CN115998961A

CN115998961A - Collagen-based polydopamine composite material with certain adhesiveness for repairing cartilage

Info

Publication number: CN115998961A
Application number: CN202111229962.0A
Authority: CN
Inventors: 李亚茹; 史艳萍; 雷英杰
Original assignee: Tianjin University of Technology
Current assignee: Tianjin University of Technology
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2023-04-25

Abstract

The invention relates to a collagen-based polydopamine composite material capable of being adhered to cartilage and bone tissues around injury and used for repairing articular cartilage and a preparation method of the composite material. The existing implant material for repairing articular cartilage, tissue engineering cartilage and matrix auxiliary autologous chondrocyte transplantation are mainly fixed in a suture mode, the fixing mode has the problems of inconvenient operation and cartilage tissue injury, and a new way is provided for solving the problems by constructing biological materials capable of adhering to cartilage and bone tissue around the injury. The invention constructs the collagen-based polydopamine composite material based on the advantages of improving the tissue adhesiveness and biocompatibility of the material, improving the cell adhesiveness of the collagen material and the like, namely improving the physicochemical property and biocompatibility of the collagen material, promoting the adhesion and proliferation of chondrocytes and providing convenience for the implantation of the material for repairing articular cartilage.

Description

Collagen-based polydopamine composite material with certain adhesiveness for repairing cartilage

Technical Field

The invention belongs to the technical field of bioengineering scaffold materials, and particularly relates to a collagen-based polydopamine composite material capable of being adhered to cartilage and bone tissues around injury.

Background

Degeneration or injury of articular cartilage is a major cause of disability in humans. Pain and swelling may be caused if not treated in time, and eventually cause the onset of osteoarthritis. Unlike most tissues, there are no vascular, neural and immune responses within the articular cartilage tissue structure, and the ability to self-repair after degeneration or injury is very limited. It is necessary to implant a suitable material to solve this problem. However, the fixation of materials in autologous chondrocyte transplantation, whether existing cartilage tissue scaffold materials, cartilage repair biomaterials or matrix-assisted autologous chondrocyte transplantation, which has been widely used, is mainly performed by suture. The suture mode has the problem of inconvenient operation and can cause cartilage tissue injury. Surgical stapling of articular cartilage is a difficult task, as it is known to be avascular, nerve-free and lymph-free, and its healing capacity is very poor. Suturing is the insertion of a surgical needle into tissue, thereby creating a passageway which is actually part of the lesion. So that the construction of new materials that adhere to the cartilage and bone tissue surrounding the injury can provide a new approach to the solution of this problem.

The field of tissue engineering scaffolds has been rapidly developed for decades to meet the great demands of people for organ and tissue repair. Collagen (COL) has good biocompatibility and biodegradability, and has abundant binding sites, which can promote cell bottom adhesion. The collagen material is a main material for matrix-assisted autologous chondrocyte transplantation and cartilage repair at present, but the adhesiveness of the tissue is not strong, so that the problem of cartilage tissue damage caused by a suture mode can be reduced by improving the adhesiveness of the tissue, and convenience is provided for matrix-assisted autologous chondrocyte transplantation and the implantation of the collagen-based cartilage repair material.

Dopamine (DA) is an amino acid in mussel adhesive protein, and has strong adhesion to various surfaces. It is mainly a tyrosine derivative in mussel podophyllotoxin. Catechol functional groups in the derivatives are the primary structure that imparts adhesion behavior to mussels. Dopamine (DA) can be oxidized and self-polymerized in alkaline solution, and a Polydopamine (PDA) layer with super-strong viscosity is formed on the surface of the material, so that super-strong adhesion on the surfaces of various materials is realized, and the super-strong adhesion performance of polydopamine is realized because catechol functional groups contained in polydopamine can form covalent bonds or non-covalent bonds (hydrogen bonds, van der Waals forces or stacking forces) with the surfaces of the substrate materials. The polydopamine improves the tissue adhesion and biocompatibility of the material. Therefore, the invention constructs the collagen-based material which damages the adhesiveness of cartilage and bone tissues through the combination of collagen and polydopamine, thereby providing convenience for the implantation of the material and reducing the problem of cartilage tissue damage caused by a suturing mode again.

Disclosure of Invention

The invention aims to construct a new material for damaging the adhesiveness of surrounding cartilage and bone tissues based on the problem of suture mode in autologous chondrocyte transplantation, and the collagen-based polydopamine composite material can provide a new way for solving the problem.

