KR101881541B1 - Surface treatment method of biodegradable polymer for improvement of osteogenesis or osseointegration - Google Patents

Abstract

The present invention provides a method for preparing a polylactic acid (PLLA) comprising the steps of: a) inducing a functional group on the surface of poly L-lactic acid (PLLA); b) binding hydroxyapatite (HAp) to the functional group-induced PLLA surface; And c) binding bone morphogenetic protein 2 (BMP-2) to the surface of the PLLA conjugated with HAp.

Description

TECHNICAL FIELD The present invention relates to a biodegradable polymer for improving osteogenesis or osseointegration, and more particularly, to a biodegradable polymer for osteogenesis or osseointegration,

The present invention relates to a method for surface treatment of biodegradable polymers for enhancing bone formation and osseointegration, and more particularly, to a method for surface treatment of biodegradable polymers, which comprises the steps of: a) inducing functional groups on the surface of poly L-lactic acid (PLLA); b) binding hydroxyapatite (HAp) to the functional group-induced PLLA surface; And c) binding bone morphogenetic protein 2 (BMP-2) to the surface of the PLLA to which HAp is bound. The present invention also provides a method for treating a biodegradable polymer.

Representative materials of medical implants are metal materials with excellent mechanical properties and processability. However, despite the excellent properties of metals, metallic implants have some problems such as stress shielding, image degradation, and implant migration. In addition, if the implant needs to be removed through reoperation after the treatment, reoperation for a large amount of nerve and blood vessels in the surrounding tissues of the treatment unit or a large number of soft tissue injuries is a great burden on the patient.

In order to overcome the disadvantages of such metallic implants, research and development of biodegradable implants has been proposed. Medical applications of these biodegradable materials have been studied since the mid-1960s mainly on polymers such as polylactic acids (PLA), polyglycolic acid (PGA), or copolymers thereof, such as PGLA (KR 10-1568146). However, due to the problem of surface modification of implants, the use of hydroxyapatite coating on the surface of expensive implants has been clinically improving the biocompatibility (Kang et al., Characterization of hydroxyapatite containing a titania layer formed by anodization coupled with blasting. Acta Odontol Scand 72: 989-998, 2014). However, there is still a problem in that the lifetime of hydroapatite is greatly reduced due to peeling of hydroapatite from the surface of the used implant.

Accordingly, the present inventors have sought to overcome the shortcomings of metallic implants and to find out a surface treatment method of biodegradable polymers having improved bone formation and osseointegration ability by modifying the surface of biodegradable polymers. As a result, a method of binding hydroxyapatite and a bone inducing factor to the surface of poly-L-lactic acid was identified.

DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for preparing polylactic acid (PLLA), which comprises the steps of: a) inducing a functional group on the surface of poly L-lactic acid (PLLA); b) binding hydroxyapatite (HAp) to the functional group-induced PLLA surface; And c) binding bone morphogenetic protein 2 (BMP-2) to the surface of the PLLA to which HAp is bound. The present invention also provides a method for treating a biodegradable polymer.

Another object of the present invention is to provide a poly-L-lactic acid (PLLA) derived functional group; Hydroxyapatite (HAp) bound to the PLLA surface; And a bone morphogenetic protein (BMP-2) bound to the PLLA surface to which the HAp is bound.

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

Accordingly, the present invention provides a method for producing a poly (L-lactic acid), comprising: a) inducing a functional group on the surface of poly L-lactic acid (PLLA); b) binding hydroxyapatite (HAp) to the functional group-induced PLLA surface; And c) binding bone morphogenetic protein 2 (BMP-2) to the surface of the PLLA to which the HAp is bound. The present invention also provides a method for treating a biodegradable polymer.

According to a preferred embodiment of the present invention, the induction of the functional group in the step a) may be performed by plasma treatment at a pressure of 0.01 to 70 mTorr for 5 to 60 seconds.

