Biz konfokal görüntüleme onun eğitim ve tavsiye için Dr Jeffrey Caplan&#39;ı teşekkür ederim. Biz de SEM yardım için Keck Elektron Mikroskopi Lab ve Dr Chaoying Ni teşekkür ederim. Bu çalışma Sağlık (NIDCD, R01DC008965 ve R01DC011377) Ulusal Sağlık Enstitüleri tarafından finanse edilmektedir. ABZ NSF Bütünleştirici Lisansüstü Eğitim ve Araştırma staj finansman için (IGERT) programı kabul eder.

1.. Biyoreaktör Meclisi (Video 1) <ol><li> Önceden belirlenmiş bir iç ve dış boyutlara sahip bir alüminyum kalıp (dairesel kalıp + boşluk pin) (Şekil 1) olun. </li><li> In: (Şekil 1 &#39;de boşluk pim ile şekillenen 12 mm, kalınlığı ~ 0.25 mm, çap) bir sabit kol ile aşama 1.1 kalıp kullanılarak, silikon membran (1.5 mm, Şekil 1: 42 mm, kalınlık çap) üretmek Bir ticari olarak temin edilebilen bir silikon elastomer kiti kullanılarak orta. </li><li> Eşleşen çıkıntı ve oluklar ile blok (0.9 cm kalınlığında) üst oyma (1.8 cm kalınlığında) ve alt, ortada: dairesel bir açıklığa (24 mm çap) sahip akrilik bir çift bloğu (Şekil 2 (4, 5)) Yap . 10. </li><li> Eşleştirilmiş akrilik bloklar arasındaki silikon membran sandviç. Sabit bir kuvvet (35 cN · m) ayarlanmış bir mikro tork tornavida kullanarak dört köşe vida ile düzeneğini sabitleyen. Sonuç olarak, bir su geçirmez, 24 mm genişliğinde ve 18 mm derinliğinde titreşim odası (Şekil 2C) oluşturulur. </li><li> Alt akrilik blokta köşe vidalar başka bir dizi titreşim odasının altında bir 3 &quot;uzun menzilli mini woofer (Şekil 2B, 8 Ω/20 W) monte edin. Bu noktada, tek bir titreşim modülü monte edilir. </li><li> Yedi ek titreşim modülleri çoğaltmak. Çubuklar halinde kesilir eşit aralıklı bir dairesel delik (: 7 cm, kalınlığı 2 cm çapında) hoparlör bazları yerleştirilerek iki sabit alüminyum çubuklar (40 cm x 10 cm x 2.5 cm) birine dördü yapıştırın. Her bir dairesel deliğe alüminyum çubuğun tarafı boyunca bir vida sokulmasıyla her bir hoparlör stabilize. </li><li> Bireysel bir hoparlör seçici tarafından hoparlörleri kontrol. Seçici üzerindeki karşılık gelen çıkışlarına sonra hoparlör vücut üzerinde pozitif ve negatif girişleri için teller takılarak seçicinin tek tek hoparlör bağlayın. Hoparlör Seçici olanak t sinyaliO fonksiyon jeneratörü, bir güç amplifikatörü geçtikten sonra, bir kez (Şekil 2E) bütün sekiz hoparlör ulaşmak için. </li><li> Bir anti-nem muhafaza içinde iki odası dizileri, hoparlör seçici ve ilgili elektronik yerleştirin. Ticari bir hücre kültürü inkübatör tüm montaj ev. </li><li> Inkübatör arka filtre düzeneği üzerinden güç amplifikatör ve hoparlör seçici bağlantı (a medikal PVC boru yoluyla) ana kablolar besleyin. </li></ol> 2.. İskele Üretimi ve Karakterizasyonu <ol><li> 15 ağırlık% &#39;lik bir konsantrasyonda kloroform içinde PCL pelet çözülür. Bir 21 G kör uçlu iğne ile kapatılmış bir 10 ml şırınga içine solüsyonu yerleştirin. </li><li> Bir programlanabilir şırınga pompa üzerine şırınga Kilit ve / ml &#39;de 1 saat akış hızını ayarlayın. </li><li> ~ 18 sm bir iğne ucu-toplayıcı için mesafe ile yatay olarak iğne arasında alüminyum folyo kaplı toplayıcı yerleştirin. </li><li> Pozitif alli Kelepçeiğne ortasında ve alüminyum kollektör için zemin timsah klibi timsah klibi, sonra 15 kV yüksek gerilim güç kaynağı gerilimini ayarlayabilirsiniz. DİKKAT: yüksek gerilim, iğne mesafe