CN216169072U - Step-by-step assembly type cartilage-bone porous bionic scaffold - Google Patents
Step-by-step assembly type cartilage-bone porous bionic scaffold Download PDFInfo
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- CN216169072U CN216169072U CN202122131497.9U CN202122131497U CN216169072U CN 216169072 U CN216169072 U CN 216169072U CN 202122131497 U CN202122131497 U CN 202122131497U CN 216169072 U CN216169072 U CN 216169072U
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Abstract
The utility model provides a cartilage phase and hard bone phase porous bionic scaffold capable of being assembled step by step. Specifically, the utility model provides a cartilage-bone composite bracket, which comprises a cartilage phase and a hard bone phase, wherein the cartilage phase is provided with a first structure, the hard bone phase is provided with a second structure, and the first structure and the second structure assemble the cartilage phase and the hard bone phase in a snap-fit manner to form a cartilage-bone composite. The cartilage-bone complex of the present invention may be used to repair cartilage-bone complex defects.
Description
Technical Field
The utility model relates to the field of biomedical tissue engineering, in particular to a step-by-step assembled cartilage-bone porous bionic scaffold.
Background
Cartilage-bone complex injury at joint parts caused by trauma, disease, degeneration and the like is a common clinical disease and has an increasing trend worldwide. The repair of cartilage-bone composite defects has great clinical requirements, and the current clinical common treatment methods comprise autologous osteochondral transplantation, allogeneic osteochondral transplantation, artificial joint replacement and the like, but have the defects of limited donor sources, foreign body rejection, no biological function and the like, the treatment effect is poor, and ideal permanent repair and reconstruction measures need to be researched urgently.
The rapid development of tissue engineering and regenerative medicine technology provides a new solution for the regenerative repair of articular cartilage complex injury. Tissue engineering is the construction of tissues in vitro to repair or replace tissues or organs that have lost function due to trauma or disease, using the basic principles and methods of life science and engineering. The bionic scaffold is used for inducing the regeneration of the in-situ cartilage-bone composite tissue to be expected to become an important means for reconstructing joint functions, but no major breakthrough exists in the field at present. The key difficulty is how to complete the preparation of the cartilage-bone composite scaffold, the bionics of microenvironment and the stepwise induction of cartilage-bone, and realize the construction of in vitro biological joints.
In view of the above, there is an urgent need in the art to develop a biomimetic scaffold suitable for cartilage-bone composite repair.
Disclosure of Invention
The utility model provides a bionic scaffold suitable for cartilage-bone composite repair.
In a first aspect of the present invention, there is provided a cartilage-bone complex comprising a cartilage phase and a hard bone phase, wherein the cartilage phase comprises a first solid phase carrier and a chondrocyte layer loaded on the first solid phase carrier, the chondrocyte layer containing chondrocytes or chondrocyte-containing particles;
the hard bone phase comprises a second solid phase carrier and a hard bone cell layer loaded on the second solid phase carrier, wherein the hard bone cell layer contains seed cells for forming a hard bone layer or particles containing the seed cells;
the hard bone phase comprises a second solid phase carrier and seed cells for forming hard bone or particles containing the seed cells loaded on the second solid phase carrier;
the cartilage phase is provided with a first structure, the hard bone phase is provided with a second structure, and the first structure and the second structure assemble the cartilage phase and the hard bone phase in a buckling mode to form a cartilage-bone compound.
In another preferred embodiment, the complex comprises a complex formed by seeding the chondrocytes or chondrocyte-containing particles onto the first solid-phase carrier and culturing the seeded chondrocytes or chondrocyte-containing particles into cartilage; and a complex formed by inoculating the hard bone-forming seed cells or the particles containing the seed cells to the second solid carrier and subjecting the second solid carrier to osteogenic culture (in the complex, chondrocytes are carried on the carrier and form a more compact integrated structure with the carrier; and the seed cells are carried on the carrier and form a more compact integrated structure with the carrier).
In another preferred embodiment, the complex comprises a complex formed by seeding the chondrocytes or the particles containing the chondrocytes on the first solid-phase carrier without chondrogenic culture, and a complex formed by seeding the seed cells forming hard bone or the particles containing the seed cells on the second solid-phase carrier without osteogenic culture.
In another preferred embodiment, said seed cells are selected from the group consisting of: bone marrow mesenchymal stem cells (BMSCs), chondrocytes, or a combination thereof.
In another preferred embodiment, the chondrocytes are selected from: elastic cartilage, hyaline cartilage, fibrocartilage tissue, or combinations thereof.
In another preferred embodiment, the chondrocytes are selected from: including ear chondrocytes, articular chondrocytes, costal chondrocytes, scapular chondrocytes, meniscal cells, or combinations thereof.
In another preferred embodiment, the chondrocyte-containing particles comprise gelated cartilage or cartilage membrane particles.
In another preferred embodiment, the cartilage gel comprises a cell population composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix encapsulates the cell population, the cartilage gel is in a gel state, and the density of the chondrocytes is at least 1.0 × 108Per ml or 1.0X 108Per gram.
In another preferred embodiment, the gel cartilage is prepared by culturing chondrocytes through gelation.
In another preferred embodiment, the adhesive rate of the gel cartilage is more than or equal to 90 percent.
In another preferred embodiment, the concentration of chondrocytes in the gel cartilage is 1.0 × 108-10×108One/ml, preferably 1.5-5X 108One per ml.
In another preferred embodiment, the gel cartilage is obtained by culturing for 2 to 5 days, preferably 2.5 to 4 days, by gelation.
In another preferred embodiment, the chondrocytes are from a mammal.
In another preferred embodiment, the chondrocytes are selected from the group consisting of ear chondrocytes, rib chondrocytes, or a combination thereof.
