Cartilage graft and construction method thereof

Abstract

The invention discloses a cartilage graft and a construction method thereof. The cartilage graft is a cartilage membrane or an injectable cartilage graft; the cartilage graft contains cartilage membranes or cartilage micro-tissues formed by in-vitro culture of cartilage cells.

Description

[0001] This application is a divisional application of the invention patent application filed on January 23, 2017, with application number 201710057997.8 and invention title "A cartilage graft and its construction method". Technical Field

[0002] This invention relates to the fields of medicine and biomedical engineering, and more specifically to a cartilage graft and a method for constructing the same. Background Technology

[0003] Cartilage defects are a common clinical condition, often resulting from congenital causes or trauma leading to deformities or absence of ear and nasal cartilage, as well as cartilage defects caused by joint trauma and degeneration. Due to the poor self-repair capacity of cartilage tissue, the repair of various cartilage injuries has always been a challenge in clinical treatment. In recent years, minimally invasive surgery has developed rapidly, and injectable filler therapy has become a research hotspot. Currently, commonly used injectable filling materials mainly include hyaluronic acid, synthetic or animal-derived collagen, and other biodegradable materials. Although these injectable materials provide satisfactory short-term results, their efficacy only lasts for 6-9 months, requiring repeated injections and potentially causing serious complications such as chronic granulomas, vascular embolism, and even blindness and cerebral infarction.

[0004] With the rise of tissue engineering technology, especially the successful in vitro and in vivo regeneration of cartilage using chondrocyte-biodegradable material composites, tissue-engineered cartilage has provided new ideas for the treatment of cartilage defects and cosmetic filler injections. Traditional tissue-engineered cartilage refers to the process of mixing cells with biodegradable scaffold materials using tissue engineering methods to form a cell-material composite, which is then transplanted or injected into specific sites in the body to form cartilage tissue, achieving the purpose of repairing cartilage defects or localized cosmetic filling and shaping. It has advantages such as simple operation, safety and effectiveness, minimally invasive or non-invasive nature, and ease of filling irregular defects, making it an important development direction for cartilage tissue engineering application research. However, it still has the following problems: 1. Insufficient number of low-generation chondrocytes: When regenerating cartilage using chondrocyte-material composites, a very high cell concentration is required (>50×10⁻⁶). 6( / ml). Therefore, when using low-generation chondrocytes, the cell count cannot meet the needs of regenerating large volumes of cartilage. To achieve the required cell count, large volumes of cartilage tissue need to be harvested, which limits donor sources and is extremely invasive. 2. High-generation chondrocytes are prone to dedifferentiation: Although the cell count can meet the requirements after large-scale in vitro expansion of chondrocytes, the resulting high-generation chondrocytes are prone to dedifferentiation and lose their ability to regenerate cartilage. 3. Difficulty in shaping: The chondrocyte-injectable material complex has a highly fluid nature, making it difficult to shape the complex after in vivo injection. 4. Uncontrollable cartilage regeneration effect: Due to the influence of a series of factors such as cell viability, material degradation rate, and individual inflammatory response to the material, the final quality and volume of regenerated cartilage cannot be precisely controlled. These problems greatly limit the widespread application of cartilage regeneration technology in the clinical repair of various cartilage defects.

[0005] Therefore, there is an urgent need in this field to provide cartilage grafts that have stable and controllable cartilage regeneration effects, are easy to shape, and have a low incidence of postoperative complications. Summary of the Invention

[0006] The present invention aims to provide a cartilage graft and a method for constructing the same.

[0007] In a first aspect of the invention, a cartilage graft is provided, the cartilage graft containing in vitro regenerated cartilage membranes or cartilage microtissue.

[0008] In another preferred embodiment, the cartilage graft contains cartilage microtissue and an injectable medium; the weight ratio of the cartilage microtissue to the injectable medium is 10:0-5; the injectable medium is selected from physiological saline, phosphate-buffered saline (PBS), hyaluronic acid, or autologous serum.

[0009] In another preferred embodiment, the in vitro regenerated cartilage membrane or cartilage microtissue is prepared by the following steps: (1) In vitro culture, expansion and passage of chondrocytes; (2) After seeding the passaged chondrocytes, chondrocytes were cultured to induce chondrocyte regeneration and obtain in vitro regenerated cartilage membranes. (3) Cut the in vitro regenerated cartilage membrane to obtain cartilage microtissue.

