CN118059315B - Method for removing residual glutaraldehyde based on indirect centrifugation - Google Patents

Abstract

The present invention belongs to the field of biomedical materials, and specifically relates to a method for removing residual glutaraldehyde based on indirect centrifugation. The specific steps of the method include: 1) placing a biological material containing glutaraldehyde in a glycine solution for neutralization treatment; 2) placing the biological material after neutralization treatment in a filter cloth, and then sealing the filter cloth and fixing it in the middle of a centrifuge tube, and removing glutaraldehyde by indirect centrifugation; 3) repeating the centrifugation operation once.

Description

A method for removing residual glutaraldehyde based on indirect centrifugation

Divisional application

This application is a divisional application based on the Chinese invention patent application with application number CN202311244739.2, application date September 25, 2023, and invention name “A method for removing residual glutaraldehyde in glutaraldehyde-cross-linked biomaterials”.

Technical Field

The invention belongs to the field of biomedical materials, and in particular relates to a method for removing residual glutaraldehyde based on indirect centrifugation.

Background Art

Biomaterials refer to a class of materials that can be used to diagnose, treat, repair or replace diseased tissues and organs or enhance the functions of organisms. They can be further divided into synthetic biomaterials and natural biomaterials. For some common natural macromolecular biomaterials, such as collagen, cellulose, chitosan, gelatin, silk fibroin and decellularized extracellular matrix, their surfaces usually have abundant characteristic groups such as hydroxyl, amino and carboxyl groups, which provide many chemical reaction sites for such materials, thus making it possible to realize the functionalization and high performance of such materials. However, after further regeneration or processing of these natural biomacromolecules, the mechanical properties of the subsequent materials obtained often do not meet the application requirements. Therefore, it is necessary to further improve the mechanical properties of the materials through physical or chemical crosslinking to achieve high performance.

Glutaraldehyde is currently considered to be one of the cross-linking agents with the best cross-linking strength. However, the residual glutaraldehyde (including free glutaraldehyde molecules and glutaraldehyde residues) in the biomaterials treated with glutaraldehyde has strong cytotoxicity, which limits its in-depth application in the field of biomaterials. At present, there are three main methods for removing residual glutaraldehyde in glutaraldehyde cross-linked biomaterials: (1) repeated washing, such as washing with aminoacetic acid, but this method can only remove the glutaraldehyde cross-linked on the surface of the material, and has limited effect on the removal of residual glutaraldehyde that has penetrated into the material. (2) heat treatment, such as heat treatment at 120°C for 12 hours, to remove glutaraldehyde from the cross-linked collagen, but this process will cause high-temperature denaturation of collagen if not properly controlled. (3) supercritical carbon dioxide extraction of residual glutaraldehyde, but the equipment cost of building a supercritical carbon dioxide platform is high, the extraction and removal process is difficult, and the high-pressure conditions also put forward higher requirements for safety. Therefore, seeking a low-cost, simple and efficient glutaraldehyde removal method will greatly promote the widespread clinical application of biomaterials in tissue repair and regeneration.

The patent with application number CN202210860705.5 and invention name “Process for treating biological tissue sheets and biological tissue sheets obtained by the process” discloses a method of washing a biological tissue sheet that has been cross-linked and fixed with glutaraldehyde with a NaCl aqueous solution for 1 to 8 times, and then mixing and reacting with a glycine solution for 3 to 6 hours to remove residual glutaraldehyde. However, the role of glycine in this method is to neutralize glutaraldehyde as a chemical neutralizing agent, but in essence, the neutralized glutaraldehyde still remains inside the biological tissue sheet, so the problem of removing glutaraldehyde from the biological tissue sheet is not really solved. Therefore, the biological tissue sheet treated by the patent method still has a certain degree of glutaraldehyde residue, resulting in the biological tissue sheet still having low biological toxicity. In addition, existing studies have shown that the product of the reaction between glycine and glutaraldehyde is glycyl glutaraldehyde, which can cause the treated biological material to turn yellow. And glycyl glutaraldehyde is unstable and easily decomposed into glycine and glutaraldehyde again. On the other hand, the glycine remaining in the biological material will be oxidized, which will also cause the material to turn yellow. Therefore, it is particularly important to remove the reaction products and residues of glycine and glutaraldehyde. Finally, the process of the patented method is time-consuming, and the soaking time with glycine alone requires 3 to 6 hours.

