KR101717233B1 - Calcium phosphate cement composition comprising zinc-bioglass nanoparticles and a kit for preparing the same - Google Patents

Abstract

The present invention relates to a calcium phosphate cement composition for regenerating dimensional tissue comprising a bioglass nanoparticle containing zinc and a kit for the preparation thereof.
The calcium phosphate cement composition containing the zinc-bioactive glass nanoparticles of the present invention promotes tooth formation differentiation of dendritic cells and promotes angiogenesis, and thus can be used as a composition for regenerating dental tissues.

Description

[0001] The present invention relates to a calcium phosphate cement composition for regenerating dimensional tissues comprising zinc-bioactive glass nanoparticles and a kit for preparing the calcium phosphate cement composition and a kit for preparing the same.

The present invention relates to a calcium phosphate cement composition for regenerating dimensional tissue comprising a zinc-containing bioglass nanoparticle and a kit for the production thereof.

Regenerative endodontics is a new field of study in modern dentistry that exploits the regenerative capacity of human dimensional cells (hDPC). hDPC has generally been studied in relation to scaffolds and reactive molecules, and is known to have self-renewal and differentiation capabilities in vitro. Scaffolds provide a suitable environment for hDPC to attach, proliferate, differentiate, and biomineralize. Various materials including polymers and minerals have been used as 3D substrates in scaffolds to serve as substrates for dimensional regeneration. Of the scaffold forms that can fill and recover defective dimensional areas, the injectable scaffolds can minimize invasive surgery.

Calcium phosphate cement (CPC) is known to be a good material for the recovery and regeneration of hard tissues such as bone and dentin. The excellent biocompatibility of calcium phosphate cement to the tissues surrounding bone and bone is due primarily to chemical composition similar to hydroxyapatite (HA), a natural mineral component. Various in vitro and in vivo experiments have already demonstrated positive effects of CPC including bone / tooth development and size / bone formation. Recent studies related to CPC have therefore focused on improving the mechanical and biological properties of CPC by mixing CPC with polymeric phases, nanoparticles, or additional components including biomolecules.

One object of the present invention is to provide a calcium phosphate cement composition comprising a powder phase and a liquid phase, which comprises a bioglass nanoparticle containing zinc.

Another object of the present invention is to provide a kit for manufacturing a calcium phosphate cement composition for regenerating dimensional tissue, the calcium phosphate cement composition comprising a calcium phosphate compound-containing powder, a curing accelerator-containing liquid and a zinc-containing living glass nanoparticle packaged in individual containers .

In order to solve the above problems, the present invention provides a calcium phosphate cement composition for regenerating dimensional tissue, which comprises biotite nanoparticles containing zinc, in a calcium phosphate cement composition comprising a powder phase and a liquid phase .

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

In the present invention, life-like glass (BG), particularly biocatalyst nanoparticle (BGN) which is a nanoparticle type of BG, was added to CPC to prepare a new bio-nanocomposite. BG is an excellent osteogenic material capable of inducing the formation of hydroxyapatite (HA) on the surface when contacted with living tissue. These HA layers are similar to natural bone matrix and provide an effective interface for cell fixation, bone differentiation and extracellular matrix degradation, and can lead to direct binding to natural bone.

Life-like glass nanoparticles (BGN) are nanofillers that can produce nanocomposites and can transport biomolecules. In addition, it can exhibit excellent biological properties such as promoting bone differentiation of stem cells, loading and delivery of drugs, and dentin remineralization of teeth. Furthermore, BGN can bind specific ions within its structure during sol-gel manufacturing. In the present invention, zinc ion, which is known as an essential trace element in bone, is used in combination with a bioactive glass structure. In the present invention, zinc may be used in conjunction with calcium phosphate compounds such as HA and tricalcium phosphate to play an important role in promoting bone cell function.

Particularly, in the present invention, the effect of the CPC / BGN substrate in the tooth formation and angiogenesis of the hDPC and the signal transmission pathway associated therewith were confirmed by mixing the nanoparticles of zinc-BGN in the CPC as the regenerating material, , It has been found that the composition can be used for regenerating dimensional tissue.

In other words, the calcium phosphate cement composition according to the present invention is characterized by promoting tooth formation differentiation and angiogenesis. In addition, the calcium phosphate cement composition of the present invention contains zinc-containing bioactive glass nanoparticles, thereby further enhancing the physiological activity of tooth formation and differentiation, exhibiting no biotoxicity, and exhibiting excellent biocompatibility, It is possible to regenerate the dimensional tissue and thus be usefully used as a composition for regenerating dimensional tissues.

The term "cement" as used in the present invention means a cured product of a paste which can be used as an alternative to the dimensional structure obtained by mixing powdery solid and liquid phases. Curing of the cement means spontaneous curing of the paste performed at room or body temperature without artificial treatment. The paste is obtained as a result of mixing the solid phase and the liquid phase.

