Auricular cartilage tissue engineering complex and its use

Abstract

The present invention provides an ear cartilage tissue engineering complex. Specifically, the complex comprises ear cartilage gel or ear cartilage membrane particles containing ear chondrocytes, and a porous framework structure, and the ear cartilage gel or ear cartilage membrane particles are loaded on the porous framework structure to form an ear cartilage gel / ear cartilage membrane particle-framework structure complex. The present invention also provides a preparation method of the ear cartilage tissue engineering complex and its use in the repair and reconstruction of articular cartilage.

Description

Technical Field

[0001] The present invention relates to the field of biomedical tissue engineering, and in particular to an ear cartilage tissue engineering complex and a preparation method thereof, and application thereof in repairing joint defects. Background Art

[0002] In recent years, with the rapid development of the economy and society, various types of articular cartilage lesions such as sports injuries, accidental injuries, and joint degenerative lesions have become increasingly common. Cartilage lesions are difficult to repair by the patient's own ability, so there is a huge clinical demand for joint repair. At present, the main treatment methods for articular cartilage lesions in clinical practice are 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 surface 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 complications in the donor site. Traditional treatment methods are generally ineffective, there is a lack of donors for autologous transplantation, and long-term complications of artificial joints are difficult to avoid.

[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. Although the in situ chondrocyte-material complex constructed by existing tissue engineering repair technology (autologous chondrocyte transplantation ACI or matrix-induced autologous chondrocyte transplantation MACI) has been gradually carried out in clinical treatment, there are still many disadvantages that are difficult to avoid: 1. The cell source is articular cartilage, and obtaining cells from the already damaged joints will inevitably increase the damage to the original joints. 2. The proliferation capacity of articular chondrocytes is limited, and the initial cell-material complex construction cycle is long, and patients need to wait for a long time. 3. The strength of the cell-material complex is low, far less than the mechanical strength level of natural cartilage.

[0004] Therefore, new approaches to joint repair are urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide an ear cartilage tissue engineering complex and a preparation method thereof, as well as use thereof in repairing joint defects.

[0006] A first aspect of the present invention provides an ear cartilage tissue engineering composite, the composite comprising:

[0007] (a) a carrier comprising a porous framework structure; and

[0008] (b) Auricular cartilage gel or auricular cartilage membrane sheet particles containing auricular chondrocytes seeded or loaded on the carrier.

[0009] In another preferred embodiment, the complex comprises a complex formed by inoculating the ear cartilage gel or ear cartilage membrane particles into the carrier and culturing them into chondrocytes (in the complex, ear chondrocytes are loaded on the carrier and form a tighter integrated structure with the carrier).

[0010] In another preferred embodiment, the complex comprises a complex formed by inoculating the ear cartilage gel or ear cartilage membrane particles into the carrier but without chondrogenic culture.

[0011] In another preferred embodiment, the ear chondrocytes are from humans or non-human mammals.

[0012] In another preferred embodiment, the auris chondrocytes are derived from autologous auris chondrocytes or allogeneic auris chondrocytes, preferably autologous auris chondrocytes.

[0013] In another preferred embodiment, the auris chondrocytes are obtained from the subject's own auris chondrocytes.

[0014] In another preferred embodiment, the subject is a human or non-human mammal.

[0015] In another preferred embodiment, the subject has a joint defect.

[0016] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0017] 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.

[0018] In another preferred embodiment, the ear 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 ear 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.

[0019] In another preferred embodiment, the ear cartilage gel is prepared by gelling and culturing ear chondrocytes.

[0020] In another preferred embodiment, the gelled culture is an in vitro culture using a gelled culture medium.

[0021] 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.

[0022] In another preferred embodiment, the adhesion rate of the ear cartilage gel is ≥90%, preferably ≥95%.

[0023] In another preferred embodiment, the concentration of chondrocytes in the ear cartilage gel is 1.0×10 8 / ml-10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0024] In another preferred embodiment, the ear cartilage gel is obtained by gelation culture for 2.5-5.5 days, preferably 3-5 days.

[0025] In another preferred embodiment, the ear chondrocytes are from humans or non-human mammals.

[0026] In another preferred embodiment, the auris chondrocytes are derived from autologous auris chondrocytes or allogeneic auris chondrocytes, preferably autologous auris chondrocytes.

[0027] In another preferred embodiment, the auris chondrocytes are obtained from the subject's own auris chondrocytes.

[0028] In another preferred embodiment, the subject is a human or non-human mammal.

[0029] In another preferred embodiment, the subject has a joint defect.

[0030] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0031] 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.

[0032] In another preferred embodiment, the ear 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 ear cartilage particles are prepared by cutting and shredding thin sheet of ear cartilage membrane, wherein the density of ear cartilage cells is at least 1.0×10 8 / ml or 1.0×10 8 Pieces / g.

