CN1410036A - Tissue engineered valve - Google Patents
Tissue engineered valve Download PDFInfo
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- CN1410036A CN1410036A CN 02145458 CN02145458A CN1410036A CN 1410036 A CN1410036 A CN 1410036A CN 02145458 CN02145458 CN 02145458 CN 02145458 A CN02145458 A CN 02145458A CN 1410036 A CN1410036 A CN 1410036A
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Abstract
A histo-engineered valve is prepared through providing the cell removed matrix as degradable scaffold, extracting vascular endothelial cells and mechanocytes as seed cells, culturing, passage, and transplating them onto the said degradable scaffold. Its advantages are high biocompatibility, normal tissue strength and self-repair nature.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to a kind of engineered valve and preparation method thereof.
Background technology
Cardiac valve replacement is Cardiac Surgery operation commonly used, and used clinically artificial valve has mechanical prosthetic valve, bioprosthetic valve two classes at present.The person that accepts the mechanical prosthetic valve needs lifelong anticoagulant to reduce thromboembolic complication, has statistics to show its 10 years survival rates only 60% in the recent period, and underlying cause of death is that valve disengaging, jam and anticoagulant cause the complication that coagulation disorders causes.Bioprosthetic valve anticoagulant throughout one's life, five year survival rate are up to 100%, but its poor durability is easy to take place calcification, need operation once more because of degeneration behind the 8-10.Along with the development of Minimally Invasive Surgery technology and the proposition of organizational project notion, novel engineered bioprosthetic valve becomes the emphasis of various countries' research at present.
Tissue engineering is used the principle and the method for life sciences and engineering, and research, exploitation are used to repair, promote or improve the biological substitution product of function and form behind various tissues of human body or the organ injury.The basic preparation method of described biological substitution product is with cell formation cell---the degradable biomaterial complex that combines with degradable biomaterial behind amplification in vitro, in order to substitute the damaged tissues organ.It has the biological tissue that formation has vitality, and it is damaged that small amounts of cells is repaired bulk, and can be by characteristics such as tissue, any plastotypes of organ defect situation.With regard to engineered cardiac valve, remain in vascular endothelial cell source at present and seek suitable biodegradable stent material two big technological difficulties.
Engineered valve comprises vascular endothelial cell and fibroblast two class seed cells, rebuild the valve experiment at present and need get big section blood vessel of patient, be separated into fibrocyte and endotheliocyte by complicated technology again, so can't break away from pattern with the repair in trauma wound.Well-known in addition somatic cell In vitro culture difficulty is very big, generally passes several generations degeneration promptly takes place, and needs a large amount of vascular endothelial cells and make up valve.To this, previously research emphasis is placed on embryonic stem cell and the telomerase mostly, but the former faces ethical objection, and the latter makes inquiries because of its possibility induced tumor rarely has.
Think at present, tissue engineering bracket material has following effect: 1) keep the integrity of organizational structure and prevent distortion, 2) suppress the hypertypic regeneration of surrounding tissue as barrier, 3) as the support of cell migration and propagation, 4) promote cell proliferation and differentiation by interaction with the cell surface cell receptor.Degradation rate and certain intensity and plasticity that ideal timbering material should possess the good cell compatibility, histocompatibility, be complementary with cell growth rate.Previously utilize polyglycolic acid synthetic high polymer degradable materials such as (PGA), but its biocompatibility and cellular affinity are not satisfactory more.
Summary of the invention
The objective of the invention is to overcome above-mentioned technological difficulties, a kind of engineered valve of external structure.The present invention extracts seed cell by number of ways, and after the gained seed cell was carried out In vitro culture, goes down to posterity and breeds, substep was planted in acellular matrix, is built into engineered valve.
The present invention adopts acellular matrix as the biodegradable stent material.Its good biocompatibility, be easy to repopulating cell, and can preserve the natural structure and the tissue intensity of normal structure.
The present invention extracts seed cell by number of ways.Described seed cell is endotheliocyte and fibroblast.
