WO2022156645A1 - 一种软骨组织工程复合物及其用途 - Google Patents
一种软骨组织工程复合物及其用途 Download PDFInfo
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- WO2022156645A1 WO2022156645A1 PCT/CN2022/072395 CN2022072395W WO2022156645A1 WO 2022156645 A1 WO2022156645 A1 WO 2022156645A1 CN 2022072395 W CN2022072395 W CN 2022072395W WO 2022156645 A1 WO2022156645 A1 WO 2022156645A1
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Definitions
- the invention relates to the field of biomedical tissue engineering, in particular, to a cartilage tissue engineering compound and a preparation method thereof, as well as its application in joint repair.
- articular cartilage lesions such as sports injuries, accidental injuries and joint degenerative diseases have become more and more common.
- the clinical treatment methods for articular cartilage lesions are mainly palliative treatment and repairing treatment.
- Palliative treatments mainly include arthroscopic debridement and chondroplasty. These treatments can clean up the uneven cartilage surface of the joint surface and remove cartilage fragments to restore the smoothness of the joint surface.
- Such methods are less traumatic. It can relieve the symptoms of patients to a certain extent, but its curative effect is limited and cannot effectively relieve the development of arthritis.
- Restorative treatment includes microfracture treatment, osteochondral transplantation, etc.
- tissue engineering is an interdisciplinary subject involving cell biology, material science, engineering and bioreactors. It uses the basic principles and methods of life science and engineering to construct the tissues needed by the human body for repairing, replacing trauma, A diseased tissue or organ that does not function.
- chondrocyte-material composite (ACI or MACI) constructed by the existing tissue engineering repair technology has been gradually developed in clinical treatment, there are still many disadvantages that cannot be avoided: 1.
- the source of cells is articular cartilage, which is already damaged. Cells are obtained from the joint parts, which will inevitably increase the damage to the original joints. 2.
- the proliferative capacity of articular cartilage cells is limited, and the construction period of the cell-material complex in the early stage is long, and the patient needs to wait for a long time.
- the strength of the cell-material composite is low, far less than the mechanical strength level of natural cartilage.
- the purpose of the present invention is to provide a kind of cartilage tissue engineering compound and its preparation method, and its application in joint repair.
- a first aspect of the present invention provides a cartilage tissue engineering composite, the composite comprising:
- the complex comprises a complex formed after the cartilage gel or perichondrium sheet particles are seeded on the carrier and cultured for chondrogenesis (in the complex, chondrocytes are It is loaded on the carrier and forms a tighter integrated structure with the carrier).
- the complex comprises a complex formed after the cartilage gel or perichondrium sheet particles are seeded on the carrier without chondrogenic culture.
- the chondrocytes are derived from human or non-human mammals.
- the chondrocytes are derived from autologous chondrocytes or allogeneic chondrocytes, preferably autologous chondrocytes.
- the chondrocytes are derived from elastic cartilage, fibrocartilage or hyaline cartilage.
- the chondrocytes are obtained from autologous chondrocytes of the subject.
- the autologous chondrocytes include elastic chondrocytes, fibrochondrocytes or hyaline chondrocytes.
- the subject is a human or a non-human mammal.
- the subject has a joint defect.
- the joint defect is an articular cartilage defect.
- 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.
- the cartilage gel includes a cell population composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix wraps the cell population, and the cartilage gel presents gel state, and the density of chondrocytes is at least 1.0 ⁇ 10 8 cells/ml or 1.0 ⁇ 10 8 cells/g.
- the cartilage gel is prepared by gelatinizing and culturing chondrocytes.
- the gelation culture is an in vitro culture with a gelation medium.
- the gelation medium contains the following components: high glucose DMEM medium containing 4-5wt% glucose, 10% FBS (v/v) and 100U/ml penicillin-streptomycin .
- the adhesion rate of the cartilage gel is ⁇ 90%, preferably ⁇ 95%.
