CN101264341A - Three-dimensional porous tissue engineering scaffold material, its preparation and application - Google Patents

Abstract

The invention relates to a three-dimensional porous tissue engineering scaffold material, its preparation and application. The material comprises polybutylene succinate PBS and polycaprolactone PCL, and its weight ratio is: polybutylene succinate 90 to 10 parts, 10 to 90 parts of polycaprolactone; preparation: put the porogen evenly in the mold, pour PBS/PCL blended chloroform solution, place the prototype of the bracket in a fume hood for at least 24 hours, and immerse in deionized water After 2-4 days, the PBS/PCL scaffold material can be obtained after vacuum drying; application: as a bone or cartilage tissue engineering cell scaffold for the repair and reconstruction of bone or cartilage tissues and organs. The inner pore structure of the scaffold material prepared by the invention is uniform, the pores are connected to each other, the pore diameter is 10-500 μm, the porosity varies between 70-91%, the scaffold system structure is stable, and the preparation process is simple.

Description

Three-dimensional porous tissue engineering scaffold material, its preparation and application

technical field

The invention belongs to the field of tissue engineering scaffold materials and preparation, in particular to a tissue engineering scaffold material using polybutylene succinate/polycaprolactone as a raw material and a tissue engineering scaffold prepared by solvent casting/particle leaching method.

Background technique

In daily life, the loss or failure of human tissues and organs is very common, which poses a serious threat to people's health and life. For a long time, human beings have been exploring and researching the use of materials and biotechnology to improve their own health. Traditional treatment methods include tissue and organ transplantation, surgical reconstruction, drug therapy, treatment with artificial substitutes and mechanical devices, etc., but these methods all have their obvious shortcomings. In the 1990s, with the development of cell biology, molecular biology, material science and related physical and chemical disciplines, tissue engineering was proposed as a new treatment method, and gradually became a very promising organ reconstruction and A medical approach to tissue regeneration. The basic principle is [Langer R, Vacanti J P. Tissue Engineering. Science, 1993, 260 (5110): 920], the tissue cells cultured in vitro are adsorbed and amplified in a biocompatible material that can be gradually degraded by the human body. On the absorbed biomaterial, a cell-biomaterial complex is formed. The biomaterial provides cells with a living three-dimensional space, which is beneficial for cells to obtain sufficient nutrients, exchange nutrients, and remove waste, so that cells can grow on the prefabricated three-dimensional shape of the scaffold. The cell-biomaterial complex is then implanted into the lesioned part of the body tissue. During the gradual degradation and absorption process of the biological scaffold, the planted cells continue to proliferate and secrete the matrix, forming new tissues and organs with functions and shapes corresponding to themselves. This vital living tissue can reconstruct the shape, structure and function of the damaged tissue and achieve permanent replacement.

Therefore, in the study of tissue engineering scaffold materials, the focus is on the selection of biodegradable materials and the preparation of porous scaffolds. In the selection of biodegradable materials, aliphatic polyester is the most promising one. At present, commercialized aliphatic polyesters mainly include polylactic acid (PLA), polyglycolide (PGA), polycaprolactone (PCL) and polyhydroxyalkanoates (PHAs), but due to their high price and mechanical Poor performance, poor processability, or some shortcomings in clinical applications limit their range of use. So far, there is no single biomaterial that can meet all the needs of tissue engineering for scaffold materials. It is necessary to integrate the advantages of existing biomaterials and organically integrate them together to prepare biocompatible and sufficient biomaterials. Space and performance support, guide and organize cell growth, tissue engineering scaffolds that can support tissue growth and ensure non-toxic degradation products in vivo. At present, the methods used to prepare porous scaffold materials at home and abroad mainly include solvent casting/particle leaching, fiber bonding, gas foaming, phase separation/emulsification, and freeze-drying.

Since the emergence of tissue engineering, a large number of scaffold materials have been used to repair various tissues and organs such as bone, cartilage, skin, tendon, nerve, blood vessel, liver, and pancreas. Among them, the research on bone and cartilage is the most, and the research on skin is the most successful. Goh et al. prepared a honeycomb-like porous PCL material with a pore size of 160-700 μm and a porosity of 48-77%. PCL scaffold materials loaded with different doses of drugs were implanted into rabbit epithelial cells, and a large number of them could grow after 2-4 weeks. Chondrocytes, formed primary cartilage tissue after 6 weeks [Goh JCH, Shao XX, Hutmacher DW, et al. Tissue engineering approach too osteochondral repair and regeneration. Journal of Mechanics in Medicine a-nd Biology, 2004, 4(4): 463 -483]. Liu Huaguo and others prepared polycaprolactone (PCL) porous scaffolds by freeze-drying/particle leaching composite method. The research results showed that the PCL scaffold prepared by composite method had high porosity (86%), uniform pore structure, good pore connectivity, The pore size can be controlled, and the scaffold structure can be controlled by adjusting the pre-freezing temperature and the particle size of the pore-forming agent [Liu Huaguo, Wang Yingjun, etc. Preparation of polycaprolactone tissue engineering scaffolds by freeze-drying/particle leaching composite method. Materials Herald, 2007, 21(2):125-131]. S.-J.Shieh et al. used sucrose to prepare shape templates, and used solvent casting/particle leaching to prepare cartilage tissue engineering carrier materials of ear-shaped PCL and PHB materials, and studied their biological characteristics in nude mice [Shy- Jou Shieh, Schinichi Terada, Joseph P. Vacanti. Tissue engineering auricular reconstruction: in vitro and in vivo studies. Biomaterials, 2004, (25): 1545-1557]. K. Fujihara et al. used PCL/CaCO ₃ to blend electrospinning at the ratio of 75:25, 25:75 (w/w) and implant osteoblasts. The absorbance of the fiber membrane is the same as that of the conventional culture plate, indicating that it has good adhesion and proliferation effect [K.Fujiharaa, M.Kotakib, S.Ramakrishna.Guided bone regeneration membrane made of polycaprolactone/calciumcarbonate composite nano-fibers.Biomaterials, 2005, (26): 4139-4147]. Teoh et al. used PCL or PCL/HA filament fibers to prepare three-dimensional porous tissue engineering carrier materials by a rapid prototyping technique-fused deposition method (FDM) for bone and cartilage tissue engineering [Teoh, Swee Hin, Hutmacher, et al. al. Methods for fabricating a filament for use in tissue engineering. USP-6,730,252. May 4, 2004.].

In domestic patents, there is a composite material of polycaprolactone, chitosan and hydroxyapatite to prepare a scaffold material with excellent performance. This method is suitable for clinically repairing various bone defects [Liu Rongfang et al. Polycaprolactone - Preparation method of chitosan network hydroxyapatite composite porous scaffold material (CN 101015712A)]. There are also artificial chest wall materials prepared by using chitin fiber and polycaprolactone through twin-screw extruder and flat vulcanizer molding [Sun Kang et al. Biodegradable polycaprolactone artificial chest wall material and its preparation method (CN 1593674A)] . The method of preparing porous polymer scaffolds for tissue engineering by combining the wax ball porogen method and thermal phase separation method can be applied to polymers including polycaprolactone, polylactic acid, polyurethane, polyanhydride, collagen, etc., and these polymers A mixture of substances and a copolymer between them [Gao Changyou et al. A method for preparing a polymer porous scaffold by combining wax ball porogen with thermally induced phase separation (CN1226336C)]. At present, the defects of the existing preparation technology of tissue engineering scaffold materials mainly exist in: in the selection of biomaterials, the mechanical strength and degradation rate of natural polymers such as collagen and chitosan are difficult to control, and large-scale production is limited; Synthetic polymers such as PGA and PLA are easy to control in terms of the macrostructure, mechanical properties and degradation cycle of the product, but there will be defects during the long-term implantation process, including bacterial infection, antigenicity, insufficient material supply and degradation. wait. So far, there is no single biomaterial that can meet all the needs of tissue engineering for scaffold materials. Therefore, combining the advantages of existing biomaterials that can be used as tissue scaffolds, organically blending them together to prepare ideal tissue engineering scaffold materials has become research focus. In the traditional preparation method, the pore size of the carrier obtained by the freeze-drying method is relatively small, and organic solvents are used in the preparation of the scaffold, which may have a certain impact on the cells; the internal structure of the scaffold obtained by the fiber bonding method is not stable and easy to It leads to planting cell damage; thermally induced phase separation technology is extremely sensitive to processing conditions, and it is difficult to precisely control the shape of scaffold materials, and there are also hidden dangers in solvent residues. Neither gas foaming technology nor electrospinning method can accurately control the size of the pores and porosity, and the latter is difficult to prepare a scaffold with a certain thickness; and newly developed methods such as three-dimensional printing technology mainly rely on computer technology support.

Polycaprolactone PCL has been proved to have good biocompatibility, and it can be blended or copolymerized with various polymers to prepare composite materials. Polybutylene succinate (PBS) is a class of aliphatic polyester developed in the early 1990s. It has low cost, good mechanical properties, excellent processing performance, good biocompatibility and bioabsorbability, etc. Advantages, it has potential advantages in the application of tissue engineering scaffold materials.

At present, there are no patent reports at home and abroad that use polybutylene succinate/polycaprolactone blend materials to prepare three-dimensional porous scaffold materials for tissue engineering by solvent casting/particle leaching.

Contents of the invention

The purpose of the present invention is to provide a three-dimensional porous tissue engineering scaffold material, its preparation and application, the method combines the advantages of existing biological materials, overcomes the shortcomings of single material performance, and the prepared tissue engineering scaffold material has good biocompatibility It can be used as a bone or cartilage tissue defect repair material and a cell scaffold material for in vitro tissue culture to meet the needs of the development of a new generation of biomaterials.

The three-dimensional porous tissue engineering scaffold material of the present invention comprises polybutylene succinate (PBS) and polycaprolactone (PCL), and its weight ratio is: polybutylene succinate 90～10 parts, 10-90 parts of polycaprolactone.

The polybutylene succinate (PBS) has a molecular weight range of 40,000 to 200,000 and a melting point of 110 to 120°C, and PCL has a molecular weight range of 1 to 200,000 and a melting point of 55 to 65°C.

The tissue engineering scaffold material has a three-dimensional porous structure with a uniform pore structure, the penetration degree of each pore is 90%-100%, the pore diameter is 10-500 μm, and the porosity varies between 70-91%.

The preparation method of the three-dimensional porous tissue engineering scaffold material of the present invention is prepared by solvent casting/particle leaching method, and the specific steps include:

(1) Completely dissolve polybutylene succinate and polycaprolactone blending raw materials in chloroform at 25-35°C, the weight ratio of raw materials is: polybutylene succinate 90-10 10 to 90 parts of polycaprolactone, stirred evenly to make a blend solution with a mass percentage concentration of 2% to 20%;

(2) Put the porogen in the diameter range of 50-500 μm evenly in the mold, pour the blend solution of polybutylene succinate and polycaprolactone, and the casting process can be repeated many times until the mold is just submerged The porogen in the prepared scaffold material;

(3) The scaffold material is placed in a fume hood for at least 24 hours to completely evaporate the solvent, and then immersed in deionized water for 2 to 4 days to leach the porogen particles, during which time the water is replaced every 6 hours;

(4) Vacuum drying to obtain a three-dimensional porous polybutylene succinate/polycaprolactone scaffold material with a sponge structure;

(5) Sterilize and pack the three-dimensional porous scaffold material.

The porogen is one or more of sodium chloride with good water solubility, glucose, ice particles, polyvinyl alcohol, polyethylene oxide and gelatin particles.

The application of the three-dimensional porous tissue engineering scaffold material of the present invention is as a bone or cartilage tissue engineering cell scaffold for the repair and reconstruction of bone or cartilage tissues and organs, and is made into a specific shape by a mold.

In the present invention, by adjusting the concentration of the blend solution and the particle size of the porogen, the pore size of the scaffold material can be controlled to be 10-500 μm, and the micropores smaller than 50 μm are caused by solvent volatilization, and the scaffold can also be formed by adjusting the concentration of the casting solution Controlled to obtain tissue engineering three-dimensional porous scaffolds with various thickness sizes and complex shapes.

