KR100791518B1 - Production and potential of bioactive glass nanofibers as a next-generation biomaterial - Google Patents

Abstract

A preparation method of bioactive glass nanofiber excellent in bioactivity and osteocyte function is provided to utilize BGNF as a novel bioactive implant material in fields of dentistry and orthopaedic hospital. A preparation method of bioactive glass nanofiber includes the steps of: mixing TEOS, calcium nitrate, and triethyl phosphate with a water-ethanol containing 0.01-1g of 0.1NHCl or 0.1NHNO3 to obtain a sol mixture; and agitating and incubating the sol mixture, adding a mixture of PVB and the sol at a ratio of 1:2-2:1 by weight, injecting 1-10ml of the sol mixture into a syringe and a metal collector for electric radiation under 0.5-2kV/cm DC field strength at the speed of 0.01-1ml/h, and heating the resulting nanofiber non-woven fabric at 600-900deg.C for 1-6 hours.

Description

Bioactive glass nanofibers (WNF) and manufacturing method thereof {Production and Potential of Bioactive Glass Nanofibers as a Next-generation Biomaterial}

1 is a schematic diagram of a bioactive glass nanofiber manufacturing process of the present invention.

2 Electron microscopy morphology of bioactive glass nanofibers (BGNF) produced by heat treatment following the electrospinning method of the present invention.

Figure 3 Electron microscopy morphology showing that the apatite hydroxide crystals deposited on the surface after 3 days in the biomimetic solution of the bioactive glass nanofibers of the present invention

Figure 4 Adult stem cells growing on the bioactive glass nanofibers of the present invention after incubation for 5 days

5 is an Alkaline Phosphatase (ALP) activity graph of adult stem cells grown on bioactive glass nanofibers of the present invention after 5 and 10 days, showing their differentiation ability into bone cells. Comparative chart using the same composition (comparison group) but with bioactive glass, not nanofiber form, and polycaprolactone, a polymer nanofiber that is nanofiber but not bioactive composition

The present invention relates to glass nanofibers (BGNF) that can be applied to a variety of new implantable biomaterials in the dental and orthopedic field, and to a method of manufacturing the same, wherein the bioactive glass nanofibers are about tens to hundreds of nanometers in size. The present invention relates to a bioactive glass nanofiber (BGNF) produced by using an electrospinning method (ES) by using sol-gel glass of a bioactive composition as a precursor and a method of manufacturing the same.

Over the past decades, bioactive materials, including calcium phosphate (apatite hydroxide and tricalcium phosphate) and glass / glass ceramics, have gained wide clinical approval in the dental and orthopedic fields. First of all, silica-based bioactive glass has been regarded as a promising bone tissue regeneration material due to its bioactivity, histocompatibility (both hard and soft tissue), osteoconductivity and osteoinduction.

Most studies have been aimed at understanding the biocompatibility mechanisms of bioactive glasses in vivo, where their biocompatibility is due to the deposition of apatite hydroxide crystals, bone-like minerals at the glass interface in direct contact with the tissue. . As such, the composition and shape of the glass are very important for the deposition of apatite hydroxide crystals on the surface. Particularly in the case of nano-shaped shape, it will show a very fast mechanism of depositing inorganic materials, which will result in excellent bioactivity.

As a domestic patent on nanofibers using electrospinning method, Korean Laid-Open Patent Publication No. 10-2005-40187 discloses nanofibers obtained from physiologically active polymers in the form of a two-dimensional or three-dimensional network, and the biological tissue is three-dimensional. At the same time, the composite support of the biomimetic nanofibers and microfibers for regenerating the nanofibers and improving the porosity to increase the surface area related to the cells to induce tissue regeneration to which the cells adhere and proliferate, and a method of manufacturing the same Described,

Korean Patent Publication No. 10-2006-38096 discloses a nonwoven fabric-type bone tissue-induced regeneration shielding membrane made of silk fibroin nanofibers prepared by electrospinning, and a method of manufacturing the same.

Korean Patent Publication No. 10-439871 consists of a polymer matrix of 10 to 90% by volume and a biodegradable nanofiber of 90 to 10% by volume of 10 to 500 nanometers in diameter with a bending strength of 290 MPa or more and a bending elastic modulus of It describes a composite material for medical devices reinforced with nanofibers of 17 GPa or more and a manufacturing method thereof.

