KR20160031682A - Method for preparing magnetic scaffold including nanoparticle with functionalized surface for bone regeneration and a magnetic scaffold obtained thereby - Google Patents

Abstract

The present invention relates to a manufacturing method of a biocompatible polymer magnetic scaffold comprising surface-functionalized nanoparticles, and to the magnetic scaffold manufactured by the manufacturing method. According to the manufacturing method of the present invention, the magnetic nanofiber scaffold has structural and magnetic properties of the evenly distributed magnetic nanoparticles by mixing the magnetic nanoparticles. Therefore, the scaffold is capable of promoting bone regeneration in a safe and effective manner on a desired region in vivo by improving mechanical properties and biological properties.

Description

TECHNICAL FIELD The present invention relates to a method for preparing a polymer magnetic scaffold for bone regeneration comprising surface-functionalized nanoparticles and a magnetic scaffold for bone regeneration prepared by the method, obtained thereby}

The present invention relates to a method for producing a biocompatible polymer magnetic scaffold including surface-functionalized nanoparticles and a magnetic scaffold prepared by the method, and more particularly, The present invention relates to a method for producing a porous magnetic scaffold in which a compatible polymer mixed solution is prepared by salt extraction, and a magnetic scaffold produced by the method.

Scaffolds for tissue engineering play an important role in providing sites for anchorage of stem cells / hepatocytes, their ability to self-replicate, and possible phagocytic differentiation. Such scaffolds are preferred to have moderate degradability, no toxicity, and active activity in connection with tissue treatment and regeneration processes. Biochemical signals provided by scaffolds such as surface chemistry and release of soluble elements have been extensively studied. Recently, bio-physics including the surface roughness, phase and substrate elasticity of the scaffold have been studied to understand the behavior of cells, especially stem cells, and to measure the ultimate potential for application to tissue engineering.

One interesting signal that is emerging in recent years is magnetic properties. Magnetic scaffold designing has attracted attention for tissue regeneration and treatment in wound and disease. In this research, the process strategy is primarily concerned with dip-coating of scaffolds in water-soluble liquid magnets containing iron oxide nanoparticles (NPs). In particular, scaffolds containing magnetic nanoparticles (NP) attract in vitro growth factors and other biomolecules. In addition, due to the ability of the bone tissue to recognize mechanical-electrical transitions, such as mechanical fragmentation of cells with increased binding of MNPs inside the scaffold and mechanical stresses induced from induced magnetic voltage leading to expression levels of multiple genes associated with bone differentiation, Increases growth and differentiation rate.

Because of their biocompatible and non-toxic properties, MNP, primarily superparamagnetic iron oxide nanoparticles (NPs), have been used in a variety of biomedical fields such as magnetic resonance imaging (MRI), drug delivery, cell and tissue targeting and fever therapy have. In particular, for heat therapy, the design of a nano-level heat-source that can be active at a long distance by application of the external alternating magnetic field is attractive. This non-invasive technique may provide localized therapy and controlled drug release leading to the growth and differentiation of specific tissues. However, the therapeutic field of magnetic fields for targeted therapy has some limitations. In this regard, the formulation of implantable biomaterials or tissue engineering scaffolds into the appropriate design of MNPs is considered one of the most attractive areas of research for the treatment and regeneration of bone-containing tissues. Scaffolds that provide magnetic activity to the patient's individual needs serve to guide and stimulate the substrate to the cells.

Recently, MNP has been introduced as a nanocomposite that can be incorporated into the polymer scaffold to provide additional magnetic properties to the scaffold. The incorporation of inorganic nanoparticles, including MNP, is considered a useful strategy for producing polymer-based bone regeneration scaffolds with properties more suitable for bone repair and regeneration in terms of both mechanical and biological aspects. MNPs incorporated into the scaffold are believed to play a number of important roles in stimulating and changing cellular responses favoring bone formation and disease treatment. This is the reason why the scaffold's usefulness can be explained by the high-temperature treatment induced by the magnetic-mechanical induction of bone cells or the temperature of cancer cells under a magnetic field.

Based on this, the present inventors have developed a magnetic biocompatible polymer scaffold incorporating MNP for bone restoration purposes. Surface-functionalized MNP was added to polycaprolactone (PCL) and a foam scaffold was produced by a salt-leaching method. The physico-chemical, mechanical, and magnetic properties of the PCL-MNP scaffolds were examined, and tissue compatibility was assessed as well as bone cell responses in rats to find usefulness in bone tissue engineering.

One object of the present invention is to provide a magnetic nanoparticle comprising: a first step of preparing magnetic nanoparticles; A second step of preparing a biocompatible polymer solution; A third step of uniformly dispersing the magnetic nanoparticles in a biocompatible polymer solution; And a fourth step of injecting a biocompatible polymer solution in which the magnetic nanoparticles are uniformly dispersed into a salt-added mold and freeze-drying the magnetic composite nanoparticles, and a fourth step of preparing the magnetic scaffold for bone regeneration.

Another object of the present invention is to provide a magnetic scaffold for bone regeneration wherein the magnetic nanoparticles produced by the above-described method are uniformly dispersed in the biocompatible polymer.

It is still another object of the present invention to provide a bone regeneration composition comprising a magnetic scaffold prepared by the above production method as an active ingredient.

According to one aspect of the present invention, there is provided a magnetic nanoparticle comprising: a first step of preparing magnetic nanoparticles; A second step of preparing a biocompatible polymer solution; A third step of uniformly dispersing the magnetic nanoparticles in a biocompatible polymer solution; And a fourth step of injecting a biocompatible polymer solution in which the magnetic nanoparticles are uniformly dispersed into a salt-added mold and freeze-drying the magnetic biocompatible polymer solution.

