KR20240034968A - Method for Manufacture of Bioink Comprising Cell-Laden Mineral Oil and Application thereof for Fabrication of Cell Construct - Google Patents

Abstract

The present invention relates to a composition for bioink containing a photocurable biocompatible polymer, stem cells, and oil, a method for manufacturing the same, and a printing method using the same.
When using the present invention, it is possible to produce a cell structure with improved mechanical stability, a cell structure capable of high moisture/nutrient absorption compared to the prior art, and a bioink based on underwater oil containing various bioactive molecules. Compared to existing methods, the initial release of growth factors is less and a cell structure capable of long-term release can be produced.

Description

Method for manufacturing bioink containing mineral oil containing cells and application thereof for fabrication of cell construct {Method for Manufacture of Bioink Comprising Cell-Laden Mineral Oil and Application thereof for Fabrication of Cell Construct}

The present invention relates to a method for producing bioink containing mineral oil containing cells and the production of a cell structure thereof. More specifically, it relates to a composition for bioink containing a photocurable biocompatible polymer, stem cells, and oil, a manufacturing method thereof, and a printing method using the same.

Bioprinting technology basically uses hydrogel-based bioink containing cells. However, cell-laden constructs made based on hydrogels show limitations in mechanical stability and cell-cell interaction, resulting in low initial cell activity. Additionally, it was difficult to ensure structural stability due to the low mechanical properties of the hydrogel. Therefore, it is necessary to produce bioink that can effectively improve mechanical stability and induce cell-cell interaction and cell activity.

The present inventors developed an oil-in-water emulsion bioink by mixing mineral oil with a photocurable collagen-based bioink and fabricated a cell structure with improved mechanical stability and biological properties. Additionally, using growth factors, it was possible to produce an underwater relic cell structure capable of forming desired tissues. It is believed that the underwater remains bioink produced through the present invention can be used to produce various cell structures and regenerate damaged tissues.

Republic of Korea Patent Publication No. 10-2017-0001444 (published on January 4, 2017)

The present inventors have made extensive research efforts to develop a bioink that can effectively improve mechanical stability and induce cell-cell interaction and cell activity. As a result, when using an oil-in-water emulsion bioink that is a photo-crosslinkable collagen-based bioink mixed with mineral oil, it effectively improves mechanical stability and promotes cell-cell interactions and cell activity. By proving that it can be derived, the present invention was completed.

Accordingly, the purpose of the present invention is to provide a composition for bioink containing a photocurable biocompatible polymer, stem cells, and oil.

Another object of the present invention is to provide a method for producing the bio-ink composition.

Another object of the present invention is to provide a printing method using the bio-ink composition.

Since cell-laden constructs produced based on conventional hydrogels have the disadvantage of low mechanical stability and cell-cell interaction, the present inventors effectively improved mechanical stability and Extensive research efforts were made to develop bioink that can induce cell-cell interaction and cell activity. As a result, manufacturing bioink containing photocurable biocompatible polymer, stem cells, and oil not only showed improved mechanical stability and high water/nutrient absorption capacity compared to collagen-based cell structures, but also activated cell-cell interactions. This was confirmed and the present invention was completed.

According to one aspect of the present invention, the present invention provides a composition for bioink containing a photocurable biocompatible polymer, stem cells, and oil.

As used herein, the term 'bio-printing' refers to an automated process of producing tissues or organs by arranging cells in a bio-material in a controlled pattern in three-dimensional space.

As used herein, the term 'bio-ink' refers to a material capable of bioprinting used in the bioprinting process defined above, and includes biomaterials containing living cells. Bioinks can include scaffold-based materials such as hydrogels, microcarriers, and decellularized matrices, as well as scaffold-free materials such as cell aggregates. there is.

In one embodiment of the present invention, the photocurable biocompatible polymer is a photocurable polymer conjugated to a biocompatible polymer.

In a specific embodiment of the present invention, the photocurable polymer is methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, hexanediol diacrylate, and hexanediol diacrylate. Methacrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, dipropylene glycol diacrylate, dipropylene glycol dimethacrylate and urethane acrylate. One or more monomers selected from the group consisting of gelatin methacryloyl (GelMA), collagen methacrylate (ColMA), hyaluronic acid methacrylate (HAMA), etc., and It is made of oligomers, but is not limited thereto.

In one embodiment of the present invention, the biocompatible polymer is hyaluronic acid, collagen, polyethylene glycol, alginate, starch, chitosan, gelatin, and dextrose. It is selected from the group consisting of dextran, cellulose, alginic acid, chondroitin sulfate, and heparin, but is not limited thereto and is used in the art as a three-dimensional structure or cell. Any biocompatible polymer that can be used for culture can be used without limitation.

In a specific embodiment of the present invention, the photocurable biocompatible polymer includes gelatin methacryloyl (GelMA), collagen methacrylate (ColMA), and hyaluronic acid methacrylate (HAMA). ), polyethylene glycol diacrylate (PEGDA), etc., but any photocurable polymer that can be used for three-dimensional culture of cell structures or cells can be used without limitation.

In one embodiment of the present invention, the stem cells are pluripotent stem cells.

In a specific embodiment of the present invention, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic germ stem cells, and germ stem cells, but are not limited thereto.

In one embodiment of the present invention, the stem cells are mesenchymal stem cells.

As used herein, the term 'mesenchymal stem cell (MSC)' refers to a stromal cell that has the ability to self-renew and exhibits multilineage differentiation.

In a specific embodiment of the present invention, the mesenchymal stem cells are mesenchymal cells derived from one or more tissues selected from the group consisting of umbilical cord, cord blood, blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. It is a stem cell (mesenchymal stromal cell), but is not limited thereto.

In a more specific embodiment of the present invention, the stem cells are adipose-derived mesenchymal stem cells.

In one embodiment of the present invention, the oil is a mixture of hydrocarbons having a carbon number of C ₁₅ to C ₅₀ extracted through refining of crude oil.

The hydrocarbons include, but are not necessarily limited to, alkanes and cycloalkanes, and may include any hydrocarbon that can be extracted during the refining process of crude oil or petroleum known in the art. In the present invention, oil may include liquid by-products generated from manufacturing gasoline and other petroleum products.

In a specific embodiment of the invention, the oil is mineral oil.

In a specific embodiment of the present invention, the oil is 3 to 40% (v/v), 3 to 30% (v/v), 3 to 25% (v/v), 3 to 24% of the total volume of the composition. (v/v), 3 to 23% (v/v), 3 to 22% (v/v), 3 to 21% (v/v), 3 to 20% (v/v), 3 to 19% (v/v), 3 to 18% (v/v), 3 to 17% (v/v), 3 to 16% (v/v), 3 to 15% (v/v), 3 to 10% (v/v), 3 to 5% (v/v), 0% (v/v), 5% (v/v), 10% (v/v), 20% (v/v), 30% (v/v), or 40% (v/v), but is not limited thereto.

In one embodiment of the present invention, the bio-ink composition may additionally include bioactive molecules.

In a specific embodiment of the present invention, the bioactive molecules include, for example, Bone morphogenetic protein 2 (BMP-2), Kartogenin (KGN), Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and insulin-like growth. It may be factor 1 (IGF-1), angiopoietin (ANG), transforming growth factor beta 1 (TGF-β1), etc., but is not limited thereto.

