A method for effectively controlling bacterial endotoxins in biological tissue materials

Abstract

The present invention relates to the field of biomaterial technology, and in particular to a method for effectively controlling bacterial endotoxins in biological tissue materials. The method comprises the following steps: a first step, transportation and preservation: collecting and removing fat from fresh biological tissue materials and placing them in a transportation and preservation solution for low-temperature transportation; a second step, gradient cleaning: placing the fresh biological tissue materials treated as above in cleaning solution I and cleaning solution II in sequence for immersion and cleaning; a third step, chemical crosslinking and fixation: placing the biological tissue materials treated as above in a chemical crosslinking solution for fixation; the method can effectively control bacterial endotoxins in biological tissue materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a method for effectively controlling bacterial endotoxins in biological tissue materials. Background Art

[0002] Endotoxins are exogenous pyrogens that activate neutrophils and other cells, causing them to release an endogenous pyrogen that acts on the body's thermoregulatory center and causes fever. The main chemical component of endotoxins is lipopolysaccharide, which is present in the cell walls of most Gram-negative bacteria, including Escherichia coli, and is released through cell lysis. Lipopolysaccharide consists of three parts: polysaccharide O antigen, core polysaccharide, and lipid A, and is located in the outer layer of the Gram-negative bacterial outer membrane. Lipid A is a lipidated glucosamine disaccharide, a unique glycolipid compound formed through pyrophosphate bonds. It has a pyrogenic effect and is the toxic component of Gram-negative bacterial endotoxins. Endotoxins generally exist in the form of aggregates, and their phosphate groups chelate with divalent metal ions to further stabilize the aggregation of endotoxin molecules, promoting the formation of larger endotoxin complexes.

[0003] Endotoxin contamination is currently widespread in biopharmaceuticals and medical devices. Trace amounts of endotoxin can enter the human body through the bloodstream and cause fever or even death. To ensure the safety of biological products and medical devices, the current standard (USP27) stipulates a limit of 0.5 EU / ml or 20 EU / device for products that directly or indirectly contact the cardiovascular and lymphatic systems. For devices that come into contact with cerebrospinal fluid, the limit is 0.06 EU / mL or 2.15 EU / device. For devices that directly or indirectly contact the intraocular environment, a lower endotoxin limit may be appropriate.

[0004] Endotoxins in medical devices primarily originate from the raw materials themselves and from external sources introduced during processing. Common methods for removing endotoxins include acid-base hydrolysis, activated carbon adsorption, ultrafiltration, ion exchange chromatography, high temperature, and oxidation, but most of these methods target the solution or container surface. Currently, there are few reports on methods for removing endotoxins from biological tissue materials. Therefore, effective endotoxin removal from biological tissue materials is crucial to the safety of medical devices containing these materials.

[0005] Only one patent related to the removal of endotoxins from biological tissue materials has been found:

[0006] Patent CN105251049 utilizes an ion buffer solution to soak and clean biomedical materials. Although this method is simple to operate, it cannot achieve a more ideal effect in removing endotoxins. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for effectively controlling bacterial endotoxins in biological tissue materials. The method performs gradient cleaning on the biological tissue materials, and ultimately makes the biological tissue material medical devices reach or even fall below the endotoxin standard.

[0008] In order to solve the above technical problems, the present invention solves them through the following technical solutions: a method for effectively controlling bacterial endotoxins in biological tissue materials, comprising the following steps: the first step, transportation and preservation: the fresh biological tissue materials collected and de-fatted are placed in a transportation and preservation solution for low-temperature transportation; the second step, gradient washing: the fresh biological tissue materials treated as above are placed in washing solution I, washing solution II, and washing solution III in sequence for immersion and washing; the third step, chemical cross-linking and fixation: the biological tissue materials treated as above are placed in a chemical cross-linking solution for fixation.

[0009] According to one embodiment, the biological tissue material includes various homologous / xenogeneic animal dermal tissues, pericardium, heart valves, blood vessel walls, etc.

[0010] According to one embodiment, the transport preservation solution is an isotonic ionic solution containing antibiotics.

[0011] According to one embodiment, the antibiotics mentioned above are preferably broad-spectrum antibiotics.

[0012] According to one embodiment, the low temperature is 0°C to 10°C (excluding 0°C).

[0013] According to one embodiment, the cleaning solution I is an isotonic ionic solution containing a surfactant.

[0014] In a preferred embodiment, the surfactant in the cleaning solution I is preferably a mild surfactant.

[0015] According to one embodiment, the surfactant in the cleaning solution I is preferably Tween 80 or Triton X-100.

