CN101766842A - Biological tissue material for artificial organs and preparation method thereof - Google Patents
Biological tissue material for artificial organs and preparation method thereof Download PDFInfo
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Abstract
本发明公开的生物组织材料是一种以猪主动脉血管为基体，以氧化海藻酸钠为交联剂制备的人工器官用生物组织材料，其制备的主要工序为：(1)将自屠宰场活杀猪体上取下的主动脉血管进行清洗，去除血迹和脂肪；(2)将清洗干净的猪主动脉血管用由缓冲液和氧化海藻酸钠交联剂配制成的交联液进行交联处理，交联体系的氧化海藻酸钠浓度为10～15g/100ml，pH值为4.0-7.4，温度为室温。经充分交联制备的人工器官用生物组织材料，既具有高分子材料富有韧性、弹性好的力学特征，又有适于周围细胞长入的天然结构，还有类似生物活性蛋白的促使细胞在其表面黏附生长的特点，预期应用于临床可作为各种软组织移植物替代材料。
The biological tissue material disclosed by the present invention is a biological tissue material for artificial organs prepared with porcine aortic blood vessels as the substrate and oxidized sodium alginate as the cross-linking agent. The main process of its preparation is: (1) The aortic blood vessel removed from the live slaughtered pig body is cleaned to remove blood and fat; (2) the cleaned pig aortic blood vessel is cross-linked with a cross-linking solution prepared by a buffer solution and an oxidized sodium alginate cross-linking agent. Linking treatment, the concentration of oxidized sodium alginate in the crosslinking system is 10-15g/100ml, the pH value is 4.0-7.4, and the temperature is room temperature. The biological tissue material for artificial organs prepared by fully cross-linking not only has the mechanical characteristics of toughness and good elasticity of polymer materials, but also has a natural structure suitable for the growth of surrounding cells, and has similar biologically active proteins to promote cells in its environment. Due to the characteristics of surface adhesion growth, it is expected to be used as a substitute material for various soft tissue grafts in clinical application.
Description
Technical field
The present invention relates to human organ and substitute artificial bio-membrane's organization material technology, more particularly, relate to a kind of human body soft tissue graft biological organization material technology, belong to technical field of biological material.
Technical background
Clinically need a large amount of soft tissue graft thing because of factors such as various diseases or traffic accident make every year, cause that such as the esophageal carcinoma organ defect needs artificial esophagus's graft, atherosclerosis causes that vascular lesion needs artificial blood vessel's graft, and the heart lobe infringement that rheumatic heart disease causes needs biological heart lobe graft.At present the soft tissue graft thing that uses is divided into biological and two kinds of synthetic.The biological material of macromolecular material synthetic has excellent mechanical property, but biocompatibility has much room for improvement, and therefore uses to be very limited.In biological material, protein between the structure of natural biological tissue source material and composition and biological tissue organ cell in the matter is similar with polysaccharide, strong with cellular affinity, the extracellular matrix support condition that can grow for cell, propagation, differentiation and function performance provides approximate in vivo tissue genesis and development, make the cell aggregation formative tissue, the control organizational structure is regulated cell phenotype, has better potential applicability in clinical practice.
But the biomaterial in natural tissues source promptly begins degraded after live body takes out, also exist group difference between different materials, should not preserve and directly use.Often need to adopt crosslinked pretreatment to solve these problems for this reason.Biological material after treatment can not only keep its good mechanical performance and complete organizational structure for a long time, but also has improved the antienzyme degradation capability, reduces even removed simultaneously the antigenicity of material again, has widened its range of application greatly.But existing cross-linking treatment method exists the inherent shortcoming of self, it is selected synthetic chemical cross-linking agent cytotoxicity height, biomaterial biocompatibility by its crosslinking Treatment is relatively poor, though and selected natural biological cross-linking agent cytotoxicity is low, biocompatibility is also better, but its price is very expensive, is difficult to practical application.
