KR101657777B1 - Organic-inorganic hybrid scaffolds comprising magnetic nanoparticles and a preparation method thereof - Google Patents

Abstract

The present invention relates to an organic-inorganic hybrid scaffold including magnetic nanoparticles and a method for producing the same. The gelatin / siloxane hybrid scaffold containing the magnetic nanoparticles of the present invention has excellent water absorption due to addition of magnetic nanoparticles, is not easily decomposed in the body, and greatly improved mechanical properties such as resistance to deformation. In addition, biocompatibility, as well as cell proliferation and osteogenesis differentiation, can be used for tissue regeneration.

Description

TECHNICAL FIELD [0001] The present invention relates to an organic-inorganic hybrid scaffold including magnetic nanoparticles and a method for manufacturing the hybrid scaffold,

The present invention relates to an organic-inorganic hybrid scaffold including magnetic nanoparticles and a method for producing the same.

Scaffolds play an important role in tissue engineering, which greatly influences the cellular and tissue reactions involved because of their coordinated physicochemical properties. Nanocomposites and hybrid scaffolds made of organic materials for osseous tissue engineering are of particular interest because they have many mechanical and biological properties essential for bone cell differentiation and osteogenesis. In addition to providing an appropriate environment for bone formation, other additional factors associated with cell attachment or growth are also required. Therefore, natural polymers having high biocompatibility and biodegradability such as gelatin, collagen, chitosan, etc. have been widely used for bone tissue regeneration.

However, high biodegradability has the advantage of rapidly releasing bioactivity in a short time after transplantation, but it dramatically reduces cell adhesion and diffusion due to abrupt changes in substrate dynamics, thus delaying new bone formation. In addition, despite their excellent biological properties, there was still a need for improved mechanical properties, particularly in conformity with natural bone applicable to rod bearing parts.
[Prior Art Literature Information]
Korean Registered Patent No. 10-1436740 (Published in September, 2014)
Korean Registered Patent No. 10-1319220 (published on Nov. 13, 2013)
BOCK, N. et al., ACTA BIOMATERIALIA (2010) Vol.6, pp.786-796

In the present invention, while studying a method of preparing a scaffold having potential utility in tissue repair and disease treatment, when a porous scaffold is prepared by adding a small amount of magnetic nanoparticles during a sol-gel process of an organic hybrid, mechanical properties It is possible to produce a magnetic scaffold which can greatly enhance bone-bioactivity by stimulating cell behavior during cell proliferation and osteogenesis differentiation, and completed the present invention.

Accordingly, it is an object of the present invention to provide a magnetic or non-magnetic hybrid scaffold with improved mechanical properties and bone-bioactivity.

It is another object of the present invention to provide a method for producing a hybrid organic-inorganic hybrid scaffold having improved mechanical properties and bone-bioactivity.

In order to solve the above problems, the present invention provides a method for producing a hybrid organic-inorganic hybrid scaffold comprising: 1) dispersing magnetic nanoparticles in a solvent; 2) adding and dispersing the magnetic nanoparticle dispersion of step 1) in a gelatin solution; 3) adding a 3- (glycidoxypropyl) trimethoxysilane and tetramethoxysilane to the dispersion of step 2) to prepare a sol; 4) adding an acidic solution to the sol of step 3) to gel; And 5) lyophilizing the gel of step 4) to produce a porous scaffold.

Hereinafter, the present invention will be described in detail.

In the present invention, a magnetic nanoparticle dispersion is added to and dispersed in a gelatin solution, 3- (glycidoxypropyl) trimethoxysilane and tetramethoxysilane are added to prepare a sol containing gelatin and siloxane, The present invention is based on the discovery that porous scaffolds exhibiting magnetic properties by magnetic nanoparticles as well as increased mechanical strength and bone-bioactivity can be produced.

The step 1 is a step of dispersing the magnetic nanoparticles in a solvent to obtain a magnetic nanoparticle dispersion.

The magnetic nanoparticles to be added to the organic / inorganic hybrid scaffold according to the present invention may include one or more metals selected from the group consisting of iron, scandium, titanium, chromium, manganese, cobalt, nickel, copper, The average particle diameter of the magnetic nanoparticles may be 1 to 20 nm. In one embodiment of the present invention, iron-containing magnetic nanoparticles were used.

In the present invention, the concentration of the magnetic nanoparticles in step 1) may be 0.01 to 3% by weight, based on the weight of the total dispersion of step 1).

In step 1) of the present invention, "solvent" include water, C _{1 -} may be daily for ₄ alcohol or their mixture.

The step 2 is a step of adding a magnetic nanoparticle dispersion of step 1) to a gelatin solution and dispersing it to obtain a gelatin solution in which magnetic nanoparticles are dispersed.

In the present invention, by previously dispersing the magnetic nanoparticles in the gelatin solution, the magnetic nanoparticles can be more uniformly distributed in the scaffold formed by the sol-gel method, thereby further improving the mechanical strength and bone-bioactivity have.

In the step 2) of the present invention, the "gelatin solution" may be one in which 1 to 10% by weight of gelatin is dissolved in water.

Step 3 is a step of preparing a gelatin-siloxane sol by adding 3- (glycidoxypropyl) trimethoxysilane and tetramethoxysilane to the dispersion of step 2).

The Si content of the sol prepared in step 3) of the present invention may be from 2 to 10% by weight based on the total weight of the sol.

In the present invention, the mixing ratio of the 3- (glycidoxypropyl) trimethoxysilane and the tetramethoxysilane may be 1: 0.1 to 1:10 by weight. In the examples of the present invention, the mixing ratio of 3- (glycidoxypropyl) trimethoxysilane and tetramethoxysilane was 1: 1.

Step 4 is a step of adding an acidic solution to cause a sol-gel reaction.

The sol-gel reaction begins with the hydrolysis of the epoxy ring present in the 3- (glycidoxypropyl) trimethoxysilane (GPTMS) molecule. Hydrolysis forms highly electrophilic functional groups on carbon, and electrophilic functional groups readily react with nucleophilic functional groups (such as amino groups) of gelatin. During the gelation of the siloxane, a polycondensation reaction of GPTMS / gelatin takes place, the -O-Si-O-network is formed and the gelatin is covalently linked to the scaffold.

Tetramethoxysilane (TMOS) not only contributes to increase the content of Si, but also increases the stability of the network. The addition of TMOS leads to the formation of -Si-O-Si- in the GPTMS / gelatin complex after the polycondensation reaction. Because TMOS has no electrophilic carbon, nucleophilic gelatin does not react directly with TMOS molecules. Instead, it is bound to the Si-O group of GPTMS, resulting in a well-aligned Si-O-Si network structure, and the gelatin is trapped between the structures.

In step 4) of the present invention, the acidic solution may be one or more selected from the group consisting of hydrochloric acid, phosphoric acid, and acetic acid.

Step 5 is a step of lyophilizing the gel of step 4) to obtain a porous scaffold. During the lyophilization process, the moisture of the gel is removed to complete the porous scaffold. The lyophilization may be performed at -20 캜 to -80 캜 for 1 to 10 hours.

In addition, the present invention can provide a magnetic organic hybrid scaffold prepared by the above-described method and formed by uniformly dispersing micropores.

The scaffolds of the present invention exhibited interconnected microporous morphology and had uniformly dispersed pores (Fig. 1A). In addition, the scaffold of the present invention has a form in which nanoparticles of 1 nm to 20 nm in size are uniformly dispersed as a whole without aggregation.

The lyophilized porous scaffold showed a uniform dispersion of relatively micropores up to 3% content of magnetic nanoparticles, but was severely hindered beyond that. Therefore, it can be seen that the content of the magnetic nanoparticles is an optimum condition for the production of nanosynthesis scaffold of gelatin / siloxane-magnetic nanoparticles.

In addition, the scaffold of the present invention exhibited superparamagnetic behavior. In addition, the saturation magnetization of the scaffold increased as the content of magnetic nanoparticles increased (FIG. 2d).

In order for the magnetic hybrid scaffold to be used for tissue reconstruction purposes, water-related properties including the absorption, swelling and degradation of water are particularly important. In this connection, it was confirmed that the scaffold of the present invention can exhibit the property that the degree of swelling and water uptake increases with increasing magnetic nanoparticle content, but does not increase the scaffold degradation rate (FIG. 3).

Furthermore, the scaffold of the present invention can be improved in mechanical properties by the addition of magnetic nanoparticles. In the embodiment of the present invention, it was confirmed that the mechanical properties can be improved as the addition amount of the magnetic nanoparticles increases (FIG. 4).

In addition, the scaffold of the present invention can exhibit excellent bone-bioactivity due to the addition of magnetic nanoparticles (FIGS. 5 to 8).

In addition, the present invention can provide a composition for bone replacement or bone substitution comprising the magnetic organic or inorganic hybrid scaffold.

The hybrid scaffold of the present invention not only exhibits excellent mechanical properties owing to the addition of magnetic nanoparticles but also exhibits excellent biocompatibility so that the regeneration rate of bone can be safely and effectively promoted at a desired site in the living body, It can be useful as a bone substitute.

