KR102494802B1 - Dentin-dental pulp regenerated strontium doped bioactive glass cement and its manufacturing method - Google Patents

Abstract

The present invention relates to dentin-dulphur regenerating strontium-doped bioactive glass cement and a manufacturing method thereof, and provides an oral drug delivery system containing the strontium-doped bioactive glass cement. The strontium-doped bioactive glass cement is surface-modified with an amine group.

Description

Dentin-dental pulp regenerated strontium doped bioactive glass cement and its manufacturing method}

The present invention relates to dentin-pulp regeneration strontium-doped bioactive glass cement and a manufacturing method thereof, and relates to an oral drug delivery system containing the strontium-doped bioactive glass cement and a manufacturing method thereof.

The delivery of drugs through the oral mucosa is very useful for drugs that are easily metabolized when administered orally, drugs with low bioavailability, and drugs that cause gastrointestinal disturbances. It is attracting attention as a new drug administration route because it is less sensitive to Therefore, not only simple oral disease treatment, but also various drugs capable of systemic delivery in small amounts can be applied to the oral mucosa.

Preparations applied to the oral mucosa are mainly liquids, troches, and ointments, but the dosage of these preparations is not constant, and the applied drug is easily lost by saliva, so that certain medicinal effects cannot be expected. In order to improve these disadvantages, an oral mucosal adhesive film has been developed (for example, RAPIDFILM, tesa Labtec GmbH). While these adhesive films can consistently show a single dosage and its efficacy, the film melts in a short time (about 3 minutes) and all the active ingredients are temporarily released locally in the oral cavity or in the digestive tract. there was

On the other hand, cardiovascular disease is a disease that occurs in the heart and major arteries and has a high mortality rate, and lowering blood lipid is particularly important for prevention and treatment. As these lipid-lowering drugs, drugs such as simvastatin are mainly used, but excessive use may cause headaches and nausea, and it is necessary to administer them at a constant concentration.

The present invention relates to dentin-dulphur regenerating strontium-doped bioactive glass cement and a method for preparing the same, and an object of the present invention is to provide a delivery system containing the bioactive glass cement that can be usefully used for drug delivery through the oral cavity.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention is an oral drug delivery system comprising strontium-doped bioactive glass nanoparticles,

The strontium-doped bioactive glass nanoparticles are surface-modified with an amine group to provide an oral drug delivery system.

In one embodiment of the present invention, the drug is characterized in that it is a drug for the treatment of cardiovascular diseases.

In another embodiment of the present invention, the drug for treating cardiovascular diseases is simvastatin.

In another embodiment of the present invention, the weight ratio of SiO ₂ :CaO:SrO of the strontium-doped bioactive glass nanoparticles is 80 to 90:5 to 15:1 to 10.

In addition, the present invention provides a method for preparing an oral drug delivery system comprising the following steps:

(a) adding an aqueous solution of strontium ions to the step of synthesizing bioactive glass nanoparticles;

(b) modifying the surface of the nanopowder obtained by heat treatment after precipitating the product of (a) with an amine group;

(c) mixing the resultant nanopowder of (b) with an aqueous phosphate solution at a ratio of greater than 0.4g/ml to 0.6g/ml and then drying; and

(d) immersing the nanopowder dried in step (c) in a solution containing a drug.

The present invention relates to a dentin-pulp regenerating strontium-doped bioactive glass cement, and provides a scaffold comprising the bioactive glass cement that can be used as an oral drug delivery system. The scaffold can effectively deliver drugs for treating cardiovascular diseases.

