KR101604372B1 - Biodegradable polymer scaffold comprising spirulina - Google Patents

Abstract

The present invention relates to biodegradable cell scaffolds comprising spirulina and a process for their preparation. The biodegradable cell scaffold comprising Spirulina according to the present invention is free from cytotoxicity and has excellent effect of increasing cell adhesion and proliferation. Therefore, it is possible to reconstitute artificial skin tissue three-dimensionally, It can be used for the development of extracellular matrix (ECM) biomaterials and can be usefully used as a cell scaffold for tissue engineering in various fields.

Description

Biodegradable polymer scaffold comprising < RTI ID = 0.0 > spirulina < / RTI &

The present invention relates to biodegradable cell scaffolds comprising spirulina and a process for their preparation.

Biotissue engineering is a series of techniques that induce normal function by restoring, regenerating, and replacing new parenchyma by transplanting a biocompatible polymer scaffold and cells that are completely absorbed in the living body and have cell affinity. This is a multi-disciplinary combination of cell cultivation, material engineering, and transplant surgery. Using these tissue engineering techniques, desired tissue can be obtained and the shape can be molded at will according to the shape of the support. In the field of tissue engineering, biological signals of cells and scaffolds are very important factors, and the role of biomaterial scaffolds in use should be to help cells to settle and culture well in vivo, Should be distributed uniformly on the support, and the separated cells should promote the regeneration of the differentiated tissue by abundantly releasing extracellular matrix (ECM) in vivo. In addition, after transplantation into the body, it should be biocompatible to absorb and decompose safely after a certain period of time.

The ideal support for effective tissue regeneration is the requirement of sufficient mechanical stress to withstand body loading, the ability to adhere and grow cells, the micro and macro structure of pores, size and shape. The scaffold plays an important role in the growth of cells that migrate from seeded cells and tissues. Most cells in the body are adherent cells. If there is no place to attach, the cells do not grow and die. Due to the characteristics of these cells, there are many difficulties in the treatment of cells. To solve this problem, supports have been introduced and widely used.

On the other hand, various biodegradable polymers have been extensively used in tissue engineering. The polymers used in the above-mentioned tissue engineering supports can be roughly classified into natural polymers and synthetic polymers, and they have suitable physico-chemical, biological and mechanical properties.

Natural polymers include collagen, hyaluronic acid, alginate, gelatin, xanthan gum, keratin, and small intestinal submucosa, which have excellent biocompatibility and low immune responses after transplantation. However, natural polymers have disadvantages in that they do not have sufficient mechanical properties when used individually.

Synthetic polymers are mainly hydrophobic polyesters such as PLA (poly (lactic acid)), PGA (poly (glycolic acid)), PLGA (poly (lactic-co-glycolic acid)) and PCL (poly (e-caprolactone)). Among them, α-hydroxy acid-based polyglycolide (PGA), polylactide (PLA), and PLGA, which is a copolymer thereof, are synthesized polymers approved by the US FDA and used as biological materials such as tissue engineering porous supports and drug delivery systems Is widely used, has high biocompatibility, biodegradability and processability. However, it is difficult to attach cells due to lack of bioactive substances and hydrophobicity, and there is a disadvantage that acid decomposition products generated during the hydrolysis process of PLGA reduce the pH around the tissue and cause inflammation.

Spirulina, on the other hand, is an algae, multicellular organelles with thin cell walls. The main ingredients in Spirulina include protein, carbohydrate, water soluble dietary fibers, antioxidant pigments (chlorophyll, carotenoids, phycocyanin), antioxidant enzymes (SOD) and gamma linolenic acid (GLA). Vitamins include beta-carotene, vitamins B1, B2, B6, B12, vitamin E, inositol, folic acid, and also contain minerals such as calcium, iron, potassium, magnesium, zinc, manganese, selenium and germanium.

Accordingly, the inventors of the present invention have conducted studies to develop a novel cell scaffold that solves the above-mentioned problems. As a result, the present inventors have found that biodegradable cell scaffolds containing spirulina have excellent cytotoxicity and increase cell adhesion and proliferation The present invention has been completed.

Domestic Patent Application Number: 10-2008-0005447

It is an object of the present invention to provide a biodegradable cell scaffold comprising spirulina and a method of producing the same.

In order to achieve the above object, the present invention provides a biodegradable cell scaffold comprising spirulina and a method of producing the same.

