Preparation method of an integrated gradual bionic gradient structure rotator cuff patch

Abstract

The present invention belongs to the field of medical materials technology and relates to a method for preparing a rotator cuff patch with an integrated, gradual biomimetic gradient structure. The present invention utilizes electrically assisted solution blow-spinning technology and thermal nanoimprinting technology to prepare a rotator cuff patch with an integrated, gradual biomimetic gradient structure. The patch has a composite layer structure, with each transition layer and adjacent micro-nanofiber membrane layers forming a continuous, unbroken structure. The internal structure and composition transitions evenly, without discrete stratification. The patch as a whole exhibits a continuous, gradual biomimetic gradient structure, successfully simulating the histological gradient characteristics of the tendon-bone transition region and effectively promoting rapid healing of the tendon-bone interface.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials and relates to a method for preparing an integrated gradually changing bionic gradient structure rotator cuff patch. Background Art

[0002] Rotator cuff tears are a common tendon injury in orthopedic surgery, with a prevalence of 15%-51%. They are most common in middle-aged and elderly individuals and those who engage in repetitive upper limb activities. Tear depth is categorized as partial tears (which do not penetrate the full thickness, resulting in decreased tendon thickness and strength) or full-thickness tears (complete rupture of the tendon). The tendon-bone interface is the primary site of injury.

[0003] The tendon-bone interface, the junction between tendon and bone, is approximately 1 mm thick and consists of four closely connected gradient zones. From tendon to bone, the following order is followed: Zone I is the tendon zone, composed primarily of highly oriented fibroblast-like tenocytes, with an extracellular matrix dominated by type I collagen. Zones II (unmineralized fibrocartilage) and III (mineralized fibrocartilage) together form the fibrocartilage zone, serving as the mineralization front and the interface between soft and hard tissues. Cells in this zone are fibrocartilage cells, whose morphology becomes hypertrophic with increasing mineralization. The extracellular matrix is rich in type II collagen, numerous proteoglycans, and leucine-rich proteins. Zone IV is the bone zone, composed of osteoblasts, osteocytes, and osteoclasts. The extracellular matrix is rich in type I collagen and contains significant calcium deposits. This gradient structure allows the tendon-bone interface to act as an "energy disperser" to avoid stress concentration at the tendon-bone junction. However, the complexity of this gradient structure makes repair of this interface extremely difficult.

[0004] Currently, the repair of rotator cuff tears mainly focuses on strengthening the physical connection between tendons and bones, and insufficient attention is paid to the reconstruction of the gradient structure of the tendon-bone interface. Clinical repair strategies include conservative treatment and surgical treatment: Conservative treatment relieves symptoms through immobilization, medication and physical therapy, but has problems such as long treatment course, easy delay in treatment, and inability to cure; surgical treatment covers two techniques: arthroscopic suture repair and patch enhancement implantation. Although suture repair is simple to operate, it has a high re-tear rate and is only suitable for small and medium-sized tears; although patch enhancement implantation can partially replace the function of the rotator cuff, due to the single structure of the existing patch, it cannot guide gradient reconstruction, resulting in the formation of scar tissue with poor mechanical properties at the repair site, lacking the stress buffering function of the natural interface, and prone to micro-damage or even rupture in the long term. Therefore, achieving gradient structure reconstruction of the tendon-bone interface is the key to solving postoperative recurrence and functional recovery.

[0005] In the research of artificial biomimetic patch implants, nanofiber membranes fabricated by electrospinning technology have shown significant potential for mimicking and constructing gradients at the tendon-bone interface due to their high controllability, high porosity, high specific surface area, and extracellular matrix-like structure. However, this technology has also exposed several challenges in practical applications. For example, during the preparation process, the difficulty of stacking the thickness of nanofiber membranes directly affects the quality and performance of integrated molding. Therefore, it is often necessary to stack multiple nanofiber membranes to achieve a gradient structure with sufficient thickness. However, the limited gradient simulation and poor integrity caused by the discontinuous rather than gradual transitions between the multiple layers of gradient structures may lead to reduced functionality, stability, and durability in vivo. In addition, low production efficiency is also a significant drawback, which greatly limits its large-scale production and clinical application. In addition to electrospinning technology, air-jet spinning, 3D printing, and integration with traditional textile technologies have to some extent overcome the thickness stacking problem faced by electrospinning and have achieved some success in improving mechanical properties, but these technologies still have limitations. If the technical accuracy is low, it is difficult to achieve accurate construction of the microstructure, which is a major drawback for the requirement of highly accurate simulation of the tendon-bone interface gradient.

[0006] Therefore, for artificial bionic patches, achieving effective and accurate tendon-bone interface gradient simulation and construction remains a difficult problem that needs to be solved urgently. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems existing in the prior art and provide a method for preparing an integrated gradually changing bionic gradient structure rotator cuff patch.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing an integrated, gradually changing, biomimetic, gradient-structured rotator cuff patch comprises: using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nanofiber membrane layer, a first transition layer, a second micro-nanofiber membrane layer, a second transition layer, a third micro-nanofiber membrane layer, a third transition layer, and a fourth micro-nanofiber membrane layer on a receiving substrate; and then using a heat-treatment nanoimprinting technique to form protrusions of an osteogenic material on the fourth micro-nanofiber membrane layer.

[0010] When preparing the i-th micro-nano fiber membrane layer, the corresponding i-th spinning solution is injected at a constant injection speed v i Push injection, i=1,2,3; when preparing the i+1th micro-nano fiber membrane layer, the corresponding i+1th spinning solution is injected at a constant speed v i+1 The process of preparing the i-th transition layer is as follows: simultaneously injecting the i-th spinning solution and the i+1-th spinning solution, and the injection speed of the i-th spinning solution is determined by v iThe speed is uniformly reduced to 0, and the injection speed of the i+1 spinning solution is uniformly increased from 0 to v i+1 , the sum of the injection speed of the i-th spinning solution and the injection speed of the i+1-th spinning solution at each moment remains unchanged; if the process of preparing the i-th transition layer is: injecting the i-th spinning solution and the i+1-th spinning solution at the same time, the injection speed of the i-th spinning solution is determined by v i The gradient drops to 0 (i.e., it first drops to a value and maintains for a certain time, then continues to drop to a value and maintains for a certain time...), and the injection speed of the i+1 spinning solution increases from 0 to v i+1 , the sum of the injection speeds of the i-th spinning solution and the i+1-th spinning solution at each moment remains unchanged. It is impossible to ensure that each transition layer and the two adjacent micro-nano fiber membrane layers constitute a continuous and unbroken structure, and the internal structure and composition changes are uniformly transitioned without discrete stratification. This is because the transition layer prepared in this way is formed by superimposing multiple subdivided layers, and there is still a broken transition between layers in essence.

[0011] The first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrocartilage; the fourth micro-nano fiber membrane layer is used to promote bone regeneration; the osteogenic material protrusions are cylindrical, with a diameter of 100-2500nm and a height of 5-2500nm. All the osteogenic material protrusions are arranged in a lattice shape, and the distance between two adjacent osteogenic material protrusions is 100-2000nm. The osteogenic material protrusions are used to further enhance the mineralization of the proximal bone side of the patch, simulate the microenvironment of natural bone tissue, enhance cell adhesion and proliferation, and promote bone regeneration and bone integration; the first micro-nano fiber membrane layer The bioperformance design is used to simulate the tendon tissue in the rotator cuff tendon-bone interface, and its fiber structure and material composition are designed to provide biomechanical properties similar to those of tendons; the bioperformance design of the second micro-nano fiber membrane layer is used to simulate the unmineralized fibrocartilage tissue in the rotator cuff tendon-bone interface, and its structural characteristics and osteogenic material concentration are designed to simulate the bioactivity of unmineralized cartilage; the bioperformance design of the third micro-nano fiber membrane layer is used to simulate the mineralized fibrocartilage tissue in the rotator cuff tendon-bone interface, and its fiber arrangement and material composition are designed to reproduce the hardness of mineralized cartilage and biological micro-nano fiber membrane; the bioperformance design of the fourth micro-nano fiber membrane layer and the osteogenic material protrusion is used to simulate the bone tissue in the rotator cuff tendon-bone interface, and its structure and osteogenic material concentration are designed to promote the growth and integration of bone tissue;

[0012] Each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition changes are uniform and transitional without showing discrete stratification.

[0013] The integrated gradual bionic gradient structure rotator cuff patch is a three-dimensional structure, and the overall structure is one or more of a cube, a cuboid, a cylinder and a polygon;

[0014] Compared with conventional patches with a single uniform structure, the multi-layer composite structure of the patch of the present invention exhibits more refined biomechanical compatibility and biodegradability in promoting tendon-bone healing and tissue regeneration, and significantly reduces the incidence of secondary tears.

[0015] Electric-assisted solution blowing spinning technology combines the advantages of air-jet spinning technology and electrospinning technology. It has the advantages of simple process, high efficiency and controllability, and wide applicability. It can achieve precise control of micro-nano-scale fibers while greatly improving production efficiency, and can achieve material accumulation of millimeter-level thickness.

[0016] An integrated gradient bionic gradient rotator cuff patch was prepared using electrically assisted solution blowing technology, achieving integrated molding. The patch as a whole presents a continuous gradient biomimetic gradient structure: in the transverse dimension, each micro-nanofiber membrane layer maintains the uniformity of fiber morphological parameters and chemical composition; in the longitudinal dimension, it exhibits a gradual change in structural parameters such as fiber diameter, orientation, porosity, and a concentration gradient change of the bone-promoting material, successfully simulating the histological gradient characteristics of the tendon-bone transition area, and ultimately achieving a biological functional simulation of the rotator cuff tendon-bone interface.

[0017] As the preferred technical solution:

[0018] As described above, in the preparation method of an integrated gradual bionic gradient structure rotator cuff patch, the average pore size of the first micro-nano fiber membrane layer is 10-50 μm to allow and facilitate the infiltration of tendon fibroblasts, the porosity is 70-85% to ensure uniform distribution of cells and nutrients, the orientation degree of the fiber arrangement is >80% to simulate the collagen arrangement of natural tendons, the fiber composition is a mixture of natural polymer materials and synthetic polymer materials to simulate the biological properties of tendon tissue, and the content of synthetic polymer materials is 10-90wt%.

[0019] As described above, a method for preparing an integrated gradual biomimetic gradient structure rotator cuff patch has an average pore size of 30-80 μm in the second micro-nano fiber membrane layer to promote the retention of chondrocytes / stem cells. Specifically, the smaller pore size and limited infiltration space can induce chondrogenesis of MSCs, thereby leading to the formation of cartilage tissue rather than bone tissue. The porosity is 80-90%, the orientation degree of fiber arrangement is 50-60% to form an anisotropic structure, and the fiber component is a synthetic polymer material.

[0020] As described above, in a method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the average pore size of the third micro-nano fiber membrane layer is 100-300 μm to facilitate vascular invasion and mineralization, the porosity is 85-95%, the orientation degree of fiber arrangement is greater than 20% and less than 30% to form a porous scaffold, and the fiber component is a mixture of synthetic polymer materials and osteogenic materials, and the content of osteogenic materials is 1-2wt%.

[0021] As described above, in the preparation method of an integrated gradual bionic gradient structure rotator cuff patch, the average pore size of the fourth micro-nano fiber membrane layer is 200-500 μm, which meets the requirements of bone tissue engineering, the porosity is >90%, the fibers are arranged in a completely random orientation, and the fiber composition is a mixture of natural polymer materials, synthetic polymer materials and osteogenic materials, the content of synthetic polymer materials is 10-90wt%, and the content of osteogenic materials is 2-5wt%.

[0022] The method for preparing the integrated gradual biomimetic gradient structure rotator cuff patch as described above, wherein the natural polymer material is one or more of type I collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, alginate, cellulose and derivatives thereof;

[0023] The synthetic polymer material is one or more of polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), polycaprolactone (PCL), polydioxanone (PDO), polylactic acid-co-caprolactone (PLCL), and polyethylene glycol (PEG). When the synthetic polymer material is PCL, it will melt after the hot nanoimprinting treatment to enhance the mechanical properties.

[0024] Osteogenic materials include but are not limited to osteogenic active drugs, osteogenic active growth factors, and inorganic osteogenic materials, such as one or more of nanohydroxyapatite, struvite nanowires, bioactive glass, and tricalcium phosphate;

[0025] The thickness of each micro-nano fiber membrane layer ranges from 0.1 to 1 mm, and the total thickness of the integrated gradual bionic gradient structure rotator cuff patch is from 0.7 to 6 mm.

[0026] The method for preparing the integrated gradual bionic gradient structure rotator cuff patch described above uses the following specific steps to form the bone-promoting material protrusions on the fourth micro-nano fiber membrane layer using the heat treatment nanoimprinting technology:

[0027] (a) Pour a photosensitive resin onto the nanopattern master and evenly spread it with a roller until it completely covers the surface of the nanopattern master. Then, cure the photosensitive resin (first using a UV light source to cure the photosensitive resin, then heating it in an oven for post-curing). After curing, a resin mold is formed on the nanopattern master. The nanopattern master and the resin mold are then separated.

[0028] (b) Electron beam evaporation technology is used to deposit osteogenic material on the surface of the resin mold;

[0029] (c) plasma treating the resin mold with the osteogenic material deposited on its surface to reduce the width of the nanopattern, thereby reducing the interfacial adhesion between the resin mold and the osteogenic material. Simultaneously, plasma treating the fourth micro-nanofiber membrane layer to promote the formation of hydroxyl groups and increase the surface energy, thereby enhancing the adhesion of the fourth micro-nanofiber membrane layer.

[0030] (d) Pressing the resin mold with the osteogenic material deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material contacts the fourth micro-nano fiber membrane layer, performing heat treatment, and removing the resin mold after the heat treatment is completed.

[0031] Thermal nanoimprinting combines a heat treatment process with a mold pattern. First, the polymer material is thermally treated to soften it. Then, under applied pressure, the nanopattern is precisely transferred while maintaining the structural integrity of the material itself. This high-precision micro-nano patterning technique not only enhances the mechanical properties of the material, but also precisely controls the micro-nanostructure of the material surface, enabling functional modification.

[0032] As described above, the method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the process parameters of the electrically assisted solution blowing spinning include: positive voltage of 10-18 kV, negative voltage of 1-3 kV, injection speed of 1-10 mL / h, air pressure of 1-10 atm, and receiving distance of 10-20 cm.

