Abstract
Over the past decade, the use of polymers as platform materials for biomedical applications including tissue engineering has been of rising interest. Recently, the use of naturally derived polysaccharides as 3-D scaffolds for tissue regeneration has shown promising material characteristics; however, due to complexities in composition, morphology, and optical properties, adequate spatial and temporal characterization of cellular behavior in these materials is lacking. Multiphoton microscopy has emerged as a viable tool for performing such quantification by permitting greater imaging depth while simultaneously minimizing un-favorable scattering and producing high-resolution optical cross sections for non-invasive analysis. Here we describe a method using endogenous contrast of cellulose nanofibers (CNF) using Second Harmonic Generation (SHG), combined with 2-photon fluorescence of Cell Tracker Orange for spatial and longitudinal imaging of cellular proliferation. Cell Tracker Orange is an ideal fluorophore to avoid the broad CNF autofluorescence allowing for segmentation of cells using a semi-automatic routine. Individual cells were identified using centroid locations for 3D cell proliferation. Overall, the methods presented are viable for investigation of cellular interactions with polysaccharide candidate biomaterials.
Highlights
Over the past decade, interest in polysaccharide-based biomaterials for biomedical applications has dramatically increased
There is fluorescence above 520 nm, 2-Photon Characterization of Polysaccharide Biomaterials the intensity is relatively weak (∼10× less than the 420 nm peak); fluorophores with emission above 520 nm are likely suitable for optimizing cellular imaging on cellulose nanofibers (CNF) films to minimize interference by CNF autofluorescence
We explored photobleaching as a potential approach to minimize the impact of CNF autofluorescnece
Summary
Interest in polysaccharide-based biomaterials for biomedical applications has dramatically increased. Common polysaccharide polymers used in biomedicine today include alginates, starches, chitosan, and cellulose nanofibers (CNF); all abundant, naturally sourced materials (Wahab and Saiful, 2016) These materials are highly attractive candidates for. 2-Photon Characterization of Polysaccharide Biomaterials biomedical applications as they are readily modifiable using surface chemistry functionalization for mechanical, optical, and biocompatibility properties Realization of these desirable and adaptable properties has led to the broad utilization in applications such as drug delivery, wound healing, and tissue engineering (Bacakova et al, 2004; Manian et al, 2008; Lin and Dufresne, 2014; Karadzic et al, 2015). Tissue engineering applications of these materials include biomedical implants, three-dimensional cell culture systems, wound healing, and tissue regeneration scaffolds Preparation of such materials requires an intricate balance of desired mechanical and biocompatibility properties. In order to vet a material’s ability to facilitate or induce such biological processes, it is necessary to quantify such attributes of the material’s interaction with biology at the cell–material interface
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