CN102989040A - Human residual ear cartilage stem cells, and method for constructing tissue engineering cartilages - Google Patents

Human residual ear cartilage stem cells, and method for constructing tissue engineering cartilages Download PDF

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CN102989040A
CN102989040A CN2011102688309A CN201110268830A CN102989040A CN 102989040 A CN102989040 A CN 102989040A CN 2011102688309 A CN2011102688309 A CN 2011102688309A CN 201110268830 A CN201110268830 A CN 201110268830A CN 102989040 A CN102989040 A CN 102989040A
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residual
credulous
cartilage
cell
osteocyte
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CN102989040B (en
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周广东
刘豫
曹谊林
刘伟
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Shanghai Guorui Life Sci & Tech Co Ltd
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Shanghai Guorui Life Sci & Tech Co Ltd
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Abstract

The invention discloses a tissue engineering cartilage graft. The tissue engineering cartilage graft contains human residual ear cartilage cells growing under a three-dimensional structure, and the human residual ear cartilage cells are passaged at the first, second, third, fourth, fifth, sixth, seventh or eighth passage. The invention also discloses an application of the human residual ear cartilage cells in the in-vitro construction of the cartilage graft.

Description

The method of the residual credulous key cell of people and structure tissue engineering bone/cartilage thereof
Technical field
The present invention relates to medical science and biomedical engineering field, relate more specifically to organization engineered cartilage that utilizes the residual credulous key cell construction of people and its production and use.
Background technology
Microtia (microtia) is the congenital diseases of a kind of sickness rate higher (about 1/3000), shows as auricle sign form disappearance, and ear field is replaced by the cartilage agglomerate (residual credulous bone) of contracture, deformity.Mainly be to get the patient to implant ear field subcutaneous after the body costicartilage is engraved as the auricle form be main to the treatment of microtia clinically at present.The major defect of the method is that normal costicartilage is caused very large wound, and cause easily that pneumothorax, thorax subside, deformity and cicatrix etc. are for district's complication.
The rise of tissue engineering technique and develop rapidly as solving this difficult problem and brought hope.The chondrocyte composite ear profile attitude biodegradable stent of using at present animal can go out accurate people's auricle form cartilage in external structure.Therefore, as long as there are enough patients to make up seed cell from the cartilage in body source, this technology is hopeful to be converted into actual clinical practice fully, rebuilds for microtia patient's external ear to bring glad tidings.
Yet it is that this technology of restriction is to the bottleneck of clinical conversion that seed cell comes source problem always.The seed cell that is used at present the structure cartilage mainly contains two kinds: 1) chondrocyte; 2) has the stem cell of cartilage differentiation potential.Because chondrocyte itself has had very strong spontaneous one-tenth cartilage ability, therefore directly make up the most stable success rate that also can guarantee of tissue engineering bone/cartilage with it; But the source of the chondrocyte in the normal human is very limited, the wound of drawing materials is large, and normal cartilage cell expansion ex vivo ability is very low, very easily wear out again after the amplification, dedifferente, thereby the forfeiture cartilage forms ability, therefore, get patient and carry out external ear cartilage regeneration shortage clinical practice feasibility from body normal cartilage cell.Has into the stem cell (such as fat stem cell or bone marrow stroma stem cell) of cartilage differentiation potential although wide material sources, the amplification ability is strong, but it is not high that these cells in vitro make up the success rate with specific modality (such as people's auricle) cartilage, and the cartilage of external structure is implanted behind the subcutaneous environment very unstable, ossified or fibrosis very easily occur, and also are unsuitable for making up auricle isotonic cartilage.
Microtia patient's residual ear belongs to discarded tissue in the external ear reconstructive operation, and cut residual ear and can not cause any damage by normal tissue, and existing studies confirm that: the residual credulous osteocyte that goes down to posterity in early days can regenerating tissues engineering cartilage.So, does does can utilize the microtia patient to make up auricle form cartilage from the residual credulous osteocyte of body carry out ear and reproduce? realize this dream, at first will solve three key issues: 1. cell quantity problem: only can separate obtaining 1-3 * 10 in a patient's the residual credulous bone 6Can individual cell with these cell amplifications to making up required cell quantity (1.5-3 * 10 of normal size people's auricle form cartilage 8Individual cell)? does 2. cell function problem: whether the residual credulous osteocyte after the amplification also has good cartilage form ability? 3. constructing technology problem: how to utilize these cells to have the cartilage of accurate people's auricle form in external structure?
In sum, this area is little in the urgent need to the searching wound of drawing materials, and have stronger multiplication capacity and cartilage and form ability, and cartilage forms stable seed cell in subcutaneous environment, and set up corresponding cartilage and make up core technology.
Summary of the invention
The present invention aim to provide that a kind of multiplication capacity is strong, cartilage directed differentiation potential strong and in external and subcutaneous environment cartilage form stable regenerating bone or cartilage seed cell (residual credulous key cell), and the method for utilizing the three-dimensional cartilage graft of this cell construction organizational project.
In a first aspect of the present invention, a kind of organization engineered cartilage graft is provided, described graft contains the residual credulous osteocyte of the people who grows under three dimensional structure; The residual credulous osteocyte of described people is to go down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations; Preferably, be the 1st, 2,3,4 or 5 generations.
In another preference, described three dimensional structure is to assemble the agglomerate (pellet) that forms or the residual credulous osteocyte of the described people of high density by the residual credulous osteocyte of described people to be inoculated in the formed cell patch of culture dish or to be formed by the medically acceptable Biodegradable material that the residual credulous osteocyte of described people is inoculated in solid; Described high density refers to 0.5 * 10 6Individual cell/cm 2-5 * 10 6Individual cell/cm 2Described medically acceptable Biodegradable material is selected from: polylactic acid (PLA), polyglycolic acid (PGA), PLGA, poly butyric, poly-anhydride, poly-phosphazo, polyamino acid, false polyamino acid, poe, polyester urethane, Merlon, Polyethylene Glycol, poly(ethylene oxide), poly-para-dioxanone, collagen, gelatin, hyaluronic acid, ammonia polyose of candy, chitosan, chitin, alginate, acellular matrix, and copolymer or mixture.
In cartilage graft provided by the invention, the residual credulous osteocyte of described people is from from body or allogeneic; Preferred microtia (microtia) patient is from the residual credulous bone of body.
Cartilage graft provided by the invention, in the cumulative volume of described graft, the residual credulous osteocyte concentration of people wherein is 2 * 10 7Individual cell/cm 3-10 * 10 7Individual cell/cm 3Preferably, be 2 * 10 7Individual cell/cm 3-7 * 10 7Individual cell/cm 3
In another preference, the defect shape that the shape of described graft need to be transplanted cartilage with human body conforms to; Preferably, the various shape such as the shape behaviour auricle of cartilage graft provided by the invention, bridge of the nose, the wing of nose, lower chin, zygomatic arch, geisoma, tubulose, rhombus, lamellar, cylinder but be not limited only to this.
In a second aspect of the present invention, a kind of preparation method of aforesaid organization engineered cartilage graft provided by the invention is provided, described method comprises step:
(1) the residual credulous osteocyte of people is carried out the plane amplification, and be passaged to for the 1st, 2,3,4,5,6,7 or 8 generations;
(2) the residual credulous osteocyte of people that the 1st, 2,3,4,5,6,7 or 8 generations was gone down to posterity forms cell patch or the residual credulous osteocyte of people that the 1st, 2,3,4,5,6,7 or 8 generations went down to posterity is mixed formation cell-biomaterial composites with the medically acceptable Biodegradable material of solid by centrifugal formation agglomerate (pellet) or with the residual credulous osteocyte high density inoculation of people that the 1st, 2,3,4,5,6,7 or 8 generations went down to posterity; With
(3) agglomerate, cell patch or complex being carried out chondrocytes in vitro induces; In cumulative volume, contain 5-50ng/ml TGF-β in the described chondrocytes in vitro induced liquid 1(Transforming Growth Factor-β 1, transforming growth factor-beta 1) and the dexamethasone (dexamethasone) of 10-100ng/ml.
Above-mentioned chondrocytes in vitro induced liquid contains the Serum-induced system at CN 200510112068.X and CN200610024205.9) in address, related content is incorporated among the present invention.
