WO2022156685A1 - 一种凝胶软骨接种于框架结构实现软骨再生的方法 - Google Patents

一种凝胶软骨接种于框架结构实现软骨再生的方法 Download PDF

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WO2022156685A1
WO2022156685A1 PCT/CN2022/072584 CN2022072584W WO2022156685A1 WO 2022156685 A1 WO2022156685 A1 WO 2022156685A1 CN 2022072584 W CN2022072584 W CN 2022072584W WO 2022156685 A1 WO2022156685 A1 WO 2022156685A1
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cartilage
gel
chondrocytes
tissue
cells
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PCT/CN2022/072584
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English (en)
French (fr)
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刘豫
周广东
慈政
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上海软馨生物科技有限公司
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Priority to US18/262,216 priority Critical patent/US20240148938A1/en
Publication of WO2022156685A1 publication Critical patent/WO2022156685A1/zh

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
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    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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Definitions

  • the invention relates to biomedical tissue engineering, in particular to a method for inoculating gel cartilage on a frame structure to realize cartilage regeneration.
  • cartilage defects caused by various types of trauma and congenital deformities have become more and more common.
  • Most cartilage defects caused by trauma or defects caused by congenital deformities are difficult to repair by the patient's own body.
  • the treatment methods for articular cartilage defects 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, remove cartilage fragments, etc., to restore the smoothness of the joint surface.
  • Such methods are less traumatic and can relieve the symptoms of patients to a certain extent, but their efficacy is limited and cannot effectively relieve the development of arthritis; restorative treatments include microfracture treatment, osteochondral transplantation, etc.
  • the cartilage defect has been repaired to a certain extent, but the larger trauma can easily lead to complications in the donor site.
  • Tissue engineering is an interdisciplinary subject involving cell biology, material science, engineering and bioreactors. Through the basic principles and methods of life science and engineering, the tissues needed by the human body are constructed to repair and replace tissues or organs that have no function due to trauma and disease. In recent years, with the progress of tissue engineering, people gradually began to study the scaffolds or tissues constructed by tissue engineering to try to repair cartilage defects. By combining with medically degradable materials, such as demineralized bone materials, the construction of cartilage tissue is expected to become a new method for tissue engineering cartilage construction. However, 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, which is not conducive to the construction of tissue engineering materials.
  • the purpose of the present invention is to provide a method for inoculating chondrocytes in a frame structure to realize cartilage regeneration.
  • a gel cartilage in a first aspect of the present invention, includes a cell population composed of chondrocytes and an extracellular matrix secreted by the chondrocytes, wherein the extracellular matrix wraps the cells population, and the gel cartilage is in a gel state, and the density of chondrocytes is at least 1.0 ⁇ 10 8 cells/ml or 1.0 ⁇ 10 8 cells/g.
  • the gel cartilage is prepared by gelatinizing and culturing chondrocytes.
  • the adhesion rate of the gel cartilage is ⁇ 90%, preferably ⁇ 95%.
  • the concentration of chondrocytes is 1.0 ⁇ 10 8 -10 ⁇ 10 8 cells/ml, preferably 1.5-5 ⁇ 10 8 cells/ml.
  • the gel cartilage is obtained by gelation culture for 2-5 days, preferably 2.5-4 days.
  • the chondrocytes are from mammals.
  • the chondrocytes are selected from elastic cartilage, fibrocartilage or hyaline cartilage.
  • the chondrocytes are selected from ear chondrocytes, costal cartilage or a combination thereof.
  • the ear chondrocytes are from autologous or allogeneic; preferably autologous human ear chondrocytes.
  • tissue engineering cartilage composite comprising:
  • the complex comprises a complex formed after the gel cartilage is seeded on the carrier and cultured for chondrogenesis (in the complex, chondrocytes are loaded on the carrier and combined with The carrier forms a tighter unitary structure).
  • the complex comprises a complex formed after the gel cartilage is seeded on the carrier without chondrogenic culture.
  • the ratio of the gel cartilage to the porous biocompatible material (or carrier) is 0.1-0.2 ml (or g) gel cartilage: 0.5-1 g porous biocompatible Material.
  • the porosity of the porous biocompatible material is ⁇ 30%, preferably ⁇ 50%, more preferably ⁇ 70%.
  • the porosity of the porous biocompatible material is 80%-95%.
  • the pore size of the porous biocompatible material is 400-800 ⁇ m.
  • the porous biocompatible material includes a biodegradable material.
  • the biodegradable material is selected from the group consisting of PCL, PGA, allogeneic bone repair material, xenogeneic bone repair material, or demineralized bone matrix.
  • the biodegradable material can also be loaded with gelatin, collagen, silk fibroin, hydrogel or a combination thereof.
  • the biodegradable material is demineralized bone matrix.
  • the demineralized bone matrix is derived from allogeneic bone or xenogeneic bone.
  • tissue engineered cartilage complex is prepared by the method described in the fourth aspect of the present invention.
