WO2008075824A1

WO2008075824A1 - Bioactive glass nanofibers and method of manufacturing the same

Info

Publication number: WO2008075824A1
Application number: PCT/KR2007/003453
Authority: WO
Inventors: Hae-Won Kim
Original assignee: Industry-Academy Cooperation Foundation, Dankook University
Priority date: 2006-12-21
Filing date: 2007-07-16
Publication date: 2008-06-26
Also published as: KR100791518B1

Abstract

Disclosed herein are bioactive glass nanofibers (BGNFs), which are manufactured using a sol-gel precursor of bioactive glass through electrospinning (ES), and a method of manufacturing the same. The bioactive glass nanofibers have a size of about a few tens to hundreds of nanometers and show excellent bioactivity and osteogenic action. Also, the bioactive glass nanofibers can be used as novel implant biomaterials in the dental and orthopedic fields.

Description

[DESCRIPTION]

[invention Title]

BIOACTIVE GLASS NANOFIBERS AND METHOD OF MANUFACTURING THE SAME [Technical Field]

The present invention relates to bioactive glass nanofibers (BGNFs) , which can be used as novel implant biomaterials in the dental and orthopedic fields, and a method of manufacturing the same, in which the bioactive glass nanofibers, having a size of about a few tens to hundreds of nanometers, are manufactured using a bioactive sol-gel glass as a precursor through electrospinning (ES) .

[Background Art]

For the past few decades, bioactive materials, including calcium phosphates (hydroxyapatite and tricalcium phosphate) and glass/glass ceramics, have been used for a wide range of clinical applications in the dental and orthopedic fields.

Among them, silica-based bioactive glass has been regarded as a promising bone-regeneration material due to its bioactivity, tissue compatibility (in both hard tissue and soft tissue) , osteoconductivity and osteoinductivity.

During this time, most studies have been focused on understanding the biocompatibility mechanism of bioactive glass in vivo, in which the biocompatibility was found to be attributable to the deposition of hydroxyapatite crystal, a bone-like inorganic material, on the glass surface, which is in direct contact with tissue. In the deposition of hydroxyapatite crystal on the surface of the bioactive glass, the composition and shape of the glass are very important. 5 Particularly, if bioactive material has a nanometer size, it will show a rapid inorganic deposition mechanism, leading to very excellent bioactivity.

Among Korean Patents relating to nanofibers based on electrospinning, Korean Patent Laid-Open Publication No. 10-

10 2005-40187 discloses a biomimetic nanofiber/microfiber composite scaffold for inducing tissue regeneration and a method for manufacturing the same, in which the nanofibers obtained from a physiologically active polymer have a two- dimensional or three-dimensional network structure and are

15 restored while tissue is three-dimensionally regenerated, such that the porosity of the scaffold is increased to increase the surface area associated with cells, and thus cells easily adhere to and proliferate in the scaffold.

Also, Korean Patent Laid-Open Publication No. 10-2006-0 38096 discloses a nonwoven membrane for inducing bone induction and regeneration, which consists of silk fibrous nanofibers, and a method for manufacturing the same.

Korean Patent Registration No. 10-439871 discloses a nanofiber-reinforced composite material for medical devices,5 which consists of 10-90 vol% of a polymer matrix and 10-90 vol% of biodegradable nanofibers having a diameter of 10-500 nanometers, and has a flexural strength of more than 290 MPa and a flexural modulus of more than 17 GPa.

Korean Patent Registration No. 10-564366 discloses a tissue-regeneration membrane based on a nanofiber nonwoven matrix and a method for manufacturing the same.

In the above-described prior technologies, nanofibers are manufactured using electrospinning, and a composition consisting only of a polymer is used, such that it can be used for the regeneration of soft tissue. However, when the prior materials are used to regenerate hard tissues, such as bones or teeth, they are greatly inferior to bioceramics such as bioactive glass with respect to physical properties, chemical properties and biological properties. Particularly, there is still no report on the preparation of nanofibers using general bioceramic material or bioactive glass, which are known to have excellent bone formation ability.

Among various types of bioactive glass, sol-gel glasses have recently been developed in advanced countries, and are generally known to show a wide range of solubility and bioactivity (showing bioactivity even at higher SiC>₂ contents) and have a high bone formation rate. Many researchers have used such sol-gel glasses in the form of powder, coating and porous materials for bone substitutes and have reported their excellent in vivo osteogenic ability, and in addition, their excellent bioactivity and cellular response. However, there is still no report of the shaping of these materials into nanoscale materials. It is known that the nanoscale biomaterials can rapidly induce various biological responses, including protein and cellular responses, and thus have an excellent ability to form tissue. [Disclosure] [Technical Problem]

The present invention has been made in order to solve the problems occurring in the prior art, and it is an object of the present invention to provide bioactive glass nanofibers (BGNFs) and a method of manufacturing the bioactive glass nanofibers using a sol-gel precursor of bioactive glass through electrospinning (ES) , in which the bioactive glass nanofibers have a size of a few tens to hundreds of nanometers and have excellent bioactivity and osteogenic action, such that they can be used as novel implant materials in the dental and orthopedic fields. [Technical Solution] To achieve the above object, the present invention provides bioactive glass nanofibers (BGNFs) and a method for manufacturing the same, in which the bioactive glass nanofibers are silica-based glass fibers showing bioactivity capable of depositing hydroxyapatite on the surface thereof in simulated body fluid (SBF) , and have a fundamental glass structure of SiO₂-CaO or SiO₂-CaO-P₂O₅, in which CaO and/or P₂O₅ is added to a fundamental framework of SiO₂, and the bioactive glass nanofibers are in the form of a nanofiber nonwoven matrix having a diameter of a few tens to hundreds of nanometers and are manufactured by making a sol from a bioactive glass composition and making a nanofiber nonwoven matrix from the sol using electrospinning. [Description of Drawings]

FIG. 1 is a flowchart showing a process for manufacturing the inventive bioactive glass nanofibers.

FTG. 2 shows the microscopic morphology of the inventive bioactive glass nanofibers (BGNFs) produced by electrospinning, followed by heat treatment.

FIG. 3 is an electron microscopic morphology showing that hydroxyapatite crystals were deposited on the surface of the inventive bioactive glass nanofibers in simulated body fluid after 3 days.

FIG. 4 is a photograph showing that adult stem cells grow on the inventive bioactive glass nanofibers after incubation for 5 days.

FIG. 5 is a graph of alkaline phosphatase (ALP) activity, expressed by adult stem cells, grown on the inventive bioactive glass nanofibers, after 5 days and 10 days, and shows the ability of the adult stem cells to differentiate into osteocytes. In FIG. 5, as comparison groups, a bioactive glass, which has the same composition of the inventive nanofibers, but is not in the form of nanofibers, and polycaprolactone, which is in the form of nanofibers, but is not a bioactive composition, were used as comparison groups. [Best Mode]

The inventive method for manufacturing bioactive glass nanofibers (BGNFs) is as follows.

As glass precursors, 1-100 g of each of tetraethyl orthosilicate (TEOS) , calcium nitrate and triethyl phosphate at a weight ratio of 0.7: 0.25: 0.05 was added to a water-ethanol mixture (molar ratio = 1:1) containing 0.01-1 g of HCl (1 N), thus preparing 10-1000 ml of a sol mixture. Herein, the molar ratio of the glass precursors to the water-ethanol mixture was adjusted to various ratios of 1.00, 0.50 and 0.25, and thus the diameter of the resulting nanofibers can be adjusted.

Also, the glass precursor composition of tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate of 0.7:0.25:0.05 was selected from the composition range showing biological activity, and this glass precursor composition can be changed without limit. However, a composition containing a silicate source, a calcium source and a phosphate source should be used.

In another method of the present invention, a sol mixture was also prepared using a composition of tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate of 0.58:0.37:0.05.

10-1000 ml of the prepared sol mixture was stirred for 6-

24 hours, and was then aged at 25 ^°C for 6-24 hours, and additionally aged at 40-70 ^°C for 12-48 hours. Then, in order to adjust the rheological properties of the aged sol so as to be suitable for the production of fibers during electrospinning, the aged sol was mixed with 10-1000 ml of 10% polyvinyl butyral

(PVB) at a weight ratio of sol: PVB ranging from 1:2 to 2:1.

Then, 1-10 ml of the sol mixture was placed in a syringe and electrospun by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/cm and an injection rate of

0.01-1 rnβ/h. Then, the formed nanofiber nonwoven matrix was heat-treated at 600-900 ^°C for 1-6 hours in air to completely remove organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNF) .

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative only, and the scope of the present invention is not limited thereto. [Mode for Invention] Example 1

As glass precursors, 100 g of each of tetraethyl orthosilicate (TEOS) , calcium nitrate and triethyl phosphate at a molar ratio of 0.7:0.25:0.05 was added to 1000 ml of a water- ethanol mixture (molar ratio= 1:1) containing 1 g of HCl (1 N), thus preparing a sol mixture. Herein, the molar ratio of the glass precursors to the water-ethanol mixture was 1.00 (weight ratio) .

