WO2022156769A1 - 一种基于大孔径的组织工程支架及其用途 - Google Patents

一种基于大孔径的组织工程支架及其用途 Download PDF

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WO2022156769A1
WO2022156769A1 PCT/CN2022/073191 CN2022073191W WO2022156769A1 WO 2022156769 A1 WO2022156769 A1 WO 2022156769A1 CN 2022073191 W CN2022073191 W CN 2022073191W WO 2022156769 A1 WO2022156769 A1 WO 2022156769A1
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tissue engineering
engineering scaffold
glue
cartilage
tissue
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PCT/CN2022/073191
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English (en)
French (fr)
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刘豫
慈政
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上海软馨生物科技有限公司
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    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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Definitions

  • the invention belongs to the field of tissue engineering, and in particular, the invention relates to a tissue engineering scaffold and a preparation method thereof.
  • Hard tissue defects including cartilage or bone defects, are increasingly common in clinical diagnosis and treatment.
  • the existing treatment methods are still mainly autologous tissue transplantation, but there are problems such as high infection risk, new donor site defects and limited donor sites.
  • advances in tissue engineering have provided new approaches for the treatment of various types of hard tissue defects.
  • scaffold materials play a crucial role in the construction of tissue engineered bone or cartilage. Therefore, it is necessary to find a material with suitable characterization, good cell compatibility and osteogenic induction activity.
  • the scaffold material has become a hot spot in bone tissue engineering at this stage.
  • the existing tissue engineering scaffolds for hard tissue repair are mostly divided into two types: one is a sponge-like porous scaffold (such as collagen sponge, polyglycolic acid/polylactic acid (PGA/PLA) scaffold, etc.); the other is a decalcified scaffold. Bone and PCL frames are represented by macroporous frame structures. Both types of brackets have pros and cons.
  • Sponge-like porous scaffolds can be constructed with different pore sizes by adjusting the solute concentration and freeze-drying parameters to meet the seeding needs of various types of cells, but the mechanical strength of such scaffolds is far from satisfactory and cannot meet the strength requirements of immediate repair; demineralized bone and PCL
  • the scaffold represented by the frame structure can have good mechanical strength and meet the mechanical strength requirements of instant repair of various parts, but its pore size is difficult to precisely control.
  • the seeding efficiency is too low, especially for materials of natural origin such as demineralized bone matrix.
  • the purpose of the present invention is to provide a tissue engineering scaffold suitable for the repair of hard tissue defects.
  • a first aspect of the present invention provides a tissue engineering scaffold, the tissue engineering scaffold comprising:
  • the degradable biological glue is selected from the group consisting of: composite gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
  • the rigid large aperture frame structure has certain hardness and mechanical strength.
  • the rigid macroporous frame structure is made of degradable biomaterials.
  • the rigid macroporous framework structure is selected from the group consisting of demineralized bone matrix, PCL framework or a combination thereof.
  • the pore size of the rigid macroporous frame structure is 300-800 ⁇ m, and the porosity is 80%-90%.
  • the rigid large-pore 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 pore size of the demineralized bone matrix is 300-800 ⁇ m, and the porosity is 80%-90%.
  • the pore size of the tissue engineering scaffold can be adjusted by the concentration of the loaded biological glue and the time of lyophilization.
  • the tissue engineering scaffold can also be loaded with chondrocyte suspension containing chondrocytes, cartilage gel or perichondral sheet particles.
  • 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 hard tissue defect.
  • the hard tissue defect includes joint defect, maxillofacial cartilage and related hard tissue defect, nasal septal defect, or a combination thereof.
  • 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 concentration (density) of chondrocytes in the chondrocyte suspension is 1.0 ⁇ 10 6 /ml to 1.0 ⁇ 10 8 /m.
  • 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 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 second aspect of the present invention provides a method for preparing the tissue engineering scaffold described in the first aspect of the present invention, the method comprising the steps of: loading or filling the bio-glue on the rigid large-pore frame structure, thereby obtaining the tissue engineering scaffolds.
  • the method includes the following steps:
  • the biological glue is medical gelatin.
  • the concentration of the biological glue solution is 0.3%-1.0%, preferably 0.6%.
  • the rigid large-pore frame structure is demineralized bone matrix.
  • the rotational speed used in the centrifugation is 500 r/min, and the centrifugation time is 2 minutes.
