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 PDF

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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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cartilage
chondrocytes
gel
complex
culture
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PCT/CN2022/072395
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English (en)
French (fr)
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周广东
刘豫
慈政
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上海软馨生物科技有限公司
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Priority to US18/262,232 priority Critical patent/US20240091407A1/en
Publication of WO2022156645A1 publication Critical patent/WO2022156645A1/zh

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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

本发明提供了一种软骨组织工程复合物。具体地，所述移植物包括含有软骨细胞的软骨凝胶或软骨膜片颗粒，和一多孔框架结构，并且所述软骨凝胶或软骨膜片颗粒负载于多孔框架结构，形成软骨凝胶/软骨膜片颗粒-框架结构复合物。本发明还提供了所述软骨组织工程移植物的制备方法及其在关节软骨的修复与重建中的用途。

Description

一种软骨组织工程复合物及其用途 技术领域
本发明涉及生物医学组织工程领域，具体地，涉及一种软骨组织工程复合物和其制备方法，及其在关节修复中的应用。
背景技术
近些年来，随着经济社会的快速发展，各类运动损伤、意外伤以及关节退行性病变等各种类型的关节软骨病变日趋多见，而软骨病变依靠患者自体能力是难以修复的，所以关节修复临床需求巨大。目前临床上对于关节软骨病变的治疗方法主要为姑息性治疗及修复性治疗。姑息性治疗主要包括关节镜下清理术和软骨成形术等，这类治疗方法可清理关节表面不平整的软骨面及移除软骨碎片等，以恢复关节面的光滑平整，此类方法创伤较小且可以一定程度缓解患者的症状，但其疗效有限，不能有效缓解关节炎的发展。修复性治疗包括微骨折治疗、骨软骨移植等，此类治疗虽使得局灶性关节软骨缺损得到一定的修复，但创伤较大易导致供区并发症。传统治疗方法总体而言疗效欠佳，自体移植缺乏供体，人工关节远期并发症难以避免。
近年来，随着组织工程学的进步，人们逐渐开始研究使用组织工程所构建的支架或组织来尝试修复软骨缺损。组织工程学是涉及细胞生物学、材料科学、工程学以及生物反应器的一门交叉学科，利用生命科学和工程学的基本原理和方法，构建人体所需要组织，用于修复、替代因创伤、疾病而无功能的组织或器官。
现有组织工程修复技术所构建的软骨细胞-材料复合物(ACI或MACI)虽已在临床治疗中逐渐开展，但仍有诸多劣势难以避免：1、细胞来源为关节软骨，在本已受损的关节部位获取细胞，难以避免的会增加原有关节的损伤。2、关节软骨细胞增殖能力有限，前期细胞-材料复合物构建周期长，患者需要等待较长时间。3、细胞-材料复合物强度低，远不及天然软骨的力学强度水平。