In one embodiment of the present invention, the composition for bioink may additionally include a photoinitiator.

As used herein, the term 'photoinitiator' refers to a substance that causes rapid crosslinking upon exposure to light.

In one embodiment of the present invention, the type of photoinitiator is not particularly limited, but an optical initiator that causes a crosslinking reaction by irradiation of ultraviolet rays or an optical initiator that causes a crosslinking reaction by irradiation of visible light can be used. there is. Non-limiting examples of suitable photoinitiators include lithium phenyl-(2,4,6-trimethyl benzoyl) phosphinate (LAP) and benzyl dimethyl ketal. ), acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclohexyl phenyl ketone ), etc. The amount of optical initiator added may vary depending on the wavelength and time of light exposed.

According to another aspect of the present invention, the present invention provides a method for producing bioink comprising the following steps:

(a) preparing a hydrogel containing a photocurable biocompatible polymer; and

(b) preparing an emulsion by mixing stem cells, photoinitiator, and oil with the hydrogel.

In one embodiment of the present invention, the hydrogel is manufactured by mixing a photocurable biocompatible polymer and an aqueous solvent.

In one embodiment of the present invention, the aqueous solvent includes, but is not limited to, purified water, phosphate buffer solution, physiological saline, etc.

In one embodiment of the present invention, an emulsion may be prepared by additionally adding a bioactive material in step (b).

The method for producing bioink of the present invention is a method for producing a composition for bioink according to another aspect of the present invention, and since the common components between them can be applied in the same way, their description is omitted to avoid complexity of the present specification. do.

In one embodiment of the present invention, the concentration of the photocurable biocompatible polymer in the aqueous solvent is about 0.1 to 20% (w/v), about 0.1 to 15% (w/v), about 0.1 to 10% ( w/v), about 0.1 to 9 % (w/v), about 0.1 to 8 % (w/v), about 0.1 to 7 % (w/v), about 0.1 to 6 % (w/v), about 0.1 to 5% (w/v), about 0.1 to 4% (w/v), about 0.1 to 3% (w/v), about 0.1 to 2% (w/v), about 1 to 20% (w) /v), about 1 to 15% (w/v), about 1 to 10% (w/v), about 1 to 9% (w/v), about 1 to 8% (w/v), about 1 to 7% (w/v), about 1 to 6% (w/v), about 1 to 5% (w/v), about 1 to 4% (w/v), about 1 to 3% (w/v) v), about 1 to 2% (w/v), about 2 to 20% (w/v), about 2 to 15% (w/v), about 2 to 10% (w/v), about 2 to 9 % (w/v), about 2 to 8 % (w/v), about 2 to 7 % (w/v), about 2 to 6 % (w/v), about 2 to 5 % (w/v) ), about 2 to 4 % (w/v), about 2 to 3 % (w/v), about 3 to 20 % (w/v), about 3 to 15 % (w/v), about 3 to 10 % (w/v), about 3 to 9 % (w/v), about 3 to 8 % (w/v), about 3 to 7 % (w/v), about 3 to 6 % (w/v) , about 3 to 5% (w/v), or about 3 to 4% (w/v), about 1% (w/v), 2% (w/v), 3% (w/v), 4 It may be %(w/v), or 5%(w/v), but is not limited thereto.

In a specific embodiment of the present invention, the step of preparing the emulsion is characterized in that it is prepared by preparing a mixture of stem cells and a photoinitiator mixed with a hydrogel containing the photocurable biocompatible polymer and mixing it with oil. do. When manufactured as above, the biocompatible polymer hydrogel containing stem cells takes the form of a homogeneous oil droplet encapsulated in oil. In other words, an oil-in-water emulsion is produced.

In a specific embodiment of the present invention, when the bioink contains a bioactive material, the step of preparing the emulsion includes (i) adding stem cells, photoinitiators, and biomaterials to the hydrogel containing the photocurable biocompatible polymer. It is prepared by preparing a mixture of active substances and mixing this mixture with oil, or (ii) a mixture containing a hydrogel containing the photocurable biocompatible polymer, stem cells, and a photoinitiator, and a bioactive substance. and a mixture of oils.

According to another aspect of the present invention, the present invention provides a method for printing bioink comprising the following steps:

(a) Printing a cell structure of a desired shape using the above-described bio-ink using a printer device including a nozzle equipped with a UV exposure device.

In one embodiment of the present invention, the UV exposure device has a UV output of 200 to 800 mW/cm ² , but is not limited thereto.

In one embodiment of the present invention, the nozzle has an inner diameter of 50-500 μm, but is not limited thereto.

In one embodiment of the present invention, the printer device outputs at a pneumatic pressure of 60 to 250 kPa, but is not limited thereto.

In one embodiment of the present invention, the printer device has a moving speed of 3 to 30 mm/s, but is not limited thereto.

In one embodiment of the present invention, the printer device prints in a temperature range of 15 to 30°C, but is not limited thereto.

The underwater remains bioink according to the present invention evaluates the size distribution, fabrication, printability, cell viability, and initial cell loss of mineral oil emulsion spheres formed based on rheological properties, and includes mineral oil and photocured collagen. It was produced by setting the concentration. The fabricated cell structure containing mineral oil showed improved mechanical stability and high water/nutrient absorption compared to the existing collagen-based cell structure. For the production of underwater relic bioink containing cells, human adipose-derived stem cells (hASC) were used as an example, and as a result, high cell growth and cytoskeleton formation were observed using immunofluorescence staining and RT- Confirmed through qPCR. In addition, a functional bioink was produced by mixing growth factors with the mineral oil of the underwater remains bioink. BMP-2 and Kartogenin were used to form bone tissue and cartilage tissue, and their effects on promoting bone tissue cartilage formation were confirmed through GAG and calcium staining, immunofluorescence staining, and RT-qPCR, respectively.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for bioink containing a photocurable biocompatible polymer, stem cells, and oil, a manufacturing method thereof, and a printing method using the same.

(b) When using the present invention, it is possible to produce a cell structure with improved mechanical stability, a cell structure capable of high moisture/nutrient absorption compared to the prior art, and an underwater oil-based bioink containing various bioactive molecules. Using , it is possible to produce a cell structure that has less initial release of growth factors and enables long-term release of growth factors compared to existing methods.

