CN102423272B - A kind of porous support with network channel and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biomaterials and regenerative medicine, in particular to a porous support with network channels and a preparation method thereof. The matrix material of the porous support of the present invention can be inorganic materials such as ceramics, glass, carbon, etc., can also be a polymer material with self-bonding, solubility, and plasticity, and can also be a composite material of inorganic and polymer materials. The preparation method mixes the matrix material, the porogen and the network substance evenly. When the matrix material is an inorganic material, the porogen and the network substance are removed by high-temperature sintering; when the matrix material is a polymer material or a polymer material When compounding with inorganic materials, the porous scaffold with network channels is prepared by normal temperature molding/particle leaching method. The porous stent of the present invention builds a network channel structure on the three-dimensional space structure, which not only increases the connectivity between the pores of the stent, but also provides a favorable space for blood vessels to grow into the stent.

Description

A porous scaffold with network channels and its preparation method

technical field

The invention belongs to the technical field of biomaterials and regenerative medicine, and in particular relates to a porous support with network channels and a preparation method thereof.

Background technique

Tissue engineering is the basic principle, basic theory, basic technology and basic method of comprehensive applied engineering and life science. The core of tissue engineering is to establish a three-dimensional complex of cells and scaffold materials, that is, a living tissue with vitality, which is used to reconstruct the shape, structure and function of the diseased tissue and achieve permanent replacement. Tissue induction does not introduce exogenous seed cells, but directly implants porous scaffolds to induce cell entry and even differentiation in the in vivo environment. Porous scaffolds play a key role in regenerative medicine research such as tissue engineering and tissue induction. It not only provides structural support for specific cells, but also acts as a template to guide tissue regeneration and control tissue structure. Therefore, screening and preparing an ideal scaffold material is very important for the development and application of regenerative medicine.

The researchers found that the seed cells inside the scaffold will quickly consume nutrients and oxygen after being implanted in the body, because the cells can only maintain survival through diffusion within 150-200 μm around the blood vessel. If the internal blood circulation cannot be established, tissue engineering And tissue induction can only be limited to the construction of thinner tissues [JeroenRouwkema, et al. Trends in Biotechnology, 2008, 26:434–441]. Therefore, the key issue in constructing a three-dimensional tissue with a certain volume is how to solve the vascularization problem of engineered tissue [GriffithLG, et al. Science .2002, 295:1009-1014.]. At present, this view has become the consensus of many tissue engineering and tissue induction scholars at home and abroad [WangL, etal. Biomaterials .2010,31:9452-9461].

