KR101685295B1 - Multiple-drug loaded scaffold and use thereof - Google Patents

Abstract

The present invention relates to a scaffold having a core-shell fiber structure comprising a drug-containing biopolymer core, a process for its preparation, and a pharmacological agent for bone regeneration comprising the scaffold carrying FGF2 and FGF18 ≪ / RTI >
The scaffold having the core-shell fiber structure of the present invention may contain two or more drugs, and the release rates of the two drugs may be varied and utilized for various purposes. In particular, when FGF2 and FGF18 are loaded, FGF2 improves cell proliferation and FGF18 induces bone differentiation, and thus has excellent effect as a pharmaceutical composition for bone regeneration.

Description

Multiple drug loaded scaffolds and uses thereof < RTI ID = 0.0 >

The present invention relates to a scaffold having a core-shell fiber structure comprising a biopolymer core containing multiple drugs, a process for producing the same, and a pharmaceutical composition for bone regeneration comprising the scaffold.

Bone regeneration scaffolds developed for therapeutic purposes for bone injuries can be used to regulate the function of stem cells / progenitor cells in vivo by providing the physical and biochemical environment in which the transplanted cells can locate themselves and be accepted in vivo (Exp I Mol et al., Exp Mol Med, 2013; 45: e57).

In addition to cell transplantation, delivery of exogenous bioactive materials with the scaffold can also be an effective treatment method, i.e., a therapeutic scaffold for loading and delivering molecules with therapeutic effects such as drugs, proteins or genes Can be used as a platform for regenerating tissues such as bone, and because of the mounted molecules, a further enhanced effect can be expected as compared with the case where a scaffold alone is implanted. Various techniques have been developed to effectively incorporate the therapeutic material into the scaffold, such as surface fixation, affinity-induced tethering, direct mixing in a scaffold, and nano / microencapsulation.

Electrospun fibers have an excellent effect as a scaffold platform for treating and regenerating tissues such as skin, muscles, nerves, cartilage and bone (Cirillo V et al., Biomaterials, 2014; 35: 8970-82) . Fibrous forms that can be controlled from hundreds to micrometers can be produced by mimicking the extracellular matrix of natural tissue in a fairly similar manner and thus may be effective in the attachment and growth of cells to be transplanted. Electrospinning techniques can also make fibers of various shapes, structures, and compositions. In particular, for drug delivery purposes, a fiber having a core-shell structure can encapsulate a considerably large amount of therapeutic molecule within the core while maintaining biological activity, and releasing the encapsulated drug can result in shell composition and / It can be adjusted by modifying the thickness. However, no electrospun fibers capable of sequentially delivering and releasing two or more drugs have been reported to date.

In the present invention, as a result of intensive efforts to develop a drug delivery scaffold for bone regeneration, a scaffold having a core-shell fiber structure including a biopolymer core containing a drug has been developed. Furthermore, the present inventors have developed a method for sequentially delivering two types of therapeutic molecules using the scaffold. That is, one molecule can be released relatively fast and the other molecule can be released relatively continuously. Such sequential therapeutic molecule delivery method can be used for tissue treatment and regeneration, which requires the activity of several molecules over time .

One object of the present invention is to provide a scaffold having a core-shell fiber structure comprising a biopolymer core containing a vitriol nanoparticle bearing a first drug and a second drug .

It is another object of the present invention to provide a pharmaceutical composition for bone regeneration comprising the scaffold.

Another object of the present invention is to provide a method for preparing vitreous silica nanoparticles, which comprises: (a) preparing a vitreous silica nanoparticle by mixing a calcium oxide precursor and a silica precursor; (b) mixing the vitrified glass nanoparticles and the first drug to prepare vitrified vitreous nanoparticles loaded with the first drug; (c) a biocompatible core component containing a living drug-containing nanoparticle and a second drug, wherein a shell component is injected into a syringe-like device external injection section and the first drug is loaded in an internal injection section, To produce core-shell fibers; And (d) recovering and drying the fiber produced.

In accordance with one aspect of the present invention, there is provided a core-shell fiber structure comprising a biopolymer core containing a vitriol nanoparticle loaded with a first drug and a second drug, Lt; / RTI >

The scaffold has a core-shell fiber structure, and the core has a structure containing (i) a vitriol-containing nanoparticle carrying a first drug, and (ii) a second drug. That is, the scaffold can transfer the first drug and the second drug at different release rates by mounting the first drug on the vitreous silica nanoparticles positioned in the core and mounting the second drug directly on the core. Also, since the scaffold includes vitrified nanoparticles and a biopolymer core, the scaffold is excellent in biocompatibility and can be used for a target disease treatment, and the target disease can be applied without limitation according to a drug to be used. Hereinafter, the configuration of the present invention will be described in detail.

The term " vitreous silica " in the present invention refers to any glass which is characteristic of biological activity, and which is not tacky in nature, but which is tacky to both hard and soft tissues when exposed to an appropriate environment such as body fluids or trishydroxymethylaminomethane buffer Lt; RTI ID = 0.0 > solid < / RTI > The vitreous glass was discovered by Hench, Bioglass ^? , Which has been used by Perioglas ^? And Novabone ^? It was used clinically as a regenerated bone with powder structure. Living vitreous is bound to bone because HCA (hydroxycarbonated apatite) layer is formed on the surface when contacted with body fluids. HCA is similar in composition to bone mineral and forms strong bonds with bone. Lifelong glaucoma dissolves safely in the human body, emits critical concentrations of silicon and calcium ions that play a role in stimulating osteocytes at the genetic level, and causes new bone regeneration even when very few active cells are present.

There are two kinds of vitreous glass, one derived from dissolution and the other derived from sol-gel. In the present invention, vitreous silica derived from a sol-gel, particularly based on silica, may be used, but is not limited thereto.

In general, the vitreous silica is mainly composed of SiO ₂ and CaO, and may further include Na ₂ O, P ₂ O _5, or a combination thereof. Particularly, the vitreous silica nanoparticles may have a Ca: Si molar ratio of 1: 4 to 1: 9, and the nanoparticles may have a diameter of 100 to 140 nm, but the present invention is not limited thereto.

The vitreous nanoparticles are loaded with the first drug, and the vitreous silica nanoparticles loaded with the drug can be incorporated into the core of the scaffold of the present invention. The vitreous nanoparticle acts as a drug substance .

In the present invention, the vitreous silica nanoparticles are used in the same meaning as mesoporous bioactive glass nanospheres (MBN). The porosity means that the nanoparticles have a number of pores. Particularly, mesoporosity means that the pores of the nanoparticles have a diameter of 2 nm or more and less than 50 nm. That is, in the present invention, the vitreous silica nanoparticles may have mesoporosity, but the number and size of the pores are not particularly limited as long as the vitreous silica nanoparticles can act as a drug delivery system.

The term " scaffold " in the present invention refers to a substance that replaces or partially replaces damaged organs or tissues in vivo, and in the present invention, The scaffold may be a biodegradable polymer material that is maintained in vivo until fully functioning and can be completely decomposed and removed, but is not limited thereto .

The term " core " in the present invention means a structure mainly located at a central portion in a scaffold having various kinds of compositions, and the term " Shell " means a structure in which, Or a structure that surrounds a portion. That is, the scaffold of the present invention has a fiber structure, and has a structural characteristic that the fiber can be distinguished into a structure of a core and a shell.

Through this structure, the core can contain (i) vitrified nanoparticles loaded with the first drug, and (ii) the second drug, and is biocompatible in vivo, Can be used. The shell encapsulates the core to encapsulate a large amount of therapeutic molecules in the core while maintaining the biological activity, and can adjust the drug release pattern by modifying the composition and thickness of the shell.

The core-shell fiber structure may include, but is not limited to, a hollow structure.

As used herein, the term " hollow structure " is intended to mean an empty hollow body, which may include a conventional hollow body, a raster-like hollow body forming a core therein, or a porous body, , But is not limited thereto.

The core of the biopolymer may be a biopolymer system that can be dissolved in an aqueous solution, such as polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, gelatin, chitosan, hyaluronic acid, dextran, And specifically may be polyethylene oxide. More specifically, it may be polyethylene oxide having a molecular weight of 50,000 to 70,000, but is not limited thereto.

The biopolymer core may further comprise vitrified nanoparticles and / or a second drug loaded with the first drug, and may further comprise at least one drug.

The shell of the fiber scaffold is a biopolymer system that can be dissolved in an organic solvent and includes polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA) ), Polydioxanone (PDO), or a combination thereof. Specifically, it may be polycaprolactone, and more specifically, it may have a molecular weight of 70,000 to 90,000, but is not limited thereto no.

The vitrified glass nanoparticles may be contained in an amount of 1 to 10 wt% based on the biopolymer core, but are not limited thereto.

The term " drug " in the present invention refers to a small molecule, chemical substance, nucleic acid, nucleic acid derivative, peptide, peptide derivative, naturally occurring protein, non-naturally occurring substance, Naturally occurring proteins, glycoproteins and steroids. Non-limiting examples of drugs include but are not limited to polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, antibody derivatives, drugs affecting metabolic function, organic compounds such as analgesics, antipyretics, anti- An antiviral compound, an antifungal compound, a cardiovascular drug, a drug affecting renal function, an electrolyte metabolite, a drug acting on the central nervous system, a chemotherapeutic compound, a receptor agonist and a receptor antagonist. The drug may also be a serum factor, including, but not limited to, extracellular molecules such as plasma proteins such as serum albumin, immunoglobulins, apogee proteins or transferrin, or proteins found on the surface of red blood cells or lymphocytes . Thus, exemplary drugs include but are not limited to small molecules, chemicals, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-naturally occurring proteins, peptide-nucleic acid (PNA), staple peptide, Antisense drugs, RNA-based silencing agents, platamers, glycoproteins, enzymes, hormones, cytokines, interferons, growth factors, blood coagulation factors, antibodies, antibody fragments, antibody derivatives, toxin- Antineoplastic agents, antimicrobial agents, antiviral agents, antifungal agents, musculoskeletal drugs, cardiovascular drugs, kidney drugs, pulmonary drugs, digestive diseases drugs, blood drugs, urinary drugs, metabolism drugs, liver drugs, nerve drugs, antidiabetic drugs , Anticancer drugs, gastric condition treatment drugs, colonic condition treatment drugs, skin condition treatment drugs and lymphatic condition treatment drugs.

In the present invention, " first drug " and / or " second drug " are applicable to any of the above-mentioned drugs that can be loaded on the scaffold of the present invention. . Depending on the first drug or second drug to be applied, the scaffold of the present invention may be used without limitation in therapeutic or regenerative uses for a variety of diseases, and the selection and combination of such drugs may be appropriately selected by those skilled in the art.

Furthermore, the scaffold of the present invention may be characterized in that the first drug and the second drug are sequentially released from the scaffold. The term " sequential release " in the present invention encompasses the complete release of one drug followed by the release of another drug, and one drug is primarily released early and the other drug slowly and continuously released have. In particular, the second drug directly loaded on the biopolymer core may be initially released at a comparatively high rate and the first drug loaded on the vitreous silica nanoparticles may be released at a relatively slow rate compared to the second drug. Therefore, the scaffold of the present invention exhibits an excellent effect for the purpose of drug delivery. In addition, the scaffold of the present invention can be used for sequentially delivering two or more drugs, so that it is possible to provide a drug delivery system more advanced than the existing drug delivery system. It can be applied in various ways for disease treatment or tissue regeneration.

The first drug may be FGF-18 (fibroblast growth factor 18), and the first drug may be FGF-18 (fibroblast growth factor 18). In addition, the first drug may be a water soluble drug or a growth factor capable of dissolving in an aqueous solution, 2 drug may be FGF-2 (fibroblast growth factor 2).

In the present invention, FGF-18 and FGF-2 refer to a protein having at least one biological activity of the corresponding protein, and FGF-18 and FGF-2 may be natural in a mature form, or in a truncated form thereof And is not limited to that form as long as it exhibits the same effect. In particular, the proliferation of osteoblasts can be enhanced by FGF-2, and osteoclast differentiation can be promoted by FGF-18.

In one specific embodiment of the present invention, an effective drug delivery system for bone regeneration capable of initially delivering FGF-2 and delivering FGF-18 later than this was established. FGF-2 is known to promote proliferation of fibroblasts and vascular endothelial cells, which play an important role in tissue regeneration. On the other hand, FGF-18 is known to promote bone formation by upregulating bone morphogenetic protein 2 (BMP2). Therefore, sequential delivery / release of FGF-2 and FGF-18 may be effective in bone therapy and regeneration. That is, FGF-2 is released early to promote the proliferation of cells necessary for bone regeneration, and to promote the differentiation of cells proliferated by FGF-18, thereby achieving a bone regeneration effect.

Specifically, for sequential delivery of the drugs, FGF-18 was first loaded on mesoporous bioactive glass nanospheres (MBN), and then the FGF-18 was loaded into the core directly loaded with FGF-2 Gt; MBN < / RTI > The MBN has an excellent effect in loading and transferring therapeutic molecules such as chemicals, proteins and genes. Furthermore, MBN can be a highly effective nanomaterials platform for regenerating hard tissue, such as bone or teeth, because nanoparticles have high surface bioactivity and excellent mineralization. In addition, Ca and Si ions can be released from the nanoparticles, thereby promoting bone differentiation of progenitor cells and stem cells.

Fig. 1 is a schematic view of one embodiment of the present invention. Fig. 1 shows a novel scaffold for bone regeneration in which MBN on which FGF-18 is mounted is mixed in core-shell electrospun fiber loaded with FGF-2. FGF-2 is released early to promote cell division and angiogenesis, and FGF-18 is released slowly and continuously to induce bone formation. In the present invention, scaffolds for bone regeneration were prepared and their physico-chemical properties such as morphology, mechanical properties, in vitro degradation and bone viability were examined. The pattern of release of the growth factor was observed using a model protein for a prolonged period of several months. The therapeutic efficacy of FGF-2 / FGF-18 transfer from fibrous scaffolds was evaluated in vivo by in vitro reactions such as growth of rat mesenchymal stem cells (Figure 22) and bone differentiation (Figures 23-25) Through the bone formation (FIG. 26 to FIG. 29), it was found that the scaffold of the present invention loaded with FGF-2 and FGF-18 had an excellent effect on bone regeneration.

