Die Autoren möchten die Start-up-Fonds und Abteilung für Chirurgie Forschungsstipendium an der Southern Illinois University, School of Medicine zu bestätigen; und das Memorial Medical Foundation Grant.

1. Chirurgische Retrieval Hinweis: Ein IRB-Zulassung erhalten, um die ACL Stumpf während ACL-Rekonstruktion Chirurgie zu sammeln. IRB-Befreiung gegeben wurde, wie die ACL-Stumpf verwendet wurden, in der Regel als Operationsmüll. 20 Patienten wurden verwendet, um die ACL-Stumpf zu sammeln. <ol><li> Verwalten allgemeine Anästhesie und präoperative Antibiotika als Per-Protokoll-Instituts für den Patienten. </li><li> Bewerben Tourniquet und pumpen 100 mg über den systolischen Blutdruck. </li><li> Situate den Patienten in der Rückenlage. Machen Sie eine horizontale anterolaterale Inzision mit Knie in 30-45 ° Beugung für ein 5-mm-Portal und legen Sie die arthroskopische Trokar und Kamera in die Tasche suprapatellaris bei 30 °. </li><li> Führen Sie eine der Routinediagnostik Arthroskopie. </li><li> Geben Sie eine Kerbe und debride die membranöse Gelenkinnenhaut über den Kreuzbandriss. </li><li> Verwenden Sie einen 4,5 mm Rasierer und Ablator die ACL Stumpf zu identifizieren. </li><li> Verwenden Sie einen Entenschnabel gerade bitter, mehrere spe sammelncimens von der verbliebenen ACL-Stumpf. </li><li> Aufrechterhaltung der Probe in normaler Kochsalzlösung in einem sterilen Behälter. </li><li> Senden Sie die Proben zur Pathologie zum Decodieren und Auslieferung an die Laboranlage. </li><li> Recover Patienten und durchzuführen, post-operative Betreuung laut Protokoll Institution. </li></ol> 2. Tissue Verdauung und HACL Isolation <ol><li> Übertragung der aus der Pathologie empfangenen ACL menschlichen Gewebes in eine Petrischale mit einer sterilen Kochsalzlösung / PBS (Phosphat-gepufferte Kochsalzlösung). </li><li> Hackfleisch mit sterilen Schere das Gewebe in 1-2 mm 3 Stücke und waschen Sie die zerkleinerte Gewebe mit Kochsalzlösung mindestens 3 Mal. </li><li> Digest des zerkleinerten Gewebes mit 0,4% Collagenase in DMEM/F-12 (50/50, 1x) Medium mit 10% fötalem Rinderserum (FBS) und 1% Penicillin / Streptomycin ergänzt für 4-6 h bei 37 ° C. </li><li> Zentrifugieren der Zellen bei 1000 × g für 5 Minuten und Resuspendieren der Zellen in dem Medium dann. </li><li> Waschen der Zellen mit dem Medium für 2-3 mal und dann seed / Kultur in T-25-Kolben für 2-3 Tage. </li></ol> 3. Cellular Expansion, Einfrieren und Auftauen <ol><li> Verwenden eines Lichtmikroskops, die Zellen für 2-3 Tage kultiviert, in einem T-25-Kolben visualisieren. </li><li> Pflegen der Zellen bei 37 ° C in DMEM/F-12 (50/50, 1x) Medium mit 10% fötalem Rinderserum (FBS) und 1% Penicillin / Streptomycin (P / S) ergänzt. Hinweis: Ändern Sie die Medien, wenn die Zellen zeigen Einhaltung und Standard-Morphologie. Verwenden Sie ein Lichtmikroskop, um die Zellen zu beobachten. </li><li> Waschen der konfluenten Zellen mit PBS am Tag 7 (~ 1,3 × 10 6 Zellen / Kolben). Hinzufügen Trypsin (0,5 g / l) und Inkubieren der Zellen bei 37 ° C für 5 min. In Medien suspendierten Zellen Trypsin zu neutralisieren. Teilen Sie die Medien-Zellgemisch zwischen drei T-25-Kolben. </li><li> Waschen der konfluenten Zellen mit PBS, fügen Trypsin (0,5 g / l) und resuspendieren sie in Gefriermedium enthält, DMSO, FBS und das Vollmedium (DMEM/F-12 Medium + 10% FBS + 1% P / S) in der Verhältnis 01.02.07. </li><li> SpeicherDie kryogene Fläschchen bei -80 ° C, wenn die Zellen innerhalb eines Monats verwendet werden und in flüssigem Stickstoff, wenn die Zellen für längere