CN105018417B - The load intrinsic stem cell of amnion freezes active amnia particle and its conditioned medium and application - Google Patents
The load intrinsic stem cell of amnion freezes active amnia particle and its conditioned medium and application Download PDFInfo
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- CN105018417B CN105018417B CN201510435208.0A CN201510435208A CN105018417B CN 105018417 B CN105018417 B CN 105018417B CN 201510435208 A CN201510435208 A CN 201510435208A CN 105018417 B CN105018417 B CN 105018417B
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Abstract
The present invention relates to organizational projects and medicine wound repair technical field, and in particular to the load intrinsic stem cell of amnion freezes active amnia particle and its conditioned medium and application.Discarded fresh amnion is prepared into particle by the present invention, is frozen in liquid nitrogen using serum-free stem cell cryopreserving liquid, long-term to retain amniotic epithelial cells activity while retaining amnion intact matrix composition, and can effectively avoid transmission.Human epidermal cell, fibroblast, the chemotactic of endothelial cell, migration can be obviously promoted by its conditioned medium for collecting preparation, zoografting discovery active amnia particle can be by adjusting inflammatory reaction, a plurality of approach such as vascularization, quickening epithelialization being promoted to improve wound healing, and amnion stroma can induce corium regeneration directly as dermal substitute, significantly improve wound healing quality.The load intrinsic stem cell of amnion prepared by the present invention freezes active amnia particle and its conditioned medium and can provide a kind of simple effective method for wound repair.
Description
Technical field
It is inherently dry thin that the present invention relates to organizational project and medicine wound repair technical field more particularly to a kind of load amnions
Born of the same parents' freezes active amnia particle and its conditioned medium and application.
Background technique
The amnion-derived placenta in childbirth production process discards tissue, and structure is similar with regular skin structure, including
Epithelial layer, basilar memebrane, compacted zone, fibroblast layer and spongy layer are free of blood vessel, nerve, lymphoid tissue.Amnion is as external application
Covering is protected for burn and corneal injury has more than 100 years history, and have commercialization both at home and abroad at present removes cell amnion
Come out (domestic Rui Ji bioamnion Jiangxi RuiJi Biotechnology Co., Ltd, it is externalDHACM, MiMedx
Group, Inc.), and it is widely used in the protection of the various acute and chronic surface of a wound.
Application of the amnion in wound repair is concentrated mainly on two aspects：It is done first is that research discovery amniotic epithelial cells have
Cell characteristics can secrete various kinds of cell/growth factor, and keratinocyte cell migration can not only be induced to promote surface of a wound epithelialization, and
And the angiogenic factors containing there are many, the granulation tissue hyperplasia of chronic wound or ulcer can be enhanced, accelerate wound healing
(Franz MG,Payne WG,Xing L,et al.The use of amnion-derived cellular cytokine
solution to improve healing in acute and chronic wound models.Eplasty.2008；8:
E21.), therefore at present researcher improves wound healing using amniotic epithelial cells or amnion-derived cell factor solution；It is another
The natural basement membrane structure of aspect amnion and extracellular matrix components can be used as dermal substitute and rebuild dermis scaffold, furthermore grind
Study carefully and also found the bioactie agent for promoting wound healing in amnion stroma containing there are many, can be epidermal cell, fibroblast
Adherency, proliferation trophic factors abundant is provided, be based on this, the present inventor be prepared in early-stage study containing epidermal cell, at
Fibrocyte sheet living skin equivalents (Huang G, Ji S, Luo P, Liu H, Zhu S, Wang G, Zhou P,
Xiao S,Xia Z.Accelerated expansion of epidermal keratinocyte and improved
dermal reconstruction achieved by engineered amniotic membrane.Cell
Transplant.2013；22(10):1831-44.), and on this basis further it is prepared for the growth of analog epidermal stem cells
Niche microenvironment amnion microcarrier and its Graftskin (Ji SZ, Xiao SC, Luo PF, Huang GF, Wang GY,
Zhu SH,et al.An epidermal stem cells niche microenvironment created by
engineered human amniotic membrane.Biomaterials2011；32(31):7801-11), this research at
Fruit also obtains Chinese patent CN201110148123.6, and entitled " simulation epidermal stem cells grow niche microenvironment
Amnion microcarrier and its Graftskin ", Authorization Notice No. CN102367434B.
