CN117357703B

CN117357703B - Collagen-based hydrogel for repairing corneal damage and its use

Info

Publication number: CN117357703B
Application number: CN202311552794.8A
Authority: CN
Inventors: 孙静; 吴凯; 范红松; 卫丹; 吴承恒; 丁杰
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2025-07-15
Anticipated expiration: 2043-11-21
Also published as: CN117357703A

Abstract

The invention provides a collagen-based hydrogel for repairing cornea injury, which is prepared by firstly preparing type I collagen into a thermodynamic self-assembled collagen hydrogel Col, and soaking the thermodynamic self-assembled collagen hydrogel Col in a polar molecular solution to prepare Cheng Jiaoyuan-based hydrogel Col-Gly. The invention also provides a collagen-based hydrogel for repairing cornea injury, which is prepared from collagen-based hydrogel Col-Gly, polar molecular solution, cross-linking agent and water. The collagen-based hydrogel is used for repairing cornea injury, and has high light transmittance and excellent mechanical strength.

Description

Collagen-based hydrogel for cornea damage repair and application

Technical Field

The invention relates to a collagen-based artificial cornea substitute, provides a collagen-based hydrogel for cornea damage repair and application thereof, and belongs to the field of biomedical materials.

Background

The cornea is the anterior most transparent tissue of the eye and plays an important role in regulating refractive function and avoiding foreign body invasion. Cornea related diseases may lead to decreased vision in patients, and in severe cases, vision loss, with risk of blindness. Cornea-shifted vegetation is considered the gold standard for recovery of vision in patients with corneal blindness. But only 1 out of every 70 patients worldwide can receive cornea transplant surgery. Therefore, development of a cornea substitute that is simple to manufacture and has good biocompatibility is very important for alleviating the problem of shortage of cornea donors.

At present, various biological materials have been developed as biomimetic scaffolds for cornea damage repair, including natural macromolecules (such as collagen, gelatin, hyaluronic acid, chitosan, alginate, etc.), synthetic polymers (such as polyethylene glycol (PEG), poly (2-hydroxyethyl methacrylate) (PHEMA), etc.), or a combination thereof. Although these materials have properties similar to those of natural cornea, only a few are clinically useful due to the complex manufacturing process, low transparency, insufficient mechanical stability after application, poor biological binding to tissues, and the like.

The invention discloses a preparation method and application of a temperature-sensitive cornea repair hydrogel with bioactivity, wherein the application number is CN202210786167.X, the preparation method comprises the following steps of S1, collecting biological tissues, S2, inactivating viruses, S3, repeatedly freezing and thawing;

S4, tissue decellularization, S5, preparing gel, namely pre-freezing the tissue obtained in the step S4 at-100 ℃ to-40 ℃ for 12-18 hours, freeze-drying, grinding the freeze-dried tissue into decellularized particles smaller than 1mm, performing enzymolysis, adjusting pH to 6.0-7.5 by alkali liquor after the enzymolysis is finished, neutralizing an isotonic solution, and adjusting concentration, and S6, adding a preservative system. The hydrogel prepared by the preparation method and the application of the thermosensitive cornea repair hydrogel with bioactivity contains abundant collagen, elastin and mucopolysaccharide, effectively inhibits the cornea lesion progress caused by physical or chemical injury, and plays a role of an object barrier by self-crosslinking under the condition of body temperature, thereby promoting regeneration and repair of damaged cornea. The invention discloses a preparation method of a cornea repair implant with bioactivity and the cornea repair implant, and solves the technical problem of lack of cornea donors. Comprises the steps of preparing a collagen swelling solution, preparing a collagen film based on the collagen swelling solution, and adding a surface functional modification substance on the surface of the collagen film in a 3D printing or coating mode under the aseptic condition so as to form the cornea repair implant with bioactivity. The surface functional modified substance is prepared from cornea matrix containing active cells of a diseased part of a patient body as a material, can improve the biological and biological stability of a high-strength collagen film, improves the physiological function of cornea, ensures that the modified collagen film has good transparency and water content after being implanted into the glasses of the patient, can recover the transparency faster in clinic, has good biocompatibility, and provides a good bracket for the keratocyte culture of a cornea transplant receptor.

