A 3D printed bionic meniscus scaffold and preparation method thereof

Abstract

The present invention discloses a 3D printed bionic meniscus scaffold and a preparation method thereof. It belongs to the field of medical prosthesis technology. The meniscus scaffold prepared by the present invention is composed of a layer of annular fiber layer and two layers of rotating grid-shaped fiber layers stacked from bottom to top, and has good mechanical properties. In biological tissue engineering technology, it can effectively avoid the adverse effects caused by the formation of a well-shaped barrel structure, which is conducive to the growth of cells in all directions, and provides a framework and metabolic site for cell adhesion, proliferation, differentiation and the final formation of new tissues. The present invention also provides a preparation method for the above-mentioned meniscus, which effectively simulates the internal collagen fiber structure of the meniscus through 3D printing technology, and has more excellent biomechanical properties, biocompatibility and the ability to promote cell secretion of extracellular matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of medical prosthesis, and in particular to a 3D printed bionic meniscus bracket and a preparation method thereof. Background Art

[0002] Meniscus injury is a common cartilage disease of the knee joint, with an average annual incidence of 0.66%, which is more common in adolescents. The meniscus has many important biomechanical functions, including load transmission, shock absorption, joint stabilization, and lubrication and nutrition of articular cartilage. In view of the important functions of the meniscus, it should be preserved and repaired as much as possible in the treatment after its injury. However, due to factors such as poor blood supply and healing ability of the meniscus, most patients currently have to adopt the method of meniscus resection (or partial resection) for treatment. Although some clinical symptoms can be relieved after surgery, meniscus defects (or partial defects) will lead to severe abnormal knee joint mechanical state, articular cartilage damage and dysfunction and other adverse consequences. In response to the above problems, foreign countries have begun to study allogeneic meniscus transplantation technology, which has gradually attracted attention and is currently considered to be the main method to solve the problem of meniscus resection. However, allogeneic meniscus transplantation technology has problems such as a small source of donors and the risk of disease transmission, so it is greatly restricted and difficult to be widely carried out.

[0003] In response to the above technical difficulties, tissue engineering technology has brought hope for the regeneration and repair of meniscus injuries. The three major elements of tissue engineering technology - seed cells, induction factors and scaffolds, play the role of "seeds", "fertilizers" and "soil" respectively. Among them, the scaffold, as a temporary substitute for the extracellular matrix, provides a three-dimensional geometric structure for engineered tissues, and provides a framework and metabolic site for cell adhesion, proliferation, differentiation and the final formation of new tissues. It is a key factor in the success of tissue engineering.

[0004] The 3D bionic meniscus structure existing in the prior art, such as patent application number: 202121399365.8, the name of the invention is: A 3D printed diamond-shaped variable-aperture PCL meniscus tissue engineering scaffold. Although it also involves a meniscus scaffold, its single structure cannot truly simulate the morphology and structure of the meniscus and the internal fiber orientation, and the single-layer diamond-shaped variable aperture design is easy to form a well-shaped barrel structure, which is not conducive to cell climbing and growth in tissue engineering technology; and the fiber density in each direction is different, which is conducive to the conduction of circumferential tension.

[0005] Therefore, how to provide a 3D printed bionic meniscus scaffold with excellent biomechanical properties and more conducive to cell growth and a preparation method thereof is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a 3D printed bionic meniscus scaffold and a preparation method thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A 3D printed bionic meniscus scaffold, the meniscus scaffold is composed of one or more fiber frame structures stacked layer by layer from bottom to top; each fiber frame structure is composed of: one annular fiber layer and two rotating grid fiber layers stacked layer by layer from bottom to top.

[0009] The beneficial effect of the above operation is that the alternating printing of a ring-shaped fiber layer and two rotating grid-shaped fiber layers can effectively avoid the adverse effects of forming a well-shaped barrel structure, which is conducive to the growth of cells in all directions.