Figure 1 shows a schematic diagram of oil-in-emulsion bioink and the production of a cell structure using the same.
Figure 2 shows optical images before and after oil-CMA mixing and the size and uniformity of oil droplets according to the number of mixing times.
Figure 3 shows optical and SEM images of the CMA/MO structure containing hASC produced using emulsion bioink.
Figure 4 is a diagram showing the survival rate of cells in a cell construct produced using the emulsion bioink of the present invention as a live-dead fluorescent staining image.
Figure 5 is a diagram comparing the CMA structure and the degree of actin filament development in CMA/MO.
Figure 6 is a diagram showing 3D images (surface and cross-sectional views) of the CMA/MO support.
Figure 7 shows optical images of CMA (3 wt/v%)/MO bioink containing various concentrations of oil (5, 10, 20, 30, and 40 v/v%).
Figure 8 is a diagram showing the average diameter of oil droplets according to the various oil concentrations.
Figures 9 and 10 show storage (storage) to evaluate the rheological properties of emulsion bioink (CMA: 3 wt/v%, MO: 0, 5, 10, 20, 30, 40 v/v%) This is a diagram showing G'), loss modulus (G''), and complex viscosity (η*).
Figure 11 is a diagram showing the change in rheological properties according to increased oil droplet size.
Figure 12 shows optical and live/dead images of the bioprinted mesh structure.
Figure 13 is a diagram showing printability according to the oil content of bioink.
Figure 14 shows the results of measuring the initial cell loss for 1 day using the MTT assay in the cell construct, depending on the oil content of the bioink of the present invention.
Figures 15 and 16 show the size of oil droplets according to CMA concentration.
Figure 17 is a diagram showing printability according to CMA concentration.
Figure 18 is a diagram showing the effect on cell proliferation according to CMA concentration.
Figure 19 is a diagram showing initial cell loss according to CMA concentration.
Figure 20 is a diagram showing mechanical sweetness, printability, cell viability, etc. according to UV intensity.
Figure 21 shows optical and SEM images of the printed cell loading configuration.
Figure 22 is a diagram showing the compressive stress-strain curve and compressive elastic modulus of the cellular structure of the present invention.
Figures 23 and 24 are diagrams showing the diffusivity of FITC-dextran of the cell construct of the present invention.
Figure 25 is a diagram showing the protein absorption capacity of the cell construct of the present invention.
Figure 26 is a diagram showing the metabolic activity of the cell construct of the present invention through cell viability test.
Figure 27 is a diagram showing the cell viability, cytoskeletal organization, and cell proliferation of the cell construct of the present invention by confirming tissue photos and gene expression rates.
Figure 28 is a diagram showing two formulation methods of the present invention.
Figures 29 and 30 are diagrams showing the release dynamics of bioactive molecules from cell structures according to the two formulation methods of the present invention.
Figures 31 and 32 are diagrams showing the biological activity according to the formulation of the cell structure containing the bioactive molecule (KGN) of the present invention.
Figure 33 is a diagram showing gene expression according to the formulation of the cell structure containing the bioactive molecule (KGN) of the present invention.
Figure 34 shows DAPI/osteopontin (OPN; red) (after 14 days of culture) and Alizarin red S (ARS) (after 14 days of culture) according to the formulation of the cell construct containing the bioactive molecule (BMP-2) of the present invention. and 21 days later) staining is shown.
Figures 35 and 36 show the OPN positive area, intensive ARS staining, and calcium deposition of the CMA/MO-BMP scaffold according to the formulation of the cell construct containing the bioactive molecule (BMP-2) of the present invention.
Figure 37 shows alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), and osteopontin (OPN) in the CMA/MO-BMP group according to the formulation of the cell construct containing the bioactive molecule (BMP-2) of the present invention. ) and osteocalcin (Ocn), showing the expression levels of osteogenesis-related genes.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating 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 by these examples according to the gist of the present invention. .

Example

Experimental methods and experimental materials

1. Production of cells and bioink

Human adipose-derived stem cells (hASC) (Lonza, USA) were used in this study to evaluate the developed bioink and cell construct. Cells were cultured in growth medium (GM) consisting of Dulbecco's Modified Eagle's Medium-low-glucose (DMEM-L; Sigma-Aldrich, USA), fetal bovine serum (10% FBS; BioWest, USA), and penicillin-streptomycin. It has been done. (1% PS, Thermo-Fisher Scientific, USA), cultured at 37°C and 5% CO ₂ , and the growth medium was changed every 2 days.

Before producing bioink, collagen was chemically modified to produce methacrylated collagen (CMA). Briefly, lyophilized collagen sponges (MSBio, South Korea) were dissolved in 0.5 M acetic acid solution at a ratio of 300 mg of collagen per 80 mL of acetic acid solution at 4°C. The solution was adjusted to pH 8-9 using NaOH solution (1 M; Sigma-Aldrich, USA). For chemical modification, methacrylic anhydrate (Sigma-Aldrich, USA) was added to the collagen solution at a density of 621 mg per 600 mg of dissolved collagen under continuous stirring at 4°C. After treatment, the obtained CMA solution was dialyzed in deionized water (DW) at 4°C using a 3.5 kDa molecular barrier tube (Spectrum Lab, Inc., USA) and then lyophilized. Freeze-dried CMA was dissolved in deionized water and mixed with 10xDMEM (Sigma-Aldrich, USA) at a ratio of 1:1 to obtain neutralized CMA hydrogel. The CMA hydrogel was then mixed with hASC ( ^2.0 did. Physical properties of bioink were measured using various concentrations of CMA (1, 2, 3, 4, 5 wt/v%) and mineral oil (MO) (5, 10, 20, 30, 40 v/v%). and biological properties were evaluated. As a control, pure CMA (3wt/v%) bioink containing hASC (2.0 × 10 ⁷ cells/mL) and 0.5% photoinitiator was used.

For bioactive molecule-laden structures, CMA containing hASC was mixed with mineral oil containing bioactive molecules (BM):

(1) KGN (28 μg/mL) to induce cartilage formation and

(2) BMP-2 (10 μg/mL), which induces osteogenesis.

Selected bioactive molecules were mixed with CMA/MO bioink using the following formulation method:

(1) mixing cell-laden CMA with a mixture of MO and BM (CMA/MO-BM);

(2) The mixture of cell-containing CMA and BM was mixed with MO (CMA-BM/MO).

Additionally, CMA bioink (CMA/MO) containing hASC mixed with BM was prepared as a control.

2. Bioprinting of cell structures

To fabricate cell constructs, the prepared CMA/MO, CMA-BM/MO, and CMA/MO-BM bioinks were dispensed using a 30G single nozzle (inner diameter: 150 μm) connected to a transparent glass nozzle, using a pneumatic dispenser (AD-3000C). , Ugin-tech, South Korea) and printed using a 3D bioprinting system (DTR3-2210 T-SG; DASA Robot, Korea) equipped with an in situ UV exposure system at controlled temperature (barrel and plate at 25°C). did. 3D cellular constructs were obtained by controlling the processing conditions, including a movement speed of 10 mm/s, pneumatic pressure of 120 kPa, and UV output of 400 mW/cm ² . The same bioprinting conditions with different pneumatic pressure (100 kPa) were used for CMA and CMA-BM bioinks. The printed 3D structures were cultured in growth medium at 37°C and 5% CO ₂ . Growth medium was changed every 2 days.

To induce osteogenic differentiation of hASCs, 10% FBS, 1% PS, 0.1 μM dexamethasone (Sigma-Aldrich, USA), 50 μM ascorbic acid (Sigma-Aldrich, USA) and 10 mM β-glycerol phosphate (Sigma-Aldrich, USA). ) was provided as osteogenic medium (OM) after 3 days of culture as a growth medium. Osteogenic medium (OM) was changed every 2 days.