Hereinafter, the configuration of the present invention will be described in detail.

The magnetic scaffold for bone regeneration according to the present invention uses mechanical and magnetic properties of magnetic nanoparticles uniformly distributed in a biocompatible polymer, and has mechanical properties including hydrophilicity, swellability of scaffold by water and mechanical strength, and mineralization , Cell adhesion, tissue compatibility, and the like, is improved by preparing a magnetic scaffold for bone regeneration.

The "magnetic nanoparticle" of the present invention is a substance having magnetic properties and is a substance in a very fine region. Magnetic nanoparticles can be applied in a variety of applications due to their structural and magnetic properties, such as MRI contrast agents, bio-diagnostic devices, anti-fake inks, and device control devices within speakers. In most practical applications where many electric devices are used, the introduction of magnetically tunable magnetic nanoparticles is convenient and cost effective.

In particular, the magnetic nanoparticles used in the bio field can bind various functional groups together for each use. Among them, bio-diagnostic reagents, DNA, RNA separation and purification, and MRI contrast agents enhance the reactivity and stability by binding functional groups such as hydroxyl group (OH), amine group (NH ₂ ) and carboxyl group (COOH) . Each functional group is selected in consideration of specific binding or stability in vivo.

Since these magnetic nanoparticles must be sensitive to the magnetic field during application, it is necessary to dispose the particles one by one. Magnetic suspensions are magnetic nanoparticles dispersed in a solvent and can be adjusted according to the strength and direction of the magnetic field. The treatment technique for preventing the magnetic nanoparticles from aggregating in the solvent during the preparation of the magnetic suspension is very important.

The magnetic nanoparticles may have a superparamagnetic property including at least one metal selected from the group consisting of iron, scandium, titanium, chromium, manganese, cobalt, nickel, copper and zinc. In one embodiment of the present invention, magnetite nanoparticles (Fe ₃ O ₄ ) were used as iron-containing magnetic nanoparticles.

In addition, the average diameter of the magnetic nanoparticles may be 5 to 20 nm, preferably 8 to 15 nm, and more preferably 10 to 11 nm.

Also, the magnetic nanoparticles are surface-modified with a surfactant, and the surfactant is selected from the group consisting of 1,2-hexadecanediol, oleic acid, oleylamine ( oleylamine, and benzyl ether may be used.

In addition, the content of the magnetic nanoparticles in the biocompatible polymer solution in which the magnetic nanoparticles are dispersed in the third step may be 1 to 20 wt%, preferably 5 to 10 wt% have.

&Quot; Biocompatibility "of the present invention is a characteristic that a biomaterial should not exhibit a rejection reaction when it comes into contact with a living body tissue or a body fluid. The biomaterial is a substance that does not exhibit cytotoxicity in vivo, biological tissue stimulation, inflammation induction, allergen induction, Characteristics. In other words, it does not negatively affect normal metabolism and physiological process by interaction with living tissue.

The "biocompatible polymer" of the present invention may be a polymeric material such as polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon, polyaramid, But are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, poly-benzyl-glutamate, polyphenyleneterephthalamide, polyaniline, polyacrylonitrile, polyethylene Polyolefins such as polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate, polylactic acid (PLA), polyglycolic acid (PGA) , Copolymers of polylactic acid and polyglycolic acid (PLGA), poly {poly (ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT) Polyesters such as polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester (POE), poly (propylene fumarate) propylene fumarate-diacrylate (PPF-DA) and poly (ethylene glycol) diacrylate (PEG-DA)

The biocompatible polymer of the present invention may preferably be polycarprolactone.

In the second step, the solvent of the biocompatible polymer solution may be at least one selected from the group consisting of acetonitrile, dichloromethane, chloroform, tetrahydrofuran and dioxane, preferably chloroform.

In the fourth step, a magnetic scaffold for bone regeneration can be prepared by using a salt extraction method and lyophilization to prepare a porous support structure.

Specifically, the salt particles suspended in a predetermined size may be poured into a plastic mold, and then the mixture may be sealed by applying pressure. The mixed solution prepared in the third step may be added dropwise and then lyophilized for 1 to 5 days. It can then be washed several times with distilled water for complete removal of the salt.

In the salt extraction method, NaCl may be used as a salt.

The salt may have an average particle diameter of 200 to 500 mu m.

In the fourth step, freeze-drying may be performed at a temperature of -90 to -60 ° C and a pressure of 3 to 8 Torr, preferably at a temperature of -75 to -64 ° C and a pressure of 4 to 5 Torr . ≪ / RTI >

The lyophilization of the fourth step may be performed using a mold selected from the group consisting of Teflon, polyethylene, ultrahigh molecular weight polyethylene, and benzyl ether.

The "scaffold" of the present invention is a support for culturing cells of human or animal tissues in vitro in the field of tissue engineering and transplanting them back into human or animal. Specifically, the scaffold is obtained by collecting a human or animal tissue, separating the cells from the tissue, culturing the scaffold in the scaffold to prepare a cell-scaffold complex, It is used to transplant in animals.

Tissue engineering techniques have been applied to almost all organs of human body such as artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessel and artificial muscle. In order to optimize the regeneration of such living tissues and organs, basically, Folds shall be provided. The basic requirements of an ideal scaffold are largely non-toxic, mechanical properties and porosity.

The non-toxic refers to the absence of blood coagulation or inflammation after transplantation of the cell-support complex into a living tissue, the mechanical properties of the support refers to the strength sufficient to support the growth of the cell, and the porosity of the support Adhesion occurs well, and sufficient space is secured between cells and cells, and oxygen and nutrients are supplied well by diffusion of body fluids, and neovascularization is smoothly performed, thereby successfully growing and differentiating cells. Basically, the cells are cultured in two dimensions. In order to cultivate them in the form of tissue or organ, a three-dimensional scaffold is necessary. These scaffolds must have numerous pores to allow cells to attach to the interior and exterior, and to have an open structure to feed the nutrients needed to grow the cells and to drain away waste products. That is, a porous three-dimensional scaffold is required.

