Aged and degenerated intervertebral discs are characterised by a significant increase in the number of senescent cells, which may be associated with the deterioration of this tissue due to their catabolic phenotype. On the other hand, carboxymethyl-lysine has been found to be accumulated with ageing in the proteins of the disc, evidencing the existence of oxidative stress in this tissue. Accordingly, here we investigated the effect of oxidative stress on the physiology of human nucleus pulposus cells. Hydrogen peroxide (H 2 O 2) at subcytotoxic concentrations transiently increased the intracellular levels of reactive oxygen species, activated the p38 MAPK, ERKs, JNKs and Akt signalling pathways and induced the nuclear translocation of NF-κΒ and Nrf2. It also provoked DNA damage and triggered a DNA repair response by activating the ATM-Chk2-p53-p21 WAF1-pRb pathway, ultimately resulting in a G1 cell cycle delay and the decrease of cells' proliferation. Prolonged exposure to H 2 O 2 led to premature cellular senescence, as characterised by the inhibition of proliferation, the enhanced senescence-associated β galactosidase staining and the over-expression of known molecular markers, without though a significant decrease in the chromosome telomere length. H 2 O 2-senescent cells were found to possess a catabolic phenotype, mainly characterised by the up-regulation of extracellular matrixdegrading enzymes (MMP-1,-2,-9 and ADAMTS-5) and the down-regulation of their inhibitors (TIMPs), as well as of several proteoglycans, including aggrecan, the major component of the nucleus pulposus. The senescent phenotype could be reversed by N-acetyl-L-cysteine, supporting the use of antioxidants for the improvement of disc physiology and the deceleration of disc degeneration.

Excess of glucocorticoids (GCs) has been reported to lead to skin atrophy and impaired wound healing. The present study investigates whether human skin fibroblasts suffer permanent damages due to a long-term exposure to GC excess. Fibroblasts obtained from patients being under GC treatment for periods over one year were cultured under standard conditions in vitro, and studied regarding pivotal parameters involved in skin homeostasis and aging, i.e. collagen production, cell proliferation, and cellular replicative lifespan. No statistical differences were observed regarding these functions compared to those of normal human skin fibroblasts. Furthermore, no differences between normal and patient-derived cells were observed regarding their sensitivity to a supra-physiological cortisol concentration. In conclusion, the prolonged exposure of human skin fibroblasts in vivo to high concentrations of exogenously-administered GC does not lead to persistent adverse effects on their physiology.

Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs--in the form of a conditioned medium (CM)--on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha-smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress-induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC-derived CM was found to be more potent than fibroblast-derived CMs and, furthermore, to inhibit alpha-smooth muscle actin expression. In conclusion, these results may indicate anti-contractile and anti-fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor-derived fibroblasts. These activities seem to persist during senescence of the target cells.

Introduction The native microenvironment of intervertebral disk (IVD) cells is characterized by adverse physicochemical conditions. Low cellularity and proliferation rate is a hallmark of the disk, possibly resulting from the absence of vascularization, which leads to the insufficient nutrition of its cells. Nutrients diffuse from the vessels of the endplates, which may sometimes be several mm away from the IVD and especially from nucleus pulposus cells. On the other hand, the IVD is confronted with several stresses, that is, mechanical load, a constant higher osmolality in comparison to other organs of the body, and oxidative stress. Our aim in the present study was to investigate the effect of the aforementioned exogenous stresses on disk cells’ proliferation and the underlying mechanisms of the observed phenomena. Materials and Methods Primary cultures of IVD cells were established. Cellular proliferation was estimated by tritiated thymidine and bromodeoxyuridine incorporation into DNA, as well as by direct cell counting. Western blot analysis was used to examine the activation of several signaling pathways. Cell cycle analysis was performed by flow cytometry. DNA damage and repair was assessed by comet assay, immunofluorescence for γH2A.X, and the host cell reactivation assay. Finally, reactive oxygen species’ production was measured using DCFH-DA. Results We showed that hyperosmotic treatment reduced nucleus pulposus cells’ proliferation by activating the G2 and G1 cell cycle checkpoints. p38 MAPK was found to participate in the manifestation of the G2 arrest under conditions of increased osmolality, since inhibition of its activity released the cells from G2 phase into mitosis. High osmolality resulted in the ATM-mediated phosphorylation of p53 on Ser15, the upregulation of p21WAF1 and the hypophosphorylation of the retinoblastoma protein in accordance to the observed G1 arrest. Furthermore, comet assay revealed the presence of DNA damage after hyperosmotic treatment, possibly attributed to the abrupt alterations in chromatin configuration observed early after exposure of the cells to this stress. Under these conditions, the histone H2A.X was phosphorylated on Ser139, a classical marker of DNA double strand breaks. In addition, when the DNA repair efficiency of the cells was directly measured by a host cell reactivation of luciferase activity assay, it was found to be significantly increased under hyperosmotic pressure. To shed light in the origin of the response, an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea were used. In contrast to urea, high salt and sorbitol were found to activate the same molecular pathways, indicating that the osmo-regulatory response of nucleus pulposus cells stems rather from cell volume alterations mediated by hypertonicity than from elevated intracellular ionic concentration. The antiproliferative effect of high osmolality was confirmed by the reduced growth factor-mediated novel DNA synthesis and ERK and Akt activation. On the o...