CN1661010A - Method for inducing fibroblast to form cartilage cells - Google Patents
Method for inducing fibroblast to form cartilage cells Download PDFInfo
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- CN1661010A CN1661010A CN 200410016514 CN200410016514A CN1661010A CN 1661010 A CN1661010 A CN 1661010A CN 200410016514 CN200410016514 CN 200410016514 CN 200410016514 A CN200410016514 A CN 200410016514A CN 1661010 A CN1661010 A CN 1661010A
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Abstract
A process for inducing fibroblast to become cartilage cell includes culturing the fibroblasts in the culture liquid contaniing the cartilage form generating protein for 3-30 days to generate cartilage cells, and separating them for culture liquid.
Description
Technical field
The present invention relates to biology and organizational engineering field, relate more specifically to induce inoblast to form chondrocyte's method.
Background technology
Cartilaginous tissue mainly plays effects such as support, protection, dispersive stress as a kind of reticular tissue.Can be divided into hyaline cartilage (joint cartilage, costicartilage, nasal cartilages, tracheal cartilage), elastic cartilage (epiglottic cartilage, credulous bone), fibrous cartilage (meniscus) three classes on the histology.They all are the single organization of only containing the chondrocyte on histology, inner no blood vessel, and nutrition mainly relies on the infiltration of tissue juice.
On structure, the chondrocyte is wrapped in the cartilage cavities, and cartilage capsule thick around it is separated chondrocyte and surrounding tissue as one natural cover for defense.Therefore, cartilaginous tissue has a little less than the antigenicity, is difficult for being discerned and attacking by body immune system and has characteristics of light immunological rejection.Early stage result of study shows, the allogeneic chondrocyte can be survived in the acceptor body and be kept secreting the function of matrix.
It is one of clinical common disease that richets is decreased, as the various articular surface illness that caused by factors such as wound, infection, autoimmunization, tumours; Auricular cartilage, nose, laryngeal cartilage support damaged, lopsided; And the wound of bone in children epiphyseal growth plate, early close, necrosis etc.Because cartilaginous tissue does not almost have self-repairing capability, so must utilize cartilage or other equivalent material to repair.
From body and allosome cartilage transplantation is present common method.It is reliable to transplant curative effect from the body cartilaginous tissue, but must be cost to sacrifice human normal tissue, and the patient will suffer the misery of once performing the operation more.In addition, the autologous tissue source is limited, and the fritter graft also has yielding problem.Allosome and heterograft have immunogenicity again.
At present,, also exist the defective of aspects such as histocompatibility, material mechanical characteristic, still can not satisfy every requirement clinically though some artificial synthetic cartilaginous tissue surrogates are arranged.The synthetic equivalent material only possesses filling, supports and keeps effect attractive in appearance, and the abiology function still can not reach truly structure and reconstruction.
Organizational engineering is that new approach has been opened up in the damaged treatment of cartilaginous tissue.The used in tissue engineering small amounts of cells is inoculated on the Biodegradable material then at amplification in vitro, is built into cell-material composite, is used for transplanting repair deficiency.
One of essential condition that makes up the organization engineered cartilage tissue is to obtain a large amount of chondrocytes that function and vigor are arranged.In high large mammal even human body, also can utilize the tissue engineering technique regenerating cartilage tissue, but these are studied applied seed cell and are the chondrocyte, and can only be with former generation or the chondrocyte of going down to posterity in early days, and the chondrocyte after external a large amount of amplification is easily taken place aging and is dedifferented, and is difficult to form in vivo cartilaginous tissue.Very limited from body chondrocyte source, allogeneic and xenogenesis chondrocyte can't stop immunological rejection again, the chondrocyte though somatomedin can increase in a large number, but its expense costliness, still uncertainly, these all make cartilage tissue engineered being difficult at clinical application to institute's formative tissue long-term effect in vivo.
Cartilage tissue engineered seed cell should possess wide material sources, it is convenient and little to the body wound to draw materials, the in-vitro multiplication ability is difficult for characteristics such as aging by force.And mainly take from from the cartilage seed cell source of body cartilaginous tissue very limited at present, very easily dedifferente in chondrocyte's vitro culture process of end differentiation eventually, amplification quantity can not satisfy needs (the VonderMark K of tissue construction, Gauss V, Vonder Mark H, et al.Relationship between cell shape and type of collagen synthesized aschondrocytes lose their cartilage phenotype in culture.Nature, 1997,267:53-532).In conjunction with developmental biology research, widely distributed dermal fibroblast and chondrocyte derives from mesodermal mesenchyme cell (Mesenchymal cells) in the body, different with the chondrocyte is that inoblast has kept the strong biological characteristics of mesodermal mesenchyme ability of cell proliferation, repeatedly goes down to posterity still to keep multiplication capacity preferably.Studies show that, inoblast can be expressed special ground substance of bone (Inaba K in specific external environment, Matsunaga S, Ishidou Y, et al.Effect oftransforming growth factor-beta on fibroblasts in ossification of theposterior longitudinal ligament.In Vivo.1996 10:445-449.).Yet be not the report that seed cell makes the chondrocyte still up to the present, with inoblast.
