Max R. Salick

3D Collagen Alignment Limits Protrusions to Enhance Breast Cancer Cell Persistence

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.

Effects of substrate mechanics on contractility of cardiomyocytes generated from human pluripotent stem cells.

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4–99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.

Characterization of thermoplastic polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by microcellular injection molding.

Polylactic acid (PLA) and thermoplastic polyurethane (TPU) are two kinds of biocompatible and biodegradable polymers that can be used in biomedical applications. PLA has rigid mechanical properties while TPU possesses flexible mechanical properties. Blended TPU/PLA tissue engineering scaffolds at different ratios for tunable properties were fabricated via twin screw extrusion and microcellular injection molding techniques for the first time. Multiple test methods were used to characterize these materials. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of the two components in the blends; differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) confirmed the immiscibility between the TPU and PLA. Scanning electron microscopy (SEM) images verified that, at the composition ratios studied, PLA was dispersed as spheres or islands inside the TPU matrix and that this phase morphology further influenced the scaffolds microstructure and surface roughness. The blends exhibited a large range of mechanical properties that covered several human tissue requirements. 3T3 fibroblast cell culture showed that the scaffolds supported cell proliferation and migration properly. Most importantly, this study demonstrated the feasibility of mass producing biocompatible PLA/TPU scaffolds with tunable microstructures, surface roughnesses, and mechanical properties that have the potential to be used as artificial scaffolds in multiple tissue engineering applications.

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity

Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user‐defined stiffness, measured the growth rate of individual cells and fit data to a thin‐shell mechanical model to extract the effective longitudinal Youngs modulus of the cell envelope of Escherichia coli (50–150 MPa), Bacillus subtilis (100–200 MPa) and Pseudomonas aeruginosa (100–200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour‐intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22‐induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Youngs modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy.

Incorporation of poly(ethylene glycol) grafted cellulose nanocrystals in poly(lactic acid) electrospun nanocomposite fibers as potential scaffolds for bone tissue engineering

Poly(ethylene glycol) (PEG)-grafted cellulose nanocrystals (CNCs) were successfully synthesized and incorporated into poly(lactic acid) (PLA) as a reinforcing filler to produce nanocomposite scaffolds consisting of CNC-g-PEG and PLA using an electrospinning technique. Morphological, thermal, mechanical, and wettability properties as well as preliminary biocompatibility using human mesenchymal stem cells (hMSCs) of PLA/CNC and PLA/CNC-g-PEG nanocomposite scaffolds were characterized and compared. The average diameter of the electrospun nanofibers decreased with increased filler loading level, due to the increased conductivity of the electrospun solutions. DSC results showed that both the glass transition temperature and cold crystallization temperature decreased progressively with higher CNC-g-PEG loading level, suggesting that improved interfacial adhesion between CNCs and PLA was achieved by grafting PEG onto the CNCs. Wettability of the electrospun nanofibers was not affected with the addition of CNCs or CNC-g-PEG and indicating that the fillers tended to stay inside of the fiber matrix under electrical field. The tensile strength of the composite fiber mats was effectively improved by the addition of up to 5% CNC-g-PEG up to 5wt.%. In addition, the cell culture results showed that PLA/CNC-g-PEG composite nanofibers exhibited improved biocompatibility to hMSCs, which revealed the potential application of this nanocomposite as the scaffolds in bone tissue engineering.

Poly(ε-caprolactone) (PCL)/cellulose nano-crystal (CNC) nanocomposites and foams

Abstract Poly(ε-caprolactone) (PCL)/cellulose nanocrystal (CNC) nanocomposites were produced via twin-screw extrusion. Microcellular nanocomposite samples were produced with microcellular injection molding using carbon dioxide (CO2) as physical blowing agent. The foaming behavior, physical properties, thermal properties, crystallization behavior, and biocompatibility were investigated. It was found that the CNCs interacted with the PCL matrix which led to a strong interface. The CNCs effectively acted as nucleation agents in microcellular injection molding. Both solid and foamed samples with higher levels of CNC content showed higher tensile moduli, complex viscosities, and storage moduli due to the reinforcement effects of CNCs. Furthermore, improvement in the foamed samples was more significant due to their fine cell structure. The addition of CNCs caused a reduction of the decomposition temperature and an increase in the glass transition temperature, crystallization temperature, and crystallinity of PCL. Moreover, the biocompatibility of the foamed nanocomposites with low CNC content was verified by 3T3 fibroblast cell culture.

Electrospinning thermoplastic polyurethane/graphene oxide scaffolds for small diameter vascular graft applications.

Fabrication of small diameter vascular grafts plays an important role in vascular tissue engineering. In this study, thermoplastic polyurethane (TPU)/graphene oxide (GO) scaffolds were fabricated via electrospinning at different GO contents as potential candidates for small diameter vascular grafts. In terms of mechanical and surface properties, the tensile strength, Youngs modulus, and hydrophilicity of the scaffolds increased with an increase of GO content while plasma treatment dramatically improved the scaffold hydrophilicity. Mouse fibroblast (3T3) and human umbilical vein endothelial cells (HUVECs) were cultured on the scaffolds separately to study their biocompatibility and potential to be used as vascular grafts. It was found that cell viability for both types of cells, fibroblast proliferation, and HUVEC attachment were the highest at a 0.5wt.% GO loading whereas oxygen plasma treatment also enhanced HUVEC viability and attachment significantly. In addition, the suture retention strength and burst pressure of tubular TPU/GO scaffolds containing 0.5wt.% GO were found to meet the requirements of human blood vessels, and endothelial cells were able to attach to the inner surface of the tubular scaffolds. Platelet adhesion tests using mice blood indicated that vascular scaffolds containing 0.5% GO had low platelet adhesion and activation. Therefore, the electrospun TPU/GO tubular scaffolds have the potential to be used in vascular tissue engineering.

Poly(ε-caprolactone) Nanofibers with a Self-Induced Nanohybrid Shish-Kebab Structure Mimicking Collagen Fibrils

A three-dimensional structure consisting of poly(ε-caprolactone) (PCL) nanofibers covered by periodically spaced PCL crystal lamellae, a self-induced nanohybrid shish-kebab (SINSK) structure, was created using electrospinning followed by a self-induced crystallization. The resulting structure that resembles the nanotopography of natural collagen nanofibrils in the extracellular matrix (ECM) of human tissues could serve as a tissue engineering scaffold. The formation mechanism of the self-induced shish-kebab structure was investigated with real-time observation of the crystallization process. Electrospun polylactic acid (PLA)/PCL nanofibers were also employed as shish elements to study the effects of different shish materials. The results show that the geometric dimensions of the shish-kebabs are highly related to the initial concentration of PCL solution. The shish material played an important role in the creation of shish-kebab structure. Cell assays with NIH 3T3 ECACC fibroblasts suggest that the nanotopography of the nanofiber surface with kebab crystals that mimic collagen fibrils facilitated the cell attachment and spreading of 3T3 fibroblasts cells.

Morphology, mechanical properties, and shape memory effects of poly(lactic acid)/ thermoplastic polyurethane blend scaffolds prepared by thermally induced phase separation

Novel blended scaffolds combining biobased polylactic acid (PLA) and thermoplastic polyurethane (TPU) were fabricated by thermally induced phase separation (TIPS) using two different solvents. Pure PLA and TPU polymer scaffolds using 1,4-dioxane as the sole solvent exhibited typical ladder-like structures, while blended PLA/TPU scaffolds using the same solvent showed a more uniform microstructure. When de-ionized water was added to the solution as a non-solvent, scaffolds with the mixed solvent showed more open cells and greater interconnectivity. In compression tests, it was found that specimens, including pure PLA, TPU, and blended scaffolds with the mixed solvent, showed a higher compressive modulus than their counterparts that used dioxane as the single solvent. Dynamic mechanical analysis (DMA) was employed to characterize the shape memory properties of the scaffolds. DMA indicated that the shape fixing ratio was highest in the PLA scaffolds, while the shape recovery ratio of the TPU scaffolds was the greatest among the specimens. More interestingly, when the mixed solvent was used, the shape memory property of the blended scaffolds displayed a similar deformation curve to the TPU scaffold. This was due to the presence of the TPU phase and similarity in structure between PLA/TPU and TPU scaffolds when mixed solvent was used. In the degradation test, the blended scaffolds showed a balanced degradation behavior in-between the more easily degraded PLA and the more stable TPU in the phosphate-buffered saline (PBS), and the addition of water to the systems accelerated the degradation process of the specimens. Cell culture results showed that all of the scaffolds had good biocompatibility.