Further, the biopolymer core of the scaffold of the present invention may further contain one or more drugs other than the first drug and the second drug, which may be appropriately selected and modified by those skilled in the art depending on the purpose of loading the drug The scaffold of the present invention can be utilized for a wide variety of purposes depending on the type and combination of drugs to be loaded.

According to another aspect of the present invention, there is provided a method for producing vitreous glass nanoparticles, comprising: (a) mixing a calcium oxide precursor and a silica precursor to produce vitrified glass nanoparticles; (b) mixing the vitrified glass nanoparticles and the first drug to prepare vitrified vitreous nanoparticles loaded with the first drug; (c) a biocompatible core component containing a living drug-containing nanoparticle and a second drug, wherein a shell component is injected into a syringe-like device external injection section and the first drug is loaded in an internal injection section, To produce core-shell fibers; And (d) recovering and drying the prepared fibers.

Drug, life glass nanoparticles, and a scaffold having a core-shell fiber structure are as described above. Hereinafter, each step of the scaffold manufacturing method will be described in detail.

Wherein the calcium oxide precursor is selected from the group consisting of tetrahydrate calcium nitrate (Ca (NO ₃ ) ₂ 4H ₂ O), calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄₎ ethoxide (calcium methoxyethoxide), or may comprise a combination thereof, wherein the silica precursor is tetraethyl ortho silicate (tetraethyl orthosilicate), TEOS (tetraethyl orthosilicate), calcium methoxy, TMOS trimethoxy orthosilicate, GPTMS (3-glycidoxypropyl) methyl diethoxysilane, 3-mercaptopropyl trimethoxysilane, GOTMS, aminophenyl trimethoxysilane, or combinations thereof. It is not.

Specifically, the lifesaving glass nanoparticles of the present invention include: 1) dissolving PEG or CTAB to prepare a template solution and adjusting the pH to 9 to 13; 2) preparing a calcium oxide-template mixed solution by adding a calcium oxide precursor to the template solution; And 3) adding a silica precursor solution to the calcium oxide-template mixed solution and sonicating and stirring to prepare a reaction product; 4) a step of centrifuging the reaction product, followed by washing, followed by drying and firing, to prepare the vitreous silica nanoparticles. However, the present invention is not limited thereto. no.

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 may be PEG or CTAB, and the template solution may be prepared by dissolving the polymer template in an anhydrous methanol solution. Also, the pH may be adjusted with NH4OH, NaOH, KOH or Tris, but is not limited thereto.

Step 2 is a step of adding a calcium oxide precursor to the template solution and stirring to form calcium oxide-template mixed solution to form calcium oxide in the template material. The calcium oxide precursor may be calcium nitrate, calcium chloride, calcium acetate, or calcium methoxyethoxide, but is not limited thereto.

In the step 3, a silica precursor solution is added to the calcium 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 may be prepared by dissolving a silica precursor in an alcohol solution of anhydrous methanol, ethanol, propanol, or isopropanol. The silica precursor may be tetraethyl orthosilicate (TEOS), trimethoxy orthosilicate (TMOS) glycidoxypropyl methyl diethoxysilane, 3-mercaptopropyl trimethoxysilane, GOTMS or aminophenyl trimethoxysilane (APTMOS). Also, the ultrasonic treatment may be performed for 15 minutes to 25 minutes at an output power of 200 W to 240 W, 10 s on / 10 s off cycle, but is not limited thereto.

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 lifesaving glass 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. Further, the firing may be carried out at 550 to 650 占 폚 for 5 to 7 hours under the atmosphere.

In one specific embodiment of the present invention, water, ethanol and 2-ethoxyethanol are used as co-solvents, hexadecyltrimethylammonium bromide (CTAB) is used as a surfactant, calcium nitrate and tetraethylorthosilicate are used to manufacture vitrified glass nanoparticles Respectively. The lifesaving glass nanoparticles were monodispersed spherical nanoparticles having mesoporosity (FIG. 2).

The step (b) is a step of mixing the vitrified nanoparticles prepared in step (a) and the first drug, but it is not limited thereto. It is possible to dissolve the first drug in a suitable solvent such as PBS, Followed by incubation at 25 to 45 ° C, specifically at 30 to 40 ° C for 1 to 15 hours, more specifically, 3 to 9 hours after dispersing the spherical glass nanoparticles. Through the above process, the first drug can be loaded on the vitreous glass nanoparticles.

In one specific embodiment of the present invention, cytochrome c (C) as the first drug was used as a model protein and mounted on the vitreous silica nanoparticles at a concentration (0 to 0.3 mg / ml). As a result, the loading amount was increased with increasing cyt C concentration, the maximum loading amount was about 0.13 mg at a concentration of 0.4 mg cytoc, and the loading capacity of the vitrified nanoparticle to cyt C was about 13% (Fig. 16) , And it was confirmed that cyt C was mounted on the mesopores of the vitreous silica nanoparticles by TEM photograph after mounting (Fig. 17). In addition, the volume of mesopores in the drug-loaded vitreous nanoparticles was significantly reduced (Fig. 18), and the band of cyt C loaded via FTIR was confirmed (Fig. 19).

The above example uses cytochrome C as a model protein, and it is not an example for confirming whether a drug can be loaded on the vitreous silica nanoparticle of the present invention. Therefore, the characteristics such as the concentration of the loaded protein, But the present invention is not limited to the above-described embodiments.

The step (c) is a step of producing a scaffold having a core-shell fiber structure, and the step of producing the core-shell fiber is a coaxial electrospinning However, any method can be used as long as it is capable of producing a fiber having a core-shell structure.

The term " syringe-like device " in the present invention includes a needle and a cylinder, such as a syringe, and in particular, an external injection unit and an internal injection unit capable of independently injecting two components. A shell component of the scaffold may be injected into the external injection portion and a core component of the scaffold may be injected into the internal injection portion. The core component may comprise a vitriol-bearing nanoparticle loaded with a first drug, and a second drug. Specifically, the first drug may be FGF18 and the second drug may be FGF2.

When performing coaxial electrospinning to produce core-shell fibers, the feed rates for the shell and core portions may be 3.0 to 5.0 ml / h and 0.3 to 0.7 ml / h, respectively .

Finally, step (d) may be, but is not limited to, recovering the fabricated fibers in a collector and drying in a vacuum.

In one specific embodiment of the present invention, polycaprolactone is used as a shell component, polyethylene oxide is used as a core component, life-like glass nanoparticles having FGF-18 loaded on the core, and scaffolds . (Fig. 3 to Fig. 10), and it was confirmed that the higher the content of vitreous silica, the better the degradation in vivo (Fig. 11) (Fig. 22 to Fig. 29).

In another aspect, the present invention includes a scaffold having a Core-Shell fiber structure, including a biopolymer core containing a vitriol nanoparticle bearing a first drug and a second drug And a pharmaceutical composition for bone regeneration. In particular, the first drug may be FGF18 and the second drug may be FGF2, but is not limited thereto.

Drug, life glass nanoparticles, and a scaffold having a core-shell fiber structure are as described above.

In the present invention, the term " bone regeneration " may mean any phenomenon that treats, alleviates, or alleviates bone damage through proliferation of damaged bone tissue or differentiation of osteoblast into osteocyte, In the present invention, in particular, the first drug and the second drug may be sequentially released to exhibit such an effect. In particular, the bone regeneration may be due to the enhancement of osteoblast proliferation through FGF-2 and / or the promotion of bone differentiation through FGF-18.

The pharmaceutical composition of the present invention can be used as a single preparation and can be used as a combined preparation containing a drug known to have a recognized bone regeneration effect and can be formulated by using a pharmaceutically acceptable carrier or excipient, ≪ / RTI > or by intrusion into a multi-dose container.

As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound being injected, without stimulating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The carrier may comprise a non-naturally occuring carrier. In addition, if necessary, other conventional additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. A diluent, a dispersant, a surfactant, a binder, a lubricant, Pills, capsules, granules or tablets, and the like.

In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of a drug, and in particular, the drug may be FGF2, FGF18 or a combination thereof, but is not limited thereto. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and is generally in the range of 0.001 to 1000 mg / kg, 200 mg / kg, more specifically 0.1 to 100 mg / kg, may be administered once a day to several times a day. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, Including but not limited to weight, general health status, sex and diet, time of administration, route of administration and fraction of the composition, duration of treatment, drugs used or co-used with the specific composition, and other factors well known in the medical arts can do.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer an amount that can achieve the maximum effect in a minimal amount without causing side effects, and can be readily determined by those skilled in the art.

The term "administering" as used herein means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is not limited to a variety of oral or parenteral routes Lt; / RTI >

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be conventionally used in the art. As a non-limiting example of such a mode of administration, the compositions may be administered orally or parenterally. The pharmaceutical composition according to the present invention can be manufactured into various formulations according to the intended administration mode.

The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.

The scaffold having the core-shell fiber structure of the present invention may contain two or more drugs, and the release rates of the two drugs may be varied and utilized for various purposes. In particular, when FGF2 and FGF18 are loaded, FGF2 improves cell proliferation and FGF18 induces bone differentiation, and thus has excellent effect as a pharmaceutical composition for bone regeneration.

FIG. 1 shows a schematic view of a new therapeutic bone (hereinafter referred to as " bone marrow bone ") in which a mesoporous bioactive glass nanosphere (MBN) loaded with fibroblast growth factor-18 (FGF-18) is mixed with a core- shell fiber scaffold equipped with fibroblast gorwth factor- Fig. 3 schematically shows the structure of a scaffold. Fig.
2 is a low magnification and high magnification photograph of MBN having a high mesoporous structure.
Figure 3 shows an SEM photograph of a core-shell fiber scaffold containing MBN at various concentrations (0, 1, 3 and 5%).
Figure 4 is a SEM cross-section photograph showing the hollow interior structure.
Figure 5 shows the FTIR analysis results of a core-shell fiber scaffold showing chemical bonds associated with the composition.
6 is a graph showing the amount of residual fiber according to MBN concentration.
Figure 7 shows the typical stress strain curves of core-shell fiber scaffolds containing various concentrations of MBN.
Figure 8 shows the tensile strength of a core-shell fiber scaffold containing various concentrations of MBN.
Figure 9 shows the modulus of a core-shell fiber scaffold containing various concentrations of MBN.
Figure 10 shows the elongation of a core-shell fiber scaffold containing various concentrations of MBN.
Figure 11 shows hydrolysis in PBS of a core-shell fiber scaffold containing 0% or 5% MBN.
Figure 12 shows the morphology of the core-shell fiber scaffold at 14 days in PBS with SEM.
Figure 13 shows the XRD pattern when the core-shell fiber scaffold was immersed in SBF for 14 days.
14 shows the FTIR spectrum when the core-shell fiber scaffold was immersed in SBF for 14 days.
15 is a SEM photograph of a core-shell fiber scaffold immersed in SBF for 14 days.
16 shows the MBN loading of the model protein cyt C according to the initial concentration. Various concentrations of cyt C were used to mount 1 mg MBN and the loading was recorded.
17 is a TEM photograph of MBN on which cyt C is mounted.
Figure 18 is a BET N ₂ absorption / release curve of MBN loaded with cyt C.
19 is an IR spectrum of MBN loaded with cyt C; MBN before cyt C was loaded was used as a control.
Figure 20 shows the release of cyt C from the core-shell fiber scaffold. Cyt C was implemented in two ways: mounted on MBN ("with MBN") or directly mounted on core ("w / o MBN").
Figure 21 shows the release of cyt C from the core-shell fiber scaffold. Cyt C was implemented in two ways: mounted on MBN ("with MBN") or directly mounted on core ("w / o MBN").
Figure 22 shows the effect of FGF-2 / FGF-18 loaded core-shell fiber scaffolds on MSC proliferation measured by CCK analysis up to 14 days. * p < 0.05, ** p < 0.01, n = 3. As a comparison group, cells cultured in a culture vessel without any additives or in a container containing FGF-2 or FGF-18 were evaluated.
FIG. 23 shows the effect of MSC bone morphogenesis on FGF-2 / FGF-18 loaded core-shell fibrous scaffolds by ALP activity until 14 days of differentiation. * p < 0.05, * p < 0.01, n = 3. As a comparison group, cells cultured in a culture vessel without any additives or in a container containing FGF-2 or FGF-18 were evaluated.
24 is an analysis of mRNA levels of bone morphogenetic genes including ALP, OPN and Col1 by quantitative RT-PCR. * p < 0.05, n = 3.
Figure 25 is an evaluation of cell mineralization by the ARS method.
Fig. 26 is a μCT photograph after 6 weeks of transplanting the scaffold into both open defect sites in the rat.
FIG. 27 shows the bone volume after 6 weeks of transplanting the scaffold into the two open defect regions of the rat. * p < 0.05, n = 3.
FIG. 28 shows the bone surface density after 6 weeks of transplanting the scaffold into the two open defect sites in the rat. * p < 0.05, n = 3.
FIG. 29 is a histological photograph of a specimen collected at 6 weeks, showing a H & E staining image on the left side and a MT staining image on the right side. (a) Control group. (b) Scaffolding alone. (c) Scaffold treatment with FGF-2 / FGF-18. The arrow indicates the defect site boundary. OB, existing bone; NB, new bone; The scale bar is 500 m; In the enlarged image, 100 ㎛

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.

In the present invention, a bone scaffold for bone regeneration was prepared by mixing mesoporous vitreous living glass nanoparticles and core-shell fiber scaffolds prepared by electrospinning, and their characteristics were analyzed. Particularly, it was confirmed whether or not a drug was loaded on the scaffold to have a bone regeneration effect.

Example  One. Mesoporous Life Sunglia  Nanoparticles and fibers having a core-shell hollow structure Scaffold  Produce

(1) Preparation of mesoporous vitreous silica nanoparticles

MBN (mesoporous bioactive glass nanosphere) was prepared as a base solution using water, ethanol and 2 - ethoxyethanol as a co - solvent and hexadecyltrimethylammonium bromide (CTAB) as a surfactant at room temperature.

Specifically, an emulsion system consisting of 150 ml of H ₂ O, 2 ml of ammonia solution, 10 ml of 2-ethoxyethanol, 20 ml of ethanol and tetrahydrate calcium nitrate (Ca (NO ₃ ) ₂ .4H ₂ O) system) was lysed with 1 g CTAB. After the mixture was stirred at room temperature for 30 minutes, tetraethyl orthosilicate was rapidly dropped into the mixture. The molar ratio of Ca: Si was set to 15: 85. The mixture was then stirred at room temperature for 4 hours. A white precipitate was obtained, filtered off, washed with distilled water and dried at 60 < 0 > C for 24 hours in air. Then, to the mixture, 40 mg of demineralized water and 20 mg of ammonium nitrate in a mixed solution was treated at 60 캜 overnight to remove remaining CTAB. The solution was centrifuged, washed with ethanol and demineralized water, and dried overnight under vacuum to obtain MBN.

(2) Preparation of fiber scaffold having core-shell hollow structure

(PEO; Mw = 60,000; Sigma-Aldrich) as a core component and polycaprolactone (PCL; Mw = 80,000) as a shell component were used as core shell hollow fiber structure scaffolds. . MBN was added to PEO at various concentrations (1, 3, and 5 wt% relative to PEO).

PCL was dissolved at 10% in a mixed solvent of chloroform and dimethylformamide in a 4: 1 ratio, and PEO was dissolved at 4% using distilled water as a solvent. Coaxial electrospinning was performed to produce PCL as the shell and PEO (+ MBN) as the core using a syringe-like device with the outer needle and the inner needle positioned in the same axis. The feed rates for the shell and core portions were 4.0 and 0.5 ml / h, respectively. Each solution loaded on the syringe was injected through a spinneret needle with a high voltage power supply (Gamma High Voltage Research, Ormond Beach) applied to the slow rotating metal collector. The distance between the needles and the collector was 15 cm and the fibers were recovered after 2 hours. The fiber agglomerates were then dried overnight under vacuum. Specifically, in order to mount proteins and growth factors in the fiber, a solution dissolved in PBS (phosphate-buffered saline) at a concentration of 100 μg / ml was mixed with a PEO solution suitable for electrospinning.

Example  2. Mesoporous Life Sunglia  A fiber having a core-shell hollow structure comprising nanoparticles Scaffold  Character analysis

(1) Analytical Apparatus and Method

The following apparatus and methods were used to analyze the characteristics of the prepared vitreous core nanoparticles and core-shell fiber scaffolds.

The morphology and nanostructure of the specimens were investigated by field emission scanning electron microscopy (SEM; Mira II LMH) and high resolution transmission electron microscopy (TEM: JEM-3010, JEOL, operating at 300 kV).

Using ATR-FTIR (attenuated total reflectance-Fourier transfrom infrared spectroscopy) at 4 cm ^-1 resolution from 4000 to 400 cm ^-1 using a Varian 640-IR spectrometer equipped with GladiATR diamond crystal accessory (PIKE Technologies) Respectively.

The thermal behavior of the specimens was investigated by thermogravimetric analysis (TGA; TGA N-1500, Scinco, operating at a heating rate of 10 ° C / min).

Mesoporous structures were analyzed by N ₂ absorption-emission measurements using an automated surface area and pore size analyzer (Quadrasorb SI, Quantachrom Instruments). The specific surface area was determined according to the BET (Brunauer-Emmett-Teller) method and the pore size distribution was confirmed using the non-local density functional theory (NLDFT) method.

The electrical properties of the surface were measured with a ζ-potential meter (Zetasizer Nano ZS, Malvern Instruments) and the ζ-potential was measured at pH 7, 25 ° C water with an applied field strength of 200 V / cm. The apparatus automatically calculates electrophoretic mobility (U), which is obtained according to the Helmholtz-Smoluchowsky formula: < RTI ID = 0.0 >

ζ = Uγ / ε, where γ is the dispersing medium viscosity and ε is the permittivity.

(2) Characterization of MBN

The MBN used as the nano component of the core-shell nanocomposite fiber was examined by TEM (FIG. 2). As a result, monodispersed spherical nanoparticles were well developed, and high-magnification photographs showed mesoporosity as a whole. The characteristics of the MBN are summarized in Table 1.

TEM 105 ± 7 nm Particle size    Laser scattering 118.4 ± 5.5 nm    Pore size 6.97 nm    Surface area 527.2 m ² / g    Pore volume 0.497 cc / g    Zeta-potential -10.2 ± 2.8 mV

The size of the nanoparticles was about 119 nm on average, the surface had a high mesoporosity, the surface area was 527.2 m ² / g, and the pore volume was 0.497 cm ³ / g. The size of the mesopores was 6.97 nm and the nanoparticles were negatively charged with a ζ-potential of -10.2 mV. MBN was mixed in the core portion of the core-shell electrospinning scaffold at various concentrations up to 5 wt.% For the PEO core material.

In the SEM photograph of the electrospun fiber, it was confirmed that the fiber structure was successfully formed in all the compositions (Fig. 3). In the electrospinning process, the inner PEO portion condensed on the wall of the outer PCL portion, hollow structure. The SEM cross-section photograph of the fibers (5% MBN) shows the hollow core structure inside (FIG. 4). The XRD pattern of the scaffold containing MBN at various concentrations shows typical PCL peaks with an amorphous pattern of MBN (Figure 5). In the TGA analysis showing the temperature-dependent weight loss pattern, it was confirmed that the nanocomposite component remained similar to that of the initially added MBN (5.014, 2.877 and 1.071 wt% for 5, 3 and 1% MBN, respectively ).

As a result, it has been confirmed that the fiber scaffold prepared through the electrospinning process has a core-shell structure, in particular, a hollow structure in the inner core, and MBN is well mixed in the fiber scaffold.

(3) Physical property test

Physical properties of electrospun fiber scaffolds of various compositions were evaluated using an Instron (Instron 3344) at a cross-head speed of 10 mm / min. For the test, a thin film having a thickness of about 150 to 200 mu m and a width x length of 30 mm x 4 mm was prepared. Tensile loads were applied to each specimen with a gauge length of 10 mm. The stress-strain curves were recorded in the test and the thickness values were recorded as the mean values of the five specimens observed in the SEM photographs of each group. The mechanical properties including tensile, elastic modulus and elongation were calculated from stress strain measurements. Four specimens were tested for each composition.

First, in order to see the stress change due to the mounting, a typical strain curve of the specimen was confirmed (Fig. 7). Mechanical parameters (including tensile strength, elastic modulus and elongation) were analyzed with the strain curves, the tensile strength measured at maximum force before breaking increased with increasing MBN content up to 5%: 0% MBN 1.8 MPa , 1% MBN 4 MPa, 3% MBN 5.9 MPa and 5% MBN 9.1 MPa (FIG. 8). The modulus of elasticity of the fiber was then measured as the initial slope of the stress-strain curve (Fig. 9). The modulus of elasticity also increased significantly with addition of MBN: 0% MBN 4.5 MPa, 3% MBN 13 MPa, 5% MBN 18.7 MPa. 10), elongation at 0% MBN was 620%, but elongation was decreased with increasing MBN content, specifically at 347, 150 and 145% for 1, 3 and 5% MBN, respectively appear.

As a result, the tensile strength and elastic modulus increased and the elongation decreased with increasing MBN content.

(4) Disassembly test

The in vitro degradability of the fibrous scaffold prepared in 37 ° C PBS (pH 7.4) was tested. Each specimen was weighed accurately and immersed in 10 ml PBS for up to 60 days. The solution was replaced every other day, and the specimen was recovered at the predetermined immersion time and rinsed with demineralized water. The dry weight was measured before and after immersion to calculate the percent decomposition. The measurements were normalized to the dry weight of the sample before immersion. Three samples of each group were evaluated, and mean values and standard deviations were recorded. After the decomposition test, the fiber morphology was observed by SEM. Also, changes in the mechanical properties of the specimens during degradation were investigated.

