Cardiovascular disease is a leading cause of death worldwide and becomes increasingly prevalent in the elderly population. Independent of etiopathogenesis, heart failure (HF) is the final common stage of numerous heart diseases. Cardiac stem cell (CSC) therapy has emerged as a promising cell-based strategy for treatment of HF. However, cell replacement is not able to fully restore a structurally damaged myocardium in advanced and end-stage HF. The objective of this project was to test the following hypotheses: that a bioengineered heart extracellular matrix (ECM) with preserved intact geometric structure could be generated using decellularization by coronary perfusion; and that autologous CSCs, to repopulate this ECM, could be isolated and expanded from the adult heart, with the caveat that autologous CSCs are depleted and impaired by both aging and chronic dilated cardiomyopathy. This will help to develop a possible therapeutic approach for advanced HF, using a combination of CSCs and engineering technique. Resident CSCs were isolated from explant-derived cells (EDCs) and expanded into cardiosphere-derived cells (CDCs) via cardiosphere formation. The CDCs expressed CSC markers (c-kit and Sca-1), pluripotent markers (Oct3/4 and Sox2), and the cardiac lineage-committed marker (Nkx2.5), and showed clonal expansion, self-renewal, and cardiomyogenic potential in vitro. In tissue engineering experiments, CDCs survived and proliferated within biomaterial alginate scaffolds for up to 7 weeks. An engineered bioartificial ECM scaffold was successfully produced from a whole rat heart using retrograde coronary perfusion and possessed an intact 3D architecture with functionally perfusable vascular network. Compared with ventricles, cultures derived from atria produced significantly higher number of c-kit+ and Sca-1+ CSCs (c-kit: 13% vs. 3.4%; Sca-1: 82% vs. 53%, respectively) and exhibited greater clonogenic and proliferative capacity. CDCs could be grown from young and aged mice, but the yield of CSCs significantly declined with age, as did cell migration and differentiation potential. In comparison to wild-type mice, atrial-CDCs from dystrophic mice showed no significant differences in CSC subpopulations and characteristics, despite confirmation of cardiac dysfunction using MRI. In conclusion, CDCs could be considered to be a viable cell candidate for cardiac therapy and may be used to treat HF at various stages, in combination with myocardial tissue engineering.

Tissue engineering is an interdisciplinary field which aims to create substitutes of damaged tissues by combining cells with biomaterials. Cells are extremely sensitive to their microenvironment and so the cell response to biomaterials can be regulated by different extrinsic stimuli and alterations of biomaterial properties. Successful implant integration into the tissue can therefore be promoted by appropriate surface roughness, chemical composition, adhesion ligand density, as well as the availability of growth factors. This thesis mainly focuses on the development of orthopedic replacements and the improvement of the currently used blood vessel prostheses. Through the study of cell-biomaterial interactions, it was demonstrated that superimposed topography with features ranging from the nano to micro scale promotes cell spreading, proliferation, and the metabolic activity of osteoblast-like cells. Moreover, when comparing the chemical composition of biomaterials for orthopedic implants, higher osteoblast densities were observed on composites with 5-15 vol. % of calcium phosphate nanoparticles, while concentrations of 25 vol. % did not support cell proliferation. Cell viability, however, was not affected. In vivo, a more intensive formation of new bone tissue, was found on samples containing...