3. Confirmation of characteristics of bioink and cell structure

3-1. Observation with light microscope, digital camera and scanning electron microscope

To visualize the 3D printed structures and dispersed oil droplets, an optical microscope (BX FM-32; Olympus, Japan) was used together with a digital camera and a SNE-3000M scanning electron microscope (SEM) (SEC Inc., Korea). used. Before observing the structure using SEM, more than 15 times the volume of the structure was placed in deionized water and incubated at 37°C for 7 days to remove the mineral oil component contained therein. The constructs were then fixed using 10% neutral buffered formalin (NBF; Sigma-Aldrich, USA) and dehydrated using successive concentrations of ethanol (50%, 60%, 70%, 80%, 90%, and 100%). and freeze-dried. The diameters of oil droplets (n = 50 or 100) and the dimensions of cellular structures (n = 4) were estimated using ImageJ software (National Institutes of Health, USA).

3-2. Observation of collagen staining

Collagen areas of CMA and CMA/MO bioink were observed with Sirius Red staining. Samples were washed with DPBS for 1 hour at 37°C in a CO ₂ environment and then incubated in Picro-Sirius Red (Sigma-Aldrich). Stained collagen was visualized using a Zeiss confocal microscope (LSM 700; Carl Zeiss, Germany) in maximum intensity projection (MIP) mode. The intensity of stained collagen was measured using ImageJ software.

3-3. Rheology

Frequency (temperature 25°C, strain 1%, and frequency range 0.1-10 Hz), stress (temperature 25°C, frequency 1 Hz, and stress range 0.1-200 Pa) sweep the conical plate. This was performed using a Bohlin Gemini HR Nano rotational rheometer (Malvern Instruments, UK) coupled to the structure (diameter 40 mm, cone angle 4°, gap 150 μm) to obtain different concentrations of CMA (1, 2, 3, 4, and 5 wt/ rheological properties (storage modulus, G', complex viscosity, η ^* , loss modulus, G'', and yield stress, τ _y ) were evaluated.

A strain of 1% was calculated using strain (temperature 25°C, frequency 1Hz, strain range 0.2-100%) and time sweep (temperature 25°C, frequency 1Hz, strain 1%) tests in the liner viscoelastic region. LVER) was selected by measuring.

The intersection of G' and G'' was evaluated to evaluate τ _y using the stress sweep results. To observe the photo-crosslinking rate, CMA (3 wt/v%)/MO (10 v/v%) bioink was incubated in an acrylic parallel-plate geometry (diameter, 40 mm) for 90 s. A time sweep (temperature 25°C, frequency 1Hz, strain 1%) was performed using an interval of 200 μm.

At 30 seconds, the bioink was exposed to UV light of various intensities (0, 75, 250, 400, 700 mW/cm ² ). All values are expressed as mean ± SD (n = 3).

The compression properties of the bioprinted constructs (6 × 6 × 4.5 mm ³ ) were evaluated using a SurTA tester (Chemilab, Korea) in the wet state in compression mode (compression speed of 0.1 mm/s). After drawing a stress-strain curve, the compressive moduli were estimated for the linear region of the curve. All values are expressed as mean ± SD (n = 4).

3-4. Wetability evaluation

FITC-dextran (4 kDa; 5 mg/mL in HBSS; Sigma-Aldrich, USA) solution was used to compare the wettability of CMA and CMA/MO structures. CMA and CMA/MO bioinks were fabricated using PDMS molds to obtain volumetric structures (5 × 10 × 1 mm ³ ) and crosslinked using UV light (400 mW/cm ² ) for 10 s. Then, the sample was added to the prepared FITC-dextran solution (20 μL) and incubated at 37°C in a CO ₂ environment. After 5, 60, and 120 min of incubation, the infiltration of the solution was visualized by fluorescence microscopy (CKX41; Olympus, Japan) (n = 4). Representative fluorescence intensities were plotted using ImageJ software.

3-5. protein absorption capacity

Protein uptake was analyzed using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo-Fisher Scientific, USA). The printed cell constructs (12 × 12 × 2.5 mm ³ ) were cultured in growth medium at 37°C in a CO ₂ environment. After incubation, the washed samples were treated with 0.1% Triton X-100. The lysate (25 μL) was then incubated with 200 μL of BCA working solution for 30 min at 37°C. A microplate reader (EpochTM; BioTek, South Korea) was used to measure the optical density (OD) of the treated specimen, and a known standard was measured at 562 nm. The amount of protein absorbed was determined using known standards. All values were expressed as mean ± SD (n = 4).

3-6. Release kinetics of bioactive substances

To evaluate the in vitro release kinetics of bioactive substances from the printed constructs, CMA-BM, CMA-BM/MO, CMA/MO-BM, KGN (28 μg/mL) and bovine serum albumin (10 μg/mL BSA ; Sigma-Aldrich, USA) was used. The printed constructs were then incubated in 1 mL DPBS. At specific incubation time points, the release profile of KGN was recorded by measuring the absorbance of the lysate at 278.4 nm using a microplate reader. The amount of KGN released was estimated using known standards. Pierce ^TM BCA Protein Assay Kit was used for BSA release kinetics. After incubation of the constructs, the lysate (25 mL) was treated with BCA working solution (200 mL) for 30 min at 37°C. The OD of released BSA was measured using a microplate reader at 562 nm. The amount of BSA released was estimated using known standards. Cumulative release profiles of KGN and BSA were plotted normalized to the initial content. All values were expressed as mean ± SD (n = 4).

4. In vitro cell response

4-1. Observation of cell viability

To observe live and dead cells, cells in the cell construct were stained with calcein AM (0.15mM; Invitrogen, USA) and ethidium homodimer-1 (2mM, Invitrogen, USA). Stained live (green) and dead (red) cells were visualized using a Zeiss confocal microscope in MIP mode. To estimate cell viability, live and dead cells were counted using ImageJ software. All values were expressed as mean ± SD (n = 4).

4-2. Observation of nuclear/F-actin formation

To observe nuclear/F-actin formation, cell constructs were incubated with diamondino-2-phenylinodole (DAPI; 1:100 in DPBS; Invitrogen, USA) and Alexa Fluor 488-conjugated phalloidin (1:100 in DPBS, Invitrogen, USA). It was dyed using . Briefly, samples were fixed with 10% NBF for 30 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min at 37°C. After incubating the specimens in DAPI/phalloidin solution for 1 hour at 37°C, cells were visualized by confocal microscopy in MIP mode. The area fraction of F-actin was measured using ImageJ software. All values are expressed as mean ± SD (n = 4).

4-3. Observation of cell proliferation rate

To estimate initial cell loss (day 1 of culture) and cell proliferation (day 7 of culture), MTT assay was performed using Cell Proliferation Kit I (Boehringer Mannheim, Germany). After washing three times with DPBS, the samples were incubated in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37°C in a CO ₂ environment. After 4 hours, purple formazan crystals were produced in metabolically active cells. The crystals were dissolved in a solubilization solution consisting of SDS. The OD of the colored solution was determined at 570 nm using a microplate reader. The metabolic cells present in the structure were determined using known cell numbers. All values were expressed as mean ± SD (n = 4).

