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Item Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells(Wiley, 2017-03) Greene, Tanja; Lin, Tsai-Yu; Andrisani, Oaurania M.; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and TechnologyPhotopolymerization techniques have been widely used to create hydrogels for biomedical applications. Visible light-based photopolymerizations are commonly initiated by type II (i.e., noncleavage-type) photoinitiator in conjunction with a coinitiator. On the other hand, type I photoinitiators (i.e., cleavage type) are rarely compatible with visible light-based initiation due to their limited molar absorbability in the visible light wavelengths. Here, we report visible light initiated orthogonal photoclick crosslinking to fabricate gelatin-norbornene and poly(ethylene glycol)-tetra-thiol hydrogels using either cleavage-type (i.e., lithium acylphosphinate, LAP) or noncleavage-type photoinitiator (i.e., eosin-Y, EY) without the use of a coinitiator. Regardless of the initiator type, the step-growth gelatin-PEG hybrid hydrogels crosslinked and degraded similarly. While both systems exhibited similar cytocompatibility for hepatic progenitor HepaRG cells, gelation initiated by noncleavage-type initiator EY afforded slightly higher degree of hepatic gene expression.Item Enzymatic crosslinking of dynamic hydrogels for in vitro cell culture(2018-04) Arkenberg, Matthew R.; Lin, Chien-ChiStiffening and softening of extracellular matrix (ECM) are critical processes governing many aspects of biological processes. The most common practice used to investigate these processes is seeding cells on two-dimensional (2D) surfaces of varying stiffness. In recent years, cell-laden three-dimensional (3D) scaffolds with controllable properties are also increasingly used. However, current 2D and 3D culture platforms do not permit spatiotemporal controls over material properties that could influence tissue processes. To address this issue, four-dimensional (4D) hydrogels (i.e., 3D materials permitting time-dependent control of matrix properties) are proposed to recapitulate dynamic changes of ECM properties. The goal of this thesis was to exploit orthogonal enzymatic reactions for on-demand stiffening and/or softening of cell-laden hydrogels. The first objective was to establish cytocompatible hydrogels permitting enzymatic crosslinking and stiffening using enzymes with orthogonal reactivity. Sortase A (SrtA) and mushroom tyrosinase (MT) were used sequentially to achieve initial gelation and on-demand stiffening. In addition, hydrogels permitting reversible stiffening through SrtA-mediated peptide ligation were established. Specifically, poly(ethylene glycol) (PEG)-peptide hydrogels were fabricated with peptide linkers containing pendent SrtA substrates. The hydrogels were stiffened through incubation with SrtA, whereas gel softening was achieved subsequently via addition of SrtA and soluble glycine substrate. The second objective was to investigate the role of dynamic matrix stiffening on pancreatic cancer cell survival, spheroid formation, and drug responsiveness. The crosslinking of PEG-peptide hydrogels was dynamically tuned to evaluate the effect of matrix stiffness on cell viability and function. Specifically, dynamic matrix stiffening inhibited cell proliferation and spheroid formation, while softening the cell-laden hydrogels led to significant increase in spheroid sizes. Matrix stiffness also altered the expression of chemoresistance markers and responsiveness of cancer cells to gemcitabine treatment. markers and responsiveness of cancer cells to gemcitabine treatment.Item Improving gelation efficiency and cytocompatibility of visible light polymerized thiol-norbornene hydrogels via addition of soluble tyrosine(RSC, 2017-03) Shih, Han; Liu, Hung-Yi; Lin, Chien-Chi; Biomedical Engineering, School of Engineering and TechnologyHydrogels immobilized with biomimetic peptides have been used widely for tissue engineering and drug delivery applications. Photopolymerization has been among the most commonly used techniques to fabricate peptide-immobilized hydrogels as it offers rapid and robust peptide immobilization within a crosslinked hydrogel network. Both chain-growth and step-growth photopolymerizations can be used to immobilize peptides within covalently crosslinked hydrogels. A previously developed visible light mediated step-growth thiol-norbornene gelation scheme has demonstrated efficient crosslinking of hydrogels composed of an inert poly(ethylene glycol)-norbornene (PEGNB) macromer and a small molecular weight bis-thiol linker, such as dithiothreitol (DTT). Compared with conventional visible light mediated chain-polymerizations where multiple initiator components are required, step-growth photopolymerized thiol-norbornene hydrogels are more cytocompatible for the in situ encapsulation of radical sensitive cells (e.g., pancreatic β-cells). This contribution explored visible light based crosslinking of various bis-cysteine containing peptides with macromer 8-arm PEGNB to form biomimetic hydrogels suitable for in situ cell encapsulation. It was found that the addition of soluble tyrosine during polymerization not only significantly accelerated gelation, but also improved the crosslinking efficiency of PEG-peptide hydrogels as evidenced by a decreased gel point and enhanced gel modulus. In addition, soluble tyrosine drastically enhanced the cytocompatibility of the resulting PEG-peptide hydrogels, as demonstrated by in situ encapsulation and culture of pancreatic MIN6 β-cells. This visible light based thiol-norbornene crosslinking mechanism provides an attractive gelation method for preparing cytocompatible PEG-peptide hydrogels for tissue engineering applications.Item Recent advances in crosslinking chemistry of biomimetic poly(ethylene glycol) hydrogels(2015-01-01) Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and TechnologyThe design and application of biomimetic hydrogels have become an important and integral part of modern tissue engineering and regenerative medicine. Many of these hydrogels are prepared from synthetic macromers (e.g., poly(ethylene glycol) or PEG) as they provide high degrees of tunability for matrix crosslinking, degradation, and modification. For a hydrogel to be considered biomimetic, it has to recapitulate key features that are found in the native extracellular matrix, such as the appropriate matrix mechanics and permeability, the ability to sequester and deliver drugs, proteins, and or nucleic acids, as well as the ability to provide receptor-mediated cell-matrix interactions and protease-mediated matrix cleavage. A variety of chemistries have been employed to impart these biomimetic features into hydrogel crosslinking. These chemistries, such as radical-mediated polymerizations, enzyme-mediated crosslinking, bio-orthogonal click reactions, and supramolecular assembly, may be different in their crosslinking mechanisms but are required to be efficient for gel crosslinking and ligand bioconjugation under aqueous reaction conditions. The prepared biomimetic hydrogels should display a diverse array of functionalities and should also be cytocompatible for in vitro cell culture and/or in situ cell encapsulation. The focus of this article is to review recent progress in the crosslinking chemistries of biomimetic hydrogels with a special emphasis on hydrogels crosslinked from poly(ethylene glycol)-based macromers.Item Silk fibroin-reinforced hydrogels for growth factor delivery and In Vitro cell culture(2016-12) Bragg, John Campbell; Lin, Chien-ChiA variety of polymers of synthetic origins (e.g., poly(ethylene glycol) or PEG) and naturally derived macromolecules (e.g., silk fibroin or gelatin) have been explored as the backbone materials for hydrogel crosslinking. Purely synthetic hydrogels are usually inert, covalently crosslinked, and have limited degradability unless degradable macromers are synthesized and incorporated into the hydrogel network. Conversely, naturally derived macromers often contain bioactive motifs that can provide biomimicry to the resulting hydrogels. However, hydrogels fabricated from a single macromer often have limitations inherent to the macromer itself. For example, to obtain high modulus PEG-based hydrogels requires an increase in macromer and crosslinker content. This is associated with an increase in radical concentration during polymerization which may cause death of encapsulated cells. Pure gelatin (G) hydrogels have weak mechanical properties and gelatin undergoes thermo-reversible physical gelation. Covalent crosslinking is usually necessary to produce stable gelatin hydrogels, particularly at physiological temperatures. The limitations of these hydrogels may be circumvented by combining them with another macromer (e.g., silk fibroin) to form hybrid hydrogels. Silk fibroin (SF) from Bombyx mori silkworms offers high mechanical strength, slow enzymatic degradability, and can easily form physical hydrogels. The first objective of this thesis was to evaluate the effect of sonication and the presence of synthetic polymer (e.g., poly (ethylene glycol) diacrylate or PEGDA) or natural macromer (e.g., gelatin) on SF physical gelation kinetics. SF physical gelation was assessed qualitatively via tilt tests. Gelation of pure SF solutions was compared to mixtures of SF and PEGDA or G, both with or without sonication of SF prior to mixing. The effect of gelatin on SF