CN110917399A - Anti-calcification method for biomaterial and biomaterial - Google Patents
Anti-calcification method for biomaterial and biomaterial Download PDFInfo
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Abstract
The application discloses a method for preventing calcification of a biomaterial and the biomaterial, wherein the method for preventing calcification of the biomaterial comprises the following steps: under the preset condition, amine compounds are used for carrying out anti-calcification treatment on the biological material, and the biological material after the anti-calcification treatment is stored in a dry state. The amino group of the amine compound can react with the aldehyde group which can initiate the calcification of the biological material, so that the aldehyde group is sealed, the fact that the residual aldehyde group can initiate the calcification of the biological material is avoided, the amine compound is used for carrying out anti-calcification treatment on the biological material, the negative influence brought by the aldehyde compound is reduced to the minimum, and therefore the amine compound has strong anti-calcification performance. Meanwhile, the biological material subjected to anti-calcification treatment is stored in a dry form, the biological material is stored in the dry form, the requirement on the storage condition is lower, the storage time is longer, and the biological material has the capability of tolerating adverse environment. Meanwhile, the biological material stored in a dry form can be directly used after being unsealed and completely packaged without rinsing and assembling, so that the application is more convenient, and the rescue time is saved.
Description
Technical Field
The application belongs to the technical field of biological material treatment, and particularly relates to an anti-calcification method of a biological material and the biological material.
Background
The biological material is a natural or artificial special functional material which is used for contacting and interacting with a living system and can carry out diagnosis, treatment, replacement, repair or induced regeneration on cells, tissues and organs of the biological material, and is also called as a biomedical material. The biological material comprises artificial synthetic material and natural material; there are single materials, composite materials, and hybrid materials that are a combination of living cells or natural tissues and inanimate materials. Biomaterials are not drugs per se, and their therapeutic approaches are essentially characterized by direct binding and interaction with the living organism.
The aldehyde compounds are used as the most common biological material tanning agents, and the safety and the reliability are proved by time. However, after the biological material tanned by the aldehyde compound is implanted into a human body, the aldehyde functional group generated by decomposition or incomplete reaction of the biological material or the residual aldehyde compound in the material is easy to become a calcified central site, and the calcification phenomenon is generated to cause the material to lose the original function. The biological material treated by the aldehyde compound is easy to calcify and decay, and is the key to be solved urgently.
Disclosure of Invention
The application provides an anti-calcification method of a biological material and the biological material, which aim to solve the technical problem that the biological material treated by aldehyde compounds is easy to calcify and decay.
In order to solve the technical problem, the application adopts a technical scheme that: an anti-calcification method for biological material, under the preset condition, using amine compound to carry out anti-calcification treatment on the biological material, and storing the biological material after anti-calcification treatment in a dry state.
According to one embodiment of the present application, the biomaterial is cross-linked with an aldehyde compound.
According to an embodiment of the present application, the amine compound is one or more of diamine compound, alcohol amine compound and/or imine compound.
According to an embodiment of the application, the amine compound comprises one or more of ethylenediamine, ethanolamine, hexamethylenediamine, octamethylenediamine, carbodiimide, lysine, arginine, asparagine or glutamine.
According to an embodiment of the present application, said anticalcification treatment of said biological material with an amine compound comprises: diluting the amine compound with a buffer solution to obtain an amine compound solution, and soaking the biological material with the amine compound solution.
According to an embodiment of the present application, the concentration of the amine compound in the amine compound solution is 0.01% to 10%.
According to an embodiment of the present application, the preset conditions include a soaking time and a treatment temperature for soaking the biological material in the amine compound solution; wherein the soaking time is 2-72 hours, and the treatment temperature is 4-58 ℃.
According to an embodiment of the present application, the preserving the biological material in a dry state after the anti-calcification treatment comprises: and (3) freeze-drying, glycerating and dehydrating and/or performing gradient dehydration on the biological material subjected to the anti-calcification treatment, and storing the biological material in a dry state.
According to an embodiment of the application, the biomaterial comprises porcine aortic valve, porcine/bovine pericardium, bovine muscle/achilles tendon, porcine bladder, porcine/bovine dermal matrix, porcine small intestine submucosa matrix, porcine/bovine peritoneum, blood vessels, xenogenic or xenogenic bone, porcine eye cornea or tympanic membrane.
In order to solve the above technical problem, another technical solution adopted by the present application is: a biomaterial which has been anticalcification treated by any of the methods described above.
The beneficial effect of this application is: according to the calcification-resisting method for the biological material, the amine compound is used for carrying out calcification-resisting treatment on the biological material, the amino group of the amine compound can react with the aldehyde group for triggering calcification of the biological material, so that the aldehyde group is sealed, the fact that the residual aldehyde group triggers calcification of the biological material is avoided, the amine compound is used for carrying out calcification-resisting treatment on the biological material, the negative influence brought by the aldehyde compound is reduced to the minimum, and therefore the calcification-resisting method has strong calcification-resisting performance. Meanwhile, the biological material subjected to anti-calcification treatment is stored in a dry form, the biological material is stored in the dry form, the requirement on the storage condition is lower, the storage time is longer, and the biological material has the capability of tolerating adverse environment. Meanwhile, the biological material stored in a dry form can be directly used after being unsealed and completely packaged without rinsing and assembling, so that the application is more convenient, and the rescue time is saved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a schematic flow diagram of an embodiment of a method of anti-calcification of a biological material of the present application;
FIG. 2 is a graph showing the results of calcium content measurement of a biomaterial according to an embodiment of the present application, which is treated by an anti-calcification method, after subcutaneous implantation in a mouse for 8 weeks;
FIG. 3 is a graph showing the results of measuring the phosphorus content of the biomaterial of the embodiment treated by the anti-calcification method of the present application after the biomaterial is subcutaneously implanted in a mouse for 8 weeks;
fig. 4 is a schematic flow chart of another embodiment of the method for anti-calcification of a biological material of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Referring to fig. 1, fig. 1 is a schematic flow chart of an embodiment of a method for anti-calcification of a biomaterial according to the present application.
An embodiment of the present application provides a method for anti-calcification of a biomaterial, the method including:
s101: under the preset condition, amine compounds are used for carrying out anti-calcification treatment on the biological material, and the biological material after the anti-calcification treatment is stored in a dry state.
In one embodiment, the biomaterial comprises a porcine aortic valve, porcine/bovine pericardium, bovine muscle/achilles tendon, porcine bladder, porcine/bovine dermal matrix, porcine small intestine submucosa matrix, porcine/bovine peritoneum, blood vessels, xenogenic or xenogenic bone, porcine eye cornea, or tympanic membrane.
In one embodiment, the biomaterial is cross-linked with an aldehyde compound before being subjected to an anti-calcification treatment. The aldehyde compounds include dialdehyde compounds such as glyoxal, and monoaldehyde compounds such as formaldehyde and acetaldehyde. The biological material after the aldehyde compound crosslinking treatment is easy to have residual aldehyde functional groups and initiate calcification. The amino group of the amine compound can react with the aldehyde group which can initiate the calcification of the biological material, so that the aldehyde group is sealed, the fact that the residual aldehyde group can initiate the calcification of the biological material is avoided, the amine compound is used for carrying out anti-calcification treatment on the biological material, the negative influence brought by the aldehyde compound is reduced to the minimum, and therefore the amine compound has strong anti-calcification performance.
In one embodiment, the amine compound is one or more of diamine compound, alcohol amine compound and/or imine compound, wherein the diamine compound has two amino groups, so that the diamine compound can not only react with residual aldehyde groups, but also react with one aldehyde group by using two amino groups, thereby enhancing the crosslinking effect.
Further, the amine compound includes one or more of ethylenediamine, ethanolamine, hexamethylenediamine, octamethylenediamine, carbodiimide, lysine, arginine, asparagine, or glutamine.
In one embodiment, the predetermined conditions include a soaking time and a treatment temperature for soaking the biological material in the amine compound solution. The soaking time is 2-72 hours, specifically, the soaking time can be 2 hours, 20 hours, 37 hours, 50 hours or 72 hours, and is not limited herein; the treatment temperature is 4 ℃ to 58 ℃, the treatment temperature is 4 ℃, 15 ℃, 31 ℃, 45 ℃ or 58 ℃, and the treatment temperature is not limited herein.
In one embodiment, the anti-calcification treatment of the biological material with an amine compound comprises: diluting the amine compound with a buffer solution to obtain an amine compound solution, and soaking the biological material with the amine compound solution.
The concentration of the amine compound in the amine compound solution is 0.01% to 10%, and specifically, the concentration of the amine compound may be 0.01%, 3%, 5%, 8%, and 10%, which is not limited herein.
Wherein the buffer system comprises PBS (phosphate buffered saline solution) and the acid-base condition is pH 7.4; MES (2 morpholinoethanesulfonic acid) at pH 5.5 under acid-base conditions; HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) at pH 7.5 under acid-base conditions; CBS (carbonate buffer solution) with an acid-base condition of pH 10.3; absolute ethyl alcohol; normal saline and ethanol/normal saline mixed solution.
The anti-calcification treatment of biological material with an amine compound further comprises: the inactivation solution is vibrated, ultrasonically treated or stirred, so that the flowing speed of the amine compound solution can be increased, and the anti-calcification treatment on the biological material can be accelerated.
In one embodiment, storing the anti-calcification treated biological material in a dry form comprises: the biological material after the anti-calcification treatment is stored in a dry state after freeze drying, glycerolysis dehydration and/or ethanol gradient dehydration. The biological material is stored in a dry state, has lower requirement on the storage condition, has longer storage time and has the capability of tolerating adverse environment. Meanwhile, the biological material stored in a dry form can be directly used after being unsealed and completely packaged without rinsing and assembling, so that the application is more convenient, and the rescue time is saved.
Wherein the glycerolysis dehydration comprises soaking the biological material in a mixed solution of high-concentration glycerol (80% -95%) and a small amount of absolute ethyl alcohol to absorb water in the biological material; the ethanol gradient dehydration comprises soaking the biological material in 75%/80% ethanol solution (ethanol mixed with normal saline) for 3-30 min, soaking in 95% ethanol solution (ethanol mixed with normal saline) for 3-30 min, and soaking in anhydrous ethanol for 3-30 min to achieve dehydration effect on the biological material; freeze-drying includes prefreezing, annealing, and resolution drying.
The following will be further explained in connection with two embodiments:
example 1:
1. anti-calcification treatment:
the amine compound is ethylenediamine, the buffer solution is PBS solution, 1.000g of ethylenediamine is weighed, 1.600g of sodium chloride, 0.040g of potassium chloride, 0.048g of monopotassium phosphate and 0.288g of disodium hydrogen phosphate are weighed, poured into a container, 200ml of water for injection is added, and the solution is dissolved and the pH value is adjusted to 7.4.
And (3) taking one pig-origin aortic valve which is crosslinked by glutaraldehyde, putting the pig-origin aortic valve into the prepared solution, and oscillating the pig-origin aortic valve for 2 hours by using a shaker at the temperature of 37 ℃ and the oscillating speed of the shaker of 100 rpm.
2. Drying:
and (3) shaking and cleaning the well-treated porcine aortic valve by using a PBS (phosphate buffer solution) solution for 10 minutes each time, then carrying out freeze drying treatment by using a freeze dryer, and boxing and storing the freeze-dried porcine aortic valve.
Example 2:
1. anti-calcification treatment:
selecting lysine as amine compound, selecting anhydrous ethanol as buffer solution, measuring 100ml of anhydrous ethanol containing 0.1% lysine, pouring into a container, adding 3 bovine pericardium with diameter of 50mm and cross-linked with glutaraldehyde, placing in a refrigerator at 4 deg.C, and refrigerating for 48 hr;
2. drying:
taking out bovine pericardium, washing with 80%/20% ethanol/normal saline solution at room temperature for 3 times for 15 min each time;
the treated bovine pericardium was also washed 3 times with HEPES for 15 minutes each, then washed 5 times with water for injection for 5 minutes each, and finally freeze-dried for storage.
The method of the two embodiments is adopted to respectively process samples, and the aldehyde content and the performance of the samples are respectively detected before and after the samples are processed, and the specific detection results are as follows:
1. resistance to thermal denaturation temperature changes (. degree. C.) before and after calcification, see in particular Table 1 below:
table 1: resistance to temperature change (DEG C) due to thermal denaturation before and after calcification
Six samples were treated by the methods of example 1 and example 2, respectively, and the P values of example 1 and example 2 were less than 0.01 compared to the non-anticalcification group, so there was no significant statistical difference; the anti-calcification treatment does not cause a reduction in the thermal denaturation temperature, so the method of the application is mild and has no substantial damage to the biological material.
2. The aldehyde content of the anticalcification treated and non-anticalcification treated biomaterials varied (nmol/g), see in particular table 2 below:
table 2: variation in aldehyde content (nmol/g) of anticalcification-treated and non-anticalcification-treated biomaterials
Serial number | Example 1 | Example 2 | Non-anticalcification |
1 | 1139.93 | 804.51 | 3688.66 |
2 | 1128.22 | 760.18 | 2922.45 |
3 | 1528.06 | 758.5 | 3140.77 |
mean | 1265.4 | 774.4 | 3250.63 |
When three samples are respectively treated by the methods of example 1 and example 2 and compared with the biological material which is not calcified, the aldehyde group content of the two groups of examples is obviously lower than that of the non-calcified group, so the anti-calcification effect of the anti-calcification method is obvious.
3. And (3) detecting the content of calcium and phosphorus after the mice are implanted subcutaneously for 8 weeks (unit mu g/mg):
referring to fig. 2 and 3, fig. 2 is a graph showing the results of measuring the calcium content of the biomaterial according to the embodiment of the present invention after the biomaterial is subcutaneously implanted in a mouse for 8 weeks; fig. 3 is a graph showing the results of measuring the phosphorus content of the biomaterial of the embodiment treated by the anti-calcification method of the present application after the biomaterial is subcutaneously implanted in a mouse for 8 weeks.
By analyzing fig. 2 and fig. 3, it can be found that, when the biological material samples are respectively processed by the methods of example 1 and example 2, and the biological material samples are implanted subcutaneously in the mouse for 8 weeks, compared with the biological material which is not calcified and is implanted subcutaneously in the mouse for 8 weeks, the biological material samples processed by the anti-calcification method of the present application have significantly reduced calcium content and phosphorus content and have stronger anti-calcification performance after being implanted subcutaneously in the mouse for 8 weeks, so that the anti-calcification method of the present application has a significant anti-calcification effect.
In summary, different from the situation of the prior art, the anti-calcification method of the biomaterial provided by the present application utilizes the amine compound to perform anti-calcification treatment on the biomaterial, the amino group of the amine compound can react with the aldehyde group which initiates calcification of the biomaterial, so as to seal the aldehyde group, thereby preventing the residual aldehyde group from initiating calcification of the biomaterial, and the amine compound is used to perform anti-calcification treatment on the biomaterial, so as to minimize the negative impact caused by the aldehyde compound, thereby having strong anti-calcification performance. Meanwhile, the biological material subjected to anti-calcification treatment is stored in a dry form, the biological material is stored in the dry form, the requirement on the storage condition is lower, the storage time is longer, and the biological material has the capability of tolerating adverse environment. Meanwhile, the biological material stored in a dry form can be directly used after being unsealed and completely packaged without rinsing and assembling, so that the application is more convenient, and the rescue time is saved.
