CN106943630A - Hyaline cartilage sample massive texture that a kind of extracorporeal culture is grown and its preparation method and application - Google Patents
Hyaline cartilage sample massive texture that a kind of extracorporeal culture is grown and its preparation method and application Download PDFInfo
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Abstract
Hyaline cartilage sample massive texture grown the invention discloses a kind of extracorporeal culture and its preparation method and application, cartilage tissue engineered reparation field.Cartilage tissue growth speed of the present invention is fast, can generate bulk hyaline cartilage sample massive texture in a short time using a small amount of cartilage cell.The preparation method rapidly and efficiently without support, cartilage cell by three-dimensional Hanging drop culture method it is pre- it is agglomerating after, be transferred in six orifice plates of low adhesion, after the self aggregation between cell micelle, hyaline cartilage massive texture generated after two weeks in culture.The tissue that in vitro culture is generated after two weeks is translucent sample, containing abundant mucopolysaccharide and Type Ⅱ collagen, and cartilage cell is presented spherical or ellipticity and is equably embedded in the lacuna being made up of extracellular matrix.With good mechanical property, while coefficient of friction is relatively low, close to natural hyaline cartilage on macroscopic property.There is potential application value in the case of clinical patient Autologous Chondrocyte supply limited amount.
Description
Technical field
The invention belongs to cartilage tissue engineered reparation field, it is related to the external artificial quick of hyaline cartilage sample massive texture
Efficient preparing technical field, is related specifically to Three-dimensional cell culture and without framework's Engineering Remediation Techniques field.
Technical background
Cartilage damage and cartilage related disorder（Such as arthritis）The quality of life of many people is affected, articular cartilage lacks
Damage is frequently due to what injury gained in sports was caused, particularly in crowd of the age more than 40 years old.There are some researches show more than 55 years old
The people for having 10% in crowd suffer from knee pain, and 25% people seriously arrives influence activity, therefore patient has to repair of cartilage
Urgent demand, various therapeutic modalities, such as gene therapy, minimal invasive operation are have also been developed therewith.But
Cartilaginous tissue does not have blood vessel, nerve fiber, lymphatic vessel, and self-repairing capability is very poor, for general adult, lesion size
More than Critical Damage size, cartilaginous tissue is just difficult to complete self-regeneration, so traditional therapeutic modality can not be played very well
Therapeutic effect.Since langer in 1993 is proposed after the concept of organizational project, tissue regeneration method reparation cartilage suffer from
The patient of arthralgia brings hope, and researchers can stimulate body itself to produce new organization by material, also can be direct
The good cartilaginous tissue of external structure is implanted to rejected region, so as to realize the purpose of repair of cartilage.
Many different types of materials are used in cartilage defect organizational project reparation field, can substantially be divided into two
Class：Natural macromolecular and artificial synthesized macromolecular.Collagen and its derivative gelatin, chitosan, chondroitin sulfate, hyaluronic acid,
Silk-fibroin, fibrin, cellulose acetate etc. belong to natural big point usually applied in the reparation of cartilage defect organizational project
Son, and PLGA, PLA, PLG, PCL, artificial synthetic polypeptide Self-Assembled be all it is cartilage tissue engineered in commonly use it is artificial
Material.Although they all solve some problems of cartilage defect repair from certain degree, it can for example, be carried at defect
For mechanical support, the environment of a good propagation, the related matrix of secretion cartilage is provided for seed cell, but is also brought perhaps
More extra insoluble problem, the toxicity of the catabolite of such as material, the degradation rate of material is difficult to and cambium
Growth rate match, portion of material promotes the extravagant adhesion of cartilage cell so that promoting the soft osteoid matrix of its eccrine fiber
Rather than hyaline cartilage sample matrix.
Just because of biomaterial brought it is extra the problem of be difficult to solve, without using the cell three-dimensional culture method of support
Application arise at the historic moment, traditional centrifugation is agglomerating（Pellet methods）Although Hanging drop culture can grow the block group of hyaline cartilage sample
The features such as knitting, but cultivate low efficiency and small tissue block size is made it possible to as an Investigating Cartilage organizational project associated biomolecule
The model of mechanism, but cannot apply in clinic.Therefore it is a kind of artificial without using the efficient extracorporeal culture of timbering material
The method of hyaline cartilage sample massive texture is extremely urgent.