Bone has the capacity to regenerate itself, however for challenging defects such as non-uniform factures, repair can be problematic. A similar challenge is presented in the repair of osteoporotic bone. Osteoporotic bone becomes increasingly porous and brittle and the risk of fracture is greatly increased. A drug mimic, poly(vinyl phosphonic acid – co – acrylic acid)(PVPA), has been incorporated into FDA approved poly(ε-caprolactone)(PCL), and aims to mimic the action of bisphosphonates to reduce the activity of osteoclasts. The PVPA polymer contains pendant phosphonic acid groups which are hypothesised to mimic the P-C-P backbone found in bisphosphonates. The PCL/PVPA scaffold has been found to have sufficient mechanical strength in order to be used as a bone void filler as well as providing a hydrophilic surface for superior cell attachment. The substrate has been found to significantly enhance the deposition of collagen, alkaline phosphatase activity and the expression of osteocalcin. Alizarin red staining revealed an increase in the rate of mineralisation in the presence of the drug mimic. The PCL/PVPA substrates have been suggested to induce osteoblast cells from a proliferative phase to a mineralisation stage. This is believed to be due to the presence of phosphorous within the scaffold which could lead to the critical concentration required for the initiation of mineralisation being reached more rapidly and effectively. The PVPA polymer has been found to mimic the action of bisphosphonates by inducing osteoclast apoptosis in vitro, and its actions of osteoclast apoptosis are comparable to that of Alendronate, a commonly administered bisphosphonate. A critical size defect model has demonstrated that the PVPA polymer has the ability to heal critical size defects; the healing potential was two fold greater than the control PCL substrate. Initial in vivo studies using a subcutaneous model demonstrated an improvement in mineralisation in the presence of PVPA. Untreated PCL/PVPA substrates displayed a high level of branched blood vessel formation, essential for healthy bone formation. However PVPA samples pre-treated with VEGF, hindered blood vessel formation and the infiltration of cells. This suggests that the PVPA alone is capable of inducing neovascularisation without the addition of VEGF. The findings suggest that the PCL/PVPA system could be used to treat challenging bone defects such as non-unions and osteoporotic fractures.

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Introduction: Mesenchymal stem cells (MSC) offer a novel cell therapy within tissue engineering and regenerative medicine (TERM)-based strategies, and the prospect of MSC therapies are widening since the discovery of MSCs within multiple locations of the body including bone marrow (BM-MSCs) and adipose tissue, (AD-MSCs). It is highly recognised that an organisms reparative and regenerative potential declines with advancing age, therefore aged patients are one of the primary target populations for TERM applications. Although information is available regarding the effects of patient age on the quality of human BM-MSCs, little and conflicting information currently exists for AD-MSCs. In addition, few studies have compared the quality of freshly isolated and expanded donor-matched BM and AD-MSCs to elucidate the more appropriate cell source. This study investigated the effect of donor age and in vitro ageing on functional behaviour (i.e. senescence state, population kinetics and differentiation potential) of donor-matched BM and AD-MSCs. Methods: The influence of donor age and in vitro ageing on mature (28-55 years) and elderly (75-86 years) donor-matched BM and AD-MSCs was assessed upon isolation (early life-span) and during extended (mid and late lifespan) timepoints through culture. During culture MSCs were characterised for cumulative population doublings (CPDs) and the expression of senescence associated marker genes, p16INK4A, p21 and p53, and transcription factor NANOG. At each lifespan telomere length was assessed along with differentiation efficiency along the osteogenic, adipogenic and chondrogenic lineages through lineage specific marker genes and histological staining. Results: Elderly BM and AD-MSCs displayed similar characteristics in terms of initial CPD number, p21, p53 and NANOG expression, telomere length and differentiation along osteogenic and adipogenic lineages. With increasing donor age there was a significant decline in p16INK4A expression within BM-MSCs, whilst expression of all chondrogenic markers significantly decreased within AD-MSCs. BM and AD-MSCs were comparable for the majority of outcome measures with the exception of chondrogenic differentiation which was superior with BM-MSCs in terms of COL2A1 expression and histological staining for proteoglycans. Donor age had a negative effect on BM-MSCs with long-term culture leading to a significantly longer PD time and decreased telomere lengths. Similar population kinetics was displayed between BM and AD-MSCs during long-term culture. Increasing culture time had effects on differentiation potential for both MSC sources with complete loss of osteogenic capacity and decreased adipogenic capacity; however chondrogenic capacity was only decreased within AD-MSCs. Differentiation potential after long-term culture between BM and AD-MSCs showed similar osteogenic and adipogenic ability yet superior chondrogenic ability was apparent within mature BM-MSCs compared to AD-MSCs, in terms of ECM deposition. Conclusions: In conclusion the source of MSCs for TERM will need to be considered depending upon the type of tissue regeneration required. The clinical outcome would be greater using MSCs during early stages of culture, as culture expansion has detrimental effects on functional properties of both BM and AD-MSCs.

Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.

Orientador: Marcos Akira d'Ávila / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-22T03:44:39Z (GMT). No. of bitstreams: 1 Salles_TaisHelenaCosta_M.pdf: 2536678 bytes, checksum: 358cfaf694adc91a715f58aabead529d (MD5) Previous issue date: 2013 / Resumo: A eletrofiação é reconhecida como uma técnica eficiente para a fabricação de microfibras e nanofibras de polímero, devido à sua versatilidade e potencial para aplicações em diversos campos. As aplicações notáveis incluem engenharia tecidual, biossensores, filtração, curativos, liberação controlada de fármacos e imobilização de enzimas. As nanofibras são geradas através da aplicação de um campo elétrico em uma solução polimérica. As fibras fiadas por este processo oferecem várias vantagens, como elevada área de superfície em relação ao volume, alta porosidade e a capacidade de manipular a composição de nanofibras, a fim de obter as propriedades e funções desejadas. Neste trabalho, a eletrofiação de blendas de gelatina com polivinilpirrolidona (PVP) para a fabricação de nanofibras foi investigada. Os polímeros foram fiados a partir de soluções contendo diversas concentrações de água e ácido acético. As soluções foram fiadas a uma tensão positiva de 29,0-29,2 kV, uma distância da ponta da agulha ao coletor de 10 cm, e uma vazão de 1 mL / h. Todas as soluções foram avaliadas quanto ao pH, condutividade elétrica, tensão superficial e viscosidade. Foram investigados os efeitos da concentração de ácido acético nas propriedades das soluções que por sua vez, influenciaram no processo de obtenção de fibras por eletrofiação. Foi observado que há uma correlação entre a concentração de ácido acético e a formação de fibras desse sistema, assim como a influência no diâmetro final das fibras. No presente estudo, uma matriz de nanofibras uniformes com diâmetro aproximado de 519, 355 e 154 nm foram produzidas via eletrofiação. A morfologia das membranas foi avaliada por Microscopia Eletrônica de Varredura (MEV). Foi realizada a análise térmica termogravimétrica (TGA) e avaliação de citotoxicidade, visando futuras aplicações em engenharia tecidual / Abstract: The electrospinning is recognized as an efficient technique for the fabrication of polymeric microfibers and nanofibers due to its versatility and potential for applications in many fields. Notable applications include tissue engineering, biosensors, filtration, wound dressings, controlled drug release and enzyme immobilization. The nanofibers are generated by applying an electric field in a polymer solution. The fibers spun by this process offers several advantages such as high surface area relative to volume, high porosity and the ability to manipulate the composition of nanofibers in order to obtain the desired properties and functions desired. In this work, the electrospinning blends of gelatin with polyvinylpyrrolidone (PVP) to fabrication nanofibers were investigated. The polymers were electrospun from solutions containing various concentrations of water and acetic acid. The solutions were electrospun at a positive voltage of 29.0 to 29.2 kV, a distance from the needle tip to the collector of 10 cm and a flow rate of 1 mL / h. All solutions were analyzed as your pH, electrical conductivity, surface tension and viscosity. We investigated the effects of acetic acid concentration on the properties of the solutions, on the other hand, influenced the process of obtaining fibers by electrospinning. It was observed that there was a correlation between the concentration of acetic acid and formation of fibers of that system, as well the influence on the final diameter of the fibers. In the present study, a matrix of nanofibers uniform with diameters of approximately 519, 355 and 154 nm had been produced by electrospinning. The morphology of the membranes was evaluated by Scanning Electron Microscopy (SEM). We made thermal analysis (TGA) and assessment of cytotoxicity, aiming future applications in tissue engineering / Mestrado / Materiais e Processos de Fabricação / Mestra em Engenharia Mecânica

