CN110452413B

CN110452413B - Collagen cross-linking agent composition and application thereof

Info

Publication number: CN110452413B
Application number: CN201910608383.3A
Authority: CN
Inventors: 戴东升; 张建伟
Original assignee: Nanjing Zhongfu Xiannong Biotechnology Co ltd
Current assignee: Nanjing Zhongfu Xiannong Biotechnology Co ltd
Priority date: 2019-07-08
Filing date: 2019-07-08
Publication date: 2021-05-18
Anticipated expiration: 2039-07-08
Also published as: CN110452413A

Abstract

The invention relates to a collagen cross-linking agent composition and application thereof. The composition comprises a photosensitive crosslinker, a chemical crosslinker, a citrate, and an imidazole. The cross-linking agent composition can be used for simultaneously carrying out chemical cross-linking and photooxidation cross-linking of the decellularized collagen and exerting a synergistic effect, so that the efficiency of cross-linking reaction is improved, and the cross-linking collagen medical material with higher quality is obtained.

Description

Collagen cross-linking agent composition and application thereof

Technical Field

The application relates to the field of medical supplies, in particular to a collagen cross-linking agent composition and application thereof.

Background

The collagen is the protein which is most widely distributed in the body of the mammal, is easy to extract and obtain, and has good biodegradability and biocompatibility. Collagen is a scaffold material for cell growth, has the effects of promoting cell adhesion and inducing cell growth and differentiation, is widely used for tissue engineering research, and is widely applied in the medical field.

The glue is widely applied due to the characteristics of excellent low immunological activity, good biocompatibility, biodegradability and the like, but unmodified collagen has the defects of poor thermal stability, excessively high degradation rate, low mechanical strength, easiness and the like, and cannot meet the use requirement in many cases. Therefore, the method of modifying the collagen is usually adopted to effectively improve the thermal stability and mechanical properties of the collagen and reduce the degradation rate of the collagen. At present, the modification research on collagen can be roughly divided into three categories, namely crosslinking modification, active group modification and polymer introduction modification.

The physical crosslinking method mainly comprises a photooxidation method, a thermal dehydrogenation method and an ultraviolet radiation method. Physical modification does not generally involve the primary structure of the protein and is primarily used for the solubilization and gelling of collagen. Physical crosslinking methods have the disadvantage of low crosslinking of the collagen and difficulty in obtaining uniform crosslinking. When the collagen solution is irradiated with ultraviolet rays, crosslinking occurs between molecules, and the viscosity increases, thereby forming a gel.

The chemical crosslinking method adopts an organic crosslinking agent or an inorganic crosslinking agent to carry out crosslinking, has higher degree of crosslinking than the modification crosslinking of a physical method, can obtain uniform crosslinking, and has good effects on adjusting and controlling various properties of collagen. However, exogenous toxic chemicals enter the collagen.

With the application research, the performance of the single cross-linking agent for cross-linking the collagen shows certain limitation, and a novel cross-linking agent which has more excellent cross-linking effect and overcomes the defects of the traditional cross-linking method is urgently needed. In the field of producing biological scaffold materials from collagen, there is also a need for scaffold materials with improved crosslinking efficiency, reduced toxic side effects and immunogenicity, as well as good tissue compatibility. The comprehensive performance of the collagen is improved through the development of the cross-linking agent, and a new power is provided for the development of medical biomaterials.

Disclosure of Invention

The invention aims to develop a cross-linking agent composition with improved performance, which is used for modification reaction of collagen and further applied to preparation of medical biomaterials.

To achieve the object, one aspect of the present invention provides a crosslinker composition comprising a photosensitive crosslinker, a chemical crosslinker, a citrate, and imidazole.

The crosslinker composition preferably further comprises a buffer system, such as a phosphate buffer system.

Preferably, the photosensitive crosslinking agent is selected from 5-aminolevulinic acid, methylene phenyl blue and the like.

Preferably, the chemical crosslinking agent is selected from aldehydes (glutaraldehyde), dicarboxylic acids, genipin, sodium alginate oxide, carbodiimide, citric acid derivatives, chitosan, polyvinyl alcohol, and the like. More preferably genipin and oxidized sodium alginate.