gelation was also evaluated quantitatively via real time in situ rheometry. Sonication accelerated gelation of SF from days to hours or minutes depending on SF concentration and sonication intensity. Both PEGDA and G were shown to accelerate SF physical gelation when added to SF and sonicated SF (SSF) solutions. The second objective was to develop a simple strategy to modulate covalently crosslinked PEG-based hydrogel properties by physically entrapping silk fibroin. The physical entrapment of silk fibroin provides an alternative method to increase gel storage modulus (G’) without the cytotoxic effect of increasing macromer and crosslinker concentration, or altering degradation kinetics by increasing co-monomer concentration. The effect of SF entrapment on gel physical and mechanical properties, as well as hydrolytic degradation and chemical gelation kinetics were characterized. SF physical crosslinking within the PEG-based network was shown to increase gel storage moduli by two days after gel fabrication. There was no change hydrolytic degradation rate associated with the increased moduli. SF entrapment did not affect gelation efficiency, but did alter gel physical properties. The third objective of this thesis was to develop a silk-gelatin in situ forming hybrid hydrogel for affinity-based growth factor sequestration and release and in vitro cell culture. SF provides mechanical strength and stability, whereas G contains bioactive motifs that can provide biomimicry to the gel network. Hydrogel G’ and its dependency on temperature, SF processing conditions, and secondary in situ chemical crosslinking (i.e., genipin crosslinking) were studied. Gelatin can be conjugated with heparin, a glycosaminoglycan, to impart growth factor (GF) binding affinity. Growth factor sequestration and release were evaluated in a pair of designed experiments. The hybrid gels were evaluated as substrates for human mesenchymal stem cell proliferation.Item Step-growth thiol-ene photopolymerization to form degradable, cytocompatible and multi-structural hydrogels(2014-01-17) Shih, Han; Lin, Chien-Chi; Xie, Dong; Bottino, MarcoHydrogels prepared from photopolymerization have been used for a variety of tissue engineering and controlled release applications. Polymeric biomaterials with high cytocompatibility, versatile degradation behaviors, and diverse material properties are particularly useful in studying cell fate processes. In recent years, step-growth thiol-ene photochemistry has been utilized to form cytocompatible hydrogels for tissue engineering applications. This radical-mediated gelation scheme utilizes norbornene functionalized multi-arm poly(ethylene glycol) (PEGNB) as the macromer and di-thiol containing molecules as the crosslinkers to form chemically crosslinked hydrogels. While the gelation mechanism was well-described in the literature, the network properties and degradation behaviors of these hydrogels have not been fully characterized. In addition, existing thiol-ene photopolymerizations often used type I photoinitiators in conjunction with an ultraviolet (UV) light source to initiate gelation. The use of cleavage type initiators and UV light often raises biosafety concerns. The first objective of this thesis was to understand the gelation and degradation properties of thiol-ene hydrogels. In this regard, two types of step-growth hydrogels were compared, namely thiol-ene hydrogels and Michael-type addition hydrogels. Between these two step-growth gel systems, it was found that thiol-ene click reactions formed hydrogels with higher crosslinking efficiency. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEGNB macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, it was found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation rate of thiol-ene hydrogels, different macromer contents and peptide crosslinkers with different amino acid sequences were used. A chymotrypsin-sensitive peptide was also used as part of the hydrogel crosslinkers to render thiol-ene hydrogels enzymatically degradable. The second objective of this thesis was to develop a visible light-mediated thiol-ene hydrogelation scheme using a type II photoinitiator, eosin-Y, as the only photoinitiator. This approach eliminates the incorporation of potentially cytotoxic co-initiator and co-monomer that are typically used with a type II initiator. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, it was found that the visible light-mediated thiol-ene hydrogels were highly cytocompatible for human mesenchymal stem cells (hMSCs) and pancreatic MIN6 beta-cells. It was also found that eosin-Y could be repeatedly excited for preparing step-growth hydrogels with multilayer structures. This new gelation chemistry may have great utilities in controlled release of multiple sensitive growth factors and encapsulation of multiple cell types for tissue regeneration.Item Thiol-norbornene photo-click hydrogels for tissue engineering applications.(Wiley, 2015-02-20) Lin, Chien-Chi; Ki, Chang Seok; Shih, Han; Department of Biomedical Engineering, Purdue School of Engineering and Technology, IUPUIThiol-norbornene (thiol-ene) photo-click hydrogels have emerged as a diverse material system for tissue engineering applications. These hydrogels are cross-linked through light mediated orthogonal reactions between multi-functional norbornene-modified macromers (e.g., poly(ethylene glycol), hyaluronic acid, gelatin) and sulfhydryl-containing linkers (e.g., dithiothreitol, PEG-dithiol, bis-cysteine peptides) using low concentration of photoinitiator. The gelation of thiol-norbornene hydrogels can be initiated by long-wave UV light or visible light without additional co-initiator or co-monomer. The cross-linking and degradation behaviors of thiol-norbornene hydrogels are controlled through material selections, whereas the biophysical and biochemical properties of the gels are easily and independently tuned owing to the orthogonal reactivity between norbornene and thiol moieties. Uniquely, the cross-linking of step-growth thiol-norbornene hydrogels is not oxygen-inhibited, therefore the gelation is much faster and highly cytocompatible compared with chain-growth polymerized hydrogels using similar gelation conditions. These hydrogels have been prepared as tunable substrates for 2D cell culture, as microgels or bulk gels for affinity-based or protease-sensitive drug delivery, and as scaffolds for 3D cell culture. Reports from different laboratories have demonstrated the broad utility of thiol-norbornene hydrogels in tissue engineering and regenerative medicine applications, including valvular and vascular tissue engineering, liver and pancreas-related tissue engineering, neural regeneration, musculoskeletal (bone and cartilage) tissue regeneration, stem cell culture and differentiation, as well as cancer cell biology. This article provides an up-to-date overview on thiol-norbornene hydrogel cross-linking and degradation mechanisms, tunable material properties, as well as the use of thiol-norbornene hydrogels in drug delivery and tissue engineering applications.Item Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture(Elsevier B.V., 2014-01) Hao, Yiting; Shih, Han; Muňoz, Zachary; Kemp, Arika; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and TechnologyWe report here a synthetically simple yet highly tunable and diverse visible light mediated thiol- vinyl gelation system for fabricating cell-instructive hydrogels. Gelation was achieved via a mixed-mode step-and-chain-growth photopolymerization using functionalized 4-arm poly(ethylene glycol) as backbone macromer, eosin-Y as photosensitizer, and di-thiol containing molecule as dual purpose co-initiator/cross-linker. N-vinylpyrrolidone (NVP) was used to accelerate gelation kinetics and to adjust the stiffness of the hydrogels. Visible light (wavelength: 400–700nm) was used to initiate rapid gelation (gel points: ~20 seconds) that reached completion within a few minutes. The major differences between current thiol-vinyl gelation and prior visible light mediated photopolymerization are that: (1) the co-initiator triethanolamine (TEOA) used in the previous systems was replaced with multifunctional thiols and (2) mixed-mode polymerized gels contain less network heterogeneity. The gelation kinetics and gel properties at the same PEG macromer concentration could be tuned by changing the identity of vinyl groups and di-thiol cross-linkers, as well as concentration of cross-linker and NVP. Specifically, acrylate-modified PEG afforded the fastest gelation rate, followed by acrylamide and methacrylate-functionalized PEG. Increasing NVP concentration also accelerated gelation and led to a higher network cross- linking density. Further, increasing di-thiol peptide concentration in the gel formulation increased hydrogel swelling and decreased gel stiffness. Due to the formation of thiol-ether-ester bonds following thiol-acrylate reaction, the gels degraded hydrolytically following a pseudo first order degradation kinetics. Degradation rate was controlled by adjusting thiol or NVP content in the polymer precursor solution. The cytocompatibility and utility of this hydrogel system were evaluated using in situ encapsulation of human mesenchymal stem cells (hMSC). Encapsulated hMSCs remained alive (>90%) throughout the duration of the study and the cells were differentiated down osteogenic lineage with varying degrees by controlling the rate and mode of gel degradation.