許多學者研究發現細胞黏著分子與細胞外基材間的交互作用會影響不同的生理過程，如血管生成、血栓形成、細胞凋亡、細胞遷移增生等現象。這些過程異常會導致臨床上的疾病，如癌細胞轉移、心肌梗塞、中風、骨質疏鬆、免疫疾病以及神經退化。
本實驗藉由原子力顯微鏡的細胞探針技術去研究小鼠胚胎纖維母細胞(NIH-3T3 fibroblast)和第一型膠原蛋白基材間的鍵結現象，其藉由改變原子力顯微鏡掃描平台的拉伸速率(0.5 μm/s、5 μm/s、25 μm/s)去研究不同施力速率(9.0 × 102 pN/s、2.2 × 104 pN/s、1.9 × 105 pN/s)對細胞拉離基材實驗的影響。同時也比較不同條件，如不同接觸時間(60 s、300 s)及不同勁度基材(1037 Pa、10930 Pa、65 GPa)，其對細胞和基材間最大附著力、做功、單一鍵結力量的影響。
實驗量測所得的細胞與基材間單一鍵結力量會隨施力速率提升而增強，並造成細胞附著力及做功的增加。而提高基材勁度於鍵結情況中也具有類似此鍵結特性的趨勢。然而，細胞與基材的接觸時間越長，造成其附著力增加，此原因是由於鍵結數目增量而非單一鍵結強度增強。
在不同施力速率、基材勁度及接觸時間下，細胞和基材分離力學資訊可以用來闡釋巨觀尺度下的一些生理現象。未來，細胞探針技術可結合螢光技術去觀察在施力過程中內層細胞骨架的變化情形，進一步完整闡明細胞與基材間分離的力學機制。

Many researchers’ investigations have found the interaction of cell adhesion molecules (CAM) and extracellular matrix (ECM) proteins could influence various physiological processes including angiogenesis, thrombosis, apoptosis, cell migration and proliferation. Abnormality in the CAM-ECM interaction can lead to clinical diseases such as metastasis, myocardial infarction, stroke, osteoporosis, inflammatory diseases, and neurodegenerative disorders.
This study uses cell probe of atomic force microscopy (AFM) to investigate the binding interaction between fibroblast and collagen type I by changing the retract speeds (0.5 μm/s, 5 μm/s, and 25 μm/s) of AFM scanner to study the effect of various loading rates (9.0 × 102 pN/s, 2.2 × 104 pN/s, and 1.9 × 105 pN/s) on cell detachment. We also compare the influences of contact time (60 s, 300s) and substrate stiffness (1037 Pa PAA, 10930 Pa PAA, and 65 GPa glass) on the maximum detachment strength, work and single binding force between cell and substrate.
The result shows the single binding strength between cells and ECM increased with the loading rate, and lead to the raise of cell maximum detachment force and work. The increase in breaking strength under higher loading rate could be related to the larger deformation of underlying cytoskeleton in higher loading rate. Increase in substrate stiffness also show similar trend in these binding characteristics. However, the increase in detachment force from longer contact time comes from increase the number of bonding instead of larger single binding strength.
This information of cell-substrate detachment mechanics under different loading rates, substrate stiffness and contact durations could be used to explain some macro level physiological events. In the future, this cell probe approach can combine fluorescent technique to observe the change of underlying cytoskeleton under loading and further elucidate the detail mechanism of cell substrate detachment.

1. Pelling, A.E. and M.A. Horton, An historical perspective on cell mechanics. Pflugers Arch, 2008. 456(1): p. 3-12.
2. Mousa, S.A., Cell adhesion molecules: potential therapeutic & diagnostic implications. Mol Biotechnol, 2008. 38(1): p. 33-40.
3. Michael, K.E. and A.J. Garcia, Cell adhesion strengthening: measurement and analysis. Methods Cell Biol, 2007. 83: p. 329-46.
4. Sniadecki, N.J. and C.S. Chen, Microfabricated silicone elastomeric post arrays for measuring traction forces of adherent cells. Methods Cell Biol, 2007. 83: p. 313-28.
5. Calderwood, D.A., Integrin activation. J Cell Sci, 2004. 117(Pt 5): p. 657-66.
6. Humphries, M.J., Integrin structure. Biochem. Soc. Trans., 2000. 28(4): p. 311-339.
7. Humphries, J.D., A. Byron, and M.J. Humphries, Integrin ligands at a glance. Journal of Cell Science, 2006. 119(19): p. 3901-3903.