The citrate is preferably sodium citrate, potassium citrate, or the like.

In a specific embodiment, the collagen crosslinker composition of the invention has the following composition: 0.05-100mg/ml of photosensitive cross-linking agent, 0.05-100mg/ml of chemical cross-linking agent, 0.05-1mol/L of citrate and 0.05-100mg/ml of imidazole.

In another aspect of the invention, the use of a cross-linker composition according to any one of claims 1 to 5 in a collagen cross-linking reaction.

Preferably, the application is actually a crosslinking method of collagen, and the crosslinking method is carried out in the following way: soaking the decellularized collagen in a cross-linking agent composition in the form of a solution; maintaining the temperature of the equilibrium system at 10-40 ℃ and the pH value of the equilibrium system at 4-9, and pre-soaking. Then, carrying out a crosslinking reaction under the conditions of illumination of an incandescent lamp and oxygen introduction under the osmotic pressure of 310-330 Osm; finally, with Na₂CO₃/NaHCO₃Adjusting pH to 6.8-7.2 with buffer solution, soaking, shaking, sealing, and storing. The pre-soaking pH is preferably 7.2-8.2.

Preferably, the pre-soaking time is 0.5-10h, and the crosslinking reaction time is 0.5-10 h.

In a further aspect of the invention, there is provided a method of producing a decellularized biological material comprising the steps of:

(1) performing cell removal treatment on the cell-containing biological material;

(2) performing a collagen crosslinking reaction in solution with the crosslinker composition of any one of claims 1 to 5;

(3) and harvesting the collagen.

Preferably, the decellularization process is performed using a detergent-enzyme digestion method, and the enzyme is trypsin or pepsin.

In contrast to the prior art, citrate is an important component of the crosslinking composition of the present invention. According to the research of the inventor, the existence of citrate in the composition can improve the stability of chemical cross-linking agents such as genipin and the like in an alkaline solution, thereby promoting the synergistic effect of the chemical cross-linking agents and the photosensitive cross-linking agents. The crosslinker composition of the invention also contains imidazole. According to the research of the inventor, the amount of the component can significantly influence the collagen modification effect.

Field of application of the invention

Hemostatic material

Collagen has been widely used as a hemostatic material because of its biocompatibility, blood absorbability, low immunogenicity, and the like. The collagen hemostatic material has a porous reticular structure, when contacting with a bleeding wound, the collagen hemostatic material rapidly adsorbs blood at the wound by utilizing the capillary action of the collagen hemostatic material, so that platelets are promoted to be condensed to generate thrombin, and the thrombin converts fibrinogen into fibrin through a re-catalysis process to solidify the blood, thereby achieving the purpose of hemostasis.

Drug delivery carrier material

The collagen is a connective tissue protein, has biocompatibility, biodegradability and low immunogenicity, and can be used as an ideal drug carrier material; the slow release of the medicine can be realized by controlling the structure of the collagen, and the collagen is crosslinked into a highly porous reticular structure, so that the mechanical strength is improved, the medicine loading rate is increased, and the biodegradation rate is slowed, thereby prolonging the release time of the medicine.

Tissue engineering scaffold material

The repair and functional reconstruction of tissue and organ defects are great challenges, and the development of tissue engineering provides a new technology for tissue regeneration and reconstruction. Collagen is often used as a tissue engineering scaffold material, seed cells are extracted from the body, the seed cells are inoculated on a collagen scaffold after in vitro amplification, and the collagen is implanted into the body after being cultured in vitro to form a functional tissue, so that the defect of the related tissue can be repaired.

Skin substitute

When the skin is seriously damaged, the skin is dead, and particularly, the burned or burnt skin is difficult to repair spontaneously. Collagen is commonly used to culture skin cells as a vehicle for the treatment of skin lesions.

Bone graft material

Collagen is an important component of bone extracellular matrix, and development of bone tissue engineering provides a new technology for bone defect repair.

Corneal graft material

Collagen protein accounts for about 70% of the dry weight of the cornea, and collagen fibers are arranged in a regular and ordered structure in the cornea. The cornea is difficult to repair or rebuild after being damaged, and researches show that normal corneal stem cells cultured and expanded in vitro are implanted into a collagen bracket to form a tissue engineering artificial cornea, so that the damaged cornea can be repaired.

