KR102794934B1 - Antioxidant gelatin hydrogel containing nanoceria for bone regeneration - Google Patents

Abstract

The present invention relates to an antioxidant gelatin hydrogel containing nanoceria for bone regeneration. The nanoceria-containing gelatin hydrogel scaffold is advantageous for bone regeneration with bone bioactivity, improved mechanical properties and excellent ROS scavenging properties, and has excellent biocompatibility, so that it can be usefully used as a nanoplatform for bone treatment.

Description

{Antioxidant gelatin hydrogel containing nanoceria for bone regeneration}

The present invention relates to an antioxidant gelatin hydrogel containing nanoceria for bone regeneration.

Bone tissue repair is generally limited and mostly requires external influences to achieve its full regenerative potential. Reactive oxygen species (ROS) have the potential to accumulate in bone defects, threatening cellular activity and hindering new bone formation. An effective approach to address this issue is to construct various scaffold platforms with osteoinductive and antioxidant properties. Here, nanoengineered biopolymer matrices with ROS scavenging properties are gaining importance. Nanoscale matrices have a dramatic effect on cell behaviors such as adhesion, proliferation, and migration. Among the nano-matrix approaches, nanoscale scaffolds play an important role because of their morphological similarity to the unique microenvironment, the extracellular matrix (ECM). Surface modification or decoration of these matrices with antioxidant nanomaterials is known to be a very promising tool to modulate the interfacial properties that determine cell fate.

Providing nano-topological features also promotes cell-to-cell and cell-to-matrix communication, thereby facilitating rapid tissue regeneration. These bio-nanomatrices are more applicable when used in an implantable form. Biological interactions occurring on the surface or subsurface control various cellular responses involved in healing and determine their overall fate during the regeneration process. Although many biomaterials with nanomaterial layering have been proposed, most of them lack adequate mechanical properties, degradation behavior, and cell-supporting properties. In addition, advanced techniques such as 3D bioprinting electrospinning or stereolithography are generally required to simultaneously deliver micro/nanotopologies to the matrix. Although these techniques provide the desired topological features, the sophisticated processing steps required pose practical challenges in designing the materials. Therefore, the design and development of uniform micro/nanotopological surfaces remains a challenging task.

Amid these growing concerns, a simple in situ method is proposed to tailor biopolymer scaffolds with nanoscale materials. The method does not require sophisticated techniques to induce nano/microtopology on the scaffolds. Nanoceria-decorated GelMA scaffolds (Ce@GelMA) provide dual micro/nano-topological features, resulting in a microscale porous matrix with nanoscale nCe clusters decorating the matrix surface.

nCe is a well-known redox nanomaterial with unique physicochemical and biological properties. The ability of nCe to switch between oxidation states (Ce ⁴⁺ /Ce ³⁺ ) promotes multienzyme-mimetic activities similar to many biological antioxidants. The excellent ROS-scavenging properties exhibited by nCe have been applied in various regenerative purposes such as treatment of oxidative stress-related diseases, anti-inflammatory therapy, tissue engineering, cancer therapy, drug delivery, and biosensing. Similar efficacy has been observed when nCe-incorporated biomaterials are used for tissue regeneration.

Against this backdrop, the present inventors employed the well-known biopolymer GelMA, which is considered a versatile biomaterial for various applications ranging from cell culture platforms to advanced bio-ink formulations. As a derivative of gelatin, GelMA has low antigenicity and biodegradability. This matrix has large cell-supporting moieties such as Arg-Gly-Asp (RGD), which makes it an ideal platform for cell growth. Since GelMA is chemically cross-linked, it remains stable under physiological conditions, and its properties can be easily tuned by manipulating the cross-linking conditions. This tunability to control the morphology and porous microstructure makes GelMA more suitable for the present invention than other biopolymer substrates.

The achievement of robust hard tissue repair due to mechanical properties achieved after direct integration of nCe into GelMA is the first in this invention. Such Ce@GelMA can synergize with the rigid biointerface of nCe in microscale GelMA. The present invention is characterized by (i) cell support provided by micro/nano topological cues, and (ii) ROS reactivity due to uniform nCe layering. That is, we demonstrate the design of a degradable biointerface made of nCe laminated onto a GelMA matrix and the capacity of the scaffold to support in vitro mineralization and ROS reactivity to induce bone regeneration.

The purpose of the present invention is to provide a bone regeneration scaffold made of a gelatin hydrogel containing nanoceria (nCe).

Another object of the present invention is to provide a method for producing a bone regeneration scaffold made of a gelatin hydrogel including nanoceria (nCe), the method comprising the following steps: (a) adding anhydrous methacrylate to gelatin to produce GelMA (gelatin methacrylate); (b) crosslinking the GelMA to produce a GelMA hydrogel; (c) immersing the GelMA hydrogel in a solution containing cerium ions; and (d) freeze-drying the resultant of step (c) to produce a Ce@GelMA scaffold.

The advantages and features of the present invention, and the methods for achieving them, will become clear with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and the present embodiments are provided only to make the disclosure of the present invention complete and to fully inform a person skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. The terms "comprises" and/or "comprising" as used in the specification do not exclude the presence or addition of one or more other components in addition to the mentioned components. Like reference numerals refer to like components throughout the specification, and "and/or" includes each and every combination of one or more of the mentioned components. Although "first", "second", etc. are used to describe various components, it is to be understood that these components are not limited by these terms. These terms are merely used to distinguish one component from another. Therefore, it should be understood that a first component mentioned below may also be a second component within the technical spirit of the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with the meaning commonly understood by those skilled in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries shall not be ideally or excessively interpreted unless explicitly specifically defined.

The present invention provides a bone regeneration scaffold made of a gelatin hydrogel containing nanoceria (nCe).

The nanoceria compound of the present invention (hereinafter referred to as “nanoceria” or “nCe”) is a nano-sized compound of ceria, which is a cerium (Ce) oxide. Ceria (CeO ₂ ) nanoparticles reversibly bind to oxygen and have free radical scavenging activity due to a change in the oxidation state between Ce ³⁺ and Ce ⁴⁺ on the surface of the CeO ₂ nanoparticles.

The gelatin of the present invention is a protein obtained by partial hydrolysis of collagen, which is a major protein component contained in connective tissues such as animal bones and cartilage, and has high biocompatibility, non-toxic biodegradability, and is used as a biocompatible polymer. The gelatin has viscosity even at low temperatures and concentrations, and when cooled to a solution state, forms a clear and elastic thermoreversible gel, but easily dissolves in an aqueous solution, and therefore, in order to improve the stability and physical properties of the hydrogel, a chemical cross-linking agent has been mainly used together in the past. In the present invention, the gelatin may include a gelatin derivative in addition to pure gelatin, and may be derived from various animals such as mammals and fish, and is not particularly limited. The gelatin of the present invention may be methacrylic acid gelatin, but is not limited thereto.

The hydrogel of the present invention is a material in which molecules interacting by chemical bonds or electrostatic attraction form a hydrophilic cross-linked polymer and absorb water corresponding to hundreds of times its dry weight. Such a hydrogel exhibits excellent biocompatibility and hydrophilic properties, and thus can be applied in various fields such as pharmaceuticals, tissue regeneration engineering, medical devices, and medicine.

The above hydrogel is known as a useful candidate material for manufacturing a tissue engineering substrate for the purpose of healing damaged human tissue. The three-dimensional structure formed by the hydrogel is also called a scaffold, and such a scaffold is used as a variety of tissue engineering materials. The above tissue engineering technology means culturing cells isolated from a patient's tissue or artificially cultured cells on a support and then transplanting them back into the body or applying them externally.

The hydrogel of the present invention may be a bioink. The bioink is meant to include a hydrogel or a mixture of hydrogel and cells used in 3D printing or bioprinting. The bioink composition may be for 3D printing or bioprinting.

Bone regeneration of the present invention means bone formation, bone differentiation or bone proliferation. The bone regeneration may preferably be filling or repair of a bone defect or fracture site. The bone defect or fracture includes a bone defect or fracture caused by an accident, tooth extraction, orthodontic treatment, osteoporosis, osteolysis, osteomalacia, bone growth disorder, osteogenesis imperfecta, bone tumor or genetic bone growth defect.

A scaffold made of a gelatin hydrogel including nanoceria (nCe) of the present invention has an excellent bone regeneration effect, and thus can be usefully used in the treatment of bone defects or fractures.

The scaffold of the present invention can have a compressive modulus of 40 to 60 kPA, preferably a compressive modulus of 45 to 55 kPA. The scaffold can have a water contact angle of 30 to 50˚, preferably a water contact angle of 35 to 45˚. The scaffold of the present invention provides a substrate suitable for bone regeneration due to improved compressive modulus and hydrophilicity, and provides an interface for rapid biological reactions with various biomolecules.

The swelling ratio of the scaffold of the present invention can be 13 to 16%, 5 to 7% and 2 to 5% in DW, PBS and growth medium, respectively. The higher the swelling ratio (swelling ratio), the higher the drug loading level between the pores can be, and when transplanted into the body, blood can be absorbed between the pores, and new growth factors and growth factors can be injected between the pores. However, if the swelling ratio is too high, pressure can be applied to the surrounding tissue. The scaffold of the present invention has an appropriate swelling ratio within the above range.

The scaffold of the present invention has a dual domain structure composed of micro-sized porous gelatin hydrogel and nano-sized nanoceria. In addition, the scaffold has excellent antioxidant activity.

In addition, the present invention provides a method for manufacturing a bone regeneration scaffold made of a gelatin hydrogel containing the nanoceria (nCe).

The method for manufacturing a scaffold for bone regeneration of the present invention comprises the following steps: (a) adding anhydrous methacrylate to gelatin to manufacture GelMA (gelatin methacrylate); (b) crosslinking the GelMA to manufacture a GelMA hydrogel; (c) immersing the GelMA hydrogel in a solution containing cerium ions; and (d) freeze-drying the resultant of step (c) to manufacture a Ce@GelMA scaffold.

The crosslinking of the present invention may be photocrosslinking using ultraviolet rays, and the ultraviolet rays may have a wavelength range of 300 to 400 nm, but is not limited thereto.

The step (a) above may additionally include a step of mixing the manufactured GelMA with a photoinitiator. The photoinitiator may be, but is not limited to, 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone.

The above freeze-drying can be performed at -60 to -90°C, preferably at -70 to -80°C, but is not limited thereto.

The present invention provides a nanoceria-containing antioxidant gelatin hydrogel for bone regeneration, wherein the nanoceria-containing gelatin hydrogel scaffold is advantageous for bone regeneration with bone bioactivity, improved mechanical properties and excellent ROS scavenging properties, and has excellent biocompatibility, so that it can be usefully used as a nanoplatform for bone treatment.

Figure 1a is a schematic diagram showing the surface modification of GelMA scaffolds, Figure 1b is a SEM image showing the surface morphology of the scaffolds, and Figure 1c is a diagram showing the SEM attached EDS signals of GelMA and Ce@GelMA showing the main characteristic peaks.
Figure 2a is a diagram showing characteristic 3D and 2D AFM images of the scaffold, and Figure 2b is a diagram showing the surface chemistry of the hybrid scaffold showing characteristic peaks analyzed using XPS.
Figure 3a shows the stress relaxation curves obtained using UTM under wet conditions, Figure 3b shows the calculated compressive moduli for the samples (GelMA and Ce@GelMA), and Figure 3c shows the water contact angles measured using the hydrogel scaffolds.
Figure 4a is a SEM image showing the formation of spherical apatite particles in the scaffold, Figure 4b is an XRD analysis of the scaffold showing characteristic diffraction peaks corresponding to planes (211), (112) and (300) similar to the bone morphology, and Figure 4c is an FT-IR spectrum of the scaffold after SBF mineralization.
Figure 5a shows images of GelMA and Ce@GelMA scaffolds treated in a non-ROS-like solution (H ₂ O) and a ROS-like solution (H ₂ O ₂ ), and Figure 5b shows the oxidase-like activity of the hybrid scaffolds confirmed by TMB analysis.
Figure 6a is a diagram showing the cell proliferation rate measured using the CCK-8 assay, and Figure 6b is a diagram showing a fluorescent image of rMSCs seeded on the scaffold stained with phalloidin (green) for f-actin and DAPI (blue) for nuclei.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the rights of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical ideas equivalent thereto.

Example 1. Synthesis of GelMA

Gelatin (bovine skin type B, Sigma) was dissolved in deionized water (DW) to a final concentration of 10% (w/v). The solution was maintained at 50°C with gentle stirring until it was completely dissolved and the solution became clear. Then, 0.60 ml of methacrylic anhydride (94%, Sigma) per 1 g of dissolved gelatin was gradually added and the solution was stirred vigorously for 1 h. The solution was centrifuged at 3500 rpm for 3 min to remove unreacted chemicals and dialyzed against DW using a 12-14 kDa dialysis membrane (Cellusep, regenerated cellulose tubular membrane (T4) MFPI).

After dialysis, the pH was maintained at ~7.4 using 1 M sodium bicarbonate solution (≥99.7%, Sigma), then rapidly frozen in liquid nitrogen and lyophilized for 10 days using a cryo-desiccator (ilshin Lab Co. Ltd, Korea). The lyophilized GelMA polymer was stored at -20°C for future use.

Example 2. Fabrication of nCe-decorated GelMA scaffolds

For the fabrication of scaffolds, GelMA hydrogel was initially formed by a simple photocrosslinking method. Specifically, 10% GelMA (w/v) was completely dissolved in DW, and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (Irgacure-2959, 98% Sigma) as a photoinitiator was added, which was dissolved in hot DW by brief sonication. Then, 1.5 mL of pre-polymer solution was transferred into a customized rectangular-shaped Teflon mold.

UV was supplied at a 10 cm irradiation distance using a 365 nm wavelength Omnicure S2000 spot UV lamp (Lumen Dynamics, Ontario, Canada). The produced hydrogels were washed repeatedly in DW to remove unreacted photoinitiator. For nCe decoration on the scaffolds, a solution containing 0.1 M cerium (III) nitrate hexahydrate (Ce(NO ₃ ) ₃ .6H ₂ 0, Sigma]) was prepared in DW by stirring.

Hydrogel discs of uniform size were punched out, immersed in the cerium ion-containing solution and maintained in a shaker for about 20 min. Then, ammonium hydroxide solution (NH ₄ OH; 28.0-30.0% NH ₃ standard, Sigma) was supplemented to maintain the reaction at an alkaline pH (~8.0). Afterwards, the hydrogel discs were immersed in the cerium solution and the reaction was continued for 2 h. The hydrogel discs were taken out from the reaction mixture, washed repeatedly with DW, and stored for 1 h to completely remove unwanted chemical residues. The resulting ceria-decorated hydrogel discs were frozen at -80 °C and lyophilized to obtain Ce@GelMA scaffolds.

Example 3. Characterization of GelMA and Ce@GelMA Scaffolds

The structure, porous morphology, and elemental composition of the freeze-dried scaffolds were investigated using field emission scanning electron microscopy (FE-SEM, Zeiss 300 Gemini, Germany) and energy dispersive spectroscopy (EDS; UltraDry, Thermo Fisher, USA) at 15 kV. The surface topology and average roughness of the scaffold surfaces after nCe decoration were evaluated using atomic force microscopy (AFM; Shimadzu SPM-9700, Japan). The average roughness observed for GelMA and Ce@GelMA is for a homogeneous area of the analyzed samples, respectively. 2D and 3D surface topology and roughness images were also obtained using AFM.

The presence and relative elemental peaks of nCe on GelMA scaffolds were analyzed using X-ray photoelectron spectroscopy (XPS; MultiLab ESCA2000, Thermo VG Scientific, UK). The analysis was performed in powder mode using a monochromatic Al Kα source at a vacuum base pressure of 2.9 × 10 ^-9 mTorr. The hydrophobic or hydrophilic behavior of the scaffolds was measured by contact angle analysis using a benchtop phoenix contact angle measurement system (SEO-PHX300, South Korea). The water contact angle of the scaffolds was analyzed using a single drop of DW loaded into a 1 mL syringe connected to the machine, mechanically sprayed onto the sample surface, and the image of the water droplet was captured using surfaceware software.

The mechanical properties of the hydrogel scaffolds were evaluated in a wet state. Initially, the hydrogel scaffolds were immersed in PBS for wetting, and the compressive modulus of the scaffolds with the dimensions of 7.0 mm × 2.0 mm was measured using a single column testing system universal testing machine (UTM; Instron 3344, USA). A 10 N static load cell was used. The compressive load was applied to each wet scaffold sample at a strain rate of 0.1 mm/min. The corresponding stress was recorded as a function of time, and the compressive modulus was evaluated using Instron Blue Hill software. Five replicate experiments were performed for each sample.

