Accumulation of zoledronic acid in rabbit intervertebral discs.

Low back pain is a major chronic musculoskeletal disorder, caused mainly due the degeneration of the intervertebral discs (IVDs). Bisphosphonates (BPs), like zoledronic acid (ZOL), are used in osteoporosis management; however, their accumulation in the IVDs and their physiological role has not been addressed so far. To this end, an SPE-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) analytical method, using on-cartridge derivatization, has been developed and validated. The analytical method presented excellent linearity (R2 > 0.992), high recoveries (67.8-82.6%), increased repeatability (0.5-9.9%) and low LOD values (21 ng g-1 in the case of IVDs) in all matrices studied. The injection of ZOL in a rabbit animal model resulted in rapid accumulation in blood plasma and the skin, followed by quick clearance. On the other hand, no ZOL was detected in nucleus pulposus, the core of the IVD, while in the peripheral annulus fibrosus a lower and delayed accumulation, as well as dispersal was found. These variations are most probably due to the avascular nature of IVD, allowing only the diffusion of small molecules in and out of the tissue, and/or to the unique physicochemical environment of IVDs. Finally, ZOL, at the concentrations found, did not affect cells' viability or the increase of reactive oxygen species (ROS).

Evaluation of senescent cells in intervertebral discs by lipofuscin staining.

Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-ß-gal staining or p16Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-ß-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.

Major traits of the senescent phenotype of nucleus pulposus intervertebral disc cells persist under the specific microenvironmental conditions of the tissue.

Intervertebral discs (IVDs) are the joints of the spine, mainly consisting of extracellular matrix (ECM) with a low number of cells embedded therein. Low cellularity stems from nutrient deprivation due to the lack of blood supply, as well as from the hypoxic and hyperosmotic conditions prevailing in the tissue. Intervertebral disc degeneration (IDD) has been firmly connected with low back pain, a major age-related disease, whereas degenerated discs have been characterized by increased proteolytic activity and accumulation of senescent cells. While the catabolic phenotype of senescent IVD cells has been documented, whether this phenotype is preserved under the harsh conditions met in the IVD milieu has never been investigated. Here we showed that a combination of low glucose, hypoxia, high osmolality and absence of serum is anti-proliferative for young disc cells. In addition, we demonstrated for the first time that classical senescence markers, namely p16INK4a, p21WAF1 and ICAM-1, remain up-regulated in senescent cells under these conditions. Finally, up-regulation of the main senescence-associated ECM degrading enzymes, i.e. MMP-1, -2 and -3 was maintained in this strict environment. Conservation of IVD cells' senescent phenotype under the actual conditions these cells are confronted with in vivo further supports their possible implication in IDD.

The differential proliferative response of fetal and adult human skin fibroblasts to TGF-ß is retained when cultured in the presence of fibronectin or collagen.

BACKGROUND: Transforming growth factor-ß is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-ß inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-ß on fetal and adult cells. METHODS: Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-ß was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. RESULTS: Fetal skin fibroblast-proliferation was inhibited by TGF-ß, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-ß on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. CONCLUSIONS: Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-ß when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. GENERAL SIGNIFICANCE: The interplay between TGF-ß and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.