Preparation method and application of hip joint polyurethane artificial glenoid lip

Abstract

The invention provides a preparation method of a hip joint polyurethane artificial glenoid lip, which comprises the following steps: carrying out 3D printing on polyurethane to prepare an annular polyurethane stent, and then modifying the surface of the annular polyurethane stent with polydopamine. According to the preparation method of the hip joint polyurethane artificial glenoid lip, the biocompatibility and cell adhesion of the hip joint polyurethane artificial glenoid lip are enhanced through a polydopamine (PDA) surface modification technology, and the hip joint polyurethane artificial glenoid lip is used for repairing acetabular glenoid lip injury and promoting fibrous cartilage regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering and orthopedic implant devices, and in particular to a preparation method and application of a polyurethane artificial labrum of a hip joint. Background Art

[0002] The acetabular labrum, a fibrous cartilage structure attached to the rim of the acetabulum, plays a vital role in the stability and sealing of the hip joint. Acetabular labral tears are the most common hip injury, especially in patients with hip impingement syndrome, and are a common cause of hip pain.

[0003] Acetabular labral tears are the most common type of hip joint lesion. With the rapid development of minimally invasive hip arthroscopy, arthroscopic labral suturing or reconstruction has become the latest treatment option. However, the use of autologous tendon transplantation for labral reconstruction has several limitations, such as limited donor tissue, decreased sensation at the donor site, risk of bleeding and infection, and post-reconstruction healing issues. Therefore, tissue-engineered artificial labrums have become a new research trend.

[0004] Acetabular labral damage can lead to decreased hip joint stability, accelerating wear and degeneration, and ultimately developing into osteoarthritis. This condition can cause patients to suffer from long-term hip pain and limited joint mobility, severely impacting their quality of life and mobility.

[0005] In response to the above problems, the present invention proposes a method for preparing a polyurethane artificial labrum of the hip joint and its application. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a polyurethane artificial labrum of the hip joint, which enhances its biocompatibility and cell adhesion through bionic design and polydopamine (PDA) surface modification technology, and is used to repair acetabular labrum damage and promote fibrocartilage regeneration.

[0007] The present invention provides a method for preparing a polyurethane artificial labrum of a hip joint, the steps comprising: preparing polyurethane into a ring-shaped polyurethane bracket by 3D printing, and then modifying the surface of the ring-shaped polyurethane bracket by polydopamine.

[0008] Furthermore, the annular polyurethane bracket has a diameter of 30 mm, a cross-section of a right triangle, a bottom width of 2.5 mm, and a height of 3 mm.

[0009] Furthermore, the molecular weight of the polyurethane is 50,000-60,000.

[0010] Furthermore, the 3D printing step includes: dissolving polyurethane at a high temperature of 200° C. for 30 minutes, extruding polyurethane through a nozzle with a diameter of 300 μm at an air pressure of 0.7 MPa and 7.0 mm / s for 3D printing, and preparing a polyurethane bracket.

[0011] Furthermore, the polydopamine modification step includes: soaking the polyurethane scaffold in a 2% polydopamine solution for 24 hours, wherein the solvent of the polydopamine solution is a 1M Tris-HCl buffer with a pH of 8.0; taking out the scaffold after it turns black; and washing away the uncross-linked dopamine with a phosphate buffer.

[0012] Furthermore, the concentration of the phosphate buffer is 0.01 M and the pH value is 7.2-7.4.

[0013] On the other hand, the polyurethane artificial labrum of the hip joint of the present invention is used in repairing acetabular labrum damage.

