This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1. Su / Yağ / Su Çift Emülsiyon Mikroküre Üretimi <ol><li> Üretim ve% 2 iyonu giderilmiş suda bir polivinil alkol (PVA) v çözeltileri% 0.5 a / hazırlar. Berrak olana kadar 50 ° C de karıştırdıktan çözüm, bu birkaç saat alabilir. Deiyonize su içinde% 2 h / h İzopropil Alkol içinde bir çözeltisi hazırlandı. </li><li> Arzu edilen hidrofilik, proteinin bir sulu çözelti hazırlayın. Aşağıdaki tablo, örnek formülasyonları sağlar. </li></ol><img alt="Tablo 1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> Tablo 1:. Örnek Protein Çözümleri aşağıdaki protein çözümleri başarıyla kapsüllü ve electrospun bu protokolü kullanarak edilmiştir. Gerektiğinde diğer hidrofilik, protein çözeltileri kullanılabilir. <ol start="3"><li> 50 ml&#39;lik bir santrifüj tüpü içine% 0.5 PVA çözeltisi 40 ml yerleştirin ve bir kenara. </li><li> 15 ml&#39;lik bir santrifüj tüpü içinde 65:35 300 mg &#39;poli 3 ml diklorometan (laktik-ko-glikolik asit) (PLGA). , Bir girdaplı karıştırıcı PLGA çözünmesini hızlandırmak için kullanılabilir. </li><li> Protein çözeltisi ve 200 ul% 2 PVA-çözeltisinden 4 ul birleştirin. PLGA solüsyonu içine dökün protein (1.4 adım). Çözümler çoğunlukla ayrı kalacaktır. </li><li> Buzlu su içeren bir deney kabı içine tüp yerleştirin. ~ 10 Watt (RMS) bir değnek sonikator kullanarak, bir üniforma kremsi beyaz emülsiyon oluşturulur kadar bir kaç saniye (5-10) için çözüm çalkalayın. </li><li> % 0.5 PVA (adım 1.3) ihtiva eden 50 ml&#39;lik bir tüp içine emülsiyonu dökün. ~ 20 saniye boyunca bir vorteks mikserde yüksek hızda çözüm karıştırın. Çözelti bulanık bir görünüm geliştirecektir. </li><li> 2 dakika boyunca 350 rpm&#39;de bir karıştırma plakası üzerinde 200 ml&#39;lik bir cam ve yere emülsiyonu aktarın. Karıştırma plakası üzerinde beher% 2 izopropil alkol 50 ml ilave edilir. Karışım sertleştirmek üzere DCM buharlaşmaya izin vermek için 1 saat içinde ve en az bir PLGA için karıştırma devam etmesini sağlar. </li><li> Transferi to santrifüj tüpleri içine mikro-küre çözeltisi. </li><li> 3 dakika boyunca 425 x g&#39;de santrifüj. Mikro küreler, bu tüpün altında toplanır ve beyaz görünür. Dikkatlice mikrosferler üzerinde tüpten çıkarın ve 500 ml&#39;lik bir şişe içinde depolar. </li><li> Tam tüpü üç çeyreği doldurulması ve sıvı içindeki mikro küreleri dağıtmak için sallama ile, iyonu giderilmiş su ile mikro küreleri durulayın. </li><li> Tekrar 1.10 ve 1.11 dört kez yineleyin. </li><li> Son durulama sonra, diğer örnekleri ile 500ml şişe içinde tekrar yer supernatant çıkarın. Daha sonra en az 24 saat boyunca liyofilize, santrifüj tüpü içinde toplanmıştır küreleri dondurun. </li><li> Bir ışık mikroskobu altında veya bir Taramalı Elektron Mikroskobu ile mikroküreler gözünüzde canlandırın. 60 um&#39;den daha büyük olmayan bir mikro-etkili üretebilmektedir içindir. Mikro-küreler çok büyük olursa, uzun sonikasyon veya girdaplama kere adım 1.6, 1.7 ya da gerekli olabilir. </li> &lt;li&gt; -20 ° C derin dondurucuda kurutulmuş mikroküreler saklayın. </li><li> Opsiyonel: Aşama 1.10 30&#39;dan 500ml şişe içinde protein miktarı test etmek için, üreticinin talimatlarına göre, bir protein deneyi kullanın. Bu mikro-kapsüllenmiş protein üretim sürecinde kullanılan ne çözeltide miktarının çıkarılması ile yüzde hesaplamak için kullanılır. </li></ol> . Mikrosferde protein yer PLGA solüsyonu 31 ml x 2 Rhodamine ug / ekleyin ve bir FITC konjüge protein kapsüllemek görselleştirmek için 1 bir örneğini göstermektedir: Not. Mikrokürelerin 2. Elektrospinning <ol><li> Önceki elektro çözeltisinin hazırlanması için, 37 ° C sıcaklıkta eritilmesi ile, iyonu giderilmiş su içinde foto-başlatıcı bir% 0.5 w / v solüsyonu oluşturmak. Bu işlem birkaç saat sürebilir. </li><li> Hyaluronik asit (Meha) metaakrilat karşı 29,% 3 (Burdick ve ark. Sentez için bakınız) w / 2% oluşturma/ hac 900 kD poli (etilen oksid) (PEO) ve% 0.05 w / v, deiyonize su içinde foto başlatıcı ağırlık solüsyonu. <ol><li> İstenen hacme için Meha ve PEO doğru miktarını hesaplayın. Örneğin, çözelti, 10 ml electrospinning Meha, 200 mg ve 300 mg PEO gerektirir. </li><li> Istenen son hacminin (9 Bu örnek için mi)% 90 deiyonize su içinde çözülür PEO. Bu işlem birkaç saat sürebilir, 37 ° C&#39;de ısıtılmış bir karıştırma plakası veya su banyosu işlemi hızlandırmak için kullanılabilir. </li><li> Sonraki Meha ekleyin ve berraklaşana kadar karıştırın çözelti üzere, bir girdaplı karıştırıcı kullanılır. Bu sadece bir kaç dakika sürer. </li><li> Son olarak geriye kalan% 10 hacim (bu, örneğin, 1 mi) doldurmak üzere% 0.5 foto başlatıcı çözeltisi ekleyin. </li></ol></li><li> 400 mg / ml kadar, istenen yoğunlukta mikro küreleri ekleyin. Bu mikrosferler, eşit çözelti içinde dağıtılır kadar vorteksli bir mikserde çözüm karıştırın. </li><li> Bir şırıngaya çözüm