According to another preferred embodiment of the present invention, the functional group of step (a) may be a hydroxyl group or a carboxyl group.

According to another preferred embodiment of the present invention, the hydroxyapatite in step b) may be mixed with dopamine in a weight ratio of 1: 1 to 1:10.

According to another preferred embodiment of the present invention, in the step b), the functional group of the PLLA surface and the HAp may be peptide-bonded.

According to another preferred embodiment of the present invention, the binding of BMP-2 in step c) may be performed through heparin.

The present invention also relates to a poly-L-lactic acid (PLLA) derived from a functional group; Bone Morphogenetic protein 2 (BMP-2) bound to the PLLA surface; And a surface-treated biodegradable polymer comprising BMP-2 bound to the surface of PLLA to which HAp is bound.

According to a preferred embodiment of the present invention, the biodegradable polymer may have an osteogenic activity of 0.05 to 0.25 M / min.

According to another preferred embodiment of the present invention, the biodegradable polymer may have an optical density (O.D.) value of 0.05 to 0.30.

DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the problems as described above. The present invention relates to a biodegradable polymer surface treatment comprising binding a hydroxyapatite to a poly-L-lactic acid surface on which a functional group is derived, The present invention can be utilized as an implant by providing a surface-treated biodegradable polymer improved in bone formation and osseointegration at the time of hard tissue fixation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a surface treatment method of a biodegradable polymer of the present invention. FIG.
2 is a graph showing cell proliferation results of Example 1 and Comparative Examples 1 to 3. FIG.
3 is a graph showing the results of measurement of ALP (Alkaline phosphatase) activity involved in bone formation promotion of Example 1 and Comparative Examples 1 to 3. FIG.
4 is a graph showing bone mineralization evaluation results of Example 1 and Comparative Examples 1 to 3. FIG.
5 shows results of ARS (Alizarin red S) staining of Example 1 and Comparative Examples 1 to 3. FIG.

Hereinafter, the present invention will be described in more detail.

As described above, since the conventional biodegradable polymer has a problem of surface modification, it has been clinically improved in biocompatibility using a simple coating of hydroxyapatite. However, a biodegradable polymer implant having improved bone formation and osseointegration at the time of hard tissue fixation has been developed And it is urgently required to develop a surface treatment method therefor.

The present invention solves the above-mentioned problems by providing a method of modifying the surface of a biodegradable polymer, poly L-lactic acid (PLLA). Thereby providing a surface-modified biodegradable polymer improved in bone formation and osseointegration at the time of hard tissue fixation.

The term "surface modification" in the present invention means a change in the chemical structure and physical structure of the particle surface. In the present invention, a structural change is caused by introducing a functional group onto the surface of the biodegradable polymer, poly L-lactic acid.

The term "poly-L-lactic acid" in the present invention means a biodegradable thermoplastic aliphatic polyester derived from renewable raw materials such as corn starch, tapioca roots and the like.

The term "hydroxyapatite " in the present invention is a substance found in teeth and bones in the body, and is often used as a filler for replacing a cut bone or as a coating agent for promoting ingrowth of bone toward an artificial implant, And an amine group.

The term "bone-inducing factor" in the present invention means a protein that can be used in combination with an osteogenic protein and directly involved in bone formation.

The present invention provides a method for preparing a polylactic acid (PLLA) comprising the steps of: a) inducing a functional group on the surface of poly L-lactic acid (PLLA); b) binding hydroxyapatite (HAp) to the functional group-induced PLLA surface; And c) binding bone morphogenetic protein 2 (BMP-2) to the surface of the PLLA to which the HAp is bound. The present invention also provides a method for treating a biodegradable polymer.

The derivation of the functional group in step a) may be performed for 5 to 60 seconds at a pressure of 0.01 to 70 mTorr in a RF generator chamber for hydroapatite attachment, preferably at a pressure of 1 to 60 mTorr The treatment is preferably performed for 10 to 50 seconds, but is not limited thereto.