The term "calcium phosphate cement " used in the present invention means a cement in which the powdery solid phase is composed of a calcium phosphate compound or a mixture of calcium and / or phosphate compounds.

The calcium phosphate cement is a material composed of an aqueous solution containing calcium phosphate particles and a substance which accelerates curing such as phosphate and phosphate as main components. When the two components are mixed at the time of the procedure and applied in a liquid state of high viscosity, Is a form of dimension substitute that regenerates damaged tissue by depositing and curing the calcium phosphate compound by the chemical reaction of the calcium phosphate compound.

The term " bioactive glass ", as used herein, was discovered by Hench and was named Bioglass ^® , which has been used clinically as a regenerated bone in powder form with Perioglas ^® and Novabone ^® products since mid 1980. Living vitreous is bound to bone because it forms a HCA (hydroxycarbonated apatite) layer on the surface when contacted with body fluids. HCA is similar in composition to bone mineral and forms strong bonds with bone. Vitality glass dissolves safely in the human body, releases critical concentrations of silicon and calcium ions that serve to stimulate bone cells at the genetic level, and causes new bone regeneration even when very few active cells are present.

While biocompatible glass is suitable for use as a recycling material, Bioglass ^® composites are not suitable for the production of porous supports. This is because it requires the use of a sintering process, which requires heating the glass to a temperature above the glass transition temperature in order to produce a localized flow. Bioglass ^® compounds crystallize immediately above the glass transition temperature, and once crystallized Bioglass ^® loses its viability. There are two kinds of vitrified glass, one derived from a solution and the other derived from a sol-gel. In the present invention, a vitrifying glass derived from a silica-based sol-gel can be used.

In general, the vitrified glass is mainly composed of SiO ₂ and CaO, and may further include Na ₂ O, P ₂ O _5, or a combination thereof. The present invention is characterized in that zinc (Zn) is contained in such vitality glass. In the present invention, the zinc-containing bio-active glass may be such that a part of calcium is replaced with zinc.

In the present invention, the content of the zinc-containing bioactive glass nanoparticles may be preferably 1 to 20% by weight of the total weight of the powder, and the zinc-containing bioactive glass nanoparticles preferably have an average particle diameter of 10 to 100 nm Lt; / RTI > The content of zinc may be preferably 0.1 to 10% by weight based on the total weight of the bioactive glass nanoparticles, and the zinc may preferably be contained in place of calcium in the bioactive glass nanoparticles.

The calcium phosphate cement of the present invention contains zinc-containing bioactive glass nanoparticles to induce formation of carbonate HA on the surface of the dental tissue, thereby promoting cell fixation and tooth formation differentiation, and increasing direct binding to natural dental tissues, Zinc can be contained to promote the function of the dimensional cell, and thus it has an excellent effect as a composition for tissue regeneration.

The zinc-containing bioactive glass nanoparticles of the present invention are preferably prepared by 1) dissolving PEG or CTAB to prepare a template solution and adjusting the pH to 9 to 13; 2) preparing a calcium oxide-zinc oxide-template mixed solution by adding a calcium oxide precursor and a zinc oxide precursor to the template solution; And 3) adding a silica precursor solution to the calcium oxide-zinc oxide-template mixed solution while ultrasonication and stirring to prepare a reaction product; 4) preparing a mesoporous bioactive glass nanoparticle by centrifuging the reaction product, followed by washing, drying and calcination.

The zinc-containing mesoporous vitreous living glass nanoparticles preferably have a molar ratio of silica (SiO ₂ ): calcium oxide (CaO) and zinc oxide (ZnO) of 60:40 to 95: 5, more preferably 85:15 Lt; / RTI >

In step 1, a self-assembled polymer template solution is prepared to prepare nanoparticles having mesopores, and the pH is adjusted to 9 to 13 to form a basic atmosphere. The self-assembled polymer template of the present invention is preferably PEG or CTAB, and the template solution can be prepared by dissolving the polymer template in an anhydrous methanol solution. The pH is preferably adjusted with NH ₄ OH, NaOH, KOH or Tris, but is not limited thereto.

Step 2 is a step of adding a calcium oxide precursor and a zinc oxide precursor to the template solution and stirring to form calcium oxide-zinc oxide-template mixed solution so as to form calcium oxide and zinc oxide in the template material. The calcium oxide precursor is preferably calcium nitrate, calcium chloride, calcium acetate or calcium methoxyethoxide, but is not limited thereto. As the zinc oxide precursor, zinc hydroxide, zinc acetate, zinc acetate hydrate, diethyl zinc, zinc nitrate, zinc nitrate hydrate, zinc carbonate, zinc acetylacetonate and zinc acetylacetonate hydrate may be used. It is not.

