Discharge of untreated industrial effluents containing heavy metals is hazardous to the environment as they are highly toxic, accumulates in the food chain and persistent in nature. Because of these adverse effects, their removal from wastewater is a substantial step in the protection of the environment and human health. Biosorption is found to be an eco-friendly, economical and lucrative separation technique in the removal of metal ions from effluent. This study explores the seperation potential of a new sorbent, fibroin (constituent of natural silk spun by Bombyx mori) for the removal of lead, chromium, copper and cobalt ions from effluent water. The biosorbent was prepared by the separation of cocoon into its constituents, fibroin and sericin. The removal of sericin from fibroin, called silk degumming, was carried out by water extraction method. Effect of temperature (55-95oC) on the kinetics and quantity of sericin removed was studied. The separation kinetics was approximated by the intraparticle diffusion model and the pseudo-second-order equation. Biosorption characteristics of fibroin for the removal of lead, chromium, copper, and cobalt ions from aqueous solution were investigated through a batch study. The effect of initial solution concentration, contact time and temperature on the sorption process was investigated. The adsorption equilibrium was described by the Langmuir isotherm. The thermodynamic parameters, the change in enthalpy (∆H) and change in entropy (∆S), were calculated by using Van’t Hoff plot. An accurate mathematical expression was used to calculate Gibbs free energy (∆G), for the adsorption of all metals on fibroin. For the kinetic data analysis, pseudo-second-order equation was modified based on the fact that the term qe in the kinetic equation should be the equilibrium uptake corresponding to the instantaneous metal concentration in the solution. In order to evaluate the rate constant k2, sorption kinetic data was fitted to the modified pseudo-second-order equation. The calculated values of rate constant k2, for the adsorption of all metals on fibroin, were used to the modified pseudo-second-order model to predict the kinetic data. A good comparison was observed between the experimental data and model calculations. The kinetic data was also fitted to the intraparticle diffusion model which showed a multi linear trend. The metal ions were desorbed from fibroin up to ten cycles of adsorption and desorption by using 0.05M ethylenediaminetetraacetic acid (EDTA). The removal of metal ions from fibroin was found to be rapid since complete desorption occurred within 15min. The uptake capacity of fibroin and adsorption/desorption kinetics remained almost the same even after ten cycles. The rate constants for both adsorption and desorption were also calculated by fitting the kinetic data to the modified pseudo-second-order model. The dynamic adsorption was studied in a flow-through column packed with fibroin for the removal of all metals. Experiments were performed in order to study the effect of influent concentration (12-75ppm), influent flow rate (0.15-0.24ml/min) and regeneration of fibroin bed (upto 4 cycles). Whereas the bed height, column diameter and amount of adsorbent packed were kept constant during this study. Fibroin bed saturated with metal ions was regenerated effectively by using 0.5M EDTA solution. After four consecutive cycles of adsorption and desorption, no change in the uptake capacity was observed. The bed depth service time model, the Thomas model and the Yoon-Nelson model were used to analyze the breakthrough data. The calculated values of Yoon-Nelson constants were used to predict the breakthrough curves. A good comparison was observed between experimental data and the Yoon-Nelson model calculations. An investigation was conducted to check if the adsorption of metal ions was carried out either by the surface of the fibroin or they were adsorbed deep inside its polymer network, and bulk was used. For this study, silk fibroin was transformed into thin films of three different thicknesses having same surface area. Batch experiments were conducted to study the thickness effect of fibroin films for the adsorption of metal ions. A constant amount of metal uptake for all three fibroin films showed that the adsorption was not a surface phenomenon, but the bulk body of the fibroin was used for this separation. Kinetic data was fitted to the modified pseudo-second-order model. The kinetic rate constant k2 was not significantly affected by the film thickness which abrogated the possibility of simple diffusion mechanism for metal sorption into fibroin. Fibroin films loaded with metal ions were desorbed once dipped in deionized water. The desorption kinetics was again described by the modified pseudo-kinetic-model. The calculated values of desorption rate constant kd were used to predict the kinetics of film desorption. A good comparison was observed between the modified pseudo-second-order model calculations and experimental desorption data of fibroin films.

Thesis (Ph. D.)--University of Michigan, 1933. / "By R. Lorimer Grant and Howard B. Lewis." Running title: Silk peptones. "Reprinted from the Journal of biological chemistry, vol. 108, no. 3 March, 1935." Bibliography: p. 673.

Thesis (Ph. D.)--University of Michigan, 1933. / "By R. Lorimer Grant and Howard B. Lewis." Running title: Silk peptones. "Reprinted from the Journal of biological chemistry, vol. 108, no. 3 March, 1935." Bibliography: p. 673.

Lower back pain and intervertebral disc degeneration represent a global socio-economical problem affecting 266 million people annually, always increasing due to aging of the population. No restorative treatments are available. In case of chronic degeneration, surgical operation with spinal fusion or total disc replacement represents the best alternative. This leads to pain relief but reduces the patient’s mobility. Moreover, follow-ups and re-intervention due to weak osteointegration are common consequences of currently used metal prostheses. For this reason, there is an urgent need to develop customized regenerative approaches aimed at the restoration of IVD function, as well as the optimization of osteointegration in actual vertebral prostheses by creating hybrid metal implants with infill materials to better induce bone ingrowth. In this work, tissue engineering-based approaches have been exploited by tuning the remarkable properties of silk fibroin for two purposes, disc restoration via in situ 3D printing technique, and improvement of osteointegration of vertebral prostheses. In situ 3D printing is the most promising strategy for the development of a personalized medicine approach aimed at the restoration of IVD. However, silk fibroin application as pristine ink in 3D printing technique is hindered by its low viscosity. For this reason, the aim of the first part of the work has been the design and development of silk fibroin-based inks in situ applications, overcoming its intrinsic limitations. Specifically, a covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing and in situ enzymatic crosslinking was designed. Two different silk fibroin molecular weights were characterized. We proved that despite the use of low concentration silk solutions, the synergistic effect of the covalent bonds with the shear forces applied in the nozzle enhanced silk secondary structure shift toward β-sheets conformation. The resultant hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. The designed process was tunable and versatile, leading to good shape fidelity and printing resolutions, making real the application of silk fibroin-based inks for in situ applications. The results obtained represent an important step for further studies on the mimicry of the whole IVD structure. 2 In the second part of the work, silk fibroin has been evaluated as candidate infill material for metal prostheses to improve bone ingrowth and osteointegration. In two independent works, silk fibroin-based foams and methacrylate silk fibroin sponges were biologically characterized and the differentiation of bone marrow-derived human mesenchymal stem cells (hBM-MSCs) toward osteogenic phenotype was studied. Silk fibroin foams have been demonstrated to induce and support cells adhesion, migration, and differentiation, and to induce early mineralization phase since day 7 during the differentiative culture. Methacrylate silk fibroin foams have been fabricated with different photo-initiator concentrations and in presence/absence of a porogen. The impact of the composition on the pore size, mechanical properties, and stem cells differentiation was deeply investigated. We demonstrated that despite all the conditions well-supported cells differentiation, the lowest photo-initiator concentration in combination with the porogen used enhanced osteogenic differentiation as confirmed by gene expression tests.

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

Silk fibroin has created a surge of interest for use as organic material due to its optical transparency, biocompatibility, biodegradability, and excellent physical properties. However, the implementation of silk films and structures into biomedical and sensing devices has been relatively low due to a lack of understanding of the mechanisms involved in such implementation. Increasing need for multifunctional high-performance organic materials has caused an emphasis on the ability of researchers to spatiotemporally pattern and control the structure and consequently functional properties of materials. Silk fibroin displays high potential for use as a controllable biomaterial that can be formed into a myriad of different structures for various applications. By implementation of an aqueous silk solution approach combining various fabrication techniques, several different pristine-silk and silk-composite membranes have been developed to investigate the importance of internal structuring. Different methods of investigation include: 1) incorporation of reinforcing nanoparticles within the silk matrix; 2) neutron reflectivity measurements of ultrathin silk films; 3) film patterning with nanoscale features followed by boundary organized surface mineralization of inorganic nanoparticles. The ultimate goal will be to provide fundamental data assisting in an increased knowledge of silk fibroin-based membranes and the effect of secondary structures on properties of interest.

This study focused on the comparison of the electrospun silk scaffolds to the electrospun silk fibroin gel scaffolds. Moreover, this study examined the differences in cross-linking effects of genipin and methanol as well as solvents on the mechanical properties and cell compatibility of the scaffolds. Silk scaffolds were electrospun from an aqueous solution or 1,1,1,3,3-hexafluoro-2-propanol (HFIP) without genipin, immediately after 8 % (wt) genipin was added to the solution, and 18 hours after genipin blended with the solution. Uniaxial tensile testing determined that the silk scaffolds electrospun from water exhibit a higher modulus and peak stress than that of the silk scaffolds electrospun from HFIP. In vitro cell culture was conducted to determine the cell compatibility of the various silk fibroin-based scaffolds. 4'-6-Diamidino-2-phenylindole (DAPI) staining and histology suggest that genipin may enhance cell compatibility, and that neither ethanol nor methanol inhibit cell interactions.

