Anti-Inflammatory Performance of Lactose-Modified Chitosan and Hyaluronic Acid Mixtures in an In Vitro Macrophage-Mediated Inflammation Osteoarthritis Model

Abstract

The development and progression of osteoarthritis (OA) is associated with macrophage-mediated inflammation that generates a broad spectrum of cytokines and reactive oxygen species (ROS). This study investigates the effects of mid-MW hyaluronic acid (HA) in combination with a lactose-modified chitosan (CTL), on pro-inflammatory molecules and metalloproteinases (MMPs) expression, using an in vitro model of macrophage-mediated inflammation. Methods. To assess chondrocyte response to HA and CTL in the presence of macrophage derived inflammatory mediators, cells were exposed to the conditioned medium (CM) of U937 activated monocytes and changes in cell viability, pro-inflammatory mediators and MMPs expression or ROS generation were analysed. Results. CTL induced changes in chondrocyte viability that are reduced by the presence of HA. The CM of activated U937 monocytes (macrophages) significantly increased gene expression of pro-inflammatory molecules and MMPs and intracellular ROS generation in human chondrocyte cultures. HA, CTL and their combinations counteracted the oxidative damage and restored gene transcription for IL-1β, TNF-α, Gal-1, MMP-3 and MMP-13 to near baseline values. Conclusions. This study suggests that HA-CTL mixture attenuated macrophage-induced inflammation, inhibited MMPs expression and exhibited anti-oxidative effects. This evidence provides an initial step toward the development of an early stage OA therapeutic treatment

In Vitro Effects of Low Doses of β-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation

“…Human monocytes U937 (Thermo Scientific, Wilmington, DE, USA) were differentiated to macrophages by the addition of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) at a final concentration of 50 ng/mL for 48 h then 1 µg/mL lipopolysaccharides (LPS, Sigma, St. Louis, MO, USA) in complete RPMI medium (containing 10% FBS, 1% P/S, 1% glutamine, all from GIBCO) for 1 h. Cells were then washed and cultivated with complete RPMI for 24 h to produce the inflammatory conditioned medium (CM), which was collected, filtered using a filter with a pore diameter of 0.22 µm (Millipore, MA, USA) and used to treat human chondrocyte cultures [ 12 ]. The differentiation of monocytes to macrophages was verified under an inverted phase-contrast microscope and confirmed with mRNA expression of the macrophage differentiation marker CD68 ( Figure S2 ).…”

“…Briefly, after washing the cells from trypsin and medium, cells were loaded for 30 min at 37 °C with 5 µM H2DCFDA in warm PBS, washed twice and placed in 60 µL of PBS. Fluorescence was measured in a BD FACSCanto™ flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at excitation and emission wavelengths of 485 and 535 nm, respectively [ 12 ]. Each experiment was performed in triplicate.…”

“…A total of 500 ng of total RNA was reversely transcribed using oligo-dT and Superscript II (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The qPCR was performed on a Rotor-Gene RG-3000A (QIAGEN, Germany) using Xpert fast SYBR (GRISP, Portugal) [ 12 ]. The expression of NF- κ B , IL-1β , IL-6 , TNF-α , Gal-1 , Gal-3 , MMP-13 , collagen type II , aggrecan and PPAR-γ was analyzed by qPCR.…”

“…It is therefore necessary to study new treatments and explore new molecules to be used alone or in combination, capable of modulating the production of oxidative and proinflammatory molecules, whilst also exerting a chondro-protective action on articular tissue [ 11 , 12 ]. One such molecule, the plant-derived bicyclic sesquiterpene β-caryophyllene (BCP), as recently reported, binds to CB2 (one of the two cannabinoid receptors, CB1 and CB2), and among its wide range of biological activities, exerts an anti-inflammatory effect [ 13 ], decreasing the expression of MMPs and IL-1β in human chondrocyte cultures [ 14 ].…”

Systematic Review of Effectiveness of Chitosan as a Biofunctionalizer of Titanium Implants

“…Although CS is a product that has awakened great interest in the area of biomedical engineering, it has poor solubility in water, which limits its use in living systems [ 33 ], where acid solutions such as acetic acid are to be used instead [ 24 , 34 , 35 ].…”