DOZ Cenevre Vakfı tarafından verilen bir hibe ile desteklenmektedir. SN Pittsburgh Doku Mühendisliği Girişimi bir Doktora Sonrası Araştırma Bursu Grant tarafından desteklenmiştir.

1. Yalıtımlı Yağ-Türetilmiş Kök Hücre (ASC) Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> Sıçan perirenal ve epididimal adipoz yalıtmak ve daha önce 6 açıklandığı gibi% 1 fetal sığır serumu (FBS) içeren steril Hank tamponlu tuz solüsyonu (HBSS) ile yıkayın. </li><li> Doku kıyma ve oda sıcaklığında 8 dakika boyunca 500 g&#39;de 50 mL tüpü ve santrifüj içine% 1 FBS içeren HBSS 25 mL içine 1-2 g transfer. </li><li> Serbest yüzen adipoz doku tabakası ve 125 mL erlen transfer toplayın ve 37 az 45 HBSS kollajenaz tip II (200 U / ml) 25 ml ile tedavi ° orbital çalkalayıcı (125 rpm) C. </li><li> Dikkatle sıvı kısmı (petrol ve yağ tabakası altında) kaldırmak ve 100 mikron ve 70 mikron naylon örgü filtre üzerinden sırayla filtre. Oda sıcaklığında 10 dakika boyunca 500 g&#39;de santrifüje süzüntü, supernatan aspiret ve 25 ml HBSS ile iki kez pelet yıkayın. </li><li> MesenPRO RS Büyüme katkısı, (100 U / penisilin G, 100 ug / mL streptomisin sülfat, ve 0.25 mg / mL Amphotericin B&#39;nin mL) antibiyotik-antimikotik ile desteklenmiş büyüme medyumu (MesenPRO RS Bazal Medium) 50 mL hücre pelletini ve 2 T75 şişeler (25 mL / şişeye) içine 2 mM L-glutamin ve pipet hücreleri. </li><li> Kültür 37 ° C (Artan Tüm deneyler için kullanılan Geçiş 2-4) de bir% 5 CO2 inkübatöründe nemlendirilmiş ASC. </li></ol> 2. Chitosan Mikroküreler (CSM) hazırlanması Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> CSM önceki protokolü kullanılarak bir iyonik 6 Koaservasyon tekniği ile birlikte, bir su-içinde-yağ emülsiyonlaştırıcı işlemle hazırlanır. Soya oluşan bir yağ fazı karışımı, 100 mL kitosan, sulu bir solüsyonu (0.5 M asetik asit içinde% 3 ağırlık / hacim kitosan 6 mL), emülsifiyeYağ, n-oktanol (1:2 v / v) ve% 5-sorbitan mono-oleat (p 80) emülsiyonlaştırıcı, karşıt yönlerde, üstten (1700 rpm) ve eş zamanlı olarak manyetik olarak karıştırıldı (1000 rpm) kullanılarak. Erken çapraz bağlama meydana gelmeden önce üzerinde oluşan miseller çözüm kalır ve dibe olamaz karıştırma temin eder bu ikili yöntemi. Dahası, içinde manyetik karıştırıcı yardımcıları misel oluşumu ve rigidization sırasında kitosan de-toplamak. </li><li> Karışım sürekli stabil bir su-içinde-yağ emülsiyonu elde edilene kadar yaklaşık 1 saat boyunca karıştırıldı. İyonik çapraz bağlama 4 saat (24 mL toplam) n-oktanol, her 15 dakika içinde% 1 w / v potasyum hidroksit ile 1.5 mL eklenmesi ile başlatılır. </li><li> Çapraz bağlama Reaksiyonun tamamlanmasından sonra, karışıma yavaş yavaş içeren CSM yağ fazı boşaltacaktır ve hemen aseton 100 mL küreler ekleyin. Yağ fazı, tamamen çıkarılana kadar aseton ile yıkanır küreler. </li><li> Bir vakum desikatörde ve anal olarak kurtarıldı küreler kurulayındaha fazla işlem olmadan yze. Ortalama partikül boyutu CSM, miligram başına yüzey alanı ve birim hacmi kübik parçacık boyutu analiz cihazı ile tespit edildi. </li><li> Sonraki deneyler için, kalıntı tuzları kaldırmak ve mutlak alkol ile sterilize etmek için steril su ile CSM üç kez yıkayın. </li></ol> 3. CSM Serbest Amino Grup Sayısını Belirleme Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> Bubins ve Ofner 7 trinitro benzensülfonik (TNBS) asit yöntemi kullanılarak iyonik çapraz sonra CSM mevcut serbest amino gruplarının sayısını belirleyin. 