Beneficial effects of the present invention:

(1) The micropore size, porosity and biodegradation rate of the scaffold can be adjusted by using blend solutions with different mass percentage concentrations, different diameters and types of porogens, and changing leaching conditions;

(2) The internal pore structure of the obtained three-dimensional porous tissue engineering scaffold material is uniform, and the pores are connected to each other. The scaffold system structure is stable and the preparation process is simple. It can be used as a bone or cartilage tissue engineering cell scaffold, and it can be made into a specific mold by using a specific mold. Shape bracket.

Description of drawings

Figure 1 is a cross-sectional optical microscope photo of the PBS/PCL three-dimensional porous tissue engineering scaffold material prepared by solvent casting/particle leaching method.

Detailed ways

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

The high polymer PBS with a molecular weight of 40,000 and a melting point of 115°C, and the high polymer PCL with a molecular weight of 80,000 and a melting point of 63°C were completely dissolved in chloroform at 25°C, and stirred evenly to make a mass percentage concentration For a 20% blend solution, uniformly place sodium chloride with a diameter of 50-200 μm in the mold, pour the PBS/PCL blend solution until it just submerges the porogen in the mold, and place the bracket material in a fume hood for 24 hours, until the solvent was completely volatilized, immersed in deionized water for 4 days to leach the sodium chloride particles, during which the water was replaced every 6 hours, and vacuum-dried to obtain a three-dimensional porous PBS/PCL scaffold with a sponge structure. The material has a pore size of 10-200 μm measured by a scanning electron microscope, a porosity of 85% measured by a liquid displacement method, a degree of pore penetration of more than 90%, an appropriate in vitro degradation period, and excellent mechanical properties. The material can be used as a bone tissue engineering carrier material for repairing and reconstructing bone damage.

Example 2

The high polymer PBS with a molecular weight of 80,000 and a melting point of 115°C, and the high polymer PCL with a molecular weight of 100,000 and a melting point of 63°C were completely dissolved in chloroform at 25°C, and stirred evenly to make a mass percentage concentration 10% blend solution, uniformly place sodium chloride with a diameter of 300-500 μm in the mold, pour PBS/PCL blend solution until just submerged in the porogen in the mold, place the bracket material in a fume hood for 24 hours, until the solvent was completely volatilized, immersed in deionized water for 4 days to leach the sodium chloride particles, during which the water was replaced every 6 hours, and vacuum-dried to obtain a three-dimensional porous PBS/PCL scaffold with a sponge structure. The material has a pore diameter of 10-500 μm measured by a scanning electron microscope, a porosity of 89% measured by a liquid displacement method, a degree of pore penetration of more than 90%, an appropriate in vitro degradation period, and excellent mechanical properties. The material can be used as a bone tissue engineering carrier material for repairing and reconstructing bone damage.

Example 3

The high polymer PBS with a molecular weight of 100,000 and a melting point of 118°C, and the high polymer PCL with a molecular weight of 150,000 and a melting point of 64°C were completely dissolved in chloroform at 30°C and stirred evenly to make a mass percentage concentration 6% blend solution, uniformly place sodium chloride with a diameter of 400-500 μm in the mold, pour PBS/PCL blend solution until just submerged in the porogen in the mold, place the bracket material in a fume hood for 24 hours, until the solvent was completely volatilized, immersed in deionized water for 4 days to leach the sodium chloride particles, during which the water was replaced every 6 hours, and vacuum-dried to obtain a three-dimensional porous PBS/PCL scaffold with a sponge structure. The pore diameter of the material measured by the scanning electron microscope is 10-500 μm, the porosity measured by the liquid displacement method is 83%, the degree of pore penetration reaches more than 90%, the in vitro degradation period is appropriate, and the mechanical properties are excellent. The material can be used as a bone tissue engineering carrier material for repairing and reconstructing bone damage.

