CN101496908A - Pearl powder artificial bone supporting material with multi-stage micro-nano structure and technique for producing the same - Google Patents

Abstract

The invention relates to a new bio-scaffold material for bone repair and a preparation method thereof. Dissolve PLGA and PLA with a mass ratio of 1:1-10:1 in chloroform, dimethyl sulfoxide, 1,4-dioxane or a mixed liquid of 1,4-dioxane and ultrapure water, Then according to the ratio of PLGA/PLA mixture: pearl powder (mass ratio) of 1:1-10:1, pearl powder treated with partial deproteinization or complete deproteinization is added to form a forming slurry. A three-dimensional scaffold with high porosity and connectivity is designed by 3D software, and then a low-temperature rapid prototyping system is used to shape the three-dimensional scaffold to obtain a scaffold with a microporous structure. The artificial bone bio-scaffold material prepared according to the above-mentioned method has a macrostructure of a channel with a pore diameter of 100-500 μm, and a microstructure of a three-dimensional scaffold structure with a micropore of 10-20 μm, wherein micron pearl powder is dispersedly distributed on the wall of the micropore. The porosity is 60%-90%, and the X, Y, and Z axes of the macroporous structure penetrate in three directions, with a penetration rate of 100%.

Description

A kind of pearl powder artificial bone scaffold material with multi-level micro-nano structure and its production process

technical field

The invention belongs to the technical field of biomedical materials, and in particular relates to a new bio-scaffold material for bone repair and a preparation method thereof.

Background of the invention

At present, there is an urgent and huge market demand for artificial bone tissue with good physiological functions and no rejection for bone defect repair in the world. In the field of artificial bone manufacturing, foreign research institutions and enterprises have invested a lot of resources in recent years and achieved a series of research results. Some artificial bone materials for defect repair have begun to enter clinical applications. At the same time, there are still many problems to be solved in the core issues of bone scaffold materials such as biocompatibility, osteoinductivity, porosity, and mechanical strength.

Pearl powder is an ideal natural bone-forming material, which has the characteristics of biocompatibility and good inductivity, and has broad application prospects. The physical and chemical properties and biological characteristics of nacre are very close to those of bone tissue. Studies have shown that there is a genetic relationship between nacre and bone, which makes nacre have good biocompatibility (Westbroek P, et al; 1998, 392 (6679):861-2). Moreover, the nacre implant can be dissolved and absorbed in the body, and has excellent biodegradation characteristics. Nacre was injected into the vertebral bone loss model in vivo, and it was observed that the nacre gradually degraded, and new mature bone trabeculae formed at the bone defect. Tightly combined with new bone, it was shown that nacre can stimulate osteoblast differentiation and new bone formation in vivo (Lamghari M, et al; Biomaterials. 2001, 22(6):555-62). The study of the mechanism shows that when the nacre degrades, calcium ions are released, which increases the local calcium ion concentration and forms the prerequisite for osteogenic mineralization. Miyamoto H et al. (Proc.Natl.Acad.Sci.USA, 1996,93(18):9657-60) think that the carbonic anhydrase-like domain of nacre matrix protein N66 can catalyze HCO3-formation, participate in the crystallization of calcium carbonate form. And Samata T et al. (FEBS Lett, 1999, 462(1-2): 225-9) found that the N16, N66 and N4 repeat regions of the matrix protein can form a structure similar to a glycine ring, which has the ability to bind Ca ²⁺ and regulate crystallization form. Nacre matrix can also produce some chemical signals and has the ability to activate osteoblasts (Almeida MJ, et al; J. Biomed. Mater. Res. 2001, 57(2): 306-12). After years of experiments and research, Lopez et al. believe that the organic components of mother of pearl contain signal molecules similar to BMPs (bone morphogenetic proteins). After entering the body, they can diffuse into the bone marrow, and then produce a chemotactic effect on osteogenic stromal cells. Activate the osteogenic cells in the bone marrow, make them differentiate, produce osteoblasts, and finally induce new bone formation. Zhang En et al. (Acta Mineralogical Sinica, 2008, 2: 112-116) used SEM and HRTEM techniques to find that the nacre layer is composed of aragonite cemented with organic matter, which is composed of flaky crystal layers, and overlaps with the organic matter layer in parallel. At the same time, on the cross-section of prism-shaped and plate-shaped aragonite crystals, there are a large number of nano-scale circular, elliptical and other shapes of organic matter, gas-liquid inclusions or holes.