The preferred external one-tenth chondrocyte induction liquid of the present invention is the serum-free induction system, in cumulative volume, serum substitute (as: the ITS premix that contains customary amount, wherein comprise insulin, transferrins, Monohydrated selenium dioxide, linoleic acid, bovine serum albumin, acetone acid, ascorbic acid phosphate) and conventional cell culture additive (such as proline, vitamin C etc.), contain the TGF-β of 5-20ng/ml simultaneously 1(Transforming Growth Factor-β 1, transforming growth factor-beta 1), the IGF-I of 10-100ng/ml (Insulin-Like Growth Factor-I, insulin like growth factor-1), and the dexamethasone of 10-100ng/ml (dexamethasone); More preferably, the TGF-β that contains 8-12ng/ml in the described one-tenth chondrocyte induction liquid 1, the IGF-I of 90-100ng/ml, and the dexamethasone of 30-50ng/ml.
In another preference, the residual credulous osteocyte of described people is from from body or allogeneic; Preferred microtia (microtia) patient is from the residual credulous bone of body.
In above-mentioned preparation method provided by the invention, the time of external one-tenth chondrocyte induction is 3-20 week; It preferably is 6-12 week.
In above-mentioned preparation method provided by the invention, in the cumulative volume of complex, the concentration of residual credulous osteocyte in complex is 2 * 10 7-10 * 10 7Individual cell/cm 3
In another preference, it is that chondrocyte mixed preferred 24-48 hour 24-168 hour afterwards with the medically acceptable Biodegradable material of solid that described serum-free for external one-tenth chondrocyte induction becomes the Applicative time of chondrocyte induction liquid.For the three-dimensional agglomerate (such as pellet) or the cell patch that need not timbering material and do carrier, only form by cell aggregation, the Applicative time that becomes chondrocyte induction liquid is after the cell aggregation thing forms in 72 hours, in preferred 24 hours.
In another preference, the application process of described serum-free medium for becoming chondrocyte induction be directly dropping at complex, pellet or cell patch, changing liquid blanking time is 24-96 hour.
In a third aspect of the present invention, provide the application of a kind of aforesaid organization engineered cartilage graft provided by the invention in making up engineered auricle graft.
In a fourth aspect of the present invention, provide the application of the residual credulous osteocyte of a kind of people in the external structure organization engineered cartilage.
In a fifth aspect of the present invention, provide the application of the residual credulous osteocyte of a kind of people in the engineered auricle graft of external structure.
In another preference, the residual credulous osteocyte of described people is from from body or allogeneic; More preferably, microtia (microtia) patient is from the residual credulous bone of body.
Accordingly, it is little to the invention provides the wound of drawing materials, and have stronger multiplication capacity and cartilage and form ability, and cartilage forms stable seed cell in subcutaneous environment, and set up corresponding cartilage structure core technology.
Description of drawings
Fig. 1 has shown that residual credulous osteocyte induces bone marrow stroma stem cell (BMSC) to become the situation of cartilage at nude mice by subcutaneous.Wherein A, D are respectively general appearance and the dyeing of II Collagen Type VI of residual credulous osteocyte and altogether cultivation group of BMSC; B, E are respectively general appearance and the dyeing of II Collagen Type VI of simple residual credulous osteocyte group; C, F are respectively general appearance and the dyeing of II Collagen Type VI of simple bone marrow stroma stem cell group.
Cellular morphology (100 *) and propagation situation when Fig. 2 has shown the amplification of the external plane of the residual credulous osteocyte of different generations; Wherein pld4 represents the residual credulous osteocyte that 1st generation increased the 4th day, by that analogy.The cellular morphology below is growth curve corresponding to identical generation.
Fig. 3 has shown the residual credulous osteocyte cartilage formational situation of different generations.P0d5 represents residual credulous osteocyte that former generation increased the 5th day and with the pellet situation of its preparation, by that analogy.
Fig. 4 has shown the three-dimensional differentiation potential of residual credulous osteocyte of the 8th generation.From left to right be followed successively by the 8th generation residual credulous osteocyte through the Alizarin red staining of osteogenic induction after 3 weeks, form pellet through become chondrocyte induction after 3 weeks II Collagen Type VI immunohistochemical staining and through becoming the oil red dyeing (200 *) after fat induced for 3 weeks.
Fig. 5 has shown that the 8th generation residual credulous osteocyte is through inducing the situation that forms cartilage.A: be pellet general appearance (n=3) that B is each group pellet weight in wet base (n=5), C is each group pellet diameter (n=5).
Fig. 6 shown residual credulous osteocyte pellet of the 8th generation through induce form cartilage substantially reach histology's (HE, Safrani n-O and dyeing of II Collagen Type VI).
What Fig. 7 had shown that residual credulous osteocyte of the 3rd generation-PGA/PLA composite body becomes 8 weeks of chondrocyte induction outward reaches II Collagen Type VI coloration result substantially; Wherein A is for substantially seeing, and B is II Collagen Type VI immunohistochemical staining, (100 *).
Fig. 8 has shown residual credulous osteocyte of the 5th generation, and the compound PGA support of bone marrow stroma stem cell (BMSC), substantially reaches HE (200 *) in nude mice by subcutaneous after 12 weeks at external one-tenth chondrocyte induction 6 Zhou Houzhi.
What Fig. 9 had shown that residual credulous osteocyte of the 3rd generation-ear supporter composite body becomes 8 weeks of chondrocyte induction outward reaches the histological stain result substantially; Wherein A is for substantially seeing, and B is HE dyeing, (100 *).
The specific embodiment
For problems of the prior art and difficulty, the cartilage that the inventor has at first investigated behind the residual credulous osteocyte amplification in vitro forms ability, the result finds unfortunately, the cartilage of residual credulous osteocyte forms ability along with subculture in vitro separately sharply descends, even the compared with normal chondrocyte descends faster, substantially, can not form the typical cartilage (Fig. 3) of homogenizing when passing to for the 3rd generation, can't satisfy the tissue engineering bone/cartilage structure to the requirement of seeding cell functions, as if this phenomenon is considered to the performance that a kind of cartilage phenotype dedifferentes usually, and the prompting that gives those skilled in the art is: carry out ear by the residual credulous osteocyte that increases and reproduce and do not possess the clinical practice feasibility.
Yet, the inventor is surprised to find that in the research process, although residual credulous osteocyte is easy to lose cartilage and forms ability, but it has superpower multiplication capacity, that is to say, although occured to dedifferente, obvious catabiosis do not occur, this point is obviously different from the chondrocyte in normal source: the normal cartilage cell begins multiplication capacity from 1st generation and just descends rapidly, when passing to for the 5th generation obviously aging, be difficult to propagation (and the ratio of the propagation that at every turn goes down to posterity is also very limited); Residual credulous osteocyte then has very strong multiplication capacity, and the 8th generation still can keep very high proliferation activity (Fig. 2) (generally passing to 3-4 for the quantitative requirement of the auricle form cartilage that substantially can satisfy the structure normal size to seed cell) even (in 1: 3 ratio) increases.
So, does whether the residual credulous osteocyte after the amplification still has cartilage form potential? strong in view of residual credulous osteocyte in-vitro multiplication ability, be difficult for the chondrocyte that the characteristic such as aging is different from differentiation and maturation fully, and with in the past the report the stem cell property class seemingly, therefore the inventor courageously supposes, the cell in residual credulous bone source is likely the stem cell that left behind in a kind of growth course.Based on this hypothesis, the inventor has carried out the multidirectional Analytical Chemical Experiment of inducing to residual credulous osteocyte, and pleasantly surprised the discovery, even amplification still has skeletonization, becomes cartilage, becomes the multi-lineage potential (Fig. 4) such as fat to the cell in the 8th generation.These results suggest, strong and the cell with similar stem cell characteristic of multi-lineage potential of really a kind of multiplication capacity of the cell in residual credulous bone source, this great discovery had no report in the past, and the inventor is referred to as residual credulous key cell for the time being.For fully confirming this great discovery, the inventor has carried out proliferation potential and the checking of many differentiation potentials to the cell in the residual credulous bone of many cases source, the result confirms, the cell in all residual credulous bones sources all has the multi-lineage potentials such as very strong proliferation potential and bone, cartilage, fat, the stem cell characteristic that shows residual credulous bone derived cell is not fortuitous phenomena, but a kind of objective reality and characteristic repeatably, the residual credulous osteocyte of these results suggest all is expected to satisfy the requirement that makes up auricle form cartilage in quantity and function aspects.
