In vitro expansion affects the response of chondrocytes to mechanical stimulation

SummaryObjectiveExpansion of autologous chondrocytes is a common step in procedures for cartilage defect repair. Subsequent dedifferentiation can alter cellular response to mechanical loading, having major consequences for the cell's behavior in vivo after reimplantation. Therefore, we examined the response of primary and expanded human articular chondrocytes to mechanical loading.MethodPrimary and expanded chondrocytes were stretched at either 0.5% or 3.0% at 0.5Hz, 2h per day, for 3 days. Gene expression levels of matrix components (aggrecan (AGC1), lubricin (PRG4), collagen type I (COL1), type II (COL2) and type X (COL10)) as well as matrix enzymes (matrix metalloproteinase 1 (MMP1), MMP3, MMP13) and SOX9 were compared to unstretched controls. To evaluate the effect of a chondrogenic environment on cellular response to stretch, redifferentiation medium was used on expanded cells.ResultsIn primary chondrocytes, stretch led to mild decreases in AGC1, COL1 and COL10 gene expression (maximum of 3.8-fold) and an up-regulation of PRG4 (2.0-fold). In expanded chondrocytes, expression was down-regulated for AGC1 (up to 21-fold), PRG4 (up to 5.0-fold), COL1 (10-fold) and COL2 (2.9-fold). Also, expression was up-regulated for MMP1 (20-fold) and MMP3 (up to 4-fold), while MMP13 was down-regulated (2.8-fold). A chondrogenic environment appeared to temper effects of stretch.DiscussionOur results show that expansion alters the response of human chondrocytes to stretch. Expanded chondrocytes greatly decrease gene expression of matrix constituents and increase expression of MMPs, whereas primary chondrocytes hardly respond. Our data could be a reference for optimization of cell sources or expansion protocols for reimplanted chondrocytes

Analysis of the intraspinal calcium dynamics and its implications on the plasticity of spiking neurons

The influx of calcium ions into the dendritic spines through the N-metyl-D-aspartate (NMDA) channels is believed to be the primary trigger for various forms of synaptic plasticity. In this paper, the authors calculate analytically the mean values of the calcium transients elicited by a spiking neuron undergoing a simple model of ionic currents and back-propagating action potentials. The relative variability of these transients, due to the stochastic nature of synaptic transmission, is further considered using a simple Markov model of NMDA receptos. One finds that both the mean value and the variability depend on the timing between pre- and postsynaptic action-potentials. These results could have implications on the expected form of synaptic-plasticity curve and can form a basis for a unified theory of spike time-dependent, and rate based plasticity.Comment: 14 pages, 10 figures. A few changes in section IV and addition of a new figur

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation

Objective: Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in invitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D invitro cultures, and its therapeutic effects in an invivo rat model of OA. Methods: Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated invitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed exvivo using contrast enhanced microCT and histology. Results: FK506 treatment of osteoarthritic chondrocytes invitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity invivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. Conclusion: FK506 protected cartilage matrix integrity invitro and invivo. Additionally, FK506 treatment invivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA