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ItemAssessing monocyte phenotype in poly(γ-glutamic acid) hydrogels formed by orthogonal thiol–norbornene chemistry(IOP, 2021-07) Kim, Min Hee; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyHydrogels with tunable properties are highly desirable in tissue engineering applications as they can serve as artificial extracellular matrix to control cellular fate processes, including adhesion, migration, differentiation, and other phenotypic changes via matrix induced mechanotransduction. Poly(γ-glutamic acid) (PGA) is an natural anionic polypeptide that has excellent biocompatibility, biodegradability, and water solubility. Moreover, the abundant carboxylic acids on PGA can be readily modified to introduce additional functionality or facilitate chemical crosslinking. PGA and its derivatives have been widely used in tissue engineering applications. However, no prior work has explored orthogonal crosslinking of PGA hydrogels by thiol-norbornene (NB) chemistry. In this study, we report the synthesis and orthogonal crosslinking of PGA-norbornene (PGANB) hydrogels. PGANB was synthesized by standard carbodiimide chemistry and crosslinked into hydrogels via either photopolymerization or enzymatic reaction. Moduli of PGA hydrogels were readily tuned by controlling thiol-NB crosslinking conditions or stoichiometric ratio of functional groups. Orthogonally crosslinked PGA hydrogels were used to evaluate the influence of mechanical cues of hydrogel substrate on the phenotype of naïve human monocytes and M0 macrophages in 3D culture. ItemDual 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 TechnologyGelatin 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. ItemEnzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells(Elsevier, 2017-01-15) Liu, Hung-Yi; Greene, Tanja; Lin, Tsai-Yu; Dawes, Camron S.; Korc, Murray; Lin, Chien- Chi; Biomedical Engineering, School of Engineering and TechnologyThe complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are 'activated' by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6h to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness. STATEMENT OF SIGNIFICANCE: Hydrogels with spatial-temporal controls over crosslinking kinetics (i.e., dynamic hydrogel) are increasingly being developed for studying mechanobiology in 3D. The general principle of designing dynamic hydrogel is to perform cell encapsulation within a hydrogel network that allows for postgelation modification in gel crosslinking density. The enzyme-mediated in situ gel stiffening is innovative because of the specificity and efficiency of enzymatic reaction. Although tyrosinase has been used for hydrogel crosslinking and in situ cell encapsulation, to the best of our knowledge tyrosinase-mediated DOPA formation has not been explored for in situ stiffening of cell-laden hydrogels. Furthermore, the current work provides a gradual matrix stiffening strategy that may more closely mimic the process of tumor development. ItemHeparinized Gelatin-Based Hydrogels for Differentiation of Induced Pluripotent Stem Cells(American Chemical Society, 2022) Arkenberg, Matthew R.; Koehler, Karl; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyChemically defined hydrogels are increasingly utilized to define the effects of extracellular matrix (ECM) components on cellular fate determination of human embryonic and induced pluripotent stem cell (hESC and hiPSCs). In particular, hydrogels cross-linked by orthogonal click chemistry, including thiol-norbornene photopolymerization and inverse electron demand Diels-Alder (iEDDA) reactions, are explored for 3D culture of hESC/hiPSCs owing to the specificity, efficiency, cytocompatibility, and modularity of the cross-linking reactions. In this work, we exploited the modularity of thiol-norbornene photopolymerization to create a biomimetic hydrogel platform for 3D culture and differentiation of hiPSCs. A cell-adhesive, protease-labile, and cross-linkable gelatin derivative, gelatin-norbornene (GelNB), was used as the backbone polymer for constructing hiPSC-laden biomimetic hydrogels. GelNB was further heparinized via the iEDDA click reaction using tetrazine-modified heparin (HepTz), creating GelNB-Hep. GelNB or GelNB-Hep was modularly cross-linked with either inert macromer poly(ethylene glycol)-tetra-thiol (PEG4SH) or another bioactive macromer-thiolated hyaluronic acid (THA). The formulations of these hydrogels were modularly tuned to afford biomimetic matrices with similar elastic moduli but varying bioactive components, enabling the understanding of each bioactive component on supporting hiPSC growth and ectodermal, mesodermal, and endodermal fate commitment under identical soluble differentiation cues. ItemHydrogel Models with Stiffness Gradients for Interrogating Pancreatic Cancer Cell Fate(MDPI, 2021-03-13) Chang, Chun-Yi; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyPancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and has seen only modest improvements in patient survival rate over the past few decades. PDAC is highly aggressive and resistant to chemotherapy, owing to the presence of a dense and hypovascularized fibrotic tissue, which is composed of stromal cells and extracellular matrices. Increase deposition and crosslinking of matrices by stromal cells lead to a heterogeneous microenvironment that aids in PDAC development. In the past decade, various hydrogel-based, in vitro tumor models have been developed to mimic and recapitulate aspects of the tumor microenvironment in PDAC. Advances in hydrogel chemistry and engineering should provide a venue for discovering new insights regarding how matrix properties govern PDAC cell growth, migration, invasion, and drug resistance. These engineered hydrogels are ideal for understanding how variation in matrix properties contributes to the progressiveness of cancer cells, including durotaxis, the directional migration of cells in response to a stiffness gradient. This review surveys the various hydrogel-based, in vitro tumor models and the methods to generate gradient stiffness for studying migration and other cancer cell fate processes in PDAC. ItemHydrolytically Degradable PEG-Based Inverse Electron Demand Diels-Alder Click Hydrogels(American Chemical Society, 2022) Dimmitt, Nathan H.; Arkenberg, Matthew R.; de Lima Perini, Mariana Moraes; Li, Jiliang; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyHydrogels cross-linked by inverse electron demand Diels-Alder (iEDDA) click chemistry are increasingly used in biomedical applications. With a few exceptions in naturally derived and chemically modified macromers, iEDDA click hydrogels exhibit long-term hydrolytic stability, and no synthetic iEDDA click hydrogels can undergo accelerated and tunable hydrolytic degradation. We have previously reported a novel method for synthesizing norbornene (NB)-functionalized multiarm poly(ethylene glycol) (PEG), where carbic anhydride (CA) was used to replace 5-norbornene-2-carboxylic acid. The new PEGNBCA-based thiol-norbornene hydrogels exhibited unexpected fast yet highly tunable hydrolytic degradation. In this contribution, we leveraged the new PEGNBCA macromer for forming iEDDA click hydrogels with [methyl]tetrazine ([m]Tz)-modified macromers, leading to the first group of synthetic iEDDA click hydrogels with highly tunable hydrolytic degradation kinetics. We further exploited Tz and mTz dual conjugation to achieve tunable hydrolytic degradation with an in vitro degradation time ranging from 2 weeks to 3 months. Finally, we demonstrated the excellent in vitro cytocompatibility and in vivo biocompatibility of the new injectable PEGNBCA-based iEDDA click cross-linked hydrogels. ItemImproving Cross-linking of Degradable Thiol-acrylate Hydrogels via Peptide Design(Office of the Vice Chancellor for Research, 2015-04-17) Bragg, John C.; Lin, Chien-ChiHydrogels fabricated from poly (ethylene glycol) (PEG) based macromers are ideal for drug delivery and tissue engineering applications. Recently, a new visible light-mediated photopolymerization scheme was developed to fabricate cytocompatible and degradable poly (ethylene glycol)-diacrylate (PEGDA) hydrogels. Co-polymerization of mono-cysteine peptides (e.g. CRGDS) with PEGDA offers the gels with cell adhesion property. However, this approach causes significant reduction in network crosslinking density, in part due to chain transfer of thiols to acrylates. The goal of the project is to improve the network cross-linking efficiency of this peptide-immobilized PEGDA hydrogel for cell culture. We hypothesized that the incorporation of bi-functional bis-cysteine peptides or silk fibroin