Osteoarthritis (OA) ist eine degenerative Erkrankung des hyalinen Knorpels. Knorpel ist ein avaskuläres Gewebe. Wenn dieser beschädigt wird, ist es schwierig, ihn zu reparieren. Der Gelenkknorpel ist verantwortlich für die glatte, reibungs- und schmerzlose Bewegung des Kniegelenks. Schwere Verluste oder die komplette Zerstörung des Gelenkknorpels führen zu hoher Reibung und Schmerzen bei der Bewegung des Kniegelenks, wie es oft in den späten Stadien der OA der Fall ist. Der komplette Gelenkersatz bleibt die ultimative Lösung. Jedoch gibt es viele andere Möglichkeitenden Knorpel über die Implantation von Stammzellen zu reparieren oder zu regenerieren, jedoch oft mit schwerwiegenden Folgen. Die Transplantation embryonaler Stammzellen kann beispielsweise zu Teratombildung führen. Die Nutzung von induzierten pluripotenten Stammzellen ermöglicht die Virusintegration in das Genom. Alternativ entstand das Konzept der Vorläufer- oder Reparaturzellen in situ. Beispielsweise fand man in späten Stadien der Osteoarthritis im menschlichen Knorpel chondrogene Vorläuferzellen mit migratorischen Fähigkeiten (CPCs). Bei Knorpelregeneration mit diesen Zellen sind bisher keine Risokofaktoren bekannt. Sie haben eine enorme Fähigkeit für die Knorpelreparatur ohne das schwerwiegende Risikofaktoren bisher bekannt waren. Allerdings bestehen noch Fragen zum Beispiel wie man CPCs in situ induziert, um das Gewebe auf physiologische Weise zu reparieren. Zweitens haben CPCs eine begrenzte Lebensdauer, zumindest in vitro. Darüber hinaus gibt es keine verfügbare optimierte Methode, um eine vollständige chondrogene Differenzierung von Stammzellen zu erreichen. Vor kurzem wurden primäre Zilien gefunden, die hilfreich für die Stammzelldifferenzierung sein könnten. Diese Zilien arbeiten als Dual-Sensor für mechanochemische Signale. Dieser Sensor wurde auch auf CPCs gefunden, bei Chondrozyten gewonnen aus Kiefergelenken (TMJ) von Discoidin-Domänen- Rezeptor-1- Knockout- Mäusen (DDR- 1 KO). OA ist nicht nur auf die großen Gelenke beschränkt, sondern wirkt sich auch auf die kleinen Gelenke wie das Kiefergelenk aus. Es ist gut bekannt, dass Chondrozyten im Gelenkknorpel keinen direkten Zell-zu-Zell-Kontakt besitzen vielmehr beruht die Kommunikation auf Zell-Matrix - Wechselwirkungen über Zellrezeptoren z.B Integrine oder DDRs. DDR -1- KO-Mäuse zeigen typische Symptome der Arthrose des Kiefergelenkknorpels. Die aus dem Kiefergelenknorpel der DDR - 1 KO Mäuse abgeleiteten Chondrozyten behielten ihre Arthroseeigenschaften. Einerseits wiesen sie eine hohe Expression von runx2 und Kollagen Typ I als typische osteogene Signaturen auf sowie andererseits eine geringe Expression von sox9, Kollagen Typ II und Aggrecan, welche relevant für die chondrogene Differenzierung sind. Die osteoarthritischen Charakteristika könnten zu einem normalen Chondrozyten- Typ umgekehrt werden über den Knockdown von runx2 oder der Exposition dieser Zellen in einer dreidimensionalen Umgebung und in Gegenwart von extrazellulärer Matrix (ECM) -Komponenten wie Laminin und Nidogen. Die Umkehr in Richtung des chondrogenen Phänotyps ist auch für die Pathogenese der Meniskusdegeneration von großer Bedeutung. Der Meniskus ist in den meisten Fällen der Ausgangspunkt für die Entwicklung von OA des Kniegelenks. Der Meniskus ist ein Bindegewebsknorpel, der als Stoßdämpfer wirkt. Hierbei verschlimmert eine Menikusschädigung die OA Pathogenese durch verstärkten Knorpelabbau. Der innere Teil des Meniskus ist avaskulärer Natur und hat eine sehr begrenzte Eigenreparaturfähigkeit. Es gibt jedoch andere Möglichkeiten, wie die teilweise Entfernung des Meniskus, die zu einer kurzfristigen Entlastung führt. Dies verhindert jedoch nicht die langfristigen Folgen, die letztlich zur Entwicklung von OA führen. Der innere Teil des menschlichen Meniskus birgt einzigartige Vorläuferzellen (MPC) und kann zur Meniskus-Regeneration über den TGFß -Signalweg aktiviert werden. Darüber hinaus wurden Maus-Meniskus-Progenitorzellen (MMPCs) in gesundem Meniskusgewebe untersucht. Diese Zellen wurden mittels immunohistochemischen Techniken ex vivo charakterisiert und behielten ihre Stammzelleigenschaften auch in vitro. Mit der Anwendung verschiedener Stammzellen zur Therapie der Knorpelregeneration sind viele kritische Konsequenzen assoziiert. Im Fokus standen deshalb gewebespezifische Zellen auch in situ Vorläuferzellen genannt, die bereits in erkranktem Knorpel vorhanden sind. Diese Zellen können sich in chondrogener Richtung entwickeln. Hierfür benötigen sie möglicherweise nur geringe Manipulationen, um daraufhin hyalinen Knorpel zu produzieren.

