Vorremmo ringraziare il Dott. Commons George e il Dr. Dean Vistnes per il loro sostegno e la collaborazione della nostra ricerca. Questo lavoro è supportato dal National Institute of Dental Research e borse di craniofacciale 1 R21 DE019274-01, R01EB009689 e RC2 DE020771-02, la Fondazione di quercia e Hagey Laboratorio di Medicina Rigenerativa di Pediatric MTL Dr. Hyun è supportato dal San Giuseppe Mercy Hospital GME .

1. Cella di isolamento e di espansione <ol><li> Tutti consenso del paziente e protocolli sperimentali sono stati esaminati e approvati dalla Stanford University Institutional Review Board (protocollo # 2188 e # 9999). </li><li> Ottenere umano tessuto adiposo sottocutaneo di procedure elettive di cui lipoaspirazione locale / anestesia generale. </li><li> Ci saranno due strati nella lipoaspirate (Figura 1A). Il surnatante contiene la maggior parte del materiale trattato cellulare. Lo strato inferiore è principalmente iniettato salina. Adiposo cellule stromali può essere raccolta da uno strato, ma la resa è molto maggiore dal supernatante. </li><li> Per isolare la frazione stromale vascolare (SVF), lavare il lipoaspirate ampiamente con uguali volumi di 1X tampone fosfato salino (PBS) contenente 2,5% betadine (x2), seguita da volume uguale di PBS 1X (x1) senza betadine. Lasciare che il lavaggio a precipitare. </li><li> Fate attenzione a mantenere sterile technique tutto il processo, come contaminazione con tessuti umani trasformati può accadere. </li><li> Aspirare lo strato inferiore e gettare dopo ogni lavaggio. </li><li> Dispensare 15 ml di tessuto adiposo in 50 ml BD Falcon tubi conici. </li><li> Digerire il tessuto adiposo con un volume uguale (15 ml) di filtrato 0,075% collagenasi di tipo II in soluzione salina bilanciata di Hank (HBSS) a 37 ° C a bagnomaria per 60 minuti con agitazione costante (circa 180 scuote / min) - ventilare ogni tubo ogni 15 min. </li><li> Neutralizzare l&#39;attività enzimatica con 15 ml di PBS contenente 10% (Fetal Bovine Serum) FBS, e centrifugare a 1000 rpm per 5 min a 4 ° C per ottenere una alta densità SVF pellet. Il pellet sarà un mix di derivate da tessuto adiposo cellule stromali ed eritrociti. </li><li> Scartare il sopranatante senza disturbare il pellet. </li><li> Risospendere il pellet in 10 ml di mezzo di crescita tradizionali (Eagle modificato da Dulbecco Medium [DMEM] / 10% FBS / 1% di penicillina-streptomicina soluzione). </li><li> Filtro sospensione<html> sione attraverso un filtro 100 micron cella in nylon per rimuovere i detriti cellulari. </li><li> Combina 3 tubi in uno pulito da 50 ml. Centrifugare a 1000 rpm per 5 min a 4 ° C. </li><li> Scartare il sopranatante senza disturbare il pellet. </li><li> Risospendere il pellet in 5 ml di mezzo di crescita tradizionali </li><li> Combina due conicals 50 ml per un 10 cm Piatto o cinque conicals 50 ml per una piastra di 15 cm. </li><li> Stabilire colture primarie notte a 37 ° C/21% di O 2, 5% di CO 2. </li><li> Dopo l&#39;incubazione per una notte, lavare i piatti estesamente con PBS per rimuovere residui non aderenti globuli rossi. Le popolazioni di cellule risultanti sono derivate da tessuto adiposo cellule stromali. </li><li> Mantenere le cellule a livello sub-confluenti livelli per evitare differenziazione spontanea a 37 ° C/21% O 2, 5% di CO 2 in terreni di coltura. Le cellule generalmente necessario dividere ogni tre o quattro giorni in terreni di crescita regolari quando piastrate al 50% di confluenza. </li></ol>ve_title &quot;&gt; 2. Scaffold Preparazione <ol><li> Impalcature sono state fatte dal Dr. Min Lee presso l&#39;Università di California, Los Angeles - Scuola di