Laufzeiten gespeichert werden. </li><li> Tauen Sie die Fläschchen in einem 37 ° C Wasserbad zur Wiederverwendung. Hinweis: In der Regel 90-95% der Zellen überleben. </li></ol> 4. Herstellung von Tissue Engineered 2-D-Milch Poly-co-Glykolsäure (PLAGA) Gerüst <ol><li> Man löst 1 g PLAGA in 12 ml Dichlormethan in einem 20 ml-Szintillations-Fläschchen und verwirbeln die Lösung für 8 h bei einer konstanten Geschwindigkeit (800 UpM). Hinweis: Detaillierte Beschreibung in Gupta et al 15. </li><li> Übertragen Sie die gelöste Lösung auf eine Glaspetrischale mit Fluorierte Schutzpapier ausgekleidet. </li><li> Legen Sie die Petrischale unter einer Vakuumhaube für 30 min. Verlassen die Petrischale bei 20 ° C über Nacht und dann bei Raumtemperatur. Platzieren Sie die Petrischalen in den Lyophilisator vollständige Verdampfung des Lösungsmittels zu gewährleisten, dünne Filme des Polymers zu erhalten. </li><li> Legen Sie das Polymer fiLMS in einem Exsikkator für 24 Stunden. </li><li> Schneiden Sie die Polymerfilme in 12 mm Durchmesser Kreisscheiben und speichern sie in einem Exsikkator, bis sie benötigt. </li></ol> 5. Rasterelektronenmikroskopie (SEM) <ol><li> Waschen Sie die Zellen (50.000 Zellen / Platte) ausgesät auf der Steuer TCPS und dem PLAGA Gerüst mit PBS 7 Tage im Anschluss an Aussaat. </li><li> Verwenden von 1,5% Glutaraldehyd in 0,1 M Cacodylatpuffer, um die Zellen zu fixieren und 2,5% OsO 4 in 0,1 M Cacodylatpuffer für Postfixierung. </li><li> Mit 0,1 M Cacodylatpuffer Waschen der fixierten Zellen. Trocknen der fixierten Zellen mit seriellen Ethanol-Dehydratisierung (50%, 70%, 80%, 90% und 100%) für 15 min jeweils, und ferner trocken in Hexamethyldisilazan (HMDS) über Nacht. </li><li> Entlüften Sie die Kammer des SEM-Sputter-Beschichtungssystem mit Knopfventil und heben Sie die obere Platte. </li><li> Legen Sie die getrockneten Proben in der Kammer. Senken Sie die Deckplatte. </li><li> Schalten Sie den Drehknopf an der Pumpe zu starten Evakuieren der Kammer. </li><li> Offene Argon Leckventil. Warten Siefür das Vakuum auf 0,05 mbar fallen zu lassen. </li><li> Bestreichen Sie die getrockneten Proben mit einer dünnen Schicht (0,13 nm) Gold / Palladium. </li><li> Liefert die Argon Leckventil in seine geschlossene Position. </li><li> Schalten Sie den Steuerknopf auf aus. </li><li> Entlüften Sie die Kammer mit Knopfventil und heben Sie die obere Platte. </li><li> Entfernen Sie die Proben und speichern sie in einem Exsikkator für 24 Stunden. </li><li> Beachten Sie die Proben unter einem Rasterelektronenmikroskop. </li></ol> 6. Immunfluoreszenzfärbungen <ol><li> Waschen Sie die Zellen (50.000 Zellen / Platte) ausgesät auf der Steuer TCPS und dem PLAGA Gerüst mit PBS 7 Tage im Anschluss an Aussaat. </li><li> Befestigen Sie die Zellen mit 70% Ethanol (kalt) für 10 min. </li><li> Die fixierten Zellen bei Raumtemperatur in PBS mit 0,05% Triton X-100 für 20 min inkubieren, mit 1% Rinderserumalbumin (BSA). </li><li> Tauchen der Proben in 1% Tween bei Raumtemperatur für 20 min. </li><li> In monoklonalen Maus-Anti-β-Aktin-Antikörper (1:400) und Inkubation der Zellen Overnight bei 4 ° C. </li><li> Mit 0,05% Tween waschen der Zellen. Hinzufügen sekundärer Antikörper (Ziege-anti-Maus-F (ab &#39;) 2-Fragment von IgG mit einer Fluoreszenzsonde, 1:400 konjugiert) zu den Zellen und Inkubation für 1 h bei Raumtemperatur. </li><li> Mit dem PBS Waschen Sie die Zellen. Färben der Zellen mit Kernfärbung und montieren mit 80% Glycerin. </li><li> Beachten Sie die Zellen unter einem konfokalen Mikroskop. </li></ol>