  • step (2) Inducing and culturing the subcultured chondrocytes obtained in step (1) in a gelation medium, thereby obtaining induced gel cartilage.
  • step (2) the subcultured chondrocytes are seeded in the culture vessel in a stacked seeding manner.
  • the concentration (m/v) of the collagenase is 0.1%-0.2%.
  • the collagenase digestion time is 6-10 hours.
  • step (2) the chondrocytes are passaged to 2-5 passages.
  • the seeding density of the chondrocytes is 0.5 ⁇ 10 6 -3 ⁇ 10 7 cells/cm 2 ; preferably, 1 ⁇ 10 6 -5 ⁇ 10 6 cells cells/cm 2 ; more preferably, 1 ⁇ 10 6 -3 ⁇ 10 6 cells/cm 2 .
  • the culture medium contains or does not contain serum.
  • the culture medium contains 5-15% (v/v) serum.
  • the serum is selected from fetal bovine serum.
  • the gelation medium is DMEM medium.
  • the DMEM medium further contains 4-5 wt% glucose, 5-20% FBS (v/v), and 50-150 U/ml penicillin.
  • the time of the induction culture is 2-5 days; preferably, it is 2.5-4 days.
  • the fourth aspect of the present invention there is provided a method for preparing the tissue engineering cartilage composite according to the third aspect of the present invention, and the gel cartilage according to the first aspect of the present invention is inoculated into a porous organism Compatible materials, in vitro cartilage culture, so as to obtain the tissue engineering cartilage complex.
  • the time for the in vitro chondrogenic culture is 2-15 days; preferably, 3-11 days; more preferably, 4-7 days.
  • tissue cartilage composite according to the second aspect of the present invention for preparing a medical product for repairing cartilage and/or hard tissue defects.
  • the cartilage and/or hard tissue defect is selected from the group consisting of articular cartilage defect, cleft lip and palate deformity, maxillofacial hard tissue defect or a combination thereof.
  • the tissue engineered cartilage composite comprises a tissue engineered cartilage graft.
  • the shape of the tissue engineered cartilage graft conforms to the shape of the defect site where cartilage needs to be transplanted in the human body.
  • the defect site is selected from articular cartilage defect, cleft lip and palate deformity, maxillofacial hard tissue or a combination thereof.
  • the shape of the tissue engineered cartilage graft is but not limited to various shapes such as human auricle, dorsum of the nose, alar of the nose, zygomatic arch, eyebrow arch, tubular, diamond, sheet, and cylinder.
  • the tissue cartilage composite according to the second aspect of the present invention is administered to a subject in need thereof thing.
  • the cartilage and/or hard tissue defect is selected from articular cartilage defect, cleft lip and palate deformity, or maxillofacial hard tissue defect.
  • Figure 1 shows the demineralized bone matrix framework.
  • Figure 2 shows the electron micrograph of the demineralized bone matrix framework, the scale bar is 1 mm.
  • Figure 3 shows a schematic diagram of chondrogel culture.
  • A is the cartilage gel cultured in the six-well plate for 3 days
  • B is the cartilage gel at the bottom of the six-well plate after aspirating the medium in the upper part of the six-well plate
  • C is the cartilage gel added with appropriate culture Injectable preparation containing cartilage gel made after the base
  • D is the perichondrium sheet cultured in six-well plate for 15 days
  • E is the schematic diagram of the perichondrium sheet with certain mechanical strength
  • F is the perichondral sheet prepared by cutting into pieces Injectable preparation containing perichondral sheet.
  • Figure 4 shows an injectable formulation containing chondrogel (culture day 3).
  • Figure 5 shows the integrated tissue-engineered cartilage prepared after the demineralized bone matrix framework seeded with the gel cartilage preparation was added to the chondrogenic medium for continued culture.
  • Figure 6 shows the electron micrographs of the as-prepared integrated tissue-engineered cartilage, and the scale bar is 200 ⁇ m.
  • Figure 7 shows a graph comparing adhesion rates of inoculated samples (cell suspension or cartilage gel) after culturing the complexes for 24 hours.
  • Figure 8 shows the comparison of the gel cartilage-demineralized bone complex (A) and the pure demineralized bone (B) grafted onto the defect site of the goat knee joint.
  • the gelatinous cartilage of the present invention has the advantages of strong adhesion, easy inoculation on a solid-phase carrier to form a complex, a high degree of complexation with the carrier in the later chondrogenic culture, immediate repair ability of the cartilage graft, excellent cartilage repair effect and the like. .
  • the present invention has been completed on this basis.
  • the inventors have also developed a composite or graft based on the gel cartilage of the present invention, that is, the gel cartilage of the present invention is seeded on a porous carrier material (also referred to as a "frame structure") to form a cartilage regeneration system. Complex.
  • a porous carrier material also referred to as a "frame structure”
  • the particle size of the gel cartilage in the present invention is significantly increased, which is beneficial to the attachment on the demineralized bone matrix material and avoids cartilage.
  • the pore size of the formed tissue-engineered bone graft was significantly reduced.
  • the tissue engineering bone graft of the present invention has an immediate repair effect on joint defects, and the microenvironment of the gel cartilage is conducive to the formation of mature cartilage tissue at the joint defect. Therefore, it is beneficial to realize cartilage regeneration and be used for various filling treatments and bone repair.
  • the present invention has been completed on this basis.
  • tissue engineering composite of the present invention As used herein, “tissue engineering composite of the present invention”, “gel cartilage-tissue engineering composite” and “tissue cartilage graft of the present invention” can be used interchangeably, all refer to the tissue described in the second aspect of the present invention Engineered cartilage complex.
  • gelation culture refers to the process of providing a special biochemical environment, enabling cells with chondrogenic differentiation potential to express and form gelatinous chondrocytes with a viscous texture and a significantly increased particle size, with chondrogenic ability.
  • cartilage (stem) cells refers to chondrocytes, cartilage stem cells, or a combination thereof.
  • 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 cells on the other hand) /ml), therefore, it is particularly suitable for preparing grafts for repairing various types of cartilage, or for cartilage transplantation or cartilage repair surgery.
  • 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 biocompatible material), and also includes Gel cartilage is loaded on a carrier material (especially a porous biocompatible material) and is a complex formed by chondrogenic culture.
  • the composite suitable for transplantation into human or animal body is the tissue engineered cartilage graft of the present invention.
  • the gel cartilage is formed by culturing in vitro for a period of time t1 under the gelation culture condition.
  • 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. ).
  • chondrocytes in a specific embodiment, are seeded on a culture medium with a gentle surface, and is induced in vitro for 2-5 days by the gelation medium of the present invention, forming a kind of free and close contact.
  • the state of the cell that is, the state of "quasi-connected" cells.
  • the chondrocytes of the present invention are cultured in vitro, and several chondrocytes are wrapped by extracellular matrix to form a structure in which cells are in relatively close contact.
  • the high concentration of chondrocytes is seeded with 1.0 ⁇ 10 7 -2.0 ⁇ 10 7 cells in a 3.5cm culture dish (eg, one well in a six-well plate), preferably , for 1.5 x 10 7 cells.
  • the cell density is 1.0 ⁇ 10 8 -10 ⁇ 10 8 cells/ml, preferably 1.5-5 ⁇ 10 8 cells/ml.
  • the gel cartilage of the present invention has a certain adhesion rate.
  • the adhesion rate of the gel cartilage of the present invention is ⁇ 90%, preferably, ⁇ 95%.
  • the gel cartilage of the present invention is differentiated from high-density cartilage gel cells.
  • the high-density chondrocytes of the present invention grow into gel cartilage through in vitro culture.
  • the particle size of the gel cartilage is significantly increased, which is more conducive to the attachment on the demineralized bone matrix.
  • the chondrocyte concentration range is preferably 1.6 ⁇ 10 6 -2.2 ⁇ 10 6 cells/cm 2 .
  • the gelation culture conditions are as follows: inoculation of high-density chondrocytes, cultured in a high-glucose medium containing 10% fetal bovine serum and 1% tertiary antibody.
  • the gel cartilage culture time is 2-5 days, and the seeding density is 1.6 ⁇ 10 6 -2.2 ⁇ 10 6 cells/cm 2 .
  • the present invention provides a specific gel material, ie, gelatinous chondrocytes, which are cultured for 3 days.
  • 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%.
  • the gel cartilage in the present invention can also be loaded on other common biological porous materials after seeding the demineralized bone, preferably, in a degradable material on, including but not limited to:
  • Degradable synthetic polymer materials such as polylactic acid (PLA), polyglycolic acid (PGA), PLGA, polyhydroxybutyric acid (PHB), polyanhydrides, polyphosphazenes, poly Amino acid (polyamino acid), pseudo-polyamino acid (pesudo-polyamino acid), polyorthoesters (polyorthoesters), polyethylene glycol, hyaluronic acid, polydioxanone, etc.;
  • PLA polylactic acid
  • PGA polyglycolic acid
  • PHB polyhydroxybutyric acid
  • polyanhydrides polyphosphazenes
  • poly Amino acid polyamino acid
  • pseudo-polyamino acid pesudo-polyamino acid
  • polyorthoesters polyorthoesters
  • polyethylene glycol hyaluronic acid
  • polydioxanone etc.
  • Natural degradable materials such as collagen, gelatin, glycosaminoglycans (GAGs), chitosan, chitin, alginate, and various Cell matrix, such as demineralized bone matrix, etc.;
  • Preferred medically acceptable biodegradable materials are solid materials or solid, liquid composite materials such as polylactic acid (PLA), polyglycolic acid (PGA), collagen, demineralized bone matrix, and the like.
  • the materials of the present invention can be prefabricated into various precise sizes and shapes to accommodate cartilage tissue constructions of different sizes and shapes.