The gelatin-siloxane scaffolds of the present invention were modified to include a small amount of magnetic nanoparticles to induce mineralization, as well as to improve cell adhesion and growth and osteo-bioactivity favoring osteogenic differentiation of cells. In addition, the hybrid scaffold of the present invention not only exhibits excellent mechanical properties owing to the addition of a small amount of magnetic nanoparticles but also exhibits excellent biocompatibility, so that the regeneration rate of bone can be safely and effectively promoted at a desired site in a living body And can be useful as bone fillers and bone substitutes.

Figure 1 shows (a) the SEM morphology of a bioactive gelatin / siloxane hybrid scaffold with magnetic nanoparticles at various concentrations (1, 2 and 3%), (b) a 3% IR spectrum of the scaffold showing the TEM image of the fold, (c) the XRD pattern of the scaffold (the appended table is the magnetite peak), and (d) the chemical bonding structure. The spectra of magnetic nanoparticles are also shown for comparison.
2 shows the result of measuring the magnetic characteristics of the hybrid scaffold. (b), (c), (d) and (d) show the result of measurement of the magnetization behavior by the SQUID in the range of small and large magnetic fields, ).
(C) the degree of swelling of the hybrid scaffold at 4 hours after immersion in distilled water; (d) a maximum of 28 days of water uptake by the hybrid scaffold; This is the result of measuring the decomposition behavior of the hybrid scaffold in PBS.
4 shows the result of measuring the mechanical properties of the hybrid scaffold. (a) is the recorded strain versus time applying a constant static compressive load, and (b) is the strain identified at 300 seconds. (c) is the dynamic modulus of elasticity of the scaffold, (c) is the storage elastic modulus E ', (d) is the loss modulus E' ) Is the tangent delta (δ) measurement result.
Figure 5 is the in vitro ability of the hybrid scaffold to form in vitro membranes immersed in 1.5x SBF for different periods. (a) is the SEM image of the scaffold after 7 days of immersion, and (b) is the measurement result of the weight change relative to the immersion time. (c) is the XRD measurement result of the 3% -magnetic nanoparticle scaffold during the immersion, and (d) is the FT-IR measurement result of the 3% -magnetic nanoparticle scaffold during the immersion.
FIG. 6 shows the results of measurement of in vitro cell behavior in a hybrid scaffold. (a) is the result of cell proliferation measured by dsDNA quantification. Cell proliferation at 14 days was significantly higher than at 7 days for all groups (* P &lt; 0.05). Significant differences were compared between groups at day 14 (marked with 'a, b, c, d'sign; p <0.05). (b) are representative SEM images of cells on days 7 and 14.
FIG. 7 shows the results of measurement of ALP activity of cells on days 7 and 14 after normalization with DNA content. Significant differences were found between groups (* P <0.05) in each period and between periods (* P <0.05) in each group.
Figure 8 shows the results of measurement of the mineralization behavior of cells identified by ARS staining. (a) is an image showing an ARS stained sample (above) and a solution (below) dissolved in different incubation periods for 0% -magnetic nanoparticles and 3% -magnetic nanoparticle scaffolds. (b) is the result of calcium content measurement at the optical density, and (c) is the SEM cell image (3% - magnetic nanoparticle scaffold) showing the appearance of cell calciumation on the 21st day. Significant differences were noted between groups (* P <0.05) at 21 days.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, and the present invention is not limited by the examples.

Manufacturing example  1: Preparation of magnetic nanoparticles

Magnetic nanoparticles were prepared according to the previously reported method (Singh RK et al., J Biomed Mater Res A., 2012, 100, 1734-1742). Briefly, first, ferrous chloride heptahydrate (FeCl ₂ .4H ₂ O) in 1M HCl was mixed with ferric chloride hexahydrate (Fe ₂ Cl ₂ .6H ₂ O) at room temperature. The mixture was added dropwise to 200 mL of 1.5 M NaOH solution with vigorous stirring. Then, the resulting precipitate was centrifuged, and the solution was taken out and separated. All steps were carried out under nitrogen gas to prevent oxidation.

Example  One: hybrid Scaffold  Produce

Gelatin (B type, bovine, Sigma), 3- (glycidoxypropyl) trimethoxysilane (GPTMS, 98%, Aldrich) and tetramethoxysilane (TMOS, Aldrich) were used as the reagents of the hybrid solution.

Gelatin was dissolved in water at a concentration of 5 wt%. Separately, magnetic nanoparticles were dispersed in distilled water at various concentrations of 1, 2, and 3 wt / wt%. Then, the magnetic nanoparticle water dispersion was added to the gelatin solution, vortexed and sonicated to obtain a homogeneous dispersion of the magnetic nanoparticles in the gelatin solution. Then, GPTMS and TMOS were added to the gelatin at a ratio of 10 wt% relative to the gelatin, while stirring vigorously for 3 hours to form a hybrid sol. 20 μL of 1M HCl was added to the slurry for use as a catalyst in the sol-gel reaction. The mixture was left overnight to allow the gelation to proceed fully.

The sample was cooled at -20 占 폚 for 3 hours and lyophilized for 24 hours (Freeze-dryer, Ilshin Lab Co., LTD) to completely remove the water of the slurry to complete the porous scaffold.

Experimental Example  One: Scaffold  Physicochemical characterization

The scaffold was coated with gold and analyzed by SEM (SEM; JSM6330F, JEOL) to observe surface morphology as well as pore morphology. The dispersion of nanoparticles in the gelatin matrix was analyzed by transmission electron microscopy (TEM; JEOL-7100). The porosity of the sample was measured by the absorbed amount of ethanol. The chemical bond structure was investigated using Fourier transform interferometry (FT-IR; Perkin-Elmer). X-ray diffraction was performed to observe the crystallographic image (XRD, Philips MRD CuK _α , 40 kV, 20 mA).

A typical porous form of a hybrid scaffold containing various magnetic nanoparticle contents was observed via SEM (Fig. 1A). In all scaffolds, the interconnected microporous morphologies were visible, and the pore dispersion was uniform. Dispersion of the magnetic nanoparticles in the gelatin / siloxane matrix was confirmed by TEM (FIG. 1B). The TEM image of the scaffold (3%; the highest content of magnetic nanoparticles) showed a dispersion of nanoparticles about 10 nm in size in the matrix, and the dispersed nanoparticles were uniformly observed throughout. The enlarged image shows that the nanoparticles (black dots) are mostly separated and more clearly visible without aggregation. This indicates that the magnetic nanoparticles are well dispersed in the gelatin / siloxane solution and that the magnetic nanoparticles are evenly dispersed to at least the hybrid scaffold of up to 3% of the magnetic nanoparticles. In fact, the lyophilized porous scaffold showed a uniform distribution of relatively micropores up to 3% content of magnetic nanoparticles, but was severely hampered beyond that. Therefore, it was confirmed that the content of the magnetic nanoparticles is an optimum condition for the production of nanosynthesis scaffold of gelatin / siloxane-magnetic nanoparticles. The prepared magnetic nanoparticles have a weak electrostatic repulsion due to their relatively weak surface charge and thus the surface must be coated with polymer or silica for uniform dispersion, but in the gelatin / siloxane hybrid solution, the uncoated magnetic nanoparticles have a stable emulsion Lt; / RTI &gt; This is believed to be due to the surface interaction of gelatin and / or siloxane. In other words, the negative charge characteristic of this medium can be used to increase the electrical repulsion of the magnetic nanoparticles.

The image of the hybrid scaffold prepared as analyzed by XRD showed characteristic peaks of the added magnetic nanoparticles (FIG. 1C). The FT-IR spectrum showed a magnetic nanoparticle band (Fe-O at 560 to 600 cm ^-1 ) in the gelatin / siloxane hybrid band in the hybrid to which the magnetic nanoparticles were added. Since the amount of nanoparticles in the scaffold was increased, the intensity of the peak also increased. The three major peaks of the band associated with gelatin were clearly observed; The main peak is CN stretching mode for amide III at 1250 cm ^-1 , C═O stretching for amide I at 1690 cm ^-1 and NH strain for amide II at 1560 cm ^-1 . The hydrolysis and polycondensation of siloxane and associated band primarily due to the reaction also Si-O-Si Si-O -Si symmetric stretch in the asymmetric stretching vibration, 798 cm ^-1 at 1093 cm ^-1, Si at 962 cm ^-1 -OH stretching and OH stretching vibrations at 3400 cm &lt; ^-1 &gt;. As a result, it can be seen that the gelatin / siloxane scaffold incorporating the magnetic nanoparticles was prepared in a typical form of a siloxane-induced organic / inorganic hybrid.

Experimental Example  2: Scaffold  Magnetic characterization

The initial magnetic response of the hybrid scaffold was determined by measuring the minimum distance the scaffold was drawn when the magnet was positioned at a certain distance. The magnetic properties of the scaffold were studied under a magnetic field at room temperature using a superconducting quantum interference device (SQUID; Quantum Design MPMS-XL7). The magnetic properties of the samples were evaluated in terms of saturation magnetization and hysteresis curve regions. Three samples were used for each condition (n = 3).

As a result of checking the magnetic properties of the hybrid scaffolds produced, the scaffolds seemed to be sensitive to external static magnetic fields. This was explained by measuring the minimum distance the magnet can stick to the scaffold (Fig. 2a). As the magnetic nanoparticle content increased, the linearly increased distance increased from 0.2 cm for 0.5% magnetic nanoparticles to 1 cm for 4% magnetic nanoparticles. This indicates that the magnetic properties can be changed depending on the content of the magnetic nanoparticles. The magnetization of the scaffold was confirmed in more detail by performing SQUID magnetometry. Figures 2b and 2c show the magnetization for the applied magnetic field in each large range (-20 kOe to +20 kOe) and in the small range (-100 Oe to +100 kOe). While the pure gelatin / siloxane scaffold showed little magnetization, the gelatin / siloxane-magnetic nanoparticle scaffold showed a sharp increase in magnetization due to the rapidly saturated small magnetic field, and the behavior was extremely low It was reversible. The magnetization behavior is generally a superficial magnetism behavior that occurs in pure magnetic nanoparticles. The saturation magnetization of the magnetic scaffold increased from 0.24 emu / g for 1% magnetic nanoparticles to 0.64 emu / g for 3% magnetic nanoparticles as the amount of magnetic nanoparticles increased (FIG. 2d). The saturation magnetization value of the scaffold was very small (70 emu / g) compared to the value of the pure magnetic nanoparticles, but this magnetization level indicates that the scaffold of the present invention generates magnetically induced heat, To be effective for use as a therapeutic scaffold such as bone cancer treatment.

Experimental Example  3: Scaffold  Water absorption and decomposition analysis

Water absorption experiments were performed by submerging the scaffolds in sterile water and measuring weight changes for 1, 3, 6, 12 and 24 hours. The percent water uptake was calculated using the following equation.

? W _u (%) = ((W _s -W _o ) / W _o ) x 100,

W _ini and W _upt are sample weights before and after each water absorption experiment. Three samples were used under each condition (n = 3). Decomposition of the scaffold was performed in phosphate buffered saline (PBS) at 37 ° C for up to 28 days. At each time interval, the samples were lyophilized overnight and weight changes recorded. Percent decomposition was calculated using the following equation.

? W _d (%) = (W _{ini -} W _deg ) / W _ini ) x 100,

W _ini and W _deg are the values recorded before and after each decomposition. Three samples were used for each condition (n = 3).

In order for the magnetic hybrid scaffold to be used for tissue restoration purposes, water-related properties, including absorption, swelling and degradation of water, are particularly important. Prior to evaluating the water-related properties of the scaffold, the level of porosity was first assessed by the ethanol absorption method. The ethanol uptake (volume%) represents the degree of porosity of the scaffold. All scaffolds showed similar levels of approximately 84-85%, indicating that the level of porosity of all scaffolds is similar (Fig. 3a). The water absorption capacity of the scaffold was then observed (Figure 3b). In all scaffolds, water uptake quickly occurred within one hour and then slowly increased. Water uptake increased from about 350% for 0% magnetic nanoparticles to about 550% for 3% magnetic nanoparticles as the magnetic nanoparticle content increased. The degree of swelling of the scaffold in water also increased from about 11% for 0% magnetic nanoparticles to about 17% for 3% magnetic nanoparticles as the magnetic nanoparticle content increased. This indicates that the scaffold to which the magnetic nanoparticles are added has a larger volume in water. Therefore, the high water absorption capacity of scaffolds with magnetic nanoparticles added is due to the increased scaffold volume, although the porosity of the scaffold is in a similar range. That is, it can be seen that the magnetic nanoparticles dispersed in the gelatin / siloxane hybrid contribute to the expansion of the matrix. The immersed magnetic nanoparticles can easily absorb water molecules due to their high hydrophilicity and negative charge properties and cause the resultant increase in electrostatic repulsion between the magnetic nanoparticles-magnetic nanoparticles and / or magnetic nanoparticle-matrix molecules .

The decomposition behavior of the scaffold was confirmed by swelling and water absorption characteristics (FIG. 3C). The degradation of the scaffold in PBS increased with time and increased from 5 to 15% at 7 days to 35 to 55% at 28 days. In addition, addition of magnetic nanoparticles did not increase degradation, but scaffolds without added magnetic nanoparticles showed the highest degradation (0%> 2% and 3%> 0%). In general, increased swelling and water-gauge appear to lead to accelerated decomposition. However, the scaffold to which the magnetic nanoparticles of the present invention are added showed a decrease in decomposition. Thus, even though magnetic nanoparticles serve to absorb very large amounts of water molecules and increase size variation, the hybrid network will remain stable without collapse due to hydrolysis.

