KR101344069B1 - A manufacturing method of Novel Fibrous Scaffold Composed of Electrospun Porous Poly(ε-caprolactone) (PCL) Fibers for Bone Tissue Engineering - Google Patents

Abstract

For bone tissue engineering studies, many different types of skeletons were fabricated using different methods. Here, new fibrous frameworks have been continuously studied for the regeneration of bone tissue defects. This porous PCL fiber-based fibrous backbone is made using the appropriate solvent of the two solvents in the electrospinning process. As a porous structure, CaP particles were easily coated on fibers after immersion in simulated body fluid (SBF) solution. Morphology, structure, CaP coating, and wettability were investigated by mass spectrometry and contact angle analysis by SEM, EDS, X-ray, and TEM techniques. The proliferation of cells in vitro and the interaction of cells in the fibrous skeleton were investigated. In addition, CaP coatings based on non-porous PCL fiber-based, porous PCL fiber-based, and porous PCL fibers, including the fibrous skeleton's ability to form bones in vivo, were also investigated. By measuring in vitro results, we found that after 12 hours of immersion in SBF (Ap-dPCL12), the fibrous scaffold based on porous PCL fibers was superior to the interaction, growth and proliferation of MG-63 osteoblasts. In vivo analysis showed that the Ap-dPCL12 fibrous framework accelerated bone formation faster than the fibrous framework based on nonporous and porous PCL fibres.

Description

A manufacturing method of Novel Fibrous Scaffold Composed of Electrospun Porous Poly (ε-caprolactone) (PCL) Fibers for Bone Tissue Engineering }

The present invention relates to a method for producing a fibrous skeleton based on porous PCL fibers using an electrospinning process.

Electrospinning technology, also known as field emission, is a method of manufacturing by using a difference in charge, and is being spotlighted as a technology for producing ultrafine polymer fibers that cannot be processed by other methods. Polymers, ceramics, composite materials, and metals This solution is a relatively simple and easy way to produce fibers with nanomelt to submicron diameters.

At the end of the capillary, the spinneret, located vertically, the electrospinning device equilibrates between gravity and surface tension and hangs in a hemispherical droplet. Applying an electric field generates a force opposite to the surface tension, and the hemispherical droplets increase in a conical shape, and when the electric field is over a certain intensity, the jet of charged polymer solution continues to eject from the Taylor cone while overcoming the surface tension. do. The spray evaporates the solvent while it is directed to the counter electrode and collects solid fibers ranging in diameter from micrometers to nanometers as they are directed to the counter electrode at high velocity.

One of the fast growing fields of application of the electrospun polymer fiber is the regenerative medicine field. Tissues targeted for regeneration include cartilage, bone, and skin tissue. The most important feature of the application of electrospun fibers in biological applications is the ability to mimic the extracellular matrix (ECM), which includes a microenvironment that can surround and fuse spermatogonia.

Such electrospinning techniques have been introduced for fibrous skeletal tissue specimens. Because of the small feature size of the electrospun fibers, the fibrous frameworks obtained from the electrospinning process generally showed high porosity and a large surface area, thus mimicking the structure of the ECM.

Nonwovens, fibrous skeletons electrospun with biodegradable polymers such as poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (lactic-co-glycolic acid) / poly (ε- Caprolactone composites are also known, and these polymer products have shown positive results in vivo, but low bone formation is still a major challenge.

To overcome these drawbacks, recent studies have included bone-like ceramics, for example fibrous frameworks or apatite coatings using hydroxyapatite, tricalcium phosphate, calcium carbonate, or even designated drugs, proteins and growths. There is an argument.

Although various fibrous skeletons are being investigated, the technique is to accelerate the deposition of Calcium Phosphate (CaP) on the fiber surface after immersion in SBF (Simulated Body Fluid) solution, and the electrospinning technique used to make the fibrous skeleton based on porous PCL fibers. Focusing on the research of the present invention proceeded.

Polymers and various porous fibers prepared different pore structures in various ways. Among them, electrospun polymer solutions and common solvents were used to make highly porous fibers. It creates many pores on the surface of the polymer material, allowing more cells to be produced, increased expression levels of certain bone formation, and better fusion with host tissues.

