||Effects of the frequency of cyclic biaxial stretching on fibroblast-seeded collagen gels
||Department of BioMedical Engineering
fibroblast-seeded collagen gels
機械性質的不足在天然聚合物所製備的人工組織的一大問題，在先前的研究當中機械力的刺激已經被證實在提高人工組織的機械性質與促進胞外基質的重塑上扮演重要的作用。本研究使用含細胞之膠原蛋白凝膠為模型，將之培養在頻率不同之動態力學刺激當中，這包括了定應變之等雙軸往復式拉伸 （CES-7%; CES-20%）以及動態單軸往復式拉伸（CSBS），自由懸浮培養與等雙軸固定拉伸則作為對照組。在六天的培養後，我們以非線性光學顯微鏡之二倍頻與雙光子吸收分別觀察膠原蛋白纖維及細胞，以組織染色觀察其細胞並且使用H&E染色影像來分析細胞的排列分布程度，也以免疫組織化學染色觀察其細胞增生、凋亡以及平滑肌動蛋白的表現，及以Western-Blot定量平滑肌動蛋白表現。我們發現，高頻率（1 Hz）的動態拉伸與低頻率（0.4 Hz）的動態拉伸對於細胞的排列程度上沒有顯著的差異，在受雙軸刺激的區域中，細胞的排列程度高低為CSBS＞CES-20%＞CES-7%；此外在受單軸刺激的區域當中，細胞的排列程度高低為CES-20%＞CSBS＞CES-7%。並且我們在非線性光學顯微鏡之二倍頻與雙光子吸收觀察膠原蛋白纖維的排列也發現，受到單軸刺激的區域膠原蛋白凝膠纖維會明顯朝著受拉伸的軸平行排列。於細胞的型態和分化上，動態拉伸（CES-7% ; CES-20%和CSBS）細胞增生的表現量較等雙軸固定拉伸培養來的不明顯，Apoptosis則在自由懸浮凝膠內的細胞有高度表現，在動態力學拉伸的情況下則較無表現。而於α-SMA的表現則為CES-20%高度表現，而等雙軸固定拉伸則無明顯表現，但於較小應變CES-7%和CSBS之動態力學培養則又再次表現α-SMA。綜合以上，動態刺激的頻率高低對於含細胞之膠原蛋白凝膠之胞外間質的微結構與細胞的表現型有關，而動態力學刺激的培養對於組織的發展與條細胞型態表現較為劇烈。
Insufficient mechanical strength is a main problem of tissue engineered grafts fabricated from natural polymers. Mechanical stimuli have been proven as an important role in turnover of artificial tissues and matrix reorganization. In this study, we used fibroblast-seeded collagen gels as a model to study the mechono-biological responses under defined cyclic biaxial mechanical stretching. The fibroblast-seeded collagen gels were cultured under five different mechanical environments: free-floating, static equibiaxial stretching, constant strain amplitude cyclic equibiaxial stretching （CES-7% and CES-20%）and cyclic strip-biaxial stretching （CSBS）. We used nonlinear optical microscopy to observe the collagen fibers and cell in the gel. Cell proliferation, apoptosis and expression of smooth muscle α actin were quantified by immunohistochemistry. Furthermore, western blot was used to quantify gene expression of collagen α-SMA. Results showed that the cell alignment were independent on the frequency of mechanical stretch. In the biaxial region of dynamic cultured environments cell alignment was CSBS＞CES-20%＞CES-7%, on the other hand in the uniaxial region of dynamic cultured environments cell alignment was CES-20%＞CSBS＞CES-7%. The image from nonlinear optical microscope illustrate that the gels cultured under dynamic cultured environments had the most collagen fiber alignment. No significance difference was observed in cell proliferation among gels cultured under dynamic cultured environments, but in the free-floating cultured condition cells would tend to apoptosis, while the expression of α-SMA showed opposite trends. In summary, the high-frequency of dynamic culture condition appeared to correlate the microstructure of the gels as well as the phenotype of the cells. Gels cultured under CES-20% regulate phenotype of fibroblast, remodeling of matrix, and turnover of cell.
