CN102989040B - The method of the residual Ear cartilage stem cell of people and structure tissue engineering bone/cartilage thereof - Google Patents

The method of the residual Ear cartilage stem cell of people and structure tissue engineering bone/cartilage thereof Download PDF

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CN102989040B
CN102989040B CN201110268830.9A CN201110268830A CN102989040B CN 102989040 B CN102989040 B CN 102989040B CN 201110268830 A CN201110268830 A CN 201110268830A CN 102989040 B CN102989040 B CN 102989040B
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cell
cartilage
people
ear cartilage
residual
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CN102989040A (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 kind of organization engineered cartilage graft, described organization engineered cartilage graft contains the residual Ear cartilage cell of the people grown under three dimensional structure; The residual Ear cartilage cell of described people went down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations.The invention also discloses the residual Ear cartilage cell of people and build application in cartilage graft in vitro.

Description

The method of the residual Ear cartilage stem 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 utilizing the residual Ear cartilage stem cell constructing 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), show as auricle mark form disappearance, ear field by contracture, deformity cartilage agglomerate (residual Ear cartilage) replace.Clinically the treatment of microtia is mainly got at present to implant ear field subcutaneous after autologous patient costicartilage is engraved as auricle form be main.The major defect of the method causes very large wound to normal costicartilage, and easily cause pneumothorax, Thoracic sink, deformity and cicatrix Deng Gong district complication.
The rise of tissue engineering technique and develop rapidly and bring hope for solving this difficult problem.The chondrocyte composite ear profile state biodegradable stent of current application animal can construct accurate people's auricle form cartilage in vitro.Therefore, as long as there is the cartilage of enough patient's autologous to build seed cell, this technology is hopeful to be converted into actual clinical practice completely, and the external ear for microtia patient is rebuild and brought glad tidings.
But it is the bottleneck of this technology of restriction to clinical conversion that seed cell carrys out source problem always.Two kinds are mainly contained at present: 1) chondrocyte for the seed cell building cartilage; 2) there is the stem cell of cartilage differentiation potential.Because chondrocyte itself has had very strong spontaneous one-tenth cartilage ability, therefore directly build with it that tissue engineering bone/cartilage is the most stable also can ensure success rate; But the chondrocyte source in normal human is very limited, wound of drawing materials is large, and normal chondrocyte amplification in vitro ability is very low, after amplification again very easily aging, dedifferente, thus lose Subchondral drilling ability, therefore, get the autologous normal chondrocyte of patient and carry out external ear cartilage regeneration shortage clinical practice feasibility.There is into the stem cell (as fat stem cell or bone marrow stroma stem cell) of cartilage differentiation potential although wide material sources, amplification ability is strong, but it is not high that these cells in vitro build the success rate with specific modality (as people's auricle) cartilage, and the cartilage of external structure is very unstable after implanting subcutaneous environment, very easily there is ossified or fibrosis, be also unsuitable for building auricle isotonic cartilage.
Belong to discarded tissue in the residual ear Postauricular flap operation outside of microtia patient, cut residual ear and can not cause any damage by normal tissue, and existing research confirms: the residual Ear cartilage cell gone down to posterity in early days can regenerating tissues engineered cartilage.So, is the residual Ear cartilage cell construction auricle form cartilage that microtia patient can be utilized autologous to carry out Postauricular flap? realize this dream, first will solve three key issues: 1. cell number problem: only can be separated in the residual Ear cartilage of a patient and obtain 1-3 × 10 6can individual cell, by these cell amplifications to cell quantity (1.5-3 × 10 built needed for normal size people auricle form cartilage 8individual cell)? 2. cell function problem: whether the residual Ear cartilage cell after amplification also has good Subchondral drilling ability? 3. constructing technology problem: the cartilage how utilizing these cells to build in vitro to have accurate people's auricle form?
In sum, this area is little in the urgent need to finding wound of drawing materials, and has stronger multiplication capacity and Subchondral drilling ability, and the seed cell that Subchondral drilling is stable in subcutaneous environment, and set up corresponding cartilage structure core technology.
Summary of the invention
The present invention aims to provide that a kind of multiplication capacity is strong, cartilage directed differentiation potential strong and in vitro and the regenerating bone or cartilage seed cell that in subcutaneous environment, Subchondral drilling is stable (residual Ear cartilage stem cell), and utilizes the method for the three-dimensional cartilage graft of this cell construction organizational project.
In a first aspect of the present invention, provide a kind of organization engineered cartilage graft, described graft contains the residual Ear cartilage cell of the people grown under three dimensional structure; The residual Ear cartilage cell of described people went 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 that the medically acceptable Biodegradable material that people's residual Ear cartilage cell described in the agglomerate (pellet) or high density that are formed by described people residual Ear cartilage cell aggregation is inoculated in cell patch that culture dish formed or is inoculated in solid by described people residual Ear cartilage cell is formed; Described high density refers to 0.5 × 10 6individual cell/cm 2-5 × 10 6individual cell/cm 2; Described medically acceptable Biodegradable material is selected from: polylactic acid (PLA), polyglycolic acid (PGA), PLGA, poly butyric, condensing model, 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, described people residual Ear cartilage cell is from autologous or allogeneic; The residual Ear cartilage of preferred microtia (microtia) autologous patient.
Cartilage graft provided by the invention, with the entire volume of described graft, the residual Ear cartilage cell concentration of people is wherein 2 × 10 7individual cell/cm 3-10 × 10 7individual cell/cm 3; Preferably, be 2 × 10 7individual cell/cm 3-7 × 10 7individual cell/cm 3.
In another preference, the shape of described graft needs the defect shape of transplanting cartilage to conform to human body; Preferably, cartilage graft provided by the invention the various shape such as shape behaviour auricle, 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, provide a kind of preparation method of organization engineered cartilage graft provided by the invention as above, described method comprises step:
(1) residual for people Ear cartilage cell is carried out plane amplification, and be passaged to for the 1st, 2,3,4,5,6,7 or 8 generations;
(2) the people's residual Ear cartilage cell gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations is mixed to form cell-biomaterial composites by the medically acceptable Biodegradable material of centrifugal formation agglomerate (pellet) or the people's residual Ear cartilage high cell densities inoculation formation cell patch gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations or the people's residual Ear cartilage cell gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations and solid; With
(3) agglomerate, cell patch or complex are carried out chondrocytes in vitro induction; With entire volume, containing 5-50ng/mlTGF-β in described chondrocytes in vitro induced liquid 1(TransformingGrowthFactor-β 1, transforming growth factor-β 1) and the dexamethasone (dexamethasone) of 10-100ng/ml.
Above-mentioned chondrocytes in vitro induced liquid, containing Serum-induced system at CN200510112068.X and CN200610024205.9) in address, related content is incorporated in the present invention.
The preferred external one-tenth chondrocyte induction liquid of the present invention is serum-free induction system, with entire volume, serum substitute (as: ITSpremix containing customary amount, wherein comprise insulin, transferrins, Monohydrated selenium dioxide, linoleic acid, bovine serum albumin, acetone acid, ascorbic acid phosphate) and regular growth cultivation additive (as proline, vitamin C etc.), the TGF-β simultaneously containing 5-20ng/ml 1(TransformingGrowthFactor-β 1, transforming growth factor-β 1), the IGF-I (Insulin-LikeGrowthFactor-I, insulin like growth factor-1) of 10-100ng/ml, and the dexamethasone of 10-100ng/ml (dexamethasone); More preferably, the TGF-β containing 8-12ng/ml in described one-tenth chondrocyte induction liquid 1, the IGF-I of 90-100ng/ml, and the dexamethasone of 30-50ng/ml.
In another preference, described people residual Ear cartilage cell is from autologous or allogeneic; The residual Ear cartilage of preferred microtia (microtia) autologous patient.
In above-mentioned preparation method provided by the invention, the time of external one-tenth chondrocyte induction is 3-20 week; It is preferably 6-12 week.
In above-mentioned preparation method provided by the invention, with the entire volume of complex, residual Ear cartilage cell concentration is in the composite 2 × 10 7-10 × 10 7individual cell/cm 3.