In another preferred embodiment, the ear chondrocytes are autologous or allogeneic; preferably autologous human ear chondrocytes.
In another preferred embodiment, the preparation of the gel cartilage comprises the following steps:
(1) providing an isolated chondrocyte, and carrying out primary culture and subculture to obtain a subcultured chondrocyte;
(2) inducing and culturing the subcultured chondrocytes obtained in the step (1) in a gelling medium to obtain induced gelled cartilage.
In another preferred example, in step (2), the subcultured chondrocytes are seeded in the culture vessel in a layered seeding manner.
In another preferred embodiment, in step (2), the chondrocytes are passaged to passage 2-5.
In another preferred example, in step (2), the culture medium contains or does not contain serum.
In another preferred example, in the step (2), the culture medium contains 5-15% (v/v) of serum.
In another preferred embodiment, the serum is selected from fetal bovine serum.
In another preferred example, in the step (2), the gelling medium is a DMEM medium.
In another preferred example, in step (2), the DMEM medium contains 4-5 wt% glucose, 5-20% FBS (v/v), 50-150U/ml streptomycin.
In another preferred example, in the step (2), the induction culture time is 2-5 days; preferably, it is 2.5 to 4 days.
In another preferred embodiment, the cartilage membrane particles comprise a cell population composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix encapsulates the cell population, and the cartilage particles are prepared by cutting a sheet-like cartilage membrane, wherein the density of the chondrocytes is at least 1.0 × 108Per ml or 1.0X 108Per gram.
In another preferred embodiment, the concentration of chondrocytes in the cartilage membrane is 1.0 × 108Per ml-10X 108One/ml, preferably 1.5-5X 108One per ml.
In another preferred embodiment, the cartilage membrane is prepared by culturing chondrocytes in a gelling medium.
In another preferred embodiment, the cartilage membrane is obtained by gelification culture for 6-30 days, preferably 7-20 days, most preferably 10-15 days.
In another preferred embodiment, the gelation culture is an in vitro culture using a gelation medium.
In another preferred embodiment, the gelling medium comprises the following components: high-glucose DMEM medium containing 4-5 wt% glucose, 10% FBS (v/v) and 100U/ml penicillin-streptomycin.
In another preferred embodiment, the thickness of the cartilage membrane is 0.2-0.25 mm.
In another preferred embodiment, the average volume of the cartilage membrane particles is 0.2. mu.l.
In another preferred embodiment, the surface area of the cartilage membrane particles is 0.05-10mm2Preferably, 1-5mm2More preferably, the average area is 1mm2。
In another preferred embodiment, the first structure is disposed on the first solid support.
In another preferred embodiment, the second structure is disposed on the second solid support.
In another preferred embodiment, the first structure and the second structure assemble the cartilage phase and the hard bone phase in a snap-fit manner to form the cartilage-bone composite.
In another preferred embodiment, the cartilage phase is formed by seeding chondrocytes or chondrocyte-containing particles on a cartilage phase solid support and then culturing the cartilage phase in a chondrogenic induction manner.
In another preferred embodiment, the hard bone phase is obtained by culturing an osteogenic induction culture after seeding seed cells forming hard bone or particles containing the seed cells on a hard bone phase solid carrier.
In another preferred embodiment, one or more of the first structure and the second structure are provided.
In another preferred embodiment, the number of the first structures and the second structures is selected from a positive integer between 1 and 5.
In another preferred embodiment, the first structure is located at the bottom end of the first solid support, and the second structure is located at the top end of the second solid support; or the first structure is positioned at the top end of the first solid phase carrier, and the second structure is positioned at the bottom end of the second solid phase carrier.
In another preferred embodiment, the first solid support and/or the second solid support comprises a porous biocompatible material.
In another preferred embodiment, the porous biocompatible material is selected from the group consisting of: PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, decalcified bone matrix and acellular matrix.
In another preferred example, the porous biocompatible material may also be loaded with gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
In another preferred embodiment, the first solid support comprises a decellularized matrix, preferably a cartilage decellularized matrix.
In another preferred embodiment, the second solid support comprises a demineralized bone matrix.
In another preferred example, the compound is a cylinder, the diameter of the cylinder is 4-8 mm, and the height of the cylinder is 4-6 mm;
the height ratio of the cartilage phase to the hard bone phase is 1: (1 to 1.5), preferably 1: 1.2.
in a second aspect of the present invention, there is provided a method for preparing a cartilage-bone complex comprising the steps of:
(a) preparing a cartilage-bone kernel grid framework: providing a split type cartilage phase and hard bone phase kernel grid framework, wherein the cartilage phase kernel grid framework is provided with a buckle protruding structure, and the hard bone phase kernel grid framework is provided with a corresponding buckle recessed structure;
(b) constructing a cartilage phase solid phase carrier: providing a cartilage acellular matrix, mixing the cartilage acellular matrix with an auxiliary agent, and perfusing the cartilage acellular matrix into a cartilage phase inner core grid framework; freeze-drying to obtain cartilage phase solid phase carrier with snap-fit protrusion structure;
(c) hard bone phase solid phase carrier construction: providing a decalcified bone matrix, mixing with an adjuvant, and pouring into the hard bone phase core grid framework; freeze-drying to obtain a hard bone phase solid phase carrier with a buckle concave structure;
(d) constructing a cartilage phase: inoculating chondrocytes to the cartilage phase solid phase carrier in vitro, and applying a chondrogenic induction culture medium in vitro to induce to construct a cartilage phase;
(e) hard bone phase construction: inoculating bone marrow mesenchymal stem cells to the hard bone phase solid phase carrier in vitro, and applying an osteogenic induction culture medium in vitro to induce and culture to construct a hard bone phase;
(f) cartilage-bone complex assembly: assembling the cartilage phase and the hard bone phase obtained in the step (d) and the step (e) through a buckling structure to construct a cartilage-bone complex.