1. Republic of Korea Patent Application No. 10-2012-0075880 2. Korea Patent Registration No. 10-1244408 3. Republic of Korea Patent No. 10-1232386

The present invention provides a fibrous framework suitable for biografting through CaP coating, electroporation, and CaP treatment and coating process based on bone conduction of electrospun porous Poly (ε-caprolactone) (PCL) fibers. Is to provide.

The present invention was prepared by dissolving Poly (ε-caprolactone) (PCL) in a solvent mixture to make a compatible biological function of the fibrous skeleton, and then forming the polymer fibrous skeleton structure of the porous surface by an electron spinning process using the same. .

The porous structure is characterized in that the precipitated CaP (Calcium Phosphate) particles.

Accelerate CaP (Calcium Phosphate) deposition on the fiber surface after immersion in SBF (Simulated Body Fluid) solution for various fibrous skeletons, focusing on the electrospinning technology used to make the fibrous skeleton based on porous PCL fibers. And reviewed.

In the electrospinning process of the present invention, PCL is dissolved by a mixture of dichloromethane (DCM) or DCM / Ace (Acetone) (10/0, 8/2, 6/4 and 5/5, v / v) solvents. . It is then delivered via a mass flow rate of 0.5 mL / h and syrince (10 mL). Commercial electrospinning setups are used with the fibrous framework described above. A suitable concentration of PCL solution for uniform fiber formation is 2% (m / v).

All electrospinning processes are performed at room temperature.

The fibrous skeleton obtained from the electrospinning step was immersed in 10 mL of 1.5 M NaOH aqueous solution for treatment in an alkaline solution, and then shaken at 37 ° C. for 24 hours at 20 RPM. The sample is taken out of NaOH solution, washed with distilled water and dried in air at room temperature for several hours.

According to the present invention, NaOH treated samples were subjected to CaP treatment to show CaP nuclei in the sample. First, the sample is soaked in CaCl ₂ solution (200mM) at 37 ° C. for 10 seconds, soaked in distilled water for 1 minute and then dried in air. Second, the sample is soaked in K ₂ HPO ₄ .3H ₂ O solution (200 mM) at 37 ° C. for 10 seconds and soaked again in distilled water for several minutes. Then dry in air for several hours. Do this step one cycle.

The preparation of the SBF solution was followed by Kokubo's. pH value was adjusted to 7.4 after preparation. The Apatite coating process soaks the fibrous framework in 10 ml SBF in a 15 ml tube. Three samples have respective conditions. After soaking, the skeleton is slowly washed with distilled water and dried until the next test.

The wettability test was investigated according to the change in contact angle with distilled water. 20 μl of distilled water drops acted on the surface of the other fibrous framework. Water droplets were taken immediately and the contact angle (θ) was recorded on the model OCA40 (Data-physics Co. Ltd., Germany).

In this way, the contact angle of each skeleton was measured three times (n = 3).

Most comparisons were performed by comparing the mass increase of samples before and after immersion in SBF solution by the following calculation.

Mass increase (%) = (Wa-Wb) / Wb × 100

Wa and Wb are dried samples before and after dipping in SBF solution, respectively. The procedure was done three times for each sample. (n = 3).

Three types of fibrous backbone, including non-porous PCL fiber based, porous PCL fiber based, and Ap-dpCL12, are delivered into cylinders (5 mm diameter x 10 mm long), pressurized for easy implantation, and sintered at 50 ° C. It is made of a 3D structure which is a predetermined three-dimensional structure.

After brief sintering, the two designated 3D skeletons obtained non-porous PCL fiber based, porous PCL fiber based, and Ap-dPCL12 fibrous frameworks.

All three types of 3D skeletons were lyophilized for 24 hours and stored at -20 ° C until used for implantation.

In another general aspect, there is provided an Ap-dPCL fibrous framework processed by the method described above.

Other functions and aspects are apparent from the following detailed description and drawings, and from the claims.

The fibrous backbone prepared in accordance with the present invention allows processing of the backbone to be biocompatible and can be used in a variety of pore size distributions, such as the ECM matrix, high bone induction and biomedical engineering, medicine and other programs.

The features and advantages represented by the facts identified above and in other studies will be clarified by the following exemplary model description given with the accompanying figures.