aktarın ve 6 inç ve 18 kalibrelik bl iliştirmekulastırınız ucu iğne. </li><li> Bir şırınga pompası şırınga ve 1.2 ml / saat olarak dağıtmak için ayarlanmış. </li><li> Toplama plakası ya da bir mandrel üzerine bir alüminyum folyo tabakası bantlayın. Bu bitmiş skafoldun kolay temizlemek ve saklanmasına olanak vermektedir. Bir dönen mandrel uyumlu lifleri oluşturmak için kullanılır. Düz bir plaka ya da hareketsiz mandrel rasgele düzenlenmiş fiberlerin sonuçlanacaktır. </li><li> Toplama aparatı ile yüksek gerilim güç kaynağından topraklama kablosu bağlayın. Iğne pozitif kablosunu bağlayın. </li><li> Iğne ucu ve toplama yüzeyi arasında 15 cm olacak şekilde şırınga pompası ve bir toplama yüzeyi ayarlayın. </li><li> Çözüm şırınganın ucunda görünür olduğunda, polimer pompalama başlatın, gerilim kaynağı açmak ve 24 kV DİKKAT gerilimini ayarlamak:. Sisteminin herhangi bir metal parçası dokunmayın üzerindeki gerilim açıldıktan sonra. Şarj ayrıca cilde elektrikli parçalardan kısa mesafelerde atlayabilir. </li><li> D kadar çözüm çalıştırınesired iskele kalınlığı elde edilir. Tam bir gerilim kaynağı ve şırınga pompası kapattığınızda. </li><li> Iskele ekli ile folyoyu çıkartın. Tamamlanan proteini içeren iskeleler -20 ° C dondurucu içinde saklanır. </li></ol> 3. Protein biyoaktivitenin test edilmesi <ol><li> Hücre kültürü ortamı hazırlayın. Ekleme,% 10 h / h Fetal Sığır Serumu Dulbecco Modified Eagle Medium,% 1 h / h, L-glutamin, ve% 1 hacim / hacim Penisilin-Streptomisin. </li><li> Bir plaka tamamen içine sığacak seçiniz cam lamelleri. </li><li> Üretici tarafından tarif edildiği gibi tedavi etmek için lamelleri 3-(trimetoksisilil) propil metakrilat kullanın. Methacrylation lamelleri iskele bağlılığı artırır. </li><li> Electrospinning önce çıkarılabilir çift taraflı bant ile electrospinner toplanma alanına metakrile lamelleri takın. Lamelleri üzerine İplik işleme ve görüntüleme kolaylaştırır. </li><li> Yukarıda tarif edildiği gibi arzu edilen kalınlığına Electrospin. </li><li> Biryıkıldıktan sonra dikkatlice mandrelinden lamelleri çıkarın elektrospinning. Net azot odasına İSKELE kaplı lamelleri yerleştirin ve tüm oksijen temizlendi olduğundan emin olun. </li><li> 15 dakika boyunca, 10 mW / cm 2, 365 nm ışık altında haznesi ve iskele yerleştirin. Uygun büyüklükte plaka içine bir yer çapraz bağlama sonrası. Iskele tarafı yukarı dönük olduğundan emin olun. </li><li> 30 dakika boyunca bir antiseptik lamba altında Yer iskelelerinin sterilize etmek. İstenen fibronektin ya da diğer protein hücre yapışmasını geliştirmek için bir kaplama olarak kullanılırsa. Kat iskelelerinin için üreticinin talimatlarına uyun. </li><li> Hasat Dorsal Kök Ganglia (DRG) gibi daha önce Hollenbeck 32 tarafından tarif. Bir DRG test edilen her iskele kaplı lamel için gerekli olacaktır. </li><li> Oyuklu bir plaka içerisinde her bir platform üzerinde ortam 100-200 ul koyun. Dikkatlice ortam damlacık her bir platform üzerinde bir DRG yerleştirin. Kalın İskele için daha fazla medya gerekli olabilir; DRG tamamen batık ve FLOA değil gerekirting. </li><li> Hücre iskeleti iskelesine yapışmasının sağlanması için 4 saat boyunca 37 ° C&#39;de iskele ve DRG inkübe edin. </li><li> Kuyunun için uygun seviyeye medyayı doldurun ve geri kuvöz içine yerleştirin. 4-6 gün inkübe devam edin. </li><li> İnkübasyon süresinden sonra, dikkatli bir şekilde, her bir oyuktan ortamını çıkarın ve hafifçe bir kez PBS ile yıkayın. / Hac% 4 paraformaldehid ağırlık ile 30 dakika boyunca hücreleri saptamak. </li><li> Nörofilament bir antikor leke kullanılarak Leke hücreleri. Bu ölçümü için nörit büyümesinin görselleştirme sağlayacaktır. DAPI ayrıca hücrelerin çekirdekleri görüntülemek için kullanılabilir. Bir örnek boyama protokolü Sundararaghavan ve arkadaşları 14 tarafından tarif edilmiştir. </li><li> Bir floresan mikroskop kullanılarak hücrelerin görselleştirmek. <ol><li> Mikroskop sahnede plaka yerleştirin. </li><li> DAPI&#39;nin için filtre ve uyarma ayarları kullanarak hücre kitlesini bulun. </li><li> Hücrenin kez genişletilmiş nevritlerle görselleştirmek için FITC&#39;ye için filtreyi geçiş olduğunu. Utoplamak ve tüm yapıyı görmek için gerekli olduğunca çok fotoğraf birleştirmek mikroskop dikiş fonksiyonu şarkı. DAPI&#39;nin, FITC&#39;ye ve parlak bir alan için tekrarlayın. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurons: Methods and Applications for the Cell Biologist. , (2003).</li><li>Boer, 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=Rat+sciatic+nerve+repair+with+a+poly-lactic-co-glycolic+acid+scaffold+and+nerve+growth+factor+releasing+microspheres.">Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres.</a> Microsurgery. 31, 293-302 (2011).</li><li>Pujic, Z., Goodhill, G. 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The authors have nothing to disclose.

Birçok çalışma, sinir hücreleri, topografik ipuçlarının (fiber hizalama) ve kimyasal uyaranlara (büyüme faktörleri) 1,2,10,11,35 yönettiği edilebileceğini göstermiştir. Elektro uyumlu lifleri oluşturmak için, basit bir yöntemdir. Büyüme faktörleri, sinir büyüme ancak sinir boruları (NC) içine dahil etmek için, uzun süreli salınımı için bir yöntem gereklidir ediyoruz. Her iki ipuçları ile daha sağlam bir sistem oluşturmak için, bu iki sinyal kombine edilmelidir. Çeşitli ...

Sinir doku mühendisliği devam eden zorluklardan biri sinirleri doğal olarak yetişen hücre dışı matris, taklit eden bir sinir kanalı (NC) yaratıyor. Araştırma hücreler, mekanik, topografik, yapışkan ve kimyasal sinyaller dahil olmak üzere 1-3 ortamında birçok faktöre yanıt göstermiştir. Bu alandaki başlıca sorunlardan biri, sinyallerin uygun kombinasyonunu belirlemek ve hücre büyümesini desteklemek için 4 uzun bir süre boyunca muhafaza ipuçlarını bir sistem imal edilir. Çevresel nöronlar yumuşak alt-tabakanın tercih ettikleri bilinmektedir hizalanır lifler tarafından yönlendirilebilir ve sinir büyüme faktörü (NGF) 5-7 yanıt. Hafta boyunca kimyasal ipuçları sağlayabilir NC&#39;ler yakın allogreftlere, sinir onarımı 8,9 güncel altın standardın için geliştirilmiş fonksiyonel iyileşmeyi sağlamak için gösterilmiştir. Malzemeler ve üretim yöntemleri, çeşitli mekanik ve topografik üretmek için kullanılabilirdiğerleri 10-13 işaret eder. Mekanik ipuçları 1,13 kritik uygulama için uygun malzeme seçimi yaparak, seçilen malzeme doğasında vardır. Üretim yöntemleri topografik ipuçları faz ayrılması, öz-montaj ve 1,13 electrospinning dahil kontrol etmek. Mikro uygulamalar, mikroflüidik, photopatterning, dağlama, tuz halindeki salamuralar, köpükler ya da gaz için, aynı zamanda 14-17 kullanılabilir. ELEKTROSPİNNİNG nedeniyle esneklik ve üretim 13,18-23 kolaylığı doku kültürü için elyaflı alt mühendisi en popüler yolu olarak ortaya çıkmıştır. Electrospun nanolifler kendini püskürtmek ve boşaltmak için 24, kısa bir aralık üzerinden germek için neden olan polimer solüsyonuna, bir yüksek voltaj uygulanarak imal edilmektedir. Bir hizalanmış iskele topraklanmış döner mandrel üzerine elyafların toplanması ile oluşturulabilir ve bağlantısız iskeleler ve durağan bir plaka 25 üzerinde toplanır. Yapışma sinyal elyaflı skafold wit kaplanması ile elde edilebilirs fibronektin veya 26 electrospinning önce HA gibi RGD gibi bir yapışma peptidi, konjuge. Onlar kontrollü salım için bir kaynak gerekiyor çünkü bu tür büyüme faktörleri gibi kimyasal sinyalleri, uzun dönemler boyunca korumak için en zor. Birçok sistem başarı çeşitli düzeylerde ile electrospun lifli ağlara kontrollü salımını eklemek için denenmiştir. Bu yöntemler harman elektrospinning, emülsiyon elektrospinning, çekirdek-kabuk elektrospinning ve protein konjugasyon 27 içerir. Buna ek olarak, elektro geleneksel olarak bu nedenle proteinin biyo kabul edilmelidir muhafaza, proteinin 28 canlılığını etkileyebilen uçucu bir çözücü içinde yapılır. Bu yaklaşım, özellikle periferik sinir büyüme için ayarlanabilir bir iskele oluşturmak için mekanik, topografik, kimyasal ve yapıştırıcı sinyalleri birleştirerek giderir. İskele mekanik tam sentetize edilmesi ile kontrol edilirMetakrilatlanmış Hyaluronik