In another preference, the described serum-free for external one-tenth chondrocyte induction becomes the Applicative time of chondrocyte induction liquid to be 24-168 hour, preferred 24-48 hour after chondrocyte mixes with the medically acceptable Biodegradable material of solid.For doing carrier without the need to timbering material, the three-dimensional agglomerate (as pellet) that formed by means of only cell aggregation or cell patch, become the Applicative time of chondrocyte induction liquid to be after cell aggregation thing is formed in 72 hours, preferably in 24 hours.
In another preference, described for becoming the application process of the serum-free medium of chondrocyte induction for directly dripping at complex, pellet or cell patch, changing liquid interval time is 24-96 hour.
In a third aspect of the present invention, provide a kind of organization engineered cartilage graft provided by the invention as above and building the application in engineered auricle graft.
In a fourth aspect of the present invention, provide the residual Ear cartilage cell of a kind of people and build application in organization engineered cartilage in vitro.
In a fifth aspect of the present invention, provide a kind of people residual Ear cartilage cell and build application in engineered auricle graft in vitro.
In another preference, described people residual Ear cartilage cell is from autologous or allogeneic; More preferably, the residual Ear cartilage of microtia (microtia) autologous patient.
Accordingly, the invention provides wound of drawing materials little, there is stronger multiplication capacity and Subchondral drilling ability, and the seed cell that Subchondral drilling is stable in subcutaneous environment, and establish corresponding cartilage structure core technology.
Accompanying drawing explanation
Fig. 1 shows residual Ear cartilage cell induction bone marrow stroma stem cell (BMSC) becomes cartilage situation at nude mice by subcutaneous.Wherein A, D are respectively the general appearance of residual Ear cartilage cell and BMSC Dual culture group and II Collagen Type VI dyes; B, E are respectively general appearance and the dyeing of II Collagen Type VI of simple residual Ear cartilage groups of cells; C, F are respectively general appearance and the dyeing of II Collagen Type VI of simple bone marrow stroma stem cell group.
Fig. 2 show different generation residual Ear cartilage cells in vitro plane amplification time cellular morphology (100 ×) and proliferative conditions; Wherein pld4 represents the residual Ear cartilage cell that 1st generation increases the 4th day, by that analogy.Be growth curve corresponding to identical generation below cellular morphology.
Fig. 3 shows different generation residual Ear cartilage cellular cartilage formational situation.P0d5 represents primary amplification to the residual Ear cartilage cell of the 5th day and the pellet situation prepared with it, by that analogy.
Fig. 4 show the 8th generation residual Ear cartilage cell three-dimensional differentiation potential.From left to right be followed successively by the 8th generation residual Ear cartilage cell through the Alizarin red staining of osteogenic induction after 3 weeks, form pellet through becoming the II Collagen Type VI immunohistochemical staining of chondrocyte induction after 3 weeks and through oil red dyeing (200 ×) of adipogenic induction after 3 weeks.
Fig. 5 show the 8th generation residual Ear cartilage cell through the situation of induced synthesis cartilage.A: be pellet general appearance (n=3), B are each group of pellet weight in wet base (n=5), and C is each group of pellet diameter (n=5).
Fig. 6 shows the 8th generation residual Ear cartilage cell pellet through the cardinal principle of induced synthesis cartilage and histology's (dyeing of HE, Safranin-O and II Collagen Type VI).
Fig. 7 shows the 3rd generation residual Ear cartilage cell-PGA/PLA composite body and becomes the chondrocyte induction cardinal principle of 8 weeks and II Collagen Type VI coloration result outward; Wherein A is for substantially to see, and B is II Collagen Type VI immunohistochemical staining, (100 ×).
Fig. 8 show the 5th generation residual Ear cartilage cell, and bone marrow stroma stem cell (BMSC) compound PGA support, becomes chondrocyte induction 6 Zhou Houzhi in the cardinal principle of nude mice by subcutaneous after 12 weeks and HE (200 ×) in vitro.
Fig. 9 show the 3rd generation residual Ear cartilage cell-ear supporter composite body become the chondrocyte induction cardinal principle of 8 weeks and histological stain result outward; Wherein A is for substantially to see, and B is HE dyeing, (100 ×).
Detailed description of the invention
For problems of the prior art and difficulty, first inventor has investigated the Subchondral drilling ability after residual Ear cartilage cell expansion ex vivo, result finds unfortunately, the Subchondral drilling ability of residual Ear cartilage cell sharply declines along with subculture in vitro separately, even compared with normal chondrocyte declines faster, substantially, the typical cartilage (Fig. 3) of homogenizing can not be formed when passing to for the 3rd generation, the requirement of tissue engineering bone/cartilage structure to seeding cell functions cannot be met, this phenomenon is considered to the performance that a kind of cartilage phenotype dedifferentes usually, the prompting giving those skilled in the art is: carry out Postauricular flap by the residual Ear cartilage cell that increases and seem not possess clinical practice feasibility.
But, in research process, inventor is surprised to find that, although residual Ear cartilage cell is easy to lose Subchondral drilling ability, but it has superpower multiplication capacity, that is, dedifferente although there occurs, do not occur obvious catabiosis, this point is obviously different from the chondrocyte of normal source: normal chondrocyte multiplication capacity from 1st generation just declines rapidly, when passing to for the 5th generation obviously aging, be difficult to breed (and the ratio of breeding of at every turn going down to posterity is also very limited); Residual Ear cartilage cell then has very strong multiplication capacity, even if (in 1: 3 ratio) amplification still can keep very high proliferation activity (Fig. 2) (generally passing to 3-4 for substantially meeting the auricle form cartilage of structure normal size to the quantitative requirement of seed cell) to the 8th generation.
So, whether does the residual Ear cartilage cell after amplification still have Subchondral drilling potential? in view of residual Ear cartilage cell proliferation in vitro ability by force, not easily the characteristic such as aging be different from the chondrocyte of differentiation and maturation completely, and it is similar with the stem cell properties reported in the past, therefore inventor courageously supposes, the cell in residual Ear cartilage source is likely the stem cell that left behind in a kind of growth course.Based on this hypothesis, inventor has carried out multidirectional differentiation-inducing experiment to residual Ear cartilage cell, and surprisingly finds, even if the cell in the 8th generation of increasing still has skeletonization, becomes cartilage, becomes the multi-lineage potentials (Fig. 4) such as fat.These results are pointed out, and a kind of really multiplication capacity of the cell in residual Ear cartilage source is strong and have the cell of the similar stem cell properties of multi-lineage potential, and this great discovery had no report in the past, and inventor is referred to as residual Ear cartilage stem cell for the time being.For fully confirming this great discovery, inventor has carried out proliferation potential and the checking of many differentiation potentials to the cell that the residual Ear cartilage of many cases is originated, result confirms, the cell in all residual Ear cartilage sources all has the multi-lineage potential such as very strong proliferation potential and bone, cartilage, fat, the stem cell properties showing residual Ear cartilage derived cell is not fortuitous phenomena, but a kind of objective reality repeatably characteristic, these results point out residual Ear cartilage cell to be all expected in quantity and function aspects meet the requirement building auricle form cartilage.
Although above-mentioned all more important discoveries, how to apply residual Ear cartilage stem cell and remain a huge challenge to build clinical applicable auricle form cartilage.Fully confirming on the basis of its stem cell properties, inventor combines induced dry-cell (as bone marrow stroma stem cell etc.) in the past and builds the experience of three-dimensional cartilage, by make repeated attempts and kinds of schemes to when optimizing and combining, establish stable residual Ear cartilage stem cell cartilage directional induction system (Fig. 5, Fig. 6), and in conjunction with three-dimensional biodegradable stent, apply the three-dimensional chondroid tissue (Fig. 7) that residual Ear cartilage stem cell constructs homogenizing maturation in vitro.Et al. Ke result shows, and the cartilage of residual Ear cartilage stem cell regenerating can maintain stable cartilage phenotype (Fig. 8) in subcutaneous environment.On this basis, further combined with accurate people's auricle form cartilage constructing technology (as computer-aided design rack forming etc.) based on animal maturation differentiation chondrocyte of previously having set up, pertinent literature comprises: LiuY, ZhangL, ZhouG, LiQ, LiuW, YuZ, LuoX, JiangT, ZhangW, CaoY.Invitroengineeringofhumanear-shapedcartilageassiste dwithCAD/CAMtechnology.Biomaterials.2010.03., 31 (8): 176-183, Patents comprises CN200710044711.9, by the PGA/PLA timbering material compound of residual for people Ear cartilage stem cell and accurate people's auricle form, and induce with the condition of above-mentioned optimization, finally construct the tissue engineering bone/cartilage (Fig. 9) with accurate people's auricle form in vitro.
