Copper Does Not Induce Tenogenic Differentiation but Promotes Migration and Increases Lysyl Oxidase Activity in Adipose-Derived Mesenchymal Stromal Cells

Abstract

Background: Copper belongs to the essential trace metals that play a key role in the course of cellular processes maintaining the whole body's homeostasis. As there is a growing interest in transplanting mesenchymal stromal cells (MSCs) into the site of injury to improve the regeneration of damaged tendons, the purpose of the study was to verify whether copper supplementation may have a positive effect on the properties of human adipose tissue-derived MSCs (hASCs) which potentially can contribute to improvement of tendon healing.

Results: Cellular respiration of hASCs decreased with increasing cupric sulfate concentrations after 5 days of incubation. The treatment with CuSO₄ did not positively affect the expression of genes associated with tenogenesis (COL1α1, COL3α1, MKX, and SCX). However, the level of COL1α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 μM CuSO₄. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 μM CuSO₄. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3.

Conclusion: Copper sulfate supplementation can have a beneficial effect on tendon regeneration not by inducing tenogenic differentiation, but by improving the recruitment of MSCs to the site of injury, where they can secrete growth factors, cytokines and chemokines, and prevent the effects of oxidative stress at the site of inflammation, as well as improve the stabilization of collagen fibers, thereby accelerating the process of tendon healing.

Figure 1

Human ASC morphology and differentiation potential. Light microscopy: (a) undifferentiated hASCs; (b) chondrogenic differentiation, HE staining of chondropellet; (c, d) adipogenic differentiation, Oil Red O staining (lipid droplets are red) of hASCs cultured in standard (c) and adipogenic (d) medium; (e, f) osteogenic differentiation, Alizarin Red staining (calcium deposits are red) of hASCs cultured in standard (e) and osteogenic (f) medium. Scale bars: 200 μm (a, b), 20 μm (c, d), and 50 μm (e, f).

Figure 2

The effect of cupric sulfate on hASC viability measured in MTT test. Data presented as means ± SEM. Analyzed using tests for related groups in comparison to the control (Ctrl); ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; n = 6‐9.

Figure 3

The effect of cupric sulfate on the COL1α1, COL3α1, MKX, and SCX expression assessed by qPCR analysis in hASCs. The expression of analyzed genes in control cells was appointed as 1. Data presented as means ± SEM. Analyzed using tests for related groups in comparison to the control; ^∗p < 0.05; n = 6.

Figure 4

The effect of cupric sulfate on the COL1α1, COL3α1, MKX, and SCX protein level assessed by immunoblotting in hASCs. Data presented as means ± SEM. Analyzed using tests for related groups in comparison to the control (Ctrl); ^∗p < 0.05; n = 11‐14.

Figure 5

The comparison of cytokine secretion profile of human tendon-derived cells and cupric sulfate-treated human adipose tissue-derived stromal cells relative to control (untreated hASCs) assessed by the Proteome Profiler. The secretion of analyzed cytokines in control cells was appointed as 1. Data presented as means.

Figure 6

The effect of cupric sulfate on migratory abilities of human adipose stromal cells. Data presented as means ± SEM. Analyzed using a t-test for related groups in comparison to the control (Ctrl). ^∗∗p < 0.01 and ^∗∗∗p < 0.001. Cells from 3 donors used in analysis.

Figure 7

The effect of cupric sulfate on copper-dependent enzyme activity in hASCs. (a) The effect of cupric sulfate on superoxide dismutase SOD1 activity. Data presented as means ± SEM; n = 3. (b) The effect of cupric sulfate on hydrogen peroxide-induced oxidative stress damage measured in MTT test. Data presented as means ± SEM. Statistical significance is indicated by letters: there is no significant difference between groups with the same letter (p > 0.05). Analyzed using tests for related groups in comparison to the control (Ctrl) + 1 mM H₂O₂; n = 6‐9. (c) The effect of cupric sulfate on lysyl oxidase activity. Data presented as means ± SEM. Analyzed using tests for related groups in comparison to the control (Ctrl); ^∗p < 0.05 and ^∗∗p < 0.01; n = 8‐9.