Experimental Example  4: Scaffold  Mechanical Characterization

The mechanical properties of the scaffold were first analyzed by measuring the static stress at the wetted scaffold. The compressive load was applied to a cylindrical sample (12 mm diameter x 6 mm thickness). The ability to resist deformation was recorded over time. Each measurement was taken on four samples and averaged over the obtained values (n = 4).

Dynamic mechanical analysis of the scaffold (DMA; MetraVib, DMA25N) was performed under parallel plate conditions. Mechanical spectroscopy was performed by dynamic frequency sweeping at a frequency range of 0.1 to 10 Hz at 37 DEG C with a strain amplitude of 5% in a linear range of viscoelasticity. We used auto-tension and auto-strain. The force was inclined in the range of 0.001 to 0.2 N, and the maximum allowable strain was set at 10%. The storage elastic modulus (E ') and the loss elastic modulus (E'') of the sample were measured. The tangent delta was calculated as the ratio of E '' / E '.

In the static compression test, the strain of the hybrid scaffold rapidly increased initially and remained almost stable. The increase in magnetic nanoparticle content reduced the strain of the scaffold and showed a high resistance to deformation under constant applied force (Figures 4a and b). The strain was reduced by 5-fold in the scaffold with 3% magnetic nanoparticles compared to the scaffold without magnetic nanoparticles.

Next, the dynamic mechanical behavior of the scaffold was observed under a constant strain at various frequencies, and the viscoelastic properties were expressed using the elastic modulus values including the storage elastic modulus (E ') and the loss elastic modulus (E''). As a result, the storage elastic modulus considered as the elastic part of the scaffold is significantly higher at 100 kPa (0% magnetic nanoparticles) to 450 kPa (3% magnetic nanoparticles) as the magnetic nanoparticle content increases (Fig. 4C). The loss modulus values considered as the viscous portion of the scaffold were also significantly greater with increasing magnetic nanoparticle content (150 kPa for 3% magnetic nanoparticles at 60 kPa for 0% magnetic nanoparticles) (Figure 4d ). The average values of the two elastic moduli are shown in FIG. 4E. In all compositions, the E 'value was higher than the E''value, and the E' increase was more noticeable in the magnetic nanoparticle added scaffolds. The tan delta determined from E '' / E 'and defined by the damping factor or loss parameter is shown in FIG. 4f, and as the magnetic nanoparticle content increases, the tangent delta . The dynamic mechanical analysis results show that the addition of magnetic nanoparticles significantly improves the storage elastic modulus of the hybrid scaffold, making it more elastic and reducing damping. The addition of magnetic nanoparticles in hybrid scaffolds greatly enhanced the mechanical properties such as elastic behavior in resistance to deformation by resisting dynamic loading conditions and compressive loading, as can be seen from static and dynamic mechanical data. This property will be effective for application to hard tissue repair of magnetic hybrid scaffolds.

Experimental Example  5: Scaffold  Biological characterization

In vitro  Ability to form apatite

The ability of the in vitro scaffold to form apatite was analyzed to assess the acellular bone-bioactivity of the hybrid scaffold. Samples were immersed in 1.5-fold stimulated bodily fluids (1.5X SBF), which is generally used to accelerate the apatite formation reaction, and observed at various time intervals of up to 14 days at 37 ° C. At each time interval, the sample was taken out, carefully washed several times with sterile water, and lyophilized. The change in weight of the scaffold during the immersion was recorded, and apatite formation was observed with SEM, XRD and FT-IR.

The bioactivity of the magnetic hybrid scaffold was first confirmed by analysis of apatite formation ability in SBF. This method has been widely used to evaluate the acellular bone-bioactivity of biomaterials developed for bone. In the present invention, 1.5X SBF was used to accelerate the apatite formation reaction and shorten the observation period. SEM images after immersing the scaffolds of various compositions in the SBF for 7 days are shown in FIG. 5A as representative images. In all scaffolds, significant mineralization appeared throughout the entire surface and covered the whole with a large apatite crystal layer. Crystal growth was observed in scaffolds with 1% magnetic nanoparticles added and scaffolds with 2% magnetic nanoparticles added to scaffolds without magnetic nanoparticles and scaffolds with 3% magnetic nanoparticles added, ), And so on. This is because the 3% magnetic nanoparticles dispersed at higher densities than the 1% and 2% magnetic nanoparticles provide more nucleation zones in the matrix and have a smaller crystal size, so that the magnetic nanoparticles dispersed in the hybrid matrix are not It can be deduced that it played an important role in nucleation. The weight of the scaffold increased relative to the immersion time due to crystal formation (Fig. 5B). The weight increased almost linearly with time, and the weight gain of the first day was about 60% after 14 days from about 10%. In addition, the weight gain was similar for all scaffolds except for scaffolds with 3% magnetic nanoparticles added at higher weights compared to others. In fact, the gelatin / siloxane hybrid composition showed excellent ability to form apatite. Also, the addition of magnetic nanoparticles affects the behavior of apatite formation. Minimal magnetic nanoparticle additions increase their ability to maintain apatite formation capacity and provide a new nucleation zone for apatite crystals when large amounts (3%) are added. The mineralized sample (3% magnetic nanoparticle as a representative sample) was further analyzed by XRD (Figure 5c) and showed a distinct peak at 2? ~ 32 ° representing the main peak of apatite. The FT-IR spectrum of the scaffold during SBF immersion showed the formation of a chemical band (phosphate band) associated with apatite. Based on the SBF experiments, the hybrid bands with magnetic nanoparticles have proven to have good apatite formation capabilities and thus have efficient bone bioactivity for use as bone regeneration scaffolds.

