TW202344276A - Hybrid hydrogel composition, preparation method and use thereof - Google Patents

Abstract

The present invention provides a hybrid hydrogel composition comprising hyaluronic acid methacryloyl, gelatin methacryloyl, and acrylate-functionalized nanosilica. The present invention also provides a method of preparing the hybrid hydrogel composition and use of the hybrid hydrogel composition in preparing a pharmaceutical composition for treating cartilage defects.

Description

Composite hydrogel composition, preparation method and use thereof

The present invention relates to a composite hydrogel composition, its preparation method and its use, and in particular to a composite hydrogel composition for tissue engineering or cell culture, its preparation method and its use.

Articular cartilage damage is a common joint disorder that affects millions of people worldwide, especially those who suffer from sports-related trauma and knee injuries. Articular cartilage lacks vascular distribution, lymphocytes, and nerves, so its self-healing ability is limited. In addition, articular cartilage damage often leads to advanced osteoarthritis, which causes severe joint pain, immobility, and joint dysfunction. Clinical treatments for the treatment of osteoarthritis have been developed to reduce pain and improve joint function. However, these treatments do not regenerate the surface of articular cartilage. Current treatments for repairing cartilage defects include microfracture, autologous chondrocyte implantation, autologous osteochondral autografts, and allograft-directed cartilage regeneration. Furthermore, standard treatment strategies have limitations and disadvantages, including those related to fibrocartilage regeneration and donor site morbidity, which result in poor mechanical properties of articular hyaline cartilage. Therefore, regeneration of cartilage-deficient tissue remains challenging today.

Tissue engineering is a multifaceted field that combines materials science and biology to replace or regenerate biological tissue. Cell sources, scaffolds, and signaling molecules are the basic elements of tissue engineering. 3D porous scaffolds can serve as templates for tissue formation, providing a suitable environment for the growth of tissues or organs. In addition, hydrogels are three-dimensional (3D) polymer networks with high water content and high porosity, which can be used for nutrient and oxygen diffusion. However, natural hydrogels, such as hyaluronic acid (HA), gelatin, etc., exhibit the composition and structure of natural extracellular matrix and show unique capabilities in regeneration and tissue engineering applications. They can be widely used in 3D cell encapsulation and surface seeding to develop biomimetic structures for cartilage tissue engineering. In addition, adipose-derived stem cells have become an alternative source of bone marrow-derived mesenchymal-stem cells. Compared with bone marrow mesenchymal stem cells, adipose stem cells can proliferate rapidly and have lower harvest risk. Adipose stem cells can be easily isolated and have the ability of multilineage differentiation. Because of these properties, adipose stem cells are considered effective as a cell source for articular cartilage regeneration.

Hyaluronic acid is an anionic biopolymer composed of repeating units of D-N-acetylglucosamine and D-glucuronic acid, which can be used as a skeleton to form proteoglycan complexes. Hyaluronic acid is considered the major extracellular matrix component in all connective tissues. Hyaluronic acid is also found primarily in cartilage tissue. A previous study showed that cell surface receptors such as cell adhesion molecule (CD44, known as homing cell adhesion molecule) interact with hyaluronic acid and mediate hyaluronic acid receptor mediated movement, thereby enhancing cell aggregation and inducing chondrogenesis and hyaline cartilage formation. The use of hyaluronic acid-based hydrogels for cartilage regeneration is becoming increasingly common as these hydrogels have demonstrated excellent biocompatibility and bioactivity. However, associated with this usage Some clinical issues are that hyaluronic acid hydrogels have poor mechanical properties, do not have a stable three-dimensional structure, and have unsatisfactory degradation rates. Fortunately, the carboxyl and hydroxyl groups of hyaluronic acid can be chemically modified through methacrylation to form cross-linked hyaluronic acid methacryloyl (HAMA) under ultraviolet (UV) light irradiation. ) hydrogel, thereby improving its chemical and mechanical properties, obtaining greater viscoelasticity, anti-swelling rate, delayed enzyme degradation (compared to unmodified hyaluronic acid), and maintaining biocompatibility. However, the applicability of hyaluronic acid-based hydrogels is still limited by their poor cell adhesion, which hinders cell proliferation. Therefore, a potential strategy has been adopted to overcome this challenge, in which HAMA composite hydrogels are combined with other natural hydrogels rich in extracellular matrix components, such as gelatin methacryloyl (GelMA). gelatin-based hydrogel).

Gelatin is a natural hydrophilic polymer produced by hydrolysis and denaturation of collagen. It has low immunogenicity due to its low content of aromatic groups. Gelatin contains various bioactive amino acid motifs (motifs), including arginine-glycine-aspartate (RGD) sequences, matrix metalloproteinase (MMP) sequences, and Cell remodeling. However, when the temperature is higher than 37°C, the thermal stability and mechanical modulus of gelatin are poor. Therefore, gelatin can also be chemically modified, that is, the amine groups (and a small amount of hydroxyl groups) on the side chains of the gelatin can be replaced by the methacrylic anhydride (methacrylic anhydride) and undergo addition when exposed to UV light. Chain reaction cross-linking to form gelatin methacrylate (GelMA) hydrogel. The amount of free amine groups in gelatin is approximately 0.3184 mmole per gram. On the other hand, a 100% degree of substitution (DS%) of GelMA should result in less than 5.0 wt% methacrylyl groups, which means that even if most functional amino acids are substituted with amine groups and hydroxyl groups, the GelMA structure <still Bag The RGD and MMP sequences including gelatin were not significantly affected. Therefore, the cell adhesion properties of gelatin are retained in GelMA hydrogel. In addition, GelMA hydrogels have excellent mechanical and tunable properties for most tissue engineering applications. A previous study showed that GelMA-based scaffolds have been successfully developed for various tissue engineering applications, such as endothelial microvascular formation, microvascular channel and endochondral bone formation, and heart valve-like structural development. Nonetheless, GelMA is still not suitable for clinical use alone due to its rapid degradation, possibly due to the presence of internal metalloproteases and the low degree of cross-linking caused by the low percentage of free amine groups in gelatin.

Some challenges in using biohydrogels for cartilage tissue engineering are that they cannot mimic the mechanical and viscoelastic behavior of native tissue and have excessively high degradation rates. Some authors in the literature have used aldehydes, transglutaminase, and tyrosinase to cross-link hydrogels for cartilage tissue engineering to reduce the high degradation rate of hydrogel scaffolds. However, the aldehyde group in the chemical cross-linking agent has a certain degree of cytotoxicity, and the cross-linking process after the cells are embedded in the biohydrogel needs to consider an appropriate reaction rate to facilitate operation. Therefore, interest in free radical photopolymerization using photoinitiators (P.I.) to cross-link composite hydrogels with improved and modulated physiological/physiochemical properties has increased significantly in recent years. In addition, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) is considered a promising photoinitiator that can transmit blue light Or UV irradiation for cross-linking. LAP showed good results in terms of cell viability in vitro.

In short, the composite hydrogel of HAMA and GelMA (hereinafter referred to as HG composite hydrogel) is considered to be a promising biomimetic material, but its environmental, mechanical, and biological properties for clinical application in cartilage defect regeneration have not yet been determined. is fully established. Therefore, although in tissue engineering , the development of a biomaterial that is suitable for adipose stem cell loading scaffolds and allows the scaffold to be directly combined with the surface of cartilage tissue is needed, but so far remains a major challenge.

In the present invention, it is revealed that the incorporation of acrylate-functionalized silica nanoparticles into HG composite hydrogel will be a promising method to develop 3D biomimetic scaffolds for cartilage tissue engineering. In a manner suitable for cartilage tissue engineering, HG composite hydrogels incorporating acryloxysilane functionalized nanosilica as a new cross-linking agent will be able to adjust physical and mechanical properties and cell activities and increase chondrogenesis. Better options for genetic expression. The hydroxyl groups on the surface of nanosilica (hereinafter referred to as nSi) can be grafted with multiple methacrylate functional groups, thereby promoting network cross-linking within the 3D structure. In addition, the surface of acrylate-functionalized nSi cross-linker has free double bonds, which can interact with the double bonds in HAMA and GelMA hydrogels to form CC bonds through addition reactions. The formation of these cross-links increases the mechanical strength of the hydrogel in a manner that mimics the viscoelastic properties of natural cartilage. The purpose of the present invention is to construct a new type of photocurable composite hydrogel system, which contains HAMA, GelMA, and a small amount of acrylate-functionalized nSi (hereinafter referred to as AFnSi) cross-linking agent to evaluate Its effect on cell survival rate and cartilage differentiation ability of human adipose stem cells. In addition, the present invention uses LAP with low cytotoxicity as a photoinitiator. Figure 1 shows the overall concept of this study, which is to embed human adipose stem cells into photo-cross-linked composite hydrogels to characterize their preparation, physical/chemical properties, and evaluate their chondrogenic differentiation ability. Figure 1a ~ Figure 1c show the synthesis of 2% (w/v) HAMA hydrogel, 10% (w/v) GelMA hydrogel, and HG-AFnSi composite hydrogel. The composite hydrogel of the HG-AFnSi system was photocured using LAP under a light source of 365~405nm, as shown in Figure 1d . The physical and chemical properties of these composite hydrogels were then verified, including swelling behavior, morphological conformation, mechanical properties, and biodegradation properties. In order to further study the cell survival rate and cartilage differentiation ability, human adipose stem cells were embedded in 2% HAMA-10% GelMA composite hydrogel with 0-1.0 (w/v) AFnSi cross-linker to examine optimal cartilage development. process ( Fig. 1e ). Furthermore, the results on the in vitro cytotoxicity and cartilage differentiation gene expression of sulfated glycosaminoglycans and type II collagen will explain why this novel composite hydrogel design has excellent potential in future cartilage tissue engineering applications ( Figure 1f ).

In the present invention, the photocurable HAMA-GelMA (HG) composite hydrogel containing a small amount of acrylate functionalized nanosilica (AFnSi) inorganic cross-linker can exhibit variable pore size, mechanical properties, high swelling rate, and extremely biocompatible acellular matrix. It is worth mentioning that the microstructural stability within this composite hydrogel is affected by the degree of cross-linking related to the amount of AFnSi and is able to withstand additional forces and large deformations, which makes the composite hydrogel stable after removing this force. Glue can return to its original shape. In other words, although the concentration or degree of cross-linking of AFnSi cross-linking agent can control the mechanical properties of the HG composite hydrogel, in the present invention, only the optimal cross-linked three-dimensional bio-hydrogel matrix can affect cell proliferation and migration. , and morphogenesis produce benefits. In addition, we developed a novel photo-crosslinked composite hydrogel of HAMA-GelMA (HG) + 0.5% (w/v) acrylate functionalized nanosilica (AFnSi) cross-linker with respect to the pore size (approximately 75 ~150μm), swelling rate (about 35~65%), and compression modulus (about 35~100kPa), the optimal hydrogel properties can improve the survival rate of human adipose stem cells and differentiate these human adipose stem cells to Perform chondrogenesis. For example, in the photo-crosslinked HG composite hydrogel with 0.5% (w/v) AFnSi cross-linker, the amount of sGAG deposition and chondrogenesis marker genes (such as SOX-9, type II collagen protein, and aggrecan (Aggrecan) gene) expression significantly increased. This bioinspired HG composite hydrogel with AFnSi cross-linker will be used in cartilage An excellent choice for generation and a promising hydrogel for future cartilage tissue regeneration research

In summary, the present invention discloses a new light-curing composite hydrogel system that can be used for cartilage tissue engineering applications. The system contains HAMA, GelMA, and 0~3.0% (w/v) acrylate functionalized nanosilica. (AFnSi) cross-linking agent. The present invention describes the preparation of related HAMA, GelMA, and AFnSi materials, and confirms their corresponding chemical structures through characterization experiments. The present invention also examines the physical and chemical properties of these composite hydrogels, including swelling behavior, morphological conformation, mechanical properties, and biodegradability. In order to further study cell survival rate and cartilage differentiation, human adipose stem cells were loaded into HAMA-GelMA (HG) composite hydrogel with a HAMA to GelMA ratio of 2:1 and 0~1.0% (w/v) AFnSi cross-linker. middle. The results show that the morphological microstructure, mechanical properties, and long degradation time of HG+0.5% AFnSi hydrogel confirm that this new cell-free matrix is the best choice to support the differentiation of human adipose stem cells. In addition, in vitro experimental results showed that compared with hyaluronic acid (HA) and HG groups, human adipose stem cells loaded in light-cured composite hydrogel HG+0.5% AFnSi not only showed a significantly increased expression of chondrogenesis marker genes ( Such as SOX-9, aggrecan, and type II collagen), it also enhances the expression of sulfated glycosaminoglycans (sGAG) and the formation of type II collagen. It can be seen that the light-cured composite hydrogel of HG+0.5% AFnSi provides a suitable environment for articular cartilage tissue engineering applications.

As used herein, the term "a" or "an" is used to describe elements and components of the invention. This is done solely for convenience and to give a general sense of the invention. The description should be understood to include one or at least one, and the singular also includes the plural unless it is obvious that it means otherwise.

The terms "about" and "approximately" as used herein indicate that a value is within a statistically significant range. This range may typically be within 20% of the given value or range, more typically still within 10% and even more typically within 5%. The allowed variations encompassed by the terms "about" and "approximately" depend on the particular system under study and can be readily understood by those skilled in the art.

The term "scaffold" used in this article refers to a three-dimensional framework structure suitable for cell growth, which is generally known as a three-dimensional scaffold. It has a large number of holes for cell attachment or seeding, and thereby guides cells in the planned three-dimensional direction. Carry out growth and differentiation to produce similar regenerative tissues or organs.

The term "treatment" as used herein refers to any amelioration of a disease or disorder (also means inhibition of a disease or amelioration of the appearance, extent, or severity of at least one of its clinical symptoms).

The term "cartilage defect" as used herein includes, but is not limited to, diseases of cartilage degeneration or cartilage defect/wear caused by age, genetic mutation or damage caused by external force, such as articular cartilage defect. Cartilage is widely found in bone articular surfaces, costal cartilage, trachea, auricles, and lumbar intervertebral discs.

Unless otherwise indicated, all numbers indicating the amounts of ingredients, reaction conditions, etc. used in this specification and the patent claims should be understood in all cases to be modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims of this invention may vary generally depending upon the desired characteristics sought to be obtained by this invention.

Therefore, the present invention provides a composite hydrogel composition comprising hyaluronic acid methacryloyl, gelatin methacryloyl, and acrylate functionalized nanoparticles. Silicon oxide. In one embodiment, the content of the acrylate functionalized nanosilica is 0.1 to 5.0% (w/v); in another embodiment In another example, the content of the acrylate functionalized nanosilica is 0.1 to 3.0% (w/v); in another embodiment, the content of the acrylate functionalized nanosilica is 0.25 to 0.75% (w/v); in yet another embodiment, the content of the acrylate functionalized nanosilica is 0.5% (w/v). In one embodiment, the volume ratio of the methacrylate hyaluronic acid to methacrylate gelatin ranges from 1:4 to 4:1; in another embodiment, the methacrylate hyaluronic acid The volume ratio of acid to methacrylate gelatin is 2:1. In one embodiment, the composite hydrogel composition further includes stem cells with chondrogenic differentiation ability. The stem cells with chondrogenic differentiation ability can be from bone marrow, placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, or milk. Mesenchymal stem cells of dental pulp, or any combination thereof; in another embodiment, the stem cells with chondrogenic differentiation ability are adipose stem cells.

The present invention also provides a method for preparing the aforementioned composite hydrogel composition, which includes the following steps: (a) preparing a methacrylate hyaluronic acid hydrogel solution; (b) preparing a methacrylate gelatin hydrogel gel solution; (c) prepare acrylate functionalized nanosilica; (d) combine the methacrylate hyaluronic acid hydrogel solution obtained in step (a) and the methyl methacryl hyaluronic acid hydrogel solution obtained in step (b) Acrylate gelatin hydrogel solution, acrylate functionalized nanosilica obtained in step (c), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (lithium phenyl -2,4,6-trimethylbenzoylphosphinate) into a composite hydrogel solution; and (e) photocuring the composite hydrogel solution obtained in step (d). In one embodiment, the concentration range of the methacrylate hyaluronic acid hydrogel solution obtained in step (a) is 0.5 to 3.0% (w/v); the methacrylate hyaluronic acid hydrogel solution obtained in step (b) The concentration range of the ester gelatin hydrogel solution is 5 to 30% (w/v); the concentration range of the acrylate functionalized nanosilica obtained in step (c) is 0.1 to 5.0% (w/v) ); and the methacrylate hyaluronic acid hydrogel solution in step (d) The volume ratio to the methacrylate gelatin hydrogel solution ranges from 1:4 to 4:1. In another embodiment, the concentration of the methacrylate hyaluronic acid hydrogel solution obtained in step (a) is 2% (w/v); the methacrylate gelatin obtained in step (b) The concentration of the hydrogel solution is 10% (w/v); the concentration of the acrylate functionalized nanosilica obtained in step (c) is 0.1 to 3.0% (w/v); and the step (c) d) The volume ratio of the methacrylate hyaluronic acid hydrogel solution and the methacrylate gelatin hydrogel solution is 2:1. In another embodiment, the content of the acrylate functionalized silicon dioxide obtained in step (c) is 0.25 to 0.75% (w/v). In yet another embodiment, the content of the acrylate functionalized nanosilica obtained in step (c) is 0.5% (w/v).

The present invention further provides the use of the aforementioned composite hydrogel composition in preparing a pharmaceutical composition for treating cartilage defects. In one embodiment, the pharmaceutical composition is a three-dimensional scaffold loaded with stem cells capable of chondrogenic differentiation. The stem cells capable of chondrogenic differentiation can be derived from bone marrow, placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, or milk. Mesenchymal stem cells of dental pulp, or any combination thereof; in another embodiment, the stem cells with chondrogenic differentiation ability are adipose stem cells. In one embodiment, the route of administration of the pharmaceutical composition includes intra-articular administration.

[Figure 1] is a schematic diagram of the experimental design and steps of the present invention, showing the in vitro study of the optimal cartilage differentiation composite hydrogel loaded with human adipose stem cells. The composite hydrogel contains hyaluronic acid methacrylate (HAMA), acrylate gelatin (GelMA), and acrylate functionalized nSi (AFnSi) cross-linkers.

[Figure 2] is a schematic diagram of the synthesis of photo-crosslinked HAMA and GelMA hydrogel. (a): Synthesis of hyaluronic acid methacrylate (HAMA) hydrogel; (b): Synthesis of gelatin methacrylate (GelMA) hydrogel.

[Figure 3] shows ¹ H-NMR spectra of HA (a), HAMA (b), gelatin (c), and GelMA (d).

[Figure 4] shows (a): Schematic diagram of the synthesis of AFnSi cross-linking agent; (b): Nanosilica, 3-acryloxypropyl silanetriol (APS), and acrylate functional FTIR spectrum of based nanosilica (AFnSi); (c): TEM image and electron beam diffraction pattern of nanosilica; (d): TEM image and electron beam diffraction pattern of AFnSi .