Advantageous effects

The cross-linking agent composition of the invention is different from the prior single photosensitive cross-linking agent and chemical cross-linking agent, and integrates the advantages of photo-oxidative cross-linking and chemical cross-linking. The invention has the unexpected discovery that photooxidative crosslinking and chemical crosslinking can be carried out simultaneously and generate a synergistic effect, thereby greatly improving the crosslinking efficiency and saving the raw materials and the reaction time. The chemical crosslinking process usually requires a relatively long crosslinking time, which is considerably shortened in combination with the photooxidative crosslinking. Especially in the presence of citrate, the synergistic effect of the photooxidative crosslinking agent and the chemical crosslinking agent is most obvious.

In addition, the invention adopts the combination of detergent, protease and the like to completely remove the cell components in the tissues, thereby completely retaining the scaffold structure of main extracellular interstitial components (cartilage scaffold, collagen fibers, elastic fibers and the like) and avoiding calcification and immune reaction caused by cells. The method of the invention provides a new method and source for constructing the tissue engineering gas tube matrix scaffold with physiological functions.

Detailed Description

EXAMPLE 1 formulation of crosslinker composition

This example prepares a crosslinker composition solution having the following formulation

Composition 1:

0.1mg/ml of genipin, 0.1mg/ml of 5-aminolevulinic acid, 0.05mol/L of sodium citrate and 0.02mg/ml of imidazole, wherein PBS is used as a buffer system, and the pH value is 8.0.

Composition 2:

0.1mg/ml sodium alginate oxide, 0.1mg/ml methylene-benzyl, 0.05mol/L sodium citrate, 0.02mg/ml imidazole, PBS as a buffer system, and pH 7.5.

Composition 3:

50mg/ml of oxidized sodium alginate, 20mg/ml of methylene-phenylene blue, 0.8mol/L of potassium citrate and PBS as a buffer system, wherein the pH value is 7.0.

Control 1:

0.2mg/ml of genipin, PBS as a buffer system and pH7.5.

Control 2:

0.2mg/ml 5-aminolevulinic acid, with PBS as the buffer system, pH 7.0.

Control 3:

0.1mg/ml sodium alginate oxide, 0.1mg/ml methylene-phenylene blue, PBS as buffer system, pH7.5.

Example 2 collagen biomaterial obtention

2.1 sclera

New Zealand white rabbits were sacrificed by air embolism, the eyeball was removed, and the sclera was separated. And then placing the soaked sclera tissue in a dodecyl dimethyl benzyl ammonium bromide solution, sequentially placing the soaked sclera tissue in a polyethylene glycol octyl phenyl ether solution for incubation, placing the incubated sclera tissue in a mixed solution of trypsin and EDTA, and placing the incubated sclera tissue in a mixed solution of Dnase-1 and Rnase-A for incubation to obtain the acellular sclera tissue. The sections were made 1cm by 1 cm. Obtaining the cell-free material.

2.2 vascular tissue

Selecting a fresh and proper bovine vein, and carrying out decellularization by adopting a multi-step detergent-enzyme digestion method. 0.25% trypsin solution for 18 hours, then placed in 0.1% SDS for 12 hours, then the tissue is immersed in 0.25% trypsin solution for 12 hours, finally treated in 25 ℃ Dispase for 12 hours, and finally the decellularized bovine jugular vein blood vessel slice is cut into 2 with the area of about 1.6cm per slice. A decellularized material 2 was obtained.

2.3 trachea

Taking adult healthy New Zealand white rabbits, performing air death by means of ear vein, and cutting the whole trachea. Washing with PBS buffer solution containing 1% antibiotic and antifungal drug for 3-4min, and dissolving in distilled water at 4 deg.C for 48 hr; soaking in 4% sodium deoxycholate distilled water solution at 37 deg.C, incubating for 4h, and continuously shaking at 40 r/min; then washing with distilled water twice to remove cell debris; soaking trachea in 2000KU/L Dnase-I normal saline at room temperature for 3h, and continuously shaking at 40r/min to dissolve cell nucleus and degrade DNA; the trachea was cut to about 1.6cm2 per area and stored in 1% antibiotic, antifungal PBS buffer at 4 ℃. A decellularized material 3 was obtained.

