KR20190123456A - Method for preparing 3D stem cell microtissue using mineralized fragmented fibers - Google Patents

Abstract

The present invention relates to a method for preparing a 3D stem cell tissue, which comprises: a step (a) of coating a surface of a fibrous particle with a polycatechol-based polymer; a step (b) of coating gelatin on the surface of the fibrous particle coated with the polycatechol-based polymer; a step (c) of dispersing the fibrous particle coated with the gelatin in an artificial body fluid and adding an inorganic salt solution thereto to acquire a mineralized fibrous particle; and a step (d) of mixing the mineralized fibrous particle with a stem cell. Accordingly, self-assembly with a cell is induced through introduction of the mineralized fibrous fiber with a similar shape to a fibrous structure of an extracellular matrix such that a 3D stem cell tissue capable of simulating a microenvironment of a tissue can be provided through interaction therebetween. Moreover, the 3D stem cell tissue can smoothly supply oxygen and nutrients through an internal space and mineral components existing on the surface can effectively control bone differentiation of the stem cell, thereby being widely used in a platform technology for screening drug, a medical cell transfer and treatment system, and a cell-based bone regeneration medical article tissue implant field and, further, being widely applied for preparation of an in-vitro 3D bone structure for replacing preclinical and clinical tests, an interaction research model between cells or between a cell and an extracellular matrix, construction of a high efficiency drug screening system and the like.

Description

Method for preparing 3D stem cell microtissue using mineralized fragmented fibers}

The present invention relates to a method for producing three-dimensional stem cell tissue using mineralized fibrous particles.

The methods of simulating three-dimensional tissues form a three-dimensional structure that is similar to the environment of human tissues, and the results can be more accurately realized when regenerated models such as anticancer research and drug screening are actually implemented. As it is expected to be judged, the research has been actively conducted, and since there are many aspects of efficient characteristics of three-dimensional tissues rather than two-dimensional tissue structures, they are widely used for high efficiency cell therapy or drug screening systems. Is considered an important requirement in the

Three-dimensional stem cell microstructure realization technology is a method to maximize stem cell self-renewal ability and differentiation ability, among which bone tissue implementation using three-dimensional microstructures effectively differentiates stem cells and at the same time finely differentiated bone tissue There are various researches related to this, because it must be able to simulate the environment and functions.

Conventional techniques known as bone differentiation control technology using microstructures, three-dimensional by culturing the microstructure produced by using the self-assembly of cells cultured in a culture medium containing L-ascorbic acid, β-glycerophosphate, etc. Method of using water-soluble osteogenic differentiation agent that regulates bone differentiation of microstructures, Surface modification of attaching active material to histological support to simulate the interaction between extracellular matrix and cells A method of inducing bone differentiation of a three-dimensional microstructure from the inside by distributing it in the microstructure in the form of micro particles is known.

However, the control of bone differentiation of conventional three-dimensional microstructures is based on the maintenance of stem cell activity and the efficiency of delivery of bone differentiation inducers, and it is dissolved in culture or surface of support to deliver materials known to be effective in inducing bone differentiation. Although various attempts have been studied, such as the method of delivering a bone differentiation inducing factor to the three-dimensional microstructure in this manner, there is a problem in that the transfer efficiency into the tissue is relatively low or the degree of bone differentiation thereof is limited. In addition, the extracellular matrix and intercellular interactions, which are essential for the microenvironment of bone tissues, are not smooth, limiting the microstructural bone structure and signaling system, and these problems require considerable time to control bone differentiation. Due to the difficulty in simulating bone structure and function, there is a problem in that it is unlikely to be applied to drug screening technology or to be used as a cell therapeutic agent such as transplantation of cells.

The present invention has been made to solve the above-mentioned problems, the present invention is to provide a method for producing a three-dimensional stem cell tissue that can effectively induce bone differentiation by mineralizing the fibrous particles with bone differentiation active material.

In addition, the present invention is to provide a three-dimensional stem cell tissue prepared according to the manufacturing method.

In order to solve the above problems, the present invention (a) coating the surface of the fibrous particles with a polycatechol-based polymer; (b) coating the surface of the fibrous particles coated with the polycatechol-based polymer with gelatin; (c) dispersing the gelatin coated fibrous particles in a simulated body fluid (SBF) and adding an inorganic salt solution to obtain mineralized fibrous particles; And (d) mixing the mineralized fibrous particles with stem cells.

According to the present invention, the method may further comprise (e) centrifuging the mixture to induce cell self-assembly.

According to the present invention, the polycatechol-based polymer may be selected from the group consisting of polydopamine, polyepinephrine, polynorepinephrine, and mixtures thereof.

According to the invention, the artificial body fluid is ^{Na +, K +, Mg 2} +, Ca 2 +, Cl - and may include _{^{SO 4 2- -, HCO 3 -}} , HPO 4 2.

According to the present invention, the inorganic salt solution is NaHCO ₃ , Na ₂ SO _{4 ,} MgCl ₂ , K ₂ HPO _{4 ·} 3H ₂ O And mixtures thereof.

According to the invention, per 1 mg of the gelatin coated fibrous particles can be dispersed in 1 to 5 mL of the simulated body fluid.

According to the present invention, an inorganic salt solution of 0.001 to 0.01 mM concentration may be added per 1 mg of the gelatin coated fibrous particles.

According to the present invention, the length of the mineralized fibrous particles may be 70 to 120 ㎛.

According to the invention, the mineralized fibrous particles may be mixed 1 to 100 ㎍ per 10000 stem cells.

In addition, the present invention provides a three-dimensional stem cell tissue prepared according to the method.

In addition, the present invention provides a bone-differentiated cell microstructure prepared through the step of culturing the three-dimensional stem cell tissue.

According to the present invention, three-dimensional stem cells capable of simulating tissue microenvironment through their interaction by inducing self-assembly with cells through the introduction of mineralized fibrous particles having a form similar to the fibrous structure of the extracellular matrix. An organization can be provided. The three-dimensional stem cell tissue according to the present invention can not only supply smooth oxygen and nutrients through the internal space, but also the mineral components present on the surface can effectively regulate bone differentiation of stem cells. It can be widely used in the field of cell delivery and treatment system, artificial tissue implant for cell based bone regenerative medicine. In addition, it can be widely applied to the production of three-dimensional bone tissue in vitro for the replacement of preclinical and clinical experiments, research model between cells and cells or extracellular matrix and intercellular interactions, and construction of high-efficiency drug screening system.