Odontoiatria. </li><li> Ponteggi PLGA sono state fabbricate da 85/15 poli (lattico-co-glicolico) (viscosità intrinseca = 0,61 dl / g, Birmingham Polymers) mediante colata solvente ed un processo di lisciviazione particolato. </li><li> PLGA / cloroformio soluzioni sono state miscelate con saccarosio diametro 200-300 micron per ottenere 92% di porosità (frazione di volume), e compressi in fogli sottili in uno stampo Teflon. </li><li> Dopo liofilizzazione overnight, ponteggi sono stati immersi in tre cambi di bidistillata (dd) H 2 O per sciogliere il saccarosio, e delicatamente rimosso dalla piastra Teflon con un fine-punta di spatola. </li><li> Dopo la percolazione particolato, tutti scaffold stati disinfettati per immersione nel 50%, 60% e 70% di etanolo per 30 minuti ciascuno, seguiti da tre risciacqui di DDH 2 O. </li><li> Tutti i ponteggi sono stati essiccati sotto una cappa a flusso laminare. </li><li> Dopo ponteggio fabbricazione, scaffolds stati rivestiti con apatite. <ol class="ualpha"><li> SBF (fluido corporeo simulato) soluzione è stata preparata con concentrazioni di ioni che erano 5 volte quello del plasma umano. Tutte le soluzioni sono sterili filtrata attraverso una membrana da 0,22 micron PES (Nalgene). Immediatamente prima del processo di rivestimento, essiccato ponteggi PLGA sono stati sottoposti a scarica a bagliore, argon-plasma etching (Harrick scientifico) per migliorare la bagnatura e l&#39;uniformità di rivestimento. </li><li> Etched ponteggi PLGA sono state poi incubate in SBF a 37 ° C all&#39;interno di una camicia d&#39;acqua incubatore per 12 ore, seguita da Mg 2 + e HCO 3 - libera SBF 2 per un&#39;altra hr 12 a 37 ° C sotto leggera agitazione. </li><li> Rivestite ponteggi PLGA stati sciacquati delicatamente con sterile DDH 2 O per lavare via l&#39;eccesso di soluzione di cloruro di sodio, essiccati in una cappa a flusso laminare e disinfettati con etanolo al 70%. </li><li> Integrità del rivestimento di apatite è stata analizzata con un microscopio elettronico a scansione (SEM). Apatite rivestito scaffolds sono stati montati su stubs SEM (Ted Pella) e ricoperti di carbonio per migliorare la conducibilità. La modalità di elettroni secondari è stata applicata durante la SEM (FEI / Phillips XL-30) osservazione. Dispersione di energia dei raggi X dello spettro è stato ottenuto per confermare la composizione elementare delle strutture apatite. </li></ol></li></ol> 3. Semina cellulare <ol><li> Giunti sub-confluenza livelli, lavare le cellule con PBS (x2) e Tripsinizzare. </li><li> Contare le cellule di quantificare per la semina </li><li> Seme sul pre-tagliati scaffold mm di diametro 4,0 con 1,5 x 10 5 cellule. <ol class="ualpha"><li> Luogo individuale impalcatura in un pozzetto di una piastra a 96 pozzetti </li><li> Risospendere le cellule in terreno di crescita regolari con una concentrazione di circa 1,5 x 10 5 cellule per 20 l di supporti </li><li> Dispensare 20 pl direttamente sul patibolo e il luogo in incubatore per colture cellulare per 30 minuti. </li><li> Dopo 30 minuti, aggiungere 200 microlitri di media e permettere alle cellule di incubaresul patibolo notte prima dell&#39;intervento </li><li> Non è necessario per assicurare l&#39;adesione delle cellule sull&#39;impalcatura come seminare un numero sufficiente di cellule garantirà che la maggioranza si attaccherà sul ponteggio. Questo è stato validato nel nostro laboratorio in vivo con bioluminescenza e l&#39;analisi istologica. </li></ol></li></ol> 4. Creazione di difetti cranica e impianto in vivo <ol><li> Anesthetize adulti (60 giorni di età) CD-1 topi nudi con cocktail anestetico. (Ketamina / xilazina / acepromazina) al 50% di