Despite recent advances in surgical technique and the development of numerous therapeutic agents, the formation post surgical adhesions (PSA) continues to cause complications for many patients. In this research, we have employed a rational system to develop a novel treatment to address this clinical need. Based on an understanding of the biochemical events that lead to PSA formation, a series of targeted polymeric biomaterials was designed to interrupt the fibrin gel matrix propagation and suppress PSA formation. Using group transfer polymerization, a series of well controlled block copolymers of polyacrylic acid and poly(ethylene glycol-methacrylate) based materials was synthesized. Subsequent functionalization with the pentapeptide Cys-Arg-Glu-Lys-Ala (CREKA) was employed to target the materials to fibrin as a marker of pro-adhesive sites. While preliminary testing of the untargeted materials verified their ability to suppress non-specific protein adsorption to model surfaces, numerous in vitro tests were conducted to study the ability to inhibit fibrin gel propagation. The ability to inhibit both the rate and quantity of fibrinogen deposition to a fibrin coated surface has been demonstrated. In addition, the rate of fibrin gel propagation and the degree of cellular attachment can modulated. Taking advantage of the systematic variation in structure facilitated by the robust synthetic methodology employed, statistical analysis was used to elucidate the structureproperty relationships governing the performance of these materials. The most important factors that lead to enhanced performance in in vitro tests are the length of PEG chain and number of peptide units conjugated to the polymer: increasing PEG chain length and increasing the number of peptides conjugated to the polymer both improve performance in all tests. The synthetic methods that have been developed, in conjunction with the experimental results, will be used to direct future studies, including cytotoxicity and animal studies.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

Background: Current research has demonstrated that aspirin and fish oil (EFA) increase plasma levels of specialized pro-resolution molecules (SPMs). This study investigates their effects on SPM precursor pools in platelet rich fibrin (PRF). Methods: Twenty healthy volunteers were randomly assigned to take aspirin; EFA or aspirin and EFA. Four hours later, SPM precursor levels were quantified using combined Liquid Chromatography tandem mass spectrometry. The differences between the groups: Aspirin (yes or no), EFA (yes or no), were analyzed by ANCOVA, testing for group differences after covarying out the baseline value. Results: There were 4 significant interactions, 1 with an aspirin effect, 2 with an EFA effect, and 64 with no difference between the groups. The significant interaction effect was found for the following lipidome: LPE(20:4), LPI(16:1), LPI(18:1), and LPI(20:3). Aspirin decreased the LPG(16:4) levels, and EFA decreased the LPE(22:5) and PG(16:0/18:0) lipidomes. Conclusions: Some SPM precursor pools in PRF were increased following supplementation.

Introdução: Os benefícios do emprego do selante de fibrina no reforço de anastomoses pancreatico-jejunais, após ressecção duodenopancreática, visando a redução da incidência de fístula pancreática pós operatória (FPPO), ainda são questionáveis. Objetivo: Avaliar a influência do emprego do selante de fibrina na anastomose pancreatico-jejunal, após duodenopancreatectomia, na incidência de fístula, bem como suas consequências clínicas e os custos hospitalares. Metodologia: Estudo retrospectivo de 62 pacientes consecutivos submetidos a duodenopancreatectomia, divididos em dois grupos: 31 pacientes utilizando o selante de fibrina (GCS) e 31 pacientes sem o emprego de selante (GSS). As variáveis estudadas foram agrupadas em epidemiológicas, clínicas, laboratoriais, com destaque para a incidência de fístula pancreática, classificada segundo a definição do International Study Group on Pancreatic Fistula, suas complicações pós operatórias catalogadas segundo a classificação de Clavien e suas repercussões na assistência e nos seus custos avaliados pelo método de absorção com rateio simples de todas as despesas, exceto a despesa com medicamentos, tratada de forma separada. Resultados: Os grupos foram homogêneos para os parâmetros epidemiológicos, clínicos, e laboratoriais e não foram registradas diferenças significativas na comparação da evolução pós operatória e dos indicadores assistenciais hospitalares. Por outro lado, os custos hospitalares foram mais elevados no GCS, em relação ao GSS (p<0,0001). Conclusão: O emprego do selante de fibrina, no reforço da anastomose pancreatico-jejunal, em pacientes submetidos a duodenopancreatectomias, nas condições estudadas, não melhorou os resultados clínicos e assistenciais e ainda aumentou os custos hospitalares. / Introduction: The benefits of fibrin sealant employment in strengthening pancreatico-jejunal anastomosis after duodenopancreatic resection, reducing the incidence of pancreatic fistula postoperative (PFPO) are still questionable. Objective: To evaluate the influence of the use of fibrin sealant in pancreatico-jejunal anastomosis after pancreaticoduodenectomy in the incidence of fistula and its clinical consequences and hospital costs. Methodology: A retrospective study of 62 consecutive patients who underwent pancreaticoduodenectomy, divided into two groups: 31 patients using fibrin sealant (GCS) and 31 patients without the sealant employment (GSS). The variables were grouped into epidemiological, clinical, laboratory, especially the incidence of pancreatic fistula classified as defined by the International Study Group on Pancreatic Fistula, their postoperative complications cataloged according to Clavien rating and its repercussions on care and its costs assessed by the absorption method with simple apportionment of all expenses except the expenditure on medicines, treated separately. Results: The groups were homogeneous for clinical, epidemiological and laboratory parameters and no significant differences were recorded in the comparison given postoperative progress and hospital assistance indicators. Moreover, hospital costs were higher in GCS, with respect to GSS (p <0.0001). Conclusion: The use of fibrin sealant in pancreatojejunal anastomosis after pancreaticoduodenectomy, in the studied conditions, did not improve the results of care and also increased hospital costs

