CN115704047A - Toxicity detection method for in-vitro cells of implant material - Google Patents

Abstract

The invention provides a toxicity detection method for in-vitro cells of an implanted material, which comprises the steps of firstly constructing a three-dimensional matrix carrier with a small pore diameter to simulate the in-vivo environment of reaction cells, then culturing the in-vitro cells of the implanted material on the three-dimensional matrix carrier, and finally carrying out toxicity detection on the in-vitro cells of the implanted material. The cells detected by the detection method are planted on the three-dimensional matrix carrier with small aperture, and the planted cells can play a normal physiological function, so that the detected data is more real, the error of in-vivo and in-vitro experimental results is reduced, an evaluation value with higher reference value is obtained, and the method has great social benefit.

Description

Toxicity detection method for in-vitro cells of implant material

Technical Field

The invention belongs to the technical field of preclinical safety detection, and particularly relates to a method for in-vitro cytotoxicity detection of an implanted material.

Background

The pre-clinical safety test of implanted material in vivo usually includes culturing the leaching solution of implanted material into fiber cell, and evaluating the safety of the implanted material based on the damage of fiber, but in this method, the leaching solution is directly contacted with fiber cell in the culture plate, and the in vivo cell is grown under certain matrix support, so that the measured data has certain distortion.

Various materials are utilized to make cells adhere to a three-dimensional space to grow, a tissue structure similar to a human body is formed, and the solution is an effective solution to play the physiological function of the cells. Patents CN102010601A, CN101624472A and CN101624473A disclose that silk fibroin can be used to prepare a scaffold material under the action of a cross-linking agent, and the scaffold material belongs to a large-aperture scaffold, is only suitable for large-scale culture of hepatocytes, and has a limited application range.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting the toxicity of in-vitro cells of an implanted material, and the method can obtain a more accurate safety evaluation value.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a method for detecting toxicity of in vitro cells of an implanted material comprises the steps of firstly constructing a three-dimensional matrix carrier with small pore size to simulate the in vivo environment of reaction cells, then culturing the in vitro cells of the implanted material on the three-dimensional matrix carrier, and finally detecting the toxicity of the in vitro cells of the implanted material, wherein the three-dimensional matrix carrier is obtained by the following steps:

1) Mixing silk fibroin solution with the mass concentration of 3% and chitosan solution with the mass concentration of 3% according to the weight ratio of 5:5 or 4:6 in a volume ratio to obtain a mixed solution;

2) Adding a crosslinking agent into the mixed solution according to the proportion relation that every 100ml of the mixed solution is added with one part of the crosslinking agent to obtain a crosslinked product; one part of the crosslinker was prepared by dissolving 1g of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide and 0.26g N-hydroxysuccinimide in 1-2ml of 95% ethanol;

3) And (3) crosslinking the crosslinked product in a refrigerator at 4 ℃ overnight, freezing the crosslinked product in a refrigerator at-20 ℃ for 24h, freezing the crosslinked product in a refrigerator at-80 ℃ for 24h, freezing the crosslinked product in a gradient manner, and drying the frozen product in a freeze dryer for 48h.

Advantageous effects

Compared with the prior art, the invention has the following advantages:

1. the cells detected by the detection method are planted on the three-dimensional matrix carrier with small aperture, and the planted cells can play a normal physiological function, so that the detected data is more real, the error of in-vivo and in-vitro experimental results is reduced, an evaluation value with higher reference value is obtained, and the method has great social benefit.

2. The three-dimensional matrix carrier prepared by the invention has better pore cross-linking degree, pore diameter uniformity and structural stability, and the conformation of the three-dimensional matrix carrier is close to the space conformation in a cell body.

Drawings

FIG. 1 is a schematic diagram of three-dimensional porous structures of different volume ratios of silk fibroin solution and chitosan solution under SEM observation.

Figure 2 is a schematic diagram of the pore size of a three-dimensional porous structure under different volume ratios of the silk fibroin solution and the chitosan solution under SEM observation.

Detailed Description

The present invention is described in detail below by way of examples, it being necessary to point out here that this example is intended to illustrate the invention further only, but not to be construed as limiting the scope of the invention, which is to be given the full breadth of the appended claims, and that those skilled in the art may make insubstantial modifications and adaptations of the invention in light of the above teachings.

Silk Fibroin (SF) is a pure natural high-molecular polymer composed of 18 kinds of amino acids, and the amino acid composition of the Silk fibroin is very similar to that of human protein. A large number of researches show that the silk fibroin not only has good cell affinity, biocompatibility, biodegradability and flexibility, but also has controllability of a three-dimensional structure, and has wide sources and low price. In recent years, silk fibroin has been studied and developed as various biomaterials, such as artificial bone, artificial blood vessel, artificial nerve, artificial dermis, drug delivery carrier, and the like.

Chitosan (CS) is a partially deacetylated product of chitin, a glycosaminoglycan analogue, capable of mimicking the extracellular matrix composed of polysaccharides and glycosaminoglycans, serving as a substrate for cell adhesion, migration and eventual tissue formation. In addition, because of its good film-forming property, toughness, air permeability and moisture retention, biodegradability, antibacterial property, and wound coagulation promoting effect, it has been widely used in artificial dermis, anti-inflammatory agent, surgical suture, bone healing treatment, artificial blood vessel, and the like.

