CN102600508B - Porcine arterial vacuum lyophilization acellular matrix, as well as preparation method and application thereof - Google Patents
Porcine arterial vacuum lyophilization acellular matrix, as well as preparation method and application thereof Download PDFInfo
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- CN102600508B CN102600508B CN201210031969.6A CN201210031969A CN102600508B CN 102600508 B CN102600508 B CN 102600508B CN 201210031969 A CN201210031969 A CN 201210031969A CN 102600508 B CN102600508 B CN 102600508B
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Abstract
The invention provides a porcine arterial vacuum lyophilization acellular matrix, as well as a preparation method and application thereof. The preparation method of the porcine arterial vacuum freeze-drying acellular matrix comprises the following steps: performing vacuum lyophilization to the porcine arterial blood vessels to obtain the best pore by adopting an optimal pre-freezing velocity; rehydrating the matrix with low-concentration detergent and performing acellular treatment; and lyophilizing the matrix and storing at normal temperature. The matrix has the advantages that the porcine aorta is used as a studying object, and a lyophilized acellular blood vessel with proper porosity, mechanical performance and immunogenicity is produced by virtue of optimized preparation conditions; animal in-vivo experiments prove the practicality of three-dimensional blood vessel clinical vascular transplantation, and experimental basis is provided to further clinical application; the preparation method has extremely low cost, so that precious medical resources reach billions of RMB can be saved assuredly; and experimental basis and guidance are provided to clinical application of tissue engineering arterial blood vessels and optimizing research and development of acellular blood vessels.
Description
Technical field
The present invention relates to field of tissue engineering technology, specifically, is a kind of porcine aorta vacuum freeze-drying acellular matrix and its preparation method and application.
Background technology
Annual newborn 150,000 the children with congenital heart disease of China.And in congenital heart disease operation, need a large amount of blood vessels or biological sticking patch substitute.Preresearch estimates only its prospect of domestic medical market is about more than one hundred million units every year.And clinical conventional artificial blood vessel and freezing preservation blood vessel of the same race are without growth property and easily cause narrow calcification pogoniasis operative treatment again, when bringing greatest misery to patient, cause the waste of a large amount of medical resources.And conventional artificial blood vessel or biological sticking patch mostly be import, price is extremely expensive.Since the eighties in 20th century, along with the development of biomedical engineering technology, various generation vascular graft be used in cardiovascular disease surgical intervention.People are finding the best substitute of blood vessel for a long time always, comprise artificial material, biomaterial and composite etc., but there is repulsion, thrombosis, endotheli ocytosis, break, the shortcoming such as calcification, its therapeutic effect is still very limited.Old friend's work still can not replace status and the effect of people's freezing preservation blood vessel of the same race completely for blood vessel graft.
Chinese patent literature CN101327338A discloses a kind of bladder acellular matrix and preparation method of remaining with bioactie agent of porous, get the bladder of pig, rabbit, Canis familiaris L. or cattle, through Digestive system, hypotonic buffer liquid, containing the high osmotic buffer of surfactant, containing the buffer of nuclease and deionized water etc., process after, then obtain aseptic bladder acellular matrix after freezing, lyophilizing and sterilizing.Chinese patent literature CN101474426A discloses a kind of vascular stroma that removes vascular tissue's inner cell and preparation method thereof; a kind of structural intergrity that can remove the cell component in vascular tissue and at utmost protect acellular matrix is provided, has comprised de-cell, remove detergent and carry out enzyme processing.Chinese patent literature CN2860403Y discloses a kind of biological type artificial blood vessel, and by be cross-linked the formed base material of animal blood vessels fixing and that go antigen to process through fixative, and the surface layer that contains anticoagulation component being bonded on base material inner surface forms.But yet there are no report about a kind of porcine aorta vacuum freeze-drying acellular matrix and its preparation method and application.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of porcine aorta vacuum freeze-drying acellular matrix is provided.
One object more of the present invention is that a kind of preparation method of porcine aorta vacuum freeze-drying acellular matrix is provided.
Another object of the present invention is that a kind of application of porcine aorta vacuum freeze-drying acellular matrix is provided.
For achieving the above object, the technical scheme that the present invention takes is: a kind of porcine aorta vacuum freeze-drying acellular matrix, the preparation method of described porcine aorta vacuum freeze-drying acellular matrix comprises the following steps: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: 0.25% trypsin PBS solution effects for lyophilizing blood vessel, then under 0.5%Tritox X-100 PBS solution room temperature, to vibrate, the vibration of PBS solution is rinsed; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
The preparation method of described porcine aorta vacuum freeze-drying acellular matrix comprises the following steps: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: lyophilizing blood vessel uses the 0.25% trypsin PBS solution that contains EDTA 0.04% at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
For realizing above-mentioned second object, the technical scheme that the present invention takes is: a kind of preparation method of porcine aorta vacuum freeze-drying acellular matrix, the preparation method of described porcine aorta vacuum freeze-drying acellular matrix is as follows: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: 0.25% trypsin PBS solution effects for lyophilizing blood vessel, then under 0.5%Tritox X-100 PBS solution room temperature, to vibrate, the vibration of PBS solution is rinsed; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
Described preparation method comprises the following steps: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: lyophilizing blood vessel uses the 0.25% trypsin PBS solution that contains EDTA 0.04% at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
For realizing above-mentioned the 3rd object, the technical scheme that the present invention takes is: the application of described porcine aorta vacuum freeze-drying acellular matrix in the biological blood vessel of preparation.
The invention has the advantages that:
1, the present invention is usingd porcine aorta as object of study, by optimizing preparation condition, make there is suitable porosity, the de-cell blood vessel of mechanical property and immunogenic lyophilizing, by animal, at body, experimental results show that the three-dimensional feasibility that builds the clinical blood vessel transplantation of blood vessel, for the clinical experimental basis that provides is further provided;
2, preparation method cost of the present invention is extremely cheap, can save undoubtedly the valuable medical resource of several hundred million units;
3, for the clinical practice of organizational project aorta vessel and the optimization of de-cell blood vessel research and development provide experimental basis and pointer.
Accompanying drawing explanation
Temperature variation curve when accompanying drawing 1 is blood vessel pre-freeze.
Accompanying drawing 2 is different rate of temperature fall hematochezia pipe analysis of porosity.
Accompanying drawing 3 is blood vessel gray value analyses under different rate of temperature fall.
Accompanying drawing 4 is blood vessel α value change curves under different pre-freeze speed.
Accompanying drawing 5 is immunogenic variations before and after the lyophilizing of porcine aorta blood vessel.
Accompanying drawing 6 is that the young pig of lyophilizing rehydration blood vessel is in body transplantation experiments.
Accompanying drawing 7 is that lyophilizing pretreatment improves the de-cell effect of porcine aorta blood vessel.
Accompanying drawing 8 is that the pretreated de-cell porcine aorta blood vessel Electronic Speculum of lyophilizing detects photo.
The specific embodiment
Below in conjunction with accompanying drawing, the specific embodiment provided by the invention is elaborated.
embodiment 1
1. the experiment measuring of blood vessel pre-freeze minimum temperature
The exploration that technique is preserved in blood vessel lyophilization is to be prepared research key issue urgently to be resolved hurrily.The present invention adopts lift Control Program for Microcomputers [Digital Research] cooling instrument to lower the temperature with fixed rate to blood vessel, be mainly in order to investigate the impact of different pre-freeze speed on blood vessel lyophilizing Storing quality, be intended to explore the process control parameter and the flow process that obtain an applicable blood vessel lyophilizing preservation.
Object is the minimum temperature that can reach during pre-freeze in freeze dryer in order to measure blood vessel, and this temperature measuring is exactly the final temperature arranging when using Control Program for Microcomputers [Digital Research] cooling instrument to blood vessel pre-freeze.Minimum by the temperature of testing known dry run medium vessels is-42.9 ℃, sees Fig. 1, temperature variation curve when Fig. 1 is blood vessel pre-freeze.
2. different pre-freeze speed hematochezia pipe porositys, grey scale change figure.
See Fig. 2 and Fig. 3, Fig. 2, Fig. 3 are respectively porosity, the grey scale change figure of whole freeze-drying process under different pre-freeze speed.Pre-freeze speed is 0.5K/min, 1K/min, and before and after the blood vessel lyophilizing of 2K/min, the rate of change of porosity is respectively 11.9%, 16.6%, 29.7%; The rate of change of average gray value is respectively 2.27%, 3.64%, and 6.36%.Rate of temperature fall is larger, changes more obviously.
3. the impact of different pre-freeze speed on vascular mechanics performance.
The mensuration of vascular mechanics performance.Arteries is used matter structure instrument to carry out tension test to it, and the square of getting width and be 7mm carries out puncture test.In test, the load of matter structure instrument adopts the power of 500N, with the speed of 1mm/min, sample is punctured and is stretched.And the blood vessel after vacuum lyophilization is soaked 2 hours in normal saline, after fully rehydration drains, the arteries of getting the same terms carries out tension test and puncture test.The variation of the pre-freeze speed lyophilization front and back vascular mechanics performance that relative analysis is different.
See Fig. 4, Fig. 4 is the α value change curve of blood vessel under different pre-freeze speed.Puncture while being 1K/min stress value and circumferential tension stress value increasing degree of pre-freeze speed is respectively 20.10% and 32.03%, and axial tensile stress reduces amplitude minimum, is-19.84%.After analysis, think that pre-freeze speed has best hole, porosity and mechanical property performance while being 1K/min.
Result shows the rate of temperature fall when controlling lyophilization, can control pore size and the porosity of internal blood vessel, also can keep the good mechanical property of blood vessel simultaneously.
4. immunogenic variation before and after the lyophilizing of porcine aorta blood vessel.
See Fig. 5, Fig. 5 is immunogenic variation before and after the lyophilizing of porcine aorta blood vessel.Indirect immunofluorescence detects respectively the variation of MHC I in lyophilizing blood vessel and fresh blood vessel, class Ⅱ antigens expression.A wherein: fresh pig aorta, before frozen dried, substantially see; B: the fresh blood vessel MHC of immuno-fluorescence assay class Ⅰ antigens situation; C: the fresh blood vessel MHC of immuno-fluorescence assay class Ⅱ antigens situation; D: fresh pig aorta, after frozen dried, substantially see; E: MHC class Ⅰ antigens situation in immuno-fluorescence assay lyophilizing blood vessel; F: MHC class Ⅱ antigens situation in immuno-fluorescence assay lyophilizing blood vessel.After the lyophilizing of result demonstration blood vessel, MHC-I, class Ⅱ antigens level obviously decline.
5. after aortic graft operation and transplanting, regularly blood vessel intervention radiography is observed angiogenic growth situation
Under the quiet combined anesthesia of gas, the blood vessel that the de-cell of lyophilizing is processed is implanted young pig descending aorta place.Along left side, the 4th intercostal enters breast, ligation 1-2 root intercostal arteries, and blocking-up descending aorta proximal part and distal end, excise one section of descending aorta, implants the lyophilizing radiation rehydration arteries of 2cm left and right, sews up continuously two anastomotic stoma, aerofluxus, open aortic occlusion clamp.Hemostasis, rinses thoracic cavity, and drum lung, closes breast.The success of operation is the key of later stage research.After transplant operation, regularly repeatedly carry out blood vessel and get involved radiography observation angiogenic growth situation.
In order to verify whether the mechanical strength of the blood vessel after frozen dried can meet application requirements, we are by the blood vessel transplantation of lyophilizing in pig body, and Substitute For Partial aorta, found that pig can long-term surviving, shows frozen dried vasoinert application.Although confirm that the blood vessel mechanical property after frozen dried decreases but can bear long-term life test, the equal long-term surviving of young pig is more than 7 months.Although blood vessel immunogenicity declines to some extent but still has calcification trend after frozen dried, also to process and further reduce its immunogenicity in conjunction with acellular.See Fig. 6, Fig. 6 is that the young pig of lyophilizing rehydration blood vessel is in body transplantation experiments.A wherein: cryodesiccated porcine aorta is processed through rehydration again; B: the blood vessel transplantation repair deficiency of frozen dried; C: after the blood vessel transplantation after frozen dried, pig survival is good; D: in the time of three months, row conduit detects, and transplanting place still keeps unimpeded (arrow indication is grafting vessel place).