In step 3, a silica precursor solution is added to the calcium oxide-zinc oxide-template mixed solution to form a desired inorganic material in the template material, and the mixture is ultrasonicated and stirred to prepare a reaction product. The silica precursor solution is preferably prepared by dissolving a silica precursor in an alcohol solution of anhydrous methanol, ethanol, propanol or isopropanol, and the silica precursor is selected from the group consisting of tetraethyl orthosilicate (TEOS), trimethoxy orthosilicate (TMOS), (3-glycidoxypropyl ) methyl diethoxysilane, 3-mercaptopropyl trimethoxysilane (MPS), γ-glycidyloxypropyl trimethoxysilane (GOTMS), or aminophenyl trimethoxysilane (APTMOS). Also, it is preferable that the ultrasonic treatment is performed for 15 to 25 minutes at an output power of 200 W to 240 W, 10 s on / 10 s off cycle.

Step 4 is a step of centrifuging the reaction product at 4500 rpm to 5500 rpm, washing, drying and calcining the reaction product to remove the template from the reaction product to obtain mesoporous vitreous silica nanoparticles. And then redispersing it several times with deionized water and several times with anhydrous ethanol after the washing. The drying may be performed at a temperature ranging from 65 ° C to 75 ° C for 10 hours to 14 hours, but is not limited thereto. The firing is preferably carried out at 550 to 650 ° C for 5 to 7 hours under the atmosphere.

In one embodiment of the present invention, the bioactive glass nanoparticles were mixed with? -TCP in an amount of 10% by weight based on? -Triccium phosphate (? -TCP), and a part of calcium was replaced with zinc % To 5.0% by weight of calcium phosphate cement. As a result, the coagulation time of the cement decreased with the increase of the zinc content, and it was confirmed that the calcium phosphate cement containing zinc-containing bioactive glass nanoparticles was morphologically very similar to the biomimetic HA (Fig. 1).

The powder phase of the calcium phosphate cement according to the present invention may be prepared by mixing at least one selected from the group consisting of amorphous calcium phosphate, dicalcium phosphate anhydrous (DCPA), tetracalcium phosphate (TTCP) But is not limited to, one or more calcium phosphate compounds selected from the group consisting of calcium phosphate, alpha-TCP, and beta -triccium phosphate. In one embodiment of the present invention, α-TCP was used.

The liquid phase of the calcium phosphate cement according to the present invention preferably contains a curing accelerator which accelerates the precipitation reaction of ionized calcium and phosphate ions to promote the formation of HA. The curing accelerator may be Na ₂ HPO ₄ , NaH ₂ PO ₄ , K ₂ HPO ₄ , NH ₄ H ₂ PO ₄ , Na ₂ SO ₄ , citric acid, maleic acid and propionic acid, but the present invention is not limited thereto.

In the present invention, the liquid phase of the cement composition may be contained in an amount of 30 to 45 parts by weight based on 100 parts by weight of the powder.

In one embodiment of the present invention, the effect of the calcium phosphate cement composition containing the zinc-containing bioglass nanoparticle produced under the above conditions on the tissue regeneration was confirmed. As a result, the cement showed no cytotoxicity, and the mineralized nodule formation of human dental cells was improved and the expression of the dental formation differentiation marker gene was increased (Fig. 2). In addition, the human dendritic cells cultured in the cement showed an increased expression of VEGF, and the cultured medium enhanced the angiogenesis of HUVEC (FIG. 3). Therefore, it was confirmed that the calcium phosphate cement of the present invention can exert an excellent effect as a composition for regenerating dimensional tissue.

The present invention also provides a kit for manufacturing a calcium phosphate cement composition for regenerating dimensional tissue, the calcium phosphate cement composition comprising a calcium phosphate compound-containing powder, a curing accelerator-containing liquid and zinc-containing bioactive glass nanoparticles packaged in individual containers.

In the present invention, the kit may be provided with instructions for using the bioactive glass nanoparticles in an amount of 0.1 to 10% by weight based on the total weight of the powder, or the bioactive glass nanoparticles may be used based on the total weight of the powder And may be quantitatively packed in an individual container in an amount of 0.1 to 10% by weight.

In the kit for manufacturing a calcium phosphate cement composition of the present invention, the components, mixing ratios, etc. of the powdery, liquid and vitrified glass nanoparticles are the same as those described in the calcium phosphate cement composition.

The kit may be prepared by dispersing bioactive glass nanoparticles in a liquid phase immediately before use and then mixing powder phases to prepare a calcium phosphate cement composition in the form of a paste and injecting the paste into a desired treatment site.

A syringe may be used in the above procedure.

In addition, the kit of the present invention may further comprise a biological protein, a drug, or a combination thereof, wherein the biological protein is bovine serum albumin, lysozyme, growth factor and the like, and examples of the drug include antibiotics and inflammatory agents.

The calcium phosphate cement composition of the present invention is capable of curing within a short period of time, promoting tooth formation differentiation and angiogenesis, and inducing rapid tissue regeneration after the procedure.

The kit of the present invention can be applied to a nanocomposite injection system for repairing and regenerating a dental tissue in tissue engineering, and can be used for dental treatment in relation to treatment of a dental tissue defect.

The calcium phosphate cement composition containing the zinc-bioactive glass nanoparticles of the present invention promotes tooth formation differentiation of dendritic cells and promotes angiogenesis, and thus can be used as a composition for regenerating dental tissues.

1 shows TEM images of Zn-BGN used in (a) CPC cementitious composites and EDS (intercalation) of Zn-BGN cement and (b) coagulation time measured in CPC / BGN cement composites with various Zn contents in BGN , and (c) SEM photographs of low- and high-strength CPC / BGN cement after solidification and SBF test.
Figure 2 shows the results of the analysis of the effect of zinc addition BG scaffolds on tooth formation differentiation in human dental cells. Cells were cultured in osteogenic supplemented medium (OS) containing 50 mg / mL ascorbic acid and 10 mM [beta] -glycerophosphate for 7 and 14 days (AD). OS and OM containing 10 ^-7 M dexamethasone were used as positive control. Cytotoxicity and ALP activity were measured by MTS (a, n = 4) and ALP activity assay (b, n = 4). The mRNA of the tooth-forming differentiation gene was examined by RT-PCR (c). Mineralized nodule formation was investigated via alizarin red staining (d). * Statistical significance is p <0.05.
Figure 3 shows the results of the analysis of the effect of BG supplemented with zinc on angiogenesis. Dimensional cells were cultured in osteogenic medium (OS) containing 50 mg / ml ascorbic acid and 10 mM? -Glycerophosphate. OM was used as a positive control for tooth formation differentiation. The mRNA expression of angiogenic cytokines was evaluated by culturing the dendritic cells for 3 days (a). Dimensional cells were cultured in serum - free medium with ZnBG for 24 hours to obtain CM. The angiogenic activity of CM in HUVEC cells was examined by tube formation analysis. HUVEC cells were cultured in CM for 18 hours (b) and counted by counting tubes (c). * Statistical significance is p <0.05.
Figure 4 shows the results of the analysis of the effect of zinc-added BG scaffolds on integrins (a, b) and downstream signaling (c). Dimensional cells were cultured in osteogenic supplemented medium (OS) containing 50 mg / mL ascorbic acid and 10 mM? -Glycerophosphate. OS and 10 ^-7 M dexamethasone were used as positive controls for 7 and 14 days (a), 45 minutes (c). Integrin mRNA was examined by RT-PCR (a). The graph shows the quantification of mRNA expression by the densitometer and shows the increase by several times compared to the control (b). * Statistical significance is p <0.05. Protein expression was examined by Western blot analysis (c).
Figure 5 shows the results of the analysis of the effect of BG supplemented with zinc on the canonical (a) and non-canonical (b) Wnt pathways. Dimensional cells were cultured in osteogenic supplemented medium (OS) containing 50 mg / mL ascorbic acid and 10 mM? -Glycerophosphate for 24 hours. OS and OM containing 10 ^-7 M dexamethasone were used as positive control. The signal path was examined by Western blot.
FIG. 6 shows the results of analysis of the effect of zinc-added BG on the phosphorylation of MAPK and the activity of the NF-kB pathway. Dimensional cells were cultured in osteogenic supplemented medium (OS) containing 50 mg / mL ascorbic acid and 10 mM? -Glycerophosphate, treated with OM containing OS and 10 ^-7 M dexamethasone for 45 minutes and used as positive control (A, b). Signal pathways were assessed via Western blot (a) and immunofluorescence staining (b).

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One: CPC / BGN  Fabrication and Characterization of Composites

CPC / BGN  Composite manufacturing

First, BGN was prepared by sol - gel method. Zinc ion addition was performed through partial calcium ion replacement. The BGN is composed of 85% SiO ₂ -15% CaO (0% Zn), 85% SiO ₂ -12.5% CaO-2.5% ZnO (2.5% Zn) and 85% SiO ₂ -10% CaO- Zn). Specifically, a polyethylene glycol (PEG) surfactant was dissolved in anhydrous methanol at pH 12.5, and then calcium nitrate tetrahydrate and zinc nitrate hexahydrate ( Aldrich) was added. TEOS (tetraethyl orthosilicate, Aldrich) was then diluted in anhydrous methanol and added dropwise to the Ca / Zn solution. During the mixing, high power sonication was performed for 20 minutes. After stirring the mixture for 24 hours, the white precipitate was allowed to settle, then thoroughly washed with water / ethanol and dried overnight. Next, heat treatment was performed at 600 ° C for 5 hours.