In sum, seed cell source deficiency is the main difficulty of the cartilage tissue engineered clinical application of restriction, so this area presses for the seed cell of developing new preparation cartilage, and with the chondrocyte and the cartilage graft of its preparation.
Summary of the invention
Purpose of the present invention just provides a kind of seed cell of cartilage graft of wide material sources, and the method for preparing chondrocyte and graft with this seed cell.
In a first aspect of the present invention, a kind of chondrocyte's of preparation method is provided, comprise step:
(a) under the condition that is fit to growth, inoblast was cultivated 3-30 days in the inoblast nutrient solution that contains 10-1000 nanograms/milliliter CKMP (especially CDMP-1), formed the chondrocyte thereby inoblast is induced;
(b) from culture, isolate the chondrocyte.
In another preference, described growth conditions is 37 ± 2 ℃, CO
25 ± 3%.
In another preference, described nutrient solution is F-12, DMEM or the RPMI-1640 that contains 0-20% (more preferably 3-15%) foetal calf serum.
In another preference, described inoblast is people's a inoblast.
In another preference, the fibroblastic density described in the step (a) is 0.5 * 10
4-5 * 10
5Individual/ml.
In another preference, described inoblast is an inoblast former generation or that go down to posterity.
In a second aspect of the present invention, provide chondrocyte with method preparation of the present invention.
In a third aspect of the present invention, a kind of organization engineered cartilage graft is provided, it comprises:
(a) pharmaceutically acceptable Biodegradable material;
(b) with the chondrocyte of the method for the invention preparation, its content is 1 * 10
5-5 * 10
7Individual cells/ml, or 1 * 10
5-5 * 10
7Individual cell/cm
3
In another preference, described pharmaceutically acceptable Biodegradable material is selected from down group: poly(lactic acid), polyglycolic acid, poly(lactic acid)-polyglycolic acid multipolymer, Pluronics, polyhydroxybutyrate, poly-acid anhydrides, poly-phosphazo, polyamino acid, false polyamino acid, poe, polyester urethane, polycarbonate, polyoxyethylene glycol, polyethylene oxide, poly-P-Dioxane ketone, collagen, gelatin, ammonia polyose of candy, chitosan, chitin, alginates, calcium alginate gel, acellular matrix, and composition thereof.
In a fourth aspect of the present invention, a kind of fibroblastic purposes is provided, it is used to prepare the chondrocyte.
In a fifth aspect of the present invention, a kind of method for preparing the organization engineered cartilage graft is provided, it comprises step: the chondrocyte of the method for the invention preparation is mixed formative tissue through engineering approaches cartilage graft with pharmaceutically acceptable Biodegradable material.
Description of drawings
Fig. 1 has shown inoblast form phase microscope observations.
A) not inducing the group cellular form is spindle shape (* 100);
B) induce the group cellular form to change (shown in the arrow) to chondroblast sample polygon by spindle shape; The square frame inner cell is a vitro culture people s-generation normal cartilage cell (* 100).
Fig. 2 has shown that the cultured in monolayer in vitro inoblast induces group (A-C) and non-I, the II of group (D-F), the immunofluorescence detected result of III Collagen Type VI of inducing.Antibody is FITC mark (green fluorescence), and nucleus is that the PI lining dyes (red fluorescence).
A) CDMP-1 induces the visible obviously II Collagen Type VI of inoblast to express, and the frame inner cell is low power finding (* 100);
B) and C) inducing cell is still expressed I, III Collagen Type VI (* 100);
D) vitro culture normal fibroblast II Collagen Type VI immunofluorescence feminine gender; E, F) normal fibroblast I, III Collagen Type VI are expressed positive.
Fig. 3 has shown the detected result of centrifuge tube being cultivated the culturing cell agglomerate.
A) Alicain Blue dyeing, the cell mass Celeste is dyed, and shows GAG secretion (* 100).
B) Masson ' s Trichrome dyeing, collagen stroma indigo plant is dyed (* 200)
Fig. 4 has shown centrifuge tube method culturing cell agglomerate II Collagen Type VI immunohistochemical staining (ABC method) detected result.
A) visible a large amount of II Collagen Type VI depositions (* 100) in cell and the matrix.
B) the local interior brown yellow granule (* 200) of back visible cell that amplifies)
Fig. 5 has shown the RT-PCR detected result to inducing cell.
A) induce group cell RT-PCR to detect Aggrecan, II collagen mRNA band of expression, do not induce the group result negative.