1 is a view showing a manufacturing process of the mineralized fibrous particles according to the present invention.
Figure 2 is a view showing the manufacturing process of the three-dimensional stem cell tissue and differentiation control principle of the three-dimensional stem cell microstructure according to the present invention.
Figure 3 (a) is a SEM image of the fibrous sheet used in the present invention and the fibrous particles prepared therefrom, (b) is an image before and after coating the polydopamine to the fibrous particles, (c) is gelatin coated The image after staining the fibrous particles (G-FF) and mineralized fibrous particles (mFF1 to mFF3) with Alizarin red reagent, (d) is an SEM image of gelatin coated fibrous particles and mineralized fibrous particles ( e) and (f) are graphs showing the length and thickness of gelatin coated fibrous particles and mineralized fibrous particles, respectively.
Figure 4 (a) is a graph showing the calcium content of the gelatin coated fibrous particles and mineralized fibrous particles, (b) is a graph showing the calcium residual amount over time when the mineralized fibrous particles are incubated in an aqueous solution (C) shows the results of X-ray diffraction analysis (XRD) on gelatin coated fibrous particles, mineralized fibrous particles, and hydroxyapatite (HAP), a human bone tissue component, and (d) Energy-dispersive X-ray spectroscopy (EDS) analysis of gelatin coated fibrous particles and mineralized fibrous particles is shown. (E) XPS (X-ray) of gelatin coated fibrous particles and mineralized fibrous particles. The results of the photoelectron spectroscopy analysis are shown.
Figure 5 (a) is carried out histological H & E staining method for the three-dimensional stem cell tissue prepared using the mineralized fibrous particles, the three-dimensional stem cell tissue prepared using gelatin coated fibrous particles (B) is a SEM and TEM image of the three-dimensional stem cell tissue, (c) is a graph showing the size of the three-dimensional stem cell tissue, (d) is a DNA for the three-dimensional stem cell tissue It is a graph showing the results of the assay, (e) shows the results of the Live / Dead assay for the three-dimensional stem cell tissue.
Figure 6 (a) is a microstructure with Alizarin red reagent for the three-dimensional stem cell tissue prepared using mineralized fibrous particles, three-dimensional stem cell tissue prepared using gelatin coated fibrous particles according to the present invention An image showing the results of internal calcium staining, (b) is an image showing the results of the Von Kossa staining, (c) and (d) is a Runt-related transcription factor 2 (Runx2) protein, a bone differentiation-specific protein marker Image showing the results of analysis of the osteoteotin (OPN) protein by immunohistochemical fluorescence staining method, (e) is a graph showing the result of quantifying calcium production in the tissue, (f) to (h) is the presence of mineralized fibrous particles In order to confirm the change in bone differentiation-related gene expression according to the amplification of the Runx2 gene, OPN gene, and Osteocalcin (OCN) gene expressed in stem cells by amplification using quantitative PCR A graph showing the result.
Figure 7 (a) is integrin α2 present in the bone cell membrane in the three-dimensional stem cell tissue prepared using mineralized fibrous particles, the three-dimensional stem cell tissue prepared using gelatin coated fibrous particles according to the present invention , α5, β1 membrane protein genes ITGα2, α5, β1 amplification of the expression level was shown to confirm the results, (b) amplified the change in the Runx2 gene, OPN gene, OCN gene expression according to the presence or absence of PSB 603 It shows the results confirmed, (c) shows the result of quantifying the amplification of Oct4, Sox2, Nanog stem cell ability related genes through qPCR.
Figure 8 shows the results of evaluating the fusion ability of the three-dimensional microstructure according to the present invention, (a) is an optical image according to the culture time of the three-dimensional microstructure fused tissue structure, (b) histological staining method (C) shows the result of Alizarin red staining, (d) shows the result of fluorescent staining by Live / Dead assay, and (e) shows the result of staining with DiO reagent. Image.
Figure 9 analyzes the bone regeneration effect of the three-dimensional stem cell microstructure implanted in bone defects in vivo (a) is a CT image of the femoral defect model, (b) and (c) quantified based on this It is a graph showing bone volume and bone thickness, (d) is a μCT picture of the skull defect model, (e) is a graph showing the bone thickness quantified based on this.

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

The present invention relates to a method for producing a three-dimensional stem cell tissue that can effectively induce bone differentiation by mineralizing the fibrous particles with the bone differentiation active material, in detail the extracellular matrix from the electrospun biodegradable polymer fiber sheet A fused three-dimensional stem is prepared by preparing fibrous particles capable of simulating fibrous structures, mineralizing the fibrous particles, and mixing them with stem cells to induce self-assembly between cell-cells and binding between cell-mineralized fibrous particles. The present invention relates to a method for producing a cell tissue. Through this, the present invention is to provide a new method for producing a three-dimensional stem cell tissue that can not only smoothly supply oxygen and nutrients through the internal space, but also effectively regulate bone differentiation of stem cells.

Therefore, the method for producing a three-dimensional stem cell tissue according to the present invention includes the following steps.

(a) coating the surface of the fibrous particles with a polycatechol-based polymer; (b) coating the surface of the fibrous particles coated with the polycatechol-based polymer with gelatin; (c) dispersing the gelatin coated fibrous particles in a simulated body fluid (SBF) and adding an inorganic salt solution to obtain mineralized fibrous particles; And (d) mixing the mineralized fibrous particles with stem cells.

In addition, the present invention may further include (e) inducing cell self-assembly by centrifuging the mixture after step (d).

First, in step (a), the surface of the fibrous particles is modified by coating the surface of the fibrous particles with a polycatechol-based polymer. In this case, the fibrous particles may be obtained by applying shear stress to the biodegradable polymer fiber sheet, the biodegradable polymer fiber sheet may be obtained by electrospinning the biodegradable polymer solution, the biodegradable polymer is polyglycolic acid (Poly glycolic acid, PGA), poly lactic acid (PLA), poly lactic-co-glycolic acid (PLGA), and polyethylene glycol (PEG) One is preferred, and poly (L-lactic acid, PLLA) may be most preferred.