[0033] In another preferred embodiment, the concentration of chondrocytes in the ear cartilage membrane is 1.0×10 8 / ml-10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0034] In another preferred embodiment, the ear cartilage membrane sheet is obtained by gelling culture for 6-30 days, preferably 7-20 days, and most preferably 10-15 days.

[0035] In another preferred embodiment, the gelled culture is an in vitro culture using a gelled culture medium.

[0036] 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.

[0037] In another preferred embodiment, the thickness of the ear cartilage membrane is 0.2-0.25 mm.

[0038] In another preferred example, the average volume of the ear cartilage membrane particles is 0.2 μl.

[0039] In another preferred embodiment, the surface area of ​​the ear cartilage membrane particles is 0.05-10 mm 2 , preferably 1-5 mm 2 , more preferably, the average area is 1 mm 2 .

[0040] In another preferred embodiment, the porous framework structure is made of a biodegradable material selected from the following group: PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, or decalcified bone matrix.

[0041] In another preferred embodiment, the framework structure may also be loaded with gelatin, collagen, silk, hydrogel or a combination thereof.

[0042] In another preferred embodiment, the frame structure is a PCL frame.

[0043] In another preferred embodiment, the framework structure is decalcified bone matrix.

[0044] In another preferred embodiment, the framework structure is an allogeneic bone repair material.

[0045] In another preferred embodiment, the allogeneic bone repair material is decalcified bone matrix.

[0046] In another preferred embodiment, the framework structure is a xenogeneic bone repair material.

[0047] In another preferred embodiment, the xenogeneic bone repair material is decalcified bone matrix.

[0048] In another preferred embodiment, the shape of the frame structure includes a cylinder, a cuboid or other specific shapes.

[0049] In another preferred embodiment, the ear cartilage gel / ear cartilage membrane particle-framework structure complex can be converted into articular cartilage under the joint microenvironment.

[0050] The second aspect of the present invention provides a method for preparing the ear cartilage tissue engineering complex described in the first aspect of the present invention, which comprises the following steps: inoculating the ear cartilage gel or ear cartilage membrane particles described in the first aspect of the present invention into a porous framework structure, and culturing chondrogenesis in vitro to obtain the ear cartilage tissue engineering complex.

[0051] In another preferred embodiment, the ear cartilage gel is inoculated into the porous framework structure by a direct filling method.

[0052] In another preferred embodiment, the ear cartilage membrane particles are inoculated into the porous framework structure by centrifugation.

[0053] In another preferred embodiment, no liquid is added to the centrifugal system of the centrifugal method, and repeated centrifugation is used to allow the ear cartilage membrane particles to enter the framework structure.

[0054] In another preferred embodiment, the chondrogenic culture is an in vitro culture using a chondrogenic culture medium.

[0055] 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.

[0056] 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.

[0057] In another preferred embodiment, the chondrogenic culture time is 3-15 days, preferably 5-11 days.

[0058] The third aspect of the present invention provides a use of the ear cartilage tissue engineering complex described in the first aspect of the present invention for preparing a medical product for repairing joint defects.

[0059] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0060] 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.

[0061] The fourth aspect of the present invention provides a method for repairing joint defects, using the ear 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.

[0062] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0063] 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.

[0064] 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

[0065] Figure 1 The structural characteristics of natural elastic cartilage and hyaline cartilage are shown. The results of HE staining, Saf-O staining, COL-II staining and Elastin staining of the two types of cartilage are shown respectively.

[0066] Figure 2 The results of the samples collected 6 months after the ear chondrocyte gel complex and articular chondrocyte gel complex were implanted in the articular cartilage. A and D are the whole articular cartilage, in which the regenerated cartilage tissue after implantation is encircled by a dotted circle and magnified in B and E; C and F are cross-sectional views of the regenerated cartilage tissue after implantation.

[0067] Figure 3 The effect of the articular cartilage microenvironment on the ectopic cartilage type of auris chondrocytes and articular chondrocytes is shown. The results of HE staining, Saf-O staining, COL-II staining and Elastin staining of the two types of regenerated cartilage are shown respectively. The bar in the figure is 150μM.

[0068] Figure 4 A general schematic diagram of a decalcified bone matrix framework is shown. The scale bar in the figure is 1 cm.

[0069] Figure 5 Electron micrographs of decalcified bone matrix frameworks are shown, with the bar representing 1 mm.

[0070] Figure 6 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).

[0071] Figure 7 A real picture of the ear cartilage gel-frame structure composite is shown. The scale bar in the picture is 1 cm.

[0072] Figure 8 The actual picture of the auricular cartilage membrane particle-frame structure complex is shown. The scale bar in the picture is 1 cm.

