KR20150007273A - Antibiotics and osteoinductive molecules-eluting implant or scaffold with enhanced osteointegration and antibacterial activity and the manufacturing method thereof - Google Patents

Abstract

Provided in the present invention are an implant or a scaffold eluting antibiotics and an osteoinductive molecules, which prevent bacterial infection after transplanting orthopedic or dental implant or scaffold, and have both osteointegration and osteoinduction activities; and a method for manufacturing the same. Provided in the present invention are an implant or scaffold having the surface modified by using a composite of a compound containing a polymer-catechol functional group. The implant or scaffold includes one or more kinds selected from antibiotics and osteoinductive molecules.

Description

TECHNICAL FIELD [0001] The present invention relates to an antibiotic and an osteogenesis promoting material-releasing implant or scaffold having improved bone fusion and antimicrobial activity, and an osteoinductive molecule-eluting implant or scaffold with enhanced osteointegration and antibacterial activity and the manufacturing method thereof.

The present invention relates to functional implants or scaffolds capable of preventing bacteria infection after orthopedic or dental implant or scaffold implantation and at the same time inducing osseointegration and osteogenesis, and a method for manufacturing the same.

An implant or a scaffold is a biomedical medical device which is implanted in vivo and exhibits a desired function. Therefore, the implant or scaffold must have mechanical strength to withstand repeated loads and transient pressures, and must meet biocompatibility, chemical compatibility, and other conditions.

Therefore, the most widely used materials among the implant or scaffold materials are titanium and titanium alloys which are highly biocompatible. Titanium is relatively lightweight compared to other metal materials due to its low specific gravity, but it can be made of an alloy with other metals or can be strengthened by proper treatment. Also, passive oxide film And has a very large corrosion resistance. In addition, osteointegration occurs when bone is implanted in the bone, and thus it is most widely used as a material for current implants or scaffolds.

Titanium and its alloys having such excellent mechanical properties, chemical stability, biocompatibility, and the like are widely used as implants or scaffolds used in orthopedic and dental fields. However, insufficient bonding between the implant or scaffold and the bone tissue loosens the implant or scaffold and eventually leads to implantation failure of the implant or scaffold. A variety of methods have been developed for surface modification and functionalization through control of calcium phosphate or hydroxyapatite coatings, biomolecules, proteins, and surface topography to improve bone fusion between the titanium surface and bone tissue. Recently, a bone morphogenic protein-2 (BMP-2) delivery system using collagen gel, sponge, fibrinogen, chitosan, etc. has been developed to improve osteoblast function. However, some of these drug delivery vehicles have shown the problem that BMP-2 is released rapidly in the short term and suddenly in the early stage.

Another major cause of implant failure in implants or scaffolds in the orthopedic or dental field is related to bacterial infections on metal surfaces. Typical titanium implants are susceptible to infection by bacteria from the patient's own skin or mucosa during insertion. When bacteria attach to the surface of the implant and grow, the bacterial cells form a thick biomembrane, which is a major obstacle to the host defense mechanism and the diffusion / penetration of the administered antibiotic. These bacterial infections cause failure of the implant and may exacerbate the patient's illness with the additional cost of implant removal.

Among these problems, in order to solve problems of rapid drug release and initial sudden release within a short period of time, heparin, which is a negatively charged linear polysaccharide, is chemically bound to a titanium surface to control the drug release rate so that BMP-2 can be slowly released (Sung Eun Kim et al., Biomaterials , 2011, 32 (2), 366-373). However, it is considered that the bacterial cell adhesion and the infection inhibiting effect are low.

In addition, Korean Patent Publication No. 2007-0068240 discloses a method for improving osteoblast function by implanting recombinant osteogenesis promoting protein into a jaw bone to rapidly differentiate undifferentiated cells around a treatment site into bone cells, There is a problem in that the above-described technique can adhere and infect bacteria on the surface of the implant. In order to solve these problems, it is necessary to add antibiotics to patients in order to prevent bacterial adhesion and infection.

Also, in order to prevent bacterial infections and improve the osseointegration efficacy, the Wilson Wang group (Biomacromolecules, 2009, 10 (6) 1603-1611; Tissue Eng Part A, 2009, 15 (2), 417-426; Biomaterials, 29 (10), 1412-1421; J Biomed Mater Res A 2008, 86 (4), 85-872) developed a titanium implant in which chitosan and its derivatives, dextran, hyaluronic acid, Has come. These titanium implants markedly increased the attachment, proliferation and alkaline phosphatase activity of osteoblast cells, while at the same time, Staphylococcus aureus ) and Staphylococcus epidermidis ). However, in the case of a titanium implant bonded with only such a polymer or peptide, there is a need to additionally take an antibiotic prescribed by a doctor in order to minimize or prevent the attachment and infection of bacterias on the surface of the implant.

Therefore, the present inventors have found that it is possible to prevent bacterial adhesion and infection that may occur after implantation of an implant or scaffold used in an orthopedic or dental clinic, and to avoid the need for additional antibiotic administration, The inventors have invented functional implants or scaffolds capable of simultaneously sustained release of antibiotics and bone formation promoting materials.

KR2007-0068240A

Biomaterials, 2011, 32 (2), 366-373, Sung Eun Kim et al.

It is an object of the present invention to provide an implant or scaffold having both osseointegration and osteogenesis efficacy and preventing bacterial infections that may occur after transplantation of orthopedic or dental implants or scaffolds, The surface of the scaffold is modified with a polymer to functionalize (activate) the surface of the implant or scaffold, and the antibiotic and the bone formation promoting substance are immobilized on the surface of the polymer-activated implant or scaffold through physical bonding, And an antibiotic, an implant for promoting osteogenesis-releasing-type implant or scaffold, and a method for manufacturing the same, which are capable of simultaneously and continuously releasing a growth promoting substance.

The present invention relates to an implant or scaffold whose surface has been modified using a complex of a compound containing a polymer-catechol functional group, wherein the implant or scaffold comprises one or two or more selected from antibiotics and bone formation promoting substances Lt; / RTI > implant or scaffold.

The present invention also relates to a process for the preparation of (a) preparing a complex of a compound containing a polymer-catechol functional group;

(b) modifying the surface of the implant or scaffold using the complex of the compound containing the polymer-catechol functional group prepared in the step (a), thereby activating the surface;

(c) immersing the surface activated implant or scaffold prepared in step (b) in a solution containing an antibiotic, and then agitating the solution at 20 to 25 ° C for 4 to 24 hours to remove the antibiotic on the surface of the implant or scaffold Immobilization; And

(d) immersing an implant or scaffold immobilized on the surface with the antibiotic prepared in step (c) in a solution containing the bone formation promoting material, stirring the mixture at 20 to 25 ° C for 4 to 24 hours, And immobilizing the bone formation promoting material on the surface of the implant or scaffold.