40 ° C&#39;de 4 saat süreyle 50 mL cam tüp içinde% 0.5 TNBS solüsyonu 1 mL mikrosferler 5 mg inkübe ve 2 saat boyunca 60 ° C&#39;de 6N HCI 3 mL ilavesi ile hidroliz. </li><li> Oda sıcaklığına kadar soğumaya örnekleri, ve deiyonize su ile 5 mL etil 10 ml ilave edilerek serbest TNBS özüeter. </li><li> ° C bir su banyosunda 15 dakika oda sıcaklığına kadar soğutulur ve kalan eter, buharlaşma ve 15 ml su ile dilüe etmek için 40 sulu faz bir 5 ml bir örnek ısıtılır. </li><li> Boş ve kitosan amino gruplarının sayısı tespit etmek CSM hazırlanması için kullanılan şekliyle kitosan olmadan TNBS çözeltisi kullanılarak bir spektrofotometre ile 345 nm&#39;de absorbans ölçün. Kitosan göre CSM serbest amino grubu ile kestirilebilir. </li></ol> 4. CSM içinde yükleniyor ASC Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> Gece steril HBSS bölümünde 2,5 sterilize CSM 5 mg dengeye ve 8 mikron gözenek büyüklüğüne membran kültür plakası eki (24-iyi plaka) ekleyin. </li><li> CSM zarı üzerine yerleşen sonra, dikkatlice HBSS aspire ve inci büyüme medya ekleme ve 700 uL içine besiyeri 300 uL ekleyinucun dışına e. </li><li> Kültürü plakası içine eklemek CSM içinde büyüme ortamı ve tohum 200 uL uygun konsantrasyonda Pastör pipetiyle ASC (1 x 04-04 Ekim x 10 4). Kültürü insert içinde orta son hacim, tohumlama sonra, 500 uL olup. </li><li> 37 azından% 5 CO2 inkübatöründe nemlendirilmiş 24 saat süreyle CSM üzerine ASC tohumlu inkübe ° C. </li></ol> 5.. CSM içinde ASC Yükleme ve Hücre Uygulanabilirlik Yüzde Belirlenmesi Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> Inkübasyondan sonra, insert membran içine göç etmiş hücreleri bozmadan steril bir 1.5 ml mikrosantrifüj tüpüne ASC yüklü CSM toplamak. </li><li> Artık orta çıkarın ve tüp taze besiyeri 250 uL ekleyin. </li><li> Her tüp 25 uL MTT [3 - (4,5-dimethylthiozole-2-il) -2,5-difeniltetrazolium bromür] eklemekçözeltisi (5 mg / mL) ve 37 azından% 5 CO2 inkübatöründe nemlendirilmiş 4 saat süre ile inkübe edilir ° C. </li><li> İnkübasyondan sonra, orta kaldırmak dimetil sülfoksit 250 uL ekleyin ve karmaşık formazan çözünürleştirmek için 2-5 dakika boyunca vorteks karışımı. </li><li> 5 dakika boyunca 2700 x g&#39;de santrifüje CSM, ve referans olarak 630 nm ile 570 nm de ölçüldü süpernatan absorbans belirler. </li><li> Uygulanabilir ASC&#39;nin bilinen numaraları elde absorbans değerine göre CSM ile ilişkili hücre numarasını belirlemek. </li></ol> 6. ASC-CSM-Gömülü Kolajen Jel Karakterizasyonu Not: aksi belirtilmediği sürece tüm işlemler oda sıcaklığında yapıldı. <ol><li> 2 N NaOH kullanılarak pH 6.8 &#39;arındırılmış Bornstein 8 ve fibrillate yöntemine göre, fare kuyruk tendonu ekstrakte tipi 1 kolajen (7.5 mg / mL) karışımı ASC-yüklü CSM (5 mg ≈ 2 x 10 hücre ihtiva eden 4). </li&gt; <li> Bir 12-iyi levhaya fibrile kolajen-ASC-CSM karışımı ekleyin ve bir 37% 5 CO2 inkübatöründe nemlendirilmiş azından 30 dakika süreyle inkübe edilir ° C. </li><li> Tam fibrilasyon sonra, 37 ° C kadar