CN109821069A - A kind of photo-crosslinking type Acellular valve and preparation method thereof and its application - Google Patents
A kind of photo-crosslinking type Acellular valve and preparation method thereof and its application Download PDFInfo
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- CN109821069A CN109821069A CN201910040192.1A CN201910040192A CN109821069A CN 109821069 A CN109821069 A CN 109821069A CN 201910040192 A CN201910040192 A CN 201910040192A CN 109821069 A CN109821069 A CN 109821069A
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- valve
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- crosslinking
- riboflavin
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Abstract
The present invention relates to a kind of photo-crosslinking type Acellular valves and preparation method thereof and its application.Above-mentioned preparation method includes: Step 1: the valve after Cell extraction is placed in riboflavin solution and is softly rocked so that riboflavin sufficiently diffuses into cellular matrix；Step 2: the valve handled through step 1 is taken out from riboflavin solution, then with remaining riboflavin in cleaning removal valve after ultraviolet light irradiation crosslinking, it can be obtained a kind of photo-crosslinking type Acellular valve.The innovative mechanical property that riboflavin and photo-crosslinking are used to handle and enhance cellular matrix valve in the present invention, and the processing can significantly improve the Young's modulus of valve as the result is shown, and can slow down its internal degradation speed.
Description
Technical field
The present invention relates to a kind of timbering materials more particularly to a kind of photo-crosslinking type Acellular valve and preparation method thereof and its
Using.
Background technique
Removing cellular matrix is to go cellular processes obtained to tissue progress Cell extraction using difference, is going cell mistake
Retain the original form of natural tissues and biomechanical property in journey as much as possible, goes cellular matrix tissue as biomimetic scaffolds material
Material is usually used in organizational project and regenerative medicine.
But due to the extracellular matrixs such as elastin laminin, proteoglycans in tissue there are still different degrees of damage, lose
Lose, cause after Cell extraction tissue there are mechanical properties decrease, it is degradable the disadvantages of.And mechanical properties decrease makes cell
Treated, and perforation, incompetence easily occur after being implanted into human body for valve；And degrade it is too fast may make implanting tissue because degradation speed
Degree regenerates far faster than tissue and implantation material early stage is caused to be decayed.
Main component due to removing cellular matrix is collagenous fibres, and the structure and space arrangement of collagenous fibres influence tissue
Mechanical property, therefore promote collagen cross-linking that can enhance its mechanical property and stability.There are many ways to promoting collagen cross-linking,
Mainly by corresponding enzyme-catalyzed cross-linking in physiological environment, such as lysyloxidase, thus make collagen obtain its natural hardness,
The elasticity of stability and organizing specific；Non- enzymatic method includes with chemical reagent, photooxidation etc., traditional pentanediol crosslinking approach
Although the mechanical property of cellular matrix can be significantly improved, there are easy calcification, easily decays and have the problems such as cytotoxicity.Cause
Cell cannot be grown in the tissue after glutaraldehyde cross-linking, it is difficult to tissue remodeling occur.
It can not only enhance the mechanical property organized after Cell extraction therefore, it is necessary to a kind of new and it can be slowed down in vivo
The preparation method of degradation speed.
Summary of the invention
In view of the above-mentioned problems, now providing a kind of photo-crosslinking type Acellular valve for aiming to solve the problem that the above problem and its preparation side
Method and its application.
Specific technical solution is as follows:
A kind of preparation method of photo-crosslinking type Acellular valve, has the feature that, includes the following steps:
Step 1: the valve after Cell extraction is placed in riboflavin solution and is softly rocked so that riboflavin sufficiently expands
Scattered entrance is gone in cellular matrix；
Step 2: the valve handled through step 1 is taken out from riboflavin solution, then with clear after ultraviolet light irradiation crosslinking
It washes away except riboflavin remaining in valve, can be obtained a kind of photo-crosslinking type Acellular valve.
Above-mentioned preparation method, also has the feature that, the mass concentration of riboflavin is in step 1 riboflavin solution
0.04%-1%.