When the implant or scaffold of the present invention includes the antibiotic and the bone formation promoting substance, since the antibiotic is continuously released from the implant or the scaffold for a long period of time, the implant or scaffold can be used for implantation or scaffold implantation, Growth, and infection, thereby significantly reducing the failure rate of implant or scaffold implantation by bacteria. In addition, there is no need to take additional antibiotics after implantation or scaffold implantation. In addition, since the material for promoting osseointegration and osteogenesis (bone formation promoting material) is continuously released for a long period of time, adhesion between the implant or scaffold and bone tissue, and osteoblast differentiation can be promoted to improve bone formation. Or scaffold transplantation, and the economic burden can be reduced because the success rate of implant or scaffold implantation can be increased.

FIG. 1 shows a method of successively immobilizing antibiotic gentamycin and bone growth promoting growth factor BMP-2 on titanium metal surfaces of implants modified by heparin treatment.
FIG. 2 is a scanning electron microscope (SEM) image of the surfaces of the titanium implants of Comparative Examples 1 to 4 and Example 1, showing (a) titanium (Comparative Example 1), (b) heparin- (E) gentamycin / BMP-2-immobilized heparin-titanium (Example 1), (b) heparin- Lt; RTI ID = 0.0 > SEM < / RTI >
FIG. 3 is a graph showing the results of a comparison between the titanium of the implant (Comparative Example 1) and the activated titanium (heparin-titanium (Comparative Example 2), gentamycin immobilized heparin-titanium (Comparative Example 3), BMP-2 immobilized heparin- Example 4), and heparin titanium (Example 1) in which gentamicin / BMP-2 was immobilized) using X-ray photoelectron spectroscopy.
FIG. 4 is a graph showing the results of drug release of gentamicin or BMP-2 from activated titanium of Comparative Examples 2 to 4 and Example 1 according to Experimental Example 2 of the present invention, wherein FIG. Shows drug release behavior of gentamycin from heparin-titanium (Comparative Example 3, GSHep-Ti) and Gentamicin / BMP-2 immobilized heparin titanium (Example 1, GS / BMP-2 Hep-Ti) FIG. 4B is a graph showing the binding of heparin-titanium (Example 1, GS / BMP-2 Hep-Ti (BMP-2 / Hep-Ti) and Gentamycin / BMP- ) Shows the drug release behavior of BMP-2.
FIG. 5 is a graph showing the results of a comparison between the results of Comparative Example 1 (Pristine Ti) and functionalized titanium (gentamycin-immobilized heparin-titanium (Comparative Example 3, GSHep-Ti), BMP-2 immobilized heparin- (Comparative example 4, BMP-2 / Hep- Ti) and gentamicin / BMP-2 is fixed heparin titanium (example 1, GS / BMP-2 Hep -Ti)) Staphylococcus aureus (Staphylococcus of aureus < / RTI > bacteria cells.
FIG. 6 is a graph showing the results of a comparison between Comparative Example 1 (Pristine Ti) and functionalized titanium (heparin-titanium (Comparative Example 2, heparinized Ti), gentamicin-fixed heparin-titanium (Comparative Example 3, GSHep- (Example 1, GS / BMP-2 Hep-Ti), BMP-2 immobilized heparin-titanium (Comparative Example 4, BMP-2 / Hep-Ti) and Gentamycin / BMP- )) To the osteoblasts.
FIG. 7 is a graph showing the results of a comparison between the results of Comparative Example 1 (Pristine Ti) and functionalized titanium (heparin-titanium (Comparative Example 2, Heparnized Ti), gentamicin-fixed heparin-titanium (Comparative Example 3, GSHep- (Example 1, GS / BMP-2 Hep-Ti), BMP-2 immobilized heparin-titanium (Comparative Example 4, BMP-2 / Hep-Ti) and Gentamycin / BMP- )) On the osteoblasts.
FIG. 8 is a graph showing the results of a comparison between the values of Pristine Ti and functionalized titanium (heparin-titanium (Comparative Example 2, Heparnized Ti), gentamicin-fixed heparin-titanium (Comparative Example 3, GSHep- (Example 1, GS / BMP-2 Hep-Ti), BMP-2 immobilized heparin-titanium (Comparative Example 4, BMP-2 / Hep-Ti) and Gentamycin / BMP- )) For 1, 3, and 7 days after osteoblast cultivation.
FIG. 9 is a graph showing the results of a comparison between Comparative Example 1 (Pristine Ti) and functionalized titanium (heparin-titanium (Comparative Example 2, Heparnized Ti), gentamicin-fixed heparin-titanium (Comparative Example 3, GSHep- (Example 1, GS / BMP-2 Hep-Ti), BMP-2 immobilized heparin-titanium (Comparative Example 4, BMP-2 / Hep-Ti) and Gentamycin / BMP- )) Showing the results of alkaline phosphatase activity after osteoblasts were cultured for 7 days, 14 days, and 21 days,
FIG. 10 is a graph showing the results of a comparison between Comparative Example 1 (Pristine Ti) and functionalized titanium (heparin-titanium (Comparative Example 2, Heparnized Ti), Gentamycin immobilized heparin-titanium (Comparative Example 3, GSHep- (Example 1, GS / BMP-2 Hep-Ti), BMP-2 immobilized heparin-titanium (Comparative Example 4, BMP-2 / Hep-Ti) and Gentamycin / BMP- )) Showing the results of calcium deposition after 21 days of osteoblast cultivation.
Figure 11 shows the results obtained by injecting the surface of heparin-dopamine PCL / PLGA scaffold surface of (a) PCL / PLGA scaffold surface of Example 2, (b) BMP-2 loaded heparin PCL / PLGA scaffold surface, and (c) BMP- The results were observed with an electron microscope.
12 is a graph showing the cell toxicity of osteoblasts to PCL / PLGA scaffolds, BMP-2 loaded heparin-PCL / PLGA scaffolds, and BMP-2 loaded heparin-dopamine-PCL / PLGA scaffolds, Was incubated in the cell culture medium and the absorbance was measured at 450 nm.
13 is a PCL / PLGA scaffolds, BMP-2 with heparin -PCL / PLGA scaffolds, BMP-2 with heparin - In order to evaluate the cell proliferation of the osteoblasts in the dopamine -PCL / PLGA scaffolds, 1 × 10 ⁵ The number of osteoblast cells in each scaffold was measured after incubation for 1, 3, and 7 days at 450 nm.
FIG. 14 shows the results of measurement of alkaline phosphatase activity as an early differentiation marker of osteoblast of PCL / PLGA scaffold, BMP-2 loaded heparin-PCL / PLGA scaffold, and BMP-2 loaded heparin-dopamine-PCL / PLGA scaffold .
FIG. 15 shows the results of measurement of calcium deposition, which is a late differentiation marker of osteoblast, with PCL / PLGA scaffold, BMP-2 loaded heparin-PCL / PLGA scaffold and BMP-2 loaded heparin-dopamine-PCL / PLGA scaffold.
Fig. 16 shows the bone morphology of the PCL / PLGA scaffold, BMP-2 loaded heparin-PCL / PLGA scaffold, and BMP-2 loaded heparin-dopamine-PCL / PLGA scaffolds. After histological analysis of each scaffold transplanted to the bone defect, hematoxylin and eosin (H & E) and Masson's trichrome (MT) staining were performed.
FIG. 17 shows the bone morphology of the PCL / PLGA scaffold, BMP-2 loaded heparin-PCL / PLGA scaffold, and BMP-2 loaded heparin-dopamine-PCL / PLGA scaffolds. The results of X-ray analysis, CT analysis, and histomorphometric analysis of each scaffold transplanted to the bone defect region were obtained.

