A cartilage tissue engineering composite based on 3D printing and its application

Abstract

The present invention provides a cartilage tissue engineering complex. Specifically, the complex includes cartilage gel or cartilage membrane particles containing chondrocytes, and a 3D-printed porous frame, and the cartilage gel or cartilage membrane is loaded on the 3D-printed porous frame to form a cartilage gel / cartilage membrane particle-3D-printed porous frame complex. After the cartilage tissue engineering complex of the present invention is transplanted into a cartilage defect, stable cartilage regeneration can be achieved at the defect. The present invention also provides a method for preparing the cartilage tissue engineering complex and its use in achieving cartilage regeneration.

Description

Technical Field

[0001] The present invention relates to the field of biomedical tissue engineering, and in particular, to a 3D-printed cartilage tissue engineering composite and its application in cartilage regeneration. Background Art

[0002] In recent years, with the rapid development of the economy and society, cartilage defects caused by various types of trauma and congenital malformations have become increasingly common, and most cartilage defects caused by trauma and congenital malformations are difficult to repair by the patient's own body. In clinical treatment, the current treatment methods for articular cartilage defects are mainly palliative treatment and restorative treatment. Palliative treatment mainly includes arthroscopic cleaning and chondroplasty. This type of treatment can clean the uneven cartilage surface of the joint and remove cartilage fragments to restore the smoothness of the joint surface. This method is less invasive and can relieve the patient's symptoms to a certain extent, but its efficacy is limited and cannot effectively relieve the development of arthritis. Restorative treatment includes microfracture treatment, osteochondral transplantation, etc. Although this type of treatment can repair focal articular cartilage defects to a certain extent, it is more traumatic and can easily lead to donor site complications.

[0003] With the advancement of tissue engineering, people have gradually begun to study the use of scaffolds or tissues constructed by tissue engineering to try to repair cartilage defects. Tissue engineering is an interdisciplinary subject involving cell biology, material science, engineering, and bioreactors. It uses the basic principles and methods of life sciences and engineering to construct tissues needed by the human body to repair and replace tissues or organs that have no function due to trauma or disease.

[0004] In recent years, with the emergence of 3D printers, 3D printing technology has also been applied in many fields, including the medical industry. Injectable cartilage produced based on tissue engineering related technologies has also been applied in medical beauty, repair and reconstruction, etc., but there are still many disadvantages.

[0005] Based on this, 3D printing-based tissue engineering technology is expected to become a new method for constructing tissue engineering cartilage. Summary of the invention

[0006] The purpose of the present invention is to provide a cartilage tissue engineering composite based on 3D printing.

[0007] A first aspect of the present invention provides a cartilage tissue engineering composite, the composite comprising:

[0008] (a) a carrier comprising a 3D printed porous framework; and

[0009] (b) Cartilage gel or cartilage membrane sheet particles containing chondrocytes seeded or loaded on the carrier.

[0010] In another preferred embodiment, the complex comprises a complex formed by inoculating the cartilage gel or cartilage membrane particles into the carrier and culturing them into chondrocytes (in the complex, chondrocytes are loaded on the carrier and form a tighter integrated structure with the carrier).

[0011] In another preferred embodiment, the complex comprises a complex formed by inoculating the cartilage gel or cartilage membrane particles into the carrier but without chondrogenic culture.

[0012] In another preferred embodiment, the chondrocytes are derived from humans or non-human mammals.

[0013] In another preferred embodiment, the chondrocytes are derived from autologous chondrocytes or allogeneic chondrocytes, preferably autologous chondrocytes.

[0014] In another preferred embodiment, the chondrocytes are derived from elastic cartilage, fibrous cartilage or hyaline cartilage.

[0015] In another preferred embodiment, the chondrocytes are obtained from the subject's autologous chondrocytes.

[0016] In another preferred embodiment, the autologous chondrocytes include elastic chondrocytes, fibrous chondrocytes or hyaline chondrocytes.

[0017] In another preferred embodiment, the subject is a human or non-human mammal.

[0018] In another preferred embodiment, the subject has a joint defect.

[0019] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0020] In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect or a combination thereof.

