Yazarlar, bu projenin daha önceki yineleme kendi teknik katkılarından dolayı Gabriel AROM ve Michael Yeranosian teşekkür etmek istiyorum. Bu proje OREF Klinisyen Bilim Adamı Yetiştirme Grant (NL) tarafından finanse edildi, HH Lee Cerrahi Araştırma Bursu (NL), Gaziler İdaresi BLR &amp; Ge Merit Değerlendirme 1 I01 BX00012601 (DM) ve Kas-İskelet Nakli Vakfı Genç Araştırmacı Ödülü (FP).

NOT: Tüm hayvan deneyleri ameliyatları başlamadan önce yerel veteriner personeli ve hayvan kullanımı komitesi tarafından onaylanmıştır. Electrospun Polikaprolakton iskelelerinin hazırlanması 1. <ol><li> A% 10 oluşturmak için 1,1,1,3,3,3-heksafluoro-2-propanol içinde granül halinde tıbbi sınıf esteri sona poli (ε-kaprolakton) tartılır ve çözünmesi PCL polimer çözeltisi ağırlık /. Homojen bir çözelti sağlamak için, en az 3 saat süreyle bir karıştırma plakası kullanılarak çözelti kaynamaya bırakılır. </li><li> PCL çözüm Electrospin iskele imalatı için son derece uyumlu PCL liflerin bir manşet oluşturmak için. <ol><li> Her zaman üzerinde fan ile bir kimyasal davlumbaz elektrospinning kurulum hazırlayın. Bu, bir gerilim kaynağı, motorlu toplama mandrel ve bir vakum portu tarafından tahrik edilen bir kaynak, PCL çözeltisi için giriş noktaları elektro işlemi için izole edilmiş bir vakum ortamı olarak hizmet etmek ve var olan büyük bir akrilik bir kutudan kutudan oluşmaktadır. Akrilik temizleyinc kutusu iyice etanol ile ve electrospun ürünün kalitesini bozabilir herhangi kirleri çıkarmak için Parafilm levhalar ile tüm yüzeyleri örtün. </li><li> Bir küt bir 10 ml şırınga yukardaki çözelti yük, yaklaşık 3 mi 18 g, 1 ½ inç iğne sona erdi. Şırınga yukarı iterek hava kabarcıklarını çıkarın. Programlanabilir şırınga pompası içine Kilit çözüm. Gerilim kaynağı tel takılması için akrilik kutunun dışında ½ iğne inç bırakarak akrilik kutuya küçük bir delikten iğne takın. </li><li> Çok hizalanmış PCL elyaflar için toplayıcı olarak 30 mm dönen torna mandrelini kullanın; alüminyum folyodan oluşan ince bir şerit ile sıkıca mandrel kapsamaktadır. Yaklaşık 15 cm uzakta şırınga iğnesi kutusunun karşı tarafında motora mandrel kilitleyin. </li><li> Vakum portuna plastik hortum takın ve davlumbaz vakum kaynağına bağlayın. Vakum kaynağı açın ve bir kapak ile akrilik kutusu kapağı. </li><li> Sve 2.5 ml / saat programlanabilir şırınga pompa infüzyon hızı. 3,450 rpm&#39;de mandrel işletmek ve bir timsah klibi kullanarak kutunun dışında iğne ucu gerilim kaynağının pozitif kurşun takmak için motorun açın. </li><li> PCL çözümünün infüzyon başladıktan sonra, gerilim kaynağı açmak ve bir 20 kV çalışma gerilimi ayarlanır. </li><li> PCL çözeltisi, 0.5 ml, homojen bir manşet oluşturmak için 12 dakika boyunca solüsyon infüzyonu. Not: Ortalama olarak, her bir kol ağzı üç ya da dört-katmanlı yapı iskeleleri bir toplam oluşturmak için kullanılabilecek iki altı şerit levha oluşturmak için yeterli electrospun malzemesine sahiptir. </li></ol></li><li> Lazer