Figure 1. PCL fibers taken with a SEM microscope. Low magnification (a), high magnification (b), using only DCM solvent (c), DCM / Ace was used as the solvent of the first PCL polymer to be dissolved.
Figure 2. SEM morphology of each of the spun non-porous (a), porous (b), fibrous framework (c) based on PCL fibers after immersion in SBF solution and corresponding EDS profiles (d)
Figure 3. XRD graph before (a) and after (a) the porous PCL fiber-based fibrous framework in SBF solution
4. Projection electron microscopy (TEM) (a) and HRTEM image (b) of Ap-dPCL fibrous framework
Figure 5. Mass change of fibrous framework based on PCL fiber and porous PCL fiber with time in SBF solution
6. Increased porosity of Ap-dPCL2, Ap-dPCL4, Ap-dPCL8, and Ap-dPCL12 fibrous skeletons cultured for 1, 3 and 5 days
Figure 7. Confocal microscopy when MG-63 osteoblasts were cultured for 1, 3, 5 days in Ap-dPCL12 fibrous skeleton
8. Optical photograph (a) of the fibrous skeleton immediately after surgery. 3D reconstruction (b), porous PCL fiber base (c), and Ap-dPCL12 fibrous framework (d) for nonporous bone regeneration μCT analysis 1 and 3 months after transplantation
9. Numerical analysis of new bone formation of bone defect regions in μCT images of nonporous, porous PCL fiber based, Ap-dPCL12 fibrous frameworks. The ratio of bone volume (BV) is obtained from μCT analysis (P <0.05) of the relative total volume (TV) of each implant.
10. H & E staining analysis of nonporous bone regeneration (a), porous PCL fibrous base (b), Ap-dPCL12 fibrous framework (c), high magnification images (d, e, f) 3 months after transplantation

The specifics of the present specification will be described in detail with reference to the drawings.

As described above, the fibrous framework composed of PCL fibers was prepared by an electrospinning process.

In the method of forming a fibrous skeleton, randomly arranged electrospun round fibers as shown in FIG.

The shape of the electrospun fibers was influenced by various parameters of the electrospinning process.

However, the effect of the solvent on the surface structure of the fibers has not been mentioned yet.

In the present invention, a large proportion of DCM / Ace solvents has been varied to degrade PCL polymers that may affect the surface structure of PCL fibers. Thus, after processing all other fixed variables, except for the volume of DCM and Ace, Figure 1 (b and c) shows that the ratio of DCM and Ace is electrospun at 100/0 and 50/50 (v / v). The difference in surface morphology between PCL fibers was drawn.

Various variable treatments of PCL fiber-based fibrous frameworks are described in detail in Table 1. When only DCM was used, the surface morphology of the PCL fibers was found to be rough as shown in FIG.

However, when using a DCM / Ace (50/50) solvent, a highly porous PCL fiber was obtained with pores of various sizes in the range of 500-1000nm as shown in Figure 1 (c).

Effect of various solvent volume ratios on the surface morphology of the fibers

N: non-porous, R / L: low roughness, R / H: high roughness, P: porous

SBF is widely used for bioinert materials and special biomimetic calcium phosphate (CaP) coatings on the surfaces of various polymers. This process is quite slow and generally takes several weeks. The surface of the suitable polymer determines the rapid deposition of CaP. Compared to biopolymer materials such as GE, PLGA and PLLA, this is an inert material. Therefore, in order to accelerate CaP deposition on the surface of the fiber, it is necessary to make or change the surface of the PCL fiber into a porous structure.

The CaP deposition process focuses on making the surface of the PCL fiber porous, and is treated with a CaP treatment for carboxylate formation on the surface of the fiber.

After 12 hours of immersion after CaP treatment and SBF solution, special deposition of CaP on the surface of PCL fiber can be seen. As shown in Table 2, the atomic state of the CaL-based fibrous backbone was immersed in SBF for 12 hours after EDS (Energy Dispersive Spectroscoy) technology was used. (%) Increased significantly (13.194%), and comparing the deposition of CaP on the surface of the non-porous PCL fiber and the porous PCL fiber, the surface of the non-porous fiber as if the small CaP particles were disturbed as shown in FIG. It can be seen deposited on. On the contrary, FIG. 2 (b) shows that CaP is deposited differently on the surface of the porous PCL fiber from the thickly covered layer of the outer surface.