4-4. Cartilage formation rate observation

To evaluate hASC chondrogenesis, quantification of glycosaminoglycan (GAG) content and Alcian blue staining were performed on cell constructs. The structure containing the cultured cells was chemically solubilized using a papain extraction solution, and the Blycan™ Sulfated Glycosaminoglycans assay kit (Biocolor Life Sciences Assays, UK) was used according to the manufacturer's protocol. Briefly, samples were incubated in papain extraction reagent (Sigma-Aldrich, USA), sodium acetate (8 mg/mL), EDTA disodium salt (0.4 mg/mL; Sigma -Aldrich, USA) and cysteine HCl (0.8 mg/mL). mL, Sigma-Aldrich, USA) was dissolved in 0.2 M sodium phosphate buffer containing 100% at 65°C for 3 hours. After decanting the supernatant, the GAGs content was stained for 30 minutes using 1,9-dimethyl-methylene blue dye reagent. The stained sample was dissolved using a dissociation reagent and the OD was measured at 656 nm using a microplate reader. GAG content was determined using known standards. All values were expressed as mean ± SD (n = 4).

To visualize GAGs formed in hASCs after chondrogenic differentiation, cell constructs were fixed with 10% NBF for 30 min and incubated in sodium acetate buffer (50mM pH 5.8, Sigma-Aldrich, USA) containing MgCl ₂ (50mM, Sigma-Aldrich, USA). USA) was incubated for 3 hours at room temperature with Alcian Blue 8GX dissolved in water. Stained samples were washed three times with DW and then visualized by light microscopy (n = 4).

To evaluate the osteogenic differentiation of hASCs in the 3D construct, calcium content was stained using Alizarin red S (ARS). After fixing the structure with 70% ethanol (Sigma-Aldrich, USA) for 1 hour at 4°C, the specimen was incubated in ARS solution (40mM, pH 4.2; Sigma-Aldrich, USA) for 1 hour at room temperature. The stained samples were washed three times with DW and then visualized under a light microscope. The constructs were then treated with 10% cetylpyridinium chloride (Sigma-Aldrich, USA) dissolved in sodium phosphate buffer (10mM, pH 7.0) for 30 min at room temperature to estimate calcium deposition. The absorbance of treated samples and known standards was measured using a microplate reader at 562 nm. Total protein content estimated using the Pierce™ BCA protein assay kit was used to normalize measured calcium deposition levels. All values were expressed as mean ± SD (n = 4).

5. Immunofluorescence

To assess the expression of FAK and differentiation markers, stem cells embedded in cell constructs were incubated with 10% NBF (fixed for 1 h), 2% BSA (blocked for 2 h), and 2% Triton X-100 (permeabilized for 2 h). . The treatment was carried out at 37°C. The processed specimens were incubated with anti-rabbit focal adhesion kinase (FAK) primary antibody (5 μg/mL; Sigma-Aldrich, USA) and anti-mouse osteopontin (OPN) primary antibody (5 μg). /mL, Invitrogen, USA) and anti-rabbit aggrecan (ACAN) primary antibodies (5 μg/mL; Abcam, USA) were treated overnight at 4°C. Samples were then stained with Alexa Fluor 594-conjugated anti-mouse secondary antibody (1:50 in DPBS; Invitrogen, USA) or Alexa Fluor 594-conjugated anti-rabbit secondary antibody (1:50 in DPBS, Invitrogen, USA). Staining was performed at 37°C for 1 hour and counterstaining with DAPI (5 μM in DPBS). Stained cells were visualized using confocal microscopy in MIP mode. Quantification of FAK+ and OPN+ areas was performed using ImageJ software. All values were expressed as mean ± SD (n = 4).

6. qRT-PCR

To estimate gene expression in cultured hASCs, qRT-PCR was performed on cell constructs using the 2 ^-ΔΔCT method. Before analysis, samples containing cells were treated with TRIzol reagent (Sigma-Aldrich) to isolate total RNA. The purity (acceptable purity range: 1.8 < OD ₂₆₀ /OD ₂₈₀ < 2.0) and concentration of the isolated RNA was evaluated using a spectrophotometer (FLX800T; Biotek, USA). After measurement, cDNA was synthesized from RNase-free DNase-treated total RNA by reverse transcription using ReverTra Ace™ qPCR RT Master Mix (Toyobo Co., Ltd., Japan). For qRT-PCR analysis using synthesized cDNA, the threshold cycle (CT) value was calculated using the StepOnePlus real-time PCR system (Applied Biosystems, USA) and Thunderbird® SYBER® qPCR mix (Toyobo Co., Ltd., Japan). was measured. Expressed gene levels were normalized to housekeeping genes using the measured CT value of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. All values were reported as mean ± SD (n = 4). Primers used are listed in Table 1 below.

Gene Source Primer sequence Left (5' - 3') Right (5' - 3') Gapdh Homo sapiens CCATGGGGAAGGTGAAGGTC AGTGATGGCATGGACTGT Itgb1 Homo sapiens CGCCCGCGGAAAAGATG AAACACCAGCAGCCGTGTAA Tln Homo sapiens CGTGCAAACCAGGCAATTCA CTCTTGGACTGCAGTGAGCA Pxn Homo sapiens TCGCTGTCGGATTTCAAGAT ATGAACCCTCCCTCGTCCT Fak Homo sapiens GCTCCCTTGCATCTTCCAGT ATTGCAGCCCTTGTCCGTTA Rhoa Homo sapiens GTCCACGGTCTGGTCTTCAG CAGCCATTGCTCAGGCAAC Rock Homo sapiens TGAAAGCCGCACTGATGGAT AAGCAGCTCTCCTGGTTGAC Yap Homo sapiens CCCTCGTTTTGCCATGAACC GTTGCTGCTGGTTGGAGTTG Taz Homo sapiens GAGAGCCGAAGCCCTTAGA CGGATTCATCTTCTGGGGCGG Runx1 Homo sapiens AACCCAGCATAGTGGTCAGC CATGGCTGCGGTAGCATTTC Acan Homo sapiens AAGCACTGGAGTTCTGTGAATCT CGGCATAGCACTTGTCCAG Col2a1 Homo sapiens GTAGAGACCCGGACCCGC ACTCTCCGAAGGGGATCTCA Col10a1 Homo sapiens CTGGGCAGAGGAAGCTTCAGA AGATGGATTCTGCGTGCTGG Sox9 Homo sapiens GTACCCGCACTTGCACAAC TCTCGCTCCTCGTTCAGAAGTC Sox5 Homo sapiens GGACTCCCACTTCTCAGCAC ATAGCTGAAGCCTGGAGGGA Sox6 Homo sapiens CCACAAAGCTCCACTGTTGC TCAGGTGTCCACCACATCG Cbfb Homo sapiens CCCGACCAGAGAAGCAAGTT GCCACAAAAGCGATTTCCGA Alp Homo sapiens GGCACCTGCCTTACTAACTCC CTTGCCACGTTGGTGTTGA Runx2 Homo sapiens CAGTGACACCATGTCAGCAA GCTCACGTCGCTCATTTTG Opn Homo sapiens AAGTTTCGCAGACCTGACATC GGGCTGTCCAATCAGAAGG Ocn Homo sapiens TGAGAGCCCTCACACTCCTC ACCTTTGCTGGACTCTGCAC

7. Statistical analysis

To evaluate the statistical analysis, Student's t-test (two groups) and ANOVA analysis along with Tukey's HSD post-hoc test (three or more groups) were performed using SPSS software (SPSS, Inc., USA). . Values of *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.