To evaluate the water absorption behavior of GelMA and Ce@GelMA, disc-shaped scaffolds (10 mm × 3 mm) were fabricated and immediately immersed in 1 ml of different solvents; DW, PBS (1X), and alpha-MEM growth media ((HyClone, Logan, UT, USA)) and stored in an incubator at 37°C for 24 h. The hydrogel discs were gently wiped with a KimWipe to remove residual solvent, and the weight value was measured. The swollen scaffolds were frozen and then lyophilized. Five matching samples were selected for each scaffold, and the swelling ratios (swelling ratios) are as follows.

Swelling ratio (%) = [wet weight of scaffold - dry weight of scaffold] / [dry weight of scaffold] x 100

Example 4. Oxidase-like activity of scaffolds

The peroxidase-like catalytically active scaffolds were characterized using a redox chemical reaction between 3,3'5,5' Tetramethylbenzidine (TMB, Sigma) and hydrogen peroxide (H ₂ O ₂ ; 30 wt% in H ₂ O). The reaction was monitored using UV-vis spectroscopy (Cary Varian UV) over a wide wavelength scan. The TMB solution was prepared in acetate buffer solution (pH = 4.1) containing 1 mM H ₂ O ₂ . The hydrogel scaffolds (5 mm) were immersed in the TMB solution for 30 min at room temperature. The characteristic absorbance peak of the mixture was then recorded at a wavelength of 652 nm.

Example 5. In vitro biomineralization of hydrogel scaffolds using SBF

The in vitro apatite-forming ability of the hydrogel scaffolds was tested in simulated body fluid (SBF) at 37°C. All chemicals were obtained from Sigma Aldrich and used without further purification. For in vitro mineralization, the hydrogel discs were immersed in SBF at 37°C for 3 days.

The apatite formation of the scaffold was analyzed using SEM (JEOL JSM 6510, Japan), and the phase and chemical bonding structures were analyzed using an X-ray diffractometer (XRD; Bruker D2 phaser) and Fourier transform infrared spectroscopy (FT-IR; Varian 640-IR spectrometer, Australia), respectively.

Example 6. Biocompatibility of Ce@GelMA scaffolds

Rat bone marrow mesenchymal stem cells (rMSCs) maintained at passages 5-6 were used in the following experiments. All assembled hydrogel scaffolds were sterilized by UV for 12 h and placed in a 48-well plate. Then, phosphate-buffered saline (PBS) was added for equilibrium swelling of the scaffolds. After removing PBS, the scaffolds were cultured using alpha-MEM media (HyClone, Logan, UT, USA) supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum, and rMSCs were seeded at a density of 8000 cells/well. After 3 days of culture, cell proliferation was analyzed using a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.)).

CCK: 200 μl aliquots of a 1:9 mixture of alpha MEM medium were added to each well, and cell viability was measured at 450 nm using a microplate reader (Molecular Devices, USA). The experiment was repeated for three replicate samples. Cell morphology was then examined using actin and nuclei stained with phalloidin and DAPI, respectively. Cells were fixed with 4% paraformaldehyde for 5 min, and 0.1% Triton X-100 surfactant and 1% bovine serum albumin were used as blocking solution. After each step, they were washed with PBS to remove residues.

Afterwards, the samples were incubated with Alexa Fluor 546-conjugated phalloidin (Invitrogen) diluted in phosphate-buffered saline (PBS) for 30 min to stain actin, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI), and fluorescence images were obtained using a light microscope (IX71, Olympus, Japan).

Experimental Example 1. Morphology and Physicochemical Analysis of Hybrid Scaffolds

Morphological analysis of the freeze-dried GelMA scaffolds revealed a porous surface structure. Ce@GelMA scaffolds were fabricated by in-situ nucleation and deposition of nCe layers on the GelMA scaffolds. The amount of cerium oxide deposited on the hybrid scaffolds varied depending on the molar concentration of the cerium source. After nCe deposition, the GelMA scaffolds were observed by FE-SEM (Fig. 1b). The surface SEM image of the scaffolds clearly showed the uniform distribution of the nCe layer. nCe appeared in the form of clusters attached to the micropores of GelMA.

The chemical composition of the GelMA scaffolds with nCe layers was evaluated using EDS attached to FE-SEM. In the GelMA scaffold, only strong peaks of carbon (35.05%) and oxygen (37.54%) were observed (Fig. 1c), whereas in the Ce@GelMA scaffold, strong peaks of cerium (34.73%) and oxygen (32.99%) were observed, confirming the presence of nCe on the GelMA surface, and its intensity indicates that it is a pure nCe phase.

