Yes / The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg−1 total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft. / This study was supported by a Research and Development Grant (Grant Number 09-10-02-03) from NHS Blood and Transplant and through WELMEC a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC, under Grant Number WT 088908/Z/09/Z

Engineering a complex tissue that exceeds 100-200 [mu]m requires a vascular connection. Methods to enhance vascularization include the delivery of angiogenic factors, and the use of scaffolds that encourage vascular ingrowth. However, these techniques rely on the host to vascularize the construct upon implantation, which is often too slow to provide nutrients to the entire construct. Hence, recent research has focused on creating de novo vascular networks prior to implantation. Such technologies would enable faster anastomosis with the host vascular system, as well as fully perfused constructs that can increase cell viability. Many techniques have been investigated to create de novo vascular networks with varying levels of success. Our approach was to utilize native vascular extracellular matrix (ECM) obtained from decellularizing highly vascularized tissue as a substrate for re-endothelialization and thus to create a three-dimensional vascular bed for ultimate use with various implant and tissue engineering applications. We have demonstrated a method of chemical decellularization that effectively removes cellular material while leaving behind an organized patent vascular network down to the capillary scale. Standard histological methods, DNA quantification, as well as vascular corrosion casting demonstrated this efficacy. Subsequent subcutaneous implantation then explantation of the scaffold at 7 and 28 days was used to assess the immunogenicity of the graft by analyzing the presence of immune cells. This scaffold was then re-endothelialized with human dermal microvascular endothelial cells (HDMECs) and conditioned with peristaltic flow for 60 hours to help improve vascular patency. Cellular distribution was determined qualitatively by first incubating the HDMECs with gold nanotracers, then imaging their presence upon implantation through ultrasound-guided photoacoustic (US/PA) imaging. Following the culture process, the scaffolds were analyzed for vascular patency through vascular corrosion casting, and cellular phenotype through histological methods---demonstrating a decrease in vascular damage. The re-endothelialized scaffolds were then assessed for functional vascular performance by perfusing whole blood through them. Results demonstrated better blood clearance in re-endothelialized scaffolds compared to scaffolds without cells. These results point to the ability of the optimized acellular (OA) scaffold to be used in future experiments focused on vascular and tissue engineering. / text

Made available in DSpace on 2014-06-11T19:28:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-02-26Bitstream added on 2014-06-13T20:57:40Z : No. of bitstreams: 1 oliveira_ca_me_arafo.pdf: 227077 bytes, checksum: bd756a932166596c8abaefd6455879f6 (MD5) / O propósito deste estudo foi analisar histologicamente a utilização da matriz dérmica acelular (AlloDermâ) associada ou não à proteína derivada da matriz do esmalte (Emdogainâ) em recessões gengivais criadas cirurgicamente em cães. Foram criados defeitos periodontais na superfície vestibular dos caninos superiores de seis cães. Estes foram submetidos à indução do acúmulo de placa por oito semanas, e após este período foram divididos e tratados em dois grupos: grupo I: matriz dérmica acelular; grupo II: matriz dérmica acelular associada à proteína derivada da matriz do esmalte, ambos com reposição coronária do retalho. Após três meses os animais foram sacrificados, os dentes retirados em bloco e processados histologicamente. Realizou-se análise histológica descritiva e histométrica, medindo-se a extensão do tecido epitelial, neoformação óssea e cementária, nível gengival, inserção conjuntiva e tamanho do defeito para os grupos I e II. Os resultados demonstraram valores estatisticamente iguais para as médias dos valores de cemento: 0.06 mm e 0.32 mm; osso: -0,75 mm e -0,86 mm; nível gengival: -2,15 mm e -3,11 mm; tamanho do defeito: 4,90 mm e 5,51 mm, respectivamente para os grupos I e II. Foi encontrada diferença estatisticamente significante em relação tecido epitelial, com médias de 2,88 mm e 2,15 mm, respectivamente para os grupos I e II. Não foi encontrada inserção conjuntiva. Com isso, pudemos concluir que o Emdogainâ não trouxe efeitos adicionais quando associado ao AlloDermâ. / The purpose of the present study was to histologically evaluate the healing of acellular dermal matrix (AlloDermâ), associated or not, a enamel matrix proteins (Emdogainâ) in gingival recessions defects created in dogs. Recessions defects were surgically created (5X7 mm) on the buccal aspect of the uppercuspids in contralateral jaw quadrants, in 6 mongrel dogs. The defects were exposed to plaque accumulation for 2 months. After they were divided into 2 groups, according to the treatment applied: Group I- acellular dermal matrix and coronally positioned flap; Group II: acellular dermal matrix, enamel matrix proteins and coronally positioned flap. After 3 months, the animals were sacrificed, and the blocks obtained were processed. Histologic and histometric analysis were performed to examine: ephithelium formation, cementum and bone regeneration, connective tissue adaptation, attachment level and defect measure. Results did not show a statistically significant difference in the cementum and bone regeneration, connective tissue adaptation, attachment level and defect measure parameters. The mean values were: 0,06 mm and 0,32 mm for cementum regeneration; -0,75 mm and -0,86 mm for bone regeneration; -2,15mm and -3,11 mm for attachment level; and 4,90 mm and 5,51 mm for defect measure, in the I and II groups, respectively. The ephithelium formation parameter was 2,88 mm and 2,15 mm, in the I and II groups, respectively, with a statistically significant difference. Within limits of this study, it can be concluded that Emdogainâ not brought additional effects when associated with AlloDermâ.