<ol>
	<li>Johnson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterior+cruciate+ligament:+A+dilemma+in+sports+medicine.">The anterior cruciate ligament: A dilemma in sports medicine.</a> Int J Sports Med. 3, 71-79 (1982).</li><li>Owings, M. F., Kozak, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+and+inpatient+procedures+in+the+United+States,+1996.">Ambulatory and inpatient procedures in the United States, 1996.</a> Vital Health Stats. 13, 1-119 (1998).</li><li>Frank, C. B., Jackson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+reconstruction+of+the+anterior+cruciate+ligament.">The science of reconstruction of the anterior cruciate ligament.</a> J Bone Joint Surg Am. 79, 1556-1576 (1997).</li><li>Miyasaka, K. C., Daniel, D. M., Stone, M. L., Hirshman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+knee+ligament+injuries+in+the+general+population.">The incidence of knee ligament injuries in the general population.</a> Am J Knee Surg. 4, 3-8 (1991).</li><li>Taylor, D. C., Posner, M., Curl, W. W., Feagin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+tears+of+the+anterior+cruciate+ligament:+over+30+year+follow-up+of+patients+treated+with+arthrotomy+and+primary+repair.">Isolated tears of the anterior cruciate ligament: over 30 year follow-up of patients treated with arthrotomy and primary repair.</a> Am J Sports Med. 37 (1), 65-71 (2009).</li><li>Liljedahl, S. O., Lindvall, N., Wetterfors, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+diagnosis+and+treatment+of+acute+ruptures+of+the+anterior+cruciate+ligament:+a+clinical+and+arthrographic+study+of+forty-eight+cases.">Early diagnosis and treatment of acute ruptures of the anterior cruciate ligament: a clinical and arthrographic study of forty-eight cases.</a> J Bone Joint Surg Am. 47, 1503-1513 (1965).</li><li>Oensten, M., Lysholdm, J., Gillquist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suture+of+fresh+ruptures+of+the+anterior+cruciate+ligament:+A+5+year+follow+up.">Suture of fresh ruptures of the anterior cruciate ligament: A 5 year follow up.</a> Acta Orthop Scan. 55, 270 (1984).</li><li>Buckley, S. L., Barrack, R. L., Alexander, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+conservatively+treated+partial+anterior+cruciate+ligament+tears.">The natural history of conservatively treated partial anterior cruciate ligament tears.</a> Am J Sports Med. 17 (2), 221-225 (1989).</li><li>Noyes, F. R., Mooar, L. A., Moorman, C. T., McGinnis, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+tears+of+the+anterior+cruciate+ligament.+Progression+to+complete+ligament+deficiency.">Partial tears of the anterior cruciate ligament. Progression to complete ligament deficiency.</a> J Bone Joint Surg Br. 71 (5), 825-833 (1989).</li><li>Bak, K., Scavenius, M., Hansen, S., Norring, K., Jensen, K. H., Jorgensen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+partial+rupture+of+the+anterior+cruciate+ligament.+Long+term+followup+of+56+cases.">Isolated partial rupture of the anterior cruciate ligament. Long term followup of 56 cases.</a> Knee Surg Sports Traumatol Arthrosc. 5 (2), 66-71 (1997).</li><li>Fritschy, D., Panoussopoulos, A., Wallensten, R., Peter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+predict+the+outcome+of+a+partial+rupture+of+the+anterior+cruciate+ligament?+A+prospective+study+of+43+cases.">Can we predict the outcome of a partial rupture of the anterior cruciate ligament? A prospective study of 43 cases.</a> Knee Surg Sports Traumatol Arthrosc. 5 (1), 2-5 (1997).</li><li>Sommerlath, K., Odensten, M., Lysholm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+late+course+of+acute+partial+anterior+cruciate+ligament+tears.+A+nine+to+15+year+follow-up+evaluation.">The late course of acute partial anterior cruciate ligament tears. A nine to 15 year follow-up evaluation.</a> Clin Orthop. , 281-2152 (1992).</li><li>Barrack, R. L., Buckley, S. L., Bruckner, J. D., Kneisl, J. S., Alexander, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+versus+complete+acute+anterior+cruciate+ligament+tears:+the+results+of+nonoperative+treatment.">Partial versus complete acute anterior cruciate ligament tears: the results of nonoperative treatment.</a> J Bone Joint Surg Br. 72 (4), 622-624 (1990).</li><li>Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+potential+of+primary+ACL+repair.">Current status and potential of primary ACL repair.</a> Clin Sports Med. 28 (1), 51-61 (2009).</li><li>Gupta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+walled+carbon+nanotube+composites+for+bone+tissue+engineering.">Single walled carbon nanotube composites for bone tissue engineering.</a> J Orthop Res. 9, 1374-1381 (2013).</li><li>Fruensgaard, S., Johannsen, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incomplete+ruptures+of+the+anterior+cruciate+ligament.">Incomplete ruptures of the anterior cruciate ligament.</a> J Bone Joint Surg Br. 71, 526-530 (1989).</li><li>Sandberg, R., Balkfors, B., Nilsson, B., Westlin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operative+versus+non-operative+treatment+of+recent+injuries+to+the+ligaments+of+the+knee.+A+prospective+randomized+study.">Operative versus non-operative treatment of recent injuries to the ligaments of the knee. A prospective randomized study.</a> J Bone Joint Surg Am. 69, 1120-1126 (1987).