Hereinafter, the present invention will be described more specifically.

The present invention relates to an implant or scaffold whose surface has been modified using a complex of a compound containing a polymer-catechol functional group, wherein the implant or scaffold comprises one or two or more selected from antibiotics and bone formation promoting substances Lt; / RTI > implant or scaffold.

More specifically, the present invention relates to a method for modifying the surface of an implant or a scaffold through a strong covalent bond or a non-covalent bond with a biocompatible polymer, and an antibiotic and / or bone fusion and bone formation capable of inhibiting bacterial adhesion and infection Wherein the antibiotic and / or the bone formation promoting substance can be released in a sustained release form simultaneously by immobilizing the bone formation promoting substance which can increase the bone formation promoting substance on the surface of the implant or scaffold modified with the polymer.

In one embodiment of the present invention, the surface of the implant or scaffold may be made of titanium and titanium alloy having excellent biocompatibility.

In another embodiment of the present invention, the surface of the implant or scaffold is selected from the group consisting of polycaprolactone (PCL); Polylactic acids (PLA), polyglycolic acid, or copolymers thereof; One or two or more kinds of biodegradable plastic (s) selected from the group consisting of polylaidoxybutyrate-co-valerate (PHBV), polyvinyl alcohol (PVA), polybutylene succinate (PBS) and polyphenylene glycol acid ≪ / RTI >

The polymer which can be used for the surface modification of the implant or scaffold is a biocompatible polymer and is a biocompatible polymer such as heparin, heparan sulfate, hyaluronic acid, alginate, polyaspartic acid [ aspartic acid], poly (acrylic acid), poly (glutamic acid), carboxymethyl cellulose, gelatin, collagen, chitosan and A derivative thereof, a derivative thereof, a glycerol chitosan, a poly-L-lysine, a polyethyleneimine, and a dendrimer having an amine group functional group at the terminal. Or two or more species, but is not limited thereto.

The dendrimer having an amine group functional group at the terminal is composed of a repeating unit (generation) bonded radially from the center, and the number of generations of the dendrimer is preferably 1 to 5.

In order to allow the polymer to form strong covalent or noncovalent bonds on the surface of implants or scaffolds, the present invention provides a method for the preparation of mussel < RTI ID = 0.0 > Adhesion properties were used. The strong adhesion properties of mussels are due to the amino acids of 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine amino acid-rich proteins found near the plaque-substrate interface . The DOPA not only participates in a reaction that causes solidification of the adhesive material, but also forms strong covalent or noncovalent bonds with the substrate. In particular, the ortho-dihydroxyphenyl functional group called catechol in the DOPA structural formula plays an important role in forming a strong covalent bond or a noncovalent bond with the substrate. Therefore, in the present invention, a compound containing a catechol functional group is used for binding the biocompatible polymer to the surface of the implant or scaffold. The compound containing the catechol functional group is preferably selected from the group consisting of 3,4-dihydroxyphenylalanine, Dopamine, 3,4-dihydroxybenzylamine, Norepinephrine, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, caffeic acid, and the like. Dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid. However, the present invention is not limited thereto.

The complex of a polymer-catechol-containing compound that binds the polymer to the surface of an implant or scaffold using a compound containing a catechol functional group and activates or functionalizes the surface of the implant or scaffold, By using a crosslinker in the compound containing the crosslinking agent.

The complex of the compound containing the polymer-catechol functional group is preferably a functional group of the amine group or the carboxy group of the polymer; And

Is preferably formed by a combination of a compound containing a catechol functional group capable of chemically bonding with any one functional group selected from the group consisting of an amine group, a carboxy group and an aldehyde functional group, and the polymer- The surface of the implant or scaffold can be modified using a complex of a compound containing a call functional group.

It is more preferable that the complex of the polymer-catechol-containing compound is prepared by the following combination. For example, a polymer having a functional group of a carboxy group such as heparin, heparan sulfate, hyaluronic acid, alginate, poly (aspartic acid), poly Poly (glutamic acid), Carboxymethyl cellulose, Gelatin, and Collagen contain a catechol having a functional group of an amine group, such as poly (acrylic acid), polyglutamic acid, Compounds such as 3,4-dihydroxyphenylalanine, Dopamine, 3,4-dihydroxybenzylamine, norepinephirine and 3,4-dihydroxyphenylalanine. Compound complexes containing polymer-catechol can be prepared through various combinations. In addition, a polymer having functional groups of an amine group such as Gelatin, Collagen, Chitosan and derivatives thereof, Glycol chitosan, Poly-L-lysine, Polyethyleneimine and Dendrimer are catechol-containing compounds having a carboxy functional group or an aldehyde functional group such as 3,4-dihydroxybenzaldehyde (3,4-dihydroxybenzaldehyde), 3,4-dihydroxybenzoic acid A compound complex containing a polymer-catechol is prepared through various combinations with 3,4-dihydroxybenzoic acid, Caffeic acid, 3,4-dihydroxyphenylacetic acid, . Table 1 below provides examples of complexes containing polymer-catechol that can be obtained through various combinations of polymers and compounds containing catechol.