The biodegradable cell scaffold comprising Spirulina according to the present invention is free from cytotoxicity and has excellent effect of increasing cell adhesion and proliferation. Therefore, it is possible to reconstitute artificial skin tissue three-dimensionally, It can be used for the development of extracellular matrix (ECM) biomaterials and can be usefully used as a cell scaffold for tissue engineering in various fields.

1 is a view of a PCL nanofiber mat through an SEM.
2 is a view of a Spirulina-PCL nanofiber mat through an SEM.
3 is a chart showing the cytotoxicity of the Spirulina-PCL nanofiber mat.
4 is a graph showing the effect of the Spirulina-PCL nanofiber mat on cell adhesion.
5 is a diagram showing cytotoxicity of Spirulina extract.
6 shows the effect of Spirulina extract on cell proliferation.
FIG. 7 is a graph showing the cell growth after 5 days of treatment with Spirulina extract (Spirulina extract concentration: A: 0.5 μg / mg, B: 1 μg / mg, C: 10 μg / : 100 μg / mg).
8 is a diagram showing cell infiltration in a Spirulina-PCL nanofiber mat.
9 is a diagram showing cell penetration in a PCL nanofiber mat.
FIG. 10 is a view of a Spirulina-PCL nanofiber mat after observing the de-saturation process through Alcian blue staining.
Fig. 11 is a view of the Spirulina-PCL nanofiber mat after H / E staining and observed through H & E staining.
FIG. 12 is a view of a Spirulina-PCL nanofiber mat after being subjected to a de-saturation process through Sirius red staining.

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

The present invention provides a biodegradable cell scaffold comprising Spirulina.

Preferably, the cell support comprises a biodegradable polymer together with spirulina.

The biodegradable polymer is selected from the group consisting of polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co- glycolide ), PLGA), diol / isocyanate aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxybutyrate), poly (hydroxyvalerate) , Polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide and derivatives or copolymers thereof, preferably polycaprolactone ( PCL).

The spirulina is preferably contained in an amount of 3 to 10% by weight based on the total weight of the biodegradable cell scaffold. When the content of spirulina is less than 3% by weight, the effect as a cell scaffold is insignificant. When it exceeds 10% by weight, the morphological stability is insufficient.

The present invention also provides a method for producing Spirulina, comprising the steps of (1) dissolving spirulina and a biodegradable polymer in an organic solvent, and (2) preparing a nanofiber mat by electrospun mixing the mixture of (1) Wherein the biodegradable cell support is a biodegradable cell support.

More specifically, a biodegradable polymer, preferably PCL, is dissolved in an organic solvent and a certain amount of spirulina is added. After sufficiently mixing the solution, the nanofiber mat is fabricated using an electrospinning apparatus. At this time, the needle is preferably 15 to 20 gauge, the distance from the collector to the collector is preferably 10 to 20 cm, and the electrospinning voltage intensity is preferably 10 to 20 kV.

The organic solvent includes tetrahydrofuran, dimethylformamide, dichloromethane, chloroform, acetone, dioxane, trifluoroethane, hexafluoroisopropane, and mixed solvents thereof, preferably tetrahydrofuran and dimethyl Formamide. More preferably, the mixing ratio of tetrahydrofuran and dimethylformamide is from 5: 10: 1 to 5: 5, more preferably 7: 3.

The biodegradable cell scaffold of the present invention can be prepared in two-dimensional and three-dimensional forms.

The biodegradable cell scaffold containing the spirulina of the present invention has no cytotoxicity and has an excellent effect of increasing cell proliferation under cell attachment and high concentration culture conditions. In addition, the biodegradable cell scaffold containing the spirulina of the present invention contributes to the stable growth of the cells in the culture environment constituted by the cell scaffold, and therefore, it can be used as a complex ECM through artificial tissue and de-aeration process . Therefore, the biodegradable cell scaffold comprising the spirulina of the present invention is useful as a cell scaffold for tissue engineering.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and experimental examples.

Example  One. Spirulina  Included Of cell support  Produce

(7: 3 (v / v) mixture of 15% by weight and 1% by weight of THF (Tetrahydrofuran): DMF (Dimethylformamide), respectively, in PCL (Polycaprolacton, Mn 80,000, Sigma USA) and Spirulina (Nature's care, Australiria) ) And mixed.