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Joni Augusto Cirelli / Banca: Elton Gonçalves Zenóbio / Resumo: O propósito deste estudo foi analisar histologicamente a utilização da matriz dérmica acelular (AlloDermâ) associada ou não à proteína derivada da matriz do esmalte (Emdogainâ) em recessões gengivais criadas cirurgicamente em cães. Foram criados defeitos periodontais na superfície vestibular dos caninos superiores de seis cães. Estes foram submetidos à indução do acúmulo de placa por oito semanas, e após este período foram divididos e tratados em dois grupos: grupo I: matriz dérmica acelular; grupo II: matriz dérmica acelular associada à proteína derivada da matriz do esmalte, ambos com reposição coronária do retalho. Após três meses os animais foram sacrificados, os dentes retirados em bloco e processados histologicamente. Realizou-se análise histológica descritiva e histométrica, medindo-se a extensão do tecido epitelial, neoformação óssea e cementária, nível gengival, inserção conjuntiva e tamanho do defeito para os grupos I e II. Os resultados demonstraram valores estatisticamente iguais para as médias dos valores de cemento: 0.06 mm e 0.32 mm; osso: -0,75 mm e -0,86 mm; nível gengival: -2,15 mm e -3,11 mm; tamanho do defeito: 4,90 mm e 5,51 mm, respectivamente para os grupos I e II. Foi encontrada diferença estatisticamente significante em relação tecido epitelial, com médias de 2,88 mm e 2,15 mm, respectivamente para os grupos I e II. Não foi encontrada inserção conjuntiva. Com isso, pudemos concluir que o Emdogainâ não trouxe efeitos adicionais quando associado ao AlloDermâ. / Abstract: The purpose of the present study was to histologically evaluate the healing of acellular dermal matrix (AlloDermâ), associated or not, a enamel matrix proteins (Emdogainâ) in gingival recessions defects created in dogs. Recessions defects were surgically created (5X7 mm) on the buccal aspect of the uppercuspids in contralateral jaw quadrants, in 6 mongrel dogs. The defects were exposed to plaque accumulation for 2 months. After they were divided into 2 groups, according to the treatment applied: Group I- acellular dermal matrix and coronally positioned flap; Group II: acellular dermal matrix, enamel matrix proteins and coronally positioned flap. After 3 months, the animals were sacrificed, and the blocks obtained were processed. Histologic and histometric analysis were performed to examine: ephithelium formation, cementum and bone regeneration, connective tissue adaptation, attachment level and defect measure. Results did not show a statistically significant difference in the cementum and bone regeneration, connective tissue adaptation, attachment level and defect measure parameters. The mean values were: 0,06 mm and 0,32 mm for cementum regeneration; -0,75 mm and -0,86 mm for bone regeneration; -2,15mm and -3,11 mm for attachment level; and 4,90 mm and 5,51 mm for defect measure, in the I and II groups, respectively. The ephithelium formation parameter was 2,88 mm and 2,15 mm, in the I and II groups, respectively, with a statistically significant difference. Within limits of this study, it can be concluded that Emdogainâ not brought additional effects when associated with AlloDermâ. / Mestre