Although how above-mentioned all more important discoveries are used residual credulous key cell and are made up clinical applicable auricle form cartilage and remain a huge challenge.Fully confirming on the basis of its stem cell characteristic, the inventor makes up the experience of three-dimensional cartilage in conjunction with induced dry-cell in the past (such as bone marrow stroma stem cell etc.), by make repeated attempts and kinds of schemes to when optimizing and combining, set up stable residual credulous key cellular cartilage directional induction system (Fig. 5, Fig. 6), and in conjunction with three-dimensional biodegradable stent, use residual credulous key cell goes out the homogenizing maturation in external structure three-dimensional chondroid tissue (Fig. 7).Body is implanted into the result and shows that the cartilage of residual credulous key cell regeneration can be kept stable cartilage phenotype (Fig. 8) in subcutaneous environment.On this basis, further combined with the accurate people's auricle form cartilage constructing technology based on the ripe differentiation of animal chondrocyte of before having set up (such as the computer-aided design rack forming etc.), pertinent literature comprises: Liu Y, Zhang L, Zhou G, Li Q, Liu W, Yu Z, Luo X, Jiang T, Zhang W, Cao Y.In vitro engineering of human ear-shaped cartilage assisted with CAD/CAM technology.Biomaterials.2010.03., 31 (8): 176-183, Patents comprises CN200710044711.9, and the residual credulous key cell of people is compound with the PGA/PLA timbering material of accurate people's auricle form, and induce with the condition of above-mentioned optimization, finally gone out the tissue engineering bone/cartilage (Fig. 9) with accurate people's auricle form in external structure.
In sum, the inventor makes up the not enough bottleneck problem in seed cell source for cartilage, find based on the accident that has very strong proliferation potential in the residual credulous osteocyte Process of in vitro, courageously hypothesis confirms first that also the cell in residual credulous bone source is the cell that a kind of multiplication capacity is strong and have the similar stem cell characteristic of multi-lineage potential, and further combined with in the past stem cell cartilage directional induction of inventor and three-dimensional cartilage constructing technology and experience, and accurately people's auricle form cartilage makes up core technology, by to the making repeated attempts and optimizing and combining of series of key techniques parameter, finally set up the external three-dimensional cartilage constructive system based on residual credulous key cell.On this basis, finished the present invention.
The residual credulous osteocyte of people
The residual credulous osteocyte of people derives from the microtia patient from the residual credulous osseous tissue of body or allosome among the present invention.Patient age and sex are not particularly limited.
Separating the method that obtains residual credulous osteocyte has multiple, in an embodiment of the present invention, described method be under the general anesthesia or local anaesthesia under cut the residual credulous bone of microtia patient, thoroughly peel off connective tissue and the perichondrium rejected around the residual credulous bone, cartilage is shredded to 1-2mm 3Fritter soaks 30-40min with 0.25% chloromycetin solution, with 0.25% trypsin+0.02%EDTA digestion 30min that vibrate in 37 ℃ of constant-temperature tables, vibrates in 37 ℃ of constant-temperature tables with 0.1% collagenase and digests 12h after the PBS flushing.Filter Digestive system with 200 mesh filter screens, collect filtrate in the 50mL centrifuge tube, the centrifugal 8min of 1800rpm, (as contain 10% hyclone with suitable culture fluid after abandoning supernatant, bFGF 10ng/ml, L-glutaminate 300 μ g/ml, vitamin C 50 μ g/ml, the DMEM culture fluid of each 100U/ml of penicillin and streptomycin) re-suspended cell precipitation, inoculation about 2 * 10 in every 10cm culture dish 6Individual living cells is in 37 ℃, 5%CO 2, 100% saturated humidity condition under cultivate, the next day change liquid.After Growth of Cells closely converges, with 0.25% trypsin+0.02%EDTA digestion, carry out passage in 1: 3 ratio.
In the present invention, preferred the 1st, 2,3,4,5,6,7 or 8 generation cell prepare tissue engineering bone/cartilage.
Although the cell in residual credulous bone source is the cell that a kind of multiplication capacity is strong and have the similar stem cell characteristic of multi-lineage potential, but the residual credulous osteocyte of people also possesses the normal mature chondrocyte simultaneously induces BMSC to become this ability of cartilage, the Coculture techniques that needs to use in the test of this ability of confirmation was also described in detail in CN200610024205.9, and related content is incorporated the present invention into.
Biodegradable material
The material that can be used for organization engineered cartilage of the present invention is medically acceptable Biodegradable material, comprises (but being not limited to):
(a) degradability synthesized polymer material, for example polylactic acid (PLA), polyglycolic acid (PGA), PLGA, poly butyric (PHB), poly-anhydride (polyanhydrides), poly-phosphazo (polyphosphazenes), polyamino acid (polyamino acid), false polyamino acid (pesudo-polyamino acid), poe (polyorthoesters), polyester urethane (polye sterurethane), Merlon (polycarbonate), Polyethylene Glycol, hyaluronic acid, poly-para-dioxanone (polydioxanone) etc.;
(b) natural degradable material, for example collagen (collagen), gelatin (gelatin), ammonia polyose of candy (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginate and various acellular matrixes etc.;
The composite of (c) composite of the copolymer of above-mentioned material or compound material, especially macromolecular material and natural material, and solid material and syringeability material.
Preferred medically acceptable Biodegradable material is solid material or solid, liquid composite material, such as polylactic acid (PLA), polyglycolic acid (PGA), collagen etc.Material among the present invention can be prefabricated into various accurate size and shapes, makes up with the cartilaginous tissue that adapts to different size and shapes.When material was the solid type material, the size and shape that can directly prefabricated one-tenth be needed also can carry out accurate plasticity to material by the model of area of computer aided and rapid shaping technique customization.
The organization engineered cartilage graft
Organization engineered cartilage graft provided by the invention contains the residual credulous osteocyte of the people who grows under three dimensional structure; The residual credulous osteocyte of described people was the 1st, 2,3,4,5,6,7 or 8 generations; It preferably is the 1st, 2,3,4 or 5 generations; It more preferably is the 1st, 2 or 3 generations.
Described three dimensional structure is (to be generally 1 * 10 by the residual credulous osteocyte gathering of described people agglomerating (pellet) or high density 6Individual cell/cm 2-5 * 10 6Individual cell/cm 2) be inoculated in the formed cell patch of culture dish; Or formed by the medically acceptable Biodegradable material that the residual credulous osteocyte of described people is inoculated in solid.
In the cumulative volume of described graft, the residual credulous osteocyte concentration of people wherein is 2 * 10 7Individual cell/cm 3-10 * 10 7Individual cell/cm 3Preferably be 2 * 10 7Individual cell/cm 3-7 * 10 7Individual cell/cm 3
The inventor finds that in experiment the cartilage of residual credulous osteocyte forms ability and descends rapidly with the amplification of going down to posterity, and the above cell of 2nd generation has been lost cartilage and formed ability.But be surprised to find that in the research, these cells have powerful multiplication capacity, in the 8th generation, still can keep very high proliferation activity even (in 1: 3 ratio) increases, substantially, can satisfy the auricle form cartilage that makes up normal size (concrete number decide on constructed particular organization and dimensional culture environment, and generally making up the auricle form at the PGA support approximately need 2 * 10 to the quantitative requirement of seed cell and pass to 3-4 generation 8Cell).Further studies confirm that, residual credulous osteocyte has the multi-lineage potential of cells and characteristic of stem, the directional induction experiment confirm, the residual credulous osteocyte of different generations all can show stronger skeletonization, become cartilage ability and one-tenth fat ability to a certain degree, the present invention proposes the concept of residual credulous key cell based on these results, and proposed thus to make up by the cartilage directional induction theory of tissue engineering bone/cartilage graft.Based on this theory, by optimization and the integration of series of key techniques parameter, set up stable residual credulous key cells in vitro cartilage and made up cultivating system.
Based on these original discoveries, in conjunction with the three-dimensional cartilage constructing technology of the existing stem cell in vitro of inventor experience, and the accurate people's auricle form cartilage constructing technology based on the ripe differentiation of animal chondrocyte of before having set up (such as the computer-aided design rack forming etc.), the residual credulous key cell of people is compound with the PGA/PLA timbering material of accurate people's auricle form, utilize the cultivating system of above-mentioned optimization, the tissue engineering bone/cartilage of the accurate people's auricle of external structure form has obtained success.The inventor has also further carried out more rigorous large scale experiment demonstration on this basis, has finally finished the present invention.
