CN107988151A - A kind of adipose tissue of external evoked vascular pedicle is into flesh method - Google Patents
A kind of adipose tissue of external evoked vascular pedicle is into flesh method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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Abstract
The present invention relates to a kind of adipose tissue of external evoked vascular pedicle into flesh method, this method utilizes the fat fascia setup action live body stent of vascular pedicle, induced maturation adipocyte in situ for main body seed cell through myogenic nutrient solution in vitro perfusion transdifferentiation into Skeletal Muscle Cell, external structure axis shape study of vascularized tissue engineering bone bone muscular tissue.The beneficial effects of the invention are as follows on the premise of at utmost body injury is mitigated, axial pattern blood vessel organizational project bone muscular tissue is cultivated, to treat muscle defect.
Description
Technical field
The invention belongs to surgical medicine technical field, and in particular to a kind of adipose tissue of external evoked vascular pedicle is into flesh
Method.
Background technology
Because a wide range of crowd's wound that the factors such as various natural calamities, unexpected injury, firearm injury often result in, and tumour are cut
Remove and the skeletal myopathy including muscular dystrophy or Duchenne-Arandisease, all there are the loss of notable musculature, it is repaired
It is clinically more stubborn problem.Clinical treatment is mainly transplanted by autologous musculature at present, as form and function
Substitute.However, the available muscle of the method is limited, Bing Duigong areas cause to damage, and cause for area's muscle volume deficiency or function funeral
Lose.Therefore there is an urgent need for the construction method that research can obtain substantial amounts of engineered musculature in the short time, for meet group,
The needs of a large amount of musculature defect repairs.As musculer tissue engineering is furtherd investigate, provided for the defect repair of musculature
Another developing direction.Musculer tissue engineering avoids body injury caused by autotransplantation, the immunological rejection of heteroplastic transplantation, and
The shortcomings of foreign body reaction of artificial substitute and infection, can obtain in the method for the even available tissue penetration of minimal amount of histocyte
, after cultured and amplified in vitro, and tissue, any plastotype of organ defect situation are pressed, carry out repair tissue defect, reach real meaning
On reconstruction and preferable form reparation.
Existing organizational project bone flesh construction method is inoculated with using only simple skeletal muscle satellite cell, although can be formed
Skeletal muscle, but no matter from the quantity or quality for forming newborn skeletal muscle cannot meet a wide range of bone of clinical repair
The needs of flesh defect.Therefore, how while organizational project bone flesh is built promote blood vessel and nerve grow into, just become bone
The key link of application is furtherd investigate and moved towards to bone muscular tissue engineering.
The content of the invention
In order to which the simple skeletal muscle satellite cell of the use for solving to mention in background technology is inoculated with, although bone can be formed
Flesh, but no matter from the quantity or quality for forming newborn skeletal muscle cannot meet that a wide range of skeletal muscle of clinical repair lacks
The technological deficiency needed is damaged, the present invention provides a kind of adipose tissue of external evoked vascular pedicle to exist into flesh method, this method
On the premise of at utmost mitigating body injury, axial pattern blood vessel organizational project bone muscular tissue is cultivated, is lacked to treat muscle
Damage.
The present invention utilizes the fat fascia setup action live body stent of vascular pedicle, in vitro, anti-using filling type biology
Device is answered to promote nutriment to exchange, and analogue body interior mechanics environment, maintain long-term surviving outside large volume fat fascia organizer.Connect
By the fat fascia tissue of myogenic culture perfusion, fat mesenchymal stem cell and ripe adipocyte in it will be induced
Differentiation becomes skeletal muscle tissue, final to obtain ripe bulk tissue engineering muscle.
The technical solution adopted in the present invention is：
A kind of adipose tissue of external evoked vascular pedicle utilizes the fat fascia group of vascular pedicle into flesh method, this method
Knit as live body stent, induced maturation adipocyte in situ is the seed cell of main body through myogenic nutrient solution in vitro perfusion transdifferentiation
Into Skeletal Muscle Cell, external structure axis shape study of vascularized tissue engineering bone bone muscular tissue.
Further, the vessel pedicle is axis shape vessel pedicle.
Further, the induced maturation adipocyte in situ is the seed cell of main body through myogenic nutrient solution in vitro perfusion
Transdifferentiation is into Skeletal Muscle Cell, and then the implementation method for being converted into skeletal muscle tissue is：The axis shape vessel pedicle is the fat
Fascia tissue provides sufficient blood supply；Using filling type bioreactor, caused by the flowing of analogue body endoskeleton muscular tissue interstitial fluid
Mechanical environment；The filling type bioreactor includes myogenic nutrient solution, by the fat fascia in the myogenic nutrient solution
Fat mesenchymal stem cell and ripe adipocyte in tissue are induced differentiation into as skeletal muscle tissue, promotion bulk tissue work
Journey muscle is ripe.
Further, the filling type bioreactor, is mainly made of reservoir, peristaltic pump and connecting pipe；It is described
Connecting pipe is connected with the axis shape vessel pedicle of the fat fascia tissue, and the peristaltic pump is connected in the connecting pipe
Pumping pressure is provided, the port for the connecting pipe being connected with the suction inlet of the peristaltic pump is inserted into the reservoir
In the myogenic nutrient solution, the another port of the connecting pipe is arranged in the reservoir.
Further, the filling type bioreactor is positioned in 37 DEG C of CO2gas incubator, by described compacted
The dynamic lasting driving of pump, makes myogenic nutrient solution continue through the fat fascia tissue.
Further, the myogenic nutrient solution in the filling type bioreactor maintained in filling process constant pH and
Oxygen concentration.
Further, the myogenic nutrient solution is described using Dulbeccos improvement Eagle culture mediums or F12 culture mediums
Dulbeccos improve in Eagle culture mediums or the F12 culture mediums containing calf serum, horse serum, penicillin or streptomysin,
Dexamethasone, hydrocortisone and 4- hydroxyethyl piperazineethanesulfonic acid buffers.
It should be noted that Dulbeccos improvement Eagle culture medium abbreviations DMEM；The English of 4- hydroxyethyl piperazineethanesulfonic acids
Entitled HEPES.
Further, the acquisition of the fat fascia tissue of the vessel pedicle is with the following method：
After the fat fascia tissue for isolating vascular pedicle, rinsed using heparin-saline, after coarse needle bioptic blood vessel,
Fat fascia tissue is placed in filling type bioreactor, detachment proximal part blood vessel.
The purpose rinsed using heparin-saline is prevention microvascular corrosion cast.
The present invention also provides a kind of method for muscle defect repair, using the fat of above-mentioned external evoked vascular pedicle
The axis shape study of vascularized tissue engineering bone bone muscular tissue that fat is organized into the acquisition of flesh method carries out muscle defect repair.
The present invention also provides a kind of method for building large volume organizational project bone tissue and tissue engineering bone/cartilage tissue, use
The adipose tissue of above-mentioned external evoked vascular pedicle into the axis shape study of vascularized tissue engineering bone bone muscular tissue that flesh method obtains into
Row structure.
The technique effect of the present invention is as follows：
The axis shape study of vascularized tissue engineering bone bone muscular tissue obtained by the method for the present invention is used for muscle defect repair, can be most
Big degree mitigates body injury, has the characteristics that following：
(1) external structure axial pattern blood vessel skeletal muscle tissue, can thoroughly avoid damage caused by autologous offer muscle；
(2) present invention by the use of large volume fat fascia as natural live body stent, break up its interior fat mesenchymal and do by induction
Cell and ripe adipocyte, can directly obtain the large volume organizational project muscle of maturation；
(3) present invention is using induction myogenic differentiations such as dexamethasone, hydrogenation cortisone, calf serum, horse serums, with tradition
The 5-azacitidine used is compared, the former control condition easily grasps, break up fully, toxicity be obviously reduced, be conducive to muscle defect
Repair.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the fat fascia surgical procedure state diagram of vascular pedicle of the present invention；
Fig. 2 is the filling type bioreactor connection diagram of the present invention.