- the concentration of chondrocytes is 1.0 ⁇ 10 8 cells/ml-10 ⁇ 10 8 cells/ml, preferably 1.5-5 ⁇ 10 8 cells/ml.
- the cartilage gel is obtained by gelation culture for 2.5-5.5 days, preferably 3-5 days.
- the chondrocytes are derived from human or non-human mammals.
- the chondrocytes are derived from autologous chondrocytes or allogeneic chondrocytes, preferably autologous chondrocytes.
- the chondrocytes are obtained from autologous chondrocytes of the subject.
- the subject is a human or a non-human mammal.
- the subject has a joint defect.
- the joint defect is an articular cartilage defect.
- 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.
- the perichondral sheet particles include a cell population composed of chondrocytes and an extracellular matrix secreted by chondrocytes, wherein the extracellular matrix wraps the cell population, and the cartilage particles are made of Thin slices of perichondrium are cut and prepared, wherein the density of chondrocytes is at least 1.0 ⁇ 10 8 cells/ml or 1.0 ⁇ 10 8 cells/g.
- the concentration of chondrocytes is 1.0 ⁇ 10 8 cells/ml-10 ⁇ 10 8 cells/ml, preferably 1.5-5 ⁇ 10 8 cells/ml.
- the perichondrium sheet is obtained by gelation culture for 6-30 days, preferably 7-20 days, and optimally 10-15 days.
- the gelation culture is an in vitro culture with a gelation medium.
- the gelation medium contains the following components: high glucose DMEM medium containing 4-5wt% glucose, 10% FBS (v/v) and 100U/ml penicillin-streptomycin .
- the thickness of the perichondrial sheet is 0.2-0.25 mm.
- the average volume of the perichondrial sheet particles is 0.2 ⁇ l.
- the surface area of the perichondral sheet particles is 0.05-10 mm 2 , preferably 1-5 mm 2 , and more preferably, the average area is 1 mm 2 .
- the porous frame structure is made of a biodegradable material selected from the group consisting of PCL, PGA, allogeneic bone repair material, xenograft bone repair material, or demineralized bone matrix.
- the framework structure can also be loaded with gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
- the frame structure is demineralized bone matrix.
- the demineralized bone matrix is derived from an allogeneic bone repair material.
- the demineralized bone matrix is derived from a xenogeneic bone repair material.
- the shape of the demineralized bone matrix includes a cylinder, a rectangular parallelepiped or other specific shapes.
- the thickness of the demineralized bone matrix is 0.3-0.8 cm, preferably, 0.4-0.6 cm, and most preferably, 0.5 cm.
- the decalcified amount of the demineralized bone matrix is 30% to 50%.
- the cartilage gel/perichondrium sheet particle-frame structure complex can generate articular cartilage in the joint microenvironment.
- the second aspect of the present invention provides a method for preparing the cartilage tissue engineering composite described in the first aspect of the present invention, comprising the following steps: adding the cartilage gel or perichondral sheet particles described in the first aspect of the present invention
- the cartilage tissue engineering complex is obtained by inoculating on the porous frame structure and culturing the cartilage tissue in vitro.
- the cartilage gel is seeded on the porous frame structure by a direct filling method.
- the perichondrial sheet particles are seeded on the porous frame structure by centrifugation.
- no liquid is added to the centrifugation system of the centrifugation method, and the perichondrial sheet particles are allowed to enter the frame structure by repeated centrifugation.
- the chondrogenic culture is in vitro culture using a chondrogenic medium.
- the chondrogenic medium has the following components: high glucose DMEM medium, serum replacement, proline, vitamin C, transforming growth factor- ⁇ 1 (TGF- ⁇ 1), insulin-like growth Factor 1 (IGF-I) and dexamethasone.
- the serum substitute is ITS premix, which contains insulin, transferrin, selenite, linoleic acid, bovine serum albumin, pyruvate, and ascorbic acid phosphate.
- the chondrogenic culture time is 3-15 days, preferably 5-11 days.
