Double-layer Janus fiber sleeve as well as preparation method and application thereof

Abstract

The invention relates to a double-layer Janus fiber sleeve and a preparation method and application thereof.Materiology, in-vitro cell experiments and in-vivo tendon injury model repair experiments are carried out through implantation, and through the repair effect of the double-layer Janus fiber sleeve on injured tendon tissue, the double-layer Janus fiber sleeve has the advantages of the anisotropic structure of the Janus structure, so that the double-layer Janus fiber sleeve has a good application prospect. Rearrangement and induced regeneration of tendon cells by an inner layer structure are achieved, and the tendon forming performance of tendons can be activated under the condition that shearing force is generated when normal tendon sliding is simulated in vitro; and the outer-layer staggered fiber structure can effectively block invasion of scar fibers, avoid excessive deposition of the scar fibers, induce new capillaries to grow into the stent to realize enrichment of nutrient substances in the blood vessels, and finally realize non-scar healing of the tendons.

Description

Technical Field

[0001] The invention relates to the field of tendon injury repair, and in particular to a double-layer Janus fiber sleeve and a preparation method and application thereof. Background Art

[0002] Tendon tissue plays an important role in connecting bone and muscle tissue and in mechanical conduction. When partial or full-thickness tear occurs, the continuity of tendon tissue will be destroyed, thereby limiting the corresponding joint and limb activities and seriously affecting the patient's quality of life. Tendon tissue follows the inflammatory phase, scar formation phase and late tendon bundle rearrangement phase. This process is regulated by endogenous and exogenous healing to achieve tissue healing. However, since tendons are vascular and cell-poor tissues, exogenous healing often dominates the repair process, bringing vascular-rich repair active tissue to the repair environment. However, excessive ingrown scar tissue accompanied by new blood vessels will lead to disordered deposition of fibers at the repair site and cause postoperative adhesions, which will eventually seriously reduce postoperative biomechanical function. Therefore, it is necessary to balance the competitive dominance of endogenous and exogenous healing, which can not only provide the repair capacity of endogenous and exogenous healing, but also avoid the ingrown scar tissue to achieve orderly regeneration of tendon tissue, and realize non-scar healing and complete regeneration of tendon tissue.

[0003] At present, physical barriers are mostly used to regulate excessive scar healing of tendon tissue and prevent postoperative adhesion, such as electrospinning fiber scaffolds, silicone sleeves, etc. However, due to the low porosity of the scaffold structure and the lack of regenerative performance, it will lead to poor postoperative biomechanical repair or even treatment failure. In order to solve the problem of lack of regenerative performance, active ingredients such as mRNA, nanomolecular drugs and various peptides are currently loaded and applied to tendon injury repair through biological tissue scaffolds. However, the addition of active ingredients still inevitably faces adverse factors such as biosafety, encapsulation efficiency and difficulties in clinical application. Therefore, chemical modification is not the most effective way to achieve tendon regeneration, and it is necessary to open up non-chemical ways to activate the repair potential of tendons. Summary of the invention

[0004] The purpose of the present invention is to provide a double-layer Janus fiber sleeve and its preparation method and application. The double-layer Janus fiber sleeve of the present invention is composed of a directional topological structure and a cross-fiber structure. The internal directional fibers induce tendon fiber regeneration under the action of shear force; the outer layer fiber staggered structure effectively blocks scar fiber invasion, avoids excessive scar fiber deposition, induces partial new blood vessel growth, and finally realizes the biological filtration effect of the outer layer fiber to achieve regeneration.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first object of the present invention is to provide a double-layer Janus fiber sleeve, which consists of a directional topological structure and a cross-fiber structure;

[0007] The directional topological structure is composed of a plurality of longitudinally parallel fibers forming a hollow cylindrical structure, and the cross-fiber structure is composed of a plurality of mutually interlaced fibers forming a hollow cylindrical structure;

[0008] The exterior of the directional topological structure is covered with a cross-fiber structure, and the directional topological structure and the cross-fiber structure are connected.

[0009] Furthermore, the length of the double-layer Janus fiber sleeve is 40-50 mm, and the diameter of the double-layer Janus fiber sleeve is 3-5 mm.

[0010] Furthermore, the diameter of a single fiber in the directional topological structure and the cross-fiber structure is 15 to 50 μm.

[0011] As a preferred technical solution, the diameter of a single fiber in the directional topological structure and the cross-fiber structure is 30 μm.