[0010] In another preferred embodiment, the chondrocyte seeding density in step (2) is 3 × 10⁻⁶. 5 cells / cm 2 -1×10 7 cells / cm 2 More preferably, 1×10 6 cells / cm 2 -5×10 6 cells / cm 2 .

[0011] In another preferred embodiment, the culture medium for the cartilage induction culture contains 1-50 ng / ml transforming growth factor beta 1 (TGFβ1), 10-200 ng / ml insulin-like growth factor (IGF), and 10-200 ng / ml dexamethasone.

[0012] More preferably, the culture medium for cartilage induction culture further contains one or more of the following substances: 1-50 ng / ml insulin, 1-50 mg / ml basic fibroblast growth factor (bFGF), 1-25 ng / ml transferrin, 1-20 mmol / ml 3-isobutyl-1-methylxanthine (IBMX), 1-50 ng / ml sodium selenite, and 5 × 10⁻⁶ mmol / ml sodium selenite. -2 -1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid, and 10-200 ng / ml vitamin C phosphate.

[0013] In another preferred embodiment, the chondrocytes are passaged for 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th generations; more preferably, they are passaged for 2nd to 5th generations.

[0014] In another preferred embodiment, the chondrocytes are derived from the patient's own body or from an allogeneic source; more preferably, they are derived from the patient's own ear cartilage, nasal septum, and rib cartilage.

[0015] In a second aspect of the invention, a method for preparing the cartilage graft provided by the present invention as described above is provided, the method comprising the steps of: (1) Expand chondrocytes in planar space and passage them to the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th generation; (2) High-density seeding of chondrocytes from passages 1, 2, 3, 4, 5, 6, 7, or 8 on cell culture dishes for chondrocyte induction culture to obtain in vitro regenerated cartilage membranes. (3) Cutting the in vitro regenerated cartilage membrane to form cartilage microtissue; (4) The in vitro regenerated cartilage membrane or cartilage microtissue is washed with physiological saline or PBS to obtain the cartilage graft provided by the present invention as described above.

[0016] In another preferred embodiment, the cut cartilage microtissue is washed with saline or PBS and then mixed with injectable media such as saline, PBS, hyaluronic acid, or autologous serum to form an injectable cartilage graft.

[0017] In a third aspect of the invention, the use of the cartilage graft provided by the present invention as described above in the construction of tissue-engineered cartilage tissue is provided.

[0018] In a fourth aspect of the invention, the use of chondrocytes in the in vitro construction of cartilage grafts is provided.

[0019] In another preferred embodiment, during the in vitro construction of cartilage grafts, in vitro cultured, expanded, and passaged chondrocytes are cultured at a rate of 3 × 10⁻⁶. 5 cells / cm 2 -1×10 7 cells / cm 2 The density (more preferably 1×10) 6 cells / cm 2 -5×10 6 cells / cm 2 Following inoculation, cartilage regeneration of cartilage membranes is induced in a culture medium containing 1-50 ng / ml TGFβ1, 10-200 ng / ml IGF, and 10-200 ng / ml dexamethasone; more preferably, the culture medium further contains one or more substances selected from the following: 1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, and 5 × 10⁻⁶ mmol / ml sodium selenite. -2 -1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid, and 10-200 ng / ml vitamin C phosphate.

[0020] In another preferred embodiment, the in vitro regenerated cartilage membrane is cut to obtain cartilage microtissue.

[0021] Accordingly, the present invention provides a cartilage graft with stable and controllable cartilage regeneration effect, easy shaping and low incidence of postoperative complications. Attached Figure Description

[0022] Figure 1 The preparation process of the cartilage graft provided by the present invention is shown; wherein, A represents auricular cartilage; B represents chondrocytes cultured in vitro; C represents in vitro regenerated perichondrium sheets; D represents cartilage microtissue formed after cutting the perichondrium sheets; E and F represent the results of injectable cartilage grafts formed by mixing cartilage microtissue with a small amount of physiological saline (approximately 10:1 ratio) and injecting them subcutaneously into the abdomen of sheep, with gross and histological findings obtained 24 weeks later; G represents tubular cartilage constructed in vitro from perichondrium sheets; H represents sheet-like cartilage formed in vivo from perichondrium sheets; and I represents tissue-engineered trachea constructed in vivo from perichondrium sheets. Detailed Implementation

