CN106421910A - adipose tissue preparation and preparation method and application thereof - Google Patents
adipose tissue preparation and preparation method and application thereof Download PDFInfo
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- CN106421910A CN106421910A CN201610614428.4A CN201610614428A CN106421910A CN 106421910 A CN106421910 A CN 106421910A CN 201610614428 A CN201610614428 A CN 201610614428A CN 106421910 A CN106421910 A CN 106421910A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3808—Endothelial cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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Abstract
the invention discloses an adipose tissue preparation, a preparation method and application thereof, wherein the adipose tissue preparation comprises adipose tissues and endothelial progenitor cells, and the preparation method of the adipose tissue preparation comprises the following steps of uniformly mixing granular fat and an endothelial progenitor cell physiological saline solution according to the proportion of 10:1, wherein the concentration of the endothelial progenitor cell physiological saline solution is 5 × 106‑1×107One per ml. The use of the above adipose tissue preparation as a filling preparation in plastic or cosmetic applications. The adipose tissue preparation is used as a filling preparation in plastic and beauty treatment, and has high survival rate in human bodies. After transplantation, the transplanted cells have a large amount of normal fat cells, clear fat cells and only a small amount of bad color, inflammatory reaction phenomena and fiber gaps; after 3 months of transplantation, the adipose tissues are light yellow, soft and glossy, and are wrapped by only a few fibers; the density of the microvascular at the central part of the transplanted fat is normal, and the revascularization is obvious.
Description
Technical field
The present invention relates to medical cosmetology formulation art and in particular to a kind of fatty tissue preparation and preparation method thereof with should
With.
Background technology
1889, the first clinical practice of Free fat was reported.Subsequently, suction lipectomy technology was in 80 years 20th century
In generation, starts appearance, makes the acquisition of fat granules become common and common, this is greatly promoted fat transplantation and fat absorption method
Combination, Autofat granule injection implantation technique also therefore developed rapidly.Clinical Application of Fat Granule Auto-graft, no
Only draw materials and be more prone to, and because having to little for acceptor site wound, do not stay cicatrix, good biocompatibility, no repel and mistake
The advantages of quick reaction, repair in various soft tissue depressions or defect in plastic surgery in recent years and be widely applied.
But, the fatty survival rate after transplanting in reality is relatively low, is typically only 30%-70%.Research finds, early in transplanting
Phase, fat graft lacks effective blood supply and is in acute ischemia anaerobic condition.Set up fully with host in graft
Blood supply before, fatty tissue can only lean on the infiltration of surrounding tissue liquid and infiltration to maintain nutrition supply, and this supply away from
From only 150～200 μm, exceed this distance and be accomplished by generating new blood vessel to provide nutrition.However, neovascularization growth delays
Slowly, daily only more than ten microns of growth, new vesselses typically will 5d just can be grown into the peripheral part of graft after the transfer, at this moment in
The fatty tissue of centre part, due to hypoxic-ischemic overlong time, there occurs necrosis, liquefaction.Therefore, in Auto fat granule
During lapsing to after transplanting, how to promote the revascularization of fat, be the key improving survival rate after fat transplantation.
Content of the invention
In view of this it is necessary to for above-mentioned problem, provide a kind of fatty tissue preparation and preparation method and application.
To achieve these goals, the present invention adopts the following technical scheme that：
Fatty tissue preparation, comprises fatty tissue and endothelial progenitor cells.
Preferably, described fatty tissue preparation is grouped into by Grainy fat tissue and endothelial progenitor cells group.
Preferably, described endothelial progenitor cells group is divided into endothelial progenitor cells normal saline solution.
Preferably, in endothelial progenitor cells normal saline, the concentration of endothelial progenitor cells is 5 × 106-1×107Individual/ml.
Preferably, the volume of Grainy fat tissue is 10 times of endothelial progenitor cells normal saline solution volume.
Preferably, the content of described endothelial progenitor cells is 5 × 105-1×106Individual endothelial progenitor cells/ml Grainy fat tissue.
Preferably, the content of described endothelial progenitor cells is 7 × 105-8×105Individual endothelial progenitor cells/ml Grainy fat tissue.
A kind of preparation method of fatty tissue preparation, by 10:1 volume ratio is by Grainy fat tissue and endothelial progenitor cells physiology
Saline solution mix homogeneously, the concentration of described endothelial progenitor cells normal saline solution is 5 × 106-1×107Individual/ml.
The application as filling preparation in shaping or beauty treatment of above-mentioned fatty tissue preparation.
Described shaping or beauty treatment include depressed deformity, mamaplasty, rich temporo and the augmentation rhinoplasty of filling facial area.
Endothelial progenitor cells (endothelial progenitor cells, EPCs) are a kind of stem cell of single pedigree,
EPCs has stem cell and vascular endothelial cell feature simultaneously, can self renewal and differentiation.When by wound, inflammation, ischemia etc.
After stimulation, it can be mobilized to peripheral blood from bone marrow (or some other tissue, organ), go back to the nest to the position damaged and break up
For vascular endothelial cell, by being integrated into already present blood vessel (angiogenesiss) or direct producing new blood vessel (blood vessel generation) and come
Play its effect.
Compared with prior art, the present invention has the advantages that：
1st, fatty tissue preparation of the present invention is used as shaping, the filling preparation in beauty treatment, and in human body, survival rate is high.By reality
Test observation, there are after transplanting a large amount of normal fat cells, adipose cell is clear, only bad color, inflammatory reaction phenomenon and fibre on a small quantity
Dimension gap；After transplanting 2-3 month, fatty tissue color is yellowish, soft, glossy, only a few fibres parcel；
2nd, after fatty tissue preparation re-injection filling of the present invention, the microvessel density in transplant fat centre is normal, blood vessel
Good fortune is obvious again.
3rd, the manufacture method of fatty tissue preparation of the present invention is simple, can prepare in a large number.
Brief description
Fig. 1 is the visual results picture of fatty tissue.
Fig. 2 is the HE coloration result picture (10 × 40 times) of fatty tissue.
Fig. 3 is the immunohistochemical staining result picture (10 × 40 times) of fatty tissue.
Wherein, figure A is matched group, and figure B is experimental group of the present invention.
Specific embodiment
In order to better illustrate the present invention, it is described further with reference to the accompanying drawings and detailed description.In the present invention
Agents useful for same or instrument all can be buied by market, and detection method of use etc. is all known in the art, will not be described here.
Embodiment 1, fatty tissue preparation
Fatty tissue preparation in the present embodiment is grouped into by following groups of following contents：
Grainy fat tissue,
5×106Individual/ml endothelial progenitor cells normal saline solution,
Wherein, the volume of Grainy fat tissue is 10 times of endothelial progenitor cells normal saline solution volume.
The preparation method of above-mentioned fatty tissue preparation is：Grainy fat tissue and endothelial progenitor cells normal saline solution are mixed all
Even, make every milliliter of Grainy fat tissue 0.1ml endothelial progenitor cells normal saline solution, in fatty tissue preparation, every milliliter of Grainy fat tissue
Containing 5 × 105The endothelial progenitor cells of individual/ml.
In the present embodiment, Grainy fat tissue is conventionally prepared.It is summarized as follows：Subcutaneous fat is placed in a centrifuge,
2000rpm, is centrifuged 3 minutes, shreds fat with shears, and the cell sieve filtration with 300 μm of aperture, obtain 300 μm of diameter
Grain fat is standby.
In the present embodiment, endothelial progenitor cells normal saline solution is prepared by the following method：
(1) separation of Cord Blood Mononuclear Cell
By Hank ' the s balanced salt solution dilution of umbilical blood sample equivalent, the people's lymph being slowly added into equivalent after mixing is thin
On born of the same parents' separating liquid, keep separating surface stable, it is to avoid its mixing；Using centrifuge swing bucket rotor, 740 × g room temperature is centrifuged
30min.Canescence cellular layer between visible lymphoblast separating liquid and blood after centrifugation, i.e. mononuclearcell (MNCs).
This confluent monolayer cells of careful collection are placed in another new centrifuge tube, and add in the PBS liquid containing 2%FBS, and 200 × g room temperature is centrifuged
5min.Supernatant is abandoned, resuspended with the PBS liquid containing 2%FBS again, 200 × g room temperature is centrifuged 5min after centrifugation.
(2) culture of endothelial progenitor cells
Abandon supernatant after the last centrifugation of mononuclearcell (MNCs), add the EGM-2 culture medium containing 10%FBS,
Soft piping and druming cell becomes single cell suspension, is inoculated in coated 6 well culture plates of early stage, is placed in carbon dioxide cell incubator
Culture, 37 DEG C, 5%CO2, humidity>95%.The cell extracting in 20ml Cord blood can be inoculated in 3 holes.After cell inoculation
24h, absorbs supernatant to remove non-attached cell and cell debriss, with fresh EG M-2 culture medium rinse cell, changes after absorption
Fresh culture is cultivated, and changes liquid daily.Cultivate to 7 days, the next day switching to, change liquid, record the initial Colony forming time；Culture
To 14 days, randomly select under 40 × inverted phase contrast microscope 3 visual field colonies quantity (with more than 50 cells as colony
Criterion).With 0.25% trypsin/EDTA had digestive transfer culture after converging to cell growth to more than 80%.By the above process
By cell culture P2 for standby
(3) collect cell
Remove culture fluid in bottle, with PBS twice, add 2ml trypsin solution, put into 37 DEG C, 5%CO2Culture
In case, after about 2 minutes, add the DMEM that 2ml contains 10% hyclone to terminate digestion, cell is counted.1000 leave the heart 5
Minute, remove supernatant culture medium, be adjusted to cell concentration 5 × 10 using normal saline5, obtain endothelial progenitor cells physiology salt water-soluble
Liquid.
