CN109078224A - A kind of repair materials of skin injury and preparation method thereof - Google Patents
A kind of repair materials of skin injury and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The invention discloses a kind of repair materials of skin injury, it is using submucous layer of small intestine film as matrix, the repair materials that compound fat mescenchymal stem cell is formed.The repairing effect of repair materials of the present invention is good, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to the fields of reparation, and in particular to a kind of repair materials of skin injury and preparation method thereof.
Background technique
Skin is the maximum organ of human body, is the natural barrier between human body and the external world, has each organizer in protective
Official from reason, change, microbiological attack ability.In addition to this, the accessory organs such as the hair follicle of skin growths, sweat gland, sebaceous glands,
Beauty is played, perspires, secrete the functions such as grease lubrication.Skin can be but clinically common big with self-healing after centainly being damaged
Often prolonged refractory or skin carries out the surface of a wound in a manner of connective tissue proliferation for Area Burn, wound, diabetic foot ulcer etc.
Healing forms scar, influences skin beauty and normal function.Clinical treatment large area or refractory skin injury, generally require
Carry out the transplanting of self or heterogenous skin.And the source for transplanting skin is often extremely limited.With the progress of organizational project, at present
Organized engineering skin listing, new selection is provided for skin injury reparation.But current organizational project skin existing on the market
Skin is expensive, and skin normal function restores limited after transplanting, it would be highly desirable to develop a kind of
Tissue engineering product.
Fat mesenchymal stem cell (Adipose derived stem cells, ADSCs) is abundance, draws materials simply
Adult stem cell, have fast breeding and lasting self-renewal capacity, under inducing culturing condition can at rouge, skeletonization, at
Cartilage differentiation.Because of its one kind for belonging to mescenchymal stem cell, II type antigen of MHC is not expressed, and there is immunoregulatory ability,
Heteroplastic transplantation does not cause immunological rejection, makes it have wide application prospect.We compare fat mesenchymal stem cell and
The mescenchymal stem cell in the sources such as umbilical cord, amnion, chorion, decidua, discovery fat mesenchymal stem cell secrete Angiogensis
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) ability it is especially strong, judgement
It is the ideal seed cell of skin injury reparation.
Small intestinal submucosa (small intestine submucosa, SIS) is one of the composed structure of small intestine, is one
The degradable natural extracellular matrix class bio-derived material of kind, it is made of one layer of fine and close connective tissue, is mainly contained
I, III collagen type has antimicrobial acivity, there is good biomechanical property and biocompatibility.Containing there are many growths
The factor: such as transforming growth factor-β (transforming growth factor- β, TGF-β), tumor necrosis factor-alpha
(tumor necrosis factor- α, TNF-α), Basic Fibroblast Growth Factor (basic fibroblast growth
Factor, bFGF) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) etc..SIS
With low immunogenicity, immune response of the receptor to bacterium and virus is not influenced；Good bio-mechanical property, cytocompatibility
Property, histocompatbility and internal degradation capability；The advantages that promoting revascularization.Therefore, SIS is applied into tendon, ligament, wing
In the wound repairs such as Guang.The preparation method of SIS: small intestinal submucosa is carried out disinfection with random order, degreasing, de- cell, is gone
After dirt processing, it is prepared.
That sees at present repairs the article and patent of full thickness skin damage using organization engineering skin, and it is attached to there are no skin
The report of neomorph.Therefore, however it remains improve the demand of the curative effect for the treatment of skin injury at present.
Summary of the invention
It to solve the above-mentioned problems, can the present invention provides repair materials of a kind of skin injury and preparation method thereof
To repair skin injury, regeneration skin accessory organ.
A kind of repair materials of skin injury of the present invention, it is characterised in that: it is using submucous layer of small intestine film as matrix, again
Close the repair materials that fat mesenchymal stem cell is formed.
Preferably, it is dry thin that 100~1000 fat mesenchymals are compounded on described every square millimeter of submucous layer of small intestine film
Born of the same parents.
Preferably, the submucous layer of small intestine film is cell free submucous layer of small intestine.
