CN114577983A

CN114577983A - Experimental method and experimental device for in-vitro degradation experiment

Info

Publication number: CN114577983A
Application number: CN202210091844.6A
Authority: CN
Inventors: 王颖; 宫海波; 展京美; 肖杰
Original assignee: Xi'an Zhuoqia New Material Technology Co ltd
Current assignee: Xi'an Zhuoqia New Material Technology Co ltd
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2022-06-03

Abstract

The invention relates to an experimental method for in vitro degradation experiments, which is characterized by comprising the following steps of: processing a sample, preparing fluid gel, embedding the sample, degrading and calculating. Wherein the making of the fluid gel is melting a main component of the gel into the fluid gel; embedding the sample, namely embedding the processed sample into the fluid gel, and forming the embedded gel embedded with the sample after curing; and the degradation is to put the embedded gel into a degradation container containing degradation liquid to be soaked for a certain time. The invention also provides an in vitro degradation experimental device, which comprises a degradation container, an embedding mould and embedding gel. The invention can be used for in vitro degradation experiments, and the gas and ions generated by degradation can be coated around the sample, so that the experimental effect is closer to the in vivo degradation.

Description

Experimental method and experimental device for in vitro degradation experiment

Technical Field

The invention relates to the field of implanted material experiments, in particular to an experimental method and an experimental device for in-vitro degradation experiments.

Background

Before the medical degradable material is applied to a human body, safety verification of animal experiments is required to be firstly carried out, and safety verification needs to be carried out in vitro before animal experiment verification is carried out. Except for conventional in vitro biocompatibility experiments, compared with the traditional medical materials, the biodegradable medical materials and products need to determine the degradation morphology and the degradation rate of the materials and products in vitro so as to provide theoretical basis and guidance for the degradation of the materials and products implanted in vivo.

At present, in-vitro degradation research of biodegradable materials mainly comprises the steps of directly soaking the materials or products in simulated body fluid or culture medium to observe degradation morphology and calculate degradation rate. The degradation product and the gas generated by degradation are dispersed, the gas directly overflows the test liquid, and cannot be gathered in a small range around the sample, a large amount of degradation gas, molecules and ions are generated particularly in an in-vitro degradation experiment of the medical magnesium alloy material, and the dispersion of the degradation product has great influence on the in-vivo degradation simulation. On the other hand, the composition of the simulated body fluid or culture medium is very different from that of the implant, so that the in vitro data obtained by the current test method is very different from the in vivo results.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an experimental method and device for an in-vitro degradation experiment, which can effectively simulate the internal environment of a material or a product implanted in a body, thereby obtaining a result similar to that in the body.

The in vitro degradation experimental method comprises the steps of processing a sample; preparing fluid gel, and melting the main components of the gel into the fluid gel; embedding a sample, embedding the processed sample into the fluid gel, and forming an embedded gel embedded with the sample after curing; degrading, namely soaking the embedded gel in a degradation container containing degradation liquid for a certain time; and measuring and calculating, namely taking the sample out of the embedding gel after degradation is finished, and observing and measuring and calculating.

Further, the method for melting the gel is to add the main components of the gel into the gel dissolving solution, heat and stir the gel to form the fluid gel.

Further, the method for embedding the test sample comprises the following steps: pouring a part of the fluid gel into an embedding mould until the sample does not sink after the sample is placed, then placing the sample, continuously pouring the fluid gel into the embedding mould until the embedding mould is full, and finally curing and demoulding to form the embedded gel embedded with the sample.

Further, during the degradation process, the temperature should be maintained at 37 ± 1 ℃.

Further, in the degradation process, a static soaking mode, a dynamic soaking mode of continuously updating the solution and a semi-static soaking mode of circularly flowing the solution can be adopted.

And further, carrying out the measurement and calculation on the sample after the degradation is finished by utilizing a weight loss method.

Further, the mass ratio of the main components of the gel to the degradation liquid is 1: 4-25.

The invention also relates to an in vitro degradation experimental device which is characterized by comprising a degradation container, wherein degradation liquid is contained in the degradation container; an embedding mold, wherein fluid gel and a sample can be sequentially added into the embedding mold, so that the sample is embedded into the gel to prepare embedded gel; the embedding gel is suspended in a degradation liquid and can be used for embedding the sample.

Further, the embedding gel is transparent or semitransparent, and the embedding thickness of the embedding gel for embedding the sample is 1-20 mm.

Further, the experimental device further comprises an internal environment simulation device for providing a constant degradation temperature and a necessary controllable circulating flow for the degradation liquid in the degradation container.

The in vitro degradation experiment performed by using the experimental device and the experimental method has the following advantages:

(1) in the in vitro degradation experiment of the invention, a sample is embedded into the permeable embedding gel, and organic molecules contained in human body are added into the degradation liquid, so that the complex composition of extracellular fluids of different tissues and the wrapping structures of different tissues during in vivo degradation are simulated, and the degradation environment of the sample is similar to that of the sample in vivo.

(2) The in vitro degradation experiment of the invention can effectively simulate the gas wrapping state generated by the degradation of the sample implanted into the human body, and particularly, when the sample is made of magnesium alloy material, the embedding gel can wrap the gas, molecules and ions generated by the degradation of the magnesium alloy material around the sample, so that the surface of the sample after the in vitro degradation experiment is closer to the in vivo degradation state. Meanwhile, the degradation rate of the in vitro degradation experiment of the invention is closer to the degradation rate of in vivo degradation.

Drawings

FIG. 1 is a flow chart of the in vitro degradation assay method of the present invention.

FIG. 2 is a schematic diagram of an in vitro degradation experimental apparatus according to the present invention.

FIGS. 3A and 3B are reference diagrams of in vitro degradation according to two embodiments of the present invention.

FIG. 3C is a reference diagram of an in vitro degradation experiment in the prior art.

Detailed Description

The technical means adopted by the invention to achieve the predetermined object of the invention are further described below with reference to the drawings and the preferred embodiments of the invention.

As shown in fig. 1, the present invention provides an in vitro degradation experimental apparatus, which comprises: degradation container 1, degradation liquid 2, embedding gel 3 and embedding mould (not shown in the figure). Wherein the degradation container 1 is filled with degradation liquid 2, and the embedded gel 3 is suspended in the degradation liquid 2. The embedding gel 3 embeds a sample 4 to be degraded in vitro as shown in fig. 1. The embedding mold is used to make an embedding gel 3 embedding a sample 4.

The degradation container 1 may include a centrifuge tube, a beaker, a flask, etc., and the present invention is not limited to the material and shape of the degradation container 1 as long as it can be used to contain the degradation liquid 2.

The degradation liquid 2 may include a simulated body fluid, a mixture of the simulated body fluid and organic molecules, a culture medium, a mixture of the culture medium and organic molecules, or the like.

Wherein the simulated body fluid comprises Hanks', PBS, SBF, m-SBF, EBSS, artificial saliva, artificial plasma, and CO by infusion₂The pH value of the degradation liquid 2 can be buffered by simulating body fluid of any solution which can be used for in vitro degradation, such as the pH value of the degradation liquid.

The culture medium comprises MEM, DMEM, BME, IMDM and other culture media which can be used for in vitro degradation; the organic molecules include any organic small molecules and organic macromolecules required by organisms, wherein the organic small molecules include vitamins, amino acids, glucose and the like, and the organic macromolecules include proteins, polysaccharides, nucleic acids, proteoglycans and the like.

Specifically, the concentration of serum protein in the degradation solution 2 in the simulated blood environment is as follows: 38-40 g/mL; the concentration of glucose was: 6.5-11 mg/mL. The concentration of glucose in the degradation solution 2 when the simulated subcutaneous implantation was: 29.5-34 mg/mL. The concentration of myofibrillar protein in the degradation solution 2 when the simulated intramuscular implantation is as follows: 200-300 mg/mL. The concentration of collagen in the degradation liquid 2 when the simulated bone is implanted is as follows: 110-147 mg/mL.

The size of the embedded gel 3 may vary depending on the size of the sample 4. The shape of the embedding gel 3 can be made into the shapes of a cylinder, a cube, a cuboid, a vertebral body, a similar bone structure and the like and any shape which can be used for in vitro degradation; the embedded gel 3 can also be combined with a bone-like structure, the bone-like structure comprises HA, TCP, beta TCP, a polymer scaffold, a composite material with various components and the like, and the bone-like structure is prepared by sintering, chemical reaction, 3D printing and other methods; the embedding gel 3 is in a transparent or translucent state, and its color is not limited.

As shown in fig. 1, the thickness of the embedding gel 3 around the test specimen 4 is an embedding thickness 34, and the embedding thickness 34 may be changed according to the corresponding in vivo implantation position in the present invention, and the present invention is not limited thereto. The embedding thickness 34 is generally in the range of 1mm to 20 mm.

The shape of the embedding mould is not limited, and the embedding mould can be made of materials such as silica gel, rubber or polyethylene. Inside the embedding mold, a marked line is carved at the half of the height of the embedding mold, and when the sample is embedded, the sample 4 is placed at the marked line of the embedding mold, thereby ensuring that the sample 4 is positioned at the center of the embedding gel 3.

Preferably, the in vitro degradation experimental apparatus of the present invention further comprises an internal environment simulation device (not shown in the figure), which can provide a constant temperature and a necessary controllable circulation flow for the degradation solution in the degradation container 1. The internal environment simulation apparatus may include a water bath tank and a circulation pump: providing a constant ambient temperature by placing the degradation container 1 in a water bath cabinet; the degradation liquid is made to flow by a circulation pump installed on the degradation vessel 1, but not limited thereto.

As shown in FIG. 2, the experimental method for in vitro degradation experiment using the in vitro degradation experimental apparatus of the present invention comprises the following steps:

s1 processing sample 4: the sample 4 may be processed into a desired shape such as a wafer, a cylinder, etc., and the shape of the sample is not limited.

S2 melting the gel: adding the main component of the gel into the gel dissolving solution, heating and stirring to melt into fluid gel.

S3 embedding the specimen, including the following two ways:

(1) and firstly pouring a part of fluid gel into the embedding mold until the sample 4 is placed, then placing the sample when the sample does not sink, then continuously pouring the other part of the fluid gel into the embedding mold until the embedding mold is full, and forming the embedding gel 3 after curing and demolding.

(2) Pouring the fluid gel into an embedding mould until the mould is full, adding the sample 4 into the fluid gel, removing the overflowing fluid gel, and forming the embedding gel 3 after curing and demoulding.

The above two modes are preferred to the mode (1), thereby reducing the waste of the fluid gel.

S4 degradation: putting the embedded gel 3 embedded with the sample 4 into a degradation container 1 containing degradation liquid 2, then putting the degradation container 1 into a water bath tank, keeping the temperature at 37 +/-1 ℃ for soaking for a period of time, and regularly observing and recording the degradation process. The soaking mode can comprise static soaking, semi-static soaking and dynamic soaking. The degradation liquid 2 is required to be kept static during static soaking, and the degradation liquid 2 is not replaced; in the semi-static soaking, the degradation liquid 2 needs to be kept flowing, but the degradation liquid 2 is not replaced, and the degradation liquid can flow by using a circulating pump, but the limitation is not taken; the dynamic soaking needs to keep the degradation liquid 2 flowing, and the degradation liquid 2 is replaced periodically.

S5, measurement: after the degradation is completed, the sample 4 embedded in the gel 3 is taken out, observed and measured for the required parameters. Wherein the degradation rate of the sample can be calculated by a weight loss method.

In the process of melting the gel at S2, the main components of the gel include agar, konjac flour, gelatin, and the like. The embedding gel 3 prepared by S2 and S3 comprises medical hydrogel, agar gel, konjac glucomannan, gelatin and other gels which can be used for embedding materials or products. During preparation, the mass ratio of the main components of the gel to the degradation liquid 2 is 1: 4-25. In order to better simulate the internal environment, the agar gel is preferably prepared in a mass ratio of agar to degradation liquid 2 of 1: 9-17; the preparation ratio of the konjac glucomannan is that the mass ratio of the konjac flour to the degradation liquid 2 is 1: 5-15; the preparation ratio of the gelatin to the degradation liquid 2 is 1: 8-20 by mass; the preparation ratio of the agar-gelatin composite gel is that the mass ratio of agar to gelatin to degradation liquid 2 is 1: 0.2-1.5: 10-30.

In the process of melting the gel at S2, the gel solution may be prepared by mixing the solution with additives. Wherein the dissolving solution comprises degradation solution 2, distilled water or other solution capable of dissolving gel; the additive is other gel or cross-linking agent different from the main component of the gel.