[Figure 5] shows the particle size distribution of nanosilica (nSi) (a) and AFnSi cross-linking agent (b).

[Figure 6] shows the XRD patterns of nanosilica (SiO2) and acrylate functionalized nanosilica.

[Figure 7] shows the swelling rate of HG hydrogels with different concentrations of AFnSi cross-linker (0, 0.1, 0.5, and 1.0% (w/v)). Error bars represent standard deviations (±SD), and **P<0.01 represents significant differences compared to HG composite hydrogel alone.

[Figure 8] is a schematic diagram of composite hydrogel scaffolds with different concentrations of AFnSi, respectively HG (a-c), HG+0.1% (w/v) AFnSi (d-f), HG+0.5% (w/v) AFnSi ( Cross-sectional SEM images and size distribution histograms of g-i), and HG+1.0% (w/v) AFnSi (j-l).

[Figure 9] shows AFnSi cross-linker with different concentrations (0, 0.1, 0.5, and 1.0% (w/v)) Compressive stress-strain curve of HG composite hydrogel. (a): Schematic diagram of a typical compression test process of HG composite hydrogel; (b): Typical curve of compressive stress (MPa) versus compressive strain (%) of HG composite hydrogel; (c): HG composite hydrogel The average Young's modulus (kPa). Error bars represent standard deviation, **p<0.01 indicates significant difference compared with HG and HG/AFnSi values.

[Figure 10] Shows the storage modulus (G'), loss modulus (G " ) (a) and loss factor (Tan δ) (b) as the angular frequency (ω; rad/s) of the photo-crosslinked HG hydrogel ) function, wherein the HG hydrogel has different concentrations of AFnSi cross-linking agent such as 0, 0.1, 0.5, and 1.0% (w/v).

[Figure 11] Storage modulus (G') as a function of temperature scan for photocrosslinked HG hydrogels with different concentrations of AFnSi cross-linker (e.g., 0, 0.1, and 0.5% (w/v)).

[Figure 12] shows (a): HG composite hydrogel with different concentrations of AFnSi cross-linker (0, 0.1, 0.5, and 1.0% (w/v)) under 2.5U/ml hyaluronidase for 30 days In vitro degradation rate; (b): Optical images of HG+0.1% AFnSi hydrogel and HG+0.5% AFnSi hydrogel immersed in PBS containing 2.5U/ml hyaluronidase on the 30th day.

[Figure 13] shows the cell viability evaluated by MTS assay in human adipose stem cells cultured with light-cured composite hydrogels HG, HG+0.5% AFnSi, and HG+1% AFnSi. The HA line alone was used as a control group. Error bars represent ±SD (n=3); *p<0.05 indicates significant difference compared with HA group.

[Figure 14] shows the fluorescence obtained by live/dead staining in human adipose stem cells cultured with light-cured composite hydrogels HG, HG+0.5% AFnSi, and HG+1% AFnSi on days 1 and 5. light image. The HA line alone was used as a control group. Scale bar is 100 μm.

[Figure 15] shows analysis by RT-qPCR of related cartilage in human adipose stem cells cultured with light-cured composite hydrogel HG and HG+0.5% AFnSi on days 1, 3, 5, and 7 Genetic/osteogenic gene expression. The HA line alone was used as a control group. The expression of chondrogenesis marker genes SOX-9 (a), aggrecan (b), and type II collagen (c) and the expression of bone formation marker gene type I collagen (d) were detected. The GAPDH line serves as a housekeeping gene. Error bars represent ±SD; ***p<0.001, **p<0.01, and *p<0.05 represent significant differences compared with the HA group. ^# p<0.05 of HG+0.5%AFnSi group indicates significant difference compared with HG group.

[Figure 16] shows that in human adipose stem cells cultured with light-cured composite hydrogels HG and HG+0.5% AFnSi, sGAG was quantified by DMMB assay and type II was quantified by ELISA on days 5 and 7, respectively. collagen and compared with HA alone as a control group. (a): total amount of sulfated glycosaminoglycans (sGAG), (b): total amount of sGAG/total amount of DNA, (c): total amount of type II collagen, and (d): type II collagen Total amount/Total amount of DNA. Error bars represent ±SD, **p<0.01 and *p<0.05 represent significant differences compared with the HA group.

[Figure 17] shows the swelling rate of HG composite hydrogels with different concentrations of AFnSi cross-linking agent such as 0, 1.0, and 3.0% (w/v).

[Figure 18] shows the HG composite hydrogel (2% (w/v) HAMA hydrogel and 15 Rheometer test results of %(w/v)GelMA hydrogel (volume ratio is 2:1).

[Figure 19] shows the cytotoxicity test results of light-cured HG composite hydrogels with different concentrations of AFnSi cross-linker such as 0%, 1%, and 3%.

[Figure 20] shows the extrusion state of water glue with different concentrations and proportions.

[Figure 21] shows the printing results of different proportions of hydrocolloids (both GelMA concentrations are 30% (w/v); HAMA concentrations are all 2% (w/v)).

[Figure 22] shows an SEM image of the surface of the hydrogel scaffold. (a) 2% (w/v) HAMA + 30% (w/v) GelMA, and the volume ratio of HAMA to GelMA is 1:3. (b) 2% (w/v) HAMA + 30% (w/v) GelMA + 0.5% (w/v) AFnSi, and the volume ratio of HAMA to GelMA is 1:3.

[Figure 23] shows an SEM image of the inside of the hydrogel scaffold. (a) 2% (w/v) HAMA + 30% (w/v) GelMA, and the volume ratio of HAMA to GelMA is 1:3. (b) 2% (w/v) HAMA + 30% (w/v) GelMA + 0.5% (w/v) AFnSi, and the volume ratio of HAMA to GelMA is 1:3.

[Figure 24] shows the live/dead staining analysis results of 3D printed hydrogel scaffolds after 1 and 3 days of culture. Ga: 2% (w/v) HAMA + 30% (w/v) GelMA, and the volume ratio of HAMA to GelMA is 1:3. Gb: 2% (w/v) HAMA + 30% (w/v) GelMA + 0.5% (w/v) AFnSi, and the volume ratio of HAMA to GelMA is 1:3.

[Figure 25] shows the chondrification results of the 3D printed hydrogel scaffold. Ga: 2% (w/v) HAMA + 30% (w/v) GelMA, and the volume ratio of HAMA to GelMA is 1:3. Gb: 2% (w/v) HAMA + 30% (w/v) GelMA + 0.5% (w/v) AFnSi, and the volume ratio of HAMA to GelMA is 1:3.

This invention may be embodied in many different forms and should not be construed as limited to the examples set forth herein. The described examples are not limited to the invention as stated in the claims. Clear range.

Example 1

Materials and methods

Material

Hyaluronic acid (molecular weight 2000 kDa) was purchased from Kikkoman (FCH-200, Japan), and pig skin gelatin (type B) and methacrylic anhydride (MA) (molecular weight 154.16 Da) were purchased from Sigma-Aldrich (USA). Photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), nanosilica (SiO ₂ ), and 3-acrylyloxypropyltrimethoxysilane ( 3-acryloxypropyl trimethoxysilane (APMS) was also obtained from Sigma-Aldrich (USA), and anhydrous sodium carbonate (Na ₂ CO ₃ ) and sodium bicarbonate (NaHCO ₃ ) were purchased from J. Baker (USA). Other reagents, such as phosphate buffer saline (PBS), DMEM (Dulbecco's Modified Eagle's Medium), fetal bovine serum (FBS), penicillin, and streptomycin, were purchased from Gibco BRL (USA). All other solvents were purchased from Merck (Germany), TEDIA (Fairfield (USA)), or JT Baker (Phillipsburg (USA)). The chemicals were all analytical/reagent grade and used without further purification.

Synthesis of HAMA Hydrogel

Hyaluronic acid methacrylate (HAMA) was produced using the previously described method (G. Camci-Unal, D. Cuttica, N. Annabi, D. Demarchi, A. Khademhosseini, Synthesis and characterization of hybrid hyaluronic acid- gelatin hydrogels, Biomacromolecules. 14 (2013) 1085-1092) (B.Teong, S.C.Wu, C.M.Chang, J.W.Chen, H.T.Chen, C.H.Chen, J.K.Chang, M.L.Ho, The stiffness of a crosslinked hyaluronan hydrogel affects its chondro- induction activity on hADSCs,Journal of Biomedical Materials Research-Part B Applied Biomaterials. 106(2018)808-816). Briefly, deionized distilled water and dimethylformamide (DMF) were stirred at a ratio of 2:1 at 37°C until the solution was dissolved to prepare 100 ml of 1 wt% hyaluronic acid (HA) solution. Then, 8 ml of methacrylic anhydride (MAA) was added to the HA solution, the pH value was maintained at 8-9 with 3N sodium hydroxide, and stirred continuously at 4°C for 24 hours. The methacrylate-modified HA solution was dialyzed against deionized distilled water for 3 days to remove unreacted methacrylate groups and used in the purification process. The HAMA product was lyophilized and stored at 4°C for further studies.