据此，关节修复亟需新方法。
发明内容
本发明的目的是在于提供一种一种软骨组织工程复合物和其制备方法，及其在关节修复中的应用。
本发明的第一方面，提供了一种软骨组织工程复合物，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。
在另一优选例中，所述的复合物包括将所述软骨凝胶或软骨膜片颗粒接种于 所述载体并经成软骨培养后所形成的复合物(在所述复合物中，软骨细胞负载于载体并与载体形成更为紧密的一体结构)。
在另一优选例中，所述的复合物包括将所述软骨凝胶或软骨膜片颗粒接种于所述载体但未经成软骨培养后所形成的复合物。
在另一优选例中，所述软骨细胞来源于人类或非人类哺乳动物。
在另一优选例中，所述软骨细胞来源于自体软骨细胞或异体软骨细胞，优选地为自体软骨细胞。
在另一优选例中，所述软骨细胞来源于弹性软骨、纤维软骨或透明软骨。
在另一优选例中，所述软骨细胞取自受试者自体软骨细胞。
在另一优选例中，所述自体软骨细胞包括弹性软骨细胞、纤维软骨细胞或透明软骨细胞。
在另一优选例中，所述受试者为人类或非人类的哺乳动物。
在另一优选例中，所述受试者具有关节缺损。
在另一优选例中，所述关节缺损为关节软骨缺损。
在另一优选例中，所述关节缺损为膝关节缺损、肘关节缺损、髋关节缺损、踝关节缺损、腕关节缺损、下颌关节缺损或其组合。
在另一优选例中，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。
在另一优选例中，所述的软骨凝胶是用软骨细胞经凝胶化培养而制备的。
在另一优选例中，所述的凝胶化培养是用凝胶化培养基进行的体外培养。
在另一优选例中，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。
在另一优选例中，所述软骨凝胶的粘附率≥90％，较佳地≥95％。
在另一优选例中，所述软骨凝胶中，软骨细胞的浓度为1.0×10 8个/ml-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
在另一优选例中，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。
在另一优选例中，所述软骨细胞来自人类或非人类哺乳动物。
在另一优选例中，所述软骨细胞来源于自体软骨细胞或异体软骨细胞，优选地为自体软骨细胞。
在另一优选例中，所述软骨细胞取自受试者自体软骨细胞。
在另一优选例中，所述受试者为人类或非人类的哺乳动物。
在另一优选例中，所述受试者具有关节缺损。
在另一优选例中，所述关节缺损为关节软骨缺损。
在另一优选例中，所述关节缺损为膝关节缺损、肘关节缺损、髋关节缺损、踝关节缺损、腕关节缺损、下颌关节缺损或其组合。
在另一优选例中，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。
在另一优选例中，所述软骨膜片中，软骨细胞的浓度为1.0×10 8个/ml-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
在另一优选例中，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。
在另一优选例中，所述的凝胶化培养是用凝胶化培养基进行的体外培养。
在另一优选例中，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。
在另一优选例中，所述软骨膜片的厚度为0.2-0.25mm。
在另一优选例中，所述软骨膜片颗粒的平均体积为0.2μl。
在另一优选例中，所述软骨膜片颗粒的表面积为0.05-10mm 2，优选地，为1-5mm 2，更优选地，平均面积为1mm 2。
在另一优选例中，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。