- the third aspect of the present invention provides a use of the cartilage tissue engineering composite described in the first aspect of the present invention for preparing a medical product for repairing joint defects.
- the joint defect is an articular cartilage defect.
- 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.
- the fourth aspect of the present invention provides a pharmaceutical composition for joint repair, the pharmaceutical composition comprising the cartilage gel or perichondrial sheet particles as described in the first aspect of the present invention and a pharmaceutically acceptable vector.
- the pharmaceutical composition is a liquid preparation.
- the pharmaceutical composition is an injection.
- the volume ratio of the cartilage gel to the pharmaceutically acceptable carrier in the pharmaceutical composition is 50% to 90%, preferably 70% to 85%.
- the volume ratio of the perichondrial sheet particles to the pharmaceutically acceptable carrier in the pharmaceutical composition is 45%-90%, preferably 60%-80%.
- a fifth aspect of the present invention provides a method for repairing a joint defect, using the cartilage tissue engineering composite described in the first aspect of the present invention to be transplanted into the defective joint of a patient to be repaired.
- the joint defect is an articular cartilage defect.
- 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.
- Figure 1 shows electron micrographs of demineralized bone matrix materials.
- the scale (bar) in the figure is 1 mm.
- Figure 2 shows a macrophotograph of the demineralized bone matrix material.
- the ruler in the figure is 1 cm.
- Figure 3 shows the schematic diagrams of ear cartilage gel and ear perichondrium sheets obtained by culturing ear chondrocytes for 3 days and 15 days.
- A-C are ear cartilage gels cultured for 3 days
- D-E are ear perichondrium sheets cultured for 15 days (D and E) and shredded ear perichondrium sheet particles (F).
- Figure 4 shows a physical image of the cartilage gel-framework composite.
- the ruler in the figure is 1 cm.
- Figure 5 shows a physical image of the perichondrial sheet particle-framework complex.
- the ruler in the figure is 1 cm.
- Figure 6 shows electron micrographs of cartilage gel-demineralized bone complexes.
- the scale bar in the figure is 200 ⁇ m.
- Figure 7 shows electron micrographs of the perichondrial sheet particle-demineralized bone complex.
- the scale bar in the figure is 200 ⁇ m.
- Figure 8 shows a graph comparing adhesion rates of seeded samples (cell suspension or cartilage gel) after culturing on demineralized bone matrix for 24 hours.
- Figure 9 shows the comparison of the gel cartilage-demineralized bone complex (A) and the pure demineralized bone (B) transplanted on the defect site of goat knee.
- cartilage tissue engineering composite for the first time, which is an integrated cartilage gel/perichondrium sheet particle-frame structure composite.
- a specific number of chondrocytes are seeded and/or plated on a flat or substantially flat culture surface, so that the seeded chondrocytes form a specific layered structure, and in a suitable
- the layered chondrocytes are cultured under gelation culture conditions, and a novel gel-like cartilage or sheet-like cartilage can be formed due to different culture time.
- the prepared cartilage tissue engineering composite can regenerate into articular cartilage at the defected joint after being transplanted into the defected joint to realize the repair of articular cartilage. reconstruction.
- the present invention has been completed.
- cartilage tissue engineering complex includes chondrogenic cultured or non-in vitro chondrogenic cultured cartilage gel-framework complexes and perichondrial sheet particle-framework complexes as described herein, herein In the invention, they can be collectively referred to as cartilage tissue engineering composites.
- seeding means seeding chondrocytes in a cell culture dish, and it can also mean seeding chondrogel/perichondrium sheet particles in a designated frame structure and making it evenly distributed, according to those skilled in the art.
- the context can understand the meaning of "inoculation” as used.
- the term “about” means that the value may vary by no more than 1% from the recited value.
- the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
- gel cartilage As used herein, “gel cartilage”, “cartilage gel”, “gel-like cartilage”, “gel-like cartilage”, “cartilage gel of the present invention” or “gel cartilage of the present invention” are interchangeable Use, all refer to the cartilage (stem) cells in the gel state of the present invention, especially the chondrocytes of a specific concentration are seeded and/or spread on a flat or substantially flat culture surface, so that the seeded chondrocytes form a layered structure , and cultured chondrocytes with layered structure under suitable gelation culture conditions to form a gelatinous cartilage culture.
