KR20160075981A

KR20160075981A - Nano-patterned patch for bone regeneration and the method for preparing the same

Info

Publication number: KR20160075981A
Application number: KR1020140184815A
Authority: KR
Inventors: 양희석; 나완근; 이민석
Original assignee: 단국대학교 천안캠퍼스 산학협력단
Priority date: 2014-12-19
Filing date: 2014-12-19
Publication date: 2016-06-30

Abstract

The present invention relates to a bone regeneration method, which comprises a first layer comprising a biodegradable polymer, and a second layer formed on one side of the first layer and including a fibrin gel, wherein a nano pattern is formed on one side or both sides of the first layer, , A method for producing the same, and a method for preventing or treating skeletal system diseases using the same.

Description

[0001] Nano-patches for bone regeneration and methods for preparing the same [0001]

The present invention relates to a bone regeneration method, which comprises a first layer comprising a biodegradable polymer, and a second layer formed on one side of the first layer and including a fibrin gel, wherein a nano pattern is formed on one side or both sides of the first layer, And a method for producing the same.

Reconstructive surgery through bone regeneration has been extensively performed in dentistry, orthopedics, and plastic surgery to restore the inherent, acquired damaged bone structure (Schwartz-Arad D. Implant Dent., 14, 131-138, 2005). Currently, the materials used for bone regeneration are classified as osteogenesis, osteoconduction, and osteoinduction depending on the osteogenesis capacity (Lee, Seo-Kyung, Journal of Korean Periodontology, 38, 125 -134, 2008).

The bone-forming material is a material that can make bone by itself by preparing cells and growth enzymes necessary for bone regeneration, and this is the autogenous bone. In contrast, the bone conduction material is absorbed after serving as a scaffold for bone regeneration, and includes various allografts, xenografts, xenografts, and the like. The bone-inducing substance is a material capable of differentiating mesenchymal cells into bone cells to produce bone. The bone-inducing substance was originally confined to the DBM (Demineralized Bone Matrix). However, recently, various growth factors have been studied and applied so that not only bone differentiation but also vascularization and macrophage function involved in bone healing . In 2001, Clokie and Sandor et al. Defined osteoactive agents as bone morphogenetic proteins (BMP) (Sandor G, Clockie C, Urist M et al. J Craniofac Surg, 12 (2), 119-127, 2001).

BMP has been approved by the US FDA and has been commercialized, and various attempts have been made to easily apply it to clinical practice. However, BMP has been reported to increase the incidence of cancer in transplanted patients, and there are side effects when using overdose.

In addition, in the case of bone regenerating materials containing calcium currently being used, the degradation is not accelerated and the engraftment with the regenerated bone is lowered, resulting in a problem that the mechanical strength of the regenerated bone is weakened. Thus, safe bone regeneration materials are still required by minimizing the implantation of artificial material.

Under these circumstances, the present inventors have made intensive studies to develop a method for effectively treating bone defects while minimizing the implantation of artificial materials. As a result, it has been found that a biodegradable polymer scaffold and a fibrin gel, It is possible to effectively treat bone defects without drug delivery or external stimulation. Thus, the present invention has been completed.

The main object of the present invention is to provide a biodegradable polymer composition comprising a first layer comprising a biodegradable polymer and a second layer formed on one side of the first layer and including fibrin gel, , And a support for bone regeneration.

It is another object of the present invention to provide a method of manufacturing a semiconductor device, which comprises: a pattern formation step of placing a mold having nanopatterns formed on one side or both sides of a first layer including a biodegradable polymer and applying pressure in the direction of the first layer from the mold direction to form a nanopattern; And a binding step of binding the first layer and the second layer containing the fibrin gel to each other, and a method for producing a support for bone regeneration.

Yet another object of the present invention is to provide a method for preventing or treating skeletal system diseases comprising the step of transplanting a scaffold for bone regeneration to a bone defect site of a subject other than a human.

According to one aspect of the present invention, there is provided a biodegradable polymer comprising a first layer comprising a biodegradable polymer and a second layer formed on one side of the first layer and including fibrin gel, And a nano pattern is formed on one side or both sides of the support.

Conventionally, materials used for bone regeneration have been reported to cause side effects such as inflammation or cancer in the implanted material, and the density of regenerated bone is so small that the mechanical strength is insufficient. Therefore, by implanting the biodegradable polymer scaffold and nano-patterned biodegradable polymer scaffold and the fibrin gel into the bone defect site, the implantation of the artificial material is minimized and the proliferation and migration of the surrounding bone tissue are promoted, It was confirmed that bone density could be increased by regeneration. That is, the inventors of the present invention were the first to confirm that there is an effect of bone regeneration by the support itself, which is not equipped with a substance or medicament constituting the bone.

