骨再生エンジニアリングに向けた知的コラーゲン計測・制御手法に関する研究開発

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(1)ȁ¡ťBtO\=otH&ÀŸůKnwJtƻœv¦ă Ĕŋ&ǭ7ŻƄǫŭ Û Ń¼ 1*ȇƬ 1ȇȂľ ý 2. Intelligent Measurement and Control of Tissue Collagen for Bone Tissue Engineering by Takeshi YASUI, Katsuya Sato, Mitsuhiko TAKAHASHI. Second-harmonic-generation (SHG) microscopy is a new tool for observing the collagen fiber in tissue in vivo. However, conventional SHG microscopy equipped with 100-fs pulse laser is insufficient to visualize collagen fiber produced by cultured cell with high contrast because of low nonlinear susceptibility (2) in low-order-structured, immature collagen. To enhance the image contrast while avoiding the sample damage, one has to increase a peak power of the laser light while maintaining the same average power as the 100-fs laser. In this paper, we enhanced the sensitive of SHG microscopy by use of a 10-fs Ti:Sapphire laser and succeeded to visualize collagen fibers produced by the cultured osteoblasts in vivo.. Key words: bone, collagen, osteoblasts, tissue engineering, second-harmonic-generation. õ'L?Gp"Ʈ98ȅƞȉuĭù"ġ Űƣ +* . &Ó 58! '#(ãŚů&ȉȈóƁõ:Ě!æ. ģı"(~Ū&Ǿ:ƴ% æÙȂȃ±ž:ǔ ®. . «|ǌő§±! 7'şŊ&ãĉ70&(. ²£&Ò7#(Æǳ" 7'4

(2) %ƟĨ5. ȂȃƜ~ćǐ'ĿƢ'Ư 2ǰß:6ǋ !ž. Ȃȃ±ž&7ȁ¡ťBtO\=otH'ǤƳċ. "ŎǏ"7ŤÑį-87ȁ('"1đ°. ĊǙ&ÒÖ! 7. ħ'Ƴ&%7ƑƗ" 6ȁ'ÓüvŬ.vȁĕ(. ȁ(ȁƨƐơKnwJt:ŦťKnwJtȂ. QOLȅQuality of lifeȐťŎ'ǆȆ:ƪ{7. ŁļǚȅȁÏǆȆ:ļƋ&i]npŉŶ7. ȁ'ǂ'Pbw[(Ļ0!Ǟȉó"ğȄƁ. #&46ȁƑƗüĐȅ¡ťȆ8! ŢÉiPS Ɛ. 1 ąêÖÚÖÚǮRNDXG^L?BtPŻƄǢ Institute of Technology and Science, Tokushima University 2 ąêÖÚÖÚǮepP_?DL?BtPŻƄǢ Institute of Health Bioscience, Tokushima University ǛƓȐ 770-8506 ąêí´ïyêũ 2-1 ąêÖÚÖÚǮRNDXG^L?BtPŻƄǢ. ġ ȁ&8ī9! ȆȂȃƜ"ȁƑƗ:. ơ2ƠƩƍƐơ5ȁƨƐơ&£±v¡£±7 #»Ƣ&%! 7ȁƨƐơ:µƎ&ÖǦť Ŧ7"(ȁƑƗ+'ȂŁļǚ±(ǝ-% ȁƐ ơ5ȁƑƗ+PIwp=Vd! 0&(Ɯ: ƖǬņǯ#!'KnwJt'þªȅȁƨƐơÒŅ £±70'ǌÐȁ'źŗ±'Ïǆ%$ȆĻ0!Ö Ī&ǐ:Ĝ 7Ĝę" 7ȁ(üĎ'.%. -16-.

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(10) ŞĆ:Į. #į- 'ƒĵSHG ŭť¯š' Ltd. ! 7. p"1LtdpTkwO:øǉ#%SHG úõ:ÒÖ7#»Ƣ&%7ĬǕ"(`pP ð 10 fs {' Ti:S qwMw1íǅ8! 7' 4

(11) %qwMwŔ:'-- SHG ǽĄǩ&ŧ 7#. f. 2f. Strong Hit!. Touch. . Light. £ǥ+'ĉŧ&À!10fs qwMw&ã!Ǆ£Ğ. Reflected and scattered light. . Non centrosysmetric material. Umwdinw#ł£Ğ@AVOdoQj"£ĞƱ. . SHG light. 2 Ultraintense pulse light. w Non centrosysmetric material. ex) anisotropic crystal, tissue collagen. 7#&46ãŝqtQřŘƙ&7`pPð:Ĭ Ź±SHG ǽĄǩ'Ȃčõ±:ƽ.'ĀSHG. . Sound (freq. = f). CW light. 7ıŻƄ"(SHG ǽĄǩ'ȁ¡ťBtO\=otH. SHG ?kwOtH:Ʈ KnwJtļǚ'»ƶ±:Ʈ. Nonlinear. Sound. fs -"àĥ&Ę6bwG`sw:Ǘ&Ö{. ƷŘ5Ĥ5&70ȁƨƐơÎǿLtdp'. Linear. (Keyboard). ãŝqtQƇ'ÚƏÙ'£Ğ&46`pPðğŮ. ǽĄǩ:ŧ !ȁüĐȅ¡ťȆǟƁ:KnwJt°Ď'. " 7"(ǹ#'ǻÍ&7ƕ. Fig. 1.. Principle of second-harmonic-generation light.. KnwJt'Ïıļǚ(gofbU[ǨȋǤ5;ļ ǚ5%7ZrgKnwJtȅKnwJt£ÙȆ" 6 8Ƶ¨ů&ǺŁǱ¾!ǯéů&×%! '4

(12) %KnwJt('ŌǪDwTw"'Ƿćã ƀċļǚȅȋǤ5;ļǚ4*Ƶ¨ůǣÀļǚȆ3 &. ,* '&. -17-.

(13) Ȃ ȊŁ'ǷƕüÚŞċ:Į! 7'0Ƿï. Ǎǲ=2mmȆřŘƙ&7`pPð:Ƽ70. &Ȃ ŷħbwG`sw:Į7cAjZſ`pPqw. h?IpRtñŒƻ:Ƒ.Ǔ.Ltdpƙ&ƭƧƏ. Mw:KnwJtÁĮťƑƗ&Śä7#ǷŴĠ. ŕŐȅrwTit 6G ŕŐȆ:ǣƙȊÙƭȅTPEFȆ. ¾Ì SHG ŭťǟƁ: !ť SHG Şūů&ŭť. ƤìŴǭƻ:ļƋh?IpRtñŒƻ'xĢ'=w. 7 )ȁƑƗ&cAjZſ`pPqwMw:Ś. j'ǎǪ:ǈķ%5ŭťȊÙƭ:ĺ¢. äÐ¾ť SHG :ŧ 7#&46ȁƑƗ&. 7#&46ƤìŴǭŌü:¹Ă. 7KnwJtļǚ'ǡĖůƻœ»Ƣ&%7ȅÇȊȆ. PMT Filter Condenser Lens. ¶â&(KnwJtÔ&ƈƝc>nktZ2ĄåƊ# ťļĐŝǆ1 SHG :ŭť7Ş&Knw. Fluorescence dye. ML Ti:Sapphire Laser. JtȂ SHG ŭť¯š:Į! 7z&ȁƑƗ& 785'ŝǆ'ÁĮš:ƛď7#ȁƑƗ5'ť. Objective Lens. Chirped Mirror. SHG (KnwJtŨĲ'1'" 7#ƺ 78. Michelson Interferometer. Relay Lens 2. Delay Relay Lens 1. Ô&1_VGHn@t[ȅĘĞ¸äȇƭȆ#' £ǲàĥƲőŏǘċŜůTkwOå . Beam Splitter. e Wedg ion Insert. ȋŁ?kwOtH»Ƣ# ŞĆ:Į! 7Ĭ 1ǤƳ%'(KnwJt£ÙÈĮ'ǷƕüÚŞċ:¥ 3. . ŧ70ƑƗĶƧ|Ƴ" 6ť 6'--'ş. Fig.. Ď"'œÝ»Ƣ%0KnwJtǭǛ£ǥ&7Į. measurement at the focal point of SHG microscopy by. ŧ%Wwp&%7#İÿ87. TPEF interferometric autocorrelator.. Experimental. setup. for. pulse. duration. ÇȌ(ƚǞö£Ğ:²£&ƱşĎ"ƻœƤ ìŴǭŌü" 6846`pPð 18.9fs " 7 #:żƾqwMw¢äųĀ'`pPðȅ=10fsȆ& Ň,!`pPðÜ&Ē6! % ţŨ(ȂŁ £ĞÄ'£ĞƱưƙ"(²£&Ʊ"% 0 " 7#ƛ 587āĲ& 100fs qwMw#Ň ,7#ȍƁõ-"`pPð:Šƅ±"! 7'" SHG ǽĄǩ'Ȃčõ±İÿ"7. Fig. 2.. Collagen-sensitive SHG microscopy.. -* ! '&) qwMwŔ&(ĻŹlw[¿İ Ti:S qwMwȅ`p Pð 10fsćŌǪ 787nmPfGZpð 103nmƘ6 ǖÂŌğ 81.8MHzȆ:ŧ Ǆ£Ğ»ÓUmwdin wȅƚǞö£ĞǦȐ-900 -6270fs2Ȇ#ł£Ğ»Ó@AV OdoQjȅƚǞö£ĞǦȐ50.8 356 fs2Ȇ&47ƚǞö £ĞƱ&46ãŝqtQřŘƙ"'`pPð:ĬŹ. Fig. 4. Pulse duration at the focal point of SHG. ±SHG ǽĄǩ'ãŝqtQȅňŏNA=0.90°. microscopy.. -18-.

