Bu çalışma UWS Araştırma Bursu ile desteklenmiştir.

1. Kitosan Yapıştırıcı Hazırlık <ol><li> Çitosan tozu, asetik asit çözelti içinde çözünür olan, asetik asit (% 2 v / v) bir stok çözelti hazırlamak için, 490 ml deiyonize su (di-H2O) için 10 ml glasiyal asetik asit ilave edin. </li><li> Asetik asit içinde Rose Bengal (RB) (% 0.01 w / v) bir stok çözeltisi hazırlanması için, photobleaching önlemek için alüminyum folyo ile sarılmış bir RB şişe içinde 5 mg ağırlığındadır. Asetik asit stok çözeltisi (bölüm 1.1 &#39;de anlatıldığı gibi) ve 49.5 mL daha sonra toz dağıtma DI-H2O 0.5 ml ilave edilir. </li><li> Çitosan solüsyonu (% 1.7 w / v) hazırlanması için, çitosan tozu (orta MW, asetilasyon derecesine% 85) 0.85 g ağırlığında ve RB asetik asit solüsyonundan 50 ml eklenir. RB kontrol olmaksızın asetik asit solüsyonu içinde çitosan çözülmesiyle hazırlanmıştır. </li><li> Çitosan karışım için bir manyetik karıştırıcı, ilave edin ve oda sıcaklığında karıştırın. Bileşenlerin tam bir çözünme sağlamak için, kitosan karıştırmaİki hafta ve 5 gün süre ile asetik asit içinde kitosan (kontrol) asetik asit içinde + RB. Çitosan çözünmüş zaman, çözelti pH ~ ~ 2.6 &#39;dan 3.9&#39; a artar. RB asidik pH içinde zayıf çözünürlüğe sahip olup, burada çözeltinin pH&#39;si ve çözünmesi için uzun bir süre içinde karıştırılmakta olan bir artış gerektirir dikkat ediniz. </li><li> 1 saat süre ile 3270 x g de santrifüjlenir ve çözümler, örneğin, bir steril 10 ml şırınga kullanılarak supernatant toplamak. Safsızlık pelet rahatsız değil emin olun. Bu adım sırasında, çözüm makroskopik kirlilikler ve çözünmeyen madde temizlenir. </li><li> Steril bir şırınga kullanarak düz perpex levha üzerinde (santrifüj) kitosan çözeltisinin enjekte. Kuru film kalınlığı, çözeltinin dağıtılan miktarı ve çözelti yayılmış olan fazla yüzey alanı arasındaki orana bağlıdır. Çözümü hava kabarcıkları serbest ve eşit levha üzerinde yayıldı emin olun. ~ 13 mikron kalınlığında ince filmler elde etmek için, 12 cm 2 yüzey üzerinde ~ 1 ml solüsyon enjektealanı (boyutları ~ 3x4 cm, Şekil 1A). </li><li> Çözeltinin photobleaching önlemek için örneğin alüminyum folyodan yapılmış bir ekranı olan bir Perspex levha, kapak. Yeterli havalandırma ekranın altında çözüm kuru garantili olduğundan emin olun. </li><li> Elde edilen film, suda çözünmez ve makroskopik olarak kabarmaz kadar oda sıcaklığında (25 ° C) ve basıncı (1 atm) kurumaya çözelti bırakın. Bu, tipik olarak 2 hafta içinde elde edilir. </li><li> Dikkatle ince ve düz spatula kullanarak filmler ayırın: bu filmlerin kolayca zarar vermeden perspex levha kapalı soyulmuş olabilir. </li><li> Bir mikrometre kullanılarak film kalınlığı ölçülür. </li><li> Temiz bir keskin makas ile dikdörtgen şeritler yapışkan kesin ve düz şekil korumak için steril cam slaytlar arasına koyun. Işık koruma için alüminyum folyo ile sarın slaytlar ve oda sıcaklığında saklayın. Veya &quot;chitosan ADHES&quot; yapıştırıcı gül &quot;olarak RB olan veya olmayan yapıştırıcılar etiketleyinive &quot;sırasıyla. </li><li> Yapışkan filmler gama radyasyonu (0.1 kGy / 24 saat için saat) 4,5 ile veya (% 90) etanol kullanılarak sterilize edilebilir yükseldi. </li></ol> 2. Yapıştırıcı Optik Zayıflama <ol><li> Bir kuvars küvet içinde gül yapışkanlı Fix. </li><li> Bir çift-kiriş