between the fixed ends matching the inner ring length of the primary hydrogel to avoid stretching it before dehydration. Subsequently, a torque is applied to the primary hydrogel using a torsion device to achieve rapid dehydration. The torsion speed is (π / 4) / s, and the torsion angle per unit length of the primary hydrogel is θ = 300° / mm.

[0082] The dehydrated hydrogel was placed in a 0.1 mM genipin solution for further cross-linking for 24 hours to obtain a cross-linked hydrogel sample, which is an ECM-like ordered hydrogel material for tendon repair.

[0083] Comparative Example 2: A method for preparing an ECM-like ordered hydrogel material for tendon repair. The difference from Example 1 is that no cells are embedded, and there is no twisting or further cross-linking. The specific preparation method includes the following steps: Step 1, preparing PLLA short rods: 400 mg of PLLAOH (molecular weight of 1 million) is dissolved in hexafluoroisopropanol to obtain a PLLA solution with a concentration of 40 mg / mL. Then, the PLLA solution with a concentration of 40 mg / mL is electrospun. The obtained electrospun wire is annealed at 100°C for 10 h. After naturally cooling to room temperature, it is annealed at 150°C for 10 h. Finally, after cooling to room temperature, high-temperature annealing is completed to obtain the annealed electrospun wire.

[0084] The annealed electrospun yarn was cut into 3mm×3mm square pieces. Water was added to an ice box, and the square pieces were laid flat on the water and frozen overnight. The next day, more water and pieces were added to freeze the ice, so that each piece had two pieces of electrospun yarn, resulting in ice blocks. The ice blocks were then sliced ​​on a freeze cutter along the direction perpendicular to the spinning to obtain PLLA short rods.

[0085] Step 2, Preparation of primary hydrogel: Take 8 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water. Mix them evenly to obtain a collagen solution with a final concentration of 5 mg / mL. Then, add the neutralized collagen solution dropwise into the groove of a custom PDMS mold, as shown in Figure 1, and place it at 37°C for crosslinking for 1 hour. Remove the primary hydrogel from the groove of the PDMS mold to obtain the primary hydrogel, denoted as COL.

[0086] Comparative Example 3: A method for preparing an ECM-like ordered hydrogel material for tendon repair. The difference from Example 1 is that no cells were embedded, and no further cross-linking was performed after dehydration. The specific preparation method includes the following steps: Step 1, preparing PLLA short rods: 400 mg of PLLAOH (molecular weight of 1 million) was dissolved in hexafluoroisopropanol to obtain a PLLA solution with a concentration of 40 mg / mL. Then, the PLLA solution with a concentration of 40 mg / mL was electrospun. The obtained electrospun wire was annealed at 100°C for 10 h. After naturally cooling to room temperature, it was annealed at 150°C for 10 h. Finally, after cooling to room temperature, high-temperature annealing was completed to obtain the annealed electrospun wire.

[0087] The annealed electrospun yarn was cut into 3mm×3mm square pieces. Water was added to an ice box, and the square pieces were laid flat on the water and frozen overnight. The next day, more water and pieces were added to freeze the ice, so that each piece had two pieces of electrospun yarn, resulting in ice blocks. The ice blocks were then sliced ​​on a freeze cutter along the direction perpendicular to the spinning to obtain PLLA short rods.

[0088] Step 2, Preparation of primary hydrogel: Take 8 mg of PLLA short rods, 800 μL of 10× PBS phosphate buffer, 130 μL of 1 mol / L NaOH solution, 6667 μL of 6 mg / mL collagen, and 410 μL of sterile water. Mix them evenly to obtain a collagen solution with a final concentration of 5 mg / mL. Then, add the neutralized collagen solution dropwise into the groove of a custom PDMS mold, as shown in Figure 1, and place it at 37℃ for crosslinking for 1 hour to obtain the primary hydrogel, denoted as T300.

[0089] Step 3: Fix the hydrogel and dehydrate it: Remove the primary hydrogel from the groove of the PDMS mold. Fix both ends of the annular primary hydrogel with paperclips, ensuring the distance between the fixed ends matches the inner ring length of the primary hydrogel to avoid stretching it before dehydration. Then, apply torque to the primary hydrogel using a torsion device to achieve rapid dehydration. The torsion speed is (π / 4) / s, and the torsion angle per unit length of the primary hydrogel is θ = 300° / mm. Obtain the dehydrated hydrogel sample.

[0090] Test 1: Mechanical characterization.

[0091] The cross-linked hydrogel samples from Examples 1-2 and Comparative Example 1 were subjected to mechanical characterization. Specifically, the cross-linked hydrogel samples from Examples 1-2 and Comparative Example 1 were subjected to tensile tests to obtain stress-strain curves, as shown in Figure 2, and the mechanical properties such as the modulus of the hydrogel were obtained.

[0092] Table 1. Mechanical characterization results of crosslinked hydrogel samples with different PLLA contents. As can be seen from the results in Figure 2 and Table 1, the higher the content of PLLA, the higher the mechanical modulus and strength of the cross-linked hydrogel sample.