Polymer-catechol-containing compound complex Polymer type Bondable catechol-containing compound Manufacturable polymer-containing catechol
Compound complex Heparin (Hep) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - Hep-DOPA
- Hep-DOPAM
- Hep-DHBA
- Hep-NENP Heparin sulfate (HS) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - HS-DOPA
- HS-DOPAM
- HS-DHBA
- HS-NENP Hyaluronic acid (HA) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - HA-DOPA
- HA-DOPAM
- HA-DHBA
- HA-NENP Alginate (AG) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - AG-DOPA
- AG-DOPAm
- AG-DHBA
- AG-NENP Polyaspartic acid (PAA) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - PAA-DOPA
- PAA-DOPAm
- PAA-DHBA
- PAA-NENP Polyacrylic acid (PAcA) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - PAcA-DOPA
- PAcA-DOPAm
- PAcA-DHBA
- PAcA-NENP Polyglutamic acid (PGA) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - PGA-DOPA
- PGA-DOPAM
- PGA-DHBA
- PGA-NENP Carboxymethyl cellulose (CMC) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP) - CMC-DOPA
- CMC-DOPAM
- CMC-DHBA
- CMC-NENP Gelatin (Gel) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP)
- 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - Gel-DOPA
- Gel-DOPAM
- Gel-DHBA
- Gel-NENP
- Gel-DHBAl
- Gel-DHBAc
- Gel-CFA
- Gel-DHPA Collagen (Col) - 3,4-dihydroxyphenylalanine (DOPA)
- Dopamine (DOPAm)
- 3,4-dihydroxybenzylamine (DHBA)
- norepinephirine (NENP)
- 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - Col-DOPA
- Col-DOPAM
- Col-DHBA
- Col-NENP
- Col-DHBAl
- Col-DHBAc
- Col-CFA
- Col-DHPA Chitosan (Chi) - 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - Chi-DHBAl
- Chi-DHBAc
- Chi-CFA
- Chi-DHPA Glycol chitosan (GC) - 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - GC-DHBAl
- GC-DHBAc
- GC-CFA
- GC-DHPA Poly-L-Lysine (PLL) - 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - PLL-DHBAl
- PLL-DHBAc
- PLL-CFA
- PLL-DHPA Polyethyleneimine (PEI) - 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - PEI-DHBAl
- PEI-DHBAc
- PEI-CFA
- PEI-DHPA Dendrimer (Den) - 3,4-dihydroxybenzaldehyde (DHBAl)
- 3,4-dihydroxybenzoic acid (DHBAc)
- Caffeic acid (CFA)
- 3,4-Dihydroxyphenylacetic acid (DHPA) - Den-DHBAl
- Den-DHBAc
- Den-CFA
- Den-DHPA

The weight average molecular weight of the polymer is preferably 1,800 to 300,000. The complex of the compound containing the polymer-catechol functional group is preferably bound to 3 to 25 moles of the compound containing the catechol functional group per mole of the polymer, but not limited thereto.

In order to activate and functionalize the surface of the implant or scaffold by bonding the compound complex containing the polymer-catechol functional group to the surface of the implant or scaffold, the base condition in which the catechol functional group can be easily oxidized and polymerized And the pH of the solution is preferably 7 to 12. It is possible to use a pH buffer such as Tris buffer, phosphate buffer (PBS) to maintain the pH of the solution under the above conditions.

Antibiotics that can be immobilized or mounted on the surface of an implant or scaffold surface activated or functionalized using the compound complex containing the polymer-catechol functional group include sisomicin, ribostamycin, vancomycin ), Tobramycin, Aztreonam, Gentamicin, Cefotiam, Ceftriaxone, Netilmicin, Ciprofloxacin, Cindamycin, ) And cytosol (Cefazolin), and more preferably one or two kinds selected from the group consisting of carboxylic acid, amine, phosphates and sulfate Functional group, but it is not limited thereto.

The concentration of the antibiotic which can be mounted or immobilized on the surface of the polymer-activated implant or scaffold is preferably in the range of 1 ng / ml to 200 mg / ml, and the implant activated with the polymer in the antibiotic concentration range The antibiotic can be mounted or immobilized on the surface of the scaffold, but the capacity to be mounted and immobilized can be determined depending on the size of the implant or scaffold. When the concentration of the antibiotic is less than 1 ng / ml, the antibacterial effect will not be exhibited. If the concentration exceeds 200 mg / ml, it may affect normal cells.

Examples of the bone formation promoting substance that can be immobilized or mounted on the surface of the surface activated or functionalized implant or scaffold using the compound complex containing the polymer-catechol functional group include bone growth promoting protein or low molecular weight bone formation Compounds may be used, and it is preferable to be an osteogenic growth factor, but the present invention is not limited thereto.

Bone morphogenetic proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP- BMP-8a, BMP-8b, BMP-10 and BMP-15 and platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor it is preferably one or more selected from the group consisting of basic fibroblast growth factor (bFGF), insulin like growth factor 1 (IGF-1) and lactoferrin.

In addition, a bisphosphonate drug family alendronate, which is a low molecular weight osteogenic compound that can be substituted for an osteogenic growth factor used for improving the osseointegration and bone formation efficacy on the surface of the surface activated or functionalized implant or scaffold of the present invention, But are not limited to, alendronate, risedronate, zoledronate, Etidronate, Clodronate, Tiludronate, Pamidronate, Olpadronate, Ibadronate; < RTI ID = 0.0 > Statin drugs such as Atrovastatin, Fluvastatin, Lovastatin, pitavastatin, pravastatin, rosuvastatin, simastatin; Selected from the group consisting of prostaglandin E2, PGE2, shinbarometin, pyrophosphate, medronate, oxidronate and oxysterol. One or two or more drugs may be mounted or immobilized.

It is preferable that the dose capable of being loaded or immobilized on the surface of the implant or scaffold activated by the bone-forming growth factor is in the range of 1 pg / ml to 3 mg / ml, and the concentration of the osteogenic growth factor is 1 pg / ml, there is no effect of osteogenesis, and when it exceeds 3 mg / ml, it is possible to cause a side effect that causes bone to be melted.

The capacity of the low molecular weight osteogenic compound capable of promoting osseointegration and bone formation to be mounted or immobilized on the surface of an implant or scaffold activated with a polymer is in the range of 1 ng / ml to 500 mg / ml desirable. If the concentration of the osteogenic growth factor is less than 1 ng / ml, the osteogenic effect is not exhibited. If the concentration exceeds 500 mg / ml, a side effect that causes the bone to be melted may occur.

In order to mount or immobilize an antibiotic or an osteogenesis promoting substance on an implant or a scaffold having a surface activated or functionalized with the polymer, it is necessary to use a weakly acidic condition in which electrostatic interactions between the polymer and the antibiotic, And a solution such as water having the above-mentioned pH range, MES buffer [2- (N-morpholino) ethanesulfonic acid], Tris buffer or phosphate buffer (PBS) It is possible to use.

The implant or scaffold of the present invention is preferably a dental or orthopedic implant or scaffold. For example, the orthopedic implant or scaffold may be a hip, knee, ankle, shoulder, elbow artificial joint, Artificial implants such as an artificial disc in the spinal region are used, and fixations such as metal screws, metal plates, and metal fixtures used for fracture treatment are included. The dental implants or scaffolds may also include fixture type implants or scaffolds, membrane type implants or scaffolds, and the like.

Therefore, when a functional implant or scaffold capable of releasing the antibiotic and the bone formation promoting material of the present invention is used in a sustained release form, the antibiotic is continuously released from the implant or scaffold for a long period of time. It is possible to prevent bacterial adhesion and infection, and there is no need to take additional antibiotics after implantation or scaffold implantation. In addition, since the substance promoting osseointegration and osteogenesis is continuously released for a long period of time, adhesion between the implant or scaffold and the bone tissue and bone formation are promoted to shorten the treatment period of the implant or scaffold, Can greatly contribute to the success of transplantation.