Preferably, the submucous layer of small intestine film is prepared as follows: being taken chitterlings intestinal segment, is cleaned, removes serous coat
Layer and muscle layer, degreasing take off cell, freeze-drying.
The degreasing is using (0.5-2): the chlorine of 2:1 is preferably used in 1 chloroform-methanol mixed liquor degreasing 4-8 hours
Imitation-carbinol mixed liquor degreasing 6 hours；The de- cell is first to be handled using the trypsin solution of 0.1%-1%, reuses 0.1-
1% SDS solution processing, is preferably first handled using 0.25% trypsin solution, and 0.3% SDS solution processing is reused.
The fat mesenchymal stem cell, which is prepared by the following method:, takes fat mesenchymal stem cell, cultivates, detection
When cell confluency degree is 80%, every milligram of total protein medium vascular endothelial growth factor content in culture supernatant, while detecting thin
The expression of antigens c D10, CD200 of born of the same parents, selection have fat mesenchymal stem cell with the following characteristics: a, convergence degree
When being 80%, in culture supernatant, every milligram of total protein medium vascular endothelial growth factor content >=300pg；B, antigen is expressed
CD10 does not express antigens c D200.
The culture medium is the DMEM/F12 culture medium for the platelet rich plasma PRP for being 2%-5% containing volume fraction, training
The condition of supporting is 37 DEG C, 5%CO2；The platelet rich plasma PRP is prepared as follows: taking blood, sodium citrate is added extremely
0.05~0.15mol/L is placed in refrigerated centrifuge and is centrifuged 15min with 4000g, collects upper liquid, as platelet rich plasma；
Preferably, sodium citrate is added to 0.1mol/L.
The fat mesenchymal stem cell is prepared as follows:
(1) adipose tissue is taken, the collagen enzyme solution digestion of 0.05-1% is carried out；
(2) postdigestive adipose tissue is taken, is centrifuged, obtains vascular stroma component；
(3) it by isolated vascular stroma component, is added after culture medium is resuspended and is cultivated, it is dry thin to obtain fat mesenchymal
Born of the same parents；The culture medium is the DMEM/F12 culture medium for the platelet rich plasma PRP for being 2%-5% containing volume fraction, the richness
Thrombocyte plasma PRP is prepared as follows: taking blood, sodium citrate is added to 0.1mol/L, is placed in refrigerated centrifuge
It is centrifuged 15min with 4000g, collects upper liquid, as platelet rich plasma；
(4) fat mesenchymal stem cell originally culture to convergence degree 80% carries out had digestive transfer culture culture, obtains filling between fat
Matter stem cell；
It is preferred that the digestion is that 1:1 is mixed by volume with digestive juice by adipose tissue in step (1), it is placed in 37 DEG C of perseverances
Revolving speed 50-250rpm digests 0.5-4h in warm shaking table；
It is preferred that the centrifugation is 150-2000g centrifugation 5min in step (2)；
It is preferred that the culture bottle is T75 culture bottle in step (3)；The condition of the culture are as follows: 37 DEG C, 2-8%CO2,
It is cultivated in saturated humidity incubator, changes liquid after 48h for the first time, every 2d is changed the liquid once later；It is preferred that in step (3), passage amplification
Ratio is 1:4-1:5.
The present invention also provides a kind of preparation methods for preparing aforementioned repair materials, it is characterised in that: steps are as follows: taking rouge
Fat mescenchymal stem cell is compound on submucous layer of small intestine film.
The present invention also provides purposes of the aforementioned repair materials in preparation skin regeneration material.