[0033] Beneficial effects:

[0034] (1) The integrated gradient bionic gradient structure rotator cuff patch prepared by the present invention has a composite layer structure, and each transition layer and the adjacent micro-nano fiber membrane layer form a continuous and unbroken structure. The internal structure and composition changes are uniformly transitioned and do not present discrete stratification. The patch as a whole presents a continuous gradient bionic gradient structure, with gradient-changing mechanical properties and tissue induction properties. It can induce the adhesion, proliferation and differentiation of tendon tissue and bone tissue in different functional layers of the patch, successfully simulate the histological gradient characteristics of the tendon-bone transition area, and realize the biological functional simulation of the rotator cuff tendon-bone interface. Compared with conventional patches with a single uniform structure, the integrated gradient bionic gradient structure rotator cuff patch prepared by the present invention can effectively promote the rapid healing of the tendon-bone interface and significantly reduce the incidence of secondary tears.

[0035] (2) The electrically assisted solution blowing spinning technology used in the present invention combines the advantages of air-jet spinning and electrospinning, and has the characteristics of simple process, high efficiency and controllability, and wide applicability. This technology not only has a continuous and stable spinning process and high production capacity, but also can accurately control the morphology and size of micro-nano-scale fibers. At the same time, due to the synergistic effect of air flow force and electric field force, this technology can handle high viscosity and high concentration spinning precursor solution, achieve efficient accumulation of materials with millimeter thickness, and significantly improve production efficiency. In addition, this technology was used to prepare an integrated gradient bionic gradient structure rotator cuff patch, realizing the integrated molding of the patch.

[0036] (3) The heat treatment nanoimprinting technology used in the present invention combines heat treatment technology, electron beam evaporation technology, plasma surface modification technology and mold patterning to achieve the precise inlay and transfer of the nanopattern of the bone-promoting material on the surface of the nanoimprint template to the surface of the fourth micro-nanofiber membrane layer of the patch, while maintaining the integrity of the material's main structure. This high-precision micro-nano patterning technology not only enhances the mechanical properties of the patch, but also accurately controls the micro-nanostructure of the material surface, further enhancing the mineralization of the bone regeneration-promoting functional layer and achieving functional modification to promote bone healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the cross-sectional SEM of the integrated gradual bionic gradient structure rotator cuff patch prepared in Example 1. DETAILED DESCRIPTION

[0038] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0039] The following are the test methods for the relevant performance indicators in each embodiment:

[0040] Pore size and porosity: A mercury intrusion instrument is used to test the pore size and porosity of the patch. A sample of at least 0.1 g is cut into pieces and placed in a low-pressure chamber to evacuate the residual gas. The mercury intrusion pressure is then gradually increased through a hydraulic system. The volume increase of mercury infiltrating the sample at different pressures is simultaneously recorded. The pore size and porosity data are obtained using the test software provided by the computer.

[0041] Orientation of fiber arrangement: High-resolution two-dimensional images of the samples were obtained using a scanning electron microscope. The fiber orientation angles were then extracted and the fiber orientation distribution was statistically analyzed using Image J software.

[0042] Peel strength: To characterize the continuity and unbroken structure of the patch's transition layers and the two adjacent micro-nanofiber membrane layers, the bonding strength between the layers of the sample was tested at room temperature using a multifunctional strength tester according to a variation of the ASTM D1876-72 (1983) T-type peel test. The patch was cut into a 20 mm x 10 mm rectangle, and the initial gauge of the instrument was adjusted according to the actual thickness of the patch. The upper and lower surfaces of one end of the patch were fixed to the lower surface of the upper sensor and the upper surface of the lower rigid material platform, respectively, using strong glue. The peel test was conducted using a 100 N sensor, a tensile speed of 10 mm / min, and a pre-tension of 0.1 N.

[0043] Energy dispersive spectroscopy analysis: Energy dispersive spectroscopy (EDS) analysis was performed on each layer of the sample using a field emission transmission electron microscope at an accelerating voltage of 200 kV.

[0044] The source information of the relevant substances in the following examples is shown in Table 1:

[0045] Table 1

[0046]

[0047] Example 1

[0048] A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0049] (1) Preparation of materials;

[0050] The first spinning solution: the solute is a mixture of type I collagen and PDLLA, the PDLLA content in the solute is 10 wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid in a volume ratio of 5:5, with a concentration of 100 mg / mL;

[0051] The second spinning solution: the solute is PDLLA, the solvent is hexafluoroisopropanol, and the concentration is 110 mg / mL;

[0052] The third spinning solution: the solute is a mixture of PDLLA and osteogenic material a, the content of osteogenic material a in the solute is 1wt%, and the solvent is hexafluoroisopropanol, with a concentration of 120mg / mL;

[0053] The fourth spinning solution: the solute is a mixture of type I collagen, PDLLA, and osteogenic material b, the content of PDLLA in the solute is 10 wt %, the content of osteogenic material b in the solute is 2 wt %, and the solvent is a mixture of hexafluoroisopropanol and acetic acid in a volume ratio of 5:5, with a concentration of 130 mg / mL;

[0054] Osteogenic material a, osteogenic material b and osteogenic material c: all are struvite nanowires;

[0055] (2) using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0056] When preparing the first micro-nanofiber membrane layer, the first spinning solution is injected at a constant injection speed v1. The process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, v1 of 1 mL / h, air pressure of 1 atm, and receiving distance of 10 cm. The first micro-nanofiber membrane layer has a thickness of 0.1 mm, an average pore size of 10 μm, a porosity of 70%, and a fiber orientation of 80%. The first micro-nanofiber membrane layer is used to guide the directional alignment of tendon fibroblasts and promote tendon differentiation and regeneration.

[0057] When preparing the first transition layer, the first spinning solution and the second spinning solution are injected simultaneously. The injection speed of the first spinning solution is uniformly reduced from v1 to 0, and the injection speed of the second spinning solution is uniformly increased from 0 to v2. The sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remains unchanged. The process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, v2 of 1 mL / h, injection time of 1 hour, air pressure of 1 atm, and receiving distance of 10 cm.

[0058] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v2; the process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, air pressure of 1 atm, and receiving distance of 10 cm; the thickness of the second micro-nano fiber membrane layer is 0.1 mm, the average pore size is 30 μm, the porosity is 80%, and the orientation degree of fiber arrangement is 53%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage;

[0059] When preparing the second transition layer, the second spinning solution and the third spinning solution are injected simultaneously. The injection speed of the second spinning solution is uniformly reduced from v2 to 0, and then uniformly increased from 0 to v3. The sum of the injection speeds of the second spinning solution and the third spinning solution at each moment remains unchanged. The process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, v3 of 1 mL / h, injection time of 1 hour, air pressure of 1 atm, and receiving distance of 10 cm.

[0060] When preparing the third micro-nanofiber membrane layer, the third spinning solution is injected at a constant injection speed v3; the process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, air pressure of 1 atm, and receiving distance of 10 cm; the thickness of the third micro-nanofiber membrane layer is 0.1 mm, the average pore size is 100 μm, the porosity is 88%, and the orientation degree of fiber arrangement is 23%; the third micro-nanofiber membrane layer is used to induce differentiation and regeneration of mineralized fibrocartilage;

[0061] When preparing the third transition layer, the third and fourth spinning solutions were injected simultaneously. The injection speed of the third spinning solution was uniformly decreased from v3 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v4. The sum of the injection speeds of the third and fourth spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 10 kV, negative voltage of 1 kV, v4 of 1 mL / h, injection time of 1 hour, air pressure of 1 atm, and receiving distance of 10 cm.

