Maintaining the Cartilage Phenotype of Late-Passage Chondrocytes Using Salidroside, TGF-β, and Sulfated Alginate for Cartilage Tissue Engineering Applications

Abstract

The limited self-repair capacity of cartilage due to its avascular and aneural nature leads to minimal regenerative ability. Autologous chondrocyte transplantation (ACT) is a popular treatment for cartilage defects but faces challenges due to chondrocyte dedifferentiation in later passages, which results in undesirable fibroblastic phenotypes. A promising treatment for cartilage injuries and diseases involves tissue engineering using cells (e.g., chondrocytes), scaffolds (e.g., Alginate Sulfate (AlgSulf)), and biochemical signals (e.g., Salidroside and TGF-β). This study focuses on investigating the effects of AlgSulf scaffolds with varying degrees of sulfation, Salidroside, and TGF-β on the proliferation, viability, and phenotype maintenance of chondrocytes. The findings demonstrate that AlgSulf films with a degree of sulfation (DS) = 2, treated with a combination of Salidroside and TGF-β, significantly enhanced chondrocyte proliferation (p < 0.001 and p < 0.0001 in P2 and P4, respectively), preserved round cell morphology, and maintained cartilage-specific gene expression (Col2, Aggrecans, and SOX9) while downregulating fibroblastic markers (Col1, MMP13, IL-1β, and IL-6). Our findings suggest the potential of this combination for enhancing cartilage regeneration in tissue engineering applications.

Keywords: Salidroside; TGF-β; alginate sulfate; cartilage; chondrocytes; tissue engineering.

Figure 1

Proliferation assay for P2 chondrocytes subjected to TGF-β only (A), Salidroside only (B), and Salidroside and TGF-β (C). (A) In the presence of TGF-β only in the media, the cells cultured on Alginate Sulfate with a DS = 2 displayed the highest increase in chondrocyte numbers during the experiment, demonstrating that a higher DS offers the most suitable medium for cell growth on both day 4 and day 7. (B) The cells cultured in the presence of Salidroside only on Alginate Sulfate with a DS = 2 illustrated a higher proliferation rate on days 4 and 7 compared to the negative control (on plastic). (C) The presence of both TGF-β and Salidroside in the media displayed a significantly higher proliferation rate on days 4 and 7. Data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA and post hoc Tukey’s tests. Statistical significances are denoted as ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; n = 3.

Figure 2

Proliferation assay for P4 chondrocytes subjected to TGF-β only (A), Salidroside only (B), and Salidroside and TGF-β (C). (A) The presence of TGF-β only in the media displayed the highest increase in chondrocyte amount during the experiment, demonstrating that a higher DS offers the most suitable medium for cell growth either on day 4 or 7. (B) Cells cultured in the presence of Salidroside only on Alginate Sulfate with a DS illustrated a higher proliferation rate on days 4 and 7 compared to the negative control. (C) The presence of both TGF-β and Salidroside in the media displayed a significantly higher proliferation rate on days 4 and 7. Data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA and post hoc Tukey’s tests. Statistical significances are denoted as * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; n = 3.

Figure 3

Live/Dead assay of P2 (Top) and P4 (Bottom) chondrocytes under various conditions. Treatments that include Salidroside, particularly when combined with TGF-β, consistently lead to higher cell viability and more enhanced protection of the chondrocyte phenotype. Alginate sulfate (DS = 2.0) optimizes the effects of Salidroside and TGF-β. The combination of Salidroside and TGF-β results in the highest cell viability and protection of chondrocytic phenotype. Scale bar = 500 μm.

Figure 4

Col2/Col1, SOX9/RUNX2, Aggrecan, SZP, and MMP13 mRNA relative quantity in P2 chondrocytes. Cells were cultured on plastic (negative control), on Pure Alginate in the presence of Salidroside and TGF-β (+ST Pure Alginate), on Alginate Sulfate of DS = 2 in the presence of Salidroside and TGF-β (+ST Alginate Sulfate DS = 2.0), on Pure Alginate in the presence of Salidroside only (+S Pure Alginate), on Alginate Sulfate of DS 2 in the presence of Salidroside only (+S Alginate Sulfate DS = 2.0), on Pure Alginate in the presence of TGF-β only (+T Pure Alginate), and on Alginate Sulfate of DS 2 in the presence of TGF-β only (+T Alginate Sulfate DS = 2.0), on days 14 and 21. (The combination of Salidroside and TGF-β, with Alginate Sulfate of DS = 2.0, led to the highest Col2/Col1 ratio, SOX9/RUNX2 ratio, and SZP and Aggrecan mRNA levels, suggesting the best condition to prevent chondrogenic dedifferentiation. In the presence of Salidroside alone, or combined with TGF-β, MMP13 mRNA levels were significantly reduced, especially on alginate sulfate.) Results are shown as a ratio to negative control. Data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA and post hoc Tukey’s tests. Statistical significances are denoted as * = p < 0.05; *** = p < 0.001; **** = p < 0.0001; n = 3.

Figure 5

Col2/Col1, SOX9/RUNX2, Aggrecan, SZP, and MMP13 mRNA relative quantity in P4 chondrocytes. Cells were cultured on plastic (negative control), on Pure Alginate in the presence of Salidroside and TGF-β (+ST Pure Alginate), on Alginate Sulfate of DS 2 in the presence of Salidroside and TGF-β (+ST Alginate Sulfate DS = 2.0), on Pure Alginate in the presence of Salidroside only (+S Pure Alginate), on Alginate Sulfate of DS = 2 in the presence of Salidroside only (S Alginate Sulfate D = 2.0), on Pure Alginate in the presence of TGF-β only (+T Pure Alginate), and on Alginate Sulfate of DS = 2.0 in the presence of TGF-β only (+T Alginate Sulfate DS = 2.0), on days 14 and 21. (The combination of Salidroside and TGF-β, with Alginate Sulfate DS = 2.0, resulted in the highest Col2/Col1 ratio, SOX9/RUNX2 ratio, SZP, and Aggrecan mRNA levels. highlighting the role of this treatment in providing a suitable chondrogenic environment for P4 chondrocytes. This treatment was also the best at downgrading the levels of MMP13 mRNA levels, especially when P4 was grown on alginate sulfate.) Results are shown as a ratio to negative control. Data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA and post hoc Tukey’s tests. Statistical significances are denoted as * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; n = 3.

Figure 6

Western blot results of Col1 and Col2 expression for P2 chondrocytes under various treatment conditions.