concentrazione (diluizione 1:1 con 0,9% NaCl) o per raccomandata regime anestetico per i protocolli delle istituzioni. Questi topi sono atimico in modo che i hASCs non provocare una reazione immunitaria. <ol class="lalpha"><li> Dosaggio: ketamina cloridrato (80 mg / kg), xilazina (2,5 mg / kg) Acepromazine (2,5 mg / kg) </li><li> Durata di entrata in vigore: circa 30 min </li></ol></li><li> Chirurgicamente drappo l&#39;animale e sterilizzare l&#39;chirurgicasito con Betadine e alcool (x3) e coprire gli occhi di topo con unguento prima di fare una incisione mediana sagittale nel cuoio capelluto dell&#39;animale per esporre l&#39;osso parietale destra. Rimuovere il pericranium dal parietale destro con raschiatura smussato (Figura 1B). </li><li> Creare un unilaterale da 4 mm a tutto spessore difetto il diritto non sutura parietale associata utilizzando una sterile diamantata punta trapano. Estrema cautela deve essere presa per non disturbare la sottostante dura mater (Figura 1B), come lo spessore del mouse cranica è &lt;0,3 millimetri. </li><li> Prima dell&#39;impianto, risciacquare scaffold con PBS sterile per impedire il trasferimento di medie fattori di crescita derivati. </li><li> Posizionare il patibolo nel difetto. </li><li> Infine, suturare la pelle chiusa e monitorare l&#39;animale per stabiliti protocolli post-operatorie. </li><li> Utilizzare analgesia stabilito come indicato dai protocolli animali vostra cura, se necessario dopo l&#39;intervento-per esempio. buprenorfina 0,1 mg / kg. </li></ol> 5. Quantificazione di Formazione osteoide <ol><li> Dopo microCT stata eseguita sul mouse, l&#39;immagine tridimensionale è stata ricostruita utilizzando software MicroView come descritto in precedenza 1. </li><li> Utilizzo di Adobe Photoshop, le immagini erano dimensionati per una altezza standard. </li><li> Utilizzando la funzione di bacchetta magica, i pixel sono stati misurati del difetto cranica. </li><li> Guarigione percentuale del difetto è stata determinata mediante successive scansioni CT misurare la superficie cranica difetto e determinare il numero di pixel e dividendolo per il numero di pixel del difetto originale </li><li> Di conseguenza, se il microCT non è disponibile, in alternativa si utilizza Adobe Photoshop e raccogliendo il calvaria a tempi determinati per istologia. </li><li> Utilizzando le macchie istologiche come anilina blu e Pentachrome che denotano la formazione osteoide, più sezioni lungo l&#39;arco del difetto deve essere macchiato </li><li> Utilizzo di Adobe Photoshop, ritagliare tutte le aree che non sononel difetto cranica </li><li> Utilizzare la funzione di bacchetta magica e determinare il numero di pixel di formazione ossea de novo nella zona del difetto e confrontare ai controlli o altre variabili. </li></ol> 6. Risultati rappresentativi Il tessuto adiposo è in grado di servire un ruolo fondamentale nella generazione di cellule progenitrici per l&#39;applicazione clinica. Tessuto adiposo ha un vantaggio unico in quanto vi è una fornitura prontamente disponibile che può essere raccolta in una procedura relativamente semplice che coinvolge minima morbilità e mortalità. Una volta che il tessuto è raccolto e raccolto, il nostro protocollo è descritto nella Figura 2. Adipose cellule stromali sono isolate attraverso una serie di fasi di lavaggio, digestione con collagenasi e centrifugazione. Una volta che le cellule vengono piastrate in coltura, possono essere espansi, inseriti in vari protocolli di differenziazione in vitro, oppure direttamente in vivo. Il cREAZIONE DI 4 mm critico-dimensione dei difetti cranica fornisce una facilmente accessibile e riproducibile nel modello