In sum, inventor builds the not enough bottleneck problem in seed cell source for cartilage, find based on the accident in residual Ear cartilage cell injuring model process with very strong proliferation potential, bold hypothesis and confirm first cell that residual Ear cartilage originate be a kind of multiplication capacity by force and there is the cell of the similar stem cell properties of multi-lineage potential, and further combined with inventor's stem cell cartilage directional induction in the past and three-dimensional cartilage constructing technology and experience, and accurately people's auricle form cartilage builds core technology, by making repeated attempts and optimizing and combining series of key techniques parameter, finally establish the external three-dimensional cartilage constructive system based on residual Ear cartilage stem cell.On this basis, the present invention is completed.
The residual Ear cartilage cell of people
In the present invention, people's residual Ear cartilage cell derived is in the residual Ear cartilage tissue of microtia autologous patient or allosome.Patient age and sex are not particularly limited.
Being separated the method obtaining residual Ear cartilage cell has multiple, in an embodiment of the present invention, described method is under general anesthesia or cuts the residual Ear cartilage of microtia patient under local anaesthesia, thoroughly peels off the connective tissue around the residual Ear cartilage of rejecting and perichondrium, by cartilage chopping to 1-2mm 3fritter, with 0.25% chloromycetin solution soak 30-40min, PBS rinse after with 0.25% trypsin+0.02%EDTA vibrate in 37 DEG C of constant-temperature tables digestion 30min, then with 0.1% collagenase vibrate in 37 DEG C of constant-temperature tables digest 12h.Digestive system is filtered with 200 mesh filter screens, collect filtrate in 50mL centrifuge tube, the centrifugal 8min of 1800rpm, to abandon after supernatant with suitable culture fluid (as containing 10% hyclone, bFGF10ng/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, inoculate about 2 × 10 in every 10cm culture dish 6individual living cells, in 37 DEG C, 5%CO 2, 100% saturated humidity condition under cultivate, the next day change liquid.After Growth of Cells closely converges, digest with 0.25% trypsin+0.02%EDTA, carry out passage in 1: 3 ratio.
In the present invention, preferably the 1st, 2,3,4,5,6,7 or 8 generation cell prepare tissue engineering bone/cartilage.
Although to be a kind of multiplication capacity strong and have the cell of the similar stem cell properties of multi-lineage potential for the cell in residual Ear cartilage source, but the residual Ear cartilage cell of people also possesses this ability that normal mature chondrocyte induction BMSC becomes cartilage simultaneously, in the test confirming this ability, need the Coculture techniques of use also described in detail in CN200610024205.9, related content is incorporated to the present invention.
Biodegradable material
The material of organization engineered cartilage used in the present invention is medically acceptable Biodegradable material, comprises (but being not limited to):
(a) degradability synthesized polymer material, such as polylactic acid (PLA), polyglycolic acid (PGA), PLGA, poly butyric (PHB), condensing model (polyanhydrides), poly-phosphazo (polyphosphazenes), polyamino acid (polyaminoacid), false polyamino acid (pesudo-polyaminoacid), poe (polyorthoesters), polyester urethane (polyesterurethane), Merlon (polycarbonate), Polyethylene Glycol, hyaluronic acid, poly-para-dioxanone (polydioxanone) etc.,
(b) natural degradable material, such as collagen (collagen), gelatin (gelatin), ammonia polyose of candy (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginate and various acellular matrixes etc.;
The composite of the copolymer of (c) above-mentioned material or compound material, especially macromolecular material and natural material, and the composite of solid material and syringeability material.
Preferably medically acceptable Biodegradable material is solid material or solid, liquid composite material, such as polylactic acid (PLA), polyglycolic acid (PGA), collagen etc.Material in the present invention can be prefabricated into various accurate size and shape, builds with the cartilaginous tissue adapting to different size and shape.When material is solid type material, directly by the prefabricated size and shape becoming to need, also accurate plasticity can be carried out by the model of area of computer aided and rapid shaping technique customization to material.
Organization engineered cartilage graft
Organization engineered cartilage graft provided by the invention contains the residual Ear cartilage cell of the people grown under three dimensional structure; The residual Ear cartilage cell of described people was the 1st, 2,3,4,5,6,7 or 8 generations; It is preferably the 1st, 2,3,4 or 5 generations; It is more preferably the 1st, 2 or 3 generations.
Described three dimensional structure (is generally 1 × 10 by described people residual Ear cartilage cell aggregation agglomerating (pellet) or high density 6individual cell/cm 2-5 × 10 6individual cell/cm 2) be inoculated in the cell patch that culture dish formed; Or the medically acceptable Biodegradable material being inoculated in solid by described people residual Ear cartilage cell is formed.
With the entire volume of described graft, the residual Ear cartilage cell concentration of people is wherein 2 × 10 7individual cell/cm 3-10 × 10 7individual cell/cm 3; Be preferably 2 × 10 7individual cell/cm 3-7 × 10 7individual cell/cm 3.
Inventor finds in an experiment, and the Subchondral drilling ability of residual Ear cartilage cell declines rapidly with amplification of going down to posterity, and cell more than 2nd generation has lost Subchondral drilling ability.But be surprised to find that in research, these cells have powerful multiplication capacity, even if (in 1: 3 ratio) amplification still can keep very high proliferation activity to the 8th generation, and to the quantitative requirement of seed cell, (concrete number, depending on constructed particular organization and dimensional culture environment, generally builds auricle form and about needs 2 × 10 on PGA support for substantially meeting the auricle form cartilage building normal size to pass to 3-4 8cell).Further research confirms, residual Ear cartilage cell has the multi-lineage potential of cells and characteristic of stem, directional induction experiment confirms, the residual Ear cartilage cell of different generation 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 Ear cartilage stem cell based on these results, and thus propose the theory being built tissue engineering bone/cartilage graft by cartilage directional induction.Based on this theory, by optimization and the integration of series of key techniques parameter, establish stable residual Ear cartilage stem cell in vitro cartilage and build cultivating system.
Based on these original discoveries, in conjunction with the existing stem cell in vitro of inventor three-dimensional cartilage constructing technology experience, and accurate people's auricle form cartilage constructing technology (as computer-aided design rack forming etc.) based on animal maturation differentiation chondrocyte of previously having set up, by the PGA/PLA timbering material compound of residual for people Ear cartilage stem cell and accurate people's auricle form, utilize the cultivating system of above-mentioned optimization, the tissue engineering bone/cartilage of external structure accurate people's auricle form obtains successfully.Inventor has also carried out more rigorous large scale experiment demonstration on this basis further, finally completes the present invention.
The residual Ear cartilage cell-biomaterial composites of people
In the present invention, the cell implantation concentrations of the residual Ear cartilage cell-biomaterial composites of people is about 2 × 10 usually 7/ ml to 7 × 10 7/ ml or higher.Material is solidity material or solid, liquid composite material, with culture fluid adjustment cell concentration, then with solidity material mixing, wherein during mixing, culture fluid and the ratio of solidity material are not particularly limited, but are as the criterion with the maximum that this solid material can adsorb culture fluid.When timbering material is special 3D shape, 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, is mixed by the described people residual Ear cartilage cell of some with pharmaceutically acceptable Biodegradable material, then through external one-tenth chondrocyte induction.Described method comprises step:
The first step, carries out plane by the cell in residual for people Ear cartilage source and cultivates amplification, and be passaged to 1-8 generation;
Second step, forms cell patch by the cell of above-mentioned amplification of going down to posterity by centrifugal formation agglomerate (pellet) or high density inoculation; Or the medically acceptable Biodegradable material of these cells and solid is mixed to form cell-biomaterial composites;
3rd step, is placed in into agglomerate, diaphragm or complex in chondrocyte induction liquid and carries out In vitro culture.
Plane in the first step method of cultivating amplification such as but not limited to, be resuspended in DMEM in high glucose culture fluid by being separated the residual Ear cartilage cell of people obtained, then obtained cell suspension is inoculated on 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; Preferably 1: 3-1: 7; Be more preferably 1: 3-1: 5) carry out passage to new culture dish, continue Secondary Culture under the same conditions.With the entire volume of described DMEM in high glucose culture fluid, preferably wherein containing 10-20%FBS, 1-10ng/mlbFGF; Inoculum concentration preferably inoculates about 0.5-2 × 10 in every 10 cm dishes 6individual living cells.
Second step the people of monolayer culture residual Ear cartilage cell reach nearly 90% converge time, digest, cell counting, when display viable count is more than 90%, centrifugally makes cell precipitation, supernatant discarded, cell dispersion; The cell of resuspended collection, centrifugal make cell aggregation formed pellet or by high cell densities inoculation formed cell patch; Also can by cell according to 2 × 10 7-10 × 10 7individual cell/cm 3density be inoculated into solid medically acceptable Biodegradable material on form complex.
In 3rd step by obtain cell patch, pellet or complex conventional culture conditions (such as but not limited to, 37 DEG C, 5%CO 2, 100% saturated humidity, conventional DMEM in high glucose culture fluid containing 10% serum) under cultivate 24-48 hour after, be replaced by into chondrocyte induction liquid, general adopt conventional become chondrocyte induction liquid, preferred serum-free becomes chondrocyte induction liquid.
Adopted in an embodiment of the invention become chondrocyte induction liquid at CN200510112068.X and CN200610024205.9 containing serum) in existingly to address, related content is incorporated in the present invention.
In another embodiment of the present invention, adopt serum-free induction system, with entire volume, serum substitute (as: ITSpremix containing customary amount, insulin-containing, transferrins, Monohydrated selenium dioxide, linoleic acid, bovine serum albumin, acetone acid, ascorbic acid phosphate) and regular growth cultivation additive (as proline, vitamin C etc.), the TGF-β simultaneously containing 5-20ng/ml 1(TransformingGrowthFactor-β 1, transforming growth factor-β 1), the IGF-I (Insulin-LikeGrowthFactor-I, insulin like growth factor-1) of 10-100ng/ml, and the dexamethasone of 10-100ng/ml (dexamethasone); More preferably, the TGF-β containing 8-12ng/ml in 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 the cytokine of other various promotion cartilage differentiation of compound or somatomedin, as BMP, CDMP etc., to strengthen chondrocyte induction efficiency and to improve extracellular matrix synthesis capability etc.
One-tenth chondrocyte induction liquid every 2-4 days that the present invention adopts changes liquid once, and the external evoked time is 3-20 week, preferred 6-12 week.
Organization engineered cartilage graft of the present invention builds in cartilage process, be mainly static culture, or add mechanical stimulation in mode that is centrifugal or vibration, also can by the mechanical environment of cartilage in the biological reactor simulation body of cartilage, similar mechanical stimulation is applied to the cartilage of external structure, promotes the maturation of cartilage and the improvement of mechanical property.
With the organization engineered cartilage that the inventive method is formed, i.e. the complex that forms of the residual Ear cartilage stem cell of people and solidity biomaterial, this complex subcutaneous or cartilage defect in implantable after external one-tenth chondrocyte induction.
In one embodiment of the invention, the residual Ear cartilage stem cell of employment and culture fluid make concentration is 5 × 10 7then the cell suspension (being not limited only to this concentration) of/ml be that main biologic bracket material forms complex (complex size, shape are determined according to the size of cartilage defect and shape) with polyglycolic acid (PGA).This complex (is not limited only to this time in thoughtful 8 weeks through chondrocytes in vitro directional induction 6, every 2 to 4 days of period changed liquid once, to ensure cytotrophy and induced efficiency), implant when vitro tissue through engineering approaches cartilage begins to take shape subcutaneous or corresponding cartilage defect.Also material can be made into the three-dimensional rack (as people's auricle form) with fine structure profile, inoculate people's residual Ear cartilage stem cell and carry out external evoked, implant cartilage defect after 6-8 week (being not limited only to this time).
Application process
After people's residual Ear cartilage cell and the solid-state biomaterial of degradable form complex, carry out into chondrocyte induction in vitro, after forming the organization engineered cartilage being applicable to cartilage defect size and shape, then the cartilage defect that implants.
Term
Term " people's residual Ear cartilage cell ", " cell in residual Ear cartilage source " or " the residual Ear cartilage stem cell of people " can exchange use, all refer to the conventional isolated cell technology using this area, what obtain from the residual Ear cartilage tissue of microtia autologous patient or allosome can secrete II Collagen Type VI (collagenII), the polygon attached cell of the cartilage specificity substrate such as glycosaminoglycans (glycosaminoglycan, GAG).
Term " separation of people's residual Ear cartilage cell " refers to the process separated from the residual ear tissue of microtia patient by residual Ear cartilage cell.
Term " amplification of people's residual Ear cartilage cell " refers to obtain a large amount of residual Ear cartilage cell and the process of a large amount of propagation in environment in vitro.
Term " one-tenth chondrocyte induction " refers to provide special biochemical environment, makes have the cellular expression cartilage phenotype of cartilage differentiation potential and possess into the process of cartilage ability.
Term " inoculation " refers to the process be uniformly distributed in by cell on three-dimensional stent material.
Term " autotransplantation " refers to required biological living material (as residual Ear cartilage cell) to take out from certain individuality and is applied to the process of same individuality again.
Term " three dimensional structure " is exactly stereochemical structure, refers to and occupies three-dimensional structure, and three-dimensional is three axles of coordinate axes, i.e. x-axis, y-axis, z-axis, wherein x represents space, left and right, and y represents upper and lower space, and z represents space, front and back.So-called three dimensions refers to the space residing for us, before and after having can be understood as-and up and down-left and right.The seed cell grown at three dimensional structure in the present invention can be the complex that the medically acceptable Biodegradable material by seed cell being inoculated in solid is formed; Also can be by the cell mass of centrifugal for seed cell formation; Or seed cell is carried out high density inoculation forms cell patch.Described seed cell is the residual Ear cartilage cell of people.
The above-mentioned feature that the present invention mentions, or the feature that embodiment is mentioned can combination in any.All features that this case description discloses can with any composition forms and use, each feature disclosed in description, anyly can provide identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
The present invention is for the significant contribution of this area: confirm or establish (one) first, under two-dimentional condition of culture, the cell in residual Ear cartilage source has very strong amplification ability, even if (in 1: 3 ratio) amplification still can keep very high proliferation activity to the 8th generation; (2), the cell in residual Ear cartilage source has the distinctive multi-lineage potential of stem cell, even if amplification still maintains the multi-lineage potentials such as good bone, cartilage, fat to the 8th generation; (3), stable residual Ear cartilage stem cell cartilage directional induction system is established; (4), establish the core technology that residual Ear cartilage stem cell in vitro builds three-dimensional cartilage, and confirm its cartilage phenotype stability in vivo in subcutaneous environment; (5) core technology building accurate people's auricle form cartilage based on people's residual Ear cartilage stem cell in vitro, is established.
Major advantage of the present invention is:
(1) can autogenous cell be applied, and once draw materials and both can obtain enough cell concentrations;
(2) process of drawing materials not damaging normal tissue completely;
(3) extracorporeal culturing method is easy to learn, easy to utilize;
(4) external one-tenth chondrocyte induction method is easy and simple to handle, becomes cartilage definite effect reliable;
(5) can by the size and shape of the prefabricated graft of cartilage defect size and shape, to reach accurate reparation;
(6) compared with other stem cell, the cartilage form that residual Ear cartilage stem cell in vitro builds more easily accurately maintains, and after implanting subcutaneous environment, cartilage phenotype remains relatively stable.
(7) the present invention set up and the chondrocyte induction system optimized not containing serum, definite ingredients, clinical practice safety is high, is convenient to standardization, is easy to form industrialization product.
The method that application the present invention determines, can obtain the residual Ear cartilage cell possessing Subchondral drilling ability in a large number, for building auricle morphology and history engineered cartilage.This tissue engineering bone/cartilage not only has good organization and learns structure, biochemistry composition and mechanical strength, and not easily shrink in external structure process, can maintain original auricle form constant, and it is stable to implant subcutaneous rear one-tenth cartilage, not easily ossified, fibrosis phenomenon occurs.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise all percentage ratio and number by weight.
Unit in percent weight in volume in the present invention is well-known to those skilled in the art, such as, refer to the weight of solute in the solution of 100 milliliters.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
In following embodiment, and DMEM in high glucose culture medium (Dulbecco ' sModificationofEagle ' sMedium, highglucose) purchased from American HyClone company, article No.: SH0495; Hyclone (FetalBovineSerum, FBS) purchased from American HyClone company, article No.: SV30087.02; Vitamin C (VitaminC) purchased from American Sigma company, article No.: A8960-5G; Trypsin Trypsin) purchased from American Amresco company, article No.: 0458; Collagenase NB4 powder (CollagenaseNB4) purchased from German Serva company, article No.: 17454; Basic fibroblast growth factor (basicfibroblastgrowthfactor, bFGF) purchased from American R & D company, article No.: 233-FB/CF; ITS universal cultivation intermixture (ITSpremix) purchased from American Sigma company, article No.: 12521; Transforming growth factor-beta 1 (TransformingGrowthFactor-β 1, TGF-β 1) purchased from American R & D company, article No.: 240-B-010; Insulin like growth factor-1 (Insulin-LikeGrowthFactor-I, IGF-I) purchased from American R & D company, article No.: 291-G1-010; Dexamethasone (dexamethasone) purchased from American Sigma company, article No.: D4902-1G; Insulin (insulin) purchased from American Sigma company, article No.: I5500; Indomethacin (indomethaein) purchased from American Sigma company, article No.: I-8280; β-phosphoglycerol purchased from American Sigma company, article No.: G9422; 1-methyl-3-isobutyl group xanthine (IBMX) purchased from American Sigma company, article No.: I5879.
Embodiment 1
The acquisition of the residual Ear cartilage stem cell of people and cultivation
Cell gets the residual Ear cartilage tissue of microtia autologous patient, donor age 8-30 year, healthy, without malignant tumor, infectious disease and autoimmune disease.
(1) draw materials: under operating room aseptic condition, after anesthesia is satisfied, routine disinfection drape, cuts the residual ear tissue of patient, is placed in 50mL sterile centrifugation tube, soaks preserve and transport with 40mL physiological saline solution.
(2) be separated residual Ear cartilage cell: in super-clean bench under aseptic condition, thoroughly peel off the connective tissue around the residual Ear cartilage of rejecting and perichondrium, by cartilage chopping to 1-2mm 3fritter, 30-40min is soaked with 0.25% chloromycetin solution, with 0.25% trypsin+0.02%EDTA (Amresco after PBS rinses, USA) vibration digestion 30min in 37 DEG C of constant-temperature tables, again with 0.1% collagenase (CollagenaseNB4, Serva, Germany) solution vibrate in 37 DEG C of constant-temperature tables digestion 12h.Filter Digestive system with 200 mesh filter screens, collect filtrate in 50mL centrifuge tube, the centrifugal 8min of 1800rpm.
(3) inoculate: to contain 10% hyclone, 10ng/mlbFGF, 300 μ g/mlL-glutamine, 50 μ g/ml vitamin Cs after abandoning supernatant, 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, in every 10cm culture dish, inoculates about 2 × 10 6individual living cells.Be placed in 37 DEG C, 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: can Fusion Strain be reached after general primary cell cultivates 7-8 days under these conditions, can Secondary Culture be continued.Culture fluid is absorbed when going down to posterity, with a small amount of PBS washing once, add the Digestive system (0.25% trypsin+0.02%EDTA (Amresco of 1.5-2.0ml, USA)), after seeing most cells kytoplasm retraction, form change circle under mirror, absorb Digestive system gently, add the appropriate DMEM conditioned medium containing serum and stop digestion, collecting cell suspension, be passaged in new culture dish in 1: 3 ratio, continue to cultivate at identical conditions, the next day change the culture fluid of 2/3rds amounts.Can Fusion Strain be reached again after general 4-5 days, can Secondary Culture be continued.With the 1st, 2,3,4 or 5 generation cell be used for experiment effect better.
Observe under inverted microscope, the results are shown in Figure 2.
Result shows, the residual Ear cartilage cell of people is spindle shape or polygon.Growth curve display is primary all keeps vigorous proliferation activity to each generation cell in the 8th generation, has no obvious aging sign.
Embodiment 2
Pellet is prepared with residual Ear cartilage stem cell
(1) collecting cell: the residual Ear cartilage cell of people of monolayer culture reaches 90% when converging, and applies 0.25% pancreatin+0.02%EDTA and digests.With cell counter counting after cell harvesting, Trypan Blue display viable count makes cell precipitation, abandoning supernatant more than the centrifugal 8min of 90%, 1800rpm, vibrating dispersion cell.
(2) prepare pellet: after collecting cell, be prepared into 0.4 × 10 so that Nostoc commune Vanch liquid is resuspended 6the cell suspension of/ml.Get this suspension of 1ml in 15ml centrifuge tube, the centrifugal 5min of 1000rpm, makes cell pellet.Slightly unscrew bottle cap, be placed in 37 DEG C, 5%CO 2, saturated humidity incubator in cultivate, change liquid (Nostoc commune Vanch liquid or become chondrocyte induction liquid) afterwards every other day.After 3 weeks, each group of pellet is drawn materials respectively and carry 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) purchased from American Sigma company, is dissolved in the PLA solution that dichloromethane makes 0.3%.
(1) cylindrical PGA/PLA support: be 4mg/ block by PGA non-woven cotton accurate weighing, is pressed into the cylinder fritter of diameter 5mm, thick 1mm with mould, after shape is fixing, drip PLA solution to saturated, air-dry.Before using, timbering material is immersed in completely in 75% ethanol and sterilizes 30 minutes.PBS rinses 3 times, then soaks 10 minutes by the DMEM culture medium containing 10% hyclone, blots rear preparation inoculating cell.
(2) people's auricle form PGA/PLA support: take PGA non-woven cotton and be about 800mg/ block, with special (such as document: LiuY, ZhangL, ZhouG, LiQ, LiuW, YuZ, LuoX, JiangT, ZhangW, CaoY.Invitroengineeringofhumanear-shapedcartilageassiste dwithCAD/CAMtechnology.Biomaterials.2010.03., disclosed in 31 (8): 176-183, disclosed in patent CN200710044711.9) the personalized auricle form of patient's personalized auricle mould compacting patient, after dimensionally stable, drip PLA solution and continue moulding in mould, after form is fixing, Auricular framework is immersed in completely in 75% ethanol and sterilizes 30 minutes.PBS rinses 3 times, then soaks 10 minutes by the DMEM culture medium containing 10% hyclone, blots rear preparation inoculating cell.
Embodiment 4
People's residual Ear cartilage cell induction bone marrow stroma stem cell builds three-dimensional cartilage
(1) acquisition of the residual Ear cartilage cell of people and cultivation: see embodiment 1;
(2) acquisition of bone marrow stroma stem cell and cultivation:
A () is drawn materials and original cuiture: under aseptic condition, extracts sheep marrow 10-20ml in 50ml centrifuge tube.Mend PBS to 45ml, the centrifugal 10min of 1800rpm, abandon supernatant, after repeated washing like this 2 times, 1 time is cleaned again with BMSC culture fluid (the low sugar DMEM culture fluid containing 10%FBS), finally resuspended with BMSC culture fluid and be inoculated in (every 2ml bone marrow inoculates 1 ware) in 10cm culture dish.Change liquid first after 5 days, when changing liquid, first wash away not adherent hemocyte with PBS.Within 2-4 days afterwards, change liquid once.