2. MATERIALS AND METHODS 6
2.1 實驗架構圖 6
2.4 Hematoxylin and eosin stain （H&E stain） 9
2.5 Angular distribution of cell orientations 9
2.6免疫組織化學染色（Immunohistochemistry, IHC） 10
2.7 TUNEL檢測 10
2.8 非線性光學顯微鏡（Nonlinear optical microscopy） 10
2.9西方墨點法（Western blot） 11
2.10酵素免疫分析法（Enzyme-linked immunosorbent assay, ELISA） 11
3. RESULTS 12
3.3 TUNEL細胞凋亡的表現 23
3.5 ELISA-TGF-β濃度 30
3.6 Western Blot 32
4. DISCUSSION 34
5. CONCLUSION 38
6. REFERENCES 39
 R. Gauvin, R. Parenteau-Bareil, D. Larouche, H. Marcoux, F. Bisson, A. Bonnet, et al., "Dynamic mechanical stimulations induce anisotropy and improve the tensile properties of engineered tissues produced without exogenous scaffolding," Acta Biomater, vol. 7, pp. 3294-301, Sep 2011.
 J. J. Hu, Y. C. Liu, G. W. Chen, M. X. Wang, and P. Y. Lee, "Development of fibroblast-seeded collagen gels under planar biaxial mechanical constraints: a biomechanical study," Biomech Model Mechanobiol, vol. 12, pp. 849-68, Oct 2013.
 B. A. Roeder, K. Kokini, J. E. Sturgis, J. P. Robinson, and S. L. Voytik-Harbin, "Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure," J Biomech Eng, vol. 124, pp. 214-22, Apr 2002.
 B. C. DiPaolo, G. Lenormand, J. J. Fredberg, and S. S. Margulies, "Stretch magnitude and frequency-dependent actin cytoskeleton remodeling in alveolar epithelia," Am J Physiol Cell Physiol, vol. 299, pp. C345-53, Aug 2010.
 E. Bell, B. Ivarsson, and C. Merrill, "Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro," Proc Natl Acad Sci U S A, vol. 76, pp. 1274-8, Mar 1979.
 E. Tamariz and F. Grinnell, "Modulation of fibroblast morphology and adhesion during collagen matrix remodeling," Mol Biol Cell, vol. 13, pp. 3915-29, Nov 2002.
 F. Grinnell and C. H. Ho, "The effect of growth factor environment on fibroblast morphological response to substrate stiffness," Biomaterials, vol. 34, pp. 965-74, Jan 2013.
 V. Knezevic, A. J. Sim, T. K. Borg, and J. W. Holmes, "Isotonic biaxial loading of fibroblast-populated collagen gels: a versatile, low-cost system for the study of mechanobiology," Biomech Model Mechanobiol, vol. 1, pp. 59-67, Jun 2002.
 B. C. Isenberg and R. T. Tranquillo, "Long-term cyclic distention enhances the mechanical properties of collagen-based media-equivalents," Ann Biomed Eng, vol. 31, pp. 937-49, Sep 2003.
 T. D. Brown, "Techniques for mechanical stimulation of cells in vitro: a review," J Biomech, vol. 33, pp. 3-14, Jan 2000.
 R. A. Gould, K. Chin, T. P. Santisakultarm, A. Dropkin, J. M. Richards, C. B. Schaffer, et al., "Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture," Acta Biomater, vol. 8, pp. 1710-9, May 2012.
 P. Pathi, T. Ma, and B. R. Locke, "Role of nutrient supply on cell growth in bioreactor design for tissue engineering of hematopoietic cells," Biotechnol Bioeng, vol. 89, pp. 743-58, Mar 30 2005.
 R. Sodian, T. Lemke, C. Fritsche, S. P. Hoerstrup, P. Fu, E. V. Potapov, et al., "Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering," Tissue Eng, vol. 8, pp. 863-70, Oct 2002.
 G. Moon du, G. Christ, J. D. Stitzel, A. Atala, and J. J. Yoo, "Cyclic mechanical preconditioning improves engineered muscle contraction," Tissue Eng Part A, vol. 14, pp. 473-82, Apr 2008.
 S. J. Kleis, S. Schreck, and R. M. Nerem, "A viscous pump bioreactor," Biotechnol Bioeng, vol. 36, pp. 771-7, Oct 20 1990.
