CN1739813A - Epiderm substitute for tissue engineering and its prepn process - Google Patents
Epiderm substitute for tissue engineering and its prepn process Download PDFInfo
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- CN1739813A CN1739813A CN 200510029692 CN200510029692A CN1739813A CN 1739813 A CN1739813 A CN 1739813A CN 200510029692 CN200510029692 CN 200510029692 CN 200510029692 A CN200510029692 A CN 200510029692A CN 1739813 A CN1739813 A CN 1739813A
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Abstract
The present invention relates to biological material and tissue engineering technology, and is especially one kind of tissue engineering epiderm substituent and its preparation process. The tissue engineering epiderm substitute features that the chitosan-gelatin film or rack possesses pores of 20-50 micron size. Compared with available technology, the present invention adopts chitosan and gelatin as the material for the cell carrying system as the cell substituent, and has the features of easy-to-obtain material, simple technological process, high ventilation and raised clinical repair effect.
Description
[technical field]
The present invention relates to biomaterial and tissue engineering technique field, specifically a kind of epiderm substitute for tissue engineering and preparation method thereof.
[background technology]
Skin is the organ of human body maximum, plays the important function of protection body.When a variety of causes causes skin injury, can cause the body worker can obstacle, heavy then crisis life.Damaged to skin histology, common clinically Therapeutic Method is transplanted for kpetrolatum gauze and split-thickness skin graft or full thick-layer skin graft are arranged, and wherein, split-thickness skin graft is the recognized standard Therapeutic Method.But, cause new skin trauma and the insufficient problem of donor up to the present also not to obtain adequate solution for this Therapeutic Method existence.
Behind the skin injury, the hypertrophic cicatrix of defect is its important complication.Studies show that,, just can reduce the cicatrization of defect, or alleviate the degree of the formation of cicatrix if the skin injury position can very fast epithelization.Along with the development of biotechnology, medical science, and the going deep into of each subject crossing, the rise of tissue engineering technique, organization engineering skin is the focus and emphasis that becomes artificial skin research.Produced a series of patent, wherein application number is that 200310108082 patent is exactly the construction method that improves the epiderm substitute of wound surface epithelization about a kind of.Yet, the chitosan one gelatin film material that uses in this patent is a kind of material of compactness, do not have breathability, particularly after adding cell, under moistening state, chitosan/gelatin generation swelling, its breathability can further reduce, especially on thicker film material, influence is more obvious, thereby influences the effect of clinical repair.
[summary of the invention]
The objective of the invention is to: on the basis of existing technology, prepare have better breathability chitosan-gelatin film material or support as the cell transfer system of epiderm substitute, increase the clinical repair effect of epiderm substitute.
The present invention adopts following method to realize:
It is 20 to 50 microns aperture that a kind of epiderm substitute for tissue engineering, chitosan-gelatin film material or support are provided with diameter.So can guarantee that the prepared epiderm substitute that goes out has air permeability and good, can strengthen the clinical repair effect.
Another innovative point of the present invention has been to provide the preparation method of epiderm substitute for tissue engineering, comprises following two kinds:
The preparation method of one, a kind of epiderm substitute for tissue engineering may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9～9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) mixed liquor is added in the ware of plane, under 35～60 ℃ condition, heated the drying and forming-film material 2～3 hours; (5) take out the film material, film is immersed in the dehydrated alcohol 20 minutes, remove ethanol; (6) EDC or 0.25% glutaraldehyde solution that adds 30mM again reacted 3 hours, carried out cross-linking reaction; (7) use a large amount of distilled water flushings then, and soaked 24 hours; (8) under 35～60 ℃ condition, heated 2～3 hours the after drying film forming; (8) beating diameter on film is 20 to 50 microns, and density is 1-10/mm
2Aperture, promptly get chitosan-gelatin film material; (9) above-mentioned chitosan-gelatin film material is placed overnight incubation in the superficial cell culture fluid, blot culture fluid then; (10) then by 1 * 10
4～1 * 10
6/ cm
2Density inoculation epidermis cell beating on the film material of via hole; (11) above-mentioned film material is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 3～15 days, get final product epiderm substitute for tissue engineering.
Two, a kind of preparation method of epiderm substitute for tissue engineering may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9～9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) with above-mentioned chitosan-gelatin mixture under-40 ℃～-70 ℃ condition pre-freeze 3-5 hour; (5) drain moisture with the freezing vacuum drier then, obtain porous support; (6) support was heated 2-3 hour under 35-60 ℃ condition, the NaOH solution soaking of reuse 1～4% is used distilled water wash 3-5 time then; (7) will add the EDC of 30mM or carry out cross-linking reaction 2～3 hours through the support of above-mentioned processing with 1～5% glutaraldehyde solution; (8) rinse well; (9) vacuum lyophilization again obtains porous chitosan-gelatin porous support; (10) above-mentioned chitosan-gelatin is propped up be placed on overnight incubation in the superficial cell culture fluid, blot culture fluid then; (11) again by 1 * 10
4～1 * 10
6/ cm
2Density inoculation epidermis cell on support; (12) above-mentioned support is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 3～15 days, get final product epiderm substitute for tissue engineering.
In the preparation method of above-mentioned epiderm substitute for tissue engineering, described culture fluid is for placing the superficial cell culture fluid.
In the end cultivate the time optimal 5-7 of being chosen as days.
The thickness of described film material is 100～600 μ m.Described backing thickness is 100～1000 μ m.Its epidermis cell substitute can be used for the skin donor site wound surface among clinical large-area burns wound surface, various skin injury wound surface, the plastic surgery, the reparation of various chronic ulcer skin injury.
The present invention compares with prior art, has used chitosan, gelatin as raw material, makes it the cell transfer system as epiderm substitute.Not only raw material is easy to get, and technology is relatively easily grasped, and the prepared epiderm substitute for tissue engineering surface that goes out is provided with aperture, and good permeability increases the clinical repair effect of epiderm substitute.
[description of drawings]
Fig. 1 be the epiderm substitute wound repairing design sketch
1 is improved epiderm substitute reparation district
2 are not improved epiderm substitute reparation district
[specific embodiment]
Below the present invention is further illustrated, this technology concerning these professional personnel still clearly.
The prepared epiderm substitute for tissue engineering of example 1 chitosan-gelatin film material
A kind of preparation method of epiderm substitute for tissue engineering may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) mixed liquor is added in the ware of plane, at 35 ℃ of heating 2 hours, drying and forming-film material; (5) take out the film material from the plane ware, film is immersed in the dehydrated alcohol 20 minutes, remove ethanol; (6) EDC that adds 30mM again reacted 3 hours, carried out cross-linking reaction; (7) use a large amount of distilled water flushings then, and soaked 24 hours; (8) in 35 ℃ of heating 2 hours, after drying film forming; (8) beating diameter on film is 20 microns, and density is 1/mm
2Aperture, promptly get chitosan-gelatin film material; (9) above-mentioned chitosan-gelatin film material is placed overnight incubation in the epidermis cell culture fluid, blot culture fluid then; (10) by 1 * 10
4/ cm
2Density inoculation epidermis cell on the film material of beating via hole; (11) above-mentioned film material is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 6 days, get final product epiderm substitute for tissue engineering.
The prepared epiderm substitute for tissue engineering of example 2 chitosan-gelatin film materials
A kind of preparation method of epiderm substitute for tissue engineering may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) mixed liquor is added in the plate, at 48 ℃ of heating 2.5 hours, drying and forming-film material; (5) film is immersed in the dehydrated alcohol 20 minutes, removes ethanol; (6) add 0.25% glutaraldehyde solution reaction 3 hours again, carry out cross-linking reaction; (7) the first then sodium borohydride solution with 2% washs, and removes unnecessary glutaraldehyde, the distilled water flushing that reuse is a large amount of, and soaked 24 hours; (8) in 48 ℃ of heating 2.5 hours, after drying film forming; (8) beating diameter on film is 35 microns, and density is 2/mm
2Aperture, promptly get chitosan-gelatin film material; (9) above-mentioned chitosan-gelatin film material is placed overnight incubation in the cuticulated epithelium culture fluid, blot culture fluid then; (10) by 1 * 10
5/ cm
2Density inoculation epidermis cell on the film material of beating via hole; (11) above-mentioned film material is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 9 days, get final product epiderm substitute for tissue engineering.
The prepared epiderm substitute for tissue engineering of example 3 chitosan-gelatin film materials
A kind of preparation method of epiderm substitute for tissue engineering may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) mixed liquor is added in the plate, at 60 ℃ of heating 3 hours, drying and forming-film material; (5) film is immersed in the dehydrated alcohol 20 minutes, removes ethanol; (6) EDC that adds 30mM again reacted 3 hours, carried out cross-linking reaction; (7) use a large amount of distilled water flushings then, and soaked 24 hours; (8) in 60 ℃ of heating 3 hours, after drying film forming; (8) beating diameter on film is 50 microns, and density is 4/mm
2Aperture, promptly get chitosan-gelatin film material; (9) above-mentioned chitosan-gelatin film material is placed overnight incubation in the epidermis cell culture fluid, blot culture fluid then; (10) by 1 * 10
6/ cm
2Density inoculation epidermis cell on the film material of beating via hole; (11) above-mentioned film material is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 15 days, get final product epiderm substitute for tissue engineering.
The prepared epiderm substitute for tissue engineering of example 4 chitosan-gelatin supports
(1) with chitosan with the dissolving of 1% acetic acid, be mixed with concentration and be 2% chitosan solution; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) with above-mentioned chitosan-gelatin mixture pre-freeze 3 hours under-40 ℃ condition; (5) drain moisture with the freezing vacuum drier then, obtain porous support; (6) support again 35 ℃ the heating 2 hours, the NaOH solution soaking of reuse 1% is used distilled water wash 3 times then; (7) will be through the support of above-mentioned processing, the EDC that adds 30mM carried out cross-linking reaction 2 hours; (8) rinse well; (9) vacuum lyophilization again obtains porous chitosan-gelatin porous support; (10) above-mentioned chitosan-gelatin is propped up be placed on overnight incubation in the epidermis cell culture fluid, blot culture fluid then; (11) by 1 * 10
4/ cm
2Density inoculation epidermis cell on support; (12) above-mentioned support is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 6 days, get final product epiderm substitute for tissue engineering.
The prepared epiderm substitute for tissue engineering of example 5 chitosan-gelatin supports
(1) with chitosan with the dissolving of 1% acetic acid, be mixed with concentration and be 2% chitosan solution; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) with above-mentioned chitosan-gelatin mixture-55 ℃ of pre-freezes 4 hours; (5) drain moisture with the freezing vacuum drier then, obtain porous support; (6) support again 48 ℃ the heating 2.5 hours, the NaOH solution soaking of reuse 3% is used distilled water wash 4 times then; (7) will carry out cross-linking reaction 2.5 hours with 2.5% glutaraldehyde solution through the support of above-mentioned processing; (8) wash with 2% sodium borohydride solution earlier, remove unnecessary glutaraldehyde, a large amount of distilled water flushing of reuse is clean; (9) vacuum lyophilization again obtains porous chitosan-gelatin porous support; (10) above-mentioned chitosan-gelatin is propped up be placed on overnight incubation in the epidermis cell culture fluid, blot culture fluid then; (11) by 1 * 10
5/ cm
2Density inoculation epidermis cell on support; (12) above-mentioned support is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 9 days, get final product epiderm substitute for tissue engineering.
The prepared epiderm substitute for tissue engineering of example 6 chitosan-gelatin supports
Chitosan with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) with above-mentioned chitosan-gelatin mixture-70 ℃ of pre-freezes 5 hours; (5) drain moisture with the freezing vacuum drier then, obtain porous support; (6) support again 60 ℃ the heating 3 hours, the NaOH solution soaking of reuse 4% is used distilled water wash 5 times then; (7) will be through the support of above-mentioned processing, the EDC that adds 30mM carried out cross-linking reaction 3 hours; (8) rinse well; (9) vacuum lyophilization again obtains porous chitosan-gelatin porous support; (10) above-mentioned chitosan-gelatin is propped up be placed on overnight incubation in the epidermis cell culture fluid, blot culture fluid then; (11) by 1 * 10
6/ cm
2Density inoculation epidermis cell on support; (12) above-mentioned support is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 15 days, get final product epiderm substitute for tissue engineering.
The application of example 7 epiderm substitute for tissue engineering in clinical
This epiderm substitute can be applied to the reparation of skin injury such as the skin donor site wound surface among clinical large-area burns wound surface, the plastic surgery, various chronic ulcers.
After split thickness skin graft is got at the patient back, stay 16 * 10cm
2The skin donor site wound surface, epiderm substitute that the technical scheme that provides according to patent application CN200310108082 makes up is provided respectively and is treated according to the epiderm substitute that the technical scheme of this patent makes up, the cell face of epidermis cell substitute is contacted with patient's wound surface, and conventional wound surface wrapping is handled.Open outer dressing after 7 days, observe the wound surface situation.The result shows the equal epithelization of wound surface that two kinds of epiderm substitutes are repaired, but, the epiderm substitute wound repairing that epiderm substitute that this patent makes up and patent application CN200310108082 make up effect compare, epithelization scope and epithelization quality all are better than the latter.
Claims (8)
1, a kind of epiderm substitute for tissue engineering is characterized in that it is 20 to 50 microns aperture that chitosan-gelatin film material or support are provided with diameter.
