A hydrogen-bonded organic framework membrane and a preparation method and application thereof

Abstract

The present application belongs to the field of material chemistry and biology technology, and particularly relates to a composite film and a preparation method and application thereof. The HOFilms material of the present application uses a mild and simple preparation method to prepare a high-quality, uniformly distributed hydrogen-bonded organic framework film, effectively retaining the biological function of the material; the production process is simple, raw materials are easy to obtain, and the material can be produced in large scale in a factory; the material can be used as a physical barrier or a support to regulate the repair microenvironment at the molecular level, and further form an integrated active Achilles tendon regeneration platform from mechanical support to biological treatment; the HOFilms material has a "wet response" intelligent property: not only can it stably exist in a wet environment in vivo, but also can utilize or respond to moisture in the environment (such as swelling, shrinking, releasing factors) to perform specific functions, thereby interacting with dynamic physiological processes; the HOFilms material itself has an active biological function, that is, improving the inflammatory microenvironment and directly promoting the regeneration of tendon cells, thereby fundamentally overcoming the healing barrier.

Description

Technical Field

[0001] This invention belongs to the fields of materials chemistry and biology, specifically relating to a composite membrane, its preparation method, and its application. Background Technology

[0002] Tendons serve as the mechanical bridge between bones and muscles, enabling the conversion between mobility and stability. Each skeletal muscle is divided into the muscle belly and the tendon. The tendon, composed of dense fibrous connective tissue, attaches the skeletal muscle to the bone. Ligaments, also composed of dense fibrous connective tissue, attach to the movable portion of the bone but restrict its range of motion to prevent injury. Generally, ligaments connect bones to bones, while tendons connect bones to muscles. However, the high tensile load on tendons makes them prone to rupture after injury, leading to pain, decreased mobility, and disability. Furthermore, due to low metabolism, poor vascular network, and reduced cell count, it is difficult to restore the structure and function of injured tendons to their natural state. Simultaneously, the activation and imbalance of the surrounding immune microenvironment create a vicious cycle of amplified inflammatory signals—abnormal tissue repair—leading to persistent inflammatory infiltration and inadequate intrinsic healing. With changes in people's lifestyles, the increasing popularity of daily outdoor sports, and the general increase in traffic accidents and strenuous exercise, Achilles tendon laceration (ATL) has become one of the more common injuries of the musculoskeletal system in clinical practice (Xu Shaoting, Liu Shuqing. Practical Orthopedics. Beijing: People's Military Medical Press, 2007, 815-817.). Clinically, it is divided into complete rupture and incomplete rupture.

[0003] Complete Achilles tendon ruptures can be classified based on the pathological findings during surgery: ① Transverse rupture (the rupture site is usually about 3cm to 5cm above the insertion point, with clean ends); ② Avulsion rupture (the Achilles tendon is avulsed at the insertion point or ruptured 1.5cm above the insertion point, with the ends often oblique and relatively clean); ③ Tear rupture (the rupture is 3cm to 4cm above the insertion point, with the ends resembling a horse's tail, varying in thickness and irregular). Achilles tendon ruptures mostly occur in young and middle-aged patients. These patients engage in high-intensity sports and require more sophisticated repair treatments. Improper treatment often leads to limited ankle joint function, weakened plantar flexor muscles, Achilles tendon adhesions, and re-rupture, thus affecting the patient's daily life and work. Radiographic and quantitative diagnostic methods are also important. Patients with severe injuries often require surgical treatment. Although arthroscopic anterior cruciate ligament reconstruction has become one of the most successful surgical techniques in the fields of orthopedics and sports medicine over the past 20 years, the failure rate and the rate of revision surgery are still as high as 10%, and the reconstructed anterior cruciate ligament is difficult to restore its original mechanical strength.

[0004] Other tendon or ligament rehabilitation surgeries, such as rotator cuff repair and Achilles tendon repair, also exhibit similar high recurrence and low healing rates. A common reason for this is the poor healing at the tendon-bone interface. Autologous grafts often lead to secondary injuries, while allogeneic grafts, xenografts, and ligament prostheses carry the risk of immune rejection. In short, tissue transplants perform poorly, requiring patients to undergo prolonged recovery periods. Some studies have used stem cells, dermal fibroblasts, and Achilles tendon fibroblasts to enhance tendon regeneration. However, immune rejection, cell sourcing, and ethical issues limit the application of exogenous cell transplantation. Furthermore, this strategy can lead to ectopic osteogenesis. Current clinical treatments, such as direct suturing, autologous transplantation, and allogeneic transplantation, suffer from poor mechanical properties, immune rejection, and donor site damage, and may not provide satisfactory long-term clinical outcomes, such as re-tears, peritendon adhesions, and limited range of motion.

[0005] The failure of conventional strategies for treating tendon injuries is primarily attributed to the low cellularity and insufficient vascularity of tendon tissue. Biomaterials, whether used alone or in combination with growth factors, stem cells, or gene regulators, can mimic the natural structure of tendons and induce appropriate biological responses. The application of biomaterials also provides researchers with a way to modify the design of biomaterials to influence cellular and molecular events during tendon repair. Currently, therapeutic platforms for tendon repair mainly include fibrous membranes, hydrogels, and decellularized materials. Fiber membranes are the most widely researched and applied biomaterials for tendon repair because they mimic the natural structure of the tendon extracellular matrix (ECM) environment (Friedman and Mooney, 2019a). However, it has been argued that a single approach is insufficient to address the various stages of tendon repair. Therefore, combining different strategies to fabricate multifunctional biomaterials is crucial for achieving complete functional recovery from tendon injuries.