The term " bone regeneration "in the present invention refers to promoting the migration or proliferation of osteocytes to a bone site requiring a defect or repair, and may be used in the same sense as bone defect treatment in this specification.

The support of the present invention means a structure which can maintain a solid form and adhere to the skin, and provides a contact surface with the implantation site. The bone regeneration support of the present invention comprises a first layer containing a biodegradable polymer and a second layer formed on one side of the first layer and including fibrin gel.

The biodegradable polymer is a polymer which is decomposed in vivo without generating harmful substances in vivo. The biodegradable polymer is capable of forming a support for the purpose of the present invention and is capable of forming nanopatterns. Can be used without. As a non-limiting example, the biodegradable polymer may be selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), poly-epsilon -caprolactone (PCL), poly-lactic acid (PLLA), polylactic acid- PLGA), polylactic acid-caprolactone copolymer (PLCL), biodegradable polycarbonate, and copolymers thereof. Since the biodegradable polymer is decomposed into water and carbon dioxide in the body, there is no need to remove the biodegradable polymer after attachment or transplantation, and the biodegradable polymer is safe and has excellent durability. The biodegradable polymer may be chemically synthesized or produced from microorganisms.

The fibrin gel constituting the fibrin gel layer is meant to include fibrinogen and thrombin. Fibrin gels are materials used in cardiovascular system operations for hemostasis, suture aids, and tissue adhesion, and can be degraded by hydrolysis or proteolysis. According to one embodiment of the present invention, the fibrin gel may contain fibrinogen and thrombin in the same volume ratio.

Since the bone regeneration support of the present invention can exhibit an excellent bone regeneration effect compared to the fibrin gel alone or the biodegradable support alone (Figs. 4A to 5B), it can be implanted in the bone defect site to treat bone defect.

Meanwhile, the conventional scaffold for grafting is composed of a substance which is not decomposed in vivo, such as polyurethane acrylate (PUA) which is a non-degradable substance, and thus can not be transplanted into a human body or an animal. There is a problem that it is necessary. There is a problem in that it may cause inflammation or the like by the non-degradable residue even if it is removed. On the other hand, it was confirmed that the bone regeneration support of the present invention can exhibit excellent biocompatibility and biodegradability by using fibrin and biodegradable polymer, and that the biodegradability is improved compared to a support having no pattern formed by forming a nanopattern 6).

In the present invention, the nanopattern is formed on the first layer containing the biodegradable polymer, and is formed of a groove and a groove in the interval of nano unit. For example, the spacing of the nanopatterns, that is, the ridges and ridges of the nanopatterns, or the distance between the grooves and the grooves may be 400 nm to 10 μm. In another example, the nanopatterns may have different spacings of the patterns. The spacing of the patterns can be adjusted by forming the spacing of the patterns formed on the mold to be used differently.

The nano pattern may be formed on one side or both sides of the first layer. When formed on both sides, since it has a larger surface area, it is excellent in bone regeneration effect and biodegradation effect.

The first layer and the second layer may be produced using any method in the art to produce a solid polymer. For example, the biodegradable polymer may be prepared by applying pressure to a polymer dissolved in an organic solvent, and then removing the organic solvent by heating, drying, or the like. The organic solvent may be chloroform, but is not limited thereto.

The first layer may be included in the support at a ratio of 0.000001 to 0.1 volume based on the volume of the second layer. If the volume ratio is more than 0.1, the biodegradability may be lowered.

The support is not affected by the size and volume of the bone defect site. That is, the support of the present invention can be manufactured and implanted irrespective of the size and volume of the bone defect site.

According to another aspect of the present invention, there is provided a method of manufacturing a semiconductor device, the method comprising: pattern formation step of placing a mold having nanopatterns formed on one side or both sides of a first layer including a biodegradable polymer and applying a pressure in a direction of the first layer in a mold direction to form a nanopattern; And a binding step of binding the first layer and the second layer comprising the fibrin gel to the support.

The biodegradable polymer, nanopattern, fibrin gel, bone regeneration, and supporter may be used as described above.

In the first step, a pattern forming step is a step of placing a mold having nanopatterns formed on one side or both sides of a first layer containing a biodegradable polymer and forming a nano pattern by applying pressure in the direction of the first layer from the mold direction . The pattern formation may be performed using capillary force lithography (CFL) by gravity and capillary force.