Accordingly, scaffolds satisfying the above-mentioned basic requirements are preferably similar to extracellular matrix in the animal body, and a method for producing a porous support such as the extracellular matrix has been studied. Representative methods include particulate leaching, emulsion freeze-drying, high pressure gas expansion, phase separation, and electrospinning.

Salt particle extraction or lyophilization may be used to produce a magnetic scaffold that meets the above requirements of the present invention.

On the other hand, the scaffold should be biocompatible and the cell should be able to attach and propagate on the scaffold to form tissue or organ equivalents. Thus, the scaffold can be considered as a substrate for cell growth in vitro or in vivo. Biocompatible, biodegradable polymers are used to prepare scaffolds with these properties.

In addition, tissue cells implanted into a scaffold made of a biodegradable polymer having the above-described porous structure form a series of tissues by continuous growth. Regeneration of a general biotissue is performed by a scaffold shape And then transplanting the tissue cells into the scaffold and growing the transplanted tissue cells.

As another aspect of the present invention, the present invention provides a magnetic nanofiber scaffold for bone regeneration wherein the magnetic nanoparticles produced by the above-described production method are uniformly dispersed in the biocompatible polymer.

The content of the magnetic nanoparticles in the prepared scaffold may be 1 to 20 wt%, preferably 5 to 10 wt%, based on the weight of the total mixed solution.

In another aspect of the present invention, there is provided a bone regeneration composition comprising the magnetic nanofiber scaffold prepared by the above-described production method as an active ingredient.

As described above, the bone regeneration scaffold is used as an active ingredient for performing bone defect treatment through transplantation to a defect site.

The scaffold for bone regeneration according to the present invention can be transplanted immediately after the mesenchymal stem cells are loaded on the scaffold or can be transplanted into the living body after proliferation of the cells on the scaffold through a certain period of culture.

The bone regeneration composition may include not only a scaffold for bone regeneration according to the present invention but also an aqueous solution for maintaining it in a state suitable for transplantation, for example, a buffer solution or an aqueous solution for injections.

The magnetic nanofiber scaffold according to the manufacturing method of the present invention increases the hydrophilicity and expandability of the scaffold to water due to the structural and magnetic characteristics of the uniformly distributed magnetic nanoparticles by incorporating the magnetic nanoparticles, And mechanical properties such as mechanical strength are improved. In addition, biological properties such as in vitro apatite formation / degradation, cell adhesion, cell penetration, biocompatibility, and in vivo bone formation ability can be improved to promote safe and effective bone regeneration at a desired site in the living body.

Fig. 1 shows the measurement results of the surface-functionalized MNP. At this time, (a) shows a TEM result and (b) shows a hysteresis loop.
Figure 2 shows a SEM image of the PCL, PCL-MNP5 and PCL-MNP10 scaffolds showing EDS mapping images showing the highly porous structure and dispersion of MNP in the scaffold.
3 shows the result of measuring the characteristics of the scaffold; (A) XRD pattern, (b) FT-IR spectrum and (c) TG analysis of PCL, PCL-MNP5 and PCL-MNP10 scaffolds.
Figure 4 shows (a) water contact angle, (b) water absorption capacity and (c) swelling volume of PCL, PCL-MNP5 and PCL-MNP10 scaffolds.
Figure 5 is the result of measuring the time-and the MNP-dependent apatite formation ability of the scaffold; (A) SEM morphology and (b) XRD patterns of PCL, PCL-MNP5 and PCL-MNP10 scaffolds submerged in SBF for 1, 3 and 7 days. In the SEM image, the scale bar is 5 μm, and the diffraction peaks starred in XRD represent characteristic bands of apatite.
Figure 6 (a) shows the magnetic field-dependent magnetization curves of the PCL, PCL-MNP5 and PCL-MNP10 scaffolds measured at ambient temperature, Figure 6 (b) It represents the enemy distance. The photograph shows the magnetic interaction between the permanent magnet and the scaffold.
Fig. 7 is a graph showing the relationship between (a) the typical compressive stress-strain curves for PCL, PCL-MNP5 and PCL-MNP10 scaffolds, (b) Mechanical properties.
Figure 8 shows the storage elastic modulus (E '), loss modulus (E "), tangent delta (E" / E') and their average values recorded in the PCL, PCL-MNP5 and PCL-MNP10 scaffolds performed during the frequency sweep Indicated by dynamic mechanical analysis.
Figure 9 shows cell adhesion to scaffolds for 1, 3, and 24 hours; (a) SEM cell morphology and (b) CCK analysis. The scale bar in the SEM is 50 탆, and * p < 0.05 and ** p < 0.01 in the one-way ANOVA test.
Figure 10 shows an increase in osteogenic cells in the scaffold; (a) SEM measurement at 14 days of culture and (b) CCK analysis of cells at 3, 7, 14 and 21 days of culture.
Figure 11 shows (a) the amount of Ca (green) and P (blue) accumulated in the cells cultured in the PCL-MNP10 scaffold for 21 days by (a) quantitative measurement of ARS on days 7, 14 and 21 of calcium accumulation, EDS mapping and EDS spectrum of Ca and P signals * p <0.05 and ** p <0.01 in the one-way ANOVA test.
Figure 12 shows the stained tissue of a scaffold implanted in the subcutaneous tissue of rats for two weeks. All groups show a mild inflammatory reaction around the scaffold and a similar regeneration pattern of thin capsules. The biological host response to the groups was minimized (F: fibrous capsules, P: polymer and R: residue).

