CN109771694A - The preparation method and application of self assembly polypeptide nano fiber water gel scaffold material - Google Patents
The preparation method and application of self assembly polypeptide nano fiber water gel scaffold material Download PDFInfo
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- CN109771694A CN109771694A CN201910212352.6A CN201910212352A CN109771694A CN 109771694 A CN109771694 A CN 109771694A CN 201910212352 A CN201910212352 A CN 201910212352A CN 109771694 A CN109771694 A CN 109771694A
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Abstract
The invention discloses a kind of preparations of self assembly polypeptide nano fiber water gel scaffold material for carrying people's umbilical cord Wharton jelly mescenchymal stem cell.The invention also discloses a kind of for carrying the tissue engineering material of people's umbilical cord Wharton jelly mescenchymal stem cell, and it includes self assembly polypeptide nano fiber water gel scaffold materials.Self assembly polypeptide nano fiber water gel scaffold material in the present invention is the hydrogel of RADA16-1 polypeptide.Self assembly polypeptide nano fiber water gel scaffold material definite ingredients of the invention, performance is stablized, and nonhazardous, good biocompatibility, reliable microenvironment can be provided for culture people's umbilical cord Wharton jelly mescenchymal stem cell, had a good application prospect in the research using the compound people's umbilical cord Wharton jelly mescenchymal stem cell of tissue engineering material.
Description
Technical field
The present invention relates to medicine, tissue engineering technique field, more particularly to a kind of carrying people umbilical cord Wharton jelly mesenchyma
The preparation method and application of the self assembly polypeptide nano fiber water gel scaffold material of stem cell.
Background technique
Source for mesenchymal stem cells is a kind of stem cell of the multi-lineage potential of non-haematological origin in mesoderm.It is exempted from
Epidemic disease source property is weak, can have become ideal in tissue engineering technique field and cellular transplantation therapy to Various Tissues Proliferation, Differentiation
Seed cell source.Mescenchymal stem cell is most found early in people's bone marrow, but there are other a small amount of cells in marrow, as hematopoiesis is thin
Born of the same parents, fat cell, fibroblast etc., and mescenchymal stem cell content is less, isolates and purifies difficulty, and with the increasing at age
Long, the quantity and Proliferation, Differentiation ability of mesenchymal stem cell are showed and are remarkably decreased.It is cannot ignoring in addition, obtain marrow
Tissue brings larger in privation and wound to donor, therefore is widely used in clinic and faces larger difficulty.
People's umbilical cord Wharton jelly mescenchymal stem cell derives from neonatal umbilical cord, belongs to medical waste tissue, do not produce to donor
Raw in privation and damage, and it is from a wealth of sources, therefore be easier to be widely applied in clinic.Huatong plastic tissue be umbilical cord arteriovenous it
Between gel-shaped interstitial, containing more collagenous fibres, clostridiopetidase A can hydrolyze the collagen fiber structure of huatong plastic tissue, but not
People's umbilical cord Wharton jelly mescenchymal stem cell is caused to damage.
With the development of biologic treatment and tissue engineering technique, a large amount of new treatment methods are obtained in scientific research level
Good effect and wide prospect.Surface is studied, the biologic bracket material of imitative nucleus pulposus is constructed using tissue engineering technique,
The microenvironment of growth can be provided for part cell, it, can not only if carrying stem cell by such biologic bracket material in this way
Guarantee the existence of stem cell, additionally it is possible to ensure stem cell in the effect of the stable maximum performance stem cell of appointed part.
It has now been found that RADA16-I (AcN-RADARADARADARADA-GG-KOSSAPTQLN-CONH2) is a kind of injectable
Self assembly polypeptide nano fiber water gel scaffold material.Nucleus pulposus can be entered by minimally invasive injection.This material, can be quick
A large amount of nanofibers are formed, composition is similar to extracellular matrix, provides good three-dimensional environment for the culture of cell.In bone, mind
Good research achievement is had been achieved with through, cardiac muscle etc..
This research explores it and is carrying people's umbilical cord China from building self assembly polypeptide nano fiber water gel scaffold material
Application in terms of logical glue mesenchymal stem cells is filled up and plays therapeutic effect using the method that Method of Tissue Engineering carries stem cell
Blank.