B), C) inducing cell RT-PCR detects and expresses I, III Collagen Type VI.
Fig. 6 has shown that the Western blotting detects the result that the II Collagen Type VI is expressed.The experimental group inoblast induces back II collagen type to be expressed as the positive, same band occurs with the normal cartilage cell; Control group is fibroblastic does not see II collagen type band of expression.
Embodiment
The inventor is through extensive and deep discovering, CKMP (cartilage-derived morphorgentic protein, CDMP) be particularly suitable as and induce inoblast to be differentiated to form chondrocyte's inducible factor, can induce inoblast effectively the body profile chondroblast.Finished the present invention on this basis.
Term
Term " purifying or isolating " refers to purifying or isolating material is substantially devoid of other cells, protein or polypeptide, for example cytokine of purifying, or isolating chondrocyte.
Term " xenotransplantation " refers to required living organisms material (as inoblast) is taken out and is applied to the method for another species object from a certain species.
Term " autotransplantation " refers to required living organisms material (as inoblast) is taken out and is applied to same patient's method from certain patient.
Term " heteroplastic transplantation " refers to required living organisms material (as the chondrocyte) is taken out the method that also is applied to again with another Different Individual of species from certain individuality.
Inoblast
Fibroblastic source is not particularly limited among the present invention, and a kind of preferred source is from the corium from body.Usually, inoblast of the present invention is from from body or allogeneic.Inoblast can be an inoblast former generation or that go down to posterity.
It all is as known in the art separating and cultivating the fibroblastic method of acquisition.A kind of preferable methods is to get the dermis of skin tissue, shred and move in the centrifuge tube, add about 10 times to 0.1% type i collagen enzymic digestion of tissue volume, 37 ℃, 2 hours, it is centrifugal that 100 order cellular filters filter the back, 1000 changeed 5 minutes, supernatant discarded adds and contains 10%FBS DMEM nutrient solution mixing cell, with 2 * 10
4/ cm
2Be inoculated in the 100mm culture dish and cultivate, when treating cytogamy to 80%-90%, go down to posterity, the density that goes down to posterity is 1 * 10
4/ cm
2Preferred the 2nd～18 generation cell be used to prepare artificial cartilage.
The preferred inoblast of one class is the s-generation～hexabasic inoblast of vitro culture.The potential that the inoblast of this moment has stronger multiplication capacity and breaks up to the chondrocyte.
Inducible factor
As used herein, " inducible factor " refer to CKMP (Cartilage-DerivedMorphogenetic Proteins, CDMPs).This is the special somatomedin of a class in the BMP family, and it brings into play important regulation in the cartilage development process, and wherein CKMP-1 (CDMP1) and CKMP-2 (CDMP2) are preferred.More preferably be CDMP1.
As used herein, " cell induction liquid " refers to contain the inoblast nutrient solution of 10-1000 nanograms/milliliter (preferably 20-500ng/ml, more preferably 50-200ng/ml) CKMP.
Except CDMP, but other factors of coupling also in the method for the invention, for example transforming growth factor-beta (transforming growth factor beta, TGF β) 1,2,3, Delicious peptide (bonemorphorgenetic protein, BMP) 2,6,7.Other induction factors of coupling cultivate, in specific cartilage cell epimatrix composition, cultivate, use retardance as: low-oxygen environment or activate specific cell signaling path, with chondrocyte's co-cultivation etc.
Induce inoblast to form the chondrocyte
Inducing step is the key of the inventive method.Usually, under the condition that is fit to growth, inoblast is being added 10-1000 nanograms/milliliter (20-500ng/ml preferably, 50-200ng/ml more preferably) cultivates 3-30 days (preferably 5-15 days) in the inoblast nutrient solution of CKMP, just can make inoblast induce the formation chondrocyte.
A kind of preferable methods is that single-layer culturing cell is induced, and carries out cell induction in the CDMP inoblast substratum (as the F-12 substratum) that contains about 10%FBS and about 10-1000ng/ml, changes nutrient solution one time in per three days, induces 3-30 days.
Another kind of preferred method is that the centrifuge tube culturing cell is induced.Inoblast is left the heart about 1500, abandon and add cell induction liquid behind the supernatant, cell mass has been shaken, be suspended in the nutrient solution, induced 3-30 days.
Through behind the inducing culture, can pass through Masson ' s Trichrome, Alcian Blue and immunohistochemical staining, RT-PCR, Western engram analysis etc. carry out qualitative and sxemiquantitative is identified, isolate the chondrocyte then from culture.