düşük vakum ayarı, 10.0 keV iniş gerilimi, 6.4 mm çalışma mesafesi ve 3.0 prob çapında işletilen bir VersaLaser Kesici 2.3 birden fazla küçük yaprak oluşturmak için PCL manşet kesti. NOT: Bu örnekte, bir bilgisayar destekli tasarım x 35 mm x 150 mm iskeleleri ile 1.5 mm arasında birden fazla yaprak elde etmek için kesici talimat için kullanılaneşit% 15 gözenek alanı 150 mikron çaplı delikler dağıttı. </li><li> Plazma iyon ivme ile PCL yüzeyinin hidrofilikliğinin ikna etmek için bir plazma temizleyici kullanarak PCL iskeleleri etch. 450 mTor ve tedavi yüksek güç (29.6 W) 30 saniye iskeleleri vakum ayarlayın. </li><li> Steril bir ortamda% 70 etanol içinde iskeleleri batırın. </li><li> Coat kollajen ile bireysel iskeleleri in vivo hücre yapışmasını ve çoğalmasını kolaylaştırmak için. <ol><li> 4 ° C&#39;de 1x PBS, 9: 1: Purecol kollajen 3 mg / mL standart solüsyon, 10x PBS ve 0.1 N NaOH 1 2.5 steril çözelti 8 seyreltilmesiyle kollajen kaplama çözeltisi oluşturur. Çözelti homojenliği sağlamak için iyice karıştırın. </li><li> Kaplama tek 1,5 mm x 35 mm x, yukarıdaki kolajen çözeltisi, bir ince tabaka filmi ile 150 um iskeleleri. Steril bir ortamda 24 saat kurumasını bekleyin. </li></ol></li><li> 5-0 Vicryl sütür kullanarak, yığın ve dört ayrı 1,5 mm x 35 mm x 150 mm iskeleler tutturmakBir Krackow implantasyon için hazır bir son 0.6 mm kalınlığında, çok katmanlı, kolajen kaplı iskele oluşturmak için dikiş kullanılmıştır. </li></ol> 2. Sıçan Cerrahi Protokolü <ol><li> Inhalasyon odasında sıçan yerleştirerek ve 2 L / dk oksijen ile% 2 izofluran sunarak anestezi neden olur. Sıçan onaylayın yeterince ayak hind basınç uygulayarak ve herhangi bir yanıt değerlendirilmesi anestezi uygulandı. </li><li> Non-steril arka masada, deri 25 mg / kg ampisilin ve 0.03 mg / kg buprenorfin enjekte. </li><li> Gözleri oftalmik merhem sürün. Operatif arka bacak kürk Klip ve üç klorheksidin alternatif scrubs ve% 70 etanol ile cerrahi siteyi hazırlık. </li><li> Hipotermi önlemek için ısıtılmış bir pad üzerinde, ameliyat masasına sıçan aktarın. Güvenli burun konisi ve 2 L / dk oksijen içinde% 2 izofluran ile prosedür aracılığıyla teslim burun konisi anestezi korumak. Maruz kalan ameliyat uzvu bırakarak, steril bir şekilde örter. </li><li&gt; Cerrahi başlamadan önce, ilk olarak ayak basıncını hind bir yanıt eksikliği ile yeterli anesthetization onaylayın. </li><li> Patella düzeyinde merkezli diz 2 cm uzunluğunda dikey kesi medial, emin olun. Kesi diz üzerine merkezli kadar yanal cildi geri çekin. </li><li> Sadece patella medial kesme ve kuadriseps ve distal tibia tubercule üzerinde patellar tendon ekleme seviyesine Musculotendinous kavşak seviyesine proksimale uzanan bir medial parapatellar artrotomi yapmak için bir neşter kullanın. Patellar veya kuadriseps tendon kesmek için değil dikkat edin. </li><li> Patellar tendon sadece lateral diz kapsülü ile 1 cm dikey kesi yaparak yanal Yayın patella. </li><li> Diz