spektrofotometre kullanarak 4 800 nm - 400 aralığında olan zayıflatılmış bir spektrum kaydedin. </li><li> Şöyle Beer yasasının geçerliliği varsayarsak, yapıştırıcı zayıflama uzunluğunu hesaplamak: I = I 0 e-Ax nerede 0 olay ışık yoğunluğu, 1 / A zayıflama uzunluğu ve x film kalınlığıdır. </li><li> Kitosan yapıştırıcı için 2.1 ile 2.3 bir kontrol olarak hizmet adımları tekrarlayın. </li></ol> 3 Vitro Uygulama In:. Bağırsak üzerine Lazer Doku Yapıştırma <ol><li> Hasat buzağı bağırsak hemen sonra -80 hayvan ötenazi ve mağaza ° C </li><li> Kullanmadan önce, defrost ve hidrat ti10 dakika için DI-H2O içine daldırarak ssue. </li><li> Operasyon mikroskobu (x10) altında # 10 bıçak kullanarak bölümleri (2 x 1 cm) içine doku ikiye bölmek. DI-H 2 O kullanarak bölümleri nemli tutun </li><li> (Uçtan uca) birbirine yakın kesi kütükleri getirin. Pamuk ipuçlarını kullanarak yüzeyden fazla suyu silin. </li><li> Bağırsak (Şekil 2) ile tam temas sağlanması microforceps ile seroza katman üzerinde kesi karşısında bir gül yapışkan bant (10 x 6 mm) yerleştirin. </li><li> Gül yapışkan bir doku yapışma modlu fiber optik (çekirdek çapı 200 um) 4 yoluna bağlı bir diyot pompalı katı hal lazer tarafından aktive edilir. </li><li> 180 mW lazer güç seviyesi ayarlayın ve Tablo 1&#39;de listelenen parametreleri kullanarak ~ 5 mm ışın spot büyüklüğü ile ~ 6 dk için yapıştırıcı ışın. Spot ışık saçmak yapıştırıcı her yerinde bitişik nokta ışın geçmeden önce ~ 5 saniye boyunca ışınlanır sağlanması. TOnun prosedürü lazer ışını yapışkan birkaç kez bütün yüzey alanı tarar garanti eder. </li><li> Doku yapıştırma gücü değerlendirmek için, mekanik kulpları kullanarak kalibre tek sütun tansiyometre (Instron, MA, ABD) için örnek bir kelepçe. 4 ayrı iki doku kütükleri kadar 22 mm / dak kulpları taşıyın. </li><li> Adım 3,5 anlatıldığı gibi doku üzerinde bir gül yapıştırıcı yerleştirin. Örnek ışın ve adım 3.8 &#39;de açıklandığı gibi doku yapıştırma gücünü ölçmek etmeyin. Bu örnek, bir kontrol olarak hizmet vermektedir. </li></ol> 4 Vivo Uygulama In:. Tibial Sinir Anastomoz <ol><li> O / izofluran 2 karışım (bundan sonra indüksiyon sırasında% 4,% 2) 5 ile Oturaklı Wistar sıçan. Subkutan intraoperatif analjezik aktivite sağlamak ve olası ağrı veya rahatsızlık hafifletmek için buprenex (0.03 mg / kg) enjekte. </li><li> Aşağıdaki cerrahi prosedür, bir ameliyat mikroskobu (x10) altında gerçekleştirilmelidir. Oblik cilt Yapsağ uyluk dorsolateral kısmında uzun ve gluteal kasları 2 yoluyla bir kas bölme yaklaşımı ile tibial sinir maruz yaklaşık 3 cm kesi. </li><li> Kısmen teşrih ve düz mikro-makas kullanarak tibial sinirin adventisya Döşeme ve steril gazlı bez veya pamuk ipuçları ile fazla su emer. </li><li> Mikro-makas ile tibial sinir kesin. </li><li> Mikro-forseps kullanarak tibial sinir altında steril bir kitosan şerit (boyutları 5 x 4 mm) yerleştirin. </li><li> Yaklaşık sinir uçlarının gül yapışkan şeridi üzerinde mikro-forseps ile uç uca. </li><li> Şeridi tam şerit lazer ışınlama sırasında sinir dönme konusunda yardımcı olabilecek bir yaka, oluşturan sinir yapışır. </li><li> ) Adımları 3.6 açıklandığı gibi lazer ile yapışkan gül etkinleştirin) ve 3.7. </li><li> Çalışan sinir yaka gereksiz yükseldi yapıştırıcı kesin. </li><li> Beş 3-0 dikişler 5 ile kaslar ve cilt