tutmak. </li><li> Sırasıyla şırınga pompa ve güç kaynağı açın; hızlı bir şekilde temiz / stabil fiber jetleri önce kuru bir kağıt havlu kullanarak iğne ucu çevreleyen kalan polimer çözüm kaldırmak ve Taylor koni 19 oluşturulmuştur. </li><li> Lifler, 250-300 um ~ bir kalınlığa Al toplayıcı üzerinde birikmesine izin verir (~ mevcut eğirme koşulları altında 7 saat). Herhangi bir artık çözücünün çıkması için 1-2 gün için bir vakumlu desikatör içinde sonuçtaki iskeleleri saklayın. </li><li> Görüntü iskelesi, tutarlı bir elyaf morfolojisi gösteren bir Tarama Elektron Mikroskobu kullanılarak, üzeri serpme altınla kaplanır. 10 </li></ol> 3.. Biyoreaktör Montaj ve Karakterizasyonu <ol><li> Dört kol (uzunluğu: silindir şeklinde bir disk (8 mm çapında) 2: zımbailk olarak, diskin dış çapı azaltmak için bir 12 mm çaplı biyopsi zımba kullanılarak bükümlenmiş PCL mat (Şekil 2A) üzerinden mm). Ardından eşit silah kesilecek nerede puan dairesel bıçak çevresinde aralıklı dört 2 mm uzunluğunda çentikler yapmak için ikinci bir 8 mm biyopsi yumruk kullanın. 8 mm yumruk attıktan sonra, dışa kollarının kenarlarını kesmek için bir neşter bıçak kullanın. Uzatılmış kollar üzerinden silikon membran oluk içine yerleştirin iskele (Video 1). Nazikçe flathead cımbız kullanarak yüzeyi basarak eklenen iskele düzleştirin. </li><li> Lazer yansıma yardımcı olmak üzere PCL iskele ince Al folyo küçük bir parça (8 mm x 2 mm, ortogonal şekil, Şekil 2B) takın. </li><li> Titreşim odası içindeki monte silikon membran / PCL iskele (aşama 1.4 &#39;de açıklandığı gibi) elde edin. Titreşim önce PCL iskele hidrat için odacık içinde 1.5 ml su ilave edilir. </li><li> Fonksiyon jeneratör kullanarak, titreşim işareti tanıtmakals sandviç akrilik haznesine (0,1 V bir tepe-tepe voltaj, Vpp olan, örneğin, 200 Hz sinüs dalgaları). Doğru her hoparlör girişindeki voltajı ölçmek için bir voltmetre kullanın. Not: fonksiyon jeneratöründen gelen Vss okuma hoparlör teslim nihai geriliminden farklı olacaktır. </li><li> Tek-nokta LDV birleştirin ve bir pan-tilt kafa tripod fiber-optik lazer sensör kafasını sabitlemek. Açı sensör kafasını o masa dik bakacak şekilde. Koaksiyel kablo üzerinden veri toplama modülüne LDV sensör kafasını bağlayın ardından USB üzerinden laptop modülü. </li><li> Silikon membran (Şekil 2B ve Şekil 3) üzerinde çeşitli önceden belirlenmiş noktalarda dik olarak bir lazer ışını Odak. </li><li> Veri toplama yazılımı kullanarak, orta zar deplasman kaydedebilirsiniz. &quot;Seçenekler&quot; menüsünden &quot;Acquisition Ayarlar&quot; ı tıklayın; sonra ölçüm modunu &quot;FFT değiştirin221;. Sonra, ana araç çubuğundaki &quot;Sürekli Ölçüm&quot; tıklatın deplasman kaydetmek için seçilen frekansta (Şekil 6D) de oluşturan tepe tıklatın. </li><li> Alt-tabaka üzerinde göreli konumun bir fonksiyonu olarak normal orta zar yer değiştirme (w 0) çizilir. Origin 8.5 veri analiz yazılımı kullanılarak yüzey titreşim profilinin 3D renk haritası oluşturun. </li></ol> 4. Titreşimli Hücre Kültürü <ol><li> Bakım MSC medya / cm 2 4,000-5,000 hücrelik bir başlangıç ​​yoğunluğunda tohumlama T150 doku kültürü şişelerinde alt kültür İnsan kemik iliğinden türetilmiş MSC. </li><li> Hücre kültürü 7-8 gün sonra (~% 85 konfluansa kadar), bu Accutase gibi bir hücre ayrıştırma maddesi ile hücreleri trypsinize, bir