The fat mesenchymal stem cell of good properties has been prepared using ad hoc approach by the present invention, is compound in SIS
On timbering material, by the synergistic effect of fat mesenchymal stem cell and SIS, skin injury can be repaired, and skin can be regenerated
Accessory organ, such as hair follicle, sweat gland, sebaceous glands, repairing effect is very good, and the reparation for clinic skin damage provides one kind
More preferably select.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
The characteristic for the hADSCs that Fig. 1 separation obtains
A1-A3, hADSCs cellular morphology, 40 ×；A1, visible attached cell after inoculation 2 days；A2, it is visible after adherent 4-5 days
More how long shuttle sample hADSCs；A3, the hADSCs of convergence degree 80% or so.B1-B3, hADSCs three-dimensional differentiation capability；B1, hADSCs
Osteoblast Differentiation；B2, hADSCs break up at rouge；B3, hADSCs are at cartilage differentiation.The double dye hADSCs streams in the channel C1-C7, FITC and PE
Formula testing result；1 κ-PE Isotype control of C1, IgG1 κ-FITC/IgG；C2, HLA-DR-FITC/CD73-PE；C3,
CD90-FITC/CD14-PE/CD34-PE；C4, CD45-FITC/CD105-PE；C5, CD44-FITC/CD166-PE；C6,
CD29-FITC/CD79α-PE；C7, CD146-FITC/CD31-PE.The experiment of D, hADSCs cell clonal formation.(n=3) E,
HADSCs passes on the amplification curve to P10 generation.(n=3)
The finished figure of the simple SIS of Fig. 2.
Fig. 3 is compounded in fat mesenchymal stem cell vital staining and electron-microscope scanning on SIS film；A, calcein-AM
The cytoplasm of the fat mesenchymal stem cell for the work that (Calcein AM) dyeing is attached on SIS, is presented bright green；Bromine second
The nucleus for the dead fat mesenchymal stem cell that coffee ingot dimer (EthD-1) dyeing is attached on SIS film is presented red.B,
Scanning electron microscopic observation: visible SIS coarse surface structure, cell, which is uniformly attached on material, to be grown, and has the extracellular base of more amount
Matter secretion.
Fig. 4 Diabetes Mellitus SD Rats full thickness dermal modeling；A, SD rat back shaving；B, each picture in left and right after skin degerming
The circle of one diameter 2cm；The full thickness dermal of a diameter 2cm is respectively cut in C and D, left and right.
The postoperative each time point skin healing of Fig. 5 is substantially seen
The postoperative each time point skin healing statistics of Fig. 6
The postoperative each time point H&E dyeing of Fig. 7；H&E coloration result shows, 28 to have not yet to see skin attached after surgery for PBS control group
Belong to the regeneration of organ；It is attached that simple human adipose mesenchymal stem cells injection is initially observed a small amount of neoplastic skin in group 21 days after surgery
Organ observed more newborn accessory organ by postoperative 28 days；28 talentes are initially observed on a small quantity simple SIS reparation group after surgery
Skin accessory organ's regeneration；And it can be observed within the SIS reparation group of compound human adipose mesenchymal stem cells 14 days after surgery a small amount of
A large amount of accessory organ's regeneration can be observed by postoperative 21 days by neoplastic skin accessory organ, and neoplastic skin is attached when arriving postoperative 28 days
Belong to organ to continue growing, and starts mature.Circle frame is the accessory organ at neoplastic skin position in figure.
Specific embodiment
Raw material, equipment used in the specific embodiment of the invention are known product, are obtained by purchase commercial product.
The preparation and its reparation of the material of the present invention of embodiment 1
1, the preparation of repair materials
S1, the adipose tissue for taking healthy volunteer's liposuction to obtain, are digested after being cleaned with injection stage physiological saline
Processing；
Specifically, the adipose tissue for taking liposuction to obtain is cleaned 3-5 times with injection stage physiological saline；By the rouge after cleaning
1:1 is mixed fat tissue by volume with digestive juice, is placed in revolving speed 150rpm in 37 DEG C of constant-temperature tables and is digested 2h.The digestive juice is
0.2% II Collagenase Type solution.
Mixed solution after S2, cancellationization is collected by centrifugation precipitating and obtains vascular stroma component SVF.