A fibroína da seda (SF) é uma proteína estrutural encontrada nos casulos do Bombyx mori e que possui propriedades potencialmente aplicáveis em fotônica. Esta matriz biocompatível é um substrato interessante para diferentes íons ou moléculas; além disso, o seu índice de refração variável permite que fótons sejam guiados neste material, possibilitando seu uso como guias de ondas biocompatíveis e reabsorvíveis, que pode ser utilizado para fornecer energia ótica para diversas aplicações, por exemplo, terapia ou imagem dentro de tecidos vivos. A sua boa adequação em sistemas ópticos deve-se principalmente a propriedades como: ser mecanicamente robusta, apresentar superfícies muito lisas, altamente transparentes (> 95%) em toda a região visível do espectro e ser modelável. Além disto, há uma característica adicional: a viabilidade de funcionalização bioquímica, o que pode conferir uma maior versatilidade a estes dispositivos. Já os íons terras raras (TR) possuem um papel amplamente conhecido no ramo da fotônica; porém, não há nenhum estudo envolvendo a produção de luz em SF dopada com íons TR, e a combinação das propriedades mecânicas e óticas desta matriz com a multifuncionalidade destes íons pode ser uma forma de se produzir dispositivos fotônicos novos e distintos. Desta forma, o presente trabalho teve como objetivo estudar a estrutura da matriz de SF na presença de diferentes íons TR (Eu3+ e Tb3+), bem como a interação existente entre os aminoácidos que constitui a matriz e estes íons. Os resultados apresentados nesta dissertação mostraram as interações TR-SF e suportam os mecanismos de transferência de energia para excitação de diferentes íons TR nesta matriz, sendo importante para futuras aplicações em fotônica / Silk fibroin (SF) is a structural protein found in Bombyx mori cocoons and has properties that are potentially applicable in photonics. This biocompatible matrix is an interesting substrate for different ions or molecules. Furthermore, its variable refractive index allows for photons to be guided in this material enabling their use as biocompatible and resorbable waveguides, which can be used to provide optical energy for various applications, e. g., therapy or imaging into living tissue. Its suitability in optical systems is mainly due to its properties such as: being mechanically robust, presenting very smooth surfaces, highly transparent (> 95%) throughout the visible region of the spectrum and being moldable. In addition, there is an extra feature: the possibility of biochemical functionalization, which may confer greater versatility to these devices. On the other hand, rare earth ions (RE) play a widely known role in the field of photonics. However, there is no studies involving the production of light in doped SF with RE ions and the combination of the mechanical and optical properties of this matrix with the multifunctionality of these ions can be a way to produce new photonic devices. Thus, the aim of the present work was to study the SF matrix structure in the presence of different RE ions (Eu3+ and Tb3+) as well as the interaction between the amino acids from the matrix and these RE ions. The results presented in this manuscript have characterized the RE-SF interactions and supported the mechanisms of energy transfer for excitation of different RE ions in this matrix being important for future applications in photonics

A calcificação ocorre pela formação de depósitos de cálcio em diferentes matrizes envolvendo fatores mecânicos, químicos e biológicos. Alguns compósitos, polímeros e proteínas são utilizados na formação de matrizes por promover maior eficiência no processo de mineralização. Estima-se que a fibroína da seda apresente também esta finalidade. A fibroína é uma proteína fibrosa extraída do casulo do bicho-da-seda (Bombyx mori), que pode ser processada como filme, membrana, esponja, pó, gel e aplicada em ossos e cartilagens, enxertos vasculares, reparação de nervos e córnea, como sistema de liberação de drogas, suturas, ligamentos, peles, tendões e substrato para cultura de células. Nesse trabalho houve a preparação de micropartículas de fibroína da seda através de dois procedimentos distintos, um por borrifamento em N&sup2 e outro por borrifamento em Na2HPO4 e o processo de calcificação realizado foi por imersão alternada de soluções tamponadas de cálcio e fosfato. As caracterizações realizadas foram Espectroscopia de Absorção no Infravermelho (FT-IR), Análise Termogravimétrica (TGA), Microscopia Eletrônica de Varredura (MEV), Espectroscopia de Energia Dispersiva (EDS) e Calorimetria Exploratória Diferencial (DSC). Os resultados obtidos mostraram que a calcificação das micropartículas de fibroína ocorre pelas duas metodologias empregadas. O teor de calcificação foi de aproximadamente 29% para micropartículas borrifadas em N&sup2 e de aproximadamente 80% para as micropartículas borrifadas em Na2HPO4. As micropartículas de fibroína calcificadas, não apresentaram transição térmica até a temperatura de 120°C, possibilitando a esterilização em autoclave a seco. / Calcification occurs by the formation of calcium deposits in different matrices involving mechanical factors, chemical and biological. Some composites, polymers, and proteins are used in forming matrices to promote higher efficiency in the process of mineralization. It is estimated that the silk fibroin also present for this purpose. The fibroin is a fibrous protein extracted from silkworm cocoon silkworm (Bombyx mori), which can be processed as film, membrane, sponge, powder, gel and applied in bone and cartilage, vascular grafts, nerve repair and corneal as a delivery system for drugs, sutures, ligaments, skins, tendons and substrate for cell culture. In this work was the preparation of microparticles of silk fibroin by two different procedures, sputter under N&sup2 and in other sputter Na2HPO4 and calcification process was performed by immersion of alternating buffered solutions of calcium and phosphate. The characterizations were performed Absorption Spectroscopy Infrared (FT-IR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and Differential Scanning Calorimetry (DSC). The results showed that the calcification of fibroin microparticles occurs by the two methodologies. The calcified fibroin microparticles showed no thermal transition temperature to 120°C, enabling autoclaving of the microparticles dry