Synthesis of GelMA hydrogel

Gelatin methacryloyl (GelMA) is also produced using the method described previously (H. Shirahama, B.H. Lee, L.P. Tan, N.J. Cho, Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis, Scientific Reports.6 ( 2016)1-11)(B.Velasco-Rodriguez,T.Diaz-vidal,L.C.Rosales-rivera,C.A.García-gonzález,C.Alvarez-lorenzo,A.Al-modlej,V.Domínguez-arca,G.Prieto ,S.Barbosa,J.F.A.Soltero Martínez,P.Taboada,Hybrid methacrylated gelatin and hyaluronic acid hydrogel scaffolds.Preparation and systematic characterization for prospective tissue engineering applications,International Journal of Molecular Sciences.22(2021)1-26.), method modification As follows: Dissolve 10g of gelatin in 100ml of 0.25M buffer solution (10w/v%) containing carbonate-bicarbonate (NaHCO3 and Na2CO3) and heat until the solution becomes clear, at which time the temperature is 50°C. Adjust the initial pH to pH 9 with 5M sodium hydroxide or 6M hydrochloric acid. Subsequently, 1 mL of liquid methacrylic anhydride (MA) was added to a 10 w/v% gelatin solution containing 10 g of gelatin under magnetic stirring at 50°C and 500 rpm. The reaction was carried out for 3 hours and then the pH was readjusted to 7.4 to stop the reaction. After filtration, the samples were dialyzed against redistilled water for 3 days, then lyophilized and stored at -80°C for later use.

Synthesis of acrylate functionalized nSi cross-linker (AFnSi)

In order to prevent the aggregation of nanoparticles and enhance the cross-linking ability, this study uses 3-acryloxypropyl silanetriol (APS) functionalized nSi as an enhanced new cross-linking agent in HG The composite hydrogel network acts as a photocurable coupling agent. First, prepare a 0.5% (w/v) 3-acryloxypropyltrimethoxysilane (APMS) solution in 100 ml of 95% ethanol and stir at 37°C. The hydrolysis reaction converts APMS to pH 4 APS within 48 hours. Finally, the pH was adjusted to 7, and the APS structure was obtained and identified using Fourier transform infrared spectroscopy (FTIR). Subsequently, 10 g of nSi powder was added to 100 ml of APS solution and stirred for 24 hours to completely form a colloidal solution. Then, the APS/nSi colloidal solution was stirred in an oil bath at 100° C. for 4 hours to perform a condensation reaction between hydroxyl groups. Then, 50 ml of ethanol was added and centrifuged at 8000 rpm for 10 min. The centrifuged pellet was collected and dried. The functional structure of the obtained acrylate functionalized nSi (AFnSi) was also identified by FTIR analysis.

Identification of synthesized HAMA and GelMA hydrogels

Proton NMR spectroscopy (Varian Gemini-200, USA) was used on hyaluronic acid, HAMA, gelatin, and GelMA hydrogels to determine the incorporation of methacrylate groups. A total of 20 mg of each sample was completely dissolved in 1 ml of deuterium oxide (D ₂ O) containing TMSP (3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid sodium) salt as an internal standard ( internal standard). Equation 1 was also used to calculate the degree of methacrylation (DoM) of HAMA hydrogel. A brief description is as follows: The degree of methacrylation of HAMA hydrogels was determined by ¹ H NMR analysis. The vinyl signal intensity of methacrylate (these signals are typically found at approximately 5.8 and 6.2 ppm) was quantified and compared to known peaks in the spectrum. Sometimes, DoM is also determined by quantifying the peak value of methyl methacrylate (at about 1.9 ppm) (SABencherif, A. Srinivasan, F. Horkay, JOHollinger, K. Matyjaszewski, NR Washburn, Influence of the degree of methacrylation on hyaluronic acid hydrogels properties,Biomaterials.29(2008)1739-1749)(M.Zhu,Y.Wang,G.Ferracci,J.Zheng,NJCho,BHLee,Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch- to-batch consistency,Scientific Reports.9(2019)1-13). The method used in this study focused on the sum of methacrylate protons at δ 6.2 ppm and δ 5.8 ppm, and compared it with the tris on the methyl group of the N-acetylglucosamine subunit at δ 2.0-2.1 ppm. Compare protons. The DoM calculation formula is shown in Equation 1 , where the numerator and denominator are the integral values of each proton. In addition, the amount of free amine groups and methacrylyl groups in GelMA hydrogel was also quantified by ¹ H-NMR analysis and used to compare with the DoM (%) value reported in the literature (H. Shirahama, BHLee, LPTan, NJCho, Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis, Scientific Reports. 6 (2016) 1-11). Basically, the quantification of methacrylamide and methacrylate groups in GelMA hydrogel is performed simultaneously. However, examining these values in comparison to the target DoM(%) of 90-100%, the signal of free amine groups in gelatin disappears (the signal can typically be found at approximately 3.0 ppm).

DoM%=[(vinyl peak)/2]/[(methyl methacrylate peak)/3)]×100%-------------( Formula 1 )

Identification of the synthesized AFnSi cross-linking agent

Collect infrared spectra (IR) of nSi, 3-acryloxypropyltrimethoxysilane (APMS), and acrylate functionalized nSi (AFnSi) using attenuated reflection (ATR) FTIR spectrum (system 2000 FT-IR, Perkin Elmer, USA) was used to determine the chemical structure of the functional groups. The transmittance readings of the sample were measured by accumulating 32 scans at a resolution of 4 cm ⁻¹ in the spectral region of 4000-400 cm ⁻¹ . A Bruker D8 advance diffractometer (Germany) was used to perform X-ray powder diffraction (XRD) to conduct phase identification of the crystalline materials of nSi and AFnSi samples. The parameter settings of the instrument are as follows: Cu-K radiation uses a graphite monochromator, voltage 40kV, current 40mA, scanning range 10°~60°, scanning speed 2°/min. Transmission electron microscopy (TEM) was also used to examine the size and morphology of these nanoparticles. That is, nSi and AFnSi suspension samples were dropped onto a copper grid coated with a carbon/formvar support film using a Pasteur pipette for TEM observation. After 15 seconds, absorb excess sample with filter paper and dry at room temperature. The copper mesh was placed in a sample holder and inserted into a 200kV Joel JEM-2100 TEM for observation.

Preparation of HG composite hydrogel

Prepare a HAMA hydrogel solution with a concentration of 2% (w/v) and a GelMA hydrogel solution with a concentration of 10% (w/v). Then, the volume ratio of HAMA hydrogel solution to GelMA hydrogel solution was 2:1, and the HG composite hydrogel solution and 0.3% (w/v) LAP were mixed at 0.1, 0.5, and 1% (w/v) respectively. ) and other different concentrations of AFnSi cross-linking agent. The composite hydrogel solution without AFnSi cross-linker was used as a control group. The composite hydrogel solution was vortexed to mix evenly, and photocured under UV light of 280-400 nm wavelength for 120 seconds using the UV curing chamber of a photocuring machine (XYZ printing (USA)). In addition, these composite hydrogel systems are represented by the following: HAMA-GelMA (HG), HAMA-GelMA+0.1% (w/v) AFnSi (HG+0.1%AFnSi), HAMA-GelMA+0.5% (w/v) AFnSi(HG+0.5%AFnSi), and HAMA-GelMA+1%(w/v)AFnSi(HG+1%AFnSi).

Characteristic analysis of HG composite hydrogel

Assessment of profit margin

In order to prepare composite hydrogel samples for swelling rate measurement, HG solutions with a volume ratio of HAMA hydrogel solution to GelMA hydrogel solution of 2:1 were mixed with different concentrations (such as 0, 0.1, 0.5, and 1.0 % (w/v) AFnSi cross-linking agent) and 0.3% (w/v) LAP were mixed into 300 μl of composite hydrogel solution, and then added to a cylindrical plastic mold. Each set of composite hydrogel solutions was then exposed to UV light of 280-400 nm for 120 seconds. After the photopolymerization step, each composite hydrogel sample containing different concentrations of AFnSi cross-linker was placed in a microcentrifuge tube (Eppendorf) containing 2 ml of PBS for 24 h to maintain equilibrium. After 24 hours, the swollen hydrogels were weighed and excess water was removed by gently blotting with Kimwipes. Each hydrogel sample was run in quadruplicate. Use the following formula ( Equation 2 ) to calculate the swelling rate of the hydrogel sample.

Swelling rate = [(W _w -W ₀ )/W ₀ ]-------------( Formula 2 )

W _w : wet weight of hydrogel sample

W ₀ : Dry weight of hydrogel sample

Microstructural Morphological Analysis

After photocuring HG composite hydrogels with different concentrations of AFnSi cross-linking agent such as 0, 0.1, 0.5, and 1.0% (w/v), the morphological characteristics of these composite hydrogels were observed. However, these composite hydrogels were prepared as previously described for use as swelling rate samples and cross-sectional areas were obtained after the freeze-drying method. After all hydrogel samples were gold-coated with a sputter coater at ambient temperature, micrographs or elemental mapping analysis were performed on all hydrogel samples using a scanning electron microscope (SEM, JEOL, Tokyo, Japan). Use ImageJ software to evaluate the features of 0, 0.1, 0.5, The average diameter of pores in HG composite hydrogels with different concentrations of AFnSi cross-linking agent such as 1.0% (w/v) and 1.0% (w/v).

Mechanical property evaluation

The compressive strength and rheological behavior of HG composite hydrogels with different concentrations of AFnSi cross-linker such as 0, 0.1, 0.5, and 1.0% (w/v) after light curing were also measured. Compression mechanical testing was performed using a universal mechanical compression testing machine (Instron 5567, USA). The speed of the crosshead is 4mm/s, and the load cell (loading cell) is 200N. When the compressive strain in the hydrogel reaches 60%, the compressive strength is calculated as the stress. Compression tests were performed in duplicate with five samples for each composite hydrogel. In addition, a hybrid rheometer (HR-2 Discovery Hybrid Rheometer-2 (TA Instruments, USA)) with a 20 mm parallel plate was used to measure the storage modulus (G ' ) and loss modulus (G " ), and loss factor (Tan δ) as a function of angular frequency (rad/s) behavior. Each composite hydrogel was added to the 0.5mm gap between the plates and maintained in equilibrium until the normal force became zero . First, a frequency sweep (ω; rad/s) was performed at a constant 1% strain at 37°C to examine the elastic properties of these composite hydrogels. In addition, the elastic properties of these composite hydrogels were also examined by using a frequency of 1rad/s and a frequency of 0.5°C/ The storage modulus (G ' ) was measured as a function of temperature and time sweep by increasing the temperature from 10 to 60°C at a heating rate of min.