[0006] The following are some core technical problems and difficulties in traditional Achilles tendon repair materials and strategies: 1. Complex material preparation processes and difficulty in achieving bioactivity. The preparation of many high-performance biomaterials (such as certain polymers or ceramics) requires high temperature, high pressure, or complex equipment, which are demanding and difficult to mass-produce. Furthermore, the bioactive factors may be destroyed during processing. 2. Mismatch between material mechanical properties and biological tissue. The Achilles tendon is a tissue that bears enormous tension. Repair materials must possess excellent mechanical properties (such as high strength and toughness) to provide sufficient mechanical support in the early stages of healing and prevent re-rupture. At the same time, the material cannot be too rigid and restrict movement. 3. Insufficient compatibility and dynamic responsiveness of implanted materials in a moist physiological environment. Traditional materials may experience performance degradation, failure, or adverse reactions in body fluid environments. 4. Insufficient inflammatory response and regenerative capacity during the repair process. A major challenge in Achilles tendon repair is the tendency for excessive inflammation after surgery, leading to pain, adhesions (growing together with surrounding tissues), and scar healing, affecting functional recovery. Simple structural support cannot solve these biological problems.

[0007] Natural hydrogels are considered to possess excellent immunogenicity and biodegradability; however, they are often limited by their poor mechanical properties, which, when combined with synthetic materials, make hydrogel-based scaffolds a dominant trend. Scaffolds encapsulating cells, cytokines, and drugs offer a promising approach to improving the tendon microenvironment by increasing tendon expression or preventing adhesions. Self-healing hydrogels can restore their original shape and function even after rupture, making them ideal for preventing peritendinous adhesions caused by synovial sheath damage. They can act as physical barriers, preventing fibroblast invasion during extrinsic healing, and as membranes to form smooth surfaces, reducing friction during tendon movement. The injectability of hydrogels facilitates the filling of irregularly repaired parts with minimal invasiveness and makes them suitable as carriers of drugs or cytokines. However, the significant forces between tendons and bones can disrupt the integrity of hydrogels, leading to rapid diffusion of drugs or cytokines. With the increasing prevalence of accelerated postoperative recovery, patients need to resume early movement after surgery. Therefore, future hydrogels for repairing tendons should consider self-healing properties when designing hydrogel parameters. The combination of self-healing and injectability of hydrogels enables the sustainable release of substances and, of course, maximizes their clinical efficacy. Functionalizing hydrogels with self-healing properties involves the use of covalent bonds, including disulfide, imine, and hydrazine bonds. However, toxic byproducts are generated during cross-linking, and measures must be taken to eliminate these toxic chemicals, such as replacing the cross-linking agent or using physical cross-linking methods instead. Regarding carriers, hydrogels in microsphere form improve loading performance and the culture microenvironment, thereby preventing cell damage during implantation. Some binding sequences, such as RGD or pDA immobilization, can also be used to modify scaffolds to enhance their loading efficacy and promote cell-scaffold adhesion.

[0008] Despite the wide range of applications in biomaterials, conventional hydrogels often exhibit low mechanical strength, severely limiting their use in load-bearing tissues such as tendons or ligaments. To overcome this issue, various strategies have been proposed to obtain hydrogels with suitable mechanical properties for tendon repair, including interpenetrating hydrogels, hybridization with other polymers or nanocomposites, and self-healing hydrogels. Combining hydrogels with fibers is another promising approach, where reinforcing fibers provide good mechanical strength while the hydrogel mimics the properties of natural ECM. The degradation behavior of hydrogels, as carriers of bioactive molecules, drugs, and cells, should also be considered. In addition to previous methods that primarily controlled hydrogel degradation by altering the structure and composition of the hydrogel, stimulus-responsive hydrogels are another promising area of ​​research. However, determining the mechanical strength of hydrogels suitable for tendon repair and how to combine mechanical properties with other material properties remains a challenge. Current research often focuses on altering degradation rates to achieve controlled delivery of drugs or biologics, while the decline in mechanical strength has been somewhat neglected. Furthermore, it is difficult to visualize the in vivo degradation of hydrogels and to observe how they respond to the complex microenvironment following tendon injury.

[0009] Therefore, there is an urgent need for a new material that is simple to prepare, has good compatibility with the implantation site microenvironment, possesses excellent mechanical properties, can resist inflammatory responses and promote regeneration, and can support and repair Achilles tendon tissue. Summary of the Invention

[0010] This invention combines WRAP films and HOF nanoparticles to obtain a hydrogen-bonded organic framework membrane (HOFilms) material for repairing Achilles tendon injuries. This membrane possesses excellent mechanical properties and good biocompatibility, improving the inflammatory microenvironment while directly promoting tendon cell regeneration, fundamentally overcoming healing obstacles and repairing Achilles tendon damage. Based on this, the invention was completed.