in vivo per testare la capacità osteogenico differenziazione dei nostri derivate da tessuto adiposo cellule stromali. Attraverso l&#39;uso di microCT, siamo in grado di seguire la formazione di tessuto scheletrico in vivo e monitorare l&#39;avanzamento dei nostri interventi (Figura 3). Il critico-dimensione dei difetti cranica non guarirà entro la durata di vita degli animali e si vede circa il 90% del difetto rimane brevetto circa 8 settimane. Lo stesso ponteggio (vedi la discussione) ha osteogeniche proprietà induttivi e ha dimostrato la capacità di indurre un po &#39;di rigenerazione ossea. I risultati tipici di posizionamento scaffold senza cellule mostra che circa un terzo del difetto avrà de novo formazione ossea a 8 settimane. Con l&#39;aumento della adipose cellule stromali, completamente due terzi o più del difetto mostrerà la rigenerazione ossea a circa 8 settimane anche se vi è Variability tra ogni animale e chirurgia. La formazione di tessuto scheletrico può essere quantificata mediante istologia e risultati tipici mostrano una eccessiva formazione di osteoide in campioni con ASC in blu di anilina e la colorazione Pentachrome (Figura 3C). Inoltre, abbiamo dimostrato che le cellule impiantate umani contribuiscono alla sottostante de novo formazione ossea attraverso l&#39;uso di hASCs GFP-marcati che mostrano colorazione in vivo dopo 2 settimane vicino alla zona di formazione ossea de novo nel difetto cranica (Figura 3D) . Inoltre, utilizziamo immunoistochimica con anticorpo umano specifico per dimostrare la sopravvivenza delle cellule umane e contributo nella zona del difetto (Figura 3E). <img alt="Figura 1" src="/files/ftp_upload/4221/4221fig1.jpg" /> Figura 1 A -. La lipoaspirate ha due strati. Lo strato superiore contiene gli adipociti e la maggior parte del prprocessa materiale cellulare, mentre lo strato inferiore contiene la soluzione salina utilizzata durante la procedura di lipoaspirazione. B - la creazione del difetto cranica attraverso una incisione sulla linea mediana pericranium per isolare l&#39;osso parietale destro, successiva creazione di un 4 mm critica difetto imprese cranica senza interruzioni della dura sottostante dura. <img alt="Figura 2" src="/files/ftp_upload/4221/4221fig2.jpg" /> Figura 2. Panoramica della raccolta e applicazione lipoaspirate dall&#39;isolamento del adipose cellule stromali alla loro espansione, la differenziazione e l&#39;uso in vitro e in vivo. <img alt="Figura 3" src="/files/ftp_upload/4221/4221fig3.jpg" /> Figura 3. A-microCT che mostra in vivo la guarigione del difetto dimensione critica cranica con l&#39;applicazione di ASC attraverso un hydroxyapatitie ponteggio (riga in basso). I controlli includono nessuna impalcatura e difetti (riga in alto) e il difetto con il posizionamento del ponteggio senza celle (riga centrale) B - Quantificazione di guarigione ossea dal microCT mostra guarigione significativamente aumentata nel gruppo ASC. C - Istologia mostrando una eccessiva formazione di osteoide del gruppo ASC (riga in basso) con blu di anilina e colorazione Pentachrome. Per anilina blu, le macchie osteoidi blu scuro e per Pentachrome, le macchie osteoidi giallo. D - hASCS marcate con GFP sono state seminate su un ponteggio e posta in un difetto cranica e sacrificati dopo 2 settimane. La colorazione è stata eseguita con un GFP anticorpo marcato per mostrare le cellule umane che contribuiscono alla rigenerazione nella zona del difetto. E -. Umano immunoistochimica dell&#39;antigene nucleare che mostra la prevalenza di cellule umane nella zona del difetto a 2 settimane <a href="http://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