[0073] Fig. 9 Shown is a comparison of the adhesion rates of seeded samples (cell suspension or cartilage gel) after 24 h of culture on decalcified bone matrix.

[0074] Fig.10 Shown is a comparison of gel cartilage-decalcified bone composite (A) and decalcified bone alone (B) transplanted into goat knee joint defects. DETAILED DESCRIPTION

[0075] After extensive and in-depth research, the inventors unexpectedly discovered and developed an ear cartilage tissue engineering complex for the first time, which is an integrated ear cartilage gel / ear cartilage membrane particle-frame structure complex. Experiments have shown that by obtaining primary ear cartilage for amplification culture, a specific number of ear 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 ear cartilage or membrane-like ear cartilage can be formed. The prepared gel-like cartilage or membrane-like cartilage is combined with a porous framework structure, and the prepared ear cartilage tissue engineering complex can be transformed and regenerated into articular cartilage at the defective joint after being transplanted into the defective joint, thereby achieving repair and reconstruction of the articular cartilage. On this basis, the present invention has been completed.

[0076] the term

[0077] 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.

[0078] As used herein, the term "ear cartilage tissue engineering complex" includes the ear cartilage gel-framework complex and the ear cartilage membrane particle-framework complex that have been cultured with or without in vitro chondrogenic culture as described herein. In the present invention, they can be collectively referred to as ear cartilage tissue engineering complexes.

[0079] As used herein, the term "inoculation" means inoculating auris chondrocytes into a cell culture dish, and may also mean inoculating auris cartilage gel / auris cartilage membrane particles into a specified framework structure and distributing them evenly. A person skilled in the art can understand the meaning of "inoculation" based on the context.

[0080] 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.).

[0081] 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".

[0082] Ear cartilage gel and its preparation

[0083] As used herein, "gel ear cartilage", "ear cartilage gel", "gelled ear cartilage", "gel-like ear cartilage", "ear cartilage gel of the present invention" or "gelled ear cartilage of the present invention" are used interchangeably and all refer to the ear cartilage (stem) cells in a gel state of the present invention, especially ear chondrocytes of a specific concentration are inoculated and / or spread on a flat or substantially flat culture surface so that the inoculated ear chondrocytes form a stacked structure, and the ear chondrocytes with a stacked structure are cultured under suitable gelling culture conditions to form a gelled ear cartilage culture.

[0084] The gel ear cartilage of the present invention is a new type of ear cartilage that is different from free ear chondrocytes, centrifugally precipitated ear chondrocytes and ear cartilage pellets. The gel ear cartilage of the present invention can be regarded as a specific form of ear cartilage between free ear chondrocytes and dense ear cartilage pellets. The gel ear cartilage of the present invention, because during the gelation culture process, the ear chondrocytes not only contact and / or interact with adjacent cells on the plane (XY plane), but also contact and / or interact with adjacent ear chondrocytes in multiple directions such as above and / or below and / or above or below, thereby promoting the ear chondrocytes to secrete and form more extracellular matrix, thereby wrapping the gelated cultured ear chondrocytes in an extracellular matrix with a certain viscosity, so that the gel ear cartilage of the present invention has both a close connection and a certain viscosity and fluidity, so that the gel ear 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 ear cartilage.

[0085] In addition, the gel ear 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 ear cartilage, or for ear cartilage transplantation or ear cartilage repair surgery.

[0086] In the present invention, the complex for repairing ear cartilage includes a complex formed by loading the gel ear cartilage of the present invention on a carrier material (especially a porous biological framework structure) without culturing into ear cartilage, and also includes a complex formed by loading the gel ear cartilage of the present invention on a carrier material (especially a porous biological framework structure) and culturing into ear cartilage.

[0087] In the present invention, the complex suitable for transplantation into the human or animal body is the auricular cartilage tissue engineering complex of the present invention, that is, the gel auricular cartilage of the present invention is loaded on a carrier material (especially a porous biological framework structure) and cultured into auricular cartilage to form a complex.

[0088] Preferably, in the present invention, the gel ear cartilage is cultured in vitro for a period of time t1 under gel culture conditions, thereby forming gel ear cartilage. Preferably, the t1 is 2.5-5.5 days, preferably 3-5 days.

[0089] In the present invention, one feature is stacking inoculation, that is, after auris chondrocytes of a specific density are inoculated in a culture container, the inoculated auris chondrocytes will form a multi-layer auris chondrocyte group stacked on each other (i.e., an auris chondrocyte group with a stacking structure) through, for example, sedimentation. 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.

[0090] Ear cartilage membrane and its preparation

[0091] As used herein, "auricular cartilage diaphragm", "membranous auricular cartilage", or "auricular cartilage diaphragm of the present invention" are used interchangeably, all referring to the auricular cartilage (stem) cells in a diaphragm state of the present invention, especially auricular chondrocytes of a specific concentration are inoculated and / or spread on a flat or substantially flat culture surface so that the inoculated auricular chondrocytes form a stacked structure, and the auricular chondrocytes with a stacked structure are cultured under appropriate culture conditions to form a membranous auricular cartilage culture.