The present invention also relates to a method of manufacturing an implant or a scaffold,

(a) preparing a complex of a compound containing a polymer-catechol functional group;

(b) modifying the surface of the implant or scaffold using the complex of the compound containing the polymer-catechol functional group prepared in the step (a), thereby activating the surface;

(c) immersing the surface-activated implant or scaffold prepared in step (b) in a solution containing an antibiotic, and then immersing the surface-activated implant or scaffold for 4 to 24 hours And immobilizing the antibiotic to the surface of the implant or scaffold; And

(d) immersing the implant or scaffold immobilized on the surface with the antibiotic prepared in step (c) in a solution containing an osteogenesis promoting substance, and then immersing the implant or scaffold for 4 to 24 hours , And immobilizing the antibiotic / bone formation promoting substance on the surface of the implant or scaffold.

The complex of the compound containing the polymer-catechol functional group in the step (a) may be a functional group of an amine group or a carboxy group of the biocompatible polymer; And

It is preferably formed by bonding with a functional group selected from the group consisting of an amine group, a carboxy group or an aldehyde functional group of a compound containing a catechol functional group.

The method for producing a complex of a compound containing a polymeric material and a catechol functional group is described in the above.

The reaction conditions in which the electrostatic interaction between the polymer, the antibiotic, the polymer and the bone formation promoting material in the step (c) and the step (d) of the method of manufacturing the implant or scaffold can easily occur are pH 5 to 6.5 (MES buffer [2- (N-morpholino) ethanesulfonic acid], Tris buffer, phosphate buffer solution (PBS) and the like are used as the solution that can be used at this time. It is possible to use the same solution.

In addition, it is preferable that after the steps (b), (c), and (d), each of the steps is washed three to four times with distilled water and then dried at 50 degrees.

The antibiotic uses the above-mentioned contents.

The concentration of the antibiotic solution that can be mounted or immobilized on the surface of the polymer-activated implant or scaffold is preferably in the range of 1 ng / ml to 200 mg / ml, and the polymer- Antibiotics can be mounted or immobilized on the scaffold surface, but the capacity to be mounted and immobilized can be determined depending on the size of the implant or scaffold. When the concentration of the antibiotic is less than 1 ng / ml, the antibacterial effect will not be exhibited. If the concentration exceeds 200 mg / ml, it may affect normal cells.

The above-mentioned bone formation promoting materials are used as described above.

The capacity of the osteogenic growth factor to be loaded or immobilized on the polymer-activated implant or scaffold surface is preferably in the range of 1 pg / ml to 3 mg / ml,

It is preferable that the dose capable of being mounted or immobilized on the surface of an implant or scaffold in which the low molecular weight bone forming compound capable of promoting osseointegration and bone formation is activated by the polymer is in the range of 1 ng / ml to 500 mg / ml Do.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

Manufacturing example  1. Preparation of heparin-dopamine complex

400 mg of heparin, 190.6 mg of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) 115 mg of N-hydrosuccinimide (NHS) was dissolved in 10 ml of MES buffer (pH 4.5) and reacted for 10 minutes. Then, 102.2 mg of Dopamine is added and reacted at 25 ℃ for about 24 hours. After the reaction, dialyzed with distilled water to remove unreacted materials and then lyophilized to prepare heparin-dopamine complex (Hep-DOPAm). The above reaction is shown in Reaction Scheme 1 below.

[Reaction Scheme 1]

The heparin-dopamine complex thus prepared was analyzed by ultraviolet and visible spectral analysis at a wavelength of 280 nm. As a result, it was confirmed that about 4.6 ± 0.8 dopamine molecules were bound to one heparin molecule.

Example 1 On the surfaces of titanium implants, heparin, gentamicin Sulfate (gentamicin sulfate) and immobilization of BMP-2

Before modifying the titanium surface of the implant, the titanium implant was placed in an ethanol solution, ultrasonically washed for about 1 hour, and dried at 50 degrees for about 24 hours. Thereafter, the titanium implant was placed in a 10 mM Tris buffer (pH 8.0) in which the heparin-dopamine complex prepared in Preparation Example 1 was dissolved at a concentration of 5 mg / ml, blocked with light, stirred overnight, Titanium implants modified with heparin-dopamine complex (heparin-titanium implants) were washed 3-4 times with distilled water and dried with flowing nitrogen gas to prepare titanium implants modified with heparin-dopamine complex. To mount the antibiotic gentamicin on the titanium implants modified with the heparin-dopamine complex, a surface-modified titanium implant with a heparin-dopamine complex was immersed in a 0.1 M MES buffer solution containing 50 mg / ml gentamycin (pH 5.6), agitated at 25 ° C and 80 rpm for about 15-24 hours, washed 3-4 times with distilled water, and dried at 50 ° C to remove gentamycin-fixed heparin-titanium implants (gentamycin-heparin - titanium implant). To further mount BMP-2, an osteogenic growth factor, gentamycin-fixed heparin-titanium implants were placed in a 0.1 M MES buffer (pH 5.6) containing 50 ng / ml of BMP-2, After washing with distilled water three to four times and drying, a heparin-titanium implant with gentamicin / BMP-2 immobilized thereon was obtained.

Comparative Example  1. Titanium Implant

Untreated titanium implants.

Comparative Example  2. Manufacture of heparin-titanium implants

Before modifying the titanium surface of the implant, the titanium implant was placed in an ethanol solution, ultrasonically washed for about 1 hour, and dried at 50 degrees for about 24 hours. Thereafter, the titanium implant was placed in a 10 mM Tris buffer (pH 8.0) in which the heparin-dopamine complex prepared in Preparation Example 1 was dissolved at a concentration of 5 mg / ml, blocked with light, stirred overnight, Titanium implants modified with heparin-dopamine complex (heparin-titanium implants) were washed 3-4 times with distilled water and dried with flowing nitrogen gas to prepare titanium implants modified with heparin-dopamine complex.

Comparative Example  3. Gentamicin  Manufacture of immobilized heparin-titanium implants

Heparin-titanium implant (gentamicin-heparin-titanium implant) in which gentamycin was immobilized was prepared in the same manner as in Example 1 except for the step of immobilizing BMP-2 on a surface-modified titanium implant with a heparin-dopamine complex Respectively.

Comparative Example  4. BMP -2-immobilized heparin-titanium implants

BMP-2 was prepared in the same manner as in Example 1 except that only the gentamicin was immobilized on the surface-modified titanium implant with the heparin-dopamine complex. Fixed heparin-titanium implants (gentamycin-heparin-titanium implants) were prepared.

Experimental Example  One.