EXAMPLE 3 crosslinking reaction

The sliced decellularized material obtained in example 2 was immersed in 20mL of the solution of example 1, and the solution was placed in an 80mL container. Maintaining the temperature of the equilibrium system at 15 ℃ and the pH value of 7.5-7.8, and soaking for 60 min. Subsequently, the crosslinking reaction was carried out for 60min under the conditions of 1000w of incandescent lamp illumination and oxygen gas introduction under the osmotic pressure of 310-330 Osm. Finally, 0.25M Na was used₂CO₃/NaHCO₃BufferAdjusting the pH value of the reaction system to 7, soaking and oscillating for 1h, and sealing and storing for later use.

Example 4 cell adhesion and cell growth experiments (biocompatibility)

Each of the cross-linked sections obtained in example 3 was seeded with 5X 10 cells of human umbilical vein endothelial cell line (CRL-2480)⁵And statically culturing for 7 days, counting cells on the surfaces of the digestive vascular sheets of the samples for 1, 4 and 7 days, carrying out HE (high-intensity electrophoresis) staining on the sections of the samples, detecting by using a scanning electron microscope and a transmission electron microscope, comparing the cell adhesion number and the combination degree of the cells on the surfaces of the vascular sheets of each treatment group, and comparing the influence of pretreatment of different cross-linking agents on the enhancement of cell adhesion and the promotion of growth conditions.

(1) Cell counting: the cell culture days 4-7, the number of cells in the experimental group is more than that in the control group (p < O.05), and no obvious difference exists between the experimental groups.

(2) And E, section HE staining result after paraffin embedding of the specimen: on the first day, cells on the surfaces of all groups of vascular sheets are densely and disorderly arranged, cell nucleuses are stacked, and parts of vascular sheets are arranged in a double-layer manner; on day 4, the blood vessel surface cells of the control group were loose, and the cells of the experimental group were connected into a sheet and arranged in a monolayer. On day 7, the control cells were still relatively small and the experimental cells were plated.

(3) Taking a 7-day sample, and displaying by a scanning electron microscope: the surface cells of the blood vessel slice of the control group are sparse, no bridging exists among cells, and the connection with the fiber structure below is not obvious. The test groups were spread in sheets with long fusiform bridges and protrusions between the cells, forming connections with the decellularized vascular sheet fibers.

(4) Transmission electron microscopy: the surface-grown endothelial cells and underlying fibrous components were observed to be present as white bands. The control cell scaffolds were able to grow endothelial cells. The cell scaffold surface of the experimental group can enhance the adhesion and adherent growth of endothelial cells. The experimental group was more effective than the control group in enhancing cell adhesion and adherent growth.

The measurement results are shown in tables 1 and 2. Wherein, the cell morphology score is: whether the cells are fragmented or intercellular bridged is examined. The best is +++ and the worst is-the worst.

TABLE 1 biocompatibility of acellular material 1

TABLE 2 biocompatibility of acellular material 2

The experimental results show that the use of the cross-linking agent composition can further improve the biocompatibility of the decellularized material compared with the single cross-linking agent, and particularly generate stronger synergistic effect on the adhesion of cells. Wherein, the synergistic effect can be obviously embodied by using citrate and imidazole as auxiliary components.

Example 5 mechanical Strength test

Determination of thermal shrinkage temperature

The material prepared in example 3 was placed in a leather thermometer, the two ends were fixed, the cross-linked section was left in a natural state without being pulled by tension, the temperature was raised at a rate of 2 ℃/min using double distilled water as a medium, and the shrinkage ratio of the collagen material was measured, and the measured thermal shrinkage temperature was the temperature at which the rate of change in shrinkage was the greatest.

Maximum tensile Strength and maximum tensile distance measurement

The cross-linked section prepared in example 3 was placed in a tensile material force gauge (INSTRON-3433), the section thickness was recorded by a VIDAS image analysis system, the tensile rate was set to 5mm/min, the tensile length and stress at the time of vascular rupture were introduced into the VIDAS system, and the maximum tensile strength of the specimen was calculated. Data are expressed as mean ± standard deviation (x ± s); the group comparison adopts t test of two samples; differences with P <0.05 were statistically significant. The results are shown in tables 3 and 4.

