KR101294963B1 - Porous scaffold prepared by using plant template and manufacturing method thereof - Google Patents

Abstract

PURPOSE: A porous scaffold and a manufacturing method thereof are provided to facilitate the attachment, differentiation, and proliferation of osteogenic cells and to improve cell metabolism. CONSTITUTION: A method for manufacturing a porous scaffold comprises: producing slurry by mixing a tracheophyte template, titanium oxide, and alcohol; collecting the slurry; and drying or sintering the slurry at 300-1800 deg. C. The temperature rising speed is 0.5-3 deg. C/min.

Description

Porous scaffold prepared by using plant template and manufacturing method

The present invention relates to a porous scaffold manufactured by using a vascular plant template and a method for producing the same, which is microporous by using the vascular plant as a template, and is capable of producing a scaffold having pores and microtunnels of various diameters for human transplantation. In addition to the production of artificial synthetic bone, it can be used as a carrier for cell therapy such as stem cells, drug delivery system of nano drugs.

Diseases such as trauma, surgery, inflammation, osteoporosis or periodontitis can result in bone loss, slow bone growth, and it is very important to improve bone growth and equip the bone anatomy. Scaffolds can be used as a substitute for lost bone structures, or as a carrier for delivery of drugs or cellular therapeutics involved in the bone regeneration process.

There is a technology for artificial bone and its manufacturing method (Korean Patent No. 10-1031121). This technology relates to a method of manufacturing artificial bones, which is made up of a composite structure of porous bodies having a large number of pores and a compact body without pores, which can compensate for the shortcomings of each component. After preparing the slurry by gelling and gelling the slurry prepared in the above step, a slurry having a different content of pore-forming agent is poured into the prepared slurry, gelled, and dried and sintered to manufacture artificial bone. However, in this case, the pore-forming agent is used to make the porous body, and the pore-forming agent does not contain a compact body, and since the pore-forming agent uses PMMA, polymer beads, naphthalene, etc., the size of the formed pores is not eco-friendly. It is relatively large and does not vary as much as ˜700 μm.

There is a description of bone prostheses with protective coatings for promoting bone mutual growth (US Application No. 08 / 521,111). This technique includes a boundary surface in contact with and attached to a living bone, a number of shallow recesses extending inwardly with respect to the recess floor, and a bone growth enhancement film over the floor and shielded by a bath relief shape. The present invention relates to an implantable bone prosthesis having a body attached to raw bone for replacing or repairing a portion of the raw bone, and hydroxyapatite is used as a film for promoting bone growth.

There is a technique related to a method for manufacturing a porous implant (Korean Patent Publication No. 10-2004-0064794). This technique comprises the steps of mixing the pure titanium (Ti) powder and hydroxyapatite (HA) powder to form a mixed powder; Mixing a binder with the mixed powder and extruding the same by an extruder to produce a molded body by injection; Removing a binder from the injection molded body; And a step of sintering the injection molded body from which the binder is removed.

There is a technique related to a porous bone-cartilage composite support for tissue engineering (Korean Patent Publication No. 10-2006-0054236). The technique comprises a tissue comprising (i) a porous bone regeneration layer comprising a biocompatible polymer and a bioactive ceramic, the ceramic being exposed to a surface, and (ii) a porous cartilage regeneration layer comprising a biocompatible elastic polymer. An engineering porous bone-cartilage composite support.

There is a technique relating to living bone-induced artificial bone (PCT / JP2004 / 00042). This technique consists of a single mass of titanium or titanium alloy, with three-dimensional mesh shapes of 100-3000 μm in diameter, with holes in succession, with holes less than 50 μm in diameter on the inner surface of the holes, with a porosity of 30-80%. A living bone-induced artificial bone comprising a porous body, an amorphous titanium oxide phase, an amorphous alkali titanate phase, and the like, and a film formed on at least a part of the hole and the surface of the hole in the porous body. It is about.

