A drug-loaded sustained-release bionic soft cartilage scaffold imitating plant phloem and a preparation method thereof

Abstract

The application discloses a kind of imitated plant phloem layer drug-loading slow-release bionic cartilage scaffold and preparation method thereof.The scaffold includes hollow porous fiber three-dimensional network formed by cross-stacked hollow porous fiber body;Including drug-loading culture solution layer filled with hollow area;Including bone marrow mesenchymal stem cells wrapped in the gap between the outside.The method comprises the following steps: three-dimensional printing of polydimethylsiloxane (PDMS) composite ink is carried out by direct writing three-dimensional printing platform to obtain hollow porous fiber three-dimensional network;After high-temperature curing, water bath heating and high-temperature high-pressure sterilization, rinse, then immerse in cell culture solution for culture, then inoculate bone marrow mesenchymal stem cells for culture, to obtain drug-loading slow-release bionic cartilage scaffold.The application can match the mechanical properties of native cartilage, and has the functions of slow-release drug to surrounding tissue to kill bacteria and anti-inflammatory, and promote the regeneration of surrounding cartilage tissue, promote the gradual repair of damaged cartilage tissue.

Description

Technical Field

[0001] This invention relates to a biomimetic cartilage scaffold, specifically to a drug-loaded sustained-release biomimetic cartilage scaffold that mimics the phloem of a plant and its preparation method. Background Technology

[0002] Osteoarthritis (OA) is the most common type of arthritis, associated with joint pain and loss of joint function. While the cause of osteoarthritis can be idiopathic, the disease is typically characterized by degenerative changes in the articular cartilage due to wear and tear or injury, including sports-related injuries. Despite its prevalence, most current treatments are merely palliative and do not prevent further deterioration of the joint condition. Ultimately, many arthritis patients will require total joint replacement surgery, an invasive end-stage treatment that involves surgically removing part of the joint and replacing it with a metal implant. However, these metal implants wear down over time, gradually losing their original replacement function. Furthermore, because metal materials generally do not conform well to the human body, they can cause a foreign body sensation during movement.

[0003] In fact, articular cartilage is an avascular connective tissue with a low degree of cellularity. Due to the lack of blood vessels, articular cartilage has a very low capacity for spontaneous repair. Therefore, there is an urgent need to design a medical scaffold for articular cartilage repair that can be implanted into the human body when the articular cartilage is initially damaged. This medical scaffold needs to have a certain degree of stability in the human body to replace some of the function of the original cartilage, while also having a certain ability to promote the repair of surrounding damaged cartilage tissue. Summary of the Invention

[0004] To address the problems existing in the background art, the present invention provides a drug-loaded sustained-release biomimetic cartilage scaffold that mimics the phloem of plants and its preparation method. While matching the mechanical properties of native cartilage, it possesses the function of sustained-release of drugs to surrounding tissues for antibacterial and anti-inflammatory effects, as well as the function of promoting the regeneration of surrounding cartilage tissue and facilitating the gradual repair of damaged cartilage tissue.

[0005] The technical solution adopted in this invention is:

[0006] I. A biomimetic cartilage scaffold with drug-loaded sustained-release structure inspired by plant phloem:

[0007] The scaffold comprises a three-dimensional network of hollow porous fibers arranged in a cross-stacking manner from bottom to top.

[0008] The scaffold comprises a drug-loaded culture medium layer filling the hollow regions of each hollow porous fiber in a three-dimensional network of hollow porous fibers;

[0009] The scaffold comprises a layer of bone marrow mesenchymal stem cells that is wrapped around the gaps between the individual hollow porous fibrous bodies of a three-dimensional network of hollow porous fibers.

[0010] The drug-loaded culture solution layer fully fills the entire hollow porous fiber three-dimensional network; at the same time, the drug-loaded culture solution layer should completely fill the entire hollow porous fiber three-dimensional network under the driving of capillary phenomenon; the bone marrow mesenchymal stem cell layer is fully filled in the gap of the hollow porous fiber three-dimensional network.

