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. 2023 Oct 28;22(1):e137803.
doi: 10.5812/ijpr-137803. eCollection 2023 Jan-Dec.

In Vitro Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Diacerein

Ali Honarpardaz  1 , Morteza Daliri Joupari  1 , Sajjad Tavakkoli  2
Affiliations

In Vitro Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Diacerein

Ali Honarpardaz et al. Iran J Pharm Res. .

Abstract

Background: Tissue engineering is the application system that tries to restore damaged tissues by different approaches, such as cellular therapy, application of cell differential factors, and various materials. One of the important goals in tissue engineering is to guide stem cells directly to the desired tissue, and researchers tried to utilize different molecules as effective factors to improve this technique.

Objectives: This study aims to demonstrate the effects of diacerein, a slow-acting drug for the treatment of osteoarthritis, on mesenchymal stem cell proliferation and evaluate its potential in the chondrogenesis process.

Methods: Stem cells were isolated from adipose tissue, characterized by flow cytometry, and cells were treated with 10-5M diacerein for three weeks. Chondrogenic gene expression of SOX9, COL2A1, ACAN, and TGFB1 were analyzed by qRT-PCR and immunocytochemistry techniques.

Results: Our results showed that diacerein increased the expression of the following genes involved in chondrogenesis: SOX9 (2.9-fold, P < 0.00), COL2A1 (2.2-fold, P < 0.00), ACAN (2.7-fold, P < 0.00), and TGFB1 (2.6-fold, P < 0.00). Immunocytochemistry results also showed increased production of collagen type II as the main protein marker for chondrocytes.

Conclusions: We observed that diacerein alone could initiate and enhance chondrogenesis, and it can be used as a differentiation factor for stem cells to chondrocyte besides its ability to inhibit IL-1β. Knowing the actual function of diacerein, it could be a good candidate for the treatment of osteoarthritis.

Keywords: Adipose Tissue; Cartilage Disease; Chondrogenesis; Diacerein; Mesenchymal Stem Cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Microscopic image of isolated HADSCs after three passages (20X)
Figure 2.
Figure 2.. Flow cytometry results of extracted HADSCs. (A) Positive markers CD73 (99.7%), CD90 (99.6%), CD44 (99.3%) and CD105 (93.1%); (B) Negative markers CD34 (0.8%) and CD45 (1.2%)
Figure 3.
Figure 3.. Results of osteogenic and adipogenic process. (A) Control, (B) Alizarin Red S staining for osteogenic process, (C) Oil Red O staining for adipogenic process (20X)
Figure 4.
Figure 4.. Viability results of HADSCs cells after 72 h by MTT assay (*** = P < 0.001). (dia 1) Diacerein with 10-4 M concentration compare to control (P < 0.001), (dia 2) diacerein with 10-5 M concentration compare to control (P = 0.865), (dia 3) diacerein with 10-6 M of diacerein compare to control (P = 0.679)
Figure 5.
Figure 5.. Alcian blue staining of cells after 3 weeks. (A) Control cell, (B) treated cells with Diacerein 10-5 M. (1) Alcian blue stain on cells, (2) threshold of stained cells by Image J software, (3) morphology of cells after 3 weeks
Figure 6.
Figure 6.. qRT-PCR analysis of genes by one-way ANOVA (*P < 0.05) (**P < 0.00). SOX9 (2.9 fold, P < 0.001), COL2A1 (2.2 fold, P < 0.001), ACAN (2.7 fold, P < 0.001), TGFB1 (2.6 fold, P < 0.001) and RUNX2 gene expression (P < 0.1)
Figure 7.
Figure 7.. ICC results after a three-week cell differentiation test. 1- DAPI staining: (A) control, (B) diacerein treatment; 2- Collagen type II expression: (A) control, (B) diacerein treatment; 3- Merged picture of DAPI and collagen type II expression. (The green color shows the collagen type II, and the blue color shows the cells nuclei)
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