bir% 5 CO 2 nemli inkübatörde 14 gün için kolajen-ASC-CSM jeller inkübe </li><li> Jel içine CSM gelen hücrelerin Yayın ve göç standart mikroskopi teknikleri kullanarak gözlenmiştir. </li></ol> 7. Temsilcisi Sonuçlar Bu çalışmada, bir kollajen jel iskele içine kitosan mikroküreler (CSM) kök hücre sunmak için bir in vitro bir strateji geliştirdik. Birörnek (çapı 175-225 mikron) ve kompozisyon Gözenekli CSM hücre taşıyıcıları (Şekil 2) olarak hazırlanmış ve kullanılmıştır. CSM&#39;den ASC kuluçkaya üzerine, hücreler CSM 2 x 10 cells/5mg 4 arasında bir konsantrasyonda bağlı. Filopodia uzatırken hücreleri, mikroküre yayılan kabil edildimikroküre gözenekli yarıklar (Şekil 3) takın. Hücre yüklenmiş CSM kollajen jel ile karıştırılır edildi sonra, hücreler hemen jeller (Şekil 4) içine göç başladı. <img alt="Tablo 1" src="/files/ftp_upload/3624/3624table1.jpg" /> Tablo 1. Bir kök hücre taşıyıcı sisteminde kitosan ve kollajen kullanımı için biyolojik avantajları. <img alt="Şekil 1" src="/files/ftp_upload/3624/3624fig1.jpg" /> Şekil 1. Şematik ASC-CSM yüklenen kollajen iskelelerinin çift kullanımı için genel bir strateji resmediyor. Şekil 2, 3, ve 4 görüntülerin yorumlanması ile yardımcı şematik içinde açıklamalı. <img alt="Şekil 2" src="/files/ftp_upload/3624/3624fig2.jpg" /> Şekil 2. Şematik kitosan mikroküreler üzerine kök hücrelerin tohumlu süreci resmediyor. Procmikron gözenek büyüklüğü membran kültür plaka insert - ess bir 8 CSM ile birlikte kültür ASC içerir. 24 saat sonra, mikroküreler ekleme kaldırılır ve bir biyomateryal matris içine almasi için hazırdır. <img alt="Şekil 3" src="/files/ftp_upload/3624/3624fig3.jpg" /> Şekil 3. ASC ile CSM yüklü morfolojik karakterizasyonu. B panelinden konfokal floresan mikroskobu ile elde edilen bir resim ile üst üste aynı görüş alanını gösterirken Panel A, ASC-yüklü CSM bir ışık mikroskobu resmediyor. ASC kalsein AM (yeşil) ile önceden yüklenmiş edildi. Panel D mikroküre (yıldız) yüklenmiştir hücreleri gösterirken Panel C, yüksüz bir mikroküre bir SEM görüntüsü bir görüntü resmediyor. Panel E TEM görüntü yüksüz mikrosfer bir kesitini göstermektedir. Gözenekler ve çatlaklar yapan çok sayıda mikroküre çapında bulunmaktadır. Panel F hücreleri ile kesitli mikroküre (oklar) eklenmiş ve CRE içine filopodia uzanan gösterirmengene. Orijinal büyütme: A &amp; B = 70X; C = 500x, D = 2,000 x; E &amp; F = 2,500 x. <img alt="Şekil 4" src="/files/ftp_upload/3624/3624fig4.jpg" /> Şekil 4. Üç boyutlu bir yapı iskelesi kollajen içine CSM gelen ASC göçü. Paneller A ve B hücreleri uzak mikroküre geçiş ve 3. günde kollajen matriks (A, oklar) içine CSM betimliyor. Panel B 12 gün sonra, benzer bir kültür gösterir. Transmisyon elektron mikroskobu (TEM) görüntüleri C, D ve C ve D E. Asteriskler tasvir hücreleri mikroküre (oklar) uzak göç ile kesitli olan bir mikroküre göstermektedir. Panel D&#39;nin yüksek büyütmede paneli E tasvir ve kollajen fibriller (ilave) hücresine bağlı filopodia gösterir. Orijinal büyütme: A &amp; B = 100x; C &amp; D = 6,000 x; E = 20.000 x, inset = 150,000 x. <img alt="Şekil 5," src="/files/ftp_upload/3624/3624fig5.jpg" /> Şekil 5.Şematik rejeneratif tıp ve ilaç teslim CSM geniş kullanım resmediyor.