Then, atomic force microscopy (AFM) was used to provide 2D and 3D images and surface roughness after nCe decoration. The average roughness observed for GelMA and Ce@GelMA is 87.52 nm and 135.09 nm, respectively, for a uniform area of the analyzed samples (Figure 2a). The results indicate that the surface roughness of GelMA increased after nCe decoration. The 3D images of the samples also showed the actual structure of the Ce@GelMA scaffolds. The GelMA surface was almost smooth without particle agglomeration on the surface, while Ce@GelMA showed nCe layers clustered in nanophase.

Next, the binding energy curves of Ce, C, and O peaks in GelMA and Ce@GelMA scaffolds were analyzed by full-range XPS analysis (Fig. 2b). While C and O elements were detected in GelMA, a characteristic Ce peak was observed only in Ce@GelMA scaffold. High-resolution full-wave XPS scan shows the presence of Ce3d oxidation state in Ce@GelMA compared to GelMA. The characteristic element peaks (C1s, N1s, O1s, Ce3d) obtained from XPS are also indicated.

For clinical application of biomaterials, satisfactory mechanical properties are required. Since the fate of stem cells depends on the stiffness of the matrix, the mechanical properties of hybrid scaffolds are an important factor to consider. Bone cells prefer a rigid matrix for growth and proliferation compared to a soft matrix. Therefore, the inventors evaluated the mechanical properties of hybrid scaffolds using uniaxial compressive tests.

All scaffold samples were soaked in PBS before use. Ce@GelMA scaffolds show an increased compressive modulus (51.85 kPa) compared with GelMA (33.08 kPa). The deposition of nCe on the GelMA surface obviously helped to improve the stress relaxation and compressive modulus of the scaffolds in the mechanical tests (Figure 3a and Figure 3b). Therefore, nCe decoration would provide a rigid matrix-like condition for rMSCs to form bone.

Next, the relative hydrophilicity of the scaffolds was analyzed using the water contact angle. The water droplet deformation on the scaffold surface analyzed by the contact angle shows a slightly lower contact angle for Ce@GelMA (40.51 ⁰ ) than that for GelMA (56.23 ⁰ ) (Figure 3c). This indicates the enhanced hydrophilicity of the GelMA scaffold after nCe decoration. The nCe decoration also improves the hydrophilicity of the scaffold, providing an interface for rapid biological responses and absorption of various biomolecules.

The water absorption capacity of the scaffolds was investigated by analyzing the swelling ratio. The analysis of the water transport of hydrogel scaffolds improves the effective application of these biomaterials for clinical applications. The swelling ratio of biopolymers varies depending on the degree of crosslinking and the type of solvent. In particular, when cells are compressed into the substrate, it affects the nutrient transport and gas exchange through the scaffold, and thus the proliferation and differentiation of cells.

We analyzed the water absorption tendency using three types of solutions (DW, PBS, and growth medium). The swelling ratios of GelMA in DW, PBS, and growth medium were 18.15%, 8.02%, and 6.02%, respectively, whereas those of Ce@GelMA were 14.01%, 6.02%, and 3.01%, respectively. The mass swelling ratio of the scaffolds after surface decoration with nCe was found to decrease due to the improvement of mechanical properties and increased surface roughness. Both scaffolds showed the maximum swelling in DW and the least swelling in growth medium. The results showed that the swelling ratio of GelMA was higher than that of Ce@GelMA.

Experimental Example 2. In vitro biomineralization of hydrogel scaffolds

The ideal substrate for bone regeneration should be similar to the bone ECM to fully achieve regenerative potential. The bone ECM is mainly composed of highly aligned collagen fibrils and hydroxyapatite minerals. Most bone regeneration strategies focus on developing materials that mimic this composition. Nano-bio-matrix supports mineralization that contributes to early bone formation. Therefore, we evaluated the acellular bone-like apatite formation ability of the scaffold in SBF.

The nCe-decorated surface was observed to exhibit remarkable bone bioactivity by forming hydroxyapatite mineral crystals within a short time upon SBF immersion. The SEM image shows spherical aggregates of apatite deposited on the scaffold surface (Fig. ^4a ). The higher mineralization ratio of Ce@GelMA compared to GelMA is attributed to the presence of nCe clusters introduced on the scaffold surface, which promote the sequential accumulation of phosphate ions ( _PO43- ) to generate calcium phosphate (CaP) crystals.