</li><li>Lamar, D. S., Bartolozzi, A. R., Freedman, K. B., Nagda, S. H., Fawcett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+modification+of+partial+tears+of+the+anterior+cruciate+ligament.">Thermal modification of partial tears of the anterior cruciate ligament.</a> Arthroscopy. 21 (7), 809-814 (2005).</li><li>Indelli, P. F., Dillingham, M. F., Fanton, G. S., Schurman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monopolar+thermal+treatment+of+symptomatic+anterior+cruciate+ligament+instability.">Monopolar thermal treatment of symptomatic anterior cruciate ligament instability.</a> Clin Orthop. 407, 138-147 (2003).</li><li>Smith, D. B., Carter, T. R., Johnson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+failure+rate+for+electrothermal+shrinkage+of+the+lax+anterior+cruciate+ligament:+a+multicenter+follow-up+past+2+years.">High failure rate for electrothermal shrinkage of the lax anterior cruciate ligament: a multicenter follow-up past 2 years.</a> Arthroscopy. 24 (6), 637-641 (2008).</li><li>Buda, R., Ferruzzi, A., Vannini, F., Zambelli, L., Di Caprio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentation+technique+with+semitendinosus+and+gracilis+tendons+in+chronic+partial+lesions+of+the+ACL:+clinical+and+arthrometric+analysis.">Augmentation technique with semitendinosus and gracilis tendons in chronic partial lesions of the ACL: clinical and arthrometric analysis.</a> Knee Surg Sports Traumatol Arthrosc. 14 (11), 1101-1107 (2006).</li><li>Sekiya, J. K., Golladay, G. J., Wojtys, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autodigestion+of+a+hamstring+anterior+cruciate+ligament+autograft+following+thermal+shrinkage:+a+case+report+and+sentinel+of+concern.">Autodigestion of a hamstring anterior cruciate ligament autograft following thermal shrinkage: a case report and sentinel of concern.</a> J Bone Joint Surg Am. 82 (10), 1454-1457 (2000).</li><li>Farquharson-Roberts, M. A., Osborne, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+rupture+of+the+anterior+cruciate+ligament+of+the+knee.">Partial rupture of the anterior cruciate ligament of the knee.</a> J Bone Joint Surg Br. 65, 32-34 (1983).</li><li>Rue, J. P., Ghodadra, N., Bach, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+tunnel+placement+in+single-bundle+anterior+cruciate+ligament+reconstruction:+a+cadaveric+study+relating+transtibial+lateralized+femoral+tunnel+position+to+the+anteromedial+and+posterolateral+bundle+femoral+origins+of+the+anterior+cruciate+ligament.">Femoral tunnel placement in single-bundle anterior cruciate ligament reconstruction: a cadaveric study relating transtibial lateralized femoral tunnel position to the anteromedial and posterolateral bundle femoral origins of the anterior cruciate ligament.</a> Am J Sports Med. 36, 73-79 (2008).</li><li>Carey, J. L., Dunn, W. R., Dahm, D. L., Zege, S. L., Spindler, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+anterior+cruciate+ligament+reconstruction+with+autograft+compared+with+allograft.">A systematic review of anterior cruciate ligament reconstruction with autograft compared with allograft.</a> J Bone Joint Surg. 91, 2242-2450 (2009).</li><li>Murray, M. M., Martin, S. D., Martin, T. L., Spector, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+changes+in+the+human+anterior+cruciate+ligament+after+rupture.">Histological changes in the human anterior cruciate ligament after rupture.</a> J Bone Joint Surg Am. 82, 1387-1397 (2000).</li><li>Andrish, J., Holmes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+on+fibroblasts+in+tissue+culture.">Effects of synovial fluid on fibroblasts in tissue culture.</a> Clin Orthop Relat Res. (138), 279-283 (1979).</li><li>Murray, M. M., Spindler, K. P., Ballard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+histologic+repair+in+a+central+wound+in+the+anterior+cruciate+ligamentwith+a+collagen-platelet-rich+plasma+scaffold.">Enhanced histologic repair in a central wound in the anterior cruciate ligamentwith a collagen-platelet-rich plasma scaffold.</a> J Orthop Res. 25, 1007-1017 (2007).</li><li>Carter, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+cruciate+ligament+thermal+shrinkage.">Anterior cruciate ligament thermal shrinkage.</a> Clin Sports Med. 21, 693-700 (2002).</li><li>Perry, J. J., Higgins, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+and+posterior+cruciate+ligament+rupture+after+thermal+treatment.">Anterior and posterior cruciate ligament rupture after thermal treatment.</a> Arthroscopy. 16, 732-736 (2000).</li><li>Cheng, M. T., Yang, H. W., Chen, T. H., Lee, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+stem+cells+from+human+cruciate+ligaments.">Isolation and characterization of multipotent stem cells from human cruciate ligaments.</a> Cell Prolif. 42 (4), 448-460 (2009).</li><li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+anterior+cruciate+ligament-derived+vascular+stem+cells.">Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells.</a> Stem Cells Dev. 21 (6), 859-872 (2012).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Das primäre Ziel dieser Studie war hACL/2D Gerüst, um die erhaltenen Zellen in einem Patch zu verwenden, um primäre Reparatur von Teil ACL Tränen zu erweitern. Konservative Behandlung von Teil ACL Tränen kann eine kurze Zeit der Ruhigstellung, Verstrebungen, einer fortschreitenden Sanierungsprogramm und regelmäßige Nachuntersuchungen 13,16,17 gehören. Doch viele Studien zeigen, dass die konservative Behandlung bei Sportlern hat mit schlechten Ergebnissen und Versagen in Zusammenhang gebracht. Buckley ...