[0011] The hollow porous fiber body is a columnar fiber structure with a porous wall and a hollow interior; each layer of the hollow porous fiber three-dimensional network includes a plurality of uniformly spaced hollow porous fiber bodies arranged in parallel, and the central axes of the hollow porous fiber bodies in each layer are located in the same plane; the single hollow porous fiber bodies between adjacent layers of the hollow porous fiber three-dimensional network are arranged in a 0° / 90° or 0° / 45° / 90° cross-stacking manner, which can enhance the mechanical properties in the fiber direction, match the mechanical properties of the cartilage scaffold with the original cartilage, and at the same time, the cross-stacking manner also provides sufficient development space for the subsequent growth and proliferation of bone marrow mesenchymal stem cells on the cartilage scaffold, avoiding the problem of low cell survival rate caused by excessive compression due to insufficient space.

[0012] The hollow porous fiber body is analogous to the plant phloem, and the porous structure of the wall of the hollow porous fiber body corresponds to the sieve tube in the plant phloem, which plays a role in slow-release of drugs, and the hollow structure in the interior corresponds to the vessel in the plant phloem, which plays a role in storage of drugs.

[0013] The outer diameter of the hollow porous fiber body is 550 microns, the inner diameter is 300 microns, and the wall thickness is 250 microns, which ensures that the culture solution can be absorbed into the interior of the hollow porous fiber body under the driving of capillary phenomenon.

[0014] The hollow porous fiber body is made of polydimethylsiloxane (PDMS) material.

[0015] The drug-loaded culture solution layer uses ground gallium nitrate particles with a particle size of less than or equal to 70 microns.

[0016] II. A preparation method of a drug-loaded slow-release bionic cartilage scaffold simulating plant phloem, comprising:

[0017] 1) Add polydimethylsiloxane (PDMS) composite ink to a direct writing three-dimensional printing platform, and perform three-dimensional printing on the direct writing three-dimensional printing platform to obtain a sample, i.e., a hollow porous fiber three-dimensional network composed of a plurality of hollow porous fiber bodies arranged in a cross-stacking manner from bottom to top.

[0018] 2) the hollow porous fiber three-dimensional network is sequentially subjected to high-temperature curing, water bath heating, and high-temperature high-pressure sterilization, then rinsed, immersed in a cell culture solution for culture, and then the bone marrow mesenchymal stem cells are inoculated into the hollow porous fiber three-dimensional network for culture until the bone marrow mesenchymal stem cells completely fill the space of the hollow porous fiber three-dimensional network, and a drug-loaded sustained-release biomimetic cartilage scaffold is obtained.

[0019] In the step 1), the dimethylsiloxane PDMS composite ink is mixed by Sylgard 184 dimethylsiloxane PDMS prepolymer, SE 1700 dimethylsiloxane PDMS prepolymer, gallium nitrate particles, and sodium bicarbonate particles in a mass ratio of 1:9:5:5.

[0020] The SE 1700 dimethylsiloxane PDMS prepolymer functions as a rheological modifier to obtain an ink with good direct writing printability, and the gallium nitrate particles and sodium bicarbonate particles complete the manufacture of the porous structure in the hollow porous fiber three-dimensional network through the combination of particle precipitation and gas foaming.

[0021] In the step 1), the direct writing three-dimensional printing platform includes a pneumatic extrusion device at the top, a coaxial nozzle following the movement of the three-axis moving platform at the bottom, and a sample collection platform directly below the coaxial nozzle, the dimethylsiloxane PDMS composite ink is installed in the coaxial nozzle, and the dimethylsiloxane PDMS composite ink is extruded from the coaxial nozzle by pneumatic extrusion through the pneumatic extrusion device, the extruded sample is on the sample collection platform directly below the coaxial nozzle, and a hollow porous fiber three-dimensional network is obtained; the inner needle in the coaxial nozzle is selected from a solid needle 27G-30G type, and the outer needle is selected from a hollow needle 18G-20G type, and the inner and outer needles cooperate to form the coaxial nozzle.