8. B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D.Watson,Molecular Biology of the Cell (Garland Publishing, New York, 1994).
9. Mittelman, M. W., in “Bacterial Adhesion: Molecular and Ecological Diversity” (M. Fletcher, Ed.), p. 89.Wiley/Liss, New York, 1996.
10. Whittaker, C.J., C.M. Klier, and P.E. Kolenbrander, Mechanisms of adhesion by oral bacteria. Annu Rev Microbiol, 1996. 50: p. 513-52.
11. Lo, C.M., M. Glogauer, M. Rossi, and J. Ferrier, Cell-substrate separation: effect of applied force and temperature. Eur Biophys J, 1998. 27(1): p. 9-17.
12. 李真甄, 細胞黏著力的量測技術, in 醫學工程研究所. 2003, 成功大學: 台南.
13. Discher, D.E., P. Janmey, and Y.L. Wang, Tissue cells feel and respond to the stiffness of their substrate. Science, 2005. 310(5751): p. 1139-43.
14. Engler, A.J., M.A. Griffin, S. Sen, C.G. Bonnemann, H.L. Sweeney, and D.E. Discher, Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J Cell Biol, 2004. 166(6): p. 877-87.
15. 張登凱, 使用原子力顯微鏡來量測細胞在不同軟硬度基材的附著力, in 醫學工程研究所. 2008, 成功大學: 台南.
16. Li, F., S.D. Redick, H.P. Erickson, and V.T. Moy, Force measurements of the alpha5beta1 integrin-fibronectin interaction. Biophys J, 2003. 84(2 Pt 1): p. 1252-62.
17. Anselme, K., Osteoblast adhesion on biomaterials. Biomaterials, 2000. 21(7): p. 667-681.
18. Owens, N.F., D. Gingell, and P.R. Rutter, Inhibition of cell adhesion by a synthetic polymer adsorbed to glass shown under defined hydrodynamic stress. J Cell Sci, 1987. 87 ( Pt 5): p. 667-75.
19. Thoumine, O. and A. Ott, Time scale dependent viscoelastic and contractile regimes in fibroblasts probed by microplate manipulation. J Cell Sci, 1997. 110 ( Pt 17): p. 2109-16.
20. Bowers, V.M., L.R. Fisher, G.W. Francis, and K.L. Williams, A micromechanical technique for monitoring cell-substrate adhesiveness: measurements of the strength of red blood cell adhesion to glass and polymer test surfaces. J Biomed Mater Res, 1989. 23(12): p. 1453-73.
21. Sato, M., N. Ohshima, and R.M. Nerem, Viscoelastic properties of cultured porcine aortic endothelial cells exposed to shear stress. J Biomech, 1996. 29(4): p. 461-7.
22. Thoumine, O. and A. Ott, Comparison of the mechanical properties of normal and transformed fibroblasts. Biorheology, 1997. 34(4-5): p. 309-26.
23. Corry, W.D. and V. Defendi, Centrifugal assessment of cell adhesion. J Biochem Biophys Methods, 1981. 4(1): p. 29-38.
24. Thoumine, O., A. Ott, and D. Louvard, Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells. Cell Motil Cytoskeleton, 1996. 33(4): p. 276-87.
25. Lotz, M.M., C.A. Burdsal, H.P. Erickson, and D.R. McClay, Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response. J Cell Biol, 1989. 109(4 Pt 1): p. 1795-805.
26. Glogauer, M., J. Ferrier, and C.A. McCulloch, Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts. Am J Physiol, 1995. 269(5 Pt 1): p. C1093-104.
27. Schmidt, C., H. Pommerenke, F. Durr, B. Nebe, and J. Rychly, Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J Biol Chem, 1998. 273(9): p. 5081-5.
28. Mege, J.L., C. Capo, A.M. Benoliel, and P. Bongrand, Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells. Cell Biophys, 1986. 8(2): p. 141-60.
29. Truskey, G.A. and T.L. Proulx, Relationship between 3T3 cell spreading and the strength of adhesion on glass and silane surfaces. Biomaterials, 1993. 14(4): p. 243-54.
30. Burmeister, J.S., J.D. Vrany, W.M. Reichert, and G.A. Truskey, Effect of fibronectin amount and conformation on the strength of endothelial cell adhesion to HEMA/EMA copolymers. J Biomed Mater Res, 1996. 30(1): p. 13-22.