Figure 1 shows a schematic diagram of the formulation of Sr-doped nano-bioactive glass cement and a schematic diagram of dentin- pulp complex regeneration therapy of nano-bioactive glass cement.
2 shows high-magnification SEM images of Sr-free nano-bioactive glass cement (NBC) and Sr-doped nano-bioactive glass cement (Sr-NBC) before and after immersion in SBF.
3 shows XRD spectra of Sr-free nano bioactive glass cement (NBC) and Sr-doped nano bioactive glass cement (Sr-NBC) before and after immersion in SBF.
4 shows ATR-FTIR spectra of Sr-free nano bioactive glass cement (NBC) and Sr-doped nano bioactive glass cement (Sr-NBC) before and after immersion in SBF.
5 (a) shows the ion secretion profile of Sr-free nano bioactive glass cement (NBC) and (b) shows the ion secretion profile of Sr-doped nano bioactive glass cement (Sr-NBC).
Figure 6 shows the biocompatibility and tooth formation of NBC and Sr-NBC using rDPSCs (in vitro). (a) Cell biocompatibility of nano-bioactive glass cement using inserts (3 μm) (b) Morphology of rDPSCs revealed by actin filaments (red) and nuclei (blue), (c and d) nano-bioactive glass The results of tooth formation by ALP activity using cement extract and ARS staining are shown.
Figure 7 shows the pulp tissue compatibility test and extrinsic tooth formation through in vivo implantation of NBC and Sr-NBC (in vivo). (a) is a schematic diagram of the experiment, (b) μCT analysis 6 weeks after implantation, (c and d) quantification analysis of new hard tissue (dentin), (e) tissue through H&E staining to show regenerated dentin Scientific analysis (f) shows odontoblast related protein expression (DMP-1 and DSPP).
Figure 8 shows dentin-pulp regeneration test through in vivo implantation of NBC and Sr-NBC (in vivo). (a) is a schematic diagram of the experiment, (b) is a μCT analysis to reveal new dentin formation 6 weeks after implantation, (c) shows the quantification of new hard tissue (dentin) in terms of volume, surface area and density, (d ) represents H&E staining for the regenerated dentin-pulp tissue complex.
9 shows the result of confirming the in vivo cell viability of NBC and Sr-NBC in dental pulp stem cells (#p3).
10 shows the result of confirming the in vivo cytotoxicity of NBC and Sr-NBC in dental pulp stem cells (#p3).
Figure 11 confirms pulp tissue compatibility after in vivo transplantation of Sr-NBC, SEM images of the root area after removing the root (a), root area (b), and bottom area (c), tooth crown after removing the root (d) and an optical image of the dimension chamber (e) of nanoparticles on the bottom surface.
12 shows the results of confirming the degree of dentin-pulp composite regeneration of Sr-NBC.

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another.

An object of the present invention is to provide an oral drug delivery system comprising strontium-doped bioactive glass nanoparticles, wherein the strontium-doped bioactive glass nanoparticles are surface-modified with an amine group.

The delivery system is prepared by including a drug in a support made of strontium-doped bioactive glass nanoparticles, and can be usefully used for drug delivery in the oral cavity.

The drug may be a drug for treating cardiovascular disease, and the drug for treating cardiovascular disease may be simvastatin. The cardiovascular disease may be a disease such as hyperlipidemia, angina pectoris, coronary artery disease, peripheral vascular disease, or stroke, but is not limited thereto.

In addition, the present invention can provide a method for preparing an oral drug delivery system comprising the following steps:

(a) adding an aqueous solution of strontium ions to the step of synthesizing bioactive glass nanoparticles;

(b) modifying the surface of the nanopowder obtained by heat treatment after precipitating the product of (a) with an amine group;

(c) mixing the resultant nanopowder of (b) with an aqueous phosphate solution at a ratio of greater than 0.4g/ml to 0.6g/ml and then drying; and

(d) immersing the nanopowder dried in step (c) in a solution containing a drug.

The bioactive glass refers to a glass-ceramic biomaterial, and bioactive glass is widely used as implants and supports for tissue regeneration due to biocompatibility. Bioactive glass can be synthesized using a sol-gel method, which is well known in the art. The chemical process of the sol-gel method is to create various kinds of inorganic networks from silicon or metal alkoxide monomer precursors. Unlike the traditional method of making inorganic glass through a melting process at a high temperature, this process has the advantage of being able to make a homogeneous inorganic oxide material with good properties such as hardness, transparency, chemical stability, controlled porosity, and thermal conductivity at room temperature. there is.

In one embodiment of the present invention, a silica precursor and an aminosilane compound are added to a solution containing a calcium oxide precursor and a surfactant as a pore-forming template, stirred to obtain bioactive glass nanoparticles, and then calcined to obtain bioactive glass nanoparticles. particles were obtained.

The calcium oxide precursor may refer to a material capable of generating a calcium oxide component of bioactive glass. The calcium oxide precursor may be any one or more compounds selected from the group consisting of calcium nitrate tetrahydrate, calcium chloride, calcium acetate, and calcium methoxyethoxide, , but is not limited thereto. In the embodiment of the present invention, calcium nitrate was used.

The surfactant serving as the pore-forming template can form aligned pores in the bioactive glass nanoparticles. The surfactant may be hexadecyltrimethyl ammonium bromide (CTAB) or polyethylene glycol (PG), but is not limited thereto. In the embodiment of the present invention, polyethylene glycol was used.

The silica precursor may refer to a material capable of generating a silica component of bioactive glass. The silica precursor is TEOS (tetraethyl orthosilicate), TES (tetraethoxysilane), TMOS (trimethoxy orthosilicate), GPTMS ((3-glycidoxypropyl)methyldiethoxysilane), MPS (3-mercaptopropyl trimethoxysilane), GOTMS (γ-glycidyloxypropyl trimethoxysilane) and APTMOS (aminophenyl) trimethoxysilane) may be any one or more compounds selected from the group consisting of, but is not limited thereto.

The bioactive glass nanoparticles are nano-level particles made of bioactive glass, and have a wider surface area and more pores to improve biodegradability and protein adsorption. In general, bioactive glass nanoparticles have SiO ₂ and CaO as main components.