[0023] To address the problems and difficulties existing in current technologies, the inventors discovered that a small number of primary chondrocytes can be rapidly expanded in vitro to achieve a geometrical increase in cell number, meeting the basic requirements for large-volume cartilage regeneration. Then, through high-density seeding combined with a special chondrogenic induction culture system, the dedifferentiation of high-generation chondrocytes is reversed, achieving large-volume cartilage regeneration in vitro and obtaining cartilage membranes. Finally, cartilage grafts are prepared using the cartilage membranes or cartilage micro-tissue obtained from their mechanical dissection. Based on this, the present invention was completed.

[0024] the term The terms "chondrocyte" and "chondrogenic cell" are used interchangeably, both referring to polygonal adherent cells that secrete cartilage-specific matrix such as type II collagen and glycosaminoglycans (GAG), obtained from autologous or allogeneic cartilage tissue using conventional cell separation techniques in the art.

[0025] The term "chondrocyte isolation" refers to the process of separating chondrocytes from tissues.

[0026] The term "chondrocyte proliferation" refers to the process of extensive proliferation of chondrocytes in an in vitro environment in order to obtain a large number of chondrocytes.

[0027] The term "chondrocyte passage" refers to the process of continuously passaged chondrocytes under in vitro culture conditions.

[0028] The term "chondrogenic culture medium" refers to a culture medium containing specific biochemical components that promotes the in vitro regeneration of cartilage tissue by chondrocytes.

[0029] The term "chondrogenic culture" refers to the process of providing a special biochemical environment that enables cells with chondrogenic potential to express the chondrogenic phenotype and acquire the ability to form chondrocytes.

[0030] The terms "cartilage sheet" and "in vitro regenerated (cartilage sheet)" are used interchangeably, both referring to sheet-like cartilage tissue formed by high-density seeding of chondrogenic cells and in vitro culture using the culture medium provided by this invention.

[0031] The term "cartilage microtissue" refers to cartilage fragments formed by mechanically cutting or shearing cartilage membranes.

[0032] The terms "injectable cartilage graft" and "injectable cartilage graft" are used interchangeably, both referring to cartilage grafts that can be transplanted into an animal via injection.

[0033] chondrocytes The source of chondrocytes in this invention is not particularly limited. They can be chondrocytes from humans or animals, derived from various cartilage tissues such as articular cartilage, costal cartilage, ear cartilage, nasal septum cartilage, and tracheal cartilage; they can also be other types of human or animal cells with cartilage differentiation potential, derived from tissues such as bone marrow, fat, muscle, and skin. A preferred source is ear cartilage, nasal cartilage, or costal cartilage from humans or animals.

[0034] The methods for isolating and obtaining chondrocytes are widely accepted and reported in the literature. A preferred method is to aseptically excise cartilage tissue under general or local anesthesia, wash it with PBS, add collagenase solution (generally 0.5-3 mg / ml, prepared with PBS or culture medium), and digest it at 37°C with shaking for 4-20 hours (depending on the source of the cartilage tissue and the progress of digestion). The chondrocyte suspension is then collected by filtration, centrifugation, washing, trypan blue staining, and microscopic counting. The viability of primary chondrocytes should generally be above 80%. Inducing stem cells to differentiate into chondrocytes is also a routine method in this field.

[0035] Methods for culturing, passaged, and cultured incubators of chondrocytes are well known in the art. A preferred method is to culture chondrocytes in a CO2 incubator. Suitable culture media include (but are not limited to): 1) F-12 medium or DMEM medium + 5%-20% fetal bovine serum; 2) F-12 medium or DMEM medium + 5%-20% autologous (or allogeneic) human serum; 3) F-12 / DMEM medium (1:1) + 2%-20% fetal bovine serum or human serum. A particularly preferred type of chondrocyte is chondrocytes isolated and cultured in vitro from passages 2 to 5. At this stage, the chondrocytes exhibit good function and viability, strong cartilage-forming ability, and are shown to express type II collagen by immunocytochemical staining, and to express type II collagen and aggrecan mRNA by RT-PCR and in situ hybridization.

