METHOD FOR PRODUCING BONE REGENERATION MATERIAL HAVING COTTON-WOOL LIKE STRUCTURE

Abstract

A method for producing a cotton-wool like material for bone regeneration using a wet spinning method. 50-80 wt % of calcium salt particles and 50-20 wt % of PDLLGA resin are put into a mixing vessel, dissolved in acetone, and stirred to produce a spinning solution with a resin concentration of 10-20 wt % in which said calcium salt particles are dispersed. The produced spinning solution is filled in a syringe, and the spinning solution filled in the syringe is injected into a collector container filled with poor solvent by extruding the spinning solution from the discharge port of an injection needle having a predetermined diameter. The spinning solution injected into the poor solvent is solidified into fibers by interdiffusion of desorption of organic solvent and penetration of poor solvent in the poor solvent solution. The fibers solidified in the poor solvent are deposited in a floating state in the collector vessel without fiber-to-fiber adhesion and collected in a cotton-wool like shape.

Claims

1. A method of producing a cotton-wool like bone regeneration material by using a wet spinning process, the method comprising: preparing a spinning solution with a resin concentration of 10-20 wt % in which calcium salt particles are dispersed by mixing the calcium salt particles of 50-80 wt % and PDLLGA resin of 50-20 wt % in a mixing container to form a mixture, and dissolving the mixture in acetone to form a dissolved mixture and stirring the dissolved mixture, and filling the spinning solution thus prepared in a syringe, injecting the spinning solution filled in the syringe into a collector container filled with a poor solvent by extruding the spinning solution from the outlet of the injection needle having a predetermined diameter, wherein the spinning solution injected into the poor solvent is solidified into fibers by interdiffusion of desorption of an organic solvent and penetration of the poor solvent, and the fibers solidified in the poor solvent are collected in cotton-wool like form by floating and depositing in the collector container without having adhesion between the fibers

2. The method according to claim 1, wherein the poor solvent is ethanol.

3. The method according to claim 1, wherein the poor solvent is water.

4. The method according to claim 1, wherein the calcium salt particles are ?-TCP particles.

5. The method according to claim 4, wherein the ?-TCP particles contain silver ions.

6. The method according to claim 4, wherein the ?-TCP particles are silver ion incorporated ?-TCP particles synthesized by substituting calcium in the crystal lattice of ?-TCP with silver ions.

7. A cotton-wool-like bone regenerating material produced from an improved wet spinning process, the cotton-wool like bone regenerating material is produced by the process of: preparing a spinning solution with a resin concentration of 10-20 wt % in which calcium salt particles are dispersed by mixing the calcium salt particles of 50-80 wt % and PDLLGA resin of 50-20 wt % in a mixing container to form a mixture, and dissolving the mixture in acetone to form a dissolved mixture and stirring the dissolved mixture, and filling the spinning solution thus prepared in a syringe, injecting the spinning solution filled in the syringe into a collector container filled with a poor solvent by extruding the spinning solution from the outlet of an injection needle having a predetermined diameter of the syringe, wherein the spinning solution injected into the poor solvent is solidified into fibers by interdiffusion of desorption of an organic solvent and penetration of the poor solvent, and the fibers solidified in the poor solvent are collected in cotton-wool like form by floating and depositing in the collector container without having adhesion between the fibers.

8. The cotton-wool like bone regeneration material of claim 7, wherein the calcium salt particles are calcium phosphate particles.

9. The cotton-wool like bone regeneration material of claim 8, wherein the calcium phosphate particles are ?-TCP particles.

10. The cotton-wool like bone regeneration material of claim 9, wherein the ?-TCP particles are silver ion incorporated ?-TCP particles synthesized by substituting calcium in the crystal lattice of ?-TCP with silver ions.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 shows a schematic diagram of the wet spinning method of this invention when ethanol is used as a poor solvent.

[0032] FIG. 2 shows a sample material collected from poor solvent in the wet spinning method (using ethanol as poor solvent) in an embodiment of the present invention.

[0033] FIG. 3 shows the wet spinning method in an embodiment of the present invention when water is used as a poor solvent.

[0034] FIG. 4 shows a cotton-wool like bone regeneration material produced by using the wet spinning method (using water as a poor solvent) in this invention example.