Specifically, fat tissue fragments postdigestive in S1 are centrifuged 5-10min with 900g, lower sediment is taken to obtain blood vessel
Matrix components SVF, wherein containing fat mesenchymal stem cell；
S3, isolated SVF is added after culture medium is resuspended and is cultivated；
It is resuspended specifically, the vascular stroma component SVF after being centrifuged in S2 is added in culture medium, with every 5mL liposuction product
The density for digesting obtained SVF 1 bottle of T75 culture bottle of inoculation is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2, saturated humidity training
It supports and is cultivated in case, change liquid after 48h for the first time, every 2d is changed the liquid once later；
The culture medium is DMEM/F12 culture medium, wherein the people's platelet rich plasma for being 2-5% containing volume fraction
PRP；
S4, it carries out had digestive transfer culture culture within fat mesenchymal stem cell originally culture 7 days or so to convergence degree 80% or so, expands
The fat mesenchymal stem cell of increasing is identified by streaming, is spare after the screening of VEGF assay in culture supernatant；
Specifically, the step of had digestive transfer culture culture described in S4 are as follows: fat mesenchymal stem cell culture convergence degree in S3
It after 80% or so, is centrifuged after being digested with digestive juice, is inoculated in after being resuspended with culture medium in new T75 culture bottle and carries out passage amplification
Culture, amplification ratio are 1:4-1:5.
Digestive juice is 0.25% trypsin solution of the EDTA containing 0.1%；
Fat mesenchymal stem cell streaming is accredited as fat mesenchymal stem cell expression CD73/CD90/CD105, positive rate
>=95%, CD14/CD34/CD45/CD79 α/HLA-DR, positive rate < 2% are not expressed.Isolated fat mesenchymal is dry thin
The property of born of the same parents is as shown in Figure 1.
Particularly, further screen following specific fat mesenchymal stem cell: expression CD10 is positive, does not express CD200；
The VEGF secreted in fat mesenchymal stem cell culture supernatant is detected by ELISA, VEGF content in every milligram of total protein
>=300pg, preferably >=500pg.
S5, preparation SIS, and the qualified fat mesenchymal stem cell of screening is inoculated on SIS；
Specifically, the step of preparing SIS described in S5:
(1) cleaning arranges: taking secondary (time interval is no more than 3h from butchering to the starting to process) removal of fresh chitterlings one small
Intestinal contents, and overturning inside and outside small intestine longitudinally split small intestine with scissors, so with physiological saline by small intestine washes clean repeatedly
Small intestine is cut into the intestinal segment for being about 10cm-15cm afterwards.
(2) it isolates SIS: firmly being struck off placenta percreta and muscle layer using spatula, until processing is that milky is translucent
Film, with brine 3 times.
(3) degreasing: water is filtered out with gauze after deionized water repeated flushing is clean, film is squeezed out.
Be immersed in chloroform-methanol mixed liquor, wherein chloroform: methanol=1:1 is placed in 6h in draught cupboard, every 60min stirring one
It is secondary, come into full contact with film with liquid.
(4) it takes off cell: the SIS after degreasing is rinsed with flowing water until without any peculiar smell.It is 0.25% that film, which is placed in concentration,
In trypsin solution, under the conditions of 4 DEG C overnight.
(5) deionized water washs removal trypsase repeatedly, is subsequently placed in 0.3% SDS and handles 4h, and washing is gone repeatedly
Except SDS.
(6) be lyophilized: cleaning is placed on pre-freeze in -80 DEG C of refrigerators, is lyophilized in vacuum freeze drier, and aluminium plastic bag heat-sealing is protected
It deposits.
(7) oxirane disinfection.(finished product SIS is as shown in Figure 2).
Inoculation step described in S5 are as follows: taking diameter is that the SIS film disk of 2cm is placed in 6 orifice plates or culture dish, and addition was flooded
The H-DMEM containing 10%PRP of SIS film sets 37 DEG C, 5%CO2, soaked overnight in saturated humidity incubator removes H-DMEM postposition
It is dried up in Biohazard Safety Equipment；It takes and screens qualified fat mesenchymal stem cell in S4 with 1 × 105A/cm2It is inoculated on SIS film,
It is placed in constant incubator and cultivates 5-7d and to fat mesenchymal stem cell cover with SIS film, obtain being compounded with fat mesenchymal dry thin
The SIS film of born of the same parents, wherein the SIS film of 100~1000 fat mesenchymal stem cells is compounded on every square millimeter.
The cell activity dyeing of the obtained SIS film for being compounded with fat mesenchymal stem cell and electron-microscope scanning are as shown in Figure 3.