The production method of the CPC powder is as follows. The α-trisodium phosphate (α-TCP) powders were each mixed with BGN powders of the above three compositions (with 10 wt% BGN for α-TCP) with or without zinc. Cementation was carried out by mixing with water so that the ratio of powder to solution in the plastic mold was 1.54.

CPC / BGN Physicochemical characterization of

For the in vitro cell test, a disc-shaped cemented specimen of 15 mm in diameter and 2 mm in thickness was stored overnight at 100% humidity, sterilized with ethanol three times, and dried. The solidification time of the cement was measured by the Gilmore needle method. The morphology of the specimens was confirmed by field emission scanning electron microscopy (FE-SEM; Tescan, Mira II LMH, Czech Republic) and the nanostructures of the specimens were analyzed by high resolution transmission electron microscopy (HR-TEM; JEM-3010, JEOL, Japan) At 300 kV. The chemical bond structure of the sample was analyzed using ATR-FTIR (attenuated total reflectance-Fourier transform infrared (Varian 640-IR, Austrailia) spectrometer equipped with GladiATR diamond crystal accessory (PIKE Technologies, USA). The cement specimen is immersed in distilled water to investigate the formation of bone-mineral-like HA. After immersion in water, specimens were identified by SEM and X-ray diffraction (XRD; Rigaku). For in vitro cell experiments, each specimen was sterilized with ethylene oxide gas and placed in each well of a 24-well plate.

Cell culture

HDPC transformed with human telomerase and immortalized was obtained from Professor Takashi Takata (Hiroshima University, Japan). The cells were cultured in α-MEM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 mg / mL streptomycin under wet, 5% CO ₂ , 37 ° C. To induce tooth formation differentiation, the cells were cultured in bone differentiation supplemented medium (OS: a-MEM, 10% FBS, 50 mg / mL ascorbic acid, 10 mM glycerophosphate). The medium was changed every two days during the incubation period.

Cell proliferation

hDPC was inoculated onto a sample at a density of 3 x 10 ⁵ per well of a 6-well plate and incubated with a microplate reader reader at a wavelength of 490 nm using a Cell Proliferation Assay Kit (Cell Titer 96 Aqueous One Solution; Promega, Madison, WI) Cell proliferation was measured.

Alkaline  Phosphatase activity

The cultured hDPSC was washed with PBS and sonicated to destroy the cells. ALP activity was assayed by measuring the activity of 0.7 M 2-aminomethyl-1-propanol (pH 10.3) and 6.7 mM MgCl ₂ Solution was measured using p-nitrophenyl phosphate (final concentration 3 mM) as a substrate. Absorbance was measured at 415 nm using an enzyme-linked immunosorbent assay reader (Beckman Coulter, Fullerton, Calif., USA).

alizarin Red  S dyeing

The cells were fixed with cold 70% ethanol for 1 hour and washed with distilled water. The cells were then stained with 40 mM Alizarin Red S (pH 4.2) for 10 minutes while gently stirring. Alizarin Red S staining level was observed through an optical microscope.

RNA  Separation and RT -PCR ( reverse transcriptase - 중합체 chain reaction )

RNA was extracted from the cells using Trizol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's instructions. 1 mg of the RNA was then reverse transcribed with AccuPower RT PreMix (Bioneer Corporation, Daejeon, South Korea) using an oligo (dT) primer (Roche Diagnostics, Mannheim, Germany). Next, 60 ng of cDNA was amplified with 1 mM of primer. Primer sequences and PCR conditions are shown in Table 1 below. The PCR products were electrophoresed on a 1.5% agarose gel stained with ethidium bromide (ETBR). Beta-actin mRNA, and the intensity of each band was quantitated using a densitometer (Quantity One; Bio-Rad, Hercules, Calif., USA) in the gel photographs.

