Gostaríamos de agradecer a John Wirth dos jardins Olbrich para fornecer graciosamente as amostras utilizadas neste projeto. Este trabalho é apoiado em parte pela nacional do coração, pulmão e Instituto de sangue (R01HL115282 a G.R.G.) Fundação Nacional de ciência (DGE1144804 J.R.G e G.R.G.) e o departamento de cirurgia da Universidade de Wisconsin e fundo Alumni (H.D.L.). Este trabalho também foi financiado em parte pela Agência de protecção ambiental (grant estrela n. º 83573701), o National Institutes of Health (R01HL093282-01A1 e UH3TR000506) e o National Science Foundation (IGERT DGE1144804).

1. decellularization do tecido de planta usando a abordagem baseada em detergente
<ol><li>
Use o fresco ou congelado F. hispida, amostras de folha. Congele não utilizadas amostras frescas em uma loja para uso futuro (até um ano) e congelador-20 ° C. Nota: Use tecido de tronco ou folha de quase qualquer planta desejada. Tempos de armazenagem prolongada podem causar danos aos tecidos.<ol>
<li>Determinar o tamanho e a forma de amostras a ser processados com base na utilização do exemplo (ou seja, amostras cortadas em tiras são adequadas para aplicações de testes mecânicas, entretanto, amostras de disco de 8 mm são úteis em aplicações de cultivo multi bem ). Corte a folha em discos de 8 mm com uma biópsia afiada, limpa soco enquanto submerso sob temperatura ambiente (20-25 ° C) água deionizada H2O. Nota: Este protocolo pode ser usado em caules e folhas de toda. No entanto, as amostras menores serão decellularize mais rápido.</li>
		<li>Incube as amostras durante 5-10 min à temperatura ambiente (20-25 ° C) água deionizada H2O em um conjunto de placa de agitação para uma configuração de baixa velocidade para lavar e/ou descongelá-los. Uso bastante água deionizada H2O para garantir que todas as amostras são completamente molhadas.</li>
	</ol>
</li>
	<li>
Preparar uma solução de 10% (p/v) Dodecil sulfato de sódio (SDS) em desionizada H2O. lugar as amostras em um recipiente adequado (um copo ou prato plástico é ideal) e adicione solução de SDS para cobrir completamente as amostras. Incube as amostras por 5 dias em temperatura ambiente (20-25 ° C) em um conjunto de placa de agitar a baixa velocidade para evitar danos às amostras. Nota: Não sobrecarregar o contêiner como isso pode retardar o processo de decellularization e levar a um tratamento desigual por SDS. Durante esta etapa, as amostras devem adquirir uma tonalidade marrom.<ol>
<li>Depois de 5 dias, substitua a solução de SDS desionizada H2O. Incubar as amostras para um adicional 10-15 min na placa de agitação para enxaguar fora qualquer solução residual de SDS.</li>
	</ol>
</li>
	<li>Prepare um tensoativo não-iônico de 1% (v/v) em solução 10% (v/v). Para fazer uma solução de 500 mL, misturar 5 mL de tensoativo não-iônico com 50 mL de água sanitária, em seguida, adicionar 445 mL de deionizada H2O. submergir amostras em solução recentemente preparada. Nota: A solução de tensoativo não-iônico/água sanitária não tem uma vida útil longa, portanto, deve ser usado dentro de 48 h de preparação.</li>
	<li>
Substitua a solução de tensoativo não-iônico/lixívia cada 24h até as amostras são completamente desmarcadas (consulte a figura 1A , para comparação visual). Então incube a amostra em água desionizada num prato de agitar por 2 min para enxaguar a excesso tensoativo não-iônico/lixívia solução. Coloque a placa de agitação para um baixo ajuste para não danificar as amostras. Nota: O tempo necessário para limpar as amostras utilizadas varia, dependendo do tipo e da espécie da planta. A descoloração completa das amostras é indicativo de decellularization completo (realizar a quantificação de DNA para garantir a limpeza completa).<ol>
<li>Lyophilize as amostras para armazená-los até um ano (flash congelamento com nitrogênio líquido é preferencial, congelar as amostras em-80 ° C também é aceitável) e armazená-los em temperatura ambiente (20-25 ° C) em baixa umidade.</li>
		<li>Reconstituir as amostras em Tris-HCl (10 mM, pH 8,5) usando o suficiente para amostras de casaco. Lave as amostras 2 - 3 vezes em media serum-free suavemente utilizando uma micropipeta antes do uso (ou seja, uso de 150-300 µ l por enxágue, em um prato bem 24 padrão). Nota: Tampão Tris-HCl e media serum-free (ou seja, DMEM, mas qualquer célula básica cultura mídia pode ser substituída) é mais eficaz na redução do impacto de viabilidade celular das amostras de SDS tratada do que aqueles tratados com desionizada H2O sozinho.</li>
	</ol>
</li>
</ol>2. preparação das amostras usando a abordagem de Decellularization livre de detergente
Nota: Os passos iniciais deste procedimento coincidem com passos 1.1-1.1.2 (veja acima).
<ol><li>Prepare uma lixívia 5% (v/v) (NaClO) e solução de bicarbonato de sódio (NaHCO3) de 3% (p/v). Aquecer a solução em uma coifa para 60-70 ° C para amostras de folha de F. hispida cortado em discos de 8 mm, agitando um prato quente. Nota: O bicarbonato de sódio pode ser substituído com carbonato de sódio (Na2CO3), ou hidróxido de sódio (NaOH). A temperatura varia muito (de temperatura de 90 ° C) e deve ser ajustada de acordo com as propriedades das amostras utilizadas.</li>
	<li>Uma vez que a solução atinja a temperatura desejada, mergulhe as amostras e reduzir a velocidade de agita para evitar danificá-los. Depois que as amostras são visivelmente limpas (consulte a figura 1B para comparação visual), retirar do banho com cuidado. Incube amostras uma vez em desionizada H2O para 1-2 min remover a solução de água sanitária em excesso. Nota: O tempo necessário para limpar as amostras pode variar amplamente. Por exemplo, corte de salsa pode ser cancelada em 10-15 min, enquanto amostras mais espessas e/ou maiores como todo folhas ou caules podem demorar horas em banhos de alta temperatura para limpar completamente.</li>
	<li>Lyophilize as amostras e armazená-los em temperatura ambiente (20-25 ° C) em baixa umidade.</li>
</ol>