The biomimetic solution (SBF) is a solution containing Na+, K+, Ca2+, Mg2+, Cl-,HCO3-, HPO42-, SO42- ions, etc., imitating human body fluids. Formation of hydroxyapatite crystals in the biomimetic solution can be expected to show the same effect in vivo.

According to one embodiment of the present invention, the weight ratio of SiO ₂ :CaO:SrO of the strontium-doped bioactive glass nanoparticles is 80-90:5-15:1-10. The most stable three-dimensional nanoporous structure is formed in the above weight ratio range. In addition, when the Si weight ratio is 90 or more, biodegradability and bioactivity may be inhibited due to a small amount of Ca, or low dentin-dimension regeneration may be exhibited due to a small amount of Sr. In addition, when the weight ratio of Ca is 15 or more, a problem may occur in making the shape or size of nanoparticles uniform due to a large amount of Ca. In addition, when the weight ratio of Sr is 10 or more, it may be difficult to maintain the shape of the nanoparticles because the connection of the glass structure is reduced.

According to one embodiment of the present invention, the zeta potential of the strontium-doped bioactive glass nanoparticles is +17 to +23 mV. According to one embodiment of the present invention, the zeta potential is +20.2±0.71 mV.

According to one embodiment of the present invention, the nano-cement is prepared by mixing the strontium bioactive glass nanoparticles in an aqueous phosphate solution at a ratio of greater than 0.4 g/ml to 0.6 g/ml. When the strontium bioactive glass nanoparticles are mixed at a ratio of 0.4 g / ml or less or 0.6 g / ml or more with respect to the aqueous phosphate solution, deposited by a chemical reaction between calcium ions and phosphate ions of the nanoparticles There is a problem in that hydroxyapatite crystals are difficult to produce or excessively produced.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention. It is obvious to those skilled in the art.

[Example]

Example 1. Preparation and analysis method of experimental materials

1-1. Where to get experiment materials

Tetraethoxysilane (TES≥99%), strontium nitrate (SN≥99%), calcium nitrate tetrahydrate (Ca(NO3)24H2O (CNT), poly(ethylene glycol) (PG, Mn=10,000), ammonium hydroxide ( 28%), anhydrous methanol (99.8%), 3-aminopropyl triethoxysilane (APTES≥98%), anhydrous toluene (99.8%) tris-hydroxymethyl aminomethane (Tris-buffer), HCl (1N), HNO3 (70%) and phosphate tablets (PBS), high level precursors for preparation of biomimetic solution (SBF) were purchased from Sigma-Aldrich Simvastatin (SV, C25H38O5≥97%) was also obtained from Sigma-Aldrich .Distilled water (Millipore Direct-Q system) was used if necessary for the experiment.

1-2. Data analysis method

Statistical analysis was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The significance level is considered as P<0.05.

Example 2. Formulation of Sr-doped nano bioactive glass cement and its properties

2-1. Synthesis method of Sr-doped nano bioactive glass cement

Sr-doped or Sr-free nano bioactive glass cements were prepared based on 85SiO2-10CaO-5SrO and 85SiO2-15CaO glass compositions (wt%). PG was used as a template for the porous structure. For Sr free and Sr-doped nanoparticles, 5 g of PG and 0.189 g of CNT or 5 g of PG, 0.126 g of CNT and 0.031 g of SN were dissolved in alkaline methanol (150 mL, pH 12.5). 0.884 g of TES was diluted in 30 mL methanol and added to the precursor solution drop-wise under high-speed stirring and pulsed ultrasound (high power). The white or blue precipitate was collected and centrifuged at 5000 rpm for 5 minutes 3 times and washed using distilled water/ethanol treated with re-dispersion, and the precipitate was kept overnight at 70°C. PG was removed by heat treatment at 600° C. in air for 5 hours (burnt off). Finally, the nanopowder was modified with surface-amine groups through coupling with APTES. The nanopowder was dispersed at 2 mg/mL in APTES/Toluene solution (2% by vol.) and refluxed at 80°C for 24 hours. The nanopowder was centrifuged at 5000 rpm for 5 min, washed/redispersed 3 times with toluene and dried overnight at 80° C. before holding in vacuum for further use. Nano bioactive glass cement was prepared by mixing bioactive glass nanopowder and 1xPBS at a P/L ratio of 0.5 g/ml. The obtained nanocement paste was transferred to a Teflon mold, formulated into a disc shape, and left to harden.

As a result, nano biocement was prepared from Sr free and Sr doped calcium silicate mesoporous bioactive glass nanoparticles functionalized with APTES, and the nanobioactive glass cement paste formed was soft enough to be injected from an arbitrary syringe (Fig. 1) It was possible to mold into different shapes. These plastic properties are essential for clinical applications and for repairing irregularly shaped defects in bones and teeth. In addition, the cement paste was set within 5 to 10 minutes in the surrounding environment, and the fully cured nano-bioactive glass cement did not collapse and maintained its geometric shape when immersed in SBF.

2-2 Characteristics measurement method of nano-bioactive glass cement

Functionality of surface morphology, nanostructure, amorphous-to-crystalline transformation and chemical structure of SBF-soaked nanobioactive glass cements were analyzed by field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) and attenuated total reflectance-Fourier transform infrared. It was investigated using spectroscopy (ATR-FTIR). Before observation by FE-SEM (Tescan, MIRA II LMH), samples were sputtered with Pt using a sputter coater (Cressington 108 Auto sputter coater). XRD measurements were performed on a Rigaku-Ultima IV using CuKα radiation (λ = 1.5418 A) and a step size of 0.02o. ATR-FTIR spectra were collected in the wavelength range of 400-2,000 cm-1 with a resolution of 4 cm-1 using a Varian 640-IR with a diamond crystal accessory (GladiATR, PIKE Technologies). The surface charge of the APTES surface-functionalized nanoparticles used in the preparation of Sr-free nano-bioactive glass cement (NBC) and Sr-doped nano-bioactive glass cement (Sr-NBC) was measured by a zeta (ζ) potential analyzer (Zetasizer Nano ZS, Malvern Instruments). The ζ potential was investigated in distilled water at pH7.4 and 25 °C with a field strength of 20 Vcm-. Ions released from NBC and Sr-NBC disks in TRIS/HCl buffer were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICPAES, Optima 4300DV, Perkin-Elmer, Waltham) at pH7.4 and 37°C. 50 mg NBC and Sr-NBC discs were immersed in 10 mL Tris buffer. At pre-determined times, the secretion medium was pipetted and centrifuged (15,000 rpm, 10 minutes) and the supernatant was collected for determination of secreted ions by ICPAES. Samples were analyzed and averages with standard deviations were reported.

2-3. Experiment result

The surface nanomorphology of NBC and Sr-NBC was visualized by SEM imaging (Fig. 2) at day 0 and after day 28 of SBF immersion. High and low magnification SEM images of NBC and Sr-NBC showed nearly identical surface nanomorphology and nanostructured cement surfaces (islands of spherical nanoparticles), hydroxyapatite (HA) nanoneedle structures after immersion in SBF for 28 days. Deformation to the surface was shown. Bioactive glass nanoparticles in powder form are generally present in a highly agglomerated state. Thus, it facilitates cementation and self-curing of the nanoparticles as they are mixed with PBS and then cured together through hydroxyapatite deposition. The hydroxyapatite was formed through a rapid reaction between calcium ions dissolved from the bioactive glass nanoparticles and phosphate ions contained in the PBS solution.

The hydroxyapatite formed by binding to the aggregated bioactive glass nanoparticles was further crystallized into entangled needle-like nanohydroxyapatite after immersion in SBF (Fig. 2).

The phases of NBC and Sr-NBC were examined by XRD on day 0 after immersion in SBF, immediately after curing, to confirm the curing reaction. The XRD spectrum showed low-intensity peaks on the graph at 2θ = 29.5 and 2θ = 31.8o corresponding to hydroxyapatite (Fig. 3a, b). This peak indicates that the nanoglass phase rapidly released Ca2+ ions, which reacted with PO ₄ ^3- ions in PBS, leading to hydroxyapatite deposition. Moreover, the XRD peaks recorded for Sr-NBC appear to have relatively higher intensities than those observed for NBC. This suggests that in the case of Sr-NBC, the amount of precipitated hydroxyapatite is higher than that of NBC, so that Sr-NBC cures faster than NBC. This suggests that Sr has a structural effect on the silica glass structure and affects its effect on the subsequent glass melting process. Replacing Ca ions with Sr ions is known to cause expansion of the silica framework structure as Sr (1,12 A°) ions have a larger ionic radius than Ca (0.99 A°). Therefore, when Ca is replaced by Sr, the linkage of the glassy structure is reduced and ceased. Thus, bioactive glass nanoparticles are more degraded with strontium substitution and show more hydroxyapatite deposition and faster curing.

The bioactivity of NBC and Sr-NBC was investigated in vitro by immersion in SBF for 28 days. XRD, FT-IR and SEM investigations confirmed the conversion of NBC and Sr-NBC to the hydroxyapatite phase. Sr-NBC showed higher bioactivity than NBC through stronger and more intense hydroxyapatite peaks in the XRD spectrum of Sr-NBC compared to NBC (Fig. 3c). This result can be explained in terms of Sr substitution, in which Ca is replaced by Sr, which increases the decomposition and ion release, thus depositing more hydroxyapatite. Moreover, the position of the XRD peak of Sr-NBC is shifted to a lower 2θ value known as Sr-substituted hydroxyapatite (Fig. 3c shows a change in the representative peak, 211).