Meanwhile, the fibrous particles surface-modified with the polycatechol-based polymer have structural characteristics similar to those of collagen, which is a major component of the extracellular matrix, so that cells can be easily attached, and pores can be formed between these fibrous particles. The strong binding between cells and cells can be alleviated, thereby enabling smooth supply of oxygen and nutrients into the three-dimensional cell globules, as well as providing a space for proliferation of cells in long-term culture.

In this case, the polycatechol-based polymer may be any one that can be easily attached to the cells, for example, polydopamine, polyepinephrine, polyepinephrine, polynorepinephrine, and mixtures thereof. Can be.

Next, in the step (b), the surface of the fibrous particles coated with the polycatechol-based polymer is coated with gelatin. This is for efficient mineralization process, the mineral formation can be promoted in the following (c) step by the gelatin.

Next, in the step (c), the gelatin-coated fibrous particles are dispersed in a simulated body fluid, followed by addition of an inorganic salt solution to obtain mineralized fibrous particles.

In this case, the simulated body fluid may include Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ , Cl ⁻ , HCO ₃ ⁻ , HPO ₄ ^2-, and SO ₄ ^2- .

In addition, the inorganic salt solution is NaHCO ₃ , Na ₂ SO _4, MgCl ₂ , K ₂ HPO ₄ 3H ₂ O And mixtures thereof.

In addition, in order to implement the effective bone differentiation control characteristics of the three-dimensional stem cell tissue, it is important to manufacture the optimal mineralized fibrous particles for the implementation of bone differentiation properties, the degree of mineralization is fibrous particles, simulated body fluids, inorganic salts It can be determined according to the mixing ratio of the solution. Therefore, it is preferable to disperse | distribute to 1-5 mL of said simulated body fluids per 1 mg of gelatin coated fibrous particles, and to add an inorganic salt solution of 0.001 to 0.01 mM concentration per 1 mg of gelatin coated fibrous particles. In particular, as can be seen from the results of the following examples, the degree of mineralization of the fibrous particles can be controlled by adjusting the concentration of the inorganic salt solution within the above-mentioned range.

Next, in step (d), the mineralized fibrous particles are mixed with stem cells, and in step (e), the cell-cell self-assembly is carried out by centrifuging the mixture. By inducing binding between cell-mineralized fibrous particles, a three-dimensional stem cell tissue according to the present invention is obtained.

In addition, the length of the mineralized fibrous particles may be 70 to 120 ㎛.

In addition, according to the content of the mineralized fibrous particles can be adjusted the average diameter of the stem cell tissue, the size of the inner space, porosity and the like. At this time, the mineralized fibrous particles are preferably mixed 1 to 100 ㎍ per 10000 stem cells.

In addition, the present invention provides a three-dimensional stem cell tissue prepared according to the method.

In addition, the present invention provides a bone-differentiated cell microstructure prepared through the step of culturing the three-dimensional stem cell tissue.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples and the like are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Electrospun Fiber Sheets and Fragments

First, an electrospun fiber sheet was prepared using an electrospinning method. Specifically, Poly (L-lactic acid) (PLLA) was dissolved in a mixture of dichloromethane and trifluoroethanol (8: 2, v / v) for 24 hours to prepare 4 wt% of a polymer solution. About 10 ml of the polymer solution was prepared. After filling the syringe, it was electrospun at a rate of 5 ml / h to the collector rotating at a speed of 100 rpm (voltage 12.8 kV). The electrospun fiber sheet was dried at room temperature for 12 hours to completely remove the remaining solvent, and the dried fiber sheet was cut into a size of about 5mm x 5mm and then 10% (v / v) ethylenediamine solution (isopropyl alcohol Base) and shear stress was applied with vigorous stirring at 37 ° C. for 30 minutes, followed by centrifugation at 4000 rpm for 10 minutes to obtain fibrous particles. The obtained fibrous particles were repeatedly washed with isopropyl alcohol and secondary distilled water, sieved (100-150 μm) and lyophilized to finally recover the fibrous particle powder (see FIG. 1).