At present, studies on scaffold materials promoting vascularization have been reported. Most of them are studies on the material surface. Kirkpatrick [UngerRE, etal. Biomaterials .2005, 26:3461-3469.] and others observed that the extracellular matrix on the surface of polyethersulfone and silk fibroin network, growth factors Under the stimulation of collagen and collagen, blood vessel-like structures can be formed. There are also domestic scholars [GuoR, etal. Biomaterials . 2011, 32: 1019-1031.] loaded the growth factor VEGF-165 on the scaffold material of collagen-chitosan to repair the deep incision of pig epidermis. After 112 days, it was found that the repaired Skin tissue has a very similar structure to normal tissue and has 80% of the tension of normal tissue. The research on the three-dimensional pore size of the scaffold is also gradually active. For example, some researchers [DrueckeD., etal. Journal of BiomedicalMaterialsResearchPartA. 75 μm) stents, it was found that the vascularization rate of these stents was affected by the pore size, and the stents with the largest pore size were the most vascularized, and the stents with the largest pore size had larger vessels at the edge and center of the material at 8 and 12 days after surgery Density and RBC velocity, and vascularization was still active at day 20. Another researcher [ KubokiY , etal. The Journal of Bone and Joint Surgery 2001, 83A (Suppl.1): S105–15.] found that the hydroxyapatite porous scaffold material with a pore size of 90-120 μm first induces cartilage formation and then ossification, while the pore size Hydroxyapatite larger than 350 μm can directly induce bone tissue formation, and its critical pore size is 300-400 μm. This may be due to the fact that the large pore size is more conducive to vascularization, thereby bringing more nutrients to the local tissue. Other researchers believe that the connectivity between the pores has a greater impact on the vascularization of the scaffold material in vivo. The pores are not connected so that the new bones cannot be connected to each other, and lack continuity and integration, and blood vessels often cannot enter these pores. blind end. For example, some researchers [MastrogiacomoM, etal. Biomaterials . 2006, 27:3230-3237] produced two kinds of porous hydroxyapatite materials with different pore diameters, porosity and intrapore communication diameters, and implanted bone marrow stromal stem cells into large Histological analysis after operation showed that the two structural materials were well vascularized, all the pores contained blood vessels, and the number of blood vessels was also large, but interestingly, all the pores in the material with larger pores were connected. The diameter of the formed blood vessel is relatively large, that is to say, the connection between pores is the bottleneck of the entry and exit of new blood vessels, which determines the lumen size of the formed blood vessel. LuJX et al [BaiF, etal. TissueEngineeringPartA .2010,16:3791-3803] have done a lot of research on the connection path of scaffold pores, they will have precise pore size and connection path of β-tricalcium phosphate (β-TCP) porous The material was implanted into animals to study the formation of blood vessels. It was found that the connecting diameter was more important than the pore size for blood vessel formation, but when the pore size was greater than 400 μm, there was no difference between the two. In summary, the scaffold structure is an extremely important morphological property of engineered tissues. The suitable surface and three-dimensional pore structure of scaffold materials are conducive to the transportation and exchange of nutrients, oxygen and metabolites, and provide channels for the growth of new blood vessels. , thus facilitating the vascularization of new tissues in tissue engineering and tissue induction techniques.

How to control the structure, size and shape of the regenerated tissue through the structure and shape design of the scaffold material, as a framework connecting cells and tissues, and guide the tissue to grow into a specific shape? The present invention envisages simulating the shape of the vascular network, directly selects some natural network-type substances or substances that can be bionically prepared for vascular network-shaped substances from nature, and then uses these network-type substances as templates to be taken out to shape the scaffold material with Porous scaffolds of vascular network-shaped channels. It not only has suitable pore size, porosity, elastic strength and degradation time; it also has biocompatibility, which is suitable for cell adhesion and matrix deposition; moreover, there are some network channels in the scaffold which are suitable for cells to adhere to along the direction of the channel. Migration guides the direction of cell growth and migration; at the same time, due to the connectivity of the channel itself, the connectivity between the scaffold holes can be increased, which is more conducive to the transfer of nutrients and the discharge of cell metabolic waste, so that the cell scaffold can maintain its own structure through its own structure. survive.

Contents of the invention

The object of the present invention is to provide a porous scaffold with network channels and a preparation method thereof.

The porous scaffold with network channels proposed by the present invention is characterized in that the scaffold contains network channels (pores) in the shape of a simulated blood vessel network, the diameter of the channels (pores) is 1 μm-5 mm, and the porosity of the scaffold is 0.1-99%. The preferred porosity range is 40-95%.

In the porous scaffold of the present invention, network channels (pores) and non-network channels (pores) can exist at the same time, wherein the diameter of the non-network channel (pore) type pores is 5-2000 μm.

In the present invention, the base material of the porous support is ceramics, glass, carbon and other inorganic non-metallic materials, including titanium oxide, aluminum oxide, calcium oxide, sodium oxide, silicon oxide, calcium phosphate series (hydroxyapatite, phosphoric acid Tricalcium, tetracalcium phosphate, calcium pyrophosphate, biphasic calcium phosphate, calcium polyphosphate), calcium sulfate, calcium carbonate, low temperature isotropic carbon and mixtures of the above materials or mixtures with additives.

The base material of the porous support of the present invention can also be any one of the following degradable polymer materials: poly DL-lactide, poly L-lactide, polyglycolide, polylactic acid-glycolic acid Copolymer, poly3-hydroxybutyrate, polyhydroxyalkanoate, polyε-caprolactone, polyε-alkyl substituted caprolactone, polyδ-valerolactone, polycarbonate, polyorthoester, Polymethyl methacrylate, polydioxanone, polydioxane, polyether ester, or any of the copolymers or blends of any of the above polyesters;

The base material of the porous stent of the present invention can also be any one of the following non-degradable polymer materials: polystyrene, polyvinyl chloride, polyacrylate, polymethacrylate, polycarbonate, nylon, polyurethane, Polyoxymethylene, polyvinyl alcohol, polyvinyl acetate, polysiloxane, or any of the copolymers or blends composed of several of them.

The matrix material of the porous scaffold in the present invention can also be a composite of one or more of inorganic materials and polymer materials in various forms.

The preparation method of the porous scaffold with network channels of the present invention is to mix the matrix material or its precursor with the template having a network structure, or mix the matrix material or its precursor with the network template and the non-network porogen simultaneously , and then remove the network template and non-network porogen, so that the precursor becomes a matrix material, and finally a porous scaffold with network channels is obtained.

The porous scaffold with network channels proposed by the present invention, when the matrix material is an inorganic material, the specific steps of preparation are as follows:

Mix inorganic materials, binders, porogens and network substances to form a uniformly dispersed mixture of inorganic materials, porogens and network substances; pour this mixture into a mold for compression molding to make inorganic materials - The mixture product of porogen-network substance, and then dry the mixture at room temperature for 0-3 days, then heat it to a high temperature of 800-1500°C, sinter to remove the binder, porogen and network substance, and obtain the obtained Inorganic porous scaffolds are required.

Inorganic materials are matrix materials, and the porogens used are divided into inorganic and organic. Inorganic porogens include high-temperature decomposable salts such as ammonium carbonate, ammonium bicarbonate, and ammonium chloride, and other decomposable compounds such as Si ₃ N ₄ and coal powder, carbon powder, etc. Organic porogens are mainly natural fibers, high molecular polymers and organic acids, such as sawdust, naphthalene, starch, and polyvinyl alcohol, polyvinyl chloride, polystyrene, polyvinyl butyral, stearic acid, methyl Methyl acrylate, methyl cellulose, urea, etc. or a mixture of several of them; the particle size of the porogen is 5-2000 μm (the preferred range is 200-600 μm), and the amount of the porogen is 1-99wt% of the mixture (preferably in the range of 40-85wt%).

The network substance used is leaf vein, loofah, silk, sugar silk net, or a mixture of several of them; the network diameter is 1 μm-5mm, and the amount of network substance is 0.00001-99wt% of the mixture (the preferred range is 0.001- 20wt%).

Since the above-mentioned network substance itself also acts as a porogen, the present invention includes not only the porous scaffold obtained by using the network substance alone, but also the porous scaffold obtained by using the network substance and other types of porogen simultaneously.

The binder used is macromolecule, liquid hydrocarbon substance and phosphate, or a mixture composed of several of them, and the amount of binder is 0.1-99wt% of the mixture. (The preferred range is 1-60wt%)

The porous scaffold with network channels proposed by the present invention, when the matrix material is a polymer material and its composite with inorganic materials, the specific steps of preparation are as follows:

(1) Disperse the polymer material or its composite with the inorganic material in solvent A to form a mixed solution, disperse the porogen in the mixed solution, and volatilize part of the solvent A while stirring to form a uniformly dispersed mixed solution- The mixture of porogen particles; then poured into a mold containing the network substance, and molded in the mold to form a mixed solution-porogen particle-network substance mixture product; then, the solvent A part is volatilized at room temperature , and then vacuum-dried to remove the residual solvent A. After the solvent A is completely removed, a molded product of the mixture is obtained; the vacuum drying temperature does not exceed the melting point or glass transition temperature of the scaffold material; the solvent A used is a soluble polymer material Solvents that do not dissolve porogens and network substances;

