CN115887769A - Method for removing endotoxin in collagen-based biological material - Google Patents

Abstract

The invention discloses a method for removing endotoxin in a collagen-based biological material, aiming at the problem that the endotoxin in the biological material is difficult to completely remove, which comprises the following steps: degreasing treatment and alkali solution treatment, wherein the sample to be treated is firstly degreased to enable the fat content to be lower than 2%, and finally, the alkali solution treatment is carried out immediately. The content of endotoxin in the collagen-based biomaterial, which is subjected to the endotoxin removal method disclosed by the invention, is lower than 0.5EU/g, and the clinical application of the implant material is facilitated.

Description

Method for removing endotoxin in collagen-based biological material

Technical Field

The invention belongs to a processing and preparation method of biomedical materials, and particularly relates to a method for removing endotoxin in a collagen-based biological material.

Background

Bacterial endotoxins are characteristic structures of the outer layer of the cell wall of gram-negative bacteria, and their active components are mainly Lipopolysaccharides (LPS). Endotoxin enters human body and may cause serious consequences such as fever, microcirculation disturbance, endotoxemia, septic shock and disseminated intravascular coagulation. Therefore, in part 2 of GB/T14233.2-2005 "inspection methods for medical infusion, blood transfusion and injection apparatus": in the biological test method, it is specified that endotoxin in transfusion, blood transfusion and injection instruments is not more than 20EU per one, and medical instruments in contact with cerebrospinal fluid and applied in the thoracic cavity are not more than 2.15EU per one. However, in the actual operation process, because the detection of endotoxin itself is easy to have a false negative result, and the endotoxin has a shielding effect (Masking effect), the endotoxin with a low dose cannot be detected, but the endotoxin with a shielding effect with a low dose can also cause a systemic inflammatory reaction, which affects the biological safety of the implant material, the practical requirement of the endotoxin of the biological material itself should be reduced as much as possible.

The removal of endotoxin is usually carried out by ultrafiltration, ion exchange, chromatography, two-phase micelle system, etc., but these methods are only suitable for treating a sample in a solution state and are expensive. CN112778412 discloses a method for preparing low endotoxin collagen, which can reduce the endotoxin content of collagen to less than 0.5EU/mL by removing endotoxin by low temperature alkali treatment, but the object of the method is collagen powder, and is not suitable for membrane material with three-dimensional mesh structure.

The endotoxin pollution of natural biological materials is mostly from animal-derived materials and the process of material selection, and at present, no ideal method can completely avoid the endotoxin pollution and no ideal method for treating the endotoxin is provided. Furthermore, the three-dimensional helical structure of collagen fibers is very prone to bind endotoxins, which are hidden in the structure and escape conventional reagent treatments, making endotoxin in such materials difficult to remove. CN105251049 discloses an ionic buffer solution for removing bacterial endotoxin in biomedical materials and application thereof, which can treat acellular dermal matrixes, amniotic membrane matrixes and small intestine submucosa extracellular matrixes, but the treatment time of the method is long (2-5 days), while the conventional acellular treatment time is only 2-4 days, the method obviously prolongs the production time and reduces the production efficiency.

Disclosure of Invention

Aiming at the problem that the endotoxin of the biological material is difficult to completely remove, the invention provides the method for removing the endotoxin of the collagen-based biological material, the fat content of the collagen-based biological material is lower than 2 percent through degreasing treatment, then the collagen-based biological material is treated by alkali solution, the endotoxin content is controlled below 0.5EU/g, the clinical application of an implant material is facilitated, the treatment time is shortened to within 1 day, and the production efficiency is improved.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for removing endotoxin from a collagen-based biomaterial, comprising: degreasing treatment and alkali solution treatment are carried out,

the sample to be treated is degreased to a fat content of less than 2% and then subjected to an alkaline treatment.

The degreasing treatment is to adopt an organic solvent to soak and oscillate a sample to be treated for multiple times;

the organic solvent is ethanol, chloroform, methanol and other solvents which can be used for removing the fat component residue of the biological material, and can be used in combination with various organic solvents, and the treatment time is 2-16h.

Or defatting with lipase.

Or degreasing with detergent.

Or degreasing with two or three of detergent, lipase and organic solvent.

The alkali solution treatment is to treat the degreased sample by adopting an alkali solution with the concentration of 0.1-2% (w/v), and the treatment time is 0.25-1h.

The alkali solution is sodium hydroxide solution or potassium hydroxide solution.

A method for removing endotoxin from a collagen-based biomaterial, comprising: degreasing the sample to be treated to make the fat content less than 1%, and then immediately performing alkali solution treatment.

The collagen-based biomaterial has an endotoxin content of less than 0.5EU/g. Preferably, the collagen-based biomaterial has an endotoxin content of less than 0.1EU/g. Before the sample to be treated is degreased, drying is firstly carried out, so that the water content of the sample to be treated is less than or equal to 10%, and preferably, the water content is 5-10%.

The drying treatment is freeze drying, air drying or heat drying treatment.