  • the material can be directly prefabricated into the required size and shape, or the material can be accurately plasticized through a model operated by computer-aided and rapid prototyping technology.
  • the preparation method of the tissue engineered cartilage graft of the present invention is simple and convenient, and the method comprises the steps:
  • the concrete steps of the adhesion rate measuring method in the present invention are as follows:
  • the assay method for DNA quantification includes the following steps:
  • gel cartilage tissue is more mature and has a certain fluidity.
  • the demineralized bone matrix material has larger pore size and better porosity.
  • the gel cartilage tissue with certain fluidity and viscosity can effectively improve the adhesion rate.
  • 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.
  • a 2.5 ⁇ 2.5cm 2 ear cartilage tissue was aseptically cut, and the mucosa and fibrous tissue on the cartilage surface were peeled off with sterile instruments. Cut the cartilage tissue into 1.5 ⁇ 1.5mm2 cartilage fragments.
  • the prepared collagenase was prepared at a concentration of 0.15%, and the cartilage fragments were added to the prepared collagenase to digest for 8 hours. After 8 hours, the collagenase solution was filtered and centrifuged to obtain ear chondrocytes, which were primary and subcultured (high glucose DMEM medium), and the cells were passaged to passage 2-5, preferably passage 3.
  • the cells were collected and resuspended, and the cells were seeded in a six-well plate (3.5cm diameter) at a cell volume of 8 ⁇ 10 6 /10ml to 30 ⁇ 10 6 /10ml per well. % glucose, 10% FBS (v/v) and 100 U/ml penicillin in DMEM medium). After culturing for 3 days, the medium in the upper part of the six-well plate was aspirated, and the cartilage gel at the bottom of the six-well plate was aggregated with tweezers (see Figure 3B). in the syringe.
  • the cartilage gel obtained after 3 days is about 0.1ml.
  • the cell density is 1.0 ⁇ 10 8 -10 ⁇ 10 8 cells/ml, preferably 1.5-5 ⁇ 10 8 /ml.
  • a decalcified bone matrix frame (as shown in Figure 1) is provided, and it is determined that the decalcified bone matrix frame has a pore size of about 400-800 ⁇ m and a porosity of about 87.3% ⁇ 3.7% (the electron microscope observation results of the decalcified bone matrix frame are as follows: shown in Figure 2).
  • the gel cartilage preparation (prepared in Example 1, with a volume of about 0.25-0.35ml) was inoculated into the above-mentioned demineralized bone matrix frame, and allowed to stand for 2 hours at 37° C., 95% humidity, and 5% carbon dioxide; after standing, the cartilage was added. The medium was further cultured for 3-11 days, and the integrated tissue engineered cartilage was obtained (Fig. 5).
  • FIG. 5 The general view of the prepared integrated tissue-engineered cartilage is shown in FIG. 5 .
  • a demineralized bone matrix framework is provided (as shown in Figure 1).
  • the gel cartilage preparation (prepared in Example 1, with a volume of about 0.25-0.35 ml) was seeded in the above-mentioned demineralized bone matrix framework, and the cells were left to stand at 37° C., 95% humidity, and 5% carbon dioxide for 2 hours for 4 passages.
  • the adhesion rate was measured by the method of the above-mentioned adhesion rate measurement. Compared with the cell suspension, the gel cartilage adhesion rate of the present invention was 92% ⁇ 2%.
  • Example 4 Animal transplantation experiment for repairing articular cartilage
  • Gel cartilage was used to inoculate demineralized bone to construct gel cartilage-demineralized bone complex, and cultured in vitro for 11 days.
  • a cartilage defect with a diameter of 7.5 mm was created on the articular surface of the knee joint of experimental animals, and a gel cartilage-demineralized bone complex was used to fill the defect.
  • the defect was filled by gel cartilage-decalcified bone complex at A, and the defect was filled by pure demineralized bone at B.
  • the defect at A is smooth and solid, surrounded by a soft tissue membrane, which has a certain elasticity and has an immediate functional repair effect.
  • the wound surface of the defect site at B is rough and only has the function of physical support, which cannot achieve immediate repair effect.
  • the experimental results show that the articular cartilage repaired by the gel cartilage-demineralized bone complex, the tissue constructed is similar to the normal cartilage tissue, which can immediately repair the joint defect and is conducive to the formation of mature cartilage tissue in the joint defect.