The content of the invention
Inventor is by researching and analysing discovery:It is thin between mescenchymal stem cell during development of mammalian embryos
Generation of born of the same parents' signal transmission to later stage hyaline cartilage has very important physiological significance, to realize a kind of external hyaline cartilage sample
The efficient structure of massive texture, should meet appropriate Micro-environmental cues stimulates to promote the related matrix of cell secretion, also to create
Make an environment for being conducive to nutriment and cell discharged waste to exchange, the cell micelle that sessile drop method is formed meet this two
Cell tight contact in individual condition, cell micelle, and due to cell micelle diameters only about 100 microns, nutriment
Exchange with cell discharged waste is very easy to, therefore, it is possible to quickly and efficiently generate block cartilaginous tissue in the short term.
The problem of existing for prior art, it is an object of the invention to provide a kind of without the efficient quick of timbering material
The external preparation method of hyaline cartilage sample massive texture, this method has speed fast --- it can form block saturating within two weeks
Bright cartilage sample massive texture, efficiently --- weight in wet base is grown for 90 ± 9.60mg using micron-sized unweighable cell micelle
Block hyaline cartilage sample massive texture, average daily growth rate is 6.43 ± 1.02mg/Day.
Hyaline cartilage sample massive texture of the present invention, including extracellular matrix and to be uniformly embedded in cartilage therein thin
Born of the same parents, the extracellular matrix contains abundant natural cartilage composition mucopolysaccharide and Type Ⅱ collagen, with being close with natural cartilage
Composition, structure and property.The hyaline cartilage sample massive texture is obtained by vitro culture, without branch in incubation
Frame.
Alternately, in above-mentioned hyaline cartilage sample massive texture, the cartilage cell shows spherical or ellipse
Round shape form, is close with natural cartilage cell.
Alternately, in above-mentioned hyaline cartilage sample massive texture, the cartilage cell is in extracellular matrix structure
Into lacuna in.
Alternately, in above-mentioned hyaline cartilage sample massive texture, the cartilage cell presents columnar arrangement
Pattern and hypertrophyization phenomenon, it is similar to natural joint hyaline cartilage cartilage cells deep columnar arrangement and hypertrophyization physiological phenomenon.
Further, the surrounding presented in the massive texture outer region cartilage cell using the cell micelle center of circle as launch point launches circle
Columnar arrangement.
Alternately, in above-mentioned hyaline cartilage sample massive texture, the hyaline cartilage sample massive texture size is big
Small is 1-2cm.
Alternately, above-mentioned hyaline cartilage sample massive texture weight in wet base is 90 ± 9.60mg.
Alternately, in above-mentioned hyaline cartilage sample massive texture, the cartilage cell is mutual by secondary self aggregation
Porous coral sample massive texture is connected between phase.
Hyaline cartilage sample massive texture proposed by the invention has and natural cartilage is close composition, structure and property
The cartilage cell included in matter, hyaline cartilage sample massive texture in substantial amounts of mucopolysaccharide and Type Ⅱ collagen, tissue show with
The spherical or ellipticity form that natural cartilage cell is close, and occur in that the cell column being close with natural cartilage hypertrophyization area
Shape arranges pattern, and macroscopic observation can be found that translucent sample is presented in tissue, and handling touch can find that it has relatively low friction system
Number and higher elastic modelling quantity.On composition：Hyaline cartilage sample massive texture of the present invention is by abundant extracellular matrix and uniform edge
Embedding therein spherical or ellipticity cartilage cell is constituted, and is confirmed through tissue section strain in extracellular matrix containing substantial amounts of viscous
Polysaccharide（GAG）And Type Ⅱ collagen（ColⅡ）.In structure：Hyaline cartilage sample massive texture of the present invention is by being uniformly distributed cartilage cell
Equably it is embedded in abundant extracellular matrix, and in obvious lacuna structure, in tissue outer region cartilage cell
The surrounding using the cell micelle center of circle as launch point is presented and launches cylindric arrangement.