The reference numerals are as follows：
1st, superficial epigastric artery；2nd, superficial epdigastric vein；3rd, femoral artery；4th, femoral vein；5th, syringe；6th, ligation；7th, reservoir；
8th, peristaltic pump；9th, connecting pipe.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical scheme will be carried out below
Detailed description.Obviously, described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all on the premise of creative work is not made
Other embodiment, belongs to the scope that the present invention is protected.
The present invention utilizes the fat fascia setup action live body stent of vascular pedicle, in vitro, anti-using filling type biology
Device is answered to promote nutriment to exchange, and analogue body interior mechanics environment, maintain long-term surviving outside large volume fat fascia organizer.Connect
By the fat fascia tissue of myogenic culture perfusion, fat mesenchymal stem cell and ripe adipocyte in it will be induced
Differentiation becomes skeletal muscle tissue, final to obtain ripe bulk tissue engineering muscle.
Specifically, the adipose tissue of external evoked vascular pedicle is realized into flesh method using following steps：
Step 1: Fatty fascial flap of the selection with clear and definite vascularity supplies area；
Step 2: as shown in Figure 1, by taking groin fat fascia tissue (i.e. fat fascia tissue) as an example, parallel abdomen is designed
Inguinal ligament notch, exposure groin fat fascia tissue, ligatures (or coagulation) bilateral femoral arterial 3 and distal end femoral vein 4, shape
Into ligation 6, fractionation of fatty fascia tissue, heparin-saline lavation is carried out from 3 proximal part of femoral artery using syringe 5, after from
Disconnected to have ligatured blood vessel, coarse needle bioptic femoral artery 3, starts fat fascia tissue and is placed in the filling type biology containing myogenic nutrient solution instead
Answer in device, the fat fascia tissue of floating is pressed into nutrient solution using vessel forceps, detachment proximal part femoral vein 4；
Step 3: configuration myogenic nutrient solution：Using DMEM or F12 culture mediums, at least contain calf serum, horse in culture medium
Serum, penicillin or streptomysin, dexamethasone, hydrocortisone and HEPES buffers；Specific myogenic nutrient solution component is seen below
Table：
Note：DMEM：Dulbeccos improves Eagle culture mediums；HEPES：4- hydroxyethyl piperazineethanesulfonic acids.
(4) as shown in Fig. 2, fat fascia tissue and filling type bioreactor are connected, by filling type bioreactor
9 one end of connecting pipe is connected with the femoral artery 3 of fat fascia tissue, and femoral artery 3 herein is axis shape blood vessel, connecting pipe 9
The other end is immersed in the myogenic nutrient solution of reservoir 7, and peristaltic pump 8 is persistently driven with the constant flow rate of 2 cc/mins, is made
Myogenic nutrient solution connecting pipe 9 through immersion from reservoir 7 sucks, and circulation is injected into femoral artery 3, and passes through fat fascia group
Other blood vessels knitted are discharged in myogenic nutrient solution；
(5) the myogenic nutrient solution in filling type bioreactor maintains constant pH and oxygen concentration in filling process.
It should be noted that pH value, which needs to maintain about 7, (is not less than 6, no more than 8), close to neutrality；Oxygen concentration approaches
The oxygen concentration of air.
Superficial epigastric artery 1 and superficial epdigastric vein 2 in Fig. 1 have the outflow of myogenic nutrient solution, the myogenic culture flowed out at this
Liquid is inputted from femoral artery 3 by connecting pipe.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all be contained
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (8)
1. a kind of adipose tissue of external evoked vascular pedicle is into flesh method；It is characterized in that, this method utilizes vascular pedicle
Fat fascia setup action live body stent, induced maturation adipocyte in situ are external through myogenic nutrient solution for the seed cell of main body
Transdifferentiation is irrigated into Skeletal Muscle Cell, external structure axis shape study of vascularized tissue engineering bone bone muscular tissue.