(14) .*($"#. %. SHG ǽĄǩ'ÞȀǣƙÇ:Ç 5 &ŽÇȌ#¿Ľ% ǣƙ#%! 7h?IpRtñŒƻ#Ltdpƙ 'ƭƧƏŕŐ:ŵ Ltdp5ŭť©Ģ SHG (KtYtLwqtQ&4!Ǳ8_t[ `Pc>pSw:ŧ ! SHG Đ£'.:ė¢Ā cCZtE@tX>tHÌǴÙÒƊ&4!ĺ¢ 87Fp_^inw# 2 Ĵ'oqwqtQ:ŧ !L tdpz'řŘPgVZ: 2 Łǈķ7#&46 200m200m ' SHG ?kwO:¹Ă"75&. (a). ĿĹ÷PXwO#'ŧ&46ÖǸƃ SHG ?kwOt Hȅ800μm800μmȆ'¹Ă1»Ƣ" 7Ňǒ'0 ¿œÝǢ:110fs qwMwŔȅlw[¿İBpa @jvc<?_wqwMw' SHG Ȇ:ŧ !¿xœ ÝǢ:ƻœ¢Ĳ74

(15) &! 7 PMT Filter Condenser Lens Sample ML Ti:Sapphire Laser. Objective Lens. 3D Stage Relay Lens 2. Chirped Mirror. (b). Relay Lens 1. e Wedg ion Insert. Beam Splitter. Block. Fig. 6. SHG images of osteoblasts-produced collagen. Galvano Mirror (X, Y). fiber by use of (a) 110-fs laser and (b) 10-fs laser. ĬĀ&ȁƨƐơŦťKnwJt'ĽÙ:ȌǜǬ&9. SHG of ML Er:F Laser. !ħƍ¤l\SotHƒĵ:Çȏ&ŽƆȉǜ "( ş'Ąù% SHG ÕƷœ88(. Fig.. 5.. Experimental. setup. for. pulse. duration. measurement at the focal point of SHG microscopy by TPEF interferometric autocorrelator.. KnwJtȁƨƐơǢ&£î! 7#:Ž! 7#ƛ 587ƆȊǜ"(SHG úõÒÖ! 68(ȁƨƐơǢ'KnwJtŖõÀz!. œÝ&ŧƐơ(h@PǼƫȁŨĲƐơĸ'ȁƨ. 7#:¸Ħ! 7#ƛ 587Ɔȋǜ"(Knw. ƐơĽƐơ MC3T3-E1 " 7PZqVUUmt_w&. JtȁƨƐơÔǢ&Ŧť8ƕƔļǚ:üĐ! 7. Ɛơ:ěƂĀǶÊÎǿLtdp:ƻœÇ. ĽÙ£7ƆȌǜ"(KnwJtƕƔļǚ×Đ. Ȏ(ȋǜǬ'ǬǶÊÎǿȁƨƐơŦťKnwJt. Ǫ7#&ƕƔļǚ'çÉ±1żƾ"7'4

(16). :10fsȅòË`sw20mWȆqwMw·* 110fs qwM. &SHG ǽĄǩ:ŧ 7#&46ȁƨƐơÎǿLt. wȅòË`sw20mWȆ:ŧ !ƻœ SHG ?kwO. dp&7KnwJt°Ď:¿x"»ƶ±7. ȅL?Q 1mm*1mmȆ:Ž! 7Ɯ:Ňǒ7#. #&Đ¬. 10fs qwMw'¥ŧ&46SHG ?kwO'KtZnP ZÖÀz8! 7#£7'ƒĵȁƨ ƐơKnwJt'ƕƔļǚ:»ƶ±". -19-.

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(18) 10fs lw[¿İ Ti:S qwMw:ŧ Ȃčõ SHG ǽ Ąǩ'ļƋƌâ%£ĞƱ&46ãŝqtQǱ ƙ"'`pPð 18.9fs :ǠĐ'ƒĵ SHG ŭ ť¯š'ȁƨƐơŦťKnwJt' SHG ?kwOtH& Đ¬5&ȁƨƐơÎǿǟƁ&7KnwJt °Ď:ħƍ¤"»ƶ±. (a). (b). (c). (d) Fig. 7. Time-series SHG images of osteoblasts-produced collagen fiber after (a) 1 week, (b) 2 weeks, (c) 3 weeks, and (d) 4 weeks.. -20-.

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