A typical hydroxyapatite spectrum was confirmed by further confirming the XRD result through the FT-IR spectrum. The FT-IR spectrum of Sr-NBC showed sharp and very intense peaks compared to NBC, thus confirming the excellent bioactivity of Sr-NBC compared to NBC (FIG. 4). The peaks at 450 cm-1, 796 cm-1 and 1057 cm-1 correspond to silicate groups derived from calcium silicate nano-bioactive glass. On the other hand, the bands at 560 cm ^-1 , 603 cm ^-1 and 1015 cm ^-1 are related to hydroxyapatite phosphate groups. The grown hydroxyapatite showed bands at 870 cm ^-1 , 1415 cm ^-1 and 1455 cm ^-1 belonging to the CO ₃ ^2- group. Thus, grown hydroxyapatite is in the carbonated form, which is important because carbonated hydroxyapatite mimics natural apatite in hard tissue.

The secretion of therapeutic ions, i.e., Sr, Ca and Si, was measured by ICP-AES after immersion in Tris-HCl buffer at pH 7.4 and 37 °C for 4 weeks to mimic physiological conditions and for biorelevance. NBC was prepared from APTES surface-functionalized mesoporous bioactive glass nanoparticles (Zeta potential +20.2 ± 0.71 mV) to release significant amounts of Ca and Si ions. It shows that Si ion release from NBC is rapid, linearly reaching 250 ppm on day 7, then gradually increasing over 3 weeks, reaching 350 ppm on day 28. The concentration of Ca ions increased rapidly during the initial time of immersion, reaching 100 ppm and then reaching 300 rpm through a steady linear increase at 28 days (Fig. 5a). Sr-NBC was prepared from APTES surface functionalized Sr-doped mesoporous bioactive glass nanoparticles (zeta potential +24.3±1.2 mV) to release significant amounts of Sr, Ca and Si ions. Sr and Ca similarly exhibited a rapid secretion profile within the first 7 days, then slowed down and leveled off (within 21 days). Ca ions initially reached 150 ppm on day 7 and completed to 220 ppm on day 28, while Sr ions reached 100 ppm on day 7 and then completed to 150 ppm on day 28. On the other hand, Si ions showed rapid secretion within the first 7 days, reached 200 ppm on day 7 and then followed an almost linear release pattern until day 28 when Si release reached 350 rpm (Fig. 5b). Sr-NBC secretes a smaller amount of Ca ions than NBC, and this is due to the fact that 5wt% CaO in the bioactive glass nanoparticle (BGn) composition (85% SiO2-15% CaO) used for the preparation of NBC was reduced to 5wt% SrO (85% SiO2-15% CaO) to produce strontium-doped bioactive glass nanoparticles (Sr-BGn).

The improved dissolution and rapid ion release of the present invention observed here is due to several factors, including 1) the mesoporous structure and nanoscale morphology of the bioactive glass prepared according to the present invention, 2) the APTES surface modification - The introduction of NH2 increases the hydrophilicity of the bioactive glass nanoparticles, resulting in improved ion release and improved decomposition compared to unmodified ones, 3) replacement of strontium to the silica glass substrate, which reduces the linkage of the glass structure and increases the decomposition rate. 4) Finally, these higher amounts of Si release observed here may be due to degradation of APTES surface residues in addition to degradation of the silica matrix itself. On the other hand, in the case of non-functionalized nanoparticles, Si, which forms the glass network, is released in a small amount because it arises only from the decomposition of the silica matrix.

Example 3. Biocompatibility and tooth formation ability of NBC and Sr-NBC

3-1. Dental pulp stem cell (DPSC) culture method

Dental pulp stem cells DPSCs were collected from incised teeth of 5-week-old male Sprague-Dawley rats. After rats were euthanized by CO2 gas, DPSCs were harvested and washed with PBS containing 1% penicillin/streptomycin. The isolated tissue was enzymatically digested for 30 minutes with a solution containing 0.08% collagenase type I and 0.2% dispase II. DPSCs were collected via centrifugation at 1500 rpm, 3 minutes and stored at 37°C in pulp stem cell medium (GM) containing α-MEM amended with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (FBS). Cultured in a humid environment with 5% CO2, odontoblast differentiation of DPSCs was investigated in odontogenic medium (OM) containing 100 nM dexamethasone, 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate.

3-2. NBC and Sr-NBC ion extraction and biocompatibility analysis method (in vitro)

Samples of nano-bioactive glass cement were tested by indirect cell culture method. Prior to experiments, nanoparticles were sterilized by EO gas. Cement discs (5 mm diameter x 2 mm height) were made in round-shaped PDMS molds and incubated overnight on a clean bench. Discs were placed on top of porous inserts (3 μm), and DPSC cells were cultured at the bottom of 24-well plates. Conditioned medium (CM) was prepared for one disc (10 mg nanoparticles), then soaked in 10 mL of dental medium overnight at 37° C., and then the medium was collected.