However it is above-mentioned independent equal in such a way that amniotic epithelial cells or de- cell amnion stroma are used to improve wound healing
It is difficult to while effectively playing the double dominant of amniotic secretion active factors and amnion natural substrates.
Up to the present, it also there have been no the active amnia being prepared into amnion while containing amniotic epithelial cells and amnion stroma
Particle constructs the research report of active dermal substitute.
Summary of the invention
Amniotic secretion active factors and amnion natural substrates can be effectively played simultaneously the purpose of the present invention is to provide a kind of
Double dominant amnion particle；Another object of the present invention is to provide the conditioned mediums containing the amnion particle, and
The amnion particle is preparing the application in dermal substitute
Main technical schemes of the invention are as follows：
The present invention is removed on fibroblast layer and the complete amnion of spongy layer reservation by removing after obtaining active amnia
Chrotoplast and amnion stroma (basilar memebrane and compacted zone) prepare the active amnia particle of microcarrier size on this basis, and adopt
Frozen in liquid nitrogen with serum-free stem cell cryopreserving liquid, it is particulate activated with long-term preservation amnion, while after half a year to amnion into
Row detects again, and selecting hepatitis virus antibody, syphilis antibody and HIV is that negative amnion carries out clinical application, can be effective
While retaining amniotic epithelial cells activity and natural substrates ingredient, transmission is avoided.
The secretion that active amnia particle prepared by the present invention containing amniotic epithelial cells not only remains amnion stem cell is special
Property, while effect of the amnion natural substrates as dermal substitute can be played, it can significantly promote wound healing, improve the surface of a wound and repair
Compound body amount.
The first aspect of the present invention, provide a kind of load intrinsic stem cell of amnion freezes active amnia particle, described
The construction method of amnion particle include the following steps：
A, the acquisition of amnion
Ratify through Hospital Ethical Committee, puerpera's informed consent, selection hepatitis virus antibody, syphilis antibody and HIV are
The fresh human placenta tissue of the negative puerpera that cuts open the belly, aseptically blunt separation chorion, removing remove fibroblast layer
And spongy layer, through penicillin containing 100U/ml, 100 μ g/ml streptomysins, 0.25 μ g/ml anphotericin phosphate
Amnion is cut into 4-10cm × 2-8cm (more excellent is 8 × 6cm) by buffered saline (PBS) 3 × 15min of soaking flushing
The sheet amnion of size.
B, the preparation of active amnia particle, freeze, recover
The sheet amnion of above-mentioned preparation is prepared into the amnion particle of 300-600 μm of size through microparticle skin cutting machine,
1000rpm is centrifuged ten minutes, supernatant is removed, with particle 1ml:5ml serum-free stem cell cryopreserving liquid (CTSTMSynth-a-Medium, life technology) it is that corresponding stem cell cryopreserving liquid is added in ratio, by the amnion containing frozen stock solution
Particle is respectively put into the cryovial of different size size (such as 3.8ml, 1.8ml), and cryovial is placed in liquid nitrogen for a long time
It saves backup.
It needs to take out above-mentioned cryovial recovery according to surface of a wound application, sets 37 DEG C of water-baths and sufficiently thaw 1 minute, PBS is rushed repeatedly
Wash centrifugation 3 times.
The second aspect of the present invention, the active amnia particle that freezes for providing the above-mentioned intrinsic stem cell of load amnion are being made
Application in standby conditioned medium.
The conditioned medium refers to the various factors of the secretion containing cell obtained after cell culture for a period of time
Culture medium.
Further, the present invention provides a kind of condition trainings for freezing active amnia particle of load intrinsic stem cell of amnion
Base is supported, preparation method is as follows：
It is immediately placed in 37 DEG C of water-baths after taking-up cryopreservation tube in liquid nitrogen sufficiently to thaw 1 minute, after PBS repeated flushing is centrifuged 3 times
Supernatant is removed, with particle 1ml：6ml DMEM culture medium is that ratio is added in six orifice plates, in 37 DEG C, 5%CO2Incubator continues to incubate
48h is educated, supernatant is collected, 1000rpm is centrifuged ten minutes, takes supernatant -80 DEG C of refrigerators after 0.2 μm of filter filters to freeze spare.
Thus to obtain active amnia particle conditions culture medium (conditioned medium, abbreviation CM) of the invention.
