KR102179902B1 - Scaffolds for differentiation of odontoblast and method for differentiation of odontoblast using the same - Google Patents

Abstract

The present invention relates to a supporter for differentiation of odontoblasts capable of promoting the induction of differentiation from neural crest stem cells (NCSC), a method for differentiation of odontoblasts using the same, and a method for manufacturing the supporter for differentiation of odontoblasts. Specifically, in the supporter for differentiation of odontoblasts according to the present invention, a porous cellulose acetate butyrate polymer matrix coating layer capable of simulating the extracelluar matrix of stem cells is formed, so that it is possible to easily induce differentiation into odontoblasts without any biochemical or chemical treatment when culturing NCSC. In addition, odontoblasts obtained by differentiation can be used as a cell therapeutic agent for regeneration of dentin or pulp tissues, thereby being able to be actively used for tooth tissue regeneration, tooth treatment, and lost tooth regeneration.

Description

Scaffolds for differentiation of odontoblast and method for differentiation of odontoblast using the same}

The present invention provides an odontoblast differentiation scaffold capable of promoting odontoblast differentiation from neural crest stem cells (NCSC), a differentiation method of odontoblasts using the same, and a scaffold for differentiation of odontoblasts. It relates to a manufacturing method. Specifically, a porous cellulose acetate butyrate polymer matrix coating layer capable of simulating the extracelluar matrix of stem cells is formed on the scaffold for differentiation of oblasts according to the present invention. When culturing neural crest stem cells among stem cells, differentiation into odontoblasts can be easily induced without any biochemical or chemical treatment. In addition, the odontoblast obtained by differentiation can be used as a cell therapy for regeneration of dentin or pulp tissue, and thus can be actively used for regeneration of tooth breakfast, tooth treatment, and regeneration of lost tooth.

In the early stages of embryogenesis, the neural plate becomes a neural crest and is a tissue from which development originates. Tooth stem cells or odontoblasts that make up human teeth are obtained from the differentiation of neural crest stem cells (NSCS). Among them, it is known to differentiate from cranial neural crest stem cells (cNSCs).

In general, differentiation from neural crest stem cells (NCSC) to odontoblasts is induced by WNTs (Wg and Int), BMP (bone morphogenic proteins), FGFs (Fibroblast growth factor), and SHH (sonic hedgehog). (Meulemans, D. & Bronner-Fraser, M. Gene-regulatory interactions in neural crest evolution and development. Dev Cell 7, 291-299).

On the other hand, in order to achieve oblastoma cells, trans-differentiation by applying cell engineering techniques including inducing cell differentiation by transplanting human cells into mouse tissues or by introducing exogenous genes into various cell types, respectively. (Ozeki, N. et al. Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of alpha 1 Integrin Expression. J Biol Chem 289, 14380-14391). However, it has not yet been successful to differentiate human pluripotent stem cells into odontoblasts in vitro.

Such differentiation is induced through biochemical and chemical stimulation, and it is difficult to efficiently control the differentiation of dentinal blasts or dental blasts with only such a conventional technique, and thus there is a limit to use as a fundamental treatment for tooth regeneration.

Under this background, development of a method capable of controlling the differentiation of odontoblasts from stem cells, especially neural crest stem cells (NCSC), has been urgently required, but the situation is still insufficient.

The inventors of the present invention use a cellulose acetate butyrate polymer among biocompatible polymers to cultivate neural crest stem cells on a substrate on which a porous matrix layer is formed, so that the differentiation of oblastoma cells is easy without expensive biochemical and chemical treatment. It was confirmed that it was induced to complete the present invention.

Korean Patent Registration No. 10-1627917 (2016.06.07. Announcement)

The inventors of the present invention have been conducting various studies to develop a method for differentiation of oblasts more effectively, using a cellulose acetate butyrate polymer among biocompatible polymers to simulate the extracelluar matrix of stem cells. When culturing the neural crest stem cells on the substrate on which the matrix layer was formed, it was confirmed that the differentiation of oblastoma cells can be induced without expensive biochemical and chemical treatment, thereby completing the present invention.