Summary of the invention
The present invention provides a kind of preparation method and applications of self assembly polypeptide nano fiber water gel scaffold material.This hair
Bright self assembly polypeptide nano fiber water gel scaffold material definite ingredients, performance is stablized, and nonhazardous, good biocompatibility,
Existence persistently can be provided to people's umbilical cord Wharton jelly mescenchymal stem cell to support, carrying stem cell using Method of Tissue Engineering
It has a good application prospect in the research of method performance therapeutic effect.
To achieve the above object, the present invention adopts the following technical scheme that:
One, self assembly polypeptide nano fiber water gel scaffold material is constructed
1, by self assembly polypeptide RADA16-I (AcN-RADARADARADARADA-GG-KOSSAPTQLN-CONH2) (purity
> 90%) polypeptide powder, is dissolved using ultrapure water, so that solution concentration is reached 1%, and use Ultrasonic Cell Disruptor probe sonication,
It dissolves it thoroughly, solution is sterilized using the disposable sterilized injecting type filter membrane that aperture is 0.22 μm then, obtains 1% certainly
Assemble polypeptide RADA16-I solution for standby.
2,1% self assembly polypeptide of 400 μ L culture mediums and 100 μ L is successively added to each aperture in the cell transwell
RADA16-I solution, then in being incubated for 1h in 37 DEG C of incubators.
3, after the small indoor solution of transwell forms gel, then to gel surface 400 μ L culture mediums are slowly added to, so
Afterwards in being incubated for 30min in 37 DEG C of incubators.Then it is carried out more every complete medium of the 30min to culture hole and gel surface
It changes, is total to the pH value twice to balance polypeptide hydrogel surface.
4, culture medium is configured to be divided into:
5, the preparation method of culture medium are as follows: after mixing each ingredient, stir sufficiently dissolution, then utilize miillpore filter positive pressure
Nutsch filter (built-in 3 Ф, 0.22 μm of filter membrane) filters, and every 90mL is divided in 100mL aseptic bottle, seals, 4 DEG C of refrigerations.And by its
PH is adjusted between 6.9~7.2.
Two, using self assembly polypeptide nano fiber water gel scaffold material, people's umbilical cord Wharton jelly mescenchymal stem cell is carried
1, people's umbilical cord Wharton jelly mescenchymal stem cell is obtained.
(1) neonatal umbilical cord used, is term birth of newborn, and the hepatitis B surface antigen of puerpera, c-hepatitis antibody, syphilis
Antibody, aids antibody immune 4 are negative healthy puerpera.
(2) umbilical cord is washed away into umbilical cord remained blood with balanced salt solution in super-clean bench, umbilical cord is cut into 4cm with operating scissors
Umbilical cord outer membrane is cut off in size segment, operating scissors stringer, carefully free 2 umbilical arteries and 1 umbilical vein and is abandoned, is taken blood vessel
Between and the huatong plastic tissue between blood vessel and outer membrane, cut into the big fine grained chippings of about 1mm3, after balanced salt solution rinses, be dipped in
Concentration is to set 37 DEG C of constant temperature digestion 18h in 0.2% collagenase solution.
(3) it when after digesting to inorganization block, is centrifuged using 2500rpm, total 10min abandons supernatant afterwards, DMEM/F12 is added to cultivate
Base and fetal calf serum make fetal calf serum concentration reach 20%, in 37 DEG C, 5%CO2 CMC model.
(4) when cell fusion reaches 80-90%, supernatant is abandoned, PBS washes cell, 0.25% trypsin digestion 5min,
1000rpm centrifugation is cultivated with the DMEM/F12 culture medium containing 10% fetal calf serum after abandoning supernatant, carries out changing liquid within every 3 days.
The preparation method of (5) 0.2% collagenase solutions is: 50ml DMEM/F12 culture is added in II Collagenase Type of 100mg
Base, after being completely dissolved, 0.22 μm of filtration sterilization.The preparation method of DMEM/F12 complete medium: 450ml DMEM/F12 culture
50ml superfine fetal calf serum is added in base, is configured to the complete medium containing 10% fetal calf serum, penicillin and streptomysin is added,
Make concentration 100u/mL penicillin, 100 μ g/m L streptomysins.