Pearl powder is used as an artificial bone material, but the key problem to be solved is that the material has poor plasticity and formability, and it is difficult to form a structure with good porosity and connectivity. However, the commonly used methods such as chemical foaming and high-temperature sintering have poor molding effects on pearl powder, and can easily lead to the inactivation of the active ingredients in pearl powder that have osteoinductive effects. Therefore, a suitable and advanced porous scaffold manufacturing process is selected, including auxiliary materials and The choice of molding method is the key issue whether the pearl powder material can be effectively used in bone repair and regeneration.

Contents of the invention

One object of the present invention is to provide a new composition for preparing artificial bone bio-scaffold materials.

Another object of the present invention is to provide a bioscaffold material for artificial bone, which has high porosity and high connectivity and good mechanical strength.

Another object of the present invention is to provide a method for preparing the above artificial bone bio-scaffold material.

As an artificial bone material, pearl powder still needs to be solved. The key problem is that the material has poor plasticity and formability, and it is difficult to form a structure with good porosity and connectivity. Polylactic acid (PLA) is a degradable polymer material formed by polymerization of lactide (LA) under certain conditions, and polylactic acid/glycolic acid copolymer (polylactide-co-glycolide, PLGA) is made of Lactide (LA) and glycolide (GA) are polymerized degradable polymer materials under certain conditions. These two materials have the characteristics of high plasticity, fast degradation, easy forming, good biocompatibility and bone conduction performance, and can be completely biodegraded, and their degradation products can enter the metabolic pathway of the human body, and finally converted into carbon dioxide and Water, these two materials have been approved by the FDA and are widely used as excipients for sustained-release preparations such as injection microcapsules, microspheres and implants, and can also be used as porous scaffolds for tissue engineering cell culture.

The inventors have found through a large number of experiments that using biocompatible polymer materials PLA and PLGA as molding auxiliary materials and nacre powder can make up for the lack of difficulty in molding, and at the same time adjust the porosity, pore size, and mechanical strength of the molding scaffold.

Therefore, the contriver has prepared a kind of novel composition, and this composition comprises PLGA, PLA and pearl powder, and in said composition, PLGA:PLA (mass ratio) is 1:1-10:1, preferably 3:1-5 : 1; PLGA/PLA mixture: pearl powder (mass ratio) is 1:1-10:1, preferably 2:1-4:1, most preferably 7:3; described pearl powder particle size is below 2000 orders, Preferably the diameter is 1-10 microns, more preferably 3-6 microns.

In the above composition, PLGA:PLA (mass ratio) is 1:1-10:1, preferably 3:1-5:1. The ratio of LA/GA in the PLGA is 90/10, 80/20, 75/25, 60/40, or 50/50, preferably LA/GA is 50/50. In the composition, PLA is added to adjust the mechanical strength of the final artificial bone bio-scaffold material, and increasing its ratio can improve the stability and mechanical performance requirements of the scaffold structure.

Dissolve the PLGA and PLA in the above ratio in an organic solvent. The organic solvent can be 1,4-dioxane or a mixed liquid of 1,4-dioxane and ultrapure water, preferably 1,4-dioxane The mixed liquid with ultrapure water, the ultrapure water in the mixed liquid accounts for about 1%-20% of the total volume, preferably 5%-10%. After dissolving PLGA and PLA in an organic solvent, add PLGA/PLA mixture: pearl powder (mass ratio) at a ratio of 1:1-10:1, preferably 2:1-4:1, most preferably 7:3 The pearl powder becomes the forming slurry for preparing the artificial bone bio-scaffold material. Wherein, the particle size of the pearl powder is below 2000 mesh, preferably 1-10 microns in diameter, more preferably 3-6 microns in diameter. In order to improve the biocompatibility of the pearl powder and reduce the immunogenicity, the above-mentioned pearl powder is preferably partially deproteinized pearl powder or completely deproteinized pearl powder. After special partial deproteinization treatment, the biocompatibility of pearl powder can be further improved, while retaining biological factors with osteoinductive activity. Completely deproteinized pearl powder retains 100nm-level regular lamellar gaps and circular hole structures, which are conducive to the diffusion of nutrients, proliferation of osteoblasts, and bone formation.