As a whole, in order to solve the defective of above-mentioned all kinds of cross-linking agent, the exploitation of still needing filters out the cross-linking agent material that can carry out desirable modification to biological tissue material for artificial organs, to prepare even more ideal biological tissue material for artificial organs.
Summary of the invention
The deficiency that exists at the biological tissue material for artificial organs of prior art, purpose of the present invention aims to provide a kind of macromolecular material synthetic biomaterial mechanical property that both had, and has again to be suitable for the natural structure that peripheral cell grows into and to impel cell at biological tissue material for artificial organs of the similar biological activity protein of its surface adhesion growth and preparation method thereof.
Can realize the biological tissue material for artificial organs of foregoing invention purpose, be the porcine aorta blood vessel of crossing with the biological cross-linking agent crosslinking Treatment of oxidized sodium alginate, in particular for the pig descending aorta blood vessel of crossing with the biological cross-linking agent crosslinking Treatment of oxidized sodium alginate.
As the porcine aorta blood vessel of crossing with the biological cross-linking agent crosslinking Treatment of oxidized sodium alginate of biological tissue material for artificial organs, its preparation method mainly comprises following manufacturing procedure:
(1) will clean removal bloodstain and fat by the aorta vessel that takes off on the pig body that kill alive from the slaughterhouse;
(2) it is that the crosslinked fluid that the oxidized sodium alginate of the buffer of 4.0-7.4 and adding is mixed with carries out crosslinking Treatment that the porcine aorta blood vessel that cleans up is used by pH value, the pH value of cross-linking system is 4.0-7.4, crosslinking temperature is a room temperature, the concentration of oxidized sodium alginate is 10～15g/100ml in the crosslinked fluid, through the full cross-linked biological tissue material for artificial organs that is prepared as.
In above-mentioned preparation scheme, taking from the bloodstain on the aorta vessel of pig live body and the cleaning of fat removes, preferably do not ooze out to there being blood and slime with normal saline flushing earlier, the back is the benzalkonium bromide aqueous solution sterilization degrease of 0.01%-0.05% with mass concentration, and benzalkonium bromide is removed in reuse phosphate buffer flushing afterwards.The benzalkonium bromide aqueous solution can add clear water by commercially available benzalkonium bromide solution (mass concentration 5%) and be mixed with.
In above-mentioned preparation scheme, the buffer of preparation oxidized sodium alginate crosslinked fluid is citric acid/sodium citrate buffer or phosphate buffer, and wherein the citric acid/sodium citrate buffer can press ratio of quality and the number of copies by the sodium citrate solution of the citric acid solution of 0.1M and 0.1M and 1: 1.8～2.0 prepares; Phosphate buffer can be by the NaCl of 0.14～0.28M, the KCl of 0.003～0.006M, the Na of 0.01～0.02M
2HPO
4KH with 0.0016～0.0032M
2PO
4Preparation.
In above-mentioned preparation scheme, the consumption of the crosslinked fluid during the effective oxidized sodium alginate crosslinked fluid of aortic blood crosslinking Treatment generally is controlled to be every 1cm
2Tissue handle with the 1ml-5ml crosslinked fluid.
In above-mentioned preparation scheme, the crosslinked of aorta vessel and oxidized sodium alginate crosslinked fluid preferably carries out crosslinking Treatment having under the condition of slight vibration, so that it is more abundant and even that crosslinked fluid can contact with the tissue that is crosslinked, normally in shaking table, carry out crosslinking Treatment.
In above-mentioned preparation scheme, the porcine aorta blood vessel is fetched into the time of carrying out crosslinking Treatment and generally is controlled to be and is not more than 6h from live to kill the pig body.
After the porcine aorta blood vessel is prepared as biological tissue material for artificial organs with the oxidized sodium alginate crosslinked fluid is crosslinked, need be stored in the alcoholic solution of 30%-50% (volume ratio of dehydrated alcohol and water is 1: 2～1: 1) standby.Material also must carry out the gamma-ray irradiation sterilization when using or carrying out experiment made on the living or cell in vitro related experiment.