Collagen is the main structural component of the primary corneal stroma and is therefore the first choice for making a corneal substitute. In vivo, collagen triple helix molecules can undergo ordered self-assembly according to endogenous cues (e.g., proteoglycans, type V collagen), ranging from nano-to micro-scale. In vitro, collagen fibers can also recombine by self-assembly through hydrogen bonding, electrostatic and hydrophilic-hydrophobic interactions at 37 ℃ and neutral pH, but this thermodynamically driven process is often uncontrollable, exhibiting poor transparency and mechanical properties. The main reason for these results is that collagen fibers are excessively aggregated during self-assembly. Although chemical crosslinking can improve the translucency and mechanical properties of collagen scaffolds to some extent, the limited mechanical integrity resulting from crosslinking may result in poor graft suture performance.

Recently, researchers have conducted studies to regulate the self-assembly behavior of collagen through various external cues (physical and chemical signals), and have expected to mimic the self-assembly behavior of collagen in vivo. For example, majumdar et al screened cyclodextrins of varying size and chemical function to modulate collagen self-assembly to create a biosynthesis that mimics the natural cornea structure. Although such hydrogels have some transparency and suture properties, the complex dehydration process and limited properties limit their use as cornea substitutes. Lei et al report a method for dynamically assembling type I collagen into a highly transparent structural material using electrochemical methods. Although this method shows good application prospects in the aspect of cornea tissue regeneration, it requires strict conditions (requiring an external electric field and a custom mold), and furthermore requires the use of the crosslinking agent glutaraldehyde, which also has a certain cytotoxicity. The basis of these methods is to actively regulate the self-assembly behavior of collagen, and to produce transparent collagen-based materials by limiting excessive aggregation of collagen.

In the field of tanning, there is a solution for dissolution regeneration. The collagen which has completed self-assembly breaks the self-assembly process under the action of external factors (such as ionic liquid and polar molecules), so that the fiber diameter changes, and the change of the fiber diameter provides possibility for designing the transparent collagen hydrogel. In addition, the novel cross-linking agent is also expected to solve the problems that the traditional cross-linking agent is large in brittleness and difficult to be used for cornea suturing.

In summary, collagen, which is one of the main components of cornea, is an ideal building block for repairing cornea defects, and how to design a collagen-based artificial cornea substitute with high light transmittance and excellent mechanics is of great importance for alleviating the problem of insufficient cornea donors.

Disclosure of Invention

The invention aims to provide a collagen-based hydrogel with high light transmittance and excellent mechanical strength for repairing cornea damage and a preparation method thereof. The collagen-based hydrogel has a transmittance of up to 95%, and has excellent toughness on the premise of keeping high transmittance after chemical crosslinking, and meets the requirements of cornea implantation suture, so that the collagen-based hydrogel has important significance in the cornea injury repair field.

The invention provides a collagen-based hydrogel for repairing cornea injury, which is prepared by firstly preparing type I collagen into a thermodynamic self-assembled collagen hydrogel Col, and soaking the thermodynamic self-assembled collagen hydrogel Col in a polar molecular solution to prepare Cheng Jiaoyuan-based hydrogel Col-Gly, wherein the volume ratio of polar molecules to water is 0-100%.

Wherein the type I collagen is derived from fetal bovine skin or other animal tissues of livestock and poultry origin;

The volume ratio of the polar molecule solution to the collagen hydrogel Col is more than 10;

The polar molecules are glycerol, urea, ca ²⁺ and ionic liquid, and the volume ratio of the polar molecules to the water is 0:5,1:4,2:3,3:2,4:1 and 5:0;

The soaking time is 1-60min.

Further preferably, the volume ratio of the glycerol to the water is 4:1, and the soaking time is 30min.

The collagen-based hydrogel Col-Gly is a collagen-based hydrogel with high transparency.

The preparation method of the thermodynamic self-assembled collagen hydrogel Col comprises the following steps:

a. Dissolving type I collagen from fetal bovine skin in 0.1-0.5M glacial acetic acid solution, wherein the concentration of type I collagen is 5-15mg/ml;

b. Adjusting pH to 7.1-8 with 1-5M sodium hydroxide solution at 2-4deg.C, centrifuging at 2-4deg.C at 3000-9000r/min to remove internal bubbles;

c. Transferring the collagen solution into a circular ring made of polytetrafluoroethylene by using a syringe after taking out, placing the circular ring in a constant temperature incubator at 30-45 ℃ and incubating for 20-60min to obtain the thermodynamic self-assembled collagen hydrogel Col, wherein the temperature in the constant temperature incubator is preferably 37 ℃.

