CN1216984C - Method of introducing cell growth factor to surface of biological polymer material - Google Patents

Abstract

The invention discloses a method for introducing cell growth factors on the surface of polymer biomaterials. In this method, the cell growth factor is physically mixed with the biomacromolecule solution in advance, and then the biomacromolecule solution mixed with the cell growth factor is stably coated on the surface of the biomaterial by using the "graft-coating" technology, so that the cell growth factor It is physically dispersed in the biomacromolecule coating to prepare an active biomaterial compounded with cell growth factors. Cell growth factors embedded in biomacromolecule coatings can stably exist on the surface of biomaterials and have good bioactivity. The method has simple operation, good repeatability and no adverse side effects, and the prepared active biological material containing the cell growth factor has a good promoting effect on the growth and activity of cells.

Description

Method for introducing cell growth factors on the surface of polymer biomaterials

                      

The present invention relates to the composite technology of polymer biomaterials and cell growth factors, in particular to the method for introducing cell growth factors on the surface of polymer biomaterials

Background technique

Tissue engineering refers to the modern treatment technology of inoculating seed cells into degradable scaffolds and transplanting the scaffolds in the body to help the human body repair damaged tissues. For the preparation of biomaterials for tissue engineering scaffolds, in addition to the surface being required to promote cell adhesion, it is also required to be able to promote cell proliferation and differentiation. The way to achieve this goal is to prepare biomaterials compounded with cell growth factors. Cell growth factors are a class of polypeptide signaling molecules that affect cell activities through intercellular signal transmission. It can promote or inhibit the proliferation, migration, differentiation and gene expression of cells. Growth factors are all polypeptide protein macromolecules. The preparation process is complicated and the price is relatively expensive. However, a small concentration of growth factors will have an important regulatory effect on the physiological activities of cells. Therefore, growth factors are widely used to regulate cell growth. process. The combination of growth factors and biomaterials is one of the important research topics in the field of tissue engineering. Bioactive materials compounded with growth factors can better promote cell proliferation and induce cell differentiation to specific tissues or organs. Therefore, in tissue engineering technology, this type of bioactive material is the development and improvement of the usual cytocompatible materials, which can better promote cell growth and differentiation, and its targeting is more specific. Combining growth factors with biological materials, such as directly coating the cell growth factor solution on the surface of biological materials, the growth factors on the surface of the material are easily lost; and the growth factor aqueous solution cannot be spread evenly on the surface of the hydrophobic polymer material. The method of chemical grafting is used to chemically immobilize growth factors on the surface of the material. On the one hand, a higher concentration of growth factor solution is required as a reaction solution, which makes the cost too high and the utilization rate of growth factors is reduced. On the other hand, chemical reactions can easily destroy growth. The native conformation of the factor thereby reducing its activity.

Contents of Invention

The purpose of the present invention is to provide a method for introducing cell growth factors on the surface of polymer biomaterials to prepare active biomaterials compounded with cell growth factors.

The method for introducing cell growth factors on the surface of polymer biomaterials of the present invention, the specific steps are:

1) Immerse the polymer biomaterial in a hydrogen peroxide solution with a concentration of 10-40%, and oxidize it under ultraviolet light irradiation, the oxidation temperature is 20-80°C, and the time is 0.1-10 hours, and macromolecular peroxidation is introduced on the surface of the material. Hydrogen group, rinse with deionized water to remove free hydrogen peroxide molecules;

2) Under the protection of nitrogen, the material is immersed in a solution of acrylic acid, methacrylic acid or other carboxyl-containing vinyl monomers with a concentration of 0.1-50v%, and in the presence of ferrous ions, the contact of the monomer on the surface of the material is initiated. Branch polymerization reaction, the concentration of ferrous ions is 0.0001-0.01 mol/L, the reaction temperature is 20-60°C, the time is 20-80 minutes, and the reaction is washed with deionized water;

3) The material is immersed in a phosphate buffer solution (pH=7.4) containing 1-50 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, at 0-40 Under the temperature of ℃, react for 1 to 24 hours to activate the carboxyl group on the surface of the material;

4) physically mixing the cell growth factor with a biomacromolecule solution with a concentration of 1-100 mg/ml, wherein the content of the cell growth factor is 1 μg-10 mg/ml;

5) The material prepared in step (3) is taken out, immersed in the biomacromolecule solution mixed with cell growth factors prepared in step (4), and the carboxyl group after the surface activation of the material is condensed with the amino group in the biomacromolecule, and the reaction The time is 1 to 24 hours. After the reaction, the material is taken out and dried directly.

The polymer biomaterial in the present invention is mainly but not limited to polylactic acid, such as polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyurethane (PU) and other commonly used materials can be used. polymeric biomaterials. The morphologies of polymeric biomaterials include two-dimensional planar membranes and three-dimensional porous scaffolds.

Said biomacromolecules include gelatin, collagen, chitosan, polylysine, polyethyleneimine, polyallylamine and other biomacromolecules containing amino groups.

Said cell growth factors include fibroblast growth factor (FGF), bone morphogenetic protein (BMP), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), epidermal growth factor (EGF) , insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and interleukin (IL), etc.

The method of the present invention is simple to operate, has good repeatability, and has no adverse side effects. The cell growth factor introduced on the surface of the material is physically embedded in the biomacromolecule coating, so that the growth factor is protected by the biomacromolecule, and its natural The conformation is maintained, so it can stably exist on the surface of biological materials with the biomacromolecule coating, and has high biological activity, which can improve the utilization efficiency of growth factors. The release of the factor to the surface of the material needs to pass through the barrier of the biomacromolecule coating, so it has a slow release effect.

Description of drawings

Fig. 1 The curve of MTT activity of chondrocytes on the surface of unmodified PLLA membrane, collagen-coated PLLA membrane, PLLA membrane compounded with BMP and PLLA membrane compounded with bFGF as a function of culture time. Seeding density 60000/well, 24-well culture plate.

Fig. 2a Optical micrographs of chondrocytes on the surface of PLLA membrane compounded with BMP. The inoculation density was 40000/cm ² , the culture time was 2 days, and hematoxylin staining was performed.

Fig. 2b Optical micrographs of chondrocytes on the surface of PLLA membrane compounded with bFGF. Seeding density 40000/cm ² . Cultured for 2 days, stained with hematoxylin.

Fig. 3 MTT activity of chondrocytes in unmodified PLLA porous scaffolds, collagen-coated PLLA porous scaffolds, PLLA porous scaffolds compounded with BMP, and PLLA porous scaffolds compounded with bFGF. The seeding density was 600×10 ⁴ /ml.

Fig. 4a Laser confocal micrographs of chondrocytes in PLLA porous scaffolds compounded with BMP. The seeding density was 600×10 ⁴ /ml, the culture time was 2 weeks, and the FDA stained.

Fig. 4b Laser confocal micrographs of chondrocytes in the PLLA porous scaffold compounded with bFGF. The seeding density was 600×10 ⁴ /ml, the culture time was 2 weeks, and the FDA stained.

Detailed ways

The present invention can be better understood by the following examples, but these examples are not intended to limit the invention.

Example 1

Introduction of bone morphogenetic protein (BMP) or basic fibroblast growth factor (bFGF) on the surface of poly-L-lactic acid (PLLA) planar membranes.

BMP or bFGF was uniformly mixed with collagen/acetic acid solution (pH<4) to obtain a collagen solution containing BMP or bFGF, wherein the concentration of the collagen solution was 2.5 mg/ml, and the concentrations of BMP and bFGF were 0.3 mg/ml and 900 mg/ml, respectively. unit/ml.

Soak the PLLA planar film in 30% hydrogen peroxide solution, and oxidize it under the irradiation of 250w ultraviolet lamp for 1 hour, and the oxidation temperature is 50°C. Rinse with deionized water to remove free hydrogen peroxide. Under the protection of nitrogen, the PLLA planar film after photooxidation was immersed in a 5v% methacrylic acid solution (containing 0.0015 mol/L ferrous ammonium sulfate), and methacrylic acid was induced on the surface of the PLLA film at 30 ° C. Grafting polymerization reaction, the reaction time is 1 hour, so as to introduce carboxyl groups on the surface of the material. Soak the PLLA membrane in 10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) in phosphate buffer (pH=7.4), the reaction temperature is 4°C , the reaction time is 4 hours, and the carboxyl groups on the surface of the material are activated during the reaction. Then the PLLA membrane was immersed in the above-mentioned collagen/acetic acid solution containing BMP or bFGF, and reacted at 4° C. for 24 hours. After the reaction, the PLLA membrane is taken out of the collagen solution, and the collagen solution physically coated on the surface of the material is retained, dried directly, and a uniform and stable collagen coating containing BMP or bFGF is obtained on the surface of the PLLA membrane.

Figure 1 shows the MTT activity of chondrocytes on the surface of PLLA membranes containing BMP or bFGF. It can be seen from the figure that the MTT activity of chondrocytes on the surface of PLLA membranes after the introduction of cell growth factors is significantly improved compared with the blank control. Figure 2a and Figure 2b are optical microscope photos of chondrocytes on the surface of the PLLA membrane containing BMP or bFGF, respectively. It can be seen from the pictures that the chondrocytes on the surface of the active PLLA membrane are evenly distributed and spread well.

Example 2

Introduction of bone morphogenetic protein (BMP) or basic fibroblast growth factor (bFGF) in PLLA porous scaffolds.

Mix BMP or bFGF with collagen/acetic acid solution (pH<4) evenly to obtain a collagen solution containing BMP or bFGF, wherein the concentration of the collagen solution is 2.5 mg/ml, and the concentrations of BMP and bFGF are 0.3 mg/ml and 0.3 mg/ml respectively. 900 units/ml.