[0010] Preferably, the annular fiber layer and the rotating grid-shaped fiber layer are both printed from molten polycaprolactone material to simulate meniscus fibers; the diameter of the printed fibers is 150 um.

[0011] Preferably, the annular fiber layer is composed of three equally divided fiber zones, namely an inner annular polycaprolactone fiber zone, a middle annular polycaprolactone fiber zone and an outer annular polycaprolactone fiber zone, and the fiber density of the three fiber zones increases successively from the inside to the outside.

[0012] The beneficial effects of the above operation are as follows: the density of the annular fiber layer gradually increases from the inside to the outside. In the outer edge of the meniscus, as the density of the annular fibers increases, the ability to absorb and conduct annular tension is enhanced, and the dispersion of axial stress is also utilized.

[0013] Preferably, the fiber spacing of the inner ring polycaprolactone fiber zone is 285-315 um, the fiber spacing of the middle ring polycaprolactone fiber zone is 190-210 um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 142.5-157.5 um.

[0014] Further preferably, the fiber spacing of the inner ring polycaprolactone fiber zone is 300 um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200 um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150 um.

[0015] Preferably, the rotating grid-shaped fiber layer is composed of polycaprolactone material simulating meniscus fibers, which are crisscross printed and rotated by 45-60°;

[0016] The beneficial effect of the above operation is that the rotating grid-shaped fiber layer presents an approximately radial distribution from the center to the edge after rotation, and has a mesh-like interlaced form, and the fiber density in each direction is the same, which is conducive to the conduction of hoop tension.

[0017] The pore size of the rotating grid-shaped fiber layer is 100-300um.

[0018] Further preferably, the rotated grid-shaped fiber layer is composed of polycaprolactone material simulating meniscus fibers, which are crisscross printed and rotated by 45°;

[0019] The pore size of the rotating grid-shaped fiber layer is 250 um.

[0020] Preferably, the inner diameter of the meniscus is 6 mm, the outer diameter is 9 mm, the distance between the inner and outer edges is 3 mm; and the thickness is 2 mm.

[0021] Another object of the present invention is to provide a method for preparing the above-mentioned 3D printed bionic meniscus scaffold, comprising the following steps:

[0022] (1) Scanning the knee meniscus using Micro-MRI to obtain scanning data;

[0023] (2) Import the scan data into the Mimics medical imaging control system software to reconstruct the medial and lateral meniscus models;

[0024] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0025] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0026] The beneficial effects of the above operation are: through digital simulation modeling of the knee meniscus and 3D bioprinting technology, a tissue engineering meniscus scaffold is constructed, which simulates the internal structural characteristics of the natural meniscus scaffold, is closer to the natural bionic structure, and has better biomechanical properties, biocompatibility and the ability to promote cell secretion of extracellular matrix than the meniscus scaffolds in the existing technology.

[0027] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The 3D printed bionic meniscus scaffold constructed by the present invention effectively simulates the internal structural characteristics of the natural meniscus scaffold, constructs an internal structure in which an annular fiber layer and two layers of rotating grid-shaped fiber layers are stacked from bottom to top, so that the ability to absorb and conduct annular tension is enhanced, and it is also beneficial to the dispersion of axial stress, so that the meniscus scaffold of the present invention has good mechanical properties; at the same time, the internal structure in which the annular fiber layer and the rotating grid-shaped fiber layer are alternately stacked can also effectively avoid the adverse effects caused by the formation of a well-shaped barrel structure, which is beneficial to the growth of cells in all directions;

[0029] (2) The composite fibrin gel and PCL material are used to prepare the 3D printed bionic meniscus scaffold of the present invention, so that the meniscus scaffold combines the advantages of the two materials, has good cell compatibility, and the ability to promote cells to secrete extracellular matrix, can well stimulate the regeneration of surrounding tissues, and gradually degrade with the growth of new tissues; it also provides the biomechanical strength required for the meniscus scaffold, and provides a suitable microenvironment for cells in terms of spatial structure and nutrients for cell growth, thereby promoting the repair and regeneration of the meniscus. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 The attached figure is: a physical picture of the 3D printed bionic meniscus scaffold;