The invention treats the collagen gel which has completed thermodynamic self-assembly through polar molecules, and changes the structure of the fiber through destroying the interaction among collagen fiber molecules, thereby obtaining excellent optical properties with structure determination, including high transparency and low haze. Because the mechanical strength of the collagen gel after polar molecular treatment can not meet the cornea suturing requirement, and the high transmittance obtained by pure polar molecular treatment can not be maintained in water for a long time, further crosslinking is needed.

The invention also provides a collagen-based hydrogel for repairing cornea damage, which is prepared from the collagen-based hydrogel Col-Gly, a polar molecule solution, a cross-linking agent and water, wherein the volume of the polar molecule solution is 30-60%, the concentration of the cross-linking agent is 2-20%, the polar molecule is glycerol, the cross-linking agent is oxazolidine, and the concentration range of the collagen-based hydrogel Col-Gly is 5-15mg/ml.

Wherein the volume of the polar molecule solution is 80%, and the concentration of the cross-linking agent is 10%.

Wherein the preparation method is soaking, and the soaking time is 2-30min.

Wherein the hydrogel contains the following compounds:

the hydrogel contains the following compounds:

The invention provides application of the collagen-based hydrogel in preparing a material for repairing cornea damage.

In the invention, the novel crosslinking agent is used for chemically crosslinking fibers in the transparent collagen hydrogel, so that the collagen-based hydrogel (Col-Gly-GA) with both high transmittance and excellent mechanics is obtained. The treatment with polar molecular glycerol significantly reduces the size of the collagen fibers, while the reduction in fiber size results in collagen gels having a transmittance of up to 95% far higher than the demand for light transmittance by artificial cornea substitutes. The use of the crosslinking agent oxazolidine ensures that the collagen-based hydrogel has better toughness while maintaining higher mechanical strength, and meets the requirements of cornea suturing. Thus being a good artificial cornea substitute suitable for cornea transplantation.

Compared with the prior art, the collagen-based hydrogel with high light transmittance and excellent mechanical strength for repairing cornea damage and the preparation method thereof have the following beneficial technical effects:

(1) The collagen-based hydrogel for repairing cornea damage provided by the invention has high light transmittance and excellent mechanical strength. After the traditional collagen hydrogel is self-assembled, the collagen fibers are excessively disordered and aggregated, so that the mechanics and the light transmittance of the collagen gel are very poor, and the requirements of cornea transplantation cannot be met. The collagen hydrogel can have a transmittance of up to 95% by soaking in glycerol solution. By testing the structure of collagen, it was found that the diameter of collagen fibers, which were approximately 100 nanometers in size, was reduced to 10 nanometers after glycerol treatment. In particular, glycerol can partially disrupt molecular interactions (such as hydrogen bonding and hydrophobic interactions) between collagen fibers, thereby breaking down the refocused collagen fibers into nanofibers. Typically, the whole process takes only a few minutes, while the size of collagen can reach the nanometer scale. Cornea is an important component of eyeball, and plays an important role in refractive adjustment, so designing a material with high light transmittance is a precondition for designing a substitute for artificial cornea. The method for improving the transmittance is the highest in collagen-based hydrogel and has important significance in the field of cornea damage repair.

(2) The collagen-based hydrogel for repairing cornea damage provided by the invention has high light transmittance and excellent mechanical strength. Another important problem faced by collagen-based hydrogels in application is their low strength and inability to meet the requirements of corneal suturing. Although the mechanical strength of the collagen gel can be improved to a great extent by using the traditional crosslinking agent such as glutaraldehyde, the collagen gel can be increased in brittleness and is easy to break, and the requirements of cornea suturing cannot be met. The novel cross-linking agent-oxazolidine is a cyclic cross-linking agent, and can interact with amino groups among collagen fibers in a covalent and non-covalent mode, so that the mechanical strength is improved, and meanwhile, the collagen hydrogel can keep higher toughness. The test shows that the breaking elongation can reach 60%, the stitching force can reach 0.5N, and the cornea stitching requirement is met. Therefore, the application prospect in artificial cornea transplantation is wide.

(3) The collagen-based hydrogel for repairing cornea damage provided by the invention has high light transmittance and excellent mechanical strength. The glycerol treatment of the self-assembled collagen gel can destroy the hydrogen bonding action among collagen fiber molecules, so that the mechanical strength of the collagen gel is reduced, the collagen gel can adapt to the surfaces of various shapes, and the shape plasticity can establish collagen-based membrane materials of various shapes. It should be noted that the cornea is a transparent tissue with a certain curvature, so the shape plastic collagen-based hydrogel provided by the invention has important application value in the aspect of designing curvature type artificial cornea substitutes.