In another preferred embodiment, the adjuvant is selected from gelatin or collagen.
In another preferred embodiment, the concentration of gelatin is selected from 1-10% (weight/volume).
In another preferred embodiment, the freeze-drying time is selected from 12 to 24 hours.
In another preferred embodiment, the temperature of the freeze-drying is-20-0 ℃.
In another preferred embodiment, in step (d), the cell suspension density of the chondrocytes is selected from 1 × 107-8×108Cells/ml; preferably, 5 × 107-1×108Cells/ml; more preferably, 7 × 107-9×107Cells/ml.
In another preferred embodiment, in the step (e), the cell suspension density of the mesenchymal stem cells is selected from 1 × 107-8×108Cells/ml; preferably, 5 × 107-1×108Cells/ml; more preferably, 2 × 107-3×107Cells/ml.
In another preferred example, in the step (a), the cartilage phase and hard bone phase core grid framework is prepared by three-dimensionally printing a PCL substrate based on a three-dimensional digital model of the cartilage phase and the hard bone phase.
In another preferred embodiment, step (b) further comprises a cross-linking treatment, comprising:
(b1) carrying out vacuum freeze drying on the cartilage phase inner core grid framework perfused with the suspension containing the cartilage acellular matrix to obtain a dry substance of the cartilage acellular matrix;
(b2) immersing the dried substance obtained in the step (b1) in a chemical cross-linking agent, and removing residual chemical cross-linking agent after cross-linking treatment at 4 ℃;
(b3) and (4) carrying out vacuum freeze drying to obtain the cartilage phase solid phase carrier with the snap-fit protrusion structure.
In another preferred embodiment, the crosslinking uses a reagent selected from the group consisting of: (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC), N-hydroxysuccinimide (NHS), glutaraldehyde, genipin, or a combination thereof.
In another preferred embodiment, the crosslinking agent is selected from EDC and/or NHS.
In another preferred embodiment, the concentration of EDC is selected from 0.2% to 1% (weight/volume).
In another preferred embodiment, the concentration of NHS is selected from 0.2% to 1% (weight/volume).
In another preferred embodiment, the time of the crosslinking treatment is selected from 18 to 36 hours.
In another preferred embodiment, in step (b1), the cartilage acellular matrix is present in the suspension in an amount of 1-10% (w/v).
In another preferred embodiment, step (c) further comprises a cross-linking treatment, comprising:
(c1) carrying out vacuum freeze drying on the hard bone phase inner core grid framework filled with the suspension containing the decalcified bone matrix to obtain a dried substance of the decalcified bone matrix;
(c2) immersing the dried product obtained in the step (c1) in a chemical cross-linking agent, and removing residual chemical cross-linking agent after cross-linking treatment at 4 ℃;
(c3) and (5) after vacuum freeze drying, obtaining the hard bone phase solid phase carrier with the snap-fit protrusion structure.
In another preferred embodiment, in step (c1), the content of the decalcified bone matrix in the suspension is 1-10% (weight/volume).
In a third aspect of the utility model, there is provided the use of a cartilage-bone complex for the manufacture of a medical product for repairing a cartilage and/or hard tissue defect.
In another preferred embodiment, the cartilage and/or hard tissue defect is selected from the group consisting of: articular cartilage defects, cleft lip and palate deformities, maxillofacial hard tissue defects, or a combination thereof.
In another preferred embodiment, the tissue engineered cartilage complex comprises a tissue engineered cartilage graft.
In another preferred embodiment, the shape of the tissue-engineered cartilage graft conforms to the shape of the defect site of the human body to which the cartilage graft is to be grafted.
In another preferred embodiment, the defect site is selected from the group consisting of an articular cartilage defect, cleft lip and palate malformation, maxillofacial hard tissue, or a combination thereof.
In another preferred embodiment, the defect site is selected from the group consisting of a knee joint defect, a temporomandibular joint defect.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Fig. 1 is a schematic structural view of a cartilage-bone complex of the present invention. Wherein, in A picture, from top to bottom include:
a chondrocyte layer 1;
a first solid support 2;
a first structure 3A;
a second structure 4A;
a second solid support 5;
a layer of scleroderma cells 6, wherein the first structure 3A is snap-fit connected to the second structure 4A, and the snap-fit structure 4A is recessed inside the second solid support 5.
In fig. B, from top to bottom:
a chondrocyte layer 1;
a first solid support 2;
a first structure 3B;
a second structure 4B;
a second solid support 5;
a layer of scleroderma cells 6, wherein the first structure 3B is snap-fit connected to the second structure 4B, the snap-fit structure 3B being recessed inside the first solid support 2.
The first solid phase carrier 2 is provided with a chondrocyte layer 1, the second solid phase carrier 5 is provided with a sclerocyte layer 6, the first solid phase carrier 2 and the chondrocyte layer 1 form a cartilage phase, and the second solid phase carrier 5 and the sclerocyte layer 6 form a hard bone phase.
Detailed Description
The inventor designs and prepares a step-by-step assembled cartilage-bone porous bionic scaffold through extensive and intensive research. The cartilage-bone porous bionic scaffold realizes the in-vitro simultaneous construction of cartilage and bone integrated biological joints. Specifically, the inventor designs a step-by-step assembly type cartilage-bone porous bionic scaffold with a buckle or tenon-and-mortise structure at the bottom end of a cartilage phase and the top end of a hard bone phase, inoculates and cultures cells on the cartilage phase and the hard bone phase scaffold in vitro, constructs mature cartilage phase and hard bone phase, and assembles a cartilage-bone porous bionic body compound through the buckle or tenon-and-mortise structure for repairing cartilage-bone composite defects. The present invention has been completed based on this finding.
Term(s) for
As used herein, "cartilage-bone complex of the utility model", "cartilage-bone biomimetic of the utility model", "cartilage-bone complex graft" are used interchangeably and refer to the cartilage-bone complex prepared according to the first aspect of the utility model.
As used herein, the term "gelation culture" refers to a process of providing a special biochemical environment for expressing cells having cartilage differentiation potential to form gelatinous chondrocytes having a thick texture and a significantly increased particle size, with chondrogenic ability.
As used herein, the term "cartilage (stem) cells" refers to chondrocytes, cartilage stem cells, or a combination thereof.
Decalcified bone matrix
The Decalcified Bone Matrix (DBM) is a bone graft material which is prepared by decalcifying allogeneic bone or xenogeneic bone and can reduce immunogenicity. The mechanical strength varies with the degree of decalcification. Has good biological property, osteoinductivity and osteoconductivity, can be biodegraded, promotes the formation of new bones and the mineralization of bone tissues, accelerates the healing of bones, can effectively repair bone injuries singly or in combination with autogenous bones, other biological materials and growth factors, and is a relatively ideal bone tissue engineering scaffold material.
Cartilage gel and preparation thereof
As used herein, "gelled cartilage", "cartilage gel", "gel-state cartilage", "gelatinous cartilage", "cartilage gel of the utility model" or "gelled cartilage of the utility model" are used interchangeably and all refer to cartilage (stem) cells of the utility model in the gel state, in particular, seeding and/or plating a specific concentration of chondrocytes onto a flat or substantially flat culture surface such that the seeded chondrocytes form a layered structure, and culturing the chondrocytes having the layered structure under suitable gelling culture conditions to form a gelatinous cartilage culture.
The gelated cartilage of the present invention is a novel type of cartilage that is different from free chondrocytes, spun-out chondrocytes and cartilage mass (pellet). The gelled cartilage of the present invention can be viewed as a particular form of cartilage between free chondrocytes and a dense mass of cartilage. The gel cartilage of the utility model has a certain viscosity and fluidity in close connection because the chondrocytes contact and/or interact with adjacent cells on a plane (X-Y plane) and contact and/or interact with adjacent chondrocytes in multiple directions such as above and/or below the chondrocytes and/or above or below the chondrocytes in multiple directions during the gelation culture process, so that the chondrocytes can secrete and form more extracellular matrix, and the gel cultured chondrocytes are wrapped in the extracellular matrix with certain viscosity, so that the gel cartilage of the utility model has certain viscosity and fluidity, and is more suitable for being seeded and loaded on various different carrier materials (especially porous carrier materials) to form a compound for repairing cartilage.
In addition, the gelated cartilage of the present invention has a gel state,on the other hand, an unusually high cell density (usually at least 1.0X 10)8One or more, e.g. 1.0X 108-10×108One/ml), therefore, the preparation method is particularly suitable for preparing grafts for repairing various types of cartilage or is used for cartilage transplantation or cartilage repair surgery.
In the present invention, the composite for repairing cartilage includes a composite formed by supporting the gel cartilage of the present invention on a carrier material (particularly, 3D printed porous frame material) without chondrogenic culture, and also includes a composite formed by supporting the gel cartilage of the present invention on a carrier material (particularly, 3D printed porous frame material) and undergoing chondrogenic culture.
In the present invention, a compound suitable for transplantation into a human or animal body is the tissue engineered cartilage graft of the present invention.
Preferably, in the present invention, the gelated cartilage is formed by culturing in vitro under the gelated culture condition for a period of time t 1. Preferably, t1 is 2.5-5.5 days, preferably 3-5 days.
In the present invention, it is a feature that after seeding chondrocytes of a specific density into a culture vessel, the seeded chondrocytes form a plurality of layers of chondrocyte populations stacked on each other (i.e., a chondrocyte population having a stacked structure) by, for example, sedimentation. Typically, the number of cells seeded in a stack of the present invention S1 is n times the number of cells S0 for 100% confluency (i.e., S1/S0 ═ n), where n is 1.5 to 20, preferably 2 to 10, more preferably 2.5 to 5, calculated on the culture area of the culture dish (or culture container) and assuming that the confluency of the cells of the plated monolayer is 100%.
Cartilage membrane and its preparation
The cartilage membrane of the present invention is prepared by extending the gelation culture time on the basis of the preparation of the cartilage gel of the present invention. That is, in the present invention, the cartilage membrane is formed by culturing the ear cartilage cells seeded and/or plated on a flat or substantially flat culture surface in vitro under the gelation culture conditions for a period of time t 2. Preferably, t2 is 6-30 days, preferably 7-20 days, and most preferably 10-15 days.
The cartilage membranes of the utility model have on the one hand an unusually high cell density (usually at least 1.0X 10)8One or more, e.g. 1.0X 108Is one (10X 10)8One/ml) and on the other hand, the thickness is thin (only 0.2-0.25mm) and the toughness is good, the particles can be cut into 'cartilage diaphragm particles' with the average volume of 0.2 mu l, and the particles are filled in a porous frame structure by a simple centrifugal mode, so the particles are particularly suitable for preparing implants for repairing various types of cartilage or used for cartilage transplantation or cartilage repair operation.
In the present invention, the composite for repairing cartilage includes a composite which is not cultured to form a cartilage by supporting the cartilage membrane particles of the present invention on a carrier material (particularly 3D printed porous frame structure), and also includes a composite which is formed by supporting the cartilage membrane particles of the present invention on a carrier material (particularly 3D printed porous frame structure) and culturing to form cartilage.
In the present invention, the compound suitable for transplantation into a human or animal body is the cartilage tissue engineering compound of the present invention, that is, the compound formed by loading the cartilage membrane particles of the present invention on a carrier material (particularly, a porous framework structure) and culturing the cartilage.
As used herein, "specific concentration" or "specific density" refers to 1.0X 10 of inoculation in a 3.5cm petri dish (e.g., one well of a six-well plate)7-2.0×107One cell, preferably, 1.5X 107And (4) cells. Performing gelation culture for different time to obtain final product with chondrocyte density of 1.0 × 108Each is-10.0 x 108The ear cartilage gel or the cartilage cells contained therein has a density of 1.0 × 108Each is-10.0 x 108Cartilage pieces per ml.
In another preferred embodiment, the gelation culture conditions are: chondrocytes of a specific density were seeded and cultured using a gelling medium, which is a high-sugar (4-5 wt% glucose) DMEM medium containing 10% fetal bovine serum and 100U/ml penicillin-streptomycin.
Cartilage and chondrocytes
Cartilage, cartilage tissue, is composed of chondrocytes and intercellular substances. The matrix in cartilage is in gel state and has high toughness. Cartilage is the connective tissue that predominates in support. The cartilage does not contain blood vessels and lymphatic vessels, and nutrients permeate from blood vessels in the cartilage membrane into intercellular substance and then nourish osteocytes.
Cartilage is classified into 3 types, namely hyaline cartilage, elastic cartilage and fibrocartilage, according to the difference in intercellular substance. The matrix of hyaline cartilage is composed of collagen fibers, fibrils and a surrounding amorphous matrix. There is a temporary scaffolding effect during the embryonic period, which is later replaced by bone. Hyaline cartilage in adults is distributed mainly in the trachea and bronchial walls, the sternal ends of ribs and the surface of bones (articular cartilage). The elastic cartilage has a matrix containing elastic fibers in addition to collagen fibers, and is largely elastic and distributed mainly in the auricle, the wall of the external auditory canal, the eustachian tube, the epiglottis, the throat and the like. The fibrous cartilage matrix has bundled collagen fibers arranged in parallel or in a cross way, and is tougher. Distributed over the intervertebral disc, glenoid, articular disc, and some tendons, ligaments, etc., to enhance the mobility and protection, support, etc.
As used herein, chondrocytes used in a tissue engineering complex for repairing articular cartilage are not limited to chondrocytes derived from articular cartilage. Preferably, it may be selected from chondrocytes derived from hyaline cartilage (e.g. articular cartilage), elastic cartilage or fibrocartilage (e.g. auricular cartilage).
In particular, the chondrocytes used for seeding in the present invention may be pre-cultured in a "gel culture" manner to obtain a state of cartilage gel or cartilage membrane particles, and then seeded with a scaffold material to perform in vitro chondrogenic culture to obtain a cartilage phase constructed in vitro.
Cartilage-bone complex and method of preparation:
the cartilage-bone composite comprises a cartilage phase and a hard bone phase, wherein the bottom end of the cartilage phase and the top end of the hard bone phase are provided with a buckle or tenon-and-mortise structure. Specifically, the cartilage phase is provided with a first structure, the hard bone phase is provided with a second structure, and the first structure and the second structure assemble the cartilage phase and the hard bone phase in a buckling mode to form a cartilage-bone composite. In a specific embodiment, after the mature cartilage and hard bone phases are constructed by seeding and culturing cells on the cartilage and hard bone phase scaffolds in vitro, the cartilage-bone composite is assembled by a snap or mortise and tenon structure.
In one embodiment of the utility model, the constructed cartilage-bone composite is a cylinder, and the diameter of the cylinder is 4-8 mm; the height of the cylinder is 4-6 mm. The height ratio of the cartilage phase to the hard bone phase is 1: (0.5 to 1.5), preferably 1: 1.2.
the height of the chondrocyte layer is 5-100 mu m; the height of the hard bone cell layer is 5-100 mu m;
the cartilage phase and the hard bone phase are connected in a buckling mode. It will be appreciated that one or more pairs of snap structures may be provided to connect the cartilage and hard bone phases. For example, 1, 2, 3, 4, 5 pairs are provided. Preferably, 1 pair of fastener structures are arranged to connect the cartilage phase and the hard bone phase.
In a particular embodiment of the utility model, a first structure 3A is provided at the bottom of the cartilaginous phase; a second structure 4A is arranged at the top end of the hard bone phase. Wherein the first structure 3A is connected with the second structure 4A in a snap-fit manner, and the second structure 4A is recessed inside the second solid phase carrier 5 (hard bone phase). The first structure 3A is arranged on the bottom surface of the cartilage phase.
In a particular embodiment of the utility model, a first structure 3B is provided at the bottom of the cartilaginous phase; the second structure 4B is arranged at the top end of the hard bone phase. Wherein the first structure 3B is connected with the second structure 4B in a snap-fit manner, and the snap-fit structure 3B is recessed inside the first solid phase carrier 2 (cartilage phase). The first structure 4B is disposed on the top surface of the cartilage phase.
Because the required induction conditions are different when the cartilage phase and the hard bone phase are constructed in vitro, the split type bracket can realize the independent culture of the cartilage phase and the hard bone phase so as to meet the requirements of different induction conditions. In addition, the assembled cartilage-bone compound can be subjected to in-vitro cartilage-bone co-culture, so that the cartilage-bone material can grow in an interface which is better close to the natural state.
The utility model provides a preparation method of a cartilage-bone compound, which comprises the following steps:
(1) preparing a cartilage-bone kernel grid framework;
(2) constructing a cartilage phase solid phase carrier;
(3) constructing a hard bone phase solid phase carrier;
(4) constructing a cartilage phase;
(5) constructing a hard bone phase;
(6) cartilage-bone complex assembly.