(b) Secondary Culture: carry out Secondary Culture when cell grows to 80% Fusion Strain.When going down to posterity, absorb culture fluid, with a small amount of PBS washing once, add the Digestive system (0.25% trypsin+0.02%EDTA (Amresco, USA)) of 1.5-2.0ml, after seeing most cells kytoplasm retraction, form change circle under mirror, absorb Digestive system gently, add the appropriate DMEM conditioned medium containing serum and stop digestion, collecting cell suspension, be passaged in new culture dish in 1: 3 ratio, continue to cultivate at identical conditions.The next day change the culture fluid of 2/3rds amounts, can Fusion Strain be reached again after general 2-5 days, can Secondary Culture be continued.Experiment effect is used for for cell better with 1-4.
(3) inoculating cell-material composite: collect bone marrow stroma stem cell (BMSC) and residual Ear cartilage stem cell, the middle BMSC of Dual culture group (n=3) mix homogeneously with 3: 1 ratios with residual Ear cartilage cell after according to 5 × 10 7individual cell/cm 3density be inoculated on cylindrical PGA support; According to 5 × 10 in simple chondrocyte group (n=3) 7individual cell/cm 3density inoculation simple residual Ear cartilage stem cell; According to 5 × 10 in simple BMSC group (n=3) 7individual cell/cm 3density inoculate simple BMSC; By cell-material composite of having inoculated in 37 DEG C, 5%CO 2, 100% humidity environment in static culture 5 hours.Slowly add the common DMEM in high glucose culture fluid containing 10%FBS of temperature in advance, continue to cultivate, every 3-4 days changes liquid once, carries out nude mice by subcutaneous Hui Zhi after 1 week.
(4) nude mice by subcutaneous heeling-in: plant in nude mice dorsal sc by the In vitro culture each group of cell-material composite of 1 week, draws materials after 10 weeks and carries out general appearance and the detection of II Collagen Type VI SABC.
A () is seen substantially:
Nude mice by subcutaneous heeling-in is after 10 weeks, and Dual culture 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 have certain elasticity; And simple BMSC group is dark yellow outward appearance, volume has obvious contraction, quality more soft (Fig. 1).
(b) II Collagen Type VI SABC: nude mice by subcutaneous heeling-in is after 10 weeks, and Dual culture group and simple chondrocyte group II expression of collagen are strong positive, 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 Ear cartilage cell has the chondrogenetic ability of induction BMSC.
Embodiment 5
The spontaneous one-tenth cartilage of people's residual Ear cartilage stem cell in vitro
(1) acquisition of the residual Ear cartilage cell of people and cultivation: draw materials, the method for original cuiture and Secondary Culture is see embodiment 1;
(2) the residual Ear cartilage stem cell II expression of collagen in plane amplification procedure: the residual Ear cartilage stem cell each generation being cultured to the 5th day carries out II Collagen Type VI immunofluorescence dyeing, fluorescence microscopy Microscopic observation, result shows: its II expression of collagen of residual Ear cartilage stem cell in plane amplification procedure increases with passage number and sharply lowers, to 2nd generation only minute quantity cell be that II Collagen Type VI is 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 Nostoc commune Vanch liquid (the DMEM in high glucose culture fluid containing 10%FBS) is resuspended 6the cell suspension of/ml.Get this suspension of 1ml in 15ml centrifuge tube, the centrifugal 5min of 1000rpm, makes cell pellet.Slightly unscrew bottle cap, be placed in 37 DEG C, 5%CO 2, saturated humidity incubator in cultivate, change liquid (Nostoc commune Vanch liquid) afterwards every other day, after 3 weeks, each group of pellet drawn materials respectively and detect.
A () is seen substantially:
Each generation (primary to the 3rd generation) residual Ear cartilage stem cell all can form spherical porcelain white cartilage sample agglomerate (pellet) that diameter is about about 2mm, wherein before 2nd generation, the residual Ear cartilage stem cell of (containing 2nd generation) becomes pellet quality pliable and tough and has certain elasticity, and the pellet quality more soft (Fig. 3) after 2nd generation.
(b) II Collagen Type VI SABC: the residual Ear cartilage pellet that stem cell becomes of (containing 2nd generation) before 2nd generation, its II Collagen Type VI positive expression, and there is typical cartilage lacuna structure; And the pellet after 2nd generation, its II Collagen Type VI is weak expression, and cartilage lacuna spline structure not obvious (Fig. 3).
Result shows, people's residual Ear cartilage stem cell only in early days (lower generation) shows certain spontaneous one-tenth cartilage ability.
Embodiment 6
People residual Ear cartilage stem cell Multidirectional Differentiation Induction experiments:
(1) acquisition of the residual Ear cartilage stem cell of people and cultivation: draw materials, the method for original cuiture and Secondary Culture is see embodiment 1;
(2) people's residual Ear cartilage stem cell osteogenic induction: the residual Ear cartilage stem cell cultivating for the 8th generation with osteogenic induction liquid, every 2-3 days changes liquid once, induces after 3 weeks and carries out Alizarin red staining.The concrete composition of osteogenic induction liquid comprises: low sugar DMEM culture medium, 10% hyclone (fetalbovineserum, FBS), 10mM dexamethasone, 10mM β-phosphoglycerol, 50mM vitamin C.Induction result: visible obviously calcium tuberosity under mirror, Alizarin red staining is positive (Fig. 4), show the 8th generation residual Ear cartilage stem cell still there is osteogenic potential.
(3) people's residual Ear cartilage stem cell becomes chondrocyte induction: by the 8th generation residual Ear cartilage stem cell be prepared into pellet, and to become chondrocyte induction liquid to cultivate, within every 2 days, change liquid once, induce after 3 weeks and carry out tissue slice and II Collagen Type VI immunohistochemical staining.Pellet preparation and In vitro culture are with reference to embodiment 4.The concrete composition of chondrocyte induction liquid is become to comprise: with entire volume, the TGF-β containing DMEM in high glucose culture medium, 10ng/ml 1the IGF-I of 100ng/ml,, the dexamethasone of 40ng/ml, one times of content ITSpremix (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.Induction result: II Collagen Type VI stained positive, visible typical cartilage lacuna spline structure (Fig. 4), show the 8th generation residual Ear cartilage stem cell still there is Chondrogenesis.
(4) people's residual Ear cartilage stem cell adipogenic induction: the residual Ear cartilage stem cell cultivating for the 8th generation with adipogenic induction liquid, every 2-3 days changes liquid once, induces after 3 weeks and carries out oil red dyeing.The concrete composition of adipogenic induction liquid comprises: low sugar DMEM culture medium, 10% hyclone (fetalbovineserum, FBS), 1 μM of dexamethasone, 0.5mM1-methyl-3-isobutyl group xanthine (IBMX), 10M insulin, 200 μMs of indomethacins (indomethaein).Induction result: under mirror, visible obviously fat drips, oil red stained positive (Fig. 4), show the 8th generation residual Ear cartilage stem cell still there is into fat potential.
Embodiment 7
Different one-tenth cartilage system induces residual Ear cartilage stem cell
(1) collecting cell: with reference to embodiment 2;
(2) pellet and experiment grouping is prepared: pellet preparation is with reference to embodiment 2, prepare 15 pellet altogether, wherein serum-free group (n=5) becomes chondrocyte induction liquid (DMEM in high glucose culture medium with serum-free, 1%1 × ITSpremix, 40g/ml proline, 10ng/mlTGF-β 1, 100ng/mlIGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs) cultivate.Containing serum group (n=5) to become chondrocyte induction liquid (DMEM in high glucose culture medium, 10%FBS, 10ng/mlTGF-β containing serum 1, 100ng/mlIGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs) cultivate.Nostoc commune Vanch group (n=5) is cultivated with the common DMEM in high glucose culture fluid containing 10%FBS.After 3 weeks, each group of pellet is drawn materials respectively, carry out general appearance and histology.