Accordingly, an object of the present invention relates to a scaffold for inducing differentiation from stem cells into oblasts.

In addition, another object of the present invention relates to a method for differentiation of stem cells into oblastoma cells, comprising the step of inducing differentiation into oblastoma cells by culturing the stem cells on a support for differentiation of oblastoma cells.

In addition, another object of the present invention relates to a method of manufacturing a scaffold for inducing differentiation from stem cells into oblasts.

Therefore, in order to achieve the above object, the present invention is a support for inducing differentiation from stem cells to oblastoma cells, the support is a substrate; And a porous thin film comprising a porous cellulose acetate butyrate polymer coating layer on the substrate.

In addition, the present invention provides a method for differentiation of stem cells into oblastoma cells, comprising the step of inducing differentiation into oblastoma cells by culturing the stem cells on a scaffold for differentiation of oblastoma cells.

In addition, the present invention comprises the steps of preparing a substrate; Forming a coating layer by coating the substrate with a cellulose acetate butyrate polymer; And the coating layer provides a method of producing a scaffold for differentiation of odontoblasts comprising the step of forming a void using a non-solvent induced phase separation (NVIPS).

The scaffold and differentiation method according to the present invention can be used without expensive chemical or biochemical stimulation (e.g., ascorbic acid, TGF-β, protein kinase, basic fibroblast growth factor, etc.) Can easily induce differentiation into oblasts. The thus-induced oblastoma cells can be usefully used as a cell therapy for the treatment of dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis and gangrene.

Figure 1 relates to a support for differentiation of oblasts according to the present invention, Figure 1 (a) is a photograph showing a state in which a non-porous cellulose acetate butyrate polymer coating layer is formed on a silicon substrate, Figure 2 (b) is a weight average molecular weight Porosity can be confirmed by SEM photographs of the 30,000 cellulose acetate butyrate polymer matrix coating layer formed thereon, and the cellulose acetate butyrate polymer matrix coating layer having a weight average molecular weight of 70,000.
FIG. 2 is a result of confirming the expression of a marker capable of confirming the expression of oblasts in order to confirm whether differentiation into oblasts is induced after culturing the neural crest stem cells on the oblast differentiation scaffold according to the present invention.
3 is a result of measuring the calcium concentration after culturing the neural crest stem cells in the oblast differentiation scaffold according to the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, "odontoblasts" are cells differentiated from pulp progenitor cells, and refer to columnar cells constituting a layer of cells in the periphery of pulp. Otoblasts are functionally responsible for directly making dentin, which is the main constituent of teeth, and these cells themselves are a component of the pulp tissue that exists inside the dentin and are arranged in the pulp cavity wall inside the tooth.

In the present invention, “neural crest stem cells (NCSC)” migrate from the rear of the neural fold during the migration stage of the developmental stage, and the neural crest stem cells It migrates to organs and is distributed. After this cycle, the neural crest stem cells differentiate into nerve cells of the peripheral nervous system, glial cells, skin melanocytes, endocrine cells, and various mesenchymal cells. It is known to be distributed in organs [Dev Dyn 2007;236:3242]. Recently, neural crest stem cells are the peripheral nerve, dorsal root ganglia, gut, hair follicle, dermal papillae, cornea, and peripheral nerves even after birth. , It has been found to be present in the dental pulp. Of these, adult human neural crest stem cells were isolated only from hair follicles, dermal papilla, and tooth substance [J Cell Biochem 2009;107:1046].

In the present invention, the "cranial neural crest stem cell (cNSC)" of "neural crest stem cells (NCSC)" is a neural crest stem cell differentiated from human embryonic stem cells. It refers to a site-specific neural crest stem cell that shows the expression pattern of a marker gene when located in the head and neck.

In the present invention, the neural crest stem cell may be a cell derived from an animal, preferably a mammal, more preferably a human.

Accordingly, the present invention is a support for inducing differentiation from stem cells to oblasts, wherein the support includes a substrate; And a porous cellulose acetate butyrate polymer matrix coating layer on the substrate.