The inset image shows the spherical apatite particles strongly attached to the scaffold surface. This enhanced roughness and surface area of the nanophase enhanced the biological activity of the hybrid scaffolds. Even in the undecorated GelMA scaffolds, a few apatite nodules were precipitated. This is due to the presence of carboxyl (COO ^- ) groups in the GelMA matrix that support mineralization. The crystallinity of the minerals precipitated in the scaffolds was later evaluated using XRD ( Figure 4b ). The characteristic peaks corresponding to (211), (112), and (300) represent the hydroxyapatite (HA) crystal phase. The calcium phosphate (CaP) crystals mineralized within GelMA exhibit sharp-wave-shaped diffraction patterns with a (002) peak.

This indicates an organic-to-inorganic mineralization phase transition where the GelMA framework directs the formation of parallel apatite crystals on the surface, making the system biomimetic. All obtained diffraction data were compared with the standard database powder diffraction file (PDF#54-0022) of the International Centre for Diffraction Data (ICDD). FT-IR spectral analysis after mineralization shows characteristic peaks associated with the CaP crystal lattice.

In addition to the characteristic amide peaks of GelMA observed at 1250 cm ^-1 and 1560 cm ^-1 , chemical bands related to phosphate (PO ₄ ) groups were observed at 575 cm ^-1 and 1020 cm ^-1 , and an additional carbonate (CO ₃ ) peak was observed at 880 cm ^-1 (Fig. 4c). These characteristic peaks indicate the presence of carbonated hydroxyapatite formed on the scaffold surface, which is similar to the natural bone composition. Therefore, the enhanced mineralization ability of Ce@GelMA is attributed to the ability of the nCe layer to act as nucleation sites for hydroxyapatite crystals, creating a promising matrix for bone tissue repair.

Experimental Example 3. ROS scavenging properties of the scaffold

An absolute balance between oxidants and antioxidants is essential for all biological systems. Oxidative stress has been identified as one of the major causes of tissue regeneration. The presence of oxidants such as reactive oxygen species (ROS) can cause damage to cellular macromolecules and lead to cell apoptosis. Traumatized or damaged bone tissue also plays a negative role in bone remodeling.

Despite the availability of bioresorbable bone matrices that support bone formation, bone regeneration is slow and in most cases, complete recovery is not achieved due to lack of antioxidant activity. Designing biomatrices with ROS scavenging properties is considered to solve these problems. nCe has the potential to scavenge harmful ROS and preserve the natural regenerative capacity of tissues. The properties of nCe have been utilized to modulate inflammation, reduce cytokine levels, and support cell function in vitro and in vivo.

Biomaterials, especially polymeric matrices containing nCe, will also have similar bioactivity. In addition to the radical scavenging properties of nCe, the presence of nCe in the matrix forms a nanopattern-like structure and maintains mechanical and surface properties, which further promotes cell growth and proliferation. Therefore, the inventors confirmed the radical scavenging efficiency of the scaffold.

Initially, simple tests were performed by treating the scaffolds under ROS and non-ROS conditions. DW was used to mimic non-ROS conditions, and H ₂ O ₂ was used for ROS conditions. When the scaffolds were incubated in DW, no change in properties was observed even after 12 h of storage. However, when incubated in H ₂ O ₂ ‹š, the Ce@GelMA scaffolds rapidly (less than 60 s) changed in color from white to tan due to the reaction between nCe and H ₂ O ₂ (Figure 5a). At the same time, the GelMA scaffolds did not show any characteristic changes. In addition, the ROS scaffolds were investigated by antioxidant assay using TMB. TMB oxidation is considered to be an indicator of oxidase-like enzyme-mimicking properties.

After incubation, the catalytic oxidation of Ce@GelMA and TMB in the presence of peroxide was confirmed by the appearance of a strong absorption peak at 652 nm, which was observed as a characteristic blue color (Fig. 5b). This color change is due to the oxidation of TMB, which is observed only in the presence _{of H2O2} and peroxidase-like substances. Since GelMA scaffolds do not react with TMB chromogen, the characteristic peak at 652 nm was not observed. The results indicate that Ce@GelMA can act as a ROS scavenger, which was not observed in GelMA (Fig. 6b). All considered, Ce@GelMA scaffolds can act as a fast-acting bionanomatrice to rapidly capture harmful ROS and promote tissue regeneration.

