Biz araştırma verdikleri destek ve işbirliği için Dr George Avam ve Dr Dean Vistnes teşekkür etmek istiyorum. Saint Joseph Mercy Hastane GME tarafından desteklenen bu çalışma Diş ve Kraniofasial Araştırma hibeleri 1 R21 DE019274-01, R01EB009689 ve RC2 DE020771-02, MTL Dr Hyun Pediatrik Rejeneratif Tıp Oak Vakfı ve Hagey Laboratuvarı Ulusal Enstitüsü tarafından desteklenmektedir .

1. Hücre İzolasyonu &amp; Genişletme <ol><li> Tüm hastanın rızası ve deneysel protokolleri Stanford Üniversitesi Kurumsal İnceleme Kurulu (Protokol # 2188 ve # 9999) tarafından gözden geçirilmiş ve onaylanmıştır. </li><li> Genel / lokal anestezi altında elektif lipoaspiration prosedürlerden İnsan subkutanöz adipoz dokusu elde edilir. </li><li> Lipoaspirate iki kat (Şekil 1A) olacaktır. Süpernatant işlenmiş hücresel malzemenin büyük bir çoğunluğu içerir. Alt tabaka daha çok tabaka ya da stromal hücreler hasat edilebilir Yağ-türetilmiştir. Salin enjekte, ancak verimi üstte yüzen madde gelen çok daha büyük olmasıdır. </li><li> Stromal vasküler fraksiyonu (SVF) izole etmek için, 1X eşit hacimlerde salin (PBS) betadin olmadan 1X PBS (x1) eşit hacmi ardından% 2.5 betadin (x2) içeren fosfat tamponlu ile yoğun lipoaspirate yıkayın. Çökelti için yıkama izin verin. </li><li> Steril te korumak için dikkatli olunişlenmiş insan dokusu ile karışması süreci boyunca chnique olabilir. </li><li> Alt tabaka aspire ve her yıkamadan sonra atın. </li><li> Tablet 50 ml BD Falcon konik tüpler içine adipoz doku 15 ml. </li><li> 37 Hank Dengeli Tuz Çözeltisi içinde süzülmüş% 0.075 Tip II kollajenaz eşit bir hacmi (15 ml) bir (HBSS) ° C su banyosu içinde 60 dakika boyunca sürekli karıştırma altında (yaklaşık 180 / dak titreşim) ile adipoz doku sindirmek - Her bir tüp her 15 dk havalandırın. </li><li> 15 ml PBS ° C olan yüksek yoğunluklu bir SVF pelet elde etmek için 4, 5 dakika için 1000 rpm&#39;de% 10 (Fetal Bovine Serum) FCS, ve santrifüj ile birlikte ihtiva eden enzim aktivitesini nötralize eder. Pelet adipoz-kaynaklı stromal hücreler ve eritrositler bir karışımı olacaktır. </li><li> Pelet aksatmadan Süpernatantı atın. </li><li> Geleneksel büyüme ortamı, 10 ml (Dulbecco Modifiye Eagle Ortamı [DMEM] /% 10 FBS /% 1 penisilin-streptomisin solüsyonu) pellet yeniden süspanse edin. </li><li> Filtre Asma<html> hücresel enkaz kaldırmak için 100 mikron naylon hücre süzgecinden sion. </li><li> Temiz bir 50 ml konik tüpler içine 3 birleştirin. 4 ° C&#39;de 5 dakika boyunca 1000 rpm&#39;de santrifüje </li><li> Pelet aksatmadan Süpernatantı atın. </li><li> Geleneksel büyüme ortam 5 ml pelet yeniden süspanse edin </li><li> Biri 10 cm tabak veya bir adet 15 cm tabak için beş 50 ml conicals başına iki 50 ml conicals birleştirin. </li><li> 37 ° C/21% O 2,% 5 CO 2 gecede primer kültürlerinde oluşturulması. </li><li> Gecelik inkübasyon sonrasında rezidüel yapışmaz kırmızı kan hücreleri çıkarmak için PBS ile yoğun tabakları yıkamak. Ortaya çıkan hücre popülasyonlarının adipoz-kaynaklı stromal hücrelerdir. </li><li> 37 ° C/21% O 2,% 5 CO büyüme ortamı içinde 2 spontan farklılaşma önlemek için alt-konfluent düzeyde hücreleri koruyun. Hücreler genellikle% 50 izdiham kaplama yaparken düzenli büyüme ortamı her üç dört gün ayırmak gerekir. </li></ol>ve_title &quot;&gt; 2. İskele Hazırlık <ol><li> Diş Hekimliği Fakültesi - İskelelerin California Üniversitesi&#39;nden Dr Min Lee, Los Angeles tarafından yapılmıştır. </li><li> PLGA iskeleleri çözücü ve bir döküm parçacık süzme işlemi ile 85/15 poli (laktik-ko-glikolik asit) (doğal viskozite = 0,61 dl / g arasında, Birmingham Polimerler) imal edilmiştir. </li><li> PLGA / kloroform çözümleri% 92 gözeneklilik (hacim fraksiyonu) ve teflon kalıp içinde ince tabakalar halinde sıkıştırılmış elde etmek için 200-300 mikron çaplı sukroz ile karıştırılmıştır. </li><li> Gecede dondurularak kurutulduktan sonra, iskelelerinin sakaroz çözmek için çift distile (dd) H 2 O üç değişiklik dalmış edildi ve nazikçe ince uçlu spatula ile teflon plaka kaldırıldı. </li><li> Parçacık süzme sonra, tüm iskeleleri GKD 2 O. üç kez durulama ile takip 30 dakika her biri için en az% 50,% 60 ve% 70 etanol içinde daldırma ile dezenfekte edildi </li><li> Tüm iskeleleri bir laminer akış kaputu altında kurutuldu. </li><li> Sonra iskele imalat, scaffolds apatit ile kaplanmıştır. <ol class="ualpha"><li> SBF (yapay vücut sıvısı) solüsyonu insan kan plazması 5 zamanlardı iyon konsantrasyonları ile hazırlanmıştır. Tüm çözümler bir 0.22 mikron PES membran (Nalgene) süzüldü steril edildi. Hemen kaplama işlemi öncesinde, PLGA iskeleleri ıslatma ve kaplama bütünlüğü geliştirmek için kızdırma deşarj, argon plazma aşındırması (Harrick Bilimsel) tabi tutuldu kurutulur. </li><li> Kabartma PLGA iskeleleri daha sonra 37 SBF inkübe edildi ° Mg 2, ardından 12 saat boyunca bir su ceketli kuluçka makinesi içinde C, + ve HCO 3-37 az 12 saat boyunca serbest SBF 2 ° C&#39;de hafif karıştırma altında. </li><li> Kaplamalı PLGA iskelelerinin bir laminer akış kaputu kurutulur ve% 70 etanol ile dezenfekte aşırı sodyum klorür çözeltisi yıkayacak steril GKD 2 O ile nazikçe yıkandı. </li><li> Apatit kaplama bütünlüğü taramalı elektron mikroskobu (SEM) ile analiz edildi. Apatit kaplı scaffolds SEM saplamaları (Ted Pella) üzerine monte edilen ve iletkenliğini artırmak için karbon ile kaplanmıştır. Ikincil elektron modu SEM (FEI / Phillips XL-30) gözlem sırasında uygulandı. Enerji dağılım X-ışını spektrumu apatit yapıları elementel bileşimi onaylamak için elde edilmiştir. </li></ol></li></ol> 3. Hücre Seeding <ol><li> Alt-izdiham düzeyleri ulaşan üzerine, PBS (x2) ile hücrelerin yıkayın ve Trypsinize. </li><li> Tohumlama için ölçmek hücreleri hesaplama </li><li> 1,5 x 10 5 hücre ile pre-cut 4.0 mm çaplı iskeleleri üzerine Tohum. <ol class="ualpha"><li> 96-kuyulu plakanın bir kuyu içine yerleştirin bireysel yapı iskelesi </li><li> Ortam 20 ul başına yaklaşık 1.5 x 10 5 hücrelerinin bir konsantrasyon ile normal büyüme ortamı içinde yeniden askıya hücrelerin </li><li> 30 dakika süreyle hücre kültürü inkübatör içine Pipet 20 doğrudan iskele üzerine ul ve yer. </li><li> 30 dakika sonra, ortam 200 ul ekle ve hücreler inkübe edincerrahi iskele bir gecede önce </li><li> Bu hücrelerin yeterli sayıda tohumlu olarak iskele üzerine hücre yapışma çoğunluğu iskele üzerine takmak sağlayacaktır sağlamak için gerekli değildir. Bu biyolüminesans ve histolojik analizi ile in vivo laboratuarımızda onaylanmıştır. </li></ol></li></ol> 4. Kalvaryum Hataları ve Vivo Ekiminde oluşturulması <ol><li> Uyutmak yetişkin (60 günlük yaşta) anestezik kokteyl ile CD-1 çıplak farelerin. (Ketamin / xylazine / Acepromazine)% 50 konsantrasyonda (% 0.9 NaCl ile 1:1 seyreltme) veya kurum protokolleri başına önerilen anestezik rejime başına. HASCs bir immun reaksiyona neden olmayacak şekilde bu fareler atimik vardır. <ol class="lalpha"><li> Dozaj: Ketamin hidroklorid (80 mg / kg), bir Xylazine (2.5 mg / kg) Acepromazine (2.5 mg / kg) </li><li> Etki süresi: yaklaşık 30 dakika </li></ol></li><li> Cerrahi hayvan asmak ve cerrahi sterilizebetadin ve alkol (x3) ile site ve önceki sağ parietal kemiğin açığa hayvanın derisi bir orta hat sagital insizyon yapma merhem ile fare gözlerinizi kapayın. Künt kazıma (Şekil 1B) ile sağ parietal kemik pericranium çıkarın. </li><li> Steril bir elmaslı trepan matkap ucu kullanarak doğru olmayan sütür ilişkili paryetal kemikte tek taraflı 4 mm tam kat defekt oluşturun. Extreme dikkatli fare kalvaryal kalınlığı gibi altta yatan duramater (Şekil 1B) rahatsız etmemek için dikkat edilmelidir &lt;.3 mm. </li><li> Takılmadan önce, orta-türevi büyüme faktörü transferini önlemek için steril PBS ile iskelelerinin yıkayın. </li><li> Defekt içine iskele yerleştirin. </li><li> Son olarak, cilt kapatıldı sütür ve kurulan postoperatif protokoller başına hayvan izlemek. </li><li> Post-operatif-örneğin gerektiği gibi hayvan bakım protokolleri yönettiği olarak kurulmuştur analjezi kullanın. Buprenorfin 0.1 mg / kg. </li></ol> 5. Osteoid Oluşumu Kantitasyonu <ol><li> MikroBT fare yapıldıktan sonra, üç-boyutlu bir görüntü daha önce tarif edildiği gibi 1 MicroView yazılımı kullanılarak yeniden inşa edildi. </li><li> Adobe Photoshop kullanarak, görüntülerin standart bir yüksekliğe büyüklükte. </li><li> Sihirli Değnek özelliğini kullanarak, piksel kalvaryal kusuru ölçüldü. </li><li> Kusur yüzdesi şifa sonraki CT taramaları kalvaryal defekt alan ölçme ve piksel sayısının belirlenmesi ve orijinal defekt en piksel sayısına bölünmesiyle tespit edildi </li><li> MikroBT kullanılamaz Sonuç olarak, eğer bir alternatif Adobe Photoshop kullanarak ve histoloji için belirlenen zaman noktalarında kalvaryal hasat edilir. </li><li> Osteoid oluşumu ifade Anilin Mavisi ve pentakrom gibi histolojik boyalar kullanarak, defekt açıklığı boyunca birden çok bölüm lekeli olmalıdır </li><li> Adobe Photoshop, olmayan tüm alanları kırpma kullanmakalvaryal defekt </li><li> Sihirli değnek özelliğini kullanın ve defekt alanındaki de novo kemik formasyonu piksel numaraları belirlemek ve denetimleri veya diğer değişkenlere karşılaştırın. </li></ol> 6. Temsilcisi Sonuçlar Yağ dokusu klinik uygulama için progenitör hücrelerin üretimi hayati bir rol hizmet etmek için bir potansiyele sahiptir. Yağ dokusu minimal morbidite ve mortalite içeren nispeten basit bir prosedür içinde hasat edilebilir bir hazır kaynağı olduğunu benzersiz bir avantaja sahiptir. Dokusu hasat edilir ve toplanır başladığında, protokol Şekil 2 &#39;de belirtilmiştir. Yağ-kaynaklı stromal hücreler bir çamaşır adım serisi, kollajenaz sindirim ve santrifüj yoluyla izole edilmiştir. Hücre kültürü içinde kaplanır sonra, bunlar, genişletilmiş, in vitro çeşitli protokoller farklılaşma içine yerleştirilmiş, ya da in vivo doğrudan yerleştirilebilir. C4 mm kritik