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Spatial control of cell gene expression by siRNA gradients in biodegradable hydrogels.

ABSTRACT: The extracellular environment exposes cells to numerous biochemical and physical signals that regulate their behavior. Strategies for generating continuous gradients of signals in biomaterials may allow for spatial control and patterning of cell behavior, and ultimately aid in the engineering of complex tissues. Short interfering RNA (siRNA) can regulate gene expression by silencing specific mRNA molecules post-transcriptionally, which may be valuable when presented in a continuous gradient for regenerative or therapeutic applications. Here, a biodegradable hydrogel system containing a gradient of siRNA is presented, and its capacity to regulate protein expression of encapsulated cells in a spatially continuous manner is demonstrated. Photocross-linkable dextran hydrogels containing a gradient of siRNA have been successfully fabricated using a dual-programmable syringe pump system, and differential gene silencing in incorporated cells that is sustained over time has been shown using green fluorescent protein as a reporter. This platform technology may be applied in tissue engineering to spatially control biologically relevant cellular processes.

SUBMITTER: Hill MC

PROVIDER: S-EPMC4406766 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Spatial control of cell gene expression by siRNA gradients in biodegradable hydrogels.

Hill Michael C MC Nguyen Minh K MK Jeon Oju O Alsberg Eben E

Advanced healthcare materials 20141222 5

The extracellular environment exposes cells to numerous biochemical and physical signals that regulate their behavior. Strategies for generating continuous gradients of signals in biomaterials may allow for spatial control and patterning of cell behavior, and ultimately aid in the engineering of complex tissues. Short interfering RNA (siRNA) can regulate gene expression by silencing specific mRNA molecules post-transcriptionally, which may be valuable when presented in a continuous gradient for ...[more]

PMID: 25530099

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Project description:Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan's five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. STATEMENT OF SIGNIFICANCE: Gradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

Dataset Information

Spatial control of cell gene expression by siRNA gradients in biodegradable hydrogels.

Publications

Spatial control of cell gene expression by siRNA gradients in biodegradable hydrogels.

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