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Nessun conflitto di interessi dichiarati.

Poiché l&#39;isolamento di adiposo cellule stromali 2 da lipoaspirate, queste cellule sono state differenziate in una varietà di linee cellulari. Il tessuto adiposo è di origine mesodermica e, quindi, cellule stromali multipotenti derivate da tessuto adiposo sarà probabilmente più efficace con l&#39;applicazione nei confronti di un lignaggio mesodermica. La capacità di generare tessuto scheletrico è particolarmente importante data la carenza di siti donatori per un innesto di osso autologo e le limitaz...

<table border="1"><tbody><tr><td> Nome del reagente: </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td></td><td></td><td></td><td> Lipoaspirate Harvest </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td></td></tr><tr><td> Sale di Hank soluzione equilibrata </td><td> Cellgro </td><td> 21-023-CV </td><td></td></tr><tr><td> Collagenasi </td><td> Sigma </td><td> C6885-500mg </td><td></td></tr><tr><td> Cella Filtro 100 micron </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> Steri-top 500 ml 0,22 micron filtro </td><td> Millipore </td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> Difetto cranica </td></tr><tr><td> Z500 Brushless MicromotorsUM50C </td><td> NSK </td><td> NSKZ500 </td><td></td></tr><tr><td> Circolare Knife 4,0 millimetri </td><td> Xemax chirurgico </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

Craniofacciale scheletrica riparazione e rigenerazione offre la promessa di formazione de novo tessuto attraverso una cella approccio che utilizza cellule staminali. Derivate da tessuto adiposo cellule stromali (CSA) hanno dimostrato di essere una fonte abbondante di cellule staminali multipotenti in grado di subire osteogenico, differenziazione condrogenico, adipogenico e miogenico. Molti studi hanno esplorato il potenziale osteogenico di queste cellule in vivo con l&#39;uso di biomateriali impalcature varie consegna cellulare. È stato dimostrato che utilizzando un osteoconduttiva, rivestimento in idrossiapatite poli (lattico-co-glicolico) (HA-PLGA) scaffold seminati con ASC, un difetto critico dimensioni cranica, un difetto che è definita dalla sua incapacità di subire spontanea guarigione sulla durata di vita dell&#39;animale, può essere efficacemente mostrare robusta rigenerazione ossea. Questo modello in vivo dimostra la base di approcci translazionale volta la rigenerazione del tessuto osseo - il cellularecomponente e matrice biologica. Questo metodo serve da modello per l&#39;applicazione clinico finale di una cellula progenitrice verso la riparazione di un difetto specifico tessuto.

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

Questo protocollo descrive l&#39;isolamento di adiposo cellule stromali di lipoaspirate e la creazione di un millimetro 4 critico imprese difetto cranica per valutare rigenerazione scheletrico.

Riparazione di un critico di dimensioni modello Difetto cranica Usando derivate da tessuto adiposo cellule stromali raccolte da Lipoaspirate

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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15.8K Views.  Stanford University. Questo protocollo descrive l'isolamento di adiposo cellule stromali di lipoaspirate e la creazione di un millimetro 4 critico imprese difetto cranica per valutare rigenerazione scheletrico.

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.

Generazione sperimentale di carcinoma-Associated fibroblasti (CAF) da fibroblasti umani mammari

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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Medicina

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.

Un modello contusivo unilaterale di lesioni del midollo spinale cervicale Uso della Impactor Infinite Horizon

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.

Creare fratture rigidamente stabilizzati per la Valutazione ossificazione intramembranosa, osteogenesi per distrazione, o guarigione di difetti critici Sized

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

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.

Un Mouse fetale Modello Skin of Wound Repair scarless

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.

Un semplice Critical dimensioni femorale Difetto Modello 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 ...

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.

Creazione e il trapianto di un foglio di cellule staminali di derivazione adiposa (ASC) in un modello diabetico di guarigione

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

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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Isolamento di cellule rigenerative di derivazione adiposa per il trattamento della disfunzione erettile a seguito di prostatectomia radicale

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Un modello murino BALB/c neonatale di enterocolite necrotizzante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

<meta charset="utf8"></head><body>Frazione vascolare stromale meccanica in idrogel di fibrina per la medicina rigenerativa

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

<meta charset="utf8"></head><body>Autore in primo piano: Esplorare il potenziale della frazione vascolare stromale derivata dal grasso per la guarigione delle ferite

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

La combinazione di frazione vascolare stromale isolata meccanicamente e idrogel di fibrina: un protocollo di elaborazione

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...