Polydatin tendon bone repair gradient bionic scaffold and preparation method thereof

Abstract

The invention discloses a polydatin tendon-bone repair gradient bionic scaffold and a preparation method thereof, a nano-hydroxyapatite (nHAP) / polycaprolactone (PCL) composite material and a biological hydrogel containing polydatin (PLD) liposome are designed and prepared by 3D printing to form a multi-partition structure, so as to meet the complex requirements of tendon-bone healing. Wherein the nHAP / PCL composite material has good mechanical performance and is matched with a tendon stress environment to serve as a mechanical framework of the stent. The introduction of nHAP can effectively promote the healing of a bone injury area, and the construction of the slow-release PLD liposome hydrogel can effectively reverse the fibrosis of a tendon-bone interface. The gradient bionic scaffold for slowly releasing the polydatin, which is constructed by a 3D printing technology, can bring a brand-new treatment scheme for tendon-bone healing.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medical materials, and discloses a rhizomary rhizomae tendon-bone repair gradient biomimetic scaffold and a preparation method thereof. BACKGROUND

[0002] Tendon-bone injury is one of the common musculoskeletal diseases in clinical practice, which refers to the injury or lesion occurring at the interface between the tendon and ligament and the bone. Patients often show symptoms such as joint pain, movement dysfunction and muscle atrophy. Common tendon-bone injuries in clinical practice include rotator cuff injury, anterior and posterior cruciate ligament injury and Achilles tendon injury, and the incidence of the injuries is increasing year by year with the popularization of sports and the acceleration of the process of social aging. Studies have shown that 18% of tendon-bone injuries occur at the tendon-bone interface (TBI), ranking third among all types of tendon-bone injuries. The tendon-bone interface (TBI) is the fibrocartilage layer between the tendon and the bone, which serves as a transition from fibrous tendon tissue to bone. The tendon bundle directly inserts into the bone, and the tendon tissue near the bone is calcified. It is characterized by the existence of four regions in a continuous gradient: tendon, fibrocartilage, calcified fibrocartilage and bone. Due to the existence of this complex gradient structure, many common tools used in surgery, such as suture anchors, cannot regenerate the insertion point, resulting in a high incidence of re-rupture. Secondly, in the healing mechanism of tendon-bone injury, endogenous healing is a very important self-repair mechanism in the process of tendon repair, which mainly relies on the function of cells inside the tendon to achieve healing. The main cell types include tendon cells, fibroblasts and inflammatory cells. However, the collagen fibers generated through this healing mechanism are often irregularly arranged, which is easy to form scar tissue. The formation of scar tissue not only affects the functional recovery of the tendon, but also may cause adhesion between the tendon and the surrounding tissue, leading to long-term dysfunction. The toughness of the scar tissue is much lower than that of the original tissue, and secondary rupture is easy to occur. Therefore, there is an urgent need for intervention means to promote the structural and functional repair of tendon-bone injury in clinical practice.

[0003] In recent decades, tissue bioengineering has become a promising strategy for skeletal muscle tissue repair and regeneration, which involves the integration of cells, scaffolds and biomolecules to create biological substitutes to repair, replace or regenerate damaged skeletal muscle tissue. Artificial synthetic scaffold materials have become a research hotspot in the field of tendon-bone healing. Artificial synthetic scaffold materials have evolved from single-layer scaffolds, multi-layer integrated scaffolds, to gradient biomimetic scaffolds. Through comprehensive comparison, gradient biomimetic scaffolds can better meet the transition between the levels of the original tendon-bone interface, and have better overall performance and higher mechanical properties. Among the degradable polymers, polycaprolactone (PCL) has good biodegradability, and its degradation products can be absorbed or metabolized by the body without toxicity and harm to the human body. As a hydrophobic semi-crystalline material, it has good toughness and thermal stability. Combined with 3D printing technology, the required structure and mechanical properties of the tendon-bone interface can be met by optimizing the printing parameters. Studies have shown that loading a certain amount of nano-hydroxyapatite (nHAP) to form a composite material can promote bone tissue repair. Hydrogels exhibit unique advantages in tissue engineering due to their high water content, high stability, and good biocompatibility. Their composition similar to the extracellular matrix can support cell proliferation, differentiation, and functional expression.

[0004] Previous studies have shown that traditional Chinese medicine polygonum cuspidatum (PLD) can reduce inflammation by inhibiting the NF-κB pathway, achieve anti-inflammatory effects, activate the Nrf2-ARE signaling pathway to play an anti-fibrosis role, significantly inhibit the transmission of IL-1β-induced macrophage NF-κB signaling pathway, reverse M1 polarization, and avoid excessive inflammatory response in the early stage of tendon-bone healing; it can promote the osteogenic effect of bone marrow mesenchymal stem cells and increase the expression of angiogenesis-specific markers. However, polygonum cuspidatum is a fat-soluble drug and is not soluble in water, and its absorption efficiency in the body is limited, so the drug delivery system needs to be optimized. Liposomes are closed vesicular structures formed by self-assembly of membrane materials such as phospholipids. The basic structure is one or more membrane-like structures composed of phospholipid bilayers, which encapsulate one or more water volumes to form concentric floating vesicles. Phospholipids are the key components of liposomes, which have a hydrophilic head group and a hydrophobic tail group. This unique structure allows phospholipids to spontaneously assemble into bilayer membrane structures in aqueous solutions. Due to its unique bilayer membrane structure, liposomes can naturally encapsulate water-soluble and fat-soluble drug molecules, making them an ideal drug delivery system.

[0005] To address the above problems, a 3D printed scaffold that can inhibit the fibrosis process of tendon-bone healing and meet certain mechanical properties is urgently needed in the industry. SUMMARY

[0006] In view of the above shortcomings, the purpose of this invention is to solve the problem that traditional scaffold materials with uniform structure and composition cannot effectively repair tendon and bone damage and inhibit fibrosis during the regeneration process. Therefore, this invention proposes a gradient biomimetic scaffold for tendon and bone repair using polydipsia glycosides prepared by 3D printing, which also takes into account the sustained-release effect of polydipsia glycosides in tendon and bone repair.

[0007] To achieve the above-mentioned technical effects, the present invention employs the following technical means: This invention first discloses a polydipsia tendon-bone repair gradient biomimetic scaffold, comprising a multi-layered partitioned structure, wherein: One side of the support is made of nHAP / PCL composite printing material; The other side of the support is a hydrogel bio-ink printing material; The overlapping area between the two materials accounts for 1 / 3 to 2 / 3 of the total length of the support.

[0008] This invention also discloses a method for preparing a gradient biomimetic scaffold for tendon-bone repair based on the above-mentioned polydipsia glycoside, comprising: By planning the printing path with dual nozzles, a multi-layered partitioned structure of the scaffold is constructed. One side is printed using nHAP / PCL composite material, and the other side is printed using hydrogel bio-ink. The printing areas of the two materials have a 1 / 3 to 2 / 3 overlap. Through this printing arrangement, a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum is obtained.

[0009] Furthermore, the preparation method specifically includes: (1) Based on the shape and size of the target tendon bone repair, a printing model is established in the modeling software, the diameter and distribution of the printing fibers are designed, and the proportion of each area is clarified; (2) Import the above model into the slicing software, set the T0 nozzle to print nHAP / PCL composite material, the printing temperature is 70-100℃, the printing speed is 3-10 mm / s, and the extrusion speed is 0.5-10 mm. 3 The T1 printhead prints hydrogel bio-ink at a printing temperature of 20-25℃ and a printing speed of 1-6 mm / s, with an extrusion speed of 3-10 mm / s. 3 / s, forming a .gCode file; (3) Cut the nHAP / PCL composite material into pieces and add it to the T0 printhead. Add the hydrogel bio-ink loaded with PLD (Polygonum cuspidatum) liposomes to the T1 printhead and import the print path control .gCode file to obtain a polygonum cuspidatum tendon bone repair gradient biomimetic scaffold.

[0010] Furthermore, the nHAP / PCL composite material described in step (2) has a filament diameter of 100-500 μm, a pore size of 400-1000 mm, and a porosity of 40-70%.

[0011] Further, the nHAP / PCL composite material described in step (2) is prepared by the following method: (2.1) Weigh 70-95 parts of PCL with a molecular weight of 2-8w and dissolve it completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%; (2.2) Weigh 5-30 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (2.3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

[0012] Further, the hydrogel bio-ink is a hydrogel bio-ink loaded with PLD liposomes, comprising: 5-30 μM PLD liposomes, 10-25 wt% GelMA (methacrylamide gelatin), and 0.5-1 wt% photoinitiator.

[0013] Further, the PLD liposomes described in step (3) are prepared by the following method: (1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 1-15mg of polydipsia glycoside, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2) Place the first solution in a rotary evaporator at 55°C and evaporate until the solvent is completely evaporated to obtain the first substance; (3) Add 10 ml of PBS to the first substance, hydrate it fully at 40-50℃, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min to obtain the supernatant, which is the PLD liposome.

[0014] Furthermore, the photoinitiator is LAP (lithium phenyl phosphate).

[0015] The present invention also discloses a polydipsia tendon-bone repair gradient biomimetic scaffold prepared according to any of the preparation methods described herein.

[0016] The beneficial effects of this invention are as follows: 1. This invention simulates the multi-level partitioned structure of the tendon-bone interface (TBI) through 3D printing gradient structures: tendon, uncalcified fibrocartilage, calcified fibrocartilage and bone, making the scaffold more integral and meeting biomechanical performance requirements.

[0017] 2. This invention, through a liposome-based sustained-release system loaded with the traditional Chinese medicine Polygonum cuspidatum glycoside, is expected to achieve anti-inflammatory, anti-fibrotic, and antioxidant effects, promoting tendon-bone interface healing. The multifunctional GelMA hydrogel can provide a rich 3D aqueous environment for cell growth and differentiation. Attached Figure Description

[0018] Figure 1 This is a demonstration image of alternating printouts from dual printheads. The white filaments are PCL, and the blue filaments are hydrogel. Figure 2 The microstructure of the polydipsia glycoside tendon-bone repair gradient biomimetic scaffold prepared in this invention is shown in Figure a, where a represents the macroscopic porous structure prepared in this invention; and b represents the microstructure of the scaffold. Figure 3 The mechanical properties of the polydipsia glycoside-based gradient biomimetic scaffold for tendon and bone repair prepared in this invention under different pore sizes are shown, where a is the tensile stress-strain curve of the scaffold; and b is the compressive stress-strain curve of the scaffold. Figure 4 The microstructure of GelMA hydrogel in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention is shown in Figure a, where a is the macroscopic porous structure of the GelMA hydrogel scaffold and b is the microstructure of the GelMA hydrogel scaffold under magnification. Figure 5 The microstructure of polydipsia liposomes in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention is shown under a transmission electron microscope. Figure 6 The images show cell viability / death staining of the hydroxyapatite PCL scaffold in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention, where a is a cell viability / death staining image of pure PCL cells and b is a cell viability / death staining image of 5% HA-PCL cells. Figure 7 A flowchart illustrating the surgical steps and stent implantation process for a New Zealand rabbit rotator cuff tear model. Detailed Implementation

[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials used in the following examples are commercially available.

[0021] Example 1

[0022] A method for preparing a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum, specifically comprising: (1) Preparation of nHAP / PCL composite material: (1.1) Weigh 95 parts of PCL with a molecular weight of 8w and dissolve them completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%. (1.2) Weigh 5 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (1.3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

[0023] (2) Preparation of hydrogel bio-ink loaded with PLD liposomes: (2.1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 15mg of resveratrol, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2.2) The first solution was placed in a rotary evaporator at 55°C and evaporated until the solvent was completely evaporated to obtain the first substance; (2.3) Add 10 ml of PBS to the first substance, hydrate it fully at 37°C, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (2.4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min, and the supernatant was obtained, which is the PLD liposome; (2.5) Take 5 μM of PLD liposomes, 10 wt% of GelMA (methacrylamide gelatin), and 0.5 wt% of LAP photoinitiator and mix them to obtain hydrogel bio-ink loaded with PLD liposomes.

[0024] (3) Based on the shape and size of the target tendon bone repair, establish a printing model in the modeling software, design the diameter and distribution of the printing fibers, and clarify the proportion of each area; (4) Import the above model into the slicing software, set the T0 nozzle to print the nHAP / PCL composite material, the printing temperature to 80℃, the printing speed to 6 mm / s, and the extrusion speed to 1 mm. 3 The obtained nHAP / PCL composite material has a filament diameter of 400 μm, a pore size of 600 mm, and a porosity of 60%; (5) T1 printhead printing hydrogel bio-ink, printing temperature 23℃, printing speed 3 mm / s, extrusion speed 3 mm 3 / s, forming a .gCode file; (6) Shred the nHAP / PCL composite material and add it to the T0 printhead. Add the hydrogel bio-ink loaded with PLD (Polygonum cuspidatum) liposomes to the T1 printhead. Import the print path control .gCode file and set the overlap range of the two materials to 1 / 3 in the modeling software to obtain a polygonum cuspidatum tendon bone repair gradient biomimetic scaffold.

[0025] Example 2

[0026] A method for preparing a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum, specifically comprising: (1) Preparation of nHAP / PCL composite material: (1.1) Weigh 90 parts of PCL with a molecular weight of 8w and dissolve them completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%. (1.2) Weigh 10 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (1.3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

[0027] (2) Preparation of hydrogel bio-ink loaded with PLD liposomes: (2.1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 10mg of polydipsia glycoside, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2.2) The first solution was placed in a rotary evaporator at 55°C and evaporated until the solvent was completely evaporated to obtain the first substance; (2.3) Add 10 ml of PBS to the first substance, hydrate it fully at 37°C, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (2.4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min, and the supernatant was obtained, which is the PLD liposome; (2.5) Take 5 μM of PLD liposomes, 10 wt% of GelMA (methacrylamide gelatin), and 0.5 wt% of LAP photoinitiator and mix them to obtain hydrogel bio-ink loaded with PLD liposomes.

[0028] (3) Based on the shape and size of the target tendon bone repair, establish a printing model in the modeling software, design the diameter and distribution of the printing fibers, and clarify the proportion of each area; (4) Import the above model into the slicing software, set the T0 nozzle to print the nHAP / PCL composite material, the printing temperature to 80℃, the printing speed to 6 mm / s, and the extrusion speed to 1 mm. 3 The obtained nHAP / PCL composite material has a filament diameter of 400 μm, a pore size of 600 mm, and a porosity of 60%; (5) T1 printhead printing hydrogel bio-ink, printing temperature 23℃, printing speed 3 mm / s, extrusion speed 3 mm 3 / s, forming a .gCode file; (6) Cut the nHAP / PCL composite material into pieces and add it to the T0 printhead. Add the hydrogel bio-ink loaded with PLD (Polygonum cuspidatum) liposomes to the T1 printhead. Import the print path control .gCode file. In the modeling software, set the final overlap range of the two materials' printing modules to 1 / 3 to obtain a polygonum cuspidatum tendon bone repair gradient biomimetic scaffold.

[0029] Example 3

[0030] A method for preparing a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum, specifically comprising: (1) Preparation of nHAP / PCL composite material: (1.1) Weigh 80 parts of PCL with a molecular weight of 8w and dissolve them completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%. (1.2) Weigh 20 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (1.3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

[0031] (2) Preparation of hydrogel bio-ink loaded with PLD liposomes: (2.1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 5mg of polygalactoside, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2.2) The first solution was placed in a rotary evaporator at 55°C and evaporated until the solvent was completely evaporated to obtain the first substance; (2.3) Add 10 ml of PBS to the first substance, hydrate it fully at 37°C, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (2.4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min, and the supernatant was obtained, which is the PLD liposome; (2.5) Take 5 μM of PLD liposomes, 10 wt% of GelMA (methacrylamide gelatin), and 0.5 wt% of LAP photoinitiator and mix them to obtain hydrogel bio-ink loaded with PLD liposomes.

[0032] (3) Based on the shape and size of the target tendon bone repair, establish a printing model in the modeling software, design the diameter and distribution of the printing fibers, and clarify the proportion of each area; (4) Import the above model into the slicing software, set the T0 nozzle to print the nHAP / PCL composite material, the printing temperature to 80℃, the printing speed to 6 mm / s, and the extrusion speed to 1 mm. 3 / s, the obtained nHAP / PCL composite material has a filament diameter of 400 μm, a pore size of 600 mm, and a porosity of 40-70%; (5) T1 printhead printing hydrogel bio-ink, printing temperature 23℃, printing speed 3 mm / s, extrusion speed 3 mm 3 / s, forming a .gCode file; (6) Cut the nHAP / PCL composite material into pieces and add it to the T0 printhead. Add the hydrogel bio-ink loaded with PLD (Polygonum cuspidatum) liposomes to the T1 printhead. Import the print path control .gCode file and set the overlap range of the printing modules of the two materials to 1 / 3 in the modeling software to obtain a polygonum cuspidatum tendon bone repair gradient biomimetic scaffold.

[0033] Example 4

[0034] A method for preparing a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum, specifically comprising: (1) Preparation of nHAP / PCL composite material: (1.1) Weigh 95 parts of PCL with a molecular weight of 8w and dissolve them completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%. (1.2) Weigh 5 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (1.3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

[0035] (2) Preparation of hydrogel bio-ink loaded with PLD liposomes: (2.1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 1mg of polydipsia glycoside, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2.2) The first solution was placed in a rotary evaporator at 55°C and evaporated until the solvent was completely evaporated to obtain the first substance; (2.3) Add 10 ml of PBS to the first substance, hydrate it fully at 37°C, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (2.4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min, and the supernatant was obtained, which is the PLD liposome; (2.5) Take 10 μM PLD liposomes, 10 wt% GelMA (methacrylamide gelatin), and 0.5 wt% LAP photoinitiator and mix them to obtain hydrogel bio-ink loaded with PLD liposomes.

[0036] (3) Based on the shape and size of the target tendon bone repair, establish a printing model in the modeling software, design the diameter and distribution of the printing fibers, and clarify the proportion of each area; (4) Import the above model into the slicing software, set the T0 nozzle to print the nHAP / PCL composite material, the printing temperature to 80℃, the printing speed to 6 mm / s, and the extrusion speed to 1 mm. 3 The obtained nHAP / PCL composite material has a filament diameter of 400 μm, a pore size of 600 mm, and a porosity of 60%; (5) T1 printhead printing hydrogel bio-ink, printing temperature 23℃, printing speed 3 mm / s, extrusion speed 3 mm 3 / s, forming a .gCode file; (6) Cut the nHAP / PCL composite material into pieces and add it to the T0 printhead. Add the hydrogel bio-ink loaded with PLD (Polygonum cuspidatum) liposomes to the T1 printhead. Import the print path control .gCode file and set the overlap range of the printing modules of the two materials to 1 / 3 in the modeling software to obtain a polygonum cuspidatum tendon bone repair gradient biomimetic scaffold.

[0037] Example 5

[0038] The proportion of nHAP in the composite material was increased to 10% according to the method of Example 1, which improved the osteogenic performance compared to the scaffold of Example 1.

[0039] Example 6

[0040] The proportion of nHAP in the composite material was increased to 20% according to the method of Example 1, which improved the osteogenic performance of the scaffold compared to Example 1.

[0041] Example 7

[0042] The PLD content in the PLD-loaded liposome hydrogel bio-ink was increased to 10 μM according to the method of Example 1, which improved the anti-fibrotic ability of the scaffold in Example 1.

[0043] Experimental Example 1 The microstructure of the polydipsia glycoside-based gradient biomimetic scaffold for tendon and bone repair prepared in this invention was observed, and details are provided below. Figure 2 ,in Figure 2 a represents the macroscopic porous structure of the polydipsia glycoside tendon-bone repair gradient biomimetic scaffold prepared in this invention; Figure 2 b represents the microstructure of the scaffold: the minimum pore size required for cell activity is 100 μm. As shown in the figure, the macroscopic porous structure of the scaffold facilitates cell diffusion, migration, and the formation of new tissues. Furthermore, the uniform distribution of nano-hydroxyapatite on the surface of the composite scaffold promotes osteogenic differentiation of cells.

[0044] Experimental Example 2 The mechanical properties of the polydipsia glycoside-based gradient biomimetic scaffold for tendon-bone repair prepared in this invention were tested, and details are available in the attached document. Figure 3 The tensile modulus is 59.5±20.7MPa and the compressive modulus is 48.65±6.9MPa, which meets the mechanical properties required for the human tendon-bone interface.

[0045] Experimental Example 3 The microstructure of the GelMA hydrogel in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention was observed, and details are provided below. Figure 4,in Figure 4 a shows the macroscopic porous structure of the 3D-printed GelMA hydrogel scaffold prepared in this invention; b shows the microscopic structure of the 3D-printed GelMA hydrogel scaffold under magnification. As shown in the figure, the printer can successfully print porous hydrogel scaffolds, which exhibit a loose and porous structure under magnification, providing a rich 3D environment for cell growth, differentiation, and migration.