Applications of
The defect repair material is customized individually according to different damage degrees such as defect area, shape, defect depth and the like, and the self condition and the requirement of the patient.
Culturing cartilage phase in vitro with chondrogenesis inducing liquid for 2-8 weeks; the hard bone phase was cultured in vitro using osteogenic induction liquid for 2-4 weeks. Because the required induction time is different when the cartilage phase and the hard bone phase are constructed in vitro, the split type bracket can realize the independent culture of the cartilage phase and the hard bone phase so as to meet the requirements of different induction time. In addition, the cartilage phase and the hard bone phase are assembled into the porous bionic scaffold with the structure similar to a 'steel bar-concrete' type, so that the problem that the natural material cannot be printed in a 3D mode to accurately control the form is solved, the biological activity and the cartilage and bone matrix microenvironment of the natural material are maintained to the greater extent, and the possibility is provided for further adding various bioactive factors to improve the regulation and control function of the composite scaffold.
Method for measuring adhesion rate
The method for measuring the adhesion rate comprises the following specific steps:
DNA quantification A1 of the inoculated sample (cell suspension or cartilage gel) was detected; detecting DNA quantification A2 after incubation of the inoculated complex (cell-scaffold complex or cartilage gel-scaffold complex) for 24 hours; the adhesion was a2/a1 × 100%.
The method for measuring the adhesion rate comprises the following steps:
an inoculated sample (such as cartilage gel or cartilage gel-3D printing double-layer porous frame compound) is taken, digested by proteinase K, the digested sample is quantitatively detected by a PicoGreen kit (Invitrogen, Carlsbad, CA, USA), the absorbance at 520nm is measured by a fluorescence microplate reader, and the DNA content is calculated according to a standard curve formula.
In the present invention, when the cartilage gel of the present invention is seeded on a support material (especially, a 3D-printed double-layered porous frame), the cartilage gel of the present invention has a certain adhesion rate, which is determined by the adhesion rate measuring method provided by the present invention. The adhesion rate of the cartilage gel or cartilage membrane particles is more than or equal to 90 percent, preferably more than or equal to 95 percent.
The main advantages of the utility model include:
(1) the design structure of the buckle can realize the step-by-step free assembly of the cartilage phase and the hard bone phase;
(2) the cells inoculated in the cartilage phase and the hard bone phase are different in types, and the split type bracket can realize that the cells of different types are respectively inoculated in the cartilage phase and the hard bone phase;
(3) the densities and the numbers of the cells inoculated in the cartilage phase and the hard bone phase are different, and the split type bracket can realize that the cells with different densities and numbers are respectively inoculated in the cartilage phase and the hard bone phase;
(4) the required culture mediums are different when the cartilage phase and the hard bone phase are constructed in vitro, and the split type bracket can realize that the different culture mediums can respectively culture the cartilage phase and the hard bone phase;
(5) the split type bracket can realize that different cell factors respectively induce the cartilage phase and the hard bone phase;
(6) the induction time required by the in vitro construction of the cartilage phase and the hard bone phase is different, and the split type bracket can realize the independent culture of the cartilage phase and the hard bone phase so as to meet the requirements of different induction time.
(7) The cartilage acellular matrix and the decalcified bone matrix can respectively provide microenvironment for cartilage regeneration and bone regeneration, realize component bionics and promote cartilage formation and bone formation;
(8) the simple cartilage acellular matrix and the decalcified bone matrix are difficult to form, gelatin or collagen in a certain proportion is compounded as an auxiliary agent, the crosslinking performance is effectively improved, and the three-dimensional porous scaffold in a certain shape can be prepared;
(9) the precise control of the pore structure and degradation rate of the stent material can be realized by regulating and controlling the compounding ratio, freeze-drying and crosslinking parameters of the material.
(10) The porous bionic scaffold with the structure of 'steel bar-concrete' solves the problem that the natural material can not be printed in a 3D mode to accurately control the form, keeps the biological activity and the cartilage and bone matrix microenvironment to the maximum extent, and provides possibility for further adding various bioactive factors to improve the regulation and control function of the composite scaffold.
The utility model will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Culture medium
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, and ascorbyl phosphate), 40 μ g/ml proline, and 10ng/ml TGF- β1100ng/ml IGF-1,40ng/ml dexamethasone and 50. mu.g/ml vitamin C.
Osteogenic induction medium:
low sugar DMEM medium, 10% Fetal Bovine Serum (FBS), 10mM dexamethasone, 10mM beta-glycerophosphate, 50mM vitamin C
The gelling medium is DMEM medium containing 4-5 wt% glucose, 10% FBS (v/v) and 100U/ml streptomycin.
Example 1: preparation of cartilage phase and hard bone phase split type core grid framework
The split three-dimensional digital model of cartilage-bone which can be assembled step by step through computer aided design is divided into cartilage phase and hard bone phase. The bottom end of the cartilage phase is provided with two buckling type protruding structures, and the top end of the hard bone phase is provided with two buckling type concave structures matched with the bottom end of the soft bone phase, so that the two parts can be tightly connected to form a complex. And adjusting parameters such as the layer width, the deposition angle, the aperture size and the like of the cartilage phase and the hard bone phase according to the structures and the mechanical characteristics of the cartilage phase and the hard bone phase. A PCL substrate is printed by a 3D printer (MAM-II Free Form contamination System) based on a three-dimensional digital model to prepare a millimeter-scale cartilage phase and hard bone phase split type inner core grid framework. By adjusting the widths of different layers, different deposition angles (-45/45 degrees, -60/60 degrees or 0/60/120 degrees) and stainless steel needles (19G, 20G or 21G) with different diameters, appropriate printing parameters such as extrusion speed, printing speed, layer height and the like are set, and the cartilage phase and hard bone phase split type inner core grid framework with different pore sizes and internal structures is prepared.