There is also a technique for ZrO ₂ scaffolds with a hydroxyapatite (HA) layer. However, in the case of the scaffold, the HA layer may peel off or wear out during implantation, and the cracks, tears and loosening from the surface of the implant may be due to the difference in mechanical properties between ZrO ₂ and HA. HA fragments can form small body bodies (sequestered bodies), which has the problem that they can interfere with osteointegration.

Other known scaffolds are made of degradable materials, which means that they are degraded after implantation into the subject. There is an advantage in that it is degradable, but in another aspect it may be a disadvantage because it is not desirable in terms of stabilization of the implant itself. In addition, some scaffolds known in the art trigger the inflammatory response or cause infection. For example, a human or animal derived bone implant scaffold may cause an allergic reaction when implanted in another animal.

On the other hand, a ceramic material layer was coated on the implant surface to stimulate the regeneration of bone tissue. However, the ceramic coating is fragile and may peel off or fall off the implant surface, which may cause the implant to not function properly.

In the present invention, by providing a scaffold having pores of various sizes and having a microporous tunnel, it serves as an excellent support for the proliferation of capillaries and the adhesion, differentiation and proliferation of osteoblasts, It is to provide a scaffold and a method of manufacturing the fluid (fluid) that can be freely passed to help cell metabolism, can also be used as a carrier for delivery of nano-drugs or cell therapy.

In the present invention, a scaffold comprising a material selected from the group consisting of metal oxides, metal phosphates, metal sulfates and metal hydroxides, wherein the scaffold provides a scaffold having a pore size of about 10 to 1,000 μm. I want to solve the problem.

In the present invention, (a) vascular plant template; And mixing a substance and an alcohol selected from the group consisting of metal oxides, metal phosphates, metal sulfates and metal hydroxides to produce a slurry; (b) recovering the resulting slurry; And (c) drying the slurry or sintering at 1,800 ° C. or lower, thereby providing a method for producing a scaffold using a tubular plant template.

Ceramic scaffolds with pores and microporous tunnels of various sizes of the present invention are made of titanium oxide having good biocompatibility and the like and thus can cause immunological problems in allogeneic bone (human bone) or distal bone (bovine bone, horse bone, etc.). It can be implanted into a subject without causing side effects such as cross infection or allergic reactions. The scaffold of the present invention also has an advantageous effect on the regeneration of tissue due to its material. In addition, the scaffold composed of interstitial fibrous walls, the water pipe, and the tissue layer of the vascular plant serves as a microporous tunnel, which is excellent in capillary growth, adhesion, differentiation, and proliferation of capillary cells. Can serve as a support In addition, various fluids in the body (fluid) can be freely passed through the cell metabolism, microporous tunnel diameter is very diverse, it is possible to proliferate capillaries of various diameters, nanocapsules, microcapsules of various sizes, etc. It can be used as a carrier for drug delivery. On the other hand, by using the plant and its charcoal, etc. as a template, there is an advantage that can utilize the agricultural waste that must be disposed of, and unlike the existing artificial or animal template is completely removed after sintering, even if the trace amount remains harmless to the human body This has the advantage of being more secure.

1 is a photograph showing a scaffold of the present invention;
2 is a photograph of a TiO ₂ scaffold made using a plant template;
3 is a photograph of a sintered TiO ₂ scaffold; And
4 is a schematic of the steps for making the scaffold of the present invention.

The present invention relates to a porous scaffold manufactured by using a plant template and a method of manufacturing the same, which is microporous by using a plant tube bundle as a template, and is capable of producing a scaffold having pores and microporous tunnels of various diameters for human transplantation. In addition to the production of artificial synthetic bone, it can be used as a carrier for cell therapy such as stem cells, drug delivery system of nano drugs.

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

The present invention provides a scaffold comprising a material selected from the group consisting of metal oxides, metal phosphates, metal sulfates and metal hydroxides, the scaffold having a pore size of about 10 to 1,000 μm.

In one embodiment of the invention, the pore size may be 10 to 1,000 μm, 10 to 500 μm.