The residual credulous osteocyte-biomaterial composites of people
The cell inoculum density of the residual credulous osteocyte-biomaterial composites of people is about 2 * 10 usually among the present invention 7/ ml to 7 * 10 7/ ml or higher.Material is solidity material or solid, liquid composite material, adjust cell concentration with culture fluid, then with the solidity material mixing, the ratio of culture fluid and solidity material is not particularly limited when wherein mixing, but is as the criterion with the maximum that this solid material can adsorb culture fluid.When timbering material is special 3D shape, such as auricle or bridge of the nose shape, calculate by the size of actual volume.
Preparation method
The preparation method of organization engineered cartilage of the present invention is easy, and the residual credulous osteocyte of the described people of some is mixed with pharmaceutically acceptable Biodegradable material, gets final product through external one-tenth chondrocyte induction again.Described method comprises step:
The first step, the cell that the residual credulous bone of people is originated carries out plane cultivation amplification, and is passaged to 1-8 generation;
Second step is inoculated the formation cell patch with the cell of the above-mentioned amplification of going down to posterity by centrifugal formation agglomerate (pellet) or high density; Or these cells are mixed with the medically acceptable Biodegradable material of solid form cell-biomaterial composites;
The 3rd goes on foot, and agglomerate, diaphragm or complex is placed into carry out In vitro culture in the chondrocyte induction liquid.
The method that amplification is cultivated on plane in the first step such as but not limited to, the residual credulous osteocyte of people that separation is obtained is resuspended in the DMEM in high glucose culture fluid, then obtained cell suspension is inoculated on the culture dish, cultivate under optimum conditions, after Growth of Cells closely converges, digestion, in proportion (such as but not limited to, 1: 2-1: 10; Preferred 1: 3-1: 7; More preferably be 1: 3-1: 5) carry out passage to new culture dish, continue under the same conditions the cultivation of going down to posterity.In the cumulative volume of described DMEM in high glucose culture fluid, preferably wherein contain 10-20%FBS, 1-10ng/ml bFGF; Inoculation about 0.5-2 * 10 in the preferred per 10 centimetres of culture dishs of inoculum concentration 6Individual living cells.
Second step reaches nearly 90% when converging at the residual credulous osteocyte of the people of monolayer culture, digests, and cell counting is when showing viable count more than 90%, centrifugal cell precipitation, supernatant discarded, the cell dispersion of making; The cell of resuspended collection centrifugally makes cell aggregation form pellet or the inoculation of cell high density is formed cell patch; Also can be with cell according to 2 * 10 7-10 * 10 7Individual cell/cm 3Density be inoculated on the medically acceptable Biodegradable material of solid and form complex.
In the 3rd step with the cell patch, pellet or the complex that obtain the cellar culture condition (such as but not limited to, 37 ℃, 5%CO 2, 100% saturated humidity, contain the conventional DMEM in high glucose culture fluid of 10% serum) the lower cultivation after 24-48 hour, be replaced by into chondrocyte induction liquid, the general employing conventionally becomes chondrocyte induction liquid get final product, preferably serum-free becomes chondrocyte induction liquid.
The serum that contains that adopts in an embodiment of the invention becomes chondrocyte induction liquid at CN200510112068.X and CN200610024205.9) in existing addressing, related content is incorporated among the present invention.
In another embodiment of the present invention, adopt the serum-free induction system, in cumulative volume, serum substitute (as: the ITS premix that contains customary amount, insulin-containing, transferrins, Monohydrated selenium dioxide, linoleic acid, bovine serum albumin, acetone acid, ascorbic acid phosphate) and conventional cell culture additive (such as proline, vitamin C etc.), contain the TGF-β of 5-20ng/ml simultaneously 1(Transforming Growth Factor-β 1, transforming growth factor-beta 1), the IGF-I of 10-100ng/ml (Insulin-Like Growth Factor-I, insulin like growth factor-1), and the dexamethasone of 10-100ng/ml (dexamethasone); More preferably, the TGF-β that contains 8-12ng/ml in the described one-tenth chondrocyte induction liquid 1, the IGF-I of 90-100ng/ml, and the dexamethasone of 30-50ng/ml.
Can preferably add in the one-tenth chondrocyte induction liquid that the present invention adopts or cytokine or the somatomedin of compound other various promotion cartilage differentiations, such as BMP, CDMP etc., to strengthen chondrocyte induction efficient and to improve extracellular matrix synthesis capability etc.
The one-tenth chondrocyte induction liquid that the present invention adopts changed liquid once in every 2-4 days, and the external evoked time is 3-20 week, preferred 6-12 week.
Organization engineered cartilage graft of the present invention makes up in the cartilage process, be mainly static culture, perhaps add mechanical stimulation in mode centrifugal or vibration, also can be by the mechanical environment of cartilage in the biological reactor simulation body of cartilage, cartilage to external structure applies similar mechanical stimulation, promotes the maturation of cartilage and the improvement of mechanical property.
The organization engineered cartilage that forms with the inventive method, the i.e. complex of the residual credulous key cell of people and solidity biomaterial formation, this complex subcutaneous or cartilage defect position in the implantable behind external one-tenth chondrocyte induction.
In one embodiment of the invention, to make concentration be 5 * 10 for the residual credulous key cell of employment and culture fluid 7The cell suspension of/ml (being not limited only to this concentration) then is master's biologic bracket material formation complex (complex size, shape are determined according to size and the shape of cartilage defect) with polyglycolic acid (PGA).This complex (is not limited only to this time through 6 thoughtful 8 weeks of chondrocytes in vitro directional induction, changed liquid once in per 2 to 4 days during this time, to guarantee cytotrophy and to induce efficient), subcutaneous or corresponding cartilage defect position implants when vitro tissue through engineering approaches cartilage begins to take shape.Also material can be made into the three-dimensional rack (such as people's auricle form) with fine structure profile, inoculate the residual credulous key cell of people and carry out external evokedly, the cartilage defect position implanted after 6-8 week (being not limited only to this time).
Application process
After the solid-state biomaterial of the residual credulous osteocyte of people and degradable forms complex, become chondrocyte induction external, form the organization engineered cartilage that is fit to the cartilage defect size and shape after, cartilage defect position again implants.
Term
Term " the residual credulous osteocyte of people ", " cell in residual credulous bone source " or " the residual credulous key cell of people " can Alternates, all refer to use the conventional isolated cell technology of this area, can secrete II Collagen Type VI (collagen II) from what the microtia patient obtained from the residual credulous osseous tissue of body or allosome, the polygon attached cell of the cartilage specificity substrate such as glycosaminoglycans (glycosaminoglycan, GAG).
Term " separation of the residual credulous osteocyte of people " refers to process that residual credulous osteocyte is separated from the residual ear tissue of microtia patient.
Term " amplification of the residual credulous osteocyte of people " refers in order to obtain a large amount of residual credulous osteocyte the process of a large amount of propagation in external environment.
Term " one-tenth chondrocyte induction " refers to the biochemical environment that provides special make the cellular expression cartilage phenotype with cartilage differentiation potential and the process that possesses into the cartilage ability.
Term " inoculation " refers to cell is uniformly distributed in process on the three-dimensional stent material.
Term " autotransplantation " refers to required biological living material (such as residual credulous osteocyte) is taken out and is applied to the process of same individuality from certain individuality.
Term " three dimensional structure " is exactly stereochemical structure, refers to occupy three-dimensional structure, and three-dimensional is three axles of coordinate axes, i.e. x axle, y axle, z axle, and wherein x represents space, the left and right sides, y represents up and down space, space before and after z represents.So-called three dimensions refers to our residing space, before and after having can be understood as-and up and down-about.Can be the complex that forms on by the medically acceptable Biodegradable material that seed cell is inoculated in solid at the seed cell of three dimensional structure growth among the present invention; It also can be the cell mass with the centrifugal formation of seed cell; Or seed cell is carried out the high density inoculation forms cell patch.Described seed cell is the residual credulous osteocyte of people.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that this case description discloses can with any composition forms and usefulness, each feature that discloses in the description can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
The present invention is for the significant contribution of this area: confirm first or set up (one), under two-dimentional condition of culture, the cell in residual credulous bone source has very strong amplification ability, and the 8th generation still can keep very high proliferation activity even (in 1: 3 ratio) increases; (2), the cell in residual credulous bone source has the distinctive multi-lineage potential of stem cell, even amplification has still kept the multi-lineage potentials such as good bone, cartilage, fat to the 8th generation; (3), set up stable residual credulous key cellular cartilage directional induction system; (4), set up residual credulous key cells in vitro and made up the core technology of three-dimensional cartilage, and confirm its in vivo cartilage phenotype stability in the subcutaneous environment; (5), set up the core technology that makes up accurate people's auricle form cartilage based on the residual credulous key cells in vitro of people.