[0021] In another preferred embodiment, the cartilage gel comprises a cell group composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix encapsulates the cell group, and the cartilage gel is in a gel state, and the density of the chondrocytes is at least 1.0×10 8 / ml or 1.0×10 8 Pieces / g.

[0022] In another preferred embodiment, the cartilage gel is prepared by culturing chondrocytes through gelation.

[0023] In another preferred embodiment, the gelled culture is an in vitro culture using a gelled culture medium.

[0024] In another preferred embodiment, the gelling medium contains the following components: high-glucose DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0025] In another preferred embodiment, the adhesion rate of the cartilage gel is ≥90%, preferably ≥95%.

[0026] In another preferred embodiment, the concentration of chondrocytes in the cartilage gel is 1.0×10 8 / ml-10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0027] In another preferred embodiment, the cartilage gel is obtained by gelation culture for 2.5-5.5 days, preferably 3-5 days.

[0028] In another preferred embodiment, the cartilage membrane particles include a cell group composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix encapsulates the cell group, and the cartilage particles are prepared by cutting and shredding the thin sheet of cartilage membrane, wherein the density of the chondrocytes is at least 1.0×10 8 / ml or 1.0×10 8 Pieces / g.

[0029] In another preferred embodiment, the concentration of chondrocytes in the cartilage membrane is 1.0×10 8 / ml-10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0030] In another preferred embodiment, the cartilage membrane sheet is obtained by gelling culture for 6-30 days, preferably 7-20 days, and most preferably 10-15 days.

[0031] In another preferred embodiment, the gelled culture is an in vitro culture using a gelled culture medium.

[0032] In another preferred embodiment, the gelling medium contains the following components: high-glucose DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0033] In another preferred embodiment, the thickness of the cartilage membrane sheet is 0.2-0.25 mm.

[0034] In another preferred embodiment, the average volume of the cartilage membrane particles is 0.2 μl.

[0035] In another preferred embodiment, the surface area of ​​the cartilage membrane particles is 0.05-10 mm 2 , preferably 1-5 mm 2 , more preferably, the average area is 1 mm 2 .

[0036] In another preferred embodiment, the 3D printed porous framework comprises an upper structure and a lower structure, the pore size of the structure is 100-300 mm; and is made of a degradable polymer material selected from the following group: polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer.

[0037] In another preferred embodiment, the 3D printed porous framework may also be loaded with gelatin, collagen, silk, hydrogel or a combination thereof.

[0038] In another preferred embodiment, the shape of the 3D printed porous frame includes a cylinder, a cuboid or other specific shapes.

[0039] The second aspect of the present invention provides a method for preparing a cartilage tissue engineering complex as described in the first aspect of the present invention, comprising the following steps: inoculating the cartilage gel or cartilage membrane particles into a 3D printed porous frame, and culturing cartilage in vitro to obtain the cartilage tissue engineering complex.

[0040] In another preferred embodiment, the cartilage gel is inoculated into the 3D printed porous framework by a direct filling method.

[0041] In another preferred embodiment, the cartilage membrane particles are inoculated into the 3D printed porous frame by centrifugation.

[0042] In another preferred embodiment, no liquid is added to the centrifugal system of the centrifugal method, and repeated centrifugation is used to allow the cartilage membrane sheet particles to enter the 3D printed porous framework.

[0043] In another preferred embodiment, the chondrogenic culture is an in vitro culture using a chondrogenic culture medium.

[0044] In another preferred embodiment, the chondrogenic culture medium has the following components: high-glucose DMEM culture medium, serum replacement, proline, vitamin C, transforming growth factor-β1 (TGF-β1), insulin-like growth factor 1 (IGF-I) and dexamethasone.

[0045] In another preferred embodiment, the serum substitute is ITS premix, which contains insulin, transferrin, selenious acid, linoleic acid, bovine serum albumin, pyruvic acid, and ascorbic acid phosphate.

[0046] In another preferred embodiment, the chondrogenic culture time is 3-15 days, preferably 5-11 days.

[0047] The third aspect of the present invention provides a use of the cartilage tissue engineering complex as described in the first aspect of the present invention for preparing a medical product for repairing joint defects.

