gms | German Medical Science

Objectives: Osteoarthritis (OA) is the leading cause of disability and chronic pain in the elderly. To date pharmacological interventions can only relieve pain, whilst cell- and compound based therapies have limited success in the regeneration of damaged tissues. Thus, there is a need for identification of novel targets for diagnostic and therapeutic approaches. The joints are innervated by calcitonin gene-related peptide (α CGRP) - and substance P (SP) positive sensory nerve fibers which are a potential source of tibial-femoral pain during OA pathogenesis. Alteration of sensory joint innervation might be partly responsible for degenerative changes which contribute to development of OA. We therefore aim to analyze the detailed effects of SP and α CGRP on the metabolism of articular chondrocytes of OA patients.

Methods: Human chondrocytes from OA patients, were expanded for 1 passage and stimulated with SP or α CGRP (10^-8M and 10^-10M) in 2D- and 3D-cell culture systems. Subsequently, proliferation was analyzed via BrdU assay, apoptosis via Caspase 3/7 activity assay and flow cytometric Annexin V measurement, senescence via ß-galactosidase assay, adhesion ability with crystal violet staining, gene expression of marker genes with qPCR and activation of signaling pathways with western blot and ELISA. GAG concentration was evaluated via DMMB assay.

Results and conclusion: Stimulation with SP (10^-8M) for 1 day resulted in a significant increase in apoptosis, in senescence and in gene expression of the pro-inflammatory genes TNF α and IL-6 and for IL-1 by trend. Stimulation with a lower SP concentration (10^-10M) increased apoptosis after 3 days stimulation, but accelerated also proliferation. α CGRP in both concentrations (10^-8M and 10^-10M) decreased adhesion ability on cell culture plastic and increased senescence. Dose-dependently α CGRP (10^-8M) induced proliferation rate, whereas α CGRP (10^-10M) increased apoptosis after 3 days. Chondrocytes moreover had increased intracellular cAMP-levels after stimulation with α CGRP. Phosphorylation of ERK1/2 was induced after stimulation with both neuropeptides, SP and α CGRP, in both concentrations. Cells cultured in fibringels for 21 days showed decreased GAG production after 7 and 21 days, when treated with α CGRP (10^-10M).