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 poly-alpha hydroxy acid (as polylactic acid PLA, polyglycolic acid PGA, polyhydroxybutyrate PHB etc.), poly-acid anhydrides (polyanhydrides), poly-phosphazo (polyphosphazenes), polyamino acid (polyamino acid), false polyamino acid (pesudo-polyamino acid), poe (polyorthoesters), polyester urethane (polyesterurethane), polycarbonate (polycarbonate), polyoxyethylene glycol, poly-P-Dioxane ketone (polydioxanone) etc.;
(b) natural degradable material, for example collagen (collagen), gelatin (gelatin), ammonia polyose of candy (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginates, calcium alginate gel etc.; Various acellular matrixes;
(c) syringeability material is as Pluronic (a kind of commercially available poly-epoxy second propylene commodity), calcium alginate gel etc.;
The matrix material of (d) matrix material of the mixture of above-mentioned materials or matrix material or multipolymer, especially macromolecular material and natural materials, and solid material and syringeability material.Multipolymer: polyglycolic acid-copolymer of poly lactic acid (lactic-co-glycolic acid, PLGA)
Preferred medically acceptable Biodegradable material is the material of syringeability, for example calcium alginate gel, Pluronic gel etc.
The cell concn of chondrocyte in the organization engineered cartilage of the present invention is about 2 * 10 usually
7/ ml to 5 * 10
7/ ml or higher.When material is the syringeability material, adjust chondrocyte's concentration with this fluent material; When material is the solidity material, adjust chondrocyte's concentration with nutrient solution, with the solidity material mixing, the ratio of nutrient solution and solidity material is not particularly limited when wherein mixing then, but with this solid material can adsorb nutrient solution maximum be as the criterion.
In organization engineered cartilage graft of the present invention, also can add or compound other various cytokines, somatomedin, various transgene component, thereby keep chondrocyte's phenotype, promote chondrocyte's growth or matrix synthesis capability etc.
The organization engineered cartilage graft
Chondrocyte with the inventive method preparation can be used for making up the organization engineered cartilage graft, and the described chondrocyte who is about to some amount mixes with pharmaceutically acceptable Biodegradable material and gets final product.
Organization engineered cartilage graft with the inventive method forms is divided into two classes, the class mixture that to be the chondrocyte constitute with the syringeability biomaterial, interior each positions of body such as that this mixture can be injected directly into is subcutaneous, cartilage defect place.Another kind of is the mixture that chondrocyte and non-syringeability biomaterial constitute, each position in the bodies such as that this mixture can directly implant is subcutaneous, cartilage defect place.
Biodegradable material is solid-state in a preference.At this moment, to make concentration be about 5 * 10 for available above-mentioned cell and nutrient solution
7The cell suspension of/ml forms mixture with biomaterial polyglycolic acid/polylactic acid bracket (PGA/PLA) then, and this mixture size, shape are determined according to the size and the shape of cartilage defect.One thoughtful three weeks of vitro culture, changed liquid once in per two to three days, to guarantee cytotrophy, each position in the bodies such as subcutaneous, the cartilage defect place of when vitro tissue through engineering approaches cartilage begins to take shape, implanting.
In another preference, Biodegradable material is syringeability material (Pluronic gel, a calcium alginate gel etc.).Adjusting cell concn with this fluent material is about 5 * 10
7/ ml is directly used in damaged reparation in subcutaneous injection or the body.
Application method
When Biodegradable material was the syringeability material, cell and biomaterial composites can directly be implanted subcutaneous or the interior cartilage defect place of body.When Biodegradable material when being solid-state, cell and biomaterial be external compound, in bio-reactor, form the organization engineered cartilage that is fit to various cartilage defects sizes and shape after, cartilage defect place again implants.
Major advantage of the present invention is:
(1) uses autogenous cell, and once draw materials and both can obtain enough cell concentrations;
(2) fibroblastic acquisition operations is simple, and is minimum to patient trauma, need not to be in hospital, and can repeat to draw materials;
(3) extracorporeal culturing method is easy to learn, and is easy to utilize.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described 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, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Inoblast is cultivated and goes down to posterity
Get the dermis of skin tissue that excises in the posthetomy under the aseptic condition, shred and move in the centrifuge tube, add about 10 times to 0.1% type i collagen enzyme (WORTHINGTON company) digestion of tissue volume, 37 ℃, 2 hours, it is centrifugal that 100 order cellular filters filter the back, 1000 changeed 5 minutes, supernatant discarded adds and contains 10%FBS DMEM nutrient solution (GIBCO) mixing cell, with 2 * 10
4/ cm
2Be inoculated in the 100mm culture dish (FALCON, FranklinLakes NJ) and cultivate, when treating cytogamy to 80%-90%, go down to posterity, the density that goes down to posterity is 1 * 10
4/ cm
2Amplification in vitro is after the s-generation, and grouping experimentizes.