2, method is resuspended using 10% sucrose water, collector's umbilical cord Wharton jelly mescenchymal stem cell will contain 1 × 10 in 20 μ L5
The suspension of personal umbilical cord Wharton jelly mescenchymal stem cell mixes rapidly with 100 μ L1% self assembly polypeptide RADA16-I solution.
3, the cell of preparation/1% self assembly polypeptide RADA16-I mixed solution is quickly placed in the small interior transwell,
400 μ L complete mediums are slowly softly added to its surface again, and the cell transwell is placed in each aperture, 400 μ L is added
In 24 orifice plates of complete medium, 10min is incubated in 37 DEG C of incubators.Then, to the complete training of culture hole and hydrogel surface
Feeding base is replaced, and 30min is incubated in 37 DEG C of incubators.Then the complete culture every 30min to culture hole and gel surface
Base is replaced, and the pH value twice to balance polypeptide hydrogel surface is total to.
4, the preparation method of complete medium is, by culture medium made from front, before use, being added in every bottle of solution
10mL fetal calf serum, as complete medium.
Specific embodiment
Combined with specific embodiments below the present invention is made further to explain.
Embodiment 1
Prepare self assembly polypeptide nano fiber water gel scaffold material.
1, by self assembly polypeptide RADA16-I (AcN-RADARADARADARADA-GG-KOSSAPTQLN-CONH2) (purity
> 90%) polypeptide powder 10mg, is dissolved using 1ml ultrapure water, so that solution concentration is reached 1%, and visit using Ultrasonic Cell Disruptor
Needle ultrasound, dissolves it thoroughly, then sterilizes solution using the disposable sterilized injecting type filter membrane that aperture is 0.22 μm, obtains
To 1% self assembly polypeptide RADA16-I solution for standby.
2,1% self assembly polypeptide of 400 μ L culture mediums and 100 μ L is successively added to each aperture in the cell transwell
RADA16-I solution, then in being incubated for 1h in 37 DEG C of incubators.
3, after the small indoor solution of transwell forms gel, then to gel surface 400 μ L culture mediums are slowly added to, so
Afterwards in being incubated for 30min in 37 DEG C of incubators.Then it is carried out more every complete medium of the 30min to culture hole and gel surface
It changes, is total to the pH value twice to balance polypeptide hydrogel surface.
4, culture medium is configured to be divided into:
5, the preparation method of culture medium are as follows: after mixing each ingredient, stir sufficiently dissolution, then utilize miillpore filter positive pressure
Nutsch filter (built-in 3 Ф, 0.22 μm of filter membrane) filters, and every 90mL is divided in 100mL aseptic bottle, seals, 4 DEG C of refrigerations.And by its
PH is adjusted between 6.9~7.2.
Embodiment 2
Collect culture people's umbilical cord Wharton jelly mescenchymal stem cell.
27 years old women, multipara, fetus term birth, Cesarean esction, taking fresh regeneration umbilical cord about 20cm is material, sterile storage
It is stored in D-Hanks liquid, 2-6 DEG C of preservation.
Umbilical cord remained blood is washed away with balanced salt solution in superclean bench, umbilical cord is cut into 5cm size segment, to every
Umbilical cord outer membrane is cut off in the equal stringer in one segment, and dissociate removal arteria umbilicalis, umbilical vein, is taken between blood vessel and between blood vessel and outer membrane
Huatong plastic tissue cuts into the big fine grained chippings of about 1mm3, and after being rinsed with balanced salt solution, being dipped in concentration is 0.2% collagenase solution
In, it sets 37 DEG C and digests 5 hours, to digestive juice at thick, 2500rpm is centrifuged 10 minutes, discards supernatant, then with 0.25% pancreas
7 DEG C of protease 3 constant temperature digestion 30 minutes, 2500rpm are centrifuged 10 minutes, are abandoned supernatant, are added DMEM/F12 culture medium and tire ox blood
Clearly, make fetal calf serum concentration 20%, 37 DEG C, 5% carbon dioxide conditions culture.Cell was in the 3rd day adherent, the 5th day use of beginning
DMEM/F12 complete medium containing 20% fetal calf serum carries out changing liquid.It changes the liquid once within every 2~3 days, is reached to cell fusion later
When to 80-90%, secondary culture is carried out.Abandon supernatant, PBS washes cell, 0.25% trypsin digestion 5 minutes, 1000rpm from
The heart carries out cell passage.It is cultivated after passage with the DMEM/F12 culture medium containing 10% fetal calf serum, carries out changing liquid within every 3 days.