uzatılmış olduğundan emin olun. İnce bir makas alın ve lateralden mediale doğru patella altında geçmektedir. Ekstansör mekanizması iki tarafında tercüme edilebilir, böylece makas birkaç kez yayıldı. </li><li> T esneme sırasındaO diz, diz ekleminin iç maruz yanal Patellayı çevirmek. Interkondiler çentik ve femur kondillerin açık görselleştirme olun. Bir neşter kullanarak, çentik ACL ve PCL transect. </li><li> 1.6 mm&#39;lik K-teli ile yük güç matkap. Interkondiler çentik ACL kökeni k-tel ucunu yerleştirin. Superolaterally delmek ve bir neşter gibi gerekli her türlü yumuşak doku çıkarılması, femur lateral üzerindeki çıkış noktasını görselleştirmek. Greft için açık geçiş sağlamak için ve bir kaç kez dışarı k-teli geçirin. </li><li> Tibia plato ACL ayak izi k-teli yerleştirin. Anterolateralinde Matkap ve anterolateral proksimal tibia üzerindeki çıkış noktasını görselleştirmek. K-telli tibia çıkar nokta tam görüntülenmiştir böylece gerekli yumuşak doku temizlemek için neşter kullanın. </li><li> Femur kemik tünelden (ideal olarak en fazla 2 santim uzunluğunda) kısaltılmış Keith iğne geçirin. K gözünden greft bir ucundan iki ameliyat dikiş ipliği ucu Konueith iğne. Femoral tünelden greft bir ucunu çekin iğne kullanın. </li><li> Tibia tünelden greft diğer ucunu geçmek önce adımı tekrarlayın. </li><li> Bir figür sekiz-dikiş ile periost veya diğer yumuşak doku çevreleyen greft femur ucunu tutturmak için 4-0 Vicryl dikiş kullanın. Manuel gerginlik uzantısı diz greft. Bir figür sekiz-dikiş ile periost veya diğer yumuşak doku çevreleyen greft tibia ucunu yapıştırın. </li><li> Figür sekiz-dikiş geçmişte her ucunda 1-2 mm bırakarak, her iki ucunda aşırı greft kesmek için bir makas kullanın. </li><li> Diz uzatın ve patellanın azaltır. 4-0 vicryl kullanarak, patellanın lateral subluksasyon önlenmesi, medial eklem kapsülü kapatmak için tek bir figür sekiz-dikiş yerleştirin. </li><li> Altta yatan kas dikiş değil özenle, bir çalışan subkütiküler 5-0 monocryl veya Vicryl dikiş ile cilt kapatın veya deri kapatıldıktan sonra herhangi bir görünür dikiş var. </li><li> Injederi altından buprenorfin ile ct sıçanlar ameliyat sonrası üç günlük bir toplam için her 12 saat. Enjeksiyon sırasında herhangi bir drenaj cerrahi sitesini veya yaranın yarılması edin. Aksama ve bazı şişme ameliyat sonrası ilk birkaç gün içinde normal, ama derhal veteriner kadrosu ile birlikte herhangi bir postoperatif endişeleri gidermek. Cerrahi kesiler tamamen iyileşmiş zaman hayvan, ameliyat sonrası 2 hafta sosyal konut dönmek olabilir. </li></ol> 3. Veri Toplama Protokolü <ol><li> Hayvanlar öldürüldükten hemen sonra, torakotomi ve ardından kapalı bir CO2 odası içinde tek tek fareleri boğmak. </li><li> Kalça ekleminde ayırarak cerrahi yeniden ve kontralateral bacaklarda hem Hasat. <ol><li> Yeniden bacaklarda için, sadece femur, tibia ve greft izole ince diseksiyon ile, arka çapraz bağ ve cerrahi kesintiye yerli ACL&#39;DEN kalıntıları