In the research complete process of preparation biological tissue material for artificial organs, the inventor is through repeated tests with to the profound understanding of cross-linking agent performance, and selected oxidized sodium alginate cross-linking agent is as the cross-linking agent of porcine aorta blood vessel.Oxidized sodium alginate is the derivant of sodium alginate, sodium alginate is a kind of natural polysaccharide of separating from Brown algae, biocompatibility, no any immunogenicity with degradability and height, do not induce the inflammatory reaction of any persistence, simultaneously because its relative low price is widely used in organizational project and medicine and other fields.With sodium metaperiodate sodium alginate is carried out oxidation modification, the sodium alginate of appropriate oxidation has not only kept excellent biological compatibility, has increased its biological stability, has also introduced active group and (CHO), can react with aminoacid or protein.
Oxidized sodium alginate has advantages such as cytotoxicity is low, little to natural tissues destruction, cellular affinity is strong as a kind of natural biological cross-linking agent.With the biological organization material that oxidized sodium alginate was handled, antigenicity is removed more thorough, and biological stability is higher, and external antienzyme degradation capability is better, and ultimate tensile strength is better.Compare with other chemical cross-linking agents, the great advantage of oxidized sodium alginate is a low cytotoxicity, and price is also cheap relatively simultaneously.Cross-linking agent cytotoxicity by the MTT test determination shows that the oxidized sodium alginate cytotoxicity is significantly less than glutaraldehyde, also less than epoxy polymer.
The present invention is a cross-linking agent with biological cross-linking agent oxidized sodium alginate, with the porcine aorta is the prepared biological tissue material for artificial organs of matrix, both had macromolecular material and be rich in the mechanical characteristics of toughness, good springiness, have again and be suitable for the natural structure that peripheral cell is grown into, the characteristics of impelling cell to grow that also have similar biological activity protein at its surface adhesion, can reduce the defective that the remainder cross-linking agent exists greatly, be well suited for as the artificial organ material.And its preparation process is simple, and specification requirement is not high, easily realizes industrialization.
Description of drawings
Accompanying drawing 1 goes cell situation (HE dyeing is amplified 100 times) for crosslinked front and back material.1-1 fresh sample wherein; 1-2 is for being 4.0 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 7 days situation under 15% the condition; 1-3 is for being 7.4 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 3 days situation under 15% the condition.
Accompanying drawing 2 is the situation of change (the Mallory trichrome stain amplifies 100 times) of the collagen fiber of crosslinked front and back material.The 2-1 fresh sample; 2-2 is for being 4.0 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 3 days situation under 15% the condition; 2-3 is for being 7.4 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 3 days situation under 15% the condition.
Accompanying drawing 3 is the situation of change (Verhoeff Garapa uniformly dyeing color method amplifies 100 times) of the elastic fiber of crosslinked front and back material.The 3-1 fresh sample; 3-2 is for being 4.0 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 3 days situation under 15% the condition; 3-3 is for being 7.4 with oxidized sodium alginate at PH, and crosslinked fluid concentration is crosslinked 3 days situation under 15% the condition.
The specific embodiment
1, biological tissue material for artificial organs prepares embodiment
Describe the specific embodiment of the present invention by the following examples, content of the present invention is described in further detail again.But of particular note, this should be interpreted as protection scope of the present invention only limits to example, be on the contrary, allly should all belong to protection scope of the present invention based on the technology that content of the present invention realized.
In following each embodiment, except that specifying, related percentage ratio is mass percent, and related umber is mass fraction.
Embodiment 1
Take out the thoracic aorta blood vessel of pig extremely of living from the slaughterhouse, put into brine ice and preserve, transfer to laboratory rapidly.Remove the fatty tissue and the reticular connective tissue of surface attachment.Wash repeatedly with a large amount of normal saline earlier, clean bloodstain and fat, till no blood and slime oozes out.Be about 0.03% benzalkonium bromide aqueous solution sterilization degrease about 45 minutes with mass concentration afterwards, the reuse phosphate buffer washes repeatedly removes clean benzalkonium bromide.The benzalkonium bromide aqueous solution is that 5% benzalkonium bromide solution adds clear water and is mixed with by commercially available mass concentration.Phosphate buffer is by the NaCl of 0.14M, the KCl of 0.003M, the Na of 0.01M
2HPO
4KH with 0.0016M
2PO
4Preparation.
