Hydrogel scaffold and manufacturing method therefor

Abstract

A poly(HEMA-co-MMA) hydrogel scaffold according to one aspect of the present invention comprises a copolymer of a 2-hydroxyethyl methacrylate (HEMA) monomer and a methyl methacrylate (MMA) monomer, wherein the molar ratio of the copolymerized 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 83:17 to 97:3.

Description

Hydrogel scaffold and method for manufacturing the same

[0001] The present invention relates to a hydrogel scaffold and a method for manufacturing the same, and more specifically, to a hydrogel scaffold capable of controlling shape and mechanical properties through self-assembly and a method for manufacturing the same.

[0002]

[0003] Cell proliferation and viability have been shown to be influenced by scaffold roughness. Therefore, recent studies have utilized various techniques, such as 3D printing, nano-micropatterning, the use of nanoparticles, and plasma treatment, to enhance cell-substrate interactions by controlling scaffold roughness. However, according to Tsang & Bhatia et al., methods such as 3D printing and nano-micropatterning use materials with unconfirmed biocompatibility and have disadvantages such as the generation of toxic byproducts, difficulties in cell integration, and long fabrication times due to non-auto-assembly. Additionally, since high-stiffness (>1000 Pa) materials such as silicon, polystyrene, and poly ε-caprolactone were used to fabricate scaffold roughness, they may not be suitable for soft tissue applications with soft stiffness (100 - 1000 Pa).

[0004] Meanwhile, fabrication methods using only poly(ethylene glycol) diacrylate (PEGDA), poly(methyl methacrylate) (PMMA), or poly(2-hydroxyethyl methacrylate) (PHEMA) have limitations in producing scaffolds with roughness, pore size, and mechanical properties suitable for soft tissue cell growth because the mechanical properties of the scaffold deteriorate rapidly due to high brittleness upon deformation.

[0005]

[0006] Depending on the purpose and method, synthetic hydrogel scaffolds may require various properties such as surface roughness, pore size, mechanical modulus, and wettability, and in order to implant them into body tissues, it is important to properly control the properties of hydrophilic and hydrophobic scaffold materials to provide an environment similar to that of the body.

[0007] One objective of the present invention is to provide a hydrogel scaffold with controlled roughness, pore size, mechanical modulus, and wettability for successful in vivo cell culture for soft tissue application by selecting and copolymerizing two types of monomers, adjusting the molar ratio of the two selected monomers, and controlling the polymerization method such as reactants, solvent, concentration, temperature, and time during polymerization.

[0008]

[0009] A poly(HEMA-co-MMA) hydrogel scaffold according to one embodiment of the present invention may comprise a copolymer of 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer.

[0010] In a more preferred embodiment, the molar ratio of the copolymerized 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 83:17 to 97:3.

[0011] In a more preferred embodiment, the scaffold may comprise a copolymer comprising a unit represented by the following chemical formula 1 and a unit represented by the following chemical formula 2.

[0012] [Chemical Formula 1]

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016] In the above chemical formula 1, x and y are each independently natural numbers greater than or equal to 1.

[0017] In a more preferred embodiment, the maximum tensile stress of the scaffold may be 5.00 kPa to 40.0 kPa.

[0018] In a more preferred embodiment, the contact angle of the scaffold surface may be 30.0° to 75.0°.

[0019] In a more preferred embodiment, the compressive modulus of the scaffold may be 15.00 kPa to 45.00 kPa.

[0020] In a more preferred embodiment, the tensile modulus of the scaffold may be 0.5 kPa to 2.5 kPa.

[0021] In a more preferred embodiment, the compression stress of the scaffold may be 250.0 kPa to 1,000 kPa.

[0022] In a more preferred embodiment, the average pore size measured for pores observed in a cross-sectional scanning electron microscope (SEM) image of one surface of the scaffold may be 1 μm to 80 μm.

[0023] A method for manufacturing a poly(HEMA-co-MMA) hydrogel scaffold according to one embodiment of the present invention may include the step of mixing a 2-hydroxyethyl methacrylate (HEMA) monomer, a methyl methacrylate (MMA) monomer, a solvent, a crosslinking agent, and an initiator to produce a copolymer of the 2-hydroxyethyl methacrylate (HEMA) monomer and the methyl methacrylate (MMA) monomer.