The active amnia particle and its conditioned medium advantage of the intrinsic stem cell of load prepared by the present invention are as follows：
1) amnion particle remains active amnia epithelial cell and complete amnion stroma structure, can not only secrete a variety of
Promote the active factors of wound healing, while its natural extracellular matrix can be used as dermal substitute induction corium and rebuild, and
Amnion particle size is 300-600 μm, and after transplanting the surface of a wound, surface of a wound blood plasma can preferably be infiltrated between amnion particle, is guaranteed on amnion
Endothelial cell survival.Furthermore the amniotic epithelial cells of research discovery recently have the multinomial differentiation potential of stem cell, and without immunogen
Property and tumorigenesis characteristic, therefore it is used for regeneration and reparation (Toda A, Okabe M, Yoshida T, the et of various tissues
al.The Potential of Amniotic Membrane/Amnion-Derived Cells for Regeneration
of Various Tissues.J PharmacolSci 2007；105(3):, including cutaneous appendages hair follicle, skin 215-228.)
Reconstruction (the Knezevic V.Differentiation potential of rat amnion.JAnat 1996 of adipose gland etc.；
189(1):1-7.Fliniaux I,Viallet JP,Dhouailly D,et al.Transformation of amnion
epithelium into skin and hair follicles.Differentiation 2004；72(9-10):558-
65.), active amnia particle prepared by the present invention is expected to play a role in cutaneous appendages are rebuild and repaired.
2) amnion particle is frozen using serum-free stem cell cryopreserving liquid, can while retaining amnion intact matrix composition,
It is long-term to retain amniotic epithelial cells activity and the conditioned medium rich in the various active factor be prepared, and avoid containing serum
Frozen stock solution saves the transmission that may cause, and furthermore can detect again to amnion tissue after half a year, and further excluding may
Existing infective virus (hepatitis virus, syphilis, inhibition of HIV), can effectively prevent transmission.
3) active amnia particle and its conditioned medium preparation method are simple in the present invention, and clinical application is convenient, Er Qieyang
Film Epithelial cell alive is easy long-term preservation, avoids amniotic epithelial cells and is separately cultured or takes off at the preparation of cell amnion stroma
Reason process, can effectively prevent cell be separately cultured or amnion stroma preparation in caused cell, matrix damage, improve active sheep
The application efficiency of film promotes laboratory and converts to clinic.
The third aspect of the present invention, the active amnia particle that freezes for providing the above-mentioned intrinsic stem cell of load amnion are being made
Application in standby wound repair graft materials or soft tissue defects filler.
The present invention provides the active amnia particles that freezes of the above-mentioned intrinsic stem cell of load amnion to prepare corium substitution
Application in object.
The present invention also provides the active amnia particle conditions culture mediums that freezes of the above-mentioned intrinsic stem cell of load amnion to exist
Application in cell culture.
The cell culture, in particular to for the application in wound repair cell culture, such as human epidermal cell, at fibre
Tie up cell, endothelial cell etc..
Experiment in vitro shows to recover after active amnia particle freezes 6 months, 2 years, after recovery in active amnia particle
Amniotic epithelial cells activity is respectively 73.4 ± 3.4%, 66.35 ± 3.83%, hence it is evident that be higher than sheet amnion (55.63 ±
2.37%, 43.93 ± 2.89%, P < 0.01).Simultaneously active amnia particle conditions culture medium can be obviously promoted human epidermal cell,
Fibroblast, the proliferation of endothelial cell, migration, hence it is evident that be higher than DMEM culture medium group (P<0.01).Furthermore diabetic mice is created
The discovery of face (db/db diabetic mice) transplantation experiments, the load intrinsic stem cell of amnion prepared by the present invention freeze active amnia
Particle can be by adjusting inflammatory reaction, a plurality of approach such as vascularization, quickening epithelialization being promoted to improve wound healing, Er Qieyang
Membrane matrix can be used as dermal substitute remodeling dermis scaffold, and not only flat appearance, softness after wound healing, shrinkage degree is light,
And there is obvious papillary structure in newborn epidermis, and amnion particle, which is gradually degraded, induces corium to rebuild, and neonatal dermal layer collagen is fine
Tie up queueing discipline, orderly, can obviously improve wound healing quality.
