A cartilage tissue engineering composite and its use

Abstract

The present invention provides a cartilage tissue engineering composite. Specifically, the graft comprises cartilage gel or cartilage membrane particles containing chondrocytes, and a porous framework structure, and the cartilage gel or cartilage membrane particles are loaded on the porous framework structure to form a cartilage gel / cartilage membrane particle-framework structure composite. The present invention also provides a method for preparing the cartilage tissue engineering graft 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, in particular to a cartilage tissue engineering complex and a preparation method thereof, and application thereof in joint repair. 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] In recent years, with the progress 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] Although the chondrocyte-material complex (ACI or MACI) constructed by existing tissue engineering repair technology 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 joint will inevitably increase the damage to the original joint. 2. The proliferation capacity of articular cartilage cells 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.

[0005] Therefore, new approaches to joint repair are urgently needed. Summary of the invention

[0006] The purpose of the present invention is to provide a cartilage tissue engineering complex and a preparation method thereof, and application thereof in joint repair.

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

[0008] (a) a carrier comprising a porous framework structure; 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 chondrocytes are from humans or non-human mammals.

[0029] In another preferred embodiment, the chondrocytes are derived from autologous chondrocytes or allogeneic chondrocytes, preferably autologous chondrocytes.

[0030] In another preferred embodiment, the chondrocytes are obtained from the subject's autologous chondrocytes.

[0031] In another preferred embodiment, the subject is a human or non-human mammal.

[0032] In another preferred embodiment, the subject has a joint defect.

[0033] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0034] 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.

[0035] 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 1.0×108 Pieces / g.

[0036] 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.

[0037] 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.

[0038] In another preferred embodiment, the gelled culture is an in vitro culture using a gelled culture medium.

[0039] 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.

[0040] In another preferred embodiment, the thickness of the cartilage membrane sheet is 0.2-0.25 mm.

[0041] In another preferred embodiment, the average volume of the cartilage membrane particles is 0.2 μl.

[0042] 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 .

[0043] 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.

[0044] In another preferred embodiment, the framework structure may also be loaded with gelatin, collagen, silk, hydrogel or a combination thereof.

[0045] In another preferred embodiment, the framework structure is decalcified bone matrix.

[0046] In another preferred embodiment, the decalcified bone matrix is ​​derived from allogeneic bone repair material.

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

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

[0049] In another preferred embodiment, the thickness of the decalcified bone matrix is ​​0.3-0.8 cm, preferably 0.4-0.6 cm, and most preferably 0.5 cm.

[0050] In another preferred embodiment, the decalcification amount of the decalcified bone matrix is ​​30% to 50%.

[0051] In another preferred embodiment, the cartilage gel / cartilage membrane particle-framework structure complex can generate articular cartilage under the joint microenvironment.

[0052] The second aspect of the present invention provides a method for preparing the cartilage tissue engineering complex described in the first aspect of the present invention, which comprises the following steps: inoculating the cartilage gel or cartilage membrane particles described in the first aspect of the present invention into a porous framework structure, and culturing cartilage in vitro to obtain the cartilage tissue engineering complex.

[0053] In another preferred embodiment, the cartilage gel is inoculated into the porous framework structure by a direct filling method.

[0054] In another preferred embodiment, the cartilage membrane particles are inoculated into the porous framework structure by centrifugation.

[0055] 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 framework structure.

[0056] In another preferred embodiment, the chondrogenic culture is an in vitro culture using a chondrogenic culture medium.

[0057] 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.

[0058] 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.

[0059] In another preferred embodiment, the chondrogenic culture time is 3-15 days, preferably 5-11 days.

[0060] The third aspect of the present invention provides a use of the cartilage tissue engineering complex described in the first aspect of the present invention for preparing a medical product for repairing joint defects.

[0061] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0062] 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.

[0063] In a fourth aspect, the present invention provides a pharmaceutical composition for joint repair, comprising cartilage gel or cartilage membrane particles and a pharmaceutically acceptable carrier.

[0064] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0065] In another preferred embodiment, the pharmaceutical composition is an injection.

[0066] In another preferred embodiment, the volume ratio of the cartilage gel to the pharmaceutically acceptable carrier in the pharmaceutical composition is 50% to 90%, preferably 70% to 85%.

[0067] In another preferred embodiment, the volume ratio of the cartilage membrane particles to the pharmaceutically acceptable carrier in the pharmaceutical composition is 45%-90%, preferably 60%-80%.

[0068] The fifth aspect of the present invention provides a method for repairing joint defects, 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.

[0069] In another preferred embodiment, the joint defect is an articular cartilage defect.

[0070] 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.