O princípio da regeneração tecidual guiada (RTG) baseia-se na utilização de membranas biocompatíveis com a finalidade de impedir a migração dos tecidos conjuntivo e epitelial para a ferida, permitindo que células do ligamento periodontal repovoem a superfície radicular e regenerem o aparato de inserção do dente. A RTG tem sido utilizada em diversas situações clínicas para facilitar o reparo de defeitos ósseos e periodontais, sendo as membranas de politetrafluoretileno expandido (e-PTFE) as mais utilizadas. O objetivo deste estudo foi avaliar a biocompatibilidade in vitro de uma nova membrana do compósito de poli(vinilideno-trifluoretileno)/titanato de bário (P(VDF-TrFE)/BT). Para isso, osteoblastos, fibroblastos e queratinócitos, células com as quais a membrana entrará em contato durante o processo de RTG, foram cultivados sobre membranas de P(VDF-TrFE)/BT e PTFE (controle). Os seguintes parâmetros foram avaliados: 1) adesão celular por contagem em hemocitômetro; 2) proliferação celular por MTT e 3) adesão e morfologia celular por fluorescência, para as linhagens osteoblástica, fibroblástica e de queratinócitos; 1) medida do conteúdo de proteína total; 2) medida da atividade de fosfatase alcalina (ALP) e 3) formação de matriz mineralizada, para as linhagens osteoblástica e fibroblástica; 1) imunolocalização de ALP por fluorescência; 2) detecção histoquímica in situ da atividade de ALP e 3) expressão dos genes Runx2, Colágeno I (COL I), Ostepontina (OPN), ALP, Sialoproteína óssea (BSP), Osteocalcina (OC), Bax, Bcl-2 e Survivina (SUR) por PCR em tempo real, para a linhagem osteoblástica; 1) expressão dos genes Periostin (PRT), Periodontal Ligament Specific 17 (PDLs17), Calcium-binding protein (S100A4), Fibromodulina (FBM), Bax, Bcl-2 e SUR por PCR em tempo real, para a linhagem fibroblástica e 1) expressão dos genes Involucrina (IVL), Queratina 1 (QRT-1), Queratina 10 (QRT-10), Queratina 14 (QRT-14), Bax, Bcl-2 e SUR por PCR em tempo real, para a linhagem de queratinócitos. Os dados quantitativos foram submetidos ao teste estatístico Mann-Whitney (nível de significância: 5%). A adesão de células osteoblásticas foi semelhante entre P(VDF-TrFE)/BT e PTFE em 30 min, 2 e 4 h (p>0,05). A proliferação de células osteoblásticas foi maior em culturas crescidas sobre o P(VDF-TrFE)/BT em 1, 7 e 10 dias (p<0,05). A epifluorescência indicou que as células osteoblásticas estavam aderidas e mais espraiadas sobre o P(VDF-TrFE)/BT em 30 min, 4 e 24 h. A imunofluorescência revelou presença de ALP apenas em culturas crescidas sobre o P(VDF-TrFE)/BT em 7 e 14 dias. O conteúdo de proteína total foi semelhante entre as duas membranas em 10 dias (p>0,05) e maior sobre o P(VDF-TrFE)/BT em 7 e 14 dias (p<0,05). Não houve diferença estatisticamente significante para atividade de ALP em 7 dias (p>0,05), entretanto em 10 e 14 dias (p<0,05) a atividade de ALP foi maior em culturas sobre o P(VDFTrFE)/ BT. A detecção histoquímica in situ revelou atividade de ALP apenas em culturas crescidas sobre o P(VDF-TrFE)/BT, tanto aos 7 quanto aos 14 dias. A formação de matriz mineralizada ocorreu apenas em culturas crescidas sobre o P(VDF-TrFE)/BT. A expressão dos genes Runx2, COL I, OPN, ALP, BSP, OC, Bax e SUR foi maior em células cultivadas sobre o P(VDF-TrFE)/BT em 7 e 14 dias (p<0,05). Não houve expressão de Bcl-2 em culturas sobre as duas membranas. A adesão de células fibroblásticas foi semelhante entre P(VDFTrFE)/ BT e PTFE em 30 min (p>0,05) e maior sobre o P(VDF-TrFE)/BT em 2 e 4 h (p<0,05). A proliferação de células fibroblásticas foi estatisticamente semelhante entre as duas membranas em 3 dias (p>0,05) e maior em culturas crescidas sobre o P(VDF-TrFE)/BT em 1 e 7 dias (p<0,05). Os ensaios de fluorescência direta revelaram que as células fibroblásticas estavam aderidas sobre as duas membranas, porém em estágios mais avançados de espraiamento sobre o P(VDF-TrFE)/BT em 30 min, 2 e 4 h. O conteúdo de proteína total foi maior sobre o P(VDF-TrFE)/BT em 10, 14 e 21 dias (p<0,05). Não se observou atividade de ALP em culturas fibroblásticas sobre as membranas de P(VDF-TrFE)/BT e PTFE aos 7 dias. Contudo, em 14 dias (p>0,05) a atividade de ALP foi semelhante entre as duas membranas e em 21 dias (p<0,05) foi estatisticamente significante em culturas crescidas sobre o P(VDF-TrFE)/BT. Aos 21 dias não se notou formação de matriz mineralizada em ambas as membranas. A expressão dos genes PRT, PDLs17, S100A4, FBM, Bax e SUR foi maior em culturas sobre o P(VDF-TrFE)/BT em 7 e 14 dias (p<0,05). A expressão de Bcl-2 foi maior em células fibroblásticas cultivadas sobre o PTFE em 7 e 14 dias (p<0,05). A adesão de queratinócitos foi semelhante entre P(VDF-TrFE)/BT e PTFE em 30 min, 2 e 4 h (p>0,05). A proliferação de queratinócitos foi estatisticamente semelhante entre as duas membranas em 4, 7, 10 e 14 dias (p>0,05) e maior sobre culturas crescidas sobre P(VDF-TrFE)/BT em 1, 17 e 21 dias (p<0,05). Por fluorescência direta notou-se que células cultivadas sobre ambas as membranas exibiam morfologia predominantemente arredondada em 30 min, 4 e 24 h. A expressão do gene IVL foi semelhante em culturas crescidas sobre as duas membranas em 7 dias (p>0,05) e maior sobre o P(VDF-TrFE)/BT em 14 dias (p<0,05). Os genes QRT-1, QRT -10 e QRT -14, Bax e SUR estavam mais expressos em culturas sobre o P(VDF-TrFE)/BT em 7 e 14 dias (p<0,05). A expressão de Bcl-2 foi maior em queratinócitos crescidos sobre o PTFE em 7 dias (p<0,05) e semelhante entre as duas membranas em 14 dias (p>0,05). Por favorecer eventos relacionados ao desenvolvimento tecidual, como a adesão, proliferação e diferenciação celular, a membrana de P(VDF-TrFE)/BT pode representar uma boa alternativa ao PTFE para procedimentos de RTG. / The principle of guided tissue regeneration (GTR) is based on the use of physical barrier to isolate periodontal defects of the gingival connective and epithelial tissues so that bone, periodontal ligament, and cementum can be regenerated from their own cells. RTG procedure has been used in many clinical situations to promote bone and periodontal regeneration. Membranes of expanded polytetrafluoroethylene (e-PTFE) are the most commonly used material for GTR. However, several membranes have been tested to be applied in GTR. The purpose of this study was to evaluate the in vitro biocompatibility of the membrane of poly(vinylidene-trifluoroethylene/barium titanate (P(VDF-TrFE)/BT) composite. For that, osteoblasts, fibroblasts and keratinocytes, cells that will interact with the membrane during RTG, were culture P(VDF-TrFE)/BT and PTFE (control) membranes for up to 21 days. The following parameters were evaluated: 1) cell adhesion by counting in hemocytometer, 2) cell proliferation and 3) cell morphology by fluorescent labeling, for osteoblastic, fibroblastic and keratinocyte lineages; 1) total protein content; 2) alkaline phosphatase (ALP) activity and 3) mineralized bone-like nodule formation, for osteblastic and fibroblastic lineages; 1) immunolocalization of ALP by fluorescent labeling; 2) histochemistry detection in situ of ALP activity and 3) gene expression of Runx2, Collagen I (COL I), Ostepontin (OPN), ALP, Bone Sialoprotein (BSP), Osteocalcin (OC), Bax, Bcl-2 and Survivin (SUR) by real-time PCR, for osteoblastic lineage; 1) gene expression of Periostin (PRT), Periodontal ligament specific (PDLs17), Calcium-binding protein (S100A4), Fibromodulin (FBM), Bax, Bcl-2 and SUR by real-time PCR, for fibroblastic lineage; 1) gene expression of Involucrin (IVL), Keratin-1 (QRT-1), Keratin-10 (QRT-10), Keratin-14 (QRT- 14), Bax, Bcl-2 and SUR days by real-time PCR, for keratinocyte lineage. Data were submitted to Mann-Whitney test (level of significance: 5%). The adhesion of osteoblastic cells was similar between P(VDF-TrFE)/BT and PTFE at 30 min, 2 and 4 h (p>0.05). Cell proliferation was higher in ostegenic cultures grown on P(VDF-TrFE)/BT at 1, 7 and 10 days (p<0.05). Epifluorescence showed that osteoblastic cells grown on P(VDF-TrFE)/BT were more adhered and spread compared to PTFE at 30 min, 4 and 24 h. Immunofluorescence revealed the presence of ALP only in osteogenic cultures grown on P(VDF-TrFE)/BT at 7 and 14 days. Total protein content was similar between the membranes at 10 days (p>0.05) and higher on the P(VDF-TrFE)/BT at 7 and 14 days (p<0.05). There was no differences in ALP activity at 7 days (p>0.05). However, ALP activity was higher in osteogenic cultures on the P(VDF-TrFE)/BT at 10 and 14 days (p<0.05). The histochemistry reveals that only ostegenic cultures grown on P(VDF-TrFE)/BT had ALP activity in situ at 7 and 14 days. Bone-like nodule formation occurred only in cultures on the P(VDF-TrFE)/BT at 21 days. Gene expression of Runx2, COL I, OPN, ALP, BSP, OC, Bax and SUR was higher in cultures grown on P(VDF-TrFE)/BT at 7 and 14 days (p<0.05). There was no expression of Bcl-2 in cultures on both membranes. The adhesion of fibroblastic cells was similar between P(VDFTrFE)/ BT and PTFE at 30 min (p>0.05) and greater in fibroblastic cultures on P(VDFTrFE)/ BT at 2 and 4 h (p<0.05). Cell proliferation was similar in fibroblastic cultures grown on both membranes at 3 days (p>0.05) and higher in cultures on P(VDF-TrFE)/BT at 1 and 7 days (p<0.05). Epifluorescence showed that fibroblastic cells grown on P(VDF-TrFE)/BT were more adhered and more spread compared to PTFE at 30 min, 4 and 24 h. Total protein content was greater on P(VDF-TrFE)/BT at 10, 14 and 21 days (p<0.05). There was no ALP activity in fibroblastic cultures grown on both membranes at 7 days. The ALP activity was similar between P(VDF-TrFE)/BT and PTFE at 14 day (p>0.05) and higher on P(VDFTrFE)/ BT at 21 days (p<0.05). Bone-like nodule formation was not observed in fibroblastic cultures on both membranes at 21 days. Gene expression of PRT, PDLs17, S100A4, FBM, Bax and SUR was higher in cultures on the P(VDF-TrFE)/BT at 7 and 14 days (p<0.05). Bcl- 2 expression was higher in fibroblastic cultures on the PTFE at 7 and 14 days (p<0.05). Keratinocyte adhesion was similar for P(VDF-TrFE)/BT and PTFE at 30 min, 2 and 4 h (p>0.05). Keratinocyte proliferation was statistically similar on both membranes at 4, 7, 10 and 14 days (p>0.05) and higher in cultures grown on P(VDF-TrFE)/BT at 1, 17 and 21 days (p<0.05). Keratinocytes displayed a round morphology and was not observed any evidence of cell spreading at 30 min, 4 and 24 h. The gene expression of IVL was similar in cultures grown on P(VDF-TrFE)/BT and PTFE at 7 days (p>0.05) and higher on the P(VDF-TrFE)/BT at 14 days (p<0.05). Genes expression of QRT-1, QRT-10, QRT-14, Bax and SUR was greater in keratinocytes grown on P(VDF-TrFE)/BT at 7 and 14 days (p<0.05). Bcl-2 expression was higher in keratinocytes cultured on PTFE at 7 days (p<0.05) and similar on both membranes at 14 days (p>0.05). Our study indicates that P(VDF-TrFE)/BT membrane promote events related to tissue development/regeneration, such as cell adhesion, proliferation and differentiation. Thus, the membrane of P(VDF-TrFE)/BT could be used as an advantageous alternative to PTFE in GTR procedures.