Cell proliferation assessment

Mesenchymal stem cells (rMSCs) derived from Brett's bone marrow were collected from the femur and tibia of Adret (180-200 g) following guidelines approved by the Dankook University Animal Ethics Committee. After centrifugation, the supernatant was collected and cultured on a normal culture medium of α-minimal essential medium (α-MEM) supplemented with 10% fetal calf serum (FBS), 100 U / ml penicillin, 100 mg / ml streptomycin 0.0 &gt; 37 C &lt; / RTI &gt; in a 5% CO ₂ humidified atmosphere. After incubation for 1 day, the medium was replaced and cultured until the cells were almost confluent. Cells in 2-3 passages after passage and maintenance in normal culture conditions were used for further testing.

For cell experiments, scaffolds were prepared in 12 mm diameter x 6 mm thickness, sterilized overnight in 70% ethanol, washed three times with PBS, and then placed in 24-well plates.

Cell proliferation was assessed at 7 and 14 days with a double-stained DNA assay kit. For this purpose, the cells were washed twice with PBS, and the cells were frozen and thawed twice for dissolution. Cell lysates were centrifuged at 1500 rpm for 10 min to remove cellular debris. Quantification of DNA was spectroscopically confirmed using a commercial dsDNA kit (Roche, Germany) in a microplate reader (Power WaveX, Bio-Tek Instruments, USA; 490 nm). A standard curve was made at a decreasing concentration of cells to indicate the number of cells. The experiment was repeated three times.

Cell morphology was also observed via SEM at each culture period (7, 14 and 21 days). Samples prepared for SEM were first washed with 0.1 M phosphate buffered saline at pH 7.4 and fixed with 2.5% glutaraldehyde (Sigma-Aldrich G400-4) in PBS. It was then washed again and kept in 0.1 M phosphate buffered saline. Various concentrations of ethanol solutions (50, 70, 90, 96 and 100% ethanol) were used to dehydrate the samples. Finally, hexamethyldisilazane (HDMS, Fluka 52620) was used for complete dehydration. Platinum was used for coating for electron microscopy.

In one embodiment of the invention, the data are presented as means ± standard deviation, and statistical analysis was performed using one-way analysis (ANOVA) followed by Fisher post-test. Statistical significance was considered at p <0.05.

After the rMSCs were cultured, they were used to evaluate the performance of the scaffold to stimulate stem cells to increase osteogenic differentiation, which is information about bone tissue engineering of the scaffold. Cell proliferation was measured after 7 and 14 days of culture through a dsDNA assay (Fig. 6A). Cell proliferation on the scaffold at day 7 was not significantly different, but there was a significant difference after 14 days. In particular, scaffolds with magnetic nanoparticles showed higher cell proliferation than scaffolds without magnetic nanoparticles (2% magnetic nanoparticles> 3% magnetic nanoparticles> 1% magnetic nanoparticles> 0% magnetic nanoparticles). The 2% magnetic nanoparticle scaffold was almost twice as much as the 0% magnetic nanoparticle scaffold. The SEM image showed the morphology of the cells directly grown on the scaffold (Figure 6b). In the hybrid scaffold, the cells were in intimate contact with the underlying substrate and showed active cellular structural expansion. Cells on magnetic nanoparticle scaffolds became longer as many highly expanded filophodia processes expanded. This was especially evident in the 2% magnetic nanoparticles and the 3% magnetic nanoparticle scaffolds, which showed considerable cell proliferation at day 14. Thus, the addition of magnetic nanoparticles in the hybrid scaffold stimulated cellular structural processes, a substantial phenomenon that cell proliferation and cell entry into the next stage of bone formation. Therefore, synthetic polymer scaffolds containing magnetic nanoparticles increased osteoblast proliferation.