According to Fig. 3 it is recognised that fat mesenchymal stem cell is successfully compound on SIS film by the present invention.
The preparation and its reparation of the material of the present invention of embodiment 2
1, the preparation of repair materials
S1, the adipose tissue for taking healthy volunteer's liposuction to obtain, are digested after being cleaned with injection stage physiological saline
Processing；
Specifically, the adipose tissue for taking liposuction to obtain is cleaned 3-5 times with injection stage physiological saline；By the rouge after cleaning
1:1 is mixed fat tissue by volume with digestive juice, is placed in revolving speed 150rpm in 37 DEG C of constant-temperature tables and is digested 2h.The digestive juice is
0.2% Type I collagen enzyme solutions.
Mixed solution after S2, cancellationization is collected by centrifugation precipitating and obtains vascular stroma component SVF.
Specifically, fat tissue fragments postdigestive in S1 are centrifuged 5-10min with 900g, lower sediment is taken to obtain blood vessel
Matrix components SVF, wherein containing fat mesenchymal stem cell；
S3, isolated SVF is added after culture medium is resuspended and is cultivated；
It is resuspended specifically, the vascular stroma component SVF after being centrifuged in S2 is added in culture medium, with every 5mL liposuction product
The density for digesting obtained SVF 1 bottle of T75 culture bottle of inoculation is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2, saturated humidity training
It supports and is cultivated in case, change liquid after 48h for the first time, every 2d is changed the liquid once later；
The culture medium is DMEM/F12 culture medium, wherein the people's platelet rich plasma for being 2-5% containing volume fraction
PRP；
S4, it carries out had digestive transfer culture culture within fat mesenchymal stem cell originally culture 7 days or so to convergence degree 80% or so, expands
The fat mesenchymal stem cell of increasing is identified by streaming, is spare after the screening of VEGF assay in culture supernatant；
Specifically, the step of had digestive transfer culture culture described in S4 are as follows: fat mesenchymal stem cell culture convergence degree in S3
It after 80% or so, is centrifuged after being digested with digestive juice, is inoculated in after being resuspended with culture medium in new T75 culture bottle and carries out passage amplification
Culture, amplification ratio are 1:4-1:5.
Digestive juice is the TrypLE of GIBCO companyTMCarry out digestion process；
Specifically, fat mesenchymal stem cell streaming described in S4 is accredited as fat mesenchymal stem cell expression CD73/
CD90/CD105, positive rate >=95% do not express CD14/CD34/CD45/CD79 α/HLA-DR, positive rate < 2%.It separates
The property of the fat mesenchymal stem cell arrived is as shown in Figure 1.
Particularly, further screen following specific fat mesenchymal stem cell: expression CD10 is positive, does not express CD200；
The VEGF secreted in fat mesenchymal stem cell culture supernatant is detected by ELISA, VEGF content in every milligram of total protein
>=300pg, preferably >=500pg.
S5, preparation SIS, and the qualified fat mesenchymal stem cell of screening is inoculated on SIS；
Specifically, the step of preparing SIS described in S5:
(1) cleaning arranges: taking secondary (time interval is no more than 3h from butchering to the starting to process) removal of fresh chitterlings one small
Intestinal contents, and overturning inside and outside small intestine longitudinally split small intestine with scissors, so with physiological saline by small intestine washes clean repeatedly
Small intestine is cut into the intestinal segment for being about 10cm-15cm afterwards.
(2) it isolates SIS: firmly being struck off placenta percreta and muscle layer using spatula, until processing is that milky is translucent
Film, with brine 3 times.
(3) degreasing: water is filtered out with gauze after deionized water repeated flushing is clean, film is squeezed out.
Be immersed in chloroform-methanol mixed liquor, wherein chloroform: methanol=1:1 is placed in 6h in draught cupboard, every 60min stirring one
It is secondary, come into full contact with film with liquid.
(4) it takes off cell: the SIS after degreasing is rinsed with flowing water until without any peculiar smell.It is 0.25% that film, which is placed in concentration,
In trypsin solution, under the conditions of 4 DEG C overnight.
(5) deionized water washs removal trypsase repeatedly, is subsequently placed in 0.3% SDS and handles 4h, and washing is gone repeatedly
Except SDS.