Tissue Engineering (Tissue Engineering) is an emerging discipline that combines cell biology, engineering and materials science into one entity. It is mainly used for constructing tissue or organ substitutes in vivo and in vitro to achieve the effects of repairing damaged tissues and maintaining or improving the form and function of organs. The tissue engineering scaffold material is a functional material which can be combined with tissue living cells and implanted into different tissues of an organism to replace corresponding defective tissues and organs.

Based on the above theoretical study, in the method for detecting toxicity of in vitro cells of an implant material of this embodiment, a three-dimensional matrix carrier with a small pore size is first constructed to simulate the in vivo environment of a reaction cell, then the in vitro cells of the implant material are cultured on the three-dimensional matrix carrier, and finally the in vitro cells of the implant material are subjected to toxicity detection, wherein the three-dimensional matrix carrier is obtained by the following method:

1) Mixing silk fibroin solution with the mass concentration of 3% and chitosan solution with the mass concentration of 3% according to the weight ratio of 5:5 or 4:6 in a volume ratio to obtain a mixed solution;

2) Adding a crosslinking agent into the mixed solution according to the proportion relation that every 100ml of the mixed solution is added with one part of the crosslinking agent to obtain a crosslinked product; one part of the crosslinker was prepared by dissolving 1g of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide and 0.26g N-hydroxysuccinimide in 1-2ml of 95% ethanol;

3) And (3) crosslinking the crosslinked product in a refrigerator at 4 ℃ overnight, freezing the crosslinked product in a refrigerator at-20 ℃ for 24h, freezing the crosslinked product in a refrigerator at-80 ℃ for 24h, freezing the crosslinked product in a gradient manner, and drying the frozen product in a freeze dryer for 48h.

Preferably, the silk fibroin solution is prepared from cocoon shells or silk. However, in addition to the selection of the three-dimensional matrix support material, the structure of the three-dimensional structure itself is also very important, which determines whether the cells can adhere well, proliferate, and differentiate on the support. As shown in the SEM image of fig. 1, all the composite scaffolds of all the ratios have interconnected mesh structures, but the pure fibroin scaffold exhibits an amorphous macroporous structure, while the pure chitosan scaffold is a poorly connected microporous structure. With the addition of chitosan, each proportional scaffold exhibited a porous network structure, as shown in fig. 2, which is a schematic diagram of a porous network structure in which 3% silk fibroin solution and 3% chitosan solution are mixed at a ratio of 5:5, 4:6, 3:7, 2:8, 1:0 and 0:1, respectively. Wherein, 5:5 and 4: the 6-ratio pore network structure is better than the other groups, showing a honeycomb-like porous structure and good pore diameter uniformity. The structure enables the three-dimensional matrix carrier to have a large surface area, can promote cells to extend into the holes, and provides corresponding surface space for cell adhesion and growth. And the connection among pores is developed, so that the migration and nutrient exchange of cells are promoted. Thus, the results of fig. 2 show that the addition of chitosan promotes the interaction of silk fibroin and chitosan, whereas too much chitosan is detrimental to the formation of cellular porous scaffolds. And 5:5 and 4: the 6 groups of scaffolds have obvious advantages in the pore cross-linking degree and the pore diameter uniformity compared with other groups, and are more suitable for the growth of cells.

The above-mentioned embodiments are only for illustrating the technical idea of the present invention, and the purpose is to make the general technical personnel understand the content of the present invention and implement the present invention, but not to limit the protection scope of the present invention. All equivalent changes and applications in different fields of the invention made according to the essence of the invention are covered by the protection scope of the invention.

Claims (2)

1. A method for detecting toxicity of in vitro cells of an implant material is characterized in that a three-dimensional matrix carrier with small pore size is firstly constructed to simulate the in vivo environment of reaction cells, then the in vitro cells of the implant material are cultured on the three-dimensional matrix carrier, and finally the in vitro cells of the implant material are subjected to toxicity detection, wherein the three-dimensional matrix carrier is obtained by the following steps:

1) Mixing silk fibroin solution with the mass concentration of 3% and chitosan solution with the mass concentration of 3% according to the weight ratio of 5:5 or 4:6 in a volume ratio to obtain a mixed solution;

2) Adding a crosslinking agent into the mixed solution according to the proportion relation that every 100ml of the mixed solution is added with one part of the crosslinking agent to obtain a crosslinked product; one part of the crosslinker was prepared by dissolving 1g of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide and 0.26g of N-hydroxysuccinimide in 1-2ml of 95% ethanol;

3) And (3) crosslinking the crosslinked product in a refrigerator at 4 ℃ overnight, freezing the crosslinked product in a refrigerator at-20 ℃ for 24h, freezing the crosslinked product in a refrigerator at-80 ℃ for 24h, freezing the crosslinked product in a gradient manner, and drying the frozen product in a freeze dryer for 48h.

2. The method for detecting the toxicity of cells implanted in vitro according to claim 1, characterized in that: the silk fibroin solution is prepared from silkworm cocoon shells or silk.

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