<ol>
	<li>Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+regenerative+medicine:+from+first+principles+to+state+of+the+art.">Tissue engineering and regenerative medicine: from first principles to state of the art.</a> Journal of Pediatric Surgery. 45 (2), 291-294 (2010).</li><li>Kim, S., 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=Survival+and+function+of+hepatocytes+on+a+novel+three-dimensional+synthetic+biodegradable+polymer+scaffold+with+an+intrinsic+network+of+channels.">Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels.</a> Annals of Surgery. 228 (1), 8-13 (1998).</li><li>Park, A., Wu, B., Griffith, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+surface+modification+and+3D+fabrication+techniques+to+prepare+patterned+poly(L-lactide)+substrates+allowing+regionally+selective+cell+adhesion.">Integration of surface modification and 3D fabrication techniques to prepare patterned poly(L-lactide) substrates allowing regionally selective cell adhesion.</a> Journal of Biomaterial Science, Polymer Edition. 9 (2), 89-110 (1998).</li><li>Steinhoff, G., 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=Tissue+engineering+of+pulmonary+heart+valves+on+allogenic+acellular+matrix+conduits:+in+vivo+restoration+of+valve+tissue.">Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue.</a> Circulation: JAMA. 102 (Suppl 3), III-50- III -55. 102 (Suppl 3), III-50-III-55 (2000).</li><li>Stock, U., 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=Tissue-engineered+valved+conduits+in+the+pulmonary+circulation.">Tissue-engineered valved conduits in the pulmonary circulation.</a> Journal of Thoracic Cardiovascular Surgery. 119 (4 Pt 1), 732-740 (2000).</li><li>Ott, H., 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nature Medicine. 14 (2), 213-221 (2008).</li><li>Guyette, J., 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=Bioengineering+Human+Myocardium+on+Native+Extracellular+Matrix.">Bioengineering Human Myocardium on Native Extracellular Matrix.</a> Circulation Research. 118 (1), 56-72 (2016).</li><li>Huerta, S., Varshney, A., Patel, P., Mayo, H., Livingston, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Mesh+Implants+for+Abdominal+Hernia+Repair:+US+Food+and+Drug+Administration+Approval+Process+and+Systematic+Review+of+Its+Efficacy.">Biological Mesh Implants for Abdominal Hernia Repair: US Food and Drug Administration Approval Process and Systematic Review of Its Efficacy.</a> JAMA Surgery. 151 (4), 374-381 (2016).</li><li>Catalano, E., Cochis, A., Varoni, E., Rimondini, L., Azzimonti, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+skin+substitutes:+an+overview.">Tissue-engineered skin substitutes: an overview.</a> Journal of Artificial Organs. 16 (4), 397-403 (2013).</li><li>Fang, B., Wan, Y., Tang, T., Gao, C., Dai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+osteoblastic+differentiation+of+human+bone+marrow+stromal+cells+on+hydroxyapatite/bacterial+cellulose+nanocomposite+scaffolds.">Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds.</a> Tissue Engineering. 15 (5), 1091-1098 (2009).</li><li>Wan, Y., 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=Biomimetic+synthesis+of+hydroxyapatite/bacterial+cellulose+nanocomposites+for+biomedical+applications.">Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications.</a> Materials Science and Engineering. 27 (4), 855-864 (2007).</li><li>Vinatier, C., 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=An+injectable+cellulose-based+hydrogel+for+the+transfer+of+autologous+nasal+chondrocytes+in+articular+cartilage+defects.">An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects.</a> Biotechnology and Bioengineering. 102 (4), 1259-1267 (2009).</li><li>Vinatier, C., 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=A+silanized+hydroxypropyl+methylcellulose+hydrogel+for+the+three-dimensional+culture+of+chondrocytes.">A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.</a> Biomaterials. 26 (33), 6643-6651 (2005).