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

The data of the present invention are expressed as mean ± standard deviation, and statistical analysis was performed using one-way ANOVA analysis. Compared to independent sample groups. p <0.05 or p <0.01 was considered to be significant.

Example  One: MNP  And PCL - MNP Scaffold  Produce

MNP was synthesized using known methods (S. Sun et al, J. Am. Soc., 2004). Specifically, MNP was prepared by reacting 3.5318 g of Fe (acac) ₃ (iron (III) acetylacetonate), 3.9123 g of 1,2-hexadecanediol, 10 ml of oleic acid, 10 ml of oleylamine ) And 40 ml of benzyl ether. The mixture was preheated to reflux at 200 [deg.] C for 30 minutes while stirring, and then heated to 300 [deg.] C in nitrogen for an additional 2 hours. The dark brown mixture was cooled at room temperature and 50 ml of ethanol was added to the precipitate. The product was collected by centrifugation at 10,000 rpm for 5 minutes, washed four times with ethanol and dried at 50 &lt; 0 &gt; C. The morphology and magnetic properties of the MNPs were measured using a transmission electron microscope (TEM; 7100, JEOL, USA) and a vibrating sample magnetometer (VSM; Quantum MPMS-XL7, USA).

To prepare the PCL-MNP scaffold, firstly 10% w / v PCL (about 80 kDa, Sigma-Aldrich, USA) was dissolved in chloroform (CHCl ₃ ) and the MNP prepared was added to the PCL solution . The concentrations of MNP were 0, 5 and 10 wt%, respectively, and named PCL, PCL-MNP5 and PCL-MNP10, respectively. The mixed solution was sonicated to make it uniform and stable. Sieved NaCl particles (200-500 μm in diameter) were poured into the cylindrical plastic molds and tightly sealed with pressure. The mixture was then dropped by one drop into a mold filled with NaCl, frozen at -70 DEG C and lyophilized for 3 days. The sample thus obtained was washed 9 times in distilled water for 10 minutes while stirring at 100 rpm to leach out the salt, and dried again.

Experimental Example  One : PCL - MNP Scaffold  Investigation of physical and chemical properties

The morphology of the scaffold was observed with a scanning electron microscope (SEM; S3000H, Hitachi, Japan) and its atomic composition was analyzed by energy dispersive spectroscopy (EDS; Bruker, USA). The image of the scaffold was measured with an X-ray diffractometer (XRD, Rigaku, Japan). Scaffolds were scanned at 40 kV and 40 mA current intensity using Cu Kα1 at a rate of 2 ° / min at a diffraction angle range of 2θ = 10 to 60 ° at step intervals of 0.02 °, 2θ. Fourier transformed infrared spectroscopy (FT-IR; Perkin-Elmer, USA) was used to observe the chemical state of the scaffold. Thermogravimetric (TGA) was performed to analyze the ratio of thermal behavior and scaffold configuration. The sample was heated from room temperature to 500 DEG C at a heating rate of 10 DEG C / min under a nitrogen atmosphere. Was measured by weight change _{- (W 0) / W 0} (W S) after immersion in a solvent in accordance with an expression of the scaffold before × 100 The ability to absorb water or ethanol is ΔW _S (%) =. W ₀ and W _S are the scaffold weights before and after each soaking. The porosity and density of the scaffold were measured using a mercury porosimeter (PM33, Quantachrome, USA). The specific surface area was analyzed by the Brunauer-Emmett-Teller (BET) method under nitrogen gas. The hydrophilicity of the scaffold was confirmed by measuring the contact angle with a Phoenix 300 analyzer. Droplet images made on the scaffold surface were obtained using a viewing system until equilibrium reached at 25 ° C. A typical photograph of water drops at equilibrium was obtained from each sample and five samples were tested for each group. The ability of the scaffold to form apatite was confirmed by immersing the scaffold in 2-fold concentrated SBF to speed up the apatite derivation process and reduce the evaluation period of the apatite forming ability of the bioactive material. Each sample (5 mm in diameter and 3 mm in thickness) was placed in 10 ml 2 × SBF and incubated at 37 ° C. for a given period (1, 3 and 7 days). Samples were collected at each time point, washed with distilled water and dried at room temperature. The apatite forming ability was analyzed using SEM and XRD.

In terms of saturation magnetization and hysteresis loops, the magnetic properties of the samples were measured by VSM applying a magnetic field of ± 20 kOe at room temperature. The VSM was measured on the basis of the reference provided on the machine (high purity nickel spheres).

 From typical TEM micrographs (FIG. 1A), spherical, well dispersed MNPs were observed with a narrow size distribution (average size of approximately 11 nm). The magnetic hysteresis curve of the synthesized MNP was measured by VSM at room temperature and shown in FIG. 1B. MNP has a saturation magnetization of 72.1 emu / g. The VSM curve passes through the origin and residual magnetization and antimagnetic properties are not observed in the VSM curve. This indicates that the superparamagnetism of MNPS is preserved at room temperature.