The technical scheme of the invention is realized through the following steps.

A method for preparing a collagen-based polydopamine composite material capable of adhering to damaged cartilage and bone tissue, comprising the following steps:

preparation of 1.10 mmol/L Tris-HCl (pH=8.5) buffer solution: 1.21g of tris (hydroxymethyl) aminomethane is weighed, 800mL of pure water is added for full dissolution, 4mol/L hydrochloric acid is added dropwise, the pH meter is used for adjusting to 8.5, and the volume is fixed to 1L.

2. Preparing a dopamine standard solution: 0.01g of dopamine was weighed out, and 5mL of 10mmol/L Tris-HCl (pH=8.5) buffer solution was added to prepare 2mg/mL dopamine solution.

3. Preparation of collagen-based-polydopamine composite (PDA-COL): preparing an air-dried collagen membrane with the size of 1cm multiplied by 1cm and 1cm multiplied by 3cm, washing the collagen membrane with pure water for three times, soaking the collagen membrane in 2mg/ml of dopamine solution, and reacting for 0h,1h,4h,12h,24h,36h and 48h respectively under the condition of normal temperature and light shielding. Soaking in pure water for 2h after taking out, washing with pure water for several times until no obvious free black substance residue is seen in the water, and drying in a freeze dryer for 12h to obtain the collagen-based-polydopamine composite material with different composite thickness and different tissue adhesion strength.

The invention has the advantages that the collagen-based polydopamine composite material for damaging cartilage and bone tissue adhesion is constructed, the material improves the tissue adhesion and biocompatibility, and solves the problem of cartilage tissue damage caused by a suture mode.

Drawings

Fig. 1: scanning Electron Microscope (SEM) photographs of the collagen-based-polydopamine composite material reacted for different times.

Fig. 2: test curves of adhesion of collagen-based-polydopamine composites and cartilage tissue sections over time.

Fig. 3: CCK8 kit detects the proliferation result graph of the cell surface of the collagen-based polydopamine material cultured for 3d by chondrocytes with different composite thickness.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Embodiment 1 as an embodiment of the preparation method of the collagen-based polydopamine composite material according to the present invention, the preparation method of the collagen-based polydopamine composite material according to the present embodiment includes the following steps:

sample pretreatment: the collagen membrane was rinsed 3 times with pure water before the experiment.

First, a 10mmol/L Tris-HCl (pH=8.5) buffer solution was prepared. 1.21g of tris (hydroxymethyl) aminomethane is weighed into a beaker, 800mL of pure water is added, the mixture is stirred with a clean glass rod until the pure water is fully dissolved, 4mol/L of hydrochloric acid is added dropwise, the mixture is adjusted to 8.5 by a PH meter, and the volume is fixed to 1L. 0.01g of dopamine was weighed and 5mL of 10mmol/L Tris-HCl (pH=8.5) buffer solution was added. Finally, the collagen membrane is soaked in the dopamine solution to react for 1h,4h,12h,24h,36h and 48h. And after the reaction, respectively taking out and placing the mixture in a freeze dryer for drying for 12 hours, thus obtaining the collagen-based polydopamine composite material with different composite thickness and different tissue adhesion strength.

Fig. 1 illustrates: the composite polydopamine fills the pores of the collagen membrane, and along with the extension of the composite time, the polydopamine fills more and more pores of the collagen membrane. a is a Scanning Electron Microscope (SEM) photograph of a pure collagen film (0 h); b is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite material (1 h); c is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite material (4 h); d is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite material (12 h); e is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite material (24 h); f is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite material (36 h); g is a Scanning Electron Microscope (SEM) photograph of the collagen-based-polydopamine composite (48 h).

Example 2

Preparation before experiment: collagen-based polydopamine composites of different composite thickness and different tissue adhesion strength and cartilage tissue sections of 1cm x 3cm in size.

After the electronic tension machine is opened in advance to enable the electronic tension machine to be stable for 5 minutes, the clamp is installed, the clamp is used for checking, the cartilage slice and the collagen-based polydopamine composite material are clamped by the clamp respectively, the cartilage slice is arranged below, the collagen-based polydopamine composite material is arranged above, the cartilage slice and the collagen-based polydopamine composite material are laminated in a staggered manner on the same vertical line, and the electronic tension machine is stretched towards two directions, wherein the speed is 0.1mm/s.

Fig. 2 illustrates: with the extension of the polydopamine coating time, the adhesion of the collagen-based polydopamine composite material to the cartilage slice is larger.