在另一优选例中，所述框架结构还可以负载有明胶、胶原、丝素、水凝胶或其组合。
在另一优选例中，所述框架结构为脱钙骨基质。
在另一优选例中，所述脱钙骨基质来源于同种异体骨修复材料。
在另一优选例中，所述脱钙骨基质来源于异种骨修复材料。
在另一优选例中，所述脱钙骨基质的形状包括圆柱体、长方体或其他特定形状。
在另一优选例中，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。
在另一优选例中，所述脱钙骨基质的脱钙量为30％～50％。
在另一优选例中，所述软骨凝胶/软骨膜片颗粒-框架结构复合物在关节微环境下可生成关节软骨。
本发明的第二方面，提供了一种制备本发明第一方面所述的软骨组织工程复合物的方法，其包括以下步骤：将本发明第一方面所述的软骨凝胶或软骨膜片颗 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。
在另一优选例中，将所述软骨凝胶采用直接填充的方法接种于多孔框架结构。
在另一优选例中，将所述软骨膜片颗粒采用离心的方法接种于多孔框架结构。
在另一优选例中，所述离心的方法的离心体系中不加入液体，采用反复离心法使软骨膜片颗粒进入框架结构。
在另一优选例中，所述的成软骨培养是使用成软骨培养基进行的体外培养。
在另一优选例中，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。
在另一优选例中，所述的血清替代物为ITS premix，其中包含胰岛素、转铁蛋白、亚硒酸、亚油酸、牛血清清蛋白、丙酮酸、抗坏血酸磷酸盐。
在另一优选例中，所述的成软骨培养时间为3-15天，优选地5-11天。
本发明的第三方面，提供了一种本发明第一方面所述的软骨组织工程复合物的用途，用于制备用于修复关节缺损的医用产品。
在另一优选例中，所述关节缺损为关节软骨缺损。
在另一优选例中，所述关节缺损为膝关节缺损、肘关节缺损、髋关节缺损、踝关节缺损、腕关节缺损、下颌关节缺损或其组合。
本发明的第四方面，提供了一种用于关节修复的药物组合物，所述药物组合物包含如本发明第一方面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。
在另一优选例中，所述药物组合物为液态制剂。
在另一优选例中，所述药物组合物为注射剂。
在另一优选例中，所述药物组合物中软骨凝胶与药学上可接受的载体的体积比为50％至90％，较佳地70％-85％。
在另一优选例中，所述药物组合物中软骨膜片颗粒与药学上可接受的载体的体积比为45％-90％，较佳地60％-80％。
本发明的第五方面，提供了一种修复关节缺损的方法，使用本发明第一方面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。
在另一优选例中，所述关节缺损为关节软骨缺损。
在另一优选例中，所述关节缺损为膝关节缺损、肘关节缺损、髋关节缺损、踝关节缺损、腕关节缺损、下颌关节缺损或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了脱钙骨基质材料电镜照片。图中标尺(bar)为1mm。
图2显示了脱钙骨基质材料大观照片。图中标尺为1cm。
图3显示了耳软骨细胞培养3天和15天所得到的耳软骨凝胶和耳软骨膜片示意图。其中A-C为3天培养的耳软骨凝胶，D-E为15天培养的耳软骨膜片(D和E)及剪碎形成的耳软骨膜片颗粒(F)。
图4显示了软骨凝胶-框架结构复合物实物图。图中标尺为1cm。
图5显示了软骨膜片颗粒-框架结构复合物实物图。图中标尺为1cm。
图6显示了软骨凝胶-脱钙骨复合体电镜照片。图中标尺为200μm。
图7显示了软骨膜片颗粒-脱钙骨复合体电镜照片。图中标尺为200μm。
图8显示了接种样品(细胞悬液或软骨凝胶)于脱钙骨基质上培养24小时后的粘附率比较图。