Pores of various sizes may exist, and because the diameters of the body and water pipes vary depending on the types of hardwoods and conifers, the range of the pores may be adjusted according to the mixing ratio of the tube bundle plants used.

As a plant template, a plant having a vascular bundle (vascular plant, tracheophyta) is used, and bamboo charcoal, bamboo, oak, oak charcoal, birch or birch charcoal, loofah, corn bar, etc. may be used. As used herein, the term vascular plant refers to a vascular plant and a vascular plant, and generically refers to a plant having a vascular bundle in the body and includes both ferns and seed plants (seed plants and genus plants).

In one embodiment of the present invention, the pore size of the scaffold may range from 10 to 1,000 μm, with at least some of the pores being interconnected or partially interconnected. In other words, the pores are not composed of only closed or closed pores. By including pores with these interconnected openings, these microporous tunnels can serve as an excellent support for capillary proliferation, adhesion, differentiation and proliferation of osteoblasts, and various fluids in the body. ) Can pass freely to help cell metabolism, proliferation of capillaries of various diameters is possible. This can prevent tissue necrosis.

In one embodiment of the invention, the compressive strength of the scaffold can be adjusted to an appropriate range. Although the compressive strength is very high, if the porosity is very low, the formation of new bone may be slow due to insufficient blood flow space, and it may not be used as a carrier for delivery because it is difficult to mount nano drugs or cells. In addition, in the case of a general porous body, blood flow space is secured due to high porosity, but there is a risk that the scaffold is destroyed due to low compressive strength. However, in the scaffold according to the present invention, because the pores of various sizes are present to an appropriate level while having a suitable level of compressive strength, it may provide a blood flow space to help form new bone. On the other hand, in the manufacture of artificial bones preferably absorbed into the bone, the compressive strength may be adjusted lower. Those skilled in the art will be able to produce scaffolds with suitable compressive strengths depending on the intended use.

In one embodiment of the invention, the material is a titanium oxide selected from the group consisting of TiO ₂ , Ti ₃ O, Ti ₂ O, Ti ₃ O ₂ , TiO, Ti ₂ O ₃ , Ti ₃ O ₅ and titanium butoxide ; Calcium phosphate; Calcium sulfate; Apatite selected from the group consisting of hydroxide apatite, silicon and magnesium substituted apatite; Zirconium dioxide; Silicon dioxide; And combinations thereof, and more specifically, TiO ₂ , titanium butoxide, calcium phosphate, calcium sulfate, hydroxyapatite, ZrO ₂ , SiO _{2, and the} like, but are not limited thereto. By using the material as a material, it can be implanted into a subject without causing side effects such as immunological problems, cross infections or allergic reactions that may occur in allogeneic bone (human bone) or xenograft (bovine bone, horse bone, etc.).

In one embodiment of the invention, the scaffold may be used as a medical implant, a drug delivery carrier or a cell delivery carrier, in particular the scaffold regenerates tissue such as bone Healing, replacement and recovery; Or for cell or drug delivery.

The invention also relates to (a) a vascular plant template; Materials selected from the group consisting of metal oxides, metal phosphates, metal sulfates and metal hydroxides; And mixing the alcohol to produce a slurry; (b) recovering the resulting slurry; And (c) drying the slurry or sintering at 1,800 ° C. or less.

In one embodiment of the present invention, the tube bundle plant template may be used as a plant having a tube bundle, specifically, bamboo, bamboo charcoal, oak, oak charcoal, birch and birch charcoal, loofah, corn bar, etc. may be used. However, it is not limited thereto. Observation of the cross-section can be used as a template for plants with well-developed voids in tissue bundles. Plants for use as templates may be used after drying.