The porous thin film is a culture support for promoting differentiation from neural crest stem cells into odontoblasts.

Specifically, it is preferable that the coating layer has pores having an average pore size of 1 to 100 μm. More preferably, it is 10-20 micrometers. The matrix coating layer in which pores are formed within the above range can induce differentiation into oblasts by simulating the structure of the extracelluar matrix of stem cells.

In addition, it is preferable that the coating layer has an average thickness of 0.1 to 100 μm. More preferably, it is 1-50 micrometers. This design can be changed as long as it is within a range suitable for simulating the structure of the extracelluar matrix of stem cells.

On the other hand, as a conventional biocompatible polymer, poly(lactic-co-glycolic acid), polystyrene, polycaprolactone, polydimethylsiloxane, polyethylene glycol ), polyethylene oxide, polylactic acid, polyglycolic acid, poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (poly(3-hydroxybutyrate-) co-3-hydroxyvalerate)), polydioxanone, polyester urethane, poly(ethylene-co-vinyl alcohol) (poly(ethylene-co-vinyl alcohol)), polyacrylic acid , Polyvinyl alcohol, polyvinylpyrrolidone, polyaniline, hyaluronic acid, alginic acid, chitin, chitosan, cellulose , Collagen (collagen), and one or more polymers selected from the group consisting of gelatin (gelatin) was used. Such biocompatible polymers should have properties such as blood affinity, biodegradability, anticoagulability, viscosity, and ability to form cell matrix.

In the present invention, a cellulose acetate butyrate polymer is used, which has the advantage of allowing cell culture on the surface.

It is preferable to use the cellulose acetate butyrate polymer having a weight average molecular weight of 5,000 to 90,000. More preferably, it is 30,000 to 70,000. When the weight average molecular weight is less than 5,000, there is a limit to the problem of phase separation during film production, and when the weight average molecular weight is more than 90,000, there is a limit in which pore formation is difficult.

In addition, the present invention provides a method for differentiation of stem cells into oblastoma cells, comprising the step of inducing differentiation into oblasts by culturing the stem cells on a support for inducing differentiation from the stem cells into oblastoma cells.

The stem cells are cranial neural crest stem cells.

The differentiation method is a method of controlling the growth and differentiation of stem cells by using the porous thin film as a culture support for stem cells by adjusting the thickness of the porous thin film and the size of the pores without biochemical and chemical treatment. Here, the biochemical and chemical treatments include, for example, a basic fibroblast growth factor, and a CHIR99021 compound as a GSK3beta antagonist.

In the present invention, the effect of culturing stem cells in a cell culture platform that mimics the structure of the extracellular matrix of oblasts using a simple cell-friendly porous thin film without such expensive treatment is expressed. . This artificially creates an environment similar to that of a tooth, so that stem cells are recognized as being contained in the tooth, and there is a causal relationship that causes differentiation to occur.

The thus prepared oblastoma cells can be usefully used as a cell therapy for the treatment of dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis and gangrene.

In the present invention, the term "cell therapy" refers to a biological property of cells by proliferating or selecting in vitro live autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. It refers to drugs used for the purpose of treatment, diagnosis and prevention through a series of methods such as changing.

The cell therapy agent using the dentinal blast according to the present invention can treat various pulp diseases through the regeneration effect of dentin or pulp tissue. For reference, pulp disease is a soft connective tissue filling the pulp cavity inside a tooth, where nerves and blood vessels are abundantly distributed, and refers to a lesion that occurs in pulp tissue that reaches the surface layer of dentin. When a physical or chemical or bacterial irritation is applied to the pulp, blood vessels in the pulp are initially enlarged and congestion can be seen (periodic congestion). There is a difference in the degree of inflammation depending on the intensity of stimulation and the presence or absence of bacterial infection, but due to the anatomical feature of the pulp being piled up by hard dentin, circulatory disorders are likely to occur when inflammation occurs, and when left as it is, the pulp is liable to necrosis. The causes of pulp disease are very diverse, but in most cases it is caused by bacterial infection due to tooth decay, perforation, fracture, crack of the tooth, and infection into the pulp through the periodontal bag. It can also cause trauma, abrasion, tooth cracking, heat and friction from dental instruments during treatment. Pulpitis caused by bacterial infection can be extended to apical disease and periodontal disease.