- the gel cartilage of the present invention is a new type of cartilage different from free chondrocytes, centrifuged chondrocytes and cartilage pellets.
- the gel cartilage of the present invention can be regarded as a specific morphological cartilage between free chondrocytes and dense cartilage mass.
- the gel cartilage of the present invention because in the process of gelation culture, the chondrocytes not only contact and/or interact with the adjacent cells on the plane (X-Y plane), but also the upper and/or lower and/or side upper or There is contact and/or interaction between adjacent chondrocytes in multiple directions below, which promotes chondrocytes to secrete and form more extracellular matrix, so as to wrap the gelatinized chondrocytes in a viscous extracellular matrix Therefore, the gel cartilage of the present invention has both close contact and certain viscosity and fluidity, so that the gel cartilage of the present invention is more suitable for seeding and loading on various carrier materials (especially porous carriers). material) to form a complex for cartilage repair.
- various carrier materials especially porous carriers. material
- the gel cartilage of the present invention has a gel state on the one hand and an unusually high cell density (usually at least 1.0 ⁇ 10 8 cells/ml or more, such as 1.0 ⁇ 10 8 to 10 ⁇ 10 8 , on the other hand) pieces/ml), therefore, it is particularly suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.
- an unusually high cell density usually at least 1.0 ⁇ 10 8 cells/ml or more, such as 1.0 ⁇ 10 8 to 10 ⁇ 10 8 , on the other hand
- the composite for repairing cartilage includes a composite without chondrogenic culture formed by loading the gel cartilage of the present invention on a carrier material (especially a porous bioframe structure), and also includes the gel of the present invention.
- Cartilage is supported on a carrier material (especially a porous bioframework) and is a complex formed by chondrogenic culture.
- the composite suitable for transplantation into human or animal body is the cartilage tissue engineering composite of the present invention, that is, the gel cartilage of the present invention is loaded on a carrier material (especially a porous bioframe structure) and cultured into chondrogenic the formed complex.
- a carrier material especially a porous bioframe structure
- the gel cartilage is formed by culturing in vitro for a period of time t1 under gelation culture conditions.
- the t1 is 2.5-5.5 days, preferably 3-5 days.
- a feature is layered seeding, that is, after a specific density of chondrocytes is seeded in a culture vessel, the seeded chondrocytes will form a multi-layered chondrocyte population stacked on each other (ie, a chondrocyte population with a stacked structure) by, for example, deposition. ).
- peripheral cartilage As used herein, “perichondral sheet”, “patch-like cartilage”, or “perichondral sheet of the present invention” are used interchangeably, and all refer to the cartilage (stem) cells of the present invention in a patch state, especially the A specific concentration of chondrocytes is seeded and/or spread on a flat or substantially flat culture surface, so that the seeded chondrocytes form a layered structure, and the chondrocytes with the layered structure are cultured under suitable culture conditions to form a membrane Cartilage cultures.
- the "perichondral sheet" of the present invention is prepared by prolonging the gelation culture time on the basis of the preparation of the "chondral gel” of the present invention. 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 gelation culture conditions, thereby forming a perichondrial sheet.
- the t2 is 6-30 days, preferably 7-20 days, and most preferably 10-15 days.
- the perichondrial sheet of the present invention has, on the one hand, an unusually high cell density (usually at least 1.0 ⁇ 10 8 cells/ml or more, such as 1.0 ⁇ 10 8 to 10 ⁇ 10 8 cells/ml), and on the other hand its Thin thickness (only 0.2-0.25mm) and good toughness, it can be chopped into "perichondral sheet particles" with an average volume of 0.2 ⁇ l, which can be filled into the porous frame structure by simple centrifugation. Therefore, it is especially suitable for the preparation of Cartilage-like grafts, or for cartilage grafting or cartilage repair surgery.