Embodiment 2, fatty tissue preparation
Fatty tissue preparation in the present embodiment is grouped into by following groups of following contents：
Grainy fat tissue,
7.5×106Individual/ml endothelial progenitor cells normal saline solution,
Wherein, the volume of Grainy fat tissue is 10 times of endothelial progenitor cells normal saline solution volume.
The preparation method of above-mentioned fatty tissue preparation is：Grainy fat tissue and endothelial progenitor cells normal saline solution are mixed all
Even, make every milliliter of Grainy fat tissue 0.1ml endothelial progenitor cells normal saline solution, in fatty tissue preparation, every milliliter of Grainy fat tissue
Containing 7.5 × 105The endothelial progenitor cells of individual/ml.
In the present embodiment, the preparation method of Grainy fat tissue and endothelial progenitor cells normal saline solution is same as Example 1.
Embodiment 3, fatty tissue preparation
Fatty tissue preparation in the present embodiment is grouped into by following groups of following contents：
Grainy fat tissue,
1×107Individual/ml endothelial progenitor cells normal saline solution,
Wherein, the volume of Grainy fat tissue is 10 times of endothelial progenitor cells normal saline solution volume.
The preparation method of above-mentioned fatty tissue preparation is：Grainy fat tissue and endothelial progenitor cells normal saline solution are mixed all
Even, make every milliliter of Grainy fat tissue 0.1ml endothelial progenitor cells normal saline solution, in fatty tissue preparation, every milliliter of Grainy fat tissue
Containing 1 × 106The endothelial progenitor cells of individual/ml.
In the present embodiment, the preparation method of Grainy fat tissue and endothelial progenitor cells normal saline solution is same as Example 1.
In order to better illustrate the effect of the present invention, zoopery is carried out to fatty tissue preparation of the present invention.Using 8 months
Age is the healthy SPF level nude mice (offer of Guangdong Province's animal experimental center) in 4-6 week, weight 15～18g, and male and female do not limit.Experiment
Animal makees lower column split：Nude mice is randomly divided into two groups of A, B, every group four, every respectively at 4 points of dorsal sc injection, every point
Injection 0.4ml fatty tissue preparation.A group is blank control group, and its fatty tissue preparation is that every milliliter of Grainy fat tissue adds 0.1ml
Normal saline.B group is experimental group, is respectively adopted the fatty tissue preparation in embodiment 2.After injection, injection 3 months after, anesthesia
Nude mice, takes each point graft to put into 10% paraformaldehyde and preserves, and carries out perusal, HE dyeing and immunohistochemical experiment.
Effect example 1, perusal
Perusal adipose tissue volume size, quality, color and luster, fibrous capsule formational situation.Result is as shown in Figure 1.By
Fig. 1 understands, A group graft quality is harder, gloomy matt it is seen that obvious fibers encapsulation phenomenon；B group is relatively soft, glossy, face
Color is yellowish, only a few fibres parcel.In sum, B group fat survival benefit is substantially better than A group fat.
Effect example 2, HE dyeing
HE dyeing is carried out to the fatty tissue after 3 months, method is as follows：Fatty tissue is put into fix 24 in fixative little
When, take out fatty tissue from fixative, using alcohol-pickled dehydration, then the fatty tissue after dehydration is put into dimethylbenzene molten
Liquid removes ethanol transparence.Transparent fatty tissue is put into embedding in the paraffin of thawing, then take out cooling at once
Solidification in bulk.Paraffin mass freezes half an hour, puts into section in microtome and obtains paraffin section, dries dewaxing dye after paraffin section
Color, then microscopy.
HE coloration result is as shown in Figure 2.As shown in Figure 2, A group adipose cell is dispersed in distribution, and fibrous connective tissue is more；B
Group fibrous connective tissue is less, and the vessel cross-section of visible distribution.In sum, B group fat survival benefit is substantially better than A
Group.