In summary, it is necessary to propose improved methods and strategies to effectively remove residual glutaraldehyde from biomaterials and improve the safety and applicability of biomaterials.

Summary of the invention

In view of this, the object of the present invention is to provide a method for removing residual glutaraldehyde in biological materials by indirect centrifugation. The specific technical scheme is as follows.

：A method for removing residual glutaraldehyde based on indirect centrifugation, the specific steps are as follows:

Step 1: placing the biomaterial containing glutaraldehyde in a glycine solution for neutralization treatment, wherein the neutralization treatment is to place the biomaterial containing glutaraldehyde in the glycine solution and immerse it in ultrasound at a power of 300-600W for 10-30 minutes, and the temperature of the ultrasound immersion is controlled to be 25-40°C;

Step 2: placing the neutralized biological material into a filter cloth, and adding a small amount of water (preferably purified water) into the filter cloth to keep the biological material moist, then sealing the filter cloth and fixing it in the middle of a centrifuge tube, and centrifuging it in an indirect centrifugal manner at a speed of 4000 to 10000 rpm for 5 to 30 minutes, and controlling the temperature of the entire centrifugal process to be 17 to 40° C.;

Step 3: Repeat the centrifugation operation in step 2 once.

Furthermore, the neutralization treatment in step 1 is to place the biological material containing glutaraldehyde in a glycine solution and soak it in ultrasound at a power of 400 W for 30 minutes or at a power of 600 W for 10 minutes.

It can be understood that the present invention adopts a short-time ultrasonic immersion method to neutralize glutaraldehyde in the biological material.

Furthermore, the molar concentration of the glycine solution in step 1 is 0.01 mol/L to 1 mol/L.

Preferably, the molar concentration of the glycine solution is 0.2 mol/L.

Furthermore, in step 2, the mixture is centrifuged at a rotation speed of 4000 rpm for 30 minutes.

Furthermore, the mesh size of the filter cloth in step 2 is 50 to 500 meshes.

Furthermore, in step 2, the amount of water added to the filter cloth is 2 to 5 ml.

Furthermore, after the indirect centrifugation operation in step 2 and step 3 is completed, the biological material in the filter cloth is taken out and rinsed with pure water.

Furthermore, the types of biomaterials include decellularized extracellular matrix, collagen sponge, gelatin, elastin or silk protein, etc. The basic structural units of these materials are amino acids, and the free amino or hydroxyl groups on the amino acids can undergo cross-linking reactions with glutaraldehyde.

It is to be understood that the method for removing residual glutaraldehyde of the present invention can also be applied to chitin, chitin, chitosan, agarose, hyaluronic acid, fibrinogen, chondroitin sulfate, alginate, aminodextran or liposome.

Furthermore, the biomaterial containing glutaraldehyde is a biomaterial that is cross-linked in a glutaraldehyde solution with a mass concentration of 0.001% to 0.1% at room temperature for 10 to 48 hours.

Furthermore, the biomaterial containing glutaraldehyde is a biomaterial that is cross-linked in a glutaraldehyde solution with a mass concentration of 0.001% to 0.1% at room temperature for 24 hours.

Furthermore, the decellularized extracellular matrix treated in the above steps is freeze-dried in a vacuum freeze dryer for storage.

Beneficial technical effects

The present invention proposes a method for completely removing residual glutaraldehyde in a biomaterial by means of indirect centrifugation. Specifically, the biomaterial containing glutaraldehyde is first soaked in glycine for a short time to neutralize the glutaraldehyde, and then the glutaraldehyde is completely removed by indirect centrifugation twice. The advantage of such operation is that, first, the short-term soaking with glycine can better prevent the biomaterial from turning yellow, and thus does not affect the subsequent clinical application of the biomaterial. The ultrasonic method can accelerate the neutralization of glutaraldehyde by glycine, so this step can complete the neutralization of most of the glutaraldehyde inside the biomaterial by glycine in a relatively short time, and at the same time, the color of the biomaterial will not become too yellow. Then, indirect centrifugation is used to completely remove the neutralized glutaraldehyde and the residual glycine together, while protecting the biomaterial from deformation and qualitative change. After two rounds of indirect mechanical centrifugation provided by the present invention, the biomaterial can almost return to its original color without deformation, and the glutaraldehyde is completely removed. The biomaterial is tested to have almost no cytotoxicity and is suitable for clinical promotion and application.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