[0036] Cartilage grafts The cartilage graft provided by the present invention can be an in vitro regenerated cartilage membrane or an injectable cartilage graft; the injectable cartilage graft contains cartilage microtissue.

[0037] The cartilage graft provided by the present invention contains chondrogenic cells; the chondrogenic cells are of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th generation; preferably the 1st, 2nd, 3rd, 4th or 5th generation; more preferably the 2nd to 5th generation.

[0038] The cartilage membrane provided by this invention is a sheet-like cartilage tissue that is regenerated in vitro by inducing culture after high-density seeding of chondrogenic cells, while the cartilage microtissue provided by this invention is obtained by cutting the cartilage membrane formed in this way.

[0039] In one embodiment of the present invention, the chondrocytes that have undergone the above-mentioned expansion and passage are used at a rate of 3 × 10⁻⁶. 5 cells / cm 2 -1×10 7 cells / cm 2 (Preferred 1×10) 6 cells / cm 2 -5×10 6 cells / cm 2 The cartilage membranes were seeded at a density suitable for in vitro cartilage induction culture, with the medium changed every 1-4 days until sheet-like cartilage tissue regenerated in vitro. The regenerated cartilage membranes were then cut or minced to form cartilage microtissue.

[0040] The aforementioned cartilage induction culture medium includes, but is not limited to, the addition of TGFβ1, IGF, and dexamethasone to conventional cell culture medium; more preferably, it also contains one or more substances selected from the following: insulin, transferrin, bFGF, IBMX, sodium selenite, proline, serum albumin, β-mercaptoethanol, L-glutamine, linoleic acid, and vitamin C phosphate.

[0041] In a preferred embodiment of the present invention, the cartilage induction culture medium is prepared by adding 1-50 ng / ml TGFβ1, 10-200 ng / ml IGF, and 10-200 ng / ml dexamethasone to the conventional cell culture medium; more preferably, one or more of the following substances are also added: 1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, and 5 × 10⁻⁶ mmol / ml sodium selenite. -2 -1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid and 10-200 ng / ml vitamin C phosphate.

[0042] The in vitro regenerated cartilage membrane provided by this invention can be directly implanted into a similar-shaped defect site in the body to obtain tissue-engineered tissue or organ; or it can be formed into a certain shape in vitro, such as, but not limited to, tubular, ear-shaped, etc., and then implanted into a similar-shaped defect site in the body to obtain tissue-engineered tissue or organ.

[0043] In one embodiment of the present invention, the in vitro regenerated cartilage membrane is supported by a silicone tube. The two membranes are rolled into a tube and placed in a centrifuge tube. The cartilage induction culture medium provided by the present invention for obtaining in vitro regenerated cartilage membranes is added and cultured for 1-10 weeks. The silicone tube is then removed, and the obtained tubular cartilage with certain mechanical strength and elasticity is reimplanted into the corresponding defect site in the body to obtain tissue-engineered tissue or organ.

[0044] The cartilage microtissue provided by this invention can be re-implanted into the defective site in the body by injection to obtain tissue-engineered tissue or organ.

[0045] The cartilage microtissue provided by this invention can also be mixed with an injectable medium to form an injectable cartilage graft. This injectable cartilage graft can then be injected back into the defect site in the body to obtain tissue-engineered tissue or organ. The weight ratio of the cartilage microtissue to the injectable medium is 10:0-5. The injectable medium includes (but is not limited to): physiological saline, PBS, autologous serum, protein fiber gel, calcium alginate gel, hyaluronic acid, polyvinyl chloride, dimethyl propylene, polypropylene-fumaric acid composite ethylene gel, polyoxypropylene, etc.; preferably, physiological saline, hyaluronic acid, or autologous serum.

[0046] This invention uses cartilage microtissue as an injectable cartilage graft. In one embodiment of this invention, the cartilage microtissue can also be mixed with a small amount of injectable medium (the weight ratio of cartilage microtissue to injectable medium is approximately 10:0-5) to form an injectable cartilage graft.