Experiment result

Example 1: Production of emulsion bio-ink ( Fabrication of an emulsion bioink )

In general, oil-water emulsions can be formed by dispersing a relatively small amount of oil in a continuous water phase using an emulsifier. We obtained cell-laden CMA/MO bioink using an oil-water mixing process to fabricate cell-laden porous structures with improved cell-cell interactions. A schematic diagram of the manufacturing process is shown in Figure 1.

In the present invention, CMA was used because it can provide an excellent bioactive environment and a GFOGER sequence that supports cell activity, and because the photocrosslinking properties of CMA can be efficient in fabricating 3D structures. The protein molecules in CMA acted as an emulsifier and the collagen was distributed more intensively around the interfacial boundaries of the emulsion spheres. The produced emulsion bioink was subjected to a 3D printing process supplemented with a UV cross-linking system to fabricate a cell-loaded mesh structure with macro and micro pores.

Figure 2 shows optical images before and after oil-CMA mixing. As shown in Figure 2, as the mixing time increased, oil droplets were dispersed uniformly in the cell-laden CMA bioink, but when mixed more than 20 times, the oil droplet size in the CMA solution was almost uniformly saturated (19.7 ± 6.5 μm). . As a result, hydrocolloid bioink could be easily obtained from a mixture of CMA and MO without additional emulsifiers.

The CMA/MO structure containing hASCs fabricated using emulsion bioink was shown in the optical and SEM images in Figure 3. As can be seen in the SEM image, it was confirmed that the macropores of the mesh structure and the micropores of the printed struts were well obtained.

To observe the cellular response to the construct, fluorescent staining pictures of cells after 3 days of culture are shown in Figure 4 [alive (green)/dead (red) and DAPI (blue)/phalloidin (green)]. The CMA scaffold is a mesh structure containing hASCs produced without using MO. As can be seen in the live-dead image of FIG. 4, cells survived well in both structures, but as shown in FIG. 5, more developed actin filaments were observed in the CMA/MO structure than in the CMA structure.

Additionally, 3D images (surface and cross-sectional views) of the CMA/MO scaffold are shown in Figure 6.

These results indicated that the cell-containing constructs fabricated using emulsion bioink were a much more suitable microenvironment for the activities of the cells contained in the constructs.

Example 2: Selection of mixing ratio of CMA and MO

2-1. Change in size of oil droplets according to mixing ratio of CMA and MO

In general, the stable formation of an emulsion is related to the mixing ratio of the oil phase and the water phase.

Figure 7 shows optical images of CMA (3 wt/v%)/MO bioink containing various concentrations of oil (5, 10, 20, 30, and 40 v/v%). As can be seen from the optical images in Figure 7, phase-separated emulsions were observed in bioinks containing 30 and 40 v/v% oil, whereas oil droplets were uniformly distributed in emulsions containing 5-20 v/v% oil. It was distributed. In addition, as a result of measuring the average diameter of the oil droplets, as shown in Figure 8, the diameter of the oil droplet decreased below 20 v/v%, but the diameter of the oil droplet decreased due to excessive oil above 30 v/v%. It increased rapidly.

2-2. Rheological properties of emulsion bioink according to mixing ratio of CMA and MO

To evaluate the rheological properties of emulsion bioink (cells: 2 × 10 ⁷ cells/mL, CMA: 3 wt/v%, MO: 0, 5, 10, 20, 30, 40 v/v%), storage Storage (G'), loss modulus (G'') and complex viscosity (η*) were analyzed using frequency and shear stress sweeps as shown in Figures 9 and 10. .

Most emulsion bioinks showed complete shear thinning behavior without depending on the volume fraction of oil, demonstrating that emulsion bioinks can be easily printed through microscale printing nozzles. Additionally, as the oil volume fraction of the emulsion bioink increased to 20 v/v%, significantly superior rheological properties, including yield stress ( _ty ), were observed, while the rheological properties were significantly lower than those of the oil in the emulsion bioink. It decreased rapidly when the volume fraction was above 30 v/v%. This phenomenon occurs because the rheological properties of the emulsion are closely related to the droplet size of the dispersed oil. According to Guerrero et al., the rheological properties of oil-in-water emulsions increased with decreasing diameter of the dispersed oil droplets due to an increase in the elastic network of protein molecules surrounding the oil droplets.

Similarly, as our results also show in Figure 11, the addition of mineral oil up to 20 v/v% improved the rheological properties, but the addition of mineral oil did not affect cell viability.

Additionally, to show the influence of time and temperature dependence on the phase separation of CMA/MO bioink, we qualitatively observed dispersed oil droplets of bioink in emulsions containing 5–20 v/v% of mineral oil. Evenly distributed oil droplets were maintained stably without phase separation even after incubation at 10, 25, and 37°C for 2 hours.

Additionally, a time sweep test (frequency of 1 Hz and strain of 1%) was performed to observe the long-term stability of the bioink. As a result, the rheological properties [CMA(3wt/v%), CMA(3wt/v%)/MO(10v/v%)] of CMA with and without MO bioink were stable up to 40 minutes.

2-3. Rheological properties of emulsion bioink according to mixing ratio of CMA and MO

To evaluate the printability and cellular activity of CMA/MO bioink containing hASCs, bioinks containing 5, 10, and 20 v/v% of oil were printed using 3D bioprinting under the following printing conditions:

Processing temperature 25℃, moving speed 10mm/s, pneumatic pressure 120 kPa, UV output 400mW/cm ²

Figure 12 shows optical and live/dead images of the mesh structure. Cells supported in the construct were alive after 3 days of culture. The printability of the bioink was calculated using optical images based on a previously reported method (Pr = L2/16A, where L is the perimeter and A is the area of the pore shape). Analysis shows that the Pr of bioinks containing 10 and 20 v/v% of MO is within the acceptable range (0.9 < Pr < 1.1), while for the oil of 5 v/v%, Pr is 0.9, as shown in Figure 13. It was less than

After fabricating the CMA/MO construct, the contained oil can be released into the culture medium and at the same time the cells contained in the construct can be separated. Figure 14 shows the results of measuring initial cell loss for 1 day using the MTT assay. As shown in the results, cell loss gradually increased as the volume fraction of oil increased. However, below 10 v/v% oil content, cell loss was statistically negligible. Based on these results, CMA/MO bioink containing 10% v/v of oil was selected to prepare cell-containing porous structures.

To observe the effect of CMA concentration on oil droplet size, printability, initial cell loss, and cell proliferation, 10 v/v% of oil was used, as shown in Figure 15. The CMA concentration (1-5 wt/v%) was changed in the bioink containing.

As can be seen from the optical images, the emulsion was obtained uniformly in the bioink containing 3-5 wt/v% CMA, but as shown in Figure 16, the bioink mixed with 1 and 2 wt/v% CMA showed a slight change in the oil droplets. It showed a wide size distribution.

The present inventors also printed mesh structures using various concentrations of CMA in bio-ink. As can be seen from the optical images, the print capability was significantly lower for 1 and 2 wt/v% CMAs: 'Pr' = 0.9 to 1.1 for CMAs 3 to 5 wt/v%, and for CMAs 1 and 2 wt/v%. For 'Pr' << 0.9 (Figure 17). However, cell survival for all printed structures was high (>90%) (Figure 18).

As shown in Figure 19, in the case of initial cell loss, higher concentrations of CMA in the cell construct induced much lower cell loss due to the relatively high modulus. However, cell loss was not statistically significant above 3wt/v% of CMA. Therefore, based on the above-mentioned printability and initial cell loss, the CMA concentration of the bio-ink was set to 3 wt/v%.

To establish UV exposure conditions for in situ cross-linking, various UV light intensities (0, 75, 250, 400, and 700 mW/cm ² ) were applied to evaluate rheological properties, printability, and cell viability.

As shown in Figure 20, as expected, higher photocrosslinking ability was observed with increasing UV intensity, showing that the storage modulus (G') of the bioink increased. At UV intensities of 400 and 700 mW/cm ² , printability was in the acceptable range, but at an intensity of 700 mW/cm ² a significant number of dead cells were observed due to the high UV output. Therefore, based on the above-mentioned printability and cell viability, UV exposure conditions (400mW/cm ² ) for the next bioprinting process were set. Additionally, the feasibility of UV conditions (400 mW/cm ² for 10 seconds) was demonstrated, and the resulting cross-linking depth of the bioink was approximately 3.8 ± 0.2 mm.

Example 3: Characterization and in vitro cell response of CMA/MO scaffolds containing cells

3-1. Characterization of CMA/MO scaffolds containing cells

Constructs containing hASC were obtained using the CMA and MO weight fractions and printing conditions set in Example 2:

(1) CMA: CMA (3 wt/v%) scaffold containing hASC and

(2) CMA/MO: CMA (3 wt/v%)/MO (10 v/v%) scaffold containing hASCs.

Figure 21 shows optical and SEM images of the printed cell loading configuration. As can be seen in the images, both mesh structures (15 × 15 × 4.5 mm ³ ) exhibit macroscale pores (average size = 454.2 ± 67.9 μm) and nanoscale collagen fibers. However, the micropores of the struts (average size = 18.3 ± 16.3 μm) were only observed in the CMA/MO structure due to the homogeneously distributed MO phase. As expected, the porosity of CMA/MO (97.9 ± 0.3%) was higher than that of the CMA structure (96.4 ± 0.3%).

Figure 23 is shown for an in situ printed structure (6 × 6 × 4.5 mm ³ ) in the wet state. Interestingly, the CMA/MO structure showed a higher compressive modulus (162.3 ± 9.5 kPa) compared to the CMA structure (117.6 ± 24.7 kPa), similar to the rheological test, and this phenomenon may be due to the oil emulsion phase. . It efficiently supported the elastic network of collagen molecules surrounding the oil droplet.

The highly porous structure is known to clearly affect the exchange of water, nutrients and digestive substances, efficiently inducing various metabolic activities. The diffusion capacity of the porous structures was quantitatively evaluated using wettability and protein absorption tests.

The wettability of the structure was measured by observing the diffusion of the FITC-conjugated dextran solution at 5, 60, and 120 minutes, as shown in Figure 23. As a result, dextran penetrated more quickly into the porous CMA/MO structure compared to the CMA scaffold, as can be seen from the quantitative intensity of dextran in Figure 24.

Additionally, as a result of measuring the protein absorption capacity, the CMA/MO scaffold showed a relatively higher protein absorption rate than the CMA scaffold, as shown in Figure 25. Based on these results, we could cautiously assume that the CMA/MO scaffold would induce efficient transport of nutrients and metabolic waste products, resulting in efficient cellular activity through the porous structure.

Next, to evaluate metabolic activity affected by the transport capacity of nutrients and metabolic waste, hASC-loaded CMA and CMA/MO bioinks were poured into a cylindrical structure (height: 10 mm, diameter: 3 mm) and the bioink was Cultured in growth medium. (GM) alone as shown in Figure 26 to observe cell viability. As a result, in CMA/MO, high cell viability (~90%) was observed at a point where the structure height, which is the distance from the structure surface, is about 2.0 ± 0.06 mm, while the distance with cell viability (~90%) was observed in the CMA structure. It was about 480.1 ± 50.0 μm. In a simple analysis, we were able to confirm that efficient metabolic exchange can occur in the CMA/MO construct compared to the CMA construct.

3-2. in vitro cell response

To observe cell activity in vitro, CMA and CMA/MO constructs containing printed cells were cultured for various periods of time.

After 3 and 7 days of cell culture, live/dead staining was performed on the bioprinted structure ((a) of Figure 27). The cells survived well in the structure, with a cell survival rate of over 92%, as shown in (b) of Figure 27. However, cell proliferation estimated using MTT analysis was much greater in the CMA/MO structure than in the CMA structure, as can be seen in (c) of Figure 27. In addition, as shown in (d) of Figure 27, F-actin and focal adhesion kinase (FAK) of hASC were observed in the printed construct by staining with DAPI/phalloidin/FAK (red). Similar to the results for cell proliferation, much more developed actin filaments (1.3-fold) and FAK (2.7-fold) were observed in the CMA/MO structure, as shown in (e) and (f) of Figure 27.

In addition, to observe cytoskeletal organization and cell proliferation in more detail, FAK/RhoA-related genes, including integrin beta 1 (Intb1), talin 1 (Tln1), paxillin (Pxn), focal adhesive kinase (Fak), and ras homolog family member, were analyzed. The expression levels of A (Rhoa), rho-associated protein kinase (Rock), yes1-related transcriptional regulator (Yap), and WW-domain-containing transcriptional regulator 1 (Taz) were measured in hASCs cultured in cell constructs (Figure 27 (g)). Based on these results, we show that the highly porous CMA/MO scaffold effectively activates the early FAK/RhoA signaling pathway due to efficient cell-to-matrix interaction, resulting in acceleration of cell growth and cytoskeletal reorganization. was confirmed.

Example 4: Application of CMA/MO scaffolds conjugated with bioactive molecules

To expand the application of CMA/MO bioinks in the field of tissue engineering, bioactive molecules in emulsion bioinks have been considered to help differentiate hASCs into cell constructs. The present inventors used two typical bioactive substances, namely, kartogenin (KGN) and bone morphogenic protein-2 (BMP-2), to induce chondrogenesis and osteogenesis in hASC. KGN is known to be able to induce chondrogenesis in mesenchymal stem cells by binding to filamin A and regulating the core-binding factor beta/RUNX1 complex. To control the release behavior, two formulation methods were chosen for the bioink containing BM, as shown in Figure 28: (1) mixing cell-laden CMA with a mixture of MO and BM (CMA/MO-BM); , (2) A mixture of cell-containing CMA and BM was mixed with MO (CMA-BM/MO).

To evaluate the release kinetics of bioactive molecules from the printed structures, we incubated the structures (12 × 12 × 2.5 mm ³ ) in DPBS at 37°C to evaluate the cumulative release of KGN and bovine serum albumin (BSA). The results are shown in Figures 29 and 30.

