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

Submitted by LAÍS GALVÃO CAETANO null (laisgalvao@iq.unesp.br) on 2017-02-06T13:41:04Z No. of bitstreams: 1 Dissertação Mestrado Laís Galvão Caetano.pdf: 2178003 bytes, checksum: 2239574ffc48afbca6a43b06084d326b (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-09T17:06:10Z (GMT) No. of bitstreams: 1 caetano_lg_me_arafo.pdf: 2178003 bytes, checksum: 2239574ffc48afbca6a43b06084d326b (MD5) / Made available in DSpace on 2017-02-09T17:06:10Z (GMT). No. of bitstreams: 1 caetano_lg_me_arafo.pdf: 2178003 bytes, checksum: 2239574ffc48afbca6a43b06084d326b (MD5) Previous issue date: 2016-12-21 / A fibroína da seda extraída dos casulos do bicho-da-sêda é um co-polímero particularmente atraente para aplicações em dispositivos fotônicos e opticoeletrônicos devido a sua transparência óptica, biocompatibilidade e propriedades mecânicas. Esta tese apresenta a obtenção de suspensões e filmes iridescentes-luminescentes de fibroína contendo nanopartículas luminescentes de carbono (“carbon dots”) obtidos a partir de carvão vegetal. As nanopartículas luminescentes de carbono apresentam biocompatibilidade, baixa toxicidade, propriedades de fotoluminescência interessantes e possibilidade de modificação da superfície. Redes de difração foram produzidas nestes filmes utilizando como molde um DVD comercial. Os materiais obtidos foram caracterizados por um conjunto de técnicas de análise: Microscopia de Força Atômica (AFM), Microscopia Eletrônica de Varredura (MEV), Microscopia Eletrônica de Transmissão (MET), Espectroscopia de absorção na região do UVVis, Espectrofotometria de absorção molecular na região do infravermelho com transformada de Fourier (FTIR) e Luminescência. Medidas de luminescência mostram que os carbon dots emitem múltiplas cores entre toda a região do visível. Planos cristalinos foram observados nas imagens de MET das nanopartículas, no qual foram asssociadas a estrutura do grafite. Pela análise de UV-Vis dos carbon dots observou-se que a absorção óptica decai gradualmente à medida em que ocorre deslocamento para o vermelho e para os filmes de fibroína observou-se um ombro referente a transição eletrônica da tirosina (aminoácido de sua composição). A partir dos espectros de FTIR observou-se que não houve mudança na estrutura da fibroína após a adição dos carbon dots. As imagens de AFM e MEV comprovam a presença dos micropadrões periódicos na superfície dos filmes. Os resultados mostraram que os filmes de fibroína contendo carbono dots tem um grande potencial para a biomedicina, principalmente devido os seus parâmetros de luminescência se extenderem em toda a região do visível e por apresentar uma matriz sustentável, com excelentes propriedades ópticas e mecânicas. / Silk fibroin extracted from silkworm cocoons is a particularly attractive copolymer for applications in photonics and optoelectronics devices due its optical transparency, biocompatibility and high mechanical strength. This thesis presents the preparation of suspensions and iridescent-luminescent films of silk fibroin containing luminescent carbon nanoparticles (carbon dots) obtained from vegetal coal. Luminescent carbon nanoparticles present high biocompatibility, low toxicity, interesting photoluminescence properties and possibility of surface modification. Diffraction gratings were produced in these films using a commercial DVD as a template. The materials obtained were characterized by a set of analysis techniques: Atomic-Force Microscopy (AFM), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), UV-Visible Spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Luminescence. Luminescence measurements show that carbon dots emit light at the entire visible region. Crystalline planes were observed in the TEM images of the nanoparticles, where upon the graphite structure was associated. By analyzing the UV-Vis spectra of the carbon dots it was observed that the optical absorption gradually decreases as the red shift occurs. In the UV-Vis spectra of the silk fibroin films it was observed a shoulder referring to the electronic transition of the tyrosine (amino acid of its composition). From the FTIR spectra it was observed that there was no change in the silk fibroin structure after addition of the carbon dots. The AFM and SEM images confirm the presence of the periodic micro-patterns on the surface of the films. The results showed that the silk fibroin films containing carbon dots have a great potential for biomedicine, mainly due to its luminescence parameters extending throughout the visible region and due to a sustainable platform with excellent optical and mechanical properties.

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

Biocompatible and biodegradable porous materials based on silk fibroin (SF), a natural protein derived from the Bombyx mori silkworm, are being extensively investigated for use in biomedical applications including mammalian cell bioprocessing, tissue engineering, and drug delivery applications. In this work, low-pressure, gaseous CO2 is used as an acidifying agent to fabricate SF hydrogels. This low-pressure CO2 acidification method is compared to an acidification method using high-pressure CO2 to demonstrate the effect of CO2 mass transfer and pressure on SF sol-gel kinetics. The