KR101957583B1 - Co-culture of stem cells and endothelial cells using porous microcarriers - Google Patents

Abstract

The present invention relates to a porous microsphere loaded with stem cells; A coculture construct for stem cells and endothelial cells, comprising a hydrogel containing endothelial cells; A method for co-culturing stem cells and endothelial cells using the same; And compositions comprising same. The stem cell and endothelial cell co-culture construct according to the present invention exhibit a high level of alkaline phosphatase activity and can exhibit a gene expression level associated with high bone formation and angiogenesis, and thus can be used as a vascularized bone graft, It is expected to be used.

Description

[0002] Co-culture of stem cells and endothelial cells using porous microcarriers [

The present invention relates to a co-culture structure of stem cells and endothelial cells using porous microspheres, and a co-culturing method of stem cells and endothelial cells using the same.

Tissue engineering is a multi-disciplinary, interdisciplinary science that aims to develop clinically effective substitutes for treating injured and diseased tissue. Complex tissue engineering is one of the major challenges in tissue engineering. Co-culture has been studied to design complex tissue structures that can better mimic the basic tissue environment through interaction of other cell types. Much effort has therefore been made to develop co-culture models for specific tissues including liver, pancreas, retina, skin, tendons, cartilage, blood vessels, nerves and bones.

In particular, bone tissue engineering is one of the most widely studied areas to meet clinical requirements. One of the challenges of bone tissue engineering is the creation of vascularized bone grafts because the bone is highly vascularized. In particular, delivery of stem cells capable of osteogenesis while enabling angiogenesis is important for vascularized bone tissue engineering. Under these circumstances, methods and systems for co-culturing cells have been developed, but most in vitro studies have been conducted in two-dimensional conditions and fail to reproduce the natural conditions of three-dimensional.

Dariima T, Jin GZ, Lee EJ, Wall IB, Kim HW. Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function. Biotechnol Lett. 2013; 35: 113543. Rouwkema J, de Boer J, Van Blitterswijk CA. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue Eng. 2006; 12: 268593.

The present invention provides a three-dimensional co-cultured structure of stem cells and endothelial cells, a method for producing the same, and a co-culture system using the same.

A first aspect of the present invention relates to a microsphere comprising a porous microspheres loaded with stem cells; And a hydrogel containing endothelial cells. The present invention also provides a coculture construct for stem cells and endothelial cells.

A second aspect of the present invention is a microsphere comprising a porous microsphere in which stem cells are dispensed; And a step of mixing a hydrogel containing endothelial cells with the endothelial cells.

A third aspect of the present invention is a method for producing stem cells and endothelial cells, comprising co-culturing a co-culture construct of stem cells and endothelial cells according to the first aspect of the present invention in a medium in which an osteogenic medium and an endothelial cell culture medium are mixed, A method for co-culture of cells is provided.

A fourth aspect of the present invention provides a composition for bone and angiogenesis comprising a coculture construct of stem cells and endothelial cells according to the first aspect of the present invention.

Hereinafter, the present invention will be described in detail.

In the case of conventional stem cells, two-dimensional adherent culture is performed in a flask with a flat bottom due to its attachment and survival characteristics. Such a culture method may result in cell loss and contamination when stem cells are reduced in their differentiation and replication ability, and when they are subcultured in an adherent manner. In addition, in order to use stem cells as a cell therapy agent, it is not economically feasible to obtain a large amount of cells by a two-dimensional culture method in which a cell size of 1.0 x 10 ⁸ cells or more is required and the cultivation area is limited. In order to use stem cells as a cell therapy agent, a culture method capable of obtaining a sufficient amount of cells is important.

In addition, when co-culturing stem cells and other types of cells, the cells should not affect each other's function, differentiation rate, and proliferation rate, and signal substance exchange between two cells cultured during cell co-culture should be smoothly performed. For this purpose, it is important to develop a culture structure for culturing two or more kinds of cells together. Preferably, the cells can be directly and advantageously interacted with signal substances between two cells cultured in cell culture, The development of a structure having the above structure is required.

