Abstract
In vitro construction is a major trend involved in cartilage regeneration and repair. Satisfactory in vitro cartilage regeneration depends on a suitable culture system. Current chondrogenic culture systems with a high content of transforming growth factor beta-1 effectively promote cartilaginous extracellular matrix (ECM) production but inhibit chondrocyte survival. As is known, inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway acts in blocking the progression of osteoarthritis by reducing chondrocyte apoptosis and cartilage destruction. However, whether inhibiting JNK signaling resists the inhibitory effect of current chondrogenic medium (CM) on cell survival and affects in vitro auricular cartilage regeneration (including cell proliferation, ECM synthesis, and degradation) has not been investigated. In order to address these issues and optimize the chondrogenic culture system, we generated a three-dimensional in vitro auricular cartilage regeneration model to investigate the effects of SP600125 (a JNK-specific inhibitor) on chondrocyte proliferation and ECM metabolism. SP600125 supplementation efficiently promoted cell proliferation at both cellular and tissue levels and canceled the negative effect of our chondrogenic culture system on cell survival. Moreover, it significantly inhibited ECM degradation by reducing the expressions of tumor necrosis factor-alpha, interleukin-1-beta, and matrix metalloproteinase 13. In addition, SP600125 inhibited ECM synthesis at both cellular and tissue levels, but this could be canceled and even reversed by adding chondrogenic factors; yet this enabled a sufficient number of chondrocytes to be retained at the same time. Thus, SP600125 had a positive effect on in vitro auricular cartilage regeneration in terms of cell proliferation and ECM degradation but a negative effect on ECM synthesis, which could be reversed by adding CM. Therefore, a combination of SP600125 and CM might help in optimizing current chondrogenic culture systems and achieve satisfactory in vitro cartilage regeneration by promoting cell proliferation, reducing ECM degradation, and enhancing ECM synthesis.
Highlights
Cartilaginous defects are among the most common human diseases
cell counting kit-8 (CCK-8) cell proliferation curves showed differences between the two groups (Figure 1B), and cell counting identified an increased number of chondrocytes in the + SP600125 group compared with the -SP600125 group (Figure 1C)
The chondrogenic culture systems used currently help in promoting extracellular matrix (ECM) production, they inhibit chondrocyte survival, resulting in a sharp decrease in the number of active cells, which is obviously unfavorable for the stability and maintenance of regenerated cartilage after implantation in vivo
Summary
Cartilaginous defects are among the most common human diseases. Damaged cartilage has poor self-repair capacity because the cartilaginous extracellular matrix (ECM) is neither vascularized nor innervated (Dhinsa and Adesida, 2012; Yin et al, 2016). To treat cartilage damage effectively, the defective area needs to be filled with repair tissue (Fini et al, 2013). In vitro construction has been an important direction and trend for cartilage regeneration because of its many advantages in avoiding cell leakage, reducing inflammatory reactions triggered by tissue culture scaffolds, and maintaining stability after transplantation in vivo (He et al, 2017), which are all of great significance for clinical applications. The chondrogenic medium (CM) currently used for cartilage regeneration contains a high content of transforming growth factor beta-1 (TGF-β1), which can effectively improve in vitro cartilage regeneration by promoting chondrocytes or cells with chondrogenic potential such as bone marrow stromal cells to generate abundant cartilaginous ECM (Blunk et al, 2002; Yang and Barabino, 2013). Overexpression of TGF-β1 was reported to induce chondrocyte apoptosis and to reduce the number of active cells (Fang et al, 2016; Zhang et al, 2018), which is obviously unfavorable for the stability and maintenance of regenerated cartilage after implantation in vivo
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