[0071] 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

[0072] Figure 1 The electron micrograph of decalcified bone matrix material is shown. The bar in the figure is 1 mm.

[0073] Figure 2 A macroscopic photograph of decalcified bone matrix material is shown. The scale bar in the figure is 1 cm.

[0074] Figure 3 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).

[0075] Figure 4 The actual picture of the cartilage gel-frame structure complex is shown. The scale bar in the picture is 1 cm.

[0076] Figure 5 The actual picture of the cartilage membrane particle-frame structure complex is shown. The scale bar in the picture is 1 cm.

[0077] Figure 6 The electron micrograph of the cartilage gel-decalcified bone complex is shown. The scale bar in the figure is 200 μm.

[0078] Figure 7 The electron micrograph of the cartilage membrane particle-decalcified bone complex is shown. The scale bar in the figure is 200 μm.

[0079] Figure 8 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.

[0080] Fig. 9 Shown is a comparison of gel cartilage-decalcified bone composite (A) and decalcified bone alone (B) transplanted into goat knee joint defects. DETAILED DESCRIPTION

[0081] 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-framework structure 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 porous framework structure, and the prepared cartilage tissue engineering complex can be 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.

[0082] the term

[0083] 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.

[0084] As used herein, the term "cartilage tissue engineering complex" includes cartilage gel-framework complex and cartilage membrane particle-framework complex that have been cultured with or without in vitro chondrogenic culture as described herein, which can be collectively referred to as cartilage tissue engineering complex in the present invention.

[0085] As used herein, the term "seeding" means inoculating chondrocytes into a cell culture dish, and may also mean inoculating cartilage gel / cartilage membrane particles into a specified framework structure and distributing them evenly. A person skilled in the art can understand the meaning of "seeding" based on the context.

[0086] 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.).

[0087] 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".

[0088] Cartilage gel and its preparation

[0089] 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.

[0090] 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.

[0091] 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 1.0×108 / 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.

[0092] 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 porous biological framework structure) without chondrogenic culture, and also includes a complex formed by loading the gel cartilage of the present invention on a carrier material (especially a porous biological framework structure) and chondrogenic culture.

[0093] 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 gel cartilage of the present invention on a carrier material (especially a porous biological framework structure) and undergoing chondrogenic culture.

[0094] 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.

[0095] 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.

[0096] Cartilage membrane sheet and its preparation

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] Cartilage and chondrocytes

[0106] 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.

[0107] 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.

[0108] The chondrocytes used in the cartilage tissue engineering complex of the present invention can be hyaline cartilage, elastic cartilage or fibrocartilage, which can be transformed into articular cartilage in the joint environment after being implanted into the joint defect of the subject.

[0109] MACI and ACI

[0110] 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.

[0111] 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.

[0112] Porous frame structure

[0113] 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.

[0114] 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.

[0115] Decalcified bone matrix

[0116] 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.

[0117] 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.

[0118] 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%.

[0119] Allogeneic bone repair materials

[0120] 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.

[0121] Xenogeneic bone repair materials

[0122] 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.

[0123] Culture medium used in the present invention

[0124] 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.

[0125] Gelation medium: DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0126] Adhesion rate

[0127] In the present invention, when the cartilage gel of the present invention is inoculated on a carrier material (especially a porous biological framework structure), 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%.

[0128] The adhesion rate in the present invention is defined as follows:

[0129] 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%.

[0130] The method for measuring the adhesion rate comprises the following steps:

[0131] The inoculated sample (such as cartilage gel or cartilage gel-frame 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 based on the standard curve formula.

[0132] The main advantages of the present invention include:

[0133] (1) The cartilage gel of the present invention is more mature than chondrocytes and has a certain fluidity.

[0134] (2) As a degradable natural material, decalcified bone matrix can be degraded in the body, has a low immune response and good biosafety.

[0135] (3) The pore size of decalcified bone matrix 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.

[0136] (4) Injectable cartilage alone cannot be shaped, and the absorption rate of injectable cartilage alone under tension conditions is high, which limits its clinical application. Using decalcified bone matrix as a framework structure, an injectable cartilage-decalcified bone matrix complex with a certain special shape can be constructed, and the cartilage absorption rate can be limited after providing mechanical support.

[0137] (5) The cell sources of the transplant of the present invention are diverse and have good expansion capacity.

[0138] (6) Compared with other tissue engineering repair methods, the cartilage gel / cartilage membrane particle-decalcified bone matrix complex can stably regenerate cartilage.

[0139] (7) The cartilage gel / cartilage membrane particle-decalcified bone matrix complex can stably regenerate cartilage and provide immediate mechanical support, with good immediate repair effect.

[0140] 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.

[0141] Example 1

[0142] Preparation of cartilage gel-decalcified bone composite

[0143] In this example, a cartilage gel-framework complex was prepared. The specific operation method is as follows:

[0144] (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.

[0145] (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;

[0146] (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;

[0147] (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);

[0148] (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 3 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 3 As shown in C;

[0149] (6) The cartilage gel preparation (prepared in step (5), with a volume of about 0.25-0.35 ml) is inoculated into the framework structure (decalcified bone matrix framework ( Figure 1 and Figure 2 ), having a pore size of about 400-800 μm and a porosity of about 87.3% ± 3.7%, and standing at 37° C., 95% humidity, and 5% carbon dioxide for 2 hours;

[0150] (7) After standing, add cartilage-forming culture medium and continue in vitro culture for 3-11 days to form a cartilage gel-frame structure complex, such as Figure 4 As shown; electron microscopy showed that the pores of decalcified bone matrix were filled with chondrocytes ( Figure 6 ).

[0151] 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-frame structure complex can be cut.

[0152] Example 2

[0153] Preparation of cartilage membrane particles-decalcified bone composite

[0154] In this example, a cartilage membrane particle-framework complex is prepared. The specific operation method is as follows:

[0155] (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;

[0156] (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;

[0157] (3) After 8 hours, the collagenase solution was filtered and centrifuged to obtain ear chondrocytes for primary and subculture;

[0158] (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;

[0159] (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 3 D), where the cell density in the cartilage membrane tissue is approximately 1.0×10 8 / ml-10×10 8 Pieces / ml;

[0160] Use forceps to pick up the cartilage membrane ( Figure 3 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 3 As shown in F;

[0161] (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 cartilage membrane particles to ensure that the frame material is completely immersed; Place the centrifuge tube containing the frame material and cartilage membrane particles in a centrifuge at 600 rpm for 2 minutes;

[0162] (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 a cartilage membrane particle-framework structure complex, such as Figure 5 As shown; electron microscopy showed that the pores of decalcified bone matrix were filled with chondrocytes ( Figure 7 ).

[0163] 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.

[0164] Comparative Example 1

[0165] Determination of adhesion rate between chondrocyte suspension and cartilage gel

[0166] Provide a demineralized bone matrix framework (such as Figure 2 The gel cartilage preparation (prepared in Example 1, 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.

[0167] 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.

[0168] 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.

[0169] like Figure 8 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.

[0170] Comparative Example 2

[0171] Animal transplantation experiment to repair articular cartilage

[0172] 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 gel cartilage-framework 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.

[0173] The repair of cartilage defects in animals was observed immediately after implantation.

[0174] The results are as follows Fig. 9 As shown:

[0175] The defect at A is smooth and solid, surrounded by soft tissue membrane, which has a certain elasticity and has excellent immediate repair effect.

[0176] The wound surface of the defect at point B is rough and only provides physical support but cannot provide immediate repair.

[0177] 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 porous framework structure; 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 comprises 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 culturing chondrocytes 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 / g, the surface area of ​​the cartilage membrane is 1-5mm 2 ; wherein the cartilage membrane sheet is obtained by culturing chondrocytes through gelation 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 cartilage gel or cartilage membrane sheets inoculated thereon; and the cartilage gel or cartilage membrane sheets are filled in the pores of the porous framework structure, wherein the cartilage gel is inoculated into the porous framework structure by a direct filling method, and the cartilage membrane sheet particles are inoculated into the porous framework structure by a centrifugal method.

2. The composite according to claim 1, characterized in that The chondrocytes include elastic chondrocytes, fibrous chondrocytes, hyaline chondrocytes or a combination thereof.

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 1, 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 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.

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

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

13. The composite according to claim 12, characterized in that The thickness of the decalcified bone matrix is ​​0.3-0.8 cm.

14. The composite according to claim 12, characterized in that The thickness of the decalcified bone matrix is ​​0.4-0.6 cm.

15. The composite according to claim 1, characterized in that The cartilage tissue engineering complex can generate articular cartilage under the joint microenvironment.

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

17. The method according to claim 16, characterized in that The cartilage membrane sheet particles are inoculated into the porous framework structure by centrifugation.

18. The method according to claim 17, 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 sheet particles to enter the framework structure.

19. The method according to claim 17, characterized in that The centrifugal speed is 600 rpm.

20. The method of claim 16, wherein: 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.

21. The method of claim 20, wherein: The chondrogenesis culture time is 3-15 days.

22. The method of claim 20, wherein: The chondrogenic culture time is 5-11 days.

23. 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.

24. The use according to claim 23, 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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