CN1234429C - Artificial skin, preparing method and application thereof - Google Patents
Artificial skin, preparing method and application thereof Download PDFInfo
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- CN1234429C CN1234429C CN 02117585 CN02117585A CN1234429C CN 1234429 C CN1234429 C CN 1234429C CN 02117585 CN02117585 CN 02117585 CN 02117585 A CN02117585 A CN 02117585A CN 1234429 C CN1234429 C CN 1234429C
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- artificial skin
- mesenchymal stem
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- bone marrow
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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
- A61L27/60—Materials for use in artificial skin
Abstract
The present invention relates to artificial skin, a preparing method thereof and an application thereof, which aims to provide artificial skin with low immunological rejection and a preparing method thereof. In order to achieve the purpose, the present invention has the technical scheme that the artificial skin is prepared by that firstly, I-shaped animal collagen is prepared into a collagen membrane; secondly, mesenchymal stem cells of autologous bone marrow are inoculated on the collagen membrane to and are cultured to obtain the artificial skin. The method for preparing the artificial skin comprises: mesenchymal stem cells of autologous bone marrow are inoculated on a collagen membrane prepared from I-shaped animal collagen and then are cultured to obtain the artificial skin. The artificial skin of the present invention provides a novel effect source of daughter cells for the clinical treatment of patients with burns, chronic ulcers and other skin defects and lays the foundation for searching artificial skin with similar physiological characteristics in clinical development.
Description
Technical field
The present invention relates to a kind of artificial skin and preparation method thereof and application.
Background technology
Skin is divided into epidermis, corium and three parts of subcutaneous fat as the organ of human body maximum.Also have hair simultaneously, refer to cutaneous appendages such as (toe) first, sebaceous gland, sweat gland.The performance of its function depends primarily on corium.Corium contains macrophage, endotheliocyte, adipose cell, dendritic cell, fibroblast etc.In these cells, fibroblast accounts for the overwhelming majority in corium, and the function performance of corium is mainly acted on.As in wound healing process, mainly be according to the synthetic collagen of fibroblast, for tissue repair provides the ultrastructure material base,, make wound obtain repairing then by a series of activities such as its propagation, contractions.
In recent years, because diseases such as burn and chronic ulcer make this field of artificial skin obtain very fast development.Especially domestic and international many researchers for development more near " artificial skin " of physiological characteristics, it is dermal substitute, use in succession from body or the fibroblastic culture technique of allosome and rebuild impaired corium, promptly not contain the substrate of cell component, comprise that collagen, fine mucin, basement membrane albumen, hyaluronic acid, polylactic acid and polyglycolic acid etc. are carrier, the inoculation dermal fibroblast, induce a series of activities such as its propagation, migration and secretory cell epimatrix, the result confirms that it can promote the wound immediate union.But owing to grow (approximately 1-2 week) in external required cultivation cycle from the body fibroblast, be not suitable for the treatment of acute wounds, and the fibroblast of inoculation allosome exists immunologic rejection and risk of disease transmission, becomes inevitable so seek a kind of more reliable seed cell source.
Distinctive biological characteristics of stem cell and potential biomedical applications thereof are worth and make it one of problem that becomes the field of biology hottest point.Especially in recent years increasing experimental evidence shows, mesenchymal stem cells MSCs (Mesenchymal stem cell as tissue stem cell a member, MSC) under certain condition, can be induced to differentiate into the cell in multiple mesoderm such as bone, cartilage, fat, muscle source, so it has certain feasibility to being divided into the corium that belongs to the mesoderm source together.
Summary of the invention
The purpose of this invention is to provide a kind of artificial skin with low immunological rejection.
A kind of artificial skin, it is obtained by following method:
1) I type animal collagen is prepared into collagem membrane;
2) autologous bone marrow mesenchymal stem cells is inoculated on the above-mentioned collagem membrane to cultivate and obtains artificial skin.
Described autologous bone marrow mesenchymal stem cells is that the mesenchymal stem cells MSCs that will take from from body obtains by the amplification in vitro cultivation.
Described I type animal collagen can come from the corium of animal heel string, animal tail tendon, animal or crystalline lens etc.The for example corium of the corium of cattle heel string, rat tail tendon, Cavia porcellus corium, pig, cattle, the crystalline lens of buphthalmos etc.
Another object of the present invention provides a kind of preparation method of artificial skin.
A kind of preparation method of artificial skin is autologous bone marrow mesenchymal stem cells to be inoculated in to cultivate on the collagem membrane that I type animal collagen makes obtain.
Described autologous bone marrow mesenchymal stem cells is that the mesenchymal stem cells MSCs that will take from from body obtains by the amplification in vitro cultivation.
The present invention utilizes the multidirectional differentiation potential from body homology mesenchymal stem cells MSCs, after its amplification in vitro cultivation, be inoculated on the collagem membrane with the preparation of the collagen composition that from animal particular organizations such as Mus, cattle, extracts, cultivate the artificial skin that forms more near physiological characteristics.It is good, not easily broken and be convenient to characteristics such as operation technique that collagem membrane of the present invention has pliability, avoided traditional inoculation from body or allosome fibroblast problem, the problem includes: the restriction of problems such as lazy weight and immunologic rejection, for skin injury patients' such as burn, chronic ulcer clinical treatment provides a kind of effective new seed cell source, explore for clinical development and a kind ofly more to lay a good foundation near the artificial skin of physiological characteristics.
Yan Zhi artificial skin was owing to all existed the immunologic rejection problem to some extent in the past, so clinical practice is limited.The present invention adopts and to combine with animal collagen from body homology mesenchymal stem cells MSCs, because MSC is from from body, separation and Culture is more or less freely, implants a little less than the reaction, so can solve the immunologic rejection that exists in the clinical skin transplantation to greatest extent.Animal collagen composition such as Mus tail is a type i collagen on the other hand, and is consistent with contained collagen composition in the application on human skin corium, thereby also do not have rejection.What is more important, because corium is depended in the performance of human body skin function, and artificial skin of the present invention is to promote MSC to the corium directed differentiation in the skin original position when using, and can be better the recovery of skin function be played a role.
The present invention will be further described below in conjunction with drawings and the specific embodiments.
Description of drawings
Fig. 1 is collagem membrane sem photograph (1500 *).
Fig. 2 for rat MSC inoculation collagem membrane after sem photograph (1000 *).
Fig. 3 is a cell proliferation situation behind the rat different densities MSC inoculation collagem membrane.
Fig. 4 is each experimental group wound shrinkage factor variation diagram.
The specific embodiment
One, the extraction of Mus tail type i collagen
1, gets 1 of 250 gram rat, cut off the Mus tail, put in 75% ethanol and soaked 30 minutes from afterbody;
2, under the aseptic condition Mus tail is cut into segment about 1.5cm, peels off fur, extract tail tendon out, in the horizontalization ware;
3, subtract broken tail tendon, immerse 0.1% acetum, put 4 ℃ of refrigerators, and jolting frequently;
4, after 48 hours, move in the sterilization centrifuge tube, 4 ℃ of 4000 rev/mins of conditions are centrifugal 30 minutes;
5, draw supernatant packing ,-20 ℃ of preservations;
6, residue joins in the 40mL0.1% acetum, recentrifuge after 24 hours, and the supernatant packing ,-20 ℃ of preservations are standby.Detecting this extract component through immunohistochemical method is type i collagen.
Two, the preparation of collagem membrane
The acid Mus tail type i collagen and the α-MEM culture fluid (α-MEM culture medium is available from Hyclone company) that contains 20% serum that with the concentration of extracting are 1mg/ml are laid in 24 orifice plates according to 3: 1 mixed, and every pore volume 0.5ml puts in 37 ℃ of incubators and observes.After 24 hours, visible 24 orifice plates of naked eyes bottom has membranoid substance to form.Thickness approaches homogeneous, the color milky white, and pliability is stronger.As shown in Figure 1, the visible collagen fiber of electron-microscope scanning are cross-linked network.
With the method for present embodiment, can also in the crystalline lens of the corium of the corium of cattle heel string, Cavia porcellus corium, pig, cattle or buphthalmos, extract type i collagen.
The separation and purification of embodiment 2, rat bone marrow mesenchymal stem cells (in strict accordance with sterile working's rules)
1, gets 250 gram left and right sides adult Wistar rats, after disconnected neck is put to death, immerse in 75% ethanol and sterilize a moment;
2, the greater trochanter of ining succession takes off the bilateral femur together;
3, reject muscle on every side, and cut greater trochanter of femur;
4, earlier insert the femur two ends respectively with 10ml syringe needle that syringe is with, respectively wear an aperture, the α-MEM culture fluid (α-MEM culture medium is available from Hyclone company) that contains 20% hyclone with 5ml syringe absorption 2ml is gone out bone marrow then, makes single cell suspension;
5, cell is placed the 10ml centrifuge tube, centrifugal 5 minutes of 1500rpm;
6, abandon supernatant, with the above-mentioned culture fluid re-suspended cell of 1ml.
7, record the nucleus number under the mirror, and by 1 * 10
6/ cm
2Cell inoculation is put 37 ℃, 5%CO in the T-25 culture bottle
2With cultivate in the incubator of saturated humidity.
8, behind the 24-48h, change culture medium, discard not adherent cell.Changed liquid once in later every 3-4 days.Cell reaches 80% and merges the back and digested 3 minutes in 37 ℃ with 0.25% trypsin and 0.02% EDTA.With 1-3 * 10
5The amplification of going down to posterity an of/bottle cell.And along with the increase pair cell of passage number carries out purification.
Can from rat marrow, isolate (8.853 ± 7.34) * 10 through said method
7Individual nucleated cell.After the cell attachment growth formed the clone, to its amplification of going down to posterity, along with the increase of passage number, cell was the spindle shape of form than homogeneous gradually.This method isolated cells is external to be passed more than 17 generations.
One, the scanning electron microscopic observation mesenchymal stem cells MSCs is to the affinity of collagem membrane
1, the 2-5 that gets the amplification in vitro cultivation is for rat bone marrow mesenchymal stem cells, after trypsin with 0.25% and 0.02% the EDTA digestion, with 2.5 * 10
5Individual cells/well is inoculated in 24 orifice plates that are covered with embodiment 1 made collagem membrane, puts 37 ℃, 5%CO
2Cultivate in the incubator of saturated humidity;
2, there is the collagem membrane of mesenchymal stem cells MSCs to take out inoculation respectively at the 1st day, the 3rd day and the 4th day, after fixing with 3% glutaraldehyde, carries out scanning electron microscopic observation.
The scanning electron microscope result shows (as shown in Figure 2), and rat MSC and Mus tail type i collagen have good affinity, and the 1st day visible mesenchymal stem cells MSCs in inoculation back is attached on the collagem membrane, by the 3rd day and the 4th day, as seen MSC is stretched to fusiformis on collagem membrane, is cross-linked with each other, and propagation in order.
Two, mtt assay is observed the propagation situation of mesenchymal stem cells MSCs on collagem membrane, and filters out best inoculum density
1, the 2-5 that gets the amplification in vitro cultivation is inoculated in 96 orifice plates and is covered with in 96 orifice plates of embodiment 1 made collagem membrane every pore volume 200 μ l with different densities for rat bone marrow mesenchymal stem cells;
2, with culture plate at 37 ℃, 5%CO
2Cultivate in the incubator of saturated humidity;
3, respectively took out a plate on the 1st, 2,3,4,5 day respectively at the inoculation back, add 20 μ lMTT solution, continued to hatch 4 hours;
4, after 4 hours, take out 96 orifice plates, inhale and abandon supernatant, add 200 μ lDMSO, vibrated 10 minutes;
5, select the 490nm wavelength, measuring each hole optical density value on the enzyme connection instrument automatically, record result, result as shown in Figure 3, rat MSC inoculation collagem membrane is after one week, its proliferation activity is compared no significant difference with the rat MSC that does not inoculate collagem membrane.The suitableeest inoculum density of growth curve showed cell is 2 * 104/cm
2
One, method
1, animal grouping and modelling
30 of Wistar rats about body weight 200 grams, are divided into 3 groups, 10 every group: (1) mesenchymal stem cells MSCs-collagem membrane group at random; (2) simple collagen film group; (3) holostrome skin excision back normal healing group (matched group).
With sodium pentobarbital (50mg/kg) intraperitoneal injection of anesthesia, back cropping, excise holostrome skin to fascia in the center, the about 2cm * 2cm of area.Have the collagem membrane of MSC and simple collagen membrane component not to be attached at the animal pattern wound surface inoculation, cover the vaseline oil yarn, adopt clinical bolus dressing to carry out sutured after, single cage is raised.The nature matched group is not then transplanted collagem membrane, and all the other handle the same.