Major advantage of the present invention is:
(1) can use autogenous cell, and once draw materials and both can obtain enough cell concentrations;
(2) process of drawing materials is not damaged normal structure fully;
(3) extracorporeal culturing method is easy to learn, and is easy to utilize;
(4) external one-tenth chondrocyte induction method is easy and simple to handle, becomes the cartilage definite effect reliable;
(5) can be by the size and shape of the prefabricated graft of cartilage defect size and shape, to reach accurate reparation;
(6) compare with other stem cell, the cartilage form that residual credulous key cells in vitro makes up is easier accurately to be kept, implant subcutaneous environment after cartilage phenotype keep relatively stable.
(7) the chondrocyte induction system of the present invention's foundation and optimization does not contain serum, definite ingredients, and clinical practice is safe, is convenient to standardization, is easy to form the industrialization product.
Use the method that the present invention determines, can obtain to possess in a large number the residual credulous osteocyte that cartilage forms ability, be used for making up auricle form tissue engineering bone/cartilage.This tissue engineering bone/cartilage not only has good organization and learns structure, Biochemical composition and mechanical strength, and be difficult in the external structure process shrinking, it is constant to keep original auricle form, and it is stable to implant subcutaneous rear one-tenth cartilage, is difficult for occuring ossified, fibrosis phenomenon.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio and umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example refers to the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Among the following embodiment, and the DMEM in high glucose culture medium (Dulbecco ' s Modification of Eagle ' s Medium, high glucose) available from U.S. HyClone company, article No.: SH0495; Hyclone (Fetal Bovine Serum, FBS) is available from U.S. HyClone company, article No.: SV30087.02; Vitamin C (Vitamin C) is available from U.S. Sigma company, article No.: A8960-5G; Trypsin Trypsin) available from U.S. Amresco company, article No.: 0458; Collagenase NB4 powder (Collagenase NB4) is available from German Serva company, article No.: 17454; Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) is available from U.S. R﹠amp; D company, article No.: 233-FB/CF; The universal cultivation intermixture of ITS (ITS premix) is available from U.S. S i gma company, article No.: 12521; Transforming growth factor-beta 1 (Transformi ng Growth Factor-β 1, TGF-β 1) is available from U.S. R﹠amp; D company, article No.: 240-B-010; Insulin like growth factor-1 (Insulin-Like Growth Factor-I, IGF-I) is available from U.S. R﹠amp; D company, article No.: 291-G1-010; Dexamethasone (dexamethasone) is available from U.S. Sigma company, article No.: D4902-1G; Insulin (insulin) is available from U.S. Sigma company, article No.: I5500; Indomethacin (indomethaein) is available from U.S. Sigma company, article No.: I-8280; β-phosphoglycerol is available from U.S. Sigma company, article No.: G9422; 1-methyl-3-isobutyl group xanthine (IBMX) is available from U.S. Sigma company, article No.: I5879.
Embodiment 1
The obtaining and cultivating of the residual credulous key cell of people
Cell is got the microtia patient from the residual credulous osseous tissue of body, donor age 8-30 year, and healthy, without malignant tumor, infectious disease and autoimmune disease.
(1) draw materials: under the operating room aseptic condition, after anesthesia was satisfied, the routine disinfection drape cut the residual ear tissue of patient, placed in the aseptic centrifuge tube of 50mL, soaked with the 40mL physiological saline solution and preserved and transportation.
(2) separate residual credulous osteocyte: under the aseptic condition, thoroughly peel off and reject residual credulous bone connective tissue and perichondrium on every side in the super-clean bench, cartilage is shredded to 1-2mm 3Fritter, soak 30-40min with 0.25% chloromycetin solution, the PBS flushing is rear with 0.25% trypsin+0.02%EDTA (Amresco, USA) vibration digestion 30min in 37 ℃ of constant-temperature tables, again with 0.1% collagenase (Collagenase NB4, Serva, Germany) solution in 37 ℃ of constant-temperature tables, vibrate digestion 12h.Filter Digestive system with 200 mesh filter screens, collect filtrate in the 50mL centrifuge tube, the centrifugal 8min of 1800rpm.
(3) inoculation: abandon behind the supernatant to contain 10% hyclone, 10ng/ml bFGF, 300 μ g/ml L-glutaminate, 50 μ g/ml vitamin Cs, the DMEM in high glucose culture fluid re-suspended cell precipitation of each 100U/ml of penicillin and streptomycin.Take a morsel cell suspension with the conventional living cell counting of trypan blue, inoculation about 2 * 10 in every 10cm culture dish 6Individual living cells.Place 37 ℃, 5%CO 2, 100% saturated humidity condition under cultivate, the next day change the culture fluid liquid of 2/3rds amounts.
(4) cultivate and go down to posterity: general primary cell is cultivated under these conditions after 7-8 days and can be reached the fusion state, can continue the cultivation of going down to posterity.Absorb culture fluid when going down to posterity, with a small amount of PBS washing once, Digestive system (0.25% trypsin+0.02%EDTA (Amresco that adds 1.5-2.0ml, USA)), see under the mirror that most cells kytoplasm retraction, form become circle after, absorb gently Digestive system, add an amount of DMEM conditioned medium that contains serum and end digestion, the collecting cell suspension, be passaged in the new culture dish in 1: 3 ratio, continue under identical condition, to cultivate, the next day change the culture fluid of 2/3rds amounts.Generally the fusion state can be reached again after 4-5 days, the cultivation of going down to posterity can be continued.With the 1st, 2,3,4 or 5 generation cell to be used for experiment effect better.
Observe under the inverted microscope, the results are shown in Figure 2.
The result shows that the residual credulous osteocyte of people is spindle shape or polygon.Growth curve shows that each generation cell all keeps vigorous proliferation activity in former 8 generations of generation to the, has no obvious aging sign.
Embodiment 2
Prepare pellet with residual credulous key cell
(1) collecting cell: the residual credulous osteocyte of the people of monolayer culture reaches 90% when converging, and uses 0.25% pancreatin+0.02%EDTA digestion.With the cell counter counting, Trypan Blue shows that viable count makes cell precipitation more than the centrifugal 8min of 90%, 1800rpm, abandoning supernatant, vibrating dispersion cell behind the cell harvesting.
(2) preparation pellet: behind the collecting cell, be prepared into 0.4 * 10 so that common culture fluid is resuspended 6The cell suspension of/ml.Get this suspension of 1ml in the 15ml centrifuge tube, the centrifugal 5min of 1000rpm makes cell pellet.Slightly unscrew bottle cap, place 37 ℃, 5%CO 2, saturated humidity incubator in cultivate, change every other day afterwards liquid (common culture fluid or one-tenth chondrocyte induction liquid).3 weeks, rear each group pellet is drawn materials respectively carried out general appearance and histology's (HE, Safranin-O, II Collagen Type VI etc.) detection.
Embodiment 3
The making of PGA/PLA degradable three-dimensional rack
Polyglycolic acid (PGA) non-woven cotton is produced by Shanghai Guorui Life Sci. ﹠ Tech. Co., Ltd., and cold drying is preserved.Polylactic acid (PLA) is dissolved in dichloromethane and makes 0.3% PLA solution available from U.S. Sigma company.
(1) cylindrical PGA/PLA support: be the 4mg/ piece with PGA non-woven cotton accurate weighing, be pressed into the cylinder fritter of diameter 5mm, thick 1mm with mould, after shape is fixing, drip PLA solution to saturated, air-dry.Before the use, timbering material is immersed in 75% ethanol sterilization 30 minutes fully.PBS flushing 3 times was soaked 10 minutes with the DMEM culture medium that contains 10% hyclone again, blotted rear preparation inoculating cell.