[0012] Furthermore, the thickness of the directional topological structure is 100-200 μm, and the thickness of the cross-fiber structure is 200-300 μm.

[0013] Furthermore, the cross-fiber structure is a grid-like structure formed by interlacing a plurality of fibers, and the angle between two intersecting fibers is in the range of 60° to 70°.

[0014] Furthermore, in the above, the pore size of the grid structure is 50-100 μm.

[0015] The second object of the present invention is to provide a method for preparing a double-layer Janus fiber sleeve, the specific steps of which are as follows:

[0016] S1. Use 3D printing software to build a pre-printing path;

[0017] S2, weigh an appropriate amount of biopolymer material and wait for the next step of printing;

[0018] S3. Print the biopolymer material obtained in step S2 using electrostatic direct writing technology to obtain a double-layer Janus fiber sleeve.

[0019] Further, in step S1, the pre-printing path is as follows:

[0020] The first 15 to 20 layers are radial fibers that divide the circumference into 20 to 30 equal parts, and the last 30 to 40 layers are cross fibers, with the angle between the two intersecting fibers ranging from 60° to 70°.

[0021] As a preferred technical solution, the pre-printing path is as follows:

[0022] The first 15 layers are radial fibers with the circumference divided into 20 equal parts, and the last 30 layers are 65° cross fibers with a total length of 50 mm.

[0023] Furthermore, in step S2, the biopolymer material is selected from any one or more of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer or polylactic acid-caprolactone copolymer.

[0024] As a preferred technical solution, the biopolymer material is polycaprolactone (PCL).

[0025] Furthermore, in step S3, the printing parameters are: rotating shaft diameter 3-7 mm, electrostatic spacing 2-3 mm, voltage 3-5 kV, material temperature control 90-110° C., and air pressure 4-6 kPa.

[0026] As a preferred technical solution, the printing parameters are: rotating shaft diameter 5 mm, electrostatic spacing 2.5 mm, voltage 3.9 kV, material temperature control 100° C., and air pressure 5 kPa.

[0027] The third object of the present invention is to provide an application of a double-layer Janus fiber sleeve, wherein the double-layer Janus fiber sleeve obtained by the above preparation method is used in the preparation of a repair material for treating tendon tissue defects.

[0028] Furthermore, the application is to prepare an implant device for treating tendon tissue defects.

[0029] As a preferred technical solution, the double-layer Janus fiber sleeve is a bionic tendon scaffold.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] The present invention implants a bionic tendon scaffold to conduct material science, in vitro cell experiments and in vivo tendon injury model repair experiments. Through the repair effect of the bionic tendon scaffold on damaged tendon tissue, the inner layer structure can rearrange and induce regeneration of tendon cells under the advantage of the structural heterogeneity of Janus, and can activate the tendon formation performance under the condition of shear force generated when normal tendon slides in vitro; the outer layer interlaced fiber structure can effectively block the invasion of scar fibers, avoid excessive scar fiber deposition, and induce new capillaries to grow into the scaffold to achieve vascular nutrient enrichment, ultimately achieving non-scar tendon healing.

[0032] In the early stage of tendon tissue healing, exogenous vascular infiltration can provide the local environment with nutrients needed for repair, remove waste and control immune response, so it is necessary to maintain a rich vascular environment around the damaged tissue and regulate the infiltration of new blood vessels. The diameter of the new capillary is about 6 to 9m, which is much smaller than the conventional diameter of fibroblasts of 50 to 80m. Therefore, by adjusting the structural gap of the biological scaffold, it is possible to selectively filter the infiltration of new capillary tissues and block the excessive early fibroblast growth, thereby achieving a selective filtering effect. As a biological function regulation means, the physical topological structure can enhance and accurately induce the fate of cell biology in the field of tissue regeneration. Tendon cells will be affected by changes in the local topological structure and show adaptive phenotypes, such as tendinogenesis and osteogenic differentiation trends. As a mechanical transmission tissue, the effect of the local stress environment, especially shear stress, can promote the repair of damaged tissues. Therefore, the topological structure combined with shear force can synergistically improve the self-repair performance of tendon tissue. The material constructed by the present invention has both biological filtration effect and biomimetic topological structure induction advantages, achieves precise behavioral regulation at the cellular level and promotes tendon tissue regeneration, and can solve the current demand for non-scar healing of tendon injuries.