  • the centrifuged centrifuge tube is refrigerated at 2-8° C. for 5-10 hours, preferably 6-8 hours.
  • the content is frozen at -20°C to -30°C for 5-10 hours, preferably 6-8 hours.
  • the biological glue-framework structure complex is vacuum lyophilized for 10-20 hours, preferably 10-16 hours, more preferably 12-14 hours.
  • the chemical cross-linking agent is selected from the group consisting of EDC, genipin, glutaraldehyde or a combination thereof.
  • the third aspect of the present invention provides a use of the tissue engineering scaffold according to the first aspect of the present invention, characterized in that the tissue engineering scaffold is used to prepare a medical product for repairing hard tissue defects.
  • the hard tissue defect includes joint defect, maxillofacial cartilage and related hard tissue defect, nasal septal defect, or a combination thereof.
  • 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 microscopy and gross photographs of demineralized bone matrix.
  • the left picture is the electron microscope image of the pores of the demineralized bone matrix, and the scale bar in the picture is 200 ⁇ m; the right picture is the general photo of the decalcified bone matrix.
  • Figure 2 shows a photograph of gelatin particles.
  • Figure 3 shows the electron microscope and gross photographs of the bioglue-decalcified bone matrix composite scaffold.
  • the left picture is the electron microscope structure diagram, the scale in the picture is 200 ⁇ m; the right picture is the general photo.
  • Figure 4 shows the regenerated cartilage-like tissue after the bioglue-demineralized bone matrix complex is loaded with chondrocytes and implanted into the experimental animal.
  • bio-glue-frame composite scaffold prepared by filling a rigid large-pore frame structure with bio-glue can be used for the repair of hard tissue defects.
  • bioglue-framework complex scaffold can effectively load the seeded cells, has good mechanical strength, and can successfully regenerate into cartilage-like tissue after implantation in vivo.
  • 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.”
  • biological glue refers to a class of degradable biological preparations that have a certain fluidity at room temperature and become solid form when the temperature is lowered (eg, to 10°C or lower).
  • the biological glue includes an agent selected from the group consisting of composite gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
  • the biological glue used is medical gelatin, and the medical gelatin is mixed with an aqueous solution to prepare an aqueous biological glue solution with a concentration of 0.3%-1.0%, preferably 0.6%.
  • the term "rigid large pore frame structure” refers to a biodegradable biomaterial frame structure having certain stiffness and mechanical strength. It can provide mechanical support when used for repairing hard tissue defects, and can be degraded in vivo without producing substances harmful to the body, the immune response of the body is low, and the biological safety is good.
  • Rigid macroporous framework structures of the present invention include, but are not limited to, demineralized bone matrix and PCL frameworks.
  • the rigid macroporous frame structure is demineralized bone matrix.
  • Decalcified bone matrix is a bone graft material that is decalcified from allogeneic or xenogeneic bone to reduce immunogenicity. It is composed of collagen, non-collagen and lower concentrations of growth factors (such as bone-forming protein, the bone-forming protein in the bone is surrounded by dense mineral components, non-decalcified bone has no ability to induce osteogenesis, decalcified bone
  • the composite natural bone graft material composed of different degrees corresponding to different mechanical strengths) is mainly derived from the skull, femoral shaft and tibial shaft of humans or animals (pig, cow, dog, rabbit, etc.).
  • 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 pore diameter of the pores of the demineralized bone matrix is 300-800 ⁇ M.
  • a chemical cross-linking agent is used to freeze-dry the biological glue-frame structure complex and then cross-link.
  • the chemical cross-linking agent is selected from EDC, genipin or glutaraldehyde.
  • the tissue engineering scaffold of the present invention can be used for hard tissue defect repair.
  • the hard tissue defects include, but are not limited to, joint defects, maxillofacial cartilage and related hard tissue defects, nasal septal defects, or combinations thereof.
  • the tissue engineering scaffold of the present invention can be loaded with cartilage gel containing chondrocytes.
  • gelled cartilage As used herein, “gelled cartilage”, “cartilage gel”, “gelatinous cartilage”, “gelatinous cartilage” are used interchangeably and all refer to cartilage (stem) cells in a gelled state, especially 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 gelation culture conditions, thereby forming Gel-like cartilage cultures.
  • the gel cartilage is a new type of cartilage different from free chondrocytes, centrifuged chondrocytes and cartilage pellets.