(4) The collagen-based hydrogel for repairing cornea damage provided by the invention has high light transmittance and excellent mechanical strength. The collagen-based hydrogel with curvature provided by the invention can keep the curvature after freeze-drying, and is interesting in that the gel film can quickly absorb water and swell when soaked in water again, and can recover to a state before freeze-drying, and the surface appearance is smooth. In the face of special conditions such as war, the rapid-to-use artificial cornea substitute has important significance for cornea transplantation operation under special conditions.

Drawings

FIG. 1 concentration screening test of polar molecular glycerol solution of the present invention

Fig. 2 is a graph showing the change in transmittance of the collagen after the self-assembly treatment with glycerol in example 1, wherein fig. a is a photograph showing the transmittance of the collagen gel during the self-assembly treatment with glycerol, and fig. B is a graph showing the change in transmittance of the collagen gel with time after the self-assembly treatment with glycerol.

FIG. 3 is a graph of the microscopic morphology of the thermodynamic self-assembled collagen hydrogel (Col) and the glycerol-treated collagen hydrogel (Col-Gly) of example 1. Wherein, graph A is the microscopic morphology of the thermodynamic self-assembled collagen gel, and mainly comprises TEM, AFM and CLSM data. Wherein, the graph B is the microcosmic appearance of the collagen after glycerol treatment and comprises TEM, AFM and CLSM data.

Fig. 4 is a view showing a mechanism of improving transmittance of a collagen hydrogel by glycerol treatment in example 1, wherein a view a polarized light microscope observes changes in crystalline regions of the collagen gel after glycerol treatment, a view B is a two-dimensional small angle scattering graph of the collagen gel before and after glycerol treatment, a view C is a statistical graph of two-dimensional small angle scattering of the collagen gel before and after glycerol treatment, and a view D is an inter-fiber size graph calculated by X-ray diffraction.

Fig. 5 is a chart showing structural analysis of the collagen gel before and after the glycerol treatment in example 1, fig. a is a circular dichroism spectrum of the collagen gel before and after the glycerol treatment, and fig. a is a fourier infrared spectrogram of the collagen gel before and after the glycerol treatment.

FIG. 6 is a schematic diagram of a collagen gel treated with chemically crosslinked glycerol in example 1, and FIG. A is a chemical reaction formula of glutaraldehyde crosslinked Col-Gly. Figure B is a chemical reaction of oxazolidine crosslinked Col-Gly.

Fig. 7 is an optical property of the collagen gel treated with chemically crosslinked glycerol in example 3, and fig. a is a change in transmittance of the collagen gel treated with chemically crosslinked glycerol, and fig. B is a change in haze value of the collagen gel treated with chemically crosslinked glycerol.

Fig. 8 is a graph showing the mechanical properties of the collagen gel treated with chemically crosslinked glycerol in example 3, a graph a showing the stress-strain curve of the collagen gel treated with chemically crosslinked glycerol, and a graph B showing the suture strength curve of the collagen gel treated with chemically crosslinked glycerol.

Fig. 9 is a graph showing the shape plasticity of the collagen-based hydrogel designed to have high light transmittance and excellent mechanical strength in example 3, and fig. a is a graph showing the freeze-drying-rehydration process of the collagen-based hydrogel.

Detailed Description

In order to clearly and fully describe the technical solutions of the various embodiments of the invention, reference should be made to the accompanying drawings, it is apparent that the described embodiments are only some embodiments of the invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on the embodiments of the present invention, are within the scope of the present invention.

In the examples below, the type I collagen involved is extracted from calves. When in use, the collagen sponge is directly dissolved in glacial acetic acid solution with a certain concentration after freeze-drying treatment, and the concentration needs to be kept fixed when in use.

Example 1

The preparation method of the collagen-based hydrogel for repairing cornea damage, which is provided by the embodiment, comprises the following steps:

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

Type I collagen from fetal bovine skin was dissolved in 0.5M glacial acetic acid at a concentration of 10mg/ml. After dissolution was complete, the pH was adjusted to 7.4 using 5M sodium hydroxide solution at 4 ℃. Then centrifuged at 6000r/min for 1min at 4℃with a high-speed centrifuge to remove internal bubbles. After removal, the collagen solution was transferred to a circular ring made of polytetrafluoroethylene using a syringe and placed in a constant temperature incubator at 37 ℃. After incubation for 30min, the ring is removed to obtain the thermodynamic self-assembled collagen hydrogel (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

Immersing the collagen hydrogel (Col) obtained in the step (1) in a glycerol solution, wherein the volume ratio of glycerol to water is 4:1, and the treatment time is 30min. After the soaking is completed, the surface residual solution is quickly washed by using up water, and the highly transparent collagen-based hydrogel named Col-Gly can be obtained.

(3) Preparation of collagen-based hydrogel (Col-Gly-OX) with both high transmittance and Excellent mechanics

And (3) immersing the high-transmittance collagen-based hydrogel (Col-Gly) obtained in the step (2) in a mixed solution consisting of glycerol, oxazolidine and water. Wherein the volume of the polar molecular solution is maintained at 50% and the concentration of the crosslinking agent is maintained at 10%. Taking out after soaking for 20min, and rapidly washing the surface residual solution with up water to obtain the collagen-based hydrogel (Col-Gly-OX) with both high transmittance and excellent mechanics.

Example 2

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

Immersing the collagen hydrogel (Col) obtained in the step (1) in a glycerol solution, wherein the volume ratio of glycerol to water is 3:2, and the treatment time is 30min. After the soaking is completed, the surface residual solution is quickly washed by using up water, and the highly transparent collagen-based hydrogel named Col-Gly can be obtained.

(3) Preparation of collagen-based hydrogel (Col-Gly-GA) with high transmittance and excellent mechanics

Example 3

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

Immersing the collagen hydrogel (Col) obtained in the step (1) in a glycerol solution, wherein the volume ratio of glycerol to water is 2:3, and the treatment time is 30min. After the soaking is completed, the surface residual solution is quickly washed by using up water, and the highly transparent collagen-based hydrogel named Col-Gly can be obtained.

Example 4

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

Immersing the collagen hydrogel (Col) obtained in the step (1) in a glycerol solution, wherein the volume ratio of glycerol to water is 1:4, and the treatment time is 30min. After the soaking is completed, the surface residual solution is quickly washed by using up water, and the highly transparent collagen-based hydrogel named Col-Gly can be obtained.

Example 5

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

And (3) immersing the high-transmittance collagen-based hydrogel (Col-Gly) obtained in the step (2) in a mixed solution consisting of glycerol, oxazolidine and water. Wherein the volume of the polar molecular solution is kept at 50% and the concentration of the cross-linking agent is kept at 5%. Taking out after soaking for 20min, and rapidly washing the surface residual solution with up water to obtain the collagen-based hydrogel (Col-Gly-OX) with both high transmittance and excellent mechanics.

Example 6

(1) Preparation of thermodynamic self-assembled hydrogels (Col)

(2) Preparation of collagen hydrogel (Col-Gly) with high transmittance

And (3) immersing the high-transmittance collagen-based hydrogel (Col-Gly) obtained in the step (2) in a mixed solution consisting of glycerol, oxazolidine and water. Wherein the volume of the polar molecular solution is maintained at 50% and the concentration of the cross-linking agent is maintained at 2%. Taking out after soaking for 20min, and rapidly washing the surface residual solution with up water to obtain the collagen-based hydrogel (Col-Gly-OX) with both high transmittance and excellent mechanics.

The invention provides a simple method for preparing collagen hydrogel with high light transmittance and excellent mechanical property, which firstly prepares non-light-transmitting collagen gel by a collagen fiber thermodynamic self-assembly mode. The specific process is to dissolve type I collagen from fetal bovine skin in glacial acetic acid solution. After dissolution was complete, the pH was adjusted to 7.4 using sodium hydroxide solution at 4 ℃. Then, the internal bubbles are removed by high-speed centrifugation, the gel is taken out and transferred into a circular ring made of polytetrafluoroethylene, and the gel is placed in a constant temperature incubator at 37 ℃, and after self-assembly is completed, the collagen hydrogel (Col) which is subjected to thermodynamic self-assembly is obtained. In order to better control the properties of the collagen hydrogel, the concentration and the self-assembly time of the prepared thermodynamic self-assembly collagen hydrogel are kept to be certain.