hemositometre, santrifüj (5 dakika için 440 x g) kullanılarak sayısı ve yeniden askıya hücre peleti içinde 4.5 x 10 6 hücre / ml &#39;lik bir konsantrasyonda taze MSC bakım ortamı. </li><li> PCL scaffo daldırın% 70 etanol O / N. ld Çözücü buharlaştırılmıştır sonra 5-8 dakika boyunca UV ışığı (254 nm) için mikrop öldürücü skafoldun iki tarafını ortaya çıkarmak. </li><li> 1 saat boyunca 37 ° C&#39;de 20 ug / ml fibronektin çözeltisi içindeki PCL iskele bekletin. Silikon membran içine fibronektin kaplı iskele yerleştirin. 1. adımda ayrıntılı olarak biyoreaktör birleştirin. </li><li> PCL güvenli iskele üzerinde düzgün bir şekilde hücre süspansiyonu 40 ul dağıtın. Hücreler titreşim bölmeye ilave bir 1.5 ml taze ortam ilave edilmeden önce 1-1.5 saat boyunca takmak için izin verin. </li><li> Kültür MSC-yüklü PCL 3 gün boyunca statik iskele ve statik kültür tamamlandıktan sonra ortam yenileyin. </li><li> Hücresel yapıları seçilen titreşim rejimleri empoze. Not: Örnek olarak, hücreler kadar 7 gün boyunca günde 12 saat boyunca ~ 40 um aw 0 ile 200 Hz&#39;de titreşim (OF), bir 1-hr-on-1-hr-off tabi tutulur. Titreşimli uyarılara maruz Yapıları Vib örnekleri ve bu kültürlerdeki olarak belirlenmişstatik aynı titreşim odalarına kırmızı olarak statik kontrollerini (İstatistik) hizmet vermektedir. </li></ol> 5.. Biyolojik Değerlendirmeleri <ol><li> Her gün, her bölmeden 200 ul hücre kültür ortamı kazan (gün 1, 3, 5 ve 7) ve (800 ul her biri) ile birlikte, aynı örnekten alikotları havuz. </li><li> MMP-1 (MMP1) ve üreticilerin prosedürü izleyerek, sırasıyla, bir MMP1 ELISA Geliştirme kiti ve bir hiyalüronan rekabetçi ELISA kiti kullanılarak HA hücresel üretim niceliğini. Öte yandan, daha önce bildirilen prosedür takip edilerek ELISA çözünür ön-elastin üretimini tahlil. 8 </li><li> 7. günde, son titreşim döngüsünün tamamlanmasından sonra, hızlı bir şekilde keskin cımbız kullanarak titreşim odalarından hücresel yapıları çıkarın ve kısa bir süre soğuk bir fosfat tamponlu tuzlu su (PBS, 4 ° C) ile yıkayın. </li><li> Canlı / ölü boyama için, propidyum iyodür (PBS içinde 1:2,000) ile yapıları ve kuluçkaya yatmakSyto-13 oda sıcaklığında 5 dakika boyunca aynı anda (PBS içerisinde 1:1000). Görüntü bir multiphoton konfokal mikroskop ile boyanmış yapılar. </li><li> Ayrı olarak, kuru buz üzerinde PBS ile durulanmıştır hücre yapıları ek dondurma ve gen analizleri için, daha önce rapor protokolü takip edilerek, toplam hücre RNA ekstrakte edin. 9 </li><li> UV-Vis spektrofotometre kullanılarak çıkarılan RNA miktarını ve kalitesini kontrol edin. 1,8-2,2 arasında A260/A280 ve A260/A230 oranları ile RNA numuneleri daha sonra QPCR analizi için kullanılmıştır. </li><li> Ters bir ticari olarak temin edilebilen bir kit kullanılarak ters transkripsiyon cDNA&#39;ya RNA (500 ng / numune) uyarlamak. </li><li> Daha önce detaylı bir prosedür izlenerek ticari olarak temin edilebilir PCR master karışımını kullanarak gerçek zamanlı bir sekans saptama sistemi üzerinde PCR reaksiyonu yapın. 8 </li><li> Ticari QPCR veri analiz yazılımı kullanılarak qPCR analiz sonuçları. Veri analizinin güvenilirliğini sağlamak için, çoklu referans genler (YWHAZ, TBP, PPIA) int olarak istihdam edilmektedirernal kontrol ve spesifik primer verimlilik değişme göz önüne alınır. 8 </li></ol>