[0014] The beneficial effects of the present invention are:

[0015] The key to this invention lies in the design of a 3D-printed polyurethane scaffold based on the anatomical structure of the glenoid labrum. This scaffold is then surface-modified with polydopamine to increase its hydrophilicity and cell adhesion. This novel scaffold has been demonstrated to promote fibrocartilage regeneration in both in vitro cell-based and in vivo minipig animal studies, demonstrating its potential for repairing acetabular labral tears and clinical applications. Currently, there are no artificial replacements for the hip labrum in clinical practice in China. This invention demonstrates that the polyurethane artificial labrum promotes labral regeneration in both in vitro cell-based and in vivo large animal studies, demonstrating its significant potential for translational applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 This is a real picture of the circular polyurethane bracket;

[0018] Figure 2 This is a photo of the polyurethane artificial labrum of the hip joint;

[0019] Figure 3 The following is a scanning electron micrograph of a donut-shaped polyurethane stent and a polyurethane artificial labrum of a hip joint;

[0020] Figure 4The electron microscope energy spectrum analysis of the annular polyurethane stent and the polyurethane artificial labrum of the hip joint;

[0021] Figure 5 Live-dead staining images of circular polyurethane scaffolds and adipose-derived mesenchymal stem cells;

[0022] Figure 6 This is a staining image of cell proliferation in a co-culture of annular polyurethane scaffold and adipose-derived mesenchymal stem cells;

[0023] Figure 7 Reconstruction diagram of polyurethane artificial labrum of hip joint;

[0024] Figure 8 This is an MRI image of the regenerated labrum 6 months after polyurethane artificial labrum reconstruction of the hip joint, in which the triangle box indicates the regenerated labrum;

[0025] Figure 9 This is a diagram showing the gross evaluation of the regenerated labrum 6 months after hip joint polyurethane artificial labrum reconstruction, where the circle represents the regenerated labrum;

[0026] Figure 10 This is a safranin-fast green-stained image of the regenerated labrum of the hip joint 6 months after polyurethane artificial labrum reconstruction. The triangle box represents the regenerated labrum. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention. It should be noted that the raw materials can be obtained commercially.

[0028] Example

[0029] The present invention provides a method for preparing a polyurethane artificial labrum of a hip joint, comprising the following steps: 3D printing polyurethane with a molecular weight of 50,000-60,000 into a ring-shaped polyurethane bracket, and then modifying the surface of the ring-shaped polyurethane bracket with polydopamine; the ring-shaped polyurethane bracket has a diameter of 30 mm, a cross-section of a right triangle, a bottom width of 2.5 mm, and a height of 3 mm.

[0030] In a further embodiment, 3D printing and polydopamine modification are performed based on the aforementioned structure, and the specific steps include:

[0031] (1) Polyurethane was dissolved at 200 °C for 30 min, and then extruded through a nozzle with a diameter of 300 μm at a pressure of 0.7 MPa and 7.0 mm / s for 3D printing to prepare a polyurethane scaffold, such as Figure 1 As shown;

[0032] (2) The polyurethane scaffold was immersed in a 2% polydopamine solution for 24 hours. The solvent of the polydopamine solution was a 1M Tris-HCl buffer solution with a pH of 8.0. After the polydopamine was cross-linked on the surface of the scaffold, the surface turned black. Figure 2 As shown, after the scaffold turns black, it is taken out and uncross-linked dopamine is washed away using a phosphate buffer solution with a concentration of 0.01 M and a pH value of 7.2-7.4.

[0033] The microstructures of the outer surface, inner surface, bottom surface and cross section of the annular polyurethane stent and the hip joint polyurethane artificial labrum were observed by scanning electron microscopy. Figure 3 As shown in the results, a large number of polydopamine particles were deposited on the surface of the polyurethane artificial labrum of the hip joint; electron microscope energy spectrum analysis (EDS) observed that the carbon (C) and oxygen (O) elements in the annular polyurethane scaffold (PU) group were significantly higher than those in the polyurethane artificial labrum of the hip joint group, while the nitrogen (N) element in the polyurethane artificial labrum of the hip joint (PUDA) group was higher than that in the annular polyurethane scaffold group, indicating that polydopamine was successfully modified on the polyurethane surface. The results are shown in the figure. Figure 4 shown.