kapatın. </li&gt; <li> Gerekirse Standart ameliyat sonrası iyileşme prosedürleri, ayrışma veya irin oluşumu ve analjezik uygulama için günlük muayene dahil takip edilmelidir. </li></ol> 5. Temsilcisi Sonuçlar Elde edilen filmler parlak, renkli gül ince ve pürüzsüz bir yüzey (Şekil 1) sahiptirler. Onlar da esnek ve neden gözyaşı veya başka görünür hasar (Şekil 1B) olmadan, küçük çaplı tüpler içine yuvarlandı olabilir. Gül yapışkanlı 530 ve 562 nm de iki emme zirvesi vardır; yeşil lazer ~ 12 mikron (Şekil 3) ve böylece güçlü 532 nm de yapıştırıcı ve gelen zayıflama uzunluğu tarafından emilir. Buna karşılık, RB olmadan kitosan film muhtemelen zayıf bir şekilde yayıldığı için lazer (1 / A ~ 162 um) zayıflar. Bu SaB anlamlı agregasyonu filmlerde oluşur spektrumları arsa görünür. Sıkıca yapıştırır yükseldiLazer ışını üzerine bağırsak (Şekil 2) 0,9 başarısızlığı tipik bir maksimum yük ± 0.4 N (n = 30) elde. Yapışma gücü yapışkan yüzey alanı, yani, 15 ile bölünmüş maksimum yük ± 6 kPa&#39;dan (n = 30) olarak tahmin edilmiştir. Işınlanmamış gül yapışkan doku daha az bağlı (2 ± 2 kPa, n = 30, eşleştirilmemiş t-testi, p &lt;10 6). Tibial sinirin in-vivo anastomoz başarılı bir şekilde yeşil lazer ile bağlantılı olarak gül yapıştırıcı uygulanması ve ardından bağ elde edildi. Bir hafta ameliyat sonrası, ameliyat sinir süreklilik ve onarım gücü 17 ± 9 kPa (n = 10). <img alt="Şekil 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> Şekil 1. A) RB-kitosan çözeltisinin düz perspex levha (levha kalınlığı ~ 5 mm) dökülmektedir. Grafik kağıt kuru dökme adım kolaylaştırmak için levha altına yerleştirilir. Suda çözünmeyen film iki hafta içinde dökümden sonra elde edilir. B) gül yapışkanlı esnek ve küçük bir silindir içine yuvarlandı olabilir. <img alt="Şekil 2," src="/files/ftp_upload/4158/4158fig2.jpg" /> Şekil 2. Gül yapışkanlı lazer ışınlama sonrası baldır bağırsak yapıştırılır. Düzgün ışınlama doku yapışma sırasında RB yapışkan uygulanır, ancak seçilen lekeler üzerinde yapışkan lazer foto-ağartma etkisini göstermek için bu resim gösterilir. <img alt="Şekil 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> Şekil 3,. Gül yapışkanın emilme tayfı (isteğe bağlı olarak birim) 530 ve 562 nm &#39;de iki adet tepe noktası gösterir. Yeşil lazer (λ = 532 nm) ve böylece güçlü bir lazer pozlama sırasında yapıştırıcı ile emilir. RB olmadan kitosan yapışkan zayıf lazer ışını emer. <table border="1"><tbody><tr><td></td><td> n </td><td><s&gt; Alanı (mm 2) Trong </td><td> Güç (W) </td><td> Süre (s) </td><td> Fluence (J / cm 2) </td><td> I (W / cm 2) </td><td> Maksimum Yük / Alan (kPa) </td></tr><tr><td> Yapıştırıcı + Lazer </td><td> 30 </td><td> 60 ± 10 </td><td> 0.18 ± 0.03 </td><td> 365 ± 5 </td><td> ~ 110 </td><td> ~ 0.9 </td><td> 15 ± 1 </td></tr><tr><td> Yapıştırıcı </td><td> 30 </td><td> 60 ± 10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2 ± 2 </td></tr></tbody></table> Tablo 1. Efsanesi. n, örneklem sayısı; Alan, gül yapışkanlı (± maksimum ortalama hata) yüzey alanı; Güç, lazer güç (± maksimum ortalama hata); Zaman, ışınlama süresi (± maksimum ortalama hata); Fluence, ortalama laser akısının, ben, tahmini ırradiance; Maksimum Yük / Alan, yapışkan yüzey alanına bölünmesiyle tamir dokusunun yetersizliği maksimum yük, (± SE) idi.

