組織工程利用體外培養的方式，使細胞分化及生長成所需要的組織及器官，進而修護受損的器官與組織，由於是體外培養因此生物反應器扮演極為重要的角色，生物反應器除了提供細胞生存環境外，還可以提供不同的機械刺激調控細胞的基因表現或是分化的路徑。
本文探討胎盤導出幹細胞遭受壓力刺激後是否會加速朝向成骨細胞分化。有鑑於前人的論文研究指出胚胎導出幹細胞 (PDMCs) 無法承受太大的壓力刺激，因此本研究主旨為設計一套低壓力且壓力精度高之生物反應器，為了得到穩定且正確的壓力值，採用回授控制系統，使得生物反應器可施加定壓或循環壓力。測試結果顯示，壓力值最大可達到300 kPa，最小值為10 kPa，頻率可達1 Hz，精度約2 kPa左右。
我們使用此生物反應器給予胚胎導出幹細胞 (PDMCs) 定常壓力 (0 kPa、10 kPa、30 kPa、50 kPa)，及添加Osteogeneic(Ost) 一群可誘導幹細胞朝向成骨細胞分化的藥物，再用Alizarin Red S stain (ASR) 檢測等方式觀察壓力對細胞分化表現的影響。而結果顯示，添加Ost後再輔以壓力刺激的確明顯在相同細胞密度下讓細胞加速朝向成骨細胞分化。未來將可使用這套系統實驗何種壓力形式或週期更能達到成骨分化的目標。

Tissue engineering aims to repair damaged tissue and organs by developing artificial tissue substitutes. To this end, cells are seeded onto three-dimensional scaffolds and cultured in vitro. When cells grow to a certain amount, the cellular scaffold is implanted into patients to repair the impaired tissue. To this end, bioreactor plays an important role. Except providing suitable biochemical environments, a variety of bioreactors have been designed to apply mechanical stimulation to facilitate cell growth, differentiation and the formation of extracellular matrix.
This thesis used a bioreactor to test the hypothesis that pressure may promote human placenta-derived multipotent cells (PDMCs) to differentiate toward osteoblasts. A self-designed bioreactor that could impose precisely hydrostatic pressure was applied. The bioreactor was able to produce both constant and sinusoidal pressure forms, with precision to 2kPa, the maximum and minimum value at 300 kPa and 10 kPa respectively, and maximum operable frequency at 1 Hz. Experiments were then conducted to test PDMCs under 0 kPa, 10 kPa, 30kPa and 50 kPa with the biochemical Osteogeneic agents added to guide the cells differentiate toward osteoblasts. The levels of cell differentiation were assessed qualitatively by using Alizarin Red S stain (ARS). Results from the experiments showed that applying constant pressure one hour a day was able to promote the differentiation of PDMCs toward osteoblasts. Bigger the pressure value with more intense ARS appeared until the enhancing effect approached a saturated condition at about 30 kPa. In the future, the bioreactor can be used to test the influence of other pressure forms on the cell differentiation as well as growth, and the results may serve as a reference for the development of bone tissue engineering.

Carver, S.E., and Heath, C.A. 1999. Semi-continuous perfusion system for delivering intermittent physiological pressure to regenerating cartilage. Tissue Engineering 5, 1-11.
Chien, C.C., Yen B.L., Lee, F.K., Lai, T.H., Chen, Y.C., Chan, S.H., Huang, H.I., 2006. In Vitro Differentiation of Human Placenta-Derived Multipotent Cells into Hepatocyte-Like Cells. Stem Cells 24, 1759 –1768.
Griffith, L.G., and Naughton, G., 2002. Tissue Engineering-current challenges and expanding opportunities. Science 295, 1009-1014.
Heyland, J., Wiegandt, K., Goepfert, C., Nagel-Heyer, S., Ilinich, E., Schumacher U. and Portner R., 2006. Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic preesure. Biotechonology Lett 28, 1641-1648.
Hodge, W.A., Fijan, R.S., Carlson, K.L., Burgess, R.G., Harris, W.H., and Mann, R.W. 1986. Contect pressures in the human hip joint measured in vivo. Proceedings of the National Academy of Sciences of the United States of America 83, 2879-2883.
Ikenoue, T., Trindade, M.C.D., Lee, M. S., Lin, Eric Y., Schurman, D.J., Goodman, S.B., and Smith, L.R., 2003. Mechanoregulation of human articular chondrodyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro. Journal of Orthopaedic Research 21, 110-116.
Jan, R., Jenneke, K.N., Elisabeth, H.B., 1995. Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro. Journal of Biomechanic. 28, 1493-1503.
Langer, R, and Vacanti, J.P., 1993. Tissue Engineering. Science 260, 920-926.
Lanza, R.P. Langer, R. Vacanti, J. and Chick, W.L., Principles of Tissue Engineering,
40
Annals of Biomedical Engineering , Vol. 27, pp. 580, 1999.
Lanza, Langer, and Vacanti, 2007. Principles of Tissue Engineering 3rd Edition. Elsevier, N.Y., Chapter 1.
Lavik, E. and Langer, R. “Tissue engineering: current state and perspectives”, Biotechnol, Vol. 65, pp. 1-8, 2004.
