Glutamate excitotoxicity, a major component of many neurodegenerative disorders, is characterized by excessive calcium influx selectively through NMDA receptors (NMDARs). However, there is a substantial uncertainty concerning why other known routes of significant calcium entry, in particular voltage-gated calcium channels (VGCCs), are not similarly toxic. Here, we report that in the majority of neurons in rat hippocampal and cortical cultures, maximal L-type VGCC activation induces much lower calcium loading than toxic NMDAR activation. Consequently, few depolarization-activated neurons exhibit calcium deregulation and cell death. Activation of alternative routes of calcium entry induced neuronal death in proportion to the degree of calcium loading. In a small subset of neurons depolarization evoked stronger calcium elevations, approaching those induced by toxic NMDA. These neurons were characterized by elevated expression of VGCCs and enhanced voltage-gated calcium currents, mitochondrial dysfunction and cell death. Preventing VGCC-dependent mitochondrial calcium loading resulted in stronger cytoplasmic calcium elevations, whereas inhibiting mitochondrial calcium clearance accelerated mitochondrial depolarization. Both observations further implicate mitochondrial dysfunction in VGCC-mediated cell death. Results indicate that neuronal vulnerability tracks the extent of calcium loading but does not appear to depend explicitly on the route of calcium entry.

The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, 3D poly(ethylene glycol) (PEG) hydrogels containing Arg-Gly-Asp (RGD), the cell adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4, and 0.8 mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels, subjected to dynamic compressive strains (0.3Hz, 18% amplitude strain) for 48h, and their response assessed by cell morphology, ECM gene expression, cell proliferation, and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8 mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e., cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell-matrix interactions mediate chondrocyte response to mechanical stimulation.

This study demonstrates gel rho(x) and loading condition influence NO, CP, and PG synthesis. Under a hypoxic environment and certain loading conditions, NO appears to have a positive effect on chondrocyte bioactivity.

We demonstrate the ability to regulate biomechanical cues through manipulations in the gel structure and chemistry and cartilage tissue engineering.