The in vitro degradation experiment carried out by using the experimental device and the experimental method has the following advantages:

(1) in the in vitro degradation experiment of the invention, a sample 4 is embedded into a permeable embedding gel 3, and organic molecules contained in human body are added into a degradation liquid 2, so that the complex composition of extracellular fluid of different tissues and the wrapping structures of different tissues during in vivo degradation are simulated, and the degradation environment of the sample 4 is similar to that of the in vivo.

(2) The in vitro degradation experiment of the invention can effectively simulate the gas wrapping state generated by degradation after the sample 4 is implanted into a human body, and particularly, when the sample 4 is made of magnesium alloy material, the embedding gel 3 can wrap the gas, molecules and ions generated by the degradation of the magnesium alloy material around the sample 4, so that the surface of the sample 4 after the in vitro degradation experiment is closer to the in vivo degradation state. Meanwhile, the degradation rate of the in vitro degradation experiment of the invention is closer to the degradation rate of in vivo degradation.

The technical scheme and effect of the invention are further illustrated by the specific examples below.

The first embodiment is as follows:

in this example, the experimental procedure for the in vitro degradation experiment is as follows:

s1 processing sample 4A: taking the medical magnesium alloy biodegradable material as a sample 4A, and processing the sample 4A into a wafer with phi of 8 multiplied by 3 mm.

S2 melt the gel: adding agar into sterile water, stirring, and heating to 80 deg.C to obtain fluid gel.

S3 embedded specimen: pouring the fluid gel into a cylindrical silica gel embedding mold with the diameter of 16mm and the height of 11mm, wherein the thickness of the fluid gel around the sample is 8mm, firstly pouring the fluid gel to a half position of a container, putting the sample 4A when the sample 4A is placed on the gel and does not sink, and continuously pouring the fluid gel until the embedding mold is full. After curing and demoulding, the embedded gel 3 embedded with the sample 4A is formed.

S4 degradation: the embedded gel 3 is placed in a degradation vessel 1 containing a degradation liquid 2, in this example the m-SBF simulant body fluid. And then the degradation container is placed into a water bath box with the temperature of 37 +/-1 ℃, the liquid is replaced every 7 days, and the degradation container is soaked for 14 days. The state of the sample 4A was observed periodically.

S5, measurement: after the degradation is completed, the sample 4A is taken out from the embedding gel 3, the surface of the sample 4A is observed, and the degradation rate is calculated by using a weight loss method.

The experimental results are as follows: as shown in fig. 3A, the sample 4A generates gas and forms small bubbles when it is degraded, and the small bubbles cannot be dispersed and gather around the sample 4A because the sample 4A is embedded by the embedding gel 3; as the degradation experiment continued, more and more small bubbles accumulated around the sample 4A within the embedding gel 3, gradually forming larger bubbles and encapsulating the sample 4A. The degradation rate in this experiment was 0.52. + -. 0.031 mm/a.

On the other hand, the same two samples as sample 4A were taken as sample 5 (not shown) and sample 6 (not shown).

The sample 5 was directly put into the same degradation vessel 1 containing the m-SBF-simulated body fluid as the degradation solution 2 as in example one while maintaining a water bath at 37 + -1 deg.C, the fluid was changed every 7 days, and the degradation rate was calculated for 14 days, wherein the outer layer of the sample 5 was not coated with the embedding gel 3.

Sample 6 was then implanted directly into the muscle for one month and the degradation rate was calculated.

The degradation rate of sample 4A was found to be close to that of sample 6 and much less than that of sample 5, as detailed in table 1.

Table 1: degradation rates of samples obtained by different degradation methods

Degradation method	Sample No. 4A	Sample No. 5	Sample No. 6
				Degradation Rate (mm/a)	0.52±0.031	2.077±0.042	0.25±0.071

The second embodiment:

the experimental method and experimental apparatus of this embodiment are substantially the same as those of the first embodiment, except that:

and selecting Hanks' solution as the degradation solution 2.

The B sample 4B is a magnesium alloy material and is processed into a cylindrical shape of Φ 2.5 × 10 mm.

C at the step of melting and gelling S2, konjak powder is added into Hanks' solution, stirred uniformly and heated to 80 ℃.

D, the embedding mold is a silica gel mold with the diameter of 12.5mm and the height of 20 mm. The embedding thickness is 10mm

E in the S4 degradation stage, changing degradation liquid 2 every two days.