The above-mentioned forming slurry can be used to prepare artificial bone bio-scaffold materials. In the low-temperature environment, the uniform polyester solution in the slurry undergoes solid-liquid phase separation/liquid-liquid phase separation to generate solvent crystallization and polyester-rich phase crystallization, and then Both phases that separate at lower temperatures solidify into solids. In the vacuum freeze-drying environment, the organic solvent volatilizes rapidly, and finally forms a composition containing PLGA, PLA and pearl powder. In the composition, PLGA:PLA (mass ratio) is 1:1-10:1, preferably 3:1-5 : 1; PLGA/PLA mixture: pearl powder (mass ratio) is 1:1-10:1, preferably 2:1-4:1, most preferably 7:3; described pearl powder particle size is below 2000 orders, Preferably the diameter is 1-10 microns, more preferably 3-6 microns.

In bone repair, the most urgent clinical needs are bone scaffold materials with large volume, specific shape and complex structure. Good porosity is the basis for rapid vascularization of tissue-engineered bone and its replacement by autologous bone. For the manufacture of large bone scaffold materials, common methods such as combustion and foaming cannot meet the requirements in terms of porosity and mechanical properties, and rapid prototyping (RPM) manufacturing technology is currently the most promising option. Due to the controllability of the rapid prototyping technology to the scaffold structure, it is possible to design the scaffold structure, and the structure not only affects the porosity, connectivity, and vascularization of the artificial bone, but also determines the mechanical properties of the material. Researchers from MIT and Princeton University in the United States have successfully used rapid prototyping equipment to manufacture artificial aggregates in recent years. The current product has completed animal experiments and entered preclinical approval (Sherwood JK, et al; Biomaterials, 2002, 23(24): 4739-51). German Seitz H et al. (J.Biomed.Mater.Res.B.Appl.Biomater; 2005, 74(2): 7828) used 3D printing technology to successfully manufacture bone materials with complex channels and pores inside, and the materials showed good Mechanical strength has now entered into industrial development.

Utilizing the above-mentioned forming slurry, the artificial bone bio-scaffold material with high porosity and connectivity can be constructed by adopting the low-temperature rapid prototyping method. The method for preparing the above-mentioned biocomposite scaffold for bone repair proposed by the present invention comprises the following steps:

(1), prepare partial deproteinized pearl powder or completely deproteinized pearl powder;

(2), preparation contains PLGA, the slurry of PLA and pearl powder;

(3) Design a three-dimensional support with high porosity and connectivity through 3D software;

(4) Using a low-temperature rapid prototyping system to shape the three-dimensional scaffold.

In step (1), the micron pearl powder with particle size below 2000 mesh is dried, and then sieved with a vibrating sieve to obtain pearl powder with a preferred diameter of 1-10 microns, more preferably 3-6 microns, Then the pearl powder is degreased, decalcified on the surface, partially deproteinized or completely deproteinized, dried and sterilized for use.

In step (2), PLGA and PLA with a mass ratio of 1:1-10:1 (preferably 3:1-5:1) are dissolved in an organic solvent selected from the group consisting of chloroform, dimethylsulfoxide , 1,4-dioxane or the mixed liquid of 1,4-dioxane and ultrapure water, preferably the mixed liquid of 1,4-dioxane and ultrapure water, and the ultrapure water in the mixed liquid accounts for about 1%-20% of the total volume, preferably 5%-10%. After dissolving PLGA and PLA in an organic solvent, add the pearl powder in step (1) according to a certain ratio to make a forming slurry for later use. The PLGA/PLA mixture: pearl powder (mass ratio) is 1:1- 10:1, preferably 2:1-4:1, most preferably 7:3;

In bone repair, the growth of tissue cells, the formation of new blood vessels, the exchange of nutrients and metabolites, and the degradation of scaffold materials all require scaffolds with high porosity and connectivity. If the porosity of the scaffold is too low and the surface area is too small, the connectivity between the pores is not good, which is not conducive to the ingrowth of the tissue; due to the small contact area with the body fluid, it is also not conducive to the degradation of the scaffold in the body. Therefore, under the premise of ensuring the stability of the scaffold structure and the required mechanical properties, the scaffold should have as high porosity and connectivity as possible. In step (3), use 3D software (such as Solidworks) to design a bracket model with high porosity and connectivity; according to actual needs, use 3D software to design a bracket model of any shape, and store the designed CAD model size as STL document. Open the model STL file with Aurora software, check and correct the STL file, and use the macroporous structure unit to perform three-dimensional filling processing on it to construct a macroporous structure with a regular pore diameter of 100-500 μm, and the XYZ axis of the macroporous structure in three directions Penetration; layering and filling processing within the ply is performed on the filled STL file; after inspection/correction, it is converted into a scan vector that can drive the subsystem of the mechanism body.