Embodiment 2
The single-layer culturing cell revulsion is induced and is formed the chondrocyte
In the present embodiment, induce the formation chondrocyte with the single-layer culturing cell revulsion.Method is as follows:
To 100 μ g/ml, adding F-12 (Gibco company) nutrient solution that contains 10%FBS, to make its final concentration be 100ng/ml, is mixed with cell induction liquid with 0.1%BSA dilution CDMP1 somatomedin (Research Diagnostics).Got the s-generation inoblast inoculation 6-12 hour, cell induction changed nutrient solution one time in per three days, induce 7 days (at 37 ℃, 5%CO
2, saturated humidity), separate obtaining the chondrocyte.
Embodiment 3
The centrifuge tube culturing cell is induced
In the present embodiment, induce the formation chondrocyte with the single-layer culturing cell revulsion.Method is as follows:
The induced liquid preparation is with embodiment 2.Collect the inoblast 1500 that was passaged to for the 6th generation and left the heart 5 minutes, abandon and add cell induction liquid behind the supernatant, cell mass has been shaken, induced acquisition chondrocyte group 7 days.
Cut into slices behind the paraffin embedding, for Masson ' s Trichrome, Alcian Blue and immunohistochemical staining etc. are identified and are used.
Embodiment 4
Induce the detection of effect
One, detection method
(a) the cell laser co-focusing detects
With control group among the embodiment 2 and experimental group cell respectively after cultivating and inducing 7 days, 95% acetone fixed 10 minutes, the PBS rinsing, 10% sheep blood serum sealing 30 minutes, the anti-I of rabbit, II, III Collagen Type VI antibody (1: 100, Chemicon company) hatches 1 hour respectively, the PBS rinsing, (the dilution in 1: 1000 of FITC goat anti-rabbit igg, Oncogene company) hatches 40 minutes, PBS rinsing, PI (Propidium iodide propidium iodide) (dilution in 1: 1000, Sigma company) incubated at room is 10 minutes, mounting after the PBS rinsing.Sample behind the mark is placed two-photon laser confocal microscope (available from Zeiss company, model LSN510), laser excitation wavelength 488nm, mirror is observed down.The gained image carries camera system input computer by mirror preserves automatic camera.
(b) cell mass II Collagen Type VI immunohistochemistry detects
After the section dewaxing entry with preparation among the embodiment 3, PBS rinsing, 3%H
2O
2The sealing endogenous peroxydase, drip 0.4% stomach en-and hatch 30min for 37 ℃, added the anti-people II of rabbit Collagen Type VI monoclonal antibody (1: 100, Chemicon company), puts wet box and spend the night for 4 ℃, drip goat antirabbit two and resist (1: 100, DAKO), hatch 30min for 37 ℃, after the PBS rinsing, the DAB colour developing.Hematorylin is redyed, the resinene mounting.Not add a resisting sample as blank.
(c) cell collagen is expressed the RT-PCR detection
Collect respectively control group and experimental group cultivate and induce 7 days after cell, detect II Collagen Type VI and aggrecan (Aggrecan) expression.Trizol (Invitrogen company) extracts total mRNA, and the mRNA that detects I, II Collagen Type VI and aggrecan expresses.According to RT-PCR test kit (Takara) operation instructions, carry out reverse transcription and pcr amplification reaction.Corresponding primer (synthetic by Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) sequence sees Table 1.Reaction conditions: 94 ℃ of sex change, 55 ℃ of annealing, 35 circulations.
Table 1. primer sequence and amplified production size
| Upstream primer | Downstream primer | The product size | |
| Aggrecan | ??5′-atgcccaagactaccagtgg | ???5′-tcctggaagctcttc?tcagt | 510bp |
| COL-I | ??5′-cttggtctcgtcacagatca | ???5′-tgttcagctttgtggacctc | 210bp |
| CoL?II | ??5′-cttgggcacctcgggctcctttag | ???5′-tccccggcactcctggcactgat | 510bp |
| COL-III | ??5’-tggacgaaatggagaa | ???5’-cgcctttaccaccaggacta | 370bp |
(d) cell II Collagen Type VI secretion Western trace detects
Collect respectively control group and experimental group cultivate and induce 7 days after cell, total cellular score is equal 1 * 10
6Individual, add three decontamination lysate 1ml, move into behind the lysing cell in the 1.5ml Eppendorftube, 10000rpm, 4 ℃ are centrifugal 5 minutes.Get supernatant, add equal-volume 2 * SDS Loading buffer, beta-mercaptoethanol 2 μ l/100 μ l.100 ℃ of boiling water sex change 10 minutes in centrifugal 2 seconds in wink, are waited to be chilled to room temperature and are promptly gone up sample.Polyacrylamide gel electrophoresis 10mA electrophoresis 3-4 hour.After electrophoresis finishes, 90v, 4 ℃ were changeed film 2 hours, and 4 ℃ of sealings of sealing damping fluid (PIERCE company) are spent the night.37 ℃ add II Collagen Type VI mouse-anti one successively and resist (1: 500, Sigma company), biotinylation two resists (1: 500, Vector Lab company), streptavidin-AP is (1: 3400, Pierce company) hybridizes, after hybridization is amplified step by step, drip 1-Step NBT/BCIP (Pierce company) colour developing liquid, carry out interpretation of result after the colour developing.