Orientador: Cecília Amélia de Carvalho Zavaglia / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-22T11:32:15Z (GMT). No. of bitstreams: 1 Rigon_GuaciradosReis_M.pdf: 2217729 bytes, checksum: 84fa9de78a4fef6ee35a155f229d49bf (MD5) Previous issue date: 2013 / Resumo: Nos últimos anos, os polímeros biorreabsorvíveis ganharam importância na área médica e odontológica, sendo utilizados em um amplo número de aplicações no corpo humano, entre elas matrizes porosas tridimensionais como suporte no crescimento de células na área da engenharia tecidual. Com o objetivo de aperfeiçoar o processo de formação de matrizes como suporte na engenharia tecidual, estudou-se a utilização do compósito formado pelo copolímero PLDLA e um tipo de nanohidroxiapatita (HA) desenvolvida no laboratório de biomateriais da FEM/UNICAMP, na formação de matrizes através do processo de rotofiação. Os compósitos foram preparados utilizando-se 5% e 10% de HA em relação ao copolímero disperso em solvente clorofórmio na presença do surfactante ácido oléico. O processo de rotofiação é um processo simples, que forma uma matriz utilizando alta velocidade de rotação durante o jateamento da solução polimérica através de um orifício central sendo desnecessária, neste caso, a utilização de campo elétrico de alta voltagem, como ocorre para o processo de eletrofiação. As matrizes foram caracterizadas pelas técnicas de microscopia eletrônica de varredura (MEV), análise termogravimétrica (TGA), calorimetria diferencial de varredura (DSC), e espectroscopia de infravermelho com transformada de Fourier (FTIR). Os resultados obtidos pela microscopia eletrônica de varredura (MEV) mostraram que houve formação de uma matriz porosa e, portanto, o compósito pode ter uma aplicação promissora como suporte para cultura de células / Abstract: In the last years, bioresorbable polymers have been receiving more importance in the medical and dentistry areas, and they have been used in a large number of applications on the human body, such as tissue engineering scaffolds. This work studies the use of a composite of poly (L-co-DL-lactic acid) (PLDLA) and nanoparticles of hydroxyapatite developed at FEM/ UNICAMP to produce membranes by Rotary Jet Spinning process. Composites were prepared with 5% and 10% of HA in a clorophormium polymer solution. Rotary Jet spinning is a simple process to produce 3D structures that does not require a high-voltage electric field, like electrospinning. The results were characterized by the following methods: scanning electron microscopy (SEM), thermogravimetry analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). The results from SEM showed that a porous membrane was obtained which could be used as scaffold in tissue engineering / Mestrado / Materiais e Processos de Fabricação / Mestra em Engenharia Mecânica

This thesis is focused on the development and evaluation of different hybrid scaffolds for the treatment of injuries in cartilage or bone. These hybrid materials were three-dimensional polycaprolactone macroporous scaffolds obtained through freeze extraction and particle leaching combined method and modified with hyaluronic acid or mineral particles. In order to facilitate the description of the obtained results, the thesis is divided in two sections dedicated to bone and cartilage tissue engineering respectively. In the case of bone tissue engineering we addressed the treatment of disorders associated with the spine that require spinal immobilization. This Thesis proposes the development of a synthetic macroporous support for intervertebral fusion as an alternative to commercial bone substitutes. Macroporous scaffolds were developed with bare polycaprolactone or its blends with polylactic acid in order to increase its mechanical properties and degradation rate. Furthermore, the scaffolds obtained were reinforced with hydroxyapatite or Bioglass®45S5 to improve their mechanical properties and turn them in bioactive scaffolds. The supports were characterized physicochemically and biologically to determine if they met the requirements of the project. Finally, materials were tested in vivo in a bone critical size defect preformed in a rabbit model against a commercial support. Cartilage engineering has been extensively studied in the last years due to the inherent limited self repair ability of this tissue. The second part of the thesis was focused in developing a construct composed by in vitro differentiated chondrocyte like cells in a hybrid scaffold for cartilage tissue engineering. Polycaprolactone hybrid substrates coated with hyaluronic acid scaffold were developed obtaining a substrate with positive influence over the development of chondrocyte phenotype in culture and able to protect the cells from excessive mechanical loading in the joint. Cell-scaffolds constructs were obtained combining hybrid scaffolds with mesenchymal stem cells and differentiating them to chondrocytes using chondrogenic culture medium combined with hypoxia, mechanical stimulus or co-culture. Finally the cellularized scaffolds were mechanically, biochemically and histologically characterized to determine the production of extracellular matrix and expression of chondrogenic markers. / Ródenas Rochina, J. (2015). Scaffold surface modifications and culture conditions as key parameters to develop cartilage and bone tissue engineering implants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48526 / TESIS