</li><li>Vinatier, C., 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=Engineering+cartilage+with+human+nasal+chondrocytes+and+a+silanized+hydroxypropyl+methylcellulose+hydrogel.">Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel.</a> Journal of Biomedical Materials Research Part A. 80 (1), 66-74 (2007).</li><li>Helenius, G., B&#228;ckdahl, H., Bodin, A., Nannmark, U., Gatenholm, P., Risberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+biocompatibility+of+bacterial+cellulose.">In vivo biocompatibility of bacterial cellulose.</a> Journal of Biomedical Materials Research Part A. 76 (2), 431-438 (2006).</li><li>Zhong, S., 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=An+aligned+nanofibrous+collagen+scaffold+by+electrospinning+and+its+effects+on+in+vitro+fibroblast+culture.">An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture.</a> Journal of Biomedical Materials Research Part A. 79 (3), 456-463 (2006).</li><li>Thomas, D., 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=A+shape-controlled+tuneable+microgel+platform+to+modulate+angiogenic+paracrine+responses+in+stem+cells.">A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.</a> Biomaterials. 35 (31), 8757-8766 (2014).</li><li>Lai, C., Zhang, S., Wang, L., Sheng, L., Zhou, Q., Xi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+microstructure+and+in+vivo+degradation+of+modified+bacterial+cellulose+sponges.">The relationship between microstructure and in vivo degradation of modified bacterial cellulose sponges.</a> Journal of Materials Chemistry B. 3 (46), 9001-9010 (2015).</li><li>M&#228;rtsonad, M., Viljantoa, J., Hurmea, T., Laippalac, P., Saukkob, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+cellulose+sponge+degradable+or+stable+as+implantation+material?+An+in+vivo+subcutaneous+study+in+the+rat.">Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.</a> Biomaterials. 20 (21), 1989-1995 (1999).</li><li>Miyamoto, T., Takahashi, S., Ito, H., Inagaki, H., Noishiki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+biocompatibility+of+cellulose+and+its+derivatives.">Tissue biocompatibility of cellulose and its derivatives.</a> Journal of Biomedical Materials Research. 23 (1), 125-133 (1989).</li><li>Entcheva, E., Bien, H., Yin, L., Chung, C., Farrell, M., Kostov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cardiac+cell+constructs+on+cellulose-based+scaffolding.">Functional cardiac cell constructs on cellulose-based scaffolding.</a> Biomaterials. 25 (26), 5753-5762 (2004).</li><li>Modulevsky, D., Cuerrier, C., Pelling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+of+Subcutaneously+Implanted+Plant-Derived+Cellulose+Biomaterials.">Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials.</a> PLoS One. 11 (6), e0157894 (2016).</li><li>McCulloh, K., Sperry, J., Adler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+transport+in+plants+obeys+Murray's+law.">Water transport in plants obeys Murray's law.</a> Nature. 421 (6926), 939-942 (2003).</li><li>Gershlak, J., 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=Crossing+kingdoms:+Using+decellularized+plants+as+perfusable+tissue+engineering+scaffolds.">Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.</a> Biomaterials. 125, 13-22 (2017).</li><li>Fontana, G., 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=Biofunctionalized+Plants+as+Diverse+Biomaterials+for+Human+Cell+Culture.">Biofunctionalized Plants as Diverse Biomaterials for Human Cell Culture.</a> Advanced Healthcare Materials. 6 (8), (2017).</li><li>Wegst, U., Bai, H., Saiz, E., Tomsia, A., Ritchie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+structural+materials.">Bioinspired structural materials.</a> Nature Materials. 14 (1), 23-36 (2015).</li><li>Gibson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hierarchical+structure+and+mechanics+of+plant+materials.">The hierarchical structure and mechanics of plant materials.</a> Journal of the Royal Society Interface. 9 (76), 2749-2766 (2012).