The preparation method of hyaline cartilage sample massive texture of the present invention is：By cartilage cell by three-dimensional Hanging drop culture side
Method it is pre- it is agglomerating after, be transferred in the culture plate of low adhesion cultivate 1-2 week, you can through cell micelle self aggregation generation hyaline cartilage sample
Massive texture,.Growth over time, body weight methods described speed is fast, few using cell quantity, and without support, distribution is waved
Effect of the effect and cell of cell extracellular matrix secretion in institutional framework structure.By pre- pockets of cartilage cell immediately
Again normal chondrocyte phenotype has been recovered（Secrete mucopolysaccharide and Type Ⅱ collagen）, reverse transcriptase-Polymerase Chain Reaction
（RT-PCR）High expression, cell micelle is presented in the hyaline cartilage related genes such as the AGG in display cartilage cell, Col II, Sox9
In cartilage cell that safranin O-fast green and Type Ⅱ collagen SABC has been secreted during pockets of is detectable viscous more
Sugar and Type Ⅱ collagen.It is pre- it is agglomerating after, in the culture plate of the low adhesion, between cell micelle occur self aggregation and two weeks with
Translucent, coefficient of friction is rapidly inside grown to by the micron order cell micelle for being initially visually difficult to differentiate low and with preferable power
Learn property.Further, the culture plate of the low adhesion is six orifice plates of low adhesion.
Alternately, in above-mentioned preparation method, the cell for being used for three-dimensional Hanging drop culture passes through two dimensional surface
Amplification is obtained.
Alternately, in above-mentioned preparation method, a diameter of 50- is obtained by the way that the pre- agglomerating operation is alternative
300 microns of cartilage cell's micelle.
Alternately, in above-mentioned preparation method, the time of the pre- agglomerating culture is 18-22 hours.
Alternately, in above-mentioned preparation method, gained hyaline cartilage sample massive texture is stored in liquid nitrogen at once
In to treat the detection and application in later stage.
Alternately, the preparation method comprises the following steps：
1）The cartilage cell that the articular hyaline cartilage of mammal source is isolated after enzymic digestion in vitro train by two dimensional surface
Support and be expanded to after P2 generations, it is 4 × 10 that concentration, which is made, in the pancreatin digestion without EDTA5Cells/ml cell suspending liquid, then will
This cell suspending liquid is inoculated on the Hanging drop culture plate in 384 holes, and the volume being inoculated with per hole is 30 μ l；
2）After being cultivated one day in Hanging drop culture plate, by adding 50ul phosphate buffer solutions（PBS）Cell micelle is collected
In 50ml centrifuge tubes, and with 300 × g centrifugal force 5 minutes, then by the cell micelle being collected into 2.3 ×
106The concentration in cells/ holes is inoculated in six orifice plates of low adhesion and cultivated；
3）A not good liquor was changed every three days, after cultivation cycle is expired for two weeks, the block group of the hyaline cartilage sample grown out is collected
Knit.
Alternately, in above-mentioned preparation method, the culture medium prescription used by whole preparation process is：Hyclone α-
MEM basal mediums, 50 μ g/ml ascorbic acid, 1% penicillin and streptomysin mixed liquor and 10% hyclone.
A kind of application of the hyaline cartilage sample massive texture grown present invention also offers extracorporeal culture, its feature
It is to use it for preparing cartilaginous tissue repair materials.
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
Beneficial effects of the present invention：
Hyaline cartilage sample massive texture prepared by the present invention has and natural joint hyaline cartilage is close composition, structure and
Property.Macroscopic observation is presented translucent, handles contact and finds that skin-friction coefficient is relatively low, and with preferable elastic modelling quantity.
Confirm that hyaline cartilage sample massive texture of the present invention contains abundant mucopolysaccharide and two using ESEM and tissue section strain
Collagen Type VI, cartilage cell is presented spherical or ellipticity and is equably embedded in the lacuna being made up of extracellular matrix.And in order to make
The preparation method that standby hyaline cartilage sample massive texture of the present invention is used possesses quickly, and required cell quantity is few and need not use
The advantages of timbering material, there is potential application value in the case of clinical patient Autologous Chondrocyte supply limited amount.
Brief description of the drawings：
Fig. 1 is that the Hanging drop culture method a in preparation method of the present invention is drop culture formation cell mass principle schematic；B is
The homogeneous drop formed in hanging-drop plates bottom；C is to form homogeneous cartilage cell group in one day.