MNPs create magnetic fields around the tissue following transplantation that provide a physical environment beneficial to the microenvironment of natural cells and tissues in the matrix scaffold. For example, the use of magnetic scaffolds in tissue engineering has been proposed as an alternative to controlling the angiogenic process in vivo. The magnetization of the PCL-MNP magnetic scaffold prepared in the present invention was confirmed. The magnetic force of the PCL-MNP scaffold is increased by the MNP dispersed in the matrix. The magnetic properties of PCL-MNP scaffolds dried at room temperature can be seen from their typical hysteresis curves measured by VSM. Figure 6a shows the magnetic field versus magnetization of the PCL-MNP scaffold. The hysteresis curves of the PCL-MNP scaffold showed similar trends to that of the MNP curve, and the magnetization values of the PCL-MNP scaffold were linearly proportional to the MNP content (1.6 and 3.1 emu at 5% and 10% MNP contents, respectively) / g). Similar to the hysteresis curve of pure MNP, the antimagnetic and residual currents were not observed in the curves of the PCL-MNP5 and PCL-MNP10 scaffolds, indicating the super magnetism of the scaffold. The magnetic moment depending on the magnetic field was close to linear up to 0.5 kOe and reached about 80% of the saturation magnetization value. This is obtained when the material can reach the maximum magnetization under a sufficient magnetic field. Therefore, it can be seen that the maximum magnetization can be easily achieved with only a small change in the external magnetic field of the magnetic scaffold. Figure 6b shows the magnetic relationship of the scaffold by measuring the distance between the permanent magnet and the magnetized scaffold. The distance of the pure PCL scaffold was 0 while the scaffolds of magnetized PCL-MNP5 and PCL-MNP10 were 4.43 and 7.23 mm, respectively. This is the distance induced by the external magnetic field of the magnetized scaffold. Additional images show the scaffold with magnets. On the basis of the magnetic properties, the PCL-MNP scaffold seems to preserve the superparamagnetic behavior with MNP even though the saturation magnetization of the magnetic scaffold is relatively small due to the very low MNP concentration. Further research is needed to elucidate the in vivo effects of magnetic scaffolds that stimulate tissue repair or disease treatment through the generated magnetic effects.

Properties of pore structure such as pore size, pore dispersion, and porosity can affect the physico-chemical properties of porous scaffolds and cell behavior. 2 is a cross-sectional view of an SEM microscope and an EDS mapping image of the scaffold. All scaffolds show a well-pored pore structure with little difference in the morphology of the pores. Macro pores larger than 250 μm are known to be suitable for permeation and proliferation of cells, whereas small pores of 100 μm limit cell penetration into pores. According to reports of the effect of pore size on osteoblastic activity, pores larger than 300 μm are preferred for the induction of osteoblasts. Thus, the pore size in the range of 250 to 500 [mu] m, as embodied in the present invention, is suitable for bone tissue engineering. The EDS mapping showed a high Fe signal in the scaffold incorporating the MNP, and the signal distribution was uniformly observed.

The porosity shows a range of 65-70%, similar to the porosity measured by the porosimeter measured using the initial ethanol replacement test. As summarized in Table 1, other properties such as bulk density and structure density were also measured by this method. The porosity of the scaffold is similar, but the density of MNP is higher than that of PCL.

The surface area of the scaffold was measured by BET analysis and increased with increasing MNP. For reference, the surface area of the scaffold was not dependent on porosity. Rather, surface area values increased with increasing MNP content. With their nano-size properties, MNPs evenly distributed in the PCL polymer matrix can be interpreted as improving the surface area of the scaffold.

In addition, the physicochemical properties of the PCL-MNP scaffold were further confirmed. The image of the scaffold was confirmed by XRD analysis (Fig. 3A). The MNP showed diffraction peaks at 2θ of about 31 °, 36 °, 43 °, 54 °, 57 °, and 63 °, which _is a typical peak of bulk magnetite (Fe ₃ O _{4 )} . The mean particle size calculated by the Scherrer equation for the strongest diffraction peak was 10.7 ± 0.019 nm, similar to the size identified by the TEM image. The magnetite peak of the PCL-MNP scaffold was more clearly observed when MNP was increased.

As shown in the FT-TR spectrum (FIG. 3b), the chemical bond structure of the scaffold showed a typical band associated with PCL and MNP, including a unique band at 578 cm ^-1 , which appears in the Fe-O bond oscillation mode of MNP. The band was more abruptly seen in the PCL-MNP scaffold by increasing the content of MNP. Typical PCL oscillation bands for C = O and CO stretching are observed at 1720 and 1293 cm ^-1 . In addition, the weak and broad OH stretching band of PCL represents an alcohol group at 3153-3640 cm ^-1 .

The thermal behavior of the scaffold was monitored by TGA (Figure 3c). The TGA curve of pure MNP shows a certain level of weight loss (about 10%), presumably due to residual organic phase. On the other hand, typical thermal degradation of pure PCL occurred at approximately 400 ° C (99% loss), and the PCL-MNP scaffold remained at a significant level of weight due to the MNP content present in the scaffold. MNP content in the PCL-MNP5 and PCL-MNP10 scaffolds was approximately 4.71 and 10.01 wt%, respectively, which was similar to the amount of MNP incorporated in the manufacture of the scaffold, considering also the weight loss of pure MNP and PCL.

The results of the hydrophilicity of the scaffold are shown in Fig. The PCL-MNP scaffold became more hydrophilic (5% MNP: 61 ° and 10% MNP: 47 °), while the pure PCL scaffold showed a high degree of hydrophobicity (contact angle 85 °). This is because of the presence of MNP, and more specifically, the carboxyl group present on the surface of MNP. In fact during the salt precipitation process, the surface of MNP will be carboxylated. Jadhav et al. Is a surfactant that captures MNP, and oleic acid electrostatically binds with salt ions to decompose the COOH group of oleic acid into COO- and H +, resulting in improved hydrophilicity of MNP. The present inventors have also found that the water dispersibility of the MNP caught in the surfactant is remarkably increased after the treatment with the sodium chloride solution (data not shown).