FIG1 is a milky white film-like decellularized extracellular matrix effect diagram of pig peritoneum after degreasing and decellularization;

FIG2 is a color diagram of the decellularized extracellular matrix after cross-linking with glutaraldehyde and soaking in a glycine solution at room temperature for 24 hours;

FIG3 is a color diagram of the decellularized extracellular matrix after cross-linking with glutaraldehyde and ultrasonic immersion in glycine solution for 30 min;

FIG4 is a diagram showing the reaction mechanism of glycine and free glutaraldehyde;

FIG5 is a chromatogram of residual glutaraldehyde detected by high performance liquid chromatography after the decellularized extracellular matrix cross-linked with glutaraldehyde was immersed in a glycine solution at room temperature for 24 hours;

FIG6 is a chromatogram of residual glutaraldehyde detected by high performance liquid chromatography after ultrasonic immersion of decellularized extracellular matrix cross-linked with glutaraldehyde in glycine solution for 30 minutes;

FIG7 is a color image of the decellularized extracellular matrix after short-term ultrasonic immersion in glycine combined with two indirect mechanical centrifugations;

FIG8 is a residual chromatogram of the decellularized extracellular matrix detected by high performance liquid chromatography after short-time ultrasonic immersion in glycine combined with two indirect mechanical centrifugations;

FIG9 is a diagram showing the cytotoxicity detection after glutaraldehyde is removed using different methods and materials;

FIG. 10 is a residual chromatogram of the collagen sponge detected by HPLC after short-time ultrasonic immersion in glycine combined with two indirect mechanical centrifugations.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Herein "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

Glossary

The "short-time ultrasonic immersion" mentioned in the present invention means that the biological material cross-linked with glutaraldehyde is immersed in a glycine solution of a specific concentration for a time period of no more than 30 minutes.

The "indirect centrifugation" mentioned in the present invention means that the material to be centrifuged is not directly placed in a centrifuge tube, but is placed in a protective container and then placed in a centrifuge tube for centrifugation.

Example 1

This example provides an example of preparing acellular extracellular matrix using porcine peritoneum as raw material

(1) Degreasing of pig peritoneum: Most of the fat attached to the pig peritoneum tissue is manually removed, and the pig peritoneum is washed with physiological saline for 3 to 8 times. The washed pig peritoneum is immersed in an organic reagent at room temperature for a certain period of time for degreasing. The degreased pig peritoneum is washed in pure water for 3 to 8 times to remove the remaining organic reagent in the pig peritoneum. The organic reagent used for degreasing the pig peritoneum can optionally include any one of acetone, isopropanol, ether, methanol, chloroform, ethyl acetate or n-hexane. The ratio of pig peritoneum to organic reagent is 1:6 to 1:14 (w/v).

(2) Decellularization of porcine peritoneum: The porcine peritoneum after defatting is immersed in a mixed alkaline solution of sodium hydroxide and sodium chloride to further remove cells, wherein the concentration of sodium hydroxide is 0.1% to 2% (w/w), and the concentration of sodium chloride is 0.5% to 2% (w/w). The porcine peritoneum is then washed 3 to 8 times in pure water until the solution is neutral. After freeze-drying, the decellularized extracellular matrix is obtained, as shown in FIG1 .

It is understandable that the materials applicable to the method of the present invention may also include collagen sponges, collagen gels, collagen films, and collagen materials obtained from other mammals.

Example 2

The decellularized extracellular matrix material (referred to as the material) treated in Example 1 was cut into 1.5 cm × 1.5 cm slices, immersed in a glutaraldehyde solution with a mass concentration of 0.001% to 0.1%, crosslinked at room temperature for 24 hours, and rinsed with pure water three times. The mechanical strength of the decellularized extracellular matrix crosslinked with glutaraldehyde was significantly enhanced. Then the decellularized extracellular matrix crosslinked with glutaraldehyde was immersed in (1): 0.2 mol/L glycine solution at room temperature for 24 hours and in (2): 0.2 mol/L glycine solution with an ultrasonic power of 400 W for 30 minutes, and the ultrasonic temperature was 25 to 40°C. The material treated by methods (1) and (2) was rinsed with pure water three times to remove most of the unreacted glycine solution on the surface. The treated decellularized extracellular matrix was freeze-dried in a vacuum freeze dryer for storage.