Referring to fig. 4, fig. 4 is a schematic flow chart of another embodiment of the anti-calcification method for biological material of the present application.
Another embodiment of the present application provides a method for anti-calcification of a biomaterial, the method including:
in another embodiment, after the anti-calcification of the biological material, the method further comprises the step of resisting oxidation of the biological material, which comprises the following specific steps:
s201: under the preset condition, the amine compound is used for carrying out anti-calcification treatment on the biological material.
The content of step S201 in this embodiment is substantially the same as the content of the corresponding step in the above embodiment, and is not described herein again.
S202: under the preset condition, the biological material is subjected to antioxidation treatment by using a reducing agent, and the biological material after the antioxidation treatment is stored in a dry state.
In another embodiment, the biomaterial comprises a porcine aortic valve, porcine/bovine pericardium, bovine muscle/achilles tendon, porcine bladder, porcine/bovine dermal matrix, porcine small intestine submucosa matrix, porcine/bovine peritoneum, blood vessels, xenogenic or xenogenic bone, porcine eye cornea, or tympanic membrane.
In another embodiment, the reducing agent comprises one or more of sodium borohydride, sodium cyanoborohydride, and/or sodium triacetoxyborohydride. Through adopting the reductant to carry out anti-oxidant treatment to biomaterial, can be so that biomaterial self has certain antioxidant capacity, under uncontrollable external environment, unpredictable's change does not take place for protection material itself that can be fine. The reducing agents have a continuous reducing effect, so that oxidation caused by the external environment can be effectively resisted in the subsequent storage, treatment and use processes of the biological material; in addition, the reducing agents have small residues on the biological materials, are harmless to human bodies, and are suitable for oxidation resistance of the biological materials.
In another embodiment, the predetermined conditions include a soaking time and a treatment temperature for the reducing solution to soak the biological material. The soaking time is 0.5-48 hours, specifically, the soaking time can be 0.5 hour, 12 hours, 24 hours, 36 hours or 48 hours, and the soaking time is not limited herein; the treatment temperature is 4 ℃ to 58 ℃, specifically, the treatment temperature is 4 ℃, 18 ℃, 31 ℃, 42 ℃ or 58 ℃, and the treatment temperature is not limited herein.
In another embodiment, the anti-oxidant treatment of the biological material with a reducing agent comprises: the reducing agent is diluted with a buffer to obtain a reducing solution, and the biological material is soaked with the reducing solution.
The concentration of the reducing agent in the reducing solution is 0.01% to 10%, and specifically, the concentration of the amine compound may be 0.01%, 3%, 5%, 8%, and 10%, which is not limited herein.
Wherein the buffer system comprises PBS (phosphate buffered saline solution) and the acid-base condition is pH 7.4; MES (2 morpholinoethanesulfonic acid) at pH 5.5 under acid-base conditions; HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) at pH 7.5 under acid-base conditions; CBS (carbonate buffer solution) with an acid-base condition of pH 10.3; absolute ethyl alcohol; normal saline and ethanol/normal saline mixed solution.
The anti-oxidative treatment of the biological material with a reducing agent further comprises: the reducing solution soaked with the biological material is oscillated, ultrasonically or stirred, so that the flow speed of the reducing solution can be increased, and the anti-oxidation treatment of the biological material can be accelerated.
In another embodiment, preserving the biological material in a dry form after the anti-oxidant treatment comprises: freeze drying the biological material after the antioxidation treatment, glycerizing dehydration and/or ethanol gradient dehydration, and storing the biological material in a dry state. The biological material is stored in a dry state, has lower requirement on the storage condition, has longer storage time and has the capability of tolerating adverse environment. Meanwhile, the biological material stored in a dry form can be directly used after being unsealed and completely packaged without rinsing and assembling, so that the application is more convenient, and the rescue time is saved.