gene The primer sequence (5'-3 ') Annealing Temp (℃) Cycle Number Product Size (bp) DSPP F: 5'-GCAAAAGTCCAGGACAGTGGGCC-3 '(SEQ ID NO: 1)
R: 5'-GTCAAATTTCCACCTCAGTTGGCCA-3 '(SEQ ID NO: 2) 61 35 342 DMP-1 F: 5'-AGAGAGAGATGGGAGAGCTGCGC-3 '(SEQ ID NO: 3)
R: 5'-TGACCTTCCATCTGCCTCCTGTTC-3 '(SEQ ID NO: 4) 61 35 202 Runx2 F: 5'-AGACCAACAGAGTCAGTGAGTGCT-3 '(SEQ ID NO: 5)
R: 5'-TGGTGTCACTGTGCTGAAGAGGC-3 '(SEQ ID NO: 6) 62 32 317 Osterix F: 5'-CTCTGCGGGACTCAACAACTCTG-3 '(SEQ ID NO: 7)
R: 5'-GAGCCATAGGGGTGTGTCATGTC-3 '(SEQ ID NO: 8) 62 32 316 Integrinα1 F: 5'-TGCTGCTGGCTCCTCACTGTTGT-3 '(SEQ ID NO: 9)
R: 5'-TAGTCTGGCGGCCACCTCTCTG-3 '(SEQ ID NO: 10) 61 35 703 Integrinα2 F: 5'-TTTCCCTGCTCTCACCGGGC-3 '(SEQ ID NO: 11)
R: 5'-ACCGGGGGACCGTAGTTGCG-3 '(SEQ ID NO: 12) 61 35 132 Integrinα3 F: 5'-CCCATCCCTGATGCCCCCGT-3 '(SEQ ID NO: 13)
R: 5'-TGCCCCCTCAGTAGTCGTCG-3 '(SEQ ID NO: 14) 61 35 415 Integrin beta 1 F: 5'-GACGCCGCGCGGAAAAGATG-3 '(SEQ ID NO: 15)
R: 5'-ACCACCCACAATTTGGCCCTGC-3 '(SEQ ID NO: 16) 61 35 159 Integrinβ3 F: 5'-CCGGCCAGATGATTCGAAGA-3 '(SEQ ID NO: 17)
R: 5'-GGGTCACACCTGGTCAGTTAGC-3 '(SEQ ID NO: 18) 61 35 329 FGF-2 F: 5'-GGCTTCTTCCTGCGCATCCA-3 '(SEQ ID NO: 19)
R: 5'-GCTCTTAGCAGACATTGGAAGA-3 '(SEQ ID NO: 20) 61 35 354 Ang-1 F: 5'-GCAGGCTCCATGCTGAACGGT-3 '(SEQ ID NO: 21)
R: 5'-ACTCCTTCCGTGCCTCTCGCA-3 '(SEQ ID NO: 22) 60 30 282 VEGF F: 5'-CACCGCCTCGGCTTGTCACAT-3 '(SEQ ID NO: 23)
R: 5'-CTGCTGTCTTGGGTGCATTGG-3 '(SEQ ID NO: 24) 60 35 404 β-actin F: 5'-CATGGATGATGATATCGCCGCG-3 '(SEQ ID NO: 25)
R: 5'-ACATGATCTGGGTCATCTTCTCG-3 '(SEQ ID NO: 26) 55 34 371

Western blot  analysis

Cell extracts were prepared by dissolving cells in ice for 30 min with protein lysis buffer (5 mM EDTA, 1 mM MgCl ₂ , 50 mM Tris HCl (pH 7.5), 0.5% Triton X-100, 2 mM phenylmethylsulfonylfluoride, and 1 mM N-ethylmaleimide). 50 μg of the protein was separated by electrophoresis on a 10% or 15% sodium dodecyl sulfate (SDS) polyacrylamide gel and electrotransferred to PVDF (polyvinylidene difluoride) membrane. The membrane was treated with blocking solution for 1 hour and p-FAK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), paxillin (Santa Cruz Biotechnology) diluted 1: (Santa Cruz Biotechnology), p-Akt (Santa Cruz Biotechnology), p-ERK (p-extracellular signal-regulated kinase, Cell Signaling Technology, Danvers , MA, USA), p-JNK (N-terminal kinase), p-p38 (Cell Signaling Technology), and NF-kB (Cell Signaling Technology) primary antibodies. The cells were then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase for 1 hour. Proteins were then visualized using an enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA).

Endothelial tube formation analysis

HUVEC (human umbilical vein endothelial cells) purchased from ATCC (University Blvd. Manassas, Va., USA) was cultured in an endothelial cell culture medium (ECM, ScienCell, Carlsbad, Calif.) At 37 ° C and 5% CO ₂ . 50 ml of ECM gel solution (Cell Biolabs, San Diego, USA) was poured into 96-well culture plates and allowed to solidify (37 DEG C, 1 hour). ZnBG was treated with hDPC for 3 days to obtain CM (condition medium). HUVEC (12 x 10 ⁴ cells / well) was inoculated on ECM gel and cultured in the CM for 18 hours at 37 ° C and 5% CO ₂ . The cells were then stained with a dye (1 mM Calcein in DMSO solution) (Cell Biolabs, San Diego, USA). Tubing formation was quantified from a high power photograph taken with a fluorescence microscope (Zeiss, Oberkochen, Germany). At least three pictures were taken in a random area of each well. A tube is defined as a linear cell junction or junction where two cells meet. The number of tubes was counted from the photograph.