Cytotoxicity was assayed by the WST test and CCK kit, in which 100 μL of the reaction mixture was transferred to a 96-well plate and absorbance was measured at 450 nm by an iMark microplate reader (BioRad, USA). Cytotoxicity was based on percentage of optical density values. 104 DPSCs were cultured in pulp stem cell medium (GM) and cultured at 37° C. for 24 hours by conditioned medium (CM). Cell viability was observed on days 1 and 3 by CCK kit. For visual inspection of the cells, the cells were subjected to immunocytochemical staining. Cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C, treated with 0.2% Triton X-100, then treated with DAPI (p36965, Invitrogen) and Phalloidin. (Alexa Flour 546, Life Technologies, Invitrogen). Cells were captured by confocal laser microscopy (CLSM; Zeiss LSM 700).

3-3. Method for analyzing odontogenic differentiation of dental pulp stem cells (DPSCs)

The odontogenic differentiation of DPSCs was investigated through alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining. 2.5x104 cells/well were cultured in a 24-well plate, and different media conditions such as growth medium (GM), tooth formation medium (OM), and conditioned medium (CM) were used. On day 21, samples were collected with 0.25% TE (trypsin and EDTA, Gipco). For the ALP assay, 0.6 mL buffer from the ALP kit was added to each sample prior to centrifugation at 10,000 rpm for 15 minutes at 4°C and then the supernatant was collected.

ALP amount was confirmed by absorbance measurement at 405 nm. The amount of dsDNA was measured at 520 nm by a dsDNA detection kit (Quant-iT Picogreen, Invitrogen), and ALP activity was normalized to dsDNA. For ARS staining, cells were treated with 40 mM ARS assay solution (pH 4.2) at room temperature for 10 minutes under gentle rotation conditions. Samples were then washed several times and captured by a camera (D1000, Canon) and a light microscope (IX71, Olympus). Finally, ARS quantification was made by 10% CPC (cetylpyridinium chloride, Sigma) in sodium phosphate (pH 7.0 and 10 mM), after which absorbance was measured at 562 nm.

3-4. Experiment result

Indirect incubation with rDPSCs showed that NBC and Sr-NBC did not show biological cytotoxicity (Fig. 6a). The nano-bioactive free cement ion extract significantly improved the viability and odontogenic ability of rDPSCs. Cells were visualized after 1 and 3 days and showed nearly similar morphology and suitable cell attachment (FIG. 6B). The secretion of ions markedly stimulated the cellular response. Cellular mineralization was tested by ARS staining. It was confirmed that Sr-NBC showed significantly more calcium deposition and promoted more bio-mineralization than NBC (Fig. 6c). ALP, an early osteogenesis/odontogenesis marker, showed the highest ALP activity in the Sr-NBC group compared to the other groups, indicating odontogenic potential.

Example 4. Biocompatibility and tooth formation of nano-bioactive glass cement

Competence (in vivo) Healthy male Sprague-Dawley (SD) rats aged 10 weeks were used in the in vivo study. Prior to transplantation, recommended animal housing conditions were provided and animals were randomly selected into 3 experimental groups: NBC and Sr-NBC and empty (control) under general anesthesia (10 mg/kg xylazine + 80 mg/kg ketamine) in each tooth. was transplanted to In this study, two different in vivo models were examined. The first is an ectopic tooth development model as a primary model to investigate tooth formation and in vivo pulp-tissue compatibility under low-odontogenic conditions. The second is a dental defect model that mimics clinically relevant real-world application conditions. This model was utilized to test the tooth development effects of NBC and Sr-NBC in a clinical-like situation.

4-1. Manufacturing method of ectopic tooth development model

In the case of ectopic odontogenesis, implantation of nano-bioactive glass cement under ectopic conditions (autologous tooth implantation on the dorsal side of the subcutaneous tissue) was performed after generating defects on the extracted first tooth, resulting in in vivo pulp tissue in severe and low odontogenic conditions. Compatibility and tooth formation were observed. Defect holes (1.35 mm in diameter) were made on the occlusal side to the right and left of the top of the extracted molar teeth to expose the dental pulp cavity. Then, 1 mg of NBC or Sr-NBC was administered into the defect cavity of each group containing 5 teeth, and the samples were implanted subcutaneously in rats. Prior to implantation, the dorsal skin was shaved and the surgical site treated with iodine and sterile alcohol, and a small subcutaneous pocket was opened through a linear skin incision.

After implantation, the skin pocket was sutured. Animals were reviewed regularly to record any signs of inflammation or infection. Implanted teeth with surrounding tissue were collected from animals euthanized under CO2 inhalation (6 weeks after surgery). Samples were fixed for 24 hours in 10% NBF (neutral buffered formalin).

4-2. Fabrication of a tooth defect model

After creating a tooth defect and applying materials according to a previously reported method, a clinically relevant in vivo tooth model was adjusted from natural teeth (Bencherif, S. A. et al., J Periodontal Implant Sci, 2013). A defect hole was created as described above until the pulp cavity was exposed. 1 mg of NBC or Sr-NBC was applied to the defect cavity in each group comprising 5 teeth. The rest of the procedure is as described above.