In vitro degradation test of hyaluronidase

These composite hydrogels were prepared as previously described in the swelling rate experiments and allowed to swell in PBS for 24 hours until equilibrium was reached. These composite hydrogels were then incubated in 1 ml of PBS buffer containing 2.6 U ml ^-1 hyaluronidase (physiological human plasma concentration) at 37°C. These composite hydrogels were collected and weighed after blotting every 5 days for 30 days. The hyaluronidase solution was replaced with fresh solution every day, and each composite hydrogel was tested in quadruplicate. The degree of degradation of the composite hydrogel was calculated by normalizing the residual hydrogel wet weight and the initial hydrogel wet weight.

Cell survival rate and chondrogenesis test of HG composite hydrogel

Isolation and culture of human adipose stem cells

This study examined the isolation of human adipose stem cells from human subcutaneous adipose tissue according to procedures described previously (SCWu, CHChen, JKChang, YCFu, CKWang, R. Eswaramoorthy, YSLin, YHWang, SYLin, GJWang, MLHo , Hyaluronan initiates chondrogenesis mainly via CD44 in human adipose-derived stem cells, Journal of Applied Physiology. 114 (2013) 1610-1618). Adipose stem cells were isolated from subcutaneous adipose tissue obtained from human patients during orthopedic surgery after obtaining informed consent from all patients and approval from the Ethics Committee of Kaohsiung Medical University Hospital (KMUH-IRB-E(II)-20150193). Briefly, 3 g of human subcutaneous adipose tissue was removed and cut into small pieces using scissors. The minced tissue was digested with 1 mg/ml type I collagenase at 37°C and ₅ % CO for 24 hours. Then, centrifuge at 1000 rpm for 5 minutes. The pellet was collected and washed twice with PBS. Afterwards, the pellet was resuspended in K-NAC medium, and cells were counted and seeded in 100 mm culture dishes. Subsequently, the adipose stem cells attached to the culture plate were kept in a 37°C 5% _CO2 incubator. The K-NAC medium used in this study is suitable for the isolation and expansion of adipose stem cells described in previous studies. The K-NAC culture medium mainly contains 25 mg bovine pituitary extract (BPE), 2.5 μg human recombinant epidermal growth factor, 2 mM N-acetyl-1-cysteine, 0.2 mM L-ascorbic acid, and Keratinocytes-SFM in 5% FBS (Gibco BRL, Rockville, MD). After 24 hours, the culture medium was changed for the first time, and PBS was used to wash away the adipose stem cells that did not adhere to the culture plate. Subsequently, the medium was replaced with fresh medium every two days. When the cells grew to nearly 90% confluence, they were subcultured for further cell research.

Cell viability analysis

According to the steps mentioned above, HG composite hydrogels with different concentrations of AFnSi cross-linking agent such as 0%, 0.5%, and 1.0% (w/v) were prepared. Briefly, 200 μl of each hydrogel sample was spread on a 24-well plate. Each hydrogel sample was cross-linked using 365nm UV light. Add 1 ml of PBS to each cross-linked hydrogel sample and incubate at 37°C and 5% CO _for 24 hours to reach the swelling equilibrium rate. Then, gently remove the PBS solution. These samples were used for cell viability and differentiation analyses.

Cell viability was determined using the CellTiter96® Aqueous Single Solution Cell Proliferation Assay (MTS) Kit (Promega, USA). In other words, HG composite hydrogels and HA hydrogels (control group) with different concentrations of AFnSi cross-linker at 0%, 0.5%, and 1.0% (w/v) were tested in human adipose stem cells by MTS analysis. ). All sample groups were cultured in DMEM with 1 × ¹⁰ cells at 37°C and 5% CO for 1, ₃ , and 5 days. Briefly, at each designated time point, the cultured medium in each sample well of the 24-well culture plate was removed and replaced with 200 μl of fresh medium and 40 μl of MTS reagent. The culture plates were cultured at 37°C, 5% _CO2 for 4 hours. Finally, 100 μl of solution was aspirated from each sample well and placed in a 96-well plate. The optical density (OD) at 490 nm was measured using a 96-well ELISA disk analyzer (Synergy H1, BioTek, Winooski, VT, USA).

In addition, HG composite hydrogels with different concentrations of AFnSi cross-linker at 0%, 0.5%, and 1.0% (w/v) were evaluated in human adipose stem cells using live/dead staining on days 1 and 5. Cytotoxicity of gel and HA hydrogel. Live/dead staining analysis was performed according to the manufacturer's protocol. Briefly, a total of 20 μl of ethidium homodimer-1 (EthD-1) and 5 μl of calcein-AM was mixed with 10 ml of PBS solution and vortexed. Then, add 500 μl of working dye solution to each well containing the sample and incubate at 37 °C, 5% CO for ₁ h. Finally, an inverted fluorescence microscope (Leica DMi8 inverted fluorescence microscope) was used to observe the living cells and dead cells in the sample.

Expression of chondrogenic marker genes

The chondrogenesis effect of HG composite hydrogel and HA hydrogel (control group) with 0% and 0.5% (w/v) AFnSi cross-linker on human adipose stem cells was determined using quantitative real-time polymerase chain reaction (quantitative real- time PCR) assay to examine the expression of chondrogenesis marker genes. Each sample was cultured with 1 × ¹⁰ cells in basal medium in a 24-well plate at 37°C, 5% CO for 1, 3, ₅ , and 7 days. At each designated time point, the HG composite hydrogel samples were collected. Total RNA extraction was performed using TRIzol reagent. The quality of the RNA was confirmed using a Thermo Scientific NanoDrop ^™ 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed into cDNA using TOOLs Easy Fast RT kit (Taipei, Taiwan). Real-time polymerase chain reactions were performed using iQ ^{™ SYBR®} Green Supermix (Bio-Rad Laboratories, Hercules, CA). The transcribed cDNA samples were analyzed for genes of interest, including SOX-9, Aggrecan, type 2 collagen, and type 1 collagen genes. Table 1 lists the primers used in this study, including the forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 2) for GADPH; the forward primer for SOX-9 Primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4); forward primer (SEQ ID NO: 5) and reverse primer (SEQ ID NO: 6) for aggrecan; for Forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8) for type 2 collagen; and forward primer (SEQ ID NO: 9) and reverse primer for type 1 collagen Primer (SEQ ID NO: 10). After completing the real-time polymerase chain reaction, a dissociation curve was generated to confirm the specificity of each PCR product. All values for the gene of interest were normalized to the performance level of glyceraldehyde-3-phosphate-dehydrogenase (GADPH) with the mean cycle threshold (Ct) using the comparative method. The experiment was repeated three times at each designated time point.

Quantification of DNA, sGAG deposition, and type II collagen synthesis

The DNA content of each sample was measured using the Hoechst dye assay and calf thymus was used as a standard curve to measure DNA content. Procedure for the sulfated glycosaminoglycan (sGAG) analytical assay using the Blyscan ^TM Kit (Biocolor Ltd., Carrickfergus, Northern Ireland) and using chondroitin solutions at different concentrations from 0 to 25 μg/μL as dimethylmethylene blue (dimethylmethylene blue, DMMB) standard for determination. Finally, optical density measurements were performed at 650 nm using an ELISA disk analyzer (Synergy H1, Biotek, Winooski, VT, USA). DMMB assay was performed to detect and quantify the amount of sGAG present in HG composite hydrogels and HA hydrogels with 0% and 0.5% (w/v) AFnSi cross-linker in human adipose stem cells. These samples were cultured with 1 × ¹⁰ cells in basal medium in 24-well plates at 37°C, ₅ % CO for 5 and 7 days. At each designated time point, these samples were collected, washed with PBS and digested using papain solution at 60 °C for 15 h. Enzyme-linked immunosorbent assay (ELISA) was also used to quantify the presence of human adipose stem cells in HG composite hydrogels and HA hydrogels with 0% and 0.5% (w/v) AFnSi cross-linker. of type II collagen. Each sample was also cultured with 1×10 ⁶ cells in 24-well plates in basal medium for 5 and 7 days. At each designated time point, these samples were collected and the type II collagen content in each sample was measured using a type II collagen assay kit (Chondrex, Redmond, WA, USA).

Statistical analysis

Statistical analysis was performed on all scored data to express mean±SD (n=3~6), t test was performed, and ^#p <0.05, *p<0.05, **p<0.01, and ***P< 0.001 represents a significant difference compared to a control or other treatment group.

result

Identification of synthesized HAMA and GelMA hydrogels

The methacrylation process involves the addition of methacrylyl groups to the amine and hydroxyl residues of gelatin and HA (see Figure 2 scheme). Hyaluronic acid methacrylate (HAMA), which has good biotolerance, is becoming increasingly popular due to its ability to gel through simple photo-cross-linking. In particular, the biocompatibility of HAMA hydrogel after light curing shows that it is not cytotoxic. In addition, seed cells can be encapsulated in HAMA hydrogel in this way, allowing the cells to temporarily live in a 3D environment. Figure 2a shows the synthesis of hyaluronic acid methacrylate (HAMA), in which the primary hydroxyl group of hyaluronic acid reacts with the methacrylate pendant group of methacrylic anhydride (MA) to form Hyaluronic acid methacrylate (HAMA) hydrogel. Methacrylamide-modified gelatin possesses both natural and synthetic properties, including cell adhesion sites and tunable mechanical properties. As shown in the schematic diagram in Figure 2b , a methacrylate gelatin (GelMA) hydrogel was successfully obtained by simply converting the acid anhydride into amine groups and hydroxyl groups in one step, and then grafting the unsaturated bonds onto the gelatin.