The plasma treatment of the present invention is a method of plasma treatment at a low pressure. Since the plasma treatment is performed in a vacuum state, when the pressure is exceeded, the ratio of the functional group induced by the plasma can be limited by the surrounding oxygen, There is a risk of generation of ozone, and there is a problem that it is possible to induce a change in physical properties of a plastic material such as PLLA which is sensitive to heat due to the problem of heat generation. Further, when the treatment time is less than or more than the above-mentioned time, there is a problem that the ratio of the carboxy group induced per unit area of the PLLA surface is decreased.

The functional group induced on the PLLA surface in step a) may be a hydroxyl group or a carboxyl group, and when the functional group is a carboxy group, a peptide bond may be formed through condensation reaction with an amine group of HAp .

The hydroxyapatite in step b) may be mixed with dopamine in a weight ratio of 1: 1 to 1:10, preferably 1: 5 to 1: 7 (weight) , And most preferably 1: 1 (weight), but is not limited thereto. In Example 1-2 of the present invention, HAp and dopamine were mixed at a weight ratio of 1: 1, respectively. In this case, dopamine plays a role of bonding HAp with the carboxy group of the surface-modified PLLA so that the proportion of dopamine is higher than that of HAp Is suitable.

The binding in the step b) may be such that the functional group of the PLLA surface and the HAp are peptide-bonded.

In step c), the binding of BMP-2 may be performed through heparin.

The present invention also relates to a poly-L-lactic acid (PLLA) derived from a functional group; Hydroxyapatite (HAp) bound to the PLLA surface; And a bone morphogenetic protein (BMP-2) bound to the PLLA surface to which the HAp is bound, to provide a biodegradable polymer surface-treated.

The surface-treated biodegradable polymer may have an osteogenic activity of 0.05 to 0.25 M / min.

In addition, the surface-treated biodegradable polymer may have an optical density (OD) value of 0.05 to 0.30.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  One. Surface-treated  Biodegradable polymers, PLLA's  Produce

Example  1-1. Functional group  Induced PLLA  Produce

PLLA specimens for in vitro use were fabricated on a 24-well plate (24-well plate) with a diameter of 14.0 mm and a thickness of 1.0 mm. PLLA specimens for in vivo (5.0 mm in length and 2.0 mm in thickness) screw. Then, to induce the functional groups of PLLA surface, the reaction was performed for 20 seconds at a pressure of 50 mTorr and 20 watt in an RF plasma generator chamber, and exposed to the atmosphere for 10 minutes immediately after removing from the chamber. As a result, hydroxyl group and carboxyl group were induced on the PLLA disc surface.

Example  1-2. Hydroxyapatite ( Hydroxyapatite , HAp )end Combined PLLA  Produce

HAp was coated with dopamine (DA) to bind HAp to the functional group-induced PLLA surface of Example 1-1. 10 mM tris buffer, pH 8.5 and 2 mg / mL were prepared by dissolving DA at room temperature. HAp was dissolved in 0.02% polyacrylic acid (PAA) at room temperature. The dopamine / hydroxyapatite (DA / HAp) powders coated with HAp were prepared by mixing the two solutions and lyophilization. At this time, HAp and dopamine were mixed at a weight ratio of 1: 1.

50 mg of DMT-MM was dissolved in 25 mL of sterilized water, and the aqueous solution and 10 surface-modified PLLA discs were reacted at room temperature for 1 hour. Then, DA / HAp was added and reacted at room temperature for 24 hours to prepare DA / HAp. After lyophilization for 24 hours at -80 ° C and 5 mTorr, the cells were sterilized by EO gas at 54 ° C for 3 hours and stored at room temperature. As a result, it was confirmed that HAp was bound through the condensation reaction between the carboxyl group formed on the PLLA surface and the amine group of DA / HAp through surface modification. 4- (4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride n-Hydrate (DMT-MM) was also used as a condensing agent in this Example.