Above-mentioned preparation method, also has the feature that, it is 5-120min that step 2 middle-ultraviolet lamp, which irradiates crosslinking time,
Ultraviolet light light intensity is 1-5mw/cm2。
Above-mentioned preparation method, also has the feature that, cleaning method in step 2 are as follows: will irradiate and be crosslinked through ultraviolet light
Valve afterwards changes liquid 1 time for every 6 hours in being cleaned 24 hours on shaking table with sterile PBS.
Above-mentioned preparation method, also has the feature that, valve is selected from porcine aorta valve or bovine pericardium.
In the present invention method of Cell extraction using the present inventor earlier application application No. is
In CN201610921956.4, entitled " a kind of for removing histiocytic kit and removing cellular processes " patent
The method of offer is handled.
The second aspect of the invention is to provide a kind of photo-crosslinking type prepared according to above-mentioned preparation method and removes cell
Valve.
The third aspect of the invention is to provide a kind of above-mentioned photo-crosslinking type Acellular valve in organizational project and regeneration doctor
Application in.
Photo-crosslinking type Acellular valve provided in the present invention can be applied to the preparation of biological support.
The beneficial effect of above scheme is:
1), the innovative mechanics that riboflavin and photo-crosslinking are used to handle and enhance cellular matrix valve in the present invention
Performance, and the processing can significantly improve the Young's modulus of valve as the result is shown, and can slow down its internal degradation speed；
2), photo-crosslinking processing method is in the mechanical property and stability that can significantly increase valve, and compared to tradition penta 2
The cross-linking treatment methods such as aldehyde, photo-crosslinking processing method will not introduce foreign substance or group, utmostly retain Cell extraction
Original institutional framework afterwards, without influencing tissue microstructure；
3), there is good bio-compatible using the biological support of photo-crosslinking type Acellular valve provided by the invention preparation
Property, in vitro culture, which is tested, shows that it is more conducive to cell growth, and the experiment of rat et al. Ke shows its relatively slow, inflammation that shows as degrading
Cellular infiltration is less and the advantages of being not susceptible to calcification；
4), using photo-crosslinking type Acellular valve provided by the invention preparation biological support have short preparation period, at
This low, time-consuming short and manageable advantage, with good potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is the ultimate tensile strength statistical chart of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention；
Fig. 2 is the elasticity modulus statistical chart of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention；
Fig. 3 is the limit tensile strain statistical chart of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention；
Fig. 4 is the histological stain photo of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention；
Fig. 5 is the micro- electromicroscopic photograph of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention；
Fig. 6 is that the Human umbilical vein endothelial cells growth of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention is bent
Line；
Fig. 7 is that the Human umbilical vein endothelial cells of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention adhere to number
Measure statistical chart；
Fig. 8 is the cell proliferation of human umbilical vein number of the photo-crosslinking type Acellular valve provided in the embodiment of the present invention
Measure statistical chart；
Fig. 9 is that the histological stain after the photo-crosslinking type Acellular valve provided in the embodiment of the present invention subcutaneously embeds shines
Piece.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art without creative labor it is obtained it is all its
His embodiment, shall fall within the protection scope of the present invention.It should be noted that in the absence of conflict, the implementation in the present invention
Feature in example and embodiment can be combined with each other.
The present invention will be further explained below with reference to the attached drawings and specific examples, but not as the limitation of the invention.
Embodiment 1
A kind of preparation of photo-crosslinking type Acellular valve, includes the following steps:
The acquisition of porcine aorta valve: Pigs Hearts is obtained under the clean conditions of slaughterhouse and is placed in the cold saline containing heparin
In and rapid transport to laboratory.In the 1h of pig death, porcine aorta valve is cut under aseptic condition, is placed in 4 DEG C containing antibiosis
12h in the DMEM in high glucose culture medium of plain (100U/ml penicillin, 250mg/ml amphotericin B and 100mg/ml streptomysin)；
Cell extraction: the Cell extraction of porcine aorta valve carries out in six orifice plates, and the displacement volume in every hole is 4ml
And at most place 3 valves, the specific method of Cell extraction is: porcine aortic valve is placed in containing 20g/L CHAPS and
In the TRIS-HCI buffer (40mmol/L, pH 7.8) of 2mmol/L TnBP, sustained oscillation is handled for 24 hours at room temperature, then with
It rinsed with sterile water 6 times, 10 minutes every time, is subsequently placed at containing 20g/L CHAPS, 2mmol/L TnBP, 10g/L ASB-
In the TRIS-HCI buffer (40mmol/L, pH 7.8) of 14 and 20g/L SB 3-10, continue concussion processing at room temperature for 24 hours,
Again by valve with PBS rinsing 4 times, each 6h is subsequently placed at containing 1mmol/L MgCl2With 100units/ml totipotent nucleus
In the TRIS-HCI buffer (50mmol/L, pH 8.0) of sour enzyme, handled for 24 hours in 37 DEG C of sustained oscillations, finally with PBS rinsing 4
Secondary reagent and cell fragment to remove remnants, each 6h are placed in antibiotic PBS, save backup in 4 DEG C；
Photo-crosslinking processing (being protected from light lower progress in room temperature): with sterile PBS preparation riboflavin solution, (mass concentration is
0.05%), and clean six orifice plate is taken, moves into 5ml riboflavin solution and 2 Acellular valves in every hole, six orifice plates is placed in and are shaken
2h is softly rocked on bed, in the valve after making riboflavin sufficiently diffuse into Cell extraction, then is drawn off and is laid in clean
Six orifice plates cover, face quartz burner is 3mw/cm with light intensity2Ultraviolet light irradiation 30min after valve is placed in six holes
It in plate, in being cleaned for 24 hours on shaking table with sterile PBS, changes within every 6 hours liquid 1 time, to clean the remaining riboflavin immersed in valve, clearly
Valve is placed in antibiotic PBS and saves backup in 4 DEG C after the photo-crosslinking of wash clean.
Mechanics properties testing
In the present invention using uniaxial tensile test to photo-crosslinking type Acellular valve (R-UV-DHV) provided by the invention into
The measurement of row mechanical property, specifically: by valve in the strip sample for being circumferentially cut into 15mm × 3mm, with electronic vernier caliper
The gauge length, width and thickness of each sample are measured, then sample is fixed on the sample folder of mechanics detector, at room temperature with 5mm/
The speed of min is at the uniform velocity stretched to sample fracture to both ends, is moistened in test process with PBS holding sample, is recorded by energy converter
The load-deformation curve of sample simultaneously calculates ultimate tensile strength, limit tensile strain and elasticity modulus.
Testing result shows: ultimate tensile is strong in the circumferential for R-UV-DHV (14.13 ± 1.589MPa) provided by the invention
Noticeably greater than only (DHV, 7.210 ± 0.6524MPa, test method are all uniaxial tensile test method (figure to the valve of removal cell to degree
1)；Meanwhile elasticity modulus is noticeably greater than DHV (15.70 ± 2.230MPa) (Fig. 2) in the circumferential；Meanwhile (64.37 ±
12.24%) limit tensile strain in the circumferential is less than the conventional valve (79.47 ± 11.84%) for only removing cell, but difference
Not statistically significant (Fig. 3).
Histology
The changes in microstructure of valve is detected in the present invention by histological stain and scanning electron microscope observation, specifically: it will
After valve is fixed for 24 hours with 4% paraformaldehyde buffer, serial dehydration, routine paraffin wax is embedded, 5 μm of slice thickness, normal with kit
(be used to evaluate the variation of valve structure with HE, Masson and EVG dyeing: Masson dyeing is mainly used to observe glue for rule dyeing
The variation of fibrinogen and EVG dyeing are used to observe the variation of elastomer).
Testing result shows: three layers of the three-decker and DHV that show R-UV-DHV provided by the invention are observed in HE dyeing
Structure is similar (Fig. 4), and Masson dyeing observation shows that the collagenous fibres in the three-decker of R-UV-DHV provided by the invention are protected
It deposits good (Fig. 4), the elastomer of the EVG dyeing observation display center R-UV-DHV provided by the invention room floor does not also reduce (figure
4), scanning electron microscopic observation shows that arrangement of collagen fibers is more neat (Fig. 5) in R-UV-DHV.