Das vordere Kreuzband (ACL) ist ein häufig verletzt intraartikuläre Bandes des Knies. Rund 200.000 (ACL) Verletzungen werden jährlich in den Vereinigten Staaten berichtet. Über 75% der Patienten mit ACL-Verletzung opt für orthopädische rekonstruktive Chirurgie 1,2,3,4. Der chirurgische Eingriff wird häufig aufgrund einer von Natur aus schlechter Heilungspotential 3 angegeben. ACL-Rekonstruktion wird typischerweise mittels eines autologen oder allogenen Sehnen erreicht. Autograft und Allograft stellen den Goldstandard für die Rekonstruktion, wie sie rühmen, hohe Erfolgsquoten und primäre Naht zu reparieren, die andere Behandlungsoption hat Ausfallraten von bis zu 94% 5,6,7 dargestellt. Teilweise Tränen der ACL repräsentieren 10% bis 28% aller ACL Tränen 8. In einer prospektiven Studie, Noyes et al. Geschätzt, dass 50% der Patienten mit teilweise Tränen, die mehr als die Hälfte der ACL ACL fortgeschritten Insuffizienz abgeschlossen, nachdem nicht-operativen Behandlung 9. Andere Studien berichten von anhaltenden Instabilität und verminderte Funktion mit weniger als 30% der Patienten in der Lage, ihre vor der Verletzung Aktivität 9,10,11,12,13 zurückzukehren. Die Behandlungsmöglichkeiten sind begrenzt und umfassen konservative Modalitäten, thermische Schrumpfung der restlichen ACL, ACL oder Wiederaufbau. Kürzlich gab, wurde erhöht Zinsen in Augmented primären Reparatur. Diese Techniken verwenden, um Biologika primäre Naht Reparaturen 14 zu verbessern. Die neuere Forschung hat versucht, ernten mesenchymalen artigen HACL Stammzellen zu Transplantat Einschränkungen zu umgehen, 31,32 aber die Gültigkeit und Wirksamkeit dieser Zellen ist noch unbekannt. Die ideale Zellquelle für das Tissue Engineering-Anwendungen scheint nicht mesechymal HACL abgeleitet Fibroblasten-Zellen sein. Die aktuelle Forschung auf die Identifizierung eines geeigneten Matrixmaterial und Zellquelle für das Gerüst entwickelt, konzentriert. Es ist das übliche Verfahren für einen Kreuzbandriss als su verworfen werdenrgical Abfälle während der Wiederherstellungschirurgie, aber diese beschädigten Band kann eine hochwertige Quelle für den Erwerb von Zellen für die Entwicklung und Verbesserung der ideale Gewebezüchtungen ACL-Ersatz. Unser Labor hat ein Protokoll für die in vitro-Expansion dieser Zellen geerntet HACL abgeleitet entwickelt. Mit einer 2D-Matrix entwickelt, mit HACL abgeleiteten Zellen infundiert haben wir einen Patch, der potenziell verstärken könnte teilweise ACL Reparatur und stärken Bänderriss entworfen.

