Genipin crosslinking decreases the mechanical wear and biochemical degradation of impacted cartilage in vitro

Abstract

High energy trauma to cartilage causes surface fissures and microstructural damage, but the degree to which this damage renders the tissue more susceptible to wear and contributes to the progression of post-traumatic osteoarthritis (PTOA) is unknown. Additionally, no treatments are currently available to strengthen cartilage after joint trauma and to protect the tissue from subsequent degradation and wear. The purposes of this study were to investigate the role of mechanical damage in the degradation and wear of cartilage, to evaluate the effects of impact and subsequent genipin crosslinking on the changes in the viscoelastic parameters of articular cartilage, and to test the hypothesis that genipin crosslinking is an effective treatment to enhance the resistance to biochemical degradation and mechanical wear. Results demonstrate that cartilage stiffness decreases after impact loading, likely due to the formation of fissures and microarchitectural damage, and is partially or fully restored by crosslinking. The wear resistance of impacted articular cartilage was diminished compared to undamaged cartilage, suggesting that mechanical damage that is directly induced by the impact may contribute to the progression of PTOA. However, the decrease in wear resistance was completely reversed by the crosslinking treatments. Additionally, the crosslinking treatments improved the resistance to collagenase digestion at the impact-damaged articular surface. These results highlight the potential therapeutic value of collagen crosslinking via genipin in the prevention of cartilage degeneration after traumatic injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:558-565, 2017.

Keywords: articular cartilage; collagen; crosslinking; genipin; post-traumatic osteoarthritis.

Figure 1

Histology (A) and immunohistochemistry (B and C) of articular cartilage after a single impact. (A) Alcian blue staining. (B) Damaged collagen. (C) Collagenase-cleaved collagen. Scale bar = 200 μm.

Figure 2

Viscoelastic parameters of articular cartilage. Indentation testing was performed pre-impact, immediately after single impact, and after treatment with the designated concentrations of genipin, all at the same locations of the cartilage surface. (A) Instantaneous stiffness; (B) Equilibrium stiffness; (C) Unloading stiffness; (D) Relaxation time constant. Data represent the mean ± SD of ratio to pre-impact data. ^** and ^***: different from 1.0 (^**p < 0.01, ^***p < 0.001). Different letters indicate statistical significance between groups (p < 0.05).

Figure 3

Hydroxyproline (HYP) released by collagenase digestion. Articular cartilage was either un-injured (No Impact), or subjected to a single impact. Specimens were then treated with the designated concentrations of genipin and sliced into 150 μm sections starting at the cartilage surface. (A) Hydroxyproline released from 150 μm thick sections from the designated cartilage depth. (B) Hydroxyproline released from the surface section of the cartilage. Data is a subset of data in A (150 on x-axis). Data represent mean ± SD of hydroxyproline content per section. Different letters indicate statistical significance between groups (p < 0.05).

Figure 4

Top: Representative coefficient of friction (COF) over the 30 min test. Bottom: The initial COF of articular cartilage surfaces. Articular cartilage was either un-injured (No Impact), or subjected to a single impact. Specimens were then treated with the designated concentrations of genipin before COF measurement. Data represent mean ± SD of COF.

Figure 5

Wear testing of articular cartilage. (A) India ink staining of articular cartilage subjected to the designated impact and genipin treatments. Images were obtained before and after wear testing and represent the maximum wear for each condition. (B) Hydroxyproline (HYP) released from articular cartilage during wear testing following the designated impact and genipin treatments. Data represent mean ± SD of hydroxyproline content per sample. (^*p < 0.05, ^**p < 0.01).