The mixture was made into a nanofiber mat using an electrospinning apparatus. At this time, the needle was 18 gauge, the distance from the collector was 15 cm, and the voltage of the electrospinning device was 15 kV. 15% by weight PCL was used as a control.

The PCL content in the solvent-removed Spirulina-PCL nanofiber mat was 93.75 wt%, and the spirulina content was 6.25 wt%.

Experimental Example  One. Spirulina  Included Of cell support  Character analysis

SEM (scanning electron microscope, Hitachi S-4300) was used to observe the manner in which the cell scaffold containing Spirulina prepared in Example 1 adheres to the cells. Cells were used in primary cultures of SD rats. More specifically, the skin of 1-day-old SD rats was cut (1 cm * 1 cm), placed in HBSS medium containing 1 mg / ml of dipase, reacted at 4 ° C for 24 hours, The dermal layer and the epidermal layer were separated. This was added again to HBSS medium containing collagenase 2 (1 mg / ml) and reacted at 4 ° C for 24 hours. Each epidermis and dermis were filtered through a cell filter with a pore size of 70 μm and cultured in high glucose-DMEM medium (10% FBS, 1% antibiotic). The Spirulina-PCL nanofiber mat or PCL nanofiber mat prepared in Example 1 was placed on a 24-well plate, and cells were seeded at a concentration of 1 × 10 ⁴ cells / well and cultured for 5 days. After 5 days, each nanofiber mat sample was washed with PBS, and 3% glutaraldehyde was added, followed by incubation at 37 DEG C for 30 minutes. Thereafter, the concentration of ethanol was increased to 25%, 50%, 75%, 90%, and 100% and dehydrated. Three drops of HMDS (hexa methyl di silazane) were dropped on each dehydrated nanofiber mat sample and allowed to react for 3 minutes and then evaporated under vacuum for 30 minutes. This was observed at a magnification of 1 to 10K. The results are shown in Fig. 1 and Fig.

As shown in Fig. 1 and Fig. 2, it was confirmed that the cells were stably attached to the Spirulina-PCL nanofiber mat of the present invention as compared with the PCL nanofiber mat.

Experimental Example  2. Spirulina  Included Of cell support  Cytotoxicity analysis

To confirm the cytotoxicity of the cell scaffolds containing the spirulina prepared in Example 1, MTT assay (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium-bromide, Sigma-Aldrich , St. Louis, MO). More specifically, the Spirulina-PCL nanofiber mat or the PCL nanofiber mat prepared in Example 1 was placed in a 24-well plate, and DMEM medium (1% antibiotic) was added for 1 day. Cells were seeded at a concentration of 3x10 ³ cells / well in a 96-well plate, followed by addition of a medium prepared by previously preparing each nanofiber sample and culturing for 1 day. Thereafter, each well was treated with MTT solution, dissolved in DMSO, and then absorbance was measured at OD 540 nm. The results are shown in Fig.

As shown in Fig. 3, the medium treated with the Spirulina-PCL nanofiber mat of the present invention showed similar values to the medium treated with the PCL nanofiber mat which was known to be already non-toxic.

EXPERIMENTAL EXAMPLE 3 Effect of Cellular Support Containing Spirulina on Cell Attachment Analysis

In order to confirm the effect of the cell scaffold containing Spirulina prepared in Example 1 on cell adhesion, the following experiment was conducted. The Spirulina-PCL nanofiber mat or the PCL nanofiber mat prepared in Example 1 was placed on a 24-well plate and cells were seeded at a concentration of ³ × 10 ⁴ cells / well. MTT assays were performed after 2, 8, and 24 hours. The MTT solution changes to purple formazan due to the metabolic activity of the cells. In this experiment, the MTT value changes according to the number of cell attachment, not the cell activity difference, within 24 hours after inoculation. The results are shown in Fig.

As shown in FIG. 4, since the result of TCPS (tissue culture polystyrene), which is a material of a cell culture plate used generally, increases with time, it is confirmed that normal cells having no defect in cell adhesion were used in the experiment. In addition, it was confirmed that the Spirulina-PCL nanofiber mat of the present invention had a more positive effect on cell attachment, as compared with the conventional PCL nanofiber mat.