(2) people's auricle form PGA/PLA support: take by weighing the about 800mg/ piece of PGA non-woven cotton, with special (document for example: Liu Y, Zhang L, Zhou G, Li Q, Liu W, Yu Z, Luo X, Jiang T, Zhang W, Cao Y.In vitro engineering of human ear-shaped cartilage assisted with CAD/CAM technology.Biomaterials.2010.03., 31 (8): 176-183 is disclosed, disclosed among the patent CN200710044711.9) the personalized auricle form of the personalized auricle mould compacting of patient patient, behind dimensionally stable, drip PLA solution and in mould, continue moulding, after form is fixing, the auricle support was immersed in 75% ethanol sterilization 30 minutes fully.PBS flushing 3 times was soaked 10 minutes with the DMEM culture medium that contains 10% hyclone again, blotted rear preparation inoculating cell.
Embodiment 4
The residual credulous osteocyte of people induces bone marrow stroma stem cell to make up three-dimensional cartilage
(1) the obtaining and cultivating of the residual credulous osteocyte of people: referring to embodiment 1;
(2) the obtaining and cultivating of bone marrow stroma stem cell:
(a) draw materials and former culture: under the aseptic condition, extract sheep marrow 10-20ml in the 50ml centrifuge tube.Mend PBS to 45ml, the centrifugal 10min of 1800rpm abandons supernatant, so after the repeated washing 2 times, clean 1 time with BMSC culture fluid (the low sugar DMEM culture fluid that contains 10%FBS) again, resuspended with the BMSC culture fluid and be inoculated in (every 2ml bone marrow is inoculated 1 ware) in the 10cm culture dish at last.Change first liquid after 5 days, elder generation is with the not adherent hemocyte of PBS flush away when changing liquid.Changed liquid once in 2-4 days afterwards.
(b) cultivation of going down to posterity: treat that cell grows to 80% cultivation of going down to posterity when merging state.When going down to posterity, absorb culture fluid, with a small amount of PBS washing once, the Digestive system (0.25% trypsin+0.02%EDTA (Amresco, USA)) that adds 1.5-2.0ml, see under the mirror that most cells kytoplasm retraction, form become circle after, absorb gently Digestive system, add an amount of DMEM conditioned medium that contains serum and end digestion, the collecting cell suspension, be passaged in the new culture dish in 1: 3 ratio, continuation is cultivated under identical condition.The next day change the culture fluid of 2/3rds amounts, generally can reach the fusion state again after 2-5 days, can continue the cultivation of going down to posterity.It is better to be used for experiment effect with 1-4 for cell.
(3) inoculating cell-material composite: collect bone marrow stroma stem cell (BMSC) and residual credulous key cell, altogether the middle BMSC of cultivation group (n=3) and residual credulous osteocyte with behind 3: 1 ratio mix homogeneously according to 5 * 10 7Individual cell/cm 3Density be inoculated on the cylindrical PGA support; In the simple chondrocyte group (n=3) according to 5 * 10 7Individual cell/cm 3The simple residual credulous key cell of density inoculation; Simple BMSC organizes in (n=3) according to 5 * 10 7Individual cell/cm 3Density inoculate simple BMSC; Cell-material composite that inoculation is good is in 37 ℃, 5%CO 2, 100% humidity environment in static culture 5 hours.Slowly add the in advance common DMEM in high glucose culture fluid that contains 10%FBS of temperature, continue to cultivate, changed liquid once in every 3-4 days, carry out nude mice by subcutaneous Hui Zhi after 1 week.
(4) nude mice by subcutaneous heeling-in: plant in the nude mice back cell-material composite of respectively organizing in 1 week of In vitro culture subcutaneous, draw materials after 10 weeks and carry out general appearance and II Collagen Type VI SABC and detect.
(a) substantially see:
After 10 weeks of nude mice by subcutaneous heeling-in, altogether cultivation group and simple chondrocyte group sample are typical cartilage sample porcelain white appearance, and original volume maintenance is constant, and quality is pliable and tough and certain elasticity arranged; And simple BMSC group is the dark yellow outward appearance, and volume has obvious contraction, and quality is soft (Fig. 1).
(b) II Collagen Type VI SABC: after 10 weeks of nude mice by subcutaneous heeling-in, cultivation group and simple chondrocyte group II expression of collagen are strong positive altogether, and have typical cartilage lacuna structure; And simple BMSC group II Collagen Type VI is negative and without typical cartilage lacuna spline structure (Fig. 1).
The above results shows: residual credulous osteocyte has the chondrogenetic ability of the BMSC of inducing.
Embodiment 5
The spontaneous one-tenth cartilage of the residual credulous key cells in vitro of people
(1) the obtaining and cultivating of the residual credulous osteocyte of people: the method for draw materials, former culture and going down to posterity being cultivated is referring to embodiment 1;
(2) the residual credulous key cell II expression of collagen in the amplification procedure of plane: the residual credulous key cell that each generation is cultured to the 5th day carries out II Collagen Type VI immunofluorescence dyeing, the fluorescence microscopy Microscopic observation, the result shows: its II expression of collagen of residual credulous key cell in the amplification procedure of plane increases and sharply downward modulation with passage number, to 2nd generation only the minute quantity cell to be the II Collagen Type VI positive, in the 3rd generation, is without obvious positive cell (Fig. 3).
(3) pellet preparation and In vitro culture: collect the cell that each generation is cultured to the 5th day, be prepared into 0.4 * 10 so that common culture fluid (the DMEM in high glucose culture fluid that contains 10%FBS) is resuspended 6The cell suspension of/ml.Get this suspension of 1ml in the 15ml centrifuge tube, the centrifugal 5min of 1000rpm makes cell pellet.Slightly unscrew bottle cap, place 37 ℃, 5%CO 2, saturated humidity incubator in cultivate, change every other day afterwards liquid (common culture fluid), after 3 weeks each group pellet is drawn materials respectively and detects.
(a) substantially see:
The residual credulous key cell of each generation (former 3 generations of generation to the) all can form the spherical porcelain white cartilage sample agglomerate (pellet) about diameter 2mm, the residual credulous key cell that wherein (contains 2nd generation) before the 2nd generation becomes the pellet quality pliable and tough and certain elasticity is arranged, and the pellet quality after the 2nd generation soft (Fig. 3).
(b) II Collagen Type VI SABC: the pellet that residual credulous key cell becomes that (contains 2nd generation) before the 2nd generation, its II Collagen Type VI positive expression, and have typical cartilage lacuna structure; And the pellet after the 2nd generation, its II Collagen Type VI is weak expression, and cartilage lacuna spline structure not obvious (Fig. 3).
The result shows that the residual credulous key cell of people is (low generation) certain spontaneous one-tenth cartilage ability of performance only in early days.
Embodiment 6
The residual credulous key Multidirectional Differentiation of Cells of people is induced experiment:
(1) the obtaining and cultivating of the residual credulous key cell of people: the method for draw materials, former culture and going down to posterity being cultivated is referring to embodiment 1;
(2) the residual credulous key cell osteogenic induction of people: cultivate the residual credulous key cell in the 8th generation with osteogenic induction liquid, changed liquid once in every 2-3 days, carry out Alizarin red staining after inducing for 3 weeks.The concrete composition of osteogenic induction liquid comprises: low sugar DMEM culture medium, 10% hyclone (fetal bovine serum, FBS), 10mM dexamethasone, 10mM β-phosphoglycerol, 50mM vitamin C.Induce the result: visible obviously calcium tuberosity under the mirror, Alizarin red staining is positive (Fig. 4), shows that the 8th generation residual credulous key cell still has osteogenic potential.
(3) the residual credulous key cell of people becomes chondrocyte induction: with the 8th generation residual credulous key cell be prepared into pellet, and to become chondrocyte induction liquid to cultivate, changed liquid once in per 2 days, carry out tissue slice and II Collagen Type VI immunohistochemical staining after inducing for 3 weeks.Pellet preparation and In vitro culture are with reference to embodiment 4.Become the concrete composition of chondrocyte induction liquid to comprise: in cumulative volume, to contain the TGF-β of DMEM in high glucose culture medium, 10ng/ml 1The IGF-I of 100ng/ml,, the dexamethasone of 40ng/ml, one times of content ITS premix (the universal cultivation intermixture of ITS, insulin-containing, transferrins, Monohydrated selenium dioxide, linoleic acid, bovine serum albumin, acetone acid, ascorbic acid phosphate), proline and vitamin C.Induce the result: II Collagen Type VI dyeing is positive, and visible typical cartilage lacuna spline structure (Fig. 4) shows that the 8th generation residual credulous key cell still has Chondrogenesis.
