CN111249524B - High-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and preparation method thereof - Google Patents
High-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention discloses a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and a preparation method thereof, wherein a polycaprolactone porous microsphere is prepared by taking gelatin as a pore-forming agent and taking a multiple emulsion-solvent volatilization method as a basis; the surface and the inner pores of the high-porosity polycaprolactone porous microsphere are communicated, the porosity of the microsphere is 75.28-90.73%, and the pore diameter of the pores is 43-217 microns. The polycaprolactone porous microsphere scaffold has high porosity, so that more space can be provided for bone tissue growth, the space occupied by polycaprolactone materials is reduced, the defect of slow degradation of polycaprolactone is overcome, and the porous microsphere has larger pore diameter and is suitable for cell ingrowth and bone tissue regeneration.
Description
Technical Field
The invention relates to the field of tissue engineering materials, in particular to a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and a preparation method thereof.
Background
In bone tissue engineering, scaffolds are in a critical position, and ideal scaffolds must have the following characteristics: the macroscopic structure is required to be adaptive to the shape of the defect; the biocompatibility is good, no toxicity exists, and cells can grow on the surface of the cell in an adhesion manner; the biodegradable material is biodegradable, and degradation products have no toxic or side effect; the porosity makes the cells have more space for adhesion growth, which is beneficial to the growth of bone tissues. The intercommunicating porous structure has many other advantages, such as: the specific surface area is increased, which is beneficial to protein adsorption, and is beneficial to the removal of metabolic waste, the transportation of nutrient substances and the transmission of biological factors.
Polycaprolactone (PCL) is an artificial polymer synthetic material, has good biocompatibility and degradability, has no toxic or side effect on human bodies, and is approved by FDA for clinical application. However, the polycaprolactone has the defects of strong crystallinity, too low degradation rate in vivo and the like, so that the application range of the material in the field of biomedicine is limited.
In the existing literature, the prepared polycaprolactone porous microsphere has low porosity and pore size, and cannot meet the requirement of an ideal stent. Porous PCL microspheres prepared by emulsion/solvent evaporation and particle leaching, such as Qingchun Zhang (Qingchun Zhang. materials Science and Engineering C,32(2012), 2589-. Porous PCL microspheres prepared from room temperature ionic liquid and camphene as liquid mold and porogen respectively, such as Seng Yeol Kim (journal of Colloid and Interface Science,465(2016)18-25), have pore diameter of only up to 100 μm.
Disclosure of Invention
The invention aims to provide a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and a preparation method thereof aiming at the defects of the prior art, the prepared polycaprolactone porous microsphere scaffold has high porosity, so that more space can be provided for bone tissue growth, meanwhile, the space occupied by polycaprolactone materials is reduced, the defect of slow degradation of polycaprolactone is overcome, and the porous microspheres have larger pore diameters and are suitable for cell growth and bone tissue regeneration.
The invention also aims to provide a preparation method of the bone tissue engineering scaffold.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration, the high-porosity polycaprolactone porous microsphere is spherical particles with interconnected surface and internal pores, the porosity of the porous microsphere is 75.28-90.73%, and the pore diameter of the porous microsphere is 43-217 μm;
wherein the pore size distribution of more than 100 μm is up to 94%.
Further, the particle size of the porous microspheres is 250-1000 μm.
Further, the relative molecular mass of the polycaprolactone is 6-10 ten thousand.
A preparation method of a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration comprises the following steps:
(1) dissolving polycaprolactone in a water-insoluble organic solvent to prepare an oil phase, wherein the concentration of the polycaprolactone in the oil phase is 0.05 g/mL; dissolving gelatin in double distilled water, packaging, refrigerating, and melting in water bath to obtain internal water phase;
(2) adding a certain amount of the internal water phase obtained in the step (1) into an oil phase, carrying out first emulsification, stirring by using a homogenizer at a stirring rotation speed of 8000-800 r/min, emulsifying into primary emulsion, adding the primary emulsion into an external water phase, carrying out second emulsification, stirring by using a stirrer at a stirring rotation speed of 600-800r/min, and emulsifying to form multiple emulsion;
(3) adding the multiple emulsion obtained in the step (2) into a polyvinyl alcohol solution and stirring to obtain polycaprolactone/gelatin microspheres;
(4) adding the microspheres prepared in the step (3) into double distilled water, stirring, repeating the stirring twice, and screening to obtain the needed polycaprolactone porous microsphere scaffold;
wherein the volume ratio of the oil phase, the internal water phase, the external water phase and the polyvinyl alcohol solution is 10 mL: 4-5mL, 50mL, 400 mL.
Further, the oil phase in the step (1) is a dichloromethane solution of polycaprolactone.
Further, the temperature of the double distilled water in the step (1) is 50 ℃, the refrigeration temperature is 4 ℃, the internal water phase is 8 wt% gelatin water solution, and the water bath temperature is 37 ℃.