[0035] FIG. 5 is an SEM photograph showing the surface topography of biodegradable fibers spun in the wet spinning method (using water as the poor solvent) in an embodiment of the present invention.

[0036] FIG. 6 shows a SEM photograph showing the surface irregularities of the spun biodegradable fiber in the wet spinning method (using water as the poor solvent) in this example.

[0037] FIG. 7 shows osteoblast adhesion to biodegradable fibers as a result of culturing osteoblasts for 6 hours, 1 day, and 3 days using cotton-wool like bone regeneration materials produced by using the wet spinning method (ethanol was used as a poor solvent) of an embodiment of the present invention.

[0038] FIG. 8 shows cells grow rapidly and steadily after 1 day of cell adhesion using cotton-wool like bone regeneration materials produced by using the wet spinning method (using ethanol as a poor solvent) in an embodiment of the present invention.

EMBODIMENTS OF THE PRESENT INVENTION

[0039] Hereinafter, embodiment of the present invention is described in detail with reference to the drawings.

Definition

[0040] <PLLGA Resin>

In the present invention, PLLGA resin refers to PLGA resin synthesized by copolymerization of lactic acid and glycolic acid containing only L-isomer. The polymerization ratio of 85:15 PLLA to PGA is called PLLGA(85:15) and the polymerization ratio of 75:25 PLLA to PGA is called PLLGA(75:25). Degradation of PLLGA can be enhanced by increasing the ratio of PGA. To dissolve PLLGA in a solvent, a chlorinated solvent such as chloroform must be used.

[0041] <PDLLGA Resin>

In the present invention, PDLLGA resin refers to PLGA resin synthesized by copolymerization of lactic acid containing D isomer and L isomer and glycolic acid. Lactic acid that is used to synthesize PLGA has a crystalline L-isomer and its optical isomer, amorphous D-isomer. PLA includes poly(L-lactic acid) (PLLA), which is composed of only the L-isomer, and poly(D-lactic acid) (PDLLA), which contains both L-isomer and D-isomer. It is possible to control the degradability of PDLLGA by changing the polymerization ratio of PDLLA to PGA. In the present invention, the amount of D-isomer in PDLLGA resin is sufficient to make the resin degradable and dissolvable in acetone by including D-isomer.

[0042] <Wet Spinning Method>

In this invention, wet spinning method refers to a method of solidifying spinning solution into fiber form by desorption of organic solvent and penetration of poor solvent. The choice of organic solvent and poor solvent affects the speed of polymer solidification and desorption/penetration of solvent. Balance of the speed of this desorption/penetration determines the form of the resulting fiber. The wet spinning method used in the present invention is modified and the conditions are set to fiberize PDLLGA resin containing calcium phosphate particles and form a cotton-wool like shape.

[0043] <Organic Solvent>

In the present invention, organic solvent is used to dissolve mixtures of PDLLGA resin and calcium phosphate particles. Chlorinated organic solvents such as chloroform have excellent solubility but are toxic. Acetone is inferior to chloroform in terms of solubility, but it is safe for living organisms because it does not contain chlorine. Since the PDLLGA resin used in the present invention is easily dissolved in a solvent, a safe non-chlorinated solvent such as acetone can be used without the need to use chloroform or other toxic chlorinated organic solvent.

[0044] <Poor Solvent>

In the present invention, poor solvent is used in the coagulation bath as the solvent that does not dissolve PDLLGA resin. It is used to collect biodegradable fibers in a cotton-wool like form. Scholarly, poor solvent is said to be a poor solvent for this solute when the solute-solvent interaction (free energy) is less than the arithmetic mean of the solute-solute and solvent-solvent interactions in a particular substance-solvent system in terms of the theory. In the present invention, poor solvent is selected by taking into account the balance of desorption and penetration between the organic solvent and poor solvent. In the present invention, ethanol or water, in which PDLLGA is insoluble, can be used suitably as a poor solvent.
In a case that ethanol is used as a poor solvent, spinning solution can be made into fibers by stirring ethanol in a collector container and stretching the fibers by the flow of poor solvent produced by stirring, as shown in FIG. 1. In this case, Hansen solubility parameter of ethanol is 26.5 ? [(MPa).sup.1/2]) and that of acetone is 20.0 [(MPa).sup.1/2]), and the degree of difference between the two is 6.5 [(MPa).sup.1/2].
In a case that water is used as a poor solvent, when the spinning solution is extruded from the nozzle, the extruded spinning solution is fiberized and floated and deposited in the collector container. In this case, Hansen solubility parameter of water is 47.8 ? [(MPa).sup.1/2] and that of acetone is 20.0 ? [(MPa).sup.1/2]), and the degree of difference between the two is 27.8 ? [(MPa).sup.1/2].
Since degree of difference of Hansen solubility parameter of water from that of acetone is considerably greater than the difference of ethanol from acetone, the rate at which acetone is desorbed from the fiber is much faster than when ethanol is used as the poor solvent. As a result, the spinning solution extruded from the nozzle rapidly is fiberized in water, so there is no need to stretch the fibers by stirring the water to make the spinning solution fibrous.

[0045] <Silver Ion Solid Soluted ?-Phase Tricalcium Phosphate>

In an embodiment of the present invention, silver ion incorporated ?-phase tricalcium phosphate refers to a ?-phase tricalcium phosphate in which the calcium sites in the crystal lattice of ?-phase tricalcium phosphate are substituted by Ag+ ion.
Silver ion incorporated ?-phase tricalcium phosphate can be prepared using the ultrasonic spray pyrolysis method. The ultrasonic spray pyrolysis method is one of the methods for synthesizing ceramic raw material powders. A sample solution is atomized by ultrasonic waves, and the droplets are introduced into a heated electric furnace to instantly remove solvent from the droplets, deposit salt, and cause pyrolysis to obtain powder (fine particles) with the desired chemical composition. Details are disclosed in JP-A2020-130417.

Example 1 (Using Ethanol as a Poor Solvent)

[0046] The following materials and equipment were used
?-phase tricalcium phosphate (Ca.sub.3 (PO).sub.42): Taihei Chemical Industry Co. ?-TCP-100.

[0047] Particle size of 1.7 mm or less was pulverized to about 4 ?m (?-TCP milled product).

PDLLGA: PDLLA:PGA (75:25) (PURASORB PDLG7507, Corbion Purac)

[0048] Ethanol: Kishida Chemical first grade, purity 99.5%.
Acetone: Wako Pure Chemicals Reagent special grade purity 99.5+%.
Size of the extrusion opening of the injection needle for spinning solution extrusion: 27 G (inner diameter 0.2 mm, outer diameter 0.4 mm)
The container is a cylindrical vessel with a diameter of 15 cm and a height of 7.5 cm, and was stirred with a magnetic stirrer using a 5 cm long stirrer (see FIG. 1). (See FIG. 1).

1. Preparation of Spinning Solution

[0049] ?-TCP and PDLLGA were mixed in a 7:3 weight ratio, dissolved in acetone, and mixed overnight to prepare a spinning solution with a polymer concentration of 17%.

2. Spinning Conditions

[0050] Extrusion speed 0.75 ml/h, stirring speed 200 rpm

3. Collection of Cotton-Wool Like Material

[0051] After wet spinning, the fibers were washed with ethanol and held overnight in ethanol to further remove the solvent. The ethanol was then removed with an absorbent sheet, and the cotton-wool like material was dried at room temperature while unraveling to obtain cotton-wool like Sample 1 (see FIG. 2).

Example 2 (Using Water as a Poor Solvent)

[0052] The following materials and equipment were used
?-phase tricalcium phosphate (Ca.sub.3(PO).sub.42): Taihei Chemical Industry Co. ?-TCP-100. Particle size of 1.7 mm or less was ground to about 4 ?m (?-TCP milled product).

PDLLGA: PDLLA:PGA (75:25) (PURASORB PDLG7507, Corbion Purac)

[0053] Pure water
Acetone: Wako Pure Chemicals Reagent special grade purity 99.5+%.
Size of the extrusion port of the injection needle for spinning solution extrusion: 33 G (inner diameter 0.07 mm, outer diameter 0.20 mm)
Poor solvent container: A cylindrical container with a diameter of 9 cm and a height of 25 cm was used (see FIG. 3).