A (), Pellet see 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 have certain elasticity, and weight in wet base reaches about 14mg; Become porcelain white spherical design containing serum group pellet, diameter reaches about 2mm, and weight in wet base is about about 3mg, and quality is more soft; Nostoc commune Vanch group pellet shape is more irregular, and diameter is 1-2mm only, and weight in wet base is about 2mg only, and quality softness is nonelastic (Fig. 5).
(b), Pellet histological examination
HE dyes: the pellet In vitro culture that the residual Ear cartilage stem cell of the 8th generation people is formed is after 3 weeks, the visible a large amount of typical cartilage lacuna spline structure of serum-free induction group, containing the Serum-induced group only visible minute quantity cartilage lacuna in edge spline structure, Nostoc commune Vanch group is without obvious lacuna structure (Fig. 6).
Safranin-O dyes: the plastidogenetic pellet In vitro culture of the residual Ear cartilage of the 8th generation people is after 3 weeks, the visible typical cartilage lacuna structure of serum-free induction group and obviously the Safranin-O positive are painted, containing Serum-induced group and Nostoc commune Vanch group all without obvious lacuna structure and Safranin-O painted (Fig. 6).
II Collagen Type VI SABC: the plastidogenetic pellet In vitro culture of the residual Ear cartilage of the 8th generation people is after 3 weeks, serum-free induction group most of region II Collagen Type VI is positive and has typical cartilage lacuna structure, containing the Serum-induced group only visible II Collagen Type VI in edge positive region; Nostoc commune Vanch group, without obvious lacuna structure, only has the point-like II expression of collagen (Fig. 6) be dispersed on a small quantity.
The above results shows: serum-free becomes chondrocyte induction system to be more conducive to residual Ear cartilage stem cell to form cartilage in vitro.
Embodiment 8
People residual Ear cartilage stem cell nude mice by subcutaneous becomes cartilage
(1) acquisition of the residual Ear cartilage stem cell of people and cultivation: see embodiment 1;
(2) residual Ear cartilage stem cell-PGA complex becomes chondrocyte induction: collect residual Ear cartilage stem cell, according to 5 × 10 7individual cell/cm 3density be inoculated into (residual Ear cartilage stem cell group, n=3) on cylindrical PGA support; In like manner, collect bone marrow stroma stem cell (BMSC) and be inoculated in (BMSC group, n=3) on cylindrical PGA support.By cell-material composite of having inoculated in 37 DEG C, 5%CO 2, 100% humidity environment in static culture 5 hours.Slowly add the common DMEM in high glucose culture fluid containing 10%FBS of temperature bath in advance, continue to cultivate.After 48 hours, change serum-free and become chondrocyte induction liquid (DMEM in high glucose culture medium, 1%1 × ITSpremix, 40 μ g/ml proline, 10ng/mlTGF-β 1, 100ng/mlIGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs), every 3-4 days changes liquid once, after cultivating 6 weeks, carries out nude mice by subcutaneous heeling-in.
(3) nude mice by subcutaneous heeling-in: plant in nude mice dorsal sc by the external one-tenth chondrocyte induction cell-material composite of 6 weeks, draws materials after 12 weeks and carries out general appearance and HE dyeing.
A () is seen substantially:
Nude mice by subcutaneous heeling-in is after 12 weeks, and residual Ear cartilage stem cell group sample is typical cartilage sample porcelain white appearance, and original volume maintenance is constant, and quality is pliable and tough, flexible; And BMSC group sample quality is hard, rough surface, skeletonization phenomenon obviously (Fig. 8);
B () HE dyes: nude mice by subcutaneous heeling-in is after 12 weeks, the visible a large amount of typical cartilage lacuna structure of residual Ear cartilage stem cell group sample, and BMSC group is ossified obviously (Fig. 8).
Result shows, cartilage graft cartilage phenotype in subcutaneous environment that residual Ear cartilage stem cell obtains is more stable.
Embodiment 9
The external evoked structure tissue engineering bone/cartilage of residual Ear cartilage stem cell compound support frame material
(1) collecting cell: with reference to embodiment 2, collect the 3rd generation residual Ear cartilage stem cell.
(2) inoculating cell-material composite: by 5 × 10 7individual cell/cm 3density by cell suspension inoculation on cylindrical PGA support or auricle shape PGA/PLA support, in 37 DEG C, 5%CO 2, 100% humidity environment in static culture 5 hours.Slowly add the common DMEM in high glucose culture fluid containing 10%FBS of temperature in advance, continue cultivation and be replaced by into chondrocyte induction liquid after 48 hours.The concrete composition of one-tenth chondrocyte induction liquid that this example adopts comprises: DMEM in high glucose culture medium, 1%1 × ITSpremix, 40 μ g/ml proline, 10ng/mlTGF-β 1, 100ng/mlIGF-I, 40ng/ml dexamethasone and 50 μ g/ml vitamin Cs.Every 3-4 days changes liquid once, draws materials and detect after 8 weeks.
A the cartilage graft of (), external structure and II Collagen Type VI immunohistochemical staining thereof check
3rd generation people residual Ear cartilage stem cell-PGA material composite is external through becoming chondrocyte induction after 8 weeks, PGA fiber is wrapped up by a large amount of cartilage matrix, show typical cartilage sample outward appearance, the white in porcelain, size and shape maintains good, quality is pliable and tough and have certain elasticity, and II expression of collagen is strong positive, proves a large amount of cartilage specificity matrix secretion (Fig. 7) further.
The normal size people auricle form cartilage of (b), external structure and histological examination thereof
3rd generation people residual Ear cartilage cell-auricle form PGA/PLA material composite is external shows cartilage sample outward appearance through becoming chondrocyte induction after 8 weeks, and size gill profile state remains good, and quality is pliable and tough and have certain elasticity.Histology shows a large amount of ripe cartilage lacuna structure, and timbering material is by a large amount of extracellular matrix parcel (Fig. 9).
Discuss
The proposition of the present invention's residual Ear cartilage stem cell theory is on the basis of the favorite outer discovery of residual Ear cartilage derived cell biological characteristics heuristic process, the bold hypothesis that a large amount of experiences in the past accumulated in conjunction with inventor propose.Based on this hypothesis, inventor verifies repeatedly through multiple-case great many of experiments, confirm the stem cell properties of residual Ear cartilage derived cell, and based on this, by optimization, the integration of series of key techniques parameter, finally establish the external three-dimensional cartilage constructing technology system based on residual Ear cartilage stem cell.Being established as of this technical system utilizes the autologous residual Ear cartilage derived cell structure tissue engineering bone/cartilage of microtia patient to carry out the cartilage defect repairs such as Postauricular flap and rebuild providing feasible clinical treatment.
It is generally acknowledged, the cell in residual Ear cartilage source belongs to chondrocyte, should have into that cartilage ability is strong, amplification in vitro easily aging a, feature of the differentiation and maturation chondrocyte such as to dedifferente, and domestic and international Most scholars also admits this viewpoint substantially.Inventor is found by systematic research, and the cell compared with normal chondrocyte in residual Ear cartilage source more easily dedifferentes, and be expanded to more than 2nd generation and substantially lose Subchondral drilling ability, this obviously limits its clinical practice feasibility.Fortunately, inventor is surprised to find that under study for action, although residual Ear cartilage cell is easy to lose Subchondral drilling ability, but it has superpower multiplication capacity, that is, dedifferente although there occurs, but there is not obvious catabiosis, even if increase, in the 8th generation, still can keep very high proliferation activity, and from this characteristic, the cell in residual Ear cartilage source is obviously different from the chondrocyte of normal differentiation maturation.