- the complex for repairing cartilage includes a complex formed by loading the perichondrial sheet particles of the present invention on a carrier material (especially a porous frame structure) without chondrogenic culture, and also includes the perichondrium of the present invention. Sheet particles are supported on a carrier material (especially a porous framework structure) and the complexes formed by chondrogenic culture.
- the composite suitable for transplantation into human or animal body is the cartilage tissue engineering composite of the present invention, that is, the perichondrial sheet particles of the present invention are loaded on a carrier material (especially a porous frame structure) and formed into cartilage The complexes formed were cultured.
- specific concentration or “specific density” refers to seeding 1.0 x 10 7 -2.0 x 10 7 cells in a 3.5 cm dish (eg, one well in a six-well plate), preferably , for 1.5 x 10 7 cells. After gelation and culture for different times, the chondrocytes with a density of 1.0 ⁇ 108-10 ⁇ 108/ml are finally formed, or the chondrocyte density is 1.0 ⁇ 108-10 ⁇ 10 8 /ml perichondral sheet.
- the gelation culture conditions are as follows: inoculation of chondrocytes of a specific density and culture in a gelation medium containing 10% fetal bovine serum and 100U/ml Penicillin-streptomycin in high glucose (4-5 wt% glucose) DMEM medium.
- chondrogenic culture refers to the use of chondrogenic medium to culture a porous framework inoculated with chondrogel or perichondral sheet particles, ultimately allowing it to form an integrated chondrogel-framework complex or cartilage
- the membrane particle-frame structure complex that is, the cartilage tissue engineering complex of the present invention, is used for transplantation in the cartilage defect of human or animal body.
- Cartilage is cartilage tissue, which is composed of chondrocytes and intercellular substance.
- the matrix in the cartilage is in a gel state and has great toughness.
- Cartilage is a supportive connective tissue.
- Cartilage does not contain blood vessels and lymphatic vessels, and nutrients penetrate into the intercellular substance from the blood vessels in the perichondrium, and then nourish bone cells.
- Cartilage can be divided into three types, namely hyaline cartilage, elastic cartilage and fibrocartilage.
- the matrix of hyaline cartilage is composed of collagen fibers, fibrils and surrounding amorphous matrix. There is a temporary scaffolding effect during the embryonic period, which is later replaced by bone.
- hyaline cartilage is found mainly in the walls of the trachea and bronchi, the sternal ends of the ribs, and the surface of the bones (articular cartilage).
- collagen fibers there are elastic fibers in the matrix of elastic cartilage.
- This cartilage is more elastic and is mainly distributed in the auricle, the wall of the external auditory canal, the Eustachian tube, the epiglottis, and the larynx.
- bundles of collagen fibers are arranged in parallel or crosswise, and are relatively tough. Distributed in the intervertebral disc, glenoid, joint disc and some tendons, ligaments, etc., to enhance the flexibility of movement and protection, support and other functions.
- the chondrocytes used in the cartilage tissue engineering composite of the present invention can be hyaline chondrocytes, elastic chondrocytes or fibrocartilage cells obtained from hyaline cartilage, elastic cartilage or fibrocartilage, and are implanted in the joint defect of the subject. Afterwards, it can be transformed into articular cartilage in the joint environment.
- MACI is the abbreviation of matrix-induced autologous chondrocyte implantation, which means "matrix-induced autologous chondrocyte transplantation", which is a technology of chondrocyte transplantation using tissue engineering technology.
- MACI is the latest and best technology for the treatment of articular cartilage defects in the world.
- ACI is the abbreviation of autologous chondrocyte implantation, which means “autologous chondrocyte transplantation”. It is one of the widely used tissue engineering techniques for the treatment of articular cartilage damage. After the advent of MACI, ACI was correspondingly called “traditional ACI” to distinguish it from MACI.
- porous frame structure refers to a structure made of a biocompatible material that has a certain number of pores on its surface and inside to facilitate the attachment of auricular cartilage gel or auricular perichondrial sheet seeded thereon. vector.