  • the gel cartilage described in the present invention can be regarded as a specific form of cartilage between free chondrocytes and dense cartilage mass.
  • the chondrocytes during the gelation culture process, not only contact and/or interact with the adjacent cells on the plane (X-Y plane), but also contact and/or interact with the cells above and/or below and/or on the plane (X-Y plane).
  • the gel cartilage of the present invention not only has close contact, but also has a 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 carrier material) to form a complex for cartilage repair.
  • 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 higher, such as 1.0 ⁇ 10 8 cells-10 ⁇ 10 8 /ml), therefore, it is especially suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.
  • 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.
  • One feature of the gel cartilage of the present invention 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 (that is, a stacked structure) through deposition, for example. chondrocyte population).
  • the tissue engineering scaffold of the present invention can be loaded with perichondrial sheet particles containing chondrocytes.
  • peripheral sheet As used herein, “perichondral sheet”, “patch-like cartilage”, or “perichondral sheet of the present invention” are used interchangeably, and all refer to cartilage (stem) cells in a patch state, especially when a particular concentration of Chondrocytes are seeded and/or plated 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 patch-like cartilage culture .
  • the "perichondrium sheet” 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 according to 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 It has a thin thickness (only 0.2-0.25mm) and good toughness, and 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 preparation Grafts to repair various types of cartilage, or for cartilage transplantation or cartilage repair surgery.
  • 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 or chondrocytes with a density of 1.0 ⁇ 108-10 ⁇ 10 are finally formed. 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.
  • the tissue engineering scaffold of the present invention After the tissue engineering scaffold of the present invention is loaded with the cartilage gel or perichondrial sheet particles, it needs to undergo chondrogenic culture to form a graft that can be used to repair hard tissue defects.
  • 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.
  • 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 present invention adopts the method of composite gelatin (or collagen, silk fibroin, hydrogel, etc.) to fill the pores of the demineralized bone matrix or PCL frame structure with gelatin to construct a new type of tissue engineering scaffold.
  • Calcium-bone scaffolds are expected to find a new method for the construction of tissue-engineered bones.
  • the tissue engineering scaffold of the present invention not only has an appropriate pore size, can effectively load the inoculated cells, but also has good mechanical strength, which can meet the mechanical strength requirements of instant repair of various parts.
  • the tissue engineering scaffold of the present invention can precisely control the pore size by adjusting the concentration ratio of biological glue or freeze-drying parameters according to the type of cells to be loaded.
  • demineralized bone matrix (as shown in Figure 1), place the demineralized bone matrix in the gelatin aqueous solution, and centrifuge for 2 minutes using a centrifuge at 500 r/min;
  • the pore size of the bioglue-decalcified bone matrix composite prepared in this example was significantly reduced (Fig. 3), and the seeded cells could be loaded more efficiently.
  • chondrocytes were seeded on the bioglue-decalcified bone matrix composite scaffold prepared in Example 1 at a concentration of 7 ⁇ 10 7 cells/ml;
  • the cell-scaffold complex was incubated in a 37°C, 95% humidity, 5% carbon dioxide incubator and then added to chondrogenesis medium (high glucose DMEM medium, 1% 1 ⁇ ITS premix ((ITS universal culture mixture, Contains insulin, transferrin, selenite, linoleic acid, bovine serum albumin, pyruvate, ascorbic acid phosphate), 40 ⁇ g/ml proline, 10ng/ml TGF- ⁇ 1, 100ng/ml IGF-1, 40ng/ ml dexamethasone and 50 ⁇ g/ml vitamin C); after 8 weeks of in vitro culture, it was replanted in vivo; after 4 weeks of in vivo regeneration, samples were taken, and the successfully regenerated cartilage-like tissue is shown in Figure 4.
  • chondrogenesis medium high glucose DMEM medium, 1% 1 ⁇ ITS premix ((ITS universal culture mixture, Contains insulin, transferrin, selenite, linoleic acid, bovine serum album
  • Example 1 of the present invention can effectively load the chondrocytes seeded therein, and provide good mechanical support at the defect, and can be successfully regenerated in vivo as cartilage-like tissue.