Made available in DSpace on 2014-06-11T19:30:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T19:00:27Z : No. of bitstreams: 1 quitzan_jg_dr_botfm.pdf: 1219663 bytes, checksum: 12cff8c4cb83129b04576882d16ebf97 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Deformidades uretrais e estenoses severas geralmente requerem técnicas de uretroplastia com tecido adicional. Dentre as diversas possibilidades, destaca-se recentemente a utilização de matrizes acelulares desenvolvidas por bioengenharia. O objetivo deste estudo foi avaliar o comportamento funcional e histológico da matriz acelular arterial heteróloga, quando implantada na uretra de coelhos. Para o preparo da matriz, a artéria aorta abdominal de suíno foi coletada e submetida a tratamento com SDS, sendo posteriormente armazenada em álcool 70º. Foram utilizados doze coelhos, machos, com peso médio de 3 kg, distribuídos por sorteio em dois grupos: (AO) – secção completa da uretra e implante de segmento tubular de matriz acelular de aorta, com 1 cm de extensão e (TT) - secção completa da uretra e sutura término-terminal. Uretrografia retrógrada foi realizada após 15 (M2) e 90 dias (M3), quando os animais foram eutanasiados. As radiografias foram analisadas quanto à presença de fístula e/ou estenose. A avaliação histopatológica por Hematoxilina-eosina, Tricrômico de Masson e Calleja caracterizou a presença de inflamação, hiperplasia epitelial, vasos sanguíneos, tecido conjuntivo, muscular e elástico nos diferentes grupos. Foram registrados 2 óbitos em cada grupo, sendo estes animais excluídos do estudo. Em M2, 36,3% de estenose foi observada nos animais do Grupo AO (p<0,05). Em M3, animais dos Grupos AO e TT apresentaram estenose, sendo que a diferença entre os grupos não foi significante (p>0,05). Maior porcetagem de fístula ocorreu no Grupo AO, em M3 (p<0,05). A avaliação macro e microscópica não nos permitiu identificar a região da matriz acelular nos animais deste grupo. Ausência de células gigantes tipo corpo estranho bem como maior presença de hiperplasia epitelial (p<0,001) e vasos sanguíneos (p<0,005) foram observadas nas lâminas... / Severe urethral deformities and stenosis usually require urethroplasty with additional tissue. Among various possibilities, use of acellular matrix is a recent advance developed by bioengineering. The aim of this study was to evaluate the histological and functional behavior of heterologous acellular aortic matrix, when implanted in the urethra of rabbits. Abdominal aorta was taken from swine, processed with SDS and stored in alcohol, in order to obtain tubular acellular matrix. Twenty-four male rabbits, weighing 3 kg, were randomly divided in two groups: (AO)- complete urethral section and replacement with a 1 cm acellular aortc matrix tubular graft and (TT)- complete urethral section and end-to-end anastomosis. A retrograde urethrogram was performed at 15 (M2) and 90 (M3) days after surgery, when the animals were euthanized. The exams were analised for presence of fistula and/or strictures. Standard histological analysis with Hematoxylin-eosin, Masson’s Trichrome and Calleja characterized the inflammation, epithelial hyperplasia, blood vessels, muscle fibers, collagen and elastic fibers content, in different groups. Two animals of each group died during the study and were excluded. In M2, 36.3% of strictures was observed in the animals of Group AO (p <0,05). In M3, strictures occurred in both groups, but this difference was not significant (p> 0,05). A greater percentage of fistula occurred in Group AO, in M3 (p <0,05). The gross and microscopic evaluations did not allow us to identify the acellular matrix region in this group. Absence of foreign body giant cells, increased presence of epithelial hyperplasia (p <0,001) and blood vessels (p <0,005) were observed. Muscle fibers were abundant in lamina propria of the animals in the Group TT (p <0,01). There was no statistical difference between the amount of elastic fibers in the two groups... (Complete abstract click electronic access below)

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22349号 / 医博第4590号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 椛島 健治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18178号 / 医博第3898号 / 新制||医||1004(附属図書館) / 31036 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮地 良樹, 教授 羽賀 博典, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

yes / Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Im-munohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrody-namic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of a-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

A matriz démica acelular, MDA, figura dentre os biomateriais que têm por objetivo restaurar defeitos mucogengivais. A correção de defeitos mucogengivais a partir de constituintes autógenos são os procedimentos mais comumente usados, no entanto, em decorrência da quantidade insuficiente de tecido doador, esses procedimentos se tornam limitados. Diante disso, o objetivo desse estudo foi avaliar, in vitro, diferentes aspectos relacionados ao cultivo prévio de fibroblastos gengivais humanos em MDA. Fibroblastos gengivais humanos foram cultivados pela técnica do explante a partir de amostras de tecido gengival queratinizado removido de três pacientes saudáveis. A MDA foi cultivada com esses fibroblastos por períodos de 14 e 21 dias para posterior análise dos eventos de: adesão celular, proliferação e viabilidade. Os resultados mostraram que em 7 dias, os fibroblastos estavam aderidos, espraiados e dispersos sobre a superfície externa da MDA, em 14 dias formavam monocamada de células de morfologia alongada e quiescentes (Ki-67 negativos) em sua maioria, sendo apenas ocasionalmente observadas no interior da MDA. Em 21 dias a monocamada exibia menor densidade celular. Os resultados sugerem que o cultivo de fibroblastos em MDA em períodos de 14 dias permite boas condições de adesão e espraiamento das células sobre a matriz, porém, a alta densidade de fibras colágenas parece ser um fator limitante à migração celular. / Acellular Dermal Matrix, ADM, is a biomaterial that has been used in periodontal procedures to treat mucogingival defects. Mucogingival defects can be corrected by autogenous grafts that are the most common procedure used in periodontology, however, because of the limited source of donor\'s tissue this procedure became limited. The aim of this investigation was to verify, in vitro, different aspects related to human gingival fibroblasts seeding on to the ADM. Human gingival fibroblasts were established from explant cultures from the connective tissue of keratinized gingiva collected from three healthy patients. ADM was seeded with gingival fibroblasts for 14 and 21 days, and then cell adherence, proliferation and viability were analyzed. Results revealed that, at day 7, fibroblasts were adherent and spreading on the ADM surface, and were unevenly distributed, forming a discontinuous single cell layer, at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. The results suggests that fibroblasts seeding on the ADM for 14 days can allow good conditions for cell adhesion and spread on the matrix, however, because of the high collagen fiber bundle density cell, migration inside the matrix was limited.