2, wound surface shrinkage factor statistics and histological examination
In hinder back 7 days, 11 days, 14 days, 21 days, 28 days with animal wound surface size overprint on transparent membrane, adopt statistical method that its wound shrinkage factor is analyzed.Each is lived and kills 2 animals in each time point simultaneously, draws materials in the wound scope.Specimen is fixed with 4% paraformaldehyde, and through paraffin embedding, section back row HE normal dyeing is observed.
Two, result
Except that inoculating 2 animals of MSC-collagem membrane group because of dying unexpectedly, all the other animals all survive.Hinder the 7-14 days interior visible experimental group wound surface in back than the matched group drying, area dwindles, and does not see traumatic infection.Credit is analysed by statistics, respectively forms mouthful shrinkage factor and changes as shown in Figure 4.As seen from the figure, different time points is respectively organized the shrinkage factor size and is arranged as MSC-collagem membrane group in order greater than simple collagen film group after hindering, simple collagen film group is greater than natural matched group, and MSC-collagem membrane group and simple collagen film group also have certain difference on the whole, illustrate that the MSC-collagem membrane more can promote wound healing to a certain extent than the simple collagen film.Histological observation shows that experimental group granulation tissue content is obviously more than natural matched group.
Embodiment 5, the propagation of human marrow mesenchymal stem cell on Mus tail collagem membrane
One, the separation and purification of human marrow mesenchymal stem cell (in strict accordance with sterile working's rules)
1, in the normal adult rib, extracts bone marrow under the aseptic condition, make single cell suspension;
2, cell is placed the 10ml centrifuge tube, centrifugal 5 minutes of 400g;
3, abandon supernatant, add the 5mlPBS re-suspended cell;
4, above-mentioned cell suspension is added in the Percoll separating medium that density is 1.073g/ml centrifugal 20 minutes of 400g in 1: 1 ratio;
5, collect cloud mononuclearcell, centrifugal 5 minutes of 400g;
6, record the nucleus number under the mirror, and by 1 * 10
6/ cm
2Cell inoculation is put 37 ℃, 5%CO in the T-25 culture bottle
2, saturated humidity incubator in cultivate;
7, behind the 24-48h, change culture medium, discard not adherent cell.Changed liquid once in later every 3-4 days.Cell reaches 80% and merges the back and digested 3 minutes in 37 ℃ with 0.25% trypsin and 0.02% EDTA.With 1-3 * 10
5The amplification of going down to posterity an of/bottle cell.And along with the increase pair cell of passage number carries out purification.
Two, the propagation of mesenchymal stem cells MSCs on collagem membrane
Above-mentioned human marrow mesenchymal stem cell is inoculated on the Mus tail collagem membrane among the embodiment 1, adopt electron-microscope scanning and mtt assay to measure cell proliferation vigor (method is with embodiment 3), the result shows, after human marrow mesenchymal stem cell is cultivated through going down to posterity, the cellular morphology homogeneous is spindle shape.After it was inoculated in Mus tail collagem membrane respectively, the scanning electron microscope result showed that people MSC is similar to rat MSC, has good affinity with Mus tail type i collagen, is stretched to fusiformis on collagem membrane, was cross-linked with each other, and bred in order.MTT result shows that also after people MSC was inoculated in one week of Mus tail collagem membrane, its proliferation activity was compared no significant difference with the people MSC that does not inoculate collagem membrane.Illustrate that the MSC between the different genera requires not have marked difference to the source of collagem membrane.
Embodiment 6, the propagation of rabbit bone marrow mescenchymal stem cell on cattle dermal collagen film
One, the separation and purification of rabbit bone marrow mescenchymal stem cell (in strict accordance with sterile working's rules)
1, extracts bone marrow in the adult certainly rabbit femoral, make single cell suspension;
2, cell is placed the 10ml centrifuge tube, centrifugal 5 minutes of 400g;
3, abandon supernatant, add the 5mlPBS re-suspended cell;
4, above-mentioned cell suspension is added on the Ficoll lymphocyte separation medium that density is 1.077g/ml centrifugal 20 minutes of 400g in 1: 1 ratio;
5, collect cloud mononuclearcell, centrifugal 5 minutes of 400g;
6, record the nucleus number under the mirror, and by 1 * 10
6/ cm
2Cell inoculation is put 37 ℃, 5%CO in the T-25 culture bottle
2, saturated humidity incubator in cultivate;
7, behind the 24-48h, change culture medium, discard not adherent cell.Changed liquid once in later every 3-4 days.Cell reaches 80% and merges the back and digested 3 minutes in 37 ℃ with 0.25% trypsin and 0.02% EDTA.With 1-3 * 10
5The amplification of going down to posterity an of/bottle cell.And along with the increase pair cell of passage number carries out purification.
