Striking a new path in reducing cartilage breakdown: combination of antioxidative therapy and chondroanabolic stimulation after blunt cartilage trauma

Abstract

Cartilage injury can trigger crucial pathomechanisms, including excessive cell death and expression of matrix-destructive enzymes, which contribute to the progression of a post-traumatic osteoarthritis (PTOA). With the intent to create a novel treatment strategy for alleviating trauma-induced cartilage damage, we complemented a promising antioxidative approach based on cell and chondroprotective N-acetyl cysteine (NAC) by chondroanabolic stimulation. Overall, three potential pro-anabolic growth factors - IGF-1, BMP7 and FGF18 - were tested comparatively with and without NAC in an ex vivo human cartilage trauma-model. For that purpose, full-thickness cartilage explants were subjected to a defined impact (0.59 J) and subsequently treated with the substances. Efficacy of the therapeutic approaches was evaluated by cell viability, as well as various catabolic and anabolic biomarkers, representing the present matrix turnover. Although monotherapy with NAC, FGF18 or BMP7 significantly prevented trauma-induced cell dead and breakdown of type II collagen, combination of NAC and one of the growth factors did not yield significant benefit as compared to NAC alone. IGF-1, which possessed only moderate cell protective and no chondroprotective qualities after cartilage trauma, even reduced NAC-mediated cell and chondroprotection. Despite significant promotion of type II collagen expression by IGF-1 and BMP7, addition of NAC completely suppressed this chondroanabolic effect. All in all, NAC and BMP7 emerged as best combination. As our findings indicate limited benefits of the simultaneous multidirectional therapy, a sequential application might circumvent adverse interferences, such as suppression of type II collagen biosynthesis, which was found to be reversed 7 days after NAC withdrawal.

Keywords: N-acetyl cysteine; bone morphogenetic protein 7; fibroblast growth factor 18; insulin-like growth factor 1; multidirectional therapy; post-traumatic osteoarthritis.

Figure 1

Effects of growth factors with NAC on trauma‐induced gene expression of ECM‐destructive enzymes. Impacted human cartilage explants were continuously treated by growth factors and NAC (2 mM or 3.5 mM). 7 days post‐trauma, gene expression levels of ECM‐destructive enzymes (A) ADAMTS‐4, (B) ADAMTS‐5, (C) MMP‐1, (D) MMP‐2, (E) MMP‐3 and (F) MMP‐13 were analysed by qRT‐PCR. T= traumatized, +I/B/F= stimulated by IGF‐1 (purple)/FGF18 (red)/BMP7 (green). Significant differences between groups were depicted as: [versus T] *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001; [versus C] c= P < 0.01; [GF versus GF] ^$ P < 0.05, ^$$$ P < 0.001, ^$$$$ P < 0.0001. T: n ≥ 20; T+N: n ≥ 13 (2 mM), n ≥ 9 (3.5); T+N+I: n ≥ 8 (2 mM), n = 4 (3.5 mM); T+N+F: n ≥ 5 (2 mM), n = 5 (3.5 mM); T+N+B: n ≥ 8 (2 mM), n = 5 (3.5 mM).

Figure 2

Effects of growth factors with NAC on trauma‐related alterations in gene expression of ECM components. Impacted human cartilage explants were continuously treated by growth factors and NAC (2 mM or 3.5 mM). 7 days post‐trauma, gene expression levels of ECM components (A) type I collagen (COL1A1), (B) type II collagen (COL2A1), (C) type X collagen (COL10A1) and (D) aggrecan (ACAN) were analysed by qRT‐PCR. T= traumatized, +I/B/F= stimulated by IGF‐1 (purple)/FGF18 (red)/BMP7 (green). Significant differences between groups were depicted as: [versus T] *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001; [versus C] c= P < 0.05; [GF versus GF] ^$ P < 0.05, ^$$P < 0.01, ^$$$$ P < 0.0001. T: n ≥ 20; T+N: n ≥ 13 (2 mM), n ≥ 9 (3.5); T+N+I: n ≥ 13 (2 mM), n = 4 (3.5 mM); T+N+F: n ≥ 6 (2 mM), n = 5 (3.5 mM); T+N+B: n ≥ 8 (2 mM), n = 5 (3.5 mM).

Figure 3

Effects of growth factors w/and w/o NAC on cell viability after cartilage trauma. Impacted human cartilage explants were continuously treated by GF w/and w/o NAC ([2 mM]: n ≥ 7; [3.5 mM]: n ≥ 4) for 7 days and subsequently analysed by Live/Dead^® Viability/Cytotoxicity Assay. C= control, T= traumatized, +I/B/F= stimulated by IGF‐1 (purple)/FGF18 (red)/BMP7 (green). Significant differences between groups were depicted as: [versus T] **P < 0.01, ****P < 0.0001; [versus C] c= P < 0.001; [versus T+N] TN= P < 0.05; [GF versus GF] ^$ P < 0.05, ^$$ P < 0.01.

Figure 4

Effects of growth factors w/or w/o NAC on OA‐associated catabolic biomarkers. (B, D) Impacted human cartilage explants were continuously treated by GF w/and w/o NAC (2 mM, n ≥ 5; or 3.5 mM, n ≥ 4). (A, C) Corresponding unimpacted controls were stimulated by GF; n ≥ 5 each. Amount of (A, B) secreted MMP‐13 and (C, D) generated type II collagen cleavage product C2C in the culture media was analysed 7 days post‐trauma by means of corresponding ELISAs. C= unimpacted control, T= traumatized, GF= growth factor, +I/B/F= stimulated by IGF‐1 (purple)/FGF18 (red)/BMP7 (green). Significant differences between groups were depicted as: [versus T] *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; [versus C] c= P < 0.05; [GF versus GF] ^$ P < 0.05, ^$$ P < 0.01, ^$$$ P < 0.001.

Figure 5

Effects of growth factors w/or w/o NAC on type II collagen synthesis as anabolic biomarker. (B) Impacted human cartilage explants were continuously treated by GF w/and w/o NAC (2 mM, n ≥ 5; or 3.5 mM, n ≥ 4). (A) Corresponding unimpacted controls were stimulated by GF; n ≥ 5 each. Amount of CPII in the culture media was analysed 7 days post‐trauma by means of ELISA. C= unimpacted control, T= traumatized, GF= growth factor, +I/B/F= stimulated by IGF‐1 (purple)/FGF18 (red)/BMP7 (green). Significant differences between groups were depicted as: [versus T] *P < 0.05, ****P < 0.0001; [versus C] c= P < 0.05; [GF versus GF] ^$$ P < 0.01, ^$$$ P < 0.001, ^$$$$ P < 0.0001.