[0092] The "ear cartilage membrane" of the present invention is prepared by prolonging the gelation culture time on the basis of the preparation of the "ear cartilage gel" of the present invention. That is, in the present invention, the ear cartilage cells 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 an ear cartilage membrane. Preferably, the t2 is 6-30 days, preferably 7-20 days, and most preferably 10-15 days.

[0093] The ear cartilage membrane 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 "ear 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.

[0094] In the present invention, the cartilage repair complex includes a complex formed by loading the ear 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 ear cartilage membrane particles of the present invention on a carrier material (especially a porous framework structure) and culturing the cartilage.

[0095] In the present invention, the complex suitable for transplantation into the human or animal body is the ear cartilage tissue engineering complex of the present invention, that is, the ear cartilage membrane particles of the present invention are loaded on a carrier material (especially a porous framework structure) and formed by chondrogenic culture.

[0096] 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 ear chondrocytes was 1.0×10 8 -10×10 8 The ear cartilage gel or the ear cartilage cell density contained is 1.0×10 8 -10×10 8 / ml of ear cartilage membrane.

[0097] 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.

[0098] As used herein, the term "chondrogenic culture" refers to the use of chondrogenic culture medium to culture a porous framework structure inoculated with ear cartilage gel or ear cartilage membrane particles, ultimately forming an integrated ear cartilage gel-framework structure complex or ear cartilage membrane particle-framework structure complex, that is, the ear cartilage tissue engineering complex of the present invention, which is used for transplantation into cartilage defects in the human body or animal body.

[0099] Cartilage and chondrocytes

[0100] 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.

[0101] 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.

[0102] The biggest difference between hyaline cartilage and elastic cartilage is that the former (i.e. hyaline cartilage) expresses abundant elastin. The structural characteristics of the two are as follows: Figure 1 shown.

[0103] In a preferred example of the present invention, autologous ear chondrocytes (elastic cartilage) taken from a subject with joint defects are prepared into ear cartilage gel or ear cartilage membrane particles through in vitro culture, and the ear cartilage gel or ear cartilage membrane particles are inoculated on a porous framework structure to prepare an ear cartilage gel-framework complex or an ear cartilage membrane particle-framework complex, and used for the repair of articular cartilage (hyaline cartilage) of joint defects.

[0104] HE staining, Saf-O staining, COL-II staining and Elastin staining

[0105] HE staining: Hematoxylin-eosin staining, referred to as HE staining, is one of the commonly used staining methods in paraffin section technology. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm purple-blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and extracellular matrix red.

[0106] Saf-O staining: also known as Safranin O staining, is a commonly used cartilage staining method. The principle of Saf-O staining is that basophilic cartilage combines with the basic dye Safranin O to show red; Safranin O is a cationic dye that combines with polyanions, and its display of cartilage tissue is based on the combination of cationic dyes and polysaccharide anion groups (chondroitin sulfate or keratan sulfate).

[0107] COL-II staining: COL-II is type II collagen, which is a high molecular weight protein. The filamentous collagen fibers are interwoven with elastin and polysaccharide proteins to form a network structure, which produces a certain mechanical strength. Type II collagen is mainly produced by chondrocytes and is mostly found in bones, joints, tendons and other tissues.

[0108] Elastin staining: Elastin is elastin, which is the main component of elastic fibers. Two forms of elastin have been found: elastin I exists in the nuchal ligament, aorta and skin, and elastin II can be obtained from cartilage. In the embodiment of the present invention, Elastin staining is used to reflect the expression of elastin in chondrocytes to distinguish different types of chondrocytes, such as ear cartilage and articular cartilage.

[0109] Cartilage transformation

[0110] As used herein, the term "cartilage conversion" refers to the conversion between different types of cartilage, that is, the conversion from one type of cartilage to another type of cartilage, or the conversion from one type of chondrocyte to another type of chondrocyte.

[0111] In the present invention, the ear chondrocytes (hyaline chondrocytes) in the ear cartilage tissue engineering graft formed by in vitro culture are transformed into articular chondrocytes (elastic chondrocytes) in the joint tissue microenvironment after being transplanted into the defective joint, and finally form articular cartilage.

[0112] MACI and ACI

[0113] MACI is the abbreviation of matrix-induced autologous chondrocyte implantation, which means "matrix-induced autologous chondrocyte transplantation". It is a technology that uses tissue engineering technology to transplant chondrocytes. MACI is currently the latest and best technology in the world for treating articular cartilage defects.