The degree of methacrylation (DoM) is a basic characteristic of the cross-linking density of the hydrogel matrix, which has a significant impact on the mechanical properties, structure, porosity, and swelling and degradation capabilities. Figure 3 illustrates verification of methacrylation of two biopolymers (HAMA, GelMA) by ¹ H NMR spectroscopy. Figure 3a and Figure 3c show the ¹ H NMR spectra of hyaluronic acid and gelatin, respectively. Figure 3b shows the ¹ H NMR spectrum of HAMA, showing methacrylyl peaks at 5.5 and 5.8 ppm. The peak at 2.16 ppm corresponds to the methyl proton of the grafted methacrylyl group (CH ₂ =CH(CH ₃ )), which gives an independent signal in the unchanged HA spectrum. HAMA with optimal DoM produces stiffer hydrogels, which have been noted to promote cell adhesion in tissue engineering applications. Based on this NMR spectrum, the DoM of HAMA was calculated to be approximately 85±10%.

Figure 3d shows that ^{the 1} H NMR spectrum of GelMA shows methacrylate peaks at ~5.5 and ~5.8 ppm, which are consistent with the acrylate protons of hydroxylysine and lysine (CH ₂ =CH(CH ₃ )). . Although both methacrylamide and methacrylate groups are present in GelMA, the content of methacrylate is much lower than that of methacrylamide. However, compared to the spectrum of unmodified gelatin, there is an increase in the peak at 1.92 ppm, which corresponds to the methyl proton of the grafted methacrylyl group (CH ₂ =CH(CH ₃ )). Therefore, quantification of methacrylamide groups in GelMA and the target DoM (%) of 90-100% confirms that the signal of free amine groups disappears in gelatin (signal can usually be found at ~3.0 ppm).

Identification of AFnSi cross-linking agent

Due to their small size and very high surface energy, nanoparticles tend to aggregate in solvents or other liquid matrices. There are some studies on surface modification through physical and chemical treatments to prevent nSi aggregation and increase the surface reactivity of nanoparticles. Among them, one of the most commonly used methods to modify nSi surfaces is chemical treatment with organosilane, which can establish strong chemical bonds between the surface of nanoparticles and polymer chains. In addition, nSi has been proven to have biosafety properties and enhance cell growth, and previous reports have indicated that silicon compounds can positively regulate cartilage extracellular matrix.

In this study, we prepared a novel acrylate-functionalized nSi (AFnSi) cross-linker to stabilize the 3D network of photo-crosslinkable HG composite hydrogels. A schematic diagram of the surface modification steps is shown in Figure 4a . It can be seen from the FTIR spectrum results in Figure 4b that the AFnSi spectrum has new bands of C=O groups at 1869cm ^-1 and 1692cm ^-1 , and a band appears at 1611cm ^-1 , showing strong C= C stretching also appears in the AFnSi spectrum, which is different from that obtained for nSi. These FTIR spectra indicate that AFnSi nanoparticles have been successfully produced. Transmission electron microscopy (TEM) was used to study the morphology of nSi and AFnSi nanoparticles, as shown in Figure 4c and Figure 4d , which show TEM images of nSi and AFnSi nanoparticle cross-linkers. The average size of the as-received nSi nanoparticles was approximately 16 nm ( Figure 5 a ). After APS surface modification on nSi, the size of AFnSi nanoparticles is also about 15 nm ( Figure 5 b ), which is not significantly different from unmodified nSi. This means that AFnSi is wrapped in the polymer chain, but the smaller APS molecules grafted onto nSi do not have much effect on its size after drying. Analysis of the crystallinity of nSi and AFnSi cross-linkers through TEM electron beam diffraction shows that they are both amorphous crystal structures, which correlates with the XRD results, as shown in Figure 6 .

Swelling rate of HG composite hydrogel

Hydrogels contain more than 90% water and are able to retain water within their three-dimensional cross-linked structure. The swelling capacity of hydrogel can be used as a criterion for judging hydrophilicity, which is determined by the size of the pores of the hydrogel. This key feature has been shown to influence cellular activity. To build a three-dimensional network, the hydrogel is cross-linked, and the degree of cross-linking affects the structure and swelling capacity of the hydrogel, as well as its integrity. Hydrogels are cross-linked hydrophilic polymer networks that do not dissolve but swell in water.

Figure 7 shows the swelling behavior of photo-crosslinked HG composite hydrogel with different concentrations of AFnSi cross-linker. The concentrations of AFnSi cross-linker incorporated into the HG composite hydrogel were 0, 0.1, 0.5, and 1% (w/v). It was observed that the swelling behavior of HG composite hydrogels with different concentrations of AFnSi cross-linker is tunable. For example, the swelling rate of the HG composite hydrogel with 0.1% (w/v) AFnSi was reduced to 60%. Similarly, the swell rate of HG composite hydrogels with 0.5% and 1% (w/v) AFnSi was significantly reduced to approximately 35% compared to HG hydrogel alone (~74%). These results were expected because increasing cross-linking concentration has been previously reported to increase cross-linking density. Therefore, HG composite hydrogels with higher AFnSi cross-linker concentration should have smaller pore sizes and less swelling than HG composite hydrogels made with lower cross-linking concentrations. These findings confirm that the swelling behavior of HG composite hydrogels can be controlled by the cross-linker concentration. These different saturation rates could be used as parameters for future evaluation of cell viability and chondrogenic capacity.

Morphological examination of HG composite hydrogel

The internal structure and morphology of scaffolds are also of considerable importance for their intended applications in tissue engineering, as they determine how the scaffold behaves in the microenvironment. Figure 8 shows the microstructure of HG composite hydrogels with different concentrations (0~1.0%) (w/v) AFnSi cross-linker. The image is from a scanning electron microscope (SEM). Although freeze-drying may create artificial holes, all HG composite hydrogels were prepared under the same experimental environment and freeze-dried at the same temperature and for the same time to eliminate any variability. All HG composite hydrogels have a honeycomb structure resulting from the connection of internal photocurable molecules (HAMA, GelMA) and AFnSi cross-linkers. However, these SEM images show that when the concentration of AFnSi cross-linker increases, the pore size decreases, which may be due to the increase in the degree of cross-linking in the HG composite hydrogel. The average diameters of the pores in the HG composite hydrogel with different concentrations of AFnSi cross-linker such as 0, 0.1, 0.5, and 1.0% (w/v) are 95±0.8μm, 86±0.6μm, and 75±0.2μm respectively. , and 76±0.2μm. There is no significant difference in the average pore diameter between HG+0.5%(w/v)AFnSi and HG+1.0%(w/v)AFnSi. This may be because adding more than 1.0% (w/v) AFnSi will cause the HG composite hydrogel system to be difficult to disperse uniformly. The pore connectivity of the HG composite hydrogel ensures its ability to undergo nutrient exchange and cell migration, indicating its potential in biomedical applications. Photocuring of HG composite hydrogel using AFnSi cross-linking agent is beneficial to the formation of porous network structures of varying degrees. In addition, the higher degree of cross-linking and uniform pores can improve the mechanical properties and suppress the problem of rapid degradation of biohydrogels, making them easier to use in tissue engineering applications.

Mechanical properties of HG composite hydrogel

A universal testing machine (Instron 5567, USA) was used to test the mechanical properties of the HG composite hydrogel under the compression model to determine the relationship between the added amount of AFnSi cross-linker and its compression properties. Figure 9a is a schematic diagram of the typical compression process of photo-crosslinked HG composite hydrogels with different concentrations (0~1.0w/v) of AFnSi cross-linker. In general, conventional hydrogels derived from natural materials are often mechanically weak and have limited use in tissue engineering and biomedical applications. Biohydrogel scaffolds with ideal mechanical compression stability and recovery are in great demand in the market. Figure 9b shows that the HG composite hydrogel doped with 0.5 and 1.0 (w/v) AFnSi cross-linker (HG+0.5%AFnSi; HG+1%AFnSi) can resist more deformation (~50% strain) and maintain Maintains up to 85% of original size after compression process. The HG composite hydrogel alone showed lower levels of stress (102kPa) and deformation (35% strain), and the maximum compressive stress of the HG+0.5% AFnSi composite hydrogel was nearly 2 times higher than that of HG alone.

In addition, the compressive modulus of the HG composite hydrogel mixed with 0.5% (w/v) AFnSi cross-linker (HG+0.5% AFnSi) also increased to about 35±1.5kPa, which is higher than that of the HG composite alone The compressive modulus of the hydrogel was three times higher ( Figure 9c ). There is no significant difference in the modulus of HG+1%AFnSi and HG+0.5%AFnSi, but the compressive stress stability of HG+0.5%AFnSi is still greater than that of HG+1.0%AFnSi cross-linker formula ( Figure 9b ). Therefore, it is inferred that adding more than 1.0% (w/v) AFnSi will lead to uneven dispersion of AFnSi in the HG composite hydrogel system.