4-3. μ-CT analysis method

NBF fixed samples were scanned by μ-CT and analyzed using Sky Scan software (Aartselaar) using X-rays at 385 μA and 65 kV in 9 μm sections under conditions of 279 ms exposure time. Second, the reconstructed images were scanned by CTAn Skyscan for hard tissue generation over the region of interest. The surface area (mm2), new hard tissue volume (%) and density (1/mm) in the defect area were determined. Finally, the 3D structures were produced and captured by CTvol Skyscan.

4-4. Tooth tissue structure by immunohistofluorescence and immunohistochemical staining

RapidCalTM solution was used to descale samples that had been fixed for 5-7 days. Samples were then dehydrated by different grades of ethanol and embedded in paraffin. Then, a 5 μm thick coronal section of the sample was cut by a semi-automatic rotating microtome and transferred to a glass slide. Specimens were visualized by light microscopy after staining by hematoxylin and eosin (H&E) and immunohistochemical staining for analysis of new bone formation. During immunofluorescence staining, tissue sections were subjected to antigen retrieval by citrate buffer for 20 min. Next, the cells were incubated overnight at 4°C with primary antibodies (DMP-1/M-20/and DSPP/M-300/, Santa Cruz Biotechnology, INC). In addition, they were treated with secondary antibodies for 1 hour at room temperature and finally captured by confocal laser microscopy.

4-5. Experiment result

Dentin-pulp composite regeneration was tested through ectopic dentin formation in which extracted teeth were implanted in subcutaneous tissue after application of nano-bioactive glass cement, in vivo pulp-tissue compatibility and harsh and less odontogenic conditions before clinical application. Tooth development was observed.

Figure 7a is a schematic diagram of a surgical procedure performed for implantation of nano-bioactive glass cement in molars for 6 weeks. 3D reconstructed images and μCT of newly formed dentin and residual nano-bioactive glass cement at the defect site were utilized to investigate the in vivo odontogenic potential of nano-bioactive glass cement. The μCT images before and after implantation are shown in Fig. 7b. New dentin formation is shown in red. Analysis of the μCT images showed a higher dentin regeneration potential in the Sr-NBC group compared to the Sham and NBC groups (Fig. 7c and d). Moreover, histological analysis by H&E staining (FIG. 7e) confirmed the formation of tertiary dentin in association with the Sr-NBC group compared to the NBC and Sham groups.

The Sr-NBC group also showed higher expression of dentin matrix protein-1 (DMP-1) and dentin-sialophosphotane (DSPP) than the NBC group (FIG. 7f). Taken together, Sr-NBC enhances pulp-tissue biocompatibility and tooth development even under heterotopic conditions, making Sr-NBC applicable to clinically relevant dental defect models.

Next, the regeneration potential of the nano-bioactive glass cement was evaluated by a clinically relevant in vivo model over 6 weeks (Fig. 8a). μCT images before and after implantation are shown in FIG. 8B. New dentin formation is shown in green. Analysis of the μCT images revealed higher dentin volume and higher dentin density in the Sr-NBC group compared to the Sham and NBC groups. Nearly similar dentin surface area was observed between NBC and Sr-NBC, but significantly larger than that of the sham group. Histological analysis by H&E staining (Fig. 8d) showed that Sr-NBCs showed better pulp-dentin tissue regeneration in vivo compared to NBCs due to Sr2+ and Ca2+ secretion. The in vivo bioactivity response of Sr-NBC was higher than that of NBC and sham groups due to the stimulatory role of Sr2+ ions on dental stem cells and their slightly larger radius than Ca2+ ions to expand the glass network and subsequent rapid degradation. Thus, high-saturation of body fluids by Sr2+ and Ca2+ occurs rapidly in Sr-NBC and precipitates hydroxyapatite faster than NBC/

Example 5. Drug loading capacity of nano bioactive glass cement

Various concentrations of simvastatin sodium salt (SVS) were prepared according to the following procedure. For preparation of 1 ml of SVS solution, a corresponding amount of SV was dissolved in 100 μL of ethanol. 150 μL of NaOH 0.1N was added to the SV ethanol solution and then incubated at 50° C. for 2 hours. The pH was adjusted to 7.2 with 0.1N HCl. Deionized water was added to the solution to make 1 mL and stored at 4°C for the next experiment. A linear calibration curve (A239=0.0144+0.0453C (R2=0.996)) was obtained by measuring the absorbance at λmax=239 nm of a series of SVS solutions (0–50 μg/mL) using a UV-visible spectrophotometer (Libra S22, Biochrom). and obtained. To determine the SVS loading capacity, NBC and Sr-NBC discs (10 mg) were immersed in 1 ml of SVS 0.25, 0.5, 1, 2 and 4 mg/ml for 2 hours at 37°C.