Cell Osteogenesis  differentiation

The activity of alkaline phosphatase (ALP) in the cells was recognized as a marker of osteogenic differentiation in the early stage. Therefore, the effect of addition of magnetic nanoparticles to osteogenic differentiation of cells was evaluated. Cell cultures were maintained for 7 and 14 days in osteogenic medium, and the samples were washed twice with PBS and treated with 1 mL of 0.2% Triton® X-100. Each sample was transferred to a 1.5-mL tube, frozen at -20 ° C for 1 hour and dissolved at 4 ° C for 30 minutes. The following samples were centrifuged at 15,000 rpm for 20 minutes at 4 ° C. Finally, the solution was analyzed for ALP. Move the supernatant to a new tube 1.5 mL 1.5 M 2- amino-2-methyl-1-propanol (Sigma A58888), 1 mM MgCl 2 and 20 mM p- nitrophenyl phosphate (SigmaP4744) a 1: 1: 1 ratio Lt; / RTI &gt; After incubation at 37 [deg.] C for 3 hours, 100 [mu] l of 1N NaOH was added to complete the reaction. The optical density was measured at 405 nm in a microplate reader using p-nitrophenol as a standard. The experiment was repeated three times.

Experiments with a representative sample of a magnetic scaffold comparing a 3% magnetic nanoparticle scaffold to a non-magnetic nanoparticle scaffold showed that both scaffolds had elevated ALP levels during 7 to 14 days of culture ). More importantly, the 3% magnetic nanoparticle scaffold showed higher ALP levels at 7 days and 14 days compared to scaffolds without magnetic nanoparticles, suggesting that magnetic scaffolds are a better substrate for osteogenic differentiation of MSCs Condition.

Cell Mineralization

Mineralization of cells in the hybrid scaffold was confirmed using Alizarin Red S (ARS) staining (Sigma Aldrich). Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. After washing, the samples were incubated in a 2% w / v ARS solution (pH 4.1 - 4.3) in sterile water at room temperature for 10 minutes. The staining solution was removed and the scaffold washed thoroughly with sterile water. The dyed samples were visualized with a digital camera. For quantification of mineralization, the samples were incubated in 10% w / v cetylpyridinium chloride (CPC) in 2% w / v ARS solution (pH 4.1 - 4.3) for 1 hour and the reaction was removed The absorption wavelength was measured at 595 nm in a microplate reader. The experiment was repeated three times.

The mineralization process is considered to be the last step of bone formation after sufficient bone matrix protein secretion. Therefore, the calcium accumulated by the cells was quantified using ARS staining. The ARS staining of the sample and the solution in which the dye was dissolved showed a more pronounced red signal in the 3% magnetic nanoparticle scaffold than in the scaffold without magnetic nanoparticles (FIG. 8A). The quantified calcium content measured in the various incubation periods (7 to 21 days) was significantly increased over time in both scaffolds, and the 3% magnetic nanoparticle scaffold more calcium than the scaffold without magnetic nanoparticles in all periods (Fig. 8C). The formation of some mineralized nodules was observed, and the morphology was easily recognizable when the cells were fully differentiated and calcified.

Claims (10)

1) dispersing the magnetic nanoparticles in a solvent;
2) adding and dispersing the magnetic nanoparticle dispersion of step 1) in a gelatin solution;
3) adding a 3- (glycidoxypropyl) trimethoxysilane and tetramethoxysilane to the dispersion of step 2) to prepare a sol;
4) adding an acidic solution to the sol of step 3) to gel; And
5) lyophilizing the gel of step 4) to prepare a porous scaffold, comprising the steps of:
Wherein the step 1) is carried out at a concentration of 0.01 to 3% by weight, based on the weight of the total dispersion of step 1), of the magnetic nanoparticles.
The method of claim 1, wherein the magnetic nanoparticles comprise at least one metal selected from the group consisting of iron, scandium, titanium, chromium, manganese, cobalt, nickel, copper and zinc.
delete The process according to claim 1, wherein the solvent of step 1) is water, C _{1 -4} alcohol or a mixed solvent thereof.
The method according to claim 1, wherein the gelatin solution in step 2) is dissolved in water in an amount of 1 to 10% by weight of gelatin.
The process according to claim 1, wherein the Si content of the sol prepared in step 3) is 2 to 10% by weight based on the total weight of the sol.
The process according to claim 1, wherein the mixing ratio of the 3- (glycidoxypropyl) trimethoxysilane and the tetramethoxysilane is 1: 0.1 to 1:10 by weight.

The method according to claim 1, wherein the acidic solution is at least one selected from the group consisting of hydrochloric acid, phosphoric acid, and acetic acid.
A magnetic hybrid scaffold prepared by the method of any one of claims 1, 2, 4 to 8 and formed by uniformly dispersing micropores.
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