[0033] Inspired by the clinical problems of scar invasion and insufficient repair performance after tendon injury, the present invention constructs an internal and external anisotropic Janus tendon repair sleeve with topological structural characteristics through a new Extruded Bio-3D Printer. In vitro, the oriented fibers inside the Janus tendon sleeve can promote the oriented attachment and migration of tendon cells, and activate the expression of mechanical force-related protein Peizo-1 and tendon-related protein Col-I and SCX in a shear force environment simulating relative movement in vivo. At the same time, in vivo, it can avoid excessive scar tissue growth, promote local extracellular matrix deposition, and accelerate the rearrangement of corresponding tendon fiber tissue, so as to achieve non-scar healing and orderly regeneration of damaged tendons in rat Achilles tendon injury models. Finally, the oriented topological structure of the medial side is used to promote the regeneration of damaged tendon tissue, and the mixed and staggered fiber structure of the lateral side is used to achieve the "biological filtration" screening effect, retaining the biological activity of vascular tissue in exogenous healing and avoiding scar growth, so as to achieve complete non-scar healing of tendons. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram showing a preview of the printing path of the bionic tendon scaffold in Example 1;

[0035] Figure 2 This is a structural diagram of the bionic tendon scaffold in Example 1;

[0036] Figure 3Figure 1 is a diagram showing the biological effects of the bionic tendon scaffold, where A is a diagram showing the structure of the bionic tendon scaffold in different observation directions, B is a schematic diagram showing the implantation and repair process of the bionic tendon scaffold in a rat tendon injury model, and C is a schematic diagram showing the mechanism and process of the bionic tendon scaffold inducing tendon tissue repair;

[0037] Figure 4 The morphological features and local morphological diagrams of the bionic tendon scaffold and the sheet scaffold, wherein A is a schematic diagram of the structure of the bionic tendon scaffold and the sheet scaffold, B is a schematic diagram of the original local oriented fiber morphology of the inner surface of the bionic tendon scaffold, C is a schematic diagram of the local morphology of the inner surface of the bionic tendon scaffold after unfolding, D is a light microscope schematic diagram of the bionic tendon scaffold and the sheet scaffold, E is a scanning electron microscope image of the inner and outer layers of the bionic tendon scaffold, and F is a scanning electron microscope image of the inner and outer layers of the bionic tendon scaffold after inoculation of cells and culture;

[0038] Figure 5 Schematic diagram of the effect of bionic tendon scaffold and sheet scaffold on the behavior pattern of tendon cells and in vitro shear simulation test, wherein A is a schematic diagram of cytoskeleton staining of bionic tendon scaffold and sheet scaffold, B is a schematic diagram of the quantitative distribution trend of surface cells of bionic tendon scaffold and sheet scaffold, C is a schematic diagram of cell migration characteristics of sheet scaffold at different time points, D is a schematic diagram of cell migration characteristics of bionic tendon scaffold at different time points, E is a schematic diagram of in vitro shear force simulation, F is a schematic diagram of the expression of mechanics and tendon formation related proteins of different groups under shear force (bar=100), and G is a schematic diagram of the quantitative analysis results of in vitro shear force simulation of different groups;

[0039] Figure 6 Schematic diagram for evaluating the in vivo repair performance of the bionic tendon scaffold, wherein A is a schematic diagram of tendon repair implantation, B is a schematic diagram of the observation of the degree of peritendinous adhesion of the injured tendon after 4 weeks, C is a schematic diagram of the gross evaluation of peritendinous adhesion, D is a HE staining diagram after implantation, E is a schematic diagram of the adhesion score of tendon adhesion and tendon healing, F is a schematic diagram of the healing score of tendon adhesion and tendon healing, G is an immunohistochemical staining diagram of different groups, and H is a schematic diagram of the expression of Col I in the peritendinous tissue of different groups after 4 weeks of evaluation;

[0040] Figure 7Schematic diagram of the evaluation of Achilles tendon motor function after repair and the evaluation of histological characteristics in the early stage of tendon healing, wherein A is a schematic diagram of gait analysis of the simple suture group 4 weeks after tendon repair, B is a schematic diagram of gait analysis of the sheet scaffold group 4 weeks after tendon repair, C is a schematic diagram of gait analysis of the bionic tendon scaffold group 4 weeks after tendon repair, D is a schematic diagram of the comparison of gait analysis areas of different groups, E is a schematic diagram of the comparison of the maximum destructive force of different groups, F is a HE staining diagram of different groups after 7 days, G is a schematic diagram of the distribution of blood vessels in the tendon observed by CD31 immunofluorescence staining in different groups, HI is a morphological analysis of CD31-labeled neovascularization in and adjacent to the tendon sheath in different groups, and J is a HE staining and CD31 immunofluorescence staining diagram of the cross-section of the tendons in different groups.