The capillary force lithography refers to a method of forming a pattern by raising a polymer to an empty space of a mold using a natural force called a capillary action. In an embodiment of the present invention, capillary force lithography is performed by placing a mold on a biodegradable polymer polymer as shown in FIG. 1A, applying gravity and capillary forces together by applying pressure, and then applying capillary force lithography The nanopattern can be formed more finely and finely. The capillary force may be, for example, capillary depression (θ> 90 °). The method of applying the pressure can be used without limitation as long as it is a method used in pattern formation in the art.

For example, the nanopattern may have the same shape as the pattern of the mold. This is due to the principle of capillary force lithography.

The first step may be one which does not use light irradiation as an example. That is, the manufacturing method may be a method other than a method using light irradiation. The production method according to the present invention is suitable for patterning a biodegradable polymer because it does not require light irradiation such as UV irradiation, and the biodegradable polymer can be easily cured by applying pressure without light irradiation.

As another example, the first step may be a PUA mold. PUA (Young? Modulus: 100-400 MPa) molds have a higher Young's modulus than PDMS (Young? Modulus: MPa) molds to produce finer patterns when manufacturing nano-sized patterns using capillary force lithography . On the other hand, in the case of a conventional PDMS mold, it is difficult to manufacture the pattern interval up to 400 nm. Accordingly, the support for bone regeneration of the present inventor may be one prepared by using a PUA mold.

The pattern forming step may be to form a nanopattern with an interval of 400 nm to 10 μm. According to the manufacturing method of the present invention, more detailed nanopatterns can be formed as compared with the general method, and thus bone regeneration effect and biodegradation ability can be further improved.

The support for bone regeneration according to the present invention has a better bone regeneration effect by promoting the migration of peripheral bone cells in accordance with the direction of the nanopattern as compared with a support having no nanopattern formed by forming a nanopattern (FIG. 3) And the resolution was also improved (FIG. 6).

And the second step means a bonding step of bonding the first layer and the second layer comprising fibrin gel. The method of combining the first layer and the second layer can be used without limitation as long as it is a method used in the art to bond or adhere two or more kinds of polymers in the art unless the first layer or the second layer is completely decomposed .

In another aspect, the present invention provides a method of preventing or treating a skeletal system disease comprising implanting the support for bone regeneration into a bone defect site of an individual.

The support may be implanted in the body using any method used in the art for implantation of the scaffold in the art without limitation.

The bone is the hardest tissue that maintains stability of the skeletal system, and the skeletal disease may mean bone disease and cartilage disease.

As a non-limiting example, the bone disease may be selected from the group consisting of systemic bone diseases including osteoporosis, nonunion / delayed union, pseudoarthrosis, osteonecrosis, Paget's disease, Including osteogenesis imperfecta, and other fractures. &Lt; Desc / Clms Page number 2 >

In the present specification, osteonecrosis can be used to mean ischemic necrosis or avascular necrosis. Jaw arthritis refers to a condition in which the bones of the fracture site are not fully united due to congestion after fracture, poor fixation, bacterial infection of the fracture site, and so on. Paget's disease is a metabolic disorder caused by the bone regeneration process in the elderly. It is a local bone disease involving a wide range of skeletal system. It is a mosaic of bone formation, which is accompanied by fibrosis and angiogenesis, and malformation and swelling. Osteoporosis is defined as a congenital rare genetic disorder in which the bone is weak without a specific cause and is fractured by sneezing or gently bumping.

For example, the cartilage diseases include osteoarthritis, cartilage partial layer defects, osteochondral defects, osteochondritis dissecans, chondromalacia, osteoarthritis, osteoarthritis, , Meniscus injury (meniscus injury), and the like.

In the present invention, the term "individual" may mean all animals including humans who have developed or are likely to develop a skeletal disease. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject suffering from or at risk of developing a skeletal system disease.

The support for bone regeneration according to the present invention can effectively treat bone defects without loading drugs or cells and stimulation from the outside and can be used as a safe implant by minimizing an artificial material. Therefore, the support for bone regeneration according to the present invention can effectively prevent or treat skeletal diseases.