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+Human+adipose-derived+stem+cells+seeded+on+a+silk+fibroin-chitosan+scaffold+enhance+wound+repair+in+a+murine+soft+tissue+injury+model.">IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model.</a> Stem Cells. 27, 250-258 (2009).</li><li>Machado, C. B., Ventura, J. M., Lemos, A. F., Ferreira, J. M., Leite, M. F., Goes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+chitosan-gelatin-chondroitin+porous+scaffold+improves+osteogenic+differentiation+of+mesenchymal+stem+cells.">3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.</a> Biomed. Mater. 2, 124-131 (2007).</li></ol>

Hiçbir rakip mali çıkarlarını var. Feragatname Burada yer alan görüşler ya da iddiaların yazarların özel görünümleri ve resmi olarak veya Savunma ya da ABD Hükümeti Bölümü görüşlerini yansıtan tefsir edilmesi değildir. Yazarlar, ABD Hükümeti çalışanları, ve bu çalışma resmi görevlerinin bir parçası olarak hazırlandı. Tüm çalışmalar ABD Ordusu Tıbbi Araştırma ve Malzeme Komutanlığı tarafından desteklenmiştir. Bu çalışma ABD Ordusu Tıbbi Araştırma ve Malzeme Komutanlığı Kurumsal Değerlendirme Kurulu ve onaylanmış protokole uygun olarak tarafından incelenen ve onaylanan bir protokol çerçevesinde yürütülmüştür.

Kök hücre tabanlı tedavinin başlıca engellerden biri tamir için belirtilen bölgelere hücrelerin teslimat için etkin yöntemler geliştiriyor. Hastadan hastaya olan değişkenlik nedeniyle, doku tipi, yaralanma boyutu ve derinliği; teslim kök hücre yöntemi bir vaka ile ayrı ayrı tespit edilmelidir. Bir matris içinde kök hücreleri gömerek ve yara yerine onlara teslim doku mühendisliği için bir sonraki mantıklı bir yaklaşım gibi görünse de, bazı teknik engeller devam etmektedir. Bu matris yapı...

