Measuring stem cell dimensionality in tissue scaffolds.

Many scaffold systems have evolved for tissue engineering and in vitro tissue models to provide a 3D (three-dimensional) microenvironment that enables cells to behave more physiologically. We hypothesized that cells would adopt morphologies with more 3D character during culture in scaffolds as compared to planar substrates. Cell shape and function are tightly linked and effects of scaffold niche properties on cell shape and dimensionality are important for directing cell function. Herein, primary human bone marrow stromal cells (hBMSCs) were cultured in 6 different scaffolds and on a planar control substrate. hBMSCs were imaged using 3D confocal microscopy, and 3D image analyses were used to assess hBMSC shape and dimensionality. A characteristic gyration tensor ellipsoid was calculated for hBMSCs in the different scaffolds which enabled hBMSC dimensionality to be classified based on shape. A "Dimensionality Matrix" was developed that showed that hBMSC shape and dimensionality were influenced by scaffold properties, and that scaffolds could drive hBMSCs into 1D, 2D or 3D shapes. In addition, the hBMSC Z-Depth was measured to determine if hBMSCs became less flat during culture in scaffolds. Z-Depth results showed that all 6 scaffolds caused an increase in cell Z-Depth compared to the 2D planar substrate. These results demonstrate that hBMSCs take on morphologies with greater 3D character in scaffolds than on a planar substrate and that scaffold properties can be adjusted to modify cell dimensionality. In addition, biomaterialists can use this measurement approach to assess and compare scaffold design modifications as they strive to create optimal cell niches that provide a 3D microenvironment.

The support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds.

Nanofiber scaffolds are effective for tissue engineering since they emulate the fibrous nanostructure of native extracellular matrix (ECM). Although electrospinning has been the most common approach for fabricating nanofiber scaffolds, airbrushing approaches have also been advanced for making nanofibers. For airbrushing, compressed gas is used to blow polymer solution through a small nozzle which shears the polymer solution into fibers. Our goals were 1) to assess the versatility of airbrushing, 2) to compare the properties of airbrushed and electrospun nanofiber scaffolds and 3) to test the ability of airbrushed nanofibers to support stem cell differentiation. The results demonstrated that airbrushing could produce nanofibers from a wide range of polymers and onto a wide range of targets. Airbrushing was safer, 10-fold faster, 100-fold less expensive to set-up and able to deposit nanofibers onto a broader range of targets than electrospinning. Airbrushing yielded nanofibers that formed loosely packed bundles of aligned nanofibers, while electrospinning produced un-aligned, single nanofibers that were tightly packed and highly entangled. Airbrushed nanofiber mats had larger pores, higher porosity and lower modulus than electrospun mats, results that were likely caused by the differences in morphology (nanofiber packing and entanglement). Airbrushed nanofiber scaffolds fabricated from 4 different polymers were each able to support osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs). Finally, the differences in airbrushed versus electrospun nanofiber morphology caused differences in hBMSC shape where cells had a smaller spread area and a smaller volume on airbrushed nanofiber scaffolds. These results highlight the advantages and disadvantages of airbrushing versus electrospinning nanofiber scaffolds and demonstrate that airbrushed nanofiber scaffolds can support stem cell differentiation.

Freeform fabricated scaffolds with roughened struts that enhance both stem cell proliferation and differentiation by controlling cell shape.

We demonstrate that freeform fabricated (FFF) scaffolds with a roughened surface topography can support hBMSC proliferation, while also inducing osteogenic differentiation, for maximized generation of calcified, bone-like tissue. Previously, hBMSCs rapidly proliferated, without osteogenic differentiation, during culture in FFF scaffolds. In contrast, hBMSCs underwent osteogenic differentiation, with slow proliferation, during culture in nanofiber scaffolds. Analysis of cell morphology showed that the topography presented by the nanofiber scaffolds drove hBMSC differentiation by guiding them into a morphology that induced osteogenic differentiation. Herein, we hypothesized that using the high-surface area architecture of FFF scaffolds to present a surface roughness that drives hBMSCs into a morphology that induces osteogenic differentiation would yield a maximum amount differentiated hBMSCs and bone-like tissue. Thus, a solvent etching method was developed that imparted a 5-fold increase in roughness to the surface of the struts of poly(ε-caprolactone) (PCL) FFF scaffolds. The etched scaffolds induced osteogenic differentiation of the hBMSCs while un-etched scaffolds did not. The etched scaffolds also supported the same high levels of hBMSC proliferation that un-etched scaffolds supported. Finally, hBMSCs on un-etched scaffolds had a large spread area, while hBMSCs on etched scaffolds has a smaller area and were more rounded, indicating that the surface roughness from the etched scaffolds dictated the morphology of the hBMSCs. The results demonstrate that FFF scaffolds with surface roughness can support hBMSC proliferation, while also inducing osteogenic differentiation, to maximize generation of calcified tissue. This work validates a rational approach to scaffold fabrication where the structure of the scaffold was designed to optimize stem cell function by controlling cell morphology.