On the basis of Yinhua's rapid prototyping system, a low-temperature molding room and temperature control system are added. Input the hierarchical information file into the Cark forming control software to automatically generate information such as coordinate paths; the motion control card controls the stepping motor drive system in the three directions of X, Y, and Z, and controls the forming position of the biomaterial forming nozzle. Under the control of the driving unit, the slurry is extruded stably. In the low-temperature forming chamber, the rapid cooling of the slurry, the homogeneous polyester solution in the slurry undergoes solid-liquid phase separation/liquid-liquid phase separation, and generates solvent crystallization and polyester-rich phases, which are then separated at lower temperatures Both phases solidify into solids. Cryostents removed from the cryogenic forming chamber are placed directly into a freeze dryer to remove solvent and support material. In the vacuum environment in the freeze dryer, the solvent in the cryo-stent is sublimated to obtain a scaffold with a microporous structure. The microporous structure of the scaffold directly depends on the two-phase structure formed during the phase separation process, the solvent crystalline phase forms the pores, the polyester-rich phase forms the pore walls, and the pearl powder is dispersedly distributed in the pore walls.

According to the artificial bone bio-scaffold material prepared by the above method, its macrostructure is a channel with an aperture of 100-500 μm, and its microstructure is a three-dimensional scaffold structure with micropores of 10-20 μm, wherein the micropore wall is dispersed with micron pearl powder, pearl powder With 100nm-level regular lamellar gap and circular hole structure. The porosity of the scaffold is 60%-90%, and the X, Y, and Z axes of the macroporous structure penetrate through three directions, with a penetration rate of 100%; the artificial bone bioscaffold material provides osteoblasts with three-dimensional space for growth and sufficient The internal and external areas are conducive to the immersion and adhesion of the cell suspension, obtaining sufficient nutrients, performing gas exchange, and discharging metabolites, so that the cells grow in the three-dimensional space of the prefabricated shape, and finally form bone tissue.

The present invention uses PLGA/PLA material as the composite material of matrix composite pearl powder and the support structure obtained with low temperature rapid prototyping technology, has the following main advantages:

1. Pearl powder has good biocompatibility and inductivity. Calcium ions generated during the degradation of pearl powder can be used by new bone tissue to promote bone tissue regeneration; the protein in the pearl layer can stimulate osteoblast differentiation and proliferation , activate osteoblast activity and participate in the formation of calcium carbonate crystals; after special partial deproteinization treatment, the biocompatibility of pearl powder is further improved, and the active factors with osteoinductive activity are retained. Completely deproteinized pearl powder retains 100nm-level regular lamellar gaps and circular hole structures, which are conducive to the diffusion of nutrients, bone cell proliferation and bone formation.

2. Adding PLGA/PLA as the binder of pearl powder makes the processing and forming of the material more convenient. The low-temperature rapid prototyping technology is used to avoid the loss of bone activity induced by pearl powder caused by other drastic methods, and the regular nanostructure in the pearl powder destroy. The porosity of the constructed scaffold is 60%-90%. The overall structure of the bracket meets the design requirements. The bracket has a regular large-hole structure and channels, and the X, Y, and Z axes of the large-hole structure are penetrated in three directions, with a 100% penetration rate;

3. Providing sufficient mechanical properties is a key requirement for a scaffold. If the scaffold cannot provide a mechanical modulus in a range of hard tissues (10-1500MPA) or soft tissues (0.4-350MPA), then the formation of any new tissue may also be difficult. Fails due to excessive deformation. The measured values of the stent structure manufactured by the present invention are respectively compressive elastic modulus 17.83±1.73MPa-25.62±2.35MPa and compressive strength 0.67±0.07MPa-1.47±0.11MPa, and the composite material has higher The strength and toughness are better than single pearl powder, and the comprehensive mechanical properties have been optimized.