 G. Vunjak-Novakovic, G. Altman, R. Horan, and D. L. Kaplan, "Tissue engineering of ligaments," Annu Rev Biomed Eng, vol. 6, pp. 131-56, 2004.
 N. L. Nerurkar, S. Sen, B. M. Baker, D. M. Elliott, and R. L. Mauck, "Dynamic culture enhances stem cell infiltration and modulates extracellular matrix production on aligned electrospun nanofibrous scaffolds," Acta Biomater, vol. 7, pp. 485-91, Feb 2011.
 G. C. Engelmayr, Jr., D. K. Hildebrand, F. W. Sutherland, J. E. Mayer, Jr., and M. S. Sacks, "A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials," Biomaterials, vol. 24, pp. 2523-32, Jun 2003.
 C. Y. Huang, K. L. Hagar, L. E. Frost, Y. Sun, and H. S. Cheung, "Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells," Stem Cells, vol. 22, pp. 313-23, 2004.
 S. Jockenhoevel, G. Zund, S. P. Hoerstrup, A. Schnell, and M. Turina, "Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties," ASAIO J, vol. 48, pp. 8-11, Jan-Feb 2002.
 A. Lichtenberg, G. Dumlu, T. Walles, M. Maringka, S. Ringes-Lichtenberg, A. Ruhparwar, et al., "A multifunctional bioreactor for three-dimensional cell (co)-culture," Biomaterials, vol. 26, pp. 555-62, Feb 2005.
 S. Thomopoulos, G. M. Fomovsky, and J. W. Holmes, "The development of structural and mechanical anisotropy in fibroblast populated collagen gels," J Biomech Eng, vol. 127, pp. 742-50, Oct 2005.
 J. J. Hu, J. D. Humphrey, and A. T. Yeh, "Characterization of engineered tissue development under biaxial stretch using nonlinear optical microscopy," Tissue Eng Part A, vol. 15, pp. 1553-64, Jul 2009.
 F. Grinnell, "Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading," Trends Cell Biol, vol. 10, pp. 362-5, Sep 2000.
 F. Grinnell, "Fibroblast biology in three-dimensional collagen matrices," Trends Cell Biol, vol. 13, pp. 264-9, May 2003.
 C. Jones and H. P. Ehrlich, "Fibroblast expression of alpha-smooth muscle actin, alpha2beta1 integrin and alphavbeta3 integrin: influence of surface rigidity," Exp Mol Pathol, vol. 91, pp. 394-9, Aug 2011.
 J. H. Wang and E. S. Grood, "The strain magnitude and contact guidance determine orientation response of fibroblasts to cyclic substrate strains," Connect Tissue Res, vol. 41, pp. 29-36, 2000.
 S. Jungbauer, H. Gao, J. P. Spatz, and R. Kemkemer, "Two characteristic regimes in frequency-dependent dynamic reorientation of fibroblasts on cyclically stretched substrates," Biophys J, vol. 95, pp. 3470-8, Oct 2008.
 B. Liu, M. J. Qu, K. R. Qin, H. Li, Z. K. Li, B. R. Shen, et al., "Role of cyclic strain frequency in regulating the alignment of vascular smooth muscle cells in vitro," Biophys J, vol. 94, pp. 1497-507, Feb 15 2008.
 H. J. Hsu, C. F. Lee, and R. Kaunas, "A dynamic stochastic model of frequency-dependent stress fiber alignment induced by cyclic stretch," PLoS One, vol. 4, p. e4853, 2009.
 A. Tondon, H. J. Hsu, and R. Kaunas, "Dependence of cyclic stretch-induced stress fiber reorientation on stretch waveform," J Biomech, vol. 45, pp. 728-35, Mar 15 2012.
 C. Neidlinger-Wilke, E. S. Grood, J.-C. Wang, R. A. Brand, and L. Claes, "Cell alignment is induced by cyclic changes in cell length: studies of cells grown in cyclically stretched substrates," J Orthop Res, vol. 19, pp. 286-93, Mar 2001.
 C. P. Ng and M. A. Swartz, "Mechanisms of interstitial flow-induced remodeling of fibroblast-collagen cultures," Ann Biomed Eng, vol. 34, pp. 446-54, Mar 2006.