Embodiment 3
The preparation of cell/polypeptide hydrogel timbering material.
1, method is resuspended using 10% sucrose water, collector's umbilical cord Wharton jelly mescenchymal stem cell will contain 1 × 105 in 20 μ L
The suspension of personal umbilical cord Wharton jelly mescenchymal stem cell mixes rapidly with 100 μ L1% self assembly polypeptide RADA16-I solution.
2, the cell of preparation/1% self assembly polypeptide RADA16-I mixed solution is quickly placed in the small interior transwell,
400 μ L complete mediums are slowly softly added to its surface again, and the cell transwell is placed in each aperture, 400 μ L is added
In 24 orifice plates of complete medium, 10min is incubated in 37 DEG C of incubators.Then, to the complete training of culture hole and hydrogel surface
Feeding base is replaced, and 30min is incubated in 37 DEG C of incubators.Then the complete culture every 30min to culture hole and gel surface
Base is replaced, and the pH value twice to balance polypeptide hydrogel surface is total to.
3, the preparation method of complete medium is, by culture medium made from front, before use, being added in every bottle of solution
10mL fetal calf serum, as complete medium.
Embodiment 4
Observe self assembly polypeptide nano fiber water gel scaffold material
1, the self assembly polypeptide nano fiber water gel scaffold material and cell that have prepared/polypeptide hydrogel is sharp respectively
30min is fixed with 2.5% glutaraldehyde.
2, it is rinsed 3 times using PBS.
3, it is dehydrated 10min every time with 30%, 50%, 70%, 80%, 90% and 100% alcohol serial dehydration respectively.
4, the drying of 1 boundary's point is carried out using the tert-butyl alcohol.
5, metal spraying is carried out to material.
6, material is observed using scanning electron microscope, amplifies 400~25000 times, voltage 20kV.
Test result
Utilize scanning electron microscope, it is seen that the fiber interweaving that self assembly polypeptide nano fiber water gel scaffold material is formed is at stagger arrangement
Orderly gap spline structure timbering material, fibre diameter about 20~40nm, aperture about 10~250nm.It will be between people's umbilical cord Wharton jelly
After mesenchymal stem cells kind is implanted into timbering material culture 15 days, visible a large amount of people's umbilical cord Wharton jelly mescenchymal stem cells, cell are observed
By the nanofiber tight adhesion in pseudopodium and bracket, three-dimensional growth state, and visible a large amount of extracellular matrix samples are showed
Secretion.
Claims (10)
1. a kind of preparation method of self assembly polypeptide nano fiber water gel scaffold material, which is characterized in that the self assembly
Polypeptide nano fiber water gel scaffold material is a kind of RADA16-I (AcN-RADARADARADARADA-GG-KOSSAPTQLN-
CONH2) hydrogel.
2. preparation method according to claim 1, which is characterized in that the self assembly polypeptide RADA16-I (AcN-
RADARADARADARADA-GG-KOSSAPTQLN-CONH2) be a kind of purity > 90% polypeptide powder, it is using ultrapure water that its is molten
Solution makes solution concentration reach 1%, and uses Ultrasonic Cell Disruptor probe sonication, dissolves it thoroughly, and solution is then utilized aperture
It is sterilized for 0.22 μm of disposable sterilized injecting type filter membrane, obtains 1% self assembly polypeptide RADA16-I solution for standby.
3. preparation method according to claim 2, which is characterized in that be successively added to each aperture in the cell transwell
1% self assembly polypeptide RADA16-I solution prepared by 400 μ L culture mediums and 100 μ L claims 2, then in 37 DEG C of incubators
Interior incubation 1h.
4. preparation method according to claim 2, which is characterized in that molten to the small interior transwell described in claim 3
After liquid forms gel, then to gel surface 400 μ L culture mediums are slowly added to, then in being incubated for 30min in 37 DEG C of incubators.Then
It is replaced every complete medium of the 30min to culture hole and gel surface, altogether twice to balance polypeptide hydrogel surface
PH value.