[0048] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0049] In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect or a combination thereof.

[0050] The fourth aspect of the present invention provides a method for repairing joint defects, comprising the steps of: using the cartilage tissue engineering complex described in the first aspect of the present invention and transplanting it into the defective joint of the patient to be repaired.

[0051] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0052] In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect or a combination thereof.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The 3D printed framework is shown. Figure 1 A is the front view, Figure 1 B is the rear view. The scale in the figure is 400mm.

[0055] Figure 2 Schematic diagrams of ear cartilage gel and ear cartilage membrane obtained by culturing ear chondrocytes for 3 days and 15 days are shown, wherein AC is ear cartilage gel cultured for 3 days, DE is ear cartilage membrane cultured for 15 days (D and E) and ear cartilage membrane particles formed by cutting (F).

[0056] Figure 3 A real picture of the cartilage gel-3D printed porous framework composite is shown. Figure 3 A is the front view, Figure 3 B Side view.

[0057] Figure 4 The actual picture of the cartilage membrane sheet-3D printed porous framework complex is shown. Figure 4A is the front view, Figure 4 B Side view. DETAILED DESCRIPTION

[0058] After extensive and in-depth research, the inventors unexpectedly discovered and developed a cartilage tissue engineering complex for the first time, which is an integrated cartilage gel / cartilage membrane particle-3D printed porous framework complex. Experiments have shown that by obtaining primary cartilage for amplification culture, a specific number of chondrocytes are inoculated and / or spread on a flat or substantially flat culture surface, so that the inoculated chondrocytes form a specific stacked structure, and the stacked chondrocytes are cultured under suitable gelation culture conditions. Due to different culture times, a novel gel-like cartilage or membrane-like cartilage can be formed. The prepared gel-like cartilage or membrane-like cartilage is combined with a 3D printed porous framework, and the prepared cartilage tissue engineering complex can be regenerated into cartilage at the defect after being transplanted into the cartilage defect, which is expected to become a new method for cartilage regeneration. On this basis, the present invention is completed.

[0059] the term

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] As used herein, the term "cartilage tissue engineering complex" includes the cartilage gel-3D printed porous framework complex and the cartilage membrane particle-3D printed porous framework complex with and without in vitro chondrogenic culture as described herein, which can be collectively referred to as cartilage tissue engineering complexes in the present invention.

[0062] As used herein, the term "seeding" means inoculating chondrocytes in a cell culture dish, and may also mean inoculating cartilage gel / cartilage membrane particles in a 3D printed porous frame and distributing them evenly. A person skilled in the art can understand the meaning of "seeding" according to the context.

[0063] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0064] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0065] Cartilage gel and its preparation

[0066] As used herein, "gel cartilage", "cartilage gel", "gel-state cartilage", "gel-like cartilage", "cartilage gel of the present invention" or "gel cartilage of the present invention" are used interchangeably and all refer to the cartilage (stem) cells in a gel state of the present invention, especially the chondrocytes of a specific concentration are seeded and / or spread on a flat or substantially flat culture surface so that the seeded chondrocytes form a stacked structure, and the chondrocytes with a stacked structure are cultured under suitable gelation culture conditions to form a gel-like cartilage culture.

[0067] The gel cartilage of the present invention is a new type of cartilage different from free chondrocytes, centrifugally precipitated chondrocytes and cartilage pellets. The gel cartilage of the present invention can be regarded as a specific form of cartilage between free chondrocytes and dense cartilage pellets. In the gel cartilage of the present invention, during the gelation culture process, chondrocytes not only contact and / or interact with adjacent cells on the plane (XY plane), but also contact and / or interact with adjacent chondrocytes in multiple directions such as above and / or below and / or above or below, thereby promoting chondrocytes to secrete and form more extracellular matrix, thereby wrapping the gelation cultured chondrocytes in an extracellular matrix with a certain viscosity, so that the gel cartilage of the present invention has both a close connection and a certain viscosity and fluidity, so that the gel cartilage of the present invention is more suitable for inoculation and loading on various different carrier materials (especially porous carrier materials), thereby forming a complex for repairing cartilage.