Orientador: Rui Seabra Ferreira / Resumo: Desenvolvido em 1990 por um grupo de pesquisadores do Centro de Estudo de Venenos e Animais Peçonhentos (CEVAP), no Estado de São Paulo, Brasil, o Biopolímero de Fibrina (BPF) possuía o principal objetivo de ser um adesivo à base de fibrina sem o uso de sangue humano, a fim de evitar a transmissão de doenças infecciosas por meio deste insumo. Após diversas pesquisas com o BPF, comprovou-se não somente sua capacidade adesiva, como também sua ação coagulante, sua ação como auxiliar no reparo ósseo e cartilaginoso e sua função como arcabouço para células-tronco mesenquimais (CTMs), devido ao fato de que o BPF possui uma estrutura tridimensional adequada. Em estudos recentes e ao exercer essa função, tal material não afetou o microambiente biológico das células, ou seja, permitiu a adesão, proliferação e diferenciação celular, e aderência e crescimento destas. Tais características, apresentadas pelo BPF, são desejáveis na maioria dos biopolímeros utilizáveis, o que ressalta a importância do aprofundamento das pesquisas com BPF e suas interações em experimentos in vivo. Assim, no capítulo 1 realizamos uma ampla revisão na literatura sobre biopolímeros de fibrina, células-tronco e reparação de tecido ósseo. No capítulo 2 é apresentado o artigo científico “Arcabouço de fibrina para células-tronco mesenquimais como potencial osteogênico”. / Abstract: Developed in 1990 by a group of researchers from the Center for the Study of Venomous and Poisonous Animals (CEVAP) in the State of São Paulo, Brazil, the Fibrin Biopolymer (GMP) had the main objective of being a fibrin-based adhesive without the use of human blood in order to avoid the transmission of infectious diseases by means of this input. After several investigations with BPF, it was verified not only its adhesive capacity, but also its coagulant action, its action as an aid in bone and cartilage repair and its function as a framework for mesenchymal stem cells (MSCs), due to the fact that the BPF has an adequate three-dimensional structure. In recent studies and in carrying out this function, such material did not affect the biological microenvironment of the cells, that is, it allowed cell adhesion, proliferation and differentiation, and adhesion and growth of these cells. These characteristics, presented by BPF, are desirable in most usable biopolymers, which underscores the importance of deepening GMP research and its interactions in in vivo experiments. Thus, in Chapter 1 we conducted a broad review in the literature on biopolymers of fibrin, stem cells and repair of bone tissue. In chapter 2 the scientific paper "Fibrin scaffold for mesenchymal stem cells as osteogenic potential" is presented. / Doutor

"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

A wide variety of techniques have been explored to synthesize small diameter tissue engineered blood vessels. Toward this end, we are exploring direct cell seeding and culture on tubular mandrels to create engineered vascular tissues. In the present study, v-shaped channels cast from polydimethyl siloxane (PDMS) were used as cell seeding wells. Fibrin microthreads placed in the chamber were used as model tubular seeding mandrels. Human mesenchymal stem cells (hMSCs) were seeded onto fibrin microthreads in v-shaped channels for 4 hours. Cell attachment to the microthreads was confirmed visually by Hoechst nuclear staining and a cell quantification assay showed that 5,114 ± 339 cells attached per 1 cm fibrin microthread sample (n = 6). Fibrin microthreads were completely degraded by hMSCs within 5 days of culture, therefore UV crosslinking was used to increase their mechanical strength and prolong the amount of time cells could be cultured on fibrin microthreads and generate tubular tissue constructs. Cell attachment was unaffected on UV-crosslinked microthreads compared to uncrosslinked microthreads, resulting in a count of 4,944 ± 210 cells per 1 cm of fibrin microthread sample (n = 3). Long term culture of the hMSCs on the UV-crosslinked fibrin microthreads showed an increase in cell number over time to 11,198 ± 582 cells per cm of microthread after 7 days with 92% cell viability (CYQUANT NF/DEAD staining) and evidence of cell proliferation. The results show that the v-well cell seeding technique was effective in promoting rapid hMSC attachment on UV-crosslinked fibrin microthreads and encouraged their growth, maintained viability and also promoted their proliferation over the culture period. In conclusion, the technique could serve as an efficient model system for rapid formation of tissue engineered vascular grafts.