Fig. 2 is a decellularized extracellular matrix after being cross-linked with glutaraldehyde and immersed in a glycine solution at room temperature for 24 hours, showing a darker yellow color. Fig. 3 is a decellularized extracellular matrix after being cross-linked with glutaraldehyde and immersed in a glycine solution for 30 minutes by ultrasound, showing a light yellow color. The reason for the yellowing of the color is that the glycyl glutaraldehyde generated by the reaction of free glutaraldehyde and glycine shows yellow. The longer the immersion time in the glycine solution, the darker the yellow color. Fig. 4 is a reaction mechanism diagram of glycine and free glutaraldehyde. After the decellularized extracellular matrix is immersed in glycine, its immersion liquid shows yellow.

The decellularized extracellular matrix obtained by the above-mentioned method steps (1) and (2) was added to physiological saline at a ratio of 6 ^cm2 /mL, and the sample was extracted by constant temperature shaking at (37±1)°C for (24±2) hours at an shaking speed of 200 rpm. After the extraction, the sample was separated from the liquid, cooled to room temperature, and the liquid was taken as the extract. The glutaraldehyde residue was tested in the extract according to DB 13/T 5127.11-2019 (Determination of toxic and harmful substances in extracts of polymer materials for implantable medical devices Part 11: Glutaraldehyde migration amount high performance liquid chromatography). The glutaraldehyde content of the decellularized extracellular matrix after immersion in (1) glycine solution at room temperature for 24 hours was 0.103 mg/L, as shown in Figure 5; the glutaraldehyde content of the decellularized extracellular matrix after ultrasonic immersion in (2) glycine solution for 30 minutes was 1.748 mg/L, as shown in Figure 6.

Cytotoxicity test: sterilize the decellularized extracellular matrix obtained by the above-mentioned method steps (1) and (2) under ultraviolet light for more than 8 hours. Then add MEM culture medium containing fetal bovine serum at a ratio of 6 ^cm2 /mL, and extract at a constant temperature of (37±1)℃ for (24±2) hours with an oscillation speed of 200rpm. After the extraction, separate the sample from the liquid, cool it to room temperature, and take the liquid as the extract. Refer to GB/T 16886.5-2017 (Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test) for in vitro cytotoxicity test of the extract, and the cytotoxicity test method refers to the CCK-8 method. Among them, the cell survival rate obtained by the test is greater than or equal to 70% and is qualified, and the cell survival rate obtained by the test is less than 70% and is unqualified. The results are shown in Figure 9. The cytotoxicity of the (1)(2) materials is shown in Figure 9 Example 2 (1) and Figure 9 Example 2 (2), respectively. It can be seen that after short-term ultrasonic immersion in glycine solution, the residual glutaraldehyde content in the decellularized matrix is higher, so the cytotoxicity is greater and the cell viability is lower.

Example 3

The decellularized extracellular matrix cross-linked with glutaraldehyde in Example 2 was soaked in 0.2 mol/L glycine solution at a power of 400W for 30 minutes, and then subjected to indirect mechanical centrifugation. The decellularized extracellular matrix was placed in a 500-mesh filter cloth, and 5 ml of pure water was further added to the filter cloth. The filter cloth was then sealed and fixed in the middle of the centrifuge tube, and the centrifugation temperature was controlled to be 17-40°C (controlling temperature centrifugation is very important for protecting biological materials), and the centrifugation speed was set to 4000-10000 rpm for 5-30 minutes, and centrifuged twice. The material after indirect mechanical centrifugation showed a lighter yellow color, close to the color of the decellularized extracellular matrix material itself, as shown in Figure 7, indicating that the glutaraldehyde neutralized by glycine was further removed. The material at this time continued to be added with physiological saline at a ratio of 6 ^cm2 /mL, and was subjected to constant temperature oscillation leaching for (24±2) hours at (37±1)°C, with an oscillation speed of 200 rpm. After the leaching was completed, the sample was separated from the liquid, cooled to room temperature, and the liquid was taken as the leaching solution. The glutaraldehyde residue test was performed with reference to DB 13/T 5127.11-2019 (Determination of toxic and hazardous substances in extracts of polymer materials for implantable medical devices Part 11: Glutaraldehyde migration capacity high performance liquid chromatography). The residual glutaraldehyde content in the decellularized extracellular matrix in this implementation was not detected, and the instrument detection limit was 0.025 mg/L, as shown in Figure 8, indicating that the residual glutaraldehyde was effectively removed after indirect mechanical centrifugation. The cytotoxicity of the material is shown in Figure 9, and the cell viability is significantly higher than that of the decellularized extracellular matrix material soaked in glycine solution for 24 hours in Example 2 (1).