will produce hydrogels with enhanced stiffness. The shear moduli of the gels were characterized via oscillatory rheometry in strain-sweep (0.1-5%) mode. Hydrolytic degradation of the gels as a function of time was also evaluated by rheometry. Cytocompatibility of the hydrogel system will be assessed by in situ encapsulation of 3T3 fibroblasts. Cell metabolic activity was determined by Alamar-Blue assay. We found that the bis-cysteine peptide enhanced gel crosslinking, as compared with mono-cysteine peptide. Incorporation of silk fibroin protein also exhibited enhancement in gel stiffness. However, the optimum concentration of incorporated silk fibroin presented an increased shear modulus compared to gels containing only the mono-cysteine peptide. Ongoing work is focused on fine-tuning gel formulations and degradation, as well as on evaluating the cytocompatibility of these visible-light cured thiol-acrylate hydrogels. ItemRecent advances in bio-orthogonal and dynamic crosslinking of biomimetic hydrogels(Royal Society of Chemistry, 2020-09-21) Arkenberg, Matthew R.; Nguyen, Han D.; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyIn recent years, dynamic, 'click' hydrogels have been applied in numerous biomedical applications. Owing to the mild, cytocompatible, and highly specific reaction kinetics, a multitude of orthogonal handles have been developed for fabricating dynamic hydrogels to facilitate '4D' cell culture. The high degree of tunability in crosslinking reactions of orthogonal 'click' chemistry has enabled a bottom-up approach to install specific biomimicry in an artificial extracellular matrix. In addition to click chemistry, highly specific enzymatic reactions are also increasingly used for network crosslinking and for spatiotemporal control of hydrogel properties. On the other hand, covalent adaptable chemistry has been used to recapitulate the viscoelastic component of biological tissues and for formulating self-healing and shear-thinning hydrogels. The common feature of these three classes of chemistry (i.e., orthogonal click chemistry, enzymatic reactions, and covalent adaptable chemistry) is that they can be carried out under ambient and aqueous conditions, a prerequisite for maintaining cell viability for in situ cell encapsulation and post-gelation modification of network properties. Due to their orthogonality, different chemistries can also be applied sequentially to provide additional biochemical and mechanical control to guide cell behavior. Herein, we review recent advances in the use of orthogonal click chemistry, enzymatic reactions, and covalent adaptable chemistry for the development of dynamically tunable and biomimetic hydrogels. ItemTargeted tissue engineering: hydrogels with linear capillary channels for axonal regeneration after spinal cord injury(Medknow Publications, 2018-04) Liu, Shengwen; Blesch, Armin; Neurological Surgery, School of MedicineSpinal cord injury (SCI) frequently results in the permanent loss of function below the level of injury due to the failure of axonal regeneration in the adult mammalian central nervous system (CNS). The limited intrinsic growth capacity of adult neurons, a lack of growth-promoting factors and the multifactorial inhibitory microenvironment around the lesion site contribute to the lack of axonal regeneration. Strategies such as transplantation of cells, delivery of bioactive compounds and gene transfer have been investigated as a means to promote axonal regrowth through the lesion, to form new synaptic connections and to improve functional outcomes. Although growth of some axonal populations can be robustly enhanced by cellular implants alone or in combination with neurotrophic factors, axons usually extend in random orientation and even reverse growth direction in the lesion site (Figure 1A) (Gros et al., 2010; Günther et al., 2015). Thus, regenerating axons often fail to approach the distal edge of the lesion site, a pre-requisite for proper contact with spared host neurons. The lack of a 3-dimensional organization in the injury site is therefore an additional barrier for successful axonal bridging. Two approaches, physical guidance through structured scaffolds and chemical guidance by growth factor gradients, have emerged as potential means to provide directional cues for axonal growth through the lesion.