4. The design of the hierarchical structure satisfies the spatial conditions for bone growth. The macropore structure is regular and round, and the pore size is 100-500 μm, which is helpful for inducing the growth of blood vessels and mature bone containing Haversian system. The pore wall of the macropore is full of pores with a diameter below 100 μm. The rounded microporous structure is helpful for the growth of cell clusters, as well as the complex and diffuse distribution of pearl powder and other growth factors to achieve controllable slow release; the thickness of the micropore wall is 1-10μm, and the regular lamellar gap of pearl powder is 100nm And the circular hole structure, which helps the diffusion of nutrients, the proliferation of osteoblasts and the formation of bone, can increase the osteoinductive activity of the scaffold

The present invention will be further described below in conjunction with specific embodiments. The protection scope of the present invention is not limited to the embodiments. Any equivalent replacement in the field implemented according to the disclosed content or principles of the present invention belongs to the protection scope of the present invention.

Description of drawings

Figure 1 is a diagram of the structure of the low-temperature rapid prototyping device and the nozzle forming process. On the basis of the rapid prototyping system of Yinhua Company, a low-temperature prototyping chamber and a temperature control system are added. The space size of the forming room is 300mm (X direction) × 320mm (Y direction) × 700mm (Z direction), the top is open, the cooling device is distributed on one side of the forming room, and the other side has an openable operation door, the lowest refrigeration The temperature is -40°C, and the constant temperature capability is ±2°C. Among them, in Figure 1 (1) is the Y-axis slider, (2) is the X-axis slider, (3) is the storage tank, (4) is the Z-axis slider, (5) is the nozzle holder, (6) ) is a feeding hose, (7) is a heat-insulating soft film, (8) is a low-temperature molding chamber, (9) is a nozzle, (10) is a formed bracket, and (11) is a workbench.

Fig. 2 is three-dimensional scaffold multilevel structure figure, wherein 2-A is overall structure; 2-B is cross-sectional macropore array structure; 2-C is macroporous structure (electron microscope); 2-D is microporous structure (electron microscope) microscope).

Figure 3 is an electron microscope image of PLGA/PLA, pearl powder composite material scaffold and New Zealand white rabbit bone marrow mesenchymal stem cells co-cultured for 10 days, showing that MSC cells proliferated in a large number in the scaffold.

Accompanying drawing 4 is PLGA/PLA, pearl powder composite scaffold and New Zealand white rabbit bone marrow mesenchymal stem cells co-cultured for 10 days, the immunohistochemical identification picture of anti-type I collagen antibody, the background of the picture is green.

Figure 5 is a picture of calcium nodules induced by alizarin red staining after 21 days of co-culture with PLGA/PLA, pearl powder composite scaffolds and New Zealand white rabbit bone marrow mesenchymal stem cells. The background in the figure is black and the spots are red calcium nodules.

Detailed ways

Example 1

Take pearl powder (Zhejiang Shanxia Lake Pearl Group), and use a vibrating sieve to sieve it with -2000 mesh and +3000 mesh to obtain pearl powder with a diameter of 1 μm-10 μm, and then degrease the pearl powder with 1:1 chloroform/methanol for 12 hours ;Soak in PBS+NaOH+NaN3 mixture at 37°C for 12h, then rinse with triple-distilled water; soak in 200g/L H ₂ O ₂ at 37°C for 6h to partially deproteinize; rinse with triple-distilled water for 6 times, and dry in a 37°C constant temperature drying oven ; Sealed packaging, cobalt 60 disinfection for use.

PLGA/PLA (3:1) was dissolved in 1,4-dioxane (Shanghai Chemical Reagent Purchase and Supply Wulian Chemical Factory, analytically pure) organic solvent according to the concentration of 15% (w/v), stirred and dissolved at 40°C, The cloud point of the formed liquid-liquid phase separation system is 28°C; add pearl powder according to the ratio of PLGA/PLA: pearl powder=70/30 (w/w) to prepare a forming slurry; store at room temperature at 30°C for future use.

Use the 3D software Solidworks to build the designed 3D structure, the CAD model size: 2×2×2cm ³ , use the Aurora software to open the 3D support model STL file, after checking and correcting the STL file, use the macroporous structural unit to make a 3D analysis of it Filling processing, constructing a macroporous structure with a regular pore diameter of 200 μm, and the XYZ axes of the macroporous structure run through in three directions; perform layering and filling processing in the filled STL file to generate a layered file (SLI, CLI) And grid file (NET) grid width: d=1mm; After inspection/correction, it is converted into a scanning vector-driven equipment molding that can drive the body subsystem of the mechanism (Yinhua company's rapid prototyping system, Figure 1). The temperature of the low-temperature molding chamber is -30°C; the speed control frequency of the nozzle is 200Hz; the scanning speed is 6mm/s.