Fig. 2 is the hyaline cartilage sample massive texture prepared by of the invention external two weeks and its imaging A under ESEM
For the hyaline cartilage sample massive texture generated after two weeks；B is hyaline cartilage sample massive texture cross section of the present invention under ESEM
Image；
The A of Fig. 3 hyaline cartilage sample massive textures of the present invention, B are safranin O-fast green section statining.C, D are Type Ⅱ collagen immune group
Weave chemistry is dyed, and slice thickness is 5 μm；
Fig. 4 is the RT-PCR gene expression analysis results of the different week old hyaline cartilage sample massive textures of the present invention, and A figures are into bone photo
The expression of correlation gene, B figures are into the expression of cartilage related gene, and C figures are the expression of hypertrophyization related gene；
Fig. 5 is the biochemical quantitative analysis results of the different week old hyaline cartilage sample massive textures of the present invention, and A figures are unit dry weight milligram
Contained GAG total amounts in sample, B figures be total collagen total amount contained by unit dry weight milligram sample, and C figures are unit dry weight milligram
Contained BMP-2 growth factor total amounts in sample, D figures be that the growth factor of TGF-β 1 contained by unit dry weight milligram sample is total
Amount.
Embodiment：
The above of embodiment by the following examples again to the present invention is described in further detail.It should manage
Solution, instantiation described herein only to explain the present invention, is not intended to limit the present invention.Do not departing from the present invention's
Any modification made within spirit and principle, and the equivalent made according to ordinary skill knowledge and customary means
Or improve, it all should include within the scope of the present invention.
Embodiment 1：
Take cartilage cell by three-dimensional Hanging drop culture method it is pre- it is agglomerating after, be transferred in the culture plate of low adhesion cultivate 1-2 week, pass through
Hyaline cartilage sample massive texture is generated after self aggregation between cell micelle.Methods described speed is fast, nothing few using cell quantity
Support is needed, the effect of the effect and cell of cell extracellular matrix secretion in institutional framework structure has been played.By pre- agglomerating
Cartilage cell recovered normal chondrocyte phenotype again immediately（Secrete mucopolysaccharide and Type Ⅱ collagen）, reverse transcriptase-
Polymerase Chain Reaction（RT-PCR）The hyaline cartilage related genes such as the AGG in display cartilage cell, Col II, Sox9 present high
Expression（Fig. 4）, the cartilage cell in cell micelle secreted safranin O-fast green and Type Ⅱ collagen during pockets of and exempted from
The detectable mucopolysaccharide of epidemic disease groupization and Type Ⅱ collagen.It is pre- it is agglomerating after, in the culture plate of the low adhesion, between cell micelle send out
It is born from aggregation and translucent, friction was rapidly grown to by the micron order cell micelle for being initially visually difficult to differentiate within two weeks
Coefficient is low and with preferable mechanical property.Further, the culture plate of the low adhesion is six orifice plates of low adhesion.
Alternately, in above-mentioned preparation method, the cell for being used for three-dimensional Hanging drop culture passes through two dimensional surface
Amplification is obtained.
Alternately, in above-mentioned preparation method, a diameter of 50- is obtained by the way that the pre- agglomerating operation is alternative
300 microns of cartilage cell's micelle.
Alternately, in above-mentioned preparation method, the time of the pre- agglomerating culture is 18-22 hours.
Alternately, in above-mentioned preparation method, gained hyaline cartilage sample massive texture is stored in liquid nitrogen at once
In to treat the detection and application in later stage.
A series of hyaline cartilage sample massive textures are made using top method, gained hyaline cartilage sample massive texture, including it is thin
Extracellular matrix and cartilage cell therein is uniformly embedded in, the extracellular matrix contains abundant natural cartilage composition mucopolysaccharide
And Type Ⅱ collagen, with the composition being close with natural cartilage, structure and property.