EXAMPLE 7 concentration screening test of polar molecular glycerol solution of the present invention

In the experiments of exploring the transmittance of glycerol solution to collagen hydrogel, a series of control experiments are carried out, and as a result, the transmittance improvement of the collagen hydrogel is found to have a certain concentration and time dependence. Specifically, the low concentration of glycerol solution (below 20%) does not affect the transmittance of the collagen hydrogel, but as the concentration of glycerol increases, the transmittance of the collagen hydrogel has a certain time dependence, probably because the increase in the concentration of glycerol accelerates the diffusion rate into the gel, resulting in a gradual increase in the transmittance over time. In addition, since glycerol has a certain viscosity, the molecular movement rate is limited to a certain extent when the concentration is 100%, resulting in a slower increase in transmittance than the 80% ratio set. Thus, 4:1 was chosen as the best preference group (see FIG. 1).

Other polar molecules such as urea and the like can also make the collagen hydrogel transparent, but the treatment mode can denature the collagen gel on one hand and destroy the structure and mechanical strength of the gel on the other hand. Glycerol was found to be the best polar molecule currently found to be highly effective in rendering collagen gels transparent.

EXAMPLE 8 screening test of the crosslinker oxazolidines of the invention

Col-Gly is derived from collagen gel treated by glycerol solution, and its properties are mainly derived from the initial collagen concentration in the collagen gel, the concentration range selected in the experiment of the invention is 5-15mg/ml, the higher the concentration is, the more stable the properties of the Col-Gly gel are, so the optimal concentration ratio of the Col-Gly gel in the invention is 15mg/ml.

The polar molecules have the optimal proportion, the collagen gel cannot be transparent due to the low-concentration polar molecular glycerol solution, and the transparent collagen gel is placed in the low-concentration polar molecular glycerol solution, so that the transmittance of the collagen gel can be reduced due to the outward diffusion of the glycerol solution in the gel. Thus, a high concentration of the polar molecular solution (i.e., glycerol solution) is an important factor in the stable transparent state of the collagen gel. The concentrations considered in the present invention are 60%,80% and 100%, and the optimal concentration in the step of preparing Col-Gly-OX is 80% when the glycerol is used in combination with the concentration of glycerol at which the collagen gel becomes transparent.

Other cross-linking agents can also realize the maintenance of the transparency and the improvement of the mechanical properties of the collagen gel, and Glutaraldehyde (GA) is one of them. However, the data in the invention already show that although glutaraldehyde treatment group (Col-Gly-GA) can improve the mechanical strength of collagen gel, the elongation at break is lower, and the requirements of cornea suturing can not be met, so that the comprehensive performance of the oxazolidine crosslinking group (Col-Gly-OX) is better.

The following analysis was made with respect to the preparation process and properties of the collagen-based hydrogels in examples 1 to 6

Test example 1 light transmittance and mechanical Property test of the gel of the present invention

1. Gel light transmittance test of the invention

Experimental materials, collagen gel (Col) completed by self-assembly, polar molecule glycerol solution.

Experimental methods the change in transmittance of the collagen gel was directly observed and tested using an ultraviolet-visible light gradiometer.

As a result of the experiment, the transmittance of the opaque collagen hydrogel (Col) was measured by first treating the collagen hydrogel with a glycerol solution, as shown in FIG. 2A, the transmittance of the collagen hydrogel was gradually increased with the increase of the treatment time, and the flower pistil of the lower fresh flower was seen, whereas the film transmittance of the collagen gel was measured by UV-Vis absorption spectroscopy (as shown in FIG. 2B), and the transmittance of the collagen was 95% or more by short-term immersion (about 15 min) of glycerol. The improvement of the transmittance meets the use requirement of the cornea implant material, and greatly increases the application of the cornea implant material in the cornea injury repair field.

2. The appearance test of the gel is that the light transmittance of the gel is improved:

experimental materials self-assembled collagen gel (Col), glycerol treatment of self-assembled collagen gel (Col-Gly). The experimental method comprises TEM test, AFM test and CLSM test.

Experimental results:

As shown in fig. 3A, the fiber size of the thermodynamic self-assembled collagen gel is about 100nm, the fibers are randomly arranged, the thick fibers are also observed by an Atomic Force Microscope (AFM), the collagen fibers can be observed in a reflection mode of a confocal microscope (CLSM), and as a result, the purely thermodynamic self-assembled hydrogel is found to have the thick and disordered fiber arrangement. In contrast, FIG. 3B shows that the fiber size of the collagen gel is significantly reduced by glycerol treatment, about 10nm, whereas only fine fibers are observed under AFM and CLSM. This reduction in fiber size will reduce light scattering and increase the transmittance of the gel.