[0024] In a more preferred embodiment, the molar ratio of the mixed 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 83:17 to 97:3.

[0025] In a more preferred embodiment, the total concentration (wt%) of the mixed 2-hydroxyethyl methacrylate monomer and methyl methacrylate monomer may be 20 wt% to 40 wt% based on the mixture.

[0026] In a more preferred embodiment, the solvent is a mixture of water and dimethylformamide (DMF), and the mixing ratio of water to dimethylformamide (DMF) may be 75:25 to 85:15 (w / w).

[0027]

[0028] One effect of the present invention is to provide a hydrogel scaffold with controlled roughness, pore size, mechanical modulus, and wettability for successful in vivo cell culture for soft tissue application.

[0029]

[0030] Figure 1 is the FT-IR analysis result of a hydrogel scaffold according to an embodiment of the present invention.

[0031] Figure 2 is a surface morphology image of a hydrogel scaffold according to an embodiment of the present invention.

[0032] Figure 3 is a graph showing the static water contact angle of a hydrogel scaffold according to an embodiment of the present invention.

[0033] Figure 4 is a graph showing the compressive stress-strain curve of a hydrogel scaffold according to an embodiment of the present invention.

[0034] Figure 5 is a graph showing the compression modulus of a hydrogel scaffold according to an embodiment of the present invention.

[0035] Figure 6 is a graph showing the tensile stress-strain curve of a hydrogel scaffold according to an embodiment of the present invention.

[0036] Figure 7 is a graph showing the tensile modulus of a hydrogel scaffold according to an embodiment of the present invention.

[0037] Figure 8 is a confocal image of cell attachment of a hydrogel scaffold according to an embodiment of the present invention.

[0038] Figure 9 shows the results of cell proliferation analysis of a hydrogel scaffold according to an embodiment of the present invention.

[0039] Figure 10 is an image showing the form in which human skin fibroblasts are attached to a hydrogel scaffold according to an embodiment of the present invention.

[0040]

[0041] Expressions such as "comprising" as used in this specification should be understood as open-ended terms implying the possibility of including other configurations.

[0042] As used herein, "preferably" and "preferably" refer to embodiments of the invention that can provide certain advantages under certain conditions. However, it is not intended to exclude other embodiments from the scope of the invention.

[0043] The numerical ranges used in this specification include lower and upper limits and all values ​​within the range, increments logically derived from the form and width of the defined range, all of which are limited values, and all possible combinations of upper and lower limits of numerical ranges limited in different forms.

[0044] Unless otherwise specifically defined in this specification, values ​​outside the numerical range that may occur due to experimental error or rounding are also included in the defined numerical range.

[0045] The meaning of 'greater than' or 'less than' as described in this specification may be replaced with the meaning of 'greater than' or 'less than'.

[0046] Meanwhile, the technical features described below relate to one embodiment that achieves the intended effect of the present invention described above.

[0047]

[0048] First, the scaffold of the present invention will be described.

[0049] The present invention relates to a hydrogel scaffold for tissue engineering, wherein the hydrogel scaffold of the present invention can control its shape and mechanical properties through self-assembly.

[0050] More preferably, the present invention relates to a scaffold for soft tissue, and more specifically, to a scaffold having mechanical properties suitable for soft tissue and an in vitro cellular response.

[0051] A hydrogel scaffold according to one embodiment of the present invention may comprise a copolymer of 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer.

[0052] The meaning of the copolymer of the above 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer is a copolymer polymerized from the 2-hydroxyethyl methacrylate (HEMA) monomer and the methyl methacrylate (MMA) monomer.

[0053] In the present invention, the copolymer thereof is named poly(HEMA-co-MMA) hydrogel scaffold.

[0054] Through research, the inventors confirmed that poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel has limitations as a scaffold because surface wettability and mechanical properties are inversely proportional to the polymer concentration.

[0055] Accordingly, the inventors were able to manufacture a scaffold in which wettability and mechanical properties were simultaneously improved and controlled by mixing hydrophobic methyl methacrylate (MMA) to overcome the limitations of such poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels.

[0056] In a more preferred embodiment, the molar ratio of the copolymerized 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 83:17 or higher, 85:15 or higher, 87:13 or higher, or 97:3 or lower, 95:5 or lower, or 92:8 or lower.

