CN1198544C - Tissue engineered esophagus - Google Patents
Tissue engineered esophagus Download PDFInfo
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- CN1198544C CN1198544C CN 02145461 CN02145461A CN1198544C CN 1198544 C CN1198544 C CN 1198544C CN 02145461 CN02145461 CN 02145461 CN 02145461 A CN02145461 A CN 02145461A CN 1198544 C CN1198544 C CN 1198544C
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- tissue engineered
- tissue
- present
- esophagus
- esophageal
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Abstract
The present invention belongs to a field of biomedical engineering, particularly to a tissue engineered esophagus and a preparation method thereof. The present invention forms the tissue engineered esophagus containing a double-layer structure of an epithelial layer and a corium layer from a support material and a seed cell according to a tissue engineering basic principle method. A vascular endothelial cell in the corium layer of the present invention directly takes part in the establishment of a capillary network and simultaneously utilizes two revascularization methods to be communicated with an acceptor blood supply in a short time, so the blood supply is provided for epidermis of the tissue engineered esophagus. The present invention alters a traditional treating mode that damage is repaired by damage and can be used for simplifying an esophagus cutting and one-period reconstruction digestive tract operation mode, medical wounds are reduced, and the present invention brings gospel for wide esophageal pathological change patients.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to a kind of tissue engineered esophageal and preparation method thereof.
Background technology
China is the country occurred frequently of esophageal carcinoma, is again the highest country of esophageal carcinoma mortality rate, and clinical Therapeutic Principle is generally through excision of breast esophageal carcinoma and first phase and rebuilds digestive tract at present.Modal esophagus succedaneum is stomach, colon and small intestinal.The operation wound of going big, coverage is wide, complication is many, wherein the incidence rate of fistula of operative incision is a kind of typically with the treatment pattern of repair in trauma wound about 3%---5%.Artificial esophagus's development is made slow progress owing to the good synthetic material of the inorganization compatibility still.
Organizational project is 21st century life sciences one a big focus.Its core content is the cell in vitro dimensional culture, and its basic skills is that seed cell and degradable biomaterial are formed complex, substitutes the damaged tissues organ.Its scientific meaning not only is to provide a kind of new Therapeutic Method for removing patient's misery, and main is the new thought that has proposed to duplicate " tissue ", " organ ", has changed the medical model that previously damages with injury repairing.
Summary of the invention
The purpose of this invention is to provide a kind of tissue engineered esophageal, change the treatment pattern of previously damaging with injury repairing.The present invention utilizes the tissue engineering skin timbering material as the biodegradable stent material according to the tissue engineering basic principles, and the esophageal tissue of epithelial layer and skin corium is arranged in external structure.Described skin corium comprises by vascular endothelial cell and fibroblast.
Tissue engineering skin timbering material of the present invention can be poly-hydroxyl second (PGA), the skin acellular matrix.Wherein the skin acellular matrix comprises allogeneic dermis acellular matrix and xenogenesis corium acellular matrix.
The biodegradable stent material pig dermis acellular matrix that the present invention adopts is available from Shanghai Dong Run biological product company.A burn case through clinic trial one over thousands of example, confirm that its histocompatibility, cell compatibility are good.
The present invention has following advantage
1. but this tissue engineered esophageal tissue is a self renewal, the living tissue of self-regeneration, and no antigen can not cause immunological rejection.
2, the present invention utilizes vascular endothelial cell to quicken the blood supply of reconstruction tissue engineered esophageal.
Complete epithelial tissue can prevent that granulation tissue hyperplasia from stopping up tube chamber, is the key of tissue engineered esophageal success.The normal structure revascularization mainly contains angiogenesis (angiogenesis), (vasculogenesis) two kinds of forms take place blood vessel.Angiogenesis is that local vascular regeneration is meant that the normal group intracellular cytokine is free to regenerating tissues generation blood capillary under stimulating.Blood vessel takes place then free to regenerating tissues generation blood capillary under some cytokine stimulates by bone marrow medium vessels endotheliocyte.The present invention utilizes above-mentioned two kinds of revascularization methods simultaneously, supplies to lead to receptor blood in the short time again, thereby supplies for the tissue engineered esophageal epithelium provides blood.
The specific embodiment
Embodiment 1
1) separates and cultivation epidermis epithelial cells
Get the strip that patient's epidermis is trimmed to wide 1-2mm, no calcium magnesium PBS rinsing twice, epidermis side is dipped in 0.5% neutral protease (DispaseII) 4 ℃ up and spends the night.After epidermis torn from corium, shred, 37 ℃ of digestion 20 minutes, the 10ml pancreatin inhibitor stopped digestion with 0.05% pancreatin-0.53mMEDTA.Behind the filtration of 150 order stainless steel filtering nets, centrifugal (1200 change 10 minutes), counting, make single cell suspension, by 3 * 10
6/ 75cm
2The density inoculated and cultured, and serum-free epidermis cell culture medium (GIBRO, USA), other adds Niu Chuiti extract and reorganization epithelical cell growth factor, at 37 ℃, 5%CO
2, to cultivate in the incubator of saturated humidity, culture fluid changed once in 3 days.When cell reaches the 60-75% density fusion, through 37 ℃ of 0.05% pancreatin digestion 10 minutes, in the cultivation of going down to posterity of 1: 3 ratio.