In the low-temperature forming chamber, the rapid cooling of the slurry, the homogeneous polyester solution in the slurry undergoes solid-liquid phase separation/liquid-liquid phase separation, and generates solvent crystallization and polyester-rich phases, which are then separated at lower temperatures Both phases solidify into solids. Cryostents removed from the cryogenic forming chamber were placed directly into a freeze dryer to remove solvent and support material (-40°C). In the vacuum environment in the freeze dryer, the solvent in the cryo-stent sublimates to obtain a scaffold with a microporous structure (Fig. 2-A, Fig. 2-B). The microporous structure of the scaffold directly depends on the two-phase structure formed during the phase separation process, the solvent crystalline phase forms the pores, the polyester-rich phase forms the pore walls, and the pearl powder is dispersedly distributed in the pore walls. The macrostructure of the scaffold has a 100-500 μm pore diameter channel (Figure 2-C), and the microstructure is a three-dimensional scaffold structure with 10-20 μm micropores (Figure 2-D), in which the micropore walls are dispersed with micron-sized pearl powder. The porosity is 79%, and the X, Y, and Z axes of the macroporous structure are penetrated in three directions, with a penetration rate of 100%; the compressive elastic modulus of the bracket is 18.71±1.61MPa and the compressive strength is 0.92±0.06MPa.

Example 2

Take pearl powder (Zhejiang Shanxia Lake Pearl Group), and sieve it with a 2000-mesh vibrating sieve to obtain pearl powder with a diameter of 3-6 μm, then degrease the pearl powder with 1:1 chloroform/methanol for 12 hours; 37 ° C PBS+NaOH Soak in +NaN3 mixed solution for 12 hours, then rinse with triple distilled water; soak in 200g/L H ₂ O ₂ at 37°C for 24 hours to completely deproteinize; rinse with triple distilled water for 6 times, and dry in a constant temperature drying oven at 37°C; seal the package, and irradiate with Co60 Disinfect and set aside.

Prepare a mixed solution of 1,4-dioxane (purchased and supplied by Shanghai Chemical Reagent Wulian Chemical Plant, analytically pure) and water, wherein the water content in the mixed solution is 5% (V _water /V _total ). PLGA/PLA (4:1) was dissolved in the mixed liquid at a concentration of 15% (w/v), stirred and dissolved at 40°C; pearls were added according to the ratio of (PLGA+PLA)/pearl powder=65/35 (w/w) Prepare the forming slurry after powdering; store at room temperature at 30°C for later use.

3D software Solidworks builds the designed 3D structure, CAD model size: 1.5×1.5×5cm ³ , opens the 3D support model STL file with Aurora software, checks and corrects the STL file, and uses macroporous structural elements to fill it in 3D Processing, constructing a macroporous structure with a regular pore diameter of 200 μm, and the XYZ axes of the macroporous structure penetrate in three directions; perform layering and filling processing on the filled STL file to generate a layer file (SLI, CLI) and Grid file (NET) grid width: d=0.5mm; after inspection/correction, it is transformed into a scan vector drive device that can drive the body subsystem of the mechanism. The temperature of the molding chamber is -30°C; the speed control frequency of the nozzle is 250Hz; the scanning speed is 5mm/s.

In the low-temperature forming chamber, solid-liquid phase separation/liquid-liquid phase separation occurs during rapid cooling of the slurry, resulting in solvent crystallization and polyester-rich phases, and then the two phases separated at lower temperatures solidify into solids. The cryo-stents taken out of the low-temperature forming chamber were first stored in liquid nitrogen for 10 min, and then placed in a freeze dryer to remove solvent and support material (-40°C). In the vacuum environment in the freeze dryer, the solvent in the cryo-stent is sublimated to obtain a scaffold with a microporous structure. The macrostructure of the scaffold has a channel with a pore size of 100 μm, and the microstructure is a three-dimensional scaffold structure with micropores of 10-20 μm, in which micron-sized pearl powder is diffusely distributed on the walls of the micropores. The porosity is 85%, and the X, Y, and Z axes of the macroporous structure are penetrated in three directions, with a penetration rate of 100%; the compressive elastic modulus of the bracket is 19.88±1.87MPa and the compressive strength is 0.98±0.08MPa.