Figure 2 (c and d) shows the CaP deposition of the non-porous PCL fiber-based and porous PCL fiber-based fibrous framework, respectively, in the EDS diagram. This shows that the peaks of Ca and P atoms based on porous PCL fibers are clearly higher than the fibrous framework based on nonporous PCL fibers.

From these results, it can be seen that the porous surface of the PCL fibers accelerates the CaP deposition than the nonporous surface.

Amount of Calcium in Fibrous Skeletons with Incubation Time of PCL Fiber-based Fibrous Skeletons in SBF

3 is an XRD graph of a porous PCL fiber-based fibrous skeleton before SBF (FIG. 3 (a)) and after SBF (FIG. 3 (b)). Before immersion, two strong peaks can be seen when PCL is 2θ = 21.4 ° and 23.7 °.

After immersion, however, two normal PCL peaks and two new apatite (AP) [6, 25] and 2θ = 31.5 ° and 42 °, corresponding to the tricalcium phosphate phase (TCP), can be seen.

XRD data indicate that the CaP coated layer is a mixture of TCP and apatite. TEM and HRTEM were used to view the internal structure of CaP deposited nonporous PCL fiber based fibrous framework.

As shown in Figure 4 (a), the electron diffraction pattern taken from the CaP layer layer on the surface of the porous PCL fibers, it was confirmed that the CaP particles are randomly arranged on the surface of the fibers randomly dispersed in an almost unstable amorphous state. In HRTEM analysis (FIG. 4 (b)), nanoparticles of 5-15 nm diameter, crystalline structure can be seen.

The wettability test results of the PCL fiber-based fibrous skeleton are shown in Table 3. Increasing the SBF submersion time from 2 to 12 hours can be seen that the water contact angle of the fibrous skeleton decreases from 96.5 ° to 50.2 °. The water contact angle of 114.2 ° indicates that the fibrous framework based on the non-porous PCL fibers exhibits a large hydrophobicity.

On the other hand, due to the porous structure of the PCL fibers, the porous PCL fiber-based fibrous framework has a contact angle of 105.7 °. The results show that the absorbency of the fibrous skeleton improves when the fibrous skeleton is porous and increases the time of immersion in the SBF solution.

Contact angle when water droplets are dropped on Ap-dPCL2, Ap-dPCL4, Ap-dPCL8, Ap-dPCL12, PCL fiber based, and porous PCL fiber based fibrous framework surfaces.

Figure 5 shows the mass changes of two non-porous PCL fiber-based and porous PCL fiber-based fibrous frameworks when immersed in SBF solution for different periods. This indicates that increasing SBF immersion time increases the mass of the skeleton. In the case of non-porous PCL fiber-based fibrous skeleton, the mass of the skeleton increased by 7.2% after 4 hours of immersion. It means rapid precipitation and homogenization of CaP. At the same time, the fibrous framework based on porous PCL fibers had a 13% increase in mass. However, after 8 hours (5-12 hours), the mass growth rate of the non-porous PCL fiber-based fibrous skeleton was low, whereas the fibrous skeleton of the porous PCL fiber was high.

MG-63 osteoblast proliferation of Ap-dPCL2, Ap-dPCL4, Ap-dPCL8 and Ap-dPCL12 fibrous frameworks is used to reduce MTT to Formazan by mitochondrial succinate dehydrogenase in complex II (succinate-ubiquinone oxidoreductase complex) I evaluated it. The level of reduction of MTT to formazan is reflected in the level of cellular metabolism.

Figure 6 compares the time intervals of Formazan absorbance when fibrous skeletons were made at different immersion times in SBF solutions. Biomimetic formation of the fibrous backbone affects cell survival and proliferation. Cell attachment and proliferation of the four types of fibrous frameworks differed threefold between 1 and 5 days. In addition, the absorbance values increased with the increase in CaP coating on the surface of the cell proliferation fibrous framework. However, a slight increase was observed between Ap-dPCL2 and Ap-dPCL4, and between Ap-dPCL8 and Ap-dPCL12. The results show that the Ap-dPCL12 fibrous backbone has the greatest impact on ECM production compared to the others.

