Simvastatin and fluvastatin attenuate trauma-induced cell death and catabolism in human cartilage

Abstract

Joint injuries are known to induce pathomechanisms that might lead to posttraumatic osteoarthritis (PTOA). In this regard, statins with their pleiotropic effects could represent potential therapeutic agents in preventing the development of PTOA. Therefore, we investigated the effects of simvastatin and fluvastatin in a drop-tower-based human ex vivo cartilage trauma model. After 7 days, a mechanical impact (0.59 J) resulted in a decrease of the cell viability and increased expression of catabolic enzymes in cartilage explants. Simvastatin and fluvastatin treatment of impacted cartilage demonstrated cell protective effects in a concentration dependent manner. Moreover, statin therapy exhibited chondroprotective effects as demonstrated by attenuated expression of MMP-2 and MMP-13 as well as subsequent breakdown of collagen type II (after impact). Further analysis indicated antioxidative properties of the statins by upregulating the gene expression of SOD2 and suppression that of NOX2 and NOX4. Despite its protective effects, simvastatin impaired the biosynthesis of collagen type II, which was confirmed during chondrogenic redifferentiation of high passage chondrocytes. However, while long-term administration of statins for 4 weeks impaired chondrogenic redifferentiation, addition of simvastatin at low concentrations for 1 week exhibited a slightly promoting effect. In conclusion, our data imply that simvastatin and fluvastatin are suitable in terms of initial harm reduction after cartilage trauma.

Keywords: anti-catabolic; antioxidative; cartilage; cell protective; fluvastatin; osteoarthritis; simvastatin; statin therapy.

FIGURE 1

Effects of statins on cell viability after cartilage trauma. Cartilage explants were traumatized and treated with simvastatin (Sim) or fluvastatin (Flu) at different concentrations (1 μM, 5 and 10 µM) for 7 days (A) Cell viability was assessed by means of a live/dead staining (B) Exemplary fluorescence images; living cells appear in green while dead cells can be distinguished by red fluorescence. Significant differences between groups were depicted as [versus T] * = p < 0.05, ** = p < 0.01 [versus C] cc = p < 0.01, ccc = p < 0.001; n ≥ 5. Blank box = unimpacted control (C), shaded boxes = traumatized cartilage explants (T).

FIGURE 2

Effects of statin treatment on trauma-induced expression of catabolic enzymes and collagen type II breakdown. Catabolic processes were assessed by means of gene expression analysis of (A) MMP13 as well as (B) quantification of MMP-13 release and (C) collagen type II breakdown product C2C. Significant differences between groups were depicted as [versus T] * = p < 0.05, ** = p < 0.01 [versus C] c = p < 0.05, cc = p < 0.01; n ≥ 4. Blank box = unimpacted control (C), shaded boxes = traumatized cartilage explants (T).

FIGURE 3

Effects of statin treatment on trauma-induced secretion and activity of gelatinases. Release of gelatinases MMP-2 and MMP-9 were measured by means of gelatin zymography as exemplarily demonstrated in (A). Corresponding statistics of zymographically detectable amounts of (B) latent (pro-) MMP-2 and (C) active MMP-2. Significant differences between groups were depicted as [versus T] * = p < 0.05; ** = p < 0.01 [versus C] c = p < 0.05; n = 5. Blank box = unimpacted control (C), shaded boxes = traumatized cartilage explants (T).

FIGURE 4

Effects of statin treatment on collagen type II expression after cartilage trauma. Expression of COL2 was determined by means of (A) gene expression analysis of COL2A1 as well as (B) quantification of CPII release. Significant differences between groups were depicted as [versus T] * = p < 0.05 [versus C] c = p < 0.05, cc = p < 0.01; n = 5. Blank box = unimpacted control (C), shaded boxes = traumatized cartilage explants (T).

FIGURE 5

Effects of statin treatment on gene expression of NADPH oxidases and SOD2 after cartilage trauma. Potential effects of statins on intracellular oxidative stress were determined by means of gene expression analysis of (A) NOX2 (B) NOX4 and (C) SOD2. Significant differences between groups were depicted as [versus T] * = p < 0.05, ** = p < 0.01 [versus C] ccc = p < 0.001; n ≥ 3. Shaded boxes = traumatized cartilage explants (T).

FIGURE 6

Effects of long-term statin treatment on chondrogenic differentiation of isolated chondrocytes (A) Chondrogenic redifferentiation of high passage chondrocytes was determined by means of proteoglycan (Safranin O) staining and collagen type II (IHC), as exemplarily demonstrated (B) Neocartilage formation was further assessed using a defined scoring system. Black scale bar = 200 μm; Significant differences between groups were depicted as [versus CDM] * = p < 0.05; ** = p < 0.01; n = 3.

FIGURE 7

Effects of short-term application of statins on chondrogenic differentiation of isolated chondrocytes (A) Exemplarily demonstrated the staining of proteoglycan (Safranin O) and collagen type II (IHC) of the redifferentiation of high passage chondrocytes (B) Chondrogenic redifferentiation was assessed by a defined scoring system. Black scale bar = 200 μm; n = 6. Significant differences between groups were depicted as [versus CDM] ** = p < 0.01; *** = p < 0.001.