Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Background. Currently, the gold standard for the microbiological diagnosis remains the culturing of preoperative aspirated joint fluid and intraoperative periprosthetic tissue samples, which give false negative results in about 7 % of cases. Lytic bacteriophages are viruses that specifically infect and lyse bacteria within their replication cycle. Aim. The aim of our study was to explore possibilities for the use of bacteriophage K for the detection of live Staphylococcus spp. bacteria in sonicate fluid of infected prosthetic joints, to possibly contribute to the development of a faster, more sensitive, specific and at the same time economical and handy method for the establishment of the right diagnosis. Material and methods. Sonicate fluid samples obtained from 104 patients with revision arthroplasty were analysed. After the optimisation two indirect phage-based methods were used: a) bioluminescence detection of bacterial intracellular ATP released by bacteriophage K mediated lysis and b) q-PCR with primers specific for bacteriophage K DNA. The results were compared with classical microbiological cultivation methods. Results. With both methods the analysis of sonicate fluid and the analysis of its over-night culture achieved 100 % specificity and predictive value, as there were no false positive results. The sensitivity of the methods was lower when analysing sonicate fluid samples directly, without cultivation. The sensitivity of qPCR detection was higher (81.25 %) compared to the sensitivity of ATP detection (62.5 %) in sonicate fluid directly as a result of 3 false negative results with the qPCR method compared to 6 false negatives with the ATP detection method. The sensitivity of the methods was significantly improved (to 94.12 %) with overnight cultivation of sonicate fluid samples prior to analysis, with no difference in detection between the methods. With both methods, with pre-cultivation of sonicate fluid samples, only one of the tested samples resulted in a false negative result. However, the same sample was negative even when tested with standard microbiological methods. In this patient, only the microbiological cultivation of the periprosthetic tissue sample was positive. The bioluminescence method took 3h with a limit of detection (LOD) in the bacterial concentration range of 10. 3. CFU/mL. The method with qPCR took 4h and had a LOD of 10. 2. CFU/mL. Conclusion. Detection of staphylococci within sonicate fluid with bacteriophage K based methods is a rapid, sensitive and specific approach