boyutlu kalvaryal defekt reation bizim adipoz-kaynaklı stromal hücrelerin osteojenik farklılaşma yeteneğini test etmek vivo modelde bir kolay erişilebilir ve tekrarlanabilir sağlar. MikroBT kullanımı sayesinde, biz in vivo iskelet doku oluşumunu takip ve müdahalelerin ilerleme (Şekil 3) takip edebiliyoruz. Kritik boyutu kalvaryal kusur hayvanın yaşam süresi içinde iyileşme olmaz ve defekt kabaca 90% 8 hafta civarında patent kalır bakın. Iskele kendisi (tartışmaya bakınız) osteojenik indüktif özellikleri vardır ve bazı kemik rejenerasyonu teşvik etmek yeteneğini göstermiştir. Hücre içermeyen iskele yerleştirme tipik sonuçlar kusur yaklaşık üçte biri 8 hafta de novo kemik formasyonuna sahip olacağını göstermektedir. Iflkenlerin olsa adipoz-kaynaklı stromal hücrelerin güçlendirme ile tam kusur üçte iki veya daha fazla 8 hafta civarında kemik rejenerasyonu gösterecektirHer hayvan ve cerrahi arasındaki ility. İskelet doku oluşumu histoloji yoluyla ölçülebilir ve tipik sonuçları Anilin Mavisi ve pentakrom boyanma (Şekil 3C) yoluyla TSK ile örneklerinde artmış osteoid oluşumu göstermek olabilir. Buna ek olarak, (Şekil 3D), implante edilmiş insan hücre kalvaryal kusur olarak de novo kemik oluşumu alan yakın 2 hafta sonra in vivo olarak boyanması göstermek GFP-etiketli hASCs kullanımı ile alt de novo kemik oluşumuna katkıda bulunduğunu göstermektedir . Buna ek olarak, hatanın alan (Şekil 3E) insan hücre hayatta kalma ve katkı göstermek için insan spesifik antikor ile immünohistokimyasal kullanır. <img alt="Şekil 1" src="/files/ftp_upload/4221/4221fig1.jpg" /> Şekil 1 A -. Lipoaspirate iki katmandan oluşmaktadır. Üst tabaka adipositler ve pr çoğunluğu içeriralt tabaka lipoaspiration işlem sırasında kullanılan salin içeren süre hücresel malzeme ocesses. B - yatan duramater kesinti olmadan sağ parietal kemik, bir 4 mm kritik büyüklükte kalvaryal defekt sonraki yaratılması izole pericranium üzerinde orta hat insizyonla kalvaryal defekt oluşturulması. <img alt="Şekil 2," src="/files/ftp_upload/4221/4221fig2.jpg" /> Şekil 2. Kendi genişleme, farklılaşma ve in vitro ve in vivo kullanıma adipoz-kaynaklı stromal hücrelerin izolasyonu gelen lipoaspirate ve hasat ve uygulama genel bakış. <img alt="Şekil 3" src="/files/ftp_upload/4221/4221fig3.jpg" /> Şekil 3,. Bir hyd yoluyla TSK uygulaması ile önemli boyut kalvaryal kusur in vivo iyileşme gösteren bir-mikroBTroxyapatitie iskelesi (Alt Satır). ASC grubunda anlamlı bir artış iyileşme gösteren mikroBT gelen kemik iyileşmesiyle Kantitasyonu - Kontroller hücreleri (Orta Sıra) Yatak olmadan iskele yerleştirme ile hiçbir iskele ve defekt (Üst satır) ve kusur vardır. C - Histoloji Anilin Mavisi ve pentakrom boyama yoluyla ASC grubu (alt satır) artmış osteoid oluşumu gösteren. Anilin Mavi için, osteoid lekeler koyu mavi ve pentakrom, osteoid lekeler sarı için. D - GFP etiketli hASCS bir iskele üzerine tohumlandıklarında ve yerleştirilen bir kalvaryal defekt içine ve 2 hafta sakrifiye edildi. Boyama kusur alanında yenilenme katkı insan hücrelerini gösteren bir GFP etiketlenmiş antikor ile yapıldı. E -. 2 hafta defekt alanında insan hücreleri yaygınlık gösteren İnsan nükleer antijen immünhistokimya <a href="http://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> .

<ol>
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Çıkar çatışması ilan etti.

Ve izolasyonu yağ olarak türetilmiş lipoaspirate dan stromal hücreleri 2, bu hücrelerin hücre soylarının çeşitli ayrışmıştır edilmiştir. Yağ dokusu mezodermal kökenlerden gelen ve bu nedenle, multipotent adipoz-kaynaklı stromal hücrelerin büyük olasılıkla bir mezodermal soy doğru uygulama ile en etkili olacaktır. İskelet doku oluşturmak için yeteneği verici bir otogreft için siteleri ve enfeksiyon, rejeksiyon ve zaman üzerinde 3 arızaları dahil sentetik malzemenin ...

<table border="1"><tbody><tr><td> Reaktif Adı: </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td></td><td></td><td></td><td> Hasat Lipoaspirate </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td></td></tr><tr><td> Hank Dengeli Tuz Çözeltisi </td><td> Cellgro </td><td> 21-023-CV </td><td></td></tr><tr><td> Kollajenaz </td><td> Sigma </td><td> C6885-500MG </td><td></td></tr><tr><td> Hücre Süzgeç 100 mikron </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> Steri-500 ml .22 mikron filtre </td><td> Millipore </td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> Kalvaryum Kusur </td></tr><tr><td> Z500 Fırçasız MicromotorsUM50C </td><td> NSK </td><td> NSKZ500 </td><td></td></tr><tr><td> Dairesel Bıçak 4,0 mm </td><td> Xemax Cerrahi </td><td> CK40 </td><td></td></tr></tbody></table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

Kraniyofasiyal iskelet onarımı ve yenilenmesi kök hücreleri kullanan bir hücre-temelli bir yaklaşım ile de novo doku oluşumu vaadi sunar. Yağ-kaynaklı stromal hücreler (TSK) osteojenik, kondrojenik adipojenik ve miyojenik farklılaşma geçiren yeteneğine multipotent kök hücrelerin bol bir kaynağı olduğu kanıtlanmıştır. Pek çok çalışma hücresel teslim için çeşitli yapı iskelesi biyomalzemelerin kullanımı ile in vivo olarak bu hücrelerin osteojenik potansiyel araştırdılar. Bu gibi olduğu gösterildi bir osteokondüktif, hidroksiapatit-kaplı poli (laktik-ko-glikolik asit) (PLGA-HA) TSK ile iskele tohumlu, bir kritik boyutlu kalvaryal kusur, kendiliğinden geçmesi için kendi yetersizlik ile tanımlanan bir bozukluk kullanılarak , hayvanın ömrü boyunca şifa etkili sağlam kemik rejenerasyon göstermek olabilir. Bu in vivo modelde kemik dokusu yenilenmesine yönelik translasyonel yaklaşım esas göstermektedir - hücreselbileşeni ve biyolojik matrisi. Bu yöntem, bir doku spesifik kusurun tamir yönelik bir projenitör hücre nihai klinik uygulama için bir model olarak hizmet vermektedir.

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Bu protokol izolasyonu açıklar adipoz-türetilmiş lipoaspirate ve iskelet rejenerasyon değerlendirmek için 4 mm kritik ölçüdeki kalvaryal defekt oluşturmadan stromal hücreler.

Lipoaspirate itibaren hasat Yağ kaynaklı stromal hücreler kullanarak Kritik ölçekli Kalvaryum Defekt Model tamiri

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

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JoVE Journal

Medicine

15.8K Görüntüleme.  Stanford University. Bu protokol izolasyonu açıklar adipoz-türetilmiş lipoaspirate ve iskelet rejenerasyon değerlendirmek için 4 mm kritik ölçüdeki kalvaryal defekt oluşturmadan stromal hücreler.

Video: Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

İnsan Meme Fibroblastlar Karsinom İlişkili Fibroblastlar Deneysel Nesil (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Sonsuz Horizon Darbeli kullanarak tek taraflı Servikal Spinal Kord Yaralanması bir Contusive Modeli

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Distraksiyon Osteogenezi, ya da Eleştirel Ölçekli Kusurların Şifa, Intramembranous kemikleşme değerlendirilmesi için Kademe Stabilize Kırıklar oluşturma

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovasküler Aort Anevrizma Onarımı Yeni Bir murin Modeli

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Histerektomi Örneklerden İlköğretim İnsan endometrial stroma hücrelerinde Kurulması İçin İki Yöntem

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Scarless Yara Repair Fare Fetal Cilt Modeli

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Farelerde Basit Kritik ölçekli Femoral Hata Modeli

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Oluşturma ve diyabetik yara iyileşmesi manken bir yağ elde edilen kök hücre (ASC) Sheet nakli

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

İnsan böbrek Perivasküler Stromal hücreler için bir roman klinik sınıf izolasyon yöntemi

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...