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CN115944781A - Cartilage tissue engineering compound based on 3D printing and application thereof - Google Patents

Cartilage tissue engineering compound based on 3D printing and application thereof Download PDF

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Publication number
CN115944781A
CN115944781A CN202111177643.XA CN202111177643A CN115944781A CN 115944781 A CN115944781 A CN 115944781A CN 202111177643 A CN202111177643 A CN 202111177643A CN 115944781 A CN115944781 A CN 115944781A
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China
Prior art keywords
cartilage
gel
chondrocytes
tissue engineering
porous frame
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CN202111177643.XA
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周广东
刘豫
张伟
慈政
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Shanghai Soft Heart Biotechnology Co ltd
Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Shanghai Soft Heart Biotechnology Co ltd
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Priority to CN202111177643.XA priority Critical patent/CN115944781A/en
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Abstract

The invention provides a cartilage tissue engineering compound. Specifically, the compound comprises cartilage gel or cartilage membrane particles containing cartilage cells and a 3D printed porous frame, and the cartilage gel or cartilage membrane is loaded on the 3D printed porous frame to form the cartilage gel/cartilage membrane particles-3D printed porous frame compound. After the cartilage tissue engineering compound is transplanted to a cartilage defect, stable cartilage regeneration can be realized at the defect. The invention also provides a preparation method of the cartilage tissue engineering compound and application of the cartilage tissue engineering compound in realizing cartilage regeneration.

Description

Cartilage tissue engineering compound based on 3D printing and application thereof
Technical Field
The invention relates to the field of biomedical tissue engineering, in particular to a cartilage tissue engineering compound based on 3D printing and application thereof in cartilage regeneration.
Background
In recent years, with the rapid development of economic society, cartilage defects caused by various traumas and congenital malformations are more and more common, and most of the defects caused by traumas and congenital malformations are difficult to repair by patients themselves. In clinical treatment, the current clinical treatment methods for articular cartilage defects mainly comprise palliative treatment and reparative treatment. Palliative treatments mainly include arthroscopic debridement, chondroplasty and the like, which can debride the cartilage surface with uneven joint surfaces and remove cartilage fragments and the like to restore smooth and flat joint surfaces, are less traumatic and can relieve the symptoms of patients to some extent, but have limited efficacy and cannot effectively relieve the development of arthritis. The reparative treatment comprises microfracture treatment, osteochondral transplantation and the like, and although the treatment can repair the focal articular cartilage defect to a certain extent, the local articular cartilage defect is easy to cause donor area complications due to large trauma.
With the progress of tissue engineering, research is gradually being conducted to try to repair cartilage defects by using scaffolds or tissues constructed by tissue engineering. Tissue engineering is a cross-discipline related to cell biology, material science, engineering and bioreactors, and utilizes the basic principles and methods of life science and engineering to construct tissues required by human bodies for repairing and replacing tissues or organs which do not work due to trauma and diseases.
In recent years, with the advent of 3D printers, 3D printing technology has also found applications in a variety of fields, including the medical industry. Injectable cartilage produced according to the related art of tissue engineering has also been applied in the fields of medical cosmetology, repair and reconstruction, etc., but still has a number of disadvantages.
Therefore, the method is expected to become a new method for constructing the tissue engineering cartilage through the 3D printing-based tissue engineering technology.
Disclosure of Invention
The invention aims to provide a cartilage tissue engineering compound based on 3D printing.
In a first aspect of the present invention, there is provided a cartilage tissue engineering complex comprising:
(a) A carrier comprising a 3D printed porous frame; and
(b) Cartilage gel or cartilage membrane particles containing cartilage cells seeded or loaded on said carrier.
In another preferred embodiment, the compound comprises a compound formed by inoculating the cartilage gel or cartilage membrane particles to the carrier and culturing the cartilage gel or cartilage membrane particles into cartilage (in the compound, chondrocytes are loaded on the carrier and form a more compact integrated structure with the carrier).
In another preferred embodiment, the complex comprises a complex formed by inoculating the cartilage gel or cartilage membrane particles to the carrier without chondrogenic culture.
In another preferred embodiment, the chondrocytes are derived from a human or non-human mammal.
In another preferred embodiment, the chondrocytes are derived from autologous chondrocytes or allogeneic chondrocytes, preferably autologous chondrocytes.
In another preferred embodiment, the chondrocytes are derived from elastic cartilage, fibrocartilage or hyaline cartilage.
In another preferred embodiment, the chondrocytes are taken from autologous chondrocytes of the subject.
In another preferred embodiment, the autologous chondrocytes comprise elastic chondrocytes, fibrochondrocytes or hyaline chondrocytes.
In another preferred embodiment, the subject is a human or non-human mammal.
In another preferred embodiment, the subject has a joint defect.
In another preferred embodiment, the articular defect is an articular cartilage defect.
In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect, or a combination thereof.
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, and the cartilage gel is in a gel state, and the density of the chondrocytes is at least 1.0 × 10 8 Per ml or 1.0X 10 8 Per gram.
In another preferred embodiment, the cartilage gel is prepared by culturing chondrocytes in a gelling medium.
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 4-5wt% glucose, 10% FBS (v/v) and 100U/ml penicillin-streptomycin.
In another preferred embodiment, the cartilage gel has an adhesion rate of 90% or more, preferably 95% or more.
In another preferred embodiment, the concentration of chondrocytes in the cartilage gel is 1.0 × 10 8 Per ml-10X 10 8 One/ml, preferably 1.5-5X 10 8 One per ml.
In another preferred embodiment, the cartilage gel is obtained by culturing for 2.5 to 5.5 days, preferably 3 to 5 days, by gelation.
In another preferred embodiment, the cartilage membrane particles comprise a cell population consisting of chondrocytes and cells secreted by chondrocytesWherein the extracellular matrix encapsulates the cell population and the cartilage particles are prepared by cutting a sheet-like sheet of cartilage membrane, wherein the density of the cartilage cells is at least 1.0 x 10 8 Per ml or 1.0X 10 8 Per gram.
In another preferred embodiment, the concentration of chondrocytes in the cartilage membrane is 1.0 × 10 8 Per ml-10X 10 8 Per ml, preferably 1.5-5X 10 8 One per ml.
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 4-5wt% glucose, 10% FBS (v/v) and 100U/ml celi-streptomycin.
In another preferred embodiment, the thickness of the cartilage membrane is 0.2-0.25mm.
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 0.05-10mm 2 Preferably, 1-5mm 2 More preferably, the average area is 1mm 2 。
In another preferred example, the 3D printed porous frame comprises an upper layer structure and a lower layer structure, the pore size of the structures is 100-300mm; and is made of a degradable high molecular material selected from the group consisting of: polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer.
In another preferred example, the 3D printed porous framework may also be loaded with gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
In another preferred example, the shape of the 3D printing porous frame includes a cylinder, a rectangular parallelepiped, or other specific shape.
In a second aspect of the present invention, there is provided a method for preparing a cartilage tissue engineering complex according to the first aspect of the present invention, comprising the steps of: and (3) inoculating the cartilage gel or cartilage membrane particles to a porous frame printed in 3D, and performing in-vitro chondrogenic culture to obtain the cartilage tissue engineering compound.
In another preferred example, the cartilage gel is seeded on a 3D printed porous frame using a direct filling method.
In another preferred example, the cartilage membrane particles are inoculated on the 3D printed porous frame by a centrifugal method.
In another preferred example, the centrifugation system of the centrifugation method is not added with liquid, and repeated centrifugation is adopted to enable cartilage membrane particles to enter the porous frame of the 3D printing.
In another preferred embodiment, the chondrogenic culture is an in vitro culture using a chondrogenic medium.
In another preferred embodiment, the chondrogenic medium has the following composition: high-glucose DMEM medium, serum replacement, proline, vitamin C, transforming growth factor-beta 1 (TGF-beta 1), insulin-like growth factor 1 (IGF-I) and dexamethasone.
In another preferred embodiment, the serum replacement is ITS premix comprising insulin, transferrin, selenious acid, linoleic acid, bovine serum albumin, pyruvic acid, ascorbyl phosphate.
In another preferred embodiment, the chondrogenic culture time is 3 to 15 days, preferably 5 to 11 days.
In a third aspect of the present invention, there is provided a use of the cartilage tissue engineering complex according to the first aspect of the present invention for the preparation of a medical product for repairing a joint defect.
In another preferred embodiment, the articular defect is an articular cartilage defect.
In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect, or a combination thereof.
In a fourth aspect of the present invention, there is provided a method of repairing a joint defect, comprising the steps of: the cartilage tissue engineering compound according to the first aspect of the present invention is used for transplantation into a defective joint of a patient to be repaired.
In another preferred embodiment, the articular defect is an articular cartilage defect.