[0041] Explanation of the accompanying figure numbers: 1. Directed topological structure, 2. Crossed fiber structure. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0043] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0044] Example 1

[0045] This embodiment provides a method for preparing a bionic tendon scaffold for preventing tendon adhesion and promoting non-scar healing, and the specific steps are as follows:

[0046] S1. Use 3D printing software to build a pre-printed path:

[0047] The printing path is as follows: the first 15 layers are radial fibers divided into 20 equal parts of the circumference, and the last 30 layers are 65° cross fibers, with a total of 50 layers printed.

[0048] The specific print path preview is as follows Figure 1 As shown, this path constructs a double-sided anisotropic structure with an inner layer of a directional topological structure 1 and an outer layer of a cross-fiber structure 2, which conforms to the characteristics of the Janus structure.

[0049] S2. Weigh an appropriate amount of biopolymer material polycaprolactone, add it into the barrel of the 3D printer, and wait for the next step of printing.

[0050] S3. The biopolymer material obtained in step S2 is printed using electrostatic direct writing technology to obtain a bionic tendon scaffold, and the printing parameters are: rotating shaft diameter 5 mm, electrostatic spacing 2.5 mm, voltage 3.9 kV, material temperature control 100° C., and air pressure 5 kPa.

[0051] like Figure 2 As shown, the total length of the bionic tendon scaffold is 50 mm, and the diameter of the bionic tendon scaffold is 5 mm. The diameter of a single fiber in the directional topological structure 1 and the cross-fiber structure 2 is 30 μm. The thickness of the directional topological structure 1 is 100 μm, and the thickness of the cross-fiber structure 2 is 200 μm.

[0052] Comparative Example 1

[0053] This embodiment provides a method for preparing a sheet-shaped stent, and the specific steps are as follows:

[0054] S1. Use 3D printing software to construct mutually perpendicular pre-printing paths, stacking 15 layers.

[0055] S2. Weigh an appropriate amount of biopolymer material polycaprolactone, add it into the barrel of the 3D printer, and wait for the next step of printing.

[0056] S3. Print the biopolymer material obtained in step S2 using electrostatic direct writing technology to obtain a sheet-shaped bracket, and the printing parameters are: rotating shaft diameter 5 mm, electrostatic spacing 2.5 mm, voltage 3.9 kV, material temperature control 100° C., and air pressure 5 kPa.

[0057] The morphological characteristics and local morphology of the bionic tendon scaffold prepared in Example 1 and the sheet scaffold prepared in Comparative Example 1 are as follows: Figure 2-4 As shown in FIG. 1 , the outer surface of the bionic tendon scaffold prepared in Example 1 is composed of interlocking PCL fibers forming a cross fiber structure 2, and the inner surface of the bionic tendon scaffold is composed of parallel longitudinal PCL fibers forming a directional topological structure 1. The original directional fiber morphology of the inner surface of the bionic tendon scaffold and the directional arrangement after the bionic tendon scaffold is unfolded are shown in FIG. Figure 4 B and 4C. As a control group, the sheet-like scaffold prepared in Comparative Example 1 is composed of oriented PCL fibers arranged in perpendicular directions to each other.

[0058] like Figure 4 D, the black arrows in the left image mark the interlaced PCL fibers in the outer surface of the biomimetic tendon scaffold, the orange arrows mark the oriented PCL fibers in the inner surface of the biomimetic tendon scaffold, and the right image shows the vertical fiber characteristics of the sheet scaffold. In contrast, the biomimetic tendon scaffold shows interlocking and longitudinally parallel PCL fibers in an optical microscope.

[0059] The microstructure of the bionic tendon scaffold was observed by SEM. Figure 4 As shown in E, the outer layer of the bionic tendon scaffold has relatively complete PCL fibers in an interlocking direction, while the interlocking PCL fibers in the inner layer of the bionic tendon scaffold have longitudinal PCL fibers scattered between them.

