Browsing by Subject "Gelatin"

Now showing 1 - 4 of 4
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    Dual Functionalization of Gelatin for Orthogonal and Dynamic Hydrogel Cross-Linking
    (American Chemical Society, 2021) Kim, Min Hee; Nguyen, Han; Chang, Chun-Yi; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    Gelatin based hydrogels are widely used in biomedical fields owing to its abundance of bioactive motifs that support cell adhesion and matrix remodeling. While inherently bioactive, unmodified gelatin exhibits temperature-dependent rheology and solubilizes at body temperature, making it unstable for three-dimensional (3D) cell culture. Therefore, the addition of chemically reactive motifs is required to render gelatin-based hydrogels with highly controllable crosslinking kinetics and tunable mechanical properties that are critical for 3D cell culture. This article provides a series of methods toward establishing orthogonally crosslinked gelatin-based hydrogels for dynamic 3D cell culture. In particular, we prepared dually functionalized gelatin macromers amenable for sequential, orthogonal covalent crosslinking. Central to this material platform is the synthesis of norbornene-functionalized gelatin (GelNB), which forms covalently crosslinked hydrogels via orthogonal thiol-norbornene click crosslinking. Using GelNB as the starting material, we further detail the methods for synthesizing gelatin macromers susceptible to hydroxyphenylacetic acid (HPA) dimerization (i.e., GelNB-HPA) and hydrazone bonding (i.e., GelNB-CH) for on-demand matrix stiffening. Finally, we outline the protocol for synthesizing a gelatin macromer capable of adjusting hydrogel stress-relaxation via boronate ester bonding (i.e., GelNB-BA). The combinations of these orthogonal chemistries affords a wide range of gelatin based hydrogels as biomimetic matrices in tissue engineering and regenerative medicine applications.
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    Modular crosslinking of gelatin based thiol-norbornene hydrogels for in vitro 3D culture of hepatic cells
    (ACS Biomaterials Science and Engineering, 2015-10-21) Greene, Tanja L.; Lin, Chien-Chi; Xie, Dong; Dai, Guoli; Yoshida, Ken
    As liver disease becomes more prevalent, the development of an in vitro culture system to study disease progression and its repair mechanisms is essential. Typically, 2D cultures are used to investigate liver cell (e.g., hepatocyte) function in vitro; however, hepatocytes lose function rapidly when they were isolated from the liver. This has promoted researchers to develop 3D scaffolds to recreate the natural microenvironment of hepatic cells. For example, gelatin-based hydrogels have been increasingly used to promote cell fate processes in 3D. Most gelatin-based systems require the use of physical gelation or non-specific chemical crosslinking. Both of these methods yield gelatin hydrogels with highly interdependent material properties (e.g., bioactivity and matrix stiffness). The purpose of this thesis research was to prepare modularly crosslinked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatic cell fate in 3D. The first objective was to establish tunable gelatin-based thiol-norbornene hydrogels and to demonstrate that the mechanical and biological properties of gelatin hydrogels can be independently adjusted. Furthermore, norbornene and heparin dual-functionalized gelatin (i.e., GelNB-Hep) was prepared and used to sequester and slowly release hepatocyte growth factor (HGF). The second objective was to investigate the viability and functions of hepatocytes encapsulated in gelatin-based hydrogels. Hepatocellular carcinoma cells, Huh7, were used as a model cell type to demonstrate the cytocompatibility of the system. The properties of GelNB hydrogels were modularly tuned to systematically evaluate the effects of matrix properties on cell viability and functions, including CYP3A4 activity and urea secretion. The last objective was to examine the effect of heparin immobilization on hepatocyte viability and functions. The conjugation of heparin onto GelNB led to suppressed Huh7 cell metabolic activity and improved hepatocellular functions. This hybrid hydrogel system should provide a promising 3D cell culture platform for studying cell fate processes.
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    Probing osteocyte function in gelatin hydrogels with tunable viscoelasticity
    (American Chemical Society, 2021) Nguyen, Han D.; Sun, Xun; Yokota, Hiroki; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and Technology
    Bone is an attractive site for metastatic cancer cells and has been considered as "soil" for promoting tumor growth. However, accumulating evidence suggests that some bone cells (e.g., osteocytes) can actually suppress cancer cell migration and invasion via direct cell-cell contact and/or through cytokine secretion. Toward designing a biomimetic niche for supporting 3D osteocyte culture, we present here a gelatin-based hydrogel system with independently tunable matrix stiffness and viscoelasticity. In particular, we synthesized a bifunctional macromer, gelatin-norbornene-boronic acid (i.e., GelNB-BA), for covalent cross-linking with multifunctional thiol linkers [e.g., four-arm poly(ethylene glycol)-thiol or PEG4SH] to form thiol-NB hydrogels. The immobilized BA moieties in the hydrogel readily formed reversible boronate ester bonds with 1,3-diols on physically entrapped poly(vinyl alcohol) (PVA). Adjusting the compositions of GelNB-BA, PEG4SH, and PVA afforded hydrogels with independently tunable elasticity and viscoelasticity. With this new dynamic hydrogel platform, we investigated matrix mechanics-induced growth and cytokine secretion of encapsulated MLO-A5 pre-osteocytes. We discovered that more compliant or viscoelastic gels promoted A5 cell growth. On the other hand, cells encapsulated in stiffer gels secreted higher amounts of pro-inflammatory cytokines and chemokines. Finally, conditioned media (CM) collected from the encapsulated MLO-A5 cells (i.e., A5-CM) strongly inhibited breast cancer cell proliferation, invasion, and expression of tumor-activating genes. This new biomimetic hydrogel platform not only serves as a versatile matrix for investigating mechano-sensing in osteocytes but also provides a means to produce powerful anti-tumor CM.