In another preferred embodiment, the joint defect is a knee joint defect, an elbow joint defect, a hip joint defect, an ankle joint defect, a wrist joint defect, a mandibular joint defect, or a combination thereof.
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 shows a 3D printing frame. Wherein fig. 1A is a front view and fig. 1B is a rear view. The scale in the figure is 400mm.
FIG. 2 shows schematic diagrams of auricular cartilage gels and auricular cartilage membranes obtained by culturing the auricular cartilage cells for 3 days and 15 days. Wherein A-C is ear cartilage gel cultured for 3 days, and D-E is ear cartilage membrane tablet (D and E) cultured for 15 days and ear cartilage membrane tablet granule (F) formed by shearing.
Figure 3 shows a physical representation of the cartilage gel-3D printed porous frame composite. Wherein fig. 3A is a front view and fig. 3B is a side view.
Figure 4 shows a physical representation of the cartilage membrane-3D printed porous frame composite. Wherein fig. 4A is a front view and fig. 4B is a side view.
Detailed Description
The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly found and developed a cartilage tissue engineering composite which is an integrated cartilage gel/cartilage membrane particle-3D printed porous frame composite. Experiments prove that by obtaining primary cartilage for amplification culture, inoculating and/or paving a certain number of chondrocytes on a flat or basically flat culture surface to ensure that the inoculated chondrocytes form a specific laminated structure, and culturing the laminated chondrocytes under proper gelation culture conditions, a novel gel-like cartilage or a membrane-like cartilage can be formed due to different culture time. The prepared gel cartilage or membrane cartilage is combined with a 3D printing porous frame, and the prepared cartilage tissue engineering compound can be regenerated into cartilage at a defect part after being transplanted into the cartilage defect part, so that the cartilage tissue engineering compound is expected to become a new method for regenerating the cartilage. On the basis of this, the present invention has been completed.
Term(s)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "cartilage tissue engineering composite" includes cartilage gel-3D printed porous frame composites and cartilage membrane particles-3D printed porous frame composites that have been cultured and not cultured in vitro as described herein, and may be collectively referred to herein as cartilage tissue engineering composites.
As used herein, the term "seeding" means seeding chondrocytes in a cell culture dish, and may also mean seeding and uniformly distributing cartilage gel/cartilage membrane particles in a 3D-printed porous frame, and the meaning of "seeding" as used will be understood by those skilled in the art from the context.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".
Cartilage gel and preparation thereof
As used herein, "gelled cartilage", "cartilage gel", "gel-state cartilage", "gel-like cartilage", "cartilage gel of the invention" or "gelled cartilage of the invention" are used interchangeably and all refer to cartilage (stem) cells of the invention in a 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, thereby forming a gelatinous cartilage culture.
The gelated cartilage of the present invention is a novel type of cartilage different from free chondrocytes, spun-sedimented 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 invention has a certain viscosity and fluidity in close connection due to the fact that in the process of gelation culture, chondrocytes not only contact and/or interact with adjacent cells on a plane (X-Y plane), but also 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, so that the chondrocytes 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 invention has certain viscosity and fluidity, and the gel cartilage of the invention is more suitable for being seeded and loaded on various carrier materials (especially porous carrier materials) to form a compound for cartilage repair.
Furthermore, the gelated cartilage of the invention has on the one hand a gelated state and on the other hand an unusually high cell density (usually at least 1.0X 10 8 One or more, e.g. 1.0X 10 8 Is one (10X 10) 8 One/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) 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) and undergoing chondrogenic culture.
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 gel cartilage of the present invention on a carrier material (particularly, 3D-printed porous frame) and culturing the gel cartilage into cartilage.
Preferably, in the present invention, the gel cartilage is formed by in vitro culturing for a period of time t1 under the gelation culture condition. 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 S1 seeded in a stack according to the invention is n times the number of cells S0 for 100% confluency (i.e. S1/S0= n), where n is 1.5-20, preferably 2-10, more preferably 2.5-5, calculated on the culture area of the culture dish (or culture container) and given a confluency of 100% of the cells of the plated monolayer.
Cartilage membrane and its preparation
As used herein, "cartilage membrane", "membrane cartilage", or "cartilage membrane of the present invention" are used interchangeably and all refer to cartilage (stem) cells of the present invention in a membrane state, in particular, by 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 culture conditions to form a membrane-shaped cartilage culture.
The cartilage membrane of the invention is prepared by prolonging the gelation culture time on the basis of the preparation of the cartilage gel. That is, in the present invention, chondrocytes seeded and/or plated on a flat or substantially flat culture surface are cultured in vitro for a period of time t2 under gelling culture conditions, thereby forming a cartilage membrane. Preferably, t2 is 6-30 days, preferably 7-20 days, and most preferably 10-15 days.
The invention relates to a cartilage membraneThe flakes have an unusually high cell density on the one hand (typically at least 1.0X 10 8 One/ml or more, e.g. 1.0X 10 8 Is one (10X 10) 8 One/ml) and on the other hand, the thickness is thin (only 0.2-0.25 mm) 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 in chondrogenesis and is formed by supporting the cartilage membrane particles of the present invention on a carrier material (particularly, a porous framework structure), and also includes a composite which is formed by supporting the cartilage membrane particles of the present invention on a carrier material (particularly, a porous framework structure) and culturing in chondrogenesis.
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 inoculations in a 3.5cm petri dish (e.g., one well in a six well plate) 7 -2.0×10 7 One cell, preferably, 1.5X 10 7 And (4) one cell. Performing gelation culture for different time to obtain final product with chondrocyte density of 1.0 × 10 8 Is one (10X 10) 8 Cartilage gel with a density of 1.0 × 10 cells/ml 8 Is one (10X 10) 8 Cartilage 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.
As used herein, the term "chondrogenic culture" refers to the culture of a porous framework structure seeded with cartilage gel or cartilage membrane particles using a chondrogenic medium, which is eventually formed into an integrated cartilage gel-framework structure complex or cartilage membrane particle-framework structure complex, i.e., a cartilage tissue engineering complex of the present invention, for transplantation into a cartilage defect of a human or animal body.
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.
The chondrocytes used in the cartilage tissue engineering compound can be hyaline chondrocytes, elastic chondrocytes or fibrocartilage cells which are taken from hyaline cartilage, elastic cartilage or fibrocartilage, and can be regenerated into cartilage to repair the defect after being implanted into the cartilage defect of a subject.
3D printing
3D printing (3 DP), a technique for constructing objects by layer-by-layer printing using bondable materials such as powdered metals or plastics based on digital model files, is one of the rapid prototyping techniques, also known as additive manufacturing. The 3D printing is usually realized by a digital technology material printer, and the printing is completed by three-dimensional design and slicing processing.
The design process of three-dimensional printing is as follows: the method is characterized in that firstly, modeling is carried out through computer modeling software, and then the built three-dimensional model is divided into sections, namely slices, layer by layer, so as to guide a printer to print layer by layer. The printer produces a solid body by reading the information of the cross-section in the document, printing the sections layer by layer with a liquid, powder or sheet material, and bonding the sections in various ways.
The porous frame for 3D printing is manufactured by a 3D printing technology, and the pore size is 100-300mm. And the porous frame of 3D printing is made by degradable macromolecular material, and the material that uses when printing promptly is degradable macromolecular material.
In another preferred embodiment, the degradable high molecular material is selected from the following group: polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer.
Culture Medium for use in the present invention
Chondrogenic medium: 1% of 1 × ITS premix ((ITS general culture mix containing insulin, transferrin, selenious acid, linoleic acid, bovine serum albumin, pyruvic acid, ascorbyl phosphate), 40 μ g/ml proline, 10ng/ml TGF-. Beta.1, 100ng/ml IGF-1,40ng/ml dexamethasone and 50 μ g/ml vitamin C).
Gelling culture medium: DMEM medium containing 4-5wt% glucose, 10% FBS (v/v) and 100U/ml streptomycin.
Rate of adhesion
In the present invention, when the cartilage gel of the present invention is seeded on a carrier material (particularly, a 3D-printed 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 is more than or equal to 90 percent, preferably more than or equal to 95 percent.
The adhesion rate in the present invention is defined as follows:
DNA quantification A1 by detection of the inoculated sample (e.g. cartilage gel); detection of DNA quantification A2 after incubation of the post-inoculation complex (e.g. cartilage gel-frame complex) for 24 hours; the adhesion rate was A2/A1 × 100%.
The method for measuring the adhesion rate comprises the following steps:
inoculated samples (such as cartilage gel or cartilage gel-3D printed porous frame composite) are taken, digested with proteinase K, the digested samples are quantitatively detected using PicoGreen kit (Invitrogen, carlsbad, CA, USA), absorbance at 520nm is measured using a fluorescence microplate reader, and DNA content is calculated according to a standard curve formula.
The main advantages of the invention include:
(1) The cartilage gel or cartilage membrane particles provided by the invention are more mature than chondrocytes and have certain fluidity.