Objetivo: O presente estudo avaliou, clinica e histomorfometricamente, a matriz dérmica acelular (MDA) como barreira física na regeneração óssea guiada. Materiais e Métodos: Inicialmente foram extraídos, em seis cães, os quatro prémolares mandibulares bilateralmente. Após um período de seis semanas, foram analisadas a quantidade e qualidade de mucosa ceratinizada (Exame Clínico 1). Duas semanas depois, os animais foram submetidos a um segundo procedimento cirúrgico (Confecção do defeito e Regeneração Óssea Guiada ROG), no qual foi criado um defeito ósseo padronizado em cada lado da mandíbula. Sobre o defeito de um hemiarco, escolhido aleatoriamente, foi posicionada e fixada a membrana absorvível de copolímeros de ácido polilático e poliglicólico e carbonato trimetileno (Grupo Controle - GC) e no defeito do hemiarco oposto, a matriz dérmica acelular - MDA (Grupo Teste - GT). Imediatamente após a sutura, foram feitas tomadas radiográficas padronizadas através de raio-X digital, as quais foram novamente realizadas 8 e 16 semanas após a cirurgia de colocação das membranas. Após quatorze semanas de cicatrização, a quantidade e qualidade da mucosa ceratinizada foram novamente avaliadas (Exame Clínico 2). Dezesseis semanas depois da ROG os animais foram sacrificados e foi então realizado o preparo histológico das peças. A análise histomorfométrica, em microscopia óptica, avaliou a quantidade e qualidade de osso neoformado. A neoformação óssea também foi quantificada através das imagens radiográficas. Resultados: Em relação aos parâmetros clínicos, o GT e o GC apresentaram aumento na EMQ e redução na AMQ. Na análise radiográfica, após o período de 16 semanas, as radiografias de subtração apresentaram ganho médio de área radiopaca em relação ao defeito inicial de 83,38 % para o GT e de 83,68% para o GC. Houve um aumento progressivo da densidade radiográfica para ambos os grupos, sem diferenças estatisticamente significantes. Histologicamente, após 16 semanas o tecido ósseo apresentou características de um osso predominantemente maduro. Histomorfometricamente, a ANO correspondeu a 58,99% da ANT no GT e a 61 % da ANT no GC. A altura óssea máxima no GT foi de 7,21 ± 0,80 mm e no GC de 7,70 ± 1,21 mm. A porcentagem de osso cortical na ANO foi de 42,47 % no GT e 38,08 % no GC. Não foram observadas diferenças estatisticamente significantes entre os grupos para nenhum destes parâmetros. Conclusão: A MDA atuou como barreira mecânica na ROG com resultados clínicos, radiográficos e histomorfométricos similares aos obtidos com a membrana absorvível. / Objective: The purpose of this study was to evaluate the acellular dermal matrix (ADM) as membrane for guided bone regeneration (GBR), comparing it to a bioabsorbable membrane. Material and Methods: In six dogs, the mandibular premolars were extracted. After 8 weeks, bone defects were surgically created bilaterally and the GBR was performed. Each side was randomly assigned to the control group (CG: bioabsorbable membrane made of glycolide and lactide copolymer) or test group (TG: ADM as a membrane). Immediately following GBR, standardized digital X-Ray radiographs were taken, and were repeated at 8 and 16 weeks post-operatively. Previously to the GBR and to euthanasia, clinical measurements of the width and thickness of the keratinized tissue (WKT and TKT, respectively) were performed. The dogs were sacrificed 16 weeks following GBR, and histomorphometric analysis was made. Area measurements of new tissue and new bone, and linear measurements of bone height were performed. Results: There were no statistically significant differences between groups for any histomorphometric measurement. Clinically, both groups showed an increase in the TKT and reduction program in the WKT. Radiographically, it could be observed an image suggestive of new bone formation in both groups 8 and 16 weeks following GBR. Conclusion: ADM acted as a mechanical barrier in GBR, with clinical, radiographic and histomorphometric results similar to those obtained with the bioabsorbable membrane.