[0011] In a first aspect, the present invention provides a hydrogen-bonded organic framework membrane (HOFilms), wherein the preparation method of the HOFilms includes the following steps: S1. Polyethylene glycol (PEG) and α-cyclodextrin (α-CD) solutions are fused to form a gel mixture; S2. Mix the gel mixture obtained in S1 with polyethylene oxide (PEO) hydrosol to obtain a thick mixture; S3. The thick mixture obtained in S2 is incubated and dried to obtain a self-supporting WRAP film. S4. Dissolve PFC-73-Ni powder in N,N-dimethylformamide (DMF), and filter the resulting mixture to obtain a homogeneous HOF solution; S5. Immerse the WRAP film obtained in S3 into the homogeneous HOF solution obtained in S4, remove the DMF by heating, and then stretch it in the reverse direction to obtain HOFilms.

[0012] Furthermore, in step S1, the concentration of polyethylene glycol in the gel mixture is selected from 1-15%, preferably 2%.

[0013] Furthermore, in step S1, the concentration of α-cyclodextrin in the gel mixture is selected from 1-50%, preferably 20%.

[0014] Furthermore, in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is selected from 1-65%, preferably 4%.

[0015] Furthermore, in step S3, the incubation temperature is selected from 40-70°C, preferably 60°C.

[0016] Furthermore, in step S4, the mass of the PFC-73-Ni powder is selected from 1-100 mg, preferably 10 mg.

[0017] Furthermore, in step S4, the volume of the N,N-dimethylformamide (DMF) solution is selected from 0.1-100 mL, preferably 1 mL.

[0018] Furthermore, in step S4, the concentration of the HOF homogenized solution is selected from 0.1-35 mg / mL, preferably 10 mg / mL. Furthermore, in step S5, the heating temperature is selected from 80-120℃, preferably 100℃.

[0019] Furthermore, the thickness of the hydrogen-bonded organic framework membrane (HOFilms) is selected from 0.1-2000µm, preferably 90µm.

[0020] In a second aspect, the present invention provides a method for preparing hydrogen-bonded organic framework membranes (HOFilms) as described in the first aspect of the present invention, the method comprising the following steps: S1. Polyethylene glycol (PEG) and α-cyclodextrin (α-CD) solutions are fused to form a gel mixture; S2. Mix the gel mixture obtained in S1 with polyethylene oxide (PEO) hydrosol to obtain a thick mixture; S3. The thick mixture obtained in S2 is incubated and dried to obtain a self-supporting WRAP film. S4. Dissolve PFC-73-Ni powder in N,N-dimethylformamide (DMF), and filter the resulting mixture to obtain a homogeneous HOF solution; S5. Immerse the WRAP film obtained in S3 into the homogeneous HOF solution obtained in S4, remove the DMF by heating, and then stretch it in the reverse direction to obtain HOFilms.

[0021] Furthermore, in step S1, the concentration of polyethylene glycol in the gel mixture is selected from 1-15%, preferably 2%.

[0022] Furthermore, in step S1, the concentration of α-cyclodextrin in the gel mixture is selected from 1-50%, preferably 20%.

[0023] Furthermore, in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is selected from 1-65%, preferably 4%.

[0024] Furthermore, in step S3, the incubation temperature is selected from 40-70°C, preferably 60°C.

[0025] Furthermore, in step S4, the mass of the PFC-73-Ni powder is selected from 1-100 mg, preferably 10 mg.

[0026] Furthermore, in step S4, the volume of the N,N-dimethylformamide (DMF) solution is selected from 0.1-100 mL, preferably 1 mL.

[0027] Furthermore, in step S4, the concentration of the HOF homogenized solution is selected from 0.1-35 mg / mL, preferably 10 mg / mL. Furthermore, in step S5, the heating temperature is selected from 80-120℃, preferably 100℃.

[0028] Furthermore, the thickness of the hydrogen-bonded organic framework membrane (HOFilms) is selected from 0.1-2000µm, preferably 90µm.

[0029] Thirdly, the present invention provides the application of hydrogen-bonded organic framework membranes (HOFilms) as described in the first aspect of the present invention in the preparation of Achilles tendon injury repair products.

[0030] Furthermore, the elongation of the hydrogen-bonded organic framework membrane HOFilms is selected from 10-600%, preferably 300%.

[0031] Furthermore, the length of the hydrogen-bonded organic framework membrane HOFilms is selected from 0.01-200 cm, preferably 1.5-2 cm.

[0032] Furthermore, the width of the hydrogen-bonded organic framework membrane HOFilms is selected from 0.01-200 cm, preferably 0.5-1 cm.

[0033] Furthermore, the preparation method of the HOFilms includes the following steps: S1. Polyethylene glycol (PEG) and α-cyclodextrin (α-CD) solutions are fused to form a gel mixture; S2. Mix the gel mixture obtained in S1 with polyethylene oxide (PEO) hydrosol to obtain a thick mixture; S3. The thick mixture obtained in S2 is incubated and dried to obtain a self-supporting WRAP film. S4. Dissolve PFC-73-Ni powder in N,N-dimethylformamide (DMF), and filter the resulting mixture to obtain a homogeneous HOF solution; S5. Immerse the WRAP film obtained in S3 into the homogeneous HOF solution obtained in S4, remove the DMF by heating, and then stretch it in the reverse direction to obtain HOFilms.