[0022] In the step 2), the high-temperature curing is specifically 15 min at a curing temperature of 80℃ in a high-temperature heating furnace; the water bath heating is specifically at least 6 hours in a hot water bath at a water bath temperature of 100℃ in a water bath kettle to form a porous structure; the rinsing after high-temperature high-pressure sterilization is specifically sterilization in a high-temperature high-pressure sterilization pot at 150℃ and 200kPa, and then rinsing at least 5 times with deionized water and a D-PBS solution.

[0023] Under the condition of water bath heating, the sodium bicarbonate particles will be decomposed by heat to produce carbon dioxide, and the porous structure in the hollow porous fiber three-dimensional network is completed through the gas foaming method.

[0024] In step 2), immersion in cell culture medium specifically involves immersing the treated hollow porous fiber three-dimensional network in a cell culture flask specifically designed for inoculating bone marrow mesenchymal stem cells under sterile conditions. Water-soluble gallium nitrate particles present in the hollow porous fiber three-dimensional network slowly release gallium ions, which have anti-inflammatory, bactericidal, and tissue regeneration-promoting effects, into the cell culture medium. The hollow porous fiber three-dimensional network then fills the cell culture medium containing the slowly released gallium ions through capillary action. Bone marrow mesenchymal stem cells are then inoculated into the hollow porous fiber three-dimensional network for culture, specifically cultured at 37°C and 5% carbon dioxide until they completely fill the gaps in the hollow porous fiber three-dimensional network.

[0025] The bottom of the culture flask has an upward convex structure, which can apply a certain external force to the hollow porous fiber three-dimensional network to promote the slow release of the drug-loaded culture medium inside the hollow porous fiber to the outside. Accompanying this process, the proliferation of bone marrow mesenchymal stem cells will be promoted to a certain extent.

[0026] The beneficial effects of this invention are:

[0027] This invention discloses a plant-inspired phloem-based drug-loaded sustained-release cartilage scaffold. Firstly, its hollow porous fiber network provides anisotropic mechanical properties, matching the mechanical characteristics of native cartilage, thus improving the scaffold's compliance with the human body and preventing excessive wear and foreign body sensation caused by mechanical property mismatch. Secondly, each hollow porous fiber in the network has a certain drug-loaded sustained-release effect. When implanted into the damaged cartilage site, the pressure from joint movement causes the drug-loaded culture medium layer within the network to gradually release, disinfecting and sterilizing surrounding tissues while promoting their repair and regeneration. Furthermore, the interlocking arrangement of fibers in the network provides ample space for cell growth. Finally, because articular cartilage has a very low degree of cellularization, its self-repair and regeneration capabilities are poor. The bone marrow mesenchymal stem cell layer surrounding the hollow porous fiber network in this invention provides sufficient cells for the repair and regeneration of surrounding tissues, playing a crucial role in this process. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the drug-loaded sustained-release plant-derived phloem cartilage scaffold of the present invention;

[0029] Figure 2 This is a schematic diagram of the direct-write coaxial 3D printing device of the present invention;

[0030] Figure 3 This is a flowchart of the preparation method of the present invention;

[0031] In the figure: 1. Hollow porous fiber body, 2. Drug-loaded culture medium layer, 3. Bone marrow mesenchymal stem cell layer, 4. Pneumatic extrusion device, 5. Polydimethylsiloxane PDMS composite ink, 6. Coaxial printhead, 7. Sample, 8. Sample collection platform. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Specific embodiments of the present invention are as follows:

[0034] like Figure 1 As shown, the hollow porous fiber three-dimensional network of the drug-loaded sustained-release plant-derived phloem cartilage scaffold adopts a seven-layer structure, with the hollow porous fiber bodies 1 between each layer stacked alternately at 0° / 90°.