Detailed description of the invention
Fig. 1 is that active amnia particle and sheet amnion are recovered after freezing 6 months, 2 years in the present invention, Hoechst/PI dyeing
Viewed under fluoroscopy amniotic epithelial cells activity afterwards.(upper row is sheet amnion, and lower row is active particles amnion)
Fig. 2 is using active amnia particle conditions culture medium in vitro culture human epidermal cell (Fig. 2A), fibroblast (figure
2B), endothelial cell (Fig. 2 C), to three kinds of cell migration abilities compared with 10%FBS group (10% fetal calf serum group) and DMEM group
Influence.
Fig. 3 is that db/db diabetic mice full thickness dermal transplants LMAM group (active amnia particle group), DMAM (is gone thin
Born of the same parents' amnion particle group) and the wound of blank control group substantially see.
Fig. 4 is db/db diabetic mice full thickness dermal post-transplantation 28 days and 60 days pathological section HE colored graphs,
Middle A is active amnia particle transplantation group (LMAM), and B is to go cell amnion particle transplantation group (DMAM), and C is blank control group, and D is
Transplanting after two months active amnia particle gradually degrade remold corium.
Specific embodiment
Now in conjunction with embodiment and attached drawing, the invention will be further described, but implementation of the invention is not limited to that.
Embodiment 1. prepares active amnia particle (LMAM) of the present invention
1, the acquisition of amnion
Ratify through Hospital Ethical Committee, puerpera's informed consent, selection hepatitis virus antibody, syphilis antibody and HIV are
The fresh human placenta tissue of the negative puerpera that cuts open the belly, aseptically blunt separation chorion, removing remove fibroblast layer
And spongy layer, through penicillin containing 100U/ml, 100 μ g/ml streptomysins, 0.25 μ g/ml anphotericin phosphate
Amnion, is cut into the sheet amnion of 8 × 6cm size by buffered saline (PBS) 3 × 15min of soaking flushing.
2, the preparation of active amnia particle, freeze, recover and determination of activity
The sheet amnion of 8 × 6cm size is prepared into the amnion particle of 300-600 μm of size through microparticle skin cutting machine, with
The particle of the sheet amnion preparation of every 8 × 6cm size is that 3.2ml serum-free stem cell cryopreserving liquid is added in a unit
(CTSTMSynth-a-Medium, life technology) it is put into cryovial (specification 3.8ml), or divide equally
1.5ml serum-free stem cell cryopreserving liquid (CTS is separately added into for two unitsTMSynth-a-Medium,life
Technology it) is put into cryovial (specification 1.8ml), cryovial is placed in liquid nitrogen and is saved backup for a long time.According to the surface of a wound
Using needing to take out above-mentioned cryovial recovery, sets 37 DEG C of water-baths and sufficiently thaw 1 minute, PBS repeated flushing is centrifuged 3 times.
Take active amnia particle fluorescence after with Hochest 33342 (10mg/ml) and PI (10mg/ml) double dyes after recovering
Microscopically observation calculates amnion cell activity.
Before the result is shown in Figure 1, respectively sheet amnion, active amnia particle freeze and after freezing 6 months, 2 years and being recovered
Expression activitiy, amniotic epithelial cells activity is respectively 73.4 ± 3.4% in active amnia particle after recovering as the result is shown,
66.35 ± 3.83%, hence it is evident that be higher than sheet amnion (55.63 ± 2.37%, 43.93 ± 2.89%, P < 0.01).
The preparation of 2. active amnia particle conditions culture medium of embodiment
1, prepared by active amnia particle conditions culture medium (conditioned medium, abbreviation CM)
It is immediately placed in 37 DEG C of water-baths after taking-up cryopreservation tube in liquid nitrogen sufficiently to thaw 1 minute, after PBS repeated flushing is centrifuged 3 times
Supernatant is removed, with particle 1ml：6ml DMEM culture medium is that ratio is added in six orifice plates, in 37 DEG C, 5%CO2Incubator continues to incubate
48h is educated, supernatant is collected, 1000rpm is centrifuged ten minutes, takes supernatant -80 DEG C of refrigerators after 0.2 μm of filter filters to freeze spare.