[0034] Furthermore, in step S1, the concentration of polyethylene glycol in the gel mixture is selected from 1-15%, preferably 2%.

[0035] Furthermore, in step S1, the concentration of α-cyclodextrin in the gel mixture is selected from 1-50%, preferably 20%.

[0036] Furthermore, in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is selected from 1-65%, preferably 4%.

[0037] Furthermore, in step S3, the incubation temperature is selected from 40-70°C, preferably 60°C.

[0038] Furthermore, in step S4, the mass of the PFC-73-Ni powder is selected from 1-100 mg, preferably 10 mg.

[0039] Furthermore, in step S4, the volume of the N,N-dimethylformamide (DMF) solution is selected from 0.1-100 mL, preferably 1 mL.

[0040] Furthermore, in step S4, the concentration of the HOF homogenized solution is selected from 0.1-35 mg / mL, preferably 10 mg / mL. Furthermore, in step S5, the heating temperature is selected from 80-120°C, preferably 100°C.

[0041] Furthermore, the thickness of the hydrogen-bonded organic framework membrane (HOFilms) is selected from 0.1-2000µm, preferably 90µm.

[0042] Beneficial effects This invention provides a novel wet-responsive hydrogen-bonded organic framework thin film material for efficiently repairing Achilles tendon injuries, with the following advantages: Simple preparation and cost-saving: High-quality, uniformly distributed hydrogen-bonded organic framework films can be prepared by using a mild and simple (solution processing) method, and the biological functions of the material can be effectively preserved in the process; the manufacturing process is simple, the raw materials are easy to obtain, and it can be mass-produced in factories. HOFilms not only serve as a physical barrier or scaffold, but also as an active treatment platform: it regulates the repair microenvironment at the molecular level, thereby realizing an integrated Achilles tendon regeneration platform that combines mechanical support with biological therapy. HOFilms materials possess "wet-responsive" intelligent properties: they can not only exist stably in the moist environment of the body, but also utilize or respond to moisture in the environment (such as swelling, contraction, and release factors) to perform specific functions, thereby interacting with dynamic physiological processes. HOFilms materials possess active biological functions—namely, improving the inflammatory microenvironment and directly promoting tendon cell regeneration, thereby fundamentally overcoming healing barriers. Attached Figure Description

[0043] Figure 1 This is a flowchart of the preparation process for HOFilms.

[0044] Figure 2 A macroscopic image of HOFilms after stretching.

[0045] Figure 3 This refers to the thickness of HOFilms after stretching.

[0046] Figure 4 The scanning electron microscope image before stretching HOFilms.

[0047] Figure 5 SEM images of HOFilms after stretching.

[0048] Figure 6 XRD patterns of HOFilms and simulated HOFs prepared by solution processing.

[0049] Figure 7 Stress-strain curves of HOFilms and WRAP films prepared by solution processing.

[0050] Figure 8 Photographs of the contact angle of HOFilms.

[0051] Figure 9 denoted as the coefficient of friction of HOFilms in a wet state.

[0052] Figure 10A wet-response shrinkage photograph of HOFilms.

[0053] Figure 11 The images show fluorescence images of macrophage markers after co-culturing Raw264.7 cells with LPS, LPS plus WRAP film, and LPS plus HOFilms, respectively.

[0054] Note: 11a shows a significant decrease in fluorescence intensity and a reduction in protein expression level of the pro-inflammatory marker iNOS; 11b shows a significant increase in expression and a higher protein expression level of the repair marker CD206.

[0055] Figure 12 Flowchart for applying HOFilms to an animal model of Achilles tendon injury and tear.

[0056] Figure 13 The injured tendon was visually evaluated 4 weeks post-surgery.

[0057] Figure 14 The results of hematoxylin-eosin (H&E) staining of the regenerated tendon tissue 4 weeks post-surgery.

[0058] Figure 15 Masson staining pathological observation of regenerated tendon tissue 4 weeks post-surgery.

[0059] Figure 16 Representative tensile stress-strain curves of tendon samples from different repair groups and the tensile strength of tendon samples after repair in different repair groups.

[0060] Note: 16a is the result of the tensile stress-strain curve; 16b is the result of the tensile strength; 16c is the result of the tensile strength. Detailed Implementation

[0061] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0063] Terminology Explanation X-ray diffraction (XRD) is a core technique in materials science for analyzing crystal structures, and its spectra contain rich information about the crystals. The horizontal axis (2θ angle) reflects the incident angle of the X-rays, measured in degrees (°); the vertical axis (intensity) represents the strength of the diffraction signal, usually expressed as counts or relative intensity.

[0064] Example 1: Preparation of hydrogen-bonded organic framework membranes (HOFilms) A. Test methods 1. Preparation of WRAP films 10 g of polyethylene glycol (PEG) was dissolved in 50 mL of deionized water. Then, 10 g of α-CD was added to the PEG solution, and the mixture was placed in a 60°C ultrasonic water bath. Incubation in the 60°C ultrasonic water bath resulted in a white mixture. After cooling to room temperature, a PEG–α-CD white gel was prepared. Subsequently, 150 mL of PEO hydrosol (4 wt%) was mixed with the white gel to obtain a thick mixture. The gel mixture was incubated at 60°C and dried to obtain a self-supporting WRAP film. 9 g of the mixture was cast onto a 90 mm diameter petri dish to form a film approximately 90 μm thick.