The confocal micrograph image of FIG. 7 confirmed that MG-63 osteoblasts were viable in Ap-dPCL12 fibrous framework after 1, 3, and 5 days of culture. Green highlights indicate viable MG-63 cells stained with DAPI (fluorescent staining reagent). In Figure 7 (a), after 1 day culture, the cells are stuck to the surface of the fiber. After 3 days, the cells grew on the surface of the skeleton, and the gap between the pores of the surface also slightly increased (Fig. 7 (b)). The cells continued to grow and spread, eventually growing to the entire surface of the skeleton after 5 days of culture (FIG. 7 (c)). This confocal image shows that MG-63 osteoblasts have been successfully adhered and diffused in Ap-dPCL12 and are very well distinguished. These results show that the Ap-dPCL12 fibrous skeleton has great potential for bone tissue engineering.

The pore formation of PCL fibers composed of fibrous skeletal and CaP coating effects was investigated in bone formation capacity in vivo. The μCT assay results in CaP coating of porous PCL fibers, which coincides with bone conduction when transplanted for one month, so bone tissue grows in the entire structure of the Ap-dPCL12 fibrous skeletal group (Figure 8. (d)). Appeared to have started.

In contrast, bones grew slightly only at the edges between the skeleton and the original bone on both the nonporous PCL fibrous and porous PCL fibrous based fibrous backbones shown in (Fig. 8. (b, c)). Due to the porous structure of the porous PCL fiber-based fibrous framework composed of PCL fibers, the growth of bone tissue on the surface was compared when comparing the non-porous PCL fiber-based fibrous framework.

Continuously, after 3 months of implantation, the density of the bone formation as shown in Figs. 8 and 9 was followed by Ap-dPCL12> Fibrous framework based on porous PCL fibers> Fibrous framework based on nonporous PCL fibers. And formed in the framework of fibrous skeletal substrates.

Histological analysis provided more biological information in the treatment of bone defect locations. It also allows simultaneous visualization of the fibrous framework, even the growth of bone tissue within the skeletal structure. After implantation for three months into three types of fibrous skeletons, bone formation was confirmed by observing the bone defects on the H & E staining micrograph. In Figures 10a and d, the bone defects were filled with very small amounts of bone tissue in the case of a non-porous PCL fiber based fibrous framework.

However, a large amount of grown bone was observed in the porous structural plate of the fibrous framework based on the porous PCL fibers as shown in Figs. 10b and e. In contrast, Figure 10 c and f had the highest percentage of defects, which was completely treated after implantation of the Ap-dPCL12 fibrous framework for 3 months. When we compare the bone defects with enlargement, we can clearly see that Ap-dPCL12 had a significant effect on the formation of new bones in bone defects.

Claims (6)

Dissolving Poly (ε-caprolactone) (PCL) in a solvent mixture to form a homogeneous PCL solution, and then forming a polymeric fiber skeleton composed of porous fibers by an electron spinning process using the same;
A second step of treating the porous fiber skeleton formed in the first step with an alkaline solution;
A third step of depositing CaP (Calcium Phosphate) on the surface of the porous fiber skeleton by treating with a CaCl ₂ solution after the second step and treating with a K ₂ HPO ₄ · 3H ₂ O solution;
Immersing in SBF (Simulated Body Fluid) solution after the third step to accelerate CaP (Calcium Phosphate) deposition on the surface of the porous fiber backbone;
Fibrous composition composed of electrospun porous Poly (ε-caprolactone) (PCL) fibers, comprising a fifth step of forming a 3D structure by pressure sintering the fibrous framework obtained from the fourth step at 50 ° C. Method for producing a skeleton.
The electrospun porous Poly (ε-caprolactone) (PCL) according to claim 1, wherein the solvent mixture is made of DCM (Dichloromethane) and Ace (Acetone), and the ratio is 1: 1 (v / v). ) Method for producing fibrous skeleton composed of fibers.
The method of producing a fibrous framework composed of electrospun porous Poly (ε-caprolactone) (PCL) fibers, characterized in that the concentration of the homogeneous PCL solution is 2% (m / v).
A fibrous skeleton composed of porous Poly (ε-caprolactone) (PCL) fibers prepared from the method of any one of claims 1 to 3.
A fibrous skeleton composed of porous Poly (ε-caprolactone) (PCL) fibers prepared from the method of claim 1 useful for cell adhesion.
6. A fibrous skeleton composed of porous Poly (ε-caprolactone) (PCL) fibers, characterized in that the cells are bone tissue cells.

Images