2, Transwell detects CM and influences on epidermal cell, fibroblast, the chemotactic of endothelial cell, migration
Using 8 μm of pore sizes Transwell migration cell (Corning, USA) detection CM to human epidermal cell, at
Fibrocyte, the chemotactic of endothelial cell, transfer ability.Respectively by human epidermal cell, fibroblast, endothelial cell with 5 × 105
The concentration of a cell/ml is inoculated in Transwell upper chamber, and 500 μ L active amnia particle conditions culture mediums accordingly are added in lower room
CM, while as a control group with 10%FBS (10% fetal calf serum), DMEM culture medium, it is cultivated for 24 hours in regular growth incubator
Afterwards, carry out cell fix, violet staining.Every hole takes 5 visuals field, photograph, analysis of accounts migration at random under 100 power microscopes
The cell quantity of cross-film.
As shown in Figure 2, it is seen that active amnia particle conditions culture medium can significant chemotactic epidermal cell, fibroblast, interior
The migration of chrotoplast, effect and 10%FBS group no significant difference, obviously higher than DMEM culture medium group (p<0.01).
3. zoopery of embodiment
Db/db diabetic mice 15 (being provided by the western Poole in Shanghai-Bi Kai experimental animal Co., Ltd) is taken, male and female are not
Limit, conventional 1% yellow Jackets intraperitoneal anesthesia, alcohol disinfecting.It is made respectively directly in every db/db diabetic mice back two sides
Diameter is the round full thickness dermal wounds of 1.2cm, and it is (real that the db/db diabetic mice surface of a wound (30) is randomly divided into LMAM group
Apply the active amnia particle group that example 1 is prepared), DMAM group (go cell amnion particle group, detailed preparation method is shown in document Ji
SZ,Xiao SC,Luo PF,Huang GF,Wang GY,Zhu SH,et al.An epidermal stem cells niche
microenvironment created by engineered human amniotic
membrane.Biomaterials2011；32(31):7801-11) and blank control group, every group of 10 surface of a wound.
LMAM, DMAM are routinely thawed after recovery, and PBS repeated flushing removes supernatant after being centrifuged 3 times, by the uniform drop coating of particle
One layer is formed on the surface of a wound, the blank control group surface of a wound is without any processing.Three groups of surface of a wound application petrolatum gauze covering protection wounds
Petrolatum gauze interrupted suture is fixed on all skin of wound with 4-0 line by face.It is postoperative carried out every 7 days observation take pictures, pass through figure
As analysis software I mage J compare each group Wound healing rate (Wound healing rate=(initial surface of a wound area-residual wound area)/
Initial surface of a wound area), and row histological observation of periodically drawing materials.
Result of study discovery, 7 days LMAM group healing speeds are higher than DMAM group (55.67 ± 4.99% after transplanting
Vs.50.98 ± 5.56%, p<And blank control group (55.67 ± 4.99%vs.35.93 ± 5.76%, p 0.05)<0.001).
3 weeks after transplanting, the LMAM group surface of a wound heals substantially, and DMAM group and blank control group also remain the surface of a wound (result is shown in Fig. 3).
Materials slice row HE dyeing in 28 days after transplanting, it is seen that LMAM group and DMAM group amnion particle fill dermal matrix, table
The covering of face new life epidermis, there is papillary structure in the newborn epidermis of LMAM group, and does not observe this in DMAM group and blank control group
Structure (Fig. 4 A, B, C).It transplants latter two month amnion particle and starts gradually to degrade and rebuild auto derma (Fig. 4 D).
The experimental results showed that higher cell activity is maintained after active amnia particle cryopreservation resuscitation prepared by the present invention,
By its collect preparation conditioned medium can be obviously promoted human epidermal cell, fibroblast, endothelial cell proliferation, move
It moves, while active amnia particle can remarkably promote the vascularization of the diabetic mice surface of a wound, accelerates wound healing, furthermore amnion base
Matter can induce corium regeneration directly as dermal substitute, and a kind of alternative viable skin can be provided for wound repair and is replaced
For object.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (10)
1. a kind of load intrinsic stem cell of amnion freezes active amnia particle, which is characterized in that the structure of the amnion particle
Construction method includes the following steps：
A, the acquisition of amnion
Selection hepatitis virus antibody, syphilis antibody and HIV are the fresh human placenta tissue of the negative puerpera that cuts open the belly, in aseptic condition
Lower blunt separation chorion, removing removal fibroblast layer and spongy layer, through penicillin containing 100U/ml, 100 μ g/ml strepto-s
PBS 3 × 15min of soaking flushing of element, 0.25 μ g/ml anphotericin, is the piece that 4-10cm wide is 2-8cm by amnion cut growth
Shape amnion；
B, the preparation of active amnia particle, freeze
Sheet amnion prepared by step A is prepared into the amnion particle of 300-600 μm of size, 1000rpm is centrifuged ten minutes, removal
Serum-free stem cell cryopreserving liquid is added in supernatant；The amnion particle being immersed in serum-free stem cell cryopreserving liquid is put into cryovial
In, cryovial is placed in liquid nitrogen and is saved；The volume ratio of amnion particle and serum-free stem cell cryopreserving liquid is 1:5.