图9显示了凝胶软骨-脱钙骨复合物(A)与单纯脱钙骨(B)移植到山羊膝关节缺损部位上的对比图。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现并开发了一种软骨组织工程复合物，该软骨组织工程复合物是一体化的软骨凝胶/软骨膜片颗粒-框架结构复合物。实验证明，通过获取原代软骨进行扩增培养，将特定数量的软骨细胞接种于和/或铺于平坦或基本平坦的培养表面，使得被接种的软骨细胞形成特定的层叠结构，并在合适的凝胶化培养条件下培养所述层叠的软骨细胞，因培养时间不同，可形成一种新颖的凝胶状软骨或膜片状软骨。将制得的凝胶状软骨或膜片状软骨与多孔框架结构结合，制备得到的软骨组织工程复合物在移植进入缺损关节后，可以在缺损的关节处再生为关节软骨，实现关节软骨的修复重建。在此基础上，完成了本发明。
术语
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，术语“软骨组织工程复合物”包括如本文所述的经体外成软骨培养的或未经体外成软骨培养软骨凝胶-框架复合物和软骨膜片颗粒-框架复合物，在本发明中，可统一称为软骨组织工程复合物。
如本文所用，术语“接种”意指将软骨细胞接种于细胞培养皿中，也可意指将软骨凝胶/软骨膜片颗粒接种于指定框架结构中并使其均匀分布，本领域技术人员根据上下文可以理解所用“接种”的含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
软骨凝胶及其制备
如本文所用，“凝胶软骨”、“软骨凝胶”、“凝胶态软骨”、“凝胶状软骨”、“本发明的软骨凝胶”或“本发明的凝胶软骨”可互换使用，皆指本发明的呈凝胶状态的软骨(干)细胞，尤其是将特定浓度的软骨细胞接种于和/或铺于平坦或基本平坦的培养表面，使得被接种的软骨细胞形成层叠结构，并在合适的凝胶化培养条件下培养具有层叠结构的软骨细胞，从而形成凝胶状软骨培养物。
本发明的凝胶软骨是不同于游离的软骨细胞、离心沉淀的软骨细胞和软骨团块(pellet)的新型软骨。本发明的凝胶软骨可视为介于游离的软骨细胞和致密的软骨团块之间的一种特定形态的软骨。本发明凝胶软骨，由于在凝胶化培养过程中，软骨细胞不仅与平面(X-Y平面)上相邻细胞存在接触和/或相互作用，而且还与其上方和/或下方和/或侧上方或下方等多方向的相邻软骨细胞存在接触和/或相互作用，从而促使软骨细胞分泌和形成更多的细胞外基质，从而将将凝胶化培养的软骨细胞包裹于具有一定粘性的细胞外基质中，从而使得本发明的凝胶软骨既具有紧密联系，而具有一定的粘性和流动性，从而使得本发明的凝胶软骨更适合接种和负载于各种不同的载体材料(尤其是多孔的载体材料)上，从而形成用于修复软骨的复合物。
此外，本发明的凝胶软骨一方面具有凝胶状态，另一方面具有异乎寻常高的细胞密度(通常至少1.0×10 8个/ml或更高，如1.0×10 8个-10×10 8个/ml)，因此，特别适合制备修复各类软骨的移植物，或用于软骨移植或软骨修复手术。
在本发明中，修复软骨的复合物包括将本发明的凝胶软骨负载于载体材料(尤其是多孔生物框架结构)所形成的未经成软骨培养的复合物，也包括将本发明的凝胶软骨负载于载体材料(尤其是多孔生物框架结构)并经成软骨培养所形成的复合物。
在本发明中，适合用于移植于人体或动物体的复合物就是本发明软骨组织工程复合物，即将本发明的凝胶软骨负载于载体材料(尤其是多孔生物框架结构)并经成软骨培养所形成的复合物。
优选地，在本发明中，在凝胶化培养条件系下，体外培养一段时间t1，从而 形成了凝胶软骨。优选地，所述的t1为2.5-5.5天，较佳地3-5天。
在本发明中，一个特征是层叠接种，即将特定密度的软骨细胞接种于培养容器后，接种的软骨细胞会通过例如沉积作用形成互相层叠的多层软骨细胞群(即具有层叠结构的软骨细胞群)。典型地，以培养皿(或培养容器)的培养面积计算，并设铺设单层细胞的汇合度为100％，则本发明的层叠接种的细胞数量S1是对于100％汇合度的细胞数量S0的n倍(即S1/S0＝n)，其中所述的n为1.5-20，较佳地2-10，更佳地2.5-5。