[0032] Figure 2 The attached figure is: a schematic diagram of the design of the annular fiber layer;

[0033] Figure 3 The attached figure is: a schematic diagram of the design of a rotating grid-shaped fiber layer;

[0034] Figure 4 The attached figure is: a schematic diagram of the fiber design inside the model;

[0035] Note: A1 is the whole model of the meniscus divided equally into 3 annular fiber layers, A2 is the whole annular fiber slice; A3 is the schematic diagram of the single annular fiber layer;

[0036] B1 is the overall model of the meniscus with a rotating square fiber layer, B2 is the overall rotating square fiber slice, and B3 is a schematic diagram of a rotating square fiber single layer;

[0037] C1-C3 are schematic diagrams of the structure of the first layer, the first two layers, and the first three layers after stacking layer by layer;

[0038] Figure 5 The attached drawings are: SEM images of the stents of Examples 1-5;

[0039] Figure 6 The attached figure is: a measurement diagram of the wire diameter and fiber spacing of the annular fiber layer of the stent in Example 4;

[0040] Figure 7 The attached figure is: a measurement diagram of the wire diameter and pore diameter of the rotating grid fiber layer of the stent in Example 4;

[0041] Figure 8 The accompanying drawings are: Compression modulus data diagram of Examples 1-5;

[0042] Fig. 9 The accompanying drawings are: a graph showing the tensile modulus data of Examples 1-5;

[0043] Fig.10 The accompanying drawings are: a graph showing the growth curves of fibrochondrocytes in four groups of scaffolds in Example 10;

[0044] Fig.11 The accompanying drawings are: GAG and DNA ratios of four groups of scaffolds in Example 10 after 1, 7, and 14 days of culture;

[0045] Figure 12-15 The attached figure shows the live and dead staining results of the PCL+FG group and the FG group at 1, 7, 14 and 21 days in Example 10. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] A 3D printed bionic meniscus bracket, the inner diameter of the meniscus is 6mm, the outer diameter is 9mm, the inner and outer edge spacing is 3mm; the thickness is 2mm, (the actual picture of the 3D printed bionic meniscus bracket is as shown in Figure 1 As shown in the attached figure); the diameter of the polycaprolactone fiber is 150um;

[0049] Annular fiber layer (the design diagram of the annular fiber layer is shown in Figure 2 The fiber spacing of the inner ring polycaprolactone fiber zone is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um; the rotating grid fiber layer (the schematic diagram of the rotating grid fiber layer design is shown in Figure 3 The aperture of the optical fiber (as shown in the attached figure) is 100um; the rotation angle is 45°.

[0050] A method for preparing a 3D printed bionic meniscus scaffold comprises the following steps:

[0051] (1) Scanning the rabbit knee meniscus using Micro-MRI to obtain scanning data;

[0052] (2) The scan data was imported into the Mimics medical imaging control system software to reconstruct the model of the medial and lateral menisci of the rabbit (the schematic diagram of the internal fiber design of the model is shown in the figure). Figure 4 as shown in the attached figure);

[0053] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0054] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0055] Example 2

[0056] A 3D printed bionic meniscus scaffold, the inner diameter of the meniscus is 6mm, the outer diameter is 9mm, the inner and outer edge spacing is 3mm; the thickness is 2mm; the diameter of the polycaprolactone fiber is 150um; the fiber spacing of the inner ring polycaprolactone fiber zone in the annular fiber layer is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um; the aperture of the rotated grid-shaped fiber layer is 150um; and the rotation angle is 45°.