[0057] The inventors investigated the properties according to various concentration ratios of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) in order to prepare a poly(HEMA-co-MMA) hydrogel scaffold having mechanical properties and in vitro cellular response suitable for soft tissues.

[0058] As a result, it was possible to manufacture a poly(HEMA-co-MMA) hydrogel scaffold in which wettability and mechanical properties were simultaneously improved and controlled at the above molar concentration ratio, and it was confirmed that the mechanical properties and morphology at the above molar concentration ratio were superior to those of the PHEMA hydrogel scaffold.

[0059] In addition, it was confirmed that the wettability characteristics of the poly(HEMA-co-MMA) hydrogel scaffold were superior to those of the hydrogel scaffold composed only of 2-hydroxyethyl methacrylate (HEMA), even though hydrophobic methyl methacrylate (MMA) was added at the above molar concentration ratio.

[0060] In addition, it was possible to manufacture an optimal scaffold that facilitates cell attachment and proliferation at the above molar concentration ratio.

[0061] In one embodiment, the copolymer may include a unit represented by the following chemical formula 1.

[0062] [Chemical Formula 1]

[0063]

[0064] In the above chemical formula 1, x and y are each independently natural numbers greater than or equal to 1, or less than or equal to 1,000,000,000.

[0065] In one embodiment, the ratio of x and y may be 83:17 or higher, 85:15 or higher, 87:13 or higher, or 97:3 or lower, 95:5 or lower, or 92:8 or lower.

[0066] In one embodiment, the copolymer may include a unit represented by the following chemical formula 2.

[0067] [Chemical Formula 2]

[0068]

[0069] The present invention may include a unit represented by Chemical Formula 2 by using pentaerythritol tetracrylate (PETA) as a crosslinking agent when polymerizing the 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer.

[0070] In a more preferred embodiment, the maximum tensile stress of the scaffold may be 5.00 kPa or more, 10.00 kPa or more, 15.00 kPa or more, 20.00 kPa or more, 25.00 kPa or more, 30.00 kPa or more, 40.00 kPa or less, 35.00 kPa or less, or 30.00 kPa or less. The present invention can adjust the maximum tensile stress to the above values ​​to enable appropriate cell attachment and proliferation.

[0071] In a more preferred embodiment, the tensile modulus of the scaffold may be 0.5 kPa or more, 1.0 kPa or more, 1.5 kPa or more, 2.5 kPa or less, or 2.0 kPa or less. The present invention can adjust the tensile modulus to the above values ​​to enable appropriate cell attachment and proliferation.

[0072] In a more preferred embodiment, the scaffold surface contact angle may be 30.0° or more, 35.0° or more, 40.0° or more, 45.0° or more, 50.0° or more, 55.0° or more, 75.0° or less, 70.0° or less, 65.0° or less, or 60.0° or less. The present invention can adjust the surface contact angle to the above values ​​to enable appropriate cell attachment and proliferation.

[0073] In a more preferred embodiment, the compression stress of the scaffold may be 250.0 kPa or more, 300.0 kPa or more, 400.0 kPa or more, 500.0 kPa or more, 600.0 kPa or more, 700.0 kPa or more, 1,000 kPa or less, or 950 kPa or less. The present invention can adjust the compression stress to the above values ​​to enable appropriate cell attachment and proliferation.

[0074] In a more preferred embodiment, the compressive modulus of the scaffold may be 15.00 kPa or more, 17.00 kPa or more, 20.00 kPa or more, 25.00 kPa or more, 30.00 kPa or more, 45.00 kPa or less, or 40.00 kPa or less. The present invention can adjust the compressive modulus to the above values ​​to enable appropriate cell attachment and proliferation.

[0075] In a more preferred embodiment, the average pore size in the scaffold may be 1 μm or more, 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, or 30 μm or less.

[0076] In the present invention, 'pore size' and 'average pore size' can be measured from a cross-sectional scanning electron microscope (SEM) image of one side of the scaffold.

[0077] Specifically, 'pore size' is the size of the pores observed on one side of the scaffold as observed from a cross-sectional scanning electron microscope (SEM) image.

[0078] The 'average pore size' is calculated by selecting 30 to 50 random pores observed on one side of the scaffold from a cross-sectional scanning electron microscope (SEM) image, measuring the pore size, and calculating the average value.

[0079] Meanwhile, the above 'pore size' and 'pore average size' can be understood as values ​​that are satisfied for pores arbitrarily selected on any one side of the scaffold.