Mineralized Fibrous  Preparation of Particles

The mineralization process was used to modify the surface of the fibrous particles. First, 30 mg of fibrous particles were dispersed in 30 ml Tris-HCl buffer (10 mM, pH 8.5), further dissolved in dopamine hydrochloride (2 mg / ml), and then stirred at 100 rpm for 20 minutes, and then reacted with secondary distilled water. After repeated washing, the mixture was further dispersed in gelatin (2 mg / ml) solution dissolved in Tris-HCl buffer, stirred for 2 hours, and then reacted. After repeated washing with secondary distilled water, freeze-drying was performed to coat polydopamine and gelatin sequentially. Fibrous particle powder was obtained.

Next, the gelatin coated 5 mg of fibrous particles were dispersed in a 10 mL SBF solution (10X, pH 4.4), followed by addition of NaHCO ₃ (0.005, 0.01, 0.04 mM) and stirring at 37 ° C. for 3 hours. Table 1), repeated washing with distilled water and then dried at 37 ℃ to obtain a mineralized fibrous particle powder (see Figure 1).

NaHCO3 Concentration Added to SBF 0.005 mM 0.01 mM 0.04 mM Sample naming mFF1 mFF2 mFF3

Mineralized Fibrous  Preparation of 3D Stem Cell Tissue Using Particles

In order to prepare a three-dimensional stem cell tissue according to the present invention using mineralized fibrous particles, a mixed solution of 10 μg fibrous particles and 40,000 Human Adipose-Derived Stem Cells (hADSCs) was prepared. Next, the fibrous particles and the mixed solution of hADSCs in a PCR tube were centrifuged at 1200 rpm for 5 minutes and incubated for 24 hours under normal cell culture conditions (37 ° C., 5% CO ₂ ). Thereafter, long-term culture in a 1.3 wt% Agarose gel-coated culture dish was performed to analyze morphological changes, proliferation, and bone differentiation (see FIGS. 1 and 2).

3D stem cell tissue Bone differentiation Mechanism  analysis

During the bone differentiation of stem cells in the human body, the gene expression level of the integrin cell membrane protein, which interacts with extracellular matrix and delivers bone differentiation and survival signals, is analyzed. It was evaluated to promote differentiation.

In addition, as the metal cations (calcium, magnesium, sodium, potassium) among the mineral components open and close the ion channel of the stem cell membrane, phosphorus oxidized component enters the cell and finally forms Adenosine to promote bone differentiation. In order to evaluate whether the mineral component of the fibrous particles promote bone differentiation, PSB 603, which is an antagonist of Adenosine, was treated to determine whether bone differentiation was inhibited. To this end, after diluting PSB 603 at a concentration of 100 nM in a cell culture and treating the microstructure including mineralized fibrous particles for a long time, the expression level of bone differentiation-related genes was relatively quantified using qPCR.

In addition, it is expected that the stem cell ability changes as the bone differentiation of the stem cells progress, the expression level of stem cell function-related genes (Oct4, Sox2, Nanog) is analyzed and the stems in the microstructure by the mineralized fibrous particles It was confirmed whether cell differentiation proceeds.

Microstructure to 3D Tissue Structure Convergence  evaluation

By confirming the fusion ability between microstructures, the possibility of formation of bone tissue structures for drug screening and the possibility of fusion with human tissues for cell therapy were evaluated. Specifically, 20 masses of microstructures were fused for several days to form a three-dimensional tissue structure, and cultured for a long time, and the change in shape was observed.

3D stem cell microstructure in vivo  goal Regenerative  evaluation

The bone differentiation-induced three-dimensional stem cell microstructures with mineralized fibrous particles were implanted into the skull and femoral defect animal models, and then the bone regeneration was confirmed. Specifically, after several weeks after implantation of dozens of microstructures, the reconstructed bone volume and thickness were confirmed by using micro-computed tomography (μCT).

Results and Discussion

Fibrous  The production of particles and Mineralization  Through the process Bone differentiation  Induction surface modification

Figure 3 (a) is a SEM image of the fibrous sheet used in the present invention and the fibrous particles prepared therefrom, (b) is an image before and after coating the polydopamine to the fibrous particles, (c) is gelatin coated After staining fibrous particles (G-FF) and mineralized fibrous particles (mFF1 to mFF3) with Alizarin red reagent, (d) is SEM images of gelatin coated fibrous particles and mineralized fibrous particles, ( e) and (f) are graphs showing the length and thickness of gelatin coated fibrous particles and mineralized fibrous particles, respectively.