[0057] A method for preparing a 3D printed bionic meniscus scaffold comprises the following steps:

[0058] (1) Scan the rabbit knee meniscus using Micro-MRI to obtain scanning data;

[0059] (2) Import the scan data into the Mimics medical imaging control system software and reconstruct the medial and lateral meniscus models of the rabbit;

[0060] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0061] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0062] Example 3

[0063] A 3D printed bionic meniscus scaffold, the inner diameter of the meniscus is 6mm, the outer diameter is 9mm, the inner and outer edge spacing is 3mm; the thickness is 2mm; the diameter of the polycaprolactone fiber is 150um; the fiber spacing of the inner ring polycaprolactone fiber zone in the annular fiber layer is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um; the aperture of the rotated grid-shaped fiber layer is 200um; and the rotation angle is 45°.

[0064] A method for preparing a 3D printed bionic meniscus scaffold comprises the following steps:

[0065] (1) Scan the rabbit knee meniscus using Micro-MRI to obtain scanning data;

[0066] (2) Import the scan data into the Mimics medical imaging control system software and reconstruct the medial and lateral meniscus models of the rabbit;

[0067] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0068] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0069] Example 4

[0070] A 3D printed bionic meniscus scaffold, the inner diameter of the meniscus is 6mm, the outer diameter is 9mm, the inner and outer edge spacing is 3mm; the thickness is 2mm; the diameter of the polycaprolactone fiber is 150um; the fiber spacing of the inner ring polycaprolactone fiber zone in the annular fiber layer is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um; the aperture of the rotated grid-shaped fiber layer is 250um; and the rotation angle is 45°.

[0071] A method for preparing a 3D printed bionic meniscus scaffold comprises the following steps:

[0072] (1) Scan the rabbit knee meniscus using Micro-MRI to obtain scanning data;

[0073] (2) Import the scan data into the Mimics medical imaging control system software and reconstruct the medial and lateral meniscus models of the rabbit;

[0074] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0075] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0076] Example 5

[0077] A 3D printed bionic meniscus scaffold, the inner diameter of the meniscus is 6mm, the outer diameter is 9mm, the inner and outer edge spacing is 3mm; the thickness is 2mm; the diameter of the polycaprolactone fiber is 150um; the fiber spacing of the inner ring polycaprolactone fiber zone in the annular fiber layer is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um; the aperture of the rotated grid-shaped fiber layer is 300um; and the rotation angle is 45°.

[0078] A method for preparing a 3D printed bionic meniscus scaffold comprises the following steps:

[0079] (1) Scan the rabbit knee meniscus using Micro-MRI to obtain scanning data;

[0080] (2) Import the scan data into the Mimics medical imaging control system software and reconstruct the medial and lateral meniscus models of the rabbit;

[0081] (3) The reconstructed model was imported into the slic3r slicing software, the internal structure of the meniscus was biomimetically designed, the meniscus scaffold with the above structure was determined, and the G code recognizable by the 3D bioprinter was exported;

[0082] (4) The reconstructed meniscus model is enlarged in proportion and simplified, and a bioprinter is used to print the meniscus scaffold according to the structure of the meniscus scaffold determined in step (3).

[0083] Example 6

[0084] Stent microstructure testing

[0085] The 3D printed bionic meniscus scaffolds prepared in Examples 1-5 were scanned using a scanning electron microscope, and the scanning SEM images thereof are shown in FIG. Figure 5 As shown in the attached figure, it can be seen from the SEM image that the surface of each stent is smooth and flat, the wire diameter is stable, the pore size distribution is uniform, and all show a clear and regular three-dimensional pore structure;

[0086] The wire diameter, pore size and fiber spacing of the annular fiber layer and the rotating grid fiber layer of the 3D printed bionic meniscus scaffold prepared in Example 4 were measured under an electron microscope. The results are as follows: Figure 6 , 7 As shown in the attached figure;

[0087] Bionic PCL meniscus scaffolds of different specifications (the annular fiber layer is the same as in Examples 1-5) with a wire diameter of 100um and 200um, a pore size of 100um, 150um, 200um, 250um and 300um, a diameter of 9mm and a thickness of 2mm were prepared respectively, and compared with the scaffolds prepared in Examples 1-5. The wire diameter and pore size of the rotating grid fiber layer of each bionic scaffold were measured under an electron microscope, and the measurement results are shown in Table 1.

[0088] Table 1: Test values ​​and design values ​​of the rotating grid fiber layer, wire diameter and pore size of the 3D printed bionic meniscus scaffold

[0089]

[0090] Result analysis: It can be seen from Table 1 that the test values ​​of the wire diameter and pore size of the rotating grid fiber layer of the prepared 3D printed bionic meniscus scaffold are basically consistent with the design values, and the maximum error is within 5%, which can meet the experimental requirements.

[0091] Example 7

[0092] Determination of porosity

[0093] Bionic PCL meniscus scaffolds of different specifications (the annular fiber layer is the same as in Examples 1-5) with a rotating grid fiber layer wire diameter of 100um and 200um, a pore size of 100um, 150um, 200um, 250um and 300um, a diameter of 9mm and a thickness of 2mm were prepared respectively, and compared with the scaffolds prepared in Examples 1-5, and the porosity of the prepared 3D printed bionic meniscus scaffolds was determined by the drainage method.

[0094] Measurement formula: Porosity = (Vtotal-Vscaffold) / Vtotal*100%;

[0095] Vtotal = total volume of the scaffold;

[0096] Vstand = change in volume of liquid.

[0097] Measurement method: Calculate the total volume (Vtotal) of the prepared stents according to their length, width and height. Immerse the stents of various specifications in a suitable measuring cylinder filled with anhydrous ethanol. After 2 hours, observe whether there are no bubbles in the stents and observe the change in the liquid volume (Vstent). Set up 4 parallel experiments for each stent of different specifications and calculate the average value. The experimental results are shown in Table 2:

[0098] Table 2: Porosity of each scaffold

[0099]

[0100] Result analysis:

[0101] The growth of cells in the scaffold requires an increase in the total area of ​​the scaffold, and also requires an appropriate pore size. Too large or too small a pore size is not conducive to cell growth. In the aforementioned "PCL scaffold porosity" experimental data, when the wire diameter and pore size change synchronously, the porosity shows a trend of changing inversely with the wire diameter. This result shows that when the wire diameter of the scaffold is reduced, a larger porosity can be obtained (of course, increasing the pore size can also increase the porosity). The reason is that when the wire diameter is small, the collapse rate of the printed scaffold fiber is relatively low. When the wire diameter and the pore size take the same value and decrease synchronously, a limit porosity of 60.75% can be obtained. This allows the porosity to be adjusted as needed within the limit range. Therefore, this project team believes that a wire diameter of 150um and an pore size of 250um are the most suitable sizes.

[0102] Example 8

[0103] Biomechanical testing

[0104] According to the unique stress characteristics of the meniscus, the axial compression modulus and tensile modulus were tested to test the mechanical properties of each group.

[0105] Compression modulus: 6 groups of tests are set up, as follows:

[0106] Experimental group 1: single rotating grid fiber scaffold: wire diameter 150um, pore size 100um;

[0107] Experimental group 2: single rotating grid fiber scaffold: wire diameter 150um, pore size 150um;

[0108] Experimental group 3: single rotating grid fiber scaffold: wire diameter 150um, pore diameter 200um;

[0109] Experimental group 4: single rotating grid fiber scaffold: wire diameter 150um, pore diameter 250um;

[0110] Experimental group 5: single rotating grid fiber scaffold: wire diameter 150um, pore diameter 300um;

[0111] Experimental group 6: rotated grid fiber + annular fiber support: the rotated grid fiber has a wire diameter of 150um and a pore size of 250um; the fiber spacing in the inner ring polycaprolactone fiber zone of the annular fiber is 300um, the fiber spacing in the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing in the outer ring polycaprolactone fiber zone is 150um.