31. Bausch, A.R., F. Ziemann, A.A. Boulbitch, K. Jacobson, and E. Sackmann, Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys J, 1998. 75(4): p. 2038-49.
32. Anselme, K., B. Lanel, C. Gentil, P. Hardouin, P.J. Marie, and M.F. Sigotluizard, Bone Organotypic Culture Method - a Model for Cytocompatibility Testing of Biomaterials. Cells and Materials, 1994. 4(2): p. 113-123.
33. Anselme, K., M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Iost, and P. Hardouin, Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses. J Biomed Mater Res, 2000. 49(2): p. 155-66.
34. Horbett, T.A., J.J. Waldburger, B.D. Ratner, and A.S. Hoffman, Cell adhesion to a series of hydrophilic-hydrophobic copolymers studied with a spinning disc apparatus. J Biomed Mater Res, 1988. 22(5): p. 383-404.
35. Garcia, A.J., P. Ducheyne, and D. Boettiger, Effect of surface reaction stage on fibronectin-mediated adhesion of osteoblast-like cells to bioactive glass. J Biomed Mater Res, 1998. 40(1): p. 48-56.
36. Yamamoto, A., S. Mishima, N. Maruyama, and M. Sumita, A new technique for direct measurement of the shear force necessary to detach a cell from a material. Biomaterials, 1998. 19(7-9): p. 871-9.
37. B. Alberts, D. Bray, J. Lewis, M. Ra!, K. Roberts, J.D. Watson, Molecular Biology of the Cell, Garland Publishing, New York, USA, 1994.
38. Addae-Mensah, K.A. and J.P. Wikswo, Measurement techniques for cellular biomechanics in vitro. Exp Biol Med (Maywood), 2008. 233(7): p. 792-809.
39. Radmacher, M., M. Fritz, C.M. Kacher, J.P. Cleveland, and P.K. Hansma, Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys J, 1996. 70(1): p. 556-67.
40. Chien, S., K.L. Sung, R. Skalak, S. Usami, and A. Tozeren, Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane. Biophys J, 1978. 24(2): p. 463-87.
41. Henon, S., G. Lenormand, A. Richert, and F. Gallet, A new determination of the shear modulus of the human erythrocyte membrane using optical tweezers. Biophys J, 1999. 76(2): p. 1145-51.
42. Hochmuth, R.M., N. Mohandas, and P.L. Blackshear, Jr., Measurement of the elastic modulus for red cell membrane using a fluid mechanical technique. Biophys J, 1973. 13(8): p. 747-62.
43. Tan, J.L., J. Tien, D.M. Pirone, D.S. Gray, K. Bhadriraju, and C.S. Chen, Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc Natl Acad Sci U S A, 2003. 100(4): p. 1484-9.
44. Yang, S. and T. Saif, Micromachined force sensors for the study of cell mechanics. Review of Scientific Instruments, 2005. 76(4): p. -.
45. Merkel, R., Force spectroscopy on single passive biomolecules and single biomolecular bonds. Physics Reports, 2001. 346(5): p. 343-385.
46. Evans, E., K. Ritchie, and R. Merkel, Sensitive Force Technique to Probe Molecular Adhesion and Structural Linkages at Biological Interfaces. Biophysical Journal, 1995. 68(6): p. 2580-2587.
47. Chu, Y.S., W.A. Thomas, O. Eder, F. Pincet, E. Perez, J.P. Thiery, and S. Dufour, Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J Cell Biol, 2004. 167(6): p. 1183-94.
48. Thoumine, O., P. Kocian, A. Kottelat, and J.J. Meister, Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis. Eur Biophys J, 2000. 29(6): p. 398-408.
49. Askenasy, N. and D.L. Farkas, Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo. Stem Cells, 2002. 20(6): p. 501-13.
50. Martin, A., M. Ashwin, S. Maria, W. Yanrong, T. Weihong, D. Randy, N. Stefan, M. Kamal, A. Kristina, and W. Ann, Using optical tweezers for measuring the interaction forces between human bone cells and implant surfaces: System design and force calibration. Review of Scientific Instruments, 2007. 78(7): p. 074302.
51. Khismatullin, D.B. and G.A. Truskey, A 3D numerical study of the effect of channel height on leukocyte deformation and adhesion in parallel-plate flow chambers. Microvasc Res, 2004. 68(3): p. 188-202.
