A method for achieving cartilage regeneration by inoculating gel cartilage into a framework structure

Abstract

The present invention provides a method for achieving cartilage regeneration by inoculating gel cartilage into a framework structure. Specifically, the chondrocytes of the present invention are prepared into gel cartilage by amplification and dense inoculation; and are inoculated into a decalcified bone matrix to form a gel cartilage-decalcified bone complex, thereby achieving cartilage regeneration and being used for various filling treatments and bone repair.

Description

Technical Field

[0001] The invention relates to biomedical tissue engineering, and in particular to a method for implementing cartilage regeneration by inoculating gel cartilage into a framework structure. Background Art

[0002] In recent years, with the rapid development of the economy and society, cartilage defects caused by various types of trauma and congenital malformations have become increasingly common. Most cartilage defects caused by trauma or congenital malformations are difficult to repair by the patient themselves. In current clinical treatment, the treatment methods for articular cartilage defects are mainly palliative treatment and restorative treatment. Palliative treatment mainly includes arthroscopic cleaning and chondroplasty, etc. This type of treatment can clean the uneven cartilage surface of the joint, remove cartilage fragments, etc., 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 alleviate the development of arthritis; restorative treatment includes microfracture treatment, osteochondral transplantation, etc. Although this type of treatment can repair focal articular cartilage defects to a certain extent, it is more traumatic and can easily lead to donor site complications.

[0003] Tissue engineering is an interdisciplinary subject involving cell biology, materials science, engineering, and bioreactors. Through the basic principles and methods of life sciences and engineering, the tissues needed by the human body are constructed to repair and replace tissues or organs that are non-functional due to trauma or disease. In recent years, with the progress of tissue engineering, people have gradually begun to study the repair of cartilage defects by scaffolds or tissues constructed by tissue engineering. By combining with medical degradable materials such as decalcified bone materials, the construction of cartilage tissue is expected to become a new method for tissue engineering cartilage construction. However, the pore size of decalcified bone matrix materials is large and the porosity is good, but the cell adhesion rate is extremely low when inoculated with chondrocyte suspension, which is not conducive to the construction of tissue engineering materials.

[0004] In summary, there is an urgent need in the art to develop a method for inoculating chondrocytes into a framework structure to form a tissue-engineered cartilage complex to achieve cartilage regeneration. Summary of the invention

[0005] The purpose of the present invention is to provide a method for achieving cartilage regeneration by inoculating chondrocytes into a framework structure.

[0006] In a first aspect of the present invention, a gel cartilage is provided, wherein the gel cartilage 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 gel cartilage 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.

[0007] In another preferred embodiment, the gel cartilage is prepared by culturing chondrocytes through gelation.

[0008] In another preferred embodiment, the adhesion rate of the gel cartilage is ≥90%, preferably ≥95%.

[0009] In another preferred embodiment, the concentration of chondrocytes in the gel cartilage is 1.0×10 8 -10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0010] In another preferred embodiment, the gel cartilage is obtained by gelling culture for 2-5 days, preferably, 2.5-4 days.

[0011] In another preferred embodiment, the chondrocytes are from mammals.

[0012] In another preferred embodiment, the chondrocytes are selected from elastic cartilage, fibrous cartilage or hyaline cartilage.

[0013] In another preferred embodiment, the chondrocytes are selected from ear chondrocytes, costal cartilage or a combination thereof.

[0014] In another preferred embodiment, the ear chondrocytes are from autologous or allogeneic sources; preferably autologous human ear chondrocytes.

[0015] In a second aspect of the present invention, a tissue engineering cartilage composite is provided, the composite comprising:

[0016] (a) a carrier comprising a porous biocompatible material; and

[0017] (b) the gel cartilage according to the first aspect of the present invention seeded or loaded on the carrier;

[0018] In another preferred embodiment, the composite comprises a composite formed by inoculating the gel cartilage into the carrier and culturing the cartilage into cartilage (in the composite, chondrocytes are loaded on the carrier and form a more compact integrated structure with the carrier).

[0019] In another preferred embodiment, the composite comprises a composite formed by inoculating the gel cartilage into the carrier but without culturing the cartilage into cartilage.

[0020] In another preferred embodiment, in the composite, the ratio of gel cartilage to porous biocompatible material (or carrier) is 0.1-0.2 ml (or g) gel cartilage: 0.5-1 g porous biocompatible material.

[0021] In another preferred embodiment, the porosity of the porous biocompatible material is ≥30%, preferably ≥50%, and more preferably ≥70%.

[0022] In another preferred embodiment, the porosity of the porous biocompatible material is 80%-95%.

[0023] In another preferred embodiment, the pore size of the porous biocompatible material is 400-800 μm.

[0024] In another preferred embodiment, the porous biocompatible material comprises a biodegradable material.

[0025] In another preferred embodiment, the biodegradable material is selected from the following group: PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, or decalcified bone matrix.

[0026] In another preferred embodiment, the biodegradable material may also be loaded with gelatin, collagen, silk, hydrogel or a combination thereof.

[0027] In another preferred embodiment, the biodegradable material is decalcified bone matrix.

[0028] In another preferred embodiment, the decalcified bone matrix comes from allogeneic bone or xenogeneic bone.

[0029] In another preferred embodiment, the tissue engineered cartilage graft is prepared by the method described in the fourth aspect of the present invention.

[0030] In a third aspect of the present invention, there is provided a method for preparing the gel cartilage according to the first aspect of the present invention, comprising the following steps:

[0031] (1) providing an isolated chondrocyte, performing primary culture and subculture, thereby obtaining subcultured chondrocytes;

[0032] (2) The subcultured chondrocytes obtained in step (1) are induced and cultured in a gelling medium to obtain induced gel cartilage.

[0033] In another preferred embodiment, in step (2), the subcultured chondrocytes are inoculated in a culture container in a stacked inoculation manner.

[0034] In another preferred embodiment, in step (2), the number of cells S1 in the stacked inoculation is n times the number of cells S0 for 100% confluence (ie, S1 / S0=n), wherein n is 1.5-20, preferably 2-10, and more preferably 2.5-5.

[0035] In another preferred embodiment, in step (1), the concentration (m / v) of the collagenase is 0.1%-0.2%.

[0036] In another preferred embodiment, in step (1), the collagenase digestion time is 6-10 hours.

[0037] In another preferred embodiment, in step (2), the chondrocytes are passaged to passage 2-5.

[0038] In another preferred embodiment, in step (2), the seeding density of the chondrocytes is 0.5×10 6 -3×10 7 cells / cm 2 ; Preferably, 1×10 6 -5×10 6 cells / cm 2 More preferably, 1×10 6 -3×10 6 cells / cm 2 .

[0039] In another preferred embodiment, in step (2), the culture medium contains or does not contain serum.

[0040] In another preferred embodiment, in step (2), the culture medium contains 5-15% (v / v) serum.

[0041] In another preferred embodiment, the serum is selected from fetal bovine serum.

[0042] In another preferred embodiment, in step (2), the gelling medium is DMEM medium.

[0043] In another preferred embodiment, in step (2), the DMEM culture medium further contains 4-5wt% glucose, 5-20% FBS (v / v), and 50-150U / ml of penicillin-streptomycin.

[0044] In another preferred embodiment, in step (2), the induction culture time is 2-5 days; preferably, 2.5-4 days.

[0045] In the fourth aspect of the present invention, a method for preparing the tissue engineering cartilage complex as described in the third aspect of the present invention is provided, wherein the gel cartilage as described in the first aspect of the present invention is inoculated into a porous biocompatible material and cultured into cartilage in vitro to obtain the tissue engineering cartilage complex.

[0046] In another preferred embodiment, the in vitro chondrogenic culture time is 2-15 days; preferably, 3-11 days; more preferably, 4-7 days.

[0047] In a fifth aspect of the present invention, there is provided a use of the tissue cartilage complex as described in the second aspect of the present invention for preparing a medical product for repairing cartilage and / or hard tissue defects.

[0048] In another preferred embodiment, the cartilage and / or hard tissue defect is selected from: articular cartilage defect, cleft lip and palate deformity, maxillofacial hard tissue defect or a combination thereof.

[0049] In another preferred embodiment, the tissue engineered cartilage complex comprises a tissue engineered cartilage graft.

[0050] In another preferred embodiment, the shape of the tissue engineering cartilage graft is consistent with the shape of the defective part of the human body where cartilage transplantation is required.

[0051] In another preferred embodiment, the defect site is selected from articular cartilage defect, cleft lip and palate deformity, maxillofacial hard tissue or a combination thereof.

[0052] In another preferred embodiment, the shape of the tissue engineering cartilage graft is a human auricle, nose dorsum, nose wing, zygomatic arch, eyebrow arch, tubular, diamond, sheet, cylinder and other shapes but not limited thereto.

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

[0054] Figure 1 The decalcified bone matrix framework is shown.

[0055] Figure 2 Electron micrographs of decalcified bone matrix frameworks are shown, scale bar 1 mm.

[0056] Figure 3 The schematic diagram of cartilage gel culture is shown. A is a cartilage gel cultured in a six-well plate for 3 days, B is a cartilage gel collected at the bottom of the six-well plate after the culture medium in the upper part of the six-well plate is removed, C is an injectable preparation containing cartilage gel prepared by adding appropriate culture medium to the cartilage gel; D is a cartilage membrane sheet cultured in a six-well plate for 15 days, E is a schematic diagram of a cartilage membrane sheet with a certain mechanical strength; F is an injectable preparation containing a cartilage membrane sheet prepared by cutting the cartilage membrane sheet into pieces.

[0057] Figure 4 An injectable formulation containing cartilage gel (culture day 3) is shown.

[0058] Figure 5The figure shows the integrated tissue-engineered cartilage prepared by seeding a decalcified bone matrix framework with a gel cartilage preparation and adding chondrogenic medium for further culture.

[0059] Figure 6 The electron micrograph of the prepared integrated tissue-engineered cartilage is shown, with the scale bar being 200 μm.

[0060] Figure 7 Shown is a comparison of the adhesion rates of seeded samples (cell suspension or cartilage gel) after 24 h of culture on the composites.

[0061] Figure 8 Shown is a comparison of gel cartilage-decalcified bone composite (A) and decalcified bone alone (B) transplanted into goat knee joint defects. DETAILED DESCRIPTION

[0062] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that 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 to form a novel gel-like cartilage. The gel-like cartilage of the present invention has the advantages of strong adhesion, easy inoculation on a solid phase carrier to form a composite, high degree of composite with the carrier in the later chondrogenic culture, immediate repair ability of the cartilage graft, and excellent cartilage repair effect. The present invention was completed on this basis.

[0063] In addition, the inventors have also developed a composite or transplant based on the gel cartilage of the present invention, that is, the gel cartilage of the present invention is seeded on a porous carrier material (also referred to as a "framework structure") to form a composite for cartilage regeneration.

[0064] By optimizing the culture medium and gelation culture time of in vitro culture, the particle size of the gel cartilage in the present invention is significantly improved compared with ordinary chondrocytes, which is conducive to the attachment on the decalcified bone matrix material, avoiding the loss of chondrocytes, and the aperture ratio of the formed tissue engineering bone graft is significantly reduced. The tissue engineering bone graft of the present invention has an immediate repair effect on joint defects, and the microenvironment of the gel cartilage is conducive to the formation of mature cartilage tissue at the joint defect. Therefore, it is conducive to achieving cartilage regeneration and is used for various filling treatments and bone repair. The present invention was completed on this basis.

[0065] the term

[0066] As used herein, "the tissue engineering complex of the present invention", "the gel cartilage-tissue engineering complex", and "the tissue cartilage graft of the present invention" are used interchangeably and all refer to the cartilage graft described in the second aspect of the present invention.

[0067] As used herein, the term "gel culture" refers to a process of providing a special biochemical environment to allow cells with chondrogenic differentiation potential to express and form gelatinous chondrocytes with a viscous texture and significantly increased particle size, thereby possessing the ability to form chondrogenic cells.

[0068] As used herein, the term "chondrocyte (stem) cell" refers to chondrocytes, cartilage stem cells, or a combination thereof.

[0069] Cartilage gel and its preparation

[0070] 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.

[0071] 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.

[0072] In addition, the gel cartilage of the present invention has a gel state on the one hand and an unusually high cell density (usually at least 1.0×10 8 / ml or higher, such as 1.0×10 8 -10×10 8 Therefore, it is particularly suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.

[0073] 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 biocompatible material) 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 biocompatible material) and chondrogenic culture.

[0074] In the present invention, the composite suitable for transplantation into the human body or animal body is the tissue engineering cartilage transplant of the present invention.

[0075] Preferably, in the present invention, the gel cartilage is formed by culturing in vitro for a period of time t1 under gelation culture conditions. Preferably, the t1 is 2.5-5.5 days, preferably 3-5 days.

[0076] 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.

[0077] In a specific embodiment, a high concentration of chondrocytes are seeded on a culture medium with a flat surface, and induced in vitro for 2-5 days by the gelling culture medium of the present invention, forming a state between free and tightly connected, that is, a "quasi-connected" cell state. Specifically, the chondrocytes of the present invention are cultured in vitro, and a plurality of chondrocytes are wrapped by the extracellular matrix, forming a structure in which the cells are in relatively tight contact.

[0078] In a specific embodiment, the high concentration of chondrocytes is inoculated in a 3.5 cm culture dish (for example, one well in a six-well plate) with 1.0×10 7 -2.0×10 7 cells, preferably 1.5×10 7 cells.

[0079] The cell density in the gel cartilage is 1.0×10 8 -10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0080] Specifically, the gel cartilage of the present invention has a certain adhesion rate. According to the adhesion rate determination method in a preferred embodiment of the present invention, the adhesion rate of the gel cartilage of the present invention is ≥90%, preferably, ≥95%.

[0081] The gel cartilage of the present invention is differentiated from high-density cartilage gel cells. The high-density cartilage cells of the present invention are cultured in vitro and grow into gel cartilage. The particle size of the gel cartilage is significantly increased, which is more conducive to the attachment to the decalcified bone matrix. The concentration range of the chondrocytes is preferably 1.6×10 6 -2.2×10 6 cells / cm 2 .

[0082] In another preferred embodiment, the gelation culture condition is: inoculating high density chondrocytes and culturing them in a high-glucose culture medium containing 10% fetal bovine serum and 1% triple antibody.

[0083] In another preferred embodiment, the gel cartilage culture time is 2-5 days, and the seeding density is 1.6×10 6 -2.2×10 6 cells / cm 2 .

[0084] The present invention provides a specific gel material, namely, gel-like chondrocytes, which is cultured for 3 days.

[0085] Decalcified bone matrix

[0086] Decalcified bone matrix (DBM) is a bone transplant material that is made of allogeneic bone or xenogeneic bone after decalcification to reduce immunogenicity. The mechanical strength corresponding to different degrees of decalcification is also different. It has good biological properties, osteoinductivity, osteoconductivity and biodegradability, promotes new bone formation and bone tissue mineralization, and thus 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.

[0087] 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%.