In preparing the scaffold, the material includes titanium oxide selected from the group consisting of TiO ₂ , Ti ₃ O, Ti ₂ O, Ti ₃ O ₂ , TiO, Ti ₂ O ₃ , Ti ₃ O ₅ and titanium butoxide; Calcium phosphate; Calcium sulfate; Apatite selected from the group consisting of hydroxide apatite, silicon and magnesium substituted apatite; Zirconium dioxide; Silicon dioxide; And any one thereof may be used, and more specifically, TiO ₂ , titanium butoxide, calcium phosphate, calcium sulfate, hydroxyapatite, ZrO ₂ , SiO _2, and the like may be used, and more specifically, TiO ₂ may be used. It may be used, but not limited to.

In one embodiment of the invention, additives such as binders, dispersants or cracking inhibitors may be used in the slurrying of each material.

As the binder, amide-based monomers such as acrylamide, agar, natural polysaccharides such as gelatin, methyl cellulose (MC), epoxy resins, and the like may be used, but are not limited thereto.

As a dispersant, polyacrylic acid sodium salt may be used, but is not limited thereto.

As the crack preventing agent, a crack preventing agent usually used in the art may be appropriately selected and used.

In one embodiment of the present invention, the step (c) can be naturally dried during sintering in accordance with the purpose of use, it can be sintered at 300 to 1,800 ℃, it can be sintered at 1,000 to 1,500 ℃. In one embodiment according to the invention, 1 hour to 10 hours, or 2 hours to 8 hours, or 3 hours to 6 hours at 1,000 to 1,500 ° C .; Specifically, 1 hour to 10 hours, or 2 hours to 8 hours, or 3 hours to 6 hours at 1,000 to 1,200 ° C; More specifically 1 hour to 10 hours, or 2 hours to 8 hours, or 3 hours to 6 hours at about 1,100 ° C .; More specifically, the sintering may be performed at about 1,100 ° C. for about 4 hours. In one embodiment of the present invention, in the step (c) if the sintering by a heat treatment method other than natural drying, the temperature increase rate may be 0.5 to 3 ℃ / min, specifically 1 to 3 ℃ / min, Specifically, it may be about 2 ° C / min. It is possible to obtain a scaffold having the most desirable physical properties in the temperature rising conditions.

The scaffold manufactured through the sintering process may be slowly cooled and recovered at room temperature. In the case of rapid cooling, there is a possibility that a scaffold having desired physical properties may not be obtained, and there is a fear of being broken.

By going through the sintering step, the plant template is burned away to form pores, leaving only the scaffold. At this time, since a plant with well-developed vacant spaces in the vascular bundle and tissue layer (genus) is used as a template, scaffolds with eco-friendly, easy to remove, and pores of various sizes can be obtained as compared with organic synthetic sponges. . On the other hand, the use of animal scaffolds may cause side effects such as immunological problems, cross-infection or allergic reactions in the body when not completely removed, whereas in the case of the vascular plant template according to the present invention, these problems may arise. There is no.

In one embodiment of the present invention, the method may further include removing the fiber component according to the type of plant used. More specifically, the fiber component may be removed through an acid or copper ammonia solution treatment step.

The manufactured scaffold can be manufactured to suit the intended use through a precision measurement process. The size and shape of the metal oxide scaffold depends on the intended use. By varying the vascular plant templates used in making the scaffolds, the size and shape of the scaffolds produced may be varied. Therefore, the scaffold can be easily customized for specific subjects and for specific uses.

The present invention provides a scaffold prepared according to the above method, the scaffold can be used as a medical implant, a drug delivery carrier or a cell delivery carrier.

The scaffold according to the present invention can be used for the filling of bone defects due to trauma, surgery, tumors, infections, osteoporosis, etc., can also be used as a frame for cell growth, for delivery of cell therapeutics such as nano drugs or stem cells. It can be used as a carrier.

More specifically, the scaffold according to the present invention may be used as a bone filling material, that is, for the purpose of filling the voids of bone, and in order to fill the bone cavity, the particle size is 0.05 to 5 mm in average diameter, specifically 0.1 to 2 mm. More specifically, it may be granulated in the range of 0.2 to 1 mm. In this case, the scaffold may be loaded with a bone forming factor, and specifically, as a bone forming factor, a bone forming protein, such as a recombinant human bone morphogenetic protein, may be used.

Biomolecules mounted on the scaffold according to the present invention include natural biomolecules, synthetic biomolecules, recombinant biomolecules and the like. Specifically, cell adhesion factors, enzymes, proteins in blood, antibodies, growth factors, hormones, DNA, RNA, RNAi, drugs, protein drugs, peptides, minerals, vitamins, bone formation factors and the like. These biomolecules may be attached to the scaffolds by immersing the scaffold in a solution containing the biomolecules or by electrochemical processes or the like, and other methods known in the art may be used.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following is to help the understanding of the present invention and does not mean to limit the scope of the present invention thereto.

Example 1 Preparation of Scaffolds Using Bamboo Charcoal

In the titania slurry synthesis, titanium butoxide (titanium butoxide, TBOT, Aldrich), ethanol (Aldrich) and ammonia water were used without purification. As water, tertiary purified water prepared by a reverse osmosis method was used. Specific synthesis process is as follows.

0.3 g of bamboo charcoal was placed in a beaker containing a mixture of 20 mL of ethanol and 0.5 mL of distilled water and sufficiently dispersed by ultrasonic wave for 10 minutes. After a day enough charcoal was left to soak in the solution. In a glovebox, 1.56 g of titanium butoxide was dissolved in 50 mL of ethanol and transferred to a separatory funnel. The bamboo charcoal template solution was stirred using a bar magnet, and the titanium butoxide solution of the separating funnel was added dropwise, followed by stirring for about 30 minutes. Stirring was added for an additional 15 minutes after the dropping was completed. It was observed that cloudyly produced titania particles were adsorbed onto the bamboo charcoal and deposited to grow. These dispersed particles were filtered using a filter paper and a white slurry was recovered. At this time, it was washed three times with 50 mL of ethanol. The white slurry obtained was placed in a drier and dried at 40 ° C. Thereafter, the dried slurry was evenly spread on the square alumina pack. After closing the lid of the alumina square bag containing the white slurry was put in a furnace (furnace) and maintained at 1,100 ℃ for 4 hours at a rate of 2 ℃ / min. At this time, all of the bamboo charcoal is burned, leaving only the titania slurry, and then slowly cooled to room temperature and recovered. The titania slurry obtained is as shown in FIG. 1.

Example 2 Preparation of Scaffolds Using Bamboo

Instead of bamboo charcoal of Example 1 using 0.3g of bamboo, titania slurry was prepared according to the same process as in Example 1.

Example 3 Preparation of Scaffolds Using Charcoal

Instead of bamboo charcoal of Example 1 using 0.3g of charcoal, titania slurry was prepared according to the same process as in Example 1.

Example 4 Preparation of Scaffolds Using Birch Charcoal

Instead of the bamboo charcoal of Example 1 using 0.3g birch charcoal, according to the same process as in Example 1 to prepare a titania slurry.

Example 5 Preparation of Scaffolds Using Scrubbers

A titania slurry was prepared according to the same procedure as in Example 1, using 0.3 g of a loofah instead of bamboo charcoal of Example 1.

Example 6 Preparation of Scaffolds Using Scrubber Charcoal

Instead of the bamboo charcoal of Example 1 using 0.3g scrubber charcoal, titania slurry was prepared according to the same process as in Example 1.

Example 7 Preparation of Scaffolds Using Corn Cobs

Instead of bamboo charcoal of Example 1, using 0.3g of corn stalks, titania slurry was prepared according to the same procedure as in Example 1.

Example 8 Preparation of Scaffolds Using Corn Charcoal

Instead of bamboo charcoal of Example 1 using 0.3g corn charcoal, titania slurry was prepared according to the same process as in Example 1.

<Experimental Method>

(1) Test Specimen Preparation

Test specimens for measuring compressive strength and porosity were prepared through the following process. 10 dense body specimens (Comparative Example 1) prepared without using a plant template, 10 specimens (Example 1) prepared according to the process of Example 1 of the present invention, and a pore-forming agent (PMMA, polymethyl methacrylate) Ten porous specimens (Comparative Example 2) prepared using were prepared. Specimens were set to width X length X height = 5 mm X 10 mm X 10 mm.

Experimental Example 1. Measurement of compressive strength

Compressive strength was measured using a TIRAtest 2810 (TIRA) UTM instrument. Compressive strength was measured at 0.5mm / min head speed. The average value of the remaining values except for the maximum value and the minimum value among five repeated measurements was obtained. The experimental results are shown in the following table.

Compressive strength measurement result Sample Comparative Example 1 Comparative Example 2 Example 1 Measured value (average value, MPa) 240.6 21.2 150.3

Experimental Example 2. Measurement of Porosity

The dry weight, the suspension weight, and the wet weight of the test specimens were measured, respectively, and the porosity was repeatedly measured five times using the Archimedes method. The measured values were averaged with the remaining values except for the maximum value and the minimum value. The results are shown in the following table.

Porosity measurement result Sample Comparative Example 1 Comparative Example 2 Example 1 Measured value (average value,%) 4.8 65.1 51.2

Experimental Example 3. Specific Surface Area Measuring Machine

The specific surface area detector (BET) can calculate the surface area per unit weight or volume by measuring the size and size distribution of pores present in the porous mass or by measuring the amount required to form a monolayer adsorbed on the sample surface. When nitrogen gas is solid, that is, the inner surface of the container or adsorption or desorption due to the temperature change without changing other conditions in a closed container, some molecules of a certain amount of gas are adsorbed or desorbed. Can be observed. After cooling the sample and blowing an inert gas such as nitrogen gas, nitrogen gas is put on the sample surface to prevent sample destruction and the surface area according to the pores of the sample can be calculated. The results of measuring the specific surface area of the scaffolds of Example 1 and Comparative Examples 1 and 2 using the specific surface area measuring device are shown in the following table.

Specific surface area measurement result Sample Example 1 Comparative Example 1 Comparative Example 1 Measured value (mean value, m ² / g) 20.33 10.2 10.1

As can be seen from the results of the above table, in the case of a compact structure having a single structure, the compressive strength is very high, but because the porosity is very low, new bone formation may be slow due to insufficient blood flow space, and it is difficult to mount nano drugs or cells for delivery. It cannot be used as a carrier. In addition, in the case of the porous body, the blood flow space is secured due to the high porosity, but there is a risk that the scaffold is destroyed due to the low compressive strength. However, in the scaffold according to the present invention, because the pores of various sizes are present to an appropriate level while having a suitable level of compressive strength, it may provide a blood flow space to help form new bone.

Claims (16)

(a) a vascular plant template; Titanium oxide; And mixing the alcohol to produce a slurry;
(b) recovering the slurry; And
(c) drying the slurry or sintering at 300 to 1,800 ° C.,
In the step (c), the temperature increase rate is characterized in that 0.5 to 3 ℃ / min,
Method for producing a scaffold using a vascular plant template.
The method of claim 1,
The tube bundle plant template of the scaffold using a tube bundle plant template, characterized in that selected from the group consisting of bamboo charcoal, bamboo, oak, oak charcoal, birch, birch charcoal, loofah, scrubber charcoal, corn stalk and corn charcoal Manufacturing method.
The method of claim 1,
The titanium oxide is prepared from a scaffold using a vascular plant template, characterized in that selected from the group consisting of TiO ₂ , Ti ₃ O, Ti ₂ O, Ti ₃ O ₂ , TiO, Ti ₂ O ₃ and Ti ₃ O ₅ . Way.
delete delete Scaffold made by the method of any one of claims 1 to 3.
Medical implant prepared using the scaffold of claim 6.
Drug delivery carrier prepared using the scaffold of claim 6.
A cell delivery carrier prepared using the scaffold of claim 6.
delete delete delete delete delete delete delete

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