(4) the residual credulous key cell of people becomes fat to induce: cultivate the residual credulous key cell in the 8th generation to become the fat induced liquid, changed liquid once in every 2-3 days, carry out oil red dyeing after inducing for 3 weeks.Become the concrete composition of fat induced liquid to comprise: low sugar DMEM culture medium, 10% hyclone (fetal bovine serum, FBS), 1 μ M dexamethasone, 0.5mM 1-methyl-3-isobutyl group xanthine (IBMX), 10M insulin, 200 μ M indomethacins (indomethaein).Induce the result: visible obviously fat drips under the mirror, and oil red dyeing is positive (Fig. 4), shows that the 8th generation residual credulous key cell still has into fat potential.
Embodiment 7
Different one-tenth cartilage systems are induced residual credulous key cell
(1) collecting cell: with reference to embodiment 2;
(2) preparation pellet and experiment grouping: pellet prepares with reference to embodiment 2, prepare altogether 15 pellet, wherein serum-free group (n=5) becomes chondrocyte induction liquid (DMEM in high glucose culture medium, 1%1 * ITSpremix with serum-free, the 40g/ml proline, 10ng/ml TGF-β 1, 100ng/ml IGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs) cultivate.Contain serum group (n=5) and become chondrocyte induction liquid (DMEM in high glucose culture medium, 10%FBS, 10ng/ml TGF-β to contain serum 1, 100ng/ml IGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs) cultivate.Common cultivation group (n=5) is cultivated with the common DMEM in high glucose culture fluid that contains 10%FBS.After 3 weeks each group pellet is drawn materials respectively, carry out general appearance and histology.
(a), Pellet sees substantially
External one-tenth chondrocyte induction is after 3 weeks, and serum-free group pellet is porcelain white spherical design, and diameter reaches about 5mm, and quality is pliable and tough and certain elasticity is arranged, and weight in wet base reaches about 14mg; Contain serum group pellet and become porcelain white spherical design, diameter reaches about 2mm, and about the about 3mg of weight in wet base, quality is soft; Common cultivation group pellet shape is more irregular, and diameter is 1-2mm only, and only about 2mg, the quality softness is nonelastic (Fig. 5) for weight in wet base.
(b), Pellet histological examination
HE dyeing: the plastidogenetic pellet In vitro culture of the residual credulous backbone of the 8th generation people is after 3 weeks, serum-free is induced the visible a large amount of typical cartilage lacuna spline structures of group, contain the only visible minute quantity cartilage lacuna in edge spline structure of Serum-induced group, common cultivation group is without obvious lacuna structure (Fig. 6).
Safranin-O dyeing: the pellet In vitro culture that the residual credulous osteocyte of the 8th generation people forms is after 3 weeks, serum-free induces group visible typical cartilage lacuna structure and the obvious Safranin-O positive painted, contains Serum-induced group and common cultivation group all without obvious lacuna structure and Safranin-O painted (Fig. 6).
II Collagen Type VI SABC: the pellet In vitro culture that the residual credulous osteocyte of the 8th generation people forms is after 3 weeks, serum-free is induced the most of regional II Collagen Type VI of group to be positive and is had typical cartilage lacuna structure, contains the only visible II Collagen Type VI in edge positive region of Serum-induced group; Common cultivation group only has the point-like II expression of collagen (Fig. 6) that is dispersed on a small quantity without obvious lacuna structure.
The above results shows: serum-free becomes the chondrocyte induction system more to be conducive to residual credulous key cell at external formation cartilage.
Embodiment 8
The residual credulous key cell nude mice by subcutaneous of people becomes cartilage
(1) the obtaining and cultivating of the residual credulous key cell of people: referring to embodiment 1;
(2) residual credulous key cell-PGA complex becomes chondrocyte induction: collect residual credulous key cell, according to 5 * 10 7Individual cell/cm 3Density be inoculated on the cylindrical PGA support (residual credulous key groups of cells, n=3); In like manner, collect bone marrow stroma stem cell (BMSC) and be inoculated on the cylindrical PGA support (the BMSC group, n=3).Cell-material composite that inoculation is good is in 37 ℃, 5%CO 2, 100% humidity environment in static culture 5 hours.The common DMEM in high glucose culture fluid that contains 10%FBS that slowly adds in advance temperature bath continues to cultivate.After 48 hours, change serum-free and become chondrocyte induction liquid (DMEM in high glucose culture medium, 1%1 * ITS premix, 40 μ g/ml proline, 10ng/ml TGF-β 1, 100ng/ml IGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs), changed liquid once in every 3-4 days, cultivated for 6 weeks after, carry out the nude mice by subcutaneous heeling-in.
(3) nude mice by subcutaneous heeling-in: the cell-material composite in external 6 weeks of one-tenth chondrocyte induction is planted in the nude mice back subcutaneous, draw materials after 12 weeks and carry out general appearance and HE dyeing.
(a) substantially see:
After 12 weeks of nude mice by subcutaneous heeling-in, residual credulous key groups of cells sample is typical cartilage sample porcelain white appearance, and original volume maintenance is constant, and quality is pliable and tough, and is flexible; And hard, the rough surface of BMSC group sample quality, the skeletonization phenomenon is (Fig. 8) obviously;
(b) HE dyeing: after 12 weeks of nude mice by subcutaneous heeling-in, the visible a large amount of typical cartilage lacuna structures of residual credulous key groups of cells sample, and BMSC group ossified obviously (Fig. 8).
The result shows that cartilage graft cartilage phenotype in subcutaneous environment that residual credulous key cell obtains is more stable.
Embodiment 9
The external evoked structure tissue engineering bone/cartilage of residual credulous key cell compound support frame material
(1) collecting cell: with reference to embodiment 2, collect residual credulous key cell of the 3rd generation.
(2) inoculating cell-material composite: by 5 * 10 7Individual cell/cm 3Density with cell suspension inoculation on cylindrical PGA support or auricle shape PGA/PLA support, in 37 ℃, 5%CO 2, 100% humidity environment in static culture 5 hours.Slowly add the in advance common DMEM in high glucose culture fluid that contains 10%FBS of temperature, continue to cultivate after 48 hours and be replaced by into chondrocyte induction liquid.The concrete composition of one-tenth chondrocyte induction liquid that this example adopts comprises: DMEM in high glucose culture medium, 1%1 * ITS premix, 40 μ g/ml proline, 10ng/mlTGF-β 1, 100ng/ml IGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs.Changed liquid once in every 3-4 days, and drew materials after 8 weeks and detect.
(a), the cartilage graft of external structure and II Collagen Type VI immunohistochemical staining thereof check
The residual credulous key cell of the 3rd generation people-PGA material composite is external through becoming chondrocyte induction after 8 weeks, the PGA fiber is by a large amount of cartilage matrix parcels, show typical cartilage sample outward appearance, be porcelain white, size and shape maintains are good, quality is pliable and tough and certain elasticity is arranged, and the II expression of collagen is strong positive, further proves a large amount of cartilage specificity matrix secretions (Fig. 7).
(b), normal size people's auricle form cartilage and the histological examination thereof of external structure
The residual credulous osteocyte of the 3rd generation people-auricle form PGA/PLA material composite is external to show cartilage sample outward appearance through becoming chondrocyte induction after 8 weeks, and big or small gill profile attitude is kept well, and quality is pliable and tough and certain elasticity arranged.The histology shows the cartilage lacuna structure of a large amount of maturations, and timbering material is by a large amount of extracellular matrix parcels (Fig. 9).
Discuss
The proposition of the residual credulous key cell theory of the present invention is on the basis of unexpected discovery in residual credulous bone derived cell biological characteristics heuristic process, the bold hypothesis that a large amount of experiences that in the past accumulated in conjunction with the inventor propose.Based on this hypothesis, the inventor verifies repeatedly through the multiple-case great many of experiments, confirmed the stem cell characteristic of residual credulous bone derived cell, and based on this, by optimization, the integration of series of key techniques parameter, finally set up the external three-dimensional cartilage constructing technology system based on residual credulous key cell.Being established as of this technical system utilized the microtia patient to make up tissue engineering bone/cartilage from the residual credulous bone derived cell of body to carry out that ear reproduces and wait cartilage defect repair and reconstruction that feasible clinical treatment is provided.