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Abstract

本发明提供了一种组织工程支架。具体地，所述组织工程支架是将可降解的生物胶状物均匀填充于硬性大孔径框架结构，制成的生物胶-框架结构复合体。本发明的组织工程支架优化了传统大孔框架结构的孔径，提高了细胞接种效率。此外，本发明还提供了所述新型组织工程支架的制备方法及其在修复硬组织缺损中的用途。

Description

一种基于大孔径的组织工程支架及其用途 技术领域
本发明属于组织工程学领域，具体地，本发明涉及一种组织工程支架及其制备方法。
背景技术
包括软骨或骨缺损在内的硬组织缺损在临床诊疗中日趋常见，现有治疗手段仍以自体组织移植为主，但存在感染风险大、产生新的供区缺损和供区有限等问题。近年来，组织工程学的进步为治疗各类硬组织缺损提供了新方法。支架材料作为组织工程三要素中的重要一环，在构建组织工程骨或软骨的过程中起到了至关重要的作用，所以寻找一种材料表征合适、细胞相容性好且具有成骨诱导活性的支架材料成为现阶段骨组织工程的热点。
现有用于硬组织修复的组织工程支架多分为两种：一种为海绵状的多孔支架(如胶原海绵，聚羟基乙酸/聚乳酸(PGA/PLA)支架等)；另一种为以脱钙骨及PCL框架为代表的大孔框架结构。这两类支架各有利弊。海绵状多孔支架可以通过调节溶质浓度及冻干参数来构建孔径不同的支架，满足各类细胞的接种需求，但这类支架力学强度多差强人意，无法满足即时修复的强度需求；脱钙骨及PCL框架结构为代表的支架可以拥有较好的力学强度，满足各类部位的即时修复的力学强度要求，但其孔径难以精准控制，孔径过大导致在接种细胞悬液时无法有效负载细胞，使得细胞接种效率过低，尤其是脱钙骨基质这类天然来源的材料，其孔径会因材料批次不同而各有差异，影响整体组织再生效果。
因此，本领域迫切需要开发一种孔径合适，且具有良好的力学强度的组织工程支架。
发明内容
本发明的目的就是提供一种适合应用于硬组织缺损修复的组织工程支架。
本发明的第一方面，提供了一种组织工程支架，所述组织工程支架包括：
(a)硬性大孔径框架结构；和
(b)负载于或填充于硬性大孔径框架结构的可降解的生物胶。
在另一优选例中，所述可降解的的生物胶选自下组：复合明胶、胶原、丝素、 水凝胶或其组合。
在另一优选例中，所述硬性大孔径框架结构具有一定硬度和力学强度。
在另一优选例中，所述硬性大孔框架结构由可降解生物材料制成。
在另一优选例中，所述硬性大孔框架结构选自下组：脱钙骨基质、PCL框架或其组合。
在另一优选例中，所述硬性大孔框架结构的孔径为300-800μm，孔隙率为80％-90％。
在另一优选例中，所述硬性大孔径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，所述脱钙骨基质的孔径为300-800μm，孔隙率为80％-90％。
在另一优选例中，所述组织工程支架的孔径大小可通过负载的生物胶浓度和冻干处理的时间调整。
在另一优选例中，所述的组织工程支架还可以负载有含有软骨细胞的软骨细胞悬液、软骨凝胶或软骨膜片颗粒。
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，所述受试者具有硬组织缺损。
在另一优选例中，所述硬组织缺损包括关节缺损、颌面部软骨及相关硬组织缺损、鼻中隔缺损、或其组合。
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，所述软骨细胞悬液中软骨细胞的浓度(密度)为1.0×10 6/ml至1.0×10 8/m。
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。
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组织工程支架的方法，所述方法包括以下步骤：将生物胶负载于或填充于硬性大孔径框架结构，从而获得所述的组织工程支架。
在另一优选例中，所述方法包括以下步骤：
(i)制备生物胶水溶液，并将所述生物胶水溶液置于离心管中；
(ii)将硬性大孔径框架结构置于含有所述生物胶水溶液的离心管中并离心；
(iii)将离心后的离心管冷藏后，取出内容物后将内容物冷冻，得到生物胶-框架结构复合体；
(iv)将所述生物胶-框架结构复合体真空冻干，得到冻干的生物胶-框架结构复合体；
(v)使用化学交联剂对所述冻干的生物胶-框架结构复合体进行交联，得到交联的生物胶-框架结构复合体；
(vi)用去离子水冲洗所述交联的生物胶-框架结构复合体，并真空冷冻干燥，得到所述组织工程支架。
在另一优选例中，所述生物胶为医用明胶。
在另一优选例中，所述生物胶水溶液的浓度为0.3％-1.0％，优选为0.6％。
在另一优选例中，所述硬性大孔径框架结构为脱钙骨基质。
在另一优选例中，所述离心采用的转速为500r/min，离心时间为2分钟。
在另一优选例中，将所述离心后的离心管于2-8℃冷藏5-10小时，优选地6-8小时。
在另一优选例中，将所述内容物于-20℃至-30℃冷冻5-10小时，优选地6-8小时。