52. Lim, C.T., E.H. Zhou, A. Li, S.R.K. Vedula, and H.X. Fu, Experimental techniques for single cell and single molecule biomechanics. Materials Science & Engineering C-Biomimetic and Supramolecular Systems, 2006. 26(8): p. 1278-1288.
53. Kuznetsova, T.G., M.N. Starodubtseva, N.I. Yegorenkov, S.A. Chizhik, and R.I. Zhdanov, Atomic force microscopy probing of cell elasticity. Micron, 2007. 38(8): p. 824-33.
54. Smith, S.B., L. Finzi, and C. Bustamante, Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science, 1992. 258(5085): p. 1122-6.
55. Santos, N.C. and M.A. Castanho, An overview of the biophysical applications of atomic force microscopy. Biophys Chem, 2004. 107(2): p. 133-49.
56. Gaboriaud, F. and Y.F. Dufrene, Atomic force microscopy of microbial cells: application to nanomechanical properties, surface forces and molecular recognition forces. Colloids Surf B Biointerfaces, 2007. 54(1): p. 10-9.
57. Benoit, M., D. Gabriel, G. Gerisch, and H.E. Gaub, Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat Cell Biol, 2000. 2(6): p. 313-7.
58. Ludwig, T., R. Kirmse, K. Poole, and U.S. Schwarz, Probing cellular microenvironments and tissue remodeling by atomic force microscopy. Pflugers Arch, 2007.
59. Franz, C.M., A. Taubenberger, P.H. Puech, and D.J. Muller, Studying integrin-mediated cell adhesion at the single-molecule level using AFM force spectroscopy. Sci STKE, 2007. 2007(406): p. pl5.
60. Puech, P.H., A. Taubenberger, F. Ulrich, M. Krieg, D.J. Muller, and C.P. Heisenberg, Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy. J Cell Sci, 2005. 118(Pt 18): p. 4199-206.
61. Swimming, escaping from a potential well, and search in cellular scale, Hsuan-Yi Chen, (in Chinese) Physics Bimonthly, August 2006.(在細胞尺度下游泳、逃出能井、以及搜尋, 陳宣毅, 物理雙月刊2006年8月, 685頁.)
62. Hutter, J.L. and J. Bechhoefer, Calibration of Atomic-Force Microscope Tips (Vol 64, Pg 1868, 1993). Review of Scientific Instruments, 1993. 64(11): p. 3342-3342.
63. Jokinen, J., E. Dadu, P. Nykvist, J. Kapyla, D.J. White, J. Ivaska, P. Vehvilainen, H. Reunanen, H. Larjava, L. Hakkinen, and J. Heino, Integrin-mediated cell adhesion to type I collagen fibrils. J Biol Chem, 2004. 279(30): p. 31956-63.
64. Bell, G.I., M. Dembo, and P. Bongrand, Cell adhesion. Competition between nonspecific repulsion and specific bonding. Biophys J, 1984. 45(6): p. 1051-64.
65. Evans, E. and K. Ritchie, Dynamic strength of molecular adhesion bonds. 1997. 72(4): p. 1541-1555.
66. Merkel, R., P. Nassoy, A. Leung, K. Ritchie, and E. Evans, Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature, 1999. 397(6714): p. 50-3.
67. Zhang, X., E. Wojcikiewicz, and V.T. Moy, Force spectroscopy of the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. Biophys J, 2002. 83(4): p. 2270-9.
68. Taubenberger, A., D.A. Cisneros, J. Friedrichs, P.H. Puech, D.J. Muller, and C.M. Franz, Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy. Mol Biol Cell, 2007. 18(5): p. 1634-44.
69. Prechtel, K., A.R. Bausch, V. Marchi-Artzner, M. Kantlehner, H. Kessler, and R. Merkel, Dynamic force spectroscopy to probe adhesion strength of living cells. Phys Rev Lett, 2002. 89(2): p. 028101.
70. Evans, E., V. Heinrich, A. Leung, and K. Kinoshita, Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton. Biophys J, 2005. 88(3): p. 2288-98.
71. Li, Q.S., G.Y. Lee, C.N. Ong, and C.T. Lim, AFM indentation study of breast cancer cells. Biochem Biophys Res Commun, 2008. 374(4): p. 609-13.