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			<td style="width:507px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">Petri-dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">08-757-103C</td>
			<td></td>
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			<td height="23" style="height:23px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP3994</td>
			<td></td>
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			<td height="25" style="height:25px;width:216px;">Collagenase</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">17018-029</td>
			<td>Store at 40C</td>
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			<td height="25" style="height:25px;width:216px;">DMEM/F-12</td>
			<td style="width:139px;">Cellgro</td>
			<td style="width:143px;">10-092-CV</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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			<td height="25" style="height:25px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">10082</td>
			<td>Store at -800C</td>
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			<td height="25" style="height:25px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-602E</td>
			<td>Store at 40C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Tubes- 15 ml</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
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			<td height="21" style="height:21px;width:216px;">T-25 flasks</td>
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			<td height="25" style="height:25px;width:216px;">Trypsin-Versene mixture</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-161E</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP231-100</td>
			<td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td>
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			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
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			<td style="width:143px;">430489</td>
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			<td height="25" style="height:25px;width:216px;">PLAGA</td>
			<td style="width:139px;">Purac Biomaterials</td>
			<td style="width:143px;">Purasorb PLG8523</td>
			<td>Store at -800C</td>
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			<td height="21" style="height:21px;width:216px;">TCPS disks</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-545-82</td>
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			<td height="23" style="height:23px;width:216px;">Dichloromethane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC36423-0010</td>
			<td>Possible cancer hazard. Store in a dry, cool place.</td>
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			<td height="40" style="height:40px;width:216px;">Bytac paper</td>
			<td style="width:139px;">Saint gobin performance plastics</td>
			<td style="width:143px;">1420652</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintillation vial</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>03-339-21G</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A7906</td>
			<td>Store at 40C</td>
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			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP337-500</td>
			<td></td>
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			<td height="46" style="height:46px;width:216px;">mouse Anti-&#946;-actin antibody (10 Ab)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A5441</td>
			<td>Store at -200C</td>
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			<td height="46" style="height:46px;width:216px;">goat-anti-mouse antibody (20 Ab)</td>
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			<td style="width:143px;">4408</td>
			<td>Store at -200C</td>
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			<td height="25" style="height:25px;width:216px;">Hoechst dye</td>
			<td style="width:139px;">Sigma Aldrich</td>
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			<td style="width:143px;">BP229-1</td>
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			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP2547-1</td>
			<td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td>
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			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC43085-1000</td>
			<td>Flammable liquid and vapor. Causes burns by all exposure routes.</td>
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			<td style="width:143px;">25-716</td>
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			<td style="width:143px;">5804R</td>
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			<td height="21" style="height:21px;width:216px;">Light Microscope</td>
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			<td height="21" style="height:21px;width:216px;">Water Bath</td>
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			<td style="width:143px;">2845</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:139px;">Labnet&#160;</td>
			<td style="width:143px;">VX-200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Petri plates</td>
			<td style="width:139px;">fisher Scientific</td>
			<td style="width:143px;">S31473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acu-Punch</td>
			<td style="width:139px;">Acuderm Inc.</td>
			<td style="width:143px;">P1225</td>
			<td>Acu-Punch was used to cut 12mm disks</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cacodylate buffer</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">97068</td>
			<td>Flammable liquid, carcinogen and irritant.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium tetraoxide</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">201030</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
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			<td height="21" style="height:21px;width:216px;">4.5mm shaver</td>
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			<td height="21" style="height:21px;width:216px;">Interference Screws</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td>stainless steel screws</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sputter Coater</td>
			<td style="width:139px;">Polaron</td>
			<td style="width:143px;">E5400</td>
			<td></td>
		</tr>
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surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

