CN100522264C - Preparation method of double-layer tissue engineering skin comprising basement membrane structure - Google Patents
Preparation method of double-layer tissue engineering skin comprising basement membrane structure Download PDFInfo
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- CN100522264C CN100522264C CNB2006100951229A CN200610095122A CN100522264C CN 100522264 C CN100522264 C CN 100522264C CN B2006100951229 A CNB2006100951229 A CN B2006100951229A CN 200610095122 A CN200610095122 A CN 200610095122A CN 100522264 C CN100522264 C CN 100522264C
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- 210000002469 basement membrane Anatomy 0.000 title claims description 37
- 238000002360 preparation method Methods 0.000 title claims description 21
- 210000001519 tissue Anatomy 0.000 title claims description 20
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 3
- 210000003491 skin Anatomy 0.000 claims description 51
- 230000008520 organization Effects 0.000 claims description 23
- 229920002101 Chitin Polymers 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- 230000003203 everyday effect Effects 0.000 claims description 5
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 4
- 108010035532 Collagen Proteins 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 102000016942 Elastin Human genes 0.000 claims description 4
- 108010014258 Elastin Proteins 0.000 claims description 4
- 229920001436 collagen Polymers 0.000 claims description 4
- 229920002549 elastin Polymers 0.000 claims description 4
- 239000004744 fabric Substances 0.000 claims description 4
- 229920002674 hyaluronan Polymers 0.000 claims description 4
- 229960003160 hyaluronic acid Drugs 0.000 claims description 4
- 210000002510 keratinocyte Anatomy 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 3
- 229910001424 calcium ion Inorganic materials 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 3
- 239000011575 calcium Substances 0.000 abstract description 3
- 229910052791 calcium Inorganic materials 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 210000000721 basilar membrane Anatomy 0.000 abstract 2
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 239000002355 dual-layer Substances 0.000 abstract 1
- 230000001681 protective effect Effects 0.000 abstract 1
- 210000002615 epidermis Anatomy 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 9
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000002734 Collagen Type VI Human genes 0.000 description 4
- 108010043741 Collagen Type VI Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000000301 hemidesmosome Anatomy 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 101100380342 Catharanthus roseus ASO gene Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000003771 matrix metalloproteinase inhibitor Substances 0.000 description 1
- 229940121386 matrix metalloproteinase inhibitor Drugs 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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Abstract
The invention relates to a method for producing dual-layer organism skin with basilar membrane. Wherein, it is formed by preparing organism skin cultivate base, preparing gel solution, cultivating breeding box and cultivating screen support; it adjusts the calcium density of cultivate base and adds external laminin to accelerate the basilar membrane formation, to strengthen the connection between organism protective skin and corium. The invention has simple process while all materials are liquid and better compatible, without hurting transplanted one; and the skin will not be separated easily; and the product has better compatibility.
Description
One, technical field
The invention belongs to organizational project, be specifically related to a kind of by adding exogenous laminin, the formation method of basement membrane between the true epidermis of promotion double-layer tissue engineering skin.
Two, background technology
Basement membrane is the important structure of thick about 0.5～1.0 a μ m between epidermis and corium, mainly is made of macromole such as IV Collagen Type VI, VII Collagen Type VI, nestin, laminin, and PAS dyeing shows aubergine homogenizing band.Basement membrane has many important biomolecules and learns functions, and the most tangible function is closely to connect epidermis and corium, for epidermis and corium provide strong combination, avoids the physiological condition lower epidermis to separate; Another significantly acts on is the polarity of determining epidermis, the barrier that provides epidermis to divide a word with a hyphen at the end of a line; In addition, in integrity that keeps skin and anti-friction performance, play irreplaceable effect.
Effort through 30 years, organization engineering skin is epidermis sheet, the dermal substitute from beginning, develops into to have double-deck artificial skin, than original simple sealed wound surface, recover the barrier function in skin injury district, to initiatively promoting the wound healing development.But it is the major defect of present double-layer tissue engineering skin that table corium is easy to separate, and its basic reason is exactly because do not make up complete basement membrane between table corium.Part Study shows, the multipotency of organization engineering skin forms alternate basement membrane at present, and this alternate basement membrane developmental immaturity, and enough adhesions can not be provided, and causes between table corium to be easy to separate.
At present, the formation mechanism of relevant basement membrane also is not very clear, and research is few; But influencing the factor of basement membrane mechanical property, mainly be the quantity of hemi desmosome, the number of anchoring filament etc. at present, and the formation of a large amount of hemi desmosomes between true epidermis just can provide the core support for the final structure of basement membrane.Therefore, in organization engineering skin research,, improve the connective stability between table corium, prevent that epidermis from partly partly separating from corium easily, just must make up complete basement membrane structure in vitro culture system for improving the performance of organization engineering skin.
In the prior art, it is open that the Chinese invention patent that a publication number is arranged is CN1795922A applies on July 5th, 2006, it is " skin basement membrane forms and facilitates agent, artificial skin to form the manufacture method of facilitating agent and artificial skin ", this method is extracted purification with various plants and is obtained matrix metallo-proteinase inhibitor and stromatin synthetics, mainly provided a kind of skin basement membrane and kept stable novel mode, though also in artificial skin, add the formation that this material promotes basement membrane.But the related material of this method is from the various plants extract, nearly 32 kinds of the kinds of plant, and complicated component, its purpose mainly is to be used for cosmetic industry, promotes basement membrane stable, promotes activating skin, prevents skin aging.And the research standard height of organization engineering skin, one-tenth is distinguished one from the other, and is nontoxic, and therefore, this method makes up the basement membrane that is used for organization engineering skin and is inapplicable.Through picking up rope, there is technical literature open about containing the preparation method of double-layer tissue engineering skin of basement membrane structure, not seeing as yet so far.
Three, summary of the invention
Purpose of the present invention just is the blank at the prior art existence, a kind of preparation method of double-layer tissue engineering skin that contains basement membrane structure is provided, make it to improve the tight switching performance between organization engineering skin epidermis and corium, prevent to show corium and in operational applications, be easy to separate, guarantee that artificial skin good stability of products, homogeneity are good.
The general plotting of the inventive method is: utilize our original compound recipe chitin organization engineering skin technology platform, influence keratinocyte and fibroblastic interaction by the calcium concentration of regulating culture medium, add exogenous laminin and promote the formation of basement membrane, thereby reach the remarkable result that improves the organization engineering skin properties of product.
For realizing the general plotting and the goal of the invention of the inventive method, the technical scheme of being taked is as follows.
A kind of preparation method of double-layer tissue engineering skin that contains basement membrane structure, it is made up of tissue engineering skin culture basigamy system, gel solution preparation, incubator cultivation and the screen cloth support four step processes of cultivating.
One) tissue engineering skin culture basigamy system
Adding concentration in the DMEM culture medium is the calcium chloride solution of 6M, regulates calcium ion concentration to 0.30～2.15mM; Adding concentration again is the laminin solution of 200 μ g/ml, regulating course mucin concentration to 1～100 μ g/ml.
Two) gel solution preparation
Acetic acid collagen, chitin, hyaluronic acid and elastin laminin are pressed the weight portion proportioning mixing of 16:2:1:1, regulate pH value to 7.2～7.4 with the 1N sodium hydroxide then.
Three) incubator is cultivated
In the gel solution of preparation, add fibroblast (1～100) * 10
5/ 20ml, mixing; Suck in the culture dish, left standstill 15 minutes, add keratinocyte suspension, (1～100) * 10 more gently
5/ 20ml gel, the last tissue engineering skin culture base 8ml that adds preparation again puts 37 ℃, 5%CO
2Cultivate in/95% air, the 90% above humidity incubator, change culture medium every day once, 8ml/ time, cultivated at least 3 days.
Four) the screen cloth support is cultivated
On the stainless steel mesh support that the Hang Zhi ≧ 3mm of organization engineering skin Yi of incubator cultivation is high, the surface of organization engineering skin is exposed in the air, carries out liquid-vapor interface and cultivate; Observe outward appearance, transmittance, the epidermal growth situation of culture every day, finish after 15 days to cultivate, obtain the sophisticated double-layer tissue engineering skin product that contains basement membrane structure; Place liquid nitrogen to preserve, standby.
In double-layer tissue engineering skin basement membrane construction method of the present invention, used raw and auxiliary material people source skin keratin forms cell, people source skin flbroblast, collagen, chitin, hyaluronic acid, elastin laminin, DMEM culture medium, calcium chloride and laminin etc., all should meet aseptic requirement.
Double-layer tissue engineering skin basement membrane construction method of the present invention, system adopts and regulates calcium concentration and the exogenous laminin of interpolation in the culture medium, promote the design concept that basement membrane structure forms between true epidermis, being connected between organization engineering skin epidermis and corium strengthened, the integrity of keeping table corium has been played important function.The inventive method has the following advantages: the one, and method is easy, and all material is liquid form, is convenient to the synthetic and production line operation of product; The 2nd, any material that is adopted all has good biological safety and biocompatibility, can not produce harm for transplanting individuality; The 3rd, the product that obtains is convenient to clinical manipulation, and epidermis is not easily separated; The 4th, consistency of product and homogeneity are good.
Four, description of drawings
Below the various dyeing of basement membrane structure compound recipe chitin organization engineering skin product that contain for the inventive method preparation Show Picture.
Fig. 1 contains basement membrane structure compound recipe chitin organization engineering skin H.E. dyeing demonstration to form (H.E. dyeing * 200) by WD epidermis and the corium under it;
Fig. 2 contains basement membrane structure compound recipe chitin organization engineering skin PAS dyeing to show that basement membrane is successive aubergine banded structure between table corium (PAS dyeing * 200);
Fig. 3 contains IV Collagen Type VI immunohistochemical staining demonstration monoclonal antibody positive reaction (IV Collagen Type VI immunohistochemical staining * 200) between basement membrane structure compound recipe chitin organization engineering skin table corium;
Fig. 4 contains basement membrane structure compound recipe chitin organization engineering skin laminin immunohistochemical staining to show multi-resistance positive reaction (laminin immunohistochemical staining * 200).
Five, the specific embodiment
Be to contain one of basement membrane structure compound recipe chitin organization engineering skin preparation example below, but the inventive method never is confined to the preparation example lifted.
At first adding concentration in the DMEM culture medium is the calcium chloride solution of 6M, regulates calcium ion concentration to 1.85mM, and adding concentration again is the laminin solution of 200 μ g/ml, regulating course mucin concentration to 20 μ g/ml, and preparation obtains the tissue engineering skin culture base; Acetic acid collagen, chitin, hyaluronic acid and elastin laminin are pressed the weight portion proportioning mixing of 16:2:1:1, regulate gel solution pH value to 7.2, preparation gel solution 20ml with the 1N sodium hydroxide; In gel solution, add fibroblast 1 * 10
6, mixing; Suck in the culture dish, after 15 minutes, add keratinocyte suspension 1 * 10 gently
6, the last tissue engineering skin culture base 8ml that adds preparation again puts 37 ℃, 5%CO
2Cultivate in/95% air, the 90% above humidity incubator, change liquid, 8ml/ time every day; Cultivate after 3 days, the capable liquid-vapor interface of the stainless steel mesh support that usefulness ≧ 3mm is high is cultivated; Observe outward appearance, transmittance, the epidermal growth situation of culture every day, finish In vitro culture after 15 days, obtain the sophisticated compound recipe chitin organization engineering skin product that contains basement membrane structure; Place liquid nitrogen to preserve, standby.
The maturation for preparing is as stated above contained basement membrane structure compound recipe chitin organization engineering skin places neutral formalin solution to fix (volume ratio of sample and fixative is 1:5), fixedly behind the 24h, dewater successively, transparent, waxdip, embedding, shop sheet, roasting sheet, carry out histological examination then respectively, comprise H.E. dyeing, PAS dyeing and immunohistochemical staining.Micro-enlarged photograph (seeing Fig. 1, Fig. 2, Fig. 3 and Fig. 4) by various dyeing demonstrations, can illustrate adopt the inventive method preparation contain basement membrane structure compound recipe chitin organization engineering skin, being connected between epidermis and corium is tight, and consistency of product and homogeneity are good.