The experiments were performed while the frequency dependence of the viscoelastic properties of all photocrosslinked HG composite hydrogels was in the linear (1% strain) region. This means that the storage modulus, loss modulus, and loss factor are approximately constant in the linear region. Figure 10 shows the effect of frequency (ω) on the dynamic characteristics at constant strain amplitude and scanning frequency from 1 to 100 Hz, and the storage modulus G' and loss modulus G" in the low angular frequency to high angular frequency region in Figure 10a shows a stable level. However, these results also show that under these conditions, the storage modulus is always higher than the loss modulus. Obviously, the loss factor is always less than 1, so all photo-crosslinked HG composite hydrogels are within 1.0 The frequency range to 100Hz is dominated by elastic response. In particular, the G' of the photo-crosslinked composite hydrogels of HG+0.5% AFnSi and HG+1.0% AFnSi is also larger in Figure 10a and Figure 10b G" range, which means that both composite hydrogels are highly elastic compared to the HG composite hydrogel alone. In other words, the loss factor (Tan δ) of the two photo-crosslinked composite hydrogels, HG+0.5% AFnSi and HG+1.0% AFnSi, is lower than other HG hydrogels in the frequency range of 1.0-100Hz, showing more significant elasticity behavior. In addition, oscillating temperature scans were performed between 20°C and 45°C with a heating rate of 0.5°C/min and a frequency of 1rad/s, and it was found that these HG composite hydrogels were thermally stable within the monitored temperature range ( Figure 11 ) .

In highly cross-linked hydrogels, the polymer chains are tightly coupled together through covalent cross-links, which may hinder chain mobility. In contrast, lower cross-link density can promote chain-to-chain movement and lead to more relaxed behavior. Therefore, an appropriate amount of cross-linking agent can provide more viscoelasticity and lower loss factor. This is why when the additional force is removed from the HG composite hydrogel with 0.5% (w/v) AFnSi cross-linker, compared with the HG composite hydrogel with 1.0% (w/v) AFnSi cross-linker ratio, it can restore its original shape with minimal energy loss ( Figure 10 ). Previous literature has pointed out that the mechanical properties of the extracellular matrix have a profound impact on the progression of various cellular functions, such as message transduction, gene expression, proliferation, differentiation, and extracellular matrix secretion. Human bones and cartilage are often subject to compressive behavior from daily use. Therefore, it is very beneficial to use composite hydrogels HG+0.5% AFnSi and HG+1.0% AFnSi as tissue engineering scaffolds in vitro and in vivo.

Study on Degradation of HG Composite Hydrogel

Biodegradability is crucial for tissue engineering scaffolds, allowing them to be modified for regeneration processes or to release encapsulated bioactive molecules. In many cases, engineered hydrogel-based scaffolds are designed to degrade in the body at about the same rate as new tissue is formed after implantation. The slow degradation process can provide a constant culture environment, which will promote cell migration and differentiation activity, providing sufficient time for tissue regeneration. Adding enzymes or chemicals to the solution can trigger hydrogel degradation. For example, hyaluronidase degrades hyaluronic acid over a short period of time. Due to this low long-term viability, it is difficult to complete the long and complex regeneration process of cartilage

The enzymatic degradation results of hyaluronidase on HG composite hydrogel are shown in Figure 12 , which can degrade the HAMA component in HG composite hydrogel. These results were studied to determine how different concentrations of AFnSi cross-linker affect the degradation rate. As expected, increasing the concentration of AFnSi cross-linker in the HG composite hydrogel from 0% to 1.0% (w/v) slowed down the degradation rate. After 30 days, the HG composite hydrogel alone showed 58% degradation at 37°C and 2.5 U/ml hyaluronidase, while the composite hydrogels of HG+0.1% AFnSi and HG+0.5% AFnSi showed respectively ~46% and ~25% degradation. In addition, the degradation rate of HG+1.0% AFnSi composite hydrogel under 37°C and 2.5 U/ml hyaluronidase enzyme degradation is very close to the degradation rate of HG composite hydrogel alone without hyaluronidase.

The enzyme degradation rate of HG composite hydrogel has been proven to be related to its swelling rate, microstructure, and cross-linking degree. For example, the degradation data confirmed the hypothesis that the degradation rate would be inversely proportional to the compression modulus of the HG composite hydrogel. Therefore, the higher cross-linking degree and compression modulus of HG+0.5% AFnSi and HG+1.0% AFnSi composite hydrogels will decrease as the degradation rate increases. However, the optimal HG cross-linked composite hydrogel system with AFnSi cross-linker should have improved stability. Qualitative to protect biohydrogels from rapid degradation while providing long-term mechanical support.

Evaluation of cell survival rate of human adipose stem cells on HG composite hydrogel

During five days of culture, cell viability was evaluated in samples of hyaluronic acid (HA) as a control group and other photocrosslinked composite hydrogels containing HG, HG+0.5% AFnSi, and HG+1.0% AFnSi. , as shown in Figure 13 . All groups were cultured in basal medium at 37°C and 5% _CO2 , and the mitochondrial conversion of tetrazolium salts to water-soluble formazan crystals in the growing cells was assessed by MTS assay. Compared with the culture method of the HA control group, the cell viability of human adipose stem cells cultured in cross-linked HG hydrogels (HG alone, HG+0.5 AFnSi, and HG+1% AFnSi) was not affected; from 0 By day 5, they were still stably maintained or increased compared with the control group. In other words, no cytotoxic changes were found between HA and HG composite hydrogels.

In addition, human adipose stem cells loaded in a three-dimensional environment were used to observe the biohydrogel in the basic culture medium through fluorescence imaging, as shown in Figure 14 . Live/Dead assay was used to determine whether these HG hydrogels would affect cell viability behavior, including photo-crosslinked HA and HG, HG+0.5% AFnSi, and HG+1.0% AFnSi. Composite hydrogel.

The results in all groups showed that the majority of human adipose stem cells were still alive on day 1, with only a few dead cells seen. After 5 days of culture, the number of dead cells increased slightly in all groups, including photo-crosslinked composite hydrogels of HA and HG, HG+0.5% AFnSi, and HG+1.0% AFnSi. In summary, human adipose stem cells are not adversely affected by culturing with HG hydrogel scaffolds with 0.5% and 1% (w/v) AFnSi cross-linker, and MTS assays confirm that these hydrogels The scaffold has good cytocompatibility required for further biological studies. Among them, the HG hydrogel with 0.5% AFnSi cross-linker can maintain a higher cell survival rate. therefore, HG hydrogel scaffolds containing 0.5% AFnSi cross-linker have low cytotoxicity and can be used to evaluate chondrogenic differentiation ability in vitro.

Chondrogenic differentiation ability of HG composite hydrogel

Cell behavior in the tissue microenvironment is strongly affected by the extracellular matrix and soluble factors. In particular, the extracellular matrix is made of fibrous proteins such as collagen, fibronectin, and elastin, which provide sufficient stiffness for the matrix in the body in the range of 1 kPa to several hundred kPa. Previous studies have also reported that adipose stem cells respond to matrix stiffness, as moderate stiffness (6-8 kPa) tends to induce better chondrogenic differentiation and causes cells to synthesize more hyaline-like cartilage matrix in the first 5 days. In addition, bioinspired hydrogel structures have been previously studied to modulate cell behavior and responses through mechanical property optimization or hydrogel stability enhancement, and nanoparticles can serve as cross-linkers when added to hydrogels. , can be used to modulate biomimetic 3D environments to recreate the complexity of native tissues for potential regenerative medicine.

The chondrogenic differentiation results of these HG composite hydrogels on gene expression and acidified glycosaminoglycan (sGAG) formation in human adipose stem cells are shown in Figures 15 and 16 . The cartilage of human adipose stem cells seeded on hyaluronic acid (HA), photo-crosslinked composite hydrogel HG and HG+0.5% AFnSi in basal medium as controls was analyzed on days 1, 3, 5, and 7. differentiation. In Figure 15 , the RT-PCR method was used to determine the relative expression levels of genes such as SOX-9, Aggrecan, type II collagen, and type I collagen genes. Look for any upregulation of these genes at various time points. SOX-9 is a marker gene for early cartilage differentiation, which was significantly up-regulated on the HG+0.5% AFnSi photo-cross-linked composite hydrogel on day 5 ( Figure 15a ). On the 7th day, the SOX-9 gene expression of HG+0.5% AFnSi increased by approximately 3.5 and 2.5 times compared with HA and HG alone. Type II collagen genes are known to be involved in the mid-to-late stages of cartilage differentiation. In fact, compared with the control group HA, the upregulation of type II collagen gene was observed in the photo-cross-linked composite hydrogel HG and HG+0.5% AFnSi from days 3 to 7, as shown in Figure 15c Show. At the same time, the aggrecan gene was also identified as a late marker gene for cartilage differentiation. The experimental data in Figure 15b shows that compared with the control group HA, the performance of aggrecan in the photo-cross-linked composite hydrogel HG and HG+0.5% AFnSi from days 3 to 7 is up-regulated. In summary, compared with the HA control group and HG alone, the photo-cross-linked composite hydrogel of HG+0.5% AFnSi showed that the expression levels of SOX-9, type II collagen, and aggrecan genes were at a higher level. The most significant increase occurred from days 3 to 7. In contrast, expression of the type 1 collagen gene is known as a fibrocartilage marker gene. When human adipose stem cells were cultured in photo-cross-linked composite hydrogels HG and HG+0.5% AFnSi, compared with the control group HA, the expression of type I collagen gene in the basal medium from day 1 to day 7 Significantly downregulated.

Furthermore, sGAG was found to be an essential extracellular matrix component in articular cartilage, and the amount of sGAG produced was found to be proportional to the degree of chondrogenesis. Therefore, the DMMB assay was used to quantify extracellular matrix production, expressed by sGAG levels. It is worth noting that in adipose stem cells cultured with photo-cross-linked composite hydrogel HG and HG+0.5% AFnSi, compared with the control group HA, the total amount of sGAG and the average amount of sGAG/DNA increased on day 5 and It was significantly higher on the 7th day, as shown in Figure 16a and Figure 16b .

Type II collagen is also an important component of articular cartilage. In order to maintain the differentiated shape of articular cartilage and related secretory activities, type II collagen provides signaling molecules. As expected, it can be seen that compared with HG and HA alone, the total amount of type II collagen and type II collagen/DNA content of the photo-crosslinked composite hydrogel of HG+0.5% AFnSi showed a significant increase. , as shown in Figure 16c and Figure 16d . Based on the proliferation results measured by MTS and the results of the cartilage differentiation gene study and the results of the sGAG and type II collagen message molecules secreted by the cartilage, it can be determined that the photo-crosslinked composite hydrogel of HG+0.5% AFnSi is not only in the basal medium When cultured, it provides an environment for the proliferation of human adipose stem cells, and can also support the production of sGAG and the secretion of type II collagen through cartilage differentiation. Overall, as demonstrated by these findings, the addition of 0.5% AFnSi cross-linker to HG composite hydrogel scaffolds significantly enhanced chondrogenic differentiation of human adipose stem cells.

Example 2

The materials and methods in this example are roughly the same as in Example 1, except that the concentration of the GelMA hydrogel solution in the preparation process of the HG composite hydrogel was adjusted to 15% (w/v) and the following tests were performed.

Swelling rate test of HG composite hydrogel

The swell rate describes the extent to which a hydrogel scaffold expands its volume. Hydrogel scaffolds with higher swell rates can stimulate cell migration and promote the transport of nutrients required for cell growth within the scaffold. However, a higher swelling ratio indicates a less cross-linked structure. In this example, HG composite hydrogel scaffolds with different concentrations of AFnSi cross-linking agent such as 0, 1.0, and 3.0% (w/v) were placed in a cylindrical plastic mold (inner diameter: 10 mm) and exposed in UV light. Then weigh the net weight of the freeze-dried hydrogel scaffold (W _d ). Then, all samples were immersed in PBS (pH=7.4) for 24 hours to reach an equilibrium swelling state. After removing the hydrogel scaffold from PBS, gently blot it dry with Kimwipes (lens paper) to remove residual liquid, and finally weigh the net weight (W _i ) of the hydrogel scaffold that has achieved swelling balance. The overflow rate is defined in terms of the percentage increase in weight.

Swelling rate (%)=(W _i -W _d )/W _d ×100%

The swelling rates of different hydrogel ratios are shown in Figure 17 . From the data, it was found that the addition of AFnSi slightly weakened the water-holding capacity of the hydrogel scaffold. This phenomenon may be due to the increased degree of cross-linking that reduces the swelling rate, or because the hydrogel scaffold is filled with AFnSi, which hinders the diffusion of water molecules into the hydrogel.

Rheological properties of HG composite hydrogel

HG composite hydrogel (2% (w/v) HAMA hydrogel with 15% (w/ v) The volume ratio of GelMA hydrogel is 2:1) Perform a rheometer test and the results are shown in Figure 18 . It can be seen from the data that the concentrations of 3% and 5% have a significant increase in strength, but at the same time the data are quite close. It is speculated that it may be caused by adding too much AFnSi dispersed in the water glue and the amount has been saturated, resulting in uneven dispersion. .

Cytotoxicity of HG composite hydrogel

Human adipose stem cells were cultured with light-cured HG composite hydrogel scaffold for 1, 3, and 5 days before XTT cell survival rate test was performed. Cells alone were used as a control group to compare with hyaluronic acid (HA) alone, HAMA, and other light-cured HG composite hydrogel scaffolds with different concentrations of AFnSi cross-linking agents such as 0%, 1%, and 3%. The results are shown in Figure 19 . As can be seen from the figure, there is no difference in the toxicity of cross-linked HAMA from other groups on the first day, but there is a gap in other groups after the third day. It is speculated that HAMA has more compound hydrogels than HA or HG. Some double bonds may result in decreased cell viability. In short, on the 5th day of culture, the data of HA, G1, G2, G3 and the control group (pure cells) were almost the same. Demonstrated no cytotoxicity.

Example 3

The materials and methods in this example are basically the same as those in Example 1. However, when preparing the HG composite hydrogel, more changes in the concentrations and ratios of HAMA and GelMA were tried, and their application in bioprinting was studied. ) field of application.

Extrusion form of HG composite hydrogel on 3D printer

The extrusion form of HG composite hydrogel on the 3D printer is shown in Figure 20. It can be seen that at the same concentration of 30% GelMA + 2% HAMA, the ratios are 2:1, 3:1 and 4:1. , the extruded water glue is linear and uninterrupted. This feature can make the extruded structure more stable.

The printing results are then used to determine the proportion to be used. As can be seen from Figure 21 , the higher the 30% GelMA proportion, the better the stackability and printing results. However, the higher the GelMA proportion, the more difficult it is to configure AFnSi when configuring water glue. scattered, so the ratio 3:1 was selected for subsequent tests.

Scanning electron microscope (SEM) image of HG composite hydrogel

Porous networks are closely related to cell migration, proliferation, and blood vessel formation. The porous network structure can help guide and promote the formation of new tissue. The larger the pore size, the more pores can allow the scaffold and surrounding tissue to grow into each other and improve the mechanical stability of the implant. And materials with high porosity enable efficient release of growth factors, such as proteins or culture fluids. After freeze-drying the 3D extruded hydrogel scaffold, the surface and internal structure of the scaffold were analyzed through scanning electron microscopy. Figures 22 and 23 show that the scaffold has a porous network structure on the surface and inside, which helps cells Growth, differentiation and attachment.

Analysis of cell survival rate of HG composite hydrogel

The hydrogel scaffold was mixed with human adipose stem cells and then 3D printed into a grid shape, and cultured for 1 or 3 days before live/dead staining analysis was performed. The results are shown in Figure 24 . The experimental results confirm that cells on the hydrogel scaffold can still survive and grow over time after 3D printing.

Chondrification results of HG composite hydrogel

The sGAG of the 3D extruded hydrogel scaffold was quantified by DMMB assay on days 5 and 7, and the degree of chondrogenesis was determined by the amount of sGAG produced. The results are shown in Figure 25 . It can be seen from the figure that the sGAG test on the 5th day and the 7th day has a good chondrification effect in the environment of the 3D extruded hydrogel scaffold added with AFnSi cross-linking agent.

One skilled in the art will quickly appreciate that the present invention can readily achieve its objectives and obtain the results and advantages mentioned, and those contained therein. The methods and compositions, their manufacturing procedures and methods and their uses in the present invention are representative of preferred embodiments, which are exemplary and are not limited to the field of the present invention. Those familiar with the art will recognize modifications and other uses. These modifications are contained in the spirit of the invention and are defined in the patent application scope. The content description and embodiments of the present invention are disclosed in detail to enable anyone skilled in the art to make and use the present invention. Even if there are various changes, modifications, and improvements, they should still be regarded as not departing from the present invention. spirit and scope.

All patents and publications mentioned in the specification are based on the general art in the field related to the invention. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The inventions suitably illustrated herein may be practiced in the absence of any requirement, or in the absence of any number of requirements, limitations or limitations not specifically disclosed herein. The terms and expressions used are for description rather than limitation of the specification, and there is no intention to use such terms and expressions to exclude any features or parts thereof that are equivalent to those shown and described. However, it should be understood that in this specification, Various changes are possible within the patentable scope of an invention. Therefore, it should be understood that although the present invention has been specifically disclosed based on the preferred embodiments and optional features, those skilled in the art will still modify and change the disclosed content, and such modifications and changes are still subject to the patent application of the present invention. within the range.

<110> Kaohsiung Medical University

<120> Composite hydrogel composition, its preparation method and its use

<130> 3859-KMU-TW

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<170> PatentIn version 3.5

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Claims (10)

A composite hydrogel composition includes hyaluronic acid methacryloyl, gelatin methacryloyl, and acrylate functionalized nanometer silica. The composition of claim 1, wherein the content of acrylate functionalized nanosilica is 0.1 to 5.0% (w/v). The composition of claim 1, wherein the volume ratio of methacrylate hyaluronic acid and methacrylate gelatin ranges from 1:4 to 4:1. The composition of claim 1, further comprising stem cells with cartilage differentiation ability. The composition of claim 4, wherein the stem cells with cartilage differentiation ability are adipose stem cells. A method for preparing the composition of claim 1, comprising the following steps: (a) Preparing a methacrylate hyaluronic acid hydrogel solution; (b) Preparing a methacrylate gelatin hydrogel solution; (c) Preparing acrylate functionalized nanosilica; (d) Combine the methacrylate hyaluronic acid hydrogel solution obtained in step (a), the methacrylate gelatin hydrogel solution obtained in step (b), and the acrylate functional group obtained in step (c) Mixed nanosilica and lithium phenyl-2,4,6-trimethylbenzoylphosphinate into a composite hydrogel solution; and (e) Light-curing the composite hydrogel solution obtained in step (d). The method of claim 6, wherein the concentration range of the methacrylate hyaluronic acid hydrogel solution obtained in step (a) is 0.5 to 3.0% (w/v); the methacrylate obtained in step (b) The concentration range of the ester gelatin hydrogel solution is 5 to 30% (w/v); the concentration range of the acrylate functionalized nanosilica obtained in step (c) is 0.1 to 5.0% (w/v) ; And in step (d), the volume ratio of the methacrylate hyaluronic acid hydrogel solution and the methacrylate gelatin hydrogel solution ranges from 1:4 to 4:1. The use of a composition according to claim 1 in preparing a pharmaceutical composition for treating cartilage defects. The use of claim 8, wherein the pharmaceutical composition is a three-dimensional scaffold loaded with stem cells having chondrogenic differentiation ability. The use of claim 9, wherein the stem cells with cartilage differentiation ability are adipose stem cells.

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