Eine Verletzung des ACL ist ein häufig auftretendes Problem in aktive Menschen. Selbst eine teilweise Tränen dieser intraartikuläre Kreuzbandriss führen zu biomechanischen Mängel, die Funktion und Stabilität beeinträchtigen. Aktuelle Optionen für die Behandlung von partiellen ACL Tränen reichen von nicht-operative, konservative Behandlung, mehrere chirurgische Optionen, wie: thermische Änderung, Single-Bundle Reparatur, komplette Rekonstruktion und Wiederaufbau des beschädigten Teil der einheimischen Band. Nur wenige Studien, wenn überhaupt, haben jede einzelne Methode für das Management konsequent überlegen gezeigt, und in vielen Fällen Patienten weiterhin anhaltende Instabilität und andere Begleiterkrankungen zu demonstrieren. Das Ziel dieser Studie ist, um eine mögliche Zellquelle für die Verwendung in der Entwicklung eines mittels Tissue Engineering Patch, der bei der Reparatur einer teilweise abgerissen ACL implementiert werden könnten. Ein neuartiges Protokoll wurde für den Ausbau von Zellen aus patie abgeleitet entwickeltnts ACL-Rekonstruktion unterzogen. Um die Zellen zu isolieren, wurde gehackt während ACL-Rekonstruktion erhalten HACL Gewebe in einer Collagenase-Lösung verdaut. Expansion wurde unter Verwendung von DMEM/F12 Medium mit 10% fötalem Rinderserum (FBS) und 1% Penicillin / Streptomycin (P / S) ergänzt. Die Zellen wurden dann bei -80 º C oder in flüssigem Stickstoff in einem Gefriermedium, bestehend aus DMSO, FBS und dem Expansionsmedium gespeichert. Nach dem Auftauen wurden die HACL abgeleiteten Zellen dann auf ein Gerüst aus Gewebezüchtungen, PLAGA (Poly Milch-co-Glykolsäure) und Steuergewebekultur-Polystyrol (TCPS) ausgesät. Nach 7 Tagen wurde SEM durchgeführt, um die zelluläre Adhäsion zu der PLAGA TCPS gegenüber der Kontrolle zu vergleichen. Zellmorphologie wurde mit Immunfluoreszenzfärbung ausgewertet. SEM (Rasterelektronenmikroskop) Mikroaufnahmen zeigten, dass Zellen wuchsen und auf beiden PLAGA und TCPS Oberflächen haften und wurden von Tag 7 über die gesamten Oberflächen konfluent. Immunfluoreszenzfärbung zeigten normale, unbelastete morphologischenal Muster auf beiden Oberflächen. Diese Technik ist vielversprechend für Anwendungen in der ACL Regeneration und Rekonstruktion.