(2) Put the above-mentioned polymer material or its composite with inorganic materials-porogen particles-network substance into the solvent B to leach the porogen particles and network substance; the solvent B used is A solvent that dissolves porogens and network substances but does not dissolve the polymer materials used or their composites with inorganic materials;

(3) Take out the above-mentioned scaffold from which the porogen particles and network substances have been leached out from the container, and after most of the solvent B volatilizes, put it into a vacuum oven for vacuum drying, completely remove the solvent B, and obtain the desired porous scaffold ; The vacuum drying temperature does not exceed the melting point or glass transition temperature of the scaffold material.

Similarly, since the above-mentioned network substance itself also acts as a porogen, the present invention includes both the simple use of the network substance to obtain a porous scaffold, and the simultaneous use of the network substance and other types of porogens. Porous scaffold.

Macromolecular materials or their composites with inorganic materials are used as matrix materials, and the porogens used are inorganic salt particles, polysaccharides, proteins, synthetic polymers, or mixtures of several of them; the particle size of porogens is The size is 5-2000 μm (the preferred range is 200-600 μm), and the amount of the porogen is 1-99wt% of the mixture (the preferred range is 80-95wt%).

The network substance used is a substance that can be made into a network and can be removed from the polymer material matrix, such as glucose, fructose, sucrose, maltose, paraffin, polyvinylpyrrolidone, or a mixture of several of them; the diameter of the network substance is 1 μm -5mm, the amount of the network substance is 0.00001-99wt% of the mixture (the preferred range is 0.001-20wt%).

The concentration range of the polymer solution used is 1-99wt%.

Solvent A used is acetone, butanone, chloroform, dichloromethane, tetrahydrofuran, benzene, toluene, xylene, ethylene glycol, cyclohexanone, dioxane, N,N-dimethylformamide, formic acid , benzyl alcohol, cyclohexane, or a mixture of several of them.

The solvent B used is any one of water, alcohol, amine, hydrocarbon and halogenated hydrocarbon, or a mixture of several of them.

The amount of solvent B is 10-1000 times of the total weight of the mixture.

In order to simulate the shape of the vascular network, the present invention directly selects some natural network-type substances from nature or materials that can be bionically prepared into a vascular network-shaped substance, and then uses these network-type substances as templates to be taken out, and shapes them in the scaffold material to prepare Porous scaffolds of vascular network-shaped channels. It not only has suitable pore size, porosity, elastic strength and degradation time, but also has biocompatibility, which is suitable for cell adhesion and matrix deposition; moreover, there are some network channels in the scaffold, which are suitable for cells to adhere to and adhere to along the direction of the channel. Migration guides the direction of cell growth and migration; at the same time, due to the connectivity of the channel itself, the connectivity between the scaffold holes can be increased, which is more conducive to the transfer of nutrients and the discharge of cell metabolic waste, so that the cell scaffold can maintain its own structure through its own structure. survive.

Description of drawings

The optical micrograph figure (×100) of Fig. 1 loofah.

Figure 2 is a scanning electron micrograph of the β-TCP three-dimensional porous scaffold with network channels obtained by using loofah as a template.

detailed description

The present invention is further described below by embodiment.

Example 1 Add 1.1g of β-TCP to 1.1g of 5% polyvinyl alcohol (PVA) aqueous solution, stir, grind, then add 0.45g of 380-550μm diameter paraffin balls and stir evenly, and then pour 0.054g of loofah (300μm in diameter) Molded in a mold, took out the scaffold, left it at room temperature for 24 hours, put it into a muffle furnace and heated it to 1050°C for sintering, and obtained a porous scaffold with network channels. A three-dimensional porous scaffold with channels (300 μm in diameter) with a pore size of about 450 μm and a porosity of about 50% was obtained.

