Generation of cell-derived three dimensional extracellular matrix substrates from two dimensional endothelial cell cultures.

Within the cellular microenvironment, extracellular matrix (ECM) proteins are critical nonsoluble signaling factors that modulate cell attachment, migration, proliferation, and differentiation. We have developed a simple method to isolate and process ECM from endothelial cell cultures to create a three-dimensional (3D) ECM substrate. Endothelial cell monolayers were chemically lysed and enzymatically digested to isolate a thin, two-dimensional (2D) ECM substrate. This thin 1.8 µm 2D ECM was collected and applied to a solid support to produce 12-16-fold thicker 3D ECM substrates with average thicknesses ranging from 21 to 29 µm. The biological activity of isolated ECM was assessed by cell culture. Neural progenitor cells were cultured on endothelial-produced ECM, and unlike the thin 2D ECM, which was quickly remodeled by cells, 3D ECM substrates remained in culture for an extended period (>7 days), suggesting that a continuous signaling cue for in vitro experiments may be provided. This simple method for creating 3D ECM substrates can be applied to a variety of cell culture models for studies aimed at identifying the signaling effects of the ECM within cellular microenvironments.

Cryopreservation of transfected primary dorsal root ganglia neurons.

Primary dorsal root ganglia (DRG) neurons are often used to investigate the relative strength of various guidance cues to promote re-growth in vitro. Current methods of neuron isolation are laborious and disposal of excess dissected cells is inefficient. Traditional immunostaining techniques are inadequate to visualize real-time neurite outgrowth in co-culture. Cryopreservation, in combination with transfection techniques, may provide a viable solution to both under-utilized tissue and insufficient methods of visualization. This study aims to qualitatively and quantitatively demonstrate successful cryopreservation of primary transfected and non-transfected DRG neurons. Fluorescent micrographs were used to assess morphology after 24h in culture and suggest similarities between freshly isolated neurons and neurons which have been transfected and/or cryopreserved. Quantitative measurements of neuron outgrowth (specifically, primary neurites, branch points and total neurite length) indicate that neuron outgrowth is not altered by cryopreservation. Transfected neurons have stunted outgrowth at 24h.

Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts.

Carbon nanofibers have exceptional theoretical mechanical properties (such as low weight-to-strength ratios) that, along with possessing nanoscale fiber dimensions similar to crystalline hydroxyapatite found in bone, suggest strong possibilities for use as an orthopedic/dental implant material. To determine, for the first time, cytocompatibility properties pertinent for bone prosthetic applications, osteoblast (bone-forming cells), fibroblast (cells contributing to callus formation and fibrous encapsulation events that result in implant loosening), chondrocyte (cartilage-forming cells), and smooth muscle cell (for comparison purposes) adhesion were determined on carbon nanofibers in the present in vitro study. Results provided evidence that, compared to conventional carbon fibers, nanometer dimension carbon fibers promoted select osteoblast adhesion. Moreover, adhesion of other cells was not influenced by carbon fiber dimensions. In fact, smooth muscle cell, fibroblast, and chondrocyte adhesion decreased with an increase in either carbon nanofiber surface energy or simultaneous change in carbon nanofiber chemistry. To determine properties that selectively enhanced osteoblast adhesion, similar cell adhesion assays were performed on polymer (specifically, poly-lactic-co-glycolic; PLGA) casts of carbon fiber compacts previously tested. Compared to PLGA casts of conventional carbon fibers, results provided the first evidence of enhanced select osteoblast adhesion on PLGA casts of nanophase carbon fibers. The summation of these results demonstrate that due to a high degree of nanometer surface roughness, carbon fibers with nanometer dimensions may be optimal materials to selectively increase osteoblast adhesion necessary for successful orthopedic/dental implant applications.