The degree of hydrolysis of the fiber scaffold was observed at 37 ° C in PBS for 30 days, and 5% and 0% MBN were compared for representative. As a result, it was confirmed that the specimen was gradually decomposed from 7 days (initial recording time) to 30 days (Fig. 11). 5% MBN showed higher degradation rate than 0% MBN, 5% MBN degraded about 15% and 0% MBN degraded 5%.

Next, the usual morphology of the sample after decomposition for 14 days was confirmed by SEM (FIG. 12), and it was also confirmed that 5% MBN was further decomposed than 0% MBN.

In conclusion, the fibrous scaffolds were degraded with time in PBS, and the higher the content of MBN, the better the decomposition was.

(5) Apatite formation ability test in SBF

The in vitro apatite formation of the fibrous scaffold at 37 ° C, pH 7.4 simulated body fluid (SBF) was investigated. SBF is similar ionic composition to human plasma ^{(142.0 mM Na +, 5.0 mM} K +, 1.5 mM Mg 2 +, 2.5 mM Ca 2 +, 147.8 mM Cl -, 4.2 mM HCO 3 -, 1.0 mM HPO 4 2 -, 0.5 has mM SO ₄ ^2-). After each sample was immersed in SBF, the formation of calcium phosphate mineral was observed. The sample was collected at each time point and washed thoroughly, and morphological changes were observed by SEM. XRD (X-ray diffraction) and FTIR were used to investigate the phase change and chemical bond structure.

The in vitro bioactivity of fibrous scaffolds was analyzed as the mean of apatite mineralization in SBF. After immersing in SBF for 14 days, samples were identified by XRD and FTIR. The XRD pattern of 5% MBN was found to have a broad peak at 2θ ~ 32 °, a characteristic of weakly crystallized apatite. However, the peak was not detected at 0% MBN (FIG. 13). Furthermore, the FTIR band at 5% MBN showed a significant level of phosphate-related chemical bonds (645 and 1200 cm ^{- 1} ) in apatite, while this band was almost absent at 0% MBN (FIG. 14). SEM photographs showed that apatite nanocrystals almost completely covering the fiber surface were formed well in 5% MBN, but this morphology was not observed in 0% MBN (FIG. 15).

In conclusion, MBN was included in the fibrous scaffolds, indicating that apatite nanocrystals were well formed in SBF, and that the in vitro bone activity was excellent.

Example  3. Identification of protein loading and transfer characteristics

Cytokine C (Sigma-Aldrich, Cat No. 12222) was used as a model protein to identify the target protein loading characteristics. First, 1 mg of MBN was completely dispersed in cytC solution (0 to 0.3 mg / ml) dissolved in PBS at various concentrations to mount cyt C in MBN, and incubated at 37 ° C for 6 hours. To quantify the drug loading, the cytochemical absorbance was measured in the supernatant using a UV-vis spectrometer (Libra S22, Biochrom, UK), centrifuged at 10,000 rpm for 5 minutes and washed once with PBS. The data were then converted to corresponding payloads determined from the standard curve. To prepare MBN / fiber scaffold with cyt C, cytN C-loaded MBN was added to the electrorheological solution. As a comparative group, cyt C was directly added to the core portion of the fiber scaffold without loading with MBN. As a result of loading test, the amount of cyt C added to the core solution was the same as the amount of cyt C loaded on MBN. 100 mg scaffolds were immersed in 5 ml of PBS at 37 占 폚 on scaffolds on which the two types of cytocytes were mounted. At different times (~65 days), the amount of cytC released was determined using a UV-vis spectrometer. PBS was replaced at each test. The cyt C loading appears to indicate the amount of cyt C loaded in the MBN for the adsorption isotherm, the drug concentration initially added to the solution. The cyt C loading was expressed according to the following mass balance equation:

_{q e = (C 0 - C} e) × (V / W), where q _e is the amount of drug (mg) uptake mBGn 1 mg, the initial and the equilibrium concentration of C ₀ and C _e are each cyt C ( mg / ml), V is the volume of the solution (ml) and W is the weight of the MBN used (mg).

q _e vs C _e After plotting the curves, a modified Langmuir isotherm model was used for curve fitting, as follows:

q _e = q _m KC _e / (1 + KC _e ), where q _m is the maximum quantity loaded and K is the unknown parameter (rate constant) that can be determined.

As a result, the loading amount was increased as the cyt C concentration was increased, and the maximum loading amount was about 0.13 mg at 0.4 mg cyt C and the loading capacity of MBN for cyt C was about 13% (FIG. 16). The data fit well to the Langmuir isotherm, shown in dashed lines. The TEM photograph of the maximum post-mount sample of cyt C shows that there is something in the meso-pore structure that is widespread, unlike the TEM photograph of the MBN that is not loaded (Fig. 17). In the BET N ₂ absorption / release curve, it was confirmed that the volume of the mesopores absorbed in the MBN loaded with cyt C was significantly reduced as compared with the MBN not loaded (Fig. 18). In the FTIR spectrum of the MBN loaded with cyt C, the cyt C-related band appeared well (Fig. 19). The characteristics of MBN loaded with cyt C are shown in Table 2 below. The pore size (3.77 nm), pore volume (0.357 cm ³ / g) and surface area (204.9 m ² / g) (-1.32 mV) decreased (compare with Table 1).

TEM 111 ± 7 nm Particle size    Laser scattering 149.6 ± 6.8 nm    Pore size 3.77 nm    Surface area 204.9 m ² / g    Pore volume 0.357 cc / g    Zeta-potential -1.32 ± 3.97 mV

Next, cyt C was loaded on MBN, fiber scaffolds were prepared, and cytochemical release of cyt C was investigated. To investigate the effect of cyt C on the release characteristics ("with MBN" group), a control ("w / o MBN") was prepared by directly mixing cyt C in the core during electrospinning. The amount of cyt C released from different fiber scaffolds was recorded over a long period of time up to 65 days (Figure 20). In the "w / o MBN" group, cyt C was released at a rapid rate (~ 7%) at the beginning (3 days), slowed down to 10 days, and rapidly released again until 20 days. Such an emission pattern was repeated throughout the test period (arrows indicate the point of time of the speed change at each step). The cyt C emission pattern of the "with MBN" group was very similar to the "w / o MBN" group. However, the recorded amount in the fast section was much smaller and the decreasing rate (slope) in the slow section was also lower in the "with MBN" group compared to the "w / o MBN" group. The cyt C release data was re-expressed as the time point at the specific release (5, 10, 15 and 20%) of cyt C (FIG. 21). The "with MBN" group took longer to emit than the "w / o MBN" group, and the time difference between the two groups increased as the emissivity increased. Thus, mixing of two growth factors, FGF-2 and FGF-18, with core-shell fibers (FGF-2) and MBN (FGF-18) results in the sequential release of FGF- It can be expected that delivery will be possible.