A durotomia acidental, na cirurgia da coluna lombar, é uma das mais comuns complicações, com uma prevalência de 1 a 17%. As fraturas da coluna também podem apresentar-se com lesão dural, chegando a 19% nas fraturas tipo explosão da coluna torácica ou lombar com lesão associada da lâmina vertebral. Procurou-se avaliar as alterações medulares que ocorreram apos a reparação de lesão dural com pontos do tipo simples, com cola de fibrina e com colágeno bovino, através de análise histológica. Manteve-se um grupo controle, sem reparação. Utilizaram-se 70 ratos da raça Wistar, sendo que 34 foram excluídos por problemas anestésicos ou intra-operatórios. Mantiveram-se nove ratos por grupo. Abordaram-se os segmentos vertebrais T8 e T9, para efetuar a lesão, que foi reparada pelos três diferentes métodos avaliados. Os animais permaneceram confinados por 24 dias, sendo submetidos à eutanásia com a coleta de material para análise histológica. O Serviço de Patologia avaliou e graduou (ausente, discreto, moderado e acentuado) os casos quanto à hiperemia, degeneração medular, necrose e infiltrado celular. De forma geral, as técnicas de reparo apresentaram resultados com maior grau de alteração, principalmente em relação ao infiltrado celular, onde todas as técnicas mostraram resultados bem piores. A sutura apresentou graus mais severos de necrose, hiperemia e infiltrado celular. A membrana de colágeno apresentou resultados com graus mais elevados de alteração em relação à hiperemia, à degeneração da substância nervosa e ao infiltrado celular. A reparação com cola de fibrina apresentou piora em relação ao grau de necrose e de infiltrado celular. A presença de alterações medulares em todos os grupos, até mesmo no grupo controle, levou a cogitar algumas possíveis causas para os resultados: as alterações medulares seriam geradas pela técnica cirúrgica ocorrendo uma lesão iatrogênica medular, mas sem repercussão clinica; os materiais estudados poderiam causar alterações no tecido neural; o simples contato do tecido neural com o meio extra-dural, após a lesão, seria a causa das alterações levando à mesma cascata de alterações que ocorre nas lesões medulares traumáticas. Apesar de todos os grupos apresentarem alterações medulares, ficou evidente que as de maior intensidade ocorreram nos grupos que utilizaram materiais de fechamento dural. Os métodos de reparação dural ainda não são os ideais, mas existe o consenso que a lesão deve ser reparada sempre na forma aguda, a fim de evitar e prevenir complicações e seqüelas. / This study aimed to evaluate spinal cord alterations after dural repair with: simple interrupted suture, collagen membrane or fibrin glue using histopathogical analysis. All activities were performed in the São Paulo University Medical School General Hospital Traumatology and Orthopedic Division. Seventy Wistar rats were used, but 34 were excluded due to anesthetic or surgery problems. Animals were kept isolated for 24 days, then killed and histological samples collected. Pathology Division assessed and graduated hyperemia, spinal cord degeneration, necrosis and cellular infiltration. Repair techniques provided higher alteration, mostly in cellular infiltration. Suture showed more severe necrosis, hyperemia and cellular infiltrates graduations. Collagen membrane provided higher alterations related to hyperemia, nerve degeneration and cellular infiltrates. Fibrin glue reparation had worse results in necrosis and cellular infiltrates graduation.Spine cord alterations in all groups, including control group, made us suppose possible reasons for the results: spine cord changes would be caused by surgical technique as iatrogenic injury, without clinical meaning; studied material would cause alterations in neural tissue; contact of neural tissue with extra dural environment, after injury, leading to the same mechanisms present in spinal cord traumatic injury. Despite all groups showed spinal cord alterations it was clear a higher intensity of injury when dural closing material were used. There is no ideal choice for dural repair material and more research is warranted to find better form of repairing dura mater.