Cell compatibility detection
The cell compatibility of valve is detected in the present invention using CCK-8 reagent, specifically: it is cut on valve with punch
Diameter be 8mm circular specimen, and be placed in Peracetic acid with sterile PBS rinsing 10min and sterilize 30min, after sterilization with
PBS is rinsed 3 times, each 10min, then is put into cell incubator and is preheated after valve to be measured is placed in 48 orifice plates, then at often to
Add 200ul Human umbilical vein endothelial cells (HUVEC) suspension (density 20x10 in sample4/ ml), orifice plate is put in cell training
It supports and is cultivated in case, changed the liquid once every other day, and detected respectively at 2h, 6h, 1 day, 4 days, 7 days, first exhaust valve (n to be measured before detection
=3) culture medium in, and with PBS rinsing 3 times, to clean nonadherent cell, then by valve to be measured transfer in clean hole,
Add culture medium 150ul so that it submerges valve, then plus 15ul CCK-8 reagent, and be incubated for 2h in cell incubator, be incubated for 2h
100ul working solution is drawn afterwards in 96 orifice plates, its absorbance is surveyed under 450nm wavelength.
Testing result shows: the cell number of glutaraldehyde cross-linking type Acellular valve (GA-DHV) is constantly in decline and becomes
Gesture, substantially equal with blank control (Control) by the 7th day, this shows that GA-DHV has biggish cytotoxicity, to being adhered to it
On the toxic effect of HUVEC, cell compatibility is poor；And the cell number of DHV and R-UV-DHV is constantly in ascendant trend,
HUVEC can be adhered to thereon and be occurred to be proliferated (Fig. 6), this shows that the cell compatibility of DHV and R-UV-DHV are significantly better than GA-
DHV, and R-UV-DHV (0.45 ± 0.06) OD value since 6h (Fig. 7) is greater than DHV (0.37 ± 0.04), until at the 4th day (Fig. 8)
This difference (R-UV-DHV (1.02 ± 0.14), DHV (0.54 ± 0.08)) statistically significant (P < 0.05), this shows R-
UV-DHV has good cell compatibility, and grows compared with cell is more advantageous to for DHV.
Histocompatbility and tissue degradation detection
Evaluated in the present invention using the subcutaneous embedding molds of female sd inbred rats of 6 week old or so valve histocompatbility and
Stability, specifically: with 10% chloraldurate to the anesthesia of SD rat abdominal cavity, abdomen preserved skin and with iodophor disinfection, by left abdomen
A stringer notch for being about 1cm is done in center, and valve is placed in capsule so that subcutaneous rat one pouch of formation by blunt separation subcutaneous tissue
In bag, three angles of 6-0Prolene nonabsorable line secure valve are used respectively, in favor of taking out implantation material, then with 4-0 silk thread
Suturing them skin, it is postoperative anaesthetized on electric blanket after restore, daily observe wound whether have swelling or infection and make to remember
Record, and respectively at postoperative 2 weeks and 4 weeks 4 rats of execution and implantation material and the tissue adjoined are taken out, do histological stain analysis.
Testing result shows: after two weeks, the inflammatory cell infiltrated in R-UV-DHV is less than implantation DHV (Fig. 9) to implantation rat,
And the tissue integrity of R-UV-DHV is significantly better than the DHV (Fig. 9) of implantation after implantation 4 weeks, this shows the tissue phase of R-UV-DHV
Capacitive is good, and tissue degradation is retarded.
The foregoing is merely preferred embodiments of the present invention, are not intended to limit embodiments of the present invention and protection model
It encloses, to those skilled in the art, should can appreciate that all with made by description of the invention and diagramatic content
Equivalent replacement and obviously change obtained scheme, should all be included within the scope of the present invention.
Claims (7)
1. a kind of preparation method of photo-crosslinking type Acellular valve, which comprises the steps of:
It is placed in riboflavin solution and softly rocks so that core yellow Step 1: being protected from light the lower valve by after Cell extraction in room temperature
Element sufficiently diffuses into cellular matrix；
Step 2: in room temperature be protected from light it is lower the valve handled through step 1 is taken out from riboflavin solution, then irradiated with ultraviolet light
Remaining riboflavin in cleaning removal valve, can be obtained a kind of photo-crosslinking type Acellular valve after crosslinking.
2. preparation method according to claim 1, which is characterized in that the matter of riboflavin in riboflavin solution described in step 1
Amount concentration is 0.04%-1%.
3. preparation method according to claim 1, which is characterized in that it is 5- that step 2 middle-ultraviolet lamp, which irradiates crosslinking time,
120min, ultraviolet light light intensity are 1-5mw/cm2。
4. preparation method according to claim 1, which is characterized in that cleaning method in step 2 are as follows: will be shone through ultraviolet light
Valve after penetrating crosslinking changes liquid 1 time for every 6 hours in being cleaned 24 hours on shaking table with sterile PBS.
5. preparation method according to claim 1-4, which is characterized in that the valve is selected from porcine aorta valve
Or bovine pericardium.
6. a kind of photo-crosslinking type Acellular valve, which is characterized in that any one of -5 preparation method preparation according to claim 1
It obtains.
7. requiring application of the 6 photo-crosslinking type Acellular valves in organizational project and regenerative medicine according to claim.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910040192.1A CN109821069A (en) | 2019-01-16 | 2019-01-16 | A kind of photo-crosslinking type Acellular valve and preparation method thereof and its application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910040192.1A CN109821069A (en) | 2019-01-16 | 2019-01-16 | A kind of photo-crosslinking type Acellular valve and preparation method thereof and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425910A (en) * | 2021-07-30 | 2021-09-24 | 中南大学湘雅二医院 | Composite cross-linked biological valve and preparation method thereof |
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|---|---|---|---|---|
| EP0779320A2 (en) * | 1989-08-02 | 1997-06-18 | University Of North Carolina At Chapel Hill | Process for cross-linking collagenous materials and resulting product |
| US6093530A (en) * | 1998-02-06 | 2000-07-25 | Sulzer Carbomedics Inc. | Non-calcific biomaterial by glutaraldehyde followed by oxidative fixation |
| CN1439431A (en) * | 2003-04-01 | 2003-09-03 | 北京市普惠生物医学工程公司 | Photo-oxidation of collagen tissue of heterogenic implant of heart |
| CN105963770A (en) * | 2016-05-09 | 2016-09-28 | 中国人民解放军第三军医大学 | Photosensitive biogel and preparation method thereof |
| US9512279B2 (en) * | 2013-12-18 | 2016-12-06 | Universite Cegy-Pontoise | Interpenetrating polymer network |
| WO2018091553A1 (en) * | 2016-11-16 | 2018-05-24 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Method for preparing a graft |
| CN108697828A (en) * | 2016-02-11 | 2018-10-23 | 生命细胞公司 | The method for preventing enzyme from degrading for stablizing the tissue products comprising collagen |
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2019
- 2019-01-16 CN CN201910040192.1A patent/CN109821069A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0779320A2 (en) * | 1989-08-02 | 1997-06-18 | University Of North Carolina At Chapel Hill | Process for cross-linking collagenous materials and resulting product |
| US6093530A (en) * | 1998-02-06 | 2000-07-25 | Sulzer Carbomedics Inc. | Non-calcific biomaterial by glutaraldehyde followed by oxidative fixation |
| CN1439431A (en) * | 2003-04-01 | 2003-09-03 | 北京市普惠生物医学工程公司 | Photo-oxidation of collagen tissue of heterogenic implant of heart |
| US9512279B2 (en) * | 2013-12-18 | 2016-12-06 | Universite Cegy-Pontoise | Interpenetrating polymer network |
| CN108697828A (en) * | 2016-02-11 | 2018-10-23 | 生命细胞公司 | The method for preventing enzyme from degrading for stablizing the tissue products comprising collagen |
| CN105963770A (en) * | 2016-05-09 | 2016-09-28 | 中国人民解放军第三军医大学 | Photosensitive biogel and preparation method thereof |
| WO2018091553A1 (en) * | 2016-11-16 | 2018-05-24 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. | Method for preparing a graft |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425910A (en) * | 2021-07-30 | 2021-09-24 | 中南大学湘雅二医院 | Composite cross-linked biological valve and preparation method thereof |
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