[0065] 2. Preparation of hydrogen-bonded organic framework membranes (HOFilms) 10 mg of PFC-73-Ni powder was dissolved in 1 mL of N,N-dimethylformamide (DMF), and the resulting mixture was filtered to obtain a homogeneous HOF solution. The WRAP film prepared in step 1 was then immersed in the HOF solution for 5 minutes and annealed at 100 °C to prepare the HOF film. Finally, the HOF film was reverse stretched by 300% to prepare HOFilms.

[0066] B. Test Results like Figure 1 The diagram shows the preparation process of HOFilms. Flexible water-responsive deformation polymer (WRAP) films were selected to spatially confine the in-situ solution-phase growth of the rigid HOF component within their porous structure. DMF was used as the solvent for the HOF casting solution. The preparation process involved immersing the WRAP film in the HOF solution at room temperature, followed by thermal removal of DMF at 100°C, ultimately yielding hydrogen-bonded organic framework membranes (HOFilms). Figure 2 and Figure 3 As shown, the hydrogen-bonded organic framework membrane (HOFilms) has an average thickness of 90 micrometers, is red in color, and can be cut to any size. Example 2 Morphology and structural characterization of hydrogen-bonded organic framework membranes (HOFilms) A. Test methods 1. Characterization of surface morphology, microstructure, and overall uniformity The surface morphology, microstructure, and overall uniformity of the thin film were observed using scanning electron microscopy (SEM) to assess its physical morphological basis as an implant material.

[0068] HOFilms were attached to the SEM sample stage using double-sided conductive adhesive, and a 3-10 nm thick layer of gold was sputtered onto the sample surface using an ion sputtering instrument. The prepared sample stage was carefully placed into the SEM sample chamber, and a vacuum was applied at 5 kV.

[0069] 2. Crystal structure characterization of thin films X-ray diffraction (XRD) was used to precisely analyze the crystal structure of the thin film to confirm its chemical composition, crystal phase purity, and crystallinity. After grinding, the sample was filled into a glass sample cell using a side-mount method. The test was conducted using a Cu Kα radiation source (λ = 1.5418 Å) with a tube voltage of 40 kV and a current of 40 mA. Data were acquired in steps of 0.02° and a scan rate of 2° / min from 5° to 50° (2θ).

[0070] B. Test Results like Figure 4 The image shown is a scanning electron microscope (SEM) image of HOFilms before stretching. Detailed structural analysis of the composite microstructure of HOFilms was performed using SEM. Compared to the WRAP film control on the left, HOFs are embedded in the WRAP films in the form of nanosheets and are tightly distributed.

[0071] like Figure 5 The image shown is a scanning electron microscope (SEM) image of HOFilms after stretching. SEM characterization after stretching reveals a well-aligned fiber bridge structure within the WRAP film, with HOF nanosheets distributed on both the surface and interior of the WRAP film. Furthermore, the HOF nanosheets did not detach from the WRAP film surface after stretching.

[0072] like Figure 6 The image shows the XRD patterns of the HOFilms and the simulated HOF prepared in Example 1. The PXRD diffraction peaks of the regenerated HOF film perfectly match the simulated single-crystal pattern. The characteristic diffraction peaks observed at 2θ values ​​of 3.97, 7.96, and 8.59 correspond to the crystal planes (020), (040), and (210), respectively. Example 3: Mechanical property characterization of hydrogen-bonded organic framework membranes (HOFilms) A. Test methods To quantitatively evaluate the mechanical strength and reliability of the WRAP film and HOFilms film prepared in Example 1 under simulated physiological conditions, a standard uniaxial tensile test was performed on them using a universal mechanical testing machine.

[0074] The WRAP and HOFilms film samples prepared in Example 1 were cut into a specified dumbbell shape and securely clamped at both ends in the upper and lower fixtures of the testing machine. The fixtures were separated at a constant rate by the control system, thereby applying a uniformly increasing tensile load along the longitudinal axis to the sample until fracture. The load (force) and displacement changes were recorded synchronously in real time. After software processing, key mechanical property parameters, including elastic modulus, tensile strength, elongation at break, and toughness, could be obtained.

[0075] B. Test Results like Figure 7 The figure shows the stress-strain curves of the HOFilms and WRAP films prepared in Example 1. The mechanical properties of HOFilms were quantitatively evaluated using a mechanical testing instrument operating at a constant deformation rate of 2 mm / s, utilizing the inherent tensile strength of the WRAP film. Analysis of the obtained stress-strain curves showed that the original WRAP film had an elongation at break of 330% and a tensile strength of 14 MPa. In contrast, the HOFilms had an elongation at break of 300% and a tensile strength of 17.5 MPa.