Example 2 Add 1.8g of hydroxyapatite to 1.1g of 5% PVA aqueous solution, stir, grind, then add 0.45g of 380-550μm diameter paraffin balls and stir evenly, then pour into a mold with 0.054g of loofah (300μm in diameter) and press it After molding, take out the scaffold, place it at room temperature for 48 hours, put it into a muffle furnace and heat it to 1100°C for sintering to obtain a three-dimensional porous scaffold with a channel (diameter of 300 μm) with a pore diameter of about 450 μm and a porosity of about 50%.

Example 3 Add 0.54g of β-TCP to 0.54g of 10% PVA aqueous solution, stir, grind, then add 0.45g of 270-380μm diameter paraffin balls and stir evenly, then pour it into a mold with 0.054g of loofah (300μm in diameter) and mold it. The scaffold was taken out, left at room temperature for 24 hours, put into a muffle furnace and heated to 1050°C for sintering to obtain a three-dimensional porous scaffold with a channel (diameter of 300 μm) with a pore diameter of about 300 μm and a porosity of about 50%.

Example 4 Add 1.8g of hydroxyapatite to 0.54g of 10% PVA aqueous solution, stir, grind, then add 0.35g of 270-380μm diameter paraffin balls and stir evenly, then pour into a mold with 0.05g of loofah (300μm in diameter) and press it Forming, taking out the scaffold, leaving it at room temperature for 24 hours, putting it into a muffle furnace for high-temperature sintering to remove the loofah, heating to 1100°C for sintering, and obtaining a porous scaffold with a porosity of 50% with network channels. A three-dimensional porous scaffold with channels (300 μm in diameter) with a pore size of about 300 μm and a porosity of 45% was obtained.

Example 5 Dissolve 2g of PLGA (85/15) in 11g of dichloromethane, 30g of sodium chloride (particle size 180 μm-280 μm) is evenly dispersed in the solution of PLGA in dichloromethane, and then pour 1g of sugar fiber ( obtained by wire drawing on a commercially available wire drawing machine, in a mold with a diameter of 10 μm), molded at room temperature, decompressed after 24 hours, washed the bracket with 200ml of deionized water, and changed the water every 0.5 hours until the aqueous solution of 0.1mol/L silver nitrate was added dropwise to In the leaching solution, no white precipitate appeared, and then vacuum-dried at a drying temperature of 20°C and a drying time of 48 hours to obtain a three-dimensional porous scaffold with a channel (diameter of 10 μm) with a pore diameter of about 200 μm and a porosity of 90%.

Example 6 Dissolve 2g of PLA in 11g of dichloromethane, then pour it into a mold loaded with 20μg of sugar fibers (10μm in diameter), press at room temperature, decompress after 24h, wash the bracket with 200ml of deionized water, and change the water every 0.5h , until the aqueous solution of 0.1mol/L silver nitrate is added dropwise to the leaching solution, no white precipitate appears, and then vacuum-dried at a drying temperature of 20°C and a drying time of 48 hours to obtain a pore size of 10 μm and a porosity of 0.1%. Three-dimensional porous scaffolds with channels (10 μm in diameter).

Example 7 Dissolve 2g of polylactic acid in 11g of dichloromethane, 75g of sodium chloride (50 μm-150 μm in particle size) is evenly dispersed in the dichloromethane solution of polylactic acid, and then pour 5 g of sugar fibers (10 μm in diameter) ), molded at room temperature, decompressed after 24 hours, washed the bracket with 200ml of deionized water, and changed the water every 0.5 hours, until the aqueous solution of 0.1mol/L silver nitrate was added dropwise to the leaching solution, and no white precipitate appeared, and then Vacuum drying was carried out at a drying temperature of 20°C and a drying time of 48 hours to obtain a three-dimensional porous scaffold with a channel (diameter of 10 μm) with a pore size of about 100 μm and a porosity of 99%.

Example 8 Disperse 2g of PLGA and 100mg of β-TCP in 11g of dichloromethane, and 26g of sodium chloride (particle size 50μm-150μm) is evenly dispersed in the solution of PLGA and β-TCP in dichloromethane, and then poured into the solution loaded with 4.5 g Sugar fiber (10 μm in diameter) mold, press at room temperature, decompress after 24 hours, wash the bracket with 200ml of deionized water, change the water every 0.5h, until the aqueous solution of 0.1mol/L silver nitrate is added dropwise to the leaching solution, No white precipitate appeared, and then vacuum-dried at a drying temperature of 20°C and a drying time of 48 hours to obtain a three-dimensional porous scaffold with a channel (diameter of 10 μm) with a pore size of about 100 μm and a porosity of 90%.

Example 9 Dissolve 2g of polystyrene in 11g of chloroform, 17g of sodium chloride (50μm-150μm in particle size) is evenly dispersed in the polystyrene chloroform solution, and then pour 1g of sugar fibers (10μm in diameter) into the In the mold, press at room temperature, decompress after 24 hours, wash the bracket with 200ml deionized water, change the water every 0.5h, until the aqueous solution of 0.1mol/L silver nitrate is added dropwise to the leachate, no white precipitation appears, and then vacuum Drying at a drying temperature of 20°C and a drying time of 48 hours to obtain a three-dimensional porous scaffold with channels (10 μm in diameter) with a pore size of about 100 μm and a porosity of 90%.

Claims (6)

1. there is a porous support for network channel, it is characterized in that this network channel diameter is 1 μm of-5mm, and the porosity of support is 0.1-99% containing network channel in described porous support; There is network channel hole and non-network access opening in support, the diameter dimension of non-network passage nibs is 5-2000 μm simultaneously; The network-like material forming network channel is vein, Retinervus Luffae Fructus or both mixing.

2. porous support according to claim 1, is characterized in that its matrix material is the mixture of one or more in titanium oxide, aluminium oxide, calcium oxide, sodium oxide, silicon oxide, calcium phosphate series, calcium sulfate, calcium carbonate, Low Temperature Isotropic Carbon.

3. one kind has the preparation method of the porous support of network channel as claimed in claim 1 or 2, it is characterized in that, host material or its precursor are mixed mutually with the template with network structure, or host material or its precursor are mixed with network template and non-network type porogen simultaneously; Then remove network template and non-network type porogen, and make precursor become host material, final acquisition has the porous support of network channel.

4. preparation method according to claim 3, is characterized in that, when being inorganic material for matrix material, concrete steps are as follows:

Inorganic material, binding agent, porogen are mixed, forms finely dispersed inorganic material, binding agent, porogen mixture; This mixture is poured in the mould that network-like material is housed and carries out compression molding, make the mixture goods of inorganic material-porogen-network-like material, then in room temperature environment, make the dry 0-3d of mixture, be heated to high temperature 800-1500 DEG C again, sintering, except no-bonder, porogen and network-like material, just obtains required inorganic porous support.

5. preparation method according to claim 4, it is characterized in that porogen used is divided into and has inorganic and organic two classes, wherein, inorganic porogen is ammonium carbonate, ammonium bicarbonate or ammonium chloride, or is Si ₃n ₄, coal dust or carbon dust; Organic porogen is the one of sawdust, naphthalene, starch, polyvinyl alcohol, polrvinyl chloride, polystyrene, polyvinyl butyral resin, stearic acid, methymethacrylate, methylcellulose, carbamide, or by formed mixture several among them; Porogen grain size is 5-2000 μm, and porogen consumption is the 1-99wt% of mixture;

Network-like material diameter is 1 μm of-5mm, and the consumption of network-like material is the 0.00001-99wt% of mixture.

6. want the preparation method described in 5 according to right, it is characterized in that binding agent used is liquefied hydrocarbon material or phosphate, or by formed mixture several among them.