As shown in Figure 29, the initial burst release of KGN was observed in the CMA-KGN construct (54.0 ± 4.4%) and the CMA-KGN/MO construct (66.3 ± 3.6%), with most being released after 14 days. This has been completed. However, a relatively low initial burst release was observed in the CMA/MO-KGN structure (29.0 ± 3.8%), and the release lasted up to 42 days of culture, showing time-dependent release kinetics. Because the ionic interaction between the carboxyl acid group of KGN and the remaining amine group of CMA is relatively weak, KGN loaded on CMA may be released earlier. However, in the case of the CMA/MO-KGN structure, KGN physically encapsulated in MO droplets was released at a relatively slow rate.

Similar results were observed for the release kinetics of BSA, as shown in Figure 30. A relatively larger initial burst release and faster release of BSA were observed in CMA-BSA and CMA-BSA/MO, whereas in CMA/MO-BSA the release of BSA lasted up to 28 days.

From the above results, we found that KGN and BSA were released in CMA-KGN/MO and CMA-BSA/MO structures with well-delayed release rates and durations compared to CMA/MO-KGN and CMA/MO-BSA scaffolds. This indicates that the bioactive molecule can act much longer while maintaining bioactivity throughout the scaffold.

To observe the biological activity of KGN contained in the constructs, CMA-KGN, CMA-KGN/MO, and CMA/MO-KGN constructs were cultured in GM to evaluate chondrogenic differentiation.

Figure 31 shows DAPI/aggrecan (ACAN, red) (7 and 14 days of cell culture) and Alcian blue (14 days of culture) staining images of the cultured construct. As shown in Figure 31, ACAN and Alcian blue staining were expressed much more in the CMA/MO-KGN scaffold than in other scaffolds.

Additionally, as shown in Figure 32, the development of glycosaminoglycan (GAG) was also detected in the CMA/MO-KGN scaffold.

Figure 33 shows more detailed gene expression observed in the scaffold, and KGN consistently released from the CMA/MO-KGN scaffold includes Runx1, aggrecan (Acan), collagen type 2 alpha-1 chain (Col2a1), and collagen type 10 alpha. The expression levels of -1 chain (Col10a1) and SRY-box transcription factors 9, 5, and 6 (Sox9, Sox5, Sox6) were significantly improved.

Additionally, to accelerate the osteogenic differentiation of hASCs in CMA-based scaffolds, BMP-2 was used as a bioactive material and three CMA-based scaffolds were fabricated.

CMA-BMP, CMA-BMP/MO and CMA/MO-BMP. Scaffolds were cultured in GM for 3 days and then cultured in osteogenic medium (OM) to support osteogenesis of hASCs, as shown in Figure S9.

Figure 34 shows DAPI/osteopontin (OPN; red) (after 14 days of culture) and Alizarin red S (ARS) (after 14 and 21 days of culture) staining.

In Figures 35 and 36, hASCs on CMA/MO-BMP scaffolds showed significantly improved OPN-positive areas, intensive ARS staining, and calcium deposition compared to other scaffolds.

Additionally, as shown in Figure 37, the expression of osteogenesis-related genes including alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (Ocn) in the CMA/MO-BMP group. The level has been adjusted upward.

Collectively, these results suggest that the initially upregulated cellular responses and continuously released bioactive molecules in the CMA/MO-based cell constructs enhanced chondrogenesis and osteogenesis of hASCs in the constructs. Therefore, the proposed cell construct could be applied to induce preferred cellular responses in various tissue types.

We proposed a new cell-laden emulsion bioink composed of methacrylated collagen and mineral oil for the fabrication of 3D cell constructs containing hASCs. The weight fractions and processing parameters of each component were carefully selected to ensure uniform distribution of oil droplets in the emulsion bioink.

Emulsion bioinks can provide an appropriate yield stress, allowing mechanically stable printed structures with hierarchical pore geometries. In vitro cell activity demonstrated that cell proliferation and cytoskeletal reorganization were significantly accelerated in cell constructs fabricated using emulsion bioink compared to normal cell-printed constructs. Additionally, to expand the potential of emulsion bioinks, two bioactive components (kartogenin and BMP-2) were physically encapsulated in oil droplets of the bioink to induce efficient chondrogenic and osteogenic differentiation of loaded hASCs. Depending on the expression level of various chondrogenic or osteogenic genes, the controlled release of bioactive molecules from the cellular constructs significantly enhanced the chondrogenic or osteogenic differentiation of hASCs in the printed structures. Based on these results, the present inventors believe that cell-printed structures using emulsion bioink can provide a biologically efficient microenvironment in which various cellular activities can be highly enhanced.

Claims (19)

A composition for bioink comprising a photocurable biocompatible polymer, stem cells, and oil.
The composition for bioink according to claim 1, wherein the photocurable biocompatible polymer is a photocurable polymer conjugated to a biocompatible polymer.
The method of claim 2, wherein the photocurable polymer is methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, hexanediol diacrylate, and hexanediol dimethacrylate. , a group consisting of polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, dipropylene glycol diacrylate, dipropylene glycol dimethacrylate, and urethane acrylate. A composition for bioink consisting of one or two or more monomers and oligomers selected from.
The method of claim 2, wherein the biocompatible polymer is hyaluronic acid, collagen, polyethylene glycol, alginate, starch, chitosan, gelatin, and dextran. ), a composition for bio-ink selected from the group consisting of cellulose, alginic acid, chondroitin sulfate, and heparin.
The method of claim 1, wherein the photocurable biocompatible polymer is gelatin methacryloyl (GelMA), collagen methacrylate (ColMA), hyaluronic acid methacrylate (HAMA), and A composition for bio-ink selected from the group consisting of polyethylene glycol diacrylate (PEGDA).
The composition for bioink according to claim 1, wherein the stem cells are pluripotent stem cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic germ stem cells, and germ stem cells.
The method of claim 1, wherein the stem cells are mesenchymal stem cells derived from one or more types of tissue selected from the group consisting of umbilical cord, cord blood, blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. cell), a composition for bioink.
The composition for bio-ink according to claim 1, wherein the stem cells are adipose-derived mesenchymal stem cells.
The composition for bio-ink according to claim 1, wherein the oil is mineral oil.
The composition for bioink according to claim 1, further comprising bioactive molecules.
The composition for bioink according to claim 1, further comprising a photoinitiator.
Method for producing bioink comprising the following steps:
(a) preparing a hydrogel containing a photocurable biocompatible polymer; and
(b) preparing an emulsion by mixing stem cells, photoinitiator, and oil with the hydrogel.
The method of claim 12, wherein the hydrogel is manufactured by mixing a photocurable biocompatible polymer and an aqueous solvent.
A printing method of bioink comprising the following steps:
(a) Printing a structure of a desired shape using the bioink of any one of claims 1 to 11 using a printer device including a nozzle equipped with a UV exposure device.
The method of printing bioink according to claim 14, wherein the UV exposure device has a UV output of 200 to 800 mW/cm ² .
The method of claim 14, wherein the nozzle has an inner diameter of 50-500 μm.
The method of printing bioink according to claim 14, wherein the printer device prints at a pneumatic pressure of 60 to 250 kPa.
The method of claim 14, wherein the printer device has a moving speed of 3 to 30 mm/s.
The method of printing bioink according to claim 14, wherein the printer device prints in a temperature range of 15 to 30°C.