1. Preparation of Spinning Solution

[0054] ?-TCP and PDLLGA were mixed in a 7:3 weight ratio, dissolved in acetone, and mixed overnight to prepare a spinning solution with a polymer concentration of 17%.

2. Spinning Conditions

[0055] Extrusion speed 0.6 ml/h

3. Collection of Cotton Shapes

[0056] The solvent acetone is replaced with water and is removed from the fiber. However, because its specific gravity is smaller than that of water, it does not accumulate at the bottom of the container but floats near the top. As a result, even after conducting spinning for a long time, the acetone does not cause the fibers to stick to each other again, and long strokes of fiber are produced (see FIG. 3). In contrast, when ethanol is used as a poor solvent, the specific gravity of acetone and ethanol is almost the same, so when the fiber is spun in ethanol, the acetone that has been removed mixes with the ethanol and floats in the ethanol. As a result, when fibers are spun for a long time in the wet spinning method, the diluted acetone makes the fibers easily stick to each other, and the dried fibers become a lumpy mass.

[0057] After wet spinning, the fiber is washed with ethanol and kept in ethanol overnight to further remove the solvent. The ethanol is then removed with an absorbent sheet, and the cotton-wool like material is dried at room temperature while unraveling to obtain cotton-wool like sample 2 (see FIG. 4).

FIG. 5 shows a SEM photograph of ?-TCP/PDLLGA fibers produced using water as the poor solvent. It is observed that filler particles are exposed on the surface of the fiber, forming an uneven topography.
FIG. 6 shows an SEM photograph of ?-TCP/PDLLGA fibers produced using water as the poor solvent. The ?-TCP/PDLLGA fibers are approximately 80-100 ?m wide and 40-50 ?m thick, with a flattened cross section with a dent on one side. According to the inventor's knowledge, the reason for this shape of the fiber is that a flow called Kalman vortex is generated when the fiber flows out of the syringe, and the center of the fiber is dented by this flow.

4. Experiment of Cell Adhesion on Cotton-Wool Like Material

[0058] Wells were filled with 1 ml of normal medium and 0.5 ml of suspension (2.4?10.sup.5 cells/ml) of mouse-derived osteoblast-like cells (MC3T3-E1) after sample 1 was blended into the medium and cultured in an incubator for 6 hours, 1 day and 3 days (CO.sub.2 concentration 5%, 37? C.). The adhesion of cells on the fibers constituting sample 1 was then observed using a scanning electron microscope. As a result of the experiment, it was observed that some cells began to adhere to the fiber surface by 1 day, and that they adhered and proliferated until they almost covered the surface in 3 days (see FIG. 7).
Wells were filled with 1 ml of normal medium and 0.5 ml of suspension (2.4?10.sup.5 cells/ml) of mouse-derived osteoblast-like cells (MC3T3-E1) after sample 1 was blended into the medium and cultured in an incubator for 6 hours, 1 day and 3 days (CO.sub.2 concentration 5%, 37? C.).

[0059] AlamarBlue? Cell Viability Reagent (Thermo Fisher Scientific, here abbreviated as ABCVR) was added to normal medium to make ABCVR solution (normal medium:ABCVR=10:1 wt %). After transferring the medium from each incubated well to a centrifuge tube, 2.0 ml of ABCVR solution was added and kept in an incubator (CO.sub.2 concentration: 5%, 37? C.) for 4 h to react. From the solution, 80 ?l was taken and transferred to a black-bottomed 96-well plate for measurement. The fluorescence intensity was then measured using a multimode plate reader (Perkin Elmer Life & Analytical Sciences, EnSpire) (excitation wavelength: 540 nm, fluorescence wavelength: 590 nm). The fluorescence intensity at 6 hours was then compared with the premature decline intensity, which was set to 1, to evaluate the metabolic activity of the cells, i.e., to determine proliferative potential.

[0060] The results clearly showed rapid and steady growth after 1 day of cell adhesion (see FIG. 8).

The experimental results confirmed that the cotton-wool like bone regeneration material consisting of thick ?-TCP/PDLLGA fibers spun by the wet spinning method of the present invention showed high proliferative potential in the osteoblast culture test.