Immune cell chemistry

The cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.1% Triton X-100. After washing with PBS buffer, the slides were blocked with 1% standard goat serum for 1 hour and then incubated with mouse monoclonal anti-human p65 antibody (Santa Cuz Biotechnology) diluted 1: 100 for 2 hours. The slides were washed with PBS and incubated with FITC (fluorescein isothiocyanate) coupled goat anti-mouse IgG (Invitrogen life technology, Grand Island, NY) for 1 hour diluted 1: Of PI (propidium iodide). The slides were enlarged 200 times with a confocal microscope (Cell Voyager, Yokogawa, Japan) and photographed.

Example  2: CPC / BGN Physicochemical characterization of

The nanocomposite mixed with CPC, Zn-BGN, was confirmed by TEM (Fig. 1A). It was confirmed that the nanosphere particles were well formed to a uniform size of ~ 80 nm. The nanoparticles showed high mesoporosity and the zinc content of 2.5% and 5% Zn-BGN in the EDS mapping of the nanoparticles was 2.48% and 5.06%, respectively. Representative EDS spectra of 5% Zn-BGN cement are shown in the inset picture.

Next, BGN was mixed with CPC and then the solidification time was measured (FIG. 1B). All specimens hardened within 40 minutes, as the content of zinc mixed in the BGN increased, reducing the coagulation time of the cement; Zn content in Zn-BGN was 0% for 37 min, 2.5% for 29 min and 5% for 25 min. As described above, it is considered that the coagulation reaction improves with increasing Zn content because Zn addition increases the surface reactivity (mainly mineral precipitation) of BGN.

The cement specimen is immersed in distilled water to observe bone-mineral-like HA formation. SEM micrographs of CPC / BGN (showing 5% Zn as a representative sample) show that CPC powder and a number of nanoparticles (BGN) surrounding the microparticles can be identified (FIG. After the immersion in water, the specimen shows a platelet crystalline form typically developed in biomimetic HA, which is completely different from that before the test (Fig. 1d).

In conclusion, the coagulation time of cement decreases with increasing Zn content, and Zn-BGN / CPC is morphologically similar to biomimetic HA.

Example  3: For cytotoxicity and tooth formation differentiation CPC / BGN Effect of

To evaluate the cytotoxic effect of cement samples, MTS analysis was performed. As shown in FIG. 2A, all the cement specimens showed no specific cytotoxicity in hDPC as compared with the control group.

ALD activity, alizarin red staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed at 7 and 14 days to investigate the effect of cement on dentin cell differentiation in hDPC. Cement containing Zn-BGN increased ALP activity and improved mineralized nodule formation in proportion to time and dose as compared to Zn-free cement (FIGS. 2b and 2d). In addition, Zn-BGN up-regulated mRNA expression of tooth-forming differentiation marker genes such as DSPP, DMP-1, Runx2 and osterix in a concentration-dependent manner compared to BN without ZN (FIG.

Taken together, the calcium phosphate cement composition of the present invention has no cytotoxicity, and promotes the differentiation of hDPC into dentin goblet cells, thus confirming that the calcium phosphate cement composition has excellent effects as a composition for tissue regeneration.

Example  4: For angiogenesis CPC / BGN Effect of

In order to test whether the cement specimen affects angiogenesis in vitro, the tube structure of the HUVEC and the blood vessels in the hDPC should be examined. MRNA expression of the neonatal factors was examined (Fig. 3). The cement containing Zn-BGN showed similar mRNA expression of FGF-2 and Ang-1 when Zn was not present, but the expression of hDPC VEGF was slightly increased at 5% Zn-BGN on the third day of culture 3a). As shown in FIG. 3B, when CM (18 hours) obtained from hDPC treated with CPC / 5% Zn-BGN for 3 days was treated, HUVEC angiogenesis was stimulated. The 5% Zn-BGN condition induced a noticeable change in endothelial cell shape with structural recombination leading to capillary-like network formation in HUVEC (FIG. 3b). In a sample containing 5% Zn-BNG, the number of tube structures per well increased significantly compared to HUVEC treated with OS or BG alone (Figure 3c).

From these results, CPC / 5% Zn-BGN increased the expression of hDPC VEGF, and the culture medium in which hDPC was cultured stimulated the angiogenesis of HUVEC. Thus, when the composition of the present invention was used for regeneration of tissue, It can be confirmed that it can be effectively induced.

Example  5: In the signaling pathway CPC / BGN Effect of

Dental differentiation is closely related to bone differentiation, and it is known that cell-matrix interactions through integrins are essential in bone differentiation. Thus, to investigate the effect of cement specimens on integrin and its downstream signaling pathways, mRNA expression of phosphorylation of integrin and its subpaths was analyzed by RT-PCR and Western blot analysis (FIG. 4). mRNA expression of integrin genes such as a1, a2, b1 and b3 was up-regulated in proportion to time and dose as compared to the absence of Zn in cement containing Zn-BGN (Fig. 4A). However, a3 was not affected by cement. In addition, integrin downstream signals including FAK, AKT, paxillin and RohA were markedly phosphorylated by Zn-BGN (FIG. 4B).