6. blood vessel carries out acellular by low concentration detergent rehydration after lyophilizing pretreatment simultaneously
0.25% trypsin PBS solution for lyophilizing blood vessel (containing EDTA 0.04%) is at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes.
Frozen dried increases blood vessel hole, when improving vascular permeability, also be more conducive to de-cell solution and enter into the de-cytosis of performance in tube wall, do not compare with the blood vessel not passing through in frozen village, the de-cell efficiency of blood vessel after frozen improves greatly, de-cell effect is ideal, even if the Cell Component of tube wall inside also can remove totally completely.Please refer to accompanying drawing 7, accompanying drawing 7 is that lyophilizing pretreatment improves the de-cell effect of porcine aorta blood vessel.A wherein: without HE dyeing before the de-cell of porcine aorta of frozen dried; B: HE dyeing before the de-cell of porcine aorta of frozen dried; C: without porcine aorta HE dyeing after de-cell of frozen dried, ductus arteriosus wall inside still remains part cell; D: through porcine aorta HE dyeing after de-cell of frozen dried, ductus arteriosus wall inside has no cell residue; E: without porcine aorta Hoechst dyeing after de-cell of frozen dried, ductus arteriosus wall inside still remains part cell; F: through porcine aorta Hoechst dyeing after de-cell of frozen dried, ductus arteriosus wall inside has no cell residue; G:DNA detection by quantitative shows that blood vessel takes off cell after frozen dried again, and residual DNA content obviously reduces.
7. blood vessel electromicroscopic photograph after fresh blood vessel, lyophilizing blood vessel and lyophilizing acellular
See Fig. 8, Fig. 8 is that the pretreated de-cell porcine aorta blood vessel Electronic Speculum of lyophilizing detects photo.A wherein: fresh blood vessel transmission electron microscope shows that a large amount of cells exist; B: after lyophilizing, transmission electron microscope shows still has cell to exist, but dead; C: lyophilizing blood vessel is through 0.25% trypsin treatment after 12 hours, and transmission electron microscope shows the acellular existence of internal blood vessel; D: fresh blood vessel scanning electron microscope shows that a large amount of cells exist; E: after lyophilizing, scanning electron microscope shows still has cell to exist, but dead; F: lyophilizing blood vessel through 0.25% trypsin treatment 12 as a child after, scanning electron microscope shows the acellular existence of internal blood vessel.
The demonstration of Electronic Speculum testing result, after lyophilizing, transmission electron microscope shows still has cell to exist, but dead; And lyophilizing blood vessel is through 0.25% trypsin treatment after 12 hours, transmission electron microscope and scanning electron microscope all show the acellular existence of internal blood vessel.
the preparation method of embodiment 2 porcine aorta vacuum freeze-drying acellular matrixes
1. lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃;
2. acellular: adopt low concentration detergent rehydration substrate simultaneously the de-cell of row process, 0.25% trypsin PBS solution lyophilizing blood vessel for (containing EDTA 0.04%) is at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes;
3. the condition that adopts step 1 is this substrate of vacuum freeze-drying again, and room temperature is preserved.
The present invention is usingd porcine aorta as object of study, and the solid of take structure blood vessel clinical transplantation carries out vacuum lyophilization as long-term goal to porcine aorta blood vessel and takes off cell lyophilizing the sequential rehydration processing of cell suspension again.By vacuum freeze-drying method for removing cells, process porcine aorta blood vessel, be intended to repair mechanism from the multiple research field such as heat and mass, low-temperature biological, cytology, molecular biology is inquired into de-cell arteritis matrix composition, preservation and transplanting.By optimizing preparation condition, make the lyophilizing blood vessel with suitable porosity and mechanical property, associating enzyme, chemical detergent further reduce the immunogenicity of blood vessel, in experiment, study Related Mechanism and the three-dimensional operating process building of the de-cell processes of blood vessel lyophilizing, feasibility by animal in the clinical blood vessel transplantation in the future of the three-dimensional structure of body experimentation blood vessel, for the clinical experimental basis that provides is further provided.Method cost is extremely cheap, can save undoubtedly the valuable medical resource of several hundred million units.
The outer three-dimensional formulation that builds biological blood vessel flow process of the de-cell porcine aorta vascular stroma preparation flow of lyophilizing and this matrix body, can be the clinical practice of organizational project aorta vessel and the optimization of de-cell blood vessel research and development provide experimental basis and pointer.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
Claims (3)
1. a porcine aorta vacuum freeze-drying acellular matrix, it is characterized in that, the preparation method of described porcine aorta vacuum freeze-drying acellular matrix comprises the following steps: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: lyophilizing blood vessel uses the 0.25% trypsin PBS solution that contains EDTA 0.04% at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
2. the preparation method of a porcine aorta vacuum freeze-drying acellular matrix, it is characterized in that, described preparation method comprises the following steps: (1) lyophilizing pretreatment: adopt pre-freeze speed 1K/min, lyophilizing pretreatment is carried out to porcine aorta blood vessel in final temperature-42.9 ℃; (2) acellular: lyophilizing blood vessel uses the 0.25% trypsin PBS solution that contains EDTA 0.04% at 37 ℃, 5%CO
2in incubator, act on 12 hours, then under 0.5%Tritox X-100 PBS solution room temperature, vibrate 4 hours, the vibration of PBS solution is rinsed 3 times, each 30 minutes; (3) condition that adopts step (1) is the substrate described in vacuum freeze-drying again, and room temperature is preserved.
3. the application of porcine aorta vacuum freeze-drying acellular matrix according to claim 1 in the biological blood vessel of preparation.
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| CN103416394A (en) * | 2012-11-01 | 2013-12-04 | 上海理工大学 | Method for drying and storage of blood vessels |
| CN109069263B (en) * | 2016-12-16 | 2021-01-29 | 厦门大开生物科技有限公司 | Porcine cornea acellular method, acellular cornea and lamellar dry cornea using method |
| CA3078625C (en) | 2017-10-09 | 2023-01-17 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN109078226A (en) * | 2018-08-07 | 2018-12-25 | 中国人民解放军第二军医大学 | A kind of de- cell porcine aorta matrix in micropore |
| CN108904884A (en) * | 2018-08-07 | 2018-11-30 | 中国人民解放军第二军医大学 | A kind of preparation method of porcine aorta acellular matrix |
| CN108949565A (en) * | 2018-09-26 | 2018-12-07 | 中国科学技术大学 | Device and method for red blood cell load freeze drying protectant |
| EP3937878A2 (en) | 2019-03-14 | 2022-01-19 | Terumo BCT Biotechnologies, LLC | Lyophilization loading tray assembly and system |
| CN111330080B (en) * | 2020-03-31 | 2021-12-07 | 江苏白衣缘生物工程有限公司 | Biomembrane for guiding regeneration of oral cavity bone and preparation method thereof |
| CN111330079B (en) * | 2020-03-31 | 2021-12-03 | 江苏白衣缘生物工程有限公司 | Artificial dura mater composite patch |
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| CN2860403Y (en) * | 2005-07-29 | 2007-01-24 | 广东冠昊生物科技有限公司 | Biotype artificial blood vessel |
| EP2031968B1 (en) * | 2006-04-21 | 2017-11-22 | Wake Forest University Health Sciences | Structurally modified acellular tissue engineering scaffolds and methods of production |
| EP2117462A1 (en) * | 2007-02-02 | 2009-11-18 | Manh-Dan Ngo | Treatment of soft tissue to increase porosity |
| CN101327338B (en) * | 2008-08-01 | 2012-07-25 | 南京大学医学院附属鼓楼医院 | Lacunaris bladder acellular matrix preserving biological activity factor and preparation |
| CN101385870B (en) * | 2008-11-03 | 2011-03-02 | 中国人民解放军第四军医大学 | Method for improving de-cellular system engineering valve/blood vessel stent |
| CN101474426B (en) * | 2009-01-14 | 2012-09-05 | 首都医科大学宣武医院 | Vascular matrix without cell in vascular tissue and preparation method thereof |
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