(2) The 3D printing porous frame can be degraded in vivo as a degradable high polymer material, so that the organism immune reaction is low and the biological safety is good.
(3) The 3D printing double-layer 3D printing porous frame material has larger pore diameter, better porosity and easy inoculation, but the cell adhesion rate is extremely low when the chondrocyte suspension is inoculated.
(4) The 3D printing double-layer 3D printing porous frame material comprises an upper layer structure and a lower layer structure, wherein the pore diameter of the upper layer structure is larger, so that inoculation is easy; and the pore size of the lower layer structure is smaller, so that the inoculated cells are prevented from leaking out.
(5) The simple cartilage gel or cartilage membrane particles cannot be formed, the cartilage absorption rate under the tension condition is high, and the clinical application is limited; according to the invention, the 3D printing porous frame is used as a frame structure, a cartilage gel/cartilage membrane particle-3D printing porous frame compound with a certain special shape can be constructed, mechanical support is provided, and the cartilage absorption rate is effectively reduced.
(6) The 3D printing porous frame can be flexibly customized into different shapes according to requirements, and the constructed cartilage gel/cartilage membrane particle-3D printing porous frame compound can be used for repairing and reconstructing different parts and different shapes.
The present invention is further illustrated by the following specific examples. The invention 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. The experimental procedures without specifying the specific conditions in the following examples are generally performed under the conditions described in the conventional conditions or under the conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.
Example 1
Preparation of 3D printing porous frame
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. The 3D printing porous frame is prepared by adjusting the width of different layers, different deposition angles (-45/45 degrees, -60/60 degrees or 0/60/120 degrees) and different diameters of stainless steel needles (19G, 20G or 21G), and setting appropriate printing parameters such as extrusion speed, printing speed, layer height and the like.
The resulting 3D printed porous frame was prepared as shown in fig. 1.
Wherein, fig. 1A is a front view, and fig. 1B is a rear view. The scale in the figure is 400mm.
Example 2
Cartilage gel-3D printing porous framework composite preparation
In this example, a cartilage gel-3D printed porous frame composite was prepared. The specific operation method comprises the following steps:
(1) Taking the soft bone tissue of the ear of the subject, aseptically cutting 2.5 × 2.5cm 2 The mucous membrane and the fibrous tissue on the surface of the cartilage are stripped by using a sterile instrument;
(2) Cutting cartilage tissue to 1.5 × 1.5mm 2 Large and small cartilage fragments; preparing 0.15% collagenase; adding cartilage fragments into prepared collagenase for digestion for 8 hours;
(3) Filtering and centrifuging the collagenase solution after 8 hours to obtain the osteocyte of the ear, and performing primary culture and subculture by using a high-glucose DMEM culture medium containing 10% FBS;
(4) After amplification, the cells were collected and resuspended at 8X 10 6 10ml to 30X 10 6 Cell amount/10 ml/well was inoculated in a six-well plate, and cultured in a gelation medium (DMEM medium containing 4-5wt% glucose, 10% FBS (v/v) and 100U/ml streptomycin);
(5) After 72 hours (3 days) of inoculation, the culture medium in the six-well plate is aspirated, the gelatinous cartilage tissue at the bottom of the six-well plate is visible (fig. 3A), the gelatinous cartilage at the bottom of the six-well plate is gathered by using forceps (fig. 2B), the yield of the gelatinous cartilage in one well is 0.1-0.2ml, and the gelatinous cartilage is collected in a 5ml syringe; the gel cartilage has cell density of 1.0 × 10 8 Per ml-10X 10 8 Per ml, preferably 1.5-5X 10 10 Each/ml. Mixing with 0.15ml culture medium to obtain preparation containing cartilage gel, as shown in FIG. 2C;
(6) A cartilage gel preparation (prepared in step (5) in a volume of about 0.25-0.35 ml) was seeded on a 3D printed porous frame (prepared in example 1, as shown in fig. 1) and allowed to stand at 37 ℃ at 95% humidity and 5% carbon dioxide for 2 hours;
(7) After standing, adding chondrogenic medium to continue culturing in vitro for 3-11 days, forming cartilage gel-3D printing porous frame composite, as shown in FIG. 3.
When the cartilage gel-3D printing porous frame composite is used for repairing joint defects, the shape and the size of cartilage needing to be repaired can be determined according to the prior MRI, CT and other auxiliary examinations.