[0035] like Figure 2 and Figure 3 As shown, the specific method for preparing a drug-loaded, sustained-release, plant-derived phloem cartilage scaffold is as follows:

[0036] 1) Add polydimethylsiloxane PDMS composite ink 5 into the direct-write 3D printing platform, and obtain sample 7 by 3D printing through the direct-write 3D printing platform, that is, a hollow porous fiber three-dimensional network composed of several hollow porous fiber bodies 1 arranged crosswise from bottom to top.

[0037] The polydimethylsiloxane (PDMS) composite ink 5 is composed of 0.6g, 5.4g, 3g, and 3g of Sylgard 184 PDMS prepolymer, SE 1700 PDMS prepolymer, gallium nitrate particles, and sodium bicarbonate particles, respectively. On one hand, PDMS possesses sufficient mechanical strength, good stability, and biocompatibility in the human body; on the other hand, the appropriate ratio of Sylgard 184 and SE 1700 also exhibits good printability. The sodium bicarbonate particles in the ink are responsible for the preparation of the porous structure, while the gallium nitrate particles are responsible for the subsequent drug-carrying and sustained-release function. The culture medium in the drug-carrying culture medium layer inside the hollow porous fiber three-dimensional network is fully absorbed into the network until it is completely filled through the capillary effect generated by the hollow structure of each fiber. At this point, the gallium nitrate particles pre-existing in the porous structure release gallium ions into the culture medium, thus enabling the culture medium to carry the drug.

[0038] The inner needle of the coaxial nozzle 6 is a solid needle of model 27G-30G, and the outer needle is a hollow needle of model 18G-20G. The inner and outer needles work together to form the coaxial nozzle 6.

[0039] 2) The hollow porous fiber three-dimensional network was cured in a high-temperature heating furnace at 80℃ for 15 minutes, then in a water bath at 100℃ for 6 hours to form a porous structure. It was then sterilized in a high-temperature, high-pressure autoclave at 150℃ and 200kPa, and rinsed five times each with deionized water and DuPont phosphate buffer solution. Finally, it was immersed in cell culture medium for inoculation. Specifically, the treated hollow porous fiber three-dimensional network was immersed in a culture flask specifically designed for inoculating bone marrow mesenchymal stem cells under aseptic conditions. In the cell culture medium, water-soluble gallium nitrate particles present in the hollow porous fiber three-dimensional network release gallium ions, which have anti-inflammatory, bactericidal, and tissue regeneration-promoting effects, into the cell culture medium. Then, the hollow porous fiber three-dimensional network fills the cell culture medium containing the gallium ions through capillary action. Bone marrow mesenchymal stem cells are then seeded into the hollow porous fiber three-dimensional network for culture. Specifically, the bone marrow mesenchymal stem cells are cultured in an environment of 37°C and 5% carbon dioxide until they completely fill the gaps in the hollow porous fiber three-dimensional network, thus obtaining a drug-loaded sustained-release biomimetic cartilage scaffold.

[0040] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomimetic cartilage scaffold with drug-loaded sustained release mimicking plant phloem, characterized in that: It includes a three-dimensional network of hollow porous fibers (1) arranged in a cross-stacking manner from bottom to top; The drug-loaded culture medium layer (2) includes the hollow regions of each hollow porous fiber body (1) that are filled in the hollow porous fiber three-dimensional network; Including the bone marrow mesenchymal stem cell layer (3) in the gaps between the various hollow porous fiber bodies (1) wrapped in the hollow porous fiber three-dimensional network; The hollow porous fiber body (1) is a columnar fiber structure with porous walls and hollow interior; each layer of the hollow porous fiber three-dimensional network includes several hollow porous fiber bodies (1) arranged in parallel with uniform intervals, and the central axis of the hollow porous fiber bodies (1) in each layer is located on the same plane; the single hollow porous fiber bodies (1) between adjacent layers of the hollow porous fiber three-dimensional network are arranged in a cross-stacking manner of 0° / 90° or 0° / 45° / 90°; the spacing between any two adjacent hollow porous fiber bodies (1) in each layer of the hollow porous fiber three-dimensional network is 400 micrometers; The hollow porous fiber body (1) is prepared using polydimethylsiloxane PDMS material; The drug loaded in the drug-loaded culture medium layer (2) is made of ground gallium nitrate particles with a particle size of less than or equal to 70 micrometers.