Experimental Example 4. Cytotoxicity and Proliferation

Advanced bone tissue therapeutics require analysis of cellular responses by adjusting the synthetic strategy and interfacial properties considered for developing biogenic nano-matrices. The role of nanotopography is important in assessing the fate of stem cells. Nanotopography affects cell adhesion and proliferation. Therefore, the proliferation rate of rMSCs initially seeded on hydrogel scaffolds was quantified using CCK-8 assay when cultured for up to 3 days.

After 3 days of culture, a significant increase in cell number was observed in Ce@GelMA compared to GelMA (Fig. 6a). The presence of cells growing on both scaffolds also confirmed their excellent biocompatibility. nCe improves cell survival, migration, proliferation, and differentiation. nCe also supports cell survival and proliferation by changing the oxygen concentration of ECM or reducing oxidative shock. The extended catalytic activity exhibited by nCe is due to its auto-regeneration ability.

The presence of higher number of cells on Ce@GelMA is due to the presence of higher Ce ⁴⁺ surface charge, which stimulates rMSC growth and proliferation. Fluorescent images of rMSCs cultured on the scaffolds were examined after staining with phalloidin and DAPI to view the cytoskeleton and nucleus, respectively. Representative images also show higher cell proliferation on Ce@GelMA compared to GelMA (Fig. 6b).

Compared with GelMA, the hybrid Ce@GelMA matrix has dual domains for cell activities, the porous structure of GelMA in the microscale range and the uniform nCe layer in the nanoscale range. The microscale region provides an ECM-like environment for cells involved in gas and nutrient exchange. The nanoscale region affects cell adhesion, proliferation and response under oxidative stress. Such Ce@GelMA can be effectively used as a bone regeneration scaffold.

Above, while the embodiments of the present invention have been described with reference to the attached drawings, it will be understood by those skilled in the art that the present invention may be implemented in other specific forms without changing the technical idea or essential features thereof. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (13)

A bone regeneration scaffold comprising a gelatin hydrogel on which nanoceria (nCe) is deposited, wherein the scaffold has an expansion ratio of 13 to 16% in DW, 5 to 7% in PBS, and 2 to 5% in a growth medium.
In the first paragraph,
A bone regeneration scaffold, characterized in that the above gelatin is methacrylic acid gelatin.
In the first paragraph,
A scaffold for bone regeneration, characterized in that the scaffold has a compressive modulus of 40 to 60 kPA.
In the first paragraph,
A scaffold for bone regeneration, characterized in that the scaffold has a water contact angle of 30 to 50 degrees.
In the first paragraph,
A scaffold for bone regeneration, characterized in that the scaffold forms apatite crystals in a simulation body fluid (SBF).
In the first paragraph,
A scaffold for bone regeneration, characterized in that the scaffold has a dual domain structure composed of micro-sized porous gelatin hydrogel and nano-sized nanoceria.
In the first paragraph,
A bone regeneration scaffold characterized in that the above bone regeneration is for filling or repairing a bone defect or fracture site.
In the first paragraph,
A bone regeneration scaffold characterized in that the scaffold has antioxidant activity.
(a) a step of manufacturing GelMA (methacrylic acid gelatin) by adding anhydrous methacrylate to gelatin;
(b) a step of cross-linking the GelMA to produce a GelMA hydrogel;
(c) a step of immersing the GelMA hydrogel in a solution containing cerium ions; and
(d) a method for producing a bone regeneration scaffold of claim 1, comprising a gelatin hydrogel on which nanoceria (nCe) is deposited, comprising a step of freeze-drying the resultant of step (c) to produce a Ce@GelMA scaffold.
In Article 9,
A manufacturing method, characterized in that the above cross-linking is photo-cross-linking using ultraviolet rays in the wavelength range of 300 to 400 nm.
In Article 9,
A manufacturing method, characterized in that the step (a) further comprises a step of mixing the manufactured GelMA with a photoinitiator.
In Article 11,
A manufacturing method, characterized in that the photoinitiator is 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone.
In Article 9,
A manufacturing method, characterized in that the above freeze-drying is performed at -60 to -90°C.