[0080] In a more preferred embodiment, in a cross-sectional scanning electron microscope (SEM) image observed on one side of the scaffold, the number of pores with a pore size of less than 1 μm may be 5% or more, or 12% or less, of the total number of pores observed on one side.

[0081] In a more preferred embodiment, in a cross-sectional scanning electron microscope (SEM) image observed on one side of the scaffold, the number of pores with a pore size of 1 μm or more and less than 5 μm may be 30% or more, 40% or more, 60% or less, or 50% or less relative to the total number of pores observed on one side.

[0082] In a more preferred embodiment, in a cross-sectional scanning electron microscope (SEM) image observed on one side of the scaffold, the number of pores with a pore size of 5 μm or more and 10 μm or less may be 10% or more, 20% or more, 35% or less, 30% or less, or 25% or less relative to the total number of pores observed on one side.

[0083] In a more preferred embodiment, in a cross-sectional scanning electron microscope (SEM) image observed on one side of the scaffold, the number of pores with a pore size greater than 10 μm and less than or equal to 50 μm may be 5% or more, 10% or more, 20% or more, 50% or less, or 45% or less relative to the total number of pores observed on one side.

[0084] The present invention can adjust the pore size to the above numerical value to enable appropriate cell attachment and proliferation.

[0085] Next, a method for manufacturing the above-mentioned poly(HEMA-co-MMA) hydrogel scaffold will be described.

[0086] First, 2-hydroxyethyl methacrylate (HEMA) monomer, methyl methacrylate (MMA) monomer, solvent, crosslinking agent, and initiator are mixed.

[0087] In a more preferred embodiment, the molar ratio of the mixed 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 83:17 or higher, 85:15 or higher, 87:13 or higher, or 97:3 or lower, 95:5 or lower, or 92:8 or lower.

[0088] In a more preferred embodiment, the total concentration (wt%) of the mixed 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer may be 20 wt% or more, 25 wt% or more, 40 wt% or less, or 35 wt% or less based on the mixture. The inventors have confirmed that excellent wettability is achieved at the above concentrations.

[0089] In a more preferred embodiment, the solvent is a mixture of water and dimethylformamide (DMF), and the mixing ratio of water to dimethylformamide (DMF) may be 75:25 to 85:15 (w / w). When using the solvent with the above mixing ratio, it is possible to manufacture a scaffold with better biocompatibility.

[0090] More preferably, the crosslinking agent may be pentaerythritol tetracrylate (PETA), and the molar concentration of the crosslinking agent may be 2.0 mol% or more, 2.5 mol% or more, 3.5 mol% or less, or 3.0 mol% or less.

[0091] The above initiator is not limited as long as it can initiate a polymerization reaction, and, for example, may be ammonium persulfate (APS).

[0092] More preferably, the molar concentration of the initiator may be 2.0 mol% or more, 2.0 mol% or more, 3.5 mol% or less, or 3.0 mol% or less.

[0093] In one embodiment, a catalyst may be further mixed during the above mixing.

[0094] In this case, the catalyst may more preferably be tetramethyl ethyl ethylene diamine (TEMED).

[0095] More preferably, the molar concentration of the catalyst may be 2.0 mol% or more, 2.0 mol% or more, 3.5 mol% or less, or 3.0 mol% or less.

[0096] In one embodiment, the polymerization reaction can be carried out at 30°C to 40°C.

[0097] After the polymerization reaction is completely finished, a washing step can be performed.

[0098]

[0099] Hereinafter, embodiments of the present invention will be described in more detail.

[0100]

[0101] Preparation of ingredients

[0102] 2-hydroxyethyl methacrylate (HEMA; Sigma-Aldrich, USA) and methyl methacrylate (MMA; USA) were used as monomers, dimethylformamide (DMF; USA) and distilled water (DW) were used as solvents, and pentaerythritol tetraacrylate (PETA; Sigma-Aldrich, USA) was prepared as a crosslinking agent.

[0103] 99% ammonium persulfate (APS; Sigma-Aldrich, USA) was prepared as an initiator, and 99% tetramethyl ethyl ethylenediamine (TEMED; Acros Organics, Belgium) was prepared as a catalyst.

[0104] All reagents except DW were purchased from Sigma-Aldrich. DW was purified using a water purification system (Di-rect-Q® water purification system, milli-Q) and tertiary purified water was used.