The surfaces of the functionalized titanium implants loaded with heparin, gentamicin and BMP-2 on the activated titanium surface of the prepared implant of Example 1 and the titanium implants of Comparative Examples 1 to 4 were observed with a scanning electron microscope, As shown in Fig. The titanium implant of Comparative Example 1 and the functionalized titanium implant (Example 1 and Comparative Examples 2 to 3) were subjected to surface element analysis using an x-ray photoelectron spectroscopy, and are shown in Fig. As shown in FIG. 3, heparin was successfully applied to the titanium implant surface by confirming an increase in nitrogen component of 3.63% and a decrease in oxygen content of 20.49% in the heparin-titanium implant compared to the unmodified titanium implant (Comparative Example 1) It was confirmed that it was reformed. In addition, compared with the heparin-titanium implant (Comparative Example 2), the heparin-titanium implant with the gentamycin-immobilized (Comparative Example 3), the heparin-titanium implant with the BMP-2 immobilized (Comparative Example 4) -2-fixed heparin titanium implant (Example 1) showed that the decrease in carbon content and the increase in nitrogen component resulted in successful loading of gentamicin and BMP-2. Table 2 below shows the surface element analysis results of titanium and functionalized titanium.

Surface element analysis of titanium and functionalized titanium Substrate C% N% O% Ti % Comparative Example 1 Pristine Ti 52.02 1.12 36.11 10.75 Comparative Example 2 Hep-Ti 76.44 4.75 15.62 3.19 Comparative Example 3 GS / Hep-Ti 75.05 5.76 16.81 2.38 Comparative Example 4 BMP-2 / Hep-Ti 75.19 5.17 17.19 2.45 Example 1 GS / BMP-2 / Hep-Ti 75.94 7.23 14.23 2.60

GS: Gentamicin sulfate

Hep: heparin

Experimental Example  2. From functionalized titanium Gentamicin  or BMP -2 drug release behavior

(Comparative Example 3), which was a functionalized titanium implant, a heparin-titanium implant with a fixed gentamycin (Comparative Example 3), a heparin-titanium implant with a fixed BMP-2 (Comparative Example 4), a heparin-titanium implant with a gentamycin / BMP- (Example 1), the functionalized titanium implants were placed in 1 ml of PBS buffer (pH 7.4) and stirred at 37 DEG C and 100 rpm to analyze the release behavior of gentamicin or BMP-2. And were measured at intervals of 1, 3, 5, and 10 hours and at 1, 3, 5, 7, 14, 21, and 28 days. I replaced it with a new buffer. The released gentamicin was analyzed by measuring the absorbance at 257 nm using an infrared and visible spectrophotometer (DU-530, Beckman Coulter ^TM , USA). The released BMP-2 was analyzed by ELISA kit (Human BMP-2 Mini ELISA Development Kit (900-M255), Peprotech, USA). The results are shown in FIG.

As shown in FIG. 4, heparin-titanium (Comparative Example 3) in which gentamicin was immobilized, heparin-titanium (Comparative Example 4) in which BMP-2 was immobilized, heparin-titanium in which gentamycin / BMP- 1), gentamycin or BMP-2 was released slightly earlier in the first day, but continued to be released thereafter for 4 weeks thereafter.

Experimental Example  3. Antimicrobial efficacy of functionalized titanium

Titanium (Comparative Example 1) and heparin-titanium (Comparative Example 3) in which functionalized titanium was fixed with gentamycin (Comparative Example 3), heparin-titanium with BMP-2 immobilized (Comparative Example 4), heparin with fixed gentamycin / BMP- To test the antibacterial effect of titanium (Example 1), each titanium was placed in TSB (Tryptic Soy Broth), which was a bacterial culture solution, and 1 x 10 ⁶ Staphylococcus aureus bacterial cells were added and agitated continuously at 37 ° C. After 6 hours, 12 hours, and 24 hours after the incubation, the absorbance of the culture solution was measured at 600 nm, and the antibacterial activity of each titanium was analyzed. The results are shown in FIG. As shown in FIG. 5, heparin-titanium (Comparative Example 4) in which titanium (Comparative Example 1) and BMP-2 were immobilized did not inhibit the growth of Staphylococcus aureus , but gentamicin-fixed heparin- Titanium (Comparative Example 3) and heparin-titanium (Example 1) immobilized with gentamicin / BMP-2 inhibited bacterial growth very effectively.

Experimental Example  4. Assessment of cytotoxicity of titanium and functionalized titanium

Titanium (Comparative Example 1), functionalized titanium heparin-titanium (Comparative Example 2), gentamycin immobilized heparin-titanium (Comparative Example 3), BMP-2 immobilized heparin-titanium (Comparative Example 4) To evaluate the cytotoxicity of osteoblast of heparin-titanium (Example 1) immobilized with mycin / BMP-2 (Example 1), each titanium was added to a cell culture medium and cultured at 37 ° C for 24 hours to obtain an extract culture. On the other hand, 1 × 10 ⁴ osteoblast MG63 cells were placed in a 96-well plate and cultured at 37 ° C. for 24 hours. The osteoblast cells were cultured for 24 hours and 48 hours, and then the culture broth was removed. For cytotoxicity, CCK-8 proliferation kit reagent was added and incubated at 37 ° C for 1 hour, and the absorbance was measured at 450 nm. As shown in FIG. 6, titanium and functionalized titanium all showed cell viability of 95% or more even after 48 hours of culture, indicating that both titanium and the functionalized titanium have no cytotoxicity to osteoblasts.

Experimental Example  5. Titanium and functionalized titanium Live / Dead  analysis

Live / dead analysis of osteoblasts of heparin-titanium, heparin-titanium, gentamycin-immobilized heparin-titanium, heparin-titanium fixed with BMP-2, and heparin-titanium fixed with gentamycin / BMP-2 For evaluation, each titanium was placed in a 48 well plate and 5 x 10 ⁴ osteoblasts were carefully placed on each titanium surface and cultured for 48 hours. After incubation for 48 hours, the cells were washed 3 to 4 times with PBS buffer, and then added with live / dead dye (Calcein, Ethidium homodimer-1 reagent, L3224 (Live / DEAD Viability / Cytotoxicity Kit for mammalian cells), Molecular Probes Min, and then analyzed with a confocal laser scanning microscope. In all groups of titanium and functionalized titanium, osteoblasts were found to be well adhered to the titanium surface very well (FIG. 7).

Experimental Example  6. Cell proliferation of titanium and functionalized titanium

Evaluation of cell proliferation on osteoblasts of titanium and functionalized titanium heparin-titanium, gentamycin-immobilized heparin-titanium, BMP-2 immobilized heparin-titanium, gentamycin / BMP-2 immobilized heparin-titanium To do this, 1 × 10 ⁵ osteoblasts were carefully placed in each titanium and cultured for 1 day, 3 days, and 7 days, after which the cells were washed with PBS buffer. After washing, CCK-8 proliferation kit reagent (Tetrazolium salt reagent, Cell Counting kit-8, Dojindo, USA) was added and incubated for 1 hour. The culture was carefully transferred to a 96-well plate and absorbance was measured at 450 nm. The osteoblasts cultured on titanium and functionalized titanium surfaces proliferated gradually after 1 day, 3 days, and 7 days (FIG. 8), confirming no difference in cell proliferation between the groups.