Introdução: Os benefícios do emprego do selante de fibrina no reforço de anastomoses pancreatico-jejunais, após ressecção duodenopancreática, visando a redução da incidência de fístula pancreática pós operatória (FPPO), ainda são questionáveis. Objetivo: Avaliar a influência do emprego do selante de fibrina na anastomose pancreatico-jejunal, após duodenopancreatectomia, na incidência de fístula, bem como suas consequências clínicas e os custos hospitalares. Metodologia: Estudo retrospectivo de 62 pacientes consecutivos submetidos a duodenopancreatectomia, divididos em dois grupos: 31 pacientes utilizando o selante de fibrina (GCS) e 31 pacientes sem o emprego de selante (GSS). As variáveis estudadas foram agrupadas em epidemiológicas, clínicas, laboratoriais, com destaque para a incidência de fístula pancreática, classificada segundo a definição do International Study Group on Pancreatic Fistula, suas complicações pós operatórias catalogadas segundo a classificação de Clavien e suas repercussões na assistência e nos seus custos avaliados pelo método de absorção com rateio simples de todas as despesas, exceto a despesa com medicamentos, tratada de forma separada. Resultados: Os grupos foram homogêneos para os parâmetros epidemiológicos, clínicos, e laboratoriais e não foram registradas diferenças significativas na comparação da evolução pós operatória e dos indicadores assistenciais hospitalares. Por outro lado, os custos hospitalares foram mais elevados no GCS, em relação ao GSS (p<0,0001). Conclusão: O emprego do selante de fibrina, no reforço da anastomose pancreatico-jejunal, em pacientes submetidos a duodenopancreatectomias, nas condições estudadas, não melhorou os resultados clínicos e assistenciais e ainda aumentou os custos hospitalares. / Introduction: The benefits of fibrin sealant employment in strengthening pancreatico-jejunal anastomosis after duodenopancreatic resection, reducing the incidence of pancreatic fistula postoperative (PFPO) are still questionable. Objective: To evaluate the influence of the use of fibrin sealant in pancreatico-jejunal anastomosis after pancreaticoduodenectomy in the incidence of fistula and its clinical consequences and hospital costs. Methodology: A retrospective study of 62 consecutive patients who underwent pancreaticoduodenectomy, divided into two groups: 31 patients using fibrin sealant (GCS) and 31 patients without the sealant employment (GSS). The variables were grouped into epidemiological, clinical, laboratory, especially the incidence of pancreatic fistula classified as defined by the International Study Group on Pancreatic Fistula, their postoperative complications cataloged according to Clavien rating and its repercussions on care and its costs assessed by the absorption method with simple apportionment of all expenses except the expenditure on medicines, treated separately. Results: The groups were homogeneous for clinical, epidemiological and laboratory parameters and no significant differences were recorded in the comparison given postoperative progress and hospital assistance indicators. Moreover, hospital costs were higher in GCS, with respect to GSS (p <0.0001). Conclusion: The use of fibrin sealant in pancreatojejunal anastomosis after pancreaticoduodenectomy, in the studied conditions, did not improve the results of care and also increased hospital costs

Peripheral nerve injuries are often associated with loss of nerve tissue and require autologous nerve grafts to provide a physical substrate for axonal growth. This thesis investigates the use of fibrin as both a tubular conduit to guide nerve regeneration and also as a matrix material to suspend various regenerative cell types within/on poly-3-hydroxybutyrate (PHB) nerve conduits. Adipose derived stem cells (ASC) are found in abundant quantities. In this thesis the ability of rat ASC to differentiate into Schwann cells was determined and a preliminary study of the neurotrophic potential of human ASC was also investigated. Rat sciatic nerve axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps and the gap was bridged using the various conduits.  At early time points the nerve grafts were harvested and investigated for axonal and Schwann cell markers.  After 16 weeks the regenerative response from sensory and motor neurons was also evaluated following retrograde labelling with Fast Blue fluorescent tracer. Stem cells were treated with a mixture of glial growth factors and after 2 weeks in vitro the expression of Schwann cell markers was analysed by immunocytochemistry and Western blotting.  ASC were cocultured with the NG108-15 neuronal cell line to determine their ability to promote neurite outgrowth.  Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  RT-PCR was used to investigate the expression of neurotrophic factors. Immunohistochemistry showed a superior nerve regeneration distance in the fibrin conduit compared with PHB. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared with an autograft group at 16 weeks. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the graft and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. In vitro studies showed that rat ASC could be differentiated to a Schwann cell phenotype. These treated cells enhanced both the number of NG108-15 cells expressing neurites and neurite length. In the same coculture model system, human superficial fat layer ASC induced significantly enhanced neurite outgrowth when compared with the deep layer fat cells. RT-PCR analysis showed ASC isolated from both layers expressed neurotrophic factors. These results indicate that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury. There was also a beneficial effect of using a fibrin matrix to seed cells within/on PHB conduits which should ultimately lead to improved functional recovery following nerve injury.  There might also be an advantage to use a simple strip of PHB rather than a conventional tube-like structure implying that single fascicle nerve grafting could be advantageous for nerve repair.  The results of in vitro experiments indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and given that human ASC express a range of neurotrophic factors they are likely to be of clinical benefit for treatment of peripheral nerve injuries.