The purpose of this study was to develop a fibrin gel system capable of serving as a three dimensional scaffold for the chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs) and to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on this system. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After chondrogenic potential of BM-MSCs was verified by pellet culture, 2 x 106 cells were pelleted and suspended in fibrinogen (80mg/ml) and then mixed with equal parts of thrombin (5 IU/ml). The specimen were then divided into four groups: aprotinin control (with aprotinin); aprotinin + transforming growth factor (TGF-beta) (with aprotinin and TGF-beta 1); amino control (with aminohexanoic acid); and amino+TGF-beta (with aminohexanoic acid and TGF- beta1). Each of these groups was further divided into three groups depending on the concentration of the inhibitor. Both of the aprotinin groups received 0.0875, 0.175, or 0.35 TIU/ml of aprotinin and both of the aminohexanoic acid groups were supplemented with 2, 4, or 8 mg/ml of aminohexanoic acid. The gels were harvested and analyzed at 7, 14, and 21 days. All of the aprotinin+TGF-beta groups exhibited a significantly higher aggrecan gene expression than control groups whereas only the amino+TGF-â group treated with 8mg/ml was significantly higher than those of the control groups. In addition, the 0.0875 and 0.175 TIU/ml aprotinin+TGF-beta groups exhibited significantly higher levels of expression than the 2 and 4 mg/ml amino+TGF-beta groups. There were no significant differences among the different concentrations of aprotinin or aminohexanoic acid with or without the treatment of TGF-beta. Similar trends were also seen when the glycosaminoglycan (GAG) content was measured and analyzed. These findings suggest that fibrin gels are a suitable environment for the chondrogenesis of BM-MSCs and that aprotinin in combination with TGF-beta1 is the optimal condition for stimulating BM-MSCs to differentiate into chondrocytes.

I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation. / text

Orientador: Rui Seabra Ferreira Junior / Resumo: Atualmente são muitos casos de pacientes que perdem estrutura óssea em acidentes ou reabsorção patológica. A bioengenharia óssea é um tratamento promissor que visa reconstruir estas estruturas sem a morbidade do enxerto autógeno. O tecido ósseo é um conjuntivo especializado com a função principal de proteção e sustentação dos tecidos moles, mas também é responsável pela produção de tipos celulares e homeostase de minerais. Sua reparação é complexa com diferentes tipos celulares e agentes quimiotáticos que funcionam de forma orquestrada até a reparação. As terapias celulares vêm sendo estudadas para promover a reparação de defeitos que o organismo por si não consegue resolver. Células menos especializadas como as células-tronco embrionárias (ESCs) possuem grande potencial terapêutico, mas são complicadas eticamente. Já as células-tronco mesenquimais (MSC) podem ser autólogas, o que minimiza o risco de imunogenicidade mas necessitam área doadora do paciente. Atualmente ainda não há consenso quanto ao uso de células tronco na terapia regenerativa pois há grandes variáveis como a melhor forma de aplicação, a quantidade correta e o melhor tipo celular para a regeneração óssea. As células produzem mediadores químicos no local enxertado, que segundo pesquisas recentes é o principal mecanismo de reparação tecidual. Estes mediadores são depositados em abundância no meio de cultura durante a cultura celular e usados na bioengenharia com a ajuda de scaffolds. Os biopolímeros de fibrina ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chapter I present a review about bone repair and its biological events, bioengineering, cells, fibrin biopolymer as scaffold and the secretoma derived from cell culture. Many patients nowadays lose bone structure in accidents or pathological reabsorption. Bone bioengineering is a promising treatment that aims to reconstruct these structures without autogenous graft morbidity. Bone tissue is a specialized connective tissue specialized in protecting and supporting soft tissues, but it is also responsible for the production of cell types and mineral homeostasis. The bone healing is a complex process where different cell types and chemotactic agents work in an orchestrated way. The cell therapies can promote the repair of defects that the body cannot solve. Less specialized cells like embryonic stem cells (ESCs) have great therapeutic potential, but are ethically complicated. In contrast, mesenchymal stem cells (MSCs) may be autologous, which minimizes the risk of immunogenicity but requires a patient's donor area. Currently there is still no consensus regarding the use of stem cells in regenerative therapy, studies uses different methods, cells and biomaterials for bone regeneration. Recent researches advocate that paracrine secretions by cells are main mechanism of tissue repair. These mediators are deposited in abundance in the culture medium during cell culture. Fibrin biopolymers (BF) are natural biomaterials to the body and can function as drug delivery of growth factors, c... (Complete abstract click electronic access below) / Doutor

The success of regenerative cardiac therapy requires reestablishing a capable blood supply via vasculature. The objective of this study was to develop an optimal scaffold formulation for de novo collateral vessel growth of aortic grafts using modified fibrin clots. This ex vivo vascular outgrowth model can be used to interrogate the complex cell or tissue interactions on the angiogenic front as vessels are formed. Based on formulation constraints, the methods used here may provide a clinically applicable option for guided collateral formation. Once understood, the methods and procedures can be tested and modified as necessary for in vivo, in situ regenerative therapy. Aortic segments from wild-type (C57BL/6J) and apolipoprotein-E deficient (ApoE) atherosclerosis-prone mice were cultured in a 3D environment created by various formulations of PEGylated fibrin. Aortic outgrowth was assessed and the optimal formulation was chosen to test the formation of de novo vascular circuits -- the first step necessary for collateral artery formation. The cultures were examined by conventional and confocal microscopy as well as by optical coherence tomography. Experiments testing the relationship between fibrin PEGylation and aortic vascular outgrowth showed that PEGylating fibrinogen prior to clot formation increased outgrowth over non-PEG control (n=6, p<.05) at lower fibrin concentrations. Lowering fibrin concentration to 10, 5, or 2.5mg/ml resulted in significantly higher outgrowth that was 1.92, 2.04, or 2.20 times that of 20mg/ml PEGylated fibrin gels. When multiple aortic segments are cultured in proximity, microvascular outgrowths visually anastamose suggesting that aorta-aorta conduits can be formed in fibrin based hydrogels. Anastomosing circuits appeared between wild-type aortic segments as well as between wild-type and atherosclerotic prone ApoE knockout segments. Fibrin gels, with or without PEGylation, form scaffolds suitable for regenerative vascular outgrowth ex vivo in normal and atherogenic environments. PEGylating fibrin prior to thrombin-initiated polymerization will allow the incorporation of growth factors or other bioactive components, making this a customizable therapy for guided collateral formation. Additionally, the incorporation of PEG itself does not limit and may actually increase the outgrowth from aortic segments in lower density gels. Finally, PEGylated fibrin gels offer an environment that will promote vascular extensions that visually anastamose, making this a viable model for ex vivo collateral formation. / text