[0068] In addition, the gel cartilage of the present invention has a gel state on the one hand and an unusually high cell density (usually at least 1.0×10 8 / ml or higher, such as 1.0×10 8 -10×10 8 Therefore, it is particularly suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.

[0069] In the present invention, the cartilage repairing complex includes a complex formed by loading the gel cartilage of the present invention on a carrier material (especially a 3D printed porous framework) and not subjected to chondrogenic culture, and also includes a complex formed by loading the gel cartilage of the present invention on a carrier material (especially a 3D printed porous framework) and subjected to chondrogenic culture.

[0070] In the present invention, the complex suitable for transplantation into the human body or animal body is the cartilage tissue engineering complex of the present invention, that is, the gel cartilage of the present invention is loaded on a carrier material (especially a 3D printed porous framework) and formed by chondrogenic culture.

[0071] Preferably, in the present invention, the gel cartilage is formed by culturing in vitro for a period of time t1 under gelling culture conditions. Preferably, the t1 is 2.5-5.5 days, preferably 3-5 days.

[0072] In the present invention, one feature is stacking inoculation, that is, after chondrocytes of a specific density are inoculated in a culture container, the inoculated chondrocytes will form a multi-layer chondrocyte group stacked on each other (i.e., a chondrocyte group with a stacking structure) through, for example, deposition. Typically, based on the culture area of ​​the culture dish (or culture container), and assuming that the confluence of the single layer of cells is 100%, the number of cells S1 of the stacking inoculation of the present invention is n times the number of cells S0 for 100% confluence (i.e., S1 / S0=n), wherein n is 1.5-20, preferably 2-10, and more preferably 2.5-5.

[0073] Cartilage membrane sheet and its preparation

[0074] As used herein, "cartilage sheet", "membranous cartilage", or "cartilage sheet of the present invention" are used interchangeably and all refer to the cartilage (stem) cells in a sheet state of the present invention, especially the chondrocytes of a specific concentration are inoculated and / or spread on a flat or substantially flat culture surface so that the inoculated chondrocytes form a stacked structure, and the chondrocytes with a stacked structure are cultured under appropriate culture conditions to form a membranous cartilage culture.

[0075] The "cartilage membrane sheet" of the present invention is prepared by prolonging the gelation culture time on the basis of the preparation of the "cartilage gel" of the present invention. That is, in the present invention, chondrocytes seeded and / or spread on a flat or substantially flat culture surface are cultured in vitro for a period of time t2 under gelation culture conditions, thereby forming a cartilage membrane sheet. Preferably, the t2 is 6-30 days, preferably 7-20 days, and most preferably 10-15 days.

[0076] On the one hand, the cartilage membrane sheet of the present invention has an unusually high cell density (usually at least 1.0×10 8 / ml or higher, such as 1.0×10 8 -10×10 8 On the one hand, it is thin (only 0.2-0.25mm) and has good toughness. It can be cut into "cartilage membrane particles" with an average volume of 0.2μl, and filled into a porous frame structure by simple centrifugation. Therefore, it is particularly suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.

[0077] In the present invention, the cartilage repair complex includes a complex formed by loading the cartilage membrane particles of the present invention on a carrier material (especially a porous framework structure) without chondrogenic culture, and also includes a complex formed by loading the cartilage membrane particles of the present invention on a carrier material (especially a porous framework structure) and culturing the cartilage.

[0078] In the present invention, the complex suitable for transplantation into the human body or animal body is the cartilage tissue engineering complex of the present invention, that is, the complex formed by loading the cartilage membrane particles of the present invention on a carrier material (especially a porous framework structure) and undergoing chondrogenic culture.

[0079] As used herein, "specific concentration" or "specific density" refers to the amount of 1.0×10 7 -2.0×10 7 cells, preferably 1.5×10 7 After gelation and culture for different time periods, the final density of chondrocytes was 1.0×10 8 -10×10 8 The density of cartilage gel or chondrocytes contained in it is 1.0×10 8 -10×10 8 / ml of cartilage membrane.

[0080] In another preferred embodiment, the gelation culture conditions are: inoculating chondrocytes at a specific density and culturing them using a gelation medium, wherein the gelation medium is a high-glucose (4-5wt% glucose) DMEM medium containing 10% fetal bovine serum and 100U / ml penicillin-streptomycin.

[0081] As used herein, the term "chondrogenic culture" refers to the use of chondrogenic culture medium to culture a porous framework structure inoculated with cartilage gel or cartilage membrane particles, ultimately forming an integrated cartilage gel-framework structure complex or cartilage membrane particle-framework structure complex, that is, the cartilage tissue engineering complex of the present invention, which is used for transplantation into cartilage defects in the human body or animal body.

[0082] Cartilage and chondrocytes

[0083] Cartilage is cartilage tissue, which is composed of chondrocytes and intercellular matrix. The matrix in cartilage is in a gel state and has great toughness. Cartilage is a connective tissue that mainly supports the bone. Cartilage does not contain blood vessels and lymphatic vessels. Nutrients penetrate from the blood vessels in the perichondrium into the intercellular matrix and then nourish the bone cells.

[0084] Cartilage can be divided into three types according to the difference in intercellular matrix, namely hyaline cartilage, elastic cartilage and fibrocartilage. The matrix of hyaline cartilage is composed of collagen fibers, fibrils and surrounding amorphous matrix. It has a temporary support function in the embryonic period, and this function is later replaced by bones. Adult hyaline cartilage is mainly distributed in the walls of the trachea and bronchus, the sternal end of the ribs and the surface of the bones (articular cartilage). In addition to collagen fibers, the matrix of elastic cartilage also contains elastic fibers. This type of cartilage has greater elasticity and is mainly distributed in the auricle, external auditory canal wall, Eustachian tube and epiglottis, larynx, etc. In the matrix of fibrocartilage, there are bundles of collagen fibers arranged in parallel or crosswise, which are relatively tough. It is distributed in the intervertebral disc, glenoid cavity, articular disc, and some tendons and ligaments to enhance the flexibility of movement and the functions of protection and support.

[0085] The chondrocytes used in the cartilage tissue engineering complex of the present invention can be hyaline cartilage, elastic cartilage or fibrous cartilage, which can be regenerated into cartilage to repair the defect after being implanted into the cartilage defect of the subject.

[0086] 3D Printing

[0087] 3D printing (3DP) is a type of rapid prototyping technology, also known as additive manufacturing. It is a technology that uses digital model files as the basis, uses powdered metal or plastic and other bondable materials to construct objects by printing layer by layer. 3D printing is usually achieved using digital technology material printers, and printing is completed after three-dimensional design and slicing.

[0088] The design process of 3D printing is: first modeling through computer modeling software, then "dividing" the built 3D model into layers of cross-sections, i.e. slices, to guide the printer to print layer by layer. The printer reads the cross-sectional information in the file, prints these cross-sections layer by layer with liquid, powder or sheet materials, and then glues the layers of cross-sections together in various ways to create a solid.

[0089] The 3D printed porous frame of the present invention is made by 3D printing technology, wherein the pore size is 100-300 mm. And the 3D printed porous frame is made of degradable polymer material, that is, the material used during printing is a degradable polymer material.

[0090] In another preferred embodiment, the degradable polymer material is selected from the following group: polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer.

[0091] Culture medium used in the present invention

[0092] Chondrogenic medium: high glucose DMEM medium, 1% 1×ITS premix (ITS universal culture mixture, containing insulin, transferrin, selenious acid, linoleic acid, bovine serum albumin, pyruvic acid, ascorbic acid phosphate), 40 μg / ml proline, 10 ng / ml TGF-β1, 100 ng / ml IGF-1, 40 ng / ml dexamethasone and 50 μg / ml vitamin C.

[0093] Gelation medium: DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0094] Adhesion rate

[0095] In the present invention, when the cartilage gel of the present invention is inoculated on a carrier material (especially a 3D printed porous frame), the cartilage gel of the present invention has a certain adhesion rate, which is determined by the adhesion rate determination method provided by the present invention. The adhesion rate of the cartilage gel of the present invention is ≥90%, preferably ≥95%.