Example 3 chondrocytes isolated from cartilage tissue were cultured in a petri dish using a mixed medium of 90% dmem medium, 10% serum, and 1% diabody. After 2 generations, the cell surface proliferation experiment of the collagen-based-polydopamine composite material is carried out.

The cell experiment steps of the collagen-based-polydopamine composite material are as follows:

1. 1 cm. Times.1 cm of the collagen-based-polydopamine composite material was sterilized.

2. Collagen-based-polydopamine composites of different composite thickness and different tissue adhesion strength were clamped into 24-well plates using sterile forceps.

3. 2.5X10 addition to 24 well plate ⁵ Culturing at a concentration of 1mL, changing the liquid of the cells after culturing for 24h, washing twice with 1mL of PBS, adding 1mL of culture medium, culturing for 48h, and then using 1mL of PB againS washing twice and adding 1mL of culture medium.

4. After incubation to 72h, collagen-based-polydopamine composites of different thickness were washed twice with 1mL of PBS and the material was transferred into a new 24-well plate using sterile forceps.

5. Under the condition of avoiding light, a mixed solution, namely 480 mu L of cck-8 reagent and 4.8mL of culture medium, is prepared according to the proportion of cck-8 to culture medium=1:10. 550 μl of the mixed solution was added to collagen-based-polydopamine composites of different composite thickness and different tissue adhesion strength. Incubate in incubator for 90 min protected from light.

After 6.90 minutes, the 24-well plate was removed from the incubator, 110. Mu.L of solution per well was transferred to a 96-well plate, and 5 wells were multiplexed.

7. The absorbance at 450mm was measured with an enzyme-labeled instrument to obtain an OD value.

Fig. 3 illustrates: the chondrocyte is cultured for 3d in the collagen-based polydopamine composite materials with different composite thicknesses and different tissue adhesion strengths, and cell proliferation experimental tests are carried out, so that the collagen-based polydopamine composite material has obvious promotion effect on adhesion and proliferation of the chondrocyte compared with a pure collagen membrane along with the extension of polydopamine coating time.

The applicant states that the above are all preferred embodiments of the invention, but the invention is not limited to the detailed methods described above, i.e. it does not mean that the invention must be practiced in dependence on the detailed methods described above. It will be apparent to those skilled in the art that various substitutions of materials and medicines, as well as the manner of operation and selection of materials and medicines, may be made in accordance with the present invention without departing from the spirit and substance of the present invention, and are intended to fall within the scope of the appended claims.

Claims

1. A collagen-based polydopamine composite material for repair of articular cartilage damage capable of adhering to cartilage and bone tissue surrounding the damage, characterized in that: the composite material is collagen material modified by polydopamine, can be adhered to cartilage and bone tissue around injury, and can be used for repairing articular cartilage injury.

2. The method for preparing the composite material, which is a collagen material modified by polydopamine composition, according to claim 1, comprising the following steps:

soaking the prepared collagen membrane in alkaline solution containing dopamine, reacting at normal temperature to polymerize the dopamine to form polydopamine, and further uniformly adsorbing and compositing the polydopamine on the collagen membrane material. And after the reaction and the compounding are finished, taking out the collagen-polydopamine composite material, fully washing the collagen-polydopamine composite material with pure water, and then freeze-drying the washed composite material to obtain the collagen-polydopamine composite material.

3. The method for preparing a collagen-based polydopamine composite material according to claim 2, wherein: the alkaline solution containing dopamine is prepared from dopamine and Tris-HCL alkaline buffer solution, the concentration of the dopamine is 2-10mg/mL, and the pH value of the Tris-HCL alkaline buffer solution is 8.5.

4. The collagen-based polydopamine composite according to claim 1, being bondable to cartilage and bone tissue surrounding an articular cartilage lesion.

5. The collagen-based polydopamine composite according to claim 4 wherein said collagen-based polydopamine composite is capable of adhering to cartilage and bone tissue surrounding an articular cartilage lesion, wherein: the adhesive strength with cartilage and bone tissue can be changed by adjusting the concentration of dopamine during the preparation of the material.

6. The collagen-based polydopamine composite according to claim 4 wherein said collagen-based polydopamine composite is capable of adhering to cartilage and bone tissue surrounding an articular cartilage lesion, wherein: the adhesive strength with cartilage and bone tissue can also be changed by adjusting the time period for preparing the material.

7. The collagen-based-polydopamine composite material according to claim 1, which can be used for repairing articular cartilage injury and constructing tissue engineering cartilage.