Table 3 comparison of mechanical properties of acellular material 1 (n ═ 20, x ± s)

Table 4 mechanical property comparison of acellular material 3 (n ═ 20, x ± s)

From the experimental results, it is known that the use of the cross-linking agent composition can further improve the mechanical strength of the decellularized material compared with the single cross-linking agent, and generate stronger synergistic effects in terms of the thermal shrinkage temperature, the maximum tensile strength and the maximum stretching distance. Wherein, the synergistic effect can be obviously embodied by using citrate and imidazole as auxiliary components.

Example 6 stability to enzymatic degradation

The cross-linked acellular material can be evaluated for resistance to enzymatic degradation in vitro, the stronger the resistance to enzymatic degradation, the lower the degradation rate of the cross-linked material. Three decellularized materials prepared in example 3 were each tested for in vitro enzymatic degradation using the following procedure: the initial mass (W) of the sample is measured₀) Thereafter, the sample was immersed in a 1.8mg/mL D-Hanks solution of collagenase type II (enzyme activity unit 1000U/mL, pH7.4) and degraded continuously at 37 ℃ for 4 hours with constant gentle shaking (60 rpm). 50 microliter 10mmol/L EDTA was added to stop the degradation, the remaining sample was dried to constant weight and the mass (W) was measured again_t). The degradation rate or percent mass loss can be calculated by the following formula:

△W％-(W₀-W_t)/W₀×100％

in the formula W₀Denotes the initial mass, W, of each sample_tThe mass after enzymatic degradation of each respective sample is indicated.

Blank represents decellularized material that was not crosslinked.

TABLE 5 comparison of the resistance of the decellularized material to enzymatic hydrolysis

The experimental result shows that the cross-linking agent composition further improves the enzymolysis resistance of the decellularized material and exerts the synergistic effect of different cross-linking agents.

Claims

1. A collagen cross-linker composition comprising one or more chemical cross-linkers, one or more photoactive cross-linkers, citrate, and imidazole, wherein the photoactive cross-linker is selected from the group consisting of 5-aminolevulinic acid, tolylene blue.

2. The collagen cross-linker composition according to claim 1, wherein the chemical cross-linker is selected from aldehydes, dicarboxylic acids, genipin, sodium alginate oxide, carbodiimide, citric acid derivatives, chitosan and polyvinyl alcohol.

3. The collagen cross-linker composition according to any one of claims 1 to 2 wherein the citrate is sodium citrate or potassium citrate.

4. The collagen cross-linker composition according to claim 3, having the following composition: 0.05-100mg/ml of photosensitive cross-linking agent, 0.05-100mg/ml of chemical cross-linking agent, 0.05-1mol/L of citrate and 0.05-100mg/ml of imidazole.

5. Use of a cross-linker composition according to any one of claims 1 to 4 in a collagen cross-linking reaction.

6. Use according to claim 5, characterized in that it is carried out as follows:

soaking the decellularized collagen in a cross-linking agent composition in the form of a solution; maintaining the temperature of the equilibrium system at 10-40 ℃ and the pH value of the equilibrium system at 4-9, and pre-soaking; then, carrying out a crosslinking reaction under the conditions of illumination of an incandescent lamp and oxygen introduction under the osmotic pressure of 310-330 Osm; finally, with Na₂CO₃/NaHCO₃Adjusting pH to 6.8-7.2 with buffer solution, soaking, shaking, sealing, and storing.

7. Use according to claim 6, wherein the pre-soaking time is 0.5-10h and the cross-linking reaction time is 0.5-10 h.

8. A method of producing a decellularized biomaterial, comprising:

(2) performing a collagen crosslinking reaction in solution with the crosslinker composition of any one of claims 1 to 4;

(3) and harvesting the collagen.

9. The method according to claim 8, wherein the decellularization process is performed using a detergent-enzyme digestion method, the enzyme is trypsin or pepsin, and the decellularized biomaterial comprises a hemostatic material, a drug release carrier material, a tissue engineering scaffold material, a skin substitute, a bone graft material, or a corneal graft material.