软骨膜片及其制备
如本文所用，“软骨膜片”、“膜片状软骨”、或“本发明的软骨膜片”可互换使用，皆指本发明的呈膜片状态的软骨(干)细胞，尤其是将特定浓度的软骨细胞接种于和/或铺于平坦或基本平坦的培养表面，使得被接种的软骨细胞形成层叠结构，并在合适的培养条件下培养具有层叠结构的软骨细胞，从而形成膜片状软骨培养物。
本发明的“软骨膜片”是在本发明所述的“软骨凝胶”的制备基础上，通过延长凝胶化培养时间所制备得到的。即，在本发明中，在凝胶化培养条件下，将接种于和/或铺于平坦或基本平坦的培养表面的软骨细胞体外培养一段时间t2，从而形成了软骨膜片。优选地，所述的t2为6-30天，较佳地7-20天，最佳地10-15天。
本发明的软骨膜片一方面具有异乎寻常高的细胞密度(通常至少1.0×10 8个/ml或更高，如1.0×10 8个-10×10 8个/ml)，另一方面它的厚度薄(仅有0.2-0.25mm)并且韧性佳，可被剪碎为平均体积0.2μl的“软骨膜片颗粒”，通过简单离心的方式填充于多孔框架结构中，因此，特别适合制备修复各类软骨的移植物，或用于软骨移植或软骨修复手术。
在本发明中，修复软骨的复合物包括将本发明的软骨膜片颗粒负载于载体材料(尤其是多孔框架结构)所形成的未经成软骨培养的复合物，也包括将本发明的软骨膜片颗粒负载于载体材料(尤其是多孔框架结构)并经成软骨培养所形成的复合物。
在本发明中，适合用于移植于人体或动物体的复合物就是本发明的软骨组织工程复合物，即将本发明的软骨膜片颗粒负载于载体材料(尤其是多孔框架结构)并经成软骨培养所形成的复合物。
如本文所用，“特定浓度”或“特定密度”是指在3.5cm的培养皿(例如，六孔板中的一个孔)中，接种1.0×10 7-2.0×10 7个细胞，较佳地，为1.5×10 7个细胞。经凝胶化培养不同的时间，最终形成所含软骨细胞密度为1.0×10 8个-10×10 8个/ml的软骨凝胶或所含软骨细胞密度为1.0×10 8个-10×10 8个/ml的软骨膜片。
在另一优选例中，所述的凝胶化培养条件为：接种特定密度的软骨细胞，使用凝胶化培养基培养，所述凝胶化培养基为含10％胎牛血清及100U/ml青-链霉素的高糖(4-5wt％葡萄糖)DMEM培养基。
如本文所用，术语“成软骨培养”是指使用成软骨培养基培养接种了软骨凝胶或软骨膜片颗粒的多孔框架结构，最终使其形成一体化的软骨凝胶-框架结构复合物或软骨膜片颗粒--框架结构复合物，即本发明的软骨组织工程复合物，用于移植于人体或动物体的软骨缺损处。
软骨和软骨细胞
软骨即软骨组织，由软骨细胞和细胞间质组成。软骨内的基质呈凝胶状态，具有较大韧性。软骨是以支持作用为主的结缔组织。软骨内不含血管和淋巴管，营养物由软骨膜内的血管中渗透到细胞间质中，再营养骨细胞。
根据细胞间质的不同可把软骨分为3种，即透明软骨、弹性软骨和纤维软骨。透明软骨的基质是由胶原纤维、原纤维和周围无定形的基质组成。在胚胎时期有临时支架作用，后来这种作用被骨代替。成人的透明软骨主要分布在气管和支气管壁中、肋骨的胸骨端和骨的表面(关节软骨)。弹性软骨的基质中除了胶原纤维还有弹性纤维，这种软骨弹性较大，主要分布在耳廓、外耳道壁、耳咽管和会厌、喉部等处。纤维软骨基质中有成束的胶原纤维平行或交叉排列，较坚韧。分布在椎间盘、关节盂、关节盘以及一些腱、韧带等处，以增强运动的灵活性和保护、支持等作用。
本发明所述的软骨组织工程复合物中所用的软骨细胞可以为取自透明软骨、弹性软骨或纤维软骨的透明软骨细胞、弹性软骨细胞或纤维软骨细胞，在植入受试者的关节缺损处后，在关节环境中可转化为关节软骨。
MACI和ACI
MACI是matrix-induced autologous chondrocyte implantation的缩写，意为“基质诱导的自体软骨细胞移植”，是利用组织工程科技手段进行软骨细胞移植的技术。MACI是目前全世界最新、最好的治疗关节软骨缺损的技术。