1000 ml of the prepared sol mixture was stirred for 24 hours, and was then aged at 25 ^°C for 24 hours, and additionally aged at 40-70 ^°C for 48 hours. In order to adjust the rheological properties of the aged sol so as to be suitable for the production of fibers during electrospinning, the aged sol was mixed with 1000 ml of 10% polyvinyl butyral (PVB) at a weight ratio of sol mixture: PVB ranging from 1:2 to 2:1. Then, 10 ml of the sol mixture was placed in a syringe and electrospun by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/ctli and an injection rate of 0.01-1 ml/h. Then, the formed nonwoven nanofiber matrix was heat-treated at 600-900 ^°C for 1-6 hours in air to completely remove organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNFs) . Example 2 As glass precursors, 10 g of each of tetraethyl orthosilicate (TEOS) , calcium nitrate and triethyl phosphate at a molar ratio of 0.7: 0.25: 0.05 was added to 100 ml of a water-ethanol mixture (molar ratio= 1:1) containing 1 g of HCl (1 N), thus preparing a sol mixture. Herein, the molar ratio of the glass precursors to the water-ethanol mixture was 0.50 (weight ratio) . 100 ml of the prepared sol mixture was stirred for 12 hours, was then aged at 25 ^°C for 12 hours, and was then additionally aged at 40-70 ^"C for 24 hours. In order to adjust the rheological properties of the aged sol so as to be suitable for the production of fibers during electrospinning, the aged sol was mixed with 100 ml of 10% polyvinyl butyral (PVB) at a weight ratio of sol mixture: PVB ranging from 1:2 to 2:1. Then, 5 ml of the sol mixture was placed in a syringe and electrospun by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/cm and an injection rate of 0.01-1 mi/h. Then, the formed nanofiber nonwoven matrix was heat-treated at 600-900 ^"C for 1-6 hours in air to completely remove the organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNFs) . Example 3

As glass precursors, 100 g of each of tetraethyl orthosilicate (TEOS) , calcium nitrate and triethyl phosphate at a molar ratio of 0.58: 0.37: 0.05 was added to 1000 ml of a water-ethanol mixture (molar ratio= 1:1) containing 1 g of HCl (1 N), thus preparing a sol mixture. Herein, the molar ratio of the glass precursors to the water-ethanol mixture was 1.00 (weight ratio) .

1000 ml of the prepared sol mixture was stirred for 24 hours, was then aged at 25 ^°C for 24 hours, and was then additionally aged at 40-70 ^°C for 48 hours. In order to adjust the rheological properties of the aged sol so as to be suitable for the production of fibers during electrospinning, the aged sol was mixed with 1000 ml of 10% polyvinyl butyral (PVB) at a weight ratio of sol mixture: PVB of 1:2 to 2:1. Then, 10 ml of the sol mixture was placed in a syringe and electrospun by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/cm and an injection rate of 0.01-1 ni^/h. Then, the formed nanofiber nonwoven matrix was heat-treated at

600-900 ^°C for 1-6 hours in air to completely remove organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNFs) . Example 4

As glass precursors, 10 g of each of tetraethyl orthosilicate (TEOS) , calcium nitrate and triethyl phosphate at a molar ratio of 0.58: 0.37: 0.05 was added to 100 ml of a water- ethanol mixture (molar ratio= 1:1) containing 1 g of HCl (1 N), thus preparing a sol mixture. Herein, the molar ratio of the glass precursors to the water-ethanol mixture was 0.50 (weight ratio) . 100 ml of the prepared sol mixture was stirred for 12 hours, was then aged at 25 ^°C for 12 hours, and was then additionally aged at 40-70 ^°C for 24 hours. In order to adjust the rheological properties of the aged sol so as to be suitable for the production of fibers during electrospinning, the aged sol was mixed with 100 ml of 10% polyvinyl butyral (PVB) at a weight ratio of sol mixture: PVB ranging from 1:2 to 2:1. Then, 5 ml of the sol mixture was placed in a syringe and electrospun by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/cπi and an injection rate of 0.01-1 Then, the formed nanofiber nonwoven matrix was heat-treated at 600-900 ^°C for 1-6 hours in air to completely remove organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNFs) .

The bioactive glass nanofibers (BGNFs) , manufactured as described above, are silica-based glass fibers showing bioactivity capable of depositing hydroxyapatite on the surface thereof in simulated body fluid (SBF) , and have a fundamental glass structure of SiO₂-CaO or Siθ₂-CaO-P₂θ₅ in which either CaO or CaO together with P₂O₅ is added to a fundamental framework of SiO₂, the bioactive glass nanofibers being in the form of a nanofiber nonwoven matrix having a size of a few tens to hundreds of nanometers . Test Examples In order to measure the morphology of the glass nanofibers manufactured in Example 1, the physical properties thereof were tested using a field emission scanning electron microscope

(FESEM; JSM6330F, JEOL) . Also, the internal characteristics of the nanofibers were observed using a transmission electron microscope (TEM; CM20, Philips) . In order to measure the bioactive characteristics of the nanofibers, the test samples were placed in simulated body fluid (a solution having an ion concentration similar to that of human body plasma) and incubated in a constant-temperature water bath at 37 ^"C, and then the changes in the surface morphology and structure of the nanofibers were analyzed with each of FESEM and TEM.

In order to examine the cell response to the bioactive glass nanofibers in the form of a nonwoven matrix, bone marrow stem cells extracted from mouse marrow were used. The cells were incubated on the nonwoven matrix for 5 days, and then the morphoLogy of the cells grown on the nonwoven matrix was observed using FESEM after cell immobilization and dehydration.

The differentiation of the stem cells into osteocytes was measured by measuring the alkaline phosphatase (ALP) activity of the cells. For this purpose, the cells were incubated for 5 days and 10 days, and then the cell layer was collected, disrupted by treatment with 0.1 % Triton X-100, and subjected to repeated freezing-thawing processes. Then, the cell lysate fraction was quantified based on the total protein content, obtained using a DC protein ana Lysis kit, and the ALP concentration of the cells was colorimetrically measured by analyzing the ALP activity using a p-nitrophenyl phosphate substrate.

The ALP analysis was performed on four repeat samples (n = 4) , and the analysis data were compared using a one-way ANOVA analysis with statistical significance at p < 0.05 and p < 0.01.

Test results

FIG. 2 shows a general morphology of the bioactive glass nanofibers (tetraethyl orthosilicate (TEOS) : calcium nitrate: triethyl phosphate = 0.7:0.25:0.05) (molar ratio), produced by electrospinning, followed by heat treatment. Specifically, FIG. 2 shows the case in which the glass precursors were used at a concenbration of 1 mole. As can be seen in FIG. 2, nanometer- sized continuous fibers were successfully produced without forming any bead. The average diameter of the fibers was measured to be 630 nm. During the heat treatment, the morphology of the nanofibers was well maintained.

FIG. 3 is a transmission electron microscopic morphology showing that hydroxyapatite crystals were deposited on the surface of the bioactive glass nanofibers (tetraethyl orthosilicate (TEOS) : calcium silicate: triethyl phosphate =

0.7:0.25:0.05) (molar ratio)), after the nanofibers were incubated in simulated body fluid for 3 days. As can be seen in FIG. 3, crystals having a size of a few nanometers were uniformly deposited on the surface of the nanofibers.

FIG. 3 shows the results of diffraction analysis, conducbed using a transmission electron microscope, in which the produced crystals show the diffraction pattern of hydroxyapatite. That is, the bioactive glass nanofibers showed an ability to form an inorganic phase of bone tissue in simulated body fluid in a very short time. This is because the nanofibers had a nanoscale structure, and thus quickly reacted with the external solution due to the rapid dissolution and re- precipitation of ions. The above test results revealed that the bioactive glass nanofibers have excellent bioactive ability, and it is generally known that the bioactive ability of biomaterials for use as bone substitutes depends on the rate of induction of hydroxyapatite crystals in simulated body solution. FIG. 4 is an electron microscope photograph showing that adult stem cells grow after they were incubated on the inventive bioactive glass nanofibers (tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7:0.25:0.05) (molar ratio) for 5 days. As can be seen in FIG. 4, the cells adhered well to the nanofiber strands, the cytoplasm spread well, and many cells proliferated. This suggests that the bioactive glass nanofibers reacted with cells in a very suitable manner.

FTG. 5 is a graphic diagram showing alkaline phosphatase (ALP) activity expressed by the adult stem cells, grown on the bioactive glass nanofibers (tetraethylorthosilicate (TEOS) : calcium nitrate: triethyl phosphate = 0.7:0.25:0.05) (molar ratio), after 10 days. The alkaline phosphatase (ALP) activity is an early-stage index for whether stem cells differentiate well into osteocytes, and it indicates osteogenic ability. In FIG. 5, the test results for the ALP activity resulting from the bioactive glass nanofibers are shown in comparison with: 1) a general bioactive glass disc, which has the same composition as that of the glass nanofibers, but is not in the form of nanofibers; and 2) a typical biopolymer polycaprolactone (PCL) , which is in the form of nanofibers, but the composition of which does not show bioactivity. As can be seen in FIG. 5, the bioactive glass nanofibers showed excellent ALP activity compared to those of the comparison groups. The bioactive glass nanofibers showed particularly good results in comparison with the biopolymer PCL, and this is considered to be because the composition of the bioactive glass is very suitable for the differentiation of stem cells into osteocytes and the formation of bone. Also, the bioactive glass nanofibers showed superiority to the bioactive glass disc, which has the same composition as that of the glass nanofibers, but are not in the form of nanofibers. This suggests that the form of nanofibers is very important for differentiation into osteocytes, and that the nanofibers have excellent osteogenic ability.

[industrial Applicability]

As described above, according to the present invention, 1) the bioactive glass composition can be successfully obtained in the form of nanofibers from a sol-gel solution using an electrospinning apparatus; 2) the size of the nanofibers can be adjusted in the range of a few tens to hundreds of nanometers by controlling the concentration of the sol-gel solution and electrospinning conditions (average diameter of the nanofibers in Example 1: 86 run) ;

3) the manufactured bioactive glass nanofibers (fibers having an average diameter of 86 nm, manufactured in Example 1) have excellent in vitro bioactivity, because hydroxyapatite is quickly deposited on the surface of the nanofibers in simulated body fluid; and

4) the bioactive glass nanofibers (fibers having an average diameter of 86 nm, manufactured in Example 1) have excellent osteogenic ability, because adult stem cells adhere well to and grow on the nanofibers and show a high rate of differentiation into osteocytes (high ALP activity) .

Accordingly, the present invention makes way for a novel type in the bone substitute material field, in that the bioactive glass nanofibers of the present invention are in the form of nanometers, unlike the prior biomaterials, consisting of micrometer-sized, bioactive glass. Also, the bioactive glass nanofibers of the present invention will be highly useful as tissue-regeneration biomaterials in the dental, orthopedic, plastic surgery and neurosurgical fieLds.

Claims

[CLAIMS] [Claim l]

Bioactive glass nanofibers (BGNF) , which are silicate glass fibers showing bioactivity capable of depositing hydroxyapatite on the surface thereof in simulated body fluid (SBF) , and have a fundamental glass structure of Siθ₂-CaO or SiO₂-CaO-P₂O₅, in which either CaO or CaO together with P₂O₅ is added to a fundamental framework of SiO₂, the bioactive glass nanofibers being in the form of a nanofiber nonwoven matrix having a size of a few tens to hundreds of nanometers. [Claim 2]

A method for manufacturing bLoactive glass nanofibers (BGNFs) , the method comprising: mixing 1-100 g of tetraethyl orthosilicate (TEOS) , as an SiO₂ source, 1-100 g of calcium nitrate, as a Ca source, and 1- 100 g of triethyl phosphate, as a P source, at a molar ratio ranging from 0.58: 0.36: 0.06 to 0.7: 0.25: 0.05; adding the mixture to 10-1000 ml of a water-ethanol mixture (molar ratio: 100:1-1:100) containing 0.01-1 g of any one compound selected from among HCl (0.1 N) and HNO₃ (0.1 N), thus preparing 10-1000 ml of a sol mixture; and stirring 10-1000 ml of the prepared sol mixture for 6-24 hours, aging the stirred mixture at 25 ^°C for 6-24 hours, and then at 40-70 ^°C for 12-48 hours, mixing the aged sol mixture with 10-1000 ml of 10 % poLyvinyl butyral (PVB) at a weight ratio of sol: PVB = 1:2-2:1 in order to adjust the rheological properties of the sol mixture so as to be suitable for production of fibers during electrospmning, placing 1-10 ml of the sol mixture in a syringe, electrospinning the sol mixture by injecting it onto a metal collector at a DC electric field strength of 0.5-2 kV/cm and an injection rate of 0.01-1 rn-β/h, and heat-treating the resulting nano fiber matrix at 600-900 ^°C for 1-6 hours in air to remove organic substances therefrom, thus manufacturing bioactive glass nanofibers (BGNFs) . [Claim 3]

The method of Claim 2, wherein the SiO₂ source is trimethyl orthosilicate (TMOS) , the Ca source is any one compound selected from among calcium chloride (CaCl₂) , calcium hydroxide (Ca(OH)₂), calcium acetate and calcium alkoxides, and the P₂Os source is selected from among phosphoric acid (H₃PO₄) and P₂O₅.