Porcine aorta blood vessel of rinsing well and crosslinked fluid place under the condition of vibration gently, carry out in the shaking table crosslinked so that crosslinked fluid be crosslinked organize abundant more with contact equably.Take out aorta vessel to carrying out crosslinking Treatment from the pig live body, the time control between this is no more than 6 hours.Crosslinked condition is: crosslinking temperature is a room temperature; The pH value of cross-linking system is about 4.0; Crosslinked fluid is that about 4.0 buffer adds the oxidized sodium alginate cross-linking agent and is mixed with by pH value, and concentration is the oxidized sodium alginate that contains in the 100ml crosslinked fluid about 15g; Crosslinking time is 3 days.Buffer in crosslinked fluid preparation: prepare the citric acid solution of 0.1M and the sodium citrate solution of 0.1M respectively, get 1 part of sodium citrate solution and add about 1.9 parts citric acid solution again and be mixed with the citric acid/sodium citrate buffer.The consumption of crosslinked fluid is every 1cm
2Tissue handle with about 1ml-5ml crosslinked fluid.
With the porcine aorta blood vessel is that matrix is the artificial esophagus biological organization material of cross-linking agent with the oxidized sodium alginate, is stored in volume ratio after crosslinked being prepared into and is in the alcoholic solution about 30% (volume ratio of dehydrated alcohol and water is 1: 2) standby.When cross-linked material will carry out experiment made on the living or cell in vitro related experiment, must carry out the gamma-ray irradiation sterilization.
The crosslinked cross-linking agent that both can save costliness can reach ideal cross-linking effect again under this condition.
Embodiment 2
What present embodiment prepared is matrix with the porcine aorta blood vessel, with the oxidized sodium alginate is the biological tissue material for artificial organs of cross-linking agent, its preparation process is substantially the same manner as Example 1, different porcine aorta blood vessel kill the descending aorta blood vessel of pig for living, the mass concentration of degreasant benzalkonium bromide aqueous solution of being used to sterilize is about 0.05%, the pH value of cross-linking system is about 4.0, the pH value of crosslinked fluid is about 4.0, and the oxidized sodium alginate concentration in the crosslinked fluid contains oxidized sodium alginate about 10g for the 100ml crosslinked fluid; Crosslinking time is 3 days.The preparation of buffer: the first citric acid solution of preparation 0.1M, prepare the sodium citrate solution of 0.1M then, get 1 part of sodium citrate solution and add about 2.1 parts of citric acid solutions again and be mixed with the citric acid/sodium citrate buffer.The consumption of crosslinked fluid is every 1cm
2Tissue handle with the 1ml-5ml crosslinked fluid.
It is standby in the alcoholic solution about 50% (volume ratio of dehydrated alcohol and water is 1: 1) that the crosslinked cross-linked material that is prepared into is stored in volume ratio.When carrying out experiment made on the living or cell in vitro related experiment, cross-linked material also must carry out the gamma-ray irradiation sterilization.
Embodiment 3
What present embodiment prepared is matrix with the porcine aorta blood vessel, with the oxidized sodium alginate is the biological tissue material for artificial organs of cross-linking agent, its preparation process is substantially the same manner as Example 1, different porcine aorta blood vessel kill the descending aorta blood vessel of pig for living, the mass concentration of degreasant benzalkonium bromide aqueous solution of being used to sterilize is about 0.04%, the pH value of cross-linking system is about 7.4, the pH value of crosslinked fluid is about 7.4, and the oxidized sodium alginate concentration in the crosslinked fluid contains oxidized sodium alginate about 15g for the 100ml crosslinked fluid; Crosslinking time is 3 days.The preparation of buffer: the NaCl of preparation 0.14M in a distilled water, the KCl of 0.003M, the Na of 0.01M
2HPO
4And the KH of 0.0016M
2PO
4, reuse HCl regulates PH to 7.4.The consumption of crosslinked fluid is every 1cm
2Tissue handle with the 1ml-5ml crosslinked fluid.