3. Mechanism test of light transmittance improvement of the gel of the invention:

Experimental materials glycerol-treated self-assembled collagen gel (Col-Gly).

The experimental method comprises the steps of polarized light microscope observation, two-dimensional small angle scattering experiment, circular dichroism experiment and infrared test.

As a result of the experiment, as shown in FIG. 4, the change of the crystal domain during the glycerol treatment of the collagen gel was observed by a polarized light microscope, and it was found by observation that the crystal domain between collagen fibers gradually decreased with the increase of the treatment time, and the change of the crystallinity of the collagen hydrogel was caused by the surface glycerol treatment. Further, two-dimensional small angle scattering found that the diffraction rings of the collagen gel were reduced after glycerol treatment, indicating that the crystal domain of Col-Gly was reduced and the crystal size was reduced, and statistical data also showed that the diffraction peaks of Col-Gly were sharpened, indicating that the arrangement of collagen fibers was more ordered.

The stabilization of the triple helix structure of collagen is important for the maintenance of the bioactivity of collagen hydrogel, so the structures of Col and Col-Gly in example 3 are characterized by infrared spectrogram and circular dichroism spectrum, and as shown in FIG. 5, it is found that the treatment of glycerol does not change the characteristic absorption peak of collagen fiber, so the triple helix structure is maintained stable.

4. The invention carries out chemical crosslinking reaction formula on collagen gel:

The collagen gel treated with glycerol alone has excellent light transmittance, but the light transmittance is unstable, and when the collagen gel is placed in water, the light transmittance is lowered, and the change is probably caused by leakage of glycerol in water, and reassembly of fibers is caused, so that the strength is lowered. In addition, a collagen gel treated with glycerol alone also causes a decrease in the mechanical strength of the collagen gel. The invention thus carries out chemical crosslinking of Col-Gly in example 1. The chemical reaction principle is shown in figure 6, glutaraldehyde undergoes Schiff base reaction through aldehyde groups and collagen amino groups to increase crosslinking. The oxazolidine can be combined with collagenous amino groups through covalent and non-covalent reactions through ring opening reaction.

The invention performs optical testing on the collagen gel after chemical crosslinking. Experimental materials are glutaraldehyde-crosslinked collagen gel, and oxazolidine-crosslinked collagen gel. The experimental method is ultraviolet and visible light absorption method. Experimental results As shown in FIG. 7, the oxazolidine crosslinked collagen hydrogels have a transmittance of up to 90% in the visible range (380 nm-800 nm) and a transmittance of about 92% at 500 nm. And the haze value test also shows that the oxazolidine crosslinked collagen-based hydrogel has a low haze value, so that the requirement of cornea transplantation is met.

5. The invention tests the mechanical property of the collagen gel after chemical crosslinking.

Experimental materials are glutaraldehyde-crosslinked collagen gel, and oxazolidine-crosslinked collagen gel.

Experimental methods stress-strain testing.

As a result of the experiment, as a substitute for artificial cornea, it is important to have a modulus of conforming to the cornea tissue and a suturing strength required for surgical suturing for cornea transplantation. The traditional glutaraldehyde crosslinked collagen gel has larger brittleness, can be easily broken, and is difficult to meet the requirements of cornea suturing, and the oxazolidine serving as a novel crosslinking agent can be combined with collagen fibers in a covalent-non-covalent mode, so that the collagen hydrogel can be endowed with excellent mechanical properties. As shown in FIG. 7A, the stress-strain curve shows that Col-Gly-GA has a high stress but an elongation at break of only 20%, i.e., shows a high brittleness. The Col-Gly-OX group stress values are relatively low, but can withstand elongation at break as high as 60%. The specific measurement of the suture strength shows that the Col-Gly-OX group can bear the suture force of about 50gf, and is an ideal material for meeting the application requirement of cornea transplantation.

6. The invention determines the applicability of the chemically crosslinked collagen gel.

The cornea is structured with a curvature, and the presence of curvature makes the cornea an important refractive tissue of the eye. The glycerol provided by the invention is used for treating the assembled collagen gel, has lower mechanical strength (shown in figure 8), and can adapt to surfaces with various shapes. As shown in fig. 9A, the collagen gel can be adapted to a rod-like structure to construct a ring-like transparent film material. In addition, the gel can also adapt to the material of the curvature base, and a transparent film material with a cornea similar shape is constructed. Interestingly, the collagen gel has the advantages that the shape of the membrane can be well preserved after freeze-drying and rehydration, and the surface of the oxazolidine treatment group is smoother, so that the collagen gel has application potential as a cornea transplantation substitute for emergency use in battlefield environment.