The split type inner core grid frame of the cartilage-bone complex shown in figure 1 has a deposition angle of 60 degrees, a stainless steel needle of 21 degrees and a pore size of 0.3 mm.
Example 2: preparation of cartilage and hard bone scaffolds
Taking gelatin granules with a certain mass (0.2g), placing the gelatin granules into deionized water with a certain volume (20ml), and shaking at a constant temperature of 37 ℃ for 2 hours to dissolve the gelatin granules to prepare gelatin solution with a concentration of 1% (m/v). The cartilage acellular matrix powder with a certain mass (0.1g) is put into 1 percent gelatin solution with a certain volume (10ml), and is precooled at low temperature of 4 ℃ and stirred for 6 hours to be fully and uniformly mixed to prepare suspension containing the cartilage acellular matrix with 1 percent.
The method comprises the following steps of putting a certain mass (0.1g) of decalcified bone matrix powder into a certain volume (10ml) of 1% gelatin solution, precooling at a low temperature of 4 ℃, stirring for 6 hours, and fully mixing uniformly to prepare suspension containing 1% of the decalcified bone matrix for constructing a hard bone phase.
The split cartilage phase and hard bone phase inner core grid frames are respectively fixed in a special cylindrical mould, certain volumes (1mL) of suspension containing 1% of cartilage acellular matrix and suspension containing 1% of decalcified bone matrix are respectively poured into the special cylindrical mould, the special cylindrical mould is frozen at the temperature of-10 ℃ for 24 hours, and after vacuum freeze drying is carried out for 48 hours, the cartilage phase and hard bone phase solid phase carriers are respectively immersed into a crosslinking solution containing 0.5% of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.3% of N-hydroxysuccinimide (NHS) (dissolved in 95% alcohol) for full crosslinking for 24 hours. And repeatedly soaking and washing the cartilage phase and hard bone phase scaffold by using deionized water for 24 hours, removing the residual chemical cross-linking agent in the three-dimensional scaffold, and performing vacuum freeze drying for 24 hours to obtain the split cartilage phase and hard bone phase solid phase carrier (namely the cartilage phase/hard bone phase scaffold).
Example 3: in vitro construction and assembly of cartilage and hard bone
3.1 cartilage phase in vitro construction
The method comprises the following steps: obtaining articular chondrocytes, inoculating cartilage phase scaffolds (with the diameter of about 0.8cm) in vitro to culture autologous articular chondrocytes, wherein the cell suspension density is 7.5X 107And/ml, in vitro applying chondrogenic induction culture medium to induce and culture for 4 weeks to obtain an integrated cartilage phase loaded with chondrocytes.
The method 2 comprises the following steps: aseptically cutting into 2.5 × 2.5cm2The ear cartilage tissue of (3) is stripped of the mucous membrane and fibrous tissue on the cartilage surface using a sterile instrument. Cutting cartilage tissue to 1.5 × 1.5mm2Large and small cartilage fragments. Collagenase is prepared at a concentration of 0.15%, and cartilage fragments are digested for 8 hours by adding to the prepared collagenase. After 8 hours, the collagenase solution is filtered and centrifuged to obtain the auricular chondrus cells, and primary and subculture (high-glucose DMEM medium) is carried out, and the cells are subcultured to 2-5 generations, preferably 3 generations.
After amplification, the cells were collected and resuspended at 8X 10 per well610ml to 30X 106/A10 ml cell volume was seeded in a six-well plate (3.5cm diameter) and cultured in a gelled medium (DMEM medium containing 4-5 wt% glucose, 10% FBS (v/v) and 100U/ml streptomycin). After 3 days of culture, the medium in the upper middle part of the six-well plate was aspirated, and the cartilage gel in the bottom of the six-well plate was collected using forceps to give a gel cartilage yield of 0.1-0.2ml in one well, which was collected in a 5ml syringe. The cartilage gel obtained after 3 days was about 0.1ml, and the cell density in the gel cartilage was 1.0X 10 as calculated by 2-fold cell expansion8-10×108One/ml, preferably 1.5-5X 108One per ml.
Mixing with 0.15ml culture medium to obtain injectable preparation containing cartilage gel.
On day 3, the viscosity of the cartilage gel is significantly increased, facilitating the combination with the load material with large pore size.
The adhesion rate was measured by the method for measuring adhesion rate described above. Compared with cell suspension, the gel cartilage adhesion rate of the utility model is 92% +/-2%.
Inoculating a gel cartilage preparation (prepared as described above, and having a volume of about 0.25-0.35ml) into the above cartilage phase scaffold, and standing at 37 deg.C, 95% humidity, and 5% carbon dioxide for 2 hours; standing, adding chondrogenic culture medium, and culturing for 3-11 days to obtain integrated cartilage phase loaded with chondrocytes.
The method 3 comprises the following steps: providing ear cartilage cells of 2-5 passages (prepared as described in method 2), collecting the cells after amplification, resuspending them, and culturing at 8X 10610ml to 30X 106The cell amount per 10 ml/well was seeded in a six-well plate and cultured in a gelling medium (DMEM medium containing 4-5 wt% glucose, 10% FBS (v/v) and 100U/ml streptomycin); after 24 hours or 48 hours of culture, replacing the fresh gelling culture medium, and continuing culturing in vitro for 15 days; the culture medium in the six-well plate was aspirated, and the auricular cartilage membrane tissue at the bottom of the six-well plate was visualized, in which the cell density in the auricular cartilage membrane tissue was about 1.0X 108Per ml-10X 108Per ml; the auricular cartilage membrane was clipped up with forceps and cut into 1X 1mm2Collecting the granules of the large ear cartilage membrane tablets into a 50ml centrifuge tube;
placing the frame material (cartilage phase bracket) to be inoculated into a centrifuge tube filled with the ear cartilage membrane particles to ensure that the frame material is completely immersed; placing the centrifuge tube filled with the frame material and the auricular cartilage diaphragm particles in a centrifuge, and centrifuging for 2 minutes at 600 revolutions per minute;
standing the inoculated frame material at 37 ℃ and 95% humidity and 5% carbon dioxide for a certain time; standing, adding chondrogenic culture medium, and culturing in vitro for 3-11 days to form cartilage membrane particle-framework structure compound.