After carefully collecting the drug solutions and centrifuging at 15,000 rpm for 5 min, their absorbance was determined at λmax=239 nm. The SVS total loading capacity was estimated from the difference between the initial and final solution concentrations of SVS.

As a result, NBC and Sr-NBC showed similar simvastatin loading capacities. When a 4 mg/mL loading solution of SVS was used, 10 mg Sr-NBC loaded about 1893 μg of SVS (i.e., 189.3 μg SVS/mg cement), whereas 10 mg NBC loaded about 1625 μg of SVS (i.e., 162.5 μSVS/mg cement). was able to load

Example 6. Confirmation of cytotoxicity (in vitro)

In this example, the effects of NBC and 5% Sr-NBC on cell viability and biocompatibility of rDPSC (# p3) were confirmed in vitro.

As shown in Figure 9, after incubating different concentrations of BGn and SrBGn (0, 5, 10, 20, 40, 80, 160, 320 mg/ml) with rDPSCs, up to 80 mg/ml nanoparticle treatment group It showed no cytotoxicity up to 3 days. For further experiments, 80 mg/ml NBC and Sr-NBC were used.

On day 3, NBC treated with rDPSC and Sr-NBC (80 mg/ml) nanoparticles were observed by LD staining. As a result, the nanoparticles showed no toxic effects and normal cell morphology. This result confirms the previous cytotoxicity results once again.

Example 7. Confirmation of tissue compatibility

In order to confirm the compatibility of the pulp tissue in the first molar defect of the rat, the tooth root was removed and the results are shown in FIG. 11 . After nanoparticle administration, tissue samples were transplanted into the dorsal hypodermis of mice. After removing the tooth roots, the teeth were checked with optical images (b-d) and SEM images (a.). For confirmation, the pulp tissue is removed and the pulp chamber can be seen at the bottom (c), the tooth crown can be checked after the root is removed (d), and the nanoparticles are covered with the dental pulp chamber at the bottom in e. can

In addition, the tissue compatibility of the dentin-pulp composite was confirmed and shown in FIG. 12 . Dentin-pulp composite regeneration was tested through ectopic dentin formation in which extracted teeth were implanted in subcutaneous tissue after material application to investigate in vivo pulp-tissue compatibility and dental formation under harsh and intense conditions prior to clinical application.

As shown in Fig. 12, 6 weeks after surgery, histological analysis by H&E staining confirmed more tertiary dentin formation associated with the SrBGn group compared to the other groups (Sham and BGn).

Although the present invention has been shown and described in relation to specific preferred embodiments in the above, the present invention can be variously modified and changed without departing from the technical features or fields of the present invention provided by the claims below. It is clear to those skilled in the art that it can be.

Claims (8)

An oral drug delivery system comprising strontium-doped bioactive glass nanoparticles,
The strontium-doped bioactive glass nanoparticles are surface-modified with amine groups through coupling with 3-aminopropyl triethoxysilane (APTES),
The weight ratio of SiO ₂ :CaO:SrO in the strontium-doped bioactive glass nanoparticles is 80 to 90:5 to 15:1 to 10,
has the shape of a disc,
An oral drug delivery system for the delivery of Simvastatin, a drug for the treatment of cardiovascular diseases.
According to claim 1,
The weight ratio of SiO ₂ :CaO:SrO in the strontium-doped bioactive glass nanoparticles is 85:10:5, oral drug delivery system.
According to claim 2,
The oral drug delivery system, wherein the simvastatin is loaded at a rate of 1893 μg with respect to 10 mg of the drug delivery system.
According to claim 3,
An oral drug delivery system having a concentration of up to 80 mg/ml.
A method for preparing an oral drug delivery system comprising strontium-doped bioactive glass nanoparticles,
(a) adding an aqueous solution of strontium ions to the step of synthesizing bioactive glass nanoparticles;
(b) modifying the surface of the nanopowder obtained by precipitating and heat-treating the product of (a) with an amine group through coupling with 3-aminopropyl triethoxysilane (APTES);
(c) mixing the resultant nanopowder of (b) with an aqueous phosphate solution at a ratio of greater than 0.4g/ml to 0.6g/ml, drying it, and formulating it into a disc shape; and
(d) immersing the disc formulated in step (c) in a solution containing Simvastatin, a drug for treating cardiovascular diseases;
The weight ratio of SiO ₂ :CaO:SrO in the strontium-doped bioactive glass nanoparticles is 80 to 90:5 to 15:1 to 10,
Method for preparing an oral drug delivery system for delivering simvastatin, a drug for treating cardiovascular disease.
According to claim 5,
The method of preparing an oral drug delivery system, wherein simvastatin is loaded at a rate of 1893 μg with respect to 10 mg of the drug delivery system.
According to claim 5,
The method of manufacturing an oral drug delivery system, wherein the oral drug delivery system has a concentration of up to 80 mg/ml.
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