da dahil olmak üzere, tüm yumuşak doku kaldırmak. </li> &lt;li&gt; kontralateral bacaklarda için, yerli ACL dışındaki tüm yumuşak doku yanı sıra ince diseksiyon ile femur ve tibia çıkarın. </li><li> Femur graft kaval kemiği kompleksinin her bir ucundan kemik ¾&#39;ünü 1 cm, ancak kaldırmak için, bu tür Dremel gibi bir döner alet kullanın. </li><li> Bu süreçte ve biyomekanik test boyunca, düzenli ve sık sık yanlış sonuçlara değiştirebilir hasat diz kuruma önlemek için normal tuzlu su ile bağ bölgeleri sprey. </li></ol></li><li> Histolojik analiz için, 48 saat süre ile 25 ° C&#39;de ayrı ayrı% 4 paraformaldehit solüsyonunda her diz düzeltin. Daha sonra, tam yumuşatma için Immunocal reaktif bir çözelti içine daldırmak diz; Bu süreç örnek kalsifik içeriğine bağlıdır ve beş gün kadar sürebilir. Örnek kalitesini düşürebilir eksik yumuşatma gibi ilerlemeyi değerlendirmek üzere günlük bireysel örnekleri kontrol edin. İstediğiniz gibi, kesit gerçekleştirmek, slayt montaj ve boyama tamamlandıktan sonra. </li><li> Perform biyomekanik test doku mühendisliği bağın fonksiyonel kapasiteyi değerlendirmek için. <ol><li> Ayrı ayrı kemiğin epifiz yaklaşık 28 G galvanizli çelik tel sararak femur ve tibia sabitleyin. Bu kemikte ziyade ilgi bağın numunenin erken çekme hatasından yanlış biyomekanik test verileri önlemektir. </li><li> Karışım polimetilmetakrilat (PMMA), kemik çimentosunun karışımı içine femur. Bunu yapmak için, iki çimento bileşenleri karıştırmak ve hemen tamamen serbestçe çıkıntılı epifiz ve ekli ligaman ile çimentolu tencerede kemik gövdesini saran, metal içine femur sabitlemek için viskoz karışımı kullanın. Spontan serbest radikal polimerizasyon yavaş yavaş bir hamur malzeme ve sonunda bir katı sertleştirilmiş matris içine karışık viskoz bileşenleri dönüştürmek için izin ver. NOT: Bu işlem birkaç dakika sürer ve el ile yapılan bir bolus sıcaklığının değerlendirerek izlenebilirGeri kalan çimento; Sıcaklık geçici ekzotermik polimerizasyon reaksiyonu sırasında artırmak ve malzeme katılaşır sonra RT azalır gerekir. </li><li> İdeal mekanik test için 20 ° fleksiyonda diz bağı korurken dışında, tibia çimentolama yukarıdaki işlemi tekrarlayın. </li><li> Bir gerilme test cihazının üzerine çimentolu femur graft tibia kompleksi monte edin ve başarısızlığa gerilim başlangıcından, zamanın bir fonksiyonu olarak kayıt yükü ve yer değiştirme için hazırlar. Bu örnekte, bir 1 kN yük hücreli bir Instron Model 5564 kullanılır. </li><li> Ön gerilimi, 0.5 N / dk&#39;lık bir rampa oranında 2 N aşı ve daha sonra 0.5 mm / sn arasında bir gerilme oranında başarısızlığa greft test edin. Sürecinde, bağ orta-madde de başarısız olduğunu ve kemik femur ve tibia güvenli olduğunu ve erken yanlış test bağın biyomekanik özelliklerini değerlendirmek olabilir, başarısız değil emin olun. </li><li> Oluşturulan yük deplasman kullanıneğrileri test ligament yetersizliği yükünü ve sertliği hesaplamak için. </li></ol></li></ol>

<ol>
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The authors declare that they have no competing financial interests.

ACL yaralanmaları şu anda yeniden inşası için sınırlı seçenekler ile ortopedik spor cerrahisinde sık görülen bir durum vardır. In vivo olarak yeniden oluşturulmasına izin olacak ACL için uygun bir doku mühendisliği vekil geliştirmek amacıyla, uygun bir hayvan modeli gereklidir. Bir atimik sıçan ACL yeniden tekrarlanabilir bir modeli kullanarak, in vivo implantasyonu olduğu gibi bu çalışmada, biyolojik olarak parçalanabilir bir mühendislik aşı imalatı tarif edilmektedir. B...

Ön çapraz bağ (ACL) yırtılması diz 1 en yaygın bağ yaralanmaları biridir. Rüptüre ACL&#39;lere cerrahi müdahale olmadan iyileşmek mümkün olduğundan, günlük yaşam yanı sıra spora katılım faaliyetlerine sınırlamalar yılda bir milyar dolar 3 tahmini maliyeti, her yıl 2 ameliyat 175,000 üzerinde hasta götürmek. Şu anda, otogreft veya allogreft tendon ya bağ rekonstrüksiyonu için kullanılır. Yüksek başarı oranları hem otogreft ve allogreft replasmanı ile elde edilebilir olsa da, ciddi komplikasyonlar bu rekonstrüksiyon seçenekleri 4 ile ilişkilidir. Otogreft dokusu, özellikle yeniden rüptür veya çok bağ yaralanma durumlarda, donör sitesi morbidite ile ilişkilidir ve arz sınırlıdır. Öte yandan, alograft doku gecikmeli aşı entegrasyonu, yan inflamatuar yanıtı, teorik enfeksiyon riski, ve sınırlı Supp ile bağlantılıdırly 5. Sentetik olmayan parçalanabilir greftler 1970 ve 1980&#39;lerde geliştirilen, ancak erken greft rüptürü, yabancı cisim reaksiyonu, osteoliz ve sinovit 6 engel edildi. Bu ciddi kaygılar sonucunda, Amerika Birleşik Devletleri&#39;nde klinik kullanıma sentetik greftler şu anda. Nedeniyle mevcut greft seçenekleri ile ve biyoloji, mühendislik ve rejeneratif tıp son gelişmeler bu sınırlamaları, ACL aşılama için bir doku mühendisliği çözeltide büyük ilgi olmuştur. Güncel doku mühendisliği stratejileri kalıcı sentetik malzeme implantasyonu 7 ile ilgili sınırlamaları kaçınarak konak doku gelişimine izin vermek için parçalanabilir biyolojik ve sentetik malzemeler kullanır. Polikaprolakton (PCL), FDA yapışma bariyer dahil olmak üzere tıbbi uygulamalarda bir dizi için onaylanmış ve 8 pansumanla olan biyolojik olarak parçalanabilir bir polimer, bu u olmuşturvasküler, kemik, kıkırdak, sinir, deri ve özofagus doku mühendisliği 5,9-16 olmak üzere geniş bir uygulama çeşitli sed. Uygun biyolojik uyumluluk, nispeten uzun bir in vivo yarı-ömrü, uygun bir mekanik mukavemet ve yüksek elastikiyet doku mühendisliği Bu polimerin popülerlik katkıda bulunur. Yara iyileşmesinin bir kemirgen modelinde, implante electrospun PCL olmayan immünojenik olduğu ve yan etkileri 13, yerel doku içine entegre olduğu gösterilmiştir. Electrospun PCL bir SEM görüntüsü, Şekil 1 &#39;de gösterilmiştir. Hem küçük hem de büyük hayvan modellerinde geçerli FDA düzenleyici standartlar, etkinliği ve güvenliği ile Amerika Birleşik Devletleri&#39;nde klinik çalışmalarda içine taşımak için PCL veya başka herhangi bir mühendislik ACL grefti için gerekli olacaktır. Ayrıca, in vivo koşullar genellikle in vitro doku mühendisliği ACL greft özelliklerini artırabilir. Fleksör digitor ile otolog Rekonstrüksiyon bir sıçan modelium longus tendonu daha önce yerel bir ACL, kesilmiş Femoral ve tibial tünel delinmiştir ve aşı geçirildi ve ameliyat dikiş ipliğinin 17-22 ile yerine tespit edildi ki, tarif edilmiştir. Bu yazıda, mühendislik ACL değiştirmeleri değerlendirilmesi için yerine otogreft-tabanlı yeniden (Şekil 2) için bu modelin bir değişiklik anlatacağız. Birçok hayvan modelleri bağ doku mühendisliği için mevcut olmasına rağmen, sıçan nedenlerle bir dizi için büyük modellere göre avantajlıdır. Bu avantajlar kolay hayvancılığı ve kullanım, daha az etik hususlar ve düşük maliyet 17,23 içerir. Buna ek olarak, fare modeli kıkırdak, tendon ve kemik doku mühendisliği 24 de dahil olmak üzere ortopedik doku rejenerasyonu için bir model olarak yaygın bir şekilde kullanılmaktadır. Özel olarak, atimik çıplak sıçan nihai implantasyon o sağlayan, hücre-aracılı bağışıklık karşılığının 25 kendi eksikliği nedeniyle seçilmiştirBu modelde f ksenogeneik donör hücreleri gelecekte daha da mühendislik greft geliştirmek için. Bu yöntemler yazıda ACL rekonstrüksiyonu bir atimik sıçan modelinde imalat ve acellular implantasyonu, biyolojik olarak parçalanabilen polimer greft tarif.