[0105]

[0106] Analysis method

[0107] [Scanning Electron Microscope: SEM]

[0108] The samples were freeze-dried and observed with an SEM (ISP, IM-60) to measure the morphology and pore size of the PHEMA and poly(HEMA-co-MMA) hydrogel scaffolds. In this case, the voltage was 10 KV and 15 KV, and all samples were observed at 3000x magnification.

[0109]

[0110] [Fourier transform infrared spectroscopy: FT-IR]

[0111] The copolymerization of poly(HEMA-co-MMA) was confirmed by analyzing functional groups using an FT-IR spectrometer (Rohde & Schwarz L128-003). A mixed powder was prepared by mixing KBr powder with freeze-dried and ground sample powder at a ratio of approximately 100:1, and a 0.1 g pellet sample was taken. Then, to measure transmittance, the infrared measurement range was set to 400–4000 cm⁻¹. -1 Set to and scanned 32 times.

[0112]

[0113] [Mechanical analysis: Mechanical test]

[0114] A general-purpose testing machine (Instron 3345) was used to analyze the physical suitability of the hydrogel. Samples for compression and tensile testing were prepared in cylindrical (diameter = 5 mm, height = 10 mm, thickness = 0.5 mm) and rectangular (width = 5 mm, length = 30 mm, thickness = 0.5 mm) shapes in accordance with ASTM D882 (Standard Test Method for Tensile Properties of Thin Plastic Sheeting). Mechanical properties were evaluated according to ISO 527 standard measurement, with compression and tensile strengths measured at least three times at a heating rate of 5 mm / min. For the tensile test, sandpaper was attached to the jig to secure the sample and prevent slippage during measurement.

[0115]

[0116] [Wettability]

[0117] To verify changes in wettability according to surface morphology and composition ratio, a contact angle tester (OCA20, Data Physics Instrument) was used to measure the contact angle according to the ASTM D5946 (Standard test method for Polymer films using water contact angle measurements) test method, with a measurement range of 10°–180° and an accuracy of 0.1°. To measure the contact angle, a slide glass solution was spin-coated and polymerized. The contact angle of the prepared specimens was measured at five points.

[0118]

[0119] [Pore Size Analysis]

[0120] For 'pore size', the scaffold part and the pore part were distinguished using an SEM instrument and imaging program for one side of the scaffold, 50 pores were selected, and the size of each pore was calculated.

[0121] The 'average pore size' was calculated by determining the average value of the 50 selected pore sizes.

[0122]

[0123] [Cell Culture and Proliferation Analysis]

[0124] Fetal human skin fibroblasts (HDF) were obtained from Genlantis (USA). HDFs were maintained at 37°C in Dulbecco’s Modified Eagle Medium (DMEM, Wellgene, Korea) containing 10% fetal bovine serum (FBS; Biological Industries, USA) and 1% penicillin-streptomycin (PS; Gibco, USA). The culture medium was replaced every 3 days, and cells were passed through to a sub-fluid level. 100,000 HDFs were seeded into each well of a 12-well cell culture plate and cultured for 24 hours; after 24 hours, the culture medium was replaced with 1 mL of DMEM and co-cultured with a scaffold using a cell culture insert (SPL, Korea). Cell proliferation activity was measured on days 1, 3, and 7 using the EZ-Cytox Cell Viability Assay Kit (Dogen, Korea). Briefly, HDFs were washed with PBS and then treated with a mixture of 50 μL of EZ-Cytox solution and 500 μL of DMEM in a cell culture incubator for 3 hours according to the manufacturer's instructions. After incubation, absorbance was analyzed at 450 nm using a SpectraMAX Plus microplate spectrophotometer (Molecular Devices, USA) (n = 3). To determine the effect of the scaffold on HDFs, co-cultured HDFs were fixed with a 4% paraformaldehyde solution, F-actin filaments were stained with Alexa Flour 488 Phalloidin (Thermo Fisher, USA), and HDF nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher, USA). The morphology of the stained HDFs was observed and analyzed using an Olympus fluorescence microscope (Olympus CKX53, Japan).