As a result of the increased hydrophilicity, the PCL-MNP scaffold exhibits excellent water absorption capacity. Water uptake was measured over a period of up to 24 hours and showed a large difference between the samples (Fig. 4B). The water uptake of the PCL-MNP scaffold quickly reached almost saturation levels within a few hours. Water uptake increased with increasing MNP content. As a result, after 24 hours, the water uptake was about 1440% for the PCL scaffold, 1870% for the PCL-MNP5 scaffold, and 2850% for the PCL-MNP10 scaffold. The water absorption capacity of the PCL-MNP10 scaffold was almost two times higher than that of the pure PCL scaffold. Therefore, the scaffold was observed to swell reliably after water absorption. As measured optically (Fig. 4c), the volume of the increased scaffold was: 5% in PCL, <8% in PCL-MNP5, <11% in PCL-MNP10 and 10% MNP in PCL Which is a twofold increase. In fact, the water absorption level is due to the pore-hammering of the water and the swelling of the scaffold. Considering a similar porosity for all scaffolds, the difference in water absorption capacity was first a result of a swollen, surface-modified property, that is, a water molecule is absorbed by a water molecule absorbed into a dispersed hydrophilic polymer network, The swelling of the folds represents the water absorption capacity of the significantly increased magnetic scaffold.

Experimental Example  2 : Scaffold  Investigation of mechanical properties

The mechanical properties of the PCL-MNP scaffold were measured by dynamic mechanical analysis (DMA, DMA25N, MetraVib, USA) under static and dynamic compression under wet conditions. Cylindrical samples (8 mm in diameter and 16 mm in height) were first fully immersed in PBS for 1 day. For statistical testing, the samples were measured by recording strain rates over time at constant compressive loads. The results obtained by averaging the values obtained by testing three different samples under the respective conditions were calculated.

The dynamic test was then performed in a flat plate environment. Mechanical spectrometry was monitored using dynamic frequency sweeps ranging from 0.5 to 10 Hz for 10 minutes at room temperature. The storage modulus (E ') and the loss modulus (E ") were recorded. The tangent delta was evaluated from the E" / E' ratio.

The mechanical behavior of the PCL-MNP scaffold was performed under static and dynamic conditions using a wet sample, as shown in Figures 7 and 8, respectively. Mechanical properties are another reason to consider scaffolding as a target for bone repair and regeneration. Figure 7a shows a typical stress-strain curves of a scaffold under a static compressive load. All three scaffolds showed similar behavior. The stress value continued to increase as the deformation increased and the rate of increase in deformation increased during compression. This generally manifests itself in porous materials in the process of densification or pore collapse under compressive load. The MNP incorporation of the scaffolds recorded an increased stress level during the entire strain range, indicating a high resistance to strain under compressive load. Figure 7a shows the initial linear region of the stress-strain curve, and the modulus of elasticity is obtained from the initial linear slope (Figure 7b). The elastic moduli of PCL, PCL-MNP5 and PCL-MNP10 were 1.2, 1.4 and 2.4 MPa, respectively, and the MNP dispersed in the matrix played an important role in hardening the scaffold. As with static mechanical testing, dynamic mechanical analysis was performed under continuous pressure amplification in the 0.5 to 10 Hz frequency range. The storage elastic modulus (E '), loss elastic modulus (E "), and tangent delta (E" / E') of the material reacting with the stress are shown in FIG. 8 . The scaffold showed almost no frequency dependency for all characteristics. E 'value was higher by several orders of magnitude than E'. Significantly, the increase in MNP content shows a consistent result of static testing, which greatly increases the storage elasticity of the scaffold. When the modulus of elasticity increased similarly to the incorporation of MNP, the tangent delta remained almost unchanged in the scaffold, which is believed to result in this change in elasticity values due to improved hydrophilicity and high expansion of the scaffold due to MNP incorporation. Although the MNP itself tends to make the PCL polymer network tighter, the increased volume in water (along the distance between the polymer chains) and internal water molecules will complement the strength.

Experimental Example  3: In vitro  Cell adhesion, growth and Mineralization  Test

MC3T3-E1 cells (ATCC; American Type Culture Collection) were cultured for 7 days in humidified air containing 5% CO ₂ and 10% fetal bovine serum (FBS, Hyclone, Thermo Scientific, (Α-Minimal Essential Medium, α-MEM, Gibco, USA) containing penicillin and 100 μl of streptomycin. 10 ⁵ cells were inoculated onto a scaffold (5 mm diameter X 3 mm high) and after 1 day, the scaffold was transferred to each well of another plate and the cell attachment rate of the scaffold was measured with a cell counting kit (CCK) did. In addition, cell proliferation was measured by the CCK method during 3, 7, 14 and 21 days of culture. In SEM observation of the cells, the sample was fixed with a 2.5% glutaraldehyde solution, dehydrated in a concentrated ethanol solution, dried at room temperature, and coated with a platinum thin film.

Mineralization of cells was assessed by Alizarin red assay (ARS; Sigma Aldrich, USA). After 14, 21 and 28 days of incubation, cells were fixed with 70% ethanol for 1 hour at 4 ° C, then immersed in 2% w / v aqueous ARS solution (pH 4.1-4.3) at room temperature for 30 minutes, , The stained sample was removed and eluted with a 10 mM sodium phosphate (pH 7) solution containing 10% w / v of cetyl pyridinium chloride (CPC) for 1 hour. Then, for the continuous quantitative evaluation of each sample solution, the total amount of intracellular mitochondrial dehydrogenase was normalized, and the absorbance of the eluent was measured using a microplate reader at an absorption wavelength of 595 nm.