[0047] Methods for constructing cartilage grafts The method for preparing cartilage grafts provided by this invention includes the following steps: The first step is the acquisition and expansion of primary cells, which involves expanding chondrocytes through planar culture and passage them to 1-8 generations; preferably, generations 1, 2, 3, 4, or 5; more preferably, generations 2-5. The second step is the preparation of cartilage membranes, which involves seeding the passaged and expanded cells into ordinary culture dishes and placing them in cartilage induction culture medium for in vitro culture to form cartilage membranes. The third step is to create a cartilage graft.

[0048] The planar culture amplification method in the first step includes, but is not limited to, resuspending the isolated chondrocytes in high-glucose DMEM culture medium, then seeding the cell suspension onto culture dishes, culturing under suitable conditions, and after the cells have nearly reached confluence, digesting and passaged to new culture dishes at a ratio (e.g., but not limited to, 1:2-1:10; preferably 1:3-1:7; more preferably 1:3-1:5), and continuing passage under the same conditions. The high-glucose DMEM culture medium preferably contains 10-20% FBS and 1-10 ng / ml bFGF based on its total volume; the seeding amount is preferably approximately 0.5-2 × 10⁶ cells per 10 cm culture dish. 6 One living cell.

[0049] The second step involves seeding chondrocytes into a culture dish and then culturing them in vitro using chondrocyte induction culture medium until a cartilage membrane is obtained. The culture medium is changed every 1-4 days, and the in vitro induction time is 1-20 weeks, preferably 2-10 weeks.

[0050] In the second step, chondrocytes are produced at a rate of 3 × 10⁻⁶. 5 cells / cm 2 -1×10 7 cells / cm 2 (Preferred 1×10) 6 cells / cm 2 -5×10 6 cells / cm 2 The cartilage induction culture medium is prepared by adding 1-50 ng / ml TGFβ1, 10-200 ng / ml IGF, and 10-200 ng / ml dexamethasone to the conventional cell culture medium; more preferably, one or more of the following substances may also be added: 1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, and 5 × 10⁻⁶ mmol / ml sodium selenite. -2 -1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid and 10-200 ng / ml vitamin C phosphate.

[0051] In one embodiment of the present invention, the cartilage membrane obtained in the second step is formed into cartilage grafts of different shapes in the third step; for example, but not limited to, forming tubular cartilage grafts with cylindrical silicone tubes as internal supports.

[0052] In another embodiment of the present invention, in the third step above, the cartilage membrane obtained in the second step is cut or shredded to form cartilage microtissue; in one embodiment, the cartilage membrane obtained in the second step is cut into 0.5-1.5 mm * 0.5-1.5 mm sizes, and then 1-10 times the volume (preferably 3-5 times the volume) of physiological saline or PBS is added. After centrifugation, the supernatant is removed to obtain cartilage microtissue, which can then be injected into the cartilage graft.

[0053] The cleaned cartilage membrane can be reimplanted, or the cleaned cartilage microtissue can be mixed with an injectable medium to form an injectable cartilage graft. The injectable medium includes, but is not limited to, physiological saline, PBS, autologous serum, protein fiber gel, calcium alginate gel, hyaluronic acid, polyvinyl chloride, dimethyl propylene, polypropylene-fumaric acid-vinyl chloride composite gel, polyoxypropylene, etc.; preferably physiological saline, hyaluronic acid, or autologous serum; the weight ratio of cartilage microtissue to injectable medium is 10:0-5.

[0054] The above-described cleaning process removes various growth factors adsorbed onto the membrane during culture, thereby eliminating immune responses (such as swelling) triggered by these growth factors. The cleaning can be repeated, for example, but not limited to, 1-5 times, preferably 2-4 times.

[0055] A specially shaped cartilage membrane or injectable cartilage graft is injected into the body to repair damaged areas or fill depressions on the body surface.

[0056] application The injectable cartilage grafts provided by this invention can be used for the repair of various cartilage defects, including (but not limited to) repair of various cartilage defects such as joints, trachea, ears, nose, etc.; as well as minimally invasive filler cosmetic surgery, including (but not limited to) rhinoplasty, chin augmentation, temporal augmentation, etc.

[0057] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0058] The main advantages of this invention are: Achieving large-volume cartilage regeneration using a small amount of autologous cartilage: Chondrocytes are obtained and expanded in large quantities using a small amount of autologous cartilage tissue (less than 100mg); through high-density seeding combined with a special chondrogenic induction system, the dedifferentiation of high-generation chondrocytes is reversed, and large-volume cartilage (greater than 10g) is regenerated in vitro with minimal donor site trauma.

[0059] In vitro cartilage regeneration is safe and controllable. In vitro regeneration of cartilage tissue using only chondrocytes, without the involvement or interference of scaffold materials, makes the cartilage regeneration technology simple, controllable, and stable. It can be derived from autologous cells, and does not contain animal-derived components such as serum during the in vitro cartilage regeneration process, making it highly safe. The cartilage regeneration process is mainly completed in vitro, allowing for standardized production and systematic pre-implantation assessment and testing, such as histological staining, collagen quantification, and sterility testing. The production process and the quality of the regenerated cartilage are controllable.

[0060] (3) Stable in vivo cartilage regeneration effect: The cartilage membrane sheets formed in vitro in the early stages of this technology contain a large amount of characteristic extracellular matrix and are already relatively mature cartilage tissue; This technology does not involve scaffold materials, thus avoiding adverse inflammatory reactions caused by materials that could affect cartilage regeneration in the body. When autologous cell-regenerated cartilage grafts are transplanted into large animal models, the volume of cartilage formed one year post-surgery is more than 80% of the original transplant volume, with stable and controllable results. Areas with insufficient cartilage formation can be completely repaired through secondary grafting.

[0061] (4) Easy to shape and can be stably maintained: Solid cartilage membranes or cartilage micro-tissues have poor fluidity, making them easy to shape and stably maintained.

[0062] (5) Low incidence of postoperative complications: Cartilage membrane grafts or cartilage micro-tissue transplants do not enter blood vessels, resulting in an extremely low incidence of complications.

[0063] (6) Long-lasting clinical effect: It can form autologous cartilage and survive for a long time, and the clinical effect can be maintained permanently.

[0064] (7) Significant advantages compared with similar technologies: The in vitro cartilage regeneration technology provided by this invention has simpler steps and more stable effects than existing technologies. Compared to injectable fillers (such as hyaluronic acid, synthetic or animal-derived collagen, etc.), the therapeutic effect is more lasting; Compared with chondrocyte-degradable material complexes, the therapeutic effect is stable and controllable, and the safety is high; Compared with existing membrane fabrication technologies, it is simpler, more stable, and safer.

[0065] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0066] The units in the weight-volume percentages of this invention are well known to those skilled in the art, for example, referring to the weight of the solute in 100 ml of solution.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0068] Five goats were used in the following examples, regardless of sex, with an average weight of 20.0 kg ± 3 kg. All animals were treated humanely in accordance with laboratory animal protection guidelines.

[0069] Example 1 Obtaining and expanding primary cells A piece of auricular cartilage, approximately 1.0 cm x 1.0 cm in size, was taken from each animal. The cartilage piece was cut into small pieces, and 0.25% trypsin (Hyclone, USA) was added. The mixture was shaken and digested for 30 minutes. Then, the animal was washed three times with PBS, and 0.25% type II collagenase (Worthington, USA) was added. The mixture was shaken and digested for 7-8 hours. The resulting digest was filtered through a 100-micron cell filter, centrifuged, and the supernatant was discarded. The precipitated cells were resuspended in high-glucose Dalberg modified Eagle medium (DMEM, Gibco, USA). The resulting cells were then cultured at 1 x 10⁻⁶ cells / mL. 4 The cells were seeded at a concentration of / cm2 on 100mm culture dishes and cultured and expanded in DMEM medium (chondrocyte culture medium) containing 10% fetal bovine serum (Hyclone, USA), 2ng / ml bFGF, 1% penicillin and streptomycin under 37℃, 5% CO2 and saturated humidity conditions. When the chondrocytes reached 70%-80% confluence, they were passaged.