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Abstract

提供了一种凝胶软骨接种于框架结构实现软骨再生的方法。具体地，软骨细胞通过扩增、密集接种制备成凝胶软骨；接种于脱钙骨基质内，制成凝胶软骨-脱钙骨复合物，实现软骨再生，用于各类充填治疗及骨修复。

Description

一种凝胶软骨接种于框架结构实现软骨再生的方法 技术领域
本发明涉及一种生物医学组织工程，尤其涉及一种凝胶软骨接种于框架结构实现软骨再生的方法。
背景技术
近些年来，随着经济社会的快速发展，各类外伤及先天畸形导致的软骨缺损日趋多见。大多数外伤导致的软骨缺损或先天畸形导致的缺损难以依靠患者自体修复。目前临床治疗中，关节软骨缺损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，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。
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。通过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。近年来，随着组织工程学的进步，人们逐渐开始研究通过组织工程所构建的支架或组织来尝试软骨缺损的修复。通过与医学可降解材料，如脱钙骨材料相结合，构建软骨组织有望成为组织工程软骨构建的新方法。然而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粘附率极低，不利于组织工程材料的构建。
综上所述，本领域迫切需要开发一种软骨细胞接种于框架结构形成组织工程软骨复合物以实现软骨再生的方法。
发明内容
本发明的目的就是提供一种软骨细胞接种于框架结构实现软骨再生的方法。
在本发明的第一方面，提供了一种凝胶软骨，所述的凝胶软骨包括软骨细 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，并且所述凝胶软骨呈凝胶态，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，所述的凝胶软骨是用软骨细胞经凝胶化培养而制备的。
在另一优选例中，所述凝胶软骨的粘附率≥90％，较佳地≥95％。
在另一优选例中，所述凝胶软骨中，软骨细胞的浓度为1.0×10 8-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
在另一优选例中，所述凝胶软骨是凝胶化培养2-5天得到的，较佳地，为2.5-4天。
在另一优选例中，所述软骨细胞来自哺乳动物。
在另一优选例中，所述软骨细胞选自弹性软骨、纤维软骨或透明软骨。
在另一优选例中，所述软骨细胞选自耳软骨细胞、肋软骨或其组合。
在另一优选例中，所述耳软骨细胞来自自体或同种异体；优选自体人耳软骨细胞。
在本发明的第二方面，提供了一种组织工程软骨复合物，所述复合物包括：
(a)载体，所述载体包括多孔生物相容性材料；和
(b)接种于或负载于所述载体的本发明第一方面所述的凝胶软骨；
在另一优选例中，所述的复合物包括将所述凝胶软骨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凝胶软骨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，所述复合物中，凝胶软骨与多孔生物相容性材料(或载体)之比为0.1-0.2ml(或g)凝胶软骨：0.5-1g多孔生物相容性材料。
在另一优选例中，所述多孔生物相容性材料的孔隙率≥30％，较佳地，≥50％，更佳地，≥70％。
在另一优选例中，所述多孔生物相容性材料的孔隙率为80％-95％。
在另一优选例中，所述多孔生物相容性材料的孔径为400-800μm。
在另一优选例中，所述多孔生物相容性材料包括生物可降解材料。
在另一优选例中，所述生物可降解材料选自下组：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，所述生物可降解材料还可以负载有明胶、胶原、丝素、水凝胶或其组合。
在另一优选例中，所述生物可降解材料为脱钙骨基质。
在另一优选例中，所述脱钙骨基质来自同种异体骨或异种骨。
在另一优选例中，所述的组织工程软骨复合物是用本发明第四方面所述的方法制备的。
在本发明的第三方面，提供了一种制备如本发明的第一方面所述的凝胶软骨的方法，包括如下步骤：
(1)提供一分离的软骨细胞,进行原代及传代培养，从而获得经传代培养的软骨细胞；
(2)对步骤(1)获得的经传代培养的软骨细胞，在凝胶化培养基中诱导培养，从而获得诱导的凝胶软骨。
在另一优选例中，步骤(2)中，经传代培养的软骨细胞以层叠接种的方式接种于培养容器中。
在另一优选例中，步骤(2)中，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。
在另一优选例中，步骤(1)中，所述胶原酶的浓度(m/v)为0.1％-0.2％。
在另一优选例中，步骤(1)中，所述胶原酶消化的时间为6-10小时。
在另一优选例中，步骤(2)中，所述软骨细胞传代至2-5代。
在另一优选例中，步骤(2)中，所述软骨细胞的接种密度为0.5×10 6-3×10 7个细胞/cm 2；较佳地，1×10 6-5×10 6个细胞/cm 2；更佳地，1×10 6-3×10 6个细胞/cm 2。
在另一优选例中，步骤(2)中，所述培养基中含有或不含有血清。
在另一优选例中，步骤(2)中，所述培养基中含有5-15％(v/v)的血清。