5. according to preparation method described in claim 3 or 4, which is characterized in that its culture medium is configured to be divided into:
6. preparation method according to claim 5, which is characterized in that the preparation method of its culture medium are as follows: mix each ingredient
After conjunction, sufficiently dissolution is stirred, is then filtered using miillpore filter positive pressure nutsch filter (built-in 3 Ф, 0.22 μm of filter membrane), every 90mL
It is divided in 100mL aseptic bottle, seals, 4 DEG C of refrigerations.And its pH is adjusted between 6.9~7.2.
7. application according to claim 1, which is characterized in that by everybody umbilical cord Wharton jelly mescenchymal stem cell of collection and
The mixing of 1% self assembly polypeptide RADA16-I solution, then carries out the preparation of cell/polypeptide hydrogel timbering material.
8. application according to claim 7, which is characterized in that method is resuspended using 10% sucrose water, collector's umbilical cord China is logical
Glue mesenchymal stem cells will contain 1 × 10 in 20 μ L5The suspension of personal umbilical cord Wharton jelly mescenchymal stem cell and 100 μ L1% are certainly
Assembling polypeptide RADA16-I solution mixes rapidly.
9. application according to claim 9, which is characterized in that the cell prepared in claim 9/1% self assembly is more
Peptide RADA16-I mixed solution is quickly placed in the small interior transwell, then 400 μ L are slowly softly added to its surface and cultivate completely
Base, and the cell transwell is placed in each aperture and is added in 24 orifice plates of 400 μ L complete mediums, it is incubated in 37 DEG C of incubators
Educate 10min.Then, the complete medium of culture hole and hydrogel surface is replaced, 30min is incubated in 37 DEG C of incubators.
Then it is replaced every complete medium of the 30min to culture hole and gel surface, altogether twice to balance polypeptide hydrogel table
The pH value in face.
10. application according to claim 9, which is characterized in that the preparation method of its complete medium is to be wanted according to right
Culture medium obtained by asking 7, before use, 10mL fetal calf serum, as complete medium are added in every bottle of solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111603482A (en) * | 2020-05-28 | 2020-09-01 | 高连如 | Mesenchymal stem cell medicine for regulating immune function and preparation method thereof |
| CN113713175A (en) * | 2021-07-27 | 2021-11-30 | 南通大学 | Method for preparing hydrogel scaffolds and use of scaffolds obtained thereby |
| CN116942889A (en) * | 2023-06-06 | 2023-10-27 | 广州图微科创生物科技有限公司 | Preparation method of hemostatic anti-adhesion polypeptide hydrogel |
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| CN104356402A (en) * | 2014-10-10 | 2015-02-18 | 孙念峰 | Functional self-assembling nano peptide hydrogel |
| CN105079783A (en) * | 2014-05-22 | 2015-11-25 | 清华大学深圳研究生院 | Pharmaceutical composition and preparation method and application thereof |
| CN106397545A (en) * | 2016-09-30 | 2017-02-15 | 暨南大学 | Hydrogel material as well as preparation method and application thereof |
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2019
- 2019-03-20 CN CN201910212352.6A patent/CN109771694A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105079783A (en) * | 2014-05-22 | 2015-11-25 | 清华大学深圳研究生院 | Pharmaceutical composition and preparation method and application thereof |
| CN104356402A (en) * | 2014-10-10 | 2015-02-18 | 孙念峰 | Functional self-assembling nano peptide hydrogel |
| CN106397545A (en) * | 2016-09-30 | 2017-02-15 | 暨南大学 | Hydrogel material as well as preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111603482A (en) * | 2020-05-28 | 2020-09-01 | 高连如 | Mesenchymal stem cell medicine for regulating immune function and preparation method thereof |
| CN113713175A (en) * | 2021-07-27 | 2021-11-30 | 南通大学 | Method for preparing hydrogel scaffolds and use of scaffolds obtained thereby |
| CN116942889A (en) * | 2023-06-06 | 2023-10-27 | 广州图微科创生物科技有限公司 | Preparation method of hemostatic anti-adhesion polypeptide hydrogel |
| CN116942889B (en) * | 2023-06-06 | 2024-02-02 | 广州图微科创生物科技有限公司 | Preparation method of hemostatic anti-adhesion polypeptide hydrogel |
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