Example  4. Confirm growth factor transfer effect

(1) Isolation and culture of rat bone marrow MSC

The male and female adult Sprague-Dawley rats (180 to 200 g) were cut into the body side and the distal wide bone and bone marrow, and α-MEM (α-minimal essential medium) containing Dysparease II and Type II cholerase . The tissues were centrifuged and resuspended in a general culture medium (α-MEM containing 10% FBS, 1% penicillin / streptomycin) and cultured in a 5% CO ₂ incubator at 37 ° C. The medium was replaced once every 3 days, and the non-adherent hematopoietic cells were removed during the exchange, and the cells were used for the experiment after passage 3. Bone production inducers including 50 ug / ml sodium ascorbate, 10 mM [beta] -glycerol phosphate and 10 nM dexamethasone were used in the test procedure to promote cellular responses.

As a test group for the cell experiment, five groups of five culture groups, namely, a culture container without any additives, an FGF-2 addition culture container, an FGF-18 addition culture container, a fibrous scaffold and a fibrous scaffold carrying FGF-2 / FGF- Respectively. MSC responses to FGF-2 / FGF-18 loaded fibrous scaffolds were compared to scaffolds without growth factors. In addition, a scaffold-free culture container was used as a control. The amounts of FGF-2 or FGF-18 were used in the same amounts as recorded in the FGF-2 and FGF-18 release tests of the fiber scaffolds.

(2) Evaluation of cell proliferation by mounting growth factors (FGF-2 and FGF-18)

Based on the cyt C loading results, two growth factors, FGF-2 and FGF-18, were loaded into the fiber scaffold. Initially, FGF-2 was treated to promote cell proliferation, and FGF-18 was used as a means to improve bone formation in the later stages. To achieve this goal, FGF-2 was directly loaded into the core of the hollow fiber for rapid delivery, and FGF-18 was loaded onto the MBN for more sustained delivery. The loading concentration of each growth factor was based on the cyt C release profile preliminary experiment. FGF-2 / FGF-18-loaded fibrous scaffolds prepared by electrospinning were used for cell and in vivo experiments.

First, MSC proliferation was first assessed to determine the biological effects of delivering FGF-2 / FGF-18 as a scaffold. MSC proliferation was confirmed by CCK analysis while MSC was cultured on a scaffold with or without FGF-2 / FGF-18 for 14 days. As a result, it was confirmed that the cell proliferation significantly increased in the scaffold carrying FGF-2 / FGF-18 in each incubation period (left side in FIG. 22). It can be seen that when MSC was cultured in a culture vessel, the cell proliferation was increased particularly when FGF-2 was added (Fig. 22, right). In view of this aspect, FGF-2 was released from the scaffold FGF-18 is not released.

(3) Confirmation of ALP activity

Next, the activity of ALP, an early bone differentiation marker, was confirmed, and bone differentiation of cells was evaluated. After culturing, the cells were collected by trypsinization and the cells were lysed with a lysis buffer. For the next enzyme reaction, an ALP reaction solution (Sigma-Aldrich) was added to the sample according to the manufacturer's instructions. The amount of sample to be added was determined according to the total protein content measured with a DC protein analysis kit (BioRad), and p-nitrophenol production was measured at 405 nm absorbance. The experiment was repeated three times for each condition.

As a result, when comparing the scaffolds with or without FGF-2 / FGF-18, it was confirmed that the FGF-2 / FGF-18-loaded group exhibits a significantly higher ALP level in all the groups ). Control experiments performed in culture vessels also showed that FGF-18 was effective in increasing ALP activity, while FGF-2 only had some effect (Figure 23 right).

(4) Confirmation of bone related gene expression

Expression of bone-associated genes including OPN (osteopontin), Col1 (collagen type 1) and ALP (alkaline phosphatase) was quantitatively confirmed by RT-PCR. Total RNA was isolated from each sample using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Next, the RNA was reverse-transcribed using a Superscript kit (Invitrogen) with a random hexamer as a primer. Then, PCR amplification was performed using Sensimix plus SYBR Mater Mix (Quantace). The comparative CT method was used to standardize and analyze the amplified PCR products of each gene with the how skipping gene GAPDH. PCR was carried out for 40 cycles of denaturation (95 ° C for 15 seconds), annealing (58 ° C for 30 seconds), and extension (72 ° C, 15 seconds), starting with an activation step of 95 ° C for 10 minutes according to the manufacturer's instructions. The primers used are shown in Table 3 below:

gene order SEQ ID NO: GAPDH (forward) 5'-GGCAAGTTCAACGGCACAGT-3 ' SEQ ID NO: 1 GAPDH (reverse) 5'-CGCTCCTGGAAGATGGTGAT-3 ' SEQ ID NO: 2 ALP (forward) 5'-ACTGGTACTCGGACAATGAG-3 ' SEQ ID NO: 3 ALP (reverse) 5'-ATCGATGTCCTTGATGTTGT-3 ' SEQ ID NO: 4 OPN (forward) 5'-CCAAGCAACTCCAATGAAAGC-3 ' SEQ ID NO: 5 OPN (reverse) 5'-TCCTCGCTCTCTGCATGGT-3 SEQ ID NO: 6 Col1 (forward) 5'-CGTGACCAAAAACCAAAAGT-3 SEQ ID NO: 7 Col1 (reverse) 5'-GGGGTGGAGGAAAGGAACAGA-3 ' SEQ ID NO: 8

As a result, the level of gene expression was significantly increased in scaffolds containing FGF-2 / FGF-18 compared to scaffolds lacking FGF-2 / FGF-18. Especially, expression of ALP and OPN genes on day 6 (Fig. 24).

(5) Confirmation of cell mineralization

Cellular mineralization was confirmed by Alizarin Red S (ARS) staining. Cells were washed with PBS and fixed with 4% formaldehyde for 10 minutes. After washing, each sample was incubated at room temperature for 10 minutes in distilled water containing 2% (w / v) ARS solution (Sigma Aldrich, pH 4.1-4.3). The staining solution was then removed and the specimens were washed with distilled water. Dyed specimens were identified by taking pictures with a digital camera (EOS 1000D, Canon).

Further, to quantify the mineralization, the specimens were incubated in a 10 mM sodium phosphate solution (pH 7) containing 10% (w / v) cetylpyridinium chloride for 1 hour and then the reaction product was eluted And the absorbance was measured at 595 nm using a microplate reader. The experiment was repeated three times.

ARS staining images showed a clear difference between the groups, that is, the scaffolds loaded with FGF-2 / FGF-18 had a darker red color than the scaffolds without growth factors (Fig. 25, left). A quantitative analysis of the eluted products confirmed statistically significant differences between the groups (Fig. 25 right).

In summary, the inventive FGF-2 / FGF-18-loaded scaffolds release FGF-2 at the early stage of transplantation to promote cell proliferation, and FGF-18 is released relatively slowly, .

Example  5. Verification of bone formation ability in vivo

(1) Transplantation in two open-defect models in rats

In vivo bone formation capacity of fibrous scaffolds was evaluated in a two-open defect model in rats. First, 10-week-old male Sprague-Dawley rats were raised in a humidified and temperature controlled environment under 12 h light / dark conditions, and feed and water were randomly provided. Anesthesia was performed intramuscularly by mixing ketamine (80 mg / kg) and xylazine (10 mg / kg). Then, the skin was cut linearly in the center of the two openings and the periosteum was lifted. Then, the saline solution was continuously sprayed and a 5 mm diameter bone defect was made in the center of each bone by a surgical knife. Scaffolds with or without FGF-2 / FGF-18 were randomly transplanted into the defect. The subcutaneous tissue was then covered with an absorbable material and the incision site was sealed with the non-absorbable material.