The Wnt signaling pathway is known to be important in dental differentiation and angiogenesis, and among the integrin sub-signals identified by Zn-BGN, the FAK activity causes canonical or The effect of Zn-BGN on Wnt signals including non-canonical pathways was investigated (Figure 5). Wnt cannonical signals such as Wnt1, Wnt3a, p-GSK-3? And? -Catenin increased in proportion to dose in hDPC treated with CPC / Zn-BGN (Fig. Similarly, non-canonical signals such as Wnt5a, CaMkII and NLk were also upregulated by Zn-BGN (Fig. 5b).

In addition, MAPK and NF-κB are known to be important signal transduction pathways in bone differentiation. Since the integrin lower signal pathway is involved in MAPK and NF-κB activity, MAPK phosphorylation and NF-κB activity are analyzed by Western blot analysis And examined through immunofluorescence (Fig. 6). Phosphorylation of ERK and p38 was higher in hDPC cultured in CPC mixed with 5% Zn-BGN than in BG mixed CPC, whereas JNK phosphorylation was not (Fig. 6A). Similarly, Western blot and concomitant photographs show that NF-κB p65 in the nucleus is significantly increased in CPC mixed with 5% Zn-BGN compared to no Zn (Fig. 6a and b).

As a result, it was confirmed that the CPC mixed with Zn-BGN of the present invention promotes tooth formation differentiation by activating the integrin and integrin lower signal pathways involved in tooth formation differentiation, and thus has an excellent effect as a composition for regenerating dental tissues.

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Claims (13)

A calcium phosphate cement composition comprising a powder phase and a liquid phase, the calcium phosphate cement composition comprising bioglass nanoparticles containing zinc ions,
The bioactive glass nanoparticles containing the zinc ion have a part of the calcium ions of the bioactive glass replaced with zinc ions,
The content of the bioactive glass nanoparticles is 1 to 10% by weight based on the total weight of the powder,
Wherein the zinc ion content is 2.5 to 5 wt% of the total weight of the bioactive glass nanoparticles.
delete delete delete delete The composition according to claim 1, wherein the bioactive glass nanoparticles have an average particle size of 10 to 100 nm.
The composition according to claim 1, wherein the zinc ion-containing bioactive glass nanoparticles are prepared by a manufacturing method comprising the steps of:
1) dissolving PEG or CTAB to prepare a template solution and adjusting the pH to 9 to 13;
2) adding a calcium precursor and a zinc precursor to the template solution to prepare a calcium ion-zinc ion-template mixed solution;
3) adding a silica precursor solution to the calcium ion-zinc ion-template mixed solution, sonicating and stirring to prepare a reaction product; And
4) Centrifugation of the reaction product, followed by washing, drying and calcination to prepare mesoporous bioactive glass nanoparticles.
The method according to claim 1, wherein the powder phase is selected from the group consisting of amorphous calcium phosphate, dicalcium phosphate anhydrous (DCPA), tetracalcium phosphate (TTCP),? Wherein the composition comprises at least one calcium phosphate compound selected from the group consisting of tricalcium phosphate,? -TCP, and? -Tricalcium phosphate.
The composition of claim 1, wherein the liquid phase comprises a cure accelerator that promotes the precipitation reaction of ionized calcium and phosphate ions to promote hydroxyapatite production.
10. The method of claim 9, wherein the curing accelerator is _{Na 2 HPO 4, NaH 2 PO} 4, K 2 HPO 4, NH 4 H 2 PO 4, Na 2 SO 4, 1 is selected from citric acid, maleic acid, and the group consisting of propionic acid Or more.
The composition according to claim 1, wherein the liquid phase of the composition is contained in an amount of 30 to 45 parts by weight per 100 parts by weight of the powder.
A liquid phase containing a calcium phosphate compound, a liquid phase containing a curing accelerator, and a life-like glass nanoparticle containing a zinc ion, each packaged in a separate container,
The bioactive glass nanoparticles containing the zinc ion have a part of the calcium ions of the bioactive glass replaced with zinc ions,
The content of the bioactive glass nanoparticles is 1 to 10% by weight based on the total weight of the powder,
Wherein the zinc ion content is 2.5 to 5 wt% of the total weight of the bioactive glass nanoparticles.
13. The method according to claim 12, wherein the bioactive glass nanoparticles are present in an amount of 0.1 to 10% by weight based on the total weight of the powdery bioactive nanoparticles, &Lt; / RTI &gt; to 10% by weight.

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