在另一优选例中，将所述生物胶-框架结构复合体真空冻干10-20小时，优选地10-16小时，更优选地12-14小时。
在另一优选例中，所述化学交联剂选自下组：EDC、京尼平、戊二醛或其组合。
本发明的第三方面，提供了一种本发明第一方面所述的组织工程支架的用途，其特征在于，所述组织工程支架用于制备硬组织缺损修复的医用产品。
在另一优选例中，所述硬组织缺损包括关节缺损、颌面部软骨及相关硬组织 缺损、鼻中隔缺损、或其组合。
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显示了脱钙骨基质电镜及大体照片。其中左图为脱钙骨基质孔隙的电镜图，图中标尺为200μm；右图为脱钙骨基质的大体照片。
图2显示了明胶颗粒照片。
图3显示了生物胶-脱钙骨基质复合体支架电镜及大体照片。其中左图为电镜结构图，图中标尺为200μm；右图为大体照片。
图4显示了生物胶-脱钙骨基质复合体负载软骨细胞后，植入实验动物体内后再生的软骨样组织。
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，将生物胶填充于硬性大孔径框架结构制备成的生物胶-框架复合体支架可用于硬组织缺损修复。实验表明，生物胶-框架复合体支架可以有效负载接种的细胞，而且具有良好的力学强度，植入体内后可成功再生为软骨样组织。
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，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”。
生物胶
如本文所用，“生物胶”是指一类在室温具有一定的流动性，在温度降低后(例如降低至10℃或更低温度)，则变为固态形式的可降解的生物制剂。
本发明的实施例中，所述生物胶包括选自下组的试剂：复合明胶、胶原、丝素、水凝胶或其组合。
在本发明的一个优选例中，所用生物胶为医用明胶，并且，将所述医用明胶与水溶液混合，制备成浓度为0.3％-1.0％，优选为0.6％的生物胶水溶液。
硬性大孔径框架结构
如本文所用，术语“硬性大孔径框架结构”是指具有一定硬度和力学强度的可降解生物材料框架结构。其在用于硬组织缺损修复时可提供力学支撑，并且在生物体内可被降解且不产生对机体有害的物质，机体免疫反应较低，生物安全性好。
本发明的硬性大孔框架结构包括(但不限于)脱钙骨基质和PCL框架。
在本发明的一个优选例中，所述硬性大孔框架结构为脱钙骨基质。
脱钙骨基质
脱钙骨基质(decalcified bone matrix，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。是一种是由胶原蛋白、非胶原蛋白以及较低浓度的生长因子(如骨形成蛋白，骨中的骨形成蛋白被致密的矿物成份包绕，未脱钙骨无诱导成骨能力，脱钙程度不同对应的机械强度也不同)等组成的复合物天然骨移植材料，主要来源于人或动物(猪、牛、狗、兔等)的颅骨、股骨干和胫骨干。
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300～800μM。
化学交联剂
本发明的组织工程支架制备过程中，使用化学交联剂将生物胶-框架结构复合体冻干后进行交联。
在本发明的一个优选例中，所述化学交联剂选自EDC、京尼平或戊二醛。
硬组织缺损。
本发明所述的组织工程支架可用于硬组织缺损修复。
所述硬组织缺损包括(但不限于)关节缺损、颌面部软骨及相关硬组织缺损、鼻中隔缺损、或其组合。
软骨凝胶及其制备
本发明的组织工程支架可以负载有含有软骨细胞的软骨凝胶。
如本文所用，“凝胶软骨”、“软骨凝胶”、“凝胶态软骨”、“凝胶状软骨”可互换使用，皆指呈凝胶状态的软骨(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。
所述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。本发明所述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。本发明所述的凝胶软骨，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，本发明所述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。
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。
本发明所述的凝胶软骨，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
本发明的组织工程支架可以负载有含有软骨细胞的软骨膜片颗粒。
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。
所述“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。