The present inventors devised a three-dimensional co-culture system of dental pulp cells (DPSC) and endothelial cells (EC) using porous microspheres and hydrogels and studied the applicability to bone tissue engineering. The embryonic stem cells and hydrogel-embedded endothelial cells were cultured in a co-culture medium consisting of osteogenic medium and endothelial cell culture medium, resulting in an increase in bone morphogenesis and angiogenesis effect compared with culture obtained by culturing only stem cells. Respectively. In addition, it was confirmed that the effect of osteogenesis and angiogenesis on the microspheres and hydrogels as a culture supporter was less than that of the co - culturing of dendritic cells and endothelial cells. In other words, it was confirmed that bone and angiogenic effects were significantly increased when co-cultured endothelial cells embedded in the hydrogel and the stem cells loaded on the porous microspheres. Thus, the co- Can be transplanted and used as a cell therapy agent. The present invention is based on this.

Stem cells can regenerate tissues or organs with cells that have self-replicating ability to make cells of the same capacity as themselves and have the ability to differentiate into specific cells, and are suitable for regenerative medicine because they can differentiate according to the characteristics of each organ after organ transplantation Do. Specifically, the stem cells may be mesenchymal stem cells. Mesenchymal stem cells are a type of adherent adult stem cells that are multipotent stem cells capable of differentiating into musculoskeletal cells such as bone, cartilage, and fat. Mesenchymal stem cells are easily cultured in vitro, are excellent in proliferative capacity and can be differentiated into desired tissues, and are used for treating degenerative arthritis, spinal cord injury, myocardial infarction, and diabetic diseases.

In the present invention, preferably, a stem cell (DPSC) can be used as a stem cell. Dimensional stem cells (DPSCs) have been studied as a source of many cell types in tissue engineering since they exhibit multi-line differentiation potential including lipogenesis, cartilage formation, osteogenesis, tooth formation and neurogenic lines.

The present invention relates to a method of embedding stem cells into porous microspheres for three-dimensional co-culture. The stem cells can be attached to the surface or pores of the porous microspheres.

The porous microspheres may be porous supports made of biodegradable polymers. Non-limiting examples of the biodegradable polymer include polycaprolactone (PCL), polylactic acid (PLA), poly-L- / D-lactide (PLDLA), polyethylene glycol (PEG), polyglycolic acid (PGA) Hydroxybutyrate (PHB), polydioxanone (PDO), polytrimethylene carbonate (PHV), or mixtures thereof.

In one embodiment of the invention, porous microspheres were prepared by mixing polycaprolactone (PCL) and poly-L- / D-lactide (PLDLA) (1: 3) with camphene.

The porous biomolecule microspheres have a pore size of 10 to 100 mu m so that the stem cells can enter and culture in the pores.

If the pore size is less than 10 mu m, the stem cells may be difficult to be loaded on the porous microspheres. If the pore size is more than 100 mu m, it may be difficult to form microspheres.

The porous microspheres may have an average diameter of 200 to 990 탆, and preferably 200 to 500 탆.

Since the porous microspheres have the sizes as described above, they can be injected into tissues using a syringe contained in the composition for bone and angiogenesis.

The stem cells can be attached to the surface or pores of the porous microspheres.

Endothelial cells are monolayer flat cells covering the lining of the heart, arteries, capillaries, veins, and lymphatic vessels. It has few cytoplasm and long elliptical nucleus in the long axis direction of blood vessel. The luminal surface of the endothelial cells is smooth, and neighboring cells overlap each other. Inside the cell body, there is a soft capsule, which is thought to transport material inside and outside the blood vessel. In particular, vascular endothelial cells play a major role in maintaining vascular homeostasis, such as vasodilation and contraction, proliferation and migration of vascular smooth muscle, and thrombogenesis and lysis.

The hydrogels may be selected from the group consisting of fibrin, fibrinogen, collagen, polyester polymers, polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid and polyhydroxybutyric acid- And a biodegradable synthetic bio-gel comprising at least one copolymer selected from the group consisting of acids. Preferably, collagen can be used.

Porous microspheres in which stem cells are dispensed; And a hydrogel containing endothelial cells may be mixed to prepare a coculture construct of stem cells and endothelial cells.