The experimental results are as follows: as shown in fig. 3B, the sample 4B in example two generates gas and forms small bubbles when it is degraded, and the small bubbles cannot be dispersed and gathered around the sample 4B because the sample 4B is embedded by the embedding gel 3; as the degradation experiment continued, more and more small bubbles accumulated around the sample 4B within the embedding gel 3, gradually forming larger bubbles and encapsulating the sample 4B.

Control experiments were set up here: as shown in FIG. 3C, the same sample 4C as the sample 4B in example two was taken, the sample 4C not coated with the embedding gel 3 was directly put into the degradation solution 2, and the degradation state was observed in the same manner as in example two in the rest of the experiment. In the control experiment, the gas generated from the sample 4C not coated with the embedding gel 3 at the initial stage of degradation was not accumulated around the sample 4C, and as the control experiment continued, no accumulation of bubbles around the sample 4C was observed.

It can be seen that, compared with the sample 4B coated with the embedded gel 3 in the second embodiment of the present invention, the gas generated from the embedded gel 3 can be effectively gathered around the sample 4B, so that the in vitro degradation experiment in the second embodiment is closer to the in vivo degradation state. On the other hand, the average in vitro degradation rate of example two was calculated to be 0.30 ± 0.018mm/a, which is not significantly different from the in vivo results.

Example three:

the experimental method and the experimental apparatus of this embodiment are substantially the same as those of the first embodiment, except that:

and the degradation liquid A2 is simulated body fluid of SBF + 10% FBS.

B sample 4D (not shown) was processed into a cylinder of 10X 10 mm.

C embedding mould is 20mm diameter high 20 mm's silica gel mould, and embedding thickness is 10 mm.

D soaking mode is the same as the embodiment.

The experimental results are as follows: the surface change of sample 4D during the in vitro degradation experiment was similar to the examples. After the degradation is finished, the sample 4D is taken out, and the calculated average degradation rate is 0.21 +/-0.047 mm/a, and the result is not obviously different from the in-vivo degradation result.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An experimental method for in vitro degradation experiments, comprising:

processing a sample;

preparing fluid gel, and melting the main components of the gel into the fluid gel;

embedding a sample, embedding the processed sample into the fluid gel, and forming an embedded gel embedded with the sample after curing;

degrading, namely soaking the embedded gel in a degradation container containing degradation liquid for a certain time;

and measuring and calculating, namely taking the sample out of the embedding gel after degradation is finished, and observing and measuring and calculating.

2. The experimental method of claim 1, wherein said method of melting said gel comprises adding said gel-forming agent to a solution of said gel, and stirring said solution with heat to form said fluid gel.

3. The assay method of claim 1, wherein the method of embedding the sample is: pouring a part of the fluid gel into an embedding mold until the sample does not sink after the sample is placed in the embedding mold, then placing the sample in the embedding mold, continuously pouring the fluid gel into the embedding mold until the embedding mold is filled with the fluid gel, and finally curing and demolding to form the embedding gel embedded with the sample.

4. The assay of claim 1 wherein the temperature is maintained at 37 ± 1 ℃ during said degradation.

5. The experimental method as claimed in claim 1, wherein during the degradation process, a static soaking mode, a dynamic soaking mode of continuously renewing solution and a semi-static soaking mode of circulating solution can be adopted.

6. The assay of claim 1 wherein said evaluation of said sample after degradation is performed by weight loss.

7. The experimental method as claimed in claim 1, wherein the mass ratio of the main component of the gel to the degradation liquid is 1: 4-25.

8. An in vitro degradation experimental device, comprising:

the degradation container is filled with degradation liquid;

an embedding mold, wherein fluid gel and a sample can be sequentially added into the embedding mold, so that the sample is embedded into the gel to prepare embedded gel;

the embedding gel is suspended in a degradation liquid, and the embedding gel can be used for embedding the sample.

9. The experimental device according to claim 8, wherein the embedding gel is transparent or translucent, and the embedding thickness of the embedding gel for embedding the test sample is 1mm to 20 mm.

10. The experimental set-up of claim 8, further comprising an internal environment simulation device for providing a constant degradation temperature and a necessarily controlled circulation flow of said degradation liquid in said degradation vessel.