Two, result
Cellular form changes: after application CDMP1 induced 3 days, the inoblast form is not seen did not have obviously change; When inducing after 7 days, the inoblast of visible spindle shape begins to change to polygon, Polygons, and is closely similar with chondrocyte's form.Cellular control unit does not see that obvious form changes (Fig. 1).
Cell collagen is expressed and observed: laser confocal microscope is observed and is found that the normal fibroblast of monolayer culture (not inducing group) is I, III Collagen Type VI luciferase expression only, does not see II Collagen Type VI fluorescencepositive cell.After application CDMP induces, II Collagen Type VI fluorescencepositive cell showed increased, but observation of cell is still expressed I, the III Collagen Type VI fluorescence positive simultaneously, and fluorescence intensity slightly weakens (Fig. 2).
Cell mass induces that back matrix is synthetic to be detected: use centrifuge tube method cultured cells agglomerate, after using CDMP1 and inducing 7 days, the Masson ' s Trichrome visible matrix that dyes is the diffusivity bright green, shows that collagen synthesizes vigorous (Fig. 3 A).The Alcian Blue visible matrix that dyes is homogeneity Celeste indigo plant and dyes, and shows that GAG deposits (Fig. 3 B) in a large number.Cell mass II Collagen Type VI immunohistochemical staining finds, brown xanchromatic size distribution in cell with extracellular matrix in; The II Collagen Type VI is expressed in that cell density is higher, the zone of matrix synthesis of densified dyeing strong (Fig. 4).Control group detects negative.
II Collagen Type VI and Aggrecan express RT-PCR and detect: the inoblast CDMP1 of monolayer culture induced back 7 days, and RT-PCR detects size and is to conform to the II Collagen Type VI of 510bp and Aggrecan amplified production with positive control chondrocyte amplification.Do not see amplification positive band (Fig. 5 A) for inducing group.Induce inoblast still can detect I, III collagen mRNA express (Fig. 5 B, C).
The II Collagen Type VI is expressed the Western engram analysis and detected: the inoblast CDMP1 of monolayer culture induced back 7 days, can detect the II collagen type and express, and is identical with positive controls chondrocytes expressed band.Do not induce the control group inoblast not see II collagen type (Fig. 6).
Embodiment 5
The preparation of tissue engineering bone/cartilage graft
With the chondrocyte of preparation in embodiment 2 or 3, respectively with 5 * 10
7The final concentration of individual cell/ml mixes, thereby makes the tissue engineering bone/cartilage graft with 30%Pluronic F-127 (a kind of commercially available poly-epoxy second propylene commodity)/DMEM.
Discuss
As the class somatomedin in the TGF-beta superfamily, people CDMP is at first gone out by clone and separate in the joint cartilage, mainly comprises two hypotypes of CDMP1 and CDMP2, BMP14 and BMP13 in the corresponding BMP of the difference family; Growth and differentiation factor in corresponding mouse BMP of the while family (Growth and Differentiation Factor, GDF).
In the whole family members of the present BMP that finds, CDMP is a most special class somatomedin relevant with growth with the generation of cartilage form, brings into play important regulation in graphic formation (patterning), cartilage generation and the long bone process of growth of bones of limbs.
Discover, CDMP1 specific expressed in embryo development procedure joint and joint cartilage form position (Tsumaki N, Tanaka K, Arikawa-Hirasawa E, et al.Role of CDMP-1 inskeletal morphogenesis:Promotion of mesenchymal cell recruitment andchondrocyte differentiation.J.Cell Biol., 1999.144:161-173); Development model (Storm EE and Kingsley DM.GDF5 coordinates bone and jointformation during digit development.Dev.Bio.1999 in the body, 209:11-27) and transgenic experiments all confirm, CDMP1 is mainly by promoting the gathering (consendation) of mesenchymal cell, improve the quantity of local cartilage precursor cell, quicken joint cartilage formation, chondrocyte's differentiation and regulate the growth of bone tissue.All can detect the expression of CDMP in the birth back and the articular cartilage tissue of growing up, prompting CDMP can continue to keep the growth of normal articular cartilage after birth; External organ culture is discovered, there are a large amount of CDMP to express in the joint cartilage of damage, under the effect of the exogenous CDMP factor, cartilage matrix is synthetic obviously to be increased, prompting CDMP can promote injury repairing process (the Ludwig E of joint cartilage, Chee KNG, Robert U, et al.Presence of cartelage-derived morphogenetic proteins in articularcartelage and enhancement of matrix replacement in vitro.Arthriti.Rheumat.1998,41:263-273).Above-mentioned studies show that, CDMP is mainly by the differentiation of matter precursor cell between regulating, and the almost all biological that has participated in cartilaginous tissue generation, growth and injury repairing is learned process, is the somatomedin that important adjusting chondrocyte breaks up.
It is generally acknowledged that dermal fibroblast is to break up whole completely opisthosoma cell, though draw materials and cultivate simple, the amplification in vitro ability is strong, no longer possesses the ability to the other types cytodifferentiation of stem-like cell, only originate as dermal tissue engineering seed cell.Yet, use CDMP 1 somatomedin among the present invention, act on the dermal fibroblast of vitro culture, but but find cell expression specificity chondrocyte matrix after 7 days: II Collagen Type VI and aggrecan, chondrocyte's sample with cellular form changes simultaneously, and this has confirmed that the diploid fibroblast of whole end differentiation still has the potentiality as the seed cell source that makes up its hetero-organization.
Cartilage cell epimatrix mainly is made up of II type collagen fiber (COLII) and proteoglycan, and wherein the main component of proteoglycan is an aggrecan.Collegen filament form network structure and have very high tensile strength, the hairbrush spline structure that is present in the aggrecan molecule in the network can make its better counter pressure support the structure of cartilaginous tissue jointly with the I type i collagen, keeping the biological property of cartilaginous tissue, therefore the expression of COLII and aggrecan is the most special mark of chondrocyte (Micky D.Tortorella at present, Michael A., et al.The Interglobular Domain of CartilageAggrecan Is Cleaved by Hemorrhagic Metalloproteinase HT-d (AtrolysinC) at the Matrix Metalloproteinase and Aggrecanase Sites.J.Bio.Chem.1998,273:5846-5850).Dermal fibroblast itself is not expressed COLII and aggrecan, under culture system effect of the present invention, the inoblast of monolayer culture is after CDMP induces 7 days, cellular form has taken place by the transformation of fusiformis to chondrocyte's sample polygon form, and immunofluorescence, Western-Blot and RT-PCR have all confirmed the expression of II collagen; RT-PCR result also observes inoblast and induces the back to express aggrecan.It is mesenchymal precursor cells gathering in the simulation cartilage form generating process, atomization that centrifuge tube is cultivated, and the research stem cell is to the extracorporeal culturing method of chondrocyte's differentiation.In order further to confirm that above-mentioned result of study, the inventor adopt centrifuge tube culture technique research inoblast to induce atomization under state of aggregation.Discover that under the CDMP1 effect, the cell mass of inducing 7 days confirms to have a large amount of acidic mucopolysaccharide depositions through A Lixinlan dyeing, the synthetic of collagen and mucopolysaccharide also observed in Masson ' s dyeing.Immunohistochemical methods, Western-Blot and RT-PCR all detect the expression of COLII and aggrecan behind the centrifuge tube inducing culture.Above-mentioned result of study and bone marrow stroma stem cell are induced the report of differentiation (the Johnstone B that conforms to the chondrocyte, Hering TM, Caplan AI, et al.In vitro chondrogenesisof bone marrow derived mesenchymal progenitor cells.Exp cell res, 1998,238:265-272.).
Find in this research that though inoblast is expressed COLII and aggrecan under the CDMP1 effect, the expression of I, III Collagen Type VI does not disappear, show that the chondrocyte through inductive inoblast and nature also has a certain distance.Lennon (Lennon D, Haynesworth SE, Arm DM, et al.Dilution of human mesenchymal stem cells with dermal fibroblasts andthe effects in vitro and in vivo osteochondrogenesis.Dev.Dyn.2000,219:50-62) wait human dermis inoblast and bone marrow stroma stem cell mixed with 25-50%, still visible cartilaginous tissue formed after centrifuge tube was cultivated and induced, and had significantly reduced the consumption of bone marrow stroma stem cell.The inventor thinks, by inducing of CDMP, might further reduce the ratio of bone marrow stroma stem cell in the cartilaginous tissue building process.Simultaneously because cartilage forms and often is the commitment in the os endochondrale process, so the inventor thinks inoblast expression specificity cartilage matrix among the present invention, can not get rid of is the stage of inoblast to the skeletonization phenotypic differentiation.Find (Gruber R inducing in the differentiation research of marrow stromal cell, Mayer C, Schulz W, et al.Stimulatory effects ofcartilage-derived morphogenetic proteins 1 and 2 on osteogenicdifferentiation of bone marrow stromal cells.Cytokine.2000,12:1630-1638), CDMP1,2 can promote that all bone marrow stroma stem cell breaks up to osteoblasts in vitro, but its osteogenesis is starkly lower than BMP-6 and OP-1; Though there is the IIA procollagen type to express, do not detect as the IIB procollagen type of ripe chondrocyte's matrix and secrete with aggrecan, somatomedin action time only is 4 days in this research.In another research, when the CDMP induction time extended to 7 days, periosteum source sexual cell had showed skeletonization and the multidirectional differentiation that becomes cartilage, and visible obviously II Collagen Type VI and aggrecan are expressed, and become the cartilage effect obviously to be better than osteogenesis.Therefore, CDMP is necessary to continue the function of its skeletonization of observation or one-tenth cartilage under different action times, show skeletonization and the dual intensity effect that becomes cartilage at different cells under same action condition.