ACI是autologous chondrocyte implantation的缩写，意为“自体软骨细胞移植”，是目前应用较广泛的治疗关节软骨损伤的组织工程技术之一。在MACI问世之后，ACI就被相应称为“传统ACI”，以便与MACI区别。
多孔框架结构
如本文所用，术语“多孔框架结构”是指其表面和内部具有一定数量的孔隙以便于使接种于其上的耳软骨凝胶或耳软骨膜片附着的由生物相容性材料制成的一载体。在本发明中，所述生物相容性材料优选生物可降解材料。
生物可降解材料是指在移植进入动物体内后，可以在体内被分解的材料。本发明的耳软骨组织工程移植物的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、脱钙骨基质或其组合，但不限于以上所述材料。其中同种异体骨修复材料和异种骨修复材料包括脱钙骨基质材料。在本发明的优选实施例中，所述耳软骨组织工程复合物的多孔框架结构为脱钙骨基质。
脱钙骨基质
本发明的优选实施例中使用的脱钙骨基质，其厚度为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％，脱钙程度合适，支持作用佳，并易于修整裁剪为合适的形状和大小。所述的脱钙骨基质的孔隙的孔径为400-800μm，易于填充软骨细胞。
脱钙骨基质(DBM)是由同种异体骨或异种骨经脱钙处理，能降低免疫原性的骨移植材料。脱钙程度不同对应的机械强度也不同。具有良好的生物学特性、骨诱导性和骨传导性和生物降解性，促进新骨形成及骨组织矿化，进而加速骨愈合，可以单独或与自体骨、其它生物材料、生长因子联合有效修复骨损伤，是比较理想的骨组织工程支架材料。然而一般的脱钙骨基质的孔径较大，接种软骨细胞悬液时细胞黏附率极低，不利于组织工程载体的构建。
在另一优选例中，本发明中所述脱钙骨基质具有400-800μm孔径，87.3％±3.7％孔隙率。
同种异体骨修复材料
同种异体骨是目前骨科最常用的骨植入材料，主要用于修复、填充骨缺损，起到固定和支撑作用。同种异体骨取自捐献的人体骨组织，“同种”表明它来自人体，“异体”表明它不是来自患者自体。供体选定后，通常在死亡24小时内，无菌条件下取得，并立即加工处理。保存方法包括新鲜冷冻和冷冻干燥两种。新鲜冷冻骨在-20℃条件下可储存1年；冷冻干燥骨经真空包装后可在室温长时间保存，且抗原性更低。与新鲜冷冻骨相比，冷冻干燥骨的力学性能会降低50％，而且环氧乙烷或大剂量γ射线照射消毒会进一步降低骨的诱导性能。
异种骨修复材料
异种骨是来源于其他物种如牛、猪等的骨修复材料。其来源广泛且价格相对低廉。但异种骨的免疫原性强，植入患者体内后容易引起免疫排斥反应。此外，异种骨无诱导间充质干细胞增殖的能力，生物活性差，需要复合其他修复材料或相关细胞因子才能达到修复效果。
本发明所用的培养基
成软骨培养基：高糖DMEM培养基，1％1×ITS premix((ITS通用型培养混合剂，含胰岛素、转铁蛋白、亚硒酸、亚油酸、牛血清蛋白、丙酮酸、抗坏血酸磷酸盐)，40μg/ml脯氨酸，10ng/ml TGF-β1，100ng/ml IGF-1,40ng/ml地塞米松和50μg/ml维生素C。
凝胶化培养基：含4-5wt％葡萄糖，10％FBS(v/v)和100U/ml青链霉素的DMEM培养基。
黏附率
在本发明中，当将本发明的软骨凝胶接种于载体材料(尤其是多孔生物框架结构)时，本发明中的软骨凝胶具有一定的粘附率，通过本发明提供的粘附率测定方法确定。本发明的软骨凝胶的粘附率≥90％，较佳的，≥95％。
本发明中的粘附率如下定义：
检测所接种样品(例如软骨凝胶)的DNA定量A1；检测接种后复合物(例如软骨凝胶-框架复合物)培养24小时后的DNA定量A2；粘附率为A2/A1*100％。
所述黏附率的测定方法包括以下步骤：