Based on this discovery, the cell that inventor courageously proposes residual Ear cartilage source is the hypothesis of the stem cell that left behind in a kind of growth course, and on this basis, the experiment of a large amount of repeatability that utilized multiple case to carry out, result shows, even if the residual Ear cartilage derived cell reaching for the 8th generation still has the multi-lineage potentials such as stronger bone, cartilage, fat, show that a kind of really ability of amplification of residual Ear cartilage derived cell is strong and have the cell of the similar stem cell properties of multi-lineage potential, therefore inventor proposes residual this theory of Ear cartilage stem cell first.
Based on the theory of residual Ear cartilage stem cell, inventor determines the main attack research direction being built cartilage by directional induction.Because inventor's long campaigns stem cell cartilage builds correlational study, possesses the experience that abundant induced dry-cell builds three-dimensional cartilage, with these experiences for instructing, by make repeated attempts and kinds of schemes to when optimizing and combining, establish stable residual Ear cartilage stem cell cartilage directional induction system, and in conjunction with three-dimensional biodegradable stent, apply the three-dimensional chondroid tissue that residual Ear cartilage stem cell constructs homogenizing maturation in vitro.On this basis, further combined with the accurate people's auricle form cartilage constructing technology based on animal maturation differentiation chondrocyte previously set up, by the PGA/PLA timbering material compound of residual for people Ear cartilage stem cell and accurate people's auricle form, and induce with the condition of above-mentioned optimization, finally construct the tissue engineering bone/cartilage with accurate people's auricle form in vitro.
In sum, residual Ear cartilage stem cell disclosed in this invention is a kind of cell of specific type, its with the differentiation and maturation chondrocyte to report in the past and other stem cell with cartilage differentiation potential all different.Apply the advantage that residual Ear cartilage stem cell constructing cartilage has its uniqueness:
First, compared with normal chondrocyte, residual Ear cartilage stem cell has very strong multiplication capacity, can meet the cell concentration built needed for larger volume cartilage (as people's auricle form cartilage) completely, and can not cause any damage by normal tissue to drawing materials of residual Ear cartilage.Therefore, the residual Ear cartilage stem cell constructing cartilage graft compared with normal chondrocyte applying patient autologous has stronger clinical practice feasibility.
Secondly, compared with the stem cell that there is cartilage differentiation potential with other, its advantage is more remarkable: 1). and Chondrogenesis is stronger: the residual Ear cartilage stem cell of lower generation (within 2 generations) can form cartilage without induction, and other stem cell with Subchondral drilling potential can also be induced to become cartilage (Fig. 1); The residual Ear cartilage stem cell of higher generation time (more than 2 generations) also more easily forms homogenizing cartilage compared with other stem cell after induction; These features may be higher relevant with the cell colony proportion in residual Ear cartilage stem cell with cartilage ability.2). in three-dimensional cartilage building process, form maintains better: be easy to occur contraction distortion in other Derived Stem Cells (as bone marrow stroma stem cell) Induction Process, and residual Ear cartilage stem cell in vitro builds in three-dimensional cartilage process and not easily shrinks, form can accurately maintain.This may with residual Ear cartilage stem cell chondrocyte induction process mesostroma produce more rapid, Subchondral drilling is relevant sooner.3). in body, in dystopy environment, cartilage stability is better: the cartilage of other Derived Stem Cells (as bone marrow stroma stem cell) external structure is easy to ossify in subcutaneous environment in vivo, and the three-dimensional cartilage that residual Ear cartilage stem cell in vitro builds is implanted subcutaneous rear cartilage phenotype and maintained comparatively stable (Fig. 8).This itself may come from cartilaginous tissue with residual Ear cartilage stem cell, external evoked after more easily to form the stable cartilage of differentiation and maturation relevant.In addition, the present invention sets up and the external structure technology optimized and be not also convenient to standardization containing the chondrocyte induction system of serum, is easy to form industrialization product.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that 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 (15)

1. an organization engineered cartilage graft, is characterized in that, it contains the residual Ear cartilage cell of the people grown under three dimensional structure; The residual Ear cartilage cell of described people went down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations;
Described organization engineered cartilage graft is prepared by following step:
(1) residual for people Ear cartilage cell is carried out plane amplification, and be passaged to for the 1st, 2,3,4,5,6,7 or 8 generations;
(2) the people's residual Ear cartilage cell gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations is mixed to form cell-biomaterial composites by the medically acceptable Biodegradable material of centrifugal formation agglomerate or the people's residual Ear cartilage high cell densities inoculation formation cell patch gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations or the people's residual Ear cartilage cell gone down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations and solid;
(3) agglomerate, cell patch or complex are carried out chondrocytes in vitro induction; With entire volume, containing 5-50ng/mlTGF-β in described chondrocytes in vitro induced liquid 1(TransformingGrowthFactor-β 1, transforming growth factor-β 1) and the dexamethasone (dexamethasone) of 10-100ng/ml; Described external one-tenth chondrocyte induction liquid is serum-free induction system.
2. cartilage graft as claimed in claim 1, it is characterized in that, described three dimensional structure is that the medically acceptable Biodegradable material that people's residual Ear cartilage cell described in the agglomerate or high density that are formed by described people residual Ear cartilage cell aggregation is inoculated in cell patch that culture dish formed or is inoculated in solid by described people residual Ear cartilage cell is formed.
3. cartilage graft as claimed in claim 1, it is characterized in that, described people residual Ear cartilage cell is from autologous or allogeneic.
4. cartilage graft as claimed in claim 3, it is characterized in that, described people residual Ear cartilage cell is from the residual Ear cartilage of microtia autologous patient.
5. the cartilage graft as described in as arbitrary in claim 1-4, it is characterized in that, with the entire volume of described graft, the residual Ear cartilage cell concentration of people is wherein 2 × 10 7individual cell/cm 3-10 × 10 7individual cell/cm 3.
6. cartilage graft as claimed in claim 5, it is characterized in that, with the entire volume of described graft, the residual Ear cartilage cell concentration of people is wherein 2 × 10 7individual cell/cm 3-7 × 10 7individual cell/cm 3.
7. cartilage graft as claimed in claim 5, is characterized in that, the shape of described graft needs the defect shape of transplanting cartilage to conform to human body.
8. cartilage graft as claimed in claim 1, is characterized in that, the time of external one-tenth chondrocyte induction is 3-20 week.
9. cartilage graft as claimed in claim 8, is characterized in that, the time of external one-tenth chondrocyte induction is 6-12 week.
10. a preparation method for the cartilage graft as described in as arbitrary in claim 1-9, described method comprises step:
(1) residual for people Ear cartilage cell is carried out plane amplification, and be passaged to for the 5th, 6,7 or 8 generations;
(2) the people's residual Ear cartilage cell gone down to posterity in the 5th, 6,7 or 8 generations is mixed to form cell-biomaterial composites by the medically acceptable Biodegradable material of centrifugal formation agglomerate or the people's residual Ear cartilage high cell densities inoculation formation cell patch gone down to posterity in the 5th, 6,7 or 8 generations or the people's residual Ear cartilage cell gone down to posterity in the 5th, 6,7 or 8 generations and solid;
(3) agglomerate, cell patch or complex are carried out chondrocytes in vitro induction; With entire volume, containing 5-50ng/mlTGF-β in described chondrocytes in vitro induced liquid 1(TransformingGrowthFactor-β 1, transforming growth factor-β 1) and the dexamethasone (dexamethasone) of 10-100ng/ml; Described external one-tenth chondrocyte induction liquid is serum-free induction system.
11. preparation methoies as claimed in claim 10, is characterized in that, the time of external one-tenth chondrocyte induction is 3-20 week.
12. preparation methoies as claimed in claim 11, is characterized in that, the time of external one-tenth chondrocyte induction is 6-12 week.
13. 1 kinds as arbitrary in claim 1-9 as described in cartilage graft building the application in engineered auricle graft.
14. 1 kinds of people's residual Ear cartilage cells build in vitro as arbitrary in claim 1-9 as described in organization engineered cartilage in application; The residual Ear cartilage cell of described people went down to posterity in the 1st, 2,3,4,5,6,7 or 8 generations.
15. apply as claimed in claim 14, it is characterized in that, described organization engineered cartilage is engineered auricle graft.
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