[0114] ACI is the abbreviation of autologous chondrocyte implantation, which means "autologous chondrocyte transplantation". It is one of the most widely used tissue engineering technologies for treating articular cartilage damage. After the advent of MACI, ACI was correspondingly called "traditional ACI" to distinguish it from MACI.

[0115] Porous frame structure

[0116] As used herein, the term "porous framework structure" refers to a carrier made of biocompatible material having a certain number of pores on its surface and inside to facilitate the attachment of the ear cartilage gel or ear cartilage membrane seeded thereon. In the present invention, the biocompatible material is preferably a biodegradable material.

[0117] Biodegradable materials refer to materials that can be decomposed in the body after being transplanted into an animal. The porous framework structure of the ear cartilage tissue engineering transplant of the present invention is made of a biodegradable material selected from the following group: PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, decalcified bone matrix or a combination thereof, but not limited to the above materials. The allogeneic bone repair material and the xenogeneic bone repair material include decalcified bone matrix material. In a preferred embodiment of the present invention, the porous framework structure of the ear cartilage tissue engineering complex is a decalcified bone matrix.

[0118] Decalcified bone matrix

[0119] The decalcified bone matrix used in the preferred embodiment of the present invention has a thickness of 0.3-0.8 cm, preferably 0.4-0.6 cm, and most preferably 0.5 cm. The decalcified amount of the decalcified bone matrix is ​​30%-50%, the decalcification degree is appropriate, the supporting effect is good, and it is easy to trim and cut into a suitable shape and size. The pore size of the pores of the decalcified bone matrix is ​​400-800 μm, which is easy to fill with chondrocytes.

[0120] Decalcified bone matrix (DBM) is a bone transplant material that is made of allogeneic bone or xenogeneic bone after decalcification to reduce immunogenicity. Different degrees of decalcification correspond to different mechanical strengths. It has good biological properties, osteoinductivity, osteoconductivity and biodegradability, promotes new bone formation and bone tissue mineralization, and accelerates bone healing. It can effectively repair bone damage alone or in combination with autologous bone, other biomaterials, and growth factors. It is an ideal bone tissue engineering scaffold material. However, the pore size of general decalcified bone matrix is ​​large, and the cell adhesion rate is extremely low when inoculated with chondrocyte suspension, which is not conducive to the construction of tissue engineering carriers.

[0121] In another preferred embodiment, the decalcified bone matrix of the present invention has a pore size of 400-800 μm and a porosity of 87.3%±3.7%.

[0122] PCL

[0123] Polycaprolactone (PCL) is a fully biodegradable polymer material. It is formed by ring-opening polymerization of ε-caprolactone in the presence of metal organic compounds (such as tetraphenyltin) as catalysts and dihydroxy or trihydroxy as initiators. It belongs to the polymeric polyester, and its molecular weight and degree of disproportionation vary with the type and amount of the starting material. It appears as a white solid powder, is non-toxic, insoluble in water, and easily soluble in a variety of polar organic solvents. PCL has good biocompatibility, good organic polymer compatibility, and good biodegradability. It can be used as a cell growth support material and is compatible with a variety of conventional plastics. It can be completely degraded in 6-12 months under natural conditions. In addition, PCL also has good shape memory temperature control properties and is widely used in the production and processing of drug carriers, plasticizers, degradable plastics, nanofiber spinning, and shaping materials.

[0124] In a preferred embodiment, the ear cartilage gel / ear cartilage membrane particles described in the present invention can be inoculated on a PCL framework structure to form an ear cartilage gel-framework complex or an ear cartilage membrane particle-framework complex for repairing articular cartilage.

[0125] Allogeneic bone repair materials

[0126] Allogeneic bone is currently the most commonly used bone implant material in orthopedics. It is mainly used to repair and fill bone defects, and plays a role in fixation and support. Allogeneic bone is taken from donated human bone tissue. "Allogeneic" indicates that it comes from the human body, and "allogeneic" indicates that it does not come from the patient's own body. After the donor is selected, it is usually obtained under sterile conditions within 24 hours of death and processed immediately. There are two preservation methods: fresh freezing and freeze drying. Fresh frozen bone can be stored for 1 year at -20°C; freeze-dried bone can be stored at room temperature for a long time after vacuum packaging, and has lower antigenicity. Compared with fresh frozen bone, the mechanical properties of freeze-dried bone will be reduced by 50%, and sterilization with ethylene oxide or high-dose gamma ray irradiation will further reduce the inductive properties of bone.

[0127] In a preferred embodiment, the ear cartilage gel / ear cartilage diaphragm particles described in the present invention can be inoculated onto a framework structure prepared from allogeneic bone (e.g., decalcified bone matrix) to form an ear cartilage gel-framework complex or an ear cartilage diaphragm particle-framework complex for repairing articular cartilage.

