Quantitative biorelevant profiling of material microstructure within 3D porous scaffolds via multiphoton fluorescence microscopy

Abstract

This study presents a novel approach, based on fluorescence multiphoton microscopy (MPM), to image and quantitatively characterize the microstructure and cell-substrate interactions within microporous scaffold substrates fabricated from synthetic biodegradable polymers. Using fluorescently dyed scaffolds fabricated from poly(DTE carbonate)/poly(DTO carbonate) blends of varying porosity and complementary green fluorescent protein-engineered fibroblasts, we reconstructed the three-dimensional distribution of the microporous and macroporous regions in 3D scaffolds, as well as cellular morphological patterns. The porosity, pore size and distribution, strut size, pore interconnectivity, and orientation of both macroscale and microscale pores of 3D scaffolds were effectively quantified and validated using complementary imaging techniques. Compared to other scaffold characterizing techniques such as confocal imaging and scanning electron microscopy (SEM), MPM enables the acquisition of images from scaffold thicknesses greater than a hundred microns with high signal-to-noise ratio, reduced bulk photobleaching, and the elimination of the need for deconvolution. In our study, the morphology and cytoskeletal organization of cells within the scaffold interior could be tracked with high resolution within the limits of penetration of MPM. Thus, MPM affords a promising integrated platform for imaging cell-material interactions within the interior of polymeric biomaterials. '

Microfibrous substrate geometry as a critical trigger for organization, self‐renewal, and differentiation of human embryonic stem cells within synthetic 3‐dimensional microenvironments

“…The 2‐D pDTEc films (2‐D Film s) were prepared by spincoating, as described previously (28). Three‐dimensional macroporous pDTEc (3‐D Porous ) scaffolds with bimodal pore size distribution were fabricated by salt leaching and rapid, selective solvent crystallization, as described previously (29). Two types of microfibrous scaffolds were fabricated with different fiber diameters by electrospinning, similar to previously described methods (27).…”

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

“…Owing to their spectral separation, DAPI/calcein double-stained cells are simultaneously excited at 1000 nm (2-PF of calcein at 500 nm, 3-PF of DAPI at 333 nm). Two-photon fluorescence was used to image and quantitatively characterize the microstructure and cell-substrate interactions within microporous scaffold substrates fabricated from synthetic biodegradable polymers [130]. The authors seeded GFP-labelled fibroblasts in Texas red-labelled poly-(DTE/DTO) carbonate blend scaffolds of varying porosity and studied the spatial distribution of micro-and macroporous regions and cell morphology patterns (e.g.…”

Bedside, Benchtop, and Bioengineering: Physicochemical Imaging Techniques in Biomineralization

“…[114] Liu et al used MPM to characterise their engineered polycarbonate scaffolds and analyse osteoblastic seeding. [115] This study demonstrated how MPM provides greater sample penetration, less photobleaching, and higher signal-to-noise ratio than conventional single photon imaging. Villa et al have shown that in vivo MPM is possible, by using it to monitor integration and ossification of donor-cell impregnated scaffold constructs in mouse calvarial defects.…”