Asit (HA). Methacrylation siteleri fotoğraf reaktif crosslinkers bağlamak için kullanılır. Çapraz malzeme artık suda çözünür ve münhasıran enzimler 29 tarafından bozuldu. Çapraz bağlama miktarı degradasyon oranı, mekanik ve malzemenin diğer fiziksel özelliklerini değiştirir. ~ 500 Pa arasında bir gerilme modülüne sahiptir methacrylation% 30 ile HA kullanılarak, sinir dokusunun doğal mekanik yakındır ve tipik olarak nöronlar 26,29 tercih edilen yumuşak bir alt-tabakayı oluşturur. Dönen mandrel üzerinde Elektrospinning topografik bir işaret sağlayacak şekilde ayarlanır lifleri oluşturmak için kullanılır. Ile birlikte mikro-elektrospinning kullanma süreleri zarfında iskele içinde kimyasal sinyaller sağlar. Kimyasal sinyali oluşturmak için kullanılan NGF içeren nörit büyüme mikroküreler desteklemek için. NGF üretimi sırasında zorlu çözücüler karşılaşmasa bu yüzden çoğu electrospun malzemelerin aksine HA su içinde çözünür. SCA, yapışkan bir sinyali eklemek içinffold fibronektin ile kaplanır. NGF fibronektin (yapıştırıcı) ile kaplanmış mikroküreler (kimyasal) bırakmadan yumuşak (mekanik) hizalanmış (topografik) lifler: tamamlandı sisteminin yukarıda açıklanan sinyalleri dört türlerini içerir. Üretim ve bu sistemin test bu protokolü tarif edilmiştir. Proses, bir su-içinde-yağ-içinde-su emülsiyonu ile çift mikrosferlerin üretimi ile başlar. Emülsiyon, bir yüzey aktif madde, polivinil alkol (PVA) ile stabilize edilir. İç su fazı protein içerir. Bu diklorometan (DCM) içinde çözüldü PLGA kabuk malzeme içeren, yağ fazına ilave edilir olarak, yüzey aktif madde DCM proteini koruyan fazlar arasında bir bariyer oluşturur. Bu emülsiyon, mikro kürelerin dış yüzeyini oluşturmak için polivinil alkol içeren bir su fazı içinde dağılmış daha uzundur. Kararlı bir emülsiyon, DCM buharlaşmasını sağlamak için karıştırılır. Durulama ve liyofılleme sonra kuru mikroküreler cont ile bırakılır, geri kalan proteini. Mikroküreler tamamlandıktan sonra onlar iskelelerinin içine electrospun hazırdır. Öncelikle elektro solüsyon hazırlanır. Solüsyonun viskozitesi uygun elyaf formasyonu için çok önemlidir. Saf HA Çözümleri Bu zorunluluğa uymayan; PEO üretebilmektedir izin vermek için bir taşıyıcı polimer olarak ilave edilir. Bu mikrosferler, mikro-dağılmış olan elyaflı yapı iskeleti sonuçlanan çözeltisi ve electrospun eklenir. Üretim işlemi tamamlandıktan sonra, protein, varlığını doğrulamak için test edilmelidir. Bunu yapmak için, NGF&#39;ye tepki veren bir birincil hücre kullanılabilir. Bu protokol, 8-10 günlük tavuk embriyolarından sırt kök gangliyon (DRG) kullanır. Hücre demetleri NGF veya boş olanlar ile dolu küreleri içeren iskelelerinin üzerine ekilir. NGF hala canlı olup olmadığını NGF içeren iskelelerde gelişmiş akson büyümesini görmelisiniz. NGF artık geçerli değilse o olacakdeğil genişletmek için nevritlerle teşvik ve denetime benzer görünmelidir. Burada tarif edilen prosedür, kesin malzeme basit değişiklikler, electrospinning yöntemi ve sistemi, çeşitli doku ve hücre tipleri için optimize edilebilir proteinleri ile, ancak, sinir destek üzerinde odaklanmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospinning growth factor releasing microspheres into fibrous scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

