A biomaterial absorbable double-layer heterogeneous membrane for promoting tendon-bone interface healing, preparation method and application

Abstract

The application discloses a kind of biological absorbable double-layer heterogeneous membranes for promoting tendon-bone interface healing, preparation method and application, belong to the field of biomedical materials.Using solution casting method, freeze-drying technology and photoetching technology, a kind of absorbable double-layer membrane with asymmetric structure is prepared by taking natural polymer material chitosan and gelatin as main raw materials.The groove orientation side of the membrane wraps tendon / ligament graft, which can effectively arrange and lengthen fibroblasts at the interface, promote cell myogenic differentiation and reduce scar hyperplasia tissue generation caused by disordered arrangement;The porous sponge side is attached to the bone tunnel, which can improve cell adhesion and proliferation capacity, and promote bone formation at the interface.The material has good flexibility, can provide certain mechanical properties for the affected area, can be degraded in vivo, and the product has no toxic and side effects on the body.The two structures jointly promote tendon-bone interface healing and reduce adverse reactions.The material has no external drug stimulation, good biological safety, low price, simple preparation process, strong operability, good adjustability for different affected areas, strong clinical execution ability, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomass absorbable bilayer heterogeneous membrane that promotes tendon-bone interface healing, its preparation method, and its application. Background Technology

[0002] Due to the diversification of sports and the improvement of people's living standards, the incidence of sports injuries is getting higher and higher. Among the many types of sports injuries, ligament and tendon injuries in various parts of the body account for a large proportion and occur in a large number of cases, affecting the health of millions of people every year. Therefore, promoting tendon and bone healing is the key to repairing tendon / ligament injuries and preventing re-tears. We are committed to minimizing surgical trauma, achieving good results, enabling patients to recover quickly, experiencing less pain, and minimizing the subsequent impact on their sports life.

[0003] In tendon / ligament repair and reconstruction surgery, poor healing of the interface between the tendon graft and the bone tunnel is a common problem. This may result in a long healing time at the junction of the two tissues, and graft dislodgement due to daily activities. In addition, excessive proliferation and abnormal arrangement of fibroblasts may occur at the affected site, forming scar tissue, which may lead to insufficient biomechanical properties of the affected site, causing re-tears or secondary damage to related tissues.

[0004] To address the above issues, research teams both domestically and internationally have proposed various approaches to promote tendon-bone interface healing and avoid various problems associated with reconstruction failure. These approaches can be broadly categorized into four types: encapsulating grafts with biomaterials and reconstructing bone tunnels; utilizing various stem cells, primarily mesenchymal stem cells (MSCs) from different sources. Besides self-regeneration, MSCs can differentiate into multiple cell types and are widely used in tissue repair and regeneration. MSCs from different sources possess varying differentiation capacities, and the direction of stem cell differentiation is regulated by cytokines. Therefore, by combining cytokine and gene overexpression strategies, various types of stem cells have the ability to improve tendon-bone healing; promoting tendon-bone healing within bone tunnels through the application of cytokines such as transforming growth factor (TGF), bone morphogenetic protein (BMP), fibroblast factor (FGF), chemokine (CXCL13), and hyaluronic acid; and utilizing mechanical stimulation to promote tendon-bone healing, commonly including low-intensity pulsed ultrasound and extracorporeal shock wave therapy. Summary of the Invention

[0005] To address potential problems during reconstructive surgery, this invention proposes a biomass-absorbable bilayer heterogeneous membrane for promoting tendon-bone interface healing, its preparation method, and its applications. This membrane possesses a bilayer asymmetric structure. The side in contact with the tendon graft features a 10μm wide uniform groove structure, which allows fibroblasts at the affected site to align along the grooves, preventing irregular arrangement and the formation of scar tissue that could negatively impact the biomechanical properties of the affected area. It also elongates the cells, inducing myoblastic differentiation of related cells at the affected site through topographic cues, thus promoting rapid tendon fiber healing. The side in contact with the bone tunnel features a freeze-dried porous sponge structure. Its porous structure effectively recruits and adheres stem cells, while the natural high-molecular-weight polysaccharide material promotes osteogenic differentiation of stem cells, thereby facilitating efficient trabecular bone ingrowth and accelerating bone tunnel closure. This invention achieves the effects of preventing scar tissue proliferation and accelerating osteogenic formation at the interface through changes in the material's topology.

[0006] This invention provides a biomass-absorbable bilayer heterogeneous membrane that promotes tendon-bone interface healing. The bilayer heterogeneous membrane has an asymmetric bilayer structure, with one side having a groove morphology and the other side having a porous sponge-like structure morphology.

[0007] The present invention provides a biomass-absorbable bilayer heterogeneous membrane for promoting tendon-bone interface healing, wherein the groove is 10 μm wide and 1 μm deep.