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Joni Augusto Cirelli / Banca: Elizabeth Pimentel Rosetti / Banca: Washington Rodrigues Camargo / Banca: Giuseppe Alexandre Romito / Resumo: O propósito deste estudo foi avaliar histológica e histometricamente o resultado da utilização das matrizes de colágeno de Pericárdio Bovino (MPB) e Dérmica Acelular (MAD) no tratamento de deiscências ósseas criadas cirurgicamente em cães. Foram criados defeitos periodontais do tipo deiscência óssea na superfície vestibular dos 2os, 3os e 4os pré-molares inferiores de seis cães. Estes dentes foram divididos em 3 grupos: grupo I: MAD; grupo II: controle e grupo III: MPB, ambos com reposição coronária do retalho. Após 70 dias 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 e nível gengival para os grupos I, II e III. Os resultados demonstraram valores estatisticamente iguais para as medianas (em mm) dos valores de epitélio: 1,49, 1,91 e 1,82; cemento: 3,18, 3,09 e 3,31 respectivamente para os grupos I, II e III. Foi encontrada diferença estatisticamente significante em relação à neoformação óssea, com medianas (em mm) de 1,23, 1,82 e 1,62, respectivamente para os grupos I, II e III. Foi observado escasso infiltrado inflamatório. Com isso, pudemos concluir que as matrizes de MPB e MAD são materiais que permitem a reparação dos tecidos periodontais. / Abstract: The goal of this investigation was to compare, histologically and histometrically, the healing process of dehiscence-type defects treated by acellular dermal matrix (AD) and bovine pericardium matrix (PB). Six mongrel dogs were used. Buccal osseous deiscences were surgically created on mesial roots of the mandibular second, third and fourth premolars. The defects were randomly assigned to one of the treatments: AD, control and PB. After 70 days of healing, the dogs were sacrificed and the blocks were processed. The histometric parameters evaluated included: gingival level, epithelial length, new cementum and new bone. No statistically significant differences were found between PB, AD and control groups for epithelial length and new cementum parameters (p<0,05). Statistically significant differences were found between PB, AD and control groups for new bone (minor for AD). The presence of a slight inflammation was observed. Withim the limits of this study, it can be concluded that PB and AD showed good biocompatibility, but did not bring a complete periodontal tissue regeneration. / Doutor

Concern over kidney disease has increased dramatically in recent years within the medical community. It is estimated that approximately one in fifteen Americans, nearly 20 million people, experience chronic kidney disease with most of those diagnosed progressing to kidney failure. The ultimate treatment available for end stage renal failure is whole kidney transplantation. However, there are very few kidneys available for patients to receive and those patients who are fortunate enough to receive an organ must remain on immunosuppressive medication for the remainder of their lives. The United States Department of Health & Human Services have reported that 18 people die every day while on the waiting list for organ donations. The treatment is fairly successful as 69% of patients who receive a kidney transplant are still alive 5 years after the transplant. Tissue engineered organs could be a promising alternative for whole organ transplantation. The overall objective is to repopulate appropriate decellularized scaffolds from pigs, which are not immunogenic, with a patient's own cells to achieve a functional organ. Therefore, there would be an inexhaustible source of organs ready for transplantation without the risk of immune rejection. The naturally obtained scaffolds devoid of immunogens are a potential matrix to create artificial kidneys. Repopulation of decellularized rat kidneys with renal progenitor cells has been reported in previous studies. This dissertation reports the scale-up of the previous technology and building of partially functional human-sized kidneys. In the first step, we investigated various cell lysing agents and developed an automated decellularization procedure for whole porcine kidney decellularization. We also developed a preservation method for native and decellularized kidneys to avoid spoilage before and after decellularization. We also developed a decontamination procedure for whole porcine kidneys. Finally, we recellularized whole porcine kidney scaffolds with renal epithelial cells and achieved partial repopulation of the renal structure.

