Recent searches
Search options
#Articular #cartilage, which is a type of #tissue found in #joints, allows for nearly frictionless motion and can absorb large loads. Unfortunately, when it is damaged, it cannot repair itself. #Tissueengineering is a promising approach to repair the damage, but it falls short of creating functional tissue.
This is because the tissue-engineered constructs do not have the same mechanical properties as native articular cartilage, which is due to the insufficient accumulation of #extracellular matrix components. To address this, researchers have been exploring the use of adenosine triphosphate (#ATP) to directly harness the underlying mechanotransduction pathways responsible.
ATP is a molecule that is released as a result of mechanical stimulation and acts as an autocrine/paracrine signaling #molecule. It acts on P2 receptors on the #plasma #membrane to promote extracellular matrix #synthesis.
However, high doses of ATP can lead to an increase in matrix #metalloproteinase 13 (MMP-13) activity and extracellular inorganic pyrophosphate (ePPi) accumulation, which can lead to undesirable effects such as #mineralization of articular cartilage. Therefore, the purpose of this study is to identify the mechanism of ATP-mediated #catabolism and to determine a therapeutic dose range to maximize the #anabolic effect.
Materials & Methods
Cell Isolation: This is the process of separating cells from a tissue sample. It is usually done using #enzymes to break down the tissue and then filtering the cells out.
3-Dimensional Culture: This is a type of #cellculture where the cells are grown in a three-dimensional environment, rather than in a flat layer. This allows the cells to interact with each other in a more natural way.
Exogenous ATP Supplementation: ATP (adenosine triphosphate) is a molecule that is important for energy production in cells. Exogenous ATP supplementation is the process of adding ATP to the cell culture from an outside source. This can help the cells to grow and function better.
MMP-13 Protein is a type of protein that is found inside cells. It was extracted from 3-D cultured constructs and then frozen and pulverized.
It was then homogenized in a buffer solution with a protease inhibitor. After that, it was centrifuged and stored at a low temperature. To measure the amount of active MMP-13, a FRET-based assay was used. This assay uses a fluorophore and quencher to measure the amount of MMP-13 that is present. To measure the amount of ECM synthesis, a range of exogenous ATP doses were used.
To measure the effect of PPi on MMP-13 activity, chondrocyte monolayer cultures were established and PPi was added to the cultures. To investigate the underlying mechanisms, inhibitors were added to the cultures. Finally, Transmission Electron #Microscopy (TEM) was used to determine the presence of CPPD #crystal accumulation in the engineered tissue constructs. Statistical analyses were then used to analyze the collected data.
The researchers found that when they added ATP to the cultures, MMP-13 activity increased in a dose-dependent manner. This means that the more ATP they added, the more MMP-13 activity increased. They also found that the levels of PPi in the media increased significantly when they added a high dose of ATP, but the levels of PPi in the tissue did not appear to be affected.
To determine the best dose of ATP to use, the researchers tested a range of doses and measured the effects on ECM synthesis (collagen and proteoglycans) and MMP-13 activity. They found that ECM synthesis and MMP-13 activity increased in response to intermediate doses of ATP, and further increased in response to higher doses of ATP.