</li><li>Hoshiba, 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=Decellularized+Extracellular+Matrix+as+an+In+vitro+Model+to+Study+the+Comprehensive+Roles+of+the+ECM+in+Stem+Cell+Differentiation.">Decellularized Extracellular Matrix as an In vitro Model to Study the Comprehensive Roles of the ECM in Stem Cell Differentiation.</a> Stem Cells International. 2016, (2016).</li><li>Guyette, J., 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=Perfusion+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li><li>Modulevsky, D. J., 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=Apple+derived+cellulose+scaffolds+for+3D+mammalian+cell+culture.">Apple derived cellulose scaffolds for 3D mammalian cell culture.</a> PLoS ONE. 9 (5), e97835 (2014).</li><li>Seba, A., Sloane, 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+Anatomical+Preparation+of+Vegetables,+by+Albertus+Seba,+F.+R.+S.+Communicated+to+the+Royal+Society+by+Sir+Hans+Sloane,+Bart.+Pr.+R.+S.+and+Col.+Med.+Lond.+Translated+from+the+German,+by+Mr.+Zolman,+F.+R.+S.">The Anatomical Preparation of Vegetables, by Albertus Seba, F. R. S. Communicated to the Royal Society by Sir Hans Sloane, Bart. Pr. R. S. and Col. Med. Lond. Translated from the German, by Mr. Zolman, F. R. S.</a> Philosophical Transactions. 36 (407), 441-444 (1775).</li><li>Parrish, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Phantom+Boutique:+A+Popular+Treatise+on+the+Art+of+Skeletonizing+Leaves+and+Seed-Vessels+and+Adapting+Them+to+Embellish+the+Home+of+Taste.">The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste.</a> The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste. , (1865).</li><li>Coffin, S., Gaudette, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aprotinin+extends+mechanical+integrity+time+of+cell-seeded+fibrin+sutures.">Aprotinin extends mechanical integrity time of cell-seeded fibrin sutures.</a> Journal of Biomedical Materials Research Part A. 104 (9), 2271-2279 (2016).</li><li>Zangala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Genomic+DNA+from+Mouse+Tails.">Isolation of Genomic DNA from Mouse Tails.</a> Journal of Visualized Experiments: JoVE. (6), e246 (2007).</li><li>Borselli, C., Cezar, C., Shvartsman, D., Vandenburgh, H., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+multifunctional+delivery+scaffold+in+the+ability+of+cultured+myoblasts+to+promote+muscle+regeneration.">The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration.</a> Biomaterials. 32 (34), 8905-8914 (2011).</li><li>Hill, E., Boontheekul, T., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+Scaffolds+to+Enhance+Transplanted+Myoblast+Survival+and+Migration.">Designing Scaffolds to Enhance Transplanted Myoblast Survival and Migration.</a> Tissue Engineering. 12 (5), 1295-1304 (2006).</li><li>Hill, E., Boontheekul, T., Mooney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+activation+of+transplanted+cells+controls+tissue+regeneration.">Regulating activation of transplanted cells controls tissue regeneration.</a> Proceedings of the National Academy of Sciences. 103 (8), 2494-2499 (2006).</li><li>Ma, J., Holden, K., Zhu, J., Pan, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Application+of+Three-Dimensional+Collagen-Scaffolds+Seeded+with+Myoblasts+to+Repair+Skeletal+Muscle+Defects.">The Application of Three-Dimensional Collagen-Scaffolds Seeded with Myoblasts to Repair Skeletal Muscle Defects.</a> Journal of Biomedicine and Biotechnology. 2011, 1-9 (2011).</li><li>Tyree, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+hydraulics:+the+ascent+of+water.">Plant hydraulics: the ascent of water.</a> Nature. 424 (6943), 923 (2003).</li><li>Raven, P., Evert, R., Eichhorn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Plants. , (2005).</li><li>Turrell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+area+of+the+internal+exposed+surface+of+dicotyledon+leaves.">The area of the internal exposed surface of dicotyledon leaves.</a> American Journal of Botany. 23 (4), 255-264 (1936).</li></ol>