Of the present invention having experimental results show that, dermal fibroblast is induced by CDMP-1, under monolayer culture and centrifuge tube culture condition, the phenomenon to chondrocyte's phenotypic differentiation has all appearred, and inoblast can be synthesized specificity chondrocyte matrix: II procollagen type and aggrecan.This result of study has not yet to see bibliographical information, and prompting is by the inducing action of CDMP, and inoblast has the possibility of originating as cartilage tissue engineered new seed cell.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.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 institute restricted portion equally.
Claims (10)
1. a method for preparing the chondrocyte is characterized in that, comprises step:
(a) under the condition that is fit to growth, inoblast was cultivated 3-30 days in the inoblast nutrient solution that contains 10-1000 nanograms/milliliter CKMP, formed the chondrocyte thereby inoblast is induced;
(b) from culture, isolate the chondrocyte.
2. the method for claim 1 is characterized in that, described growth conditions is 37 ± 2 ℃, CO
25 ± 3%.
3. the method for claim 1 is characterized in that, described nutrient solution is the F-12 that contains the 0-20% foetal calf serum, DMEM or RPMI-1640.
4. the method for claim 1 is characterized in that, described inoblast is people's a inoblast.
5. the method for claim 1 is characterized in that, the fibroblastic density described in the step (a) is 0.5 * 10
4-5 * 10
5Individual/ml.
6. the method for claim 1 is characterized in that, described inoblast is an inoblast former generation or that go down to posterity.
7. a chondrocyte is characterized in that, it prepares with the described method of claim 1.
8. organization engineered cartilage graft is characterized in that it comprises:
(a) pharmaceutically acceptable Biodegradable material;
(b) chondrocyte of the described method preparation of claim 1, its content is 1 * 10
5-5 * 10
7Individual cells/ml, or 1 * 10
5-5 * 10
7Individual cell/cm
3
9. graft as claimed in claim 8, it is characterized in that, described pharmaceutically acceptable Biodegradable material is selected from down group: poly(lactic acid), polyglycolic acid, poly(lactic acid)-polyglycolic acid multipolymer, Pluronics, polyhydroxybutyrate, poly-acid anhydrides, poly-phosphazo, polyamino acid, false polyamino acid, poe, polyester urethane, polycarbonate, polyoxyethylene glycol, polyethylene oxide, poly-P-Dioxane ketone, collagen, gelatin, ammonia polyose of candy, chitosan, chitin, alginates, calcium alginate gel, acellular matrix, and composition thereof.
10. a fibroblastic purposes is characterized in that, is used to prepare the chondrocyte.
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| CNB2004100165142A CN100482784C (en) | 2004-02-25 | 2004-02-25 | Method for inducing fibroblast to form cartilage cells |
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|---|---|---|---|
| CNB2004100165142A CN100482784C (en) | 2004-02-25 | 2004-02-25 | Method for inducing fibroblast to form cartilage cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748095A (en) * | 2008-11-28 | 2010-06-23 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| CN107267448A (en) * | 2017-07-28 | 2017-10-20 | 陈强 | A kind of induced fibroblast is the method for in vivo bioreactor |
| CN114540285A (en) * | 2017-06-25 | 2022-05-27 | 科.东股份公司 | Method for preparing transplantable cartilage tissue |
| CN116024164A (en) * | 2015-10-21 | 2023-04-28 | 京都府公立大学法人 | Method for producing cells |
-
2004
- 2004-02-25 CN CNB2004100165142A patent/CN100482784C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748095A (en) * | 2008-11-28 | 2010-06-23 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| CN101748095B (en) * | 2008-11-28 | 2013-05-08 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| CN116024164A (en) * | 2015-10-21 | 2023-04-28 | 京都府公立大学法人 | Method for producing cells |
| CN114540285A (en) * | 2017-06-25 | 2022-05-27 | 科.东股份公司 | Method for preparing transplantable cartilage tissue |
| CN107267448A (en) * | 2017-07-28 | 2017-10-20 | 陈强 | A kind of induced fibroblast is the method for in vivo bioreactor |
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| Publication number | Publication date |
|---|---|
| CN100482784C (en) | 2009-04-29 |
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