The coculture construct of stem cells and endothelial cells of the present invention may have a three-dimensional shape in which three-dimensional microspheres are embedded in the hydrogel.

The co-culturing method of stem cells and endothelial cells includes co-culturing the co-culture constructs of stem cells and endothelial cells in a medium in which an osteogenic medium and an endothelial cell culture medium are mixed.

The osteogenic medium may be one comprising α-MEM (α-minimum essential medium eagle), fetal bovine serum (FBS), penicillin / streptomycin, ascorbic acid, β-glycerophosphate and dexamethasone But is not limited to. The endothelial cell culture medium may include, but is not limited to, a vascular cell basal medium (ATCC, USA) and an endothelial cell growth kit (ATCC, USA).

Preferably, the weight ratio of the osteogenic medium and the endothelial cell culture in the co-culture medium may be 5 to 15: 1. The composition of the culture medium and the ratio of cells are important parameters in optimizing co-culture of different types of cells in the same medium. Conventionally, 1: 1 cell ratios have often been reported to enable both bone formation and angiogenic functions in many co-culture studies. For example, Kaigler D. et al. Co-cultured MSC / EC in a 1: 1 mixture of growth medium / EM and confirmed better bone formation activity [Kaigler D. et al., FASEB J. 2005]. However, Ma et al. Reported that co-culture of MSC / EC in osteogenic medium is optimal for osteogenesis and angiogenesis, but can not induce bone formation in a 1: 1 mixture of OM / EM [Ma J. et al., Tissue Eng Part C Methods. 2011].

In the example of the present invention, the medium in which the osteogenic medium and the endothelial cell medium are mixed at a ratio of 10: 1 not only maintains high viability of the endothelial cells but also promotes the osteogenic activity of the stem cells (Figs. 1B and 6) (FIG. 2B), indicating that the angiogenic activity of the DPSC-micro support / HUVEC-gel construct can be increased (FIG. 6).

The three-dimensional co-culture construct of stem cells and endothelial cells of the present invention may exhibit a higher basic phosphatase activity (ALP) than a single cultured cell. In addition, the level of expression of genes associated with osteogenesis and angiogenesis may be higher than in monocultured cells. In the example of the present invention, 3D co-culture of DPSC and HUVEC confirmed that expression of both osteogenic factors (type I collagen and ALP) and angiogenesis (vWF and VE-cadherin) genes was significantly increased. Bone morphogenetic protein 2 (BMP2), which can secrete high levels of vascular endothelial growth factor (VEGF), which increases survival and differentiation of angiogenic cells, and angiogenic cells, which can promote osteogenic differentiation, . Major dissolving factors such as vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) are involved in the communication between the osteogenic lineage and the angiogenic lineage, and can stimulate osteogenic and angiogenic genes.

The three-dimensional co-culture structure of stem cells and endothelial cells of the present invention can be utilized as a vascularized bone graft.

The present invention provides a composition for bone and angiogenesis comprising a coculture construct for stem cells and endothelial cells.

The co-culture structure may have a size of 200 to 400 mu m so as to allow blood injection.

The composition may be administered to injured bone tissue. As can be seen in FIG. 7, HDSPC loaded microspheres with HUVEC-embedded hydrogel can be administered to damaged bone tissue to form vascularized bone tissue.

The composition may be in the form of an injectable vasoconstrictor.

Conventional stem cell culture supporters are difficult to inject directly into the blood vessels because of their large size, thus making it difficult to regenerate the damaged tissue. The structure of the present invention is not only high in blood vessels and osteogenesis effects compared with other supports but also can be used for regeneration of injured tissues since blood vessel infusion is possible because of its micrometer size. Particularly, a microsphere and a hydrogel-containing structure can be prepared as a casting form, and bone and angiogenesis can be induced in a damaged region when administered to injured bone tissue.

The present invention provides a co-culture construct capable of simultaneously culturing stem cells and endothelial cells while enhancing the functions of cells. The three-dimensional co-culture structure of stem cells and endothelial cells of the present invention can be obtained by culturing single- Exhibit higher basic phosphatase activity (ALP) than cells, and may exhibit higher levels of expression of genes related to osteogenesis and angiogenesis than monocultured cells. Therefore, the three-dimensional co-culture structure of stem cells and endothelial cells according to the present invention is expected to be highly applicable to bone tissue engineering as a vascularized bone graft.