[0125]

[0126] [Cell Fixation and Dehydrogenation for Scanning Electron Microscopy Analysis]

[0127] HDF cells were fixed and dehydrated to verify the cell attachment pattern on the hydrogel scaffold. Before cell fixation, cells were washed twice with cold 0.1 M PBS to remove residual culture medium and serum residues. Cells were fixed overnight in 0.1 mL of 2.5% glutaraldehyde PBS. After fixation, all residual fixative was removed, and cells were washed twice with cold PBS for 5 minutes. The fixed cells were treated with graded concentrations of ethanol (30%, 50%, 75%, 95%, 100%) for dehydration. Dehydration solution was added every 5 minutes, and the samples were immediately freeze-dried using a freeze-dryer (LP-10, Ilshin Biobase, South Korea) to minimize degradation of the hydrogel scaffold.

[0128]

[0129] [Statistical analysis]

[0130] GraphPad Prism version 7.00 (GraphPad, USA) was used for all statistical analyses. Data were expressed as mean ± standard deviation from at least three independent experiments. Statistical significance was analyzed using one-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc tests. A p-value of < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). All data were graphically retransmitted using Origin 8.0 software (Origin Lab Co., USA).

[0131]

[0132] Scaffold manufacturing

[0133] <Comparative Example>

[0134] PHEMA Hydrogel Scaffold Manufacturing

[0135] Samples with HEMA monomer concentrations of 50 wt%, 40 wt%, 30 wt%, and 20 wt% were prepared and designated as HC50, HC40, HC30, and HC20, respectively. The solvent ratio was DW : DMF = 80 : 20 (w / w), and the crosslinking agent PETA (2.8 mol%), initiator APS (3.0 mol%), and catalyst TEMED (3.0 mol%) were added to the solution based on their molar ratios in the total solution. Subsequently, the homogeneous solution was poured into a Petri dish (90x15 mm, SPL Life Science) and polymerized at 37°C for 1 hour. After polymerization was complete, the unreacted solution was removed by washing twice with ethanol and DW.

[0136]

[0137] Analysis of optimal monomer concentration

[0138] The optimal concentration of the monomer was analyzed using the PHEMA hydrogel scaffold prepared in the above comparative example.

[0139] As a result of the analysis according to HEMA monomer concentration, the contact angles were measured as 81.2 ± 4.6° (HC50), 46.4 ± 2.3° (HC40), 21.1 ± 1.6° (HC30), and 43.2 ± 2.2° (HC20), respectively, confirming that HC30 had the best wettability.

[0140] HC50 exhibits a Cassie-Baxter model that is homogeneous and pore-closed, unaffected by micro / nano structures; HC40 exhibits Wenzel-Cassie-Baxter model characteristics that are influenced by the nanostructure surface as polymer chain aggregation begins; HC30 exhibits the most hydrophilic Wenzel model characteristics, with nano and micro pores observable as aggregation progresses; and HC20 shows that the Cassie-Baxter-Wenzel characteristics are influenced by the microstructure as hydrophobicity increases and phase separation intensifies.

[0141] Such changes in surface morphology imply the possibility of freely controlling surface properties even when using the same material, and it was confirmed that this can be utilized for various purposes, such as inducing cell attachment and detachment by using HEMA concentration ratios.

[0142] As a result of the mechanical property analysis, H50 could not be measured due to cracking, and the compressive strength and modulus values ​​of HC 40, HC 30, and HC 20 were measured as 2.45 ± 0.42 MPa / 94 ± 4.71 KPa, 1.80 ± 0.21 MPa / 28.41 ± 1.42 KPa, 1.08 ± 0.34 MPa, and 15.14 ± 0.75 KPa, respectively. These results show that the mechanical properties of the hydrogel become stronger as the concentration of HEMA increases.

[0143] In addition, it was confirmed that tensile strength decreased as the polymer concentration decreased. This is considered to be a phenomenon of material property deterioration caused by the weakening of the bonding force of polymer chains due to phase separation.

[0144] Meanwhile, pore analysis results showed that for HC40, the average pore size was 0.17 μm, and the pore volume percentage of pores with a size of less than 1 μm was measured as 100% of the total number of pores, and for HC50, the average pore size was 0.50 μm, and the pore volume percentage of pores with a size of less than 1 μm was 92% of the total number of pores, and the pore number percentage of pores with a size of less than 5 μm was measured as 100%.

[0145] In conclusion, it was confirmed that the most optimal monomer concentration is 30 wt%.