Soak the PLLA porous stent in 30% hydrogen peroxide solution, release the air in the stent by vacuuming, then press the hydrogen peroxide solution into the stent to restore the pressure, and oxidize it for 1 hour under the irradiation of 250w UV lamp , oxidized to a temperature of 50°C. The hydrogen peroxide solution in the scaffold was removed by centrifugation. Press deionized water into the inside of the scaffold, then remove it by centrifugation, and repeat it several times to clean the hydrogen peroxide in the porous scaffold. Under the protection of nitrogen, the photooxidized PLLA support was immersed in a 10v% methacrylic acid solution (containing 0.0015 mol/L ferrous ammonium sulfate), and the methacrylic acid solution was pressed into the porous support. Initiate the graft polymerization reaction of methacrylic acid on the internal surface of the PLLA porous scaffold at ℃, and the reaction time is 1 hour, so as to introduce carboxyl groups on the surface of the material. Deionized water is pressed into the inside of the scaffold, and then removed by centrifugation, repeated several times to clean the unreacted methacrylic acid monomer in the porous scaffold. The PLLA porous scaffold was immersed in 10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in phosphate buffer (pH=7.4), and the liquid was pressed into the bracket. The reaction temperature is 4° C., the reaction time is 4 hours, and the carboxyl groups on the surface of the material are activated during the reaction. The EDAC solution in the porous scaffold was removed by centrifugation, the porous scaffold was immersed in the above-mentioned collagen/acetic acid solution containing BMP or bFGF, and the liquid was pressed into the scaffold, and reacted at 4°C for 24 hours. After the reaction, the PLLA porous scaffold is taken out from the collagen solution, dried, and a uniform and stable collagen coating containing BMP or bFGF is obtained inside the PLLA porous scaffold, thereby preparing an active porous scaffold containing cell growth factors.

Figure 3 shows the MTT activity of chondrocytes inside the PLLA porous scaffold containing cell growth factors. It can be seen from the figure that the activity of chondrocytes inside the scaffold is significantly improved after the introduction of cell growth factors. Laser confocal microscopy photos showed (Fig. 4a, Fig. 4b) that the chondrocytes inside the PLLA scaffold containing cell growth factors were evenly distributed and spread well. The above results show that the PLLA porous scaffold material introduced with cell growth factor BMP or bFGF has excellent cell compatibility.

Claims (4)

1. A method for introducing cell growth factors on the surface of a polymer biomaterial, comprising the following steps: 1) Immerse the polymer biomaterial in a hydrogen peroxide solution with a concentration of 10-40%, and oxidize it under ultraviolet light irradiation, the oxidation temperature is 20-80°C, and the time is 0.1-10 hours, and macromolecular peroxidation is introduced on the surface of the material. Hydrogen group, rinse with deionized water to remove free hydrogen peroxide molecules; 2) Under the protection of nitrogen, the material is immersed in a solution of acrylic acid, methacrylic acid or other carboxyl-containing vinyl monomers with a concentration of 0.1-50v%, and in the presence of ferrous ions, the contact of the monomer on the surface of the material is initiated. Branch polymerization reaction, the concentration of ferrous ions is 0.0001-0.01 mol/L, the reaction temperature is 20-60°C, the time is 20-80 minutes, and the reaction is washed with deionized water; 3) Soak the material in a phosphate buffer solution with a pH of 7.4 containing 1-50 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 0-40°C Under high temperature, react for 1 to 24 hours to activate the carboxyl groups on the surface of the material; 4) physically mixing the cell growth factor with a biomacromolecule solution with a concentration of 1-100 mg/ml, wherein the content of the cell growth factor is 1 μg-10 mg/ml; 5) The material prepared in step (3) is taken out, immersed in the biomacromolecule solution mixed with cell growth factors prepared in step (4), and the carboxyl group after the surface activation of the material is condensed with the amino group in the biomacromolecule, and the reaction The time is 1 to 24 hours. After the reaction, the material is taken out and dried directly. 2. the method for introducing cell growth factors on the polymer biomaterial surface according to claim 1, characterized in that said polymer biomaterial is selected from polyglycolic acid, lactic acid-glycolic acid copolymer, polycaprolactone or any of the polyurethane polymer biomaterials. 3. the method for introducing cell growth factors on the polymer biomaterial surface according to claim 1, characterized in that said biomacromolecules are selected from gelatin, collagen, chitosan, polylysine, polyethylene Imine or polyallylamine Any of biomacromolecules containing amino groups. 4. the method for introducing cell growth factors on the polymer biomaterial surface according to claim 1, characterized in that said cell growth factors are selected from fibroblast growth factor, bone morphogenetic protein, transforming growth factor β, Any of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor, tumor necrosis factor, or interleukin.

Images