The shape of the fibrous particles fragmented from the lyophilized fibrous particle powder was confirmed through (a) of FIG. 3, and the polydopamine-coated fibrous particles, which are the connecting materials between the fibrous particles, were brown in FIG. 3 (c) qualitatively determine the calcium content of the gelatinous fibrous particles to facilitate mineralization by electrostatic attraction and the fibrous particles whose mineralization is controlled by the concentration of NaHCO ₃ . It was confirmed that the mineral is evenly attached to the surface of the fibrous particles through (d) of Figure 4, the diameter of the fibrous particles while maintaining the length of about 60 μm through (e) and (f) of Figure 4 It was confirmed that it thickened more than twice.

Fibrous  Particle Mineralization  analysis

Figure 4 (a) is a graph showing the calcium content of the gelatin-coated fibrous particles and mineralized fibrous particles, (b) is a graph showing the amount of calcium remaining over time when the mineralized fibrous particles are incubated in an aqueous solution (C) shows the results of X-ray diffraction analysis (XRD) on gelatin coated fibrous particles, mineralized fibrous particles, and hydroxyapatite (HAP), a human bone tissue component, and (d) Energy-dispersive X-ray spectroscopy (EDS) analysis of gelatin coated fibrous particles and mineralized fibrous particles is shown. (E) XPS (X-ray) of gelatin coated fibrous particles and mineralized fibrous particles. The results of the photoelectron spectroscopy analysis are shown.

This confirmed that the degree of mineralization is controlled by the NaHCO ₃ concentration, it was confirmed that the presence of about 250 μg of calcium in 1 mg of the fibrous particles (Fig. 4 (a)). In addition, even after 14 days in the aqueous solution it was confirmed that the mineral is maintained on the surface of the fibrous particles (Fig. 4 (b)).

In addition, it was confirmed that the mineralized fibrous particles have surface characteristics similar to those of the hydroxyapatite (HAP) component of human bone tissue (FIG. 4 (c)), and the mineralized fibrous particles mainly contain calcium and phosphorus components (FIG. 4). (D)), the presence of calcium and phosphorus on the surface of the mineralized fibrous particles was confirmed (Fig. 4 (e)).

Mineralization Fibrous  Preparation of 3D Microstructure Using Particles

Figure 5 (a) is carried out histological H & E staining method for the three-dimensional stem cell tissue prepared using the mineralized fibrous particles, the three-dimensional stem cell tissue prepared using gelatin coated fibrous particles (B) is a SEM and TEM image of the three-dimensional stem cell tissue, (c) is a graph showing the size of the three-dimensional stem cell tissue, (d) is a DNA for the three-dimensional stem cell tissue It is a graph showing the results of the assay, (e) shows the results of the Live / Dead assay for the three-dimensional stem cell tissue.

Through this, it was confirmed that the fibrous particles and the cells are evenly mixed and distributed, it was confirmed qualitatively that the interaction between the two is also made smoothly (Fig. 5 (a)). In addition, it was confirmed that the distribution and interaction of the stem cells and mineralized fibrous particles in the microstructure is evenly made (Fig. 5 (b)), the size of the tissue has an initial area of about 0.5 mm ² and then long-term culture After it was confirmed that the agglomeration to about 0.3 mm ² (Fig. 5 (c)). In addition, as a result of performing the DNA assay, it was confirmed that the amount of DNA was almost maintained in the fused three-dimensional micro-organisms during the culture for 7 days, and it was confirmed that the cells survived stably in the tissues (FIG. 5 (d). )), Live / Dead assay confirmed that most of the cells participating in the formation of fused three-dimensional stem cell microstructures survived (FIG. 5 (e)).

3D microstructure Bone differentiation  control

Figure 6 (a) is a microstructure with Alizarin red reagent for the three-dimensional stem cell tissue prepared using mineralized fibrous particles, three-dimensional stem cell tissue prepared using gelatin coated fibrous particles according to the present invention An image showing the results of internal calcium staining, (b) is an image showing the results of the Von Kossa staining, (c) and (d) is a Runt-related transcription factor 2 (Runx2) protein, a bone differentiation-specific protein marker Image showing the results of analysis of the osteoteotin (OPN) protein by immunohistochemical fluorescence staining method, (e) is a graph showing the result of quantifying calcium production in the tissue, (f) to (h) is the presence of mineralized fibrous particles In order to confirm the change in bone differentiation-related gene expression according to the amplification of the Runx2 gene, OPN gene, and Osteocalcin (OCN) gene expressed in stem cells by amplification using quantitative PCR A graph showing the result.