[0008] This invention also provides a method for preparing a biomass-absorbable bilayer heterogeneous membrane that promotes tendon-bone interface healing. The membrane is prepared using natural polymer chitosan and gelatin as the main raw materials via maskless photolithography, casting, and vacuum freeze-drying. The specific steps are as follows: (1) Using maskless photolithography, uniformly arranged grooves 10 μm wide and approximately 1 μm deep are etched on a single-crystal silicon wafer. The grooves are then copied into a polymer mold using polydimethylsiloxane for film preparation; (2) Chitosan is dissolved in acetic acid solution at room temperature, and gelatin is added... Dissolved in distilled water at 0℃, and prepared as a 1.5% (w / v) homogeneous solution. The two solutions were mixed in equal proportions in a 40℃ water bath, and glycerol was added. The mixture was stirred thoroughly to prepare a casting solution. (3) The casting solution prepared in step (2) was uniformly cast into the mold prepared in step (1), placed in a 65℃ oven to dry for 5 hours, and then cast the casting solution on the surface a second time. The mold was immediately placed at -20℃ and frozen overnight. Then it was freeze-dried under vacuum at -70℃ for 72 hours to form an asymmetric double-layer film with two different morphologies.

[0009] The present invention provides a method for preparing a biomass absorbable bilayer heterogeneous membrane that promotes tendon-bone interface healing. In step (2), the concentration of the acetic acid solution is 2% acetic acid solution, and the amount of glycerol added is 30% of the total mass of solid materials.

[0010] The present invention provides the application of a biomass-absorbable bilayer heterogeneous membrane for promoting tendon-bone interface healing. The micron-grooved surface of the bilayer heterogeneous membrane can guide the directional alignment of fibroblasts, and the porous sponge-like structure surface can efficiently adhere to cells and promote the differentiation of stem cells into bone at the affected site. By promoting osteoogenesis and inhibiting the formation of scar tissue, it promotes tendon-bone interface healing more efficiently.

[0011] Beneficial technical effects of the present invention:

[0012] Preliminary experimental results show that the biomass-absorbable bilayer heterogeneous membrane prepared in this invention has good biocompatibility, enabling cells to adhere and proliferate on its surface. The hydrophobic side of the micron-level grooves effectively prevents cell intrusion, while the hydrophilic side promotes cell adhesion and strong proliferation. After cell culture, the cytoskeleton can be observed under a microscope to show that the cells are arranged and elongated along the grooves on the micron-level groove side, while the cells are distributed in multiple layers on the porous side, resulting in a significant increase in cell number. Attached Figure Description

[0013] Figure 1 The images shown are scanning electron microscope (SEM) images of the thin film surface prepared in Example 1, wherein (a) is a scanning electron microscope image of the groove morphology surface and (b) is a scanning electron microscope image of the porous sponge-like structure surface.

[0014] Figure 2 A scanning electron microscope image of the cross-section of the thin film prepared in Example 1;

[0015] Figure 3 The effects of three morphologies on cell viability were investigated in Example 2 and the comparative example;

[0016] Figure 4 The effects of micron-groove morphology on cytoskeleton distribution were investigated in Example 2 and the comparative example. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0018] Example 1:

[0019] (1) Using maskless photolithography, uniformly arranged grooves with a width of 10 μm and a depth of about 1 μm are etched on a single crystal silicon wafer. Then, polydimethylsiloxane is used to copy the grooves into a polymer mold for thin film preparation.

[0020] (2) Dissolve 0.27g of chitosan powder in 18ml of 2% low-concentration acetic acid solution at room temperature, and dissolve 0.03g of gelatin particles in 2ml of distilled water at 80℃ to prepare 1.5% (w / v) homogeneous solutions respectively. Mix the two solutions in a 40℃ water bath, add 90μl of glycerol, and stir thoroughly to prepare a casting solution.

[0021] (3) The prepared casting solution is uniformly cast into a micron groove mold, placed in a 65°C oven to dry for 5 hours, and then cast onto the surface a second time. The casting solution is then immediately placed at -20°C and frozen overnight. Finally, it is freeze-dried under vacuum at -70°C for 72 hours to form an asymmetric double-layer structure film with two different morphologies.

[0022] The prepared thin film was sputtered with gold onto its surface and cross-sectional fracture surfaces using an ion sputtering apparatus. The results were then observed under a scanning electron microscope. Figure 1 As can be seen, one side of the micron-groove mold exhibits a uniform groove structure, while the porous side of the freeze-dried mold has continuous and dense channels. Figure 2 The interface between the two structures is shown to be completely and tightly integrated.

[0023] Example 2:

[0024] (1) Using maskless photolithography, uniformly arranged grooves with a width of 10 μm and a depth of about 1 μm are etched on a single crystal silicon wafer. Then, polydimethylsiloxane is used to copy the grooves into a polymer mold for thin film preparation.