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Aqui, dois métodos de decellularize de tecidos vegetais são descritos. Os resultados apresentados aqui, juntamente com os resultados de estudos prévios25, sugerem que os protocolos apresentados são prováveis aplicável a um amplo espectro de espécies de plantas e podem ser executadas em ambos os caules e folhas. Estes procedimentos são simples e não requer equipamento especializado, então decellularization de planta pode ser realizada na maioria dos laboratórios. Vale ressaltar que, apó...

Engenharia de tecidos surgiu na década de 1980 para criar tecidos vivos substitutos e potencialmente endereço importantes órgãos e tecidos escassez1. Uma estratégia tem andaimes usados para estimular e orientar o corpo para regenerar tecidos ou órgãos. Embora avançado abordagens de fabricação, tais como impressão 3-d produziram andaimes com propriedades físicas originais, a capacidade de fabricar andaimes com uma variada gama de propriedades físicas e biológicas realizáveis permanece um desafio2 , 3. Além disso, devido à falta de uma rede vascular funcional, estas técnicas têm sido limitadas em regenerar tecidos 3-dimensional. A utilização de tecidos animais e humanos decellularized como andaimes tem auxiliado em contornar este problema,4,5,6,7. No entanto, custo elevado, lotes de variabilidade e disponibilidade limitada podem limitar a utilização generalizada de animal decellularized moldes8. Também existem preocupações sobre a transmissão de doença potencial aos pacientes e uma reação imunológica de alguns tecidos de mamíferos decellularized9.
Celulose, derivada da planta e fontes bacterianas, tem sido extensivamente usado para gerar biomateriais para uma ampla gama de aplicações na medicina regenerativa. Alguns exemplos incluem: osso10,11, cartilagem12,13,14 e15de cicatrização de feridas. Andaimes que são compostas de celulose têm um benefício adicional em que eles são duráveis e resistentes a ser discriminadas em células de mamíferos. Isto é devido ao fato de que células de mamíferos não produz as enzimas necessárias para quebrar as moléculas de celulose. Em comparação, andaimes produziram usando macromoléculas da matriz extracelular, como o colágeno, são facilmente quebrados16 em podem não ser adequados para aplicações de longo prazo. Andaimes de colágeno podem ser estabilizadas por químicos do cross-linking. No entanto, há um trade-off devido a toxicidade inerente dos cross-linkers que afetam a biocompatibilidade dos andaimes17. Por outro lado, a celulose tem o potencial de permanecem presentes no local da implantação por períodos prolongados de tempo porque é impermeável à degradação enzimática por pilhas mamíferas18,19,20. Isto pode ser alterado por meio do ajuste da taxa de degradação através de pré-tratamento de hidrólise e co entrega dos andaimes com cellulases21. A biocompatibilidade de celulose de origem vegetal decellularized andaimes na vivo também foi demonstrada em um estudo feito em ratos22.
Através de centenas de milhões de anos de evolução, as plantas refinaram sua estrutura e composição para aumentar a eficiência do transporte de fluidos e retenção. Planta vasculares vasos minimizam resistência hidráulica, ramificando em vasos menores, semelhantes da vasculatura mamíferos de acordo com Murray lei23. Depois da decellularization, complexa rede da planta de vasos e poros interconectados é mantida. Tendo em conta o vasto número de espécies de plantas distintas prontamente disponíveis, andaimes de origem vegetal têm potencial para superar as limitações de projeto atualmente afetando andaimes em tecido engenharia24,25. Por exemplo, Modulevsky et al demonstraram que angiogênese e célula de migração ocorreu quando apple decellularized tecido foi implantado por via subcutânea na parte de trás de um rato de22. Da mesma forma, Gershlak et al mostrou que as células endoteliais poderiam ser crescidas dentro da vasculatura de folhas decellularized24. Em um experimento separado, Gershlak et al também foram capazes de mostrar que cardiomyocytes poderiam ser cultivadas na superfície das folhas e foram capazes de contrato24.
Plantas também incluem a organização complexa do celular à escala macroscópica, que é difícil de alcançar, mesmo com as mais avançadas técnicas de fabricação desenvolvidas até à data. A complexa concepção hierárquica de tecidos vegetais torna-los mais forte do que a soma dos seus constituintes,26. Plantas possuem uma infinidade de propriedades mecânicas diferentes, que vão desde componentes rígidos e duros como hastes, para os muito mais flexíveis e maleáveis, tais como folhas de27. Folhas variam dependendo da espécie em termos de tamanho, forma, quebrar a força, o grau de vascularização e podem levar a diferentes graus de Hidrofilia. Em geral, essas propriedades da planta sugerem que plantas decellularized podem servir como únicos e altamente funcionais de dispositivos médicos, incluindo como andaimes de engenharia de tecidos.
Este protocolo centra-se em dois métodos de decellularize de tecidos vegetais, tais como folhas e caules, para utilização como andaimes em engenharia de tecidos. O primeiro método é uma técnica baseada em detergente que usa uma série de banhos para retirar DNA e matéria celular, que foi adaptada de uma técnica amplamente utilizada decellularize mamíferos e vegetais tecidos6,22,25 ,28,29,30. O segundo método é livre de detergente e é uma adaptação de um protocolo de "esqueletonização", geralmente usado para remover os tecidos moles de folhas31. Trabalhos anteriores mostraram que ferver as folhas em uma solução de água sanitária e bicarbonato de sódio facilitou a separação da vasculatura do tecido mole circundante31. Esta técnica pode ser citada para experiências realizadas nodia 17 e18 séculos, tais como o trabalho de Albertus Seba32 e Edward Parrish33 . Estas experiências centradas em torno de deixando matéria vegetal, como folhas e frutos, submersos em água por longos períodos de tempo (semanas ou meses) e permitindo que os tecidos mais macios à deterioração embora naturalmente. Aqui, a abordagem de "esqueletonização" é adaptada para usar condições mais leves, tais como tempos de incubação mais longos a temperaturas mais baixas, para remover resíduos celulares e para não perturbar significativamente a estrutura de tecido mole. Para os experimentos detalhados neste documento, foram utilizados três tipos de plantas: Ficus hispida, Pachira aquatica e uma espécie de Garcinia. Resultados de DNA quantificação, ensaios mecânicos e impacto sobre a atividade metabólica celular de ambos os métodos são descritos.