(6) be lyophilized: cleaning is placed on pre-freeze in -80 DEG C of refrigerators, is lyophilized in vacuum freeze drier, and aluminium plastic bag heat-sealing is protected
It deposits.
(7) oxirane disinfection.
Inoculation step described in S5 are as follows: taking diameter is that the SIS film disk of 2cm is placed in 6 orifice plates or culture dish, and addition was flooded
The H-DMEM containing 10%PRP of SIS film sets 37 DEG C, 5%CO2, soaked overnight in saturated humidity incubator removes H-DMEM postposition
It is dried up in Biohazard Safety Equipment；It takes and screens qualified fat mesenchymal stem cell in S4 with 1 × 105A/cm2It is inoculated on SIS film,
It is placed in constant incubator and cultivates 5-7d and to fat mesenchymal stem cell cover with SIS film, obtain being compounded with 100 on every square millimeter
The SIS film of~1000 fat mesenchymal stem cells.
Beneficial effects of the present invention are proved below by way of the mode of experimental example:
The reparative experiment of the material of the present invention of experimental example 1
1, skin injury animal model modeling
The modeling of skin injury animal model is carried out by two-step method, the first step passes through High fat diet and streptozotocin STZ
Rat diabetes model is made in injection, and second step cuts the full thickness skin that 2 diameters are 2cm with operating scissors at left and right sides of rat back
The success of defect, i.e. modeling.
Specifically, selecting 12 week old cleaning grades or SPF grades of SD rats, weight 200g or so is raised one week after quarantine is qualified
Environment is adapted to, is then raised 3 weeks using high lipid food.The injection streptozotocin of 30mg/kg weight is pressed from rat tail vein
STZ, injecting latter all rat tail vein blood samplings with blood glucose meter can be detected rat blood sugar raising, be higher than 16.7mmol/L with blood glucose
For modeling success, eliminate not at mould rat.Blood sampling detects a blood glucose weekly later, confirms rat diabetes model stability.STZ
Next step skin injury modeling is carried out after persistently carrying out after injection High fat diet 12 weeks.
Specifically, being rejected on Sterile surgery platform big with the successful Diabetes Mellitus SD Rats of the aforementioned modeling of chloral hydrate anesthesia
Hair exposure operative site in mouse back is mould in rat back or so two using the disk of diameter 2cm with iodophor disinfection operative site
The circle that side is 2cm with two internal diameters of oiliness stroke.Rat back full thickness skin i.e. modeling is cut along circle inside with operating scissors
Full thickness skin damages successfully, as shown in Figure 4.
2, the reparation of skin injury
Fat mesenchymal stem cell: preparing according to the method for embodiment 1, have convergence degree be 80% when, culture supernatant
In, every milligram of total protein medium vascular endothelial growth factor content >=300pg, and expression antigens c D10, antigens c D200 is not expressed
Characteristic.
The not simple SIS film of compound fat mescenchymal stem cell: it prepares according to the method for embodiment 1.
The SIS film reparation of compound fat mescenchymal stem cell: it prepares according to the method for embodiment 1.
To verify the effect that the SIS film of compound fat mescenchymal stem cell repairs skin injury, we damage full thickness skin
Wound model rat is divided into four groups, it may be assumed that A group, PBS control group, the 100 μ L PBS solution of multi-point injection around the surface of a wound；B group, merely
Fat mesenchymal stem cell injection group, multi-point injection 1 × 10 around the surface of a wound6ADSCs/100μL PBS；C group, not compound fat
The simple SIS film reparation group of mescenchymal stem cell, the surface of a wound stick the SIS film of diameter 2cm；With D group, compound fat mesenchyma is done thin
The SIS film reparation group of born of the same parents, the surface of a wound stick the SIS film for being compounded with the diameter 2cm of ADSCs.The 3rd day after surgery, the 7th day, the 14th day,
It observes and takes pictures within 21st day, the 28th day and measure wound healing situation.The 7th day after surgery, the 14th day, the 21st day, the 28th day time
Point puts to death 8 SD rats respectively, and materials surface of a wound skin is fixed, and H&E is dyed after paraffin embedding.