It is generally acknowledged that the cell in residual credulous bone source belongs to chondrocyte, should have into the feature of the differentiation and maturation chondrocytes such as the cartilage ability is strong, amplification in vitro easily wears out, dedifferente, Most scholars is also admitted this viewpoint substantially both at home and abroad.The inventor finds by systematic research, and easier the dedifferenting of cell compared with normal chondrocyte in residual credulous bone source is expanded to and substantially loses cartilage formation ability more than the 2nd generation, and this has obviously limited its clinical practice feasibility.Fortunately, the inventor is surprised to find that under study for action, although residual credulous osteocyte is easy to lose cartilage and forms ability, but it has superpower multiplication capacity, that is to say, although occured to dedifferente, obvious catabiosis does not but appear, even amplification still can keep very high proliferation activity to the 8th generation, from this characteristic, the cell in residual credulous bone source is obviously different from the chondrocyte of normal differentiation maturation.
Based on this discovery, the cell that the inventor has courageously proposed residual credulous bone source is the hypothesis of the stem cell that left behind in a kind of growth course, and on this basis, utilize a plurality of cases to carry out the experiment of a large amount of repeatability, the result shows, still have the multi-lineage potentials such as stronger bone, cartilage, fat even reach the residual credulous bone derived cell in the 8th generation, show the cell that really a kind of ability of amplification of residual credulous bone derived cell is strong and have the similar stem cell characteristic of multi-lineage potential, so residual credulous this theory of key cell has been proposed first the inventor.
Based on the theory of residual credulous key cell, the inventor has determined to make up by directional induction the main attack research direction of cartilage.Because the inventor is engaged in for a long time the stem cell cartilage and makes up correlational study, possessed the experience that abundant induced dry-cell makes up three-dimensional cartilage, take these experiences as guidance, by make repeated attempts and kinds of schemes to when optimizing and combining, set up stable residual credulous key cellular cartilage directional induction system, and in conjunction with three-dimensional biodegradable stent, use residual credulous key cell goes out the homogenizing maturation in external structure three-dimensional chondroid tissue.On this basis, further combined with the accurate people's auricle form cartilage constructing technology based on the ripe differentiation of animal chondrocyte of before having set up, the residual credulous key cell of people is compound with the PGA/PLA timbering material of accurate people's auricle form, and induce with the condition of above-mentioned optimization, finally gone out the tissue engineering bone/cartilage with accurate people's auricle form in external structure.
In sum, the cell that residual credulous key cell disclosed in this invention is a kind of specific type, it is all different with differentiation and maturation chondrocyte and other stem cell with cartilage differentiation potential reported in the past.Use residual credulous key cell construction cartilage its unique advantage arranged:
At first, compare with the normal cartilage cell, residual credulous key cell has very strong multiplication capacity, can satisfy to make up the required cell concentration of larger volume cartilage (such as people's auricle form cartilage) fully, and can not cause any damage by normal tissue to drawing materials of residual credulous bone.Therefore, use patient and have stronger clinical practice feasibility from the residual credulous key cell construction cartilage graft compared with normal chondrocyte of body.
Secondly, compare with other stem cell with cartilage differentiation potential, its advantage is more remarkable: 1). and Chondrogenesis is stronger: the residual credulous key cell of low generation (in 2 generations) can form cartilage without inducing, and can also induce other stem cell with cartilage formation potential to become cartilage (Fig. 1); The residual credulous key cell of higher generation time (more than 2 generations) after inducing also than the easier formation homogenizing of other stem cell cartilage; These features may with residual credulous key cell in to have into a cell colony proportion of cartilage ability higher relevant.2). form is kept better in the three-dimensional cartilage building process: be easy to occur contraction distortion in other Derived Stem Cells (such as bone marrow stroma stem cell) Induction Process, and residual credulous key cells in vitro makes up difficult contraction the in the three-dimensional cartilage process, and form can accurately be kept.This may be with residual credulous key cell be more rapid in the generation of chondrocyte induction process mesostroma, cartilage formation is faster relevant.3). in the body in the dystopy environment cartilage stability better: the cartilage of other Derived Stem Cells (such as bone marrow stroma stem cell) external structure is easy to ossify in the subcutaneous environment in vivo, and the three-dimensional cartilage that residual credulous key cells in vitro makes up is implanted subcutaneous rear cartilage phenotype and kept comparatively stable (Fig. 8).This may with residual credulous key cell itself come from cartilaginous tissue, external evoked after the stable cartilage of easier formation differentiation and maturation relevant.In addition, the present invention sets up and the external structure technology of optimizing and the chondrocyte induction system that does not contain serum also are convenient to standardization, is easy to form the industrialization product.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. an organization engineered cartilage graft is characterized in that, it contains the residual credulous osteocyte of the people who grows under three dimensional structure; The residual credulous osteocyte of described people is to go down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations; Preferred the 1st, 2,3,4 or 5 generations.
2. cartilage graft as claimed in claim 1, it is characterized in that described three dimensional structure is to assemble the agglomerate that forms or the residual credulous osteocyte of the described people of high density by the residual credulous osteocyte of described people to be inoculated in the formed cell patch of culture dish or to be formed by the medically acceptable Biodegradable material that the residual credulous osteocyte of described people is inoculated in solid.
3. cartilage graft as claimed in claim 1 is characterized in that, the residual credulous osteocyte of described people is from from body or allogeneic; Preferred microtia patient is from the residual credulous bone of body.
4. such as the arbitrary described cartilage graft of claim 1-3, it is characterized in that in the cumulative volume of described graft, the residual credulous osteocyte concentration of people wherein is 2 * 10 7Individual cell/cm 3-10 * 10 7Individual cell/cm 3Preferred 2 * 10 7Individual cell/cm 3-7 * 10 7Individual cell/cm 3
5. cartilage graft as claimed in claim 4 is characterized in that, the defect shape that the shape of described graft need to be transplanted cartilage with human body conforms to.
6. preparation method such as the arbitrary described cartilage graft of claim 1-5, described method comprises step:
(1) the residual credulous osteocyte of people is carried out the plane amplification, and be passaged to for the 1st, 2,3,4,5,6,7 or 8 generations;
(2) the residual credulous osteocyte of people that the 1st, 2,3,4,5,6,7 or 8 generations was gone down to posterity forms cell patch or the residual credulous osteocyte of people that the 1st, 2,3,4,5,6,7 or 8 generations went down to posterity is mixed formation cell-biomaterial composites with the medically acceptable Biodegradable material of solid by centrifugal formation agglomerate or with the residual credulous osteocyte high density inoculation of people that the 1st, 2,3,4,5,6,7 or 8 generations went down to posterity;
(3) agglomerate, cell patch or complex being carried out chondrocytes in vitro induces; In cumulative volume, contain 5-50ng/ml TGF-β in the described chondrocytes in vitro induced liquid 1(Transforming Growth Factor-β 1, transforming growth factor-beta 1) and the dexamethasone (dexamethasone) of 10-100ng/ml.
7. preparation method as claimed in claim 6 is characterized in that, the time of external one-tenth chondrocyte induction is 3-20 week; Preferred 6-12 week.
8. one kind such as the application of the arbitrary described cartilage graft of claim 1-5 in making up engineered auricle graft.
9. the application of the residual credulous osteocyte of people in the external structure organization engineered cartilage.
10. the application of the residual credulous osteocyte of people in the engineered auricle graft of external structure.
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CN105797154A (en) * 2014-12-31 2016-07-27 中国科学院上海生命科学研究院 Cartilaginous stem cell separation method and use thereof
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CN112370573A (en) * 2020-11-04 2021-02-19 陕西佰傲干细胞再生医学有限公司 Cartilage membrane and preparation method thereof
CN114848915A (en) * 2021-01-20 2022-08-05 上海软馨生物科技有限公司 Ear cartilage tissue engineering compound and application thereof
CN115581811A (en) * 2022-11-03 2023-01-10 上海交通大学医学院附属第九人民医院 Autologous tissue engineering living cell meibomian plate substitute and preparation method and application thereof
CN115581811B (en) * 2022-11-03 2024-04-02 上海交通大学医学院附属第九人民医院 Autologous tissue engineering living cell meibomian substitute and preparation method and application thereof

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