Korean Patent Publication No. 10-564366 discloses a tissue regeneration shielding membrane using a nanofiber nonwoven fabric and a method of manufacturing the same.

The conventional technique as described above is a technique for producing nanofibers using the electrospinning method, but can be used for soft tissue regeneration by using only the composition of the polymer, but as a hard tissue regeneration material such as bone or tooth, It is much inferior to bioceramic such as bioactive glass. In particular, there have been no reports of producing nanofibers using bioceramic or bioactive glass, which are generally known to be excellent in bone formation ability.

Among the various types of bioactive glasses, sol-gel based glass has recently been developed in developed countries, which, in comparison with molten glass, not only has a broad range of solubility and bioactivity, but also (higher SiO ₂ content). Bioactivity), bone formation rate is also known to be very fast. Many researchers have used these sol-gel glasses in the form of powders, coatings and porous bodies for bone substitutes, and have reported on their good in vivo bone formation ability as well as their excellent bioactivity and cellular response. However, there are no reports of these in nano form. It is known that by shaping the biomaterial into nano units, it is possible to rapidly induce various biological reactions including protein and cellular reactions, and eventually increase the ability to form tissues.

In order to solve the above problems, the present invention relates to a bioactive glass nanofibers (BGNF) prepared by using an electrospinning method (ES) by using a sol-gel precursor of bioactive glass, and a method of manufacturing the same Glass nanofibers range in size from tens to hundreds of nanometers, and have excellent bioactivity and osteocytic activity, which can be widely used as new biomaterials for implantation in the dental and orthopedic fields (BGNF). And it is a technical problem to be achieved by the present invention to provide a manufacturing method thereof.

The present invention to attain the object is a biomimetic solution (simulated body fluid: SBF) in a silica based glass to indicate the bioactivity ability to deposit the hydroxide apatite, the basic skeleton of SiO ₂ CaO (or P ₂ O ₅ may be added to form a basic glass structure of SiO ₂ -CaO or SiO ₂ -CaO-P ₂ O ₅ , which is nanofibrous (usually tens to hundreds of nanometers in diameter). Bioactive glass nano to make sol of bioactive composition and make it into non-woven fabric of nanofiber using electrospinning method to manufacture bioactive glass nanofiber (BGNF) in the form of nanofiber nonwoven matrix A fiber (BGNF) and a method for producing the same.

Bioactive glass nanofibers (BGNF) manufacturing method is as follows.

Water-ethanol mixture containing 0.01-1 g of HCl (1 N) at 1-100 g of tetraethyl orthosilicate (TEOS), calcium nitrate, and triethyl phosphate, respectively, at a weight ratio of 0.7: 0.25: 0.05 Molar ratio = 1: 1) to 10-1000 ml to make 10-1000 ml of the prepared sol mixture. At this time, the molar ratio of the glass precursors to water + ethanol was differently adjusted to glass precursor / water + ethanol = 1.00, 0.50, 0.25, it was shown that the diameter of the final nanofibers can be adjusted.

The composition of the glass precursor is tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7: 0.25: 0.05, which is an area showing bioactivity, and the composition can be changed. However, silicate raw material, calcium raw material, and phosphate raw material were used.

Another method of the present invention was also prepared using a composition of tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.58: 0.37: 0.05.

10 to 1000 ml of the prepared sol mixture was stirred for 6 to 24 hours, and then aged at 25 ° C. for 6 to 24 hours and further at 40 to 70 ° C. for 12 to 48 hours, and then the aged sol was used to produce fibers during electrospinning. In order to adjust the rheological properties of the sol to suitably, 10 to 1000 ml of 10% polyvinyl-butyral (PVB) is mixed with the weight of sol: PVB = 1: 2 to 2: 1, and then 1 to 10 ml of the sol mixture. Take the sample into a syringe and inject into a metal collector under a DC electric field strength of 0.5 to 2 kV / cm and an injection rate of 0.01 to 1 ml / h and electrospin the formed nanofiber nonwoven fabric at 600 to 900 ° C. for 1 to 6 hours. Heat treatment in air produced bioactive glass nanofibers (BGNF) from which organic sources were completely removed.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1

A water-ethanol mixture comprising 1 g of HCl (1N) in 100 g each of tetraethyl orthosilicate (TEOS), calcium nitrate, and triethyl phosphate at a molar ratio of 0.7: 0.25: 0.05 as the glass precursor (molar ratio = 1: 1) The glass precursor, a sol mixture prepared by adding to 1000 ml, of the molar ratio of water + ethanol was prepared as glass precursor / water + ethanol = 1.00 (by weight),

After stirring 1000 ml of the prepared glass precursor sol mixture for 24 hours, the mixture was aged at 25 ° C. for 24 hours and further at 40 to 70 ° C. for 48 hours, and then the sol was flowed so as to be suitable for generating fibers during electrospinning. In order to control the chemical properties, 1000 ml of 10% polyvinyl-butyral (PVB) and a sol mixture: PVB = 1: 2 ~ 2: 1 were mixed by weight, and then 10 ml of the sol mixture was taken into a syringe, and 0.5 ~ The nanofiber nonwoven fabric formed by electrospinning by injection into a metal collector under a DC electric field strength of 2 kV / cm and an injection rate of 0.01-1 ml / h was heat treated in air at 600-900 ° C. for 1-6 hours to provide an organic source. Completely removed bioactive glass nanofibers (BGNF) were prepared.

Example 2

A water-ethanol mixture comprising 0.1 g of tetraethyl orthosilicate (TEOS), calcium nitrate, and triethyl phosphate at a molar ratio of 0.7: 0.25: 0.05 each of 10 g each of HCl (1N) (molar ratio = 1) : 1) The molar ratio of water precursor to ethanol is obtained by adding the glass precursor, which is a sol mixture added to 100 ml, to glass precursor / water + ethanol = 0.50 (by weight),

100 ml of the prepared glass precursor sol mixture was stirred for 12 hours, and then aged at 25 ° C. for 12 hours and further at 40 to 70 ° C. for 24 hours, and then the flow of the sol was adjusted so that the matured sol was suitable for generating fibers during electrospinning. In order to control the chemical properties, 100 ml of 10% polyvinyl-butyral (PVB) and a sol mixture: PVB = 1: 2 to 2: 1 were mixed by weight, 5 ml of the sol mixture was taken into a syringe, and 0.5 to An organic source was prepared by heat-treating nanofiber nonwoven fabric formed by injecting into a metal collector under a DC electric field strength of 2 kV / cm and an injection rate of 0.01 to 1 ml / h in air at 600 to 900 ° C for 1 to 6 hours. To completely remove the bioactive glass nanofibers (BGNF).

Example 3

Water-ethanol mixture comprising 1 g of HCl (1N) in 100 g each of tetraethyl orthosilicate (TEOS), calcium nitrate, and triethyl phosphate at a molar ratio of 0.58: 0.37: 0.05 as the glass precursor (molar ratio = 1: 1) The glass precursor, a sol mixture prepared by adding to 1000 ml, of the molar ratio of water + ethanol was prepared as glass precursor / water + ethanol = 1.00 (by weight),

After stirring 1000 ml of the prepared glass precursor sol mixture for 24 hours, the mixture was aged at 25 ° C. for 24 hours and further at 40 to 70 ° C. for 48 hours. In order to control the chemical properties, 1000 ml of 10% polyvinyl-butyral (PVB) and a sol mixture: PVB = 1: 2 ~ 2: 1 were mixed by weight, and then 10 ml of the sol mixture was taken into a syringe, and 0.5 ~ The nanofiber nonwoven fabric formed by electrospinning by injection into a metal collector under a DC electric field strength of 2 kV / cm and an injection rate of 0.01-1 ml / h was heat treated in air at 600-900 ° C. for 1-6 hours to provide an organic source. Completely removed bioactive glass nanofibers (BGNF) were prepared.

Example 4

Water-ethanol mixture comprising 0.1 g of tetraethyl orthosilicate (TEOS), calcium nitrate, and triethyl phosphate in a molar ratio of 0.58: 0.37: 0.05 each 10 g of HCl (1N) (molar ratio = 1) : 1) The molar ratio of water precursor to ethanol was prepared by adding 100 ml of glass compound, which was prepared as glass precursor / water + ethanol = 0.50 (by weight),

100 ml of the prepared glass precursor sol compound was stirred for 12 hours, and then aged at 25 ° C. for 12 hours and further at 40 to 70 ° C. for 24 hours, and then the sol was flowed so as to be suitable for producing fibers during electrospinning. To control the chemical properties, 100 ml of 10% polyvinyl-butyral (PVB) and a sol compound: PVB = 1: 2 to 2: 1 were mixed, and 5 ml of the sol mixture was taken into a syringe and 0.5 to The nanofiber nonwoven fabric formed by electrospinning by injecting into a metal collector under a DC electric field strength of 2 kV / cm and an injection rate of 0.01 to 1 ml / h was heat treated in air at 600 to 900 ° C for 1 to 6 hours to provide an organic source. Completely removed bioactive glass nanofibers (BGNF) were prepared.