The in vitro cells responding to the magnetic scaffold were evaluated for cell adhesion. Figure 9a shows SEM micrographs of cell-attached PCL and PCL-MNP scaffolds at 1, 3 and 24 hours after cell inoculation. Granule-like MC3T3-E1 cells appeared well at 3 hours after inoculation in the PCL-MNP5 and PCL-MNP10 scaffolds, and these cells were found to be more abundant in the nanocomposite scaffold than in the pure PCL scaffold. CCK measurements were performed as shown in Figure 9B to assess cell adhesion levels. In the PCL-MNP5 and PCL-MNP10 scaffolds, cell adhesion rapidly increased at 3 hours after cell inoculation and increased sharply up to 24 hours, although there was no significant difference in cell attachment rate over time in pure PCL scaffolds. At 3 hours, the level of cell adhesion at PCL-MNP10 was approximately 1.4 times higher than that of the pure PCl scaffold.

 Cell morphogenesis, proliferation and differentiation in vitro were further studied. Figure 10a shows SEM micrographs of cells grown in the scaffold for 14 days. All scaffolds showed a large number of cell products and cells throughout the scaffold. There was a slight difference in cell growth morphology among the scaffolds. The proliferation of cultured cells in the scaffold was monitored as shown in Figure 10b for up to 21 days. Cells grew rapidly in all scaffolds up to 14 days. The number of cells did not increase in the long-term culture of the cells from 14 days to 21 days. It is believed that this is related to the possibility of cell aggregation on the scaffold surface and / or conversion from cell proliferation to differentiation. Interestingly, especially after 7 days, the cell proliferation rate was higher in the pure PCL than in the PCL-MNP scaffold, with large differences between 14 and 21 days (1.3 to 1.5-fold difference). Thus indicating that cell proliferation is higher in pure PCL for 7-14 days. It is shown that the cells experience a more active process of osteoblast differentiation in the PCL-MNP scaffold and can experience faster conversion from proliferation to differentiation.

As a final stage of osteoblast differentiation, cell mineralization is considered important. The present inventors analyzed the mineralization behavior of cells in the scaffold by a method of quantifying calcium precipitation. For this purpose, staining was performed with ARS which selectively binds to calcium. Figure 11A shows the level of calcium precipitation in cells cultured in each scaffold via ARS quantification. On days 7 and 14, there was little difference between the scaffolds, while there was a large difference in the relatively long incubation period of 21 days. The calcium level in the PCL-MNP10 scaffold was about 2.8-fold higher than that of the pure PCL scaffold. On day 21, calcium precipitation in the PCL-MNP10 scaffold was analyzed by EDS (Figure 11b). The EDS mapping showed high signals of Ca (green) and P (blue), and the Ca / P ratio was 1.7, similar to the stoichiometric HA ratio. These results demonstrate that cell mineralization has been greatly enhanced in the magnetic scaffold and also shows that surface-functionalized MNP in the scaffold affects the final stage of osteogenesis and mineralization of cells. Although the present invention is evaluating the last stage of bone formation, further investigation of bone formation behavior including expression of osteogenic genes and protein synthesis is needed to explain improved cell mineralization. Bone differentiation cells can produce sufficient levels of bone matrix proteins important for the mineralization of cells. In this respect, it is necessary to discuss the basis of the improvement of cell proliferation and osteogenesis differentiation by magnetic scaffold. The significant increase in cell adhesion in the PCL-MNP magnetic scaffold is thought to be due to the hydrophilicity of the scaffold, which probably enhances the affinity of the protein and cell. Modification of the hydrophilicity of the PCL surface has been reported to increase early cell adhesion. Such enhanced early cell attachment will then affect matrix production for cell proliferation, differentiation, and cell mineralization. In addition, along with improved hydrophilicity, stimuli associated with magnetism in cell behavior can be improved. It has been reported that the role of MNP in MNPs incorporated into biomaterials and scaffolds affects cell proliferation and osteogenesis differentiation in vitro. MNP has been proposed as a kind of single magnetic domain on the nanoscale leading to significant changes in the ion channel of the cell membrane, which may affect the behavior of cell proliferation and differentiation. Nanoscale-generated magnetic effects can be enhanced by increasing the content of MNP, which will have a stronger impact on outcome in vitro.

After immersing in SBF, the in vitro cell-free mineralization behavior of the PCL-MNP scaffold was evaluated. Figure 5 shows the SEM morphology and XRD pattern of the scaffold after being immersed in SBF. The PCL-MNP5 and PCL-MNP10 scaffolds began to form mineral nanocrystals on their surface at day 1. On the third day, the entire surface was covered with a mineral phase. After 7 days, the mineral crystals grew considerably (Fig. 5A). On the contrary, the pure PCL scaffold began to form minerals on day 3 and covered less surface than on PCL-MNP on day 7. The SEM results based on the characteristic peaks of HA crystals (2 &amp;thetas; 26 DEG and 32 DEG) were confirmed by the XRD pattern. It is clear that the incorporation of MNP increases the mineralization behavior of the scaffold in SBF. This is due to the carboxylated MNP surface dispersed inside the scaffold. Calcium ions inside the medium will adhere better to the negative charge scaffold surface, and as a result will attract phosphate ions to form mineral crystal nuclei. On the other hand, there was no cell involved in SBF, and it was somewhat limited due to non - dynamic conditions. As a result, cell - free mineralization made it possible to predict the surface reaction possibility and bone bioactivity of magnetic scaffold useful for bone regeneration.