在另一优选例中，所述血清选自胎牛血清。
在另一优选例中，步骤(2)中，所述凝胶化培养基为DMEM培养基。
在另一优选例中，步骤(2)中，所述DMEM培养基还含有4-5wt％葡萄糖，5-20％FBS(v/v)，50-150U/ml的青链霉素。
在另一优选例中，步骤(2)中，所述诱导培养的时间为2-5天；较佳地，为2.5-4天。
在本发明的第四方面，提供了一种制备如本发明的第三方面所述的组织工程软骨复合物的方法，将如本发明的第一方面所述的的凝胶软骨接种于多孔生物相容性材料，体外成软骨培养，从而获得所述的组织工程软骨复合物。
在另一优选例中，所述体外成软骨培养的时间为2-15天；较佳地，3-11天；更佳地，4-7天。
在本发明的第五方面，提供了一种如本发明的第二方面所述的组织软骨复合物的用途，用于制备用于修复软骨和/或硬组织缺损的医用产品。
在另一优选例中，所述软骨和/或硬组织缺损选自：关节软骨缺损、唇腭裂畸形、颌面部硬组织缺损或其组合。
在另一优选例中，所述组织工程软骨复合物包含组织工程软骨移植物。
在另一优选例中，所述组织工程软骨移植物的形状与人体需要移植软骨的缺损部位形状相符。
在另一优选例中，所述缺损部位选自关节软骨缺损、唇腭裂畸形、颌面部硬组织或其组合。
在另一优选例中，所述组织工程软骨移植物的形状为人耳廓、鼻背、鼻翼、颧弓、眉弓、管状、菱形、片状、圆柱等多种形状但不仅限于此。
在本发明的第六方面，提供了一种修复软骨和/或硬组织缺损相关疾病的方法，其特征在于，向有需要的受试者施用如本发明的第二方面所述的组织软骨复合物。
在另一优选例中，所述软骨和/或硬组织缺损选自关节软骨缺损、唇腭裂畸形、或颌面部硬组织缺损。
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显示了脱钙骨基质框架。
图2显示了脱钙骨基质框架的电镜图，比例尺为1mm。
图3显示了软骨凝胶培养的示意图。其中，A为六孔板中培养3天的软骨凝胶，B为吸去六孔板中上部的培养基后，聚集六孔板底部的软骨凝胶物，C为软骨凝胶中加入适当培养基后制成的含软骨凝胶的可注射制剂；D为六孔板中培养15天的软骨膜片，E为有一定机械强度的软骨膜片示意图；F为软骨膜片剪碎后制备的含软骨膜片的可注射制剂。
图4显示了含软骨凝胶(培养第3天)的可注射制剂。
图5显示了接种有凝胶软骨制剂的脱钙骨基质框架，在加入成软骨培养基继续培养后所制备的一体化的组织工程化软骨。
图6显示了所制备的一体化的组织工程化软骨的电镜图，比例尺为200μm。
图7显示了接种样品(细胞悬液或软骨凝胶)于复合物上培养24小时后的粘附率比较图。
图8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
本发明人通过广泛而深入的研究，首次意外地发现，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，可形成一种新颖的凝胶状软骨。本发明的凝胶状软骨具有粘附力强、易于接种于固相载体形成复合物、在后期的成软骨培养中与载体复合程度高、软骨移植物具有即时修复能力、软骨修复效果优异等优点。在此基础上完成了本发明。
此外，本发明人还开发了基于本发明凝胶软骨的复合物或移植物，即将本发明凝胶软骨接种于多孔的载体材料(也可称为“框架结构”)，形成用于软骨 再生的复合物。
通过优化体外培养的培养基、凝胶化培养的时间，与普通软骨细胞相比，本发明中的凝胶软骨的粒径大小显著提高，有利于在脱钙骨基质材料上的附着，避免软骨细胞的流失，形成的组织工程骨移植物的孔径率显著缩小。本发明的组织工程骨移植物对于关节缺损起到即时修复效果，凝胶软骨的微环境有利于关节缺损处形成成熟的软骨组织。因此有利于实现软骨再生，用于各类充填治疗及骨修复。在此基础上完成了本发明。
术语
如本文所用，“本发明的组织工程复合物”、“凝胶软骨-组织工程复合体”、“本发明的组织软骨移植物”可互换使用，皆指本发明第二方面所述的组织工程软骨复合物。
如本文所用，术语“凝胶化培养”指提供特殊的生化环境，使具有软骨分化潜能的细胞表达形成质地粘稠、粒径显著增加的凝胶状软骨细胞，具备成软骨能力的过程。
如本文所用，术语“软骨(干)细胞”指软骨细胞、软骨干细胞或其组合。
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 8-10×10 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(尤其是多孔生物相容性材料)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(尤其是多孔生物相容性材料)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组织工程软骨移植物。
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t 1，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。
在一具体实施例中，高浓度的软骨细胞接种在表面平缓的培养介质上，通过本发明中的凝胶化培养基，体外诱导2-5天，形成了一种介于游离和紧密联系之间的状态，即“准联系”的细胞状态。具体地，本发明的软骨细胞通过体外培养，通过细胞外基质包裹若干个软骨细胞，形成了一种细胞相对紧密接触的结构。
在一具体实施例中，所述高浓度的软骨细胞为在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。
