Marian F. Young

The Determination of Stem Cell Fate by 3D Scaffold Structures through the Control of Cell Shape

Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.

The Effect of 3D Hydrogel Scaffold Modulus on Osteoblast Differentiation and Mineralization Revealed by Combinatorial Screening

Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus ( approximately 10 kPa to approximately 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of approximately 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.

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.

Nanofiber scaffold gradients for interfacial tissue engineering

We have designed a 2-spinnerette device that can directly electrospin nanofiber scaffolds containing a gradient in composition that can be used to engineer interfacial tissues such as ligament and tendon. Two types of nanofibers are simultaneously electrospun in an overlapping pattern to create a nonwoven mat of nanofibers containing a composition gradient. The approach is an advance over previous methods due to its versatility – gradients can be formed from any materials that can be electrospun. A dye was used to characterize the 2-spinnerette approach and applicability to tissue engineering was demonstrated by fabricating nanofibers with gradients in amorphous calcium phosphate nanoparticles (nACP). Adhesion and proliferation of osteogenic cells (MC3T3-E1 murine pre-osteoblasts) on gradients was enhanced on the regions of the gradients that contained higher nACP content yielding a graded osteoblast response. Since increases in soluble calcium and phosphate ions stimulate osteoblast function, we measured their release and observed significant release from nanofibers containing nACP. The nanofiber-nACP gradients fabricated herein can be applied to generate tissues with osteoblast gradients such as ligaments or tendons. In conclusion, these results introduce a versatile approach for fabricating nanofiber gradients that can have application for engineering graded tissues.

Fabricating Gradient Hydrogel Scaffolds for 3D Cell Culture

Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.