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1.
Biomacromolecules ; 17(12): 3872-3882, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27936722

RESUMO

Photopolymerized biomimetic hydrogels with adaptable properties have been widely used for cell and tissue engineering applications. As a widely adopted gel cross-linking method, photopolymerization provides experimenters on-demand and spatial-temporal controls in gelation kinetics. Long wavelength ultraviolet (UV) light initiated photopolymerization is among the most popular methods in the fabrication of cell-laden hydrogels owing to its rapid and relatively mild gelation conditions. The use of UV light, however, still causes concerns regarding its potential negative impacts on cells. Alternatively, visible light based photopolymerization can be used to cross-link cell-laden hydrogels. The majority of visible light based gelation schemes involve photoinitiator, co-initiator, and comonomer. This multicomponent initiation system creates added challenges for optimizing hydrogel formulations. Here, we report a co-initiator/comonomer-free visible light initiated thiol-norbornene photopolymerization scheme to prepare modular biomimetic hydrogels suitable for in situ cell encapsulation. Eosin-Y was used as the sole initiator to initiate modular gelation between synthetic macromers (e.g., thiolated poly(vinyl alcohol) or poly(ethylene glycol)) and functionalized extracellular matrices (ECMs) including norbornene-functionalized gelatin (GelNB) or thiolated hyaluronic acid (THA). These components are modularly cross-linked to afford bioinert (i.e., purely synthetic), bioactive (i.e., using gelatin), and biomimetic (i.e., using gelatin and hyaluronic acid) hydrogels. The stiffness of the hydrogels can be easily tuned without affecting the contents of the bioactive components. Furthermore, the use of naturally derived biomacromolecules (e.g., gelatin and HA) renders these hydrogels susceptible to enzyme-mediated degradation. In addition to demonstrating efficient and tunable visible light mediated gelation, we also utilized this biomimetic modular gelation system to formulate artificial tumor niche and to study the effects of cell density and gel modulus on the formation of pancreatic ductal adenocarcinoma (PDAC) spheroids.


Assuntos
Carcinoma Ductal Pancreático/patologia , Reagentes de Ligações Cruzadas/química , Hidrogéis/química , Norbornanos/química , Neoplasias Pancreáticas/patologia , Esferoides Celulares/patologia , Compostos de Sulfidrila/química , Contagem de Células , Sobrevivência Celular , Humanos , Luz , Polimerização , Engenharia Tecidual , Células Tumorais Cultivadas
2.
Acta Biomater ; 48: 258-269, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27769941

RESUMO

The 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.


Assuntos
Hidrogéis/química , Monofenol Mono-Oxigenase/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Metaloproteinases da Matriz/metabolismo , Miofibroblastos/citologia , Norbornanos/química , Células Estreladas do Pâncreas/citologia , Células Estreladas do Pâncreas/metabolismo , Peptídeos/química , Polietilenoglicóis/química , Polimerização , Proteólise , Compostos de Sulfidrila/química
3.
ACS Biomater Sci Eng ; 1(12): 1314-1323, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33429678

RESUMO

Gelatin-based hydrogels are increasingly used to promote cell fate processes in 3D. Here, we report the use of orthogonal thiol-norbornene photochemistry to prepare modularly cross-linked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatocellular carcinoma cell fate in vitro. In addition to demonstrating the ability to independently tune the mechanical and biological properties of modular gelatin-norbornene (GelNB) hydrogels, we also determined that network cross-linking density plays a key role in the mechanisms of proteolytic gel degradation. During in vitro degradation studies, GelNB hydrogels with lower cross-linking density degraded faster and followed a surface erosion mechanism, whereas dense GelNB hydrogels degraded in a bulk degradation mechanism. Hepatocellular carcinoma cells, Huh7, were encapsulated and grown in GelNB hydrogels with modularly tuned stiffness, bioactive motifs, and heparin content. We systematically evaluated the effect of matrix properties on cell viability and functions in vitro, including CYP3A4 activity and urea secretion. We found that encapsulated Huh7 cells exhibited higher cellular metabolic activity when encapsulated in modular GelNB hydrogels composed of higher gelatin contents or gels with lower stiffness. Interestingly, altering gelatin content and matrix stiffness did not significantly affect hepatocyte-specific cellular functions. To improve cellular function, we prepared norbornene and heparin dual-functionalized gelatin through a two-step synthesis protocol. Heparin-functionalized GelNB (i.e., GelNB-Hep) hydrogels were able to sequester and slowly release hepatocyte growth factor (HGF) in vitro. Finally, the conjugation of heparin on GelNB led to suppressed Huh7 cell metabolic activity and improved CYP3A4 activity and urea secretion.

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