No abstract

Small pore sizes inherent to electrospun matrices can hinder efficient cellular ingrowth. To facilitate infiltration while retaining its extracellular matrix-like character, electrospinning was combined with salt leaching to produce a scaffold having deliberate, engineered delaminations. We made elegant use of a specific randomizing component of the electrospinning process, the Taylor Cone and the falling fiber beneath it, to produce a uniform, well-spread distribution of salt particles. After 3 weeks of culture, up to 4 mm of cellular infiltration was observed, along with cellular coverage of up to 70% within the delaminations. To our knowledge, this represents the first observation of extensive cellular infiltration of electrospun matrices. Infiltration appears to be driven primarily by localized proliferation rather than coordinated cellular locomotion. Cells also moved from the salt-generated porosity into the surrounding electrospun fiber matrix. Given that the details of salt deposition (amount, size, and number density) are far from optimized, the result provides a convincing illustration of the ability of mammalian cells to interact with appropriately tailored electrospun matrices. These layered structures can be precisely fabricated by varying the deposition interval and particle size conceivably to produce in vivo-like gradients in porosity such that the resulting scaffolds better resemble the desired final structure.

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

Malignant gliomas are the most common tumors originating within the central nervous system and account for over 15,000 deaths annually in the United States. The median survival for glioblastoma, the most common and aggressive of these tumors, is only 14 months. Therapeutic strategies targeting glioma cells migrating away from the tumor core are currently hampered by the difficulty of reproducing migration in the neural parenchyma in vitro. We utilized a tissue engineering approach to develop a physiologically relevant model of glioma cell migration. This revealed that glioma cells display dramatic differences in migration when challenged by random versus aligned electrospun poly-epsilon-caprolactone nanofibers. Cells on aligned fibers migrated at an effective velocity of 4.2 +/- 0.39 microm/h compared to 0.8 +/- 0.08 microm/h on random fibers, closely matching in vivo models and prior observations of glioma spread in white versus gray matter. Cells on random fibers exhibited extension along multiple fiber axes that prevented net motion; aligned fibers promoted a fusiform morphology better suited to infiltration. Time-lapse microscopy revealed that the motion of individual cells was complex and was influenced by cell cycle and local topography. Glioma stem cell-containing neurospheres seeded on random fibers did not show cell detachment and retained their original shape; on aligned fibers, cells detached and migrated in the fiber direction over a distance sixfold greater than the perpendicular direction. This chemically and physically flexible model allows time-lapse analysis of glioma cell migration while recapitulating in vivo cell morphology, potentially allowing identification of physiological mediators and pharmacological inhibitors of invasion.

Glioblastoma multiforme (GBM), one of the deadliest forms of human cancer, is characterized by its high infiltration capacity, partially regulated by the neural extracellular matrix (ECM). A major limitation in developing effective treatments is the lack of in vitro models that mimic features of GBM migration highways. Ideally, these models would permit tunable control of mechanics and chemistry to allow the unique role of each of these components to be examined. To address this need, we developed aligned nanofiber biomaterials via core–shell electrospinning that permit systematic study of mechanical and chemical influences on cell adhesion and migration. These models mimic the topography of white matter tracts, a major GBM migration ‘highway’. To independently investigate the influence of chemistry and mechanics on GBM behaviors, nanofiber mechanics were modulated by using different polymers (i.e., gelatin, poly(ethersulfone), poly(dimethylsiloxane)) in the ‘core’ while employing a common poly(ε-caprolactone) (PCL) ‘shell’ to conserve surface chemistry. These materials revealed GBM sensitivity to nanofiber mechanics, with single cell morphology (Feret diameter), migration speed, focal adhesion kinase (FAK) and myosin light chain 2 (MLC2) expression all showing a strong dependence on nanofiber modulus. Similarly, modulating nanofiber chemistry using extracellular matrix molecules (i.e., hyaluronic acid (HA), collagen, and Matrigel) in the ‘shell’ material with a common PCL ‘core’ to conserve mechanical properties revealed GBM sensitivity to HA; specifically, a negative effect on migration. This system, which mimics the topographical features of white matter tracts, should allow further examination of the complex interplay of mechanics, chemistry, and topography in regulating brain tumor behaviors.