In order to evaluate the uniform distribution of mineralized fibrous particles in the fused three-dimensional microstructure, the results of staining calcium inside the microstructure with Alizarin red reagent resulted in very even distribution of the fibrous particles and the production of additional calcium components during long-term culture. Qualitatively confirmed that this was done (Fig. 6 (a)). Likewise, even distribution and morphology of the mineralized fibrous particles inside the microstructure could be more clearly confirmed through Von Kossa staining (FIG. 6 (b)).

In addition, in order to qualitatively determine the degree of bone differentiation of the fused three-dimensional microstructure by mineralized fibrous particles, osteotension specific protein markers, Runt-related transcription factor 2 (Runx2) and Osteopontin (OPN) proteins, were used. As a result of analysis by immunohistochemical fluorescence staining, it was qualitatively confirmed that both proteins are distinctly produced in the microstructure containing mineralized fibrous particles (Fig. 6 (c), (d)).

In addition, in order to quantitatively determine the degree of bone differentiation of the fused three-dimensional microstructure by the mineralized fibrous particles, as a result of quantifying calcium production in the tissue, the mineralized fibrous particles in the existing microstructure were included during long-term culture. It was confirmed that a greater amount of calcium was produced than the amount of calcium present (FIG. 6E).

In addition, in order to confirm the change in gene expression related to bone differentiation according to the presence or absence of mineralized fibrous particles, the amplification of the Runx2 gene, OPN gene, and Osteocalcin (OCN) gene expressed in stem cells was confirmed by amplification using quantitative PCR. It was confirmed that a relatively large amount of osteogenic differentiation gene was expressed in the microstructure containing the oxidized fibrous particles (Fig. 6 (f), (g), (h)), these results by immunohistochemical staining It was confirmed that the degree of protein production is similar to the analysis results.

Figure 7 (a) is integrin α2 present in the bone cell membrane in the three-dimensional stem cell tissue prepared using mineralized fibrous particles, the three-dimensional stem cell tissue prepared using gelatin coated fibrous particles according to the present invention , α5, β1 membrane protein genes ITGα2, α5, β1 amplification of the expression level was confirmed by the results, and (b) amplified the changes in the Runx2 gene, OPN gene, OCN gene expression according to the presence or absence of PSB 603 antagonists It shows the results confirmed, (c) shows the results of quantification by amplifying the gene associated with Oct4, Sox2, Nanog stem cell ability through qPCR.

As a result of amplifying the expression level of the integrin α2, α5, β1 membrane protein genes ITGα2, α5, β1 in the bone cell membranes, it was confirmed that the ITGα5 gene was relatively expressed in the microstructure containing mineralized fibrous particles. Bar (Fig. 7 (a)), it could be inferred that the mineralized fibrous particles promote bone differentiation through integrin α5 biomechanism.

In addition, as a result of amplifying the change in the expression of Runx2 gene, OPN gene, and OCN gene with or without PSB 603 antagonist, the expression level of gene was relatively low. It was confirmed to promote differentiation (FIG. 7B).

In addition, amplification and quantification of Oct4, Sox2, and Nanog stem cell-related genes through qPCR showed that there was no change between groups in the initial culture, but relatively small amount in microstructures containing mineralized fibrous particles during long-term culture. It was confirmed that the stem cell ability of the gene (Fig. 7 (c)), which shows that the differentiation progressed from the stem cells to mature bone cells by mineralized fibrous particles.

3D microstructure Convergence  evaluation

Figure 8 shows the results of evaluating the fusion ability of the three-dimensional microstructure according to the present invention, (a) is an optical image according to the culture time of the three-dimensional microstructure fused tissue structure, (b) histological staining method (C) shows the result of Alizarin red staining, (d) shows the result of fluorescence staining by Live / Dead assay, and (e) shows the result of staining with DiO reagent. Image.