[0046] Test Example 4 Transmission electron microscopy was performed on the polydipsia liposomes in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention, and their microstructure is as follows: Figure 5 As shown: The prepared drug-loaded liposomes are nano-sized particles with uniform morphology and a particle size of 85.61±3.25nm.

[0047] Experimental Example 5 The cell viability / death staining of the hydroxyapatite PCL scaffold in the polydipsia tendon-bone repair gradient biomimetic scaffold prepared in this invention was observed. See details below. Figure 6 As shown in the figure, most bone marrow mesenchymal stem cells can survive and proliferate on both types of scaffolds, and the 5% HA-PCL scaffold can significantly promote cell adhesion.

[0048] Experimental Example 6 The polydipsia tendon-bone repair gradient biomimetic scaffold prepared according to this invention was applied in a New Zealand rabbit rotator cuff tear model surgery, based on... Figure 7 The results show that in the rotator cuff tear model, the tendon is completely severed, the insertion point of the humerus and tendon is completely destroyed, and the 2mm bone marrow duct drilled from the insertion point can be completely inserted into the scaffold, indicating that the rotator cuff tear model was successfully created.

[0049] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum, comprising a multi-layered partitioned structure, wherein: One side of the support is made of nHAP / PCL composite printing material; The other side of the support is a hydrogel bio-ink printing material; The overlapping area between the two materials accounts for 1 / 3 to 2 / 3 of the total length of the support.

2. A method for preparing the polydipsia glycoside tendon-bone repair gradient biomimetic scaffold according to claim 1, comprising: By planning the printing path with dual nozzles, a multi-layered partitioned structure of the scaffold is constructed. One side is printed using nHAP / PCL composite material, and the other side is printed using hydrogel bio-ink. The printing areas of the two materials have a 1 / 3 to 2 / 3 overlap. Through this printing arrangement, a gradient biomimetic scaffold for tendon and bone repair using Polygonum cuspidatum is obtained.

3. The preparation method according to claim 2, wherein: The printing conditions for the nHAP / PCL composite material are as follows: Printing temperature: 70-100℃; printing speed: 3-10 mm / s; extrusion speed: 0.5-10 mm / s. 3 / s.

4. The preparation method according to claim 3, wherein: The printing filament has a diameter of 100-500 μm, a pore size of 400-1000 mm, and a porosity of 40-70%.

5. The preparation method according to claim 2, wherein: The printing conditions for the hydrogel bio-ink are as follows: Printing temperature: 20-25℃; printing speed: 1-6 mm / s; extrusion speed: 3-10 mm / s. 3 / s.

6. The preparation method according to claim 2, wherein: The nHAP / PCL composite material was prepared by the following method: (1) Weigh 70-95 parts of PCL and dissolve them completely in dichloromethane solution to obtain a PCL solution with a mass-volume ratio of 50%; (2) Weigh 5-30 parts of nHAP and add them to the PCL solution to form a composite material emulsion; (3) The solvent dichloromethane in the composite emulsion is completely evaporated to obtain the nHAP / PCL composite material.

7. The preparation method according to claim 2, wherein: The hydrogel bio-ink is a hydrogel bio-ink loaded with PLD liposomes, comprising: 5-30 μM PLD liposomes, 10-25 wt% GelMA, and 0.5-1 wt% photoinitiator.

8. The preparation method according to claim 7, wherein: The PLD liposomes were prepared by the following method: (1) Weigh 33mg of soybean lecithin, 10mg of cholesterol, and 1-15mg of polydipsia glycoside, and dissolve them completely in 15ml of chloroform to obtain the first solution; (2) Place the first solution in a rotary evaporator at 55°C and evaporate until the solvent is completely evaporated to obtain the first substance; (3) Add 10 ml of PBS to the first substance, hydrate it fully at 40-50℃, and then place it in an ice-water bath for 100 W sonication for 15 min to obtain the second solution; (4) The second solution was centrifuged at 5000 rpm and 4℃ for 30 min to obtain the supernatant, which is the PLD liposome.

9. The preparation method according to claim 7, wherein: The photoinitiator is LAP.

10. A polydactyly-based gradient biomimetic scaffold for tendon and bone repair prepared by any one of the preparation methods described in claims 2 to 9.

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