85 üzerinde% protein kapsülleme çapında mikroküreler ± 14 mikron 50 sürekli üretilen ve iskeleler içine electrospun edilmiştir. Ebat üç ayrı üretim serisi ikinci mikro-örnekleri görüntüleme ile saptanmıştır. Ticari laboratuar yazılımı kullanılarak ölçülür bir optik mikroskop ve uzunlukları üzerinde tutulmuş ve görüntüler. Şekil 1 boyut dağılımının bir histogramı gösterir. Encapsulation oranı da üretim sürecinde kaçan protein miktarının tarafından, üç a...

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Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Lifli iskelelerinin içine Microspheres ibra Elektroeğirme Büyüme Faktörü

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K Görüntüleme.  Wayne State University. This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Video: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Elektrospinning Temelleri: Optimizasyon Çözümü ve Cihazlar Parametreleri

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Biyomühendislik

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Elastomerik PGS Arter Doku Mühendisliği Yapı iskeleleri

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Akson Streç Büyüme: Nöronal Büyüme Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

İnsan Pankreas Kanseri Hücrelerinde Epidermal Büyüme Faktörü kullanarak Gen Teslimat ve Transfeksiyon Jelatin Tabanlı Engineered Nanovectors Reseptör-hedefli

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Fare Embriyonik kök hücrelerden Mühendislik Sinir Dokular için Poli electrospun fibröz iskeleleri (gliserol-dodecanedioate)

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Korelatif Mikroskopisi kullanılarak sağlam hücrelerde Epidermal Büyüme Faktörü Alıcısının stokiyometrisinden incelenmesi

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

Heparin Binding Domain osteojenik Büyüme Faktörü Teslim polielektrolit Kompleksi

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

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 ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Üç boyutlu Poly(ε-caprolactone) İskele kontrol edilebilir türleri morfoloji ile Electrospinning yazma doku mühendisliği uygulamaları için erime

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Decellularized matris kıkırdak elde edilen iskele imalatı

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...