[0112] The above-mentioned stent was made into a cylindrical shape with a diameter of 9 mm and a height of 2 mm. The compression rate was 0.1 mm / min, the maximum compression volume was 70%, and a compression test was performed using a universal material testing machine to calculate the compression modulus. The experimental results are as follows: Figure 8 As shown in the attached figure.

[0113] Tensile modulus: The bionic scaffold was prepared into a dumbbell-shaped standard tensile specimen with a length of 75 mm, an inner length of 40 mm, an outer width of 10 mm, an inner width of 5 mm, and a thickness of 2 mm. The tensile rate was 50 mm / min. A compression test was performed using a universal material testing machine to calculate the tensile modulus. The experimental results are shown in Fig. 9 As shown in the attached figure.

[0114] Result analysis:

[0115] When the wire diameter is 150um, the axial compression modulus of the stent gradually decreases with the increase of the pore size. The compression modulus of the composite annular designed scaffold with an pore size of 250um (designed scaffold, annular fiber average pore size 215um) is similar to that of the mesh scaffold with an pore size of 100um. This shows that adding annular fiber design can significantly improve the axial compression modulus.

[0116] Depend on Fig. 9 It can be seen that when the wire diameter is 150um, the tensile modulus gradually decreases with the increase of the pore size. The tensile modulus of the designed scaffold (designed scaffold with an average pore size of 215um) is slightly worse than that of the scaffold with an pore size of 200um in terms of the circumferential tensile modulus and radial tensile modulus.

[0117] Example 9

[0118] Preparation of composite fibrin gel

[0119] (1) Preparation of gelatin / sodium alginate gel solution: Weigh 6 g gelatin and 1.2 g sodium alginate respectively, add physiological saline, stir and mix, dilute to 40 ml, and sterilize by pasteurization. Prepare gelatin / sodium alginate gel solution and store in a refrigerator at 4°C.

[0120] (2) Preparation of fibrinogen solution: Weigh 100 mg of fibrinogen powder, irradiate under ultraviolet light for 30 minutes, add 1 ml of 37°C saline, stir to mix, and prepare fibrinogen solution. Store in a refrigerator at 4°C.

[0121] (3) Take 800 μl of the above gelatin / sodium alginate gel solution and 200 μl of the fibrinogen solution, mix them evenly to make a composite fibrin gel, and place it in an incubator for later use.

[0122] Example 10

[0123] In order to explore the effects of different scaffold structures and scaffold materials on the growth of meniscus fibrochondrocytes, three groups of scaffolds were set up based on the 3D bionic meniscus scaffold prepared in Example 4 and the composite fibrin gel prepared in Example 9. Control experiments were carried out, with 3 parallel experiments in each group. P3 generation rabbit MFC cells were injected into the three groups of scaffolds to observe the effects of different scaffold materials on cell growth.

[0124] Experimental group 1: PCL scaffold, (PCL scaffold prepared in Example 4 + cells);

[0125] Experimental group 2: FG scaffold, (composite fibrin gel scaffold + cells);

[0126] Experimental group 3: PCL+FG scaffold, (PCL scaffold prepared in Example 4 + composite fibrin gel + cells);

[0127] The preparation of the three groups of scaffolds and their cell inoculation and culture are as follows:

[0128] (1) PCL scaffold preparation and cell inoculation and culture

[0129] The PCL meniscus scaffold prepared in Example 4 was placed in a 24-well plate. The P3 generation rabbit MFC cell suspension was slowly added dropwise to each scaffold so that the cell content in each scaffold was 1.0×10 6 Place the 24-well plate in a 37°C, 5% CO 2 Culture the cells in a cell culture incubator for 4 h. After 4 h, slowly add cell culture medium along the well wall and replace the culture medium every day.