Pulp diseases showing such various causes and symptoms are included in the scope of the present application, and examples of pulp diseases include, but are not limited to, dentin hypersensitivity, pulp congestion, dentitis, dimensional degeneration, pulp necrosis and gangrene.

When the expression is measured through analysis methods such as FACS, real-time PCR, and immunofluorescence staining, the oblasts are expressed in early odontoblasts rather than genes expressed in late phase of odontoblast maturation (early odontoblastic). It can be seen that a gene called fate) is expressed more, and the expression of such a gene is a marker that can confirm differentiation into oblastoma cells, and may be known markers. As an example, such a marker may be one that expresses one or more genes selected from the group consisting of RUNX2, OPN, OSX, DMP1, DSPP, MMP20, and ALP, preferably RUNX2, OPN, DMP1, DSPP, MMP20 , And ALP genes may be expressed.

In addition, the present invention provides a method of manufacturing the scaffold for differentiation of the oblastoma cells. Specifically, the method of manufacturing the scaffold for differentiation of oblastoma cells comprises the steps of: (a) preparing a substrate; (b) forming a coating layer by coating the substrate with a cellulose acetate butyrate polymer; And (c) forming voids in the coating layer using a non-solvent induced phase separation (NVIPS).

The substrate used in step (a) may be a silicon substrate, but any substrate that can be used in the art is not limited thereto.

In the step (b), the cellulose acetate butyrate polymer is coated on the substrate by dip-coating, spin-coating, spray-coating, or a combination thereof. A method including can be used, and spin coating is preferably used.

The step (c) is a step for preparing a matrix structure by forming voids in the polymer coating layer of cellulose acetate butyrate. Membrane manufacturing methods include melting and phase separation methods, and melting methods include extraction and stretching methods, and phase separation methods include non-solvent induced phase separation (NVIPS) and thermally induced phase separation (TIPS, Thermally- Induced Phase Separation).

Among them, in the present invention, a void is formed using a non-solvent induced phase separation (NVIPS). The non-solvent induced phase separation method is widely used because it is easy to control the size of pores and can use various materials.

When performing the non-solvent induced phase separation method, the pore size and the amount of pores can be controlled by controlling the relative humidity in the chamber, and the higher the relative humidity, the higher the pore and the porosity. In the chamber, a potassium chloride (KCl) solution may be provided and performed.

At this time, it is preferable to perform it under conditions of 50 to 70% relative humidity. When the relative humidity is less than 50%, the porosity is too low to form an extracellular matrix structure, and when the relative humidity exceeds 70%, the pores are too large to reduce the adhesion of cells and the porosity is too high to form an extracellular matrix structure. There is a limit that cannot be performed, so it is better to perform it within the above range.

Therefore, the scaffold and differentiation method according to the present invention can be obtained from stem cells to oblasts through a cell-friendly porous thin film modified with a simple surface structure without expensive chemical or biochemical stimulation (e.g., ascorbic acid, TGF-β, protein kinase, etc.). Can easily induce differentiation. The thus-induced oblastoma cells can be usefully used as a cell therapy for the treatment of dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis and gangrene.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Preparation Example: Preparation of a scaffold for inducing differentiation from stem cells into oblasts

To prepare a porous thin film that is a support for inducing differentiation into oblasts, first, cellulose acetate butyrate (CAB) (Mn = 30,000 and 70,000) was dissolved in acetone to prepare an acetone solution (30 mg/mL). A square-shaped silicon wafer substrate having a size of 2 cm x 2 cm was cleaned by ultrasonic treatment in acetone solution, and dried in an oven to prepare. In the chamber, a cellulose acetate butyrate solution was spin-coated on a silicon wafer substrate at a speed of 4,000 rpm for 40 seconds to form a non-porous coating layer (see FIG. 1(a)).

Next, a saturated KCl solution was placed in the spin coating chamber, and a non-solvent induced phase separation method was performed to form a void. At this time, it was performed under conditions of 0%, 50%, and 70% relative humidity.