Two, the propagation of mesenchymal stem cells MSCs on collagem membrane
The mesenchymal stem cells MSCs in above-mentioned rabbit source is inoculated on the cattle dermal collagen film, adopt electron-microscope scanning and mtt assay to measure cell proliferation vigor (method is with embodiment 3), the result shows, after the mesenchymal stem cells MSCs of rabbit is cultivated through going down to posterity, the cellular morphology homogeneous is spindle shape.After it was inoculated in collagem membrane, the scanning electron microscope result showed that rabbit MSC and cattle dermal collagen film have good affinity, are stretched to fusiformis on collagem membrane, was cross-linked with each other, and bred in order.MTT result shows that also rabbit MSC inoculation cattle dermal collagen film is after one week, and its proliferation activity is compared no significant difference with the rabbit MSC that does not inoculate collagem membrane.Illustrate that so long as type i collagen, the collagem membrane of separate sources is little to the growth effect of MSC.
Claims (10)
1, a kind of artificial skin, it is obtained by following method:
1) I type animal collagen is prepared into collagem membrane;
2) autologous bone marrow mesenchymal stem cells is inoculated on the above-mentioned collagem membrane to cultivate and obtains artificial skin.
2, artificial skin according to claim 1 is characterized in that: described autologous bone marrow mesenchymal stem cells is that the mesenchymal stem cells MSCs that will take from from body obtains by the amplification in vitro cultivation.
3, artificial skin according to claim 1 and 2 is characterized in that: described I type animal collagen comes from the heel string of animal.
4, artificial skin according to claim 1 and 2 is characterized in that: described I type animal collagen comes from the animal tail tendon.
5, artificial skin according to claim 1 and 2 is characterized in that: described I type animal collagen comes from the corium of animal.
6, artificial skin according to claim 1 and 2 is characterized in that: described I type animal collagen comes from the animal crystalline lens.
7, a kind of preparation method of artificial skin is autologous bone marrow mesenchymal stem cells to be inoculated in to cultivate on the collagem membrane that I type animal collagen makes obtain.
8, method according to claim 7 is characterized in that: described autologous bone marrow mesenchymal stem cells is that the mesenchymal stem cells MSCs that will take from from body obtains by the amplification in vitro cultivation.
9, the application of the artificial skin of claim 1 in the seed cell of preparation treatment fire victim skin injury.
10, the application of the artificial skin of claim 1 in the damaged seed cell of preparation treatment chronic ulcer patient skin.
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| CN 02117585 CN1234429C (en) | 2002-05-09 | 2002-05-09 | Artificial skin, preparing method and application thereof |
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| CN 02117585 CN1234429C (en) | 2002-05-09 | 2002-05-09 | Artificial skin, preparing method and application thereof |
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| CN1234429C true CN1234429C (en) | 2006-01-04 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100366303C (en) * | 2004-12-24 | 2008-02-06 | 于海鹰 | Preparation and application of anti blocking biomembrane for tendon and ligament |
| CN100402097C (en) * | 2006-07-24 | 2008-07-16 | 暨南大学 | Skin wound repairing agar/collagen dressing and its prepn and application |
| RU2010109698A (en) * | 2007-09-19 | 2011-09-27 | Плуристем Лтд. (Il) | ADHESIVE CELLS OF FATTY TISSUE OR PLACENTA AND THEIR USE FOR MEDICAL PURPOSES |
| CN102161981B (en) * | 2010-02-23 | 2013-01-02 | 中国人民解放军总医院第一附属医院 | Method for jointly inducing bone marrow mesenchymal stem cells into sweat gland cells by recombinant protein |
| CN102552880A (en) * | 2011-12-14 | 2012-07-11 | 南方医科大学 | Biological preparation for promoting skin wound to be healed |
| CN105596372A (en) * | 2015-12-15 | 2016-05-25 | 天津卫硕生物科技有限公司 | Method for treating various severe skin damages by using autogeneic bone marrow-derived mesenchymal stem cells (BMSCs) |
| CN106967678A (en) * | 2017-02-21 | 2017-07-21 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method for the collagem membrane for being inoculated with autologous bone marrow mesenchymal stem cells |
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