Effect example 3, SABC
To and 3 months after fatty tissue carry out immunohistochemical experiment, method is as follows：
Paraffin section (preparation method is with HE colouring method) is placed in 20 minutes in 65 DEG C of baking ovens, PBS rinses.Deca about 50
μ l3%H2O2In section, room temperature stands 10 minutes, and PBS rinses.Section is placed in EDTA solution, puts into heating in pressure cooker,
Timing 3 minutes after pressure cooker snifting valve jet.Take out section rewarming 1 hour, PBS rinses.Using the inspection of two step method SABC
Test agent box (BOSTER), one anti-(rabbit igg) of Deca suitably dilution to specifications, 20-37 DEG C is incubated 1～2 hour or 4 DEG C
Overnight.Washed 2 minutes with 0.01M PBS, Deca polymerization HRP labelling anti-rabbit IgG (SV-0002), 37 DEG C be incubated 30 minutes, PBS or
TBS rinses 2 minutes.Take 1ml distilled water, in reagent adding box, each 1 of A, B, C reagent, adds to section after mixing.Color development at room temperature, mirror
The lower control response time, general 5-30 minute.Distilled water wash.Haematoxylin is slightly redyed, is dehydrated, transparent, mounting, observation.
Result is as shown in Figure 3.In figure positive position shows to be blood capillary cross section at this.Count micro- under every group of 20 visuals field
The number of blood vessel, statistical result is as shown in table 1.
Table 1, blood capillary number statistical table
| Group | Each visual field blood capillary cross section average (individual) |
| A group | 1±0.976 |
| B group | 4±1.523 |
As shown in Table 1, the extremely notable (P of B group and A group difference<0.001).Each visual field blood capillary cross section average of B group is
More than 4, each visual field blood capillary cross section average of A group is 1 about, the blood capillary that B group fatty tissue is formed in vivo
Number is four times of A group it was demonstrated that the present invention program is conducive to fat to survive in vivo.
Embodiment described above only have expressed the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformation can also be made and improve, these broadly fall into the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of fatty tissue preparation it is characterised in that：Comprise fatty tissue and endothelial progenitor cells.
2. fatty tissue preparation according to claim 1 it is characterised in that：Described fatty tissue preparation by Grainy fat tissue and
Endothelial progenitor cells group is grouped into.
3. fatty tissue preparation according to claim 1 it is characterised in that：Described endothelial progenitor cells group is divided into endothelium ancestral thin
Born of the same parents' normal saline solution.
4. fatty tissue preparation according to claim 1 it is characterised in that：In endothelial progenitor cells normal saline, endothelium ancestral is thin
The concentration of born of the same parents is 5 × 106-1×107Individual/ml.
5. the fatty tissue preparation according to any one of claim 2-4 it is characterised in that：The volume of Grainy fat tissue is endothelium
10 times of CFU-GM normal saline solution volume.
6. fatty tissue preparation according to claim 1 it is characterised in that：The content of described endothelial progenitor cells is 5 × 105-
1×106Individual endothelial progenitor cells/ml Grainy fat tissue.
7. fatty tissue preparation according to claim 1 it is characterised in that：The content of described endothelial progenitor cells is 7 × 105-
8×105Individual endothelial progenitor cells/ml Grainy fat tissue.
8. a kind of preparation method of fatty tissue preparation it is characterised in that：By 10:1 volume ratio is by Grainy fat tissue and endothelium
CFU-GM normal saline solution mix homogeneously, the concentration of described endothelial progenitor cells normal saline solution is 5 × 106-1×107Individual/
ml.
9. the application as filling preparation in shaping or beauty treatment of the fatty tissue preparation described in any one of claim 1-7.
10. according to claim 9 application it is characterised in that：Described shaping or beauty treatment include the depression of filling facial area
Deformity, mamaplasty, rich temporo and augmentation rhinoplasty.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229785A (en) * | 2019-06-17 | 2019-09-13 | 白晋 | A method of fat transfer success rate is promoted using endothelial progenitor cell |
| CN116474172A (en) * | 2023-04-17 | 2023-07-25 | 广州市麦施缔医疗科技有限公司 | Epidermal stem cell and fat mixed injection method and breast augmentation and hip augmentation regeneration method |
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| CN1912109A (en) * | 2005-08-09 | 2007-02-14 | 中国人民解放军军事医学科学院野战输血研究所 | Structural method and application of tissue engineering adipose tissue |
| CN102058905A (en) * | 2009-11-18 | 2011-05-18 | 四川大学 | Composite fat granule and preparation method thereof |
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2016
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| CN1912109A (en) * | 2005-08-09 | 2007-02-14 | 中国人民解放军军事医学科学院野战输血研究所 | Structural method and application of tissue engineering adipose tissue |
| CN102058905A (en) * | 2009-11-18 | 2011-05-18 | 四川大学 | Composite fat granule and preparation method thereof |
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Cited By (2)
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|---|---|---|---|---|
| CN110229785A (en) * | 2019-06-17 | 2019-09-13 | 白晋 | A method of fat transfer success rate is promoted using endothelial progenitor cell |
| CN116474172A (en) * | 2023-04-17 | 2023-07-25 | 广州市麦施缔医疗科技有限公司 | Epidermal stem cell and fat mixed injection method and breast augmentation and hip augmentation regeneration method |
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