<table border="1" ><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Hanks BalancedSalt Çözüm (HBSS) </td><td> Gibco </td><td> 14175 </td><td> Sarf </td></tr><tr><td> Sığır fetüs serumu </td><td> Hyclone </td><td> SH30071.03 </td><td> Sarf </td></tr><tr><td> Kollajenaz Tip II </td><td> Sigma-Aldrich </td><td> C6685 </td><td> Sarf </td></tr><tr><td> 70 mikron naylon gözenekli filtre </td><td> BD Biosciences </td><td> 352350 </td><td> Sarf </td></tr><tr><td> 100 mikron naylon gözenekli filtre </td><td> BD Biosciences </td><td> 352360 </td><td> Sarf </td></tr><tr><td> MesenPRO Büyüme Orta Sistemi </td><td> Invitrogen </td><td> 12746-012 </td><td> Sarf </td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030 </td><td> Sarf </td></tr><tr><td> T75 Doku Kültürü Flask </td><td> BD Biosciences </td><td> 137787 </td><td> Sarf </td></tr><tr><td> Chitosan </td><td> Sigma-Aldrich </td><td> 448869 </td><td> Sarf </td></tr><tr><td> Asetik asit </td><td> Sigma-Aldrich </td><td> 320099 </td><td> Sarf </td></tr><tr><td> N-oktanol </td><td> Acros Organics </td><td> 150630025 </td><td> Sarf </td></tr><tr><td> Sorbitan-Mono-oleat </td><td> Sigma-Aldrich </td><td> S6760 </td><td> Sarf </td></tr><tr><td> Potasyum hidroksit </td><td> Sigma-Aldrich </td><td> P1767 </td><td> Sarf </td></tr><tr><td> Aseton </td><td> Fisher Scientific </td><td> L-4859 </td><td> Sarf </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich </td><td> 270741 </td><td> Sarf </td></tr><tr><td> Trinitro benzensülfonik asit </td><td> Sigma-Aldrich </td><td> P2297 </td><td> Sarf </td></tr><tr><td> Hidroklorik asit </td><td> Sigma-Aldrich </td><td> 320331 </td><td> Sarf </td></tr><tr><td> Etil eter </td><td> Sigma-Aldrich </td><td> 472-484 </td><td> Sarf </td></tr><tr><td> 8-um Doku Kültürü Plaka Uçlar </td><td> BD Biosciences </td><td> 353097 </td><td> Sarf </td></tr><tr><td> 1.5 ml Mikrosantrifüj Tüpler </td><td> Balıkçı </td><td> 05-408-129 </td><td> Sarf </td></tr><tr><td> MTT Reaktif </td><td> Invitrogen </td><td> M6494 </td><td> Sarf </td></tr><tr><td> Dimetil Sulfoxide </td><td> Sigma-Aldrich </td><td> D8779 </td><td> Sarf </td></tr><tr><td> Qtracker Hücre Etiketleme Kiti (Q tracker 655) </td><td> Molecular Probes </td><td> Q2502PMP </td><td> Sarf </td></tr><tr><td> 1 Kollajen Tip </td><td> Travigen </td><td> 3447-020-01 </td><td> Sarf </td></tr><tr><td> Sodyum Hidroksit </td><td> Sigma-Aldrich </td><td> S8045 </td><td> Sarf </td></tr><tr><td> 12-Peki Doku Kültürü Tabaklar </td><td> BD Biosciences </td><td> 353043 </td><td> Sarf </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5417R </td><td> Ekipman </td></tr><tr><td> Orbital Çalkalayıcı </td><td> New Brunswick Bilimsel </td><td> C24 </td><td> Ekipman </td&gt; </tr><tr><td> Hava-% 5 CO2 ile nemlendirilmiş Kuluçka </td><td> Thermo Scientific </td><td> Model 370 </td><td> Ekipman </td></tr><tr><td> Tepegöz Karıştırıcı </td><td> IKA </td><td> Visc6000 </td><td> Ekipman </td></tr><tr><td> Manyetik Karıştırıcı </td><td> Corning </td><td> PC-210 </td><td> Ekipman </td></tr><tr><td> Vakum Desikatör </td><td> - </td><td> - </td><td> Ekipman </td></tr><tr><td> Parçacık Boyut Analiz Cihazı </td><td> Malvern </td><td> STP2000 Spraytec </td><td> Ekipman </td></tr><tr><td> Su Banyosu </td><td> Fisher Scientific </td><td> Isotemp210 </td><td> Ekipman </td&gt; </tr><tr><td> Spektrofotometre </td><td> Beckman </td><td> Beckman Coulter DU800UV/Visible Spektrofotometre </td><td> Ekipman </td></tr><tr><td> Girdap </td><td> Diagger </td><td> 3030a </td><td> Ekipman </td></tr><tr><td> Mikroplaka Okuyucu </td><td> Molecular Devices </td><td> SpectraMax M2 </td><td> Ekipman </td></tr><tr><td> Floresan / Işık Mikroskobu </td><td> Olympus </td><td> IX71 </td><td> Ekipman </td></tr><tr><td> Konfokal Mikroskop </td><td> Olympus </td><td> Konfokal Mikroskop FV-500 Lazer </td><td> Ekipman </td></tr><tr><td> Taramalı Elektron Mikroskobu </td><td> Carl Zeiss MicroImaging </td><td> Leo 435 VP </td><td> Ekipman </td></tr><tr><td> Transmisyon Elektron Mikroskobu </td><td> JEOL </td><td> JEOL 1230 </td><td> Ekipman </td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