[0146]

[0147] <Example 1>

[0148] Based on the analysis results above, a hydrogel scaffold was prepared in the same manner as the comparative example above, except that the monomer concentration having the best characteristics was fixed at 30 wt% and the molar ratio of HEMA and MMA was mixed at 95:5.

[0149]

[0150] <Example 2>

[0151] In the above Example 1, a poly(HEMA-co-MMA) hydrogel scaffold was prepared in the same manner as in the above Example 1, except that the molar ratio of HEMA and MMA was mixed at 90:10.

[0152]

[0153] <Example 3>

[0154] In the above Example 1, a poly(HEMA-co-MMA) hydrogel scaffold was prepared in the same manner as in the above Example 1, except that the molar ratio of HEMA and MMA was mixed at 85:15.

[0155]

[0156] <Example 4>

[0157] In the above Example 1, a poly(HEMA-co-MMA) hydrogel scaffold was prepared in the same manner as in the above Example 1, except that the molar ratio of HEMA and MMA was mixed at 80:20.

[0158]

[0159] FT-IR analysis results

[0160] According to Figure 1, it was confirmed that polymerization was successfully carried out depending on the ratio of HEMA and MMA.

[0161]

[0162] SEM analysis results

[0163] Figure 2(a) is an SEM image of Example 1, Figure 2(b) is an SEM image of Example 2, Figure 2(c) is an SEM image of Example 3, and Figure 2(d) is an SEM image of Example 4.

[0164] According to Figure 2, it was confirmed that in Examples 1 and 2, the aggregated polymer chains are connected, and in Examples 3 and 4, as the amount of MMA increases, phase separation occurs due to hydrophilicity and hydrophobicity, causing the spacing between aggregated particles to widen and the size of the aggregated particles to become irregular.

[0165] As a result, it was confirmed that when the MMA content is reduced below a certain concentration, PMMA can act as a secondary crosslinking agent for PHEMA due to hydrogen bonding between acrylic acid and hydroxyl groups, but when it is increased above a certain concentration, it causes rapid phase separation.

[0166]

[0167] Surface contact angle measurement results

[0168] Table 1 and Figure 3 below show the results of measuring the surface contact angles for the scaffolds of Examples 1 to 4.

[0169]

[0170] Surface contact angle Example 1 36.9 ± 1.8° Example 2 57.0 ± 2.7° Example 3 65.9 ± 3.4° Example 4 74.7 ± 3.7°

[0171]

[0172] According to the above measurement results, it can be seen that hydrophobic properties appear in the concentration ratio of the poly(HEMA-co-MMA) hydrogel scaffold as PMMA increases. Meanwhile, it is determined that the superior wettability of the poly(HEMA-co-MMA) hydrogel scaffold compared to the HC50 hydrogel scaffold composed solely of HEMA, despite the addition of hydrophobic MMA, is due to the morphological characteristics of the poly(HEMA-co-MMA) hydrogel scaffold.

[0173] As a result, according to Table 1 and Figures 2 to 3, it was confirmed that it is possible to artificially manufacture a poly(HEMA-co-MMA) hydrogel scaffold that exhibits superior wettability compared to using only HEMA through additional interactions when an appropriate amount of MMA is injected.

[0174]

[0175] Mechanical property analysis results

[0176] Table 2 and Figures 4 to 7 below show the results of the mechanical property analysis for the scaffolds of the examples.

[0177]

[0178] Compressive Strength (KPa) Compression Modulus (KPa) Maximum Tensile Strength (KPa) Tensile Modulus (KPa) Example 1: 32.4 ± 52.5 34.2 ± 1.7 30.5 ± 2.1 1.6 ± 0.1 Example 2: 761.2 ± 24.2 38.8 ± 1.9 30.6 ± 3.7 2.0 ± 0.1 Example 3: 355.2 ± 50.4 18.1 ± 1.0 8.74 ± 1.5 00.60 ± 0.03 Example 4: 254.2 ± 36.4 16.1 ± 0.8 4.4 ± 1.3 0.5 ± 0.1

[0179]

[0180] According to the measurement results above, it was confirmed that the interaction between HEMA and MMA in Examples 1 and 2 resulted in superior mechanical properties compared to HC30.

[0181]

[0182] Pore ​​analysis results

[0183] Table 3 below shows the pore analysis results for the scaffold of the example.