Example  1-3. HAp Combined PLLA  On the surface Bone factor ( Bone Morphogenetic protein  2, BMP- 2) Combined PLLA  Produce

In order to bind BMP-2 to the HAp-bound PLLA surface of Example 1-2, 10 PLLA / HAp discs prepared by the method of Example 1-2 were dissolved in 25 ml of sterilized water and 50 ml of DMT- The reaction was allowed to proceed at room temperature for 1 hour. Hep was added, reacted at 4 ° C for 24 hours, and washed three times with DPBS. 50 ng of BMP-2 was dissolved in 25 ml of DPBS, and the mixture was reacted at 4 ° C for 24 hours. -80 ° C, 5 mTorr, and stored at -20 ° C for 24 hours.

Comparative Example  One.

In vitro plLA specimens (14.0 mm in diameter, 1.0 mm in thickness) were prepared for 24-well culture plates (24 well plates) and PLLA swatches (5.0 mm in length and 2.0 mm in thickness) a screw was prepared.

Comparative Example  2. Bone factor ( Bone Morphogenetic protein  2, BMP- 2) Combined PLLA  Produce

In order to prepare PLLA / BMP-2 having BMP-2 bound to the functional group-induced PLLA surface of Example 1-1, carboxyl group induced on the surface of PLLA disc and amine group of ethylenediamine (ED) were bound. At this time, 4- (4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride n-Hydrate (DMT-MM) was used as a condensing agent. After dissolving 50 mL of DMT-MM in 25 mL of sterilized water (DW), 10 PLLA discs were reacted at room temperature for 1 hour. 1 mL of EA was dissolved in 25 mL of DW and reacted for 24 hours. The cells were lyophilized for 24 hours at -80 ° C and 5 mTorr and sterilized with ethylene oxide (EO) gas at 54 ° C for 3 hours. 50 mg of DMT-MM was dissolved in 25 mL of sterilized water (DW), and reacted at room temperature for 1 hour. Then, 50 mg of Heparin (Hep) was added and reacted at 4 ° C for 24 hours. 50 ng of BMP-2 was dissolved in 100 μl of Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) and reacted for 24 hours. -80 ° C, 5 mTorr, and stored at -20 ° C for 24 hours.

Comparative Example  3.

Hydroxyapatite (HAp) -coupled PLLA was prepared in the same manner as in Example 1-2.

Experimental Example

The MG-63 cell line was cultured in a 24-well plate at 37 ° C and 5% CO 2. Cells were used at 5 × 10 3 cells / well. The culture medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in Dulbecco's modified eagle medium (DMEM). The culture medium was changed every 3 days. The same applies to all of the following Experimental Examples 1 to 3.

Experimental Example  1. Evaluation of Cell Suitability by Cell Differentiation Measurement

In order to confirm the toxicity of Example 1 and Comparative Examples 1 to 3, MG-63 cells were seeded in the discs of Example 1 and Comparative Examples 1 to 3. Cell proliferation was confirmed using cell counting kit 8 (CCK-8) at day 1, day 3, and day 7. CCK-8 was added dropwise to each well and cultured at 37 ° C and 5% CO ₂ for 1 hour. The culture broth was transferred to a microplate at 100 nm intervals and measured at 450 nm in an ELISA instrument. The experiment was repeated 3 times.

As shown in Fig. 2, there was no significant difference in the cell proliferation results in the discs of Examples 2 to 4 and Comparative Example 1 at the 1st day and 3rd day, but the cell proliferation results in the disc of Example 4 at the 7th day It was confirmed that it increased about twice as much as that of Comparative Example 1, and increased by 0.1 or more as compared with Examples 2 and 3, respectively.

Experimental Example  2. Alkaline 포스화제  ( ALP ) activity  Assessment of bone differentiation by measurement

To measure the ALP activity of Example 1 and Comparative Examples 1 to 3, MG-63 cells were cultured on the discs of Example 1 and Comparative Examples 1 to 3. ALP activity was measured using the ALP assay kit on days 7, 14, and 21. ALP is a major mediator of osteogenesis and is highly expressed from 7 to 14 days.