取接种样品(例如软骨凝胶或软骨凝胶-框架复合物)，使用蛋白酶K消化，消化后的样品使用使用PicoGreen试剂盒(Invitrogen,Carlsbad,CA,USA)定量检测，使用荧光酶标仪测定520nm的吸光度，依据标准曲线公式计算出DNA含量。
本发明的主要优点包括：
(1)本发明的软骨凝胶较软骨细胞而言更加成熟，且具有一定的流动性。
(2)脱钙骨基质作为可降解的天然材料，可在体内降解，机体免疫反应较低，生物安全性好。
(3)脱钙骨基质材料孔径较大，孔隙率较好，但接种软骨细胞悬液时细胞黏附率极低，使用具有一定流动性且较为粘稠的软骨凝胶样组织可有效提高黏附率。
(4)单纯可注射软骨无法成型，张力条件下单纯可注射软骨吸收率较高，临床应用受限，使用脱钙骨基质作为框架结构，可构建一定特殊形状的可注射软骨-脱钙骨基质复合物，且提供力学支撑后有限降低软骨吸收率。
(5)本发明的移植物的细胞来源多样，扩增能力好。
(6)相较于其他组织工程修复方法，软骨凝胶/软骨膜片颗粒-脱钙骨基质复合物可稳定再生软骨。
(7)软骨凝胶/软骨膜片颗粒-脱钙骨基质复合物可稳定再生软骨可提供即时力学支撑，即时修复效果好。
下面，通过具体的实施例对本发明做进一步说明。下面结合具体实施例，进 一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
实施例1
软骨凝胶-脱钙骨复合物制备
在本实施例中，制备软骨凝胶-框架复合物。具体操作方法如下：
(1)取受试者自体部分耳软骨组织，无菌切取2.5×2.5cm 2的软骨组织,使用无菌器械将软骨表面的黏膜及纤维组织剥离；
(2)将软骨组织剪为1.5×1.5mm 2大小软骨碎片；配置浓度为0.15％胶原酶；将软骨碎片加入配置好的胶原酶中消化8小时；
(3)8小时后将胶原酶溶液过滤离心得到耳软骨细胞，使用含有10％的FBS的高糖DMEM培养基进行原代及传代培养；
(4)扩增后收集细胞重悬，按照8×10 6/10ml至30×10 6/10ml/孔的细胞量接种于六孔板中，以凝胶化培养基(含4-5wt％葡萄糖，10％FBS(v/v)和100U/ml青链霉素的DMEM培养基)培养；
(5)在接种72小时(3天)后将六孔板中培养基吸去，可见六孔板底部的凝胶状软骨组织(图3A)，使用镊子将六孔板底部的凝胶状软骨聚集(图3B)，一个孔的凝胶软骨产量为0.1-0.2ml，收集至5ml注射器中；该凝胶软骨中，细胞密度为1.0×10 8个/ml-10×10 8个/ml，较佳地1.5-5×10 10个/ml。将其与0.15ml培养基混合，制成含软骨凝胶的制剂，如图3C所示；
(6)将软骨凝胶制剂(步骤(5)制备，体积约0.25-0.35ml)，接种于框架结构(脱钙骨基质框架(图1和图2所示)，具有约400-800μm孔径，约87.3％±3.7％孔隙率)中，于37℃、95％湿度、5％二氧化碳静置2小时；
(7)静置后加入成软骨培养基继续体外培养3-11天，形成软骨凝胶-框架结构复合物，如图4所示；电镜观察可见，脱钙骨基质的孔隙被软骨细胞填充(图6)。
当用于关节缺损修复时，可根据前期MRI、CT等辅助检查确定所需修复的软骨形状、大小切割软骨凝胶-框架结构复合物。
实施例2
软骨膜片颗粒-脱钙骨复合物制备
在本实施例中，制备软骨膜片颗粒-框架复合物。具体操作方法如下：
(1)无菌切取2.5×2.5cm 2的耳软骨组织；使用无菌器械将软骨表面的黏膜及纤维组织剥离；
(2)将软骨组织剪为1.5×1.5mm 2大小软骨碎片；配置浓度为0.15％胶原酶； 将软骨碎片加入配置好的胶原酶中消化8小时；
(3)8小时后将胶原酶溶液过滤离心得到耳软骨细胞，进行原代及传代培养；
(4)扩增后收集细胞重悬，按照8×10 6/10ml至30×10 6/10ml/孔的细胞量接种于六孔板中，以凝胶化培养基(含4-5wt％葡萄糖，10％FBS(v/v)和100U/ml青链霉素的DMEM培养基)培养；培养24小时或48小时后更换新鲜凝胶化培养基，继续体外培养至15天；