[0093] Mechanical properties were tested on the crosslinked hydrogel sample of Example 1 and the primary hydrogel TC300-1PL of Comparative Example 2, and the results are shown in Figure 4.

[0094] As can be seen from Figure 4, the primary hydrogel TC300-1PL of Comparative Example 2 was not twisted or further crosslinked. The hydrogel prepared from it has much worse mechanical properties than the hydrogel obtained by twisting and further crosslinking in Example 1.

[0095] Mechanical properties were tested on the crosslinked hydrogel sample of Example 1 and the dehydrated hydrogel sample of Comparative Example 3, and the results are shown in Figure 5.

[0096] As can be seen from Figure 5, the hydrogel sample of Comparative Example 3 was not further cross-linked after dehydration. The mechanical properties of the hydrogel prepared from it were much worse than those of the hydrogel obtained by further cross-linking treatment in Example 1.

[0097] Test 2: Biocompatibility characterization.

[0098] Biocompatibility characterization was performed on the cross-linked hydrogel tissue cells in Examples 3 and 4.

[0099] The specific experimental method is as follows: wash the sample with PBS and add it to a multi-well plate; add about 500 μL of dye to each well; wrap the multi-well plate with aluminum foil and place it in an incubator for 45 min; take it out and soak each well in PBS for 1 min to 2 min and then wash it twice (to wash away the surrounding fluorescence so that the fluorescence only appears in the cells and prevent interference); cover the light and prepare for observation under a fluorescence microscope; observe under a fluorescence microscope and the results are shown in Figure 3.

[0100] Dye formulation: 1 mL PBS + 2 μL dead (EthD-1) + 0.5 μL life (Calcein AM); where PBS represents phosphate-buffered saline, pH=7.4; dead (EthD-1) represents ethidium bromide homodimer-1 (dead cell dye); and life (Calcein AM) represents calcein acetylated methyl ester (live cell dye).

[0101] Detection principle: Calcein AM enters the cell and reacts with enzymes in the living cell to form Calcein fluorescent molecules, which remain inside the cell. Therefore, the live cells are detected by Calcein fluorescent molecules (ex / em ~495nm / ~515nm), emitting green fluorescence. EthD-1 cannot pass through the cell membrane of the living cell and can only enter the dead cell. After binding with gene fragments inside the cell, it emits fluorescence (ex / em ~495nm / ~635nm), and the dead cells are detected by emitting red fluorescence.

[0102] In Figure 3, green represents live cells and red represents dead cells. As can be seen from Figure 3, the material system of Example 4 (TC300-2PL) can support the survival of bone marrow mesenchymal stem cells for at least 7 days, with a survival rate >99%. Similarly, the material system of Example 3 (TC300-1PL) can support the survival of bone marrow mesenchymal stem cells for at least 7 days, with a survival rate >99%.

[0103] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an ECM-inspired ordered hydrogel material for tendon repair, characterized in that, Includes the following steps: PLLA electrospun fibers were annealed and then cryo-sliced ​​to obtain PLLA short rods. The annealing process was carried out twice at 100℃ to 150℃. The PLLA short rods and matrix raw material solution were mixed and added to a mold for the first crosslinking treatment to obtain a primary hydrogel. The primary hydrogel was twisted to obtain a dehydrated hydrogel. The dehydrated hydrogel and crosslinking agent were subjected to a second crosslinking treatment to obtain an ECM-like ordered hydrogel material for tendon repair.

2. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The specific operation of the annealing process is as follows: the first annealing process is carried out at 100℃~110℃; and the second annealing process is carried out at 140℃~150℃.

3. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 2, characterized in that, The first and second annealing processes each take 8 to 10 hours.

4. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The specific operation of cryo-slicing is as follows: the annealed electrospun fibers are divided into small pieces, placed in water and frozen, and then the frozen ice blocks containing electrospun fibers are sliced ​​along the direction perpendicular to the spinning to obtain PLLA short rods.

5. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The matrix raw material solution was prepared by mixing collagen, PBS buffer, NaOH and water; the concentration of collagen was 5 mg / mL.

6. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The conditions for the first crosslinking treatment are: temperature 35℃~37℃, time 1h~2h.

7. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The specific operation of the torsion treatment is as follows: fix both ends of the primary hydrogel, apply torque to the fixed primary hydrogel, where the torsion angle is 300° / mm, and obtain a dehydrated hydrogel.

8. The method for preparing the ECM-like ordered hydrogel material for tendon repair according to claim 1, characterized in that, The crosslinking agent is a 0.1 mM genipin solution; the specific operation of the second crosslinking treatment is as follows: the dehydrated hydrogel is immersed in the crosslinking agent and soaked at 37°C for 16-24 hours to obtain an ECM-like ordered hydrogel material for tendon repair.

9. An ECM-inspired ordered hydrogel material for tendon repair, characterized in that, It is prepared by the method of any one of claims 1 to 8 for the preparation of ECM-like ordered hydrogel material for tendon repair.

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