[0062] When preparing the fourth micro-nano fiber membrane layer, the fourth spinning solution is injected at a constant injection speed v4; the process parameters include: positive voltage of 10 kV, negative voltage of 1 kV, air pressure of 1 atm, and receiving distance of 10 cm; the fourth micro-nano fiber membrane layer has a thickness of 0.1 mm, an average pore size of 200 μm, a porosity of 90%, and completely randomly oriented fibers; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0063] (3) forming protrusions of osteogenic material c on the fourth micro-nanofiber membrane layer using heat treatment nanoimprinting technology;

[0064] (3.1) Pour a photosensitive resin onto the nano-patterned master and evenly spread it with a roller until it completely covers the surface of the nano-patterned master. Then, cure the photosensitive resin to form a resin mold on the nano-patterned master. Separate the nano-patterned master from the resin mold.

[0065] (3.2) Depositing osteogenic material c on the surface of the resin mold using electron beam evaporation technology;

[0066] (3.3) Plasma treatment is performed on the resin mold with the osteogenic material c deposited on the surface, and the fourth micro-nanofiber membrane layer is also plasma treated;

[0067] (3.4) Pressing a resin mold with osteogenic material c deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material c contacts the fourth micro-nano fiber membrane layer, and then performing heat treatment. After the heat treatment, the resin mold is removed to obtain an integrated gradual bionic gradient structure rotator cuff patch.

[0068] The resulting integrated, gradient-bionic gradient rotator cuff patch has a total thickness of 0.7 mm. The osteogenic material c protrusions on the fourth micro-nanofiber membrane layer are cylindrical, 100 nm in diameter, and 5 nm in height. All of the osteogenic material c protrusions are arranged in a lattice pattern, with a spacing of 100 nm between adjacent osteogenic material c protrusions. Each transition layer and its two adjacent micro-nanofiber membrane layers form a continuous, unbroken structure, with uniform transitions in internal structure and composition without discrete stratification (see cross-sectional SEM image of the integrated, gradient-bionic gradient rotator cuff patch). Figure 1 shown);

[0069] The peel strength of the integrated bionic gradient structure rotator cuff patch is 48.3 mN / mm. The energy dispersion spectrum analysis of each layer of the integrated bionic gradient structure rotator cuff patch is shown in Table 2:

[0070] Table 2

[0071]

[0072] As can be seen from Table 2, the first micro-nanofiber membrane layer is dominated by type I collagen and has a high N content, and PDLLA slightly increases the C ratio; the PDLLA content in the first transition layer increases, C increases, and N decreases; the solute in the second micro-nanofiber membrane layer contains only PDLLA, and the C and O ratios are close to the theoretical values; due to the introduction of Mg, P, and N signals by trace struvite nanowires in the second transition layer, PDLLA still dominates C and O; the content of struvite nanowires (osteogenic material a) in the third micro-nanofiber membrane layer increases, the Mg and P signals are significantly enhanced, and the C ratio decreases; the increase in type I collagen in the third transition layer enhances N, and the increase in struvite nanowires (osteogenic material b) leads to increased Mg and P; the fourth micro-nanofiber membrane layer has the highest proportion of type I collagen, a significant increase in N, and struvite nanowires (osteogenic material c) continuously provide Mg and P signals.

[0073] Example 2

[0074] A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0075] (1) Preparation of materials;

[0076] The first spinning solution: the solute is a mixture of gelatin and PLGA, the PLGA content in the solute is 30wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid in a volume ratio of 7:3, with a concentration of 125mg / mL;

[0077] The second spinning solution: the solute is PLGA, the solvent is hexafluoroisopropanol, and the concentration is 135 mg / mL; the third spinning solution: the solute is a mixture of PLGA and osteogenic material a, the content of osteogenic material a in the solute is 1 wt%, and the solvent is hexafluoroisopropanol, and the concentration is 145 mg / mL;

[0078] The fourth spinning solution: the solute is a mixture of gelatin, PLGA and osteogenic material b, the content of PLGA in the solute is 30wt%, the content of osteogenic material b in the solute is 2wt%, and the solvent is hexafluoroisopropanol and acetic acid in a volume ratio of 7:3;

[0079] mixture at a concentration of 155 mg / mL;

[0080] Osteogenic material a, osteogenic material b, and osteogenic material c: all are nanohydroxyapatite;

[0081] (2) using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0082] When preparing the first micro-nanofiber membrane layer, the first spinning solution was injected at a constant injection speed v1. The process parameters included: positive voltage of 12 kV, negative voltage of 1 kV, v1 of 3 mL / h, air pressure of 3 atm, and receiving distance of 13 cm. The first micro-nanofiber membrane layer had a thickness of 0.3 mm, an average pore size of 23 μm, a porosity of 75%, and a fiber orientation of 83%. The first micro-nanofiber membrane layer was used to guide the directional alignment of tendon fibroblasts, promoting tendon differentiation and regeneration.

[0083] When preparing the first transition layer, the first spinning solution and the second spinning solution were injected simultaneously. The injection speed of the first spinning solution was uniformly decreased from v1 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v2. The sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remained unchanged. The process parameters included: positive voltage of 12 kV, negative voltage of 1 kV, v2 of 3 mL / h, injection time of 1 hour, air pressure of 3 atm, and receiving distance of 13 cm.

[0084] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v2; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm; the second micro-nano fiber membrane layer has a thickness of 0.3 mm, an average pore size of 45 μm, a porosity of 82%, and a fiber orientation degree of 60%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage;

[0085] When preparing the second transition layer, the second spinning solution and the third spinning solution were injected simultaneously. The injection speed of the second spinning solution was uniformly decreased from v2 to 0, and then uniformly increased from 0 to v3. The sum of the injection speeds of the second and third spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 12 kV, negative voltage of 1 kV, v3 of 3 mL / h, injection time of 1 hour, air pressure of 3 atm, and receiving distance of 13 cm.

[0086] When preparing the third micro-nanofiber membrane layer, the third spinning solution was injected at a constant injection speed v3; the process parameters included: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm; the third micro-nanofiber membrane layer had a thickness of 0.3 mm, an average pore size of 135 μm, a porosity of 85%, and a fiber orientation of 30%; the third micro-nanofiber membrane layer was used to induce differentiation and regeneration of mineralized fibrocartilage;

[0087] When preparing the third transition layer, the third and fourth spinning solutions were injected simultaneously. The injection speed of the third spinning solution was uniformly decreased from v3 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v4. The sum of the injection speeds of the third and fourth spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 12 kV, negative voltage of 1 kV, v4 of 3 mL / h, injection time of 1 hour, air pressure of 3 atm, and receiving distance of 13 cm.

[0088] When preparing the fourth micro-nanofiber membrane layer, the fourth spinning solution is injected at a constant injection speed v4. The process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm. The fourth micro-nanofiber membrane layer has a thickness of 0.3 mm, an average pore size of 271 μm, a porosity of 91%, and completely randomly oriented fibers. The fourth micro-nanofiber membrane layer is used to promote bone regeneration.

[0089] (3) forming protrusions of osteogenic material c on the fourth micro-nanofiber membrane layer using heat treatment nanoimprinting technology;

[0090] (3.1) Pour a photosensitive resin onto the nano-patterned master and evenly spread it with a roller until it completely covers the surface of the nano-patterned master. Then, cure the photosensitive resin to form a resin mold on the nano-patterned master. Separate the nano-patterned master from the resin mold.

[0091] (3.2) Depositing osteogenic material c on the surface of the resin mold using electron beam evaporation technology;

[0092] (3.3) Plasma treatment is performed on the resin mold with the osteogenic material c deposited on the surface, and the fourth micro-nanofiber membrane layer is also plasma treated;

[0093] (3.4) Pressing a resin mold with osteogenic material c deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material c contacts the fourth micro-nano fiber membrane layer, and then performing heat treatment. After the heat treatment, the resin mold is removed to obtain an integrated gradual bionic gradient structure rotator cuff patch.

[0094] The resulting integrated, biomimetic, gradient-structured rotator cuff patch has a total thickness of 2.1 mm. The osteogenic material c projections on the fourth micro-nanofiber membrane layer are cylindrical, 500 nm in diameter, and 100 nm in height. All of the osteogenic material c projections are arranged in a lattice pattern, with a spacing of 500 nm between adjacent ones. Each transition layer and its two adjacent micro-nanofiber membrane layers form a continuous, unbroken structure, with uniform transitions in internal structure and composition, without discrete layers.

[0095] The peel strength of the integrated bionic gradient structure rotator cuff patch is 51.4 mN / mm. The energy dispersion spectrum analysis of each layer of the integrated bionic gradient structure rotator cuff patch is shown in Table 3:

[0096] Table 3

[0097]

[0098] As can be seen from Table 3, in the first micro-nanofiber membrane layer, gelatin as the main component contributes N element, and the presence of PLGA increases the proportion of C element; in the first transition layer, with the increase of PLGA content, the proportion of C and O elements increases, and the N content decreases due to the decrease of gelatin proportion; the solute of the second micro-nanofiber membrane layer contains only PLGA, and the ratio of C and O elements is close to 1:1 (due to the alternating arrangement of LA and GA units); the second transition layer has Ca and P signals due to the introduction of a small amount of nanohydroxyapatite (osteogenic material a), but C and O elements are not significantly different. The contribution of PLGA to the Ca and P elements was still the main one; in the third micro-nanofiber membrane layer, the proportion of nanohydroxyapatite (osteogenic material a) further increased, the Ca and P signals were significantly enhanced, and the proportion of C element decreased; in the third transition layer, the recovery of gelatin component caused the N element signal to rebound, and the continuous addition of nanohydroxyapatite (osteogenic material b) further increased the Ca and P contents; in the fourth micro-nanofiber membrane layer, the proportion of gelatin reached the highest level, the N content increased significantly, while nanohydroxyapatite (osteogenic material c) continued to provide Ca and P signals.

[0099] Example 3

[0100] A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0101] (1) Preparation of materials;

[0102] The first spinning solution: the solute is a mixture of chitosan and PCL, the PCL content in the solute is 50wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution (the volume fraction of acetic acid is 80%) with a volume ratio of 7:3 and a concentration of 150mg / mL;

[0103] The second spinning solution: the solute is PCL, the solvent is hexafluoroisopropanol, and the concentration is 160 mg / mL;

[0104] The third spinning solution: the solute is a mixture of PCL and osteogenic material a, the content of osteogenic material a in the solute is 2wt%, and the solvent is hexafluoroisopropanol, with a concentration of 170mg / mL;

[0105] The fourth spinning solution: the solute is a mixture of chitosan, PCL, and osteogenic material b, with a PCL content of 50 wt% and an osteogenic material b content of 3 wt%. The solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution (the volume fraction of acetic acid is 80%) in a volume ratio of 7:3, with a concentration of 180 mg / mL.

[0106] Osteogenic material a, osteogenic material b, and osteogenic material c: all are nanohydroxyapatite;

[0107] (2) using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0108] When preparing the first micro-nanofiber membrane layer, the first spinning solution was injected at a constant injection speed v1. The process parameters included: positive voltage of 14 kV, negative voltage of 2 kV, v1 of 5 mL / h, air pressure of 5 atm, and receiving distance of 15 cm. The first micro-nanofiber membrane layer had a thickness of 0.5 mm, an average pore size of 31 μm, a porosity of 80%, and a fiber orientation of 82%. The first micro-nanofiber membrane layer was used to guide the directional alignment of tendon fibroblasts, promoting tendon differentiation and regeneration.

[0109] When preparing the first transition layer, the first spinning solution and the second spinning solution were injected simultaneously. The injection speed of the first spinning solution was uniformly decreased from v1 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v2. The sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remained unchanged. The process parameters included: positive voltage of 14 kV, negative voltage of 2 kV, v2 of 5 mL / h, injection time of 1 hour, air pressure of 5 atm, and receiving distance of 15 cm.

[0110] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v2; the process parameters include: positive voltage of 14 kV, negative voltage of 2 kV, air pressure of 5 atm, and receiving distance of 15 cm; the thickness of the second micro-nano fiber membrane layer is 0.5 mm, the average pore size is 58 μm, the porosity is 85%, and the orientation degree of fiber arrangement is 56%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage;

[0111] When preparing the second transition layer, the second spinning solution and the third spinning solution were injected simultaneously. The injection speed of the second spinning solution was uniformly decreased from v2 to 0, and then uniformly increased from 0 to v3. The sum of the injection speeds of the second and third spinning solutions remained constant at each moment. The process parameters included: positive voltage of 14 kV, negative voltage of 2 kV, v3 of 5 mL / h, injection time of 1 hour, air pressure of 5 atm, and receiving distance of 15 cm.

[0112] When preparing the third micro-nanofiber membrane layer, the third spinning solution was injected at a constant injection speed v3. The process parameters included: positive voltage of 14 kV, negative voltage of 2 kV, air pressure of 5 atm, and receiving distance of 15 cm. The third micro-nanofiber membrane layer had a thickness of 0.5 mm, an average pore size of 186 μm, a porosity of 95%, and a fiber orientation of 23%. The third micro-nanofiber membrane layer was used to induce differentiation and regeneration of mineralized fibrocartilage.

[0113] When preparing the third transition layer, the third and fourth spinning solutions were injected simultaneously. The injection speed of the third spinning solution was uniformly decreased from v3 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v4. The sum of the injection speeds of the third and fourth spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 14 kV, negative voltage of 2 kV, v4 of 5 mL / h, injection time of 1 hour, air pressure of 5 atm, and receiving distance of 15 cm.

[0114] When preparing the fourth micro-nanofiber membrane layer, the fourth spinning solution is injected at a constant injection speed v4. The process parameters include: positive voltage of 14 kV, negative voltage of 2 kV, air pressure of 5 atm, and receiving distance of 15 cm. The fourth micro-nanofiber membrane layer has a thickness of 0.5 mm, an average pore size of 359 μm, a porosity of 92%, and completely randomly oriented fibers. The fourth micro-nanofiber membrane layer is used to promote bone regeneration.

[0115] (3) forming protrusions of osteogenic material c on the fourth micro-nanofiber membrane layer using heat treatment nanoimprinting technology;

[0116] (3.1) Pour a photosensitive resin onto the nano-patterned master and evenly spread it with a roller until it completely covers the surface of the nano-patterned master. Then, cure the photosensitive resin to form a resin mold on the nano-patterned master. Separate the nano-patterned master from the resin mold.