As shown in Fig. 5~Fig. 7, compared with PBS control group, 3 days and 7 day time point, compared with PBS control group, fat
Mescenchymal stem cell reparation group, SIS reparation group and SIS compound fat mescenchymal stem cell reparation group wound healing faster, in morning
Phase (3 days, 7 days and 14 day time points) SIS compound fat mescenchymal stem cell group wound healing is most fast.H&E slice the results show that
It is repaired compared to more simple fat mesenchymal stem cell and SIS, is compounded with the SIS reparation group skin healing of fat mesenchymal stem cell
More neoplastic skin accessory organs (hair follicle, sweat gland, sebaceous glands) is observed at position, and occur neoplastic skin accessory organ when
Between it is early, there is skin accessory organ (hair follicle, sweat gland, sebaceous glands) within 14 days after repair.
The experiment results show that the present invention is compounded with the SIS film of specific fat mesenchymal stem cell, function admirable is modified, is led to
Fat mesenchymal stem cell and the synergistic effect of SIS are crossed, can quickly repair skin injury, and skin accessory organ can be regenerated,
Such as hair follicle, sweat gland, sebaceous glands, repairing effect are very good.
To sum up, material of the present invention repairs the excellent effect of skin, provides one kind newly for reparations of clinic skin damage
Selection, potential applicability in clinical practice are excellent.
Claims (10)
1. a kind of repair materials of skin injury, it is characterised in that: it is using submucous layer of small intestine film as matrix, between compound fat
The repair materials that mesenchymal stem cells are formed.
2. repair materials according to claim 1, it is characterised in that: described every square millimeter of submucous layer of small intestine film upper multiple
100~1000 fat mesenchymal stem cells are closed.
3. repair materials according to claim 1, it is characterised in that: the submucous layer of small intestine film is cell free small intestine
Submucosa.
4. repair materials according to claim 3, it is characterised in that: the submucous layer of small intestine film is made as follows
It is standby: to take chitterlings intestinal segment, clean, remove placenta percreta and muscle layer, degreasing takes off cell, freeze-drying.
5. repair materials according to claim 3, it is characterised in that: the degreasing is using (0.5-2): 1 chloroform-first
Alcohol mixed liquor degreasing 4-8 hours, chloroform-methanol mixed liquor degreasing 6 hours for preferably using 2:1；The de- cell is first to use
The trypsin solution of 0.1%-1% is handled, and reuses the SDS solution processing of 0.1-1%, preferably first molten using 0.25% pancreatin
Liquid processing reuses 0.3% SDS solution processing.
6. repair materials according to claim 1, it is characterised in that: the fat mesenchymal stem cell is as follows
Be prepared: taking fat mesenchymal stem cell, cultivate, when detection cell confluency degree is 80%, in culture supernatant every milligram it is total
Albumen medium vascular endothelial growth factor content, while the expression of antigens c D10, CD200 of cell is detected, selection has tool
There is the fat mesenchymal stem cell of following feature: when a, convergence degree are 80%, in culture supernatant, every milligram of total protein medium vessels
Endothelial growth factors content >=300pg；B, antigens c D10 is expressed, does not express antigens c D200.
7. repair materials according to claim 6, it is characterised in that: it is 2%- that the culture medium, which is containing volume fraction,
The DMEM/F12 culture medium of 5% platelet rich plasma PRP, condition of culture are 37 DEG C, 5%CO2；The platelet rich plasma
PRP is prepared as follows: take blood, sodium citrate be added to 0.05~0.15mol/L, be placed in refrigerated centrifuge with
4000g is centrifuged 15min, collects upper liquid, as platelet rich plasma；Preferably, sodium citrate is added to 0.1mol/L.