The present invention passes through to extract the marrow blood endothelial cell, vein, arteries vascular endothelial cell, and the fatty tissue capillary endothelial cell obtains the endotheliocyte seed cell; By extracting marrow fibroblast, the subcutaneous connective tissue fibroblast obtains the fibroblast seed cell.With the external cell culture and going down to posterity of carrying out of gained seed cell, after reaching requirement, step inoculation is in the biodegradable stent material, the inoculation fibroblast is made fibroblast-timbering material complex earlier, invents engineered valve in composite surface inoculation vascular endothelial cell construction cost again.
Acellular matrix of the present invention can be allogeneic and xenogenesis valvular tissue acellular matrix, pericardial tissue acellular matrix and vascular tissue's acellular matrix.
Vascular endothelial cell of the present invention can derive from bone marrow, vein blood vessel, arteries and fatty tissue.It can also be vascular endothelial cell through genetic modification, transformation.
Fibroblast of the present invention can derive from bone marrow and subcutaneous connective tissue.It can also be fibroblast through genetic modification, transformation.
The engineered valve of the present invention has following characteristics:
Organizational structure with normal valvular tissue, the surface covers for vascular endothelial cell, and the centre is to be the connective tissue of main component with the fibroblast, is the living tissue with metabolic function.
The specific embodiment
Embodiment
Extract seed cell (following each method can separately or be united employing)
The extraction of vascular endothelial cell
The extracting method of derived from bone marrow vascular endothelial cell: extract receptor autologous 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.
The extracting method of vein blood vessel, arteries source vascular endothelial cell: 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.
The extracting method of fatty tissue source vascular endothelial cell: 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 like this.0.25---0.5% pancreatin or 0.5% neutral protease (DispaseII), 37 ℃, 5%CO2 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.
Fibroblastic extraction
The fibroblastic extracting method of derived from bone marrow: above-mentioned (A1.1) crossed the pillar cells (CD31 feminine gender) put in the culture dish in addition and cultivate, the DMED culture fluid, fibroblast more easily grows, and utilizes the differential adherent method can obtain fibroblast.
Subcutaneous connective tissue fibroblastic extracting method of originating: the corium behind the epidermis of will tearing is cut into fragment, collagenase with 0.2% filters through 150 order stainless steel filtering nets 37 ℃ of digestion 2 hours, with 1200r/min centrifugal 5 minutes, supernatant discarded adds no calcium magnesium PBS and cleans 3 times.Add the DMEM culture fluid 10ml that contains 10% hyclone, mixing.
Cell culture and going down to posterity:
Make single cell suspension behind the cell counting that said method is obtained, press 1-2 * 106/75cm
2The density inoculated and cultured contains the DMEM culture fluid of 10% hyclone, and 37 ℃, 5%CO
2, 100% relative humidity is cultivated, and culture fluid changed once in 3 days.Cell fusion does not have calcium magnesium PBS flushing with 10ml during to 60%-75% density, through 37 ℃ of digestion of 0.25% pancreatin 5 minutes, adds the DMEM culture fluid that contains 10% hyclone and stops digestion, moves 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, cell fusion is to 60%-75% density, and repeated transmission is commissioned to train foster.
The preparation acellular matrix
Get donor valve, pericardium or vascular tissue, put into following solution in turn and handle: 1, the distilled water rinsing of pre-cooling; 2, continue in 5% hydrochloric acid-10% sodium chloride solution to stir 4 ℃; 3, chloroform: methanol (1: 1), 25 ℃; 4, Hydrazoic acid,sodium salt, 25 ℃; 5, hydrogen peroxide soaks; 6, phosphate buffer, 25 ℃; Through lyophilization, behind the oxirane disinfection, standby again.Described donor can derive from allogeneic or heteroplasm.
Prepare engineered valve:
Be gathered into fibrocyte earlier, it is inoculated on the above-mentioned timbering material, inoculum density is 1 * 10
5---10
8/ CM.Culture fluid changed once in 3 days, cultivated 7-10 days, make up fibroblast-timbering material complex.
Collect vascular endothelial cell, it is seeded in fibroblast-timbering material composite surface, preparation vascular endothelial cell-fibroblast-timbering material complex is sewed up plastotype with it by valve shape again, makes engineered valve.