Experiment summarization shows that the collagen-based artificial cornea substitute for cornea damage repair provided by the invention has the advantages of simple preparation, excellent optical performance (high transmittance and low haze value), higher mechanical performance and capability of meeting the requirements of cornea suturing. Meanwhile, the shape adaptability of the collagen membrane also enables the collagen membrane to meet the design requirement of a cornea curvature structure, and particularly, the shape retention characteristic of the collagen membrane after freeze-drying and rehydration is hopeful to be used as a cornea implantation substitute in emergency situations such as battlefield and the like. These properties are important for solving the inadequacy of the cornea donor, and for developing collagen-based artificial cornea substitutes for cornea damage repair.

Those of ordinary skill in the art will recognize that the embodiments described herein are for the purpose of aiding the reader in understanding the principles of the present invention and should be understood that the scope of the invention is not limited to such specific statements and embodiments. Those of ordinary skill in the art can make various other specific modifications and combinations from the teachings of the present disclosure without departing from the spirit thereof, and such modifications and combinations remain within the scope of the present disclosure.

Claims

1. A collagen-based hydrogel for repairing corneal damage, characterized in that: type I collagen is first prepared into a thermodynamically self-assembled collagen hydrogel Col, which is then immersed in a polar molecule solution to prepare a collagen-based hydrogel Col-Gly;

The type I collagen is derived from fetal bovine skin;

The volume ratio of the polar molecule solution to the collagen hydrogel Col is greater than 10;

The polar molecule immersed in the polar molecule solution is glycerol, and the volume ratio of the polar molecule to water is 1:4-4:1;

The immersion time in the polar molecule solution is 1-60 minutes;

The preparation method of the thermodynamically self-assembled collagen hydrogel Col is as follows:

a. Dissolve type I collagen from fetal bovine skin in 0.1-0.5M glacial acetic acid solution, with the concentration of type I collagen being 5-15mg/ml;

b. After the type I collagen solution is completely dissolved, adjust the pH to 7.1-8 using a 1-5M sodium hydroxide solution at 2-4°C; then centrifuge at 3000-9000r/min at 2-4°C to remove internal bubbles;

c. After taking it out, use a syringe to transfer the collagen solution into a ring made of polytetrafluoroethylene, and place it in a constant temperature incubator at 30°C-45°C. Incubate for 20-60 minutes to obtain the thermodynamically self-assembled collagen hydrogel Col;

The collagen-based hydrogel Col-Gly is immersed in a mixed solution consisting of polar molecules, a cross-linking agent and water to prepare a collagen-based hydrogel Col-Gly-OX; wherein the polar molecule is glycerol and the cross-linking agent is oxazolidine.

2. The collagen-based hydrogel for corneal damage repair according to claim 1 is characterized in that: the polar molecule immersed in the polar molecule solution is propylene glycol, and the volume ratio of propylene glycol to water is 4:1; the immersion time in the polar molecule solution is 30 minutes.

3. The collagen-based hydrogel for corneal damage repair according to claim 1, characterized in that the type I collagen is replaced by animal tissues derived from other livestock and poultry sources.

4. The collagen-based hydrogel for repairing corneal damage according to claim 1, characterized in that the temperature in the constant temperature incubator is 37°C.

5. The collagen-based hydrogel for corneal damage repair according to claim 1 is characterized in that the volume of the polar molecule solution in the mixed solution is 50-60% or 80%, the concentration of the cross-linking agent is 2%-20%; and the concentration range of the collagen-based hydrogel Col-Gly is 5-15 mg/ml.

6. The collagen-based hydrogel for repairing corneal damage according to claim 5, characterized in that the volume of the polar molecule solution in the mixed solution is 80%, and the concentration of the cross-linking agent is 10%.

7. The collagen-based hydrogel for corneal damage repair according to claim 1, characterized in that the immersion time in the mixed solution consisting of polar molecules, cross-linking agent and water is 2-30 minutes.

8. The collagen-based hydrogel for corneal damage repair according to any one of claims 1 to 7, characterized in that the hydrogel contains the compound:

.

9. Use of the collagen-based hydrogel according to any one of claims 1 to 8 in preparing a material for repairing corneal damage.