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Fluorescent microparticles for sensing cell microenvironment oxygen levels within 3D scaffolds.

ABSTRACT: We present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automated and non-invasive sensor analysis method based on fluorescence microscopy to measure oxygen concentration in a hydrogel scaffold. We demonstrate that the microparticles are non-cytotoxic and that their response is comparable to that of a traditional dissolved oxygen meter. Microparticle size (5-40 microm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Two methods of calibration were evaluated and revealed that the sensor system enables characterization of a range of hypoxic to hyperoxic conditions relevant to cell and tissue biology (i.e., pO(2) 10-160 mmHg). The calibration analysis also revealed that the microparticles have a high fraction of quenched luminophore (0.90+/-0.02), indicating that the reported approach provides significant advantages for sensor performance. This study thus reports a versatile oxygen-sensing technology that enables future correlations of local oxygen concentration with individual cell response in cultured engineered tissues.

SUBMITTER: Acosta MA

PROVIDER: S-EPMC2861433 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Fluorescent microparticles for sensing cell microenvironment oxygen levels within 3D scaffolds.

Acosta Miguel A MA Ymele-Leki Patrick P Kostov Yordan V YV Leach Jennie B JB

Biomaterials 20090314 17

We present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automa ...[more]

PMID: 19285719

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Project description:High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-β1 (TGF-β1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-β1 (pTGF-β1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-β1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

Dataset Information

Fluorescent microparticles for sensing cell microenvironment oxygen levels within 3D scaffolds.

Publications

Fluorescent microparticles for sensing cell microenvironment oxygen levels within 3D scaffolds.

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