[0070] Example 2 Preparation of cartilage membrane 1 The P4 generation chondrocytes obtained in Example 1 were digested, centrifuged, and resuspended at 1x10⁻⁶. 6 / cm2 Inoculate the culture medium at the specified concentrations onto ordinary culture dishes and use chondrocyte induction medium (1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, 5 × 10⁻⁶ ppm). -2 The culture medium consisted of 1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid, 10-200 ng / ml vitamin C phosphate, 10 ng / ml dexamethasone, 5 ng / ml TGFβ1, and 5 ng / ml IGF, with daily medium changes. After 4 weeks of in vitro culture, a 100 mm diameter perichondrial flap was peeled off from the culture dish for systematic pre-implantation evaluation and testing, such as wet weight measurement, histological staining, collagen quantification, and sterility testing. The results showed that the wet weight of the perichondrial flap was relatively small, indicating the formation of a thin perichondrial flap tissue.

[0071] Example 3 Preparation of cartilage membrane 2 The P4 generation chondrocytes obtained in Example 1 were digested, centrifuged, and resuspended at 2 x 10⁻⁶. 6 / cm 2 Inoculate the culture medium at the specified concentrations onto ordinary culture dishes and use chondrocyte induction medium (1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, 5 × 10⁻⁶ ppm). -2 The culture medium consisted of 1-1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid, 10-200 ng / ml vitamin C phosphate, 10 ng / ml dexamethasone, 5 ng / ml TGFβ1, and 5 ng / ml IGF, with daily medium changes. After 4 weeks of in vitro culture, a 100 mm diameter perichondrial flap was peeled off from the culture dish for systematic pre-implantation evaluation and testing, such as wet weight measurement, histological staining, collagen quantification, and sterility testing. The results showed that the wet weight of the perichondrial flap was relatively high, indicating the formation of a thick perichondrial flap tissue.

[0072] Example 4 Cartilage membranes were prepared by in vitro culture of cartilage containing only TGFβ1, IGF, and dexamethasone. The P4 generation chondrocytes obtained in Example 1 were digested, centrifuged, and resuspended at 5 x 10⁻⁶. 6 / cm 2The cartilage membrane was inoculated onto ordinary culture dishes and cultured in cartilage induction medium (1-50 ng / ml TGFβ1, 5 ng / ml IGF, and 10 ng / ml dexamethasone), with the medium changed daily. After 4 weeks of in vitro culture, a 100 mm diameter cartilage membrane could be peeled off from the culture dish for systematic pre-implantation evaluation and testing, such as: volume and wet weight measurement, histological staining, GAG and collagen quantification, and sterility testing. The results showed that the cartilage membrane formed by in vitro culture using this induction medium met the implantation requirements, although the volume, wet weight, GAG, and collagen content were relatively low.

[0073] Example 5 In vitro construction of tubular cartilage using perichondrial sheets Two cartilage membranes obtained in Example 2 were stacked and placed at 37°C, 5% CO2, and saturated humidity for 30 minutes. Using a silicone tube as an internal support, the two membranes were rolled into a tube and placed in a 50ml centrifuge tube. 40ml of cartilage induction culture medium was added (1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, 5 × 10⁻⁶ ppm). -2 The medium was 1-1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid, 10-200 ng / ml vitamin C phosphate, 10 ng / ml dexamethasone, 5 ng / ml TGFβ1, and 5 ng / ml IGF. The medium was changed daily. After 8 weeks of in vitro culture, tubular cartilage with certain mechanical strength and elasticity could be obtained. See Appendix. Figure 1 G in the text.

[0074] Example 6 Perichondrial membranes form sheet-like cartilage within the body, constructing tissue-engineered tracheas. The autologous cartilage membrane obtained in Example 2 was implanted subcutaneously into the abdomen of a sheep. Eight weeks post-surgery, a sheet of cartilage approximately 90 mm in diameter was obtained. After prevascularization and epithelialization, it could be used to construct a functional tissue-engineered trachea in vivo. (See Appendix) Figure 1 H in the text.

[0075] Example 7 Preparation and cleaning of injectable cartilage microtissue The cartilage membrane sheet obtained in Example 2 was cut into 1.0 mm pieces. For a sample measuring 1.0 mm, add 3 times the volume of physiological saline or PBS, centrifuge at 1000 rpm for 5 minutes, and aspirate the supernatant. Repeat the above washing procedure 3 times.

[0076] Example 8 In vivo injection of pure cartilage microtissue The cleaned cartilage microtissue obtained in Example 3 was injected subcutaneously into the abdomen as an injectable cartilage graft.

[0077] Example 9 In vivo injection of pure cartilage microtissue The cleaned cartilage microtissue obtained in Example 3 was mixed with a small amount of hyaluronic acid or physiological saline (approximately a 10:1 ratio) to form an injectable cartilage graft, which was injected subcutaneously into the abdomen. Samples were collected and observed after 24 weeks. The results showed that, grossly, porcelain-white, relatively hard cartilage-like tissue was visible. Histological examination with HE staining further confirmed that it was homogeneous, mature cartilage-like tissue, with uniformly distributed specific cartilage lacunar structures and a large amount of extracellular matrix. See Appendix. Figure 1 E and F in the example.

[0078] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A cartilage graft, characterized in that, The cartilage graft contains in vitro regenerated cartilage membranes or cartilage microtissue; wherein the in vitro regenerated cartilage membranes or cartilage microtissue are prepared by the following steps: (1) In vitro culture, expansion and passage of chondrocytes; (2) After seeding the passaged chondrocytes, chondrocytes were cultured to induce chondrocyte regeneration and obtain in vitro regenerated cartilage membranes. (3) Cut the in vitro regenerated cartilage membrane to obtain cartilage microtissue.

2. The cartilage graft as described in claim 1, characterized in that, The cartilage graft contains cartilage microtissue and an injectable medium; the weight ratio of the cartilage microtissue to the injectable medium is 10:0-5; the injectable medium is selected from physiological saline, phosphate-buffered saline (PBS), hyaluronic acid, or autologous serum.

3. The cartilage graft as described in claim 1, characterized in that, The chondrocyte seeding density in step (2) is 3 × 10⁻⁶. 5 cells / cm 2 -1×10 7 cells / cm 2 ; Preferably 1×10 6 cells / cm 2 -5×10 6 cells / cm 2 .

4. The cartilage graft as described in claim 1, characterized in that, The culture medium for cartilage induction culture contains 1-50 ng / ml transforming growth factor (TGFβ1), 10-200 ng / ml insulin-like growth factor (IGF), and 10-200 ng / ml dexamethasone.

5. The cartilage graft as described in claim 4, characterized in that, The culture medium for cartilage induction culture also contains one or more of the following substances: 1-50 ng / ml insulin, 1-50 mg / ml bFGF, 1-25 ng / ml transferrin, 1-20 mmol / ml IBMX, 1-50 ng / ml sodium selenite, and 5 × 10⁻⁶ mmol / ml sodium selenite. -2 -1 mg / ml proline, 1-100 mg / ml serum albumin, 1-20 mg / ml β-mercaptoethanol, 1-50 mg / ml L-glutamine, 1-20 μg / ml linoleic acid and 10-200 ng / ml vitamin C phosphate.

6. The cartilage graft as described in claim 1, characterized in that, The chondrocytes are passaged through generations 1, 2, 3, 4, 5, 6, 7, or 8; preferably generations 2 to 5.

7. The cartilage graft as described in claim 1, characterized in that, The chondrocytes are derived from the patient's own body or from an allogeneic source; preferably, they are derived from the patient's own ear cartilage, nasal septum, or rib cartilage.

8. A method for preparing a cartilage graft as described in any one of claims 1-7, characterized in that, The method includes the following steps: (1) Expand chondrocytes in planar space and passage them to the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th generation; (2) High-density seeding of chondrocytes from passages 1, 2, 3, 4, 5, 6, 7, or 8 on cell culture dishes for chondrocyte induction culture to obtain in vitro regenerated cartilage membranes. (3) Cutting the in vitro regenerated cartilage membrane to form cartilage microtissue; (4) The in vitro regenerated cartilage membrane or cartilage microtissue is washed with physiological saline or PBS to obtain the cartilage graft as described in any one of claims 1-7.

9. The method as described in claim 8, characterized in that, The method further includes: washing the cut cartilage microtissue with physiological saline or PBS, and then mixing it with injectable media such as physiological saline, PBS, hyaluronic acid, or autologous serum to form an injectable cartilage graft.

10. The use of a cartilage graft as described in any one of claims 1-7 in the construction of tissue-engineered cartilage tissue.

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