[0096] The adhesion rate in the present invention is defined as follows:

[0097] The DNA quantification of the inoculated sample (eg, cartilage gel) is detected as A1; the DNA quantification of the inoculated complex (eg, cartilage gel-frame complex) after culturing for 24 hours is detected as A2; and the adhesion rate is A2 / A1*100%.

[0098] The method for measuring the adhesion rate comprises the following steps:

[0099] The inoculated sample (such as cartilage gel or cartilage gel-3D printed porous framework complex) was digested with proteinase K. The digested sample was quantitatively detected using a PicoGreen kit (Invitrogen, Carlsbad, CA, USA). The absorbance at 520 nm was measured using a fluorescent microplate reader, and the DNA content was calculated according to the standard curve formula.

[0100] The main advantages of the present invention include:

[0101] (1) The cartilage gel or cartilage membrane particles provided by the present invention are more mature than chondrocytes and have a certain fluidity.

[0102] (2) As a degradable polymer material, the 3D printed porous framework can be degraded in the body, has a low immune response and good biosafety.

[0103] (3) 3D printing The double-layer 3D printed porous framework material has a large pore size, good porosity, and is easy to inoculate, but the cell adhesion rate is extremely low when inoculated with chondrocyte suspension. The present invention uses cartilage tissue with a certain fluidity and relatively viscosity to effectively improve the adhesion rate.

[0104] (4) The 3D printed double-layer 3D printed porous framework material of the present invention includes an upper and lower layer structure, wherein the pore size of the upper layer structure is larger and easy to be inoculated; while the pore size of the lower layer structure is smaller to prevent the inoculated cells from leaking out.

[0105] (5) Simple cartilage gel or cartilage membrane particles cannot be formed, and the cartilage absorption rate is high under tension conditions, which limits their clinical application. The present invention uses a 3D printed porous frame as a frame structure to construct a cartilage gel / cartilage membrane particle-3D printed porous frame complex with a certain special shape, which provides mechanical support while effectively reducing the cartilage absorption rate.

[0106] (6) The 3D printed porous framework of the present invention can be flexibly customized into different shapes according to needs, and the constructed cartilage gel / cartilage membrane particle-3D printed porous framework complex can be used for repair and reconstruction of different parts and different shapes.

[0107] Below, the present invention is further described by specific examples. Below in conjunction with specific examples, the present invention is further described. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on the conditions described in the normal conditions, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, the materials and reagents used in the examples of the present invention are all commercially available products.

[0108] Example 1

[0109] Preparation of 3D printed porous framework

[0110] Based on the three-dimensional digital model, PCL substrate was printed by 3D printer (MAM-Ⅱ Free Form Fabrication System) to prepare millimeter-scale cartilage phase and bone phase split core mesh framework. By adjusting different layer widths, different deposition angles (-45° / 45°, -60° / 60° or 0° / 60° / 120°) and stainless steel needles of different diameters (19G, 20G or 21G), setting appropriate extrusion speed, printing speed and layer height and other printing parameters, 3D printed porous frameworks were prepared.

[0111] The prepared 3D printed porous framework is as follows Figure 1 shown.

[0112] in, Figure 1 A is the front view, Figure 1 B is the rear view. The scale in the figure is 400mm.

[0113] Example 2

[0114] Preparation of cartilage gel-3D printed porous framework composite

[0115] In this example, a cartilage gel-3D printed porous framework complex is prepared. The specific operation method is as follows:

[0116] (1) Take part of the subject's own ear cartilage tissue and cut a 2.5×2.5cm 2 Use sterile instruments to peel off the mucosa and fibrous tissue on the surface of the cartilage.