2) dermal fibroblast separates and cultivates
Separating digesting
With above-mentioned corium fragment, 37 ℃ of collagenases 0.2% digestion 2 hours is filtered through 150 order stainless steel filtering nets, and with 1200r/min centrifugal 5 minutes, supernatant discarded added no calcium magnesium PBS cleaning 3 times.Add the DMEM culture fluid 10ml that contains 10% hyclone, mixing, trypan blue dyeing counting.
Former be commissioned to train foster
Make single cell suspension behind the counting, press 1-2 * 10
6/ 75cm
2The density inoculated and cultured contains the DMEM culture fluid of 10% hyclone, at 37 ℃, and 5%CO
2, to cultivate in the incubator of 100% relative humidity, culture fluid changed once in 3 days.When cell fusion arrives 60%-75% density, there is not calcium magnesium PBS flushing with 10ml, through 37 ℃ of digestion of 0.25% pancreatin 5 minutes, add the DMEM culture fluid that contains 10% hyclone and stop digesting, move in the centrifuge tube centrifugal 5 minutes of 1200r/min.Abandoning supernatant adds the DMEM culture fluid 10ml contain 10% hyclone, mixing, and counting is in the cultivation of going down to posterity of 1: 3 ratio.Change culture fluid 2-3 day one time, during to 60%-75% density, repeated transmission is commissioned to train foster up to cell fusion.
3) endotheliocyte extracts
Following extracting method can be separately or use in conjunction.
The derived from bone marrow vascular endothelial cell extracts
Extract bone marrow 5-10ml, PBS cleans, centrifugal speed 1500g * 10min.Ficoll separates (5ml cell suspension+Ficoll 5ml, centrifugal speed 200g * 25min), extract nucleated cell, twice of PBS+EDTA cleaning.Add the CD31 antibody that indicates magnetic bead and place 15min for 6-12 ℃; Per 108 cells+5-10ccPBS cleans twice.MACS separates: MS+/RS+ post 500ulPBS in advance soaks into, and PBS500ul cleaned the filter post 3 times after cell suspension was crossed post.1mlPBS flushing filter post gets the CD31+ cell.
Vein blood vessel, arteries source vascular endothelial cell extract:
Extracting vein blood pipe, arteries 5-10CM, the two ends ligation, perfusion is through 0.25---0.5% pancreatin or 0.5% neutral protease (DispaseII), 37 ℃, 5%CO in the tube chamber
2, placed 5-10 minute in the incubator of saturated humidity, decontrol an end, pour out content, add the DMEM culture fluid that contains 10% hyclone and stop digestion, move in the centrifuge tube centrifugal 5 minutes of 1200r/min.PBS cleans twice.
Fatty tissue source vascular endothelial cell extracts:
The fatty tissue that extracts is moved in the centrifuge tube centrifugal 5 minutes of 1200r/min.Abandoning supernatant adds the DMEM culture fluid 10ml that contains 10% hyclone, mixing.Blood capillary in the isolated adipose tissue.0.25---0.5% pancreatin or 0.5% neutral protease (DispaseII), 37 ℃, 5%CO
2, placed 5-10 minute in the incubator of saturated humidity.Add the DMEM culture fluid that contains 10% hyclone and stop digestion, move in the centrifuge tube centrifugal 5 minutes of 1200r/min.PBS cleans twice.
4) make up tissue engineered esophageal
Collect cultured vascular endothelial cells and dermal fibroblast, it is inoculated on the tissue engineering skin timbering material, be built into vascular endothelial cell, fibrocyte-acellular dermal complex.Inoculum density is 1 * 10
5---10
8Culture fluid changed once in 3 days, and incubation time is 7-10 days.
Form double-decker: collect epidermis cell, it is seeded in vascular endothelial cell, fibroblast-biodegradable stent material composite surface.Form gas-liquid interface in composite surface after one week, promote the further differentiation of vascular endothelial cell.
Claims (6)
1, a kind of tissue engineered esophageal, it is characterized in that constituting and contain epithelial layer and the double-deck tissue engineered esophageal of skin corium by timbering material and seed cell, described timbering material is an acellular matrix, and described seed cell comprises fibroblast, vascular endothelial cell, epidermis epithelial cells.
2, tissue engineered esophageal according to claim 1 is characterized in that described timbering material is the pig dermis acellular matrix.
3, tissue engineered esophageal according to claim 1 is characterized in that described epidermis epithelial cells has substituted the structure that the esophagus epithelial cell is used for the esophagus epithelium.
4, tissue engineered esophageal according to claim 1 is characterized in that described vascular endothelial cell extracts from bone marrow, or vein blood vessel or fatty tissue.
5, tissue engineered esophageal according to claim 1 is characterized in that described skin corium comprises vascular endothelial cell.
6, tissue engineered esophageal according to claim 1 is characterized in that described vascular endothelial cell is used for the reconstruction of capillary network under the mucosa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02145461 CN1198544C (en) | 2002-11-15 | 2002-11-15 | Tissue engineered esophagus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02145461 CN1198544C (en) | 2002-11-15 | 2002-11-15 | Tissue engineered esophagus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1410034A CN1410034A (en) | 2003-04-16 |
| CN1198544C true CN1198544C (en) | 2005-04-27 |
Family
ID=4750894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02145461 Expired - Fee Related CN1198544C (en) | 2002-11-15 | 2002-11-15 | Tissue engineered esophagus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1198544C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7927414B2 (en) * | 2008-09-05 | 2011-04-19 | Ethicon, Inc. | Method of manufacturing acellular matrix glue |
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2002
- 2002-11-15 CN CN 02145461 patent/CN1198544C/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| CN1410034A (en) | 2003-04-16 |
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