[0076] Example 4: Coefficient of friction of hydrogen-bonded organic framework membranes (HOFilms) in a wet state. A. Test methods First, ensure the surface of the thin film sample to be tested is clean, flat, and dry, and firmly fix it on the sample stage of the contact angle measuring instrument. Then, in a constant temperature and humidity environment, use a microsyringe to gently deposit a 2 μL droplet of ultrapure water onto the film surface, and immediately capture its side profile image using a high-speed camera.

[0077] The lubrication properties of hydrogen-bonded organic framework films (HOFilms) were characterized using a high-temperature micro-dynamic wear tester (Optimal Instruments, USA). The prepared patch was placed on the sample stage, and deionized water was sprayed onto the sample surface to keep it moist. The friction frequency was set to 1 Hz, the amplitude to 4 mm, the load to 1 N, and the running time to 1500 s. The experimental data were recorded.

[0078] B. Test Results like Figure 8As shown, the film surface exhibits significant hydrophilicity, with an average contact angle of 32.4°. This value is far below the critical point of 90° for hydrophilicity and hydrophobicity, indicating that the material surface possesses high surface energy and good water wettability. This strong hydrophilicity usually stems from the presence of abundant polar functional groups or a certain degree of microscopic roughness on the surface, which is beneficial for the spreading and wetting of aqueous solutions.

[0079] like Figure 9 As shown, in a humid environment, the average coefficient of friction of the material is significantly reduced to approximately 0.3, and the friction force curve is stable. The adsorption layer or fluid film formed by water molecules at the friction interface effectively isolates the contact surfaces, reduces the direct contact area, and thus plays a lubricating role, significantly reducing frictional resistance and material wear.

[0080] Example 5: Wet-response shrinkage properties of hydrogen-bonded organic framework membranes (HOFilms) A. Test methods The wet-response shrinkage characteristics of hydrogen-bonded organic framework membranes (HOFilms) were measured using a digital camera, recording the rapid shrinkage process of the membranes upon contact with water. HOFilms were cut into 2.0 cm × 0.5 cm rectangles, wrapped around glass rods, and fixed at both ends with bio-adhesive. Deionized water was then sprayed onto the membrane surface, and the shrinkage state of HOFilms was observed.

[0081] B. Test Results like Figure 10 As shown, in the dry state (OS), the film maintains its initial flat shape, with gaps between it and the underlying cylindrical glass rod substrate, failing to form a tight bond. After spraying deionized water onto the film surface, the film rapidly undergoes uneven directional shrinkage. This shrinkage process drives the film to generate sufficient internal stress, enabling it to overcome its own rigidity and adaptively wrap around and tightly adhere to the curved surface of the glass rod.

[0082] Example 6 Anti-inflammatory properties of hydrogen-bonded organic framework membranes A. Experimental Method: RAW264.7 cells in the logarithmic growth phase were placed at an appropriate density (e.g., 5 × 10⁻⁶). 4 Seeds were placed in each well onto coverslips. Cells were cultured at 37°C in a 5% CO2 incubator until fully adherent. An inflammatory cell model was induced using LPS (100 ng / mL). HOFilms membrane extract was added to the culture medium and co-cultured with the cells, followed by aspiration of the medium. 4% paraformaldehyde was added for fixation. Blocking buffer (5% BSA or 10% normal goat serum) was added and incubated at room temperature. Primary antibody dilutions were prepared (diluted with blocking buffer): iNOS, recommended dilution ratio 1:100 - 1:200; CD206, recommended dilution ratio 1:200 - 1:500.

[0083] After aspirating the blocking solution, add the diluted primary antibody (approximately 50-100 µL) to the coverslip. Incubate in a humidified chamber at 4°C. After incubation, aspirate the primary antibody and wash with PBST (PBS + 0.05% Tween-20). Add the secondary antibody mixture and continue incubation. After incubation, aspirate the secondary antibody. Add DAPI (1 µg / mL) to stain the nucleus, aspirate excess liquid, and seal the coverslip onto the slide with an anti-fluorescence quenching mounting medium, sealing the edges. After the mounting medium has solidified, acquire images using a laser confocal microscope.

[0084] B. The experimental results were used to investigate the immunomodulatory function of HOFilms membranes, and macrophage phenotypes were analyzed by immunofluorescence staining. For example... Figure 11 As shown, compared with the control group, macrophages co-cultured with HOFilms membranes exhibited significantly reduced fluorescence intensity of the pro-inflammatory marker iNOS and a 33% decrease in protein expression level. Figure 11 a), while the expression of the repair marker CD206 was significantly enhanced, with its protein expression level increasing by 20% ( Figure 11 (b) indicates that HOFilms membranes can effectively guide macrophages to polarize towards the anti-inflammatory M2 phenotype.

[0085] Example 7: Application of hydrogen-bonded organic framework membranes in a rat model of Achilles tendon rupture. A. Test methods All animal experiments were conducted in accordance with the approval of the Animal Experiment Ethics Committee of Shanghai University. SD rats (weighing approximately 200 grams) provided by Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd. were randomly divided into four groups of three rats each. A full-thickness Achilles tendon defect model was established by disinfecting the rat hind limbs. After anesthesia, the right Achilles tendon was exposed under aseptic conditions and completely transected 1 cm from its insertion point on the calcaneus. End-to-end anastomosis was performed using a modified Kessler technique (5-0 polypropylene sutures). Postoperatively, the rats were divided into four groups: 1) Normal group: No surgical intervention was performed; 2) Defect group: Unrepaired Achilles tendon defect; 3) WRAP group: WRAP membrane was implanted at the Achilles tendon defect site; 4) HOFilms group: Bio-HOFilms were implanted at the Achilles tendon defect site.