2. a kind of load intrinsic stem cell of amnion according to claim 1 freezes active amnia particle, which is characterized in that
The construction method of the amnion particle is further comprising the steps of：
C, it recovers
The cryovial recovery for taking out step B, sets 37 DEG C of water-baths and sufficiently thaws 1 minute, PBS repeated flushing is centrifuged 3 times.
3. a kind of load intrinsic stem cell of amnion according to claim 1 freezes active amnia particle, which is characterized in that
In the step A, the size of sheet amnion is 8 × 6cm.
4. a kind of load intrinsic stem cell of amnion according to claim 3 freezes active amnia particle, which is characterized in that
In the step B, with the particle of the sheet amnion preparation of every 8 × 6cm size for a unit, 3.2ml serum-free is added
Stem cell cryopreserving liquid is put into the cryovial that specification is 3.8ml.
5. a kind of load intrinsic stem cell of amnion according to claim 3 freezes active amnia particle, which is characterized in that
In the step B, two units are divided into the particle of the sheet amnion preparation of every 8 × 6cm size, are separately added into
1.5ml serum-free stem cell cryopreserving liquid is put into the cryovial that specification is 1.8ml.
6. a kind of load intrinsic stem cell of amnion freezes active amnia particle conditions culture medium, which is characterized in that the item
The preparation method of part culture medium is：
After taking out cryovial as described in claim 1, be immediately placed in 37 DEG C of water-baths and sufficiently thaw 1 minute, PBS repeated flushing from
Supernatant is removed after the heart 3 times, with amnion particle 1ml：6ml DMEM culture medium is that ratio is added in six orifice plates, in 37 DEG C, 5%CO2
Incubator continues to be incubated for 48h, collects supernatant, and 1000rpm is centrifuged ten minutes, takes supernatant -80 DEG C of ice after 0.2 μm of filter filters
Case freezes spare.
7. the active amnia particle that freezes of the load intrinsic stem cell of amnion is transplanted in preparation wound repair as described in claim 1
Application in material.
8. the active amnia particle that freezes of the load intrinsic stem cell of amnion is filled out preparing soft tissue defects as described in claim 1
Fill the application in object.
9. the active amnia particle conditions culture medium that freezes of the load intrinsic stem cell of amnion is trained in cell as claimed in claim 6
Application in supporting.
10. load the intrinsic stem cell of amnion as claimed in claim 6 freezes active amnia particle conditions culture medium in the surface of a wound
Application in reparation.
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| CN108812643B (en) * | 2018-07-18 | 2021-09-21 | 银丰生物工程集团有限公司 | Preparation and cryopreservation method and application of human placental chorionic villus tissue |
| CN110484491B (en) * | 2019-08-20 | 2022-03-18 | 北京京蒙高科干细胞技术有限公司 | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof |
| CN111718894B (en) * | 2020-06-28 | 2022-05-13 | 上海交通大学医学院附属第九人民医院 | Method for promoting diabetic wound healing by jointly utilizing hFDSPCs-CM and HA |
| CN113521397B (en) * | 2021-08-09 | 2022-04-05 | 唐山市工人医院 | Preparation method of anti-adhesion membrane for preventing and treating myofascial adhesion and anti-adhesion membrane |
| CN114377208A (en) * | 2022-01-29 | 2022-04-22 | 中国人民解放军海军军医大学第一附属医院 | Micro active dermis substitute and construction method thereof |
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| CN102367434A (en) * | 2011-06-03 | 2012-03-07 | 中国人民解放军第二军医大学 | Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof |
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| WO2012112410A2 (en) * | 2011-02-14 | 2012-08-23 | Mimedx Group Inc. | Micronized placental tissue compositions and methods for making and using the same |
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| CN102367434A (en) * | 2011-06-03 | 2012-03-07 | 中国人民解放军第二军医大学 | Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof |
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