Figure 1 shows the results of HDPSC osteogenesis by ALP staining after culturing in different culture media for 7 days to determine optimal cell culture conditions (osteogenic differentiation is shown in purple color).
Figure 2 is the result of HUVEC survival analysis by live / dead staining after incubation in different culture media for 7 days to determine optimal cell culture conditions (live cells are shown as green fluorescence , Dead cells are indicated by red fluorescence).
Figure 3 shows SEM and fluorescence images of HDPSC growth on microspheres recorded for 10 days. A is a SEM image of a cell / microsphere structure; B is the box area of A at high magnification; C and D are fluorescence images of cell / microsphere structures stained with pallaidin (green) and DAPI (expanded map of the map) coupled with Alexa Fluor 488.
FIG. 4 is a phase contrast and fluorescence image of cell growth and survival at day 14 in the HDPSC, HUVEC, and HDPSC / HUVEC groups. AC HDPSC group; DF HDPSC / HUVEC group; GI HUVEC County. A, D, G culture 1 day; B, E, H culture 14 days; C, F, I Culture 14 days of live / dead staining (live cells (green), dead cells (red)).
Figure 5 shows osteogenic differentiation on day 14 as determined by ALP activity. AC: ALP staining; A: HDPSC group; B: HDPSC / HUVEC group; C: HUVEC group; D: The result of quantifying ALP activity using the ALP assay kit. Statistical significance ( ^* p <0.05; HDPSC / HUVEC vs. HDPSC or HUVEC, n = 3)
Fig. 6 shows the expression of qPCR bone formation and angiogenic genes at 14 days in each group. Statistical significance of the indicated groups ^{(* p <0.05; HDPSC /} HUVEC against HDPSC, ^* p <0.05; HDPSC for HUVEC, n = 3)
Figure 7 is a schematic representation of a possible future application to vascular bone tissue engineering of HDPSC loaded microspheres with HUVEC-containing hydrogel.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Cell preparation

Human Dimension Stem Cells (DPSCs) can be obtained from Nam S et al., Nam S. et al., J Tissue Eng. 2011; 812547]. The cells were grown in alpha-denatured Eagle's medium (alpha-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA). The culture medium was changed every 2 to 3 days.

Human umbilical vein endothelial cells (HUVEC; ATCC, USA) were cultured in a 5% CO ₂ humidified incubator at 37 ° C in a vascular cell basal medium (ATCC, USA) supplemented with Endothelial Cell Growth Kit-VEGF (ATCC, USA). The culture medium was changed every 2 to 3 days.

Preparation of Porous Microspheres

Polycaprolactone (PCL, Mw = 80 kDa, Sigma-Aldrich) mixed with poly-L / D-lactide (PLDLA, L-lactide: D, L-lactide = 70:30, Sigma-Aldrich, USA) Were prepared with porous microspheres as described in previous studies (Jin GZ, Kim HW. Tissue Eng Regen Med. 2016; 13: 23541). Briefly, 10% (w / v) PCL / PLDLA (1: 3) was dissolved in chloroform and mixed with 60% (w / v) camphene. The mixed solution was dropped into a water pool containing 2% polyvinyl alcohol with gentle stirring at 450 rpm at 4 占 폚. It was then filtered, washed and lyophilized to obtain porous microspheres.

Preparation Example 1: Preparation of microspheres loaded with stem cells

Before the cells were dispensed, the microspheres were sterilized with 70% ethanol for 2 hours and washed three times with phosphate buffered saline (PBS). One milliliter DPSC (2 x 10 ⁶ ) was added to 10 mg pre-wet microspheres pre-wetted with alpha-MEM supplemented with 10% FBS and 1% penicillin / streptomycin for 12 hours to obtain a DPSC loaded microsphere construct.