Das Arbeitsmodell für die chirurgische Wiederherstellung, Gewebe Verdauung und Isolierung des menschlichen vorderen Kreuzbandes (HACL) abgeleiteten Zellen ist in Abbildung 1 dargestellt. Die aus den Explantaten migriert und an die T-25-Kolben eingehalten Zellen. Diese Zellen wurden für 3 Tage und dann wurden unter einem Lichtmikroskop (2) sichtbar gemacht. Ein Monolayer wurde von Tag 7 erhalten. Die Anwesenheit von gesunden, lebensfähigen Zellen zeigten die erfolgreichen Abruf und Ku...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

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

Für zukünftige Anwendungen als Patch zu Teil Tränen des vorderen Kreuzbandes (ACL) zu reparieren, wurden menschliche ACL abgeleitet von Zellen während rekonstruktive Verfahren, in vitro erweitert und Tissue Engineering Scaffolds gewachsen erhalten Gewebe isoliert. Zelladhäsion und Morphologie wurde dann durchgeführt, um die Biokompatibilität auf Gerüstoberfläche bestätigen.

Chirurgische Retrieval, Isolierung und<em&gt; In-vitro-</em&gt; Erweiterung des menschlichen vorderen Kreuzbandes abgeleiteten Zellen für Tissue Engineering Anwendungen

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

surgical-retrieval-isolation-vitro-expansion-human-anterior-cruciate

Research

JoVE Journal

Bioengineering

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

11.4K Aufrufe.  Southern Illinois University School of Medicine.  Für zukünftige Anwendungen als Patch zu Teil Tränen des vorderen Kreuzbandes (ACL) zu reparieren, wurden menschliche ACL abgeleitet von Zellen während rekonstruktive Verfahren, in vitro erweitert und Tissue Engineering Scaffolds gewachsen erhalten Gewebe isoliert. Zelladhäsion und Morphologie wurde dann durchgeführt, um die Biokompatibilität auf Gerüstoberfläche bestätigen. 

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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Forschung

Biotechnik

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

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

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

Athymischen Rattenmodell für die Bewertung von Engineered vorderen Kreuzbandes Grafts

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

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Test auf Augenreizung (EIT) zur Kennzeichnung der Gefahr von Augen Reizt Chemicals mit rekonstruierter menschlicher Hornhaut-ähnlichen Epithelial (RhCE) Gewebemodell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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

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

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

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

Anwendungen<em&gt; In Vivo</em&gt; Funktionstest der Ratte Vorderer Schienbeingewebe Skeletal Muscle Repair Engineered für die Bewertung

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

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

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

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

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

Elektrospinnen Schreiben von dreidimensionalen Poly(ε-caprolactone) Gerüste mit steuerbaren Morphologien für Tissue Engineering Anwendungen zu schmelzen

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

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolierung von mesenchymalen Stammzellen aus menschlichen alveoläre Periost und Effekte von Vitamin D auf knochenbildenden Aktivität Periost-abgeleitete Zellen

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Zwei Methoden zur Decellularization von Pflanzengewebe für Tissue-Engineering-Anwendungen

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

Herstellung von autologem plättchenreichem Plasma zur Förderung der In-vitro-Expansion humaner Fibroblasten

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Anterior Segment Organ Culture Platform zur Verfolgung von Open Globe Verletzungen und therapeutischer Leistung

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasten-abgeleitetes Human Engineered Connective Tissue für Screening-Anwendungen

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...