The collagen-based biomaterial is a solid material, and comprises a membrane-shaped porcine small intestine submucosa, a basement membrane, dermis, pericardium, peritoneum, or three-dimensional ligament or achilles tendon.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects:

the technical principle of the invention lies in that the hydrophilicity of the material is enhanced, the permeability is increased, the cell membrane is damaged and most of the biological load is removed by effectively removing lipid components; the subsequent alkali solution treatment can reach the aim of further and completely removing endotoxin. Although the separate degreasing and alkali treatment are common methods in the cell removal treatment, the invention can completely remove the endotoxin by adjusting the process parameters and continuously applying the process parameters and the alkali treatment, does not prolong the production period and ensures the production efficiency.

Detailed Description

The method for removing endotoxin from a collagen-based biomaterial according to the present invention will be described in detail with reference to the following specific examples. The advantages and features of the present invention will become more apparent from the following description.

Because the raw materials of animal-derived materials and the material taking process can not avoid the pollution of endotoxin, the thorough removal of the endotoxin of the acellular matrix biological materials is always a difficult problem for processing the materials. Although the conventional decellularization treatment can remove cells or biological loads including endotoxin to a certain extent, the conventional decellularization treatment does not disclose the requirement of the degreasing treatment, does not disclose the detection value of the endotoxin, and cannot guarantee the treatment effect of the endotoxin due to the interference of the conventional detection means.

The method for removing the endotoxin from the collagen-based biomaterial is characterized in that the collagen-based biomaterial is a solid material and comprises a membrane-shaped porcine small intestine submucosa, a basement membrane, dermis, pericardium, peritoneum and the like, or three-dimensional bone tissues, ligaments, achilles tendons and the like. The endotoxin removal method comprises degreasing treatment and alkali solution treatment which are applied in a combined manner, and the specific endotoxin removal method comprises the following steps:

s1: degreasing treatment: firstly, drying a sample to be treated: drying the sample to be processed by adopting a freeze drying, air drying or heat drying mode to ensure that the water content is less than or equal to 10 percent,

then, treating the dried sample to be treated by using organic solvents such as ethanol, trichloromethane or methanol and the like for 2-16h;

or directly degreasing the sample to be treated by adopting lipase; the fat content of the sample to be treated is lower than 2%;

or degreasing with detergent;

or degreasing with two or three of detergent, lipase and organic solvent;

preferably, the fat content of the sample to be treated is less than 1%.

S2: and (3) treating the degreased sample by using an alkali solution with the concentration of 0.1-2% (w/v), wherein the treatment time is 0.25-1h, and the alkali solution is preferably a sodium hydroxide solution or a potassium hydroxide solution.

The collagen-based biomaterial treated by the endotoxin removal method has the endotoxin content of less than 0.5EU/g, and further has the endotoxin content of less than 0.1EU/g.

The invention is further characterized in the following by specific examples

Example 1

Because the porcine small intestine submucosa is derived from the small intestine, the content of endotoxin is relatively high, and the difficulty in removing the endotoxin is high, the porcine small intestine submucosa material is selected as an experimental material. The small intestine submucosa was separated from the freshly obtained porcine small intestine, briefly washed with peroxyacetic acid and phosphate buffer, sterilized and freeze-dried to sample 1.

After the sample 1 was degreased 10 times by washing with a chloroform/methanol (3, 1,v/v) mixed solution, the content of fat was 1.2% and the sample was designated as sample 2, and the organic solvent residue was removed by stepwise rehydration. Then, the sample was treated with 0.2% sodium hydroxide solution for 60min, rinsed with water for injection, and freeze-dried to obtain sample 3.

Sample 1 was treated with 0.2% sodium hydroxide solution for 60min, rinsed with water for injection and freeze-dried to give sample 4.

And respectively preparing the multilayer samples into patches to be detected. Cutting the patch into small pieces, and leaching with water for 2 hr respectively. The endotoxin content of the patch was measured using dynamic photometry and the results were converted as shown in table 1:

TABLE 1 measurement of endotoxin content of samples 1-4

	Endotoxin content (EU/g)
		Sample 1	786.98
Sample 2	4.98
		Sample 3	0.05
Sample 4	467.32

In view of the problem of false negative in endotoxin detection, in order to further clarify the endotoxin removal effect, this example further performed semi-quantitative analysis on endotoxin residues by macrophage activation. The higher the amount of TNF- α secreted, the more severe the inflammatory response the implant may cause, indirectly reflecting the higher the endotoxin content.

Taking THP-1 mononuclear macrophage in logarithmic phase of growth according to 1 × 10 ⁴ The density of individual cells/well was plated onto 24-well cell culture plates and incubated overnight at 37 ℃. Cells after overnight culture were replaced with fresh medium. Each set of samples was cut to 1X 1cm ² Is placed in an upper chamber of a transwell and is connected with a pressure-sensitive adhesivetranswell was placed in a well of a plate, 0.5mL of cell culture medium was added to the upper chamber, and after 24hr of co-culture, cell culture supernatant was aspirated, centrifuged, and TNF-. Alpha.content was measured by ELISA. Cells were counted by adding Alamar Blue reagent and staining for viable cells. LPS is used as a positive control substance, and a conventional complete culture medium is used as a negative control substance. The results of TNF-. Alpha.content of each test sample are shown in Table 2.

TABLE 2 TNF-alpha content results of the test samples

The results in Table 2 show that endotoxin in the raw material can be removed more completely by the degreasing and alkali treatment method disclosed by the invention.

Example 2

The porcine small intestine submucosa is selected as the experimental material. The freshly obtained porcine small intestine submucosa was briefly washed with peroxyacetic acid and phosphate buffer, sterilized and freeze-dried to sample 5.

Treating the sample 5 with lipase at low temperature for 12h, carrying out freeze drying, rinsing with chloroform for 2 times (each time for 0.5 h), detecting that the fat content is 2.0%, obtaining a sample 6, and carrying out stepwise rehydration to remove organic solvent residues. Then, the sample was treated with 1.5% sodium hydroxide solution for 15min, rinsed with water for injection, and freeze-dried to obtain sample 7.

Sample 5 was treated with 1.5% sodium hydroxide solution for 15min, rinsed with water for injection and freeze-dried to give sample 8.

And respectively preparing the multilayer samples into patches to be detected. Cutting the patch into small pieces, and leaching with water for 2h respectively in endotoxin detection. The endotoxin content of the patch was measured using dynamic photometry and the results were converted as shown in table 3:

TABLE 3 measurement of endotoxin content of samples 5-8

	Endotoxin content (EU/g)
		Sample 5	786.98
Sample 6	5.30
		Sample 7	0.38
Sample 8	345.61

Example 3

The porcine small intestine submucosa was decellularized using the method disclosed in example 1 of CN110960731A, and the residual amount of endotoxin was compared to the method of the present invention.

(1) Material taking and pretreatment: separating the lower mucous membrane layer of the small intestine of the pig, immediately putting ultrapure water at about 4 ℃ for cleaning for 3 times, and removing surface fat and fiber by using an operation tool to avoid contacting pollutants as much as possible; washing with 10% NaCl solution by shaking for 1h, repeating for 3 times, and washing with ultrapure water for 4 times;

(2) Carrying out decellularization treatment on the pretreated porcine small intestine submucosa according to the treatment scheme shown in the table 4;

TABLE 4 treatment protocol for step (2)

The endotoxin content of sample 9 was 1.66EU/g and that of sample 10 was 0.08EU/g as determined by dynamic photometry. Thus, although the conventional decellularization treatment also includes a separate degreasing treatment and a separate alkali treatment, the fat content of the defatted material is not required, and the endotoxin treatment in the present application requires that the fat content after the degreasing treatment is less than 2%, and immediately thereafter the alkali treatment is performed, not only is the treatment time shortened, but also the endotoxin content is reduced to less than 0.5EU/g. Further explaining the invention, the aim of removing endotoxin more completely can be achieved by adjusting the process parameters and the process flow, the production period is not prolonged additionally, and the production efficiency is ensured.

The embodiments of the present invention have been described in detail with reference to specific examples, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.

Claims (10)

1. A method for removing endotoxin from a collagen-based biomaterial, comprising: degreasing treatment and alkali solution treatment are carried out,

and (3) degreasing the sample to be treated to enable the fat content of the sample to be treated to be lower than 2%, and then carrying out alkali solution treatment.

2. The method for removing endotoxin from a collagen-based biomaterial according to claim 1, wherein the degreasing treatment is a treatment of the sample to be treated with an organic solvent.

3. The method for removing endotoxin from a collagen-based biomaterial according to claim 2, wherein the organic solvent is ethanol, chloroform or methanol, and the treatment time is 2 to 16 hours.

4. The method for removing endotoxin from a collagen-based biomaterial according to claim 1, wherein the degreasing treatment is a treatment of the sample to be treated with one or more of lipase, detergent, and organic solvent.

5. The method for removing endotoxin from a collagen-based biomaterial according to claim 1, wherein the alkali treatment is a treatment of the degreased sample with an alkali solution at a concentration of 0.1% to 2% (w/v) for a period of 0.25 to 1 hour.

6. The method for removing endotoxin from a collagen-based biomaterial according to claim 1 or 5, wherein the alkali solution is a sodium hydroxide solution or a potassium hydroxide solution.

7. The method of removing endotoxin from a collagen-based biomaterial according to claim 1, comprising: degreasing treatment and alkali solution treatment are carried out,

degreasing the sample to be treated to enable the fat content of the sample to be less than 1%, and then performing alkali solution treatment.

8. The method for removing endotoxin from a collagen-based biomaterial according to claim 1, wherein the endotoxin content of the collagen-based biomaterial is less than 0.5EU/g, preferably the endotoxin content of the collagen-based biomaterial is less than 0.1EU/g.

9. The method for removing endotoxin from a collagen-based biomaterial according to claim 1 or 3, wherein the sample to be treated is first dried to reduce the water content of the sample to be treated to 10% or less before being treated with the organic solvent.

10. The method for removing endotoxin from a collagen-based biomaterial according to claim 1, wherein the collagen-based biomaterial is a solid material comprising a membranous porcine small intestine submucosa, a basement membrane, dermis, pericardium, peritoneum, or a three-dimensional ligament or achilles tendon.