Master Final Project - Tendons & Ligaments

Gap junctions knock-down and their role in 3D micro-tendons compaction

Lissa Verhoeven

Background

The underlying mechanisms of how basic functional tissue remodelling is altered to result in tendinopathy is poorly understood. Tendons are repeatedly subjected to mechanical load, resulting in remodelling of the tissue and changes in tenocyte metabolism. This mechanical stimulation induces a signal, which is transmitted between tenocytes via intercellular junction communication. Therefore, it is proposed that the intercellular communication network between tenocytes, consisting of Connexin 43 (Cx43) among others, plays an important role during remodelling.Alterations in the intercellular communication network may be one of the underlying mechanisms that drives the tendon to transition from basic functional remodelling into tendinopathy. One of the important roles of intercellular communication through gap junctions during remodelling in tendons is to promotes (micro-)tissue compaction. Two hypotheses for the underlying mechanism of this process were proposed previously:

1. Contracting cells ‘recruit’ neighbouring cells through gap junctions, synchronizing and/or increasing cellular contraction
2. Cells communicating through gap junctions can reorganize the surrounding (collagen) extracellular matrix more efficiently, resulting in faster (micro-)tissue compaction

Goal

The aim of the research in this chapter was to determine if one or a combined effect of both hypotheses explains why intercellular communication through gap junctions promotes (micro-) tissue compaction.

Method

18α-GA is used to block cellular communication (e.g. gap junction proteins). CytoD and Marimastat were used for inhibiting F-actin polymerization and collagen cleavage, respectively, and compared with the effect of inhibiting intercellular communication through gap junctions using 18α-GA. The tissue were made by adding a cell-gel mixture to the system, where after the inhibitors were added at t = 1 day or t = 4 days. Of all tissues, the compaction, intracellular actin-fibre network, αSMA production and MMP2 activity were assessed.

Results

The main results were that 1) intercellular communication via gap junctions could be inhibited in 3D tissue using 18α-GA; 2) Disruption of cellular stress fibres 1 day and 4 days after making the tissue significantly reduced tissue compaction and increased the expression of αSMA. Furthermore, inhibiting cellular communication appeared to increase this effect; 3) Inhibiting cellular communication resulted in more round and less elongated cells and an increase in the cleavage of collagen fibres; 4) Inhibiting the cleavage of collagen fibres resulted in local anisotropy of stress fibres and elongation of the cells; 5) Disruption of stress fibres at cell-induced compaction resulted in tissue expansion.

Conclusion

A combined effect of both the hypothesis appears to explain the underlying mechanism of how intercellular communication through gap junctions promotes (micro-)tissue compaction. A new hypothesis is proposed; gap junction communication induces cellular micro-contractions resulting in a more efficient reorganization of the surrounding (collagen) extracellular matrix and subsequently in faster (micro-)tissue compaction.