[0086] The Achilles tendon defect was sutured using 6-0 non-absorbable sutures. The rats were euthanized four weeks post-surgery, and the harvested Achilles tendons were collected for histological and biomechanical analysis.

[0087] B. Test Results Based on the excellent anti-inflammatory and mechanical properties of HOFilms film, a bilateral tendon defect model was established in rats. The sham-operated control group received only surgical suturing, while the experimental group had the defect sites wrapped with either WRAP film or HOFilms film, respectively. Figure 12 ).

[0088] like Figure 13 The image shows the results of a macroscopic evaluation of the injured tendon at 4 weeks post-surgery. The macroscopic assessment at four weeks post-surgery revealed significant pathological changes in the untreated defect: the tendon exhibited marked edema, increased circumference, erythematous discoloration, and an irregular surface. In stark contrast, the tendon treated with the HOFilm film not only significantly reduced inflammation, but also achieved a degree of physiological length restoration comparable to the uninjured control group (control group), while maintaining a smooth surface morphology and free of peritendinous adhesions.

[0089] To further analyze histopathological progress, hematoxylin and eosin (H&E) staining was used to assess inflammatory status, cell arrangement, and collagen tissue structure. Figure 14 The image shows the pathological observation results of hematoxylin-eosin (H&E) staining of regenerated tendon tissue at 4 weeks of age. Healthy tendons exhibited a neat and orderly cell arrangement without inflammatory infiltration; while the sham-operated group showed significant inflammatory response and collagen matrix destruction. Both WRAP film and HOFilms film interventions reduced inflammatory cell infiltration compared to the sham-operated group, but HOFilms film achieved superior repair while maintaining significantly lower residual inflammation compared to the other methods. Hierarchical collagen tissue forms an anisotropic structure through tightly cross-linked parallel fiber bundles, which is a key source of its tensile strength. Masson's trichrome staining method can quantify collagen density, arrangement integrity, and the degree of pathological vacuolation; blue represents collagen fibers, and red represents sarcoplasmic components (…). Figure 15 Analysis confirmed that 4 weeks post-surgery, the tendons in the sham surgery group exhibited severe fibrous disorder, reduced filling density, and significant cystic degeneration, while the HOFilm film induced a near-physiological fiber arrangement with minimal structural defects.

[0090] Example 8: Representative tensile stress-strain tests of tendon samples from different repair groups A. Test methods The Achilles tendon tissue from Example 7 was cut and its two ends were securely clamped in the upper and lower fixtures of the testing machine. The control system caused the fixtures to separate at a constant rate, thereby applying a uniformly increasing tensile load along the longitudinal axis to the sample until fracture. The load (force) and displacement changes were recorded simultaneously in real time during this process. After software processing, key mechanical property parameters, including elastic modulus, tensile strength, elongation at break, and toughness, could be obtained.

[0091] B. Test Results like Figure 16 As shown, representative tensile stress-strain curves of tendon samples from different repair groups and the post-repair tensile strength results of tendon samples from different repair groups are presented. This is used to assess the biomechanical integrity of the healed tendon. Mechanical test results indicate that HOFilms film exhibits excellent mechanical recovery efficacy in repairing tissue defects. The curves of normal tissue show the highest strength and toughness. The curves of simple defective tissue show a significant decrease in both strength and stiffness. After repair with WRAP film, the curve position is significantly higher than that of the defective group, indicating that it can partially restore the mechanical properties of the tissue. After repair with HOFilms film, its stress-strain curve is closest to that of normal tissue, especially its load-bearing capacity (area under the curve) at higher strains is stronger, indicating a superior repair effect. It not only improves strength but may also better restore the extensibility or toughness of the tissue. Figure 16 a). Stress statistics show that, compared with normal tissue, the strength of the simple defect group is significantly reduced ( p <0.0001), although there was an improvement after WRAP repair ( p = 0.0005 vs. defect group) but still significantly lower than normal levels ( p <0.0001)( Figure 16 b). Quantitative analysis of tensile strength further confirmed this difference; in contrast, HOFilms films significantly enhanced the strength of the defective tissue ( p = 0.0002), and its final strength was no statistically different from that of normal tissue, and was significantly better than that of the WRAP group ( p = 0.0048) Figure 16 c). These data collectively demonstrate that HOFilms films can almost completely restore the tensile strength of damaged tissue to its physiological state, and their mechanical repair effect is significantly better than that of WRAP films.

Claims

1. A hydrogen-bonded organic framework membrane (HOFilms), wherein the preparation method of the HOFilms comprises the following steps: S1. Polyethylene glycol (PEG) and α-cyclodextrin (α-CD) solutions are fused to form a gel mixture; S2. Mix the gel mixture obtained in S1 with polyethylene oxide (PEO) hydrosol to obtain a thick mixture; S3. The thick mixture obtained in S2 is incubated and dried to obtain a self-supporting WRAP film. S4. Dissolve PFC-73-Ni powder in N,N-dimethylformamide (DMF), and filter the resulting mixture to obtain a homogeneous HOF solution; S5. Immerse the WRAP film obtained in S3 into the homogeneous HOF solution obtained in S4, remove the DMF by heating, and then stretch it in the reverse direction to obtain HOFilms.