FIG. 1A shows a process for producing a biodegradable support (BNP) having nanopatterns according to an embodiment of the present invention, and FIG. 1B shows formation of nanopatterns of a biodegradable support having nanopatterns formed thereon.
FIG. 2 shows a process of manufacturing a support (BNP-FG) combined with a fibrin gel according to an embodiment of the present invention, and a process of implanting the bone-deficient animal model.
Fig. 3 shows the effect of promoting cell migration by the bone regeneration support.
FIG. 4A shows the bone regeneration effect in the bone defect model, and FIG. 4B shows the numerical value of FIG. 4A.
FIG. 5A shows the bone regeneration region by Goldner trichrome staining, and FIG. 5B shows the FIG.
FIG. 6 shows the excellent biodegradability of a biodegradable support (BNP) having nanopatterns according to an embodiment of the present invention.
In the figure, FG refers to fibrin gel, BFP refers to a biodegradable flat patch on which no nanopattern is formed, and BNP refers to a biodegradable nanopatterned patch on which a nanopattern is formed. The results of the above experiment were expressed as mean ± standard deviation, and statistical analysis was carried out through ANOVA and p <0.05 was considered significant (*)

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1. Biodegradable support with nanopattern ( BNP )

First, a PDMS (Polydimethylsiloxane) solution, a SYLGARD 184 (SILICONE ELASTOMER BASE, Dow corning) and a curing solution (SYLGARD 184, SILICONE ELASTOMER CURING AGENT, Dow corning) were stirred at a ratio of 10: 1 The PDMS cushion was prepared by curing in a 60 ° C oven for 12 hours or more by pouring into a 100 mm diameter Patri dish.

On the other hand, as shown in FIG. 1A, on the top of a cover glass (glass is also shown in the drawing) prepared by immersing in isopropyl alcohol and cleanly cleaned using an ultrasonic sonicator for 30 minutes, 30 μl of polymer dissolved in chloroform at a concentration of 15% w / v was applied. Thereafter, the prepared PDMS cushion was fixed above and below the cover glass to uniformly position the polymer solution on the cover glass. A weight of 500 g was placed on the top of the cover glass, and the solvent contained in the polymer was absorbed into the PDMS at room temperature for 10 minutes. After the absorption of the solvent was adequately carried out for 10 minutes, only the upper PDMS was removed, and chloroform remaining in the PLGA solution was vaporized and removed on the hot plate at 120 DEG C for 10 minutes to prepare a PLGA patch (represented by PLGA in the drawing).

On the other hand, a PUA mold was manufactured by the following process: 100 P of PUA solution was applied to a silicon wafer master having a nano pattern, and a polyethylene terephthalate (PET) film was fixed on the solution to spread the solution evenly. The silicon wafer master with the PET film attached thereto was exposed to UV light for 40 seconds and removed, and the PET film was removed from the silicon wafer master. The silicon wafer master was then removed and then treated overnight with UV light.

The prepared PUA mold was fixed on a PLGA patch, and a PDMS cushion was fixed thereon. Thereafter, a weight of 1200 g was placed on the top and pressure was applied for 15 minutes (capillary force lithography). After the weight was removed, the solvent was removed from the vacuum oven for another 24 hours to prepare a biodegradable nanopatterned patch (BNP) having a nanopattern.

To confirm that the pattern was well formed, the BNP was observed with an optical microscope.

FIG. 1 (b) shows the PUA mold, and FIG. 1 (b) shows the BNP produced by the observation under an optical microscope. When pattern formation is successful, iridescence appears as shown in (a) and (b) above. Therefore, it was confirmed that the pattern of the same size as the PUA mold was formed on the whole BNP by the above-mentioned manufacturing method.

The BNP was sputter coated with platinum (Pt) to a thickness of 5 nm and SEM (scanning electronic microscopy) images were obtained using a Hitachi S-4800 microscope (Hitachi, Tokyo, Japan).

Figure 1 (b) shows an SEM image of BNP. Unlike (d) and (c), which showed no patterned PLGA, it was confirmed that the pattern was uniformly formed on the surface.

Example 2. Fibrin gel and Combined Support ( BNP - FG ) Produce

A tubular mold was placed on the BNP prepared in Example 1 in accordance with the size of a mouse skull diameter of 4 mm to be used as a bone defect model. Fibrin gel and thrombin solution were mixed in a ratio of 1: 1 (v / v) (Fig. 2). The height of the support (BNP-FG) combined with the fibrin gel was 1 mm.

Experimental Example One. BNP - FG Observation of cell migration

In order to confirm whether BNP-FG promotes cell migration, a rat osteoblast was isolated from the mouse bone of a newborn mouse and cultured on a support for bone regeneration prepared in Example 2 Respectively. The results of observation for 48 hours are shown in Fig.