Experimental Example  7. Alkalinity of titanium and functionalized titanium Phosphatase  Activity

The effect of alkaline phosphatase activity, which is an early differentiation marker of osteoblast, was evaluated by analyzing the activity of titanium and functionalized titanium. Carefully place 1 x 10 ⁵ osteoblasts on each titanium and incubate for 7, 14, and 21 days. After incubation, the cells are washed with PBS buffer, and 1 × RIPA buffer is added to the cells and sonicated at 0 ° C and 110 watts for 1 minute. The lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C. After centrifugation, the supernatant was incubated with the p-nitrophenylphosphate solution at 37 ° C for 30 minutes, and the reaction was terminated with 500 μl of 1 N NaOH. The absorbance at 405 nm was measured, 9. As shown in FIG. 9, heparin-titanium with titanium, heparin-titanium, and gentamicin-immobilized heparin-titanium showed similar alkaline phosphatase activity to osteoblasts and showed no difference. In addition, heparin-titanium in which BMP-2 was immobilized and heparin-titanium in which gentamycin / BMP-2 was immobilized had similar alkaline phosphatase activities. However, heparin-titanium (Example 1) in which BMP-2 was immobilized (Comparative Example 4) and Gentamycin / BMP-2 was immobilized was made of titanium (Comparative Example 1), heparin-titanium (Comparative Example 2) Showed significantly higher alkaline phosphatase activity as compared to heparin-titanium (Comparative Example 3) in which gentamicin was immobilized.

Experimental Example  8. Calcium deposition of titanium and functionalized titanium

The osteointegration efficacy of Example 1 and Comparative Examples 1 to 4 titanium was evaluated by measuring and analyzing calcium deposition as a late differentiation marker of osteoblast. Carefully place 1 x 10 ⁵ osteoblasts on each titanium and incubate for 21 days. After incubation, the cells are washed with PBS buffer and carefully scraped off the cells from each titanium surface. The scraped cells were centrifuged for 1 minute at 13,500 rpm and 0.1% Triton X-100 solution was added to the cells. Ultrasonic wave is applied for about 1 minute at 0 degree to crush the cell membrane, and centrifugation is performed. The supernatant was separated and analyzed for absorbance at 612 nm using a QuantiChrom ^™ Calcium Assay kit. As shown in FIG. 10, the degree of calcium deposition of titanium (Comparative Example 1), heparin-titanium (Comparative Example 2), and gentamicin-immobilized heparin-titanium (Comparative Example 3) were similar and statistically significant I did. In addition, heparin-titanium (Comparative Example 4) in which BMP-2 was immobilized and heparin-titanium (Example 1) in which gentamycin / BMP-2 was immobilized had similar degree of calcium deposition. However, heparin-titanium with BMP-2 immobilized heparin-titanium and gentamycin / BMP-2 immobilized has significantly higher calcium deposition than titanium, heparin-titanium, and gentamycin-immobilized heparin- Respectively.

Thus, as shown in the above Experimental Example, the gentamicin / BMP-2 immobilized heparin-titanium implant of Example 1, which is a functional implant in which the titanium surface of the implant is modified to fix the antibiotic and the bone formation promoting substance, Not only prevented bacterial infections but also had excellent effects on osseointegration and osteogenesis.

Example  2. PCL / PLGA Scaffold  On the surface BMP -2 immobilization

Before modifying the surface of the PCL / PLGA scaffold, the scaffold was placed in an ethanol solution, ultrasonically washed for about 1 hour, and dried at 50 degrees for about 24 hours. Thereafter, the PCL / PLGA scaffold was placed in a 10 mM Tris buffer (pH 8.0) in which the heparin-dopamine complex prepared in Preparation Example 1 was dissolved at a concentration of 5 mg / ml, and the mixture was shaken overnight After that, the surface-modified PCL / PLGA scaffold (heparin-dopamine-PCL / PLGA scaffold) modified with heparin-dopamine complex was washed 3-4 times with distilled water and dried with flowing nitrogen gas to prepare heparin-dopamine complex A surface modified PCL / PLGA scaffold was prepared. To mount BMP-2, an osteogenic growth factor, a surface-modified PCL / PLGA scaffold with a heparin-dopamine complex was added to a 0.1 M MES buffer (pH 5.6) containing 50 ng / ml of BMP-2 , Stirred overnight, washed 3-4 times with distilled water, and dried to obtain a heparin-dopamine-PCL / PLGA scaffold in which BMP-2 was immobilized.

Experimental Example  9. PCL / PLGA And functionalized PCL / PLGA Scaffold  Surface observation

The functionalized PCL / PLGA scaffold carrying BMP-2 on heparin and heparin-dopamine scaffolds on the activated PCL / PLGA scaffold surface of the scaffold of Example 2 was observed with a scanning electron microscope. As a result, 11, the surface of the PCL / PLGA scaffold with heparin-treated BMP-2 was rough and the surface of PCL / PLGA scaffold with heparin-dopamine BMP-2 appeared smooth. Indicating that the heparin-dopamine complex scaffold is suitable for loading BMP-2.

Experimental Example  10. PCL / PLGA And functionalized PCL / PLGA Scaffold  Cytotoxicity Assessment

To evaluate the cytotoxicity of PCL / PLGA and BMP-2 immobilized heparin-PCL / PLGA, BMP-2 immobilized heparin-dopamine-PCL / PLGA scaffolds on osteoblast cells, each scaffold was placed in cell culture medium And cultured at 37 ° C for 24 hours to obtain an extract culture. On the other hand, 1 × 10 ⁴ osteoblast MG63 cells were placed in a 96-well plate and cultured at 37 ° C. for 24 hours. The osteoblast cells were cultured for 24 hours and 48 hours, and then the culture broth was removed. For cytotoxicity, CCK-8 proliferation kit reagent was added and incubated at 37 ° C for 1 hour, and the absorbance was measured at 450 nm. As shown in FIG. 12, both PCL / PLGA and functionalized PCL / PLGA scaffolds showed cell viability of 90% or more even after 48 hours of culture. This result indicates that both PCL / PLGA and functionalized PCL / PLGA scaffolds Lt; RTI ID = 0.0 > cytotoxic < / RTI >

Experimental Example  11. PCL / PLGA And functionalized PCL / PLGA Scaffold  Cell proliferation

PCL / PLGA and the BMP-2 loaded PCL / PLGA in heparin -PCL / PLGA, a BMP-2 is fixed heparin - In order to evaluate the cell proliferation of the osteoblasts in the dopamine -PCL / PLGA scaffolds, 1 × 10 ⁵ of Osteoblast cells were carefully placed in each scaffold and cultured for 1, 3, and 7 days, and then the cells were washed with PBS buffer. After washing, CCK-8 proliferation kit reagent (Tetrazolium salt reagent, Cell Counting kit-8, Dojindo, USA) was added and incubated for 1 hour. The culture was carefully transferred to a 96-well plate and absorbance was measured at 450 nm. The osteoblasts cultured on PCL / PLGA and functionalized PCL / PLGA gradually proliferated after one day, three days, and seven days (Fig. 13), and the difference in cell proliferation between the groups was observed on day 7 It was confirmed that the heparin-dopamine-PCL / PLGA scaffold in which BMP-2 was immobilized effectively increased.

Experimental Example 12. PCL / PLGA and Functionalized PCL / PLGA Alkaline phosphatase in scaffold Activity

The osteogenic activity of PCL / PLGA and functionalized PCL / PLGA scaffolds was assessed by measuring and analyzing alkaline phosphatase activity, an early differentiation marker of osteoblast. 1 × 10 ⁵ osteoblasts are carefully placed on each PCL / PLGA and cultured for 3, 10, and 14 days. After incubation, the cells were washed with PBS buffer, and 1 × RIPA buffer was added to the cells and sonicated at 110 ° C for 1 minute at 0 ° C. The lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C. After centrifugation, the supernatant was incubated with the p-nitrophenylphosphate solution at 37 ° C for 30 minutes, and the reaction was terminated with 500 μl of 1 N NaOH. The absorbance at 405 nm was measured, Respectively. As shown in FIG. 14, PCL / PLGA and BMP-2-loaded heparin-PCL / PLGA scaffolds showed similar alkaline phosphatase activity to osteoblasts and showed no difference. However, the heparin-dopamine-PCL / PLGA scaffold with immobilized BMP-2 exhibited significantly higher alkaline phosphatase activity than the PCL / PLGA scaffold.

Experimental Example  13. PCL / PLGA And functionalized PCL / PLGA Scaffold  Calcium deposition

The osteogenic effects of PCL / PLGA and BMP-2-fixed heparin-dopamine-PCL / PLGA scaffolds were evaluated by measuring calcium deposition as a late differentiation marker of osteoblast. 1 × 10 ⁵ osteoblasts were carefully placed on each scaffold and incubated for 21 days. After incubation, the cells were washed with PBS buffer and carefully removed from each scaffold with Tris-EDTA solution. The detached cells were centrifuged for 1 minute at 13,500 rpm, and 0.1% Triton X-100 solution was added to the cells. Ultrasonic waves were applied for about 1 minute at 0 degree to crush the cell membrane, followed by centrifugation. The supernatant was separated and analyzed for absorbance at 612 nm using the QuantiChrom ™ Calcium Assay kit. As shown in FIG. 15, the degree of calcium deposition of PCL / PLGA and BMP-2-loaded heparin-PCL / PLGA scaffolds was similar and statistically not significantly different. However, heparin-dopamine-PCL / PLGA scaffolds in which BMP-2 was immobilized exhibited significantly higher levels of calcium deposition than PCL / PLGA scaffolds.

Experimental Example  14. Histology and histomorphological analysis

X-rays of each scaffold implanted in a 8 mm bony defect in the tibia of rats were taken and half of each sample was processed for histological and histomorphological analysis. Each tissue was fixed in 10% buffered formaldehyde and decalcified in 10% formic acid. Both sides were trimmed at right angles to the sagittal suture including the equator of the bone defect and embedded in paraffin. Blocks were cut to a thickness of 6 μm and stained with hematoxylin and eosin (H & E) and Masson's trichrome (MT). Histologically, the functional PCL / PLGA scaffold group showed less foreign body reaction and inflammation, progressed to early osteogenesis, and postoperative recovery and bone regeneration were observed. In the case of PCL / PLGA scaffolds, the foreign body reaction and the inflammatory reaction were severe at the beginning, and the scaffold remained at 8 weeks, and scaffold remained (Fig. 16). Histopathological analysis showed that functionalized PCL / PLGA scaffolds were significantly higher than PCL / PLGA scaffolds at 8 weeks ( P <0.001). At the 8th week of bone formation, the heparin-dopamine PCL-PLGA scaffold group with BMP-2 was found to be higher than that of the other groups by more than 80% (Fig. 17).

Thus, as shown in the above Experimental Example, the heparin-dopamine PCL / PLGA scaffold in which BMP-2 immobilized in Example 2, which is a functional scaffold in which the surface of the PCL / PLGA scaffold was modified to immobilize an osteogenesis promoting substance, It has been shown that it has an excellent effect on bone formation effect.

Claims (9)

As scaffolds whose surface has been modified using chemical conjugates of heparin-dopamine,
The scaffold is derived from BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP- (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), insulin like growth factor-1 growth factor 1 (IGF-1), and lactoferrin.
Wherein the surface of the scaffold is made of PCL / PLGA. The scaffold of claim 1, wherein the bone formation promoting material is contained at a concentration of 1 pg / ml to 3 mg / ml. The scaffold of claim 1, wherein the scaffold is a dental or orthopedic scaffold. [4] The scaffold of claim 3, wherein the dental scaffold is a fixture type or a membrane type. [5] The orthopedic scaffold of claim 3, wherein the orthopedic scaffold includes a prosthetic joint including an artificial joint including a hip, a knee, an ankle, a shoulder or an elbow, or a spinal area artificial disc; And
Wherein the scaffold is selected from the group consisting of a metal screw, a metal plate, or a fixture including a metal fixture used in fracture treatment. I) forming a chemical conjugate of heparin-dopamine;
II) washing and drying the PCL / PLGA scaffold and pretreating it;
III) modifying the surface of the PCL / PLGA scaffold pretreated in step II) with a chemical compound of heparin-dopamine formed in step I); And
IV) BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a and BMP-4 were added to the PCL / PLGA scaffold surface- (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (BGF) -8b, BMP-10 and BMP- immobilizing one or more kinds of bone formation promoting substances selected from the group consisting of bFGF, insulin like growth factor 1 (IGF-1) and lactoferrin, A method for manufacturing a PCL / PLGA scaffold. [7] The method of claim 6, wherein washing and drying in step II) are performed by ultrasonic washing in an ethanol solution for 1 hour and then drying at 50 &lt; 0 &gt; C for 24 hours. [7] The method of claim 6, wherein the pretreatment of the PCL / PLGA scaffold is performed in step III) by adding 10 mM Tris buffer solution (pH 8.0) in which the chemical conjugate of heparin-dopamine is dissolved at a concentration of 5 mg / ml And stirring the mixture in a light-shielded atmosphere, followed by washing with distilled water and drying. 7. The method of claim 6, wherein the immobilization of the bone formation promoting material in step IV) is performed by immersing the PCL / PLGA scaffold surface-modified in step III) in a 0.1 M MES buffer solution containing 50 ng / (pH 5.6) and stirred, followed by washing with distilled water and drying. The method for producing a functionalized PCL / PLGA scaffold according to claim 1,

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