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Description
Introduction.
Osteoarthritis (OA) is a highly prevalent musculoskeletal disease characterized by a chronic inflammation and joint disability. OA is characterized by cartilage degradation and bone overgrowth. Current pharmacological approaches have a merely symptomatic effect and do not delay disease progression.
Several studies have pointed out that chondrocyte senesce contribute to the pathogenesis of OA. Senescent cells secrete cytokines and proteases, that produce inflammation, decrease cartilage regeneration, and promote OA progression. Therefore, the development of an OA therapy aimed to decrease senescence could be a promising alternative for OA control.
The objective of this work is to develop a controlled release system designed to reduce the senescent cells present in OA patients’ cartilage and to increase their apoptosis to promote a healthy cartilage homeostasis.
Methodology.
Systems development: Polymeric systems loaded with a senolytic drug were prepared and characterized in terms of drug loading (DL) and loading efficiency (LE).
Osteoarthritic chondrocytes isolation: Cartilage samples obtained from patients with OA who underwent total knee replacement surgery were extracted. Samples were washed and digested with 1 mg/mL pronase and with 1 mg/mL collagenase P following the procedure described by Scotece et al1. The resulting chondrocyte pellet was resuspended in culture medium and seeded in 24-well plates.
Assessment of chondrocyte senescence, caspase-3 activity and LDH secretion: Primary chondrocytes were seeded at 62,500 cells/well in 24-well plates. Then cells were stimulated with TNF-α (10 ng/mL) and treated with the senolytic drug or with the drug-loaded delivery systems for 24 hours. Non-stimulated cells were used as negative control. Cell senescence was evaluated by analysing chondrocytes β-galactosidase activity using the senescence cells histochemical staining kit (Sigma Aldrich). Caspase-3 activity was quantified by the caspase 3 Assay kit (Sigma Aldrich) and LDH secretion was assessed by the cytotoxicity detection kit (Roche) following the manufacturer’s protocols.
Results.
The polymeric delivery systems were characterized by a high drug LE (110.0 ± 3.6) indicating their suitability to incorporate the therapeutic molecule.
The treatment with TNF- α produced an increase in the number of senescent cells as expected. On the other hand, the samples of stimulated chondrocytes treated with the senolytic drug or with the polymeric systems presented a decrease in the number of senescent cells. These findings indicate an anti-senescence effect of the developed systems.</div>
The caspase-3 activity is highly related to the lactate dehydrogenase (LDH) release. In this sense, the stimulation with TNF-α also led to an increase in the chondrocytes’ caspase-3 activity and LDH release. The treatment with the drug alone or loaded in the systems further increased the caspase-3 activity and the LDH release of the cells compared to unstimulated cells.
Conclusions.
The developed polymeric systems allowed to reduce senescence and to increase the apoptosis of OA chondrocytes. Similar activity profile was found for both, the senolytic agent alone and the developed drug delivery system.
References.
1.Scotece, M. et al., Cel. Physiology and Biochemistry. 49, 2414-2426 (2018).
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