[0088] Tissue engineered cartilage grafts

[0089] Pure gel cartilage cannot be formed, and the absorption rate of pure gel cartilage under tension conditions is high, which limits its clinical application. Using decalcified bone matrix as a framework structure can construct a gel cartilage-decalcified bone matrix complex with a certain special shape, and after providing mechanical support, the cartilage absorption rate can be limited.

[0090] It should be understood that in addition to the decalcified bone matrix used in the specific embodiments, the gel cartilage in the present invention can also be loaded on other common biological porous materials after being inoculated on the decalcified bone, preferably on degradable materials, including but not limited to:

[0091] (a) Degradable synthetic polymer materials, such as polylactic acid (PLA), polyglycolic acid (PGA), PLGA, polyhydroxybutyric acid (PHB), polyanhydrides, polyphosphazenes, polyamino acid, pesudo-polyamino acid, polyorthoesters, polyethylene glycol, hyaluronic acid, polydioxanone, etc.;

[0092] (b) Natural biodegradable materials, such as collagen, gelatin, glycosaminoglycan (GAGs), chitosan, chitin, alginate, and various decellularized matrices, such as decalcified bone matrix;

[0093] (c) Copolymers or composite materials of the above materials, especially composite materials of polymer materials and natural materials, and composite materials of solid materials and injectable materials.

[0094] Preferred medically acceptable biodegradable materials are solid materials or solid and liquid composite materials, such as polylactic acid (PLA), polyglycolic acid (PGA), collagen, decalcified bone matrix, etc. The materials in the present invention can be prefabricated into various precise sizes and shapes to adapt to the construction of cartilage tissues of different sizes and shapes. When the material is a solid material, the material can be directly prefabricated into the required size and shape, or the material can be precisely plasticized by a model operated by computer-aided and rapid prototyping technology.

[0095] Preparation method

[0096] The method for preparing the tissue engineered cartilage graft of the present invention is simple and convenient, and the method comprises the steps of:

[0097] (1) Cultivation of chondrocytes;

[0098] (2) Induced culture of gel cartilage;

[0099] (3) Inoculation of tissue engineering vectors.

[0100] Adhesion rate determination method

[0101] The specific steps of the adhesion rate determination method in the present invention are as follows:

[0102] The DNA quantification of the inoculated sample (cell suspension or cartilage gel) is measured A1; the DNA quantification of the inoculated complex (cell-scaffold complex or cartilage gel-scaffold complex) after culturing for 24 hours is measured A2; the adhesion rate is A2 / A1×100%.

[0103] The method for determining DNA quantification includes the following steps:

[0104] The inoculated sample (eg, 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.

[0105] The main advantages of the present invention include

[0106] (1) Gel cartilage tissue is more mature than chondrocytes and has a certain fluidity.

[0107] (2) As a natural material that can be degraded in the body, decalcified bone matrix has a low immune response and good biosafety.

[0108] (3) The decalcified bone matrix material has a larger pore size and better porosity. The gel cartilage tissue with a certain fluidity and relatively viscosity can effectively improve the adhesion rate.

[0109] (4) Pure gel cartilage cannot be formed, and the absorption rate of pure gel cartilage under tension conditions is high, which limits its clinical application. Using decalcified bone matrix to construct a gel cartilage-decalcified bone matrix complex can provide mechanical support and reduce the cartilage absorption rate.

[0110] (5) The decalcified bone can be customized into different shapes according to needs, and the gel cartilage-decalcified bone complex can be used for the repair and reconstruction of different parts and shapes.

[0111] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0112] Culture medium

[0113] 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.

[0114] Gelation medium: DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

[0115] Example 1: In vitro culture of gel cartilage

[0116] Sterile cut 2.5×2.5cm 2 Use sterile instruments to peel off the mucosa and fibrous tissue on the surface of the cartilage. Cut the cartilage tissue into 1.5×1.5mm 2 Large and small cartilage fragments. Prepare collagenase at a concentration of 0.15%, add the cartilage fragments to the prepared collagenase and digest for 8 hours. After 8 hours, filter and centrifuge the collagenase solution to obtain ear chondrocytes, and perform primary and subculture (high-glucose DMEM medium), and subculture the cells to the 2nd to 5th generation, preferably the 3rd generation.

[0117] After expansion, the cells were collected and resuspended at 8×10 6 / 10ml to 30×10 6 The cells were inoculated in a six-well plate (3.5 cm diameter) with a volume of 100 ml and cultured in a gelling medium (DMEM medium containing 4-5 wt% glucose, 10% FBS (v / v) and 100 U / ml penicillin-streptomycin). After 3 days of culture, the medium in the upper part of the six-well plate was aspirated and the cartilage gel at the bottom of the six-well plate was aggregated using tweezers (see Figure 3 B), The yield of gel cartilage from one well is 0.1-0.2 ml, which is collected into a 5 ml syringe.

[0118] After 3 days, the cartilage gel was about 0.1 ml. Based on the 2-fold cell expansion, the cell density in the gel cartilage was 1.0×10 8 -10×10 8 / ml, preferably 1.5-5×10 8 Pieces / ml.

[0119] It was mixed with 0.15 ml of culture medium to prepare an injectable preparation containing cartilage gel (see Figure 3 C. Figure 4 ).

[0120] like Figure 3 As shown, on the third day, the viscosity of the cartilage gel increased significantly, making it easier to combine with the load material with a large pore size.

[0121] In particular, when the cartilage gel on day 3 was cultured for a further 15 days, a sheet-like cartilage membrane was obtained which was thin and had good mechanical strength.

[0122] Example 2: In vitro construction of gel cartilage tissue

[0123] Provide a demineralized bone matrix framework (such as Figure 1 As shown), it was determined that the decalcified bone matrix framework had a pore size of about 400-800 μm and a porosity of about 87.3%±3.7% (the electron microscopic observation results of the decalcified bone matrix framework are shown in Figure 2 shown).

[0124] 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 frame and allowed to stand for 2 hours at 37°C, 95% humidity, and 5% carbon dioxide; after standing, the cartilage-forming culture medium was added and cultured for 3-11 days to obtain integrated tissue-engineered cartilage ( Figure 5 ).

[0125] result

[0126] The obtained integrated tissue-engineered cartilage is generally Figure 5 shown.

[0127] The electron microscopy results are as follows Figure 6 As shown, it was shown that the pores of decalcified bone matrix were basically effectively filled after inoculation of gel cartilage.

[0128] Example 3: Adhesion rate determination

[0129] Provide a demineralized bone matrix framework (such as Figure 1 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 frame, and the cells were kept at 37°C, 95% humidity, and 5% carbon dioxide for 2 hours and passaged 4 times.

[0130] like Figure 7 As shown, the adhesion rate was measured by the above-mentioned adhesion rate measurement method. Compared with the cell suspension, the adhesion rate of the gel cartilage of the present invention was 92%±2%.

[0131] Example 4: Animal transplantation experiment for repairing articular cartilage

[0132] The gel cartilage was inoculated into decalcified bone to construct a gel cartilage-decalcified bone complex, which was cultured in vitro for 11 days.

[0133] A cartilage defect with a diameter of 7.5 mm was created on the articular surface of the knee joint of the experimental animals, and the defect was filled with a gel cartilage-decalcified bone complex.

[0134] like Figure 8 As shown, the defect at A was filled with a gel cartilage-decalcified bone complex, and the defect at B was filled with simple decalcified bone.

[0135] Observe the wound surface:

[0136] The defect at point A is smooth and solid, surrounded by a soft tissue membrane with a certain degree of elasticity, and has an immediate functional repair effect.

[0137] The wound surface of the defect at point B is rough and only provides physical support, but cannot provide immediate repair effects.

[0138] The experimental results show that by repairing articular cartilage with a gel cartilage-decalcified bone complex, the constructed tissue is similar to the morphology of normal cartilage tissue, which can have an immediate repair effect on joint defects and is conducive to the formation of mature cartilage tissue at the joint defect.

[0139] 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 gel cartilage, characterized in that: The gel cartilage 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 gel cartilage 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; and the gel cartilage is obtained by gelling chondrocytes for 2-5 days, and the adhesion rate of the gel cartilage is ≥90%; wherein the chondrocytes are from the subject's own body; the culture medium used for the gelling culture is a DMEM culture medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

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

3. The gel cartilage according to claim 1, characterized in that The concentration of chondrocytes in the gel cartilage is 1.5-5×10 8 Pieces / ml.

4. The gel cartilage according to claim 1, characterized in that The gel cartilage is obtained by gelling culture for 2.5-4 days.

5. The gel cartilage according to claim 1, characterized in that: The chondrocytes are derived from mammals.

6. The gel cartilage according to claim 1, characterized in that The chondrocytes are selected from elastic cartilage, fibrous cartilage or hyaline cartilage.

7. The gel cartilage according to claim 1, characterized in that The chondrocytes are selected from auris chondrocytes, costal chondrocytes or a combination thereof.

8. A tissue engineering cartilage composite, characterized in that: The compound comprises: (a) a carrier comprising a porous biocompatible material; and (b) The gel cartilage according to claim 1 seeded or loaded on the carrier.

9. The tissue engineering cartilage composite according to claim 8, characterized in that: In the composite, the ratio of gel cartilage to porous biocompatible material is 0.1-0.2 ml or 0.1-0.2 g gel cartilage: 0.5-1 g porous biocompatible material.

10. The tissue engineering cartilage composite according to claim 8, characterized in that: The porosity of the porous biocompatible material is ≥50%.

11. The tissue engineering cartilage composite according to claim 8, characterized in that: The porosity of the porous biocompatible material is ≥70%.

12. The tissue engineering cartilage composite according to claim 8, characterized in that: The pore size of the porous biocompatible material is 400-800 μm.

13. The tissue engineering cartilage composite according to claim 8, characterized in that: The porous biocompatible material includes a biodegradable material.

14. The tissue engineering cartilage composite according to claim 13, characterized in that: The biodegradable material is selected from the group consisting of PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, or decalcified bone matrix.

15. The tissue engineering cartilage composite according to claim 13, characterized in that: The biodegradable material is also loaded with gelatin, collagen, silk, hydrogel or a combination thereof.

16. A method for preparing the gel cartilage as claimed in claim 1, characterized in that: The steps include: (1) providing an isolated chondrocyte, performing primary culture and subculture, thereby obtaining subcultured chondrocytes; (2) The subcultured chondrocytes obtained in step (1) are induced and cultured in a gelling medium for 2-5 days to obtain induced gel cartilage; wherein the gelling medium is a DMEM medium containing 4-5wt% glucose, 10% FBS (v / v) and 100U / ml penicillin-streptomycin.

17. The method according to claim 16, characterized in that In step (2), the subcultured chondrocytes are inoculated in a culture container in a layered inoculation manner.

18. The method according to claim 17, characterized in that In step (2), the number of cells S1 in the stacked inoculation is n times the number of cells S0 for 100% confluence, that is, S1 / S0=n, where n is 1.5-20.

19. The method according to claim 18, characterized in that The n is 2-10.

20. The method of claim 18, wherein: The n is 2.5-5.

21. The method of claim 16, wherein: In step (2), the induction culture time is 2.5-4 days.

22. A method for preparing the tissue engineering cartilage composite according to claim 8, characterized in that: The gel cartilage as claimed in claim 1 is inoculated into a porous biocompatible material and cultured into chondrogenesis in vitro to obtain the tissue engineering cartilage complex.

23. A use of the tissue engineering cartilage composite as claimed in claim 8, characterized in that: Used to prepare medical products for repairing cartilage and / or hard tissue defects.

Images