[0060] The bionic tendon scaffold obtained in Example 1 was implanted to conduct material science, in vitro cell experiments and in vivo tendon injury model repair experiments. The performance and potential of the bionic tendon scaffold in promoting non-scar healing of tendons were verified through in vitro cell experiments and in vivo animal experiments (see Model Figure 3 )

[0061] Experimental Example 1 Study on the influence of the bionic tendon scaffold provided by the present invention on the behavior pattern of tendon cells

[0062] This example provides an in vitro cell seeding experimental method, which is performed using the bionic tendon scaffold prepared in Example 1, and the specific steps are as follows:

[0063] Culture primary tendon cells, select cells of the 2nd to 3rd generation, and start cell seeding experiments when the cell fusion rate reaches 60% to 70%. 5 Pieces / cm 2 The cells were resuspended at a density of 1000 cells / mL and seeded on the inner and outer fiber network surfaces of the bionic tendon scaffold. After culturing for 7 days, the cells were ready for subsequent experiments.

[0064] like Figure 4 As shown in F, tenocytes were inoculated in the outer layer and cultured for 7 days and observed by SEM, showing that the tenocytes attached to the PCL fibers on the outer surface but did not penetrate the underlying fiber layer. In addition, the PCL fibers on the outer surface became thinner than before implantation, resulting in cell degradation and erosion on the PCL fibers, as marked by the black arrows, and the tenocytes were blocked by the interlocking network of the outer layer. Tenocytes were inoculated in the inner layer and cultured for 7 days and observed by SEM. Tenocytes were mainly recruited and attached to the surface of the longitudinal PCL fibers, and the overall morphology of the tenocytes was arranged along the longitudinal direction, as shown by the orange arrows.

[0065] Experimental Example 2 Study on the influence of bionic tendon scaffolds and sheet scaffolds on the behavior patterns of tendon cells

[0066] Tendon cells were inoculated on the bionic tendon scaffold obtained in Example 1 or the sheet scaffold prepared in Comparative Example 1 and cultured for 7 days.

[0067] like Figure 5As shown in A, tenocytes showed random and non-directional cell distribution on the culture plate without scaffold inoculation. After inoculation with the scaffold, tenocytes grew and attached along the PCL fibers and showed different directions. Then, the degree of orientation of tenocytes in different culture systems was analyzed and counted. Compared with the chaotic cell arrangement on the culture plate without scaffold inoculation, both the sheet scaffold and the bionic tendon scaffold allowed the tenocytes to appear in a more directional arrangement ( Figure 5 B).

[0068] Subsequently, cells were cultured on the inner surface of the sheet scaffold and the biomimetic tendon scaffold for different periods of time (5 days and 10 days) to observe the cell migration characteristics. Figure 5 As shown in C and 5D , tenocytes migrated and extended mainly along the longitudinal PCL fibers and then gradually moved toward the interlaced PCL fibers on both sides.

[0069] Experimental Example 3: In vitro shear simulation test of bionic tendon scaffold and sheet scaffold

[0070] Shear force within the tendon is the main loading mechanism for redistributing load. Cells within the tendon are mechanically sensitive to the shear stress caused by sliding of collagen fibers in the tendon, which can regulate the stiffness and balance of the tendon.

[0071] First, tendon cells were seeded in the inner layer of the bionic tendon scaffold and cultured for 10 days. Then, the bionic tendon scaffold with cell growth was placed in a shear stress device, using continuous medium flow to simulate a shear environment ( Figure 5 E).

[0072] To further clarify the effect of shear stress on tenocytes, the expression of genes such as Peizo1, ColI, and SCX, which respond to mechanical stress and are related to tendon regeneration, was also studied by immunofluorescence staining in this study. Figure 5 F. Compared with the unstimulated bionic tendon scaffold and the sheet scaffold, the fluorescence intensity of Peizo1, ColI and SCX increased after the bionic tendon scaffold was stimulated by shear force ( Figure 5 G).

[0073] Experimental Example 4 Evaluation of the recovery of tendon function after surgery on the bionic tendon scaffold provided by the present invention

[0074] This embodiment provides an in vivo tendon injury model repair experimental method, which is performed using the bionic tendon scaffold prepared in Example 1 and the sheet scaffold prepared in Comparative Example 1. The specific steps are as follows:

[0075] The Achilles tendon tissue of rats was exposed, the abdomen of the Achilles tendon was cut with a blade, the tendon sheath stumps on both sides of the tendon were cleaned and retained about 2 cm, and the modified Kessler method was used for in situ suture using 9-0 silk thread, and a simple bionic tendon scaffold (bionic tendon scaffold group) or a sheet scaffold (sheet scaffold group) was wrapped, and the incision was closed layer by layer. After 4 weeks of feeding under a standard feeding environment, subsequent in vivo testing experiments were performed.

[0076] The Achilles tendon tissue of rats was exposed, the abdomen of the Achilles tendon was cut with a blade, the tendon sheath stumps on both sides of the tendon were cleaned and about 2 cm was retained, and the incision was closed layer by layer by using the modified Kessler method with 9-0 silk thread. After 4 weeks of feeding under standard feeding conditions, subsequent in vivo detection experiments were performed (simple suture group).

[0077] like Figure 6 As shown in A, the ruptured tendon was sutured and then wrapped with a bionic tendon scaffold or a sheet scaffold. Four weeks after surgery, the adhesion of the tissue around the bionic tendon scaffold was significantly improved compared with the other groups. In contrast, denser fibrotic tissue was observed around the tendon tissue in the direct suture group and the sheet scaffold group ( Figure 6 B). The adhesion score of the bionic tendon scaffold was also much lower than that of the other two groups, and was statistically different from that of the control group ( Figure 6 C).

[0078] Hematoxylin and eosin (H&E) were then used to observe the adhesion on the tendon surface and the repair condition of the repair area. Figure 6 As shown in D, the results of tendon pathological section staining in the bionic tendon scaffold group showed less surface adhesion, while the results of the simple suture group showed more scar tissue deposition and invasion of the repair area. In addition, the corresponding adhesion histological evaluation showed that the adhesion score of the bionic tendon scaffold group was lower than that of the other two groups, with a statistically significant difference ( Figure 6 E), and the bionic tendon scaffold group showed higher healing scores ( Figure 6 F). Figure 6 G and H show that the deposition of type I collagen in the bionic tendon scaffold is more and statistically significant, which may be related to the fact that the oriented fibers in the bionic tendon scaffold promote the dispersion and migration of local cells. In contrast, the scores of the simple suture group and the sheet scaffold group were lower, and the deposition of type I collagen on the fibers was also less. In addition, the expression of type I and type III collagen in different groups was analyzed by immunofluorescence staining, among which the percentage of type I collagen expression in the bionic tendon scaffold was significantly higher than that in the other two groups. The type I collagen fibers in the bionic tendon scaffold group were arranged in a dense parallel array and contained almost no type III collagen.

[0079] Experimental Example 5 Evaluation of Achilles tendon motor function after repair of the bionic tendon scaffold provided by the present invention

[0080] In this study, the Catwalk gait analysis system was used to evaluate the postoperative biomechanical function of the repaired tendon. Figure 7 AC shows that the bionic tendon scaffold group has a larger footprint attachment area and better foot shape display. The footprint attachment area of ​​the bionic tendon scaffold group is significantly larger than that of the sheet scaffold group and the simple suture group ( Figure 7 D). This indicates that the bionic tendon scaffold can promote the recovery of lower limb function after tendon repair in rats.

[0081] like Figure 7 As shown in E, the maximum rupture force of the bionic tendon scaffold group in the biomechanical test was greater than that of the other two groups ( Figure 7 E).

[0082] Experimental Example 6 Evaluation of the histological characteristics of the early stage of tendon healing of the bionic tendon scaffold provided by the present invention

[0083] To further determine the effects of different types of patches on collagen production at the tendon healing site, immunofluorescence staining was performed on sections of the tendon injury site 7 days after surgery. Figure 7 F and Figure 7 G shows that the adhesion between the tendon and the scaffold was less in the cross-sectional and longitudinal sections of the bionic tendon scaffold group compared with the simple suture group.

[0084] Furthermore, in the cross-section of the bionic tendon scaffold, a small gap was shown around the tendon and the scaffold, and in the longitudinal section of the bionic tendon scaffold, less attachment was also shown around the tendon and the scaffold.

[0085] There are many angiogenesis processes in the bionic tendon scaffold. CD31 immunofluorescence staining was used to observe and analyze the angiogenesis markers, such as Figure 7 HJ. Finally, quantitative analysis of angiogenesis also showed that the biomimetic tendon scaffold could induce early angiogenesis, which almost existed in the internal scaffold and did not overgrow into the tendon tissue. The results showed that the biomimetic tendon scaffold was able to support the tissue space for new blood vessel formation and avoid blood vessels growing directly into the tendon.