Multipotent kök hücreleri rejeneratif tıp 1-3 alanında çok faydalı olduğu kanıtlanmıştır. Bununla birlikte, doku rejenerasyonu için etkili bir şekilde, bu hücrelerin kullanmak için, değişken bir dizi dikkate alınmalıdır. Bu değişkenler şunlardır: toplam hacmi ve implantasyon sitenin yüzey alanı, doku mekanik özellikleri ve damarlanma miktarını ve hücre dışı matris bileşenlerini kapsamaktadır doku mikro,. Ana doku taklit eden bir mekanik mukavemet korurken Bu nedenle, bu hücrelerin sağlamak için kullanılan materyallerin belirlenmiş bir kimyasal bileşimi ile biyo-uyumlu olmalıdır. Bu malzemeler ayrıca oksijen ve besin hücrelere tutunur ve çoğalırlar için elverişli bir mikro sağlamak için geçirgen olması gerekir. Chitosan, mükemmel biyouyumluluk bir katyonik polisakkarit, kolayca kimyasal olarak değiştirilmiş ve in vivo mac in ile bağlamak için yüksek afinite edilebilirromolecules 4-5. Chitosan, hücre yapışması, göçü ve çoğalması için bir substrat olarak işlev sağlayan, ekstraselüler matriksin glikozaminoglikan kısmı taklit eder. Bu çalışmada, üç boyutlu bir yapı iskelesi 6 göre kollajen bir içine adipoz-türevi kök hücreleri (Artan) sunmak üzere mikrosferler şeklinde kitosan kullanmaktadır. İdeal bir hücreden mikrosfer oranı inkübasyon süresi ve yüklenmesi olabilir hücrelerin sayısını elde etmek için hücre yoğunluğu açısından değerlendirildi. ASC kitosan mikroküreler (CSM) üzerine ekildikten sonra, bir kollajen iskele gömülü olan ve uzun süre kültüründe muhafaza edilebilir. Özetle, bu çalışmada hassas bir üç boyutlu yapı iskelesi biyomalzemenin içinde kök hücreleri sunmak için bir yöntem sağlar.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Mevcut kök hücre tedavilerinde büyük bir engel konak dokularına bu hücrelerin sunmak için en etkili yöntem belirlemektir. Adipoz kaynaklı kök hücreler kendi multipotency korumasını sağlarken Burada, verimli ve yaklaşım, basit bir kitosan tabanlı dağıtım yöntemi açıklar.

Kök Hücre yüklü Chitosan Mikrokürelerin teslimi için bir Kollajen Hidrojel oluşturma

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K Görüntüleme.  United States Army Institute of Surgical Research. Mevcut kök hücre tedavilerinde büyük bir engel konak dokularına bu hücrelerin sunmak için en etkili yöntem belirlemektir. Adipoz kaynaklı kök hücreler kendi multipotency korumasını sağlarken Burada, verimli ve yaklaşım, basit bir kitosan tabanlı dağıtım yöntemi açıklar.

Video: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipik Kollajen Ben Assay: 3-Boyutlu Bağlamında Hücre Davranışı değerlendirin A Dövülebilir Platformu

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Biyomühendislik

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Cilt Tip I Kolajen hazırlanması için geliştirilmiş bir yöntem

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Doğallığı Kollajen Tellerinin Görüntüleme<em&gt; In vivo</em&gt; Ve<em&gt; Ex vivo</em&gt; Kafesli Kollajen Mimetik Peptidlerinin Fotoğraf-tetiklenen Hybridization yoluyla

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Için Amelogenin-kitosan hidrojelin geliştirilmesi<em&gt; In Vitro</emBir Yoğun Arayüzü ile&gt; Emaye büyütme

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscale Vortex destekli Sıralı Moleküler Teslimat için Electroporator

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Rekombinant kolajen peptid Microcarriers hücre genişletme ve hücre teslim sistemi bir biyoreaktör olarak potansiyel kullanım için ben Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Bir böbrek korteks ekstraselüler matrisi elde edilen hidrojel imalatı

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) &amp; crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling &amp; degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p&#8804;0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

Diyabetik Yaraların Hızlandırılmış İyileşmesi İçin Doksiksin Yüklü Kollajen-Kitosan Kompozit İskele

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Periferik Nöromühendislik için Grafen Bazlı 3D Biyohibrid Hidrojel Biyomürekkebin Hazırlanması ve Karakterizasyonu

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Doku Mühendisliği Uygulamaları için Boyut Kontrollü ve Emülsiyonsuz Kitosan-Genipin Mikrojellerinin İmalatı

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...