Claims (1)
1, a kind of preparation method of double-layer tissue engineering skin that contains basement membrane structure is characterized in that:
1) it is made up of tissue engineering skin culture basigamy system, gel solution preparation, incubator cultivation and the screen cloth support four step processes of cultivating;
2) tissue engineering skin culture basigamy system is that adding concentration is the calcium chloride solution of 6M in the DMEM culture medium, regulates calcium ion concentration to 0.30～2.15mM; Adding concentration again is the laminin solution of 200 μ g/ml, regulating course mucin concentration to 1～100 μ g/ml;
3) the gel solution preparation is with acetic acid collagen, chitin, hyaluronic acid and the elastin laminin weight portion proportioning mixing by 16:2:1:1, regulates pH value to 7.2～7.4 with the 1N sodium hydroxide then;
4) the incubator cultivation is to add fibroblast (1～100) * 10 in the gel solution of preparation
5/ 20ml, mixing; Suck in the culture dish, left standstill 15 minutes, add keratinocyte suspension, (1～100) * 10 more gently
5/ 20ml gel, the last tissue engineering skin culture base 8ml that adds preparation again puts 37 ℃, 5%CO
2Cultivate in/95% air, the 90% above humidity incubator, change culture medium every day once, 8ml/ time, cultivated at least 3 days;
5) to cultivate be on the high stainless steel mesh support of the Hang Zhi ≧ 3mm of organization engineering skin Yi that incubator is cultivated to the screen cloth support, the surface of organization engineering skin is exposed in the air, carrying out liquid-vapor interface cultivates, finish after 15 days to cultivate, obtain the sophisticated double-layer tissue engineering skin product that contains basement membrane structure.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100951229A CN100522264C (en) | 2006-09-14 | 2006-09-14 | Preparation method of double-layer tissue engineering skin comprising basement membrane structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100951229A CN100522264C (en) | 2006-09-14 | 2006-09-14 | Preparation method of double-layer tissue engineering skin comprising basement membrane structure |
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| CN1923301A CN1923301A (en) | 2007-03-07 |
| CN100522264C true CN100522264C (en) | 2009-08-05 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773688B (en) * | 2010-02-05 | 2013-10-23 | 中国人民解放军第四军医大学 | Preparation method of tissue engineering skin containing appendant organs |
| CN101879329A (en) * | 2010-06-21 | 2010-11-10 | 蒋明军 | Artificial skin containing basilar membrane and construction method thereof |
| CN101955908B (en) * | 2010-07-22 | 2013-01-23 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
| FR2976001B1 (en) * | 2011-05-30 | 2014-10-31 | Oreal | RECONSTRUCTED SCALP LEATHER MODEL AND METHOD FOR SCREENING ACTIVE MOLECULES |
| CN109550080A (en) * | 2019-01-24 | 2019-04-02 | 中国人民解放军陆军特色医学中心 | A kind of artificial bilayer's skin and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310027A (en) * | 2001-02-06 | 2001-08-29 | 中国人民解放军第三军医大学第三附属医院 | Preparation of compound artificial skin |
| CN1468634A (en) * | 2002-07-15 | 2004-01-21 | 上海组织工程研究与开发中心 | Double-layered artificial skin and its prepn process |
| CN1607012A (en) * | 2003-10-13 | 2005-04-20 | 刘凯 | Method for preparing human body tissue engineering skin |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310027A (en) * | 2001-02-06 | 2001-08-29 | 中国人民解放军第三军医大学第三附属医院 | Preparation of compound artificial skin |
| CN1468634A (en) * | 2002-07-15 | 2004-01-21 | 上海组织工程研究与开发中心 | Double-layered artificial skin and its prepn process |
| CN1607012A (en) * | 2003-10-13 | 2005-04-20 | 刘凯 | Method for preparing human body tissue engineering skin |
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