[0130] (2) Preparation of composite fibrin gel scaffolds and cell inoculation and culture

[0131] The P3 generation rabbit MFC cell suspension was fully mixed with the composite fibrin gel to make the cell concentration in the gel 1.0×10 7 100ul of composite fibrin gel containing cells was used to prepare composite fibrin gel scaffolds, and the cell content in each scaffold was 1.0×10 6 The scaffolds prepared above were placed in 10 ml (3% Cacl 2 )+100ul thrombin in a culture dish, cross-link for 5 minutes, place the prepared composite fibrin gel scaffold in a 24-well plate, slowly add cell culture medium along the well wall, and incubate at 37°C and 5% CO 2 Culture the cells in a cell culture incubator with a controlled environment. Change the culture medium every 24 hours.

[0132] (3) Preparation of PCL-composite fibrin gel scaffolds and cell inoculation and culture

[0133] The P3 generation rabbit MFC cell suspension was fully mixed with the composite fibrin gel to make the cell concentration in the gel 1.0×10 7 100ul of the composite fibrin gel containing cells was injected into the PCL meniscus scaffold prepared in Example 4, so that the cell content in each scaffold was 1.0×10 6 Then the above scaffold was placed in 10 ml (3% Cacl 2 )+100ul thrombin in a culture dish, cross-link for 5min, place the prepared PCL-composite fibrin gel scaffold in a 24-well plate, slowly add cell culture medium along the well wall, and incubate at 37°C and 5% CO 2 Culture the cells in a cell culture incubator with a controlled environment. Change the culture medium every 24 hours.

[0134] Note: Thrombin storage solution: 100 UN bottled thrombin powder (Cat. No.: T6884, Sigma) is dissolved in 1 ml 0.1% BSA solution, mixed and dissolved to prepare 100 UN / ml (20X) storage solution.

[0135] Preparation of 0.1% BSA: Dissolve 10 mg BSA powder in 10 ml sterile PBS and mix well to dissolve.

[0136] 3% calcium chloride solution: Weigh 3g of calcium chloride powder, dilute to 100ml, stir to mix, filter through a 0.22μm filter and set aside.

[0137] Effects of the second and third groups of scaffolds on the proliferation of meniscus fibrochondrocytes

[0138] The PCL scaffold (PCL), composite fibrin gel scaffold (FG) and PCL-composite fibrin gel scaffold (PCL+FG) were placed in a 24-well plate. The flat plate group was used as the control group (2D). A total of 4 groups were used to measure the proliferation of cells in the scaffolds of each group on days 1, 4, 7, 14 and 21. The growth curves are shown in Figure 2. Fig.10 As shown in the attached figure.

[0139] Analysis of results: The 2D group had the fastest proliferation rate in the first 7 days; the FG group was similar to the 2D group and could proliferate rapidly at around 7 days, but both groups showed a decreasing trend after 7 days. The PCL group basically did not proliferate in the first 4 days, and began to proliferate slowly after the 4th day, reaching a peak on the 14th day, and then began to decline. In the 21-day proliferation experiment observation, the PCL+FG group showed an overall trend of slow proliferation. The proliferation rate was slower than the other three groups in the first 14 days, but it could reach about twice the initial concentration on the 21st day. It can be seen that the PCL composite gel group is more suitable for long-term cell growth and can significantly promote cell proliferation.

[0140] 3. Effects of three groups of scaffolds on the GAG ​​content of single cells in meniscus fibrocartilage

[0141] The PCL scaffold (PCL), composite fibrin gel scaffold (FG) and PCL-composite fibrin gel scaffold (PCL+FG) were cultured in four groups. The flat group was used as the control group (2D). The GAG ​​and DNA content of each group of scaffolds was measured after 1, 7, and 14 days of culture. The GAG / DNA ratio was used as the standard. The experimental results are shown in Fig.11 As shown in the attached figure.

[0142] DNA measurement method: The DNA in each group of scaffolds was extracted using a genomic DNA extraction kit, and then the DNA content was measured using the Nanodrop One micro-nucleic acid protein concentration meter.