It is standby in the alcoholic solution about 50% (volume ratio of dehydrated alcohol and water is 1: 1) that the crosslinked cross-linked material that is prepared into is stored in volume ratio.When carrying out experiment made on the living or cell in vitro related experiment, cross-linked material also must carry out the gamma-ray irradiation sterilization.
Embodiment 4
What present embodiment prepared is matrix with the porcine aorta blood vessel, with the oxidized sodium alginate is the biological tissue material for artificial organs of cross-linking agent, its preparation process is substantially the same manner as Example 1, different porcine aorta blood vessel kill the descending aorta blood vessel of pig for living, the mass concentration of degreasant benzalkonium bromide aqueous solution of being used to sterilize is about 0.05%, the pH value of cross-linking system is about 7.4, the pH value of crosslinked fluid is about 7.4, and the oxidized sodium alginate concentration in the crosslinked fluid contains oxidized sodium alginate about 10g for the 100ml crosslinked fluid; Crosslinking time is 3 days.The preparation of buffer: the NaCl of preparation 0.14M in a distilled water, the KCl of 0.003M, the Na of 0.01M
2HPO
4And the KH of 0.0016M
2PO
4, reuse HCl regulates PH to 7.4.The consumption of crosslinked fluid is every 1cm
2Tissue handle with the 1ml-5ml crosslinked fluid.
It is standby in the alcoholic solution about 50% (volume ratio of dehydrated alcohol and water is 1: 1) that the crosslinked cross-linked material that is prepared into is stored in volume ratio.When carrying out experiment made on the living or cell in vitro related experiment, cross-linked material also must carry out the gamma-ray irradiation sterilization.
2, biological tissue material for artificial organs testing performance index embodiment
(1) degree of cross linking test implementation example
The degree of cross linking is an important indicator of reflection crosslinking degree, and it is represented by the ratio of the free amino group content in the free amino group content in the crosslinked back tissue and the uncrosslinked tissue.Because the crosslinked of oxidized sodium alginate will consume free amino groups a large amount of in the tissue, so the degree of cross linking is big more, free amino group residual in the tissue is just few more.The degree of cross linking that above-mentioned biological tissue material for artificial organs is prepared the cross-linked material of embodiment preparation is measured, and adopts ninhydrin method to carry out.
The degree of cross linking of cross-linked material under the table 1-different condition
As can be seen from the above table, in the identical time,, under concentration 15% condition, crosslinked the fastest at pH4.0.Four kinds of crosslinked conditions all can reach the higher degree of cross linking after 3 days.The high more illustrative material of the degree of cross linking is crosslinked good more, and the free amino group in the biological tissue is removed thorough more, and the antigenicity of biological tissue is removed also thorough more, and it is weak more that material is implanted the immunological rejection that produces behind the live body.
(2) Mechanics Performance Testing embodiment
As the bioengineered tissue material that artificial organ is used, the mechanical property of tissue is a crucial index.The mechanical property that above-mentioned biological tissue material for artificial organs is prepared the cross-linked material of embodiment preparation is measured, and adopts the 302instron universal material experimental machine of INSTRON company to measure.
The mechanical property of cross-linked material under the table 2-different condition
We can see from top data, more crosslinked mechanical properties that can improve material, and modulus wherein, maximum tensional stress all is significantly improved, and this is wherein with at PH7.4, the sample increase rate maximum of handling under the condition of crosslinked fluid concentration 15%.And, reduce to some extent in the strain at maximum stress place.Tissue after this explanation is crosslinked shows slightly stiff, and the elasticity of fresh tissue is better, and soft.