<table border="0" cellpadding="0" cellspacing="0" width="888">
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			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
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			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
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			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
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			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
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			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
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			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
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	</tbody>
</table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Tek bir cerrah tarafından 92 sıçan ameliyatları bizim deneyim, yara tamamlanmasına kesiden ameliyat süresi ortalama 4.7 dakika bir standart sapma ile, 16.9 dakika idi. Hayvanlar öldürüldükten hemen sonra, fareler 356 ± 23 g ağırlığında. Tüm sıçanlar iyi cerrahi tolere, ve hiçbir komplikasyon yaşadı. Ameliyattan hemen sonra, sıçanlar operatif ekstremitede ağırlık vermeye kaydetti, ama hafif bir topallama sergilendi. Bir hafta sonrası ameliyat, tüm sıçanlar kayda değer topallayarak yürüye...

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Engineered Ön Çapraz Bağ Greftlerinin Değerlendirme Atimik Rat Modeli

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

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Bioengineering

10.3K Görüntüleme.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Video: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Doku Değerlendirilmesinde Manyetik Rezonans Elastography Metodoloji Construct Büyüme Tasarlandı

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Biyomühendislik

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Cerrahi Alma, İzolasyon ve<em&gt; In vitro</em&gt; Doku Mühendisliği Uygulamaları için İnsan Ön Çapraz Bağ-türevli Hücreleri Genişleme

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Doku Engineered Vasküler Greftlerinin ve sonraki İmplantasyonunda Cerrahi Tekniği<em&gt; İn Vivo</em&gt; İzleme

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Bir Fare Modeli Doku mühendisliği Kapların Üretimi ve Tırnak

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3D Doku mühendisliği İnsan Özofagus Mukozal Modeli Üretimi, Karakterizasyonu ve Potansiyel Kullanımları

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Polarize Sinir Doku Engineered 3D İpek-kolajen bazlı Modeli

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Uygulamaları<em&gt; İn Vivo</em&gt; Doku değerlendirilmesi için Rat Tibialis Anterior Fonksiyonel Test İskelet Kası Onarım Engineered

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

3D Baskılı Kılavuzları ve Halka İstifleme Yöntemiyle Engineered Vasküler Greft ölçeklendirme

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Egzersiz Koşullandırılmış Serum İle Ligament Yapılarının Tedavisi: Bir Translasyonel Doku Mühendisliği Modeli

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Yüksek üretilen iş kullanımı otomatik Microbioreactor sistemi modeli Igg1 CHO hücrelerdeki imalatı için

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...