Experimental Example  4 : In vivo  Organizational suitability test

The body experiment was approved by Dankook University animal experiment ethics committee in Korea. Three 10-week-old male Spraque-Dawley rats weighing 350 grams were used. The scaffold groups tested were transplanted with PCL, PCL-MNP5 and PCL-MNP10, and four samples per group (5 mm diameter × 3 mm height). Prior to implantation, the scaffold was sterilized with ethylene oxide gas. The animals were anesthetized by intramuscular injection of 80 mg / kg ketamine and 10 mg / kg xylazine to body mass. The skin of the mouse was shaved and sterilization of the surgical site was made with povidone and 70% ethanol. Using a # 10 bladder mounted on a bard-parker scalpel, a 2 cm long linear incision was made in the skin. Four small subcutaneous implants were prepared by dissecting and dissecting from each rat's vertebrae using the Halsted-mosquito hemostatic forceps. The scaffold was inserted into the prepared area away from the incision. The incision was later sealed with 4-0 nonabsorbable monofilament suture material (Dafilon (R), B. Braun, Germany). During and after the surgery, the mice were kept warm and observed until recovered from the anesthesia. Rats were placed in one cage. The animals were maintained in a cage for 12 hours day / 12 hours night schedule and provided free-standard pelleted food and water.

Two weeks after transplantation, the animals were sacrificed by cervical rupture. Tissue samples for histological analysis were immediately immersed in 4% formaldehyde solution at room temperature and dehydrated in step ethanol. The specimens were planted on paraffin in the form of a split. The paraffin block was subdivided into 5 [mu] m thickness along the longitudinal axis using a rotary microtome. The slides were routinely stained with hematoxylin and eosin (HE) or Masson's trichrome (MT) staining, and then biocompatibility and angiogenesis were microscopically observed. Point-indexed histologic scores from all dyed slides (1), mild (2), intermediate (3), and severe (4) indicate that inflammatory response, fibrous capsule thickness, vascularity and fibroblast proliferation .

The histocompatibility of the scaffold was evaluated as a preliminary study of the histocompatibility of magnetic scaffolds after subcutaneous implantation at the site of administration for 2 weeks. After the anesthesia, there was no postoperative recovery, and all animals showed normal recovery without material-related complications and remained steady during the study. Two weeks after the operation, samples surrounding the tissue were taken and observed under a microscope, showing no signs of redness or inflammation. Histological photographs of the samples are shown in Fig. 12, and the tissue responses including the tendency of fibrocyte encapsulation, inflammation reaction, microvascular production, and abundant granulation tissue in the fibroblasts using a microscope are shown in Table 2 Respectively. No significant differences were observed in the three scaffold groups. Mild to normal fibroblast and vascular cell proliferation were observed in all samples. Encapsulation of the fibrous tissue is seen in the minimally degraded scaffold. All scaffolds were filled with fibroblasts. On the other hand, mild inflammatory reactions occurred in only a small portion of the sample, and overall there was no rejection after animal implantation. Newly formed organizations showed good integration into adjacent organizations in all groups. Organized fibrous tissue forms a space between the scaffold and adjacent tissues and muscles. Fibroblasts are present in adjacent fiber capsules around these scaffold systems.

The PCL-MNP magnetic scaffolds showed good histocompatibility in the rat subcutaneous tissue model for 2 weeks and a deeper in vitro study is needed to demonstrate the efficacy of the magnetic scaffold by using it as a bone regeneration model for a longer implantation period . Although this issue remains to be investigated further, the utility of magnetic scaffolds for bone repair and regeneration is evident in the early adherence, proliferation and mineralization as well as in water affinity and swelling behavior and in vivo cellular responses to scaffolds in improved mechanical properties As a result.

Claims (14)

A first step of preparing magnetic nanoparticles;
A second step of preparing a biocompatible polymer solution;
A third step of uniformly dispersing the magnetic nanoparticles in a biocompatible polymer solution; And
And a fourth step of injecting a biocompatible polymer solution in which the magnetic nanoparticles are uniformly dispersed into a salt-added mold and freeze-drying the magnetic nanoparticle.
The method according to claim 1,
Wherein the magnetic nanoparticles are surface-modified with a surfactant. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
3. The method of claim 2,
The surfactant is at least one selected from the group consisting of 1,2-hexadecanediol, oleic acid, oleylamine, and benzyl ether. Wherein the bone scaffold is a bone scaffold.
The method according to claim 1,
Wherein the magnetic nanoparticles have superparamagnetism including at least one metal selected from the group consisting of iron, scandium, titanium, chromium, manganese, cobalt, nickel, copper and zinc. &Lt; / RTI &gt;
The method according to claim 1,
Wherein the magnetic nanoparticles have a diameter of 9 to 13 nm.
The method according to claim 1,
Wherein the content of the magnetic nanoparticles in the biocompatible polymer solution in which the magnetic nanoparticles are dispersed in the third step is 5 to 10 wt% based on the weight of the whole mixed solution.
The biocompatible polymer of claim 1, wherein the biocompatible polymer comprises a poly (lactide-co-glycolide), a poly (lactic acid), a poly (glycolic acid), and a poly (caprolactone) Wherein the bone scaffold is at least one selected from the group consisting of bone scaffolds and bone scaffolds.
The method according to claim 1,
Wherein the solvent of the biocompatible polymer solution in the second step is at least one selected from the group consisting of acetonitrile, dichloromethane, chloroform, tetrahydrofuran and dioxane.
The method according to claim 1,
Wherein the salt of the fourth step is NaCl. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
The method according to claim 1,
Wherein the salt of the fourth step has an average particle diameter of 200 to 500 mu m.
The method according to claim 1,
Wherein the freeze-drying in the fourth step is performed at a temperature of -90 to -60 ° C and a pressure of 3 to 8 Torr.
The method according to claim 1,
Wherein the material of the mold in the fourth step is at least one selected from the group consisting of Teflon, polyethylene, and ultrahigh molecular weight polyethylene.
A magnetic scaffold for bone regeneration wherein the magnetic nanoparticles produced by the method of any one of claims 1 to 12 are uniformly dispersed in the biocompatible polymer.
A bone regeneration composition comprising the magnetic scaffold of claim 13 as an active ingredient.

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