External stresses surrounding bone can stimulate heat shock proteins (HSPs), which are involved in anti-apoptosis, cell proliferation, and differentiation. In vitro stress modulation and HSP induction may be critical factors for enhancing bone regeneration. We investigated whether applying individual or combinatorial stress conditioning (thermal, tensile, and biochemical) and effective HSP modulation could induce in vitro responses in preosteoblasts indicating mitogenic/osteogenic/angiogenic/anti-osteoclastic effects. A preosteoblast cell line (MC3T3-E1) was exposed to conditioning protocols utilizing thermal stress applied with a water bath, tensile stress using a Flexcellâ„¢ bioreactor, and biochemical stress with the addition of growth factors (GFs) (i.e. transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2)). Furthermore, the role of HSP70 in osteogenesis under normal conditions and in response to heat was investigated by transfecting preosteoblasts with HSP70 small interfering RNA alone or in combination with thermal stress and measuring cellular response. Heating at 44°C (for 8 minutes) rapidly induced osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factors (VEGF), and cyclooxygenase 2 (COX-2) mRNA at 8 hour post-heating (PH). The addition of GFs with heating induced OPG and VEGF genes more than heating or GF addition alone. OPN, OCN, and OPG secretions increased with the addition of GFs. However, matrix metalloproteinase-9 (MMP-9) secretion was inhibited by heating, with more significant declines associates with GF inclusion. Equibiaxial tension (5%, 0.2 Hz, 10 seconds tension/10 seconds rest, 6 days) with GFs enhanced proliferation than tension or GF addition alone. MMP-9 secretion decreased in response to tension alone or more with GFs. Tension (1-5%, 24 hours) with GFs induced prostaglandin E synthase 2 (PGES-2), OPG, and VEGF genes more than tension or GFs alone. Combinatorial conditioning with thermal stress (44°C, 8 minutes) and tension (3%, 0.2 Hz, 10 seconds tension/10 seconds rest, 4 hours for HSP gene and 24 hours for VEGF secretion and MMP-9 gene) induced HSP27 and HSP70, secretion of VEGF (protein), and suppression of MMP-9 (gene) more than heating or tension alone. HSP70 silencing followed by heating (44°C, 8 minutes) enhanced expression of HSP27. Mitogenic activity was inhibited by heating with more significant decrease occurring by heating and HSP70 silencing. At 10 hours PH, TGF-β1, MMP-9, and ALP mRNA decreased in response to heating and HSP70 silencing. At 48 hours PH, heating following HSP70-silencing induced VEGF secretion significantly. In conclusion, effective application of individual or combinatorial conditioning utilizing heating, tension, and GFs could be beneficial as a bone healing-strategy by rapidly inducing stress proteins (HSPs), angiogenic factor (e.g. VEGF), anti-osteoclastogenic cytokines (e.g. OPG), and bone matrix proteins (e.g. OPN and OCN) with anti-resorptive activity by inhibiting MMP-9. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/77212 |
| Date | 29 October 2010 |
| Creators | Chung, Eunna |
| Contributors | Biomedical Engineering, Rylander, M. Nichole, Dahlgren, Linda A., Morgan, Abby W., Goldstein, Aaron S., Freeman, Joseph W. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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