[0117] (3.2) Depositing osteogenic material c on the surface of the resin mold using electron beam evaporation technology;

[0118] (3.3) Plasma treatment is performed on the resin mold with the osteogenic material c deposited on the surface, and the fourth micro-nanofiber membrane layer is also plasma treated;

[0119] (3.4) Pressing a resin mold with osteogenic material c deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material c contacts the fourth micro-nano fiber membrane layer, and then performing heat treatment. After the heat treatment, the resin mold is removed to obtain an integrated gradual bionic gradient structure rotator cuff patch.

[0120] The resulting integrated, biomimetic, gradient-structured rotator cuff patch has a total thickness of 3.5 mm. The osteogenic material c projections on the fourth micro-nanofiber membrane layer are cylindrical, 800 nm in diameter and 200 nm in height. All of the osteogenic material c projections are arranged in a lattice pattern, with an 800 nm spacing between adjacent ones. Each transition layer and its two adjacent micro-nanofiber membrane layers form a continuous, unbroken structure, with uniform transitions in internal structure and composition, without discrete layers.

[0121] The peel strength of the integrated bionic gradient structure rotator cuff patch is 61.7 mN / mm. The energy dispersion spectrum analysis of each layer of the integrated bionic gradient structure rotator cuff patch is shown in Table 4:

[0122] Table 4

[0123]

[0124] As can be seen from Table 4, in the first micro-nano fiber membrane layer, chitosan, as one of the main components, provides N element, while the presence of PCL significantly increases the proportion of C element; in the first transition layer, with the increase of PCL content, the proportion of C element further increases, while the N content decreases due to the decrease of chitosan proportion; in the second micro-nano fiber membrane layer, since the solute contains only PCL, C element occupies a dominant position, and its C to O element ratio is close to the theoretical value (about 69:31); in the second transition layer, due to the introduction of a small amount of nano-hydroxyapatite (osteogenic material a), it begins to show In the third micro-nanofiber membrane layer, the Ca and P signals were found, but the C and O elements still mainly came from PCL; in the third micro-nanofiber membrane layer, with the increase of nanohydroxyapatite (osteogenetic material a) content, the Ca and P signals were significantly enhanced; in the third transition layer, the recovery of chitosan composition caused the trace N signal to rebound, and at the same time, the increase of the proportion of nanohydroxyapatite (osteogenetic material b) further enhanced the Ca and P signals; in the fourth micro-nanofiber membrane layer, the combined effect of chitosan and nanohydroxyapatite (osteogenetic material c) caused the N element as well as the Ca and P contents to rise synchronously.

[0125] Example 4

[0126] A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0127] Spinning solution 1: The solute is a mixture of silk fibroin and PDO, the PDO content in the solute is 70 wt%, and the solvent is a mixture of hexafluoroisopropanol and formic acid in a volume ratio of 8:2, with a concentration of 175 mg / mL;

[0128] The second spinning solution: the solute is PDO, the solvent is hexafluoroisopropanol, and the concentration is 185 mg / mL;

[0129] The third spinning solution: the solute is a mixture of PDO and osteogenic material a, the content of osteogenic material a in the solute is 2wt%, and the solvent is hexafluoroisopropanol, with a concentration of 195mg / mL;

[0130] The fourth spinning solution: the solute is a mixture of silk fibroin, PDO and osteogenic material b, the content of PDO in the solute is 70wt%, the content of osteogenic material b in the solute is 4wt%, and the solvent is a mixture of hexafluoroisopropanol and formic acid in a volume ratio of 8:2, with a concentration of 205mg / mL;

[0131] Osteogenic material a, osteogenic material b and osteogenic material c: all are bioactive glass;

[0132] (2) using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0133] When preparing the first micro-nanofiber membrane layer, the first spinning solution was injected at a constant injection speed v1. The process parameters included: positive voltage of 16 kV, negative voltage of 2 kV, v1 of 7 mL / h, air pressure of 7 atm, and receiving distance of 17 cm. The first micro-nanofiber membrane layer had a thickness of 0.7 mm, an average pore size of 42 μm, a porosity of 83%, and a fiber orientation of 90%. The first micro-nanofiber membrane layer was used to guide the directional alignment of tendon fibroblasts, promoting tendon differentiation and regeneration.

[0134] When preparing the first transition layer, the first spinning solution and the second spinning solution were injected simultaneously. The injection speed of the first spinning solution was uniformly decreased from v1 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v2. The sum of the injection speeds of the first and second spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 16 kV, negative voltage of 2 kV, v2 of 7 mL / h, injection time of 1 hour, air pressure of 7 atm, and receiving distance of 17 cm.

[0135] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v2; the process parameters include: positive voltage of 16 kV, negative voltage of 2 kV, air pressure of 7 atm, and receiving distance of 17 cm; the second micro-nano fiber membrane layer has a thickness of 0.7 mm, an average pore size of 73 μm, a porosity of 87%, and a fiber orientation degree of 50%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage;

[0136] When preparing the second transition layer, the second spinning solution and the third spinning solution were injected simultaneously. The injection speed of the second spinning solution was uniformly decreased from v2 to 0, and then uniformly increased from 0 to v3. The sum of the injection speeds of the second and third spinning solutions remained constant at each moment. The process parameters included: positive voltage of 16 kV, negative voltage of 2 kV, v3 of 7 mL / h, injection time of 1 hour, air pressure of 7 atm, and receiving distance of 17 cm.

[0137] When preparing the third micro-nanofiber membrane layer, the third spinning solution was injected at a constant injection speed v3. The process parameters included: positive voltage of 16 kV, negative voltage of 2 kV, air pressure of 7 atm, and receiving distance of 17 cm. The third micro-nanofiber membrane layer had a thickness of 0.7 mm, an average pore size of 247 μm, a porosity of 90%, and a fiber orientation of 25%. The third micro-nanofiber membrane layer was used to induce differentiation and regeneration of mineralized fibrocartilage.

[0138] When preparing the third transition layer, the third and fourth spinning solutions were injected simultaneously. The injection speed of the third spinning solution was uniformly decreased from v3 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v4. The sum of the injection speeds of the third and fourth spinning solutions remained constant at each moment. The process parameters included: positive voltage of 16 kV, negative voltage of 2 kV, v4 of 7 mL / h, injection time of 1 hour, air pressure of 7 atm, and receiving distance of 17 cm.

[0139] When preparing the fourth micro-nanofiber membrane layer, the fourth spinning solution is injected at a constant injection speed v4. The process parameters include: positive voltage of 16 kV, negative voltage of 2 kV, air pressure of 7 atm, and receiving distance of 17 cm. The fourth micro-nanofiber membrane layer has a thickness of 0.7 mm, an average pore size of 420 μm, a porosity of 94%, and completely randomly oriented fibers. The fourth micro-nanofiber membrane layer is used to promote bone regeneration.

[0140] (3) forming protrusions of osteogenic material c on the fourth micro-nanofiber membrane layer using heat treatment nanoimprinting technology;

[0141] (3.1) Pour a photosensitive resin onto the nano-patterned master and evenly spread it with a roller until it completely covers the surface of the nano-patterned master. Then, cure the photosensitive resin to form a resin mold on the nano-patterned master. Separate the nano-patterned master from the resin mold.

[0142] (3.2) Depositing osteogenic material c on the surface of the resin mold using electron beam evaporation technology;

[0143] (3.3) Plasma treatment is performed on the resin mold with the osteogenic material c deposited on the surface, and the fourth micro-nanofiber membrane layer is also plasma treated;

[0144] (3.4) Pressing a resin mold with osteogenic material c deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material c contacts the fourth micro-nano fiber membrane layer, and then performing heat treatment. After the heat treatment, the resin mold is removed to obtain an integrated gradual bionic gradient structure rotator cuff patch.

[0145] The resulting integrated, biomimetic, gradient-structured rotator cuff patch has a total thickness of 4.9 mm. The osteogenic material c projections on the fourth micro-nanofiber membrane layer are cylindrical, 1500 nm in diameter and 1200 nm in height. All of the osteogenic material c projections are arranged in a lattice pattern, with a spacing of 1500 nm between adjacent ones. Each transition layer and its two adjacent micro-nanofiber membrane layers form a continuous, unbroken structure, with uniform transitions in internal structure and composition, without discrete layers.

[0146] The peel strength of the integrated bionic gradient structure rotator cuff patch is 59.6 mN / mm. The energy dispersion spectrum analysis of each layer of the integrated bionic gradient structure rotator cuff patch is shown in Table 5:

[0147] Table 5

[0148]

[0149] As can be seen from Table 5, in the first micro-nanofiber membrane layer, silk fibroin provides N as a nitrogen-containing component, while PDO dominates the C and O element ratios due to its high content; in the first transition layer, as the proportion of PDO increases further, the C element ratio increases significantly, while the N content decreases due to the decrease in the proportion of silk fibroin; in the second micro-nanofiber membrane layer, since the solute only contains PDO, its C and O element ratio is close to the theoretical value of 3:2; in the second transition layer, due to the introduction of trace bioactive glass (osteogenizing material a), Si, Ca, and P element signals begin to appear, but C , O elements still mainly come from PDO; in the third micro-nanofiber membrane layer, with the increase of the proportion of bioactive glass (osteogenizing material a), the Si, Ca, and P element signals are significantly enhanced; in the third transition layer, the proportion of bioactive glass continues to increase, dominating the changes in Si, Ca, and P elements, while the presence of silk fibroin provides trace N elements; in the fourth micro-nanofiber membrane layer, the proportion of bioactive glass (osteogenizing material c) reaches the highest level, and the Si, Ca, and P element signals are significantly enhanced, while the composition of silk fibroin slightly increases the N element content.

[0150] Example 5

[0151] A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0152] (1) Preparation of materials;

[0153] The first spinning solution: the solute is a mixture of hyaluronic acid and PLCL, the content of PLCL in the solute is 90wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution (the volume fraction of acetic acid is 90%) in a volume ratio of 7:3, with a concentration of 200mg / mL;

[0154] The second spinning solution: the solute is PLCL, the solvent is hexafluoroisopropanol, and the concentration is 210 mg / mL;

[0155] The third spinning solution: the solute is a mixture of PLCL and osteogenic material a, the content of osteogenic material a in the solute is 2wt%, and the solvent is hexafluoroisopropanol with a concentration of 220mg / mL;

[0156] The fourth spinning solution: the solute is a mixture of hyaluronic acid, PLCL, and osteogenic material b, the content of PLCL in the solute is 90wt%, the content of osteogenic material b in the solute is 5wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution (the volume fraction of acetic acid is 90%) in a volume ratio of 7:3, with a concentration of 230mg / mL;

[0157] Osteogenic material a, osteogenic material b and osteogenic material c: all are tricalcium phosphate;

[0158] (2) using an electrically assisted solution blowing spinning technique to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0159] When preparing the first micro-nanofiber membrane layer, the first spinning solution is injected at a constant injection speed v1. The process parameters include: positive voltage of 18 kV, negative voltage of 3 kV, v1 of 10 mL / h, air pressure of 10 atm, and receiving distance of 20 cm. The first micro-nanofiber membrane layer has a thickness of 1 mm, an average pore size of 50 μm, a porosity of 85%, and a fiber orientation degree of 88%. The first micro-nanofiber membrane layer is used to guide the directional alignment of tendon fibroblasts and promote tendon differentiation and regeneration.

[0160] When preparing the first transition layer, the first spinning solution and the second spinning solution were injected simultaneously. The injection speed of the first spinning solution was uniformly decreased from v1 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v2. The sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remained unchanged. The process parameters included: positive voltage of 18 kV, negative voltage of 3 kV, v2 of 10 mL / h, injection time of 1 hour, air pressure of 10 atm, and receiving distance of 20 cm.

[0161] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v2; the process parameters include: positive voltage of 18 kV, negative voltage of 3 kV, air pressure of 10 atm, and receiving distance of 20 cm; the second micro-nano fiber membrane layer has a thickness of 1 mm, an average pore size of 80 μm, a porosity of 90%, and a fiber orientation degree of 58%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage;

[0162] When preparing the second transition layer, the second spinning solution and the third spinning solution were injected simultaneously. The injection speed of the second spinning solution was uniformly decreased from v2 to 0, and then uniformly increased from 0 to v3. The sum of the injection speeds of the second and third spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 18 kV, negative voltage of 3 kV, v3 of 10 mL / h, injection time of 1 hour, air pressure of 10 atm, and receiving distance of 20 cm.

[0163] When preparing the third micro-nanofiber membrane layer, the third spinning solution was injected at a constant injection speed v3; the process parameters included: positive voltage of 18 kV, negative voltage of 3 kV, air pressure of 10 atm, and receiving distance of 20 cm; the third micro-nanofiber membrane layer had a thickness of 1 mm, an average pore size of 300 μm, a porosity of 92%, and a fiber orientation of 20%; the third micro-nanofiber membrane layer was used to induce differentiation and regeneration of mineralized fibrocartilage;

[0164] When preparing the third transition layer, the third and fourth spinning solutions were injected simultaneously. The injection speed of the third spinning solution was uniformly decreased from v3 to 0, and the injection speed of the second spinning solution was uniformly increased from 0 to v4. The sum of the injection speeds of the third and fourth spinning solutions at each moment remained unchanged. The process parameters included: positive voltage of 18 kV, negative voltage of 3 kV, v4 of 10 mL / h, injection time of 1 hour, air pressure of 10 atm, and receiving distance of 20 cm.

[0165] When preparing the fourth micro-nano fiber membrane layer, the fourth spinning solution is injected at a constant injection speed v4; the process parameters include: positive voltage of 18 kV, negative voltage of 3 kV, air pressure of 10 atm, and receiving distance of 20 cm; the fourth micro-nano fiber membrane layer has a thickness of 1 mm, an average pore size of 500 μm, a porosity of 93%, and completely randomly oriented fibers; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0166] (3) forming protrusions of osteogenic material c on the fourth micro-nanofiber membrane layer using heat treatment nanoimprinting technology;

[0167] (3.1) Pour a photosensitive resin onto the nano-patterned master and evenly spread it with a roller until it completely covers the surface of the nano-patterned master. Then, cure the photosensitive resin to form a resin mold on the nano-patterned master. Separate the nano-patterned master from the resin mold.

[0168] (3.2) Depositing osteogenic material c on the surface of the resin mold using electron beam evaporation technology;

[0169] (3.3) Plasma treatment is performed on the resin mold with the osteogenic material c deposited on the surface, and the fourth micro-nanofiber membrane layer is also plasma treated;

[0170] (3.4) Pressing a resin mold with osteogenic material c deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material c contacts the fourth micro-nano fiber membrane layer, and then performing heat treatment. After the heat treatment, the resin mold is removed to obtain an integrated gradual bionic gradient structure rotator cuff patch.

[0171] The resulting integrated, biomimetic, gradient-structured rotator cuff patch has a total thickness of 6 mm. The osteogenic material c projections on the fourth micro-nanofiber membrane layer are cylindrical, 2500 nm in diameter and 2500 nm in height. All of the osteogenic material c projections are arranged in a lattice pattern, with a spacing of 2000 nm between adjacent ones. Each transition layer and its two adjacent micro-nanofiber membrane layers form a continuous, unbroken structure, with uniform transitions in internal structure and composition, without discrete layers.

[0172] The peel strength of the integrated bionic gradient structure rotator cuff patch is 55.8 mN / mm. The energy dispersion spectrum analysis of each layer of the integrated bionic gradient structure rotator cuff patch is shown in Table 6:

[0173] Table 6

[0174]

[0175] As can be seen from Table 6, in the first micro-nanofiber membrane layer, PLCL dominated the C and O element ratios due to its high content, while HA provided a trace amount of N. In the first transition layer, as the PLCL content increased, the C element ratio increased, while the N content decreased due to the decreased HA content. In the second micro-nanofiber membrane layer, since the solute contained only PLCL, the C:O element ratio was close to the theoretical value of 3:2. Due to the introduction of a trace amount of tricalcium phosphate (osteogenizing material a), Ca and P element signals began to appear in the second transition layer, but the C and O elements still mainly originated from PLCL. In the third micro-nanofiber membrane layer, as the tricalcium phosphate (osteogenizing material a) content increased, the Ca and P signals significantly increased, and the C element ratio decreased accordingly. In the third transition layer, the tricalcium phosphate content further increased, resulting in a significant increase in the Ca and P contents, while HA contributed a trace amount of N. In the fourth micro-nanofiber membrane layer, the tricalcium phosphate (osteogenizing material c) content reached the highest level, the Ca and P signal intensities were the strongest, and the C element ratio further decreased.

[0176] Comparative Example 1

[0177] A method for preparing a patch differs from Example 1 only in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as in Example 1.

[0178] The peel strength of the final patch was 15.6 mN / mm.

[0179] Comparative Example 2

[0180] A method for preparing a patch differs from Example 2 only in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as in Example 2.

[0181] The peel strength of the final patch was 16.9 mN / mm.

[0182] Comparative Example 3

[0183] A method for preparing a patch, which differs from Example 3 only in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as in Example 3.

[0184] The peel strength of the final patch was 21.8 mN / mm.

[0185] Comparative Example 4

[0186] A method for preparing a patch, which differs from Example 4 only in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as in Example 4.

[0187] The peel strength of the final patch was 20.9 mN / mm.

[0188] Comparative Example 5

[0189] A method for preparing a patch, which differs from Example 5 only in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as in Example 5.

[0190] The peel strength of the final patch was 18.9 mN / mm.

[0191] Comparing Comparative Examples 1-5 with Examples 1-5, the peel strength of the patches significantly decreased. This demonstrates that the presence of the transition layer plays a key role in enhancing interlayer adhesion. By achieving a uniform transition in the material composition and structure of each layer, it effectively avoids interlayer discreteness and significantly improves the overall mechanical properties of the patch. In contrast, the direct preparation of adjacent micro-nanofiber layers in the comparative examples lacks a compositional transition, resulting in weak interlayer adhesion and greater susceptibility to interlayer separation under external forces, significantly reducing the patch's peel strength and hindering the long-lasting and stable repair effect of the rotator cuff patch in practical applications.

Claims

1. A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, characterized in that: After sequentially forming a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate by using an electrically assisted solution blown spinning technique, a bone promoting material protrusion is formed on the fourth micro-nano fiber membrane layer by using a heat treatment nanoimprinting technique; When preparing the i-th micro-nano fiber membrane layer, the corresponding i-th spinning solution is injected at a constant injection speed v i Perform bolus injection, i = 1, 2, 3; When preparing the i+1th micro-nano fiber membrane layer, the corresponding i+1th spinning solution is injected at a constant speed v i+1 Give a bolus injection; The process of preparing the i-th transition layer is as follows: simultaneously injecting the i-th spinning solution and the i+1-th spinning solution, and the injection speed of the i-th spinning solution is determined by v i The speed is uniformly reduced to 0, and the injection speed of the i+1 spinning solution is uniformly increased from 0 to v i+1 , the sum of the injection speed of the i-th spinning solution and the injection speed of the i+1-th spinning solution at each moment remains unchanged; The first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrocartilage; and the fourth micro-nano fiber membrane layer is used to promote bone regeneration. The osteogenic material protrusions are cylindrical, with a diameter of 100-2500nm and a height of 5-2500nm. All osteogenic material protrusions are arranged in a lattice, and the distance between two adjacent osteogenic material protrusions is 100-2000nm. Each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change evenly and transitionally without showing discrete stratification; The specific steps of forming the bone-promoting material protrusions on the fourth micro-nanofiber membrane layer using the heat treatment nanoimprinting technology are as follows: (a) pouring a photosensitive resin onto a nano-pattern master, evenly spreading it with a roller until it completely covers the surface of the nano-pattern master, curing the photosensitive resin to form a resin mold on the nano-pattern master, and separating the nano-pattern master from the resin mold; (b) depositing osteogenic material on the surface of the resin mold using electron beam evaporation technology; (c) performing plasma treatment on the resin mold on which the osteogenic material is deposited, and simultaneously performing plasma treatment on the fourth micro-nanofiber membrane layer; (d) Pressing the resin mold with the osteogenic material deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material contacts the fourth micro-nano fiber membrane layer, performing heat treatment, and removing the resin mold after the heat treatment is completed.

2. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the first micro-nano fiber membrane layer is 10-50 μm, the porosity is 70-85%, the orientation degree of the fiber arrangement is >80%, the fiber component is a mixture of natural polymer materials and synthetic polymer materials, and the content of the synthetic polymer material is 10-90wt%.

3. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the second micro-nano fiber membrane layer is 30-80 μm, the porosity is 80-90%, the orientation degree of the fiber arrangement is 50-60%, and the fiber component is a synthetic polymer material.

4. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the third micro-nano fiber membrane layer is 100-300 μm, the porosity is 85-95%, the orientation degree of the fiber arrangement is greater than 20% and less than 30%, the fiber component is a mixture of synthetic polymer materials and osteogenic materials, and the content of osteogenic materials is 1-2wt%.

5. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the fourth micro-nano fiber membrane layer is 200-500 μm, the porosity is >90%, the fibers are completely randomly oriented, and the fiber composition is a mixture of natural polymer materials, synthetic polymer materials and osteogenic materials, the content of synthetic polymer materials is 10-90wt%, and the content of osteogenic materials is 2-5wt%.

6. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The thickness of each micro-nano fiber membrane layer ranges from 0.1 to 1 mm, and the total thickness of the integrated gradual bionic gradient structure rotator cuff patch is from 0.7 to 6 mm.

7. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The process parameters of electric-assisted solution blowing spinning include: positive voltage of 10-18 kV, negative voltage of 1-3 kV, injection speed of 1-10 mL / h, air pressure of 1-10 atm, and receiving distance of 10-20 cm.

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