8. repair materials according to claim 6, it is characterised in that: the fat mesenchymal stem cell is as follows
Preparation:
(1) adipose tissue is taken, the collagen enzyme solution digestion of 0.05-1% is carried out；
(2) postdigestive adipose tissue is taken, is centrifuged, obtains vascular stroma component；
(3) it by isolated vascular stroma component, is added after culture medium is resuspended and is cultivated, obtain fat mesenchymal stem cell；Institute
State the DMEM/F12 culture medium that culture medium is the platelet rich plasma PRP for being 2%-5% containing volume fraction, the rich platelet
Blood plasma PRP is prepared as follows: taking blood, sodium citrate is added to 0.1mol/L, is placed in refrigerated centrifuge with 4000g
It is centrifuged 15min, collects upper liquid, as platelet rich plasma；
(4) fat mesenchymal stem cell originally culture to convergence degree 80% carries out had digestive transfer culture culture, and it is dry to obtain fat mesenchymal
Cell；
It is preferred that the digestion is that 1:1 is mixed by volume with digestive juice by adipose tissue in step (1), it is placed in 37 DEG C of constant temperature and shakes
Revolving speed 50-250rpm digests 0.5-4h in bed；
It is preferred that the centrifugation is 150-2000g centrifugation 5min in step (2)；
It is preferred that the culture bottle is T75 culture bottle in step (3)；The condition of the culture are as follows: 37 DEG C, 2-8%CO2, it is saturated wet
It is cultivated in degree incubator, changes liquid after 48h for the first time, every 2d is changed the liquid once later；It is preferred that in step (3), passage amplification ratio is
1:4-1:5。
9. a kind of preparation method for preparing repair materials described in claim 1~8 any one, it is characterised in that: steps are as follows:
Fat mesenchymal stem cell is taken, is compound on submucous layer of small intestine film.
10. purposes of the repair materials described in claim 1~8 any one in preparation skin regeneration material.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808808A (en) * | 2020-08-04 | 2020-10-23 | 北京臻溪谷医学研究中心（有限合伙） | Culture method of mesenchymal stem cells on acellular allogeneic dermal scaffold and application of mesenchymal stem cells |
| CN114949358A (en) * | 2022-05-23 | 2022-08-30 | 徐晓明 | Composite material for deep wound repair and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101366977A (en) * | 2008-09-03 | 2009-02-18 | 陕西瑞盛生物科技有限公司 | Tissue mending material with biological activity and preparation method thereof |
| CN102367435A (en) * | 2011-11-04 | 2012-03-07 | 四川新生命干细胞科技股份有限公司 | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material, preparation method thereof and application |
| CN105154391A (en) * | 2015-10-22 | 2015-12-16 | 四川新生命干细胞科技股份有限公司 | Method for isolating and culturing endothelium from human adipose tissue for clone formation of cells |
| CN107913290A (en) * | 2017-12-22 | 2018-04-17 | 北京再生生物科技研究院有限公司 | A kind of compound cells preparation, preparation method and its usage |
-
2018
- 2018-09-14 CN CN201811075436.1A patent/CN109078224A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101366977A (en) * | 2008-09-03 | 2009-02-18 | 陕西瑞盛生物科技有限公司 | Tissue mending material with biological activity and preparation method thereof |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material, preparation method thereof and application |
| CN102367435A (en) * | 2011-11-04 | 2012-03-07 | 四川新生命干细胞科技股份有限公司 | Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells |
| CN105154391A (en) * | 2015-10-22 | 2015-12-16 | 四川新生命干细胞科技股份有限公司 | Method for isolating and culturing endothelium from human adipose tissue for clone formation of cells |
| CN107913290A (en) * | 2017-12-22 | 2018-04-17 | 北京再生生物科技研究院有限公司 | A kind of compound cells preparation, preparation method and its usage |
Non-Patent Citations (2)
| Title |
|---|
| WEE KIAT ONG ET AL.: "Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots", 《STEM CELL REPORTS》 * |
| 韩超: "不同浓度同种异体富血小板血浆提取液对脂肪干细胞体外生长状况的影响", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808808A (en) * | 2020-08-04 | 2020-10-23 | 北京臻溪谷医学研究中心（有限合伙） | Culture method of mesenchymal stem cells on acellular allogeneic dermal scaffold and application of mesenchymal stem cells |
| CN114949358A (en) * | 2022-05-23 | 2022-08-30 | 徐晓明 | Composite material for deep wound repair and preparation method thereof |
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Application publication date: 20181225 |