[0016] According to one embodiment, the concentration of the surfactant in the cleaning solution I is 0.001% to 0.01%.

[0017] According to one embodiment, the cleaning solution II is an isotonic ionic solution containing a surfactant.

[0018] According to one embodiment, the surfactant in the cleaning solution II is preferably an ionic surfactant.

[0019] In a preferred embodiment, the above-mentioned ionic surfactant is preferably sodium lauryl sulfate.

[0020] According to one embodiment, the concentration of the surfactant in the cleaning solution II is 0.1% to 1.0%.

[0021] According to one embodiment, the cleaning solution III is an isotonic ionic solution.

[0022] According to one embodiment, the cations in the isotonic ion solutions in the cleaning solution I, the cleaning solution II, and the cleaning solution III are selected from one of the following: sodium ions and potassium ions.

[0023] According to one embodiment, the anions in the isotonic ion solutions in the cleaning solution I, the cleaning solution II, and the cleaning solution III are selected from one of the following: chloride ion; phosphate ion; hydrogen phosphate ion; dihydrogen phosphate ion; bicarbonate ion; and sulfate ion.

[0024] According to one embodiment, the chemical cross-linking solution is preferably glutaraldehyde.

[0025] Compared with the prior art, the present invention removes endotoxins from biological tissue materials by adding a gradient cleaning process, which has the following advantages:

[0026] 1. Different from the prior art, in one embodiment of the present invention, antibiotics are added during the transportation of fresh biological tissue materials to inhibit bacterial growth and reduce the difficulty of subsequent removal of bacteria and endotoxins.

[0027] 2. Different from the prior art, one embodiment of the present invention adopts a gradient cleaning method to remove, lyse and inhibit bacteria contained in biological tissue materials multiple times, in multiple aspects and in depth. First, the biological tissue material is placed in a cleaning solution I containing a low concentration of surfactant. The low concentration of surfactant in the treatment solution can change the relative affinity of the bacterial surface to water, making the bacteria better dispersed in water and easier to elute. Then, the biological tissue material is placed in a cleaning solution II containing a high concentration of surfactant. The high concentration of surfactant in the treatment solution can lyse bacterial cells and denature proteins, causing most bacteria to lyse and die. Finally, the treated biological tissue material is placed in an isotonic ionic solution for cleaning, which is beneficial for removing the above-mentioned bacterial endotoxins, nucleic acids and proteins in biological tissue cells, phospholipids on cell membranes and other components that cause pyrogens, immunity and calcification after lysis, thereby increasing the safety and durability of the material.

[0028] 3. Because the cell membranes of fresh biological tissue materials contain phospholipids, which are one of the main factors causing calcification in biological tissue materials, unlike the prior art, one embodiment of the present invention contains a high concentration of surfactant in cleaning solution II, which not only lyses bacteria but also cells carried by the fresh biological tissue itself, thereby clearing antigens from animal-derived biological materials. The embodiments of this application can achieve other advantageous technical effects not listed here, which may be partially described below and are predictable and understandable to those skilled in the art after reading this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Gram-negative bacterial cell wall structure and lipid A chemical formula structure.

[0030] Figure 2 Schematic diagram of HE staining of the control group.

[0031] Figure 3 Schematic diagram of HE staining of the experimental group. DETAILED DESCRIPTION

[0032] The following description and accompanying drawings illustrate various embodiments of the present invention. A person skilled in the relevant art will be able to implement other embodiments of the present invention without one or more of the details described herein. Therefore, it is not the applicant's intention to limit or in any way restrict the scope of the appended claims to the detailed description. Although the implementation process of each step is described in detail below with reference to the accompanying drawings, the steps and step order described, as well as their terminology, should not be considered necessary to implement all embodiments of the present invention.

[0033] Likewise, it is understood that the phrases and terms used herein are for descriptive purposes and should not be considered restrictive. The use of "include," "comprising," or "having" and variations thereof herein is intended to encompass the items listed thereafter and their equivalents as well as additional items.

[0034] The present application will be described in more detail below with reference to different embodiments and examples of several aspects of the application.

[0035] This embodiment provides a method for effectively controlling bacterial endotoxins in biological tissue materials, which includes the following steps: the first step, transportation and preservation: the fresh biological tissue material collected and defatted is placed in a transportation and preservation solution for low-temperature transportation; the second step, gradient cleaning: the fresh biological tissue material that has undergone the above treatment is placed in cleaning solution I, cleaning solution II, and cleaning solution III for immersion and cleaning in sequence; the third step, chemical cross-linking and fixation: the biological tissue material that has undergone the above treatment is placed in a chemical cross-linking solution for fixation.