Further, in the step (2), the amount of the inner water phase is 4-5mL, the outer water phase is 1 wt% of polyvinyl alcohol aqueous solution, the temperature is 4 ℃, the time of the first emulsification stirring is 40s, and the time of the second emulsification stirring is 60 s.
Further, the concentration of the polyvinyl alcohol solution in the step (3) is 0.1 wt%, and the temperature is 4 ℃.
Further, the stirring time of the multiple emulsion added with the polyvinyl alcohol solution in the step (3) is 8 hours, and the rotating speed is 250 r/min.
Further, the temperature of the double distilled water in the step (4) is 50 ℃, and the stirring speed is 250 r/min; the standard sieve pore size used for the sieving is 250 and 1000 μm.
Compared with the prior art, the invention has the beneficial effects that:
the polycaprolactone porous microspheres prepared under the synergistic effect of specific concentration, rotation speed and proportion by a multiple emulsion-solvent volatilization method have high porosity and larger pore size, and the pores of the microspheres are mutually communicated so as to be beneficial to the growth of cells and tissues, the transmission of oxygen and nutrition and the discharge of metabolites.
The polycaprolactone porous microspheres are particle-like, and can meet the clinical filling requirement of irregular bone cavities.
The porous structure in the polycaprolactone porous microsphere enables the microsphere to have a large specific surface area, and pores are communicated with each other, so that cells can grow into the microsphere.
The polycaprolactone porous microspheres have high porosity which can reach 91 percent, the high porosity means that more space can be provided for bone tissues to grow in, and on the other hand, the high porosity means that the consumption of polycaprolactone raw materials is low, so that the problem of slow degradation of polycaprolactone is solved to a certain extent.
The pore diameter of the polycaprolactone porous microsphere can reach 40-220 microns, the pore diameter distribution of the polycaprolactone porous microsphere is up to 94% when the pore diameter is larger than 100 microns, favorable conditions are provided for the growth of cells and bone tissues, and the growth of the cells and the bone tissues is facilitated.
Drawings
FIG. 1 is a field emission scanning electron microscope picture of the polycaprolactone porous microspheres of the invention.
FIG. 2 is a microscopic picture of the polycaprolactone porous microsphere after being inoculated with bone marrow mesenchymal stem cells for one week and being subjected to fluorescent staining.
FIG. 3 is a field emission scanning electron microscope picture of the polycaprolactone porous microsphere inoculated with mesenchymal stem cells for one week.
Detailed Description
In order to better understand the technical content of the present invention, specific embodiments are described below with reference to the accompanying drawings.
Polycaprolactone used in the following examples was purchased from Jinan Dai Ting Bio-engineering Ltd and had a relative molecular mass of 6-10 ten thousand.
[ PREPARATION ]
The preparation methods of the following examples were all prepared using the same method, namely:
(1) dissolving polycaprolactone in dichloromethane to prepare a polycaprolactone solution with the concentration of 0.05g/mL, and taking 10mL as an oil phase; dissolving 4g of gelatin in 50mL of 50 ℃ double distilled water, subpackaging and refrigerating at 4 ℃ for solidification, and when in use, putting the subpackaged and refrigerated gelatin into a water bath at 37 ℃ for melting, and taking a certain amount as an internal water phase;
(2) adding the internal water phase obtained in the step (1) into an oil phase, carrying out first emulsification stirring at a certain rotating speed by a homogenizer for 40s continuously to emulsify the internal water phase into primary emulsion, adding the primary emulsion into 50mL of 1 wt% polyvinyl alcohol solution with the temperature of 4 ℃, carrying out second emulsification stirring at a certain rotating speed by a stirrer for 60s continuously to emulsify the primary emulsion to form multiple emulsion;
(3) adding the multiple emulsion obtained in the step (2) into 400mL of 0.1 wt% polyvinyl alcohol solution at 4 ℃, stirring for 8 hours at the rotating speed of 250r/min, standing, removing supernatant, and leaving microspheres to precipitate to obtain polycaprolactone/gelatin microspheres;
(4) and (3) adding 50 ℃ double-distilled water into the microspheres prepared in the step (3), stirring at the rotating speed of 250r/min until the water temperature is reduced to room temperature, repeating the stirring twice, screening by using a sieve with the pore diameter of 1000 microns and 250 microns, and freeze-drying to obtain the needed polycaprolactone porous microsphere scaffold.
The variables in the preparation of examples 1-10 are shown in table 1:
TABLE 1
[ characterizations ] of
As shown in FIG. 1, the field emission scanning electron microscope photograph of the prepared polycaprolactone porous microsphere shows that the microsphere has a loose porous structure and the pores are communicated with each other.
The porosity of the polycaprolactone porous microspheres prepared in examples 1 to 10 was measured by a pressure pump, and the pore size of the polycaprolactone porous microspheres was measured by taking a picture with a microscope, and the results are shown in table 2:
TABLE 2
As can be seen from Table 2, the polycaprolactone porous microspheres prepared under the synergistic effect of specific concentration, rotation speed and proportion have high porosity and larger pore size, the porosity can reach up to 90.73%, the pore size can reach up to 217 μm, and the pore size distribution of more than 100 μm is up to 94%, so that favorable conditions are provided for the growth of cells and bone tissues.