[0117] (2) Cut the cartilage tissue into 1.5×1.5 mm 2 Large and small cartilage fragments; prepared with 0.15% collagenase; added cartilage fragments into the prepared collagenase and digested for 8 hours;

[0118] (3) After 8 hours, the collagenase solution was filtered and centrifuged to obtain ear chondrocytes, and primary and subcultured cells were cultured using high-glucose DMEM medium containing 10% FBS;

[0119] (4) After amplification, the cells were collected and resuspended at 8×10 6 / 10ml to 30×10 6 / 10ml / well of cells were seeded in a six-well plate and cultured in a gelling medium (DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin);

[0120] (5) After 72 hours (3 days) of inoculation, the culture medium in the six-well plate was aspirated and gelatinous cartilage tissue was visible at the bottom of the six-well plate ( Figure 3 A), using forceps to collect the gelatinous cartilage at the bottom of the six-well plate ( Figure 2 B) The yield of gel cartilage in one well is 0.1-0.2 ml, which is collected into a 5 ml syringe. The cell density in the gel cartilage is 1.0×10 8 / ml-10×10 8 / ml, preferably 1.5-5×10 10 It was mixed with 0.15 ml of culture medium to prepare a preparation containing cartilage gel, such as Figure 2 As shown in C;

[0121] (6) The cartilage gel preparation (prepared in step (5), with a volume of about 0.25-0.35 ml) was inoculated into a 3D printed porous frame (prepared in Example 1, such as Figure 1 As shown), stand at 37°C, 95% humidity, 5% carbon dioxide for 2 hours;

[0122] (7) After standing, add chondrogenic culture medium and continue in vitro culture for 3-11 days to form a cartilage gel-3D printed porous framework complex, such as Figure 3 shown.

[0123] When used for repairing joint defects, the shape and size of the cartilage to be repaired can be determined based on previous auxiliary examinations such as MRI and CT, and the cartilage gel-3D printed porous framework complex can be cut.

[0124] Example 3

[0125] Preparation of cartilage membrane particle-3D printed porous framework composite

[0126] In this example, a cartilage membrane particle-3D printed porous framework complex is prepared. The specific operation method is as follows:

[0127] (1) Aseptically cut 2.5×2.5cm 2 ear cartilage tissue; use sterile instruments to peel off the mucosa and fibrous tissue on the surface of the cartilage;

[0128] (2) Cut the cartilage tissue into 1.5×1.5 mm 2 Large and small cartilage fragments; prepared with 0.15% collagenase; added cartilage fragments into the prepared collagenase and digested for 8 hours;

[0129] (3) After 8 hours, the collagenase solution was filtered and centrifuged to obtain ear chondrocytes for primary and subculture;

[0130] (4) After amplification, the cells were collected and resuspended at 8×10 6 / 10ml to 30×10 6 / 10ml / well of cells were inoculated into a six-well plate and cultured in a gelling medium (DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin); after culturing for 24 hours or 48 hours, fresh gelling medium was replaced and the in vitro culture was continued for up to 15 days;

[0131] (6) After the culture medium in the six-well plate is removed, the cartilage membrane tissue at the bottom of the six-well plate can be seen ( Figure 2 D), where the cell density in the cartilage membrane tissue is approximately 1.0×10 8 / ml-10×10 8 Pieces / ml;

[0132] Use forceps to pick up the cartilage membrane ( Figure 2 E), cut it into 1×1mm 2 After the cartilage membrane pieces are granulated to the desired size, collect them into a 50 ml centrifuge tube. Figure 2 As shown in F;

[0133] (7) The 3D printed porous framework to be inoculated (prepared in Example 1, such as Figure 1 and Figure 2 The frame is placed in a centrifuge tube containing cartilage membrane particles, ensuring that the frame is completely immersed; the centrifuge tube containing the frame and cartilage membrane particles is placed in a centrifuge, centrifuged at 600 rpm for 2 minutes;

[0134] (8) The inoculated 3D printed porous framework is placed at 37° C., 95% humidity, and 5% carbon dioxide for a certain period of time; after the period of placement, a chondrogenic culture medium is added and the culture is continued in vitro for 3-11 days to form a cartilage membrane particle-3D printed porous framework complex, such as Figure 4 shown.

[0135] When used for repairing joint defects, the shape and size of the cartilage to be repaired can be determined based on previous auxiliary examinations such as MRI and CT, and the ear cartilage membrane particle-frame structure complex can be cut.

[0136] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A cartilage tissue engineering composite, characterized in that: The compound comprises: (a) a carrier comprising a 3D printed porous framework; and (b) cartilage gel or cartilage membrane sheet particles containing chondrocytes seeded or loaded on the carrier, wherein the chondrocytes are taken from the subject's autologous chondrocytes; Furthermore, the composite is a composite formed by inoculating the cartilage gel or cartilage membrane particles into the carrier and culturing the cartilage, wherein the chondrocytes are loaded on the carrier and form a more compact integrated structure with the carrier; The cartilage gel comprises a cell group composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix encapsulates the cell group, and the cartilage gel is in a gel state, and the density of the chondrocytes is at least 1.0×10 8 / ml or 1.0×10 8 / g; wherein the cartilage gel is obtained by gelation culture for 2.5-5.5 days, and the adhesion rate of the cartilage gel is ≥90%; The cartilage membrane particles include a cell group composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix wraps the cell group, and the cartilage particles are prepared by cutting and shredding the thin sheet of cartilage membrane, wherein the density of the chondrocytes is at least 1.0×10 8 / ml or 1.0×10 8 The surface area of ​​the cartilage membrane particles is 1-5 mm 2 ; wherein the cartilage membrane sheet is obtained by gelling culture for 6-30 days; Wherein, the gel culture is an in vitro culture carried out using a gel culture medium, wherein the gel culture medium contains the following components: a high-glucose DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin; The 3D printed porous framework comprises an upper structure and a lower structure, wherein the pore size of the structure is 100-300 mm; and is made of a degradable polymer material selected from the group consisting of polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer; The cartilage gel or cartilage membrane particles are filled in the pores of the 3D printed porous framework, wherein the cartilage gel is filled in the 3D printed porous framework by a direct filling method.

2. The composite according to claim 1, characterized in that The chondrocytes are derived from elastic cartilage, fibrous cartilage or hyaline cartilage.

3. The composite according to claim 1, characterized in that The concentration of chondrocytes in the cartilage gel is 1.0×10 8 / ml-10×10 8 Pieces / ml.

4. The composite according to claim 1, characterized in that The adhesion rate of the cartilage gel is ≥95%.

5. The composite according to claim 1, characterized in that The cartilage gel is obtained by gelation culture for 3-5 days.

6. The composite according to claim 1, characterized in that The concentration of chondrocytes in the cartilage membrane is 1.0×10 8 / ml-10×10 8 Pieces / ml.

7. The composite according to claim 1, characterized in that The cartilage membrane sheet is obtained by gelling culture for 7-20 days.

8. The composite according to claim 7, characterized in that The cartilage membrane sheet is obtained by gelling culture for 10-15 days.

9. The composite according to claim 1, characterized in that The thickness of the cartilage membrane sheet is 0.2-0.25 mm.

10. The composite according to claim 1, characterized in that The 3D printed porous framework is also loaded with gelatin, collagen, silk or a combination thereof.

11. A method for preparing the cartilage tissue engineering composite according to claim 1, comprising the following steps: The cartilage gel or the cartilage membrane particles are filled into a 3D printed porous frame and cultured into cartilage in vitro to obtain the cartilage tissue engineering complex.

12. The method according to claim 11, characterized in that The cartilage gel is filled into the 3D printed porous framework by a direct filling method.

13. The method according to claim 11, characterized in that The cartilage membrane sheet particles are filled into the 3D printed porous frame by centrifugation.

14. The method according to claim 13, characterized in that In the centrifugal method, no liquid is added to the centrifugal system, and repeated centrifugation is used to allow the cartilage membrane particles to enter the 3D printed porous framework.

15. The method according to claim 11, characterized in that The chondrogenic culture is an in vitro culture using a chondrogenic medium, wherein the chondrogenic medium has the following components: high-glucose DMEM medium, serum replacement, proline, vitamin C, transforming growth factor-β1 (TGF-β1), insulin-like growth factor 1 (IGF-I) and dexamethasone.

16. The method according to claim 11, characterized in that The chondrogenesis culture time is 3-15 days.

17. The method according to claim 11, characterized in that The chondrogenic culture time is 5-11 days.

18. A use of the cartilage tissue engineering composite as claimed in claim 1, characterized in that: Used to prepare medical products for repairing joint defects.

19. The use according to claim 18, characterized in that The joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect or a combination thereof.

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