Example 4

This example provides a glutaraldehyde removal result of a glutaraldehyde-crosslinked collagen sponge.

The collagen sponge was treated according to the short-time ultrasonic immersion of glycine in Example 2 and the mechanical centrifugation method of Example 3. After centrifugation, it was rinsed with pure water 3 times to remove most of the unreacted glycine solution on the surface. The treated collagen sponge was freeze-dried in a vacuum freeze dryer for easy storage. Then, the high performance liquid chromatography test and cytotoxicity test were performed according to the standard method of the above example. The result showed that no residual glutaraldehyde was detected, and the instrument detection limit was 0.025 mg/L, as shown in Figures 9 and 10.

Example 5

This example provides the residual amount of glutaraldehyde in the decellularized extracellular matrix under different ultrasonic immersion and centrifugation operations.

Table 1 Glutaraldehyde residues in different ultrasonic immersion operations

Ultrasonic power Ultrasound time Glutaraldehyde Residue 300W 30min 2.146mg/L 400W 30min 1.748mg/L 600W 10min 1.836mg/L

From the above experiments, it can be seen that after 30 minutes of ultrasound at a power of 400 W, the residual amount of glutaraldehyde is the least, so the material under this ultrasound parameter is selected to continue indirect mechanical centrifugation.

Table 2 Glutaraldehyde residues at different centrifugation times

Centrifugation times Centrifugation parameters Glutaraldehyde Residue 1 time 4000rpm/30min 0.153mg/L 2 times 4000rpm/30min ≤0.025mg/L

It can be seen from the above experiment that after the second centrifugation, almost no glutaraldehyde remains, so the method of the present invention finally determines to perform two mechanical centrifugations.

Table 3 Glutaraldehyde residues at different centrifugation parameters

From the above experiment, it can be seen that after centrifugation at 4000 rpm for 30 min, the residual amount of glutaraldehyde is the least, so this centrifugation parameter is selected as the optimal operation.

The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims (5)

1. A method for removing residual glutaraldehyde in biological materials based on indirect centrifugation, characterized in that the specific steps are as follows: Step 1: placing the biomaterial containing glutaraldehyde in a glycine solution for neutralization treatment, wherein the neutralization treatment is placing the biomaterial containing glutaraldehyde in a glycine solution with a molar concentration of 0.2 mol/L and immersing it in ultrasound at a power of 400 W for 30 minutes or at a power of 600 W for 10 minutes, and controlling the temperature of ultrasound immersion to be 25-40° C.; the type of the biomaterial is a decellularized extracellular matrix sheet, and the decellularized extracellular matrix sheet after treatment is light yellow; Step 2: Put the decellularized extracellular matrix sheet that has completed the neutralization treatment into a filter cloth, add a small amount of water into the filter cloth to keep the decellularized extracellular matrix sheet moist, then seal the filter cloth and fix it in the middle of the centrifuge tube, centrifuge it at a speed of 4000 rpm for 30 minutes, and control the temperature of the entire centrifugation process to be 17-40°C; Step 3: Repeat the centrifugation operation of step 2 once; the color of the decellularized extracellular matrix sheet after indirect mechanical centrifugation is close to the color of the decellularized extracellular matrix sheet itself. 2. The method according to claim 1, characterized in that the mesh size of the filter cloth in step 2 is 50 to 500 meshes. 3. The method according to claim 1, wherein the amount of water added to the filter cloth in step 2 is 2 to 5 mL. 4. The method according to claim 1, characterized in that after the indirect centrifugation operation in step 2 and step 3 is completed, the biological material in the filter cloth is taken out and rinsed with pure water. 5. The method according to claim 1, characterized in that the biomaterial containing glutaraldehyde is a biomaterial cross-linked in a glutaraldehyde solution with a mass concentration of 0.001% to 0.1% at room temperature for 10 to 48 hours.