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma Life Science</td>
			<td style="width:136px;">75746-1KG</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">Triton X-100</td>
			<td style="width:111px;">MP Biomedicals, LLC</td>
			<td style="width:136px;">807426</td>
			<td style="width:277px;">Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Concentrated bleach (8.25% sodium hypochlorite)</td>
			<td style="width:111px;">Clorox</td>
			<td style="width:136px;">Item #: 31009</td>
			<td style="width:277px;">Standard concentrated bleach.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium bicarbonate</td>
			<td style="width:111px;">Acros Organics</td>
			<td style="width:136px;">217120010</td>
			<td style="width:277px;">Can be substituted with sodium hydroxide or sodium carbonate.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">8 mm Biopunch</td>
			<td style="width:111px;">HealthLink</td>
			<td style="width:136px;">15111-80</td>
			<td style="width:277px;">Cuts samples that fit well in 24 well plate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Belly Dancer-Shake table</td>
			<td style="width:111px;">Stovall Life Sciences</td>
			<td style="width:136px;">BDRAA115S</td>
			<td style="width:277px;">Use low speeds to not damage tissues. Can use any model/brand of shake table.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Isotemp hot/stir plate</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any style/brand of hot/stir plate.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Beaker</td>
			<td style="width:111px;">Any</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any size beaker as long as it will fit your samples and not overcrowd them.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Tris Hydrochloride</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">BP153-500</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DMEM</td>
			<td style="width:111px;">Corning</td>
			<td style="width:136px;">MT50003PC</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Quant-iT Picogreen dsDNA assay</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:136px;">P11496</td>
			<td style="width:277px;">Can use any dsDNA quantification mehtod on hand.</td>
		</tr>
	</tbody>
</table>