[0036] Step 1: Obtain fresh biological tissue materials;

[0037] Step 2: Place the biological tissue material in a transport preservation solution and transport it at low temperature;

[0038] Step 3: Place the above biological tissue material in cleaning solution I for soaking and cleaning;

[0039] Step 4: Soak the biological tissue material after cleaning with cleaning solution I in cleaning solution II for cleaning;

[0040] Step 5: Place the biological tissue material cleaned with cleaning solution II in cleaning solution III

[0041] Medium soak cleaning;

[0042] Step 6: Fix the biological tissue material processed in the above steps in a chemical crosslinking agent solution.

[0043] According to the method for effectively controlling bacterial endotoxins in biological tissue materials of the present invention,

[0044] In step 1: obtaining fresh biological tissue materials, such as bovine pericardium, porcine pericardium, porcine heart valve, etc.

[0045] In step 2: the biological tissue material is transported at low temperature, and the transport preservation solution is an isotonic ion solution containing penicillin, streptomycin, etc., and the low temperature is 0°C to 10°C (excluding 0°C).

[0046] In step 3: the immersion cleaning in cleaning solution I is to immerse the biological tissue material in an isotonic ionic solution containing 0.001% to 0.01% of Tween 80, Triton X-100 or other surfactants for 0.5 to 1 hour, and then shake (80 to 120 rpm) and clean it 2 to 4 times.

[0047] In step 4, the cleaning solution II is to immerse the biological tissue material cleaned with cleaning solution I in an isotonic ionic solution containing 0.1% to 1.0% sodium lauryl sulfate surfactant for 0.5 to 1 hour, and then shake (80 to 120 rpm) and clean it 2 to 4 times.

[0048] In step 5, the cleaning solution III is to immerse the biological tissue material that has been cleaned with the cleaning solution II in an isotonic ion solution such as physiological saline or PBS for 0.5 to 1 hour, and then shake (80 to 120 rpm) and clean it 2 to 4 times.

[0049] In step 6: the chemical cross-linking solution mainly comprises formaldehyde, glutaraldehyde, etc. Specific embodiment one:

[0051] Detection of initial contaminating bacteria and bacterial endotoxins in bovine pericardial materials

[0052] Methods: Twenty pieces of pericardium from healthy male and female yellow cattle aged about 2 years old in Ningbo were collected, wiped to remove fat, and divided into two groups, 10 pieces in each group. The control group: the bovine pericardium was collected and processed, transported at low temperature, and simply cleaned. After cleaning, 3 pieces were randomly taken out for detection of the number of contaminated bacteria, and the count was N1. The remaining 7 pieces of bovine pericardium were fixed with glutaraldehyde and cut into 50mm×60mm pieces for endotoxin detection. The experimental group: the bovine pericardium was collected and processed; in the first step, it was placed in physiological saline containing 100IU penicillin and 100μg / mL streptomycin for low temperature transportation; in the second step, the above-mentioned cleaning was carried out. The remaining bovine pericardium is then soaked in 0.1% sodium lauryl sulfate saline for 30 minutes, then washed twice with shaking (80 rpm). Three slices are removed for bacterial count testing, with the count being N2. Third, the cleaned bovine pericardium is soaked in 0.1% sodium lauryl sulfate saline for 30 minutes, then washed twice with shaking (80 rpm). Fourth, the twice-washed bovine pericardium is soaked in saline for 30 minutes, then washed twice with shaking (80 rpm). Fifth, the remaining bovine pericardium is fixed in glutaraldehyde solution. After fixation, it is cut into 50 mm × 60 mm slices for endotoxin testing.

[0053] Result analysis: After the bovine pericardium biological tissue materials were treated by different methods, the number of contaminating bacteria in the experimental group was 4.1×10 3 cfu / cm 2 The number of contaminating bacteria in the control group was 9.2×10 5 cfu / cm 2 There was a significant difference between the two groups; the endotoxin in the experimental group (50mm×60mm) was no more than 2.15EU / piece, that is, no more than 0.072EU / cm 2 The endotoxin content of the control group (50mm×60mm) was greater than 20EU / piece, that is, greater than 0.67EU / cm 2 , with significant differences.

[0054] Conclusion: Polysorbate 80 can promote the elution of contaminating bacteria in biological tissue materials. The treatment method of this invention can effectively control endotoxins in biological tissue materials. Specific embodiment two:

[0056] Detection of initial contamination bacteria, cell residues and bacterial endotoxins in bovine pericardial materials

[0057] Methods: Twenty pieces of pericardium from healthy male and female yellow cattle aged about 2 years old in Henan Province were collected, wiped to remove fat, and divided into two groups, 10 pieces in each group. Control group: After the bovine pericardium was collected and processed, it was transported at low temperature and simply cleaned. After cleaning, 3 pieces were randomly taken out for detection of the number of contaminated bacteria, and the count was N1. The remaining 7 pieces of bovine pericardium were fixed with glutaraldehyde, and after fixation, they were cut into 100mm×40mm pieces for endotoxin detection and cut into 10mm×10mm pieces for HE staining to observe the cell residues; Experimental group: After the bovine pericardium was collected and processed; in the first step, it was placed in a 500IU penicillin and 100μg / mL streptavidin solution. The bovine pericardium is then transported at low temperature in a saline solution containing 0.01% Tween 80. Second, the bovine pericardium is soaked in a saline solution containing 0.01% Tween 80 for 1 hour, then washed four times with shaking (120 rpm). Three slices are removed for bacterial count analysis, with the count being N2. Third, the remaining bovine pericardium after washing is soaked in a saline solution containing 1.0% sodium dodecyl sulfate for 1 hour, then washed four times with shaking (120 rpm). Fourth, the bovine pericardium, which has undergone the second wash, is soaked in saline solution for 1 hour, then washed four times with shaking (120 rpm). Fifth, the bovine pericardium is fixed in a glutaraldehyde solution. After fixation, the slices are cut into 100 mm × 40 mm pieces for endotoxin testing and 10 mm × 10 mm pieces for HE staining to observe residual cells.

[0058] Result analysis: After the bovine pericardium biological tissue materials were treated by different methods, the number of contaminating bacteria in the experimental group was 3.99×10 3 cfu / cm 2 The number of contaminating bacteria in the control group was 9.13×10 5 cfu / cm 2 There was a significant difference between the two groups; the endotoxin in the experimental group (100mm×40mm) was no more than 2.15EU / piece, that is, no more than 0.054EU / cm 2 The endotoxin content of the control group (100mm×40mm) was greater than 20EU / piece, that is, greater than 0.50EU / cm 2 , with significant differences. Figure 2 and Figure 3 It can be seen that the bovine pericardium in the control group had more and clearly visible cell nuclei after HE staining, while the bovine pericardium in the experimental group had no visible cell nuclei, indicating that the cells were completely removed.

[0059] Conclusion: Low concentration of surfactant can promote the elution of contaminating bacteria in biological tissue materials, and high concentration of surfactant can effectively remove cells in biological tissue materials. The treatment method of this invention can effectively control endotoxins in biological tissue materials.

[0060] In summary, the above content is only a preferred embodiment of the present invention. For ordinary technicians in this field, according to the concept of the present invention, there may be changes in the specific implementation method and application scope. The content of this specification should not be understood as limiting the present invention.

Claims

1. A method for effectively controlling bacterial endotoxins in biological tissue materials, comprising the following steps: a first step, transportation and preservation: collecting and removing fat from fresh biological tissue materials and placing them in a transportation and preservation solution for low-temperature transportation; a second step, gradient washing: soaking and washing the above-mentioned fresh biological tissue materials in washing solution I, washing solution II, and washing solution III in sequence; a third step, chemical crosslinking and fixation: fixing the biological tissue materials after the above-mentioned treatments in a chemical crosslinking solution; the washing solution I and the washing solution II are both isotonic ionic solutions containing surfactants, the concentration of the surfactant in the washing solution I is lower than the concentration of the surfactant in the washing solution II, the surfactant concentration in the washing solution I is 0.001% to 0.01%, the surfactant concentration in the washing solution II is 0.1% to 1.0%, and the washing solution III is an isotonic ionic solution.

2. The method for effectively controlling bacterial endotoxins in biological tissue materials according to claim 1, characterized in that: The biological tissue materials include dermal tissues, pericardium, heart valves, and blood vessel walls of various homologous / xenogeneic animals.

3. The method for effectively controlling bacterial endotoxins in biological tissue materials according to claim 1, characterized in that: The transport preservation solution is an isotonic ion solution containing antibiotics.

4. The method for effectively controlling bacterial endotoxins in biological tissue materials according to claim 1, characterized in that: The cations in the isotonic ionic solution are selected from one of the following: sodium ions and potassium ions.

5. The method for effectively controlling bacterial endotoxins in biological tissue materials according to claim 1, characterized in that: The anions in the isotonic ionic solution are selected from one of the following: chloride ion; phosphate ion; hydrogen phosphate ion; dihydrogen phosphate ion; bicarbonate ion; and sulfate ion.

Images