[0184]

[0185] Average pore size (μm) Pore distribution (Number %) Less than 1μm 1μm or more Less than 5μm 5μm or more 10μm or less Over 10μm 50μm or less Example 1 23.190668341444 Example 27.3483210462024 Example 3 4.670041050328 Example 4 4.6423860266

[0186]

[0187] Biocompatibility analysis results

[0188] Figures 8 and 9 show confocal images of cell attachment and cell proliferation results of the hydrogel scaffolds of Examples 1 to 4.

[0189] As a result of the analysis, the co-cultured HDFs had high viability under experimental conditions and did not exhibit significant cell morphology, toxicity, or cell proliferation activity in the scaffolds of Examples 1 to 4.

[0190] In addition, under scaffold co-culture conditions, the morphology of HDF confirmed a well-extended cytoskeleton and proliferation.

[0191]

[0192] Cell adhesion and morphology

[0193] Figure 10 shows SEM images regarding HDF cell adhesion and morphology in the hydrogel scaffolds of Examples 1 to 4.

[0194] Cells were cultured on a scaffold for one day, then immediately fixed and dehydrated. Prior to observation, HDF cells attached to the scaffold were freeze-dried and imaged using a 750x SEM to analyze the cell attachment patterns under different scaffold conditions.

[0195] Analysis results confirmed that the surface and pore size of the scaffold provide a biocompatible structure for cell adhesion and proliferation.

[0196]

[0197] However, in the case of the scaffold of Example 4, it was confirmed that HDF cells did not attach to the scaffold despite it being a non-cytotoxic substance. It is believed that this was because the environment was difficult for cells to attach and grow due to the irregular surface and narrow pore size.

[0198]

[0199] As a result, the present invention identified appropriate concentrations of HEMA and MMA capable of producing synthetic hydrogel scaffolds with improved mechanical properties and suitable biocompatibility through hydrophilic-hydrophobic interactions and hydrogen bonding compared to PHEMA hydrogel scaffolds, and confirmed that poly(HEMA-co-MMA) hydrogel scaffolds with the ability to attach or detach cells were produced through various surface morphologies prepared by phase separation of HEMA and MMA.

Claims

It comprises a copolymer of 1,2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer, and The molar ratio of the copolymerized 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer is 83:17 to 97:3, Scaffold.

2. In Paragraph 1, The scaffold comprises a copolymer comprising a unit represented by the following chemical formula 1 and a unit represented by the following chemical formula 2. Scaffold: [Chemical Formula 1] [Chemical Formula 2] In the above chemical formula 1, x and y are each independently natural numbers greater than or equal to 1.

3. In Paragraph 1, The maximum tensile stress of the above scaffold is 5.00 kPa to 40.0 kPa, Scaffold.

4. In Paragraph 1, The above scaffold surface contact angle is 30.0° to 75.0°, Scaffold.

5. In Paragraph 1, The compressive modulus of the above scaffold is 15.00 kPa to 45.00 kPa, Scaffold.

6. In Paragraph 1, The tensile modulus of the above scaffold is 0.5 kPa to 2.5 kPa, Scaffold.

7. In Paragraph 1, The compression stress of the above scaffold is 250.0 kPa to 1,000 kPa, Scaffold.

8. In Paragraph 1, The average pore size measured for the pores observed in the cross-sectional scanning electron microscope (SEM) image of one surface of the above scaffold is 1 μm to 80 μm, Scaffold.

9. In the method for manufacturing a scaffold according to claim 1, The method comprises the step of preparing a copolymer of 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer by mixing 2-hydroxyethyl methacrylate (HEMA) monomer, methyl methacrylate (MMA) monomer, a solvent, a crosslinking agent, and an initiator. The molar ratio of the 2-hydroxyethyl methacrylate (HEMA) monomer and methyl methacrylate (MMA) monomer mixed above is 83:17 to 97:3, Method for manufacturing a scaffold.

10. In Paragraph 9, The total concentration (wt%) of the 2-hydroxyethyl methacrylate monomer and methyl methacrylate monomer mixed above is 20 wt% to 40 wt% based on the mixture, Method for manufacturing a scaffold.

11. In Paragraph 9, The above solvent is a mixture of water and dimethylformamide (DMF), and The above water:dimethylformamide (DMF) mixing ratio is 75:25 to 85:15 (w / w), Method for manufacturing a scaffold.

Images