本发明所述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。
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。
本发明的组织工程支架在负载了所述的软骨凝胶或软骨膜片颗粒后，需要经过成软骨培养，形成可用于修复硬组织缺损的移植物。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。
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。
本发明的有益效果：
(1)本发明采用复合明胶(或胶原、丝素、水凝胶等)的方式，将脱钙骨基质或PCL框架结构的孔隙由明胶填充，构建新型组织工程支架，改变孔径后的新型脱钙骨支架有望为组织工程骨的构建找到一种新的方法。
(2)本发明的组织工程支架不仅具有大小适当的孔径，可以有效负载接种的细胞，而且具有良好的力学强度，可以满足各类部位的即时修复的力学强度要求。
(3)本发明的组织工程支架可根据所需负载的细胞种类，通过调节生物胶浓度比例或冻干参数精准控制孔径。
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
生物胶-脱钙骨基质复合体支架的制备
按照以下步骤制备生物胶-脱钙骨基质复合体支架：
1.将医用明胶颗粒置于离心管中，加入去离子水，放置于37℃摇床中，配成浓度为0.6％的明胶水溶液；
2.提供一脱钙骨基质(如图1所示)，将脱钙骨基质置于明胶水溶液中后，使用离心机500r/min，离心2分钟；
3.将离心后的装有脱钙骨基质与明胶溶液的离心管置于4℃冰箱8小时；
4.从4℃冰箱中将离心管取出，取出生物胶-脱钙骨基质复合体，放于-20冰箱冷冻8小时；
5.使用真空干燥机将冷冻好的生物胶-脱钙骨基质复合体冻干12小时；
6.将脱钙骨-明胶支架浸入化学交联剂(EDC、京尼平或戊二醛)中交联，交联完成后用去离子水反复冲洗、浸泡，去除残余的化学交联剂，再经真空冷冻干燥获得最终的生物胶-脱钙骨基质复合体支架(如图3所示)。
与单纯的脱钙骨基质(图1)相比，本实施例中制得的生物胶-脱钙骨基质复合体的孔径显著缩小(图3)，可以更有效地负载接种的细胞。
实施例2
生物胶-脱钙骨基质复合体支架的应用
首先将软骨细胞按7×10 7细胞/ml的浓度接种于实施例1中制备的生物胶-脱钙骨基质复合体支架；
然后将细胞-支架复合物于37℃、95％湿度、5％二氧化碳培养箱中孵育后加入成软骨培养基(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)；体外培养8周后，回植体内；体内再生4周后取材，成功再生的软骨样组织如图4所示。
结果显示，使用本发明实施例1中制备的生物胶-脱钙骨基质复合体支架有效地负载了接种于其中的软骨细胞，并在缺损处提供了良好的力学支撑，在体内可成功再生为软骨样组织。
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 (10)

  1. 一种组织工程支架，其特征在于，所述组织工程支架包括：
    (a)硬性大孔径框架结构；和
    (b)负载于或填充于硬性大孔径框架结构的可降解的生物胶。
  2. 如权利要求1所述的组织工程支架，其特征在于，所述可降解的的生物胶选自下组：复合明胶、胶原、丝素、水凝胶或其组合。
  3. 如权利要求1所述的组织工程支架，其特征在于，所述硬性大孔框架结构选自下组：脱钙骨基质、PCL框架或其组合。
  4. 如权利要求1所述的组织工程支架，其特征在于，所述硬性大孔框架结构的孔径为300-800μM，孔隙率为80％-90％。
  5. 如权利要求1所述的组织工程支架，其特征在于，所述组织工程支架的孔径大小可通过负载的生物胶浓度和冻干处理的时间调整。
  6. 如权利要求1所述的组织工程支架，其特征在于，所述的组织工程支架还可以负载有含有软骨细胞的软骨细胞悬液、软骨凝胶或软骨膜片颗粒。
  7. 如权利要求6所述的组织工程支架，其特征在于，所述软骨细胞悬液中软骨细胞的浓度(密度)为1.0×10 6/ml至1.0×10 8/m。
  8. 一种制备如权利要求1所述的组织工程支架的方法，其特征在于，所述方法包括以下步骤：
    (i)制备生物胶水溶液，并将所述生物胶水溶液置于离心管中；
    (ii)将硬性大孔径框架结构置于含有所述生物胶水溶液的离心管中并离心；
    (iii)将离心后的离心管冷藏后，取出内容物后将内容物冷冻，得到生物胶-框架结构复合体；
    (iv)将所述生物胶-框架结构复合体真空冻干，得到冻干的生物胶-框架结构复合体；
    (v)使用化学交联剂对所述冻干的生物胶-框架结构复合体进行交联，得到交联的生物胶-框架结构复合体；
    (vi)用去离子水冲洗所述交联的生物胶-框架结构复合体，并真空冷冻干燥，得到所述组织工程支架。
  9. 一种如权利要求1所述组织工程支架的用途，其特征在于，所述组织工程支架用于制备硬组织缺损修复的医用产品。