(6)吸去六孔板中的培养基，可见六孔板底部的软骨膜片组织(图3D)，其中软骨膜片组织中的细胞密度约为1.0×10 8个/ml-10×10 8个/ml；
使用镊子将软骨膜片夹起(图3E)，将其切割为1×1mm 2大小的软骨膜片颗粒后，收集至50ml离心管，如图3F所示；
(7)将需要接种的框架材料(脱钙骨基质框架(图4和图5))置于装有软骨膜片颗粒的离心管中，确保框架材料被完全浸没过；将装有框架材料及软骨膜片颗粒的离心管放于离心机中，600转/分钟，离心2分钟；
(8)将接种好的框架材料于37℃、95％湿度、5％二氧化碳静置一定时间；静置后加入成软骨培养基继续体外培养3-11天，形成软骨膜片颗粒-框架结构复合物，如图5所示；电镜观察可见，脱钙骨基质的孔隙被软骨细胞填充(图7)。
当用于关节缺损修复时，可根据前期MRI、CT等辅助检查确定所需修复的软骨形状、大小切割耳软骨膜片颗粒-框架结构复合物。
对比例1
软骨细胞悬液与软骨凝胶的黏附率测定
提供一脱钙骨基质框架(如图2所示)。将凝胶软骨制剂(实施例1制备，体积约0.25-0.35ml)接种于上述脱钙骨基质框架中，接种前测定凝胶软骨制剂的DNA含量。
取原代培养的软骨细胞于37℃、95％湿度、5％二氧化碳传代4次，加入细胞培养基制备成细胞悬液，并测定制备的软骨细胞悬液的DNA含量。将细胞悬液接种于上述脱钙骨基质框架中。
将接种好的软骨凝胶-脱钙骨基质复合物与软骨细胞悬液-脱钙骨基质复合物于37℃、95％湿度、5％二氧化碳的培养箱中静置24小时。分别取样两种复合物，测定其DNA含量。
如图8所示，通过说明书中所述的粘附率测定方法计算粘附率。与细胞悬液相比，本发明的凝胶软骨粘附率为92％±2％，约为细胞悬液黏附率的3倍。
对比例2
修复关节软骨的动物移植实验
在实验动物膝关节的关节面制造直径7.5mm的软骨缺损，分别将实施例1中 制备的凝胶软骨-框架复合物(凝胶软骨-脱钙骨复合体)与单纯脱钙骨基质基质植入A缺损处和B缺损处。
植入后立即观察动物体内软骨缺损的修复情况。
结果如图9所示：
A处缺损部位光滑，质实，周围包绕软组织膜，有一定弹性，具有优异的即时修复效果
B处缺损部位创面粗糙，仅有物理支撑作用，无法起到即使修复作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 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。
  2. 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。
  3. 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。
  4. 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％。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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％。
  11. 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。
  12. 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。
  13. 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。
  14. 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。
  15. 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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