Luo, Z.J., and Seedhom, B.B., 2007 Light and low-frequency pulsatile hydrostatic pressure enhances extracellular matrix formation by bone marrow mesenchymal cells: an in-vitro study with special reference to cartilage repair. Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine 221, 499-507.
Mader, S., and Callian, P., 2001. Understanding human anatomy & physiology. McGraw-Hill, Taipei, 247.
Makoto, W., Mark, A.A., Joseph, R., and Bauer, E.S., 1997. Effect of pressure on cultured smooth muscle cells. Journal of Life Sciences 61, 987-996.
Maul, T.M., Hamilton, D.W., Nieponice, A., Soletti, L., Vorp D.A., 2007. A new experimental system for the extended application of cyclic hydrostatic pressure to cell culture. Jornal of Biomechanical Engineering 129, 110-116.
Miyanishi, K., Trindade, M.C.D., Lindsey, D.P., Beaupre, G.S., Carter, D.R., Goodman, S.B., Schurman, D.J., and Smith, R.L., 2006. Effects of hydrostatic pressure and transforming growth factor-s3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Engineering 12, 1419-1428.
Mizuno, S., Tateishi, T., Ushida, T., and Glowacki, J., 2002. Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture. Journal of Cellular Physiology 193, 319-327.
Mizuno, S., Watanabe, S., and Takagi, T., 2004. Hydrostatic fluid pressure promotes cellularity and proliferation of human dermal fibroblasts in a three-dimensional
41
collagen gel/sponge. Biochemical Engineering Journal 20, 203-208.
Nagatomi, J., Arulanandam, B.P., Meunier A., and Bizios, R., 2002. Effect of cyclic pressure on bone marrow cell cultures. Journal of Biomechanical Engineering 124, 319-327.
Nagatomi, J., Arulanadam, B.P., Metzger, D.W., Meunier A., and Bizios R., 2003. Cyclic pressure affects osteoblast functions pertinent to osteogensis. Annals of Biomedical Engineering 31, 917-923.
Saxena, A.K., 2005. Tissue engineering: resent concepts and strategies. Journal of Indian Association of Pediatric Surgeons 10, 14-19.
Seyedin, S.M., Thompson, A.Y., Bentz, H., Rosen, D.M., McPherson, J.M., Conti, A., Siegel, N.R., Galluppi, G.R., and Piez, K.A., 1986. Cartilage-inducing factor-A. Apparent identity to transforming growth factor-beta. Journal of Biological Chemistry 261, 5693-5695.
Shieh, S.J., Terada, S. and Vacanti, J.P., 2004. Tissue engineering auricular reconstruction: in vitro and in vivo studies. Biomaterials 25, 1545-1557.
Thambyah, A., Pereira, B.P., Wyss, U., 2005. Estimation of bone-on-bone contact forces in the tibiofemoral joint during walking. The Knee 12, 383-388.
Ute, H., Michael, S., Christian, D., Niki, I., Joachim, H., Thorsten, G., Bodo, K., 2001. Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering. Journal of Biomechanic 34, 941-949.
Wang, E.A., Rosen, V., D’Alessandro, J.S., Bauduy, M., Cordes, P., Harada, T., Israel, D.I., Hewick, R.M., Kerns, K.M., and LaPan, P., 1990. Recombinant human bone morphogenetic protein induces bone formation. Proceedings of the National Academy of Sciences 87, 2220-2224.
Yen, B.L., Huang, H.I., Chien, C.C., Jui, H.Y., Ko, B.S., Yao, M., Shun, C.T., Yen,
42
M.L., Lee, M.C., Chen, C.Y., 2005. Isolation of multipotent cells from human term placenta. Stem Cells 23, 3-9
Yen, M.L., Chien, C.C., Chiu, I.M., Huang, H.I., Chen, Y.C., Hu, H.I., Yen, B.L., 2007. Multilineage differentiation and characterization of the human fetal osteoblastic 1.19 cell line: A possible in vitro model of human mesenchymal progenitors. Stem Cells 25, 125-131
Yen, B.L., Chien, C.C., Chen, Y.C., Chen, J.T., Huang, J.S., Lee, F.K., Huang, H.I., 2008.Placenta-derived multiotent cells differentiate into neuronal and glial cells in vitro. Tissue Engineering 14, 9-17
Yu, V., Damek-Poprawa, M., Nicoll, S.B., Akintoye, S.O., 2009. Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cell. Biochemical and Biophysical Research Communications. 386, 661-665.
Zhang, W.J., Liu, W., and Cao, Y., 2007. Tissue engineering of blood vessel. Journal of Cellular and Molecular Medicine 11, 945-957.
Zhang, M., Wang, J.J., Chen, Y.J., 2006. Effect of mechanical pressure on intracellular calcium release channel and cytoskeletal structure in rabbit mandibular condylar chondrocytes. Life Sciences. 78, 2480-2487.
蘇鴻麟，「再生醫學綜論」，科學發展，Vol. 414, pp.6-11, 2008
劉貴助，2006，細胞在注流式生物反應器之生長研究，中央大學機械工程學系碩士論文。
許峻偉，2008，靜水壓力式生物反應器之設計與製作及壓力對骨髓幹細胞增生影響之研究，中央大學機械工程學系碩士論文。
廖士慶，2009，設計與製作回授控制壓力式生物反應器以探討壓力對骨髓幹細胞增生與型態之影響，中央大學機械工程學系碩士論文。