Wherein the glycerolysis dehydration comprises soaking the biological material in a mixed solution of high-concentration glycerol (80% -95%) and a small amount of absolute ethyl alcohol to absorb water in the biological material; the ethanol gradient dehydration comprises soaking the biological material in 75%/80% ethanol solution (ethanol mixed with normal saline) for 3-30 min, soaking in 95% ethanol solution (ethanol mixed with normal saline) for 3-30 min, and soaking in anhydrous ethanol for 3-30 min to achieve dehydration effect on the biological material; freeze-drying includes prefreezing, annealing, and resolution drying.
Generally, the processing steps of the biological material are aldehyde crosslinking, anti-calcification and anti-oxidation treatment in sequence. The biological material is subjected to anti-calcification treatment by the method, and the amine compound is used for carrying out anti-calcification treatment on the biological material, so that the negative influence brought by the aldehyde compound is minimized, and the biological material has stronger anti-calcification performance. The biological material is subjected to anti-oxidation treatment by the method, so that the biological material has certain anti-oxidation capacity, and the material can be well protected from unpredictable change in an uncontrollable external environment. The biological material which is subjected to anti-calcification and anti-oxidation treatment and stored in a dry state has stable performance and is convenient to store.
In another embodiment of the present application, a biomaterial is provided, and the biomaterial is processed by the anti-calcification method in any one of the above embodiments, so that the amino group of the amine compound can react with the aldehyde group that initiates calcification of the biomaterial, thereby blocking the aldehyde group and preventing the residual aldehyde group from initiating calcification of the biomaterial. Wherein the biomaterial comprises porcine aortic valve, porcine/bovine pericardium, bovine muscle/achilles tendon, porcine bladder, porcine/bovine dermal matrix, porcine small intestine submucosa matrix, porcine/bovine peritoneum, blood vessel, xenogenic or xenogenic bone, porcine eye cornea or tympanic membrane.
The above description is only an example of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings, or which are directly or indirectly applied to other related technical fields, are intended to be included within the scope of the present application.
Claims (10)
1. A method for anti-calcification of a biological material,
under the preset condition, amine compounds are used for carrying out anti-calcification treatment on the biological material, and the biological material after the anti-calcification treatment is stored in a dry state.
2. The method according to claim 1, wherein the biomaterial is cross-linked with an aldehyde compound.
3. The method according to claim 2, wherein the amine compound is one or more of a diamine compound, an alcohol amine compound, and/or an imine compound.
4. The method of claim 3, wherein the amine compound comprises one or more of ethylenediamine, ethanolamine, hexamethylenediamine, octamethylenediamine, carbodiimide, lysine, arginine, asparagine, or glutamine.
5. The method of claim 2, wherein said anticalcification of said biological material with an amine compound comprises:
diluting the amine compound with a buffer solution to obtain an amine compound solution, and soaking the biological material with the amine compound solution.
6. The method according to claim 5, wherein the concentration of the amine compound in the amine compound solution is 0.01% to 10%.
7. The method according to claim 5, wherein the preset conditions comprise a soaking time and a treatment temperature for soaking the biological material in the amine compound solution; wherein the soaking time is 2-72 hours, and the treatment temperature is 4-58 ℃.
8. The method of claim 1, wherein the preserving the anti-calcification treated biological material in a dry form comprises:
and (3) freeze-drying, glycerating and dehydrating and/or performing gradient dehydration on the biological material subjected to the anti-calcification treatment, and storing the biological material in a dry state.
9. The method of claim 1, wherein the biological material comprises porcine aortic valve, porcine/bovine pericardium, bovine muscle/achilles tendon, porcine bladder, porcine/bovine dermal matrix, porcine small intestine submucosa matrix, porcine/bovine peritoneum, blood vessels, xenogenic or xenogenic bone, porcine eye cornea, or tympanic membrane.
10. A biomaterial, characterized in that it has been subjected to an anti-calcification treatment by a method as claimed in any one of claims 1 to 9.
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