At 0 hour (h) in Fig. 3, the outermost side of the white dotted line indicates the side on which the cells were cultured, and the inside side refers to the side where the cells were not cultured. According to the pattern direction formed on the inner surface (indicated by a white arrow (↔)), Vertical indicates that the horizontal patterns are arranged vertically, and Parallel indicates that the vertical patterns are arranged horizontally. And a case where no pattern was formed on the inner surface was expressed as Flat. As a negative control, TCPS (Tissue Culture Polystyrene), which is generally used for cell culture, was used.

As a result, BNP-FG has the effect of improving the cell migration ability without any drug treatment or physical stimulation, and it has been confirmed that the cell migration rate is 10 times or more higher than that in the case of no pattern (Flat).

In addition, it did not interfere with cell proliferation even when cultured in BNP-FG as in the case of the existing material (TCPS).

Experimental Example 2. Implanted in animal bone defect model BNP - FG Check the effect of

A bone defect model with a diameter of 4 mm was made in the mouse skull (Fig. 2), and the BNP-FG prepared in Example 2 was transplanted. After 8 weeks, the degree of bone regeneration was analyzed using micro-CT.

As a result, as shown in FIG. 4A and FIG. 4B, BNP-FG conjugated with a biodegradable support (BNP) having a nanopattern formed on the fibrin gel was not transplanted with fibrin gel (FG) , The bone regeneration effect was about 80%. This is 2.5 times better than BFP-FG without nanopattern.

The bone regeneration site was confirmed by Goldner trichrome staining (FIG. 5A and FIG. 5B), and the results were similar to those of the CT results.

The results were expressed as mean ± SD. Statistical analysis was performed using ANOVA and p <0.05 was considered significant (*).

These results indicate that the bone regeneration support BNP-FG of the present invention, to which a biodegradable support having a nanopattern formed in the fibrin gel, is bonded, is excellent in bone regeneration.

Experimental Example 3. Support ( BNP ) Resolution

BIP (Biodegradable Nanopatterned Patch) of Example 1 and BIP (Biodegradable Flat Patch) prepared in the same manner were immersed in a solution containing enzyme (trypsin, 0.05%) at the same weight, except that the nanopattern was not formed , And the weight change of each support was measured at intervals of one week.

As a result, as shown in FIG. 6, it was confirmed that the resolution increased after the 2nd week compared to the case where the pattern was formed (BNP) and the case where the pattern was not formed (BFP). Accordingly, since the biocompatibility is excellent, the bone regeneration support according to the present invention can be effectively used for implantation in the body.

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 above-described embodiments are to be considered in all respects as illustrative 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 scope of the present invention as defined by the appended claims.

Claims

1. A bone regeneration support for bone regeneration comprising a first layer comprising a biodegradable polymer and a second layer formed on one side of the first layer and including fibrin gel and having nanopatterns formed on one side or both sides of the first layer, .

The method according to claim 1,
Wherein the biodegradable polymer is selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), poly-epsilon -caprolactone (PCL), poly-lactic acid (PLLA), polylactic acid- Wherein the support is at least one member selected from the group consisting of caprolactone copolymer (PLCL), biodegradable polycarbonate, and copolymers thereof.

The method according to claim 1,
Wherein the fibrin gel comprises fibrinogen and thrombin in the same volume ratio.

The method according to claim 1,
Wherein the nano-pattern interval is 400 nm to 10 mu m.

The method according to claim 1,
Wherein the support is implanted in a bone defect site.

A pattern forming step of placing a mold having nanopatterns formed on one side or both sides of a first layer including a biodegradable polymer and applying a pressure in a direction of the first layer in a mold direction to form a nanopattern; And
A method for manufacturing a support for bone regeneration according to claim 1, comprising a binding step of binding the first layer and a second layer comprising fibrin gel.

The method according to claim 6,
Wherein the fibrin gel comprises fibrinogen and thrombin in the same volume ratio.

The method according to claim 6,
Wherein the pattern formation step uses capillary force lithography.

The method according to claim 6,
Wherein the pattern formation step forms a nanopattern at intervals of 400 nm to 10 μm.

A method for preventing or treating a skeletal system disease, comprising transplanting a scaffold for bone regeneration according to any one of claims 1 to 5 to a bone defect site of an individual other than a human.

11. The method of claim 10,
The skeletal system diseases include osteoporosis, osteoarthritis, pseudoarthrosis, osteonecrosis, Paget's disease, osteogenesis imperfecta, fractures, osteoarthritis, cartilage part Wherein the disease is any one selected from the group consisting of osteochondral dissociation, osteochondral defect, osteochondritis dissecans, chondromalacia, and meniscus injury. A method of preventing or treating a disease.