Embodiment 2：
1）Prepare cartilage cell's micelle that diameter is about 100 μm：Cartilage cell is isolated from mammal joint hyaline cartilage,
And be inoculated in two-dimentional culture dish and be expanded to P2In generation, it is 4 × 10 to obtain concentration through pancreatin digestion5Cells/ml cell suspending liquid,
Then this cell suspending liquid is inoculated in the Hanging drop culture plate in 384 holes（Perfecta3D®）On, the volume being inoculated with per hole is 30 μ
l。
Cell suspending liquid is inoculated in hanging-drop plates and formed after drop, due to the effect of gravity, the cell in culture medium
Culture plate material polystyrene is not readily accessible to, therefore is always maintained at the state of suspension, because iuntercellular is adhered to each other and gravity
Effect, cell start aggregation it is agglomerating.After one day, it is about 100 μm that several diameters not waited to dozens of are formed in single hole
Cartilage cell's micelle, by having obvious mucopolysaccharide and two in safranin O-fast green and formal cell micelle of Type Ⅱ collagen immunostaining
Collagen Type VI, and RT-PCR results also demonstrate that cartilage cell after disengaging two dimensional surface is agglomerating, its hyaline cartilage related gene table
Reach（AGG、ColⅡ、Sox9）Conspicuousness height expression is presented, the aggregation of cell is agglomerating rapidly to have recovered cartilage cell's correlation table
Type.
2）After being cultivated one day in Hanging drop culture plate, by adding 50ul phosphate buffer solutions（PBS）By cell micelle
Be collected in 50ml centrifuge tubes, and with 300 × g centrifugal force 5 minutes, then by the cell micelle being collected into 2.3 ×
106The concentration in cells/ holes is inoculated in six orifice plates of low adhesion（Beaver®）In cultivate.
After cell micelle is collected into, in order to prevent in follow-up culture, cell is migrated from micelle to culture plate table
Face, we have selected six orifice plates of low adhesion.On the one hand can effectively suppress cell migration goes out cell micelle to six orifice plates of low adhesion,
Still further aspect can also be effectively facilitated the secondary self aggregation between cell micelle.The secondary self aggregation of cell mass causes cell
Group can connect into porous coral sample massive texture from each other, give attachment point and the space of the growth of cell micelle.
3）A not good liquor was changed every three days, after cultivation cycle is expired for two weeks, the hyaline cartilage sample block grown out is collected
Shape tissue, and be stored in liquid nitrogen to treat the detection and application in later stage at once.Culture medium prescription used by whole preparation process is：
Hyclone α-MEM basal mediums, 50 μ g/ml ascorbic acid, 1% penicillin and streptomysin mixed liquor and 10% tire ox blood
Clearly.
Start the fast-growth in low adhesion sheet after cell micelle self aggregation, because cell micelle original dimension is smaller, energy
Effective exchange of nutriment and cell discharged waste is enough effectively facilitated, the growth rate of cell is very fast.Keeping fast breeder
After speed two weeks, cell micelle starts to reduce growth rate, and starts in outer region hypertrophyization cell and columnar arrangement occur
Pattern.
Electron scanning Electronic Speculum (as shown in Figure 2) confirm the hyaline cartilage sample massive texture be by abundant extracellular matrix and
Uniformly inlay therein spherical or ellipticity cartilage cell to be constituted, and in obvious lacuna structure, contaminated through histotomy
Color is (as shown in Figure 3) to be confirmed to contain substantial amounts of mucopolysaccharide in extracellular matrix（GAG）And Type Ⅱ collagen（ColⅡ）, it is outer in tissue
The surrounding that all region cartilage cells are presented using the cell micelle center of circle as launch point launches cylindric arrangement.
Gained hyaline cartilage sample massive texture has and natural cartilage is close composition, structure and property, it is transparent soft
The cartilage cell included in bone sample massive texture in substantial amounts of mucopolysaccharide and Type Ⅱ collagen, tissue shows thin with natural cartilage
The spherical or ellipticity form that born of the same parents are close, and occur in that the cell columnar arrangement figure being close with natural cartilage hypertrophyization area
Case, macroscopic observation can be found that translucent sample is presented in tissue, and handling to touch can find that it has relatively low coefficient of friction and higher
Elastic modelling quantity.On composition：Hyaline cartilage sample massive texture of the present invention is by abundant extracellular matrix and uniformly inlays therein
Spherical or ellipticity cartilage cell is constituted, and confirms to contain substantial amounts of mucopolysaccharide in extracellular matrix through tissue section strain
（GAG）And Type Ⅱ collagen（ColⅡ）.In structure：Hyaline cartilage sample massive texture of the present invention is uniform by being uniformly distributed cartilage cell
Ground is embedded in abundant extracellular matrix, and in obvious lacuna structure, is presented in tissue outer region cartilage cell
Launch cylindric arrangement using the cell micelle center of circle as the surrounding of launch point.