Experimental Example 1: Culture of microspheres loaded with stem cells and cell adhesion and growth behavior analysis

The DPSC loaded microsphere construct was incubated in a sway at 45 rpm at 3 rpm for 6 hours using MyLab SLRM-3 Intelli-Mixer (SLRM-3, Seouplin Bioscience, South Korea). Thereafter, the construct was placed in a spinner flask (S-flask 4500-1 L, TAITEC, Japan) containing 70 ml of? -MEM together with 10% FBS culture medium and incubated at 37 ° C in a 5% CO ₂ incubator for 10 days Lt; / RTI &gt; At this time, the stirring speed was 30 rpm.

The DPSC loaded microsphere structure was fixed with 2.5% (v / v) glutaraldehyde and dehydrated with a graded series of ethanol (75, 90, 95 and 100%) and treated with hexamethyldisilazane solution Coated with gold, and observed with a scanning electron microscope (SEM, Hitachi S-3000H) and an optical microscope. The results are shown in Fig.

Cell distribution images on the microspheres were observed with an Alexa Fluor 488-conjugated Paloidin (Invitrogen, USA) staining using an inverted fluorescence microscope. The construct was fixed with 4% paraformaldehyde for 10 minutes and then incubated with 20 nM Alexa Fluor 488-conjugated phalloidin diluted in PBS for 30 minutes. Cell nuclei were counterstained for 5 min with 4 ', 6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using an inverted fluorescence microscope equipped with a DP-72 digital camera (Olympus Co., Tokyo, Japan). The results are shown in Fig.

The porous PCL / PLDLA microspheres have pores with a highly open channel shape with an average diameter of 303 μm. Figures 3A and 3B show SEM micrographs of the HDPSC / microsphere structure on day 10 after agitation-culture at 30 rpm. The cells grew elongated on the spherical microsphere surface. Some cells were observed to migrate into the pores. Cell images were also observed by fluorescence microscopy after co-staining of actin filaments (green with Alexa Fluor 488-conjugated paloidine) and nuclei (blue with DAPI). Paloidin staining results of actin filaments further support cell attachment and growth on the microsphere surface (Figures 3C and 3D).

Example 1: Preparation of DPSC / Microsphere-HUVEC / collagen hydrogel structure

Collagen hydrogel was prepared according to Jin GZ et al., Jin GZ, Kim HW. Tissue Eng Regen Med. 2016; 13: 23541). HUVEC (5 × 10 ⁵ ) was mixed with 2% collagen to prepare a HUVEC embedded hydrogel. 10 mg of DPSC / microspheres cultured for 10 days were mixed with 2 mL of HUVECs / collagen hydrogel to prepare a mixture of DPSC / microsphere and HUVEC / collagen hydrogel.

Comparative Example 1: Preparation of hydrogel containing HUVEC not mixed with DPSC / microsphere and DPSC / microsphere mixed with only hydrogel without HUVEC

For comparison, a DPSC loaded microsphere structure mixed with only hydrogels without HUVEC was prepared.

In addition, a hydrogel containing HUVEC was prepared, which was not mixed with the DPSC loaded microsphere structure. Specifically, the HUVEC hydrogel was poured into a polydimethylsiloxane mold (8 mm in diameter and 2 mm in thickness) and polymerized at 37 캜 for 30 minutes in a humidified incubator. After gelation, the hydrogel was incubated with optimal cell culture medium for 14 days. The culture medium was changed every 2 days.

ALP activity measurement

Cellular ALP activity was measured as an early osteogenic marker. First, osteoblast differentiation of cells on each sample was observed using an ALP color development kit (Takara, Japan) according to the manufacturer's instructions. For this purpose, the sample was fixed and reacted with the ALP reaction substrate for 1 hour. Violet stained samples were observed under an optical microscope. Next, for quantification of ALP activity, samples were added to cell lysis buffer (10% Triton X-100, 1 M Tris-HCl, 0.5 M NaCl and 0.5 M EDTA).

Total protein content was determined using a commercial DC protein assay kit (Bio-Rad, Hercules, USA). Bovine serum albumin (Sigma) was used as a reference curve for protein analysis. The amount of each sample used in the enzyme reaction was determined by standardizing the total protein content. ALP activity was measured using an ALP assay kit (procedure number ALP-10, Sigma). The ρ-nitrophenol produced by the reaction was then determined based on the absorbance at 405 nm using an ELISA plate reader.

Experimental Example 2: Optimization of co-culturing conditions

To determine the optimal cell culture medium for co-culture of DPSC and HUVEC, which allows DPSC bone formation and maintains HUVEC viability, DPSC or HUVEC are mixed with 1 ml of 4 different culture media as described in Table 1 below To 1 x 10 &lt; ⁴ &gt; cells / 24-well, and cultured for 7 days. The culture medium was changed every 2 days. The experiment was repeated 3 times independently to ensure optimized co-culture conditions.

Badge classification Constituent Osteogenesis medium (OM) α-MEM + 10% FBS + 1% penicillin / streptomycin + 50 μg / ml ascorbic acid + 10 mM β-glycerophosphate + 100 nM dexamethasone Endothelial cell medium (EM) Vascular cell basal medium + endothelial cell growth kit MIX 1 A 10: 1 mixture of OM and EM MIX 2 A 1: 1 mixture of OM and EM

ALP staining analysis was used to evaluate qualitatively the bone formation ability of DPSC on day 7 and the results are shown in FIG. Cells showed the darkest color in OM. In MIX 1, cell staining appeared to be slightly brighter than in OM, but was significantly darker in MIX 2. There was no ALP staining in EM.

In addition, viability of HUVEC was evaluated qualitatively on day 7 by live dead staining analysis and the results are shown in Fig. As a result, the number of dead cells in OM was significantly increased, and in contrast, the cells showed similar high viability in MIX 1, MIX 2, and EM. Thus, it can be seen that HUVEC survives and bone formation of DPSC increases in MIX1, a 10: 1 mixture of osteogenic medium (OM) / endothelial cell medium (EM).

Experimental Example 3: Cell growth and survival analysis

Cell viability was analyzed by applying a live / dead assay kit (Molecular Probes, Reduced Biohazard Viability / Cytotoxicity Kit, USA) according to the manufacturer's instructions for the constructs cultured for 14 days. The samples were incubated at room temperature for 30 minutes in a live / dead assay staining solution and then observed under an inverted fluorescence microscope equipped with a DP-72 digital camera (DP2-BSW, Olympus, Tokyo, Japan) The results are shown in Fig. In Figure 4, live cells were labeled with green fluorescence and dead cells were labeled with red fluorescence.

In both the DPSC and DPSC / HUVEC groups, DPSC began to migrate from the microspheres to the collagen gel on day 1, and the cells appeared elongated in the radial direction (FIGS. 4A and 4D). However, cells of the HUVEC group showed a restricted process (Fig. 4G). After 14 days, both the DPSC and DPSC / HUVEC groups showed strong proliferation of cells from the microspheres into the collagen gel (FIGS. 4B and 4E). In contrast, cells of the HUVEC group showed a small round shape (Fig. 4H).

The live / dead assay was used to analyze the viability of the cells on day 14 in three different groups (FIGS. 4C, 4F and 4I). Cells present in the hydrogel can be easily observed under a microscope. Cells are a mixture of DPSCs that migrated from the microspheres and HUVECs that were initially present. The results showed that some dead cells were observed in all groups but most cells were alive.

Experimental Example 4: Evaluation of ALP activity

The possibility of osteogenesis of the construct was evaluated using ALP activity as a functional index, and the results are shown in Fig. At day 14, ALP staining appeared to be more intense in the cells in the microspheres than in the hydrogel, because the cells in the microspheres were DPSC, but the cells in the hydrogel were mainly HUVEC with migrated DPSC . In particular, ALP stained images of the samples showed darker staining in the DPSC / HUVEC group than in the DPSC group (FIGS. 5A and 5B). On the other hand, ALP staining was not observed in the HUVEC group (Fig. 5C). The ALP enzyme activity was significantly higher in the DPSC / HUVEC group than in the DPSC group after 14 days of culture, and the ALP enzyme activity of the HUVEC group was the lowest among them (FIG. 5D).

Experimental Example 5: Quantitative Real-time Polymerase Chain Reaction (qPCR) Analysis

The construct was incubated with optimal cell culture medium for 14 days. Osteogenic and angiogenic gene expression was analyzed using a Rotor-Gene RG-3000A qPCR instrument (Australia). The total RNA of the sample was extracted using TRIzol reagent (Invitrogen). First strand cDNA was synthesized from total RNA (1 μg) according to the manufacturer's instructions using a SuperScript first strand synthesis system for real-time PCR (Invitrogen, USA). The reaction mixture was made up to 50 mu l. Real-time PCR was performed using SYBR GreenER qPCR SuperMix reagent (Invitrogen, USA). The relative transcript amount was calculated using the 2 ^-ΔΔCt method with β-actin as an endogenous reference gene amplified from the sample. The primer sequences of the genes are summarized in Table 2 below.

On day 14, expression of osteogenic factors (type I collagen and ALP) and angiogenic genes (vWF and VE-cadherin) was analyzed using qPCR (Fig. 6). Type I collagen gene expression was significantly higher in the DPSC / HUVEC group than in the other two groups and lowest in the HUVEC group. Expression of the ALP gene was similar to that of type I collagen in the order of DPSC / HUVEC> DPSC> HUVEC. Expression of angiogenic genes including vWF and VE-cadherin was significantly higher in the DPSC / HUVEC group than in the HUVEC group and lowest in the DPSC group: DPSC / HUVEC> HUVEC> DPSC.

Statistical analysis

Data is expressed as mean ± 1 standard deviation. Statistical analysis was performed using one-way ANOVA analysis and post hoc LSD test. p <0.05 was considered statistically significant.

Claims (17)

Porous microspheres loaded with stem cells; And a hydrogel containing endothelial cells, wherein the stem cell and endothelial cell co-
Wherein the structure is a three-dimensional microsphere embedded in a hydrogel, the structure having a three-dimensional shape. The structure according to claim 1, wherein the porous microspheres have an average diameter of 200 to 990 m. The structure according to claim 1, wherein the porous microspheres have a pore size of 10 to 100 탆 so that the stem cells can be cultured in the pores. The construct of claim 1, wherein the stem cells are attached within the surface or pores of the porous microspheres. delete The method of claim 1, wherein the microspheres are selected from the group consisting of polycaprolactone (PCL), polylactic acid (PLA), poly-L- / D-lactide (PLDLA), polyethylene glycol (PEG), polyglycolic acid (PHB), polydioxanone (PDO), polytrimethylene carbonate (PHV), or a mixture thereof. The construct of claim 1, wherein the stem cell is a pulp stem cell. The composition of claim 1, wherein the hydrogel is selected from the group consisting of fibrin, fibrinogen, collagen, polyester polymer, polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid- glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid And polyhydroxybutyric acid-valeric acid, wherein the biodegradable synthetic bio-gel is a biodegradable synthetic bio-gel. Porous microspheres in which stem cells are dispensed; And a hydrogel containing endothelial cells, wherein the stem cell and the endothelial cell are mixed with each other,
Wherein the structure has three-dimensional microspheres embedded in a hydrogel, and has a three-dimensional shape. Culturing the stem cell and endothelial cell coculture construct of any one of claims 1 to 4 and 6 to 8 in a medium in which an osteogenic medium and an endothelial cell culture medium are mixed, A method for co-culturing cells and endothelial cells. The osteogenic medium of claim 10, wherein the osteogenic medium is selected from the group consisting of alpha-minimal essential medium eagle, Fetal Bovine Serum (FBS), penicillin / streptomycin, ascorbic acid, beta -glycerophosphate and dexamethasone &Lt; / RTI &gt; 11. The method according to claim 10, wherein the endothelial cell culture medium comprises a vascular cell basal medium (ATCC, USA) and an endothelial cell growth kit (ATCC, USA). Way. 11. The co-culture method according to claim 10, wherein the weight ratio of the osteogenic medium and the endothelial cell medium in the co-culture medium is 5 to 15: 1. A composition for bone and blood vessel formation comprising a coculture construct of the stem cell and the endothelial cell according to any one of claims 1 to 4 and 6 to 8. 15. The composition of claim 14, wherein the co-cultured construct has a size of 200-400 microns to enable vascularization. 15. The composition of claim 14, wherein the composition is administered to injured bone tissue. 15. The composition of claim 14, wherein the composition is in the form of a pouch capable of vascularization.

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