Indiana University-Purdue University Indianapolis (IUPUI) / Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an
essential role in directing their differentiation to the chondrogenic lineage. A better
understanding of the mechanisms that allow MSCs to respond to mechanical stimuli
is important to improving cartilage tissue engineering and regenerative medicine.
Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in
joints. However, little is known about the underlying mechanisms that facilitate HP
mechanotransduction. Understanding the signaling pathways in MSCs in transducing
HP to a beneficial biologic response and their interrelationship were the focus of this
thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity
were investigated in conjunction with HP application.
Intracellular Ca++ concentration was previously shown to be changed with HP
application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations
were observed with 10 minutes loading of HP. Additionally a problem with the biore
actor design was discovered. First the gel was floating around in the bioreactor even
without loading. After stabilizing the gel and stopping it from floating, there were
still about 16 µm of movement and deformation in the system. The movement and
deformation was analyzed for the gel structure and different parts of the bioreactor.
Furthermore, we investigated the role of FAK in early and late chondrogenesis
and also its involvement in HP mechanotransduction. A FAK inhibitor was used on
MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis.
However, when low doses of FAK inhibitor added to the MSC culture from day 21 to
42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation
increased. The beneficial effect of HP was suppressed with overnight addition of the
FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransd
ucation by MSCs.
Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduc
tion was also explored. The results indicated that overnight sirtuin1 inhibition in
creased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally,
the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure
and inhibitor application. These two together demonstrated that sirtuin1 inhibition
enhances chondrogenesis.
In this research we have investigated the role of Ca++ signaling, FAK involvement,
and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understand
ings about the mechanisms regulating the chondrogenesis with respect to HP could
have important implications for cartilage tissue engineering and regenerative studies.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/17954 |
| Date | 12 1900 |
| Creators | Hosseini, Seyedeh Ghazaleh |
| Contributors | Wagner, Diane R., Na, Sungsoo, Ji, Julie |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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