Bioactive glass nanofibers (BGNF) prepared as described above is biomimetic solution (simulated body fluid: SBF) in a silica based glass to indicate the ability of biological activity which can be deposited hydroxide apatite, a basic skeleton of SiO ₂ CaO (or P ₂ O ₅ , which may be accompanied) is added to form a basic glass structure of SiO ₂ -CaO or SiO ₂ -CaO-P ₂ O ₅ , which is nanofiber-shaped (usually a diameter It has physical properties of tens to hundreds of nanometers) nonfiber nonwoven matrix.

Experimental Example

The morphology of the nanofiber glass prepared in Example 1 was tested for physical properties using field emission scanning electron microscopy (FESEM, JSM6330F, JEOL). In addition, the properties inside the nanofibers were observed using a transmission electron microscope (TEM, CM20, Philips).

The bioactive properties of the nanocomposites were measured by placing samples in a simulated body fluid (ie, solutions containing ionic concentrations similar to those of human plasma) and storing them in a 37 ° C thermostatic bath. Analysis was by FESEM and TEM.

In order to know the cellular response to non-woven bioactive glass nanofibers, bone marrow stem cells extracted from rat bone marrow were evaluated. After culturing the cells on the nonwoven fabric for 5 days, the morphology of the cells grown on the nonwoven fabric was observed using FESEM after cell fixation and dehydration.

Differentiation into osteoblasts was measured by measuring alkaline phosphatase (ALP) activity of adult stem cells. After 5 and 10 days of incubation, the cell layers were collected, ruptured by treatment with 0.1% Triton X-100, and further freeze-thaw procedures were followed to quantify the cell lysate aliquots with the total protein content obtained from the DC protein assay kit. Afterwards, ALP activity was measured colorimetrically by analysis using p-nitrophenyl phosphate substrate.

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

Experiment result

In Figure 2, the general morphology of the bioactive glass nanofibers (tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7: 0.25: 0.05) (molar contrast ratio) produced by electrospinning followed by heat treatment is shown. do. The case where the glass precursor of 1 mol concentration is used typically is shown. It can be seen that nano sized continuous fibers were successfully produced without forming any beads. The average diameter of the fibers was measured at 630 nm. During the heat treatment, the nanofiber morphology was well maintained.

In FIG. 3, apatite hydroxide crystals were formed on the surface after 3 days in a biomimetic solution of bioactive glass nanofibers (tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7: 0.25: 0.05) (molar contrast ratio). Transmission electron microscope morphology showing deposition. It can be seen that grains of nanometer size are uniformly deposited on the surface of the nanofibers.

3 shows the diffraction analysis of the transmission electron microscope, which shows that the resulting grains show the diffraction pattern of the apatite hydroxide. That is, the bioactive glass nanofibers showed the ability to form mineral phase of bone tissue in a very fast time in the biomimetic solution. This is because the structure of the nanofibers was made in nano units and reacted quickly with the external solution, which is due to the rapid ion dissolution and reprecipitation thereof.

The results of this experiment indicate that the bioactive glass nanofibers have excellent bioactivity, and the bioactivity of biomaterials, which are commonly used as bone substitutes, depends on how quickly the apatite hydroxide crystals are induced in the biomimetic solution. Known.

FIG. 4 shows adult stem cells growing on the bioactive glass nanofibers of the present invention (tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7: 0.25: 0.05) (molar ratio) after incubation for 5 days. An electron micrograph showing the appearance.

Since the cells adhere well to the nanofiber strands, the cytoplasm is well spread, and many cells are proliferated, the bioactive glass nanofibers can be judged to be very suitable reactions with the cells.

FIG. 5 shows Alkaline Phosphatase (ALP) expressing adult stem cells grown on bioactive glass nanofibers (tetraethyl orthosilicate (TEOS): calcium nitrate: triethyl phosphate = 0.7: 0.25: 0.05) (molar contrast ratio) after 10 days. A graph showing activity, which is an early stage indicator of whether stem cells differentiate into osteocytes, showing bone formation ability.

