Exploring the role of directly coupled alternating electric fields on chondrocyte morphology and redifferentiation capacity with a focus on sex differences

Abstract

Purpose: In cell-based therapies addressing articular cartilage lesions, a central challenge is to avoid the formation of fibrous cartilage resulting from dedifferentiation processes. Electrical stimulation emerges as a promising approach for promoting chondrocytic redifferentiation. This study investigated the effects of varying electric fields on morphological changes and the redifferentiation capacity of human chondrocytes with regard to alterations in sex.

Methods: Chondrocytes, isolated from the articular cartilage of male and female patients undergoing total knee replacement, were exposed to alternating electric fields of varying strengths ranging from 0.8 to 1.2, 15 to 20 and 100 to 140 V/m. Afterwards, cell morphology and viability, as well as the deposition of collagen (Col) 1 and 2, were evaluated.

Results: Following electrical stimulation, in particular at 15-20 V/m, an increase in the Col2/Col1 ratio and an elevated proportion of rounded, chondrocyte-like cell morphology were observed, indicating a promoting effect on the redifferentiation of chondrocytes. Comparative analysis between both sexes revealed that chondrocytes from female donors exhibit higher Col1 synthesis rates, a decreased Col2/Col1 ratio, and a higher proportion of elongated, fibroblast-like cells compared to chondrocytes derived from male donors.

Conclusion: Our in vitro study suggests that chondrocytes from male donors are more prone to re-differentiate after electrical stimulation.

Level of evidence: N/A.

Keywords: articular cartilage lesion; cell‐based therapy; chondrocyte redifferentiation; directly coupled electrical stimulation.

Figure 1

Schematic overview of the experimental design and the electric stimulation setup consisting of two cylindrical electrodes made of a titanium alloy with an insulator in between. The electrical fields within the cell culture were coupled in via a function generator, on which the input voltages and frequency were set. Created in BioRender. Jonitz‐Heincke (2025). https://BioRender.com/z6jx4ix.

Figure 2

Human chondrocytes were exposed to alternating electric fields of 0.8–1.2, 15–20 and 100–140 V/m for 3 days. Afterwards, cellular morphology was analysed. For this purpose, cells were stained with Calcein AM, and images were captured. The images were analysed using CellProfiler, and the cells were classified into rounded and elongated cells, with form factor as the criterion. (a) Data were presented as individual values with mean and standard deviations. One‐way confidence interval (ANOVA) was conducted for statistical analysis with multiple comparisons using Tukey's multiple comparisons test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (b, c) Heatmaps indicate the proportion of cartilage cells (in %) in the corresponding form factor area. The form factor is displayed as the corresponding upper limit of a specific range, for example, 0.3 corresponds to a range of 0.2–0.299(…), and so on. As an exception, 0.2 corresponds to a range of 0.0–0.199(…), and 1.0 corresponds to a range of 0.9 and 1.0.

Figure 3

Human chondrocytes were exposed to alternating electric fields of 0.8–1.2, 15–20 and 100–140 V/m for 3 days. Afterward, cellular viability via WST‐1 assay was analysed. (a) Comparison of cell metabolic activity between different groups without sex‐based distinction. (b) Comparison of cell metabolic activity both within and between chondrocytes from male and female donors. The optical density (OD) data were presented as individual values (male [n = 3]; female [n = 3]) with means and standard deviations. Statistical analysis was conducted by (a) paired t‐test, and (b) Wilcoxon matched‐pairs signed rank test (comparison within the same sex) and Mann–Whitney test (comparison between male and female), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 4

Human chondrocytes were exposed to alternating electric fields of 0.8–1.2, 15–20 and 100–140 for 3 days. Subsequently, the synthesis of collagen (Col)2 and Col1 was assessed in the supernatant and normalized to total protein content. (a) Comparison of Col2 synthesis rates between different groups. (b) Comparison of Col1 synthesis between different groups; (c) Col2/Col1 ratio was calculated and compared between different groups. (d) Comparison of Col2 synthesis both within and between chondrocytes from male and female donors. (e) Comparison of Col1 synthesis both within and between chondrocytes from male and female donors. (f) The Col2/Col1 ratio was calculated separately for males and females and then compared both within and between chondrocytes from male and female donors. All data were presented as individual values with mean and standard deviations. Statistical analysis was conducted using a paired t‐test. An unpaired t‐test was performed to compare males and females. *p ≤ 0.05.