Experimental Example  4. Spirulina  Cytotoxicity analysis of extracts

MTT assay and BrdU assay (Roche-applied science) were performed to confirm cytotoxicity of Spirulina extract itself. First, Spirulina was dissolved in water at a concentration of 5 g / l and then stirred at room temperature for 24 hours. Thereafter, undissolved residues were removed using a centrifuge, and then lyophilized to obtain a Spirulina extract. Cells were seeded at a density of ³ × 10 ³ cells / well in a 96-well plate and cultured for 1 day. Each well was exchanged with DMEM medium containing Spirulina extract at a concentration (0-100 μg / mg), and MTT assay and BrdU assay (Roche-applied science) were performed after 1, 3 and 5 days. The results are shown in Fig. 5 and Fig. 6, respectively.

As shown in FIG. 5, the MTT assay confirmed inhibition of the decrease in cell activity in cells treated with Spirulina extract, confirming that Spirulina extract had a positive effect on the tissue structure.

In addition, as shown in FIG. 6, the results of the BrdU assay showed that the cells treated with Spirulina extract showed higher values than those of the cells treated with Spirulina extract, confirming that the Spirulina extract promoted cell division.

In addition, the Spirulina extract was treated in the same manner, and after 5 days, the MTT assay was performed and the cells were observed. The results are shown in Fig.

As shown in Fig. 7, it was confirmed that all of the cells treated with various concentrations of Spirulina extract (0.5-100 μg / mg) were normally grown well.

Experimental Example  5. Spirulina  Included On the cell support  Cell penetration analysis for

In order to confirm cell penetration of the cell scaffold containing Spirulina prepared in Example 1, the following experiment was conducted. First, the Spirulina-PCL nanofiber mat or PCL nanofiber mat prepared in Example 1 was placed on a 24-well plate, and the cells were seeded at a concentration of 1 x 10 ⁴ cells / well and then cultured for 5 days. After the incubation, 4% paraformaldehyde was added, and the cells were fixed by incubating for 20 minutes. The sample was stained with H & E, then frozen with OCT and cut to a thickness of 40 [mu] m using an ultra-thin separator. This was observed with a reversed phase microscope. The results are shown in Fig. 8 and Fig.

As shown in Fig. 8 and Fig. 9, it was confirmed that the cells were stably infiltrated into the Spirulina-PCL nanofiber mat of the present invention.

Experimental Example  6. Spirulina  Included Of cell support  De-saturation analysis

In order to carry out the de-fatization analysis of the cell scaffold comprising Spirulina prepared in Example 1, the following experiment was carried out. First, the Spirulina-PCL nanofiber mat or PCL nanofiber mat prepared in Example 1 was placed on a 24-well plate, and the cells were seeded at a concentration of 1 x 10 ⁴ cells / well and then cultured for 5 days. After completion of the culture, the medium was removed and washed with PBS. Thereafter, the process of freezing for 20 seconds using liquid nitrogen and dissolving at room temperature for 5 minutes was repeated five times. The sample was treated with 0.1% (w / v) NH ₄ OH for 1-2 minutes and then washed with distilled water to remove debris such as cytoplasm. Finally, Spirulina-PCL nanofibers were recovered by freeze-drying and then observed with Alcian blue staining, H & E staining and Sirius red staining. The results are shown in Figs. 10 to 12, respectively.

As shown in FIGS. 10 to 12, it was confirmed that GAG remained in the nanofiber even after the de-saturation process, and the cells were almost completely removed. In addition, it was confirmed that the cell nucleus was removed by decolletage, and only collagen remained.

As a result, it was confirmed that the cells were well distributed on the Spirulina-PCL nanofibers of the present invention and actively produced the extracellular matrix. The Spirulina-PCL nanofibers were used as cell supports to form artificial tissues And it is confirmed that it is possible to manufacture a complex ECM that can be used as an insert in a human body when the cell is removed.

Claims (4)

(1) dissolving spirulina and polycaprolactone (PCL) in a mixed solvent of tetrahydrofuran and dimethylformamide mixed at a volume ratio (v / v) of 6 to 8: 2 to 4; And
(2) preparing a nanofiber mat by electrospinning the mixture of step (1) under a voltage of 10 to 20 kV,
Wherein the spirulina is comprised between 6 and 6.5 wt% based on the total weight of the biodegradable cell support. ≪ RTI ID = 0.0 > 8. < / RTI > delete delete The method of claim 1, wherein the mixed solvent is mixed with tetrahydrofuran and dimethylformamide in a volume ratio (v / v) of 7: 3.

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