Through this, the approximate size and shape of the three-dimensional microstructure fused tissue structure could be confirmed. Specifically, a large structure having a diameter of about 3 mm was initially formed. (FIG. 8A). In addition, as a result of confirming the internal structure of the fused tissue structure using histological staining method, it was confirmed that the junction between the microstructures was partially formed in the state of empty space in the early stage, and fused in a dense form during long-term culture. It was confirmed that the filling the space as a whole (Fig. 8 (b)). In addition, the analysis of the internal calcium distribution through Alizarin red staining confirmed that the bone differentiation activity persists after fusion (Fig. 8 (c)).

In addition, as a result of fluorescence staining by the Live / Dead assay to qualitatively analyze the fusion surface and the junction surface of the three-dimensional tissue structure, it can be confirmed that empty spaces remain between the junctions initially, similar to the histological staining results. However, as the fusion progressed during the long-term culture, it was confirmed that the empty space between the junctions disappeared, and the structure of each of the microstructures remaining on the surface after fusion was confirmed (FIG. 8 (d)).

In addition, five microstructures stained with green fluorescent color by DiO staining reagent and five microstructures stained with red fluorescent color by DiD reagent to qualitatively confirm the formation of three-dimensional tissue structure by fusion of microstructures. As a result of the fluorescence image of the change in the junction region by fusion, as the interval between the microstructures stained with different colors became narrower as the primary passes, the cells were mixed and thus the cells were moved between the microstructures. It was qualitatively demonstrated that dimensional tissue structure fusion took place (FIG. 8E).

3D microstructure in vivo  goal Regenerative  evaluation

Figure 9 analyzes the bone regeneration effect of the three-dimensional stem cell microstructures implanted in bone defects in vivo (a) is a CT image of the femoral defect model, (b) and (c) quantified based on this It is a graph showing bone volume and bone thickness, (d) is a μCT picture of the skull defect model, (e) is a graph showing the bone thickness quantified based on this.

Through this, it was confirmed that both the femoral defect model and the skull defect model increase the regeneration effect of the bone defect by the three-dimensional stem cell tissue containing mineralized fibrous particles according to the present invention.

Claims (10)

(a) coating the surface of the fibrous particles with a polycatechol-based polymer;
(b) coating the surface of the fibrous particles coated with the polycatechol-based polymer with gelatin;
(c) dispersing the gelatin coated fibrous particles in a simulated body fluid (SBF) and adding an inorganic salt solution to obtain mineralized fibrous particles; And
(d) mixing the mineralized fibrous particles with stem cells; a method for producing a three-dimensional stem cell tissue comprising a. The method of claim 1,
(e) centrifuging the mixture to induce cell self-assembly; the method of manufacturing a three-dimensional stem cell tissue further comprising. The method of claim 1,
The polycatechol-based polymer is a polydopamine (polydopamine), polyepinephrine (polyepinephrine), polynorepinephrine (polynorepinephrine) and a method for producing a three-dimensional stem cell tissue, characterized in that selected from the group consisting of a mixture thereof. The method of claim 1,
The artificial body fluid is ^{Na +, K +, Mg 2+} , Ca 2+, Cl -, HCO 3 -, HPO 4 method of producing a three-dimensional organization stem cells comprising the ^2- and SO ₄ ^2-. The method of claim 1,
The artificial body fluid is ^{Na +, K +, Mg 2+} , Ca 2+, Cl -, HCO 3 -, HPO 4 method of producing a three-dimensional organization stem cells comprising the ^2- and SO ₄ ^2-. The method of claim 1,
Method for producing a three-dimensional stem cell tissue, characterized in that dispersed in 1 to 5 mL of the simulated body fluid per 1 mg of the gelatin coated fibrous particles. The method of claim 1,
Method for producing a three-dimensional stem cell tissue, characterized in that the addition of inorganic salt solution of 0.001 to 0.01 mM concentration per 1 mg gelatin coated fibrous particles. The method of claim 1,
The length of the mineralized fibrous particles is a method for producing a three-dimensional stem cell tissue, characterized in that 70 to 120 ㎛. The method of claim 1,
The mineralized fibrous particles are a method for producing a three-dimensional stem cell tissue, characterized in that 1 to 100 ㎍ mixed per 10000 stem cells. Three-dimensional stem cell tissue prepared according to the method of claim 1.

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