2, a kind of preparation method of epiderm substitute for tissue engineering is characterized in that may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9～9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) mixed liquor is added in the ware of plane, under 35～60 ℃ condition, heated the drying and forming-film material 2～3 hours; (5) take out the film material, and film is immersed in the dehydrated alcohol 20 minutes, remove ethanol; (6) EDC or 0.25% glutaraldehyde solution that adds 30mM again reacted 3 hours; (7) use a large amount of distilled water flushings then, and soaked 24 hours; (8) under 35～60 ℃ condition, heated 2～3 hours the after drying film forming; (8) beating diameter on film is 20 to 50 microns, and density is 1-10/mm
2Aperture, promptly get chitosan-gelatin film material; (9) above-mentioned chitosan-gelatin film material is placed overnight incubation in the superficial cell culture fluid, blot culture fluid then; (10) by 1 * 10
4～1 * 10
6/ cm
2Density inoculation epidermis cell beating on the film material of via hole; (11) above-mentioned film material is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 3～15 days, get final product epiderm substitute for tissue engineering.
3, a kind of preparation method of epiderm substitute for tissue engineering is characterized in that may further comprise the steps: (1) with the dissolving of 1% acetic acid, is mixed with concentration and is 2% chitosan solution with chitosan; (2) gelatin dissolved in distilled water is mixed with concentration 4% gelatin solution; (3) be mixing in 1: 9～9: 1 by weight with chitosan solution and gelatin solution, and under 37 ℃ condition, mixed 24 hours; (4) with above-mentioned chitosan-gelatin mixture-40 ℃～-70 ℃ pre-freeze 3-5 hour; (5) drain moisture with the freezing vacuum drier then, obtain porous support; (6) support was heated 2-3 hour under 35-60 ℃ condition, the NaOH solution soaking of reuse 1～4% is used distilled water wash 3-5 time then; (7) will add the EDC of 30mM or carry out cross-linking reaction 2～3 hours through the support of above-mentioned processing with 1～5% glutaraldehyde solution; (8) rinse well; (9) vacuum lyophilization again obtains porous chitosan-gelatin porous support; (10) above-mentioned chitosan-gelatin is propped up be placed on overnight incubation in the superficial cell culture fluid, blot culture fluid then; (11) then by 1 * 10
4～1 * 10
6/ cm
2Density inoculation epidermis cell on support; (12) above-mentioned support is cultivated with the superficial cell culture fluid of serum-free, changed liquid once in per 3 days, incubation time is 3～15 days, get final product epiderm substitute for tissue engineering.
4,, it is characterized in that described culture fluid is that superficial cell is cultivated culture fluid according to the preparation method of claim 2 or 3 described a kind of epiderm substitute for tissue engineering.
5,, it is characterized in that described incubation time is 5-7 days according to the preparation method of claim 2 or 3 described a kind of epiderm substitute for tissue engineering.
6, the preparation method of a kind of epiderm substitute for tissue engineering according to claim 2, the thickness that it is characterized in that described film material are 100～600 μ m.
7, the preparation method of a kind of epiderm substitute for tissue engineering according to claim 3 is characterized in that described backing thickness is 100～1000 μ m.
8, a kind of epiderm substitute for tissue engineering according to claim 1 is characterized in that it can be used for the skin donor site wound surface among clinical large-area burns wound surface, skin injury wound surface, the plastic surgery, the reparation of various chronic ulcer skin injury.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510029692 CN1739813A (en) | 2005-09-15 | 2005-09-15 | Epiderm substitute for tissue engineering and its prepn process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510029692 CN1739813A (en) | 2005-09-15 | 2005-09-15 | Epiderm substitute for tissue engineering and its prepn process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1739813A true CN1739813A (en) | 2006-03-01 |
Family
ID=36092434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510029692 Pending CN1739813A (en) | 2005-09-15 | 2005-09-15 | Epiderm substitute for tissue engineering and its prepn process |
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| Country | Link |
|---|---|
| CN (1) | CN1739813A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101927035A (en) * | 2010-07-20 | 2010-12-29 | 西南大学 | Method for preparing artificial skin using shell substrate as raw material |
| CN102016009A (en) * | 2008-04-14 | 2011-04-13 | Ucl商业股份有限公司 | Membrane |
| CN103721294A (en) * | 2013-12-27 | 2014-04-16 | 太原理工大学 | Quick construction preparation method of human epidermal tissues |
-
2005
- 2005-09-15 CN CN 200510029692 patent/CN1739813A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016009A (en) * | 2008-04-14 | 2011-04-13 | Ucl商业股份有限公司 | Membrane |
| CN102016009B (en) * | 2008-04-14 | 2015-05-20 | Ucl商业股份有限公司 | Membrane |
| CN101927035A (en) * | 2010-07-20 | 2010-12-29 | 西南大学 | Method for preparing artificial skin using shell substrate as raw material |
| CN103721294A (en) * | 2013-12-27 | 2014-04-16 | 太原理工大学 | Quick construction preparation method of human epidermal tissues |
| CN103721294B (en) * | 2013-12-27 | 2015-05-13 | 太原理工大学 | Quick construction preparation method of human epidermal tissues |
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