[0128] Xenogeneic bone repair materials

[0129] Xenogeneic bone is a bone repair material derived from other species such as cattle and pigs. It is widely available and relatively cheap. However, xenogeneic bone has strong immunogenicity and is prone to cause immune rejection after implantation in patients. In addition, xenogeneic bone has no ability to induce the proliferation of mesenchymal stem cells and has poor biological activity. It needs to be combined with other repair materials or related cytokines to achieve a repair effect.

[0130] In a preferred embodiment, the ear cartilage gel / ear cartilage diaphragm particles described in the present invention can be inoculated onto a framework structure prepared from xenogeneic bone (such as decalcified bone matrix) to form an ear cartilage gel-framework complex or an ear cartilage diaphragm particle-framework complex for repairing articular cartilage.

[0131] Culture medium used in the present invention

[0132] 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.

[0133] Gelation medium: DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0134] Adhesion rate

[0135] In the present invention, when the ear cartilage gel of the present invention is inoculated on a carrier material (especially a porous biological framework structure), the ear 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 ear cartilage gel of the present invention is ≥90%, preferably, ≥95%.

[0136] The adhesion rate in the present invention is defined as follows:

[0137] The DNA quantification of the inoculated sample (eg, ear cartilage gel) is detected A1; the DNA quantification of the inoculated complex (eg, ear cartilage gel-frame complex) after culturing for 24 hours is detected A2; the adhesion rate is A2 / A1*100%.

[0138] The method for measuring the adhesion rate comprises the following steps:

[0139] Take the inoculated sample (such as ear cartilage gel or ear cartilage gel-frame complex), digest it with proteinase K, and use the PicoGreen kit (Invitrogen, Carlsbad, CA, USA) for quantitative detection of the digested sample. Use a fluorescent microplate reader to measure the absorbance at 520 nm, and calculate the DNA content according to the standard curve formula.

[0140] The main advantages of the present invention include:

[0141] (1) Ear cartilage gel is more mature than chondrocytes and has a certain fluidity.

[0142] (2) The cell source is ear cartilage, and there is no need to obtain the material from the joint. There will be no secondary damage to the patient's joint area. In addition, ear cartilage cells will be transformed into articular cartilage cells in the joint environment.

[0143] (3) The expansion capacity of ear cartilage is significantly better than that of articular cartilage, and the waiting period for patients is short.

[0144] (4) The selected framework structures are mostly natural materials or materials whose degradation products are neutral, with moderate degradation rates in the body, low immune responses, and good biosafety.

[0145] (5) The pore size of the framework material is large and the porosity is good, but the cell adhesion rate is extremely low when inoculated with chondrocyte suspension. The use of cartilage gel-like tissue with a certain fluidity and relatively viscosity can effectively improve the adhesion rate.

[0146] (6) Compared with other tissue engineering repair methods, the auricular cartilage gel / auricular cartilage membrane particle-framework structure complex can stably regenerate cartilage.

[0147] (7) The ear cartilage gel / ear cartilage membrane particle-frame structure complex can stably regenerate cartilage and provide immediate mechanical support, with good immediate repair effect

[0148] 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 used to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), 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.

[0149] Example 1

[0150] Study on the transformation of different types of cartilage in the microenvironment of natural articular cartilage

[0151] According to the difference in the structure of the extracellular matrix, cartilage tissue is divided into three types: elastic cartilage, hyaline cartilage, and fibrous cartilage. Among them, ear cartilage is elastic cartilage, and articular cartilage is hyaline cartilage. The biggest difference between the two is that the former expresses abundant elastin (see Figure 1 In order to study the fate of different types of cartilage in the natural articular cartilage microenvironment, the following experiment was conducted.

[0152] (1) Isolation and culture of two types of chondrocytes: After the goat is anesthetized, a portion of the auricle is removed under sterile conditions, and the cartilage of the non-weight-bearing area of ​​the joint is scraped. Under sterile conditions, the pieces are cut into 1 mm × 1 mm sizes and prepared with 0.15% collagenase; the cartilage fragments are added to the prepared collagenase and digested for 8 hours; after 8 hours, the collagenase solution is filtered and centrifuged to obtain ear chondrocytes, which are cultured in primary and subculture stages to expand to the required cell quantity.

[0153] (2) Preparation of Pluronic thermosensitive gel: Mix pluronic powder and H-DMEM in a ratio of 3:10 at room temperature, then stir in a 4°C refrigerator overnight, and finally sterilize by high temperature for use.

[0154] (3) Preparation of cell-gel mixture: The cells were collected by digestion with 0.25% trypsin, counted, and 100×10 6 The cells were fully mixed with Pluronic thermosensitive gel, placed in an incubator for 30 min, washed twice with PBS and the supernatant was discarded. The cell pellet was precooled in an ice box. 6After adding 1 ml of gel to the cells and mixing thoroughly, use a 1 ml syringe with a 20 ml needle to extract the cell-gel mixture. Hold the syringe in your hand and let the gel quickly become semi-solid to keep the cells evenly distributed in the gel.