[0025] (2) Dissolve 0.15g of chitosan powder in 10ml of 2% low-concentration acetic acid solution at room temperature, and dissolve 0.15g of gelatin particles in 10ml of distilled water at 80℃ to prepare 1.5% (w / v) homogeneous solutions respectively. Mix the two solutions in a 40℃ water bath, add 90μl of glycerol, and stir thoroughly to prepare a casting solution.

[0026] (3) The prepared casting solution is uniformly cast into a micron groove mold, placed in a 65°C oven to dry for 5 hours, and then cast onto the surface a second time. The casting solution is then immediately placed at -20°C and frozen overnight. Finally, it is freeze-dried under vacuum at -70°C for 72 hours to form an asymmetric double-layer structure film with two different morphologies.

[0027] Comparative Example

[0028] (1) Dissolve 0.15g of chitosan powder in 10ml of 2% low-concentration acetic acid solution at room temperature, and dissolve 0.15g of gelatin particles in 10ml of distilled water at 80℃ to prepare 1.5% (w / v) homogeneous solutions respectively. Mix the two solutions in a 40℃ water bath, add 90μl of glycerol, and stir thoroughly to prepare a casting solution.

[0029] (2) The prepared casting solution is uniformly cast into a micron groove mold and placed in a 65°C oven to dry for 24 hours to form a biofilm. One side of the biofilm has a template-like groove morphology, and the other side has a dense and smooth morphology.

[0030] Rat osteoblasts were co-cultured with membranes of three different morphologies (grooved, porous sponge-like, and dense smooth) to investigate the effects of these morphologies on cell proliferation. The membranes of the three morphologies were plated onto the bottom of 24-well plates, sterilized with UV light, and then seeded with 1000 weeks of rat osteoblasts per well. After culturing in complete culture medium for 1, 3, and 7 days, live and dead cells were stained using a Calcein / PI cell viability and cytotoxicity assay kit to observe biocompatibility and cell proliferation. Figure 3 As shown, all three morphologies possess the ability to accommodate cell adhesion and proliferation, exhibiting good biocompatibility. After 7 days of culture, the cell number gradually increased. Comparison among the materials revealed that the porous sponge-like morphology exhibited the highest cell proliferation on its surface, while the grooved surface showed the lowest. After 7 days of culture, cytoskeleton staining of the grooved morphology was performed using phalloidin and DAPI to observe cell arrangement. Figure 4 The results show that the surface of the micron-grooves can cause cells to align in a specific orientation.

Claims

1. A biomass-absorbable bilayer heterogeneous membrane for promoting tendon-bone interface healing, characterized in that: The bilayer heterogeneous membrane has an asymmetrical bilayer structure. The side that contacts the tendon graft has a groove-like morphology, which allows fibroblasts in the affected area to arrange themselves along the groove, avoiding irregular arrangement that could lead to scar tissue proliferation and affect the biomechanical properties of the affected area. It also elongates the cells and induces myoblastic differentiation of the cells in the affected area through topographic cues, enabling the tendon fibers to heal rapidly. The other side that contacts the bone tunnel has a porous sponge-like structure. Its porous structure effectively recruits and adheres stem cells, and the natural high-molecular-weight chitosan promotes osteogenic differentiation of stem cells, enabling efficient ingrowth of trabecular bone and accelerating the closure of the bone tunnel in the affected area. The groove is 10 μm wide and 1 μm deep; This bilayer heterogeneous membrane is prepared using natural polymer chitosan and gelatin as the main raw materials through maskless photolithography, casting, and vacuum freeze-drying techniques. The specific steps are as follows: (1) Using maskless photolithography, uniformly arranged grooves with a width of 10 μm and a depth of 1 μm are etched on a single crystal silicon wafer. Then, polydimethylsiloxane is used to copy the grooves into a polymer mold for thin film preparation. (2) Chitosan was dissolved in 2% acetic acid solution at room temperature, and gelatin was dissolved in distilled water at 80°C to prepare uniform solutions of 1.5% w / v. The two solutions were mixed in equal proportions in a water bath at 40°C, and glycerol, accounting for 30% of the total mass of solid materials, was added and stirred thoroughly to prepare a casting solution. (3) The casting solution prepared in step (2) is uniformly cast into the mold prepared in step (1), placed in an oven at 65°C for 5 hours to dry, and then cast the casting solution on the surface a second time. Immediately place it at -20°C to freeze overnight, and then freeze-dry it under vacuum at -70°C for 72 hours to form an asymmetric double membrane with two different morphologies. The micron groove surface of the asymmetric double membrane can guide the directional arrangement of fibroblasts, and the porous sponge-like structure surface can efficiently adhere to cells and promote the differentiation of stem cells into bone in the affected area. By promoting osteoogenesis and inhibiting the formation of scar tissue, it can more efficiently promote the healing of the tendon-bone interface.

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