<ol>
	<li>Titze, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical-Stress+in+Phonation.">Mechanical-Stress in Phonation.</a> J. Voice. 8, 99-105 (1994).</li><li>Titze, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Relation+Between+Subglottal+Pressure+and+Fundamental-Frequency+in+Phonation.">On the Relation Between Subglottal Pressure and Fundamental-Frequency in Phonation.</a> J. Acoust. Soc. Am. 85, 901-906 (1989).</li><li>Gray, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+the+vocal+folds.">Cellular physiology of the vocal folds.</a> Otolaryngol. Clin. N. Am. 33, 679-698 (2000).</li><li>Thibeault, S. L., Gray, S. D., Bless, D. M., Chan, R. W., Ford, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+and+rheologic+characterization+of+vocal+fold+scarring.">Histologic and rheologic characterization of vocal fold scarring.</a> J. Voice. 16, 96-104 (2002).</li><li>Hansen, J. K., Thibeault, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+and+review+of+the+literature:+Vocal+fold+scarring.">Current understanding and review of the literature: Vocal fold scarring.</a> J. Voice. 20, 110-120 (2006).</li><li>Pittenger, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Hanson, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Mesenchymal+Stem+Cells+From+Human+Vocal+Fold+Fibroblasts.">Characterization of Mesenchymal Stem Cells From Human Vocal Fold Fibroblasts.</a> Laryngoscope. 120, 546-551 (2010).</li><li>Tong, Z., Duncan, R. L., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+the+behaviors+of+mesenchymal+stem+cells+via+the+combination+of+high-frequency+vibratory+stimulations+and+fibrous+scaffolds.">Modulating the behaviors of mesenchymal stem cells via the combination of high-frequency vibratory stimulations and fibrous scaffolds.</a> Tissue Eng. Part A. 19, 1862-1878 (2013).</li><li>Tong, Z., Sant, S., Khademhosseini, A., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Fibroblastic+Differentiation+of+Mesenchymal+Stem+Cells+Via+the+Combination+of+Fibrous+Scaffolds+and+Connective+Tissue+Growth+Factor.">Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor.</a> Tissue Eng. Part A. 17, 2773-2785 (2011).</li><li>Jones, D. L., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+place+like+home:+anatomy+and+function+of+the+stem+cell+niche.">No place like home: anatomy and function of the stem cell niche.</a> Nat. Rev. Mol. Cell Biol. 9, 11-21 (1038).</li><li>Wang, J. H., Thampatty, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+Adult+and+Stem+Cells.">Mechanobiology of Adult and Stem Cells.</a> Int. Rev. of Cell Mol. Biol. 271, 301-346 (2008).</li><li>Doroski, D. M., Levenston, M. E., Temenoff, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+tensile+culture+promotes+fibroblastic+differentiation+of+marrow+stromal+cells+encapsulated+in+poly+(ethylene+glycol)-based+hydrogels.">Cyclic tensile culture promotes fibroblastic differentiation of marrow stromal cells encapsulated in poly (ethylene glycol)-based hydrogels.</a> Tissue Eng. Part A. 16, 3457-3466 (2010).</li><li>Kim, B. S., Nikolovski, J., Bonadio, J., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+strain+regulates+the+development+of+engineered+smooth+muscle+tissue.">Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.</a> Nat. Biotechnol. 17, 979-983 (1999).</li><li>Webb, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+strain+increases+fibroblast+proliferation,+matrix+accumulation,+and+elastic+modulus+of+fibroblast-seeded+polyurethane+constructs.">Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs.</a> J. Biomech. 39, 1136-1144 (2006).