3.2 hard bone phase in vitro construction
(1) Acquisition of BMSC cells: 3-5 ml of bone marrow is taken from the anterior superior iliac spine of a patient through puncture, and is placed on PercoII separating medium (the density is 1.073g/L) for gradient density centrifugation, wherein the ratio of the bone marrow to the separating medium is 1: 2. 2550r/min for 30 min, and the middle cloudy cell layer was aspirated and washed 1 time with Phosphate Buffered Saline (PBS). Centrifuging at 1550r/min, discarding the supernatant to obtain nucleated cells at 2X 107Cells/cm2Inoculating culture dish, in vitro cell amplification, and osteogenesis inducing with osteogenesis culture solution.
After primary cell inoculation, liquid is changed within 48 hours, after 80-90% of cells are fused, 0.25% of pancreatin is adopted for digestion, and the cell size is 2 multiplied by 103Cells/cm2Subculturing at 37 deg.C with 5% CO2The incubator was grown to passage 3, and cells were collected and counted.
(2) After the in vitro induction culture of the cartilage phase scaffold for 1 week, the hard bone phase scaffold (the diameter is about 8mm) is inoculated in vitro and cultured to the autologous bone marrow mesenchymal stem cells of the 3 rd generation, the cell suspension density is 2.5 multiplied by 107And/ml, in vitro using osteogenic induction medium for induction culture for 3 weeks.
3.3 Assembly
After relatively mature cartilage tissues and bone tissues are constructed in vitro, the protruding buckling structures of the cartilage phase and the concave structures matched with the hard bone phase are assembled to construct a cartilage-bone complex for repairing osteochondral composite defects.
The cartilage-bone complex of the present invention is shown in fig. 1.
Discussion of the related Art
The support materials for cartilage and bone repair and the preparation method thereof are various, and the cartilage-bone integrated bionic support in the prior art can be used for the instant repair of cartilage composite defects, but the repair range is limited. When a large area of osteochondral composite defect is needed, the existing cartilage-bone integrated bionic scaffold is difficult to well complete the repair. The repair of osteochondral complex defects needs to be realized by tissue engineering methods and in-vitro framework biological joints by scaffold complex autologous cells. At this time, the application of the existing integrated bracket is greatly limited for the following reasons:
(1) the types and the number of the cells inoculated in the cartilage phase and the hard bone phase are different;
(2) the types of culture media and cytokines required by the in vitro construction of the cartilage phase and the hard bone phase are different;
(3) the time required for the in vitro construction of the cartilage and hard bone phases is different, etc.
The above processes are difficult to realize accurate regulation and control on the existing integrated bracket. However, the step-by-step assembly type cartilage-bone porous bionic scaffold respectively provides microenvironment for cartilage regeneration and bone regeneration, realizes component bionic, and promotes cartilage formation and bone formation; through the assembly, the integrated culture of the cartilage-bone scaffold material in vitro is realized. The bionic scaffold provided by the utility model maintains the biological activity and the cartilage and bone matrix microenvironment to a greater extent, and provides possibility for further adding various bioactive factors to improve the regulation and control functions of the composite scaffold.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A cartilage-bone complex comprising a cartilage phase and a hard bone phase, wherein the cartilage phase comprises a first solid phase carrier and a chondrocyte layer loaded on the first solid phase carrier, wherein the chondrocyte layer comprises chondrocytes or chondrocyte-containing particles;
the hard bone phase comprises a second solid phase carrier and a hard bone cell layer loaded on the second solid phase carrier, wherein the hard bone cell layer contains seed cells for forming a hard bone layer or particles containing the seed cells;
the cartilage phase is provided with a first structure, the hard bone phase is provided with a second structure, and the first structure and the second structure assemble the cartilage phase and the hard bone phase in a buckling mode to form a cartilage-bone compound.
2. The complex of claim 1, wherein the seed cell is selected from the group consisting of: bone marrow mesenchymal stem cells (BMSCs), chondrocytes, or a combination thereof.
3. The complex of claim 1, wherein the chondrocyte is selected from the group consisting of: elastic cartilage, hyaline cartilage, fibrocartilage tissue, or combinations thereof.
4. The complex of claim 1, wherein said first structure is disposed on said first solid support and said second structure is disposed on said second solid support.
5. The composite of claim 1, wherein the first and second structures assemble the cartilage phase and the hard bone phase in a snap-fit manner to form the cartilage-bone composite.
6. The complex of claim 1, wherein the first structure is located at the bottom end of a first solid support and the second structure is located at the top end of a second solid support; or the first structure is positioned at the top end of the first solid phase carrier, and the second structure is positioned at the bottom end of the second solid phase carrier.
7. The composite of claim 1, wherein the number of first structures and second structures is selected from a positive integer between 1 and 5.
8. The complex of claim 1, wherein the first solid support comprises an acellular matrix.
9. The complex of claim 1, wherein the second solid support comprises a demineralized bone matrix.
10. The composite of claim 1, wherein the composite is a cylinder, wherein the cylinder has a diameter of 4 to 8mm and a height of 4 to 6 mm;
the height ratio of the cartilage phase to the hard bone phase is 1: (1-1.5).
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