- the biocompatible material is preferably a biodegradable material.
- Biodegradable materials refer to materials that can be decomposed in the body after being implanted into an animal.
- the porous frame structure of the ear cartilage tissue engineering graft of the present invention is made of a biodegradable material selected from the group consisting of PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, demineralized bone matrix or a combination thereof , but not limited to the materials described above.
- the allogeneic bone repair material and the xenogeneic bone repair material include demineralized bone matrix material.
- the porous framework structure of the ear cartilage tissue engineering composite is demineralized bone matrix.
- the demineralized bone matrix used in the preferred embodiment of the present invention has a thickness of 0.3-0.8 cm, preferably 0.4-0.6 cm, and most preferably 0.5 cm.
- the decalcified bone matrix has a decalcification amount of 30% to 50%, the decalcification degree is suitable, the support function is good, and it is easy to be trimmed and cut into a suitable shape and size.
- the pores of the demineralized bone matrix have a pore diameter of 400-800 ⁇ m, which is easy to fill with chondrocytes.
- Demineralized bone matrix is a bone graft material that is decalcified from allogeneic or xenogeneic bone to reduce immunogenicity. Different degrees of decalcification correspond to different mechanical strengths. It has good biological properties, osteoinductivity, osteoconductivity and biodegradability, promotes new bone formation and bone tissue mineralization, thereby accelerating bone healing, and can be effectively repaired alone or in combination with autologous bone, other biomaterials, and growth factors It is an ideal scaffold material for bone tissue engineering. However, the pore size of general demineralized bone matrix is large, and the cell adhesion rate is extremely low when inoculated with chondrocyte suspension, which is not conducive to the construction of tissue engineering carriers.
- the demineralized bone matrix in the present invention has a pore size of 400-800 ⁇ m and a porosity of 87.3% ⁇ 3.7%.
- Allogeneic bone is the most commonly used bone implant material in orthopaedics. It is mainly used to repair and fill bone defects, and play a role of fixation and support. Allogeneic bone is obtained from donated human bone tissue, with "syngeneic” indicating that it came from the human body, and "allogeneic” indicating that it was not from the patient's own body. After the donor is selected, it is usually obtained within 24 hours of death under sterile conditions and processed immediately. Preservation methods include fresh freezing and freeze drying. Fresh frozen bone can be stored for 1 year at -20°C; freeze-dried bone can be stored at room temperature for a long time after vacuum packaging, and has lower antigenicity. Compared with fresh frozen bone, the mechanical properties of freeze-dried bone were reduced by 50%, and sterilization by ethylene oxide or high-dose gamma irradiation further reduced the inductive properties of bone.
- Xenografts are bone repair materials derived from other species such as cattle and pigs. It is widely available and relatively inexpensive. However, xenogeneic bone is highly immunogenic, and it is easy to cause immune rejection after implantation in patients. In addition, the xenogeneic bone has no ability to induce the proliferation of mesenchymal stem cells, and has poor biological activity. It needs to be compounded with other repair materials or related cytokines to achieve the repair effect.
- the culture medium used in the present invention is a culture medium used in the present invention.
- Chondrogenic medium high glucose DMEM medium, 1% 1 ⁇ ITS premix ((ITS universal culture mixture, containing insulin, transferrin, selenite, linoleic acid, bovine serum albumin, pyruvate, ascorbic acid phosphate) salt), 40 ⁇ g/ml proline, 10 ng/ml TGF- ⁇ 1, 100 ng/ml IGF-1, 40 ng/ml dexamethasone and 50 ⁇ g/ml vitamin C.
- ITS premix (ITS universal culture mixture, containing insulin, transferrin, selenite, linoleic acid, bovine serum albumin, pyruvate, ascorbic acid phosphate) salt)
- 40 ⁇ g/ml proline 10 ng/ml TGF- ⁇ 1, 100 ng/ml IGF-1, 40 ng/ml dexamethasone and 50 ⁇ g/ml vitamin C.
- Gelling medium DMEM medium containing 4-5 wt% glucose, 10% FBS (v/v) and 100 U/ml penicillin.
- the cartilage gel of the present invention when the cartilage gel of the present invention is seeded on a carrier material (especially a porous bioframe structure), the cartilage gel of the present invention has a certain adhesion rate, which is determined by the adhesion rate provided by the present invention. method is determined.
- the adhesion rate of the cartilage gel of the present invention is ⁇ 90%, preferably ⁇ 95%.
- the adhesion rate in the present invention is defined as follows:
- Detection of DNA quantification A1 of inoculated samples eg, chondrogel
- detection of DNA quantification A2 after 24 hours of incubation of inoculated complexes eg, chondrogel-framework complex
- adhesion rate A2/A1*100% e.g., chondrogel-framework complex
- the method for measuring the adhesion rate comprises the following steps:
- the cartilage gel of the present invention is more mature than chondrocytes and has a certain fluidity.
- Decalcified bone matrix as a degradable natural material, can be degraded in the body, the immune response of the body is low, and the biological safety is good.
- the demineralized bone matrix material has large pore size and good porosity, but the cell adhesion rate is extremely low when inoculated with chondrocyte suspension.
- the use of a certain fluidity and relatively viscous cartilage gel-like tissue can effectively improve the adhesion rate. .
- the cell source of the graft of the present invention is diverse, and the expansion ability is good.
- the cartilage gel/perichondrium sheet particle-decalcified bone matrix composite can stably regenerate cartilage.
- the cartilage gel/perichondral sheet particle-demineralized bone matrix composite can stabilize the regeneration of cartilage and provide instant mechanical support, and the instant repair effect is good.
- the cartilage gel preparation (prepared in step (5), the volume is about 0.25-0.35ml) is seeded on the frame structure (decalcified bone matrix frame (shown in Figure 1 and Figure 2), with a pore size of about 400-800 ⁇ m, about 87.3% ⁇ 3.7% porosity), stand at 37°C, 95% humidity, 5% carbon dioxide for 2 hours;
- the cartilage gel-frame structure complex When used for joint defect repair, the cartilage gel-frame structure complex can be cut to determine the shape and size of the cartilage to be repaired according to auxiliary examinations such as MRI and CT in the early stage.
- perichondrial sheet particle-framework complexes were prepared.
- the specific operation method is as follows:
- Fig. 3D Aspirate the medium in the six-well plate, and the perichondrium sheet tissue at the bottom of the six-well plate can be seen (Fig. 3D).
- the cell density in the perichondrium sheet tissue is about 1.0 ⁇ 108 cells/ml-10 ⁇ 10 8 /ml;
- the shape and size of the cartilage to be repaired can be determined according to auxiliary examinations such as MRI and CT in the early stage.
- a demineralized bone matrix framework is provided (as shown in Figure 2).
- the gel cartilage preparation (prepared in Example 1, with a volume of about 0.25-0.35 ml) was inoculated into the above-mentioned demineralized bone matrix framework, and the DNA content of the gel cartilage preparation was determined before inoculation.
- the primary cultured chondrocytes were passaged 4 times at 37°C, 95% humidity, 5% carbon dioxide, added to cell culture medium to prepare a cell suspension, and the DNA content of the prepared chondrocyte suspension was determined.
- the cell suspension was seeded in the demineralized bone matrix framework described above.
- the seeded cartilage gel-demineralized bone matrix complex and chondrocyte suspension-decalcified bone matrix complex were left standing for 24 hours in an incubator at 37° C., 95% humidity, and 5% carbon dioxide.
- the two complexes were sampled separately and their DNA content was determined.
- the adhesion rate was calculated by the adhesion rate measurement method described in the specification.
- the gel cartilage adhesion rate of the present invention is 92% ⁇ 2%, which is about 3 times that of the cell suspension.
- a cartilage defect with a diameter of 7.5 mm was produced on the articular surface of the knee joint of experimental animals, and the gel cartilage-frame complex (gel cartilage-decalcified bone complex) prepared in Example 1 and the pure decalcified bone matrix were implanted respectively. into the A defect and the B defect.
- the defect at A is smooth and solid, surrounded by a soft tissue membrane, with a certain elasticity, with excellent immediate repair effect
- the wound surface of the defect at B is rough and only has the function of physical support, which cannot be repaired.
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Abstract
Description
Claims (15)
- 一种软骨组织工程复合物,其特征在于,所述复合物包括:(a)载体,所述载体包括多孔框架结构;和(b)接种于或负载于所述载体的含有软骨细胞的软骨凝胶或软骨膜片颗粒。
- 如权利要求1所述的复合物,其特征在于,所述软骨细胞包括弹性软骨细胞、纤维软骨细胞、透明软骨细胞或其组合。
- 如权利要求1所述的复合物,其特征在于,所述的软骨凝胶包括软骨细胞构成的细胞群以及软骨细胞所分泌的细胞外基质,其中所述细胞外基质包裹所述的细胞群,并且所述软骨凝胶呈凝胶态,并且软骨细胞的密度为至少1.0×10 8个/ml或1.0×10 8个/g。
- 如权利要求3所述的复合物,其特征在于,所述软骨凝胶的粘附率≥90%,较佳地≥95%。
- 如权利要求3所述的复合物,其特征在于,所述软骨凝胶是凝胶化培养2.5-5.5天,较佳地3-5天得到的。
- 如权利要求1所述的复合物,其特征在于,所述软骨膜片颗粒包括软骨细胞构成的细胞群以及软骨细胞所分泌的细胞外基质,其中所述细胞外基质包裹所述的细胞群,并且所述软骨颗粒是由薄片状的软骨膜片剪碎制得,其中软骨细胞的密度为至少1.0×10 8个/ml或1.0×10 8个/g。
- 如权利要求6所述的复合物,其特征在于,所述软骨膜片是凝胶化培养6-30天,较佳地7-20天,最佳地10-15天得到的。
- 如权利要求5或权利要求6所述的复合物,其特征在于,所述的凝胶化培养是用凝胶化培养基进行的体外培养,所述的凝胶化培养基含有以下组分:含4-5wt%葡萄糖的高糖DMEM培养基、10%FBS(v/v)和100U/ml青-链霉素。
- 如权利要求1所述的复合物,其特征在于,所述多孔框架结构是由选自下组的生物可降解材料制成:PCL、PGA、同种异体骨修复材料、异种骨修复材料、或脱钙骨基质。
- 如权利要求9所述的复合物,其特征在于,所述的多孔框架结构为脱钙骨基质,所述脱钙骨基质的厚度为0.3~0.8cm,较佳地,0.4~0.6cm,最佳地,0.5cm,所述脱钙骨基质的脱钙量为30%~50%。
- 一种制备如权利要求1所述的软骨组织工程复合物的方法,其包括以下步骤:将权利要求3所述的软骨凝胶或权利要求6所述的软骨膜片颗粒接种于多孔框架结构,经体外成软骨培养,从而获得所述的软骨组织工程复合物。
- 如权利要求11所述的方法,其特征在于,所述的成软骨培养是使用成软骨培养基进行的体外培养,所述的成软骨培养基具有以下组分:高糖DMEM培养基、血清替代物、脯氨酸、维生素C、转化生长因子-β1(TGF-β1)、胰岛素样 生长因子1(IGF-I)和地塞米松。
- 一种如权利要求1所述的软骨组织工程复合物的用途,其特征在于,用于制备用于修复关节缺损的医用产品。
- 一种用于关节修复的药物组合物,所述药物组合物包含如权利要求1中所述的软骨凝胶或软骨膜片颗粒以及药学上可接受的载体。
- 一种修复关节缺损的方法,其特征在于,使用如权利要求1所述的软骨组织工程复合物,移植入待修复患者的缺损关节内。
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