[0143] GAG measurement method: The sample was placed in a 1.5 ml EP tube and the GAG ​​quantitative detection kit was used for the total content of tissue glycosaminoglycan (GAG) DMMB colorimetric quantitative detection kit.

[0144] Result analysis: Fig.11 As shown in the attached figure, there was no significant difference between the groups on the first day, but on the 7th and 14th days, the PCL+FG group had significantly higher GAG secretion values ​​per cell than the other three groups. In the 2D group and the PCL group, there was no significant increase in GAG secretion per cell. In the FG group, GAG secretion per cell increased slowly. In summary, the PCL+FG group can significantly promote the secretion of GAG and the synthesis of extracellular matrix of the meniscus cells.

[0145] 3. Live and dead cell staining of PCL+FG group and FG group scaffolds

[0146] The PCL+FG group and the FG group were cultured for 1, 7, 14, and 21 days, and cell live and dead staining was performed. 5ul.16Mm PI storage solution was added to 10ml PBS and mixed to obtain 8uM PI solution; 5μl 4mM Calcein AM storage solution was added to the above PI solution and vortexed to mix. Incubate at room temperature in the dark for 20min, wash once in PBS, observe and take pictures under a fluorescence microscope, and the experimental results are shown in the figure. Figure 12-15 As shown in the attached figure.

[0147] Results: The number of living cells in the two groups of scaffolds increased significantly. The ratio of living and dead cells in the two groups of scaffolds was basically the same, indicating that the PCL+FG group scaffold had good compatibility with implanted cells.

[0148] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0149] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A 3D printed bionic meniscus scaffold, It is characterized in that The meniscus support is composed of one or more groups of fiber frame structures stacked layer by layer from bottom to top; each group of fiber frame structures is composed of: one annular fiber layer and two rotating grid fiber layers stacked layer by layer from bottom to top; The annular fiber layer and the rotating grid-shaped fiber layer are both printed from molten polycaprolactone material to simulate meniscus fibers; the printed fiber diameter is 150um; The annular fiber layer is composed of three equally divided fiber regions, namely an inner annular polycaprolactone fiber region, a middle annular polycaprolactone fiber region and an outer annular polycaprolactone fiber region, and the fiber density of the three fiber regions increases from the inside to the outside; The fiber spacing of the inner ring polycaprolactone fiber zone is 285-315um, the fiber spacing of the middle ring polycaprolactone fiber zone is 190-210um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 142.5-157.5um; The rotating grid-shaped fiber layer is made of polycaprolactone material simulating meniscus fibers, printed in a criss-cross pattern, and rotated 45-60 degrees; The pore size of the rotating grid-shaped fiber layer is 100-300um.

2. A 3D printed bionic meniscus scaffold according to any one of claim 1, It is characterized in that The fiber spacing of the inner ring polycaprolactone fiber zone is 300um, the fiber spacing of the middle ring polycaprolactone fiber zone is 200um, and the fiber spacing of the outer ring polycaprolactone fiber zone is 150um.

3. A 3D printed bionic meniscus scaffold according to claim 1, It is characterized in that The rotating grid-shaped fiber layer is made of polycaprolactone material simulating meniscus fibers, printed in a criss-cross pattern, and rotated 45 degrees; The pore size of the rotating grid-shaped fiber layer is 250 um.

4. A 3D printed bionic meniscus scaffold according to claim 1, It is characterized in that The inner diameter of the meniscus is 6 mm, the outer diameter is 9 mm, the distance between the inner and outer edges is 3 mm, and the thickness is 2 mm.

5. The 3D printed bionic meniscus scaffold according to any one of claims 1 to 4, It is characterized in that The fibers of the scaffold also contain a composite fibrin gel; The composite fibrin gel is prepared by mixing gelatin, sodium alginate and fibrinogen.

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