2. The biomimetic cartilage scaffold with drug-loaded sustained release based on the plant phloem layer according to claim 1, characterized in that: The hollow porous fiber body (1) has an outer diameter of 550 micrometers, an inner diameter of 300 micrometers, and a wall thickness of 250 micrometers.

3. The method for preparing a biomimetic cartilage scaffold with drug-loaded sustained release based on any one of claims 1-2, characterized in that, include: 1) Add polydimethylsiloxane PDMS composite ink (5) into the direct writing 3D printing platform, and obtain the sample (7) by 3D printing through the direct writing 3D printing platform, that is, a hollow porous fiber three-dimensional network composed of several hollow porous fiber bodies (1) arranged crosswise from bottom to top. 2) After the hollow porous fiber three-dimensional network is subjected to high temperature curing, water bath heating and high temperature and high pressure sterilization in sequence, it is rinsed and then immersed in cell culture medium for culture. Then, bone marrow mesenchymal stem cells are seeded into the hollow porous fiber three-dimensional network for culture until the bone marrow mesenchymal stem cells completely fill the gaps of the hollow porous fiber three-dimensional network to obtain a drug-loaded sustained-release biomimetic cartilage scaffold. In step 2), high-temperature curing specifically involves curing at 80°C for 15 minutes in a high-temperature heating furnace; water bath heating specifically involves bathing in a water bath at 100°C for at least 6 hours to form a porous structure; and rinsing after high-temperature and high-pressure sterilization specifically involves sterilizing in a high-temperature and high-pressure sterilizer at 150°C and 200 kPa, and then rinsing at least 5 times with deionized water and Durham phosphate buffer solution respectively.

4. The method for preparing the biomimetic cartilage scaffold with drug-loaded sustained release in the phloem layer according to claim 3, characterized in that: In step 1), the polydimethylsiloxane PDMS composite ink (5) is a mixture of Sylgard 184 polydimethylsiloxane PDMS prepolymer, SE 1700 polydimethylsiloxane PDMS prepolymer, gallium nitrate particles and sodium bicarbonate particles in a mass ratio of 1:9:5:

5.

5. The method for preparing the biomimetic cartilage scaffold with drug-loaded sustained release in the phloem layer according to claim 3, characterized in that: In step 1), the direct-write 3D printing platform includes a pneumatic extrusion device (4) at the top, a coaxial nozzle (6) at the bottom, and a sample collection platform (8) directly below it. The dimethylsiloxane PDMS composite ink (5) is installed in the coaxial nozzle (6). The dimethylsiloxane PDMS composite ink (5) is extruded from the coaxial nozzle (6) by the pneumatic extrusion device (4) in the form of air pressure extrusion. The extruded sample (7) is placed on the sample collection platform (8) directly below the coaxial nozzle (6) to obtain a hollow porous fiber three-dimensional network.

6. The method for preparing the biomimetic cartilage scaffold with drug-loaded sustained release in the phloem layer according to claim 4, characterized in that: In step 2), immersion in cell culture medium specifically involves immersing the treated hollow porous fiber three-dimensional network in cell culture medium in a culture flask under sterile conditions. Gallium nitrate particles present in the hollow porous fiber three-dimensional network slowly release gallium ions into the cell culture medium, and then the hollow porous fiber three-dimensional network is filled with the cell culture medium containing slowly released gallium ions. Bone marrow mesenchymal stem cells are seeded into the hollow porous fiber three-dimensional network for culture, specifically cultured in an environment of 37°C and 5% carbon dioxide until they completely fill the gaps in the hollow porous fiber three-dimensional network. The bottom of the culture flask has an upward convex structure.

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