2. the adipose tissue of external evoked vascular pedicle according to claim 1 is into flesh method, it is characterised in that the blood
Guan Di is axis shape vessel pedicle.
3. the adipose tissue of external evoked vascular pedicle according to claim 2 is into flesh method, it is characterised in that the original
Position induced maturation adipocyte for main body seed cell through myogenic nutrient solution in vitro perfusion transdifferentiation into Skeletal Muscle Cell, and then
Being converted into the implementation method of skeletal muscle tissue is：The axis shape vessel pedicle provides sufficient blood supply for the fat fascia tissue；Profit
With filling type bioreactor, mechanical environment caused by the flowing of analogue body endoskeleton muscular tissue interstitial fluid；The filling type biology
Reactor includes myogenic nutrient solution, by the fat mesenchymal stem cell in the fat fascia tissue in the myogenic nutrient solution
Induced differentiation into ripe adipocyte as skeletal muscle tissue, promotion bulk tissue engineering muscle maturation.
4. the adipose tissue of external evoked vascular pedicle according to claim 3 is into flesh method, it is characterised in that the filling
Note formula bioreactor, is mainly made of reservoir, peristaltic pump and connecting pipe；The connecting pipe and the fat fascia group
The axis shape vessel pedicle knitted is connected, and the peristaltic pump is connected in the connecting pipe and provides pumping pressure, with the wriggling
The port for the connecting pipe that the suction inlet of pump is connected is inserted into the myogenic nutrient solution in the reservoir, the company
The another port in adapter road is arranged in the reservoir.
5. the adipose tissue of external evoked vascular pedicle according to claim 4 is into flesh method, it is characterised in that the filling
Note formula bioreactor is positioned in 37 DEG C of CO2gas incubator, is persistently driven by the peristaltic pump, makes myogenic culture
Liquid continues through the fat fascia tissue.
6. the adipose tissue of external evoked vascular pedicle according to claim 5 is into flesh method, it is characterised in that the filling
Myogenic nutrient solution in note formula bioreactor maintains constant pH and oxygen concentration in filling process.
7. the adipose tissue of external evoked vascular pedicle according to claim 6 is into flesh method, it is characterised in that the life
Flesh nutrient solution improves Eagle culture mediums using Dulbeccos improvement Eagle culture mediums or F12 culture mediums, the Dulbeccos
Or contain calf serum, horse serum, penicillin or streptomysin, dexamethasone, hydrocortisone and 4- in the F12 culture mediums
Hydroxyethyl piperazineethanesulfonic acid buffer.
8. the adipose tissue of external evoked vascular pedicle according to claim 7 is into flesh method, it is characterised in that the blood
The acquisition of the fat fascia tissue of Guan Di is with the following method：
After the fat fascia tissue for isolating vascular pedicle, rinsed using heparin-saline, after coarse needle bioptic blood vessel, by fat
Fat fascia tissue is placed in filling type bioreactor, detachment proximal part blood vessel.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214195A (en) * | 2021-12-15 | 2022-03-22 | 中国科学院大连化学物理研究所 | Mold for in-vitro construction of large-size vascularized muscle bundle and use method thereof |
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| CN101443023A (en) * | 2005-05-25 | 2009-05-27 | 再生医疗技术公司 | Method for treating angiocarpy disease using fat tissue source cell |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214195A (en) * | 2021-12-15 | 2022-03-22 | 中国科学院大连化学物理研究所 | Mold for in-vitro construction of large-size vascularized muscle bundle and use method thereof |
| CN114214195B (en) * | 2021-12-15 | 2023-11-03 | 中国科学院大连化学物理研究所 | Mold for in-vitro construction of large-size vascularized muscle bundles and application method thereof |
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