</li><li>Kim, Y. J., Sah, R. L. Y., Grodzinsky, A. J., Plaas, A. H. K., Sandy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Regulation+of+Cartilage+Biosynthetic+Behavior+-+Physical+Stimuli..">Mechanical Regulation of Cartilage Biosynthetic Behavior - Physical Stimuli..</a> Arch. Biochem. Biophys. 311, 1-12 (1994).</li><li>Titze, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+bioreactor+for+engineering+vocal+fold+tissues+under+combined+tensile+and+vibrational+stresses.">Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses.</a> J. Biomech. 37, 1521-1529 (2004).</li><li>Kutty, J. K., Webb, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibration+stimulates+vocal+mucosa-like+matrix+expression+by+hydrogel-encapsulated+fibroblasts.">Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts.</a> J. Tissue Eng. Regen. Med. 4, 62-72 (2010).</li><li>Farran, A. J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Characterization+of+a+Dynamic+Vibrational+Culture+System.">Design and Characterization of a Dynamic Vibrational Culture System.</a> J. Tissue Eng. Regen. Med. , (2011).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Theobald, P., Tyrer, J., Lepper, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+scanning+vibrometer+in+mapping+ultrasound+fields.">The application of scanning vibrometer in mapping ultrasound fields.</a> J. Phys.: Conf. Ser. 1, 167-173 (2004).</li><li>Brown, W. S., Morris, R. J., Hollien, H., Howell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speaking+Fundamental-Frequency+Characteristics+as+a+Function+of+Age+and+Professional.">Speaking Fundamental-Frequency Characteristics as a Function of Age and Professional.</a> J. Voice. 5, 310-315 (1991).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanotransduction:+putting+all+the+pieces+together+again.">Cellular mechanotransduction: putting all the pieces together again.</a> Faseb J. 20, 811-827 (2006).</li><li>Titze, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+bioreactor+for+engineering+vocal+fold+tissues+under+combined+tensile+and+vibrational+stresses.">Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses.</a> J. Biomech. 37, 1521-1529 (2004).</li><li>Moore, J., Thibeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+Into+the+Role+of+Elastin+in+Vocal+Fold+Health+and+Disease.">Insights Into the Role of Elastin in Vocal Fold Health and Disease.</a> J. Voice. 26, 269-275 (2012).</li><li>Thibeault, S. L., Bless, D. M., Gray, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+protein+alterations+in+rabbit+vocal+fold+with+scar.">Interstitial protein alterations in rabbit vocal fold with scar.</a> J. Voice. 17, 377-383 (2003).</li><li>Branski, R. C., Verdolini, K., Sandulache, V., Rosen, C. A., Hebda, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocal+fold+wound+healing:+A+review+for+clinicians.">Vocal fold wound healing: A review for clinicians.</a> J. Voice. 20, 432-442 (2006).</li><li>Clark, I. A., Swingler, T. E., Sampieri, C. L., Edwards, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+matrix+metalloproteinases+and+their+inhibitors.">The regulation of matrix metalloproteinases and their inhibitors.</a> Int. J. Biochem. Cell B. 40, 1362-1378 (2008).</li><li>Silver, F. H., Horvath, I., Foran, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelasticity+of+the+vessel+wall:+The+role+of+collagen+and+elastic+fibers.">Viscoelasticity of the vessel wall: The role of collagen and elastic fibers.</a> Crit. Rev. Biomed. Eng. 29, 279-301 (2001).</li><li>Kutty, J. K., Webb, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+Therapies+for+the+Vocal+Fold+Lamina+Propria.">Tissue Engineering Therapies for the Vocal Fold Lamina Propria.</a> Tissue Eng. Part B: Rev. 15, 249-262 (2009).</li></ol>

Hiçbir rakip mali çıkarları var.

In vitro fonksiyonel vokal kord dokuların başarılı mühendislik multipotent hücrelerinin davranışlarını aracılık etmek bir vokal kord benzeri mikroçevresinin rekreasyon gerektirir. Genellikle doku veya organ yapıları da gerçekleştirmek için gerekli işlevleri yansıttığı kabul edilmektedir. Vokal kord dokuları için 22, fonasyon sırasında ortaya çıkan yüksek frekanslı titreşimler doku olgunlaşması için önemli olduğu önerilmiştir. Vokal kord biyoreaktör kültürlü ...

Bir epitel tabakası oluşan insan vokal kord, lamina propria (LP) ve vocalis kas, ses üretimi için akustik dalgalar halinde akciğerlerden hava akışını dönüştüren özel bir yumuşak doku. 1. Vokal kıvrımlar, normal fonasyon sırasında düzenli salınım, değişen temel frekanslarda% 30&#39;a varan suşları sergileyen 100-300 Hz arasında. 2 Adult vokal kat LP yüzeysel (SLP) oluşan bir degrade yapı, bir ara (TDP) ve derin (DLP) tabakasıdır. Bundan başka sınıflandırma grupları epitelyum ve mukoza tabakası olarak SLP ve ses ligamana İLP DLP ve bir araya getirir. 3 bağ olgun kolajen ve elastin lifleri ile zenginleştirilmiştir ise SLP tabaka, seyrek dağılmış kollajen lifleri ile öncelikle bir amorf matrisi içermektedir yeterli gücü sağlamak. 4. yapısı ve doğan vokal kıvrım mekaniği kendi olgun meslektaşları arasında büyük ölçüde değişebilir. Her ne kadar mekanizmasıvokal kord gelişimini ve olgunlaşmasını düzenleyen s henüz tam deneysel kanıt seslendirme kaynaklı mekanik stres tanımlayan rollerine işaret etti, anlaşılamamıştır. Sesli taciz, enfeksiyonlar, kimyasal maddelere ve cerrahi prosedürler dahil olmak üzere birçok tıbbi durumlar, vokal kat zarar verebilir. Vokal kord bozuklukları ABD nüfusunun yaklaşık% 3-9 etkiler. Vokal kord hastalıkları için güncel tedavi yöntemleri 5 sınırlıdır ve bir kök hücre tabanlı doku mühendisliği yaklaşımı vokal kord fonksiyonunu yeniden kazandırmak için umut verici bir strateji olarak ortaya çıkmıştır. Mezenkimal kök hücreler (MKH) vokal kord doku mühendisliği için birincil vokal kord fibroblastlar için uygun bir alternatiftir. 6-9 Kök hücre kaderinin şartname ve sonraki doku gelişimi mekanik durumdur ki onlar ikamet belirli bir niş, aracılık bir önemli bir faktördür. 10. Mekanik kuvvetler doku morfonogenezi a temel düzenleyicileridirlernd özellikle rutin bir doku mühendisliği açısından bakıldığında yükleme. 11 maruz kalan dokular için homeostazı, bu maruz kalma fizyolojik olarak uygun mekanik uyarılarla hücre farklılaşması ve dokuya özel matris yeniden kök teşvik için olduğu gösterilmiştir. 12-15 Doku kültürü biyoreaktörler in vitro hücre veya doku büyümesi için arzu edilen fizyolojik bir ortam simüle etmek için tasarlanmıştır. Vokal kord doku mühendisliği için, phonating vokal kıvrımların mekanik ortamını yeniden oluşturmak için özellikle önemlidir. İdeal vokal kord biyoreaktör etkili frekans, genlik ve titreşim süresi boyunca uyduruk kontrol sağlayan, kültür hücreleri titreşimli ipuçları teslim etmelidir. Titze ve arkadaşları matriks proteinlerinin hücresel üretimini teşvik etmek için yüksek frekanslı (20-200 Hz) salınımları ile statik esneme birleştiren bir vokal kord biyoreaktörünü (T1 biyoreaktör) 16 geliştirmiştir. Istimalg Bu biyoreaktör, Webb ve arkadaşları 17 hyaluronik asit (HA)-tabanlı hidrojel içinde kültüre dermal fibroblastlar üzerinde 10 günlük, 100-Hz titreşimlerin etkilerini inceledi. Titreşime maruz yapılar HA sintaz-2 (var2) statik kontrollere göre, dekorinin, fibromodulin ve matris metaloproteinaz-1 (MMP1), bir yüksek ifade sergiledi. Uyarıcı etkileri bağımlı zaman olduğu bulunmuştur. Daha yakın zamanlarda, grubumuz 18, bir güç yükselticisi, bir fonksiyon jeneratörü, kapalı bir hoparlör ve ekli hücrelere salınımlı hava transfer eden bir çevresel olarak sabitlenmiş silikon membran kullanılan bir ses kat biyoreaktör (J1 biyoreaktör) toplandı. J1 biyoreaktör içinde yetiştirilir Neonatal sünnet derisi fibroblast kadar% 0.05 &#39;lik bir düzlem suşu ile, 60, 110 ya da 300 Hz&#39;de titreşim 1 saat tabi tutulmuştur. QPCR sonuçlar, bazı ECM genlerin ekspresyon orta çeşitli titreşim frekanslarına karşılık olarak değiştirilmiş olduğu ileri sürülmüştürve genlikleri. Bu biyoreaktör tasarımlar, ilginç ederken, çeşitli sınırlamalar var. Örneğin, T1 sistem ulaşılabilir maksimum frekansları sınırlayarak, mekanik bağlanma için konektörler ve bar bir sayıda gerektirir. Ayrıca, hücreler veri yorumlama karmaşık istenmeyen mekanik çalkalama ve sıvı pertürbasyon tabi tutulabilir. J1 biyoreaktör, diğer yandan, nispeten düşük enerji dönüşüm verimliliği sergiler ve kullanıcı dostu değildir. Buna ek olarak, titreşim sıklıkla temel silikon zardan hücre yüklü yapıları ayırır. J1 sürüm olarak aynı prensibe göre dizayn Burada bildirilen J2 vokal kord biyoreaktör, tutarlılık ve tekrarlanabilirlik için optimize edilmiştir. Fonasyon taklit eden titreşimler MSC nüfuslu lifli poli (ε-kaprolakton) (PCL) iskeleleri effectiv olan tek tek monte titreşim odalarına aerodinamik oluşturulurely güvenli. Lazer Doppler Titreşim Ölçer (LDV) kullanıcı membran / iskele montaj titreşim profilini doğrulamak sağlar. Bizim tanıtımda, MSC 7 gün boyunca 12 saat günlük bir toplam için 1 saatlik-on-1-hr-kapalı (OF) model ile 200 Hz sinüzoidal titreşimlere maruz kalmaktadır. Dayatılan titreşimli ipuçlarını hücresel yanıtlar sistematik olarak incelenmiştir. Genel olarak, J2 vokal kord dinamik hücre kültürü çalışmaları, yüksek verim ve tekrarlanabilir şekilde yapılacak sağlayan, en kullanıcı dostu özellikleri sağlar.

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="16">
			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

construction and characterization of a novel vocal fold bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Elektrospinning ile imal PCL iskeleleri mikron büyüklüğünde interstisyel gözenekler ve 4.7 um (Şekil 4A) arasında bir ortalama çapa sahip rastgele bir biçimde dolanmış elyaflar içerir. Daha yüksek bir büyütme oranında nano ölçekli oluklar ve gözenekler tek tek liflerin (Şekil 4B) üzerinde görülebilir. Fibronektinle iskelelerinin kaplama hidrofilikliğin geliştirir ve ilk hücre yapışmasını / PCL iskelesi (yayınlanmamış gözlem) yayılan kolaylaştırır. ...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Bir Roman Vokal Fold Bioreactor İnşaatı ve Karakterizasyonu

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Research

JoVE Journal

Bioengineering

9.1K Görüntüleme.  University of Delaware. A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Video: Construction and Characterization of a Novel Vocal Fold Bioreactor

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Pulsatif Biyoreaktör kullanarak Mühendislik Biyolojik Tabanlı Vasküler Greftler

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Biyomühendislik

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sentetik, Multi-Layer, Self-Salınımlı Vokal Fold Model Fabrikasyon

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Doku Mühendisliği için iki eksenli Mekanik Yükleniyor Biyoreaktör tasarımı

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Bir Benchtop Biyoreaktör Operasyonu

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Model Vocal Fold Polip'in Neden Olduğu Üç Boyutlu Akış Ayrımının Araştırılması

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Lökosit-trombosit Zengin Fibrin, Yeni Bir Biyomateryalinin Karakterizasyonu

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Çeşitli mikrobiyal Toplulukları Yüksek Yoğunluklu Yetiştirme için Yeni Bir Biyoreaktör

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Kinetik Büyüme Testleri için Sıcaklık, Işık ve pH İzleme ile Bir Bench Ölçekli Algal Fotosentetik Biyoreaktörün Kurulması ve Kurulması

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Sinir Büyümesi Faktörü Kapsüllemesi için Yeni HDL&#39;yi taklit eden Nanopartiküllerin Hazırlanması ve Karakterizasyonu

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...