Example 3

Take the composite material scaffold prepared in Example 1 and New Zealand white rabbit bone marrow mesenchymal stem cells (MSCs), and co-culture them in a three-dimensional cell/tissue culture system (RCCS ^™ , NASA). After culturing for 10 days, fix with glutaraldehyde at 4°C, and After fixation in acetic acid at 4°C for 1 h, double staining with uranyl acetate-lead citrate, the structure was observed with a scanning electron microscope (Figure 3). It shows that the cells grow, proliferate, and migrate well in the three-dimensional scaffold structure. The cells stretch in a certain direction in the three-dimensional space, and form a network structure in the pores, leaving channels for nutrients to pass through.

Example 4

Take the composite material scaffold prepared in Example 1 and New Zealand white rabbit bone marrow mesenchymal stem cells (MSCs), and co-culture them in a three-dimensional cell/tissue culture system (RCCS ^TM NASA). Marin solution was fixed for 30 minutes, immunohistochemical identification was carried out with FITC-labeled anti-type I collagen antibody, and fluorescence microscope observation (Figure 4) showed that more than 90% of the cells and structures expressed green fluorescence. Type I collagen accounts for about 90% of the organic content of bone tissue. Studies have shown that MSC cells in the scaffold differentiate into osteoblasts and synthesize important components of bone.

Example 5

Take the composite material scaffold prepared in Example 1 and New Zealand White Rabbit Bone Marrow Mesenchymal Stem Cells (MSCs), and co-culture them in a three-dimensional cell/tissue culture system (RCCS ^™ NASA). After culturing for 21 days, take out the scaffold and fix with 4% paraformaldehyde After 30 minutes, the induced calcium nodules were stained with alizarin red, and a large number of red calcium nodules appeared at the site of cell aggregation in the scaffold, and the scaffold showed significant osteoinductive activity (Fig. 5).

Claims (10)

1, a kind of compositions that contains PLGA, PLA and Margarita powder is characterized in that: PLGA:PLA in the described compositions (mass ratio) is 1:1-10:1; The PLGA/PLA mixture: Margarita powder (mass ratio) is 1:1-10:1, and described Margarita powder diameter is the 1-10 micron.

2, compositions according to claim 1 is characterized in that: Margarita powder is part deproteinization or complete Deproteinated Margarita powder in the described compositions.

3, the artificial bone biologic bracket material of preparation of compositions according to claim 1 is characterized in that: described timbering material is that macrostructure has 100-500 μ m aperture passage, and micro structure is the three-dimensional rack structure with 10-20 μ m micropore.

4, according to according to right 3 described artificial bone biologic bracket materials, it is characterized in that: the porosity of described timbering material is 60%-90%.

5, according to according to right 3 described artificial bone biologic bracket materials, it is characterized in that: the X of the macroporous structure of described timbering material, Y, three directions of Z axle connect, and have 100% perforation rate.

6, according to according to right 3 described artificial bone biologic bracket materials, it is characterized in that: the modulus of elasticity in comperssion of described timbering material is 17.83 ± 1.73MPa-25.62 ± 2.35MPa.

7, according to according to right 3 described artificial bone biologic bracket materials, it is characterized in that: the comprcssive strength 0.67 ± 0.07MPa-1.47 ± 0.11MPa of described timbering material.

8, a kind of method for preparing artificial bone biologic bracket material as claimed in claim 3 comprises the steps:

(1) antigen Margarita powder or complete Deproteinization Margarita powder are removed in preparation;

(2) preparation contains the slurry of PLGA, PLA and Margarita powder;

(3) has the three-dimensional rack of high porosity and connection rate by the 3D software design;

(4) utilize the low temperature rapid prototyping system to make the three-dimensional rack molding.

9, method according to claim 8, it is characterized in that: the process of step (2) is for being that PLGA and the PLA of 1:1-10:1 is dissolved in the organic solvent with mass ratio, described organic solvent is selected from chloroform, dimethyl sulfoxide, 1,4-dioxane or 1, the mixing material of 4-dioxane and ultra-pure water, then according to the PLGA/PLA mixture: Margarita powder (mass ratio) is made the shaping slurry for the Margarita powder in the ratio adding step (1) of 1:1-6:1.

10, method according to claim 8 is characterized in that: the low temperature rapid prototyping system described in the step (4) installs low temperature moulding chamber and temperature control system additional on the basis of Yin Hua company rapid prototyping system.

Images