[0034] Figure 3 Outer means outside; Inner means inner surface; Bottom means bottom surface; Section means section.

[0035] The biocompatibility, proliferation and differentiation ability of the polyurethane / polydopamine artificial labrum scaffold were tested by in vitro cell experiments. Rat adipose-derived mesenchymal stem cells (ADSCs) were extracted and cultured adherently (complete medium containing 10% serum, 37°C, 5% CO2). ADSCs passaged to P2 were inoculated onto the surface of the PUDA scaffold. After culturing for 1, 3, and 7 days, the biocompatibility of the scaffold was evaluated using a live-dead staining kit (KeyGEN BioTECH).

[0036] The operation process is as follows: on the 1st, 3rd, and 7th day of culture, the culture medium was discarded, the scaffolds were washed with 1×PBS (pH 7.2-7.4, 0.01M), and the scaffolds were stained with a solution containing 2μM chloroacetyl methyl ester and 8μM propidium iodide at room temperature in the dark for 30 minutes, followed by washing with PBS to terminate the staining. The stained scaffolds were imaged using a laser confocal microscope (TCS-SP8STED, Leica), with green indicating live cells and red indicating dead cells. Figure 5 As shown in Figure 2, the biocompatibility of the annular polyurethane scaffold and the hip joint polyurethane artificial labrum is good ( Figure 5 ).

[0037] EdU (5-Ethynyl-2'-deoxyuridine) is a thymidine nucleoside analog that can replace thymidine (T) and be incorporated into replicating DNA molecules during cell proliferation. The specific reaction of fluorescent dyes can quickly detect cell DNA replication activity and can quickly and accurately detect cell proliferation ability.

[0038] Cells in logarithmic growth phase were taken and plated at 4×10 3 ~1×10 5 Cells were seeded on the surface of the scaffold and cultured to the normal growth stage. EdU solution was diluted with complete culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50μM EdU culture medium; 1mL of 50μM EdU culture medium was added to each well and incubated for 2 hours, and the culture medium was discarded; cells were washed with PBS. 1mL of cell fixative was added to each well and incubated at room temperature for 30 minutes, and the fixative was discarded; 1mL of 2mg / mL glycine was added to each well, and after incubation on a decolorizing shaker for 5 minutes, the glycine solution was discarded; PBS was washed for 5 minutes; 1mL of immunostaining permeabilization solution (0.5% TritonX-100 in PBS, Beyotime Biotechnology-Immunostaining Permeabilization Solution) was added to each well and decolorized and incubated on a shaker for 10 minutes; PBS was washed for 5 minutes. 1mL of 1X was added to each well The staining reaction solution was incubated in a decolorizing shaker for 30 minutes at room temperature in the dark, and then the staining reaction solution was discarded. After Hoechst33342 stained the nucleus, the stained scaffold was imaged using a laser confocal microscope (TCS-SP8 STED, Leica). Blue indicated all cells, and red indicated proliferating cells. Figure 6 As shown, the proliferation and differentiation abilities of the PUDA scaffold group were stronger.

[0039] The effect of polyurethane / polydopamine artificial labrum scaffolds on promoting fibrocartilage regeneration was tested in an in vivo minipig animal model. The specific steps are as follows:

[0040] The miniature pig was anesthetized and the anesthetic gas outlet tube was connected to a large animal ventilator to administer inhalation anesthesia. The surgery was performed on the miniature pig under general anesthesia. The skin, subcutaneous tissue, and joint capsule in front of the right hip joint were incised layer by layer to expose the hip joint. Part of the glenoid labrum was removed from the posterior and superior aspect of the acetabular rim. Three holes were made in the acetabular rim, and three 10 mm long and 2 mm diameter anchors with wires were inserted. The polyurethane / polydopamine scaffold was sutured and fixed to the acetabular rim to achieve glenoid labrum reconstruction ( Figure 7 ), Figure 7 The dark black strip is a polyurethane artificial labrum, which is fixed to the acetabular rim by wire anchors. Figure 8 ), general assessment ( Figure 9 ) Labrum regeneration can be seen, and safranin fast green staining can show proteoglycan deposition in the regenerated labrum ( Figure 10 ). Figure 8 The median labrum is cartilage, which can be observed through MRI. The red triangle in the image is the labrum. Figure 9 This image was taken six months after the acetabulum was removed from the body to observe the regenerated labrum. Figure 10 The medial labrum is cartilage tissue, which is composed of chondrocytes, cartilage matrix, and fibers. Cartilage tissue and its surrounding perichondrium constitute cartilage. Cartilage is classified into hyaline cartilage, elastic cartilage, and fibrocartilage based on the cellulose content of the matrix. Numerous cartilage staining methods exist, including toluidine blue, alcian blue, and safranin fast green. The modified safranin fast green cartilage staining method relies on the principle that basophilic cartilage combines with the basic dye safranin O to produce a red color, while eosinophilic bone combines with the acidic dye fast green to produce a green or blue color. This contrasts sharply with the red color of cartilage, thus distinguishing cartilage from bone. Safranin O staining is approximately proportional to anion concentration and indirectly reflects the content and distribution of proteoglycans in the matrix. When cartilage is damaged, glycoproteins are released from the cartilage, resulting in uneven distribution of matrix components and weak or no staining with safranin O. Image analysis software allows for quantitative analysis of the cartilage matrix stained with safranin O. Fast green binds to collagen fibers and is resistant to fading. The red triangle area in the figure is the labrum, where Safranin O deposition can be observed.

[0041] The present invention designs a 3D-printed polyurethane scaffold by simulating the anatomical structure of the labrum, and uses polydopamine to modify its surface to increase the hydrophilicity and cell adhesion of the scaffold. The effect of this new scaffold in promoting fibrocartilage regeneration has been verified in in vitro cell experiments and in vivo miniature pig animal experiments, respectively. It is expected to be used for the repair of acetabular labral tears and clinical applications.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a polyurethane artificial labrum of a hip joint, characterized in that: The steps include: Polyurethane is made into a circular polyurethane stent by 3D printing, and then the surface of the circular polyurethane stent is modified with polydopamine.

2. The method for preparing a polyurethane artificial labrum of a hip joint according to claim 1, characterized in that: The annular polyurethane bracket has a diameter of 30 mm and a cross section of a right triangle with a bottom width of 2.5 mm and a height of 3 mm.

3. The method for preparing a polyurethane artificial labrum of a hip joint according to claim 1, characterized in that: The molecular weight of the polyurethane is 50,000-60,000.

4. The method for preparing a polyurethane artificial labrum of a hip joint according to claim 1, characterized in that: The 3D printing step includes: dissolving polyurethane at a high temperature of 200° C. for 30 minutes, extruding polyurethane through a nozzle with a diameter of 300 μm at an air pressure of 0.7 MPa and 7.0 mm / s for 3D printing, and preparing a polyurethane bracket.

5. The method for preparing a polyurethane artificial labrum of a hip joint according to claim 1, characterized in that: The polydopamine modification step includes: soaking the polyurethane scaffold in a 2% polydopamine solution for 24 hours, wherein the solvent of the polydopamine solution is a 1M Tris-HCl buffer solution with a pH of 8.0, taking out the scaffold after it turns black, and washing away the uncross-linked dopamine with a phosphate buffer solution.

6. The method for preparing a polyurethane artificial labrum of a hip joint according to claim 5, characterized in that: The concentration of the phosphate buffer is 0.01 M, and the pH value is 7.2-7.

4.

7. Use of the artificial labrum prepared by the method for preparing a polyurethane artificial labrum of a hip joint according to any one of claims 1 to 6 in repairing acetabular labrum injuries.

8. The use according to claim 7, characterized in that The artificial labrum is fixed by suturing and used, and induces host cells to migrate and differentiate into fibrocartilage tissue.

Images