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Çıkar çatışması ilan etti.

Asidik pH RB dağılmasından uzamış kitosan çözeltisi karıştırma gerektirir ama yapıştırıcı imalatı, basit bir kuru-dökme işlemi dayanmaktadır yükseldi. Bu bir suda çözünmeyen filmi haline gelene kadar çözüm kadar kuruması için önemlidir. Ağırlık su içeriği kurutulmuş film 6 içinde ~% 10 olduğu zaman meydana gelir. Çözünmeyen filmler genellikle sıcaklık ve basınç (~ 25 ° C ve ~ 1 atm), standart şartlar az 2 hafta kuru döküm sonra elde edilir. Bunun için lazer sa?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Rose Bengal </td><td> Sigma-Aldrich </td><td> 632-69-9 </td><td></td></tr><tr><td> Kitosan (orta MW) </td><td> Sigma-Aldrich </td><td> 10318AJ </td><td></td></tr><tr><td> Asetik sirke asidi </td><td> Sigma-Aldrich </td><td> 08050051 </td><td> % 2 DI su, v / v </td></tr><tr><td> Manyetik karıştırıcı </td><td> Heidolph </td><td> MR Hei-Mix S </td><td></td></tr><tr><td> Santrifüj </td><td> Beckman Coulter </td><td> Allegra X-12R </td><td></td></tr><tr><td> Spektrofotometre </td><td> Varian </td><td> Cary 4000 UV-Görünür </td><td></td></tr><tr><td> Lazer </td><td> CNI Lazer </td><td> MGL-532 </td><td></td></tr><tr><td> Mikrometre </td><td> Mitutoyo </td><td> Serisi 227 </td><td></td></tr><tr><td> Cerrahi mikroskop </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

Fotokimyasal doku bağlama (PTB) iki doku kenarları 1,2 arasında gül bengal (RB) bir çözeltisi uygulanarak elde edilir ve doku onarımı, sütürsüz için bir tekniktir. Bunlar daha sonra seçici RB tarafından emilir bir lazer tarafından ışınlanır. Çıkan fotokimyasal reaksiyonlar sözde minimum ısı üretimi 3 dokuda kollajen liflerin çapraz. Bu raporda, RB lazer aktive olan bir roman doku yapıştırıcısı imal ince kitosan filmlerde dahil edilmiştir. Kitosan ve ~ 0.1 ağırlıkça% RB içeren esas Yapıştırıcı film, bir katı hal lazeri (λ = 532 nm, Fluence ~ 110 J / cm 2, spot büyüklüğü ~ 0.5 cm) tarafından imal ve buzağı bağırsak ve sıçan tibia sinirlere yapıştırılır . Kişisel bir bilgisayar ile ara-yüzeyi kurulan bir tek sütun gerilimölçer, yapışma direnci test etmek için kullanılır. 15 bir güç ± 6 kPa, (n = 30) ile sıkıca bağırsak RB-kitosan yapıştırır. Yapışma gücü 2 &amp; düşerplusmn; 2 kPa (n = 30) lazer yapıştırıcı uygulanan değilken. Tibial sinir anastomoz da dikişler kullanımı olmaksızın yapılabilir. Bir roman kitosan yapışkan olduğu tahvil fotokimyasal doku imal edilmiştir ve dikiş gerektirmez.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Dikişler genellikle cerrahi işlemler sırasında doku onarımı için gereklidir. Onlar invaziv ve doku zarar verebilir Ancak, bunların uygulama sorunlu olabilir. Bir roman doku yapıştırıcı üretim ve uygulama yöntemleri burada raporlanır. Bu yapışkan film lazer devreye girer ve sütürlerin kullanımı gerektirmez.

Fabrikasyon ve Lazer Doku Onarımı Rose Bengal-kitosan Film Uygulamaları

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

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Research

JoVE Journal

Bioengineering

12.1K Görüntüleme.  University of Western Sydney, NSW Australia. Dikişler genellikle cerrahi işlemler sırasında doku onarımı için gereklidir. Onlar invaziv ve doku zarar verebilir Ancak, bunların uygulama sorunlu olabilir. Bir roman doku yapıştırıcı üretim ve uygulama yöntemleri burada raporlanır. Bu yapışkan film lazer devreye girer ve sütürlerin kullanımı gerektirmez.

Video: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Miyokard Doku Mühendisliği Biyolojik Türetilmiş Enjektabl Malzeme imalatı

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Biyomühendislik

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D ve 3D Ortamlarda Sinir Progenitör Hücreleri Elektrik Alanı kontrollü Yönetmen Göç

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Elektrik Stimülasyonu murin myoblast Progenitör Hücreler ve Uygulama Mühendislik İskelet Kası Dokular

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Üç boyutlu Mürekkep Püskürtmeli Baskı Teknolojisi Kullanımı İnsan Kıkırdak Doku Fabrikasyon

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Elektrospinning Süreci Uyarlama bir Tri-katmanlı için üç Benzersiz ortamlar sağlamak için<em&gt; In Vitro</em&gt; Havayolu Wall Modeli

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Sıkıştırılmış Kolajen kıyılmış Doku: Tek aşamalı Rekonstrüktif Onarım için Cep içeren Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Vasküler kaynaklı mikroakışkan Ağların görüntü kılavuzluğunda, Lazer tabanlı Fabrikasyon

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

İmalat ve Macrostructure içeren optik doku hayaletler karakterizasyonu

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Vasküler Görselleştirme için Çift Raster Taramalı Fotoakoustik Küçük Hayvan Görüntüleyici

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...