  10. 如权利要求9所述的用途，其特征在于，所述硬组织缺损包括关节缺损、颌面部软骨及相关硬组织缺损、鼻中隔缺损、或其组合。
PCT/CN2022/073191 2021-01-22 2022-01-21 一种基于大孔径的组织工程支架及其用途 WO2022156769A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115382021A (zh) * 2022-09-22 2022-11-25 诺一迈尔（苏州）医学科技有限公司 一种复合人工软骨支架及其制备方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030229400A1 (en) * 2002-05-13 2003-12-11 Koichi Masuda Tissue engineered osteochondral implant
CN103877615A (zh) * 2014-03-18 2014-06-25 北京大学第三医院 一种软骨组织工程支架及其制备方法
CN104606717A (zh) * 2014-12-09 2015-05-13 邹仕放 一种多孔β-TCP陶瓷/明胶复合材料制备方法
KR20160095677A (ko) * 2015-02-03 2016-08-12 아주대학교산학협력단 연골결손 치료용 조성물 및 인공연골 제조방법
CN106730026A (zh) * 2017-03-01 2017-05-31 北京大学第三医院 一种组织工程软骨复合支架及制备方法
CN108342356A (zh) * 2017-01-23 2018-07-31 曹谊林 一种软骨移植物及其构建方法
CN109432495A (zh) * 2018-12-27 2019-03-08 南京师范大学 一种矿化骨组织工程支架及其制备方法
US20190184061A1 (en) * 2016-08-02 2019-06-20 Atems Co,. Ltd. Composition for cartilage regeneration and preparing thereof
CN110090323A (zh) * 2019-05-08 2019-08-06 武汉理工大学 一种β-磷酸三钙多孔陶瓷载药支架及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101091084B1 (ko) * 2008-11-07 2011-12-09 한국과학기술연구원 연골 손상의 치료를 위한 세포군집체-하이드로겔-고분자 지지체 복합체, 이의 제조방법 및 이를 유효성분으로 함유하는 연골 손상 치료용 조성물

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030229400A1 (en) * 2002-05-13 2003-12-11 Koichi Masuda Tissue engineered osteochondral implant
CN103877615A (zh) * 2014-03-18 2014-06-25 北京大学第三医院 一种软骨组织工程支架及其制备方法
CN104606717A (zh) * 2014-12-09 2015-05-13 邹仕放 一种多孔β-TCP陶瓷/明胶复合材料制备方法
KR20160095677A (ko) * 2015-02-03 2016-08-12 아주대학교산학협력단 연골결손 치료용 조성물 및 인공연골 제조방법
US20190184061A1 (en) * 2016-08-02 2019-06-20 Atems Co,. Ltd. Composition for cartilage regeneration and preparing thereof
CN108342356A (zh) * 2017-01-23 2018-07-31 曹谊林 一种软骨移植物及其构建方法
CN106730026A (zh) * 2017-03-01 2017-05-31 北京大学第三医院 一种组织工程软骨复合支架及制备方法
CN109432495A (zh) * 2018-12-27 2019-03-08 南京师范大学 一种矿化骨组织工程支架及其制备方法
CN110090323A (zh) * 2019-05-08 2019-08-06 武汉理工大学 一种β-磷酸三钙多孔陶瓷载药支架及其制备方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115382021A (zh) * 2022-09-22 2022-11-25 诺一迈尔（苏州）医学科技有限公司 一种复合人工软骨支架及其制备方法
CN115382021B (zh) * 2022-09-22 2024-01-05 诺一迈尔（苏州）医学科技有限公司 一种复合人工软骨支架及其制备方法

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