As shown in FIG. 3, there was no significant difference between the results of Example 1 and Comparative Example 1 in the case of Day 7, and in Comparative Example 1 in case of Day 21, The results of Example 1 were about twice as much as those of Comparative Example 1 and about 0.05 to 0.1 as compared to Comparative Example 2 and Comparative Example 3 in the case of the 14th day. The results showed that the biodegradable polymer surface-treated with HAp and BMP-2 showed the best bone formation ability at 14 days.

Experimental Example  3. Calcium deposition assay  through bone mineralization  evaluation

To evaluate bone mineralization through the calcium deposition assay of Example 1 and Comparative Examples 1 to 3, cultured cells were used for 7 days, 14 days and 21 days. Cells were washed 3 times with DPBS without Ca2 +, Mg2 +. Cells were fixed with 4% formalin 1 mL / well for 20 minutes, washed 3 times with sterile DW 1 mL / well, and dried for 2 minutes. 40 mM alizarin red S (ARS) solution was added dropwise at 500? L / well and shaded for 45 minutes at room temperature. Thereafter, the cells were washed with DW 1 mL / well for 3 minutes and repeated 5 times. After washing, the cells were observed. Cetylpyridinium chloride was dissolved in DBPS for quantification and 10 w / v% solution (10% CPC buffer) was used. 10% CPC buffer was added at 1 mL / well and reacted for 1 hour. Then, the cells were transferred to a microplate at 100? L and measured at 550 nm in an ELISA instrument.

As shown in FIG. 4, it was confirmed that as the cell culture time increased, the amount of the calcium form of the surface-treated Example 1 was increased as compared with Comparative Examples 1 to 3. Particularly, it was confirmed that the amount of calcium form of Example 1 increased more than twice that of Comparative Example 1 in the case of the 14th day. FIG. 5 shows that the calcium amount of the cells can be visually confirmed through the results of staining in Example 1 and Comparative Examples 1 to 3. As the cell culture time was increased, the discs of Example 1 and Comparative Examples 1 to 3 were stained It was confirmed that the area occupied was widened. In particular, in Example 1, it was confirmed that the dyed area was wider than in Comparative Examples 1 to 3 in all cases.

4 and 5, it can be seen that the biodegradable polymer surface-treated with HAp and BMP-2 has the effect of strengthening the bones.

Claims (9)

a) inducing a functional group on the surface of poly L-lactic acid (PLLA);
b) coating hydroxyapatite with dopamine by mixing hydroxyapatite (HAp) and dopamine (DA);
c) coupling dopamine / hydroxyapatite (DA / HAp) coated with dopamine to the functional group-induced PLLA surface;
d) introducing heparin (Hep) into the PLLA surface by reacting the surface of the PLLA conjugated with DA / HAp with an aqueous solution of heparin; And
e) immersing the heparin-introduced PLLA surface in an aqueous solution having Bone Morphogenetic protein 2 (BMP-2) dissolved therein to induce binding of heparin to BMP-2; Processing method.
The method according to claim 1, wherein the induction of the functional group in step (a) is carried out by plasma treatment at a pressure of 0.01 to 70 mTorr for 5 to 60 seconds.
The method according to claim 1, wherein the functional group in step a) is a hydroxyl group or a carboxyl group.
delete The method according to claim 1, wherein in step (c), the functional group of the PLLA surface and the HAp are peptide-bonded.
delete A biodegradable polymer surface-treated by the method of claim 1.
The biodegradable polymer according to claim 7, wherein the biodegradable polymer has an osteogenic activity of 0.05 to 0.25 M / min.
The surface-treated biodegradable polymer according to claim 7, wherein the biodegradable polymer has an optical density (OD) value of 0.05 to 0.30.

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