two methods for decellularization of plant tissues for tissue engineering applications

Os enxertos autólogos, sintéticos e derivados de animais usados atualmente como andaimes para substituição de tecido têm limitações devido à baixa disponibilidade, pobre biocompatibilidade e custo. Tecidos vegetais têm características favoráveis que as tornam excepcionalmente adequado para usar como andaimes, tais como elevada área superficial, transporte de água excelente e retenção, porosidade interconectada, preexistente redes vasculares e uma vasta gama de mecânica Propriedades. Dois métodos bem sucedidos de decellularization planta para aplicações de engenharia de tecido são descritos aqui. O primeiro método é baseado em banhos de detergente para remover matéria celular, que é semelhante aos métodos estabelecidos anteriormente usados para limpar tecidos de mamíferos. O segundo é um método livre de detergente, adaptado de um protocolo que isola a vasculatura de folha e envolve o uso de uma lixívia aquecida e o banho de sal para limpar as folhas e caules. Ambos os métodos produzem andaimes com propriedades mecânicas comparáveis e baixo impacto metabólico celular, permitindo que o usuário selecione o protocolo que melhor se adapte às sua aplicação pretendida.

Ambos os métodos renderam andaimes que foram adequados para a cultura de células e tecidos, aplicações de engenharia. A Figura 1 mostra o fluxo de trabalho geral do processo de decellularization, usando uma folha intacta para o método baseado em detergente e cortadas amostras (8 mm de diâmetro) para o método livre de detergente. Decellularization bem sucedido dos tecidos Ficus hispida , ambos os métodos a seguir rendeu amostras claras e intac...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

Aqui apresentamos, e contraste dois protocolos usados para decellularize de tecidos vegetais: uma abordagem baseada em detergente e uma abordagem livre de detergente. Ambos os métodos deixam a matriz extracelular dos tecidos da planta utilizada, que pode então ser utilizada como andaimes para aplicações de engenharia de tecidos.

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

two-methods-for-decellularization-plant-tissues-for-tissue

Research

JoVE Journal

Bioengineering

14.5K visualizações.  University of Wisconsin-Madison. Aqui apresentamos, e contraste dois protocolos usados para decellularize de tecidos vegetais: uma abordagem baseada em detergente e uma abordagem livre de detergente. Ambos os métodos deixam a matriz extracelular dos tecidos da planta utilizada, que pode então ser utilizada como andaimes para aplicações de engenharia de tecidos.

Vídeo: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengenharia

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decelularização e Recellularization de Fígados inteiros

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

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

PGS elastomérico Andaimes em Engenharia de Tecidos Arterial

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

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

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

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Recuperação cirúrgica, isolamento e<em&gt; In vitro</em&gt; Expansão de humanos anteriores Cruciate células do ligamento derivados para aplicações em engenharia de tecidos

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

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Cápsulas à base de plantas de extração de para aplicações de microencapsulação

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...