 D. Karamichos, N. Lakshman, and W. M. Petroll, "Regulation of corneal fibroblast morphology and collagen reorganization by extracellular matrix mechanical properties," Invest Ophthalmol Vis Sci, vol. 48, pp. 5030-7, Nov 2007.
 P. D. Arora, N. Narani, and C. A. McCulloch, "The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts," Am J Pathol, vol. 154, pp. 871-82, Mar 1999.
 F. Grinnell, "Fibroblasts, myofibroblasts, and wound contraction," J Cell Biol, vol. 124, pp. 401-4, Feb 1994.
 B. Hinz and G. Gabbiani, "Mechanisms of force generation and transmission by myofibroblasts," Curr Opin Biotechnol, vol. 14, pp. 538-46, Oct 2003.
 B. Li and J. H. Wang, "Fibroblasts and myofibroblasts in wound healing: force generation and measurement," J Tissue Viability, vol. 20, pp. 108-20, Nov 2011.
 F. Grinnell, M. Zhu, M. A. Carlson, and J. M. Abrams, "Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue," Exp Cell Res, vol. 248, pp. 608-19, May 1 1999.
 S. Nakagawa, P. Pawelek, and F. Grinnell, "Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity," J Invest Dermatol, vol. 93, pp. 792-8, Dec 1989.
 C. P. Ng, B. Hinz, and M. A. Swartz, "Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro," J Cell Sci, vol. 118, pp. 4731-9, Oct 15 2005.
 G. H. Altman, R. L. Horan, I. Martin, J. Farhadi, P. R. Stark, V. Volloch, et al., "Cell differentiation by mechanical stress," FASEB J, vol. 16, pp. 270-2, Feb 2002.
 K. Y. Arai, M. Sugimoto, K. Ito, Y. Ogura, N. Akutsu, S. Amano, et al., "Repeated folding stress-induced morphological changes in the dermal equivalent," Skin Res Technol, vol. 20, pp. 399-408, Nov 2014.
 Y. Meng, X. Han, L. Huang, D. Bai, H. Yu, Y. He, et al., "Orthodontic mechanical tension effects on the myofibroblast expression of alpha-smooth muscle actin," Angle Orthod, vol. 80, pp. 912-8, Sep 2010.
 B. Kinner, J. M. Zaleskas, and M. Spector, "Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells," Exp Cell Res, vol. 278, pp. 72-83, Aug 1 2002.
 P. D. Arora and C. A. McCulloch, "Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts," J Cell Physiol, vol. 159, pp. 161-75, Apr 1994.
 M. E. Blaauboer, T. H. Smit, R. Hanemaaijer, R. Stoop, and V. Everts, "Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts," Biochem Biophys Res Commun, vol. 404, pp. 23-7, Jan 7 2011.
 S. Nakagawa, P. Pawelek, and F. Grinnell, "Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness," Exp Cell Res, vol. 182, pp. 572-82, Jun 1989.
 A. Desmouliere, L. Rubbia-Brandt, A. Abdiu, T. Walz, A. Macieira-Coelho, and G. Gabbiani, "Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon," Exp Cell Res, vol. 201, pp. 64-73, Jul 1992.
 R. Montesano and L. Orci, "Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing," Proc Natl Acad Sci U S A, vol. 85, pp. 4894-7, Jul 1988.
 B. Hinz, G. Celetta, J. J. Tomasek, G. Gabbiani, and C. Chaponnier, "Alpha-smooth muscle actin expression upregulates fibroblast contractile activity," Mol Biol Cell, vol. 12, pp. 2730-41, Sep 2001.
 F. Grinnell and W. M. Petroll, "Cell motility and mechanics in three-dimensional collagen matrices," Annu Rev Cell Dev Biol, vol. 26, pp. 335-61, 2010.
 R. A. Brown, "In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models?," Exp Cell Res, vol. 319, pp. 2460-9, Oct 1 2013.
 J. H. Wang and B. P. Thampatty, "Mechanobiology of adult and stem cells," Int Rev Cell Mol Biol, vol. 271, pp. 301-46, 2008.
 J. H. Wang, B. P. Thampatty, J. S. Lin, and H. J. Im, "Mechanoregulation of gene expression in fibroblasts," Gene, vol. 391, pp. 1-15, Apr 15 2007.