Example 3
Cartilage membrane particle-3D printing porous frame composite preparation
In this example, cartilage membrane particle-3D printed porous frame composites were prepared. The specific operation method comprises the following steps:
(1) Aseptically cutting into 2.5 × 2.5cm 2 The soft bone tissue of the ear; using a sterile instrument to strip the mucous membrane and the fibrous tissue on the surface of the cartilage;
(2) Cutting cartilage tissue to 1.5 × 1.5mm 2 Large and small cartilage fragments; preparing 0.15% collagenase; adding cartilage fragments into prepared collagenase for digestion for 8 hours;
(3) Filtering and centrifuging the collagenase solution after 8 hours to obtain the ear cartilage cells, and performing primary culture and subculture;
(4) After amplification, the cells were collected and resuspended at 8X 10 6 10ml to 30X 10 6 Cell amount/10 ml/well was inoculated in a six-well plate, and cultured in a gelation medium (DMEM medium containing 4-5wt% 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;
(6) The culture medium in the six-well plate was aspirated, and the cartilage membrane tissue at the bottom of the six-well plate was visualized (FIG. 2D), wherein the cell density in the cartilage membrane tissue was about 1.0X 10 8 Per ml-10X 10 8 Per ml;
the cartilage membrane was clamped up using forceps (FIG. 3E) and cut to 1X 1mm 2 After the size of the cartilage membrane particles, collect into a 50ml centrifuge tube, as shown in fig. 2F;
(7) 3D printed porous frames (prepared in example 1, as shown in FIGS. 1 and 2) to be seeded were placed in centrifuge tubes containing cartilage membrane particles to ensure that the frames were completely submerged; placing the centrifuge tube with the frame and the cartilage membrane particles in a centrifuge, and centrifuging for 2 minutes at 600 revolutions per minute;
(8) Standing the inoculated 3D printing porous frame at 37 ℃ and 95% humidity and 5% carbon dioxide for a certain time; after standing, adding chondrogenic culture medium to continue culturing in vitro for 3-11 days, and forming cartilage membrane particle-3D printing porous frame composite, as shown in figure 4.
When the compound is used for repairing joint defects, the shape and the size of cartilage needing to be repaired can be determined according to the prior MRI, CT and other auxiliary examinations.
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 tissue engineering complex, wherein the complex comprises:
(a) A carrier comprising a 3D printed porous frame; and
(b) Cartilage gel or cartilage membrane particles containing cartilage cells seeded or loaded on said carrier.
2. The complex of claim 1, wherein the chondrocytes are derived from elastic cartilage, fibrocartilage or hyaline cartilage.
3. The composite of claim 1, wherein said cartilage gel comprises a population of chondrocytes and extracellular matrix secreted by chondrocytes, wherein said extracellular matrix encapsulates said population of chondrocytes and said cartilage gel is in a gel state and wherein chondrocytes have a density of at least 1.0 x 10 8 Per ml or 1.0X 10 8 Per gram.
4. The composite of claim 1, wherein said cartilage membrane particles comprise a population of chondrocyte cells and extracellular matrix secreted by the chondrocyte cells, wherein said extracellular matrix encapsulates said population of cells, and wherein said cartilage particles are prepared by shearing a sheet-like cartilage membrane having a chondrocyte density of at least 1.0 x 10 8 Per ml or 1.0X 10 8 Per gram.
5. The composite of claim 1, wherein the 3D printed porous frame comprises an upper layer and a lower layer, the structures having a pore size of 100-300mm; and is made of a degradable high molecular material selected from the group consisting of: polyglycolide, polylactide, polycaprolactone, or glycolide-lactide copolymer.
6. The composite of claim 1, wherein the 3D printed porous framework may also be loaded with gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
7. A method of preparing the cartilage tissue engineering complex of claim 1, comprising the steps of: inoculating the cartilage gel of claim 3 or the cartilage membrane particles of claim 4 on a 3D printed porous frame, and culturing in vitro to form cartilage, thereby obtaining the cartilage tissue engineering compound.
8. The method of claim 7, wherein the cartilage gel is seeded onto the 3D printed porous frame using a direct fill method.
9. The method of claim 7, wherein the cartilage membrane particles are seeded onto the 3D printed porous frame using centrifugation.
10. Use of the cartilage tissue engineering complex according to claim 1 for the preparation of a medical product for the repair of joint defects.
CN202111177643.XA 2021-10-09 2021-10-09 Cartilage tissue engineering compound based on 3D printing and application thereof Pending CN115944781A (en)

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