2. The hydrogen-bonded organic framework membrane HOFilms as described in claim 1, wherein in step S1, the concentration of polyethylene glycol in the gel mixture is selected from 1-15%, and the concentration of α-cyclodextrin in the gel mixture is selected from 1-50%; in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is selected from 1-65%; in step S3, the incubation temperature is selected from 40-70°C; in step S4, the mass of the PFC-73-Ni powder is selected from 1-100 mg; the volume of the N,N-dimethylformamide (DMF) solution is selected from 0.1-100 mL, and the concentration of the HOF homogenized solution is selected from 0.1-35 mg / mL; in step S5, the heating temperature is selected from 80-120°C; and the thickness of the hydrogen-bonded organic framework membrane HOFilms is selected from 0.1-2000 µm.

3. The hydrogen-bonded organic framework membrane HOFilms as described in claim 1, wherein in step S1, the concentration of polyethylene glycol in the gel mixture is 2%, the concentration of α-cyclodextrin in the gel mixture is 20%, in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is 4%; in step S3, the incubation temperature is 60°C; in step S4, the mass of the PFC-73-Ni powder is selected from 10 mg; the volume of the N,N-dimethylformamide (DMF) solution is 1 mL, and the concentration of the HOF homogenized solution is 10 mg / mL; in step S5, the heating temperature is 100°C; and the thickness of the hydrogen-bonded organic framework membrane HOFilms is 90 µm.

4. The hydrogen-bonded organic framework membrane (HOFilms) as described in claim 1, wherein the stretching ratio of the hydrogen-bonded organic framework membrane (HOFilms) is 300%; the length of the hydrogen-bonded organic framework membrane (HOFilms) is 1.5-2 cm; and the width of the hydrogen-bonded organic framework membrane (HOFilms) is 0.5-1 cm.

5. A method for preparing hydrogen-bonded organic framework membranes (HOFilms), the method comprising the following steps: S1. Polyethylene glycol (PEG) and α-cyclodextrin (α-CD) solutions are fused to form a gel mixture; S2. Mix the gel mixture obtained in S1 with polyethylene oxide (PEO) hydrosol to obtain a thick mixture; S3. The thick mixture obtained in S2 is incubated and dried to obtain a self-supporting WRAP film. S4. Dissolve PFC-73-Ni powder in N,N-dimethylformamide (DMF), and filter the resulting mixture to obtain a homogeneous HOF solution; S5. Immerse the WRAP film obtained in S3 into the homogeneous HOF solution obtained in S4, remove the DMF by heating, and then stretch it in the reverse direction to obtain HOFilms.

6. The preparation method according to claim 5, wherein in step S1, the concentration of polyethylene glycol in the gel mixture is selected from 1-15%, and the concentration of α-cyclodextrin in the gel mixture is selected from 1-50%; in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is selected from 1-65%; in step S3, the incubation temperature is selected from 40-70℃; in step S4, the mass of the PFC-73-Ni powder is selected from 1-100 mg; the volume of the N,N-dimethylformamide (DMF) solution is selected from 0.1-100 mL, and the concentration of the HOF homogenized solution is selected from 0.1-35 mg / mL; in step S5, the heating temperature is selected from 80-120℃; and the thickness of the hydrogen-bonded organic framework membrane HOFilms is selected from 0.1-2000 µm.

7. The preparation method according to claim 5, wherein in step S1, the concentration of polyethylene glycol in the gel mixture is 2%, the concentration of α-cyclodextrin in the gel mixture is 20%, in step S2, the mass percentage of the polyethylene oxide (PEO) hydrosol is 4%; in step S3, the incubation temperature is 60°C; in step S4, the mass of the PFC-73-Ni powder is selected from 10 mg; the volume of the N,N-dimethylformamide (DMF) solution is 1 mL, and the concentration of the HOF homogenized solution is 10 mg / mL; in step S5, the heating temperature is 100°C; and the thickness of the hydrogen-bonded organic framework membrane HOFilms is 90 µm.

8. The preparation method according to claim 5, wherein the elongation of the hydrogen-bonded organic framework membrane HOFilms is 300%; the length of the hydrogen-bonded organic framework membrane HOFilms is 1.5-2 cm; and the width of the hydrogen-bonded organic framework membrane HOFilms is 0.5-1 cm.

9. The application of the hydrogen-bonded organic framework membrane HOFilms as described in claim 1 in the preparation of Achilles tendon injury repair products.

10. The application as described in claim 9, wherein the elongation of the hydrogen-bonded organic framework membrane HOFilms is 300%; the length of the hydrogen-bonded organic framework membrane HOFilms is 1.5-2 cm; the width of the hydrogen-bonded organic framework membrane HOFilms is 0.5-1 cm; and the thickness of the hydrogen-bonded organic framework membrane HOFilms is 90 µm.

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