Example 4

The high polymer PBS with a molecular weight of 150,000 and a melting point of 117°C, and the high polymer PCL with a molecular weight of 10,000 and a melting point of 60°C were completely dissolved in chloroform at 35°C, and stirred evenly to make a mass percentage concentration For a 4% blend solution, put sodium chloride with a diameter of 100-300 μm evenly in the mold, pour the PBS/PCL blend solution until it just submerges the porogen in the mold, and place the bracket material in a fume hood for 24 hours, until the solvent was completely volatilized, immersed in deionized water for 4 days to leach the sodium chloride particles, during which the water was replaced every 6 hours, and vacuum-dried to obtain a three-dimensional porous PBS/PCL scaffold with a sponge structure. The material has a pore size of 10-300 μm measured by a scanning electron microscope, a porosity of 83% measured by a liquid displacement method, a degree of pore penetration of more than 90%, an appropriate in vitro degradation period, and excellent mechanical properties. The material can be used as a bone tissue engineering carrier material for repairing and reconstructing bone damage.

Example 5

The high polymer PBS with a molecular weight of 200,000 and a melting point of 118°C, and the high polymer PCL with a molecular weight of 200,000 and a melting point of 64°C were completely dissolved in chloroform at 35°C, and stirred evenly to make a mass percentage concentration 2% blend solution, uniformly place sodium chloride with a diameter of 300-500 μm in the mold, pour PBS/PCL blend solution until just submerged in the porogen in the mold, place the bracket material in a fume hood for 24 hours, until the solvent was completely volatilized, immersed in deionized water for 4 days to leach the sodium chloride particles, during which the water was replaced every 6 hours, and vacuum-dried to obtain a three-dimensional porous PBS/PCL scaffold with a sponge structure. The material has a pore size of 10-500 μm measured by a scanning electron microscope, a porosity of 76% measured by a liquid displacement method, a degree of pore penetration of more than 90%, an appropriate in vitro degradation period, and excellent mechanical properties. The material can be used as a bone tissue engineering carrier material for repairing and reconstructing bone damage.

Claims (7)

1. A three-dimensional porous tissue engineering scaffold material, characterized in that: the material comprises polybutylene succinate PBS and polycaprolactone PCL, and its weight ratio is: polybutylene succinate 90～ 10 parts, 10-90 parts of polycaprolactone. 2. The three-dimensional porous tissue engineering scaffold material according to claim 1, characterized in that: the polybutylene succinate has a molecular weight range of 40,000 to 200,000 and a melting point of 110 to 120°C; the molecular weight range of PCL is 1 to 200,000, and the melting point is 55 to 65°C. 3. The three-dimensional porous tissue engineering scaffold material according to claim 1 or 2, characterized in that: the tissue engineering scaffold material has a three-dimensional porous structure, the pore structure is uniform, and the degree of penetration between the pores is 90% to 100%. The pore diameter is 10-500 μm, and the porosity varies between 70-91%. 4. A method for preparing a three-dimensional porous tissue engineering scaffold material, which is prepared by solvent casting/particle leaching, and the specific steps include: (1) Completely dissolve polybutylene succinate and polycaprolactone blending raw materials in chloroform at 25-35°C, the weight ratio of raw materials is: polybutylene succinate 90-10 10 to 90 parts of polycaprolactone, stirred evenly to make a blend solution with a mass percentage concentration of 2% to 20%; (2) uniformly placing porogens in the range of 50-500 μm in diameter in the mold, pouring a blended solution of polybutylene succinate and polycaprolactone to prepare a scaffold material; (3) The scaffold material is placed in a fume hood for at least 24 hours to completely evaporate the solvent, and then immersed in deionized water for 2 to 4 days to leach the porogen particles; (4) Vacuum drying, sterilization and packing. 5. the preparation method of three-dimensional porous tissue engineering scaffold material according to claim 4 is characterized in that: described porogen is sodium chloride, glucose, ice particle, polyvinyl alcohol, polyethylene oxide and gelatin particle one or several. 6. The preparation method of the three-dimensional porous tissue engineering scaffold material according to claim 4, characterized in that: the water is changed every 6 hours during the immersion in deionized water in the step (3). 7. The application of a three-dimensional porous tissue engineering scaffold material, which is used as a bone or cartilage tissue engineering cell scaffold for the repair and reconstruction of bone or cartilage tissues and organs.

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