Nesta tese, foram produzidas e estudadas membranas de gelatina e quitosana com nanopartículas de prata, visando obter sinergia em suas propriedades para aplicações em engenharia de tecidos. Os materiais foram escolhidos por suas propriedades e aplicações similares. A gelatina é um polipeptídio biocompatível e biodegradável, obtido do colágeno de animais. A quitosana, um polímero encontrado na parede celular de alguns organismos do reino fungi, pode ser obtida da desacetilação da quitina. É biocompatível, biodegradável, e pode ter efeito antimicrobiano. Nanopartículas de prata também têm efeito antimicrobiano. As membranas foram preparadas por método de casting, e as nanopartículas de prata (AgNPs) foram sintetizadas com o método de Turkevich adaptado (TK-AgNPs) e outro similar com adição de ácido tânico (AT-AgNPs). Membranas com diferentes composições foram fabricadas: de gelatina apenas, de quitosana, e blendas de gelatina com quitosana (GQ), com adição de diferentes concentrações de AT-AgNPs ou TK-AgNPs, reticuladas com glutaraldeído (Gta) ou não. O objetivo era identificar uma composição com boa capacidade antimicrobiana e baixa toxicidade para células humanas. As membranas eram visualmente homogêneas, e a incorporação de nanopartículas foi comprovada por difração de raios X e espectroscopia no infravermelho. As AT-AgNPs eram menores e mais monodispersas do que as TK-AgNPs. As membranas contendo apenas quitosana e gelatina não apresentaram atividade antimicrobiana. As que continham AgNPs foram tóxicas para a bactéria Gram- positiva Staphylococcus aureus segundo testes de halo de inibição, com toxicidade dependente da concentração de AgNPs. As mais eficazes foram as membranas quitosana/TK- AgNPs/Gta, GQ 0,05% AT-AgNPs e GQ 0,1% AT-AgNPs. Nenhuma das membranas apresentou toxicidade contra células de fibroblastos humano. Tais resultados confirmam a possibilidade de aplicação dos materiais estudados como curativos para regeneração tecidual. / In this thesis, gelatin and chitosan membranes with silver nanoparticles were produced and studied, aiming at obtaining synergy in their properties for tissue engineering applications. The materials were chosen for their similar properties and applications. Gelatin is a biocompatible and biodegradable polypeptide derived from animal collagen. Chitosan, a polymer found in the cell wall of some fungi, can be obtained from the deacetylation of chitin. It is biocompatible, biodegradable, and may have antimicrobial effect. Silver nanoparticles also have antimicrobial effect. The membranes were prepared using the casting method, and the silver nanoparticles (AgNPs) were synthesized with an adapted Turkevich method (TK- AgNPs) and a similar one with addition of tannic acid (AT-AgNPs). Membranes with different compositions were made: gelatin only, chitosan, and blends of gelatin and chitosan (GQ), with the addition of different concentrations of AT-AgNPs or TK-AgNPs, crosslinked with glutaraldehyde (Gta) or not. The objective was to identify a composition with good antimicrobial capacity and low toxicity to human cells. The membranes were visually homogeneous, and the incorporation of nanoparticles was confirmed by X-ray diffraction and infrared spectroscopy. The AT-AgNPs were smaller and more monodisperse than the TK- AgNPs. Membranes containing only chitosan and gelatin had no antimicrobial activity. Those containing AgNPs were toxic to the Gram-positive Staphylococcus aureus bacterium according to halo tests, with AgNP concentration-dependent toxicity. The most effective were the chitosan/TK-AgNPs/Gta membranes, 0.05% GQ AT-AgNPs and 0.1% GQ AT-AgNPs. None of the membranes showed toxicity against human fibroblast cells. These results confirm the possibility of application of the membranes as curatives for tissue regeneration.

No description available.

Regeneration of large bone defects presents a critical challenge to orthopaedic clinicians as the current treatment strategies are severely limited. Tissue engineering has therefore emerged as a promising alternative to bone grafting techniques. This approach features the delivery of bioactive agents such as stem cells, genes, or proteins using biomaterial delivery systems which together stimulate endogenous repair mechanisms to regenerate the tissue. Because bone is a highly mechanosensitive tissue which responds and adapts dynamically to its mechanical environment, application of mechanical stimuli may enhance endogenous tissue repair. While mechanical loading has been shown to stimulate bone fracture healing, the ability of loading to enhance large bone defect regeneration has not been evaluated. The goal of this thesis was to evaluate the ability of sustained osteogenic growth factor delivery and functional biomechanical loading to stimulate vascularized repair of large bone defects in a rat segmental defect model. First, we evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. We determined the dose-response relationship between dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bone regeneration in a hybrid alginate-based protein delivery system and compared with the current clinically-used collagen sponge. The hybrid delivery system improved bone formation and reduced the effective dose due to its sustained delivery properties in vivo. Next, we tested the hypothesis that transfer of compressive ambulatory loads during segmental defect repair enhances bone formation and subsequent limb regeneration. We found that delayed application of axial loads enhanced bone regeneration by altering bone formation, tissue differentiation and remodeling, and local strain distribution. Finally, we evaluated the hypothesis that in vivo mechanical loading can enhance neovascular growth to influence bone formation. We found that early mechanical loading disrupted neovascular growth, resulting in impaired bone healing, while delayed loading induced vascular remodeling and enhanced bone formation. Together, this thesis presents the effects of dose and delivery system on BMP-mediated bone regeneration and demonstrates for the first time the effects of in vivo mechanical loading on vascularized regeneration of large bone defects.

Thesis--University of Oklahoma. / Includes bibliographical references (leaves 51-55).

This thesis presents strategies for constructing multi-functional biomaterials based on hyaluronan (HA) derivatives for various biomedical applications, such as drug delivery, tissue regeneration, and imaging biomaterials. The aim of this study is to improve the functionalities of HA biomaterials as well as simplify the preparation procedures.  Native HA polymer contains D-glucuronic acid residue with a carboxyl group per disaccharide unit that can be easily modified by carbodiimide-mediated amidation reaction. Therefore, we have designed a series of orthogonal groups (hydrazide, carbazate, aldehyde, and thiol) that can be linked to HA under mild conditions using the carbodiimide chemistry. Multiple functionalities can be introduced to the obtained HA derivatives via chemoselective “click”-type transformations.   The modified HA derivatives were used for the preparation of either nanogel particles (NPs) or bulk hydrogels. Due to “click” character of the reactions used, structural HA transformations were performed with high fidelity on different scales including molecular (polymers), nanometer (NPs), and a visible scale (bulk hydrogels). By linking pyrene or camptothecin to hydrophilic HA backbone, amphiphilic polymers were obtained and utilized as drug delivery carriers or prodrugs, respectively. Subsequently, physically loaded drug (doxorubicin) could be released upon degradation of HA carriers, while the chemically linked camptothecin was released intact by a thiol-triggered cleavage reaction. Bisphosphonated HA (HA-BP) polymers were prepared to induce hydrogel scaffold bio-mineralization for bone regeneration application. Moreover, we could recruit strong binding capacity of bisphosphonate (BP) groups to calcium ions for the formation of physically crosslinked HA-BP gel upon simple mixing of the polymer and calcium phosphate nanoparticle components. This gel was more stable in vivo compared to hydrazone crosslinked HA gels. Furthermore, the hydrogel composed of fluorine-19 (19F) linked HA polymer was successfully observed by both 1H and 19F MR imaging.         In conclusion, the presented herein study describes new approaches for building up multi-functional biomaterials from the HA-based blocks. The utilization of carbodiimide and click chemistries along with the enzymatic degradation of HA allowed simple and efficient interconversion between HA macromolecules, nanoparticles and macroscopic hydrogels. These HA-based biomaterials show high potential for use in the fields of drug delivery, bone regeneration, and imaging techniques.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 176-207).