Tendon and ligament injuries are common orthopedic problems that have an enormous impact on the quality of life of affected patients. Despite the frequency at which these injuries occur, current treatments are unable to restore native function to the damaged tissue. Because of this, reinjury is common. It is well known that mechanical stimulation is beneficial for promoting tendon and ligament development and tissue homeostasis; however, the specific mechanisms remain unclear. The transcription factor scleraxis (Scx) is an interesting candidate for mediating the tendon and ligament mechanoresponse, as it has been shown that Scx expression is induced by cyclic mechanical strain in tenocytes and is required for mechanically-induced stem cell tenogenesis. Moreover, Scx expression is increased in adult tendons following exercise. The studies described in this dissertation therefore focus on the combined role of Scx and mechanical stimulation in two contexts: 1) influencing ligament cell differentiation and 2) regulating adult tenocyte behavior.
In the first study, transient Scx overexpression combined with mechanical strain in a 3D collagen hydrogel model was investigated as a means of deriving mature ligament cells from stem cells for use in ligament tissue engineering. Scx overexpression in C3H10T1/2 cells cultured in collagen hydrogels under static strain resulted in increased construct contraction and cell elongation, but no concurrent increase in the expression of ligament-related genes or production of glycosaminoglycans (GAG). When combined with low levels of cyclic strain, Scx overexpression resulted in increased mechanical properties of the tissue constructs, increased GAG production, and increased expression of ligament-related genes compared to cyclic strain alone. Together, these results demonstrate that Scx overexpression combined with cyclic strain can induce ligament cell differentiation and suggest that Scx does so by improving the mechanosensitivity of cells to cyclic strain.
In the second study, the role of Scx in adult tenocyte mechanotransduction was explored using RNA-sequencing (RNA-seq) and small interfering RNA (siRNA) technologies. Equine tenocytes were exposed to siRNA targeting Scx or a control siRNA and maintained under cyclic mechanical strain prior to being submitted for RNA-seq. Comparison of the resulting transcriptomes revealed that Scx knockdown decreased the expression of several genes encoding important focal adhesion adaptor proteins. Correspondingly, Scx-depleted tenocytes showed abnormally long focal adhesions, decreased cytoskeletal stiffness, and an impaired ability to migrate on soft surfaces. This suggests that Scx regulates the tenocyte mechanoresponse by promoting the expression of focal adhesion-related genes.
Combined, the results of these studies support a role for Scx in tendon and ligament cell mechanotransduction and identify the regulation of genes related to maintaining the cell-extracellular matrix connection and cytoskeletal dynamics as a potential mechanism. These findings enhance our understanding of how mechanical stimulation influences cell behavior and provide new research directions and methodologies for future studies of tendon and ligament mechanobiology. / Ph. D. / Tendon and ligament injuries are very common, but current treatments are unable to completely repair the damaged tissue. We know that exercise plays an important role during the development of tendons and ligaments and in keeping them healthy during adulthood. Despite this, we do not understand exactly how either of these processes occur. A tendon and ligament related protein called scleraxis (Scx) appears to be important in translating mechanical strain into a cellular response. As such, Scx could be useful for making ligament cells from stem cells, which could then be used in custom-engineered, patient-specific tissue grafts for surgical repair of torn ligaments. Studying what Scx does in adult tendon cells could also help us to understand how tendon cells sense and respond to exercise or physical changes in their environment.
To explore whether or not Scx can promote stem cell differentiation, we generated stem cells with increased levels of Scx and put them into 3D collagen constructs. When the constructs were held under static tension, cells with increased Scx became longer and were better able to organize the collagen compared to normal cells. When we exercised the constructs, cells with increased Scx also had higher levels of ligament-related genes and resulted in stiffer constructs compared to normal cells. Increasing Scx expression in combination with mechanical strain could therefore be a useful way to make ligament cells that can be used in engineered replacement tissues.
To explore the function of Scx in adult tendon cells, we created tendon cells with decreased levels of Scx and exposed them to mechanical strain. We then generated a database of all the genes affected by the decrease in Scx. Analysis of this database revealed that decreasing the levels of Scx also decreased the expression of genes associated with several pathways involved in linking the internal skeleton of the cell to the extracellular environment. This suggested that Scx helps facilitate the ability of a tendon cell to sense and respond to its surroundings. To evaluate this, we examined the ability of tendon cells with decreased Scx to migrate on different surfaces, the stiffness of the internal skeleton, and the structure of the protein complexes responsible for anchoring cells to a surface. As predicted by our gene database, we found that decreasing Scx levels also decreases the stiffness of the cell’s internal skeleton, changes the shape of the anchoring complexes, and impairs the ability of tendon cells to migrate on soft surfaces. These results show that Scx governs tendon cell function by affecting the cell’s ability to interact with its local environment.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/86631 |
| Date | 12 June 2018 |
| Creators | Nichols, Anne Elizabeth Carmack |
| Contributors | Biomedical and Veterinary Sciences, Dahlgren, Linda A., Johnson, Sally E., Whittington, Abby R., Harris, Thurl E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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