Example 1: Confirmation of a porous thin film through SEM measurement

In order to confirm the formation of voids in the thin film prepared in Preparation Example, an SEM photograph was measured. As a result of the measurement, when the relative humidity was 0%, the thin film was not formed with pores, but when the relative humidity was 70%, it was confirmed that the pores were formed (see FIG. 1(b)).

Example 2: Confirmation of the differentiation of oblasts by confirming the expression markers of oblasts genes

Neural crest stem cells were obtained from human embryonic stem cells, and 100,000 neural crest stem cells were attached to the thin film prepared in the above Preparation Example, and then cultured for 30 days in 5% carbon dioxide and 37°C temperature maintained culture conditions. . During the additional culture period, the medium is exchanged every other day, and the medium is used by adding B27 and N-2 supplement to Neurobasal medium manufactured by Thermo Fisher Scientific. In order to confirm whether differentiation from neural crest stem cells to oblastoma cells has been induced after culture, real-time whether RUNX2, OPN, OSX, DMP1, DSPP, MMP20, and ALP genes are expressed as markers that can confirm the expression of oblastoma cells. It was analyzed using PCR (see Fig. 2).

As a result of the analysis, it was confirmed that among the markers known to be expressed in early oblasts, gene expression was improved in all of RUNX2, OPN, DMP1, DSPP, MMP20, and ALP, which represent oblastic expression except for OSX (Osterix).

This suggests that the porous thin film according to the present invention induced the differentiation of neural crest stem cells into oblasts.

Example 3: Confirmation of oblast differentiation through calcium concentration confirmation

Otoblasts are known to deposit calcium in the medium. Thus, by measuring the calcium concentration after culturing the neural crest stem cells on the odontoblast differentiation scaffold according to the present invention, it was again confirmed whether the differentiation into the oblastoma cells was induced (see FIG. 3).

As a result of the measurement, it was confirmed that the cells cultured on the support for differentiation of oblasts according to the present invention released a lot of calcium ions compared to cells cultured in Geltrex (Mock).

This suggests that the porous thin film according to the present invention induces differentiation from neural crest stem cells into oblasts.

In the scaffold for differentiation of oblasts according to the present invention, a porous cellulose acetate butyrate polymer matrix coating layer capable of simulating the extracelluar matrix of stem cells is formed, so that the scaffold has a neural crest on the scaffold. It is an excellent invention in that it can easily induce differentiation into odontoblasts without any biochemical or chemical treatment when culturing neural crest stem cells (NCSC). In addition, the odontoblast obtained by differentiation can be used as a cell therapy for regeneration of dentin or pulp tissue, and thus can be actively used for regeneration of tooth breakfast, tooth treatment, and regeneration of lost tooth.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

As a support for inducing differentiation from neural crest stem cells to oblasts,
The support is a substrate; And a porous cellulose acetate butyrate polymer matrix coating layer having a weight average molecular weight of 30,000 to 70,000 on the substrate,
The coating layer is a porous thin film, characterized in that the average pore size (average pore size) is 10 to 20㎛, the average thickness is 1 to 50㎛, the support for differentiation of oblastoma cells.
delete delete delete The method of claim 1,
The matrix coating layer is characterized in that it has an extracellular matrix structure of the oocytes, the support for oblast differentiation.
A method for differentiation of stem cells into oblasts comprising the step of inducing differentiation into oblasts by culturing neural crest stem cells on the scaffold for differentiation of oblasts of claim 1.
delete The method of claim 6,
The differentiation method is characterized in that the growth and differentiation of stem cells are controlled by using the porous thin film as a culture support for stem cells by adjusting the thickness and the size of the pores of the porous thin film.
(a) preparing a substrate;
(b) forming a coating layer by coating the substrate with a cellulose acetate butyrate polymer; And
(c) forming voids in the coating layer using a non-solvent induced phase separation (NVIPS) under 50 to 70% relative humidity conditions;
Containing, the method for producing a scaffold for oblast differentiation. delete

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