The engineered valvular tissue that the present invention makes up has normal valvular tissue organizational structure, but is self renewal, and the living tissue of self-regeneration, and no antigen can not cause immunological rejection.
Claims (8)
1, a kind of engineered valve is characterized in that preparing as follows: extract seed cell by number of ways, after carrying out In vitro culture, go down to posterity and breeding, substep is planted in the biodegradable stent material, is built into engineered valve.
2, by the described engineered valve of claim 1, it is characterized in that described biodegradable stent material is an acellular matrix.
3, by the described engineered valve of claim 1, it is characterized in that described seed cell is vascular endothelial cell and fibroblast.
4, by claim 1 and 2 described engineered valves, it is characterized in that described acellular matrix is allogeneic and xenogenesis valvular tissue acellular matrix, pericardial tissue acellular matrix and vascular tissue's acellular matrix.
5, by claim 1 and 3 described engineered valves, it is characterized in that described vascular endothelial cell derives from bone marrow, vein blood vessel, arteries and fatty tissue.
6, by claim 1 and 3 described engineered valves, it is characterized in that described fibroblast derives from bone marrow and subcutaneous connective tissue.
7,, it is characterized in that described valve preparation method is that the inoculation fibroblast is made fibroblast on timbering material earlier by the described engineered valve of claim 1---timbering material complex, again in composite surface inoculation vascular endothelial cell.
8, organizational project valve according to claim 1 is characterized in that the seed cell inoculum density is 1 * 10
5---10
8/ CM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02145458 CN1410036A (en) | 2002-11-15 | 2002-11-15 | Tissue engineered valve |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02145458 CN1410036A (en) | 2002-11-15 | 2002-11-15 | Tissue engineered valve |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1410036A true CN1410036A (en) | 2003-04-16 |
Family
ID=4750891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02145458 Pending CN1410036A (en) | 2002-11-15 | 2002-11-15 | Tissue engineered valve |
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| CN (1) | CN1410036A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294254C (en) * | 2005-01-21 | 2007-01-10 | 周建业 | Method for removing cells for heterogeneous cardiovascular transplant |
| CN101073678B (en) * | 2007-06-26 | 2010-11-10 | 中国人民解放军第二军医大学 | Tissue engineering venous valve and its production |
| CN102143765A (en) * | 2008-09-05 | 2011-08-03 | 伊西康公司 | Acellular matrix glue |
| CN104940997A (en) * | 2014-03-27 | 2015-09-30 | 复旦大学 | Human tissue-engineered cardiac muscle tissue |
| CN106726009A (en) * | 2017-03-06 | 2017-05-31 | 董念国 | Sustainer intervenes valve manufacture method |
| CN107106285A (en) * | 2014-11-13 | 2017-08-29 | 国立研究开发法人国立循环器病研究中心 | Connective tissue body formation base material and base material take out utensil |
-
2002
- 2002-11-15 CN CN 02145458 patent/CN1410036A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294254C (en) * | 2005-01-21 | 2007-01-10 | 周建业 | Method for removing cells for heterogeneous cardiovascular transplant |
| CN101073678B (en) * | 2007-06-26 | 2010-11-10 | 中国人民解放军第二军医大学 | Tissue engineering venous valve and its production |
| CN102143765A (en) * | 2008-09-05 | 2011-08-03 | 伊西康公司 | Acellular matrix glue |
| US8858698B2 (en) | 2008-09-05 | 2014-10-14 | Mentor Worldwide Llc | Acellular matrix glue |
| CN102143765B (en) * | 2008-09-05 | 2014-11-19 | 伊西康公司 | Acellular matrix glue |
| CN104940997A (en) * | 2014-03-27 | 2015-09-30 | 复旦大学 | Human tissue-engineered cardiac muscle tissue |
| CN107106285A (en) * | 2014-11-13 | 2017-08-29 | 国立研究开发法人国立循环器病研究中心 | Connective tissue body formation base material and base material take out utensil |
| CN106726009A (en) * | 2017-03-06 | 2017-05-31 | 董念国 | Sustainer intervenes valve manufacture method |
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