[ application test ]
1. Cell adhesion growth assay
The porous polycaprolactone microspheres prepared in example 3 were soaked in 75% ethanol overnight and washed thoroughly with sterile PBS the following day. Transferring the aseptic polycaprolactone porous microspheres into a 24-hole cell culture plate, and inoculating 2 x 10 mesenchymal stem cells into each hole4And (4) respectively. After 1 week of culture, fixation with 4% paraformaldehyde for 30min, incubation with 0.25% Triton X-100 at room temperature for 15min, staining with phalloidin under dark conditions at 37 deg.C for 30min, staining with DAPI for 30s, washing with PBS, and photographing under inverted fluorescence microscope for observation.
Fig. 2 is a fluorescent staining picture of the polycaprolactone porous microsphere after the bone marrow mesenchymal stem cells are inoculated for one week, and it is found that the cells can be adhered to and grow on the surface of the polycaprolactone porous microsphere, which indicates that the polycaprolactone porous microsphere has good cytological characteristics.
2. Cell growth into the well test
The porous polycaprolactone microspheres prepared in example 3 were soaked in 75% ethanol overnight and washed thoroughly with sterile PBS the following day. Transferring the aseptic polycaprolactone porous microspheres into a 24-hole cell culture plate, and inoculating 2 x 10 mesenchymal stem cells into each hole4And (4) respectively. After 1 week of culture, fixing with 2.5% glutaraldehyde for 30min, dehydrating with 30%, 50%, 70%, 90% ethanol gradient, dehydrating with anhydrous ethanol for three times, drying, spraying gold, and observing by taking pictures under a field emission scanning electron microscope.
Fig. 3 is a scanning electron microscope image of the field emission after the mesenchymal stem cells are inoculated in the polycaprolactone porous microsphere for one week, and it is found that the cells can grow into the pores of the microsphere (indicated by white arrows), which indicates that the pores of the polycaprolactone porous microsphere can accommodate the growth of the cells.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.
Claims (10)
1. A high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration is characterized in that: the high-porosity polycaprolactone porous microsphere is spherical particles with mutually communicated surface and internal pores, the porosity of the porous microsphere is 75.28-90.73%, and the pore diameter of the porous microsphere is 43-217 mu m;
wherein the pore size distribution of more than 100 μm is up to 94%.
2. The high porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 1, characterized in that: the particle size of the porous microsphere is 250-1000 mu m.
3. The high porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 1, characterized in that: the relative molecular mass of the polycaprolactone is 6-10 ten thousand.
4. A method for preparing a high porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to any one of claims 1 to 3, comprising the following steps:
(1) dissolving polycaprolactone in a water-insoluble organic solvent to prepare an oil phase, wherein the concentration of the polycaprolactone in the oil phase is 0.05 g/mL; dissolving gelatin in double distilled water, packaging, refrigerating, and melting in water bath to obtain internal water phase;
(2) adding a certain amount of the internal water phase obtained in the step (1) into an oil phase, carrying out first emulsification, stirring by using a homogenizer at a stirring rotation speed of 8000-800 r/min, emulsifying into primary emulsion, adding the primary emulsion into an external water phase, carrying out second emulsification, stirring by using a stirrer at a stirring rotation speed of 600-800r/min, and emulsifying to form multiple emulsion;
(3) adding the multiple emulsion obtained in the step (2) into a polyvinyl alcohol solution and stirring to obtain polycaprolactone/gelatin microspheres;
(4) adding the microspheres prepared in the step (3) into double distilled water, stirring, repeating the stirring twice, and screening to obtain the needed polycaprolactone porous microsphere scaffold;
wherein the volume ratio of the oil phase, the internal water phase, the external water phase and the polyvinyl alcohol solution is 10 mL: 4-5mL, 50mL, 400 mL.
5. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: the oil phase in the step (1) is a dichloromethane solution of polycaprolactone.
6. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: the temperature of the double distilled water in the step (1) is 50 ℃, the refrigeration temperature is 4 ℃, the internal water phase is 8 wt% of gelatin water solution, and the water bath temperature is 37 ℃.
7. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: in the step (2), the amount of the inner water phase is 4-5mL, the outer water phase is 1 wt% of polyvinyl alcohol aqueous solution, the temperature is 4 ℃, the time of the first emulsification stirring is 40s, and the time of the second emulsification stirring is 60 s.
8. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: the concentration of the polyvinyl alcohol solution in the step (3) is 0.1 wt%, and the temperature is 4 ℃.
9. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: and (4) adding the multiple emulsion into the polyvinyl alcohol solution in the step (3), and then stirring for 8 hours at a rotating speed of 250 r/min.
10. The preparation method of the high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration according to claim 4, wherein the preparation method comprises the following steps: the temperature of the double distilled water in the step (4) is 50 ℃, and the stirring speed is 250 r/min; the standard sieve pore size used for the sieving is 250 and 1000 μm.
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