所述凝胶软骨中，细胞密度为1.0×10 8-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
具体地，本发明中的凝胶软骨具有一定的粘附率，通过本发明一优选例中 的粘附率测定方法，本发明的凝胶软骨的粘附率≥90％，较佳的，≥95％。
本发明的凝胶软骨由高密度软骨凝胶细胞分化而来。本发明的高密度软骨细胞通过体外培养，生长成凝胶软骨。凝胶软骨的粒径大小显著提高，更有利于在脱钙骨基质上的附着。所述的软骨细胞浓度范围优选为1.6×10 6-2.2×10 6个细胞/cm 2。
在另一优选例中，所述的凝胶化培养条件为：接种高密度的软骨细胞，使用含10％胎牛血清及1％三抗的高糖培养基中培养。
在另一优选例中，所述的凝胶软骨培养时间为2-5天，接种密度为1.6×10 6-2.2×10 6个细胞/cm 2。
本发明提供了一种通过培养3天的特定的凝胶材料即凝胶状的软骨细胞。
脱钙骨基质
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粘附率极低，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。
组织工程化软骨移植物
单纯凝胶软骨无法成型，张力条件下单纯凝胶软骨吸收率较高，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凝胶软骨-脱钙骨基质复合体，且提供力学支撑后有限降低软骨吸收率。
应理解，除了具体实施例中使用的脱钙骨基质，本发明中的凝胶软骨接种在脱钙骨后，还可负载在其他常见的生物的多孔材料上，较佳地，在可降解材料上，包括但不限于：
(a)可降解性合成高分子材料，例如聚乳酸(PLA)、聚羟基乙酸(PGA)、PLGA、 聚羟基丁酸(PHB)、聚酸酐(polyanhydrides)、聚偶磷氮(polyphosphazenes)、聚氨基酸(polyamino acid)、假聚氨基酸(pesudo-polyamino acid)、聚原酸醋(polyorthoesters)、聚乙二醇、透明质酸、聚对二氧六环酮(polydioxanone)等；
(b)天然可降解材料，例如胶原(collagen)、明胶(gelatin)、糖氨聚糖(glycosaminoglycan,GAGs)、壳聚糖(chitosan)、甲壳素(chitin)、海藻酸盐，以及各种脱细胞基质，如脱钙骨基质等；
(c)上述材料的共聚物或复合型材料，尤其是高分子材料与天然材料的复合材料，以及固体材料与可注射性材料的复合材料。
优选的医学上可接受的生物可降解材料是固体材料或固、液体复合材料，例如聚乳酸(PLA)、聚起基乙酸(PGA)、胶原、脱钙骨基质等。本发明中的材料可预制成各种精确的大小与形状，以适应不同大小和形状的软骨组织构建。当材料为固体型材料时，可以直接将材料预制成需要的大小与形状，也可以通过计算机辅助及快速成型技术运制的模型对材料进行精确的塑性。
制备方法
本发明的组织工程化软骨移植物的制备方法简便，所述方法包括步骤：
(1)软骨细胞的培养；
(2)诱导培养凝胶软骨；
(3)接种组织工程载体。
粘附率测定方法
本发明中的粘附率测定方法的具体步骤如下：
检测所接种样品(细胞悬液或软骨凝胶)的DNA定量A1；检测接种后复合物(细胞-支架复合物或软骨凝胶-支架复合物)培养24小时后的DNA定量A2；粘附率为A2/A1×100％。
DNA定量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)凝胶软骨组织较软骨细胞而言更加成熟，且具有一定的流动性。
(2)脱钙骨基质作为可在体内降解的天然材料，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，具有一定流动性且较为粘稠的凝胶软骨组织可有效提高粘附率。
(4)单纯凝胶软骨无法成型，张力条件下单纯凝胶软骨吸收率较高，临床应用受限，使用脱钙骨基质构建凝胶软骨-脱钙骨基质复合体，可提供力学支撑，降低软骨吸收率。
(5)根据需求将脱钙骨定制为不同形状，构建凝胶软骨-脱钙骨复合体可用于不同部位、不同形状的修复重建。
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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)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，否则百分比和份数是重量百分比和重量份数。
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-β 1，100ng/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。
实施例1：凝胶软骨的体外培养
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。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。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(高糖DMEM培养基)，细胞传代至 第2-5代，优选第3代。
扩增后收集细胞重悬，按每孔8×10 6/10ml至30×10 6/10ml的细胞量接种于六孔板(3.5cm直径)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3天后，将六孔板中上部的培养基吸去，使用镊子将六孔板底部的软骨凝胶聚集(见图3B)，一个孔的凝胶软骨产量为0.1-0.2ml，收集至5ml注射器中。