[0155] (4) Implantation of three chondrocyte gel complexes into the articular cartilage microenvironment: After the goats were anesthetized, the bare joint below the surgical side was wrapped with a sterile towel. The knee joint was extended and flexed to determine the position of the patellar activity. A longitudinal arc incision of about 8-10 cm was made along the posterior edge of the patellar ligament, with the upper end reaching about 2-3 cm above the upper edge of the patella and the lower end reaching 1 cm below the tibia tubercle. The skin was lifted inward, and the subcutaneous, superficial fascia, and deep fascia were cut in layers with large blood vessel and meningeal scissors. The joint capsule and synovium were opened along the posterior edge of the fascia, and care was taken not to damage the fascia. The pulley area was fully exposed, and a circular defect of the entire cartilage layer was created with a 5 mm diameter trephine. Different chondrocyte gel mixtures were injected into the defect. The cysts were cultured in vivo for six months.

[0156] (5) Sample collection results: Six months after in vivo sampling, it was found that both the ear chondrocyte gel complex and the articular chondrocyte gel complex formed translucent cartilage tissue in the articular cartilage microenvironment (see Figure 2 AC, DF), and no elastin expression (see Figure 3 ).

[0157] Example 2

[0158] Preparation of ear cartilage gel-frame structure composite

[0159] In this example, an ear cartilage gel-frame complex was prepared. The specific operation method is as follows:

[0160] (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.

[0161] (2) Cut the ear cartilage tissue into 1.5×1.5mm 2 Large and small cartilage fragments; prepared with 0.15% collagenase; added cartilage fragments into the prepared collagenase and digested for 8 hours;

[0162] (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;

[0163] (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);

[0164] (5) After 72 hours (3 days) of inoculation, the culture medium in the six-well plate was aspirated and gelatinous ear cartilage tissue was visible at the bottom of the six-well plate ( Figure 6 A), using forceps to collect the gelatinous ear cartilage on the bottom of a six-well plate ( Figure 6 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 ear cartilage gel, such as Figure 6 As shown in C;

[0165] (6) The ear cartilage gel preparation (prepared in step (5), with a volume of about 0.25-0.35 ml) was inoculated into the framework structure (decalcified bone matrix framework ( Figure 4 and Figure 5 ), having a pore size of about 400-800 μm and a porosity of about 87.3% ± 3.7%, at 37° C., 95% humidity, and 5% carbon dioxide for 2 hours;

[0166] (7) After standing, add chondrogenic culture medium and continue in vitro culture for 3-11 days to form an ear cartilage gel-frame structure complex. Figure 7 As shown;

[0167] 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 gel-frame structure complex can be cut.

[0168] Example 3

[0169] Preparation of auricular cartilage membrane particle-frame structure complex

[0170] In this example, an ear cartilage membrane particle-framework complex is prepared. The specific operation method is as follows:

[0171] (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;

[0172] (2) Cut the ear cartilage tissue into 1.5×1.5mm 2Large and small cartilage fragments; prepared with 0.15% collagenase; added cartilage fragments into the prepared collagenase and digested for 8 hours;

[0173] (3) After 8 hours, the collagenase solution was filtered and centrifuged to obtain ear chondrocytes for primary and subculture;

[0174] (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;

[0175] (6) After the culture medium in the six-well plate is removed, the ear cartilage membrane tissue at the bottom of the six-well plate can be seen ( Figure 6 D), the cell density in the ear cartilage membrane tissue is about 1.0×10 8 / ml-10×10 8 Pieces / ml;

[0176] Use forceps to pick up the ear cartilage membrane ( Figure 6 E), cut it into 1×1mm 2 After the ear cartilage membrane pieces are granulated to the desired size, collect them into a 50 ml centrifuge tube. Figure 6 As shown in F;

[0177] (7) Place the framework material (decalcified bone matrix framework) to be inoculated Figure 4 and Figure 5 )) Place the frame material in a centrifuge tube containing ear cartilage membrane particles to ensure that the frame material is completely immersed; Place the centrifuge tube containing the frame material and ear cartilage membrane particles in a centrifuge at 600 rpm for 2 minutes;

[0178] (8) The inoculated framework material is placed at 37° C., 95% humidity, and 5% carbon dioxide for a certain period of time; after the placement, a chondrogenic culture medium is added and the culture is continued in vitro for 3-11 days to form an ear cartilage membrane particle-framework structure complex, such as Figure 8 As shown;

[0179] 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 auricular cartilage membrane particle-framework structure complex can be cut.

[0180] Comparative Example 1

[0181] Determination of adhesion rate between chondrocyte suspension and cartilage gel

[0182] Provide a demineralized bone matrix framework (such as Figure 4 The gel cartilage preparation (prepared in Example 2, with a volume of about 0.25-0.35 ml) was inoculated into the above decalcified bone matrix framework, and the DNA content of the gel cartilage preparation was determined before inoculation.

[0183] The primary cultured chondrocytes were passaged 4 times at 37°C, 95% humidity, and 5% carbon dioxide, and cell culture medium was added to prepare a cell suspension, and the DNA content of the prepared chondrocyte suspension was determined. The cell suspension was inoculated into the above decalcified bone matrix frame.

[0184] The inoculated cartilage gel-decalcified bone matrix complex and chondrocyte suspension-decalcified bone matrix complex were placed in an incubator at 37°C, 95% humidity, and 5% carbon dioxide for 24 hours. The two complexes were sampled and their DNA content was determined.

[0185] like Fig. 9 As shown, the adhesion rate was calculated by the adhesion rate determination method described in the specification. Compared with the cell suspension, the adhesion rate of the gel cartilage of the present invention was 92%±2%, which is about 3 times that of the cell suspension.

[0186] Comparative Example 2

[0187] Animal transplantation experiment to repair articular cartilage

[0188] A cartilage defect with a diameter of 7.5 mm was made on the articular surface of the knee joint of the experimental animal, and the ear cartilage gel-frame complex (gel cartilage-decalcified bone complex) prepared in Example 1 and the simple decalcified bone matrix were implanted into the defect A and the defect B, respectively.

[0189] The repair of cartilage defects in animals was observed immediately after implantation.

[0190] The results are as follows Fig.10 As shown:

[0191] The defect at point A is smooth and solid, surrounded by a soft tissue membrane with a certain degree of elasticity, and has an excellent immediate repair effect.

[0192] The wound surface of the defect at point B is rough and only provides physical support but cannot provide immediate repair.

[0193] 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. An ear cartilage tissue engineering composite, characterized in that: The compound comprises: (a) a carrier comprising a porous framework structure; and (b) ear cartilage gel or ear cartilage membrane sheet particles containing ear chondrocytes seeded or loaded on the carrier, wherein the ear chondrocytes are derived from the subject's autologous ear chondrocytes; Furthermore, the composite is a composite formed by inoculating the ear cartilage gel or ear cartilage membrane particles into the carrier and culturing the carrier into cartilage, wherein the ear chondrocytes are loaded on the carrier and form a more compact integrated structure with the carrier; The ear cartilage gel comprises a cell group composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix wraps the cell group, and the ear 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 ear cartilage gel is obtained by gelation culture for 2.5-5.5 days, and the adhesion rate of the ear cartilage gel is ≥90%; The ear 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 ear cartilage membrane particles are prepared by cutting and shredding thin sheet-like ear cartilage membranes, wherein the density of the ear cartilage cells is at least 1.0×10 8 / ml or 1.0×10 8 The surface area of ​​the ear cartilage membrane particles is 1-5 mm 2 ; wherein the ear cartilage membrane piece 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 porous framework structure refers to a carrier made of biocompatible material having a certain number of pores on its surface and inside to facilitate the attachment of ear cartilage gel or ear cartilage membrane particles inoculated thereon; and the ear cartilage gel or ear cartilage membrane particles are filled in the pores of the porous framework structure, wherein the ear cartilage gel is inoculated into the porous framework structure by a direct filling method.

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

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

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

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

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

7. The composite according to claim 1, characterized in that The ear cartilage membrane piece is obtained by gelling culture for 10-15 days.

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

9. The composite according to claim 1, characterized in that The porous framework structure is made of a biodegradable material selected from the group consisting of PCL, PGA, allogeneic bone repair materials, xenogeneic bone repair materials, or decalcified bone matrix.

10. The composite according to claim 1, characterized in that The porous framework structure is a decalcified bone matrix.

11. The composite according to claim 9 or 10, characterized in that The framework structure is also loaded with gelatin, collagen, silk or a combination thereof.

12. The composite according to claim 1, characterized in that The ear cartilage tissue engineering complex can be transformed into articular cartilage under the joint microenvironment.

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

14. The method according to claim 13, characterized in that The ear cartilage membrane particles are filled into the porous framework structure by centrifugation.

15. The method according to claim 14, characterized in that In the centrifugal method, no liquid is added to the centrifugal system, and repeated centrifugation is used to allow the ear cartilage membrane particles to enter the framework structure.

16. The method according to claim 13, 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.

17. The method according to claim 13, characterized in that The chondrogenesis culture time is 3-15 days.

18. The method according to claim 13, characterized in that The chondrogenic culture time is 5-11 days.

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

20. The use according to claim 19, 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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