Embodiment 3：
Take cartilage cell by three-dimensional Hanging drop culture method it is pre- it is agglomerating after, be transferred in the culture plate of low adhesion cultivate two weeks, pass through
Hyaline cartilage sample massive texture is generated after self aggregation between cell micelle.As shown in figure 5, whole biopsy tissues region is all divided
Red and brown has not been caught, implys that the presence of a large amount of GAG and Type Ⅱ collagen.Meanwhile, growth factor B MP-2 and TGF-β 1
In the presence of also being confirmed by immunofluorescence dyeing, there is substantial amounts of green fluorescence in the region in figure where cell.Further, it is quantitative to survey
Section the result more than result verification of examination, in the class cartilaginous tissue of all week old, we all detect GAG, collagen,
BMP-2, TGF-β 1 presence.
Alternately, in above-mentioned preparation method, hyaline cartilage massive texture is successfully prepared.
Alternately, in above-mentioned preparation method, this hyaline cartilage contains Type Ⅱ collagen and GAG, while also containing
1 two kinds of growth factors of BMP-2 and TGF-β.
A series of hyaline cartilage sample massive textures are made using top method, gained hyaline cartilage sample massive texture, including it is thin
Extracellular matrix and cartilage cell therein is uniformly embedded in, the extracellular matrix contains abundant natural cartilage composition mucopolysaccharide
And Type Ⅱ collagen, with the composition being close with natural cartilage, structure and property.
Embodiment 4：
Take cartilage cell by three-dimensional Hanging drop culture method it is pre- it is agglomerating after, be transferred in the culture plate of low adhesion cultivate two weeks, pass through
Hyaline cartilage sample massive texture is generated after self aggregation between cell micelle.As shown in figure 5, in the class cartilaginous tissue of 1mg dry weights,
GAG content is not changed in different week old class cartilaginous tissues, is all 180 μ g or so；The class cartilaginous tissue of 1mg dry weights
In, total collagen content is to increase over time the significant increase trend of presentation one；The class cartilaginous tissue of 1mg dry weights
In, the content of BMP-2 growth factors increases over time one significant downward trend of presentation；The class cartilage group of 1 mg dry weights
In knitting, the content of TGF-β 1 was reduced to conspicuousness before this, was then just increased to conspicuousness.
Alternately, by this patent method, at two weeks, successfully artificial culture went out hyaline cartilage tissue；
Alternately, such hyaline cartilage tissue, containing abundant GAG, collagen content, 1 mg dry weights contain respectively
180 μ g GAG and 200 μ g collagens.
Alternately, the hyaline cartilage tissue of this patent artificial incubation contain BMP-2 and the growth of 1 two kinds of TGF-β because
Son, 1 mg dry weights contain 30 pg BMP-2 and 100 pg TGF-βs 1. respectively
A series of hyaline cartilage sample massive textures are made using top method, gained hyaline cartilage sample massive texture, including it is extracellular
Matrix and cartilage cell therein is uniformly embedded in, the extracellular matrix contains abundant natural cartilage composition mucopolysaccharide and two
Collagen Type VI, with the composition being close with natural cartilage, structure and property.
The preferred embodiments of the present invention are the foregoing is only, for the purpose of the present invention, are merely illustrative, and it is non-limiting
's；Those of ordinary skill in the art understand, in patent requirements limited range of the present invention, it can be carried out it is many change,
Modification, or even equivalent change, but fall within protection scope of the present invention.
Claims (10)
1. the hyaline cartilage sample massive texture that a kind of extracorporeal culture is grown, it is characterised in that including extracellular matrix and uniformly
Cartilage cell therein is embedded in, the extracellular matrix contains abundant natural cartilage composition mucopolysaccharide and Type Ⅱ collagen.
2. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described soft
Osteocyte shows spherical or ellipticity form.
3. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described soft
Osteocyte is in the lacuna that extracellular matrix is constituted.
4. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described soft
Osteocyte presents columnar arrangement pattern and hypertrophyization phenomenon.
5. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described
Bright cartilage sample massive texture size is 1-2cm.
6. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described
The surrounding that massive texture outer region cartilage cell is presented using the cell micelle center of circle as launch point launches cylindric arrangement.
7. the hyaline cartilage sample massive texture that extracorporeal culture according to claim 1 is grown, it is characterised in that described soft
Osteocyte connects into porous coral sample massive texture by secondary self aggregation from each other.
8. the preparation method for the hyaline cartilage sample massive texture that a kind of extracorporeal culture is grown, it is characterised in that by cartilage cell
By three-dimensional Hanging drop culture method it is pre- it is agglomerating after, be transferred in the culture plate of low adhesion cultivate two weeks, through between cell micelle
Hyaline cartilage sample massive texture is generated after self aggregation.
9. preparation method according to claim 8, it is characterised in that specific steps include：
1）The cartilage cell that the articular hyaline cartilage of mammal source is isolated after enzymic digestion in vitro train by two dimensional surface
Support and be expanded to after P2 generations, it is 4 × 10 that concentration, which is made, in the pancreatin digestion without EDTA5Cells/ml cell suspending liquid, then will
This cell suspending liquid is inoculated on the Hanging drop culture plate in 384 holes, and the volume being inoculated with per hole is 30 μ l；
2）After being cultivated one day in Hanging drop culture plate, by adding 50ul phosphate buffer solutions（PBS）Cell micelle is collected
In 50ml centrifuge tubes, and with 300 × g centrifugal force 5 minutes, then by the cell micelle being collected into 2.3 ×
106The concentration in cells/ holes is inoculated in six orifice plates of low adhesion and cultivated；
3）A not good liquor was changed every three days, after cultivation cycle is expired for two weeks, the block group of the hyaline cartilage sample grown out is collected
Knit.
10. a kind of application for the hyaline cartilage sample massive texture that extracorporeal culture is grown, it is characterised in that use it for preparing soft
Osseous tissue renovating material.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114891736A (en) * | 2022-07-01 | 2022-08-12 | 中晶生物技术股份有限公司 | Method for enhancing growth and characteristic maintenance of chondrocytes by three-dimensionally cultured mesenchymal stem cell exosomes and application of method |
| CN115317672A (en) * | 2022-08-27 | 2022-11-11 | 四川大学 | Bionic bone cartilage integrated repair implant and preparation method and application thereof |
| WO2023160588A1 (en) * | 2022-02-25 | 2023-08-31 | 上海软馨生物科技有限公司 | Tissue engineering cartilage particle graft and preparation method therefor |
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| CN102526806A (en) * | 2012-01-20 | 2012-07-04 | 陕西博鸿生物科技有限公司 | Tissue engineering cartilage and preparation method thereof |
| US20140199408A1 (en) * | 1999-08-17 | 2014-07-17 | Immunopath Profile, Inc. | Therapeutic stem cell nutrient composition and uses thereof |
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| US20140199408A1 (en) * | 1999-08-17 | 2014-07-17 | Immunopath Profile, Inc. | Therapeutic stem cell nutrient composition and uses thereof |
| CN102526806A (en) * | 2012-01-20 | 2012-07-04 | 陕西博鸿生物科技有限公司 | Tissue engineering cartilage and preparation method thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023160588A1 (en) * | 2022-02-25 | 2023-08-31 | 上海软馨生物科技有限公司 | Tissue engineering cartilage particle graft and preparation method therefor |
| CN114891736A (en) * | 2022-07-01 | 2022-08-12 | 中晶生物技术股份有限公司 | Method for enhancing growth and characteristic maintenance of chondrocytes by three-dimensionally cultured mesenchymal stem cell exosomes and application of method |
| CN115317672A (en) * | 2022-08-27 | 2022-11-11 | 四川大学 | Bionic bone cartilage integrated repair implant and preparation method and application thereof |
| CN115317672B (en) * | 2022-08-27 | 2023-11-14 | 四川大学 | Bionic bone cartilage integrated repair implant, and preparation method and application thereof |
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