3天后得到的软骨凝胶为0.1ml左右，按细胞扩增2倍计算，该凝胶软骨中，细胞密度为1.0×10 8-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
将其与0.15ml培养基混合制成含软骨凝胶的可注射制剂(见图3C、图4)。
如图3所示，第3天，软骨凝胶的粘稠度显著提升，便于与孔径大的负载材料结合。
特别地，当第3天的软骨凝胶继续培养到第15天时，得到了一种片状的软骨膜片，厚度薄并且机械强度佳。
实施例2：凝胶软骨组织的体外构建
提供一脱钙骨基质框架(如图1所示)，经测定，该脱钙骨基质框架具有约400-800μm孔径，约87.3％±3.7％孔隙率(脱钙骨基质框架的电镜观察结果如图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，于37℃、95％湿度、5％二氧化碳静置2小时；静置后加入成软骨培养基继续培养3-11天，得到一体化的组织工程化软骨(图5)。
结果
所制得的一体化的组织工程化软骨的大体如图5所示。
电镜观察结果如图6所示，表明接种凝胶软骨后脱钙骨基质的孔被基本有效填充。
实施例3：粘附率测定
提供一脱钙骨基质框架(如图1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，细胞于37℃、95％湿度、5％二氧化碳静置2小时传代4次。
如图7所示，通过上述粘附率测定的方法测定粘附率。与细胞悬液相比， 本发明的凝胶软骨粘附率为92％±2％。
实施例4：修复关节软骨的动物移植实验
使用凝胶软骨接种于脱钙骨中，构建凝胶软骨-脱钙骨复合体，并体外继续培养11天。
在实验动物膝关节的关节面制造直径7.5mm的软骨缺损，使用凝胶软骨-脱钙骨复合体填补缺损。
如图8所示，其中A处通过凝胶软骨-脱钙骨复合体填补缺损，B处通过单纯脱钙骨填补缺损。
观察创面：
A处缺损部位光滑，质实，周围包绕软组织膜，有一定弹性，具有即时功能性修复效果。
B处缺损部位创面粗糙，仅有物理支撑作用，无法起到即时修复效果。
实验结果说明，通过凝胶软骨-脱钙骨复合体修复关节软骨，构建的组织与正常软骨组织形态相近，可对于关节缺损起到即时修复效果，有利于关节缺损处形成成熟的软骨组织。
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 (17)

  1. 一种凝胶软骨，其特征在于，所述的凝胶软骨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，并且所述凝胶软骨呈凝胶态，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。
  2. 如权利要求1所述的凝胶软骨，其特征在于，所述的凝胶软骨是用软骨细胞经凝胶化培养而制备的。
  3. 如权利要求1所述的凝胶软骨，其特征在于，所述凝胶软骨的粘附率≥90％。
  4. 如权利要求1所述的凝胶软骨，其特征在于，所述凝胶软骨中，软骨细胞的浓度为1.0×10 8-10×10 8个/ml，较佳地1.5-5×10 8个/ml。
  5. 如权利要求1所述的凝胶软骨，其特征在于，所述凝胶软骨是凝胶化培养2-5天得到的，较佳地，为2.5-4天。
  6. 如权利要求1所述的凝胶软骨，其特征在于，所述软骨细胞选自弹性软骨、纤维软骨或透明软骨。
  7. 一种组织工程软骨复合物，其特征在于，所述复合物包括：
    (a)载体，所述载体包括多孔生物相容性材料；和
    (b)接种于或负载于所述载体的权利要求1所述的凝胶软骨。
  8. 如权利要求7所述的组织工程软骨复合物，其特征在于，所述多孔生物相容性材料的孔隙率≥30％，较佳地，≥50％，更佳地，≥70％。
  9. 如权利要求7所述的组织工程软骨复合物，其特征在于，所述多孔生物相容性材料的孔径为400-800μm。
  10. 如权利要求7所述的组织工程软骨复合物，其特征在于，所述多孔生物相容性材料包括生物可降解材料。
  11. 如权利要求10所述的组织工程软骨复合物，其特征在于，所述生物可降解材料选自下组：PCL、PGA、同种异体骨修复材料、异种骨修复材料、或脱钙骨基质。
  12. 一种制备如权利要求1所述的凝胶软骨的方法，其特征在于，包括如 下步骤：
    (1)提供一分离的软骨细胞,进行原代及传代培养，从而获得经传代培养的软骨细胞；
    (2)对步骤(1)获得的经传代培养的软骨细胞，在凝胶化培养基中诱导培养，从而获得诱导的凝胶软骨。
  13. 如权利要求12所述的方法，其特征在于，步骤(2)中，经传代培养的软骨细胞以层叠接种的方式接种于培养容器中。
  14. 一种制备如权利要求7所述的组织工程软骨复合物的方法，其特征在于，将如权利要求1所述的凝胶软骨接种于多孔生物相容性材料，体外成软骨培养，从而获得所述的组织工程软骨复合物。
  15. 一种如权利要求7所述的组织软骨复合物的用途，其特征在于，用于制备用于修复软骨和/或硬组织缺损的医用产品。
  16. 一种修复软骨和/或硬组织缺损相关疾病的方法，其特征在于，向有需要的受试者施用如权利要求7所述的组织软骨复合物。
  17. 如权利要求16所述的方法，其特征在于，所述软骨和/或硬组织缺损选自关节软骨缺损、唇腭裂畸形、或颌面部硬组织缺损。
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