Experimental results show the superiority of the bioactive glass nanofibers,

1) general bioactive glass substrates having the same composition but not in nanofiber form;

2) The form is shown in comparison with polycaprolactone (PCL), a representative biopolymer whose nanofibers do not show bioactivity in composition. Bioactive glass nanofibers were found to show significantly better ALP activity than the control groups.

In particular, it shows a particularly excellent result compared with PCL, a biopolymer, because the composition of the bioactive glass is very suitable for differentiation and bone formation of bone cells. In addition, the same composition, but not in nanofiber form, compared with bioactive glass substrates, the advantage of the nanofiber form is very important for differentiation into osteocytes, and it can be said that it has a good bone formation ability. have.

The present invention as described above

1) Bioactive glass compositions can be successfully obtained in the form of nanofibers using sol-gel solutions and electrospinning devices.

2) The size of the nanofibers can be obtained from several tens of nanometers to several hundred nanometers by controlling the concentration of the sol-gel solution and the electro-nano spinning conditions (average diameter of Example 1 is 86 nm fiber),

3) It was confirmed that the prepared bioactive glass nanofibers (average diameter of 86 nm fiber in Example 1) showed excellent in vitro bioactivity by showing surface deposition of fast apatite hydroxide in the biomimetic solution.

4) Adult stem cells adhere well and grow on bioactive glass nanofibers (average diameter of 86 nm fiber), and have excellent bone formation ability by showing differentiation rate (ALP activity) into excellent bone cells. Bar,

The present invention is believed to have opened the way for a new type in the field of bone substitute material, unlike the conventional bio-material for implantation made of micro-active bioactive glass, in the form of nano-units. Biomaterials are expected to play a major role in the fields of dentistry, orthopedics, plastic surgery and neurosurgery.

Claims (3)

In the bioactive glass nanofibers (BGNF), biomimetic solution (simulated body fluid: SBF) as a silica-based glass to indicate the ability of biological activity which can be deposited hydroxide apatite in, the basic skeleton of SiO ₂ CaO, Or CaO and P ₂ O ₅ added together to form a basic glass structure of SiO ₂ -CaO or SiO ₂ -CaO-P ₂ O ₅ , which is nanofiber-shaped (usually tens to hundreds of nanometers in diameter) Bioactive glass nanofibers (BGNF), characterized in that they are a nonfiber nonwoven matrix. In the method of producing bioactive glass nanofibers BGNF), Tetraethyl orthosilicate (TEOS) as SiO2 raw material, calcium nitrate as Ca raw material, and triethyl phosphate as mole number P1 from 0.58: 0.36: 0.06 to 0.7: 0.25: 0.05 By mixing 10-1000 ml of a sol mixture prepared by adding 10-1000 ml of a water-ethanol mixture (relative to the number of moles = 100: 1-1: 100) containing 0.01-1 g of any one compound selected from 0.1NHCl and 0.1NHNO3 , 10 to 1000 ml of the prepared sol mixture was stirred for 6 to 24 hours and then aged at 25 ° C. for 6 to 24 hours and further at 40 to 70 ° C. for 12 to 48 hours. In order to suitably adjust the rheological properties of the sol, 10 to 1000 ml of 10% polyvinyl-butyral (PVB) is mixed with the weight of sol: PVB = 1: 2 to 2: 1, and then 1 to 10 ml of the sol mixture. The nanofiber nonwoven fabric formed by injecting into a metal collector under a DC electric field strength of 0.5 to 2 kV / cm and an injection rate of 0.01 to 1 ml / h was electrospun for 1 to 6 hours at 600 to 900 ° C. A method of producing bioactive glass nanofibers (BGNF) in which the organic source is completely removed by heat treatment in air. The initial precursor of glass according to claim 2 is trimethyl orthosilicate (TMOS) as SiO2 raw material, calcium chloride (CaCl2), calcium hydroxide (Ca (OH) 2), calcium acetate (calcium acetate), calcium-alkoxide as CaO raw material A method for producing a bioactive glass nanofiber (BGNF), characterized in that any one selected from the group (calcium alkoxides), and any one selected from phosphoric acid (H3PO4), P2O5 as a P2O5 raw material.

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