(3) cytotoxicity test implementation example:
Reach ideal clinical effectiveness, also must make material that good cell compatibility is arranged.Above-mentioned biological tissue material for artificial organs being prepared the Cytotoxic measurement of cross-linked material that embodiment prepares, adopt the tetrazolium salts colorimetry, serves as to detect cell with the L-929 cell, and crosslinked material is carried out cell toxicity test.The result is as follows:
The cytotoxicity of cross-linked material under the table 3-different condition
Annotate: condition 1 is pH4.0, concentration 15%; Condition 2 is pH4.0, concentration 10%; Condition 3 is pH7.4, concentration 15%; Condition 4 is pH7.4, concentration 10%.
Cytotoxicity is classified as, and 0～1 grade is qualified, so the cell compatibility of this cross-linked material is qualified.
3, biological tissue material for artificial organs Micro-Structure Analysis embodiment
Go cell situation, collagen fiber situation of change, elastic fiber situation of change etc. also very important after biological organization material is crosslinked to cross-linked material.The variation of these situations can obtain by the light microscopic photo relative analysis to the crosslinked front and back of bioengineered tissue material.
(1) crosslinked front and back material removes cell situation Micro-Structure Analysis embodiment
The crosslinked back of porcine aorta blood vessel material goes the cell situation to see the light microscopic photo shown in accompanying drawing 1-1,1-2, the 1-3.Can find out that from the light microscopic photo structure of flesh tissue is very complete, have much to have the nuclear cell of purple.At condition PH is 7.4, and crosslinked fluid concentration is that crosslinked three days sample has been removed the part cell in 15% the crosslinked fluid.And be 4.0 at condition PH, crosslinked fluid concentration is in 15% the crosslinked fluid, and crosslinked three days sample, the cell component of the overwhelming majority have been removed, and the removal effect of pair cell is more obvious.Cell is that tissue produces one of antigenic main source, and its removal is thorough more, and the immunogenicity that crosslinked tissue produces is just more little.
(2) the collagen fiber situation of change Micro-Structure Analysis embodiment of crosslinked front and back material
The collagen fiber situation of change of the crosslinked back of porcine aorta blood vessel material is seen the light microscopic photo shown in accompanying drawing 2-1,2-2, the 2-3.Section adopts the Mallory trichrome staining to make.Can find out that from the light microscopic photo collagen fiber of fresh sample are very complete.And under four conditions the crosslinked cross-linked material that obtained in three days in the crosslinked fluid, it is all very complete that collagen fiber are preserved, and is not damaged.In addition, also can find out at PH to be crosslinked under 4.0 the condition, its effect of removing cell is stronger.
(3) the elastic fiber situation of change Micro-Structure Analysis embodiment of crosslinked front and back material
The elastic fiber situation of change of the crosslinked back of porcine aorta blood vessel material is seen the light microscopic photo shown in accompanying drawing 3-1,3-2, the 3-3.Section adopts Verhoeff Garapa uniformly dyeing color method to make.Can find out that from the light microscopic photo elastic fibers of fresh sample is very complete.And in four condition crosslinked fluids crosslinked three days sample, it is all very complete that elastic fibers is preserved.
For by the biological organization material handled, we wish under the situation of not destroying collagen fiber and elastic fibers microstructure strongly, and some can produce immunoreactive bioactive substance to remove cell etc.Can find out that from the situation of top several figure the situation in cell place to go is better in the tissue after crosslinked, and microstructure preserves better, collagen fiber and elastic fibers all do not have destroyed.
Claims (10)
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| CN106310386A (en) * | 2016-09-30 | 2017-01-11 | 四川大学 | Biological tissue material for artificial organs, and preparation method of biological tissue material |
| CN115814162A (en) * | 2023-02-06 | 2023-03-21 | 四川大学 | Functional biological heart valve material and preparation method and application thereof |
| CN116173302A (en) * | 2023-02-06 | 2023-05-30 | 四川大学 | Non-glutaraldehyde crosslinked heart valve and preparation method and application thereof |
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| CN116173302A (en) * | 2023-02-06 | 2023-05-30 | 四川大学 | Non-glutaraldehyde crosslinked heart valve and preparation method and application thereof |
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| CN101766842B (en) | 2013-11-06 |
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