CN102475910A - Esophageal tissue constructed by tissue engineering - Google Patents
Esophageal tissue constructed by tissue engineering Download PDFInfo
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- CN102475910A CN102475910A CN2010105535620A CN201010553562A CN102475910A CN 102475910 A CN102475910 A CN 102475910A CN 2010105535620 A CN2010105535620 A CN 2010105535620A CN 201010553562 A CN201010553562 A CN 201010553562A CN 102475910 A CN102475910 A CN 102475910A
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Abstract
The invention belongs to the field of biomedical engineering and specifically relates to an esophageal tissue constructed by tissue engineering. A tissue-engineered esophagus containing an epithelial layer and a dermal layer is consisted of scaffold materials and seed cells according to the basic principles and methods of tissue engineering. Vascular endothelial cells in the dermal layer directly participate in the establishment of a capillary network, and two revascularization methods are used to quickly communicate the capillary network in the dermal layer with the blood supply channel of a receptor, thus providing blood to the epithelium of the tissue-engineered esophagus. The esophageal tissue of the invention changes a former therapeutic mode of repairing damage by damage, and can be used for simplifying esophagectomy and one-stage reconstruction of digestive tracts, thus reducing medical trauma and benefiting the majority of patients with esophageal diseases.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to a kind of esophageal tissue that makes up through organizational project.
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 anastomotic stoma expectorant 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 for removing less patient suffering a kind of new Therapeutic Method to be provided, and main is to have proposed to duplicate " tissue ", " organ " and new thought, changed the medical model that previously damages with injury repairing.
Summary of the invention
The objective of the invention is deficiency, a kind of esophageal tissue that makes up through organizational project is provided, change the treatment pattern of the past with the injury repairing damage to above-mentioned prior art.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.
The tissue engineering skin timbering material that the present invention adopted can be to gather polyglycolic acid (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, 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 successful key of tissue engineered esophageal.The normal structure revascularization mainly contains angiogenesis, 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 keratin cell
Get the strip that patient's epidermis is trimmed to wide 1-2mm, no calcium magnesium PBS rinsing twice, epidermis side is dipped in the 0.5 % neutral protease 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-0.53mM EDTA.Behind the filtration of 150 order stainless steel filtering nets, centrifugal (1200 change 10 minutes), counting, process 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 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 digestion of the collagenase of 0.2 % 2 hours, filter through 15O order stainless steel filtering net, with 1200r/min centrifugal 5 minutes, supernatant discarded added no calcium magnesium PBS cleaning 3 times.Add the DMEM culture fluid 1Oml that contains 10 % hyclones, mixing, trypan blue dyeing counting.
Former be commissioned to train foster
Process single cell suspension behind the counting, press 1-2 * 10
6/ 75cm
2The density inoculated and cultured contains the DMEM culture fluid of 10 % hyclones, at 37 ℃, and 5 % C
O2, to cultivate in the incubator of 100 % relative humiditys, culture fluid changed once in 3 days.When cell fusion during to 60 %-75 % density; Do not have calcium magnesium PBS flushing with 10ml,, add the DMEM culture fluid that contains 10 % hyclones and stop digestion through 37 ℃ of digestion of 0.25 % pancreatin 5 minutes; Move in the centrifuge tube centrifugal 5 minutes of 1200r/min.Abandoning supernatant adds the DMEM culture fluid 10ml contain 10 % hyclones, mixing, and counting is in the cultivation of going down to posterity of 1: 3 ratio.Changed culture fluid on the 2nd-3 one time, when cell fusion to 60 %-75 % density, repeated transmission is commissioned to train foster.
3) endotheliocyte extracts
Following method for distilling can be separately or Combined application.
The derived from bone marrow vascular endothelial cell extracts
Extract bone marrow 5-10ml, PBS cleans, centrifugal speed 1 500g * l0min.(PBS+EDIA cleans twice for 5ml cell suspension+Ficoll 5ml, centrifugal speed 200 g * 25min), extract nucleated cell in the Ficoll separation.Add the CD31 antibody that indicates magnetic bead and place 15min for 6-12 ℃; Per 10
8Cell+5-1OccPBS cleans twice.MACS separates: MS +/the 500ulPBS infiltration in advance of RS+post, PBS500ul cleaned the filter post 3 times after cell suspension was crossed post.LmlPBS 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, 37oC, 5 % C0 in the tube chamber
2, placed in the incubator of saturated humidity 5-10 minutes, decontrol an end, pour out content, add the DMEM culture fluid that contains 10 % hyclones 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 % hyclones, mixing.Blood capillary in the isolated adipose tissue.0.25-0.5 % pancreatin or 0.5 % neutral protease, 37 ℃, 5 % CO
2, placed 5-10 minute in the incubator of saturated humidity.Add the DMEM culture fluid that contains 10 % hyclones and stop digestion, move in the centrifuge tube centrifugal 5 minutes of 1200r/min.PBS cleans twice.
4) make up tissue engineered esophageal and 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 one 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 one biodegradable stent material composite surface.Form gas one liquid interface in composite surface after one week, promote the further differentiation of vascular endothelial cell.
Claims (1)
1. tissue engineered esophageal is characterized in that being made up of timbering material and seed cell and contains epithelial layer and the double-deck tissue engineered esophageal of skin corium; Timbering material comprises biomaterial and synthetic material, and timbering material can be an acellular matrix, and timbering material can be the pig dermis acellular matrix; Seed cell comprises fibroblast, vascular endothelial cell, epidermis epithelial cells; Vascular endothelial cell extracts from bone marrow, or vein blood vessel or arteries or fatty tissue; Skin corium comprises vascular endothelial cell; Vascular endothelial cell is used for the reconstruction of capillary network under the open-minded film.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105535620A CN102475910A (en) | 2010-11-22 | 2010-11-22 | Esophageal tissue constructed by tissue engineering |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105535620A CN102475910A (en) | 2010-11-22 | 2010-11-22 | Esophageal tissue constructed by tissue engineering |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102475910A true CN102475910A (en) | 2012-05-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105535620A Pending CN102475910A (en) | 2010-11-22 | 2010-11-22 | Esophageal tissue constructed by tissue engineering |
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| CN (1) | CN102475910A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104473706A (en) * | 2014-12-09 | 2015-04-01 | 金华市人民医院 | Biodegradable composite type tubular urethral stent and preparation method |
| CN113679889A (en) * | 2021-07-20 | 2021-11-23 | 杭州贤石生物科技有限公司 | Acellular matrix composite material and preparation method and application thereof |
-
2010
- 2010-11-22 CN CN2010105535620A patent/CN102475910A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104473706A (en) * | 2014-12-09 | 2015-04-01 | 金华市人民医院 | Biodegradable composite type tubular urethral stent and preparation method |
| CN113679889A (en) * | 2021-07-20 | 2021-11-23 | 杭州贤石生物科技有限公司 | Acellular matrix composite material and preparation method and application thereof |
| CN113679889B (en) * | 2021-07-20 | 2022-10-25 | 杭州贤石生物科技有限公司 | Acellular matrix composite material and preparation method and application thereof |
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| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120530 |