[0086] The bionic tendon scaffold obtained in Example 1 was implanted to carry out material science, in vitro cell experiments and in vivo tendon injury model repair experiments. Through the repair effect of the bionic tendon scaffold on the damaged tendon tissue, the inner layer structure can achieve rearrangement and induced regeneration of tendon cells under the advantage of the structural heterogeneity of Janus, and can activate the tendon formation performance under the condition of shear force generated when simulating normal tendon sliding in vitro; the outer layer staggered fiber structure can effectively block the invasion of scar fibers, avoid excessive scar fiber deposition, and induce new capillaries to grow into the scaffold to achieve vascular nutrient enrichment, and finally achieve non-scar healing of the tendon.

[0087] The present invention successfully prepared for the first time a bionic tendon scaffold with an anisotropic structure, which is used to induce tendon repair and prevent scar tissue from infiltrating during the repair process. Under shear force simulation, the internal directional topographic structure can enhance the biological behavior of tendon cells, including adhesion, migration and orientation. In addition, the outer layer of staggered fibers can prevent scar formation and allow vascular tissue to infiltrate, providing a local angiogenesis environment. Therefore, the research of the present invention strongly proves that the bionic tendon scaffold provided by the present invention is an innovative new method for tendon repair. This method focuses on regenerating the structural anisotropic Janus characteristic advantages between the inner and outer double membranes, which is a new strategy in this field.

[0088] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A double-layer Janus fiber casing, characterized in that: The double-layer Janus fiber sleeve consists of a directional topological structure (1) and a cross-fiber structure (2); The directional topological structure (1) is composed of a plurality of longitudinally parallel fibers forming a hollow cylindrical structure, and the cross-fiber structure (2) is composed of a plurality of mutually interlaced fibers forming a hollow cylindrical structure; The directional topological structure (1) is covered with a cross-fiber structure (2) on the outside, and the directional topological structure (1) and the cross-fiber structure (2) are connected.

2. A double-layer Janus fiber sleeve according to claim 1, characterized in that: The length of the double-layer Janus fiber sleeve is 40-50 mm, and the diameter of the double-layer Janus fiber sleeve is 3-5 mm.

3. A double-layer Janus fiber sleeve according to claim 1, characterized in that: The diameter of a single fiber in the oriented topological structure (1) and the cross-fiber structure (2) is 15 to 50 μm; The thickness of the oriented topological structure (1) is 100 to 200 μm, and the thickness of the cross-fiber structure (2) is 200 to 300 μm.

4. A double-layer Janus fiber sleeve according to claim 1, characterized in that: The cross-fiber structure (2) is a grid-like structure formed by interlacing a plurality of fibers, and the angle between two intersecting fibers is in the range of 60° to 70°.

5. A double-layer Janus fiber sleeve according to claim 4, characterized in that: The pore size of the grid structure is 50-100 μm.

6. A method for preparing a double-layer Janus fiber sleeve as claimed in any one of claims 1 to 3, characterized in that: The specific steps are as follows: S1. Use 3D printing software to build a pre-printing path; S2, weigh an appropriate amount of biopolymer material and wait for the next step of printing; S3. Print the biopolymer material obtained in step S2 using electrostatic direct writing technology to obtain a double-layer Janus fiber sleeve.

7. The method for preparing a double-layer Janus fiber sleeve according to claim 6, characterized in that: In step S1, the pre-printing path is as follows: The first 15 to 20 layers are radial fibers that divide the circumference into 20 to 30 equal parts, and the last 30 to 40 layers are cross fibers, with the angle between the two intersecting fibers ranging from 60° to 70°.

8. The method for preparing a double-layer Janus fiber sleeve according to claim 6, characterized in that: In step S2, the biopolymer material is selected from any one or more of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer or polylactic acid-caprolactone copolymer; In step S3, the printing parameters are: rotating shaft diameter 3-7 mm, electrostatic spacing 2-3 mm, voltage 3-5 kV, material temperature control 90-110° C., and air pressure 4-6 kPa.

9. Application of a double-layer Janus fiber casing, characterized in that: Use of the double-layer Janus fiber sleeve obtained by the preparation method as described in any one of claims 6 to 8 in preparing a repair material for treating tendon tissue defects.

10. The use of a double-layer Janus fiber sleeve according to claim 9, characterized in that: The application is to prepare an implant device for treating tendon tissue defects.

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