(2) Analysis of bone formation

After the transplantation, the rats were each raised in a cage and observed for side effects such as infection and inflammation during the experiment. Six weeks after transplantation, the rats were sacrificed to obtain both openings, the skin was incised, and tissues including the surgical site and surrounding tissue were recovered. The tissues were fixed with 10% buffered neutralized formalin for 24 hours at room temperature and subjected to microcomputed tomography (" CT ") analysis and tissue analysis.

After fixation, μCT pictures were taken to analyze new bone formation. Specifically, the recovered specimen was scanned with a 12.56 μm camera pixel size using a μCT scanner (Skyscan 1176; Skyscan, Aartselaar), with a 1 mm aluminum filter, 0.5 ° rotation step and 180 ° rotation angle, Respectively. The cylindrical region of interest (ROI) was located at the center of the single defect completely covered with the new bone. Percentage (%) of new bone volume and surface density (1 mm ^-1 ) of newly formed bone in each ROI were determined by using CTAn software (Skyscan) to specify thresholds for total bone content (including cancellous bone and cortical bone) .

Following μCT imaging, specimens were prepared for histological analysis. The fixed sample was decalcified with RapidCal ( ^TM) solution (BBC Chemical Co., Stanwood, WA), dehydrated gradually (70% to 100%) and then embedded with paraffin. The center of the circular defect was cut into 5 ㎛ thick and defined as representative tissue. Slides were stained with H & E (hematoxylin & eosin) in a conventional manner and examined using an optical microscope (IX71, Olympus, Tokyo).

The fibrous scaffolds (with or without FGF-2 / FGF-18) were transplanted for 6 weeks in the open defect areas of the rats and the osteogenic potential was assessed. , And the opaque portion indicates hard tissue formation (Figure 26). In the control group, hard tissues were hardly formed in the defect area except for a small level of bone at the edge, whereas the two scaffold groups formed a hard tissue toward the defect site, and some tissues were formed at the center there was. Comparing the scaffold groups, it can be seen that when FGF-2 / FGF-18 was loaded, the hard tissue growing inside increased considerably.

The μCT photographs were quantified to analyze bone indices including bone volume and bone surface density (FIGS. 27 and 28). Bone volume was significantly increased in the scaffold, especially in the growth factor-loaded scaffolds: control group 13%, scaffold alone 22%, FGF-2 / FGF-18 scaffold 35% or more. Bone surface density was also the same.

The specimens collected at 6 weeks were stained with H & E and MT (Masson's trichrome), and histological photographs were taken (FIG. 29). As a result, inflammation was scarcely observed in all groups. In the low magnification photographs, the control group showed almost no bone formation toward the defect site (FIG. 29A). In both scaffolds (FIGS. 29B and 29C) (Fig. 29 (b) and 29 (c)). This tendency was further observed in MT staining photographs. In high-magnification photographs, new bone area can be observed in a scaffold carrying FGF-2 / FGF-18, and calcification with multiple osteogenic cells and various blood vessels And bone cells surrounded by the bone matrix. The new bone showed an immature woven bone structure and a mature lamellar structure was not seen.

In conclusion, the fibrous scaffold carrying the FGF-2 / FGF-18 of the present invention showed excellent effect on bone regeneration in vivo without inducing inflammation when transplanted into the bone defect model.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> Multiple-drug loaded scaffolds and use thereof <130> KPA150109-KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 1 ggcaagttca acggcacagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 2 cgctcctgga agatggtgat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP-F <400> 3 actggtactc ggacaatgag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP-R <400> 4 atcgatgtcc ttgatgttgt 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OPN-F <400> 5 ccaagcaact ccaatgaaag c 21 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> OPN-R <400> 6 tcctcgctct ctgcatggt 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1-F <400> 7 cgtgaccaaa aaccaaaagt 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Col1-R <400> 8 ggggtggagg aaaggaacag a 21

Claims (23)

A scaffold having a Core-Shell fiber structure comprising a biopolymer core containing a vitriol nanoparticle loaded with FGF-18 as a first drug and FGF-2 as a second drug,
And polyethylene oxide (PEO) as a component of the biopolymer core,
And polycaprolactone (PCL) as a component of the shell of the scaffold,
And sequentially discharges the first drug and the second drug.
delete The scaffold of claim 1, wherein the vitreous silica nanoparticles have a Ca: Si molar ratio of 1: 4 to 1: 9.
The scaffold of claim 1, wherein the vitreous silica nanoparticles have a diameter of 100 to 140 nm.
The scaffold of claim 1, wherein the core-shell fiber structure has a hollow structure therein.
delete delete The scaffold of claim 1, wherein the vitrified glass nanoparticles are comprised between 1 and 10 weight percent of the biopolymer core.
The scaffold of claim 1, wherein the polyethylene oxide has a molecular weight of from 50,000 to 70,000.
The scaffold of claim 1, wherein the polycaprolactone has a molecular weight of from 70,000 to 90,000.
delete The scaffold of claim 1, wherein the biopolymer core further comprises at least one drug.
A pharmaceutical composition for bone regeneration comprising the scaffold of any one of claims 1, 3 to 5, 8 to 10, and 12.
(a) mixing the calcium oxide precursor and the silica precursor to produce vitrified glass nanoparticles;
(b) mixing the vitrified glass nanoparticles with FGF-18 as a first drug to prepare a vitreous glass nanoparticle loaded with a first drug;
(c) injecting a shell component into a syringe-like device external injection section, and injecting the biomolecule core with bioactive glass nanoparticles loaded with the first drug into an internal injection section and FGF-2 as a second drug Forming core-shell fibers by injecting a Core component; And
(d) recovering and drying the prepared fibers, the method comprising the steps of:
Wherein the biopolymer core component comprises polyethylene oxide (PEO)
The shell component of the scaffold comprises polycaprolactone (PCL)
Wherein the first drug and the second drug are sequentially released.
The method of claim 14, wherein the calcium oxide precursor is selected from the group consisting of tetrahydrate calcium nitrate (Ca (NO ₃ ) ₂ 4H ₂ O), calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄ ), and calcium methoxyethoxide calcium methoxyethoxide). &lt; / RTI &gt;
15. The method of claim 14, wherein the silica precursor is selected from the group consisting of tetraethyl orthosilicate, tetraethyl orthosilicate (TEOS), trimethoxy orthosilicate (TMOS), 3-glycidoxypropyl methyl diethoxysilane, 3-mercaptopropyl trimethoxysilane (MPS) GOTMS (? -Glycidyloxypropyl trimethoxysilane), and APTMOS (aminophenyl trimethoxysilane).
15. The production method according to claim 14, wherein a molar ratio of Ca: Si of the vitrified glass nanoparticles is 1: 4 to 1: 9.
15. The production method according to claim 14, wherein the biopolymer core component is dissolved in distilled water and injected, and the shell component is dissolved in a mixed solution of chloroform and dimethylformamide to be injected.
delete delete 15. The method of claim 14, wherein the biopolymer core further comprises at least one drug.
15. The method of claim 14, wherein the step of fabricating the core-shell fibers is performed by performing coaxial electrospinning.
23. The method of claim 22, wherein the feed rates for the shell and core portions of the coaxial electrospinning are 3.0 to 5.0 ml / h and 0.3 to 0.7 ml / h, respectively.

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