Effects of regenerative radioelectric asymmetric conveyer treatment on human normal and osteoarthritic chondrocytes exposed to IL-1β. A biochemical and morphological study

Abstract

Purpose: Osteoarthritis (OA) is a degenerative disease characterized by a progressive loss of articular cartilage extracellular matrix and is due to functional impairments occurring in chondrocytes. In previous works, we highlighted that Regenerative Tissue Optimization (TO-RGN) treatment with radioelectric asymmetric conveyer (REAC) technology influenced the gene expression profiles controlling stem cell differentiation and the pluripotency of human skin-derived fibroblasts in vitro. Since interleukin-1 beta signaling has been implicated in the induction and progression of this disease (through metalloproteinase-3 synthesis and nitric oxide production), we investigated whether REAC TO-RGN might influence the biochemical and morphological changes induced by interleukin-1 beta in normal and OA chondrocytes.

Methods: The induction of metalloproteinase-3 and proteoglycan synthesis was evaluated by a solid-phase enzyme-amplified sensitivity immunoassay, and nitric oxide production was evaluated with the Griess method. Ultrastructural features were observed by transmission electron microscopy.

Results: REAC TO-RGN treatment decreased nitric oxide and metalloproteinase-3 production in normal and OA chondrocytes, while inducing an increase in proteoglycan synthesis. OA chondrocytes were more affected by REAC TO-RGN treatment than were normal chondrocytes. Ultrastructural changes confirmed that REAC TO-RGN may counteract the negative effects of interleukin-1 beta incubation.

Conclusion: The results of this in vitro study suggest that REAC TO-RGN treatment may represent a new, promising approach for the management of OA.

Keywords: REAC TO-RGN treatment; human chondrocytes ultrastructure; metalloproteinase; nitric oxide; proteoglycans.

Figure 1

Effects of incubation of human normal and OA chondrocytes, at basal conditions; with REAC TO-RGN treatment alone; with IL-1β (5 ng/mL) alone; or coincubation with IL-1β and REAC TO-RGN treatment for 48 h in the culture medium. (A) The quantity of PG (ng/10⁶); (B) the MMP-3 level (ng/10⁶ cells); and (C) the NO production (ng/10⁶ cells). Note: Data are expressed as mean values ± SD. Abbreviations: OA, osteoarthritis; REAC, radioelectric asymmetric conveyer technology; TO-RGN, Regenerative Tissue Optimization; IL, interleukin; PG, proteoglycan; MMP, metalloproteinase; NO, nitric oxide.

Figure 2

TEM micrographs of human OA chondrocytes. (A) Basal conditions: The cell shows an euchromatic nucleus (N), a reduction in cytoplasmic components, such as rough endoplasmic reticulum (RER) and mitochondria (M). The plasma membrane presents cytoplasmic processes. (B) Incubation with REAC TO-RNG: The nucleus (N) is euchromatic; the cytoplasm shows a significant increase of the presence of rough endoplasmic reticulum and mitochondria (M). (C) Incubation with IL-1b: The cytoplasm shows a diffuse vacuolization (V) and it contains a reduced quantity of typical organelles, such as Golgi bodies, rough endoplasmic reticulum and mitochondria (M). (D) Incubation with REAC TO-RNG + IL-1β: The cell partially restores its morphology. The nucleus (N) is euchromatic, the cytoplasm shows a restored organization: a much reduced number of vacuoles (V) is present, rough endoplasmic reticulum is abundant and mitochondria (M) are well shaped. The plasma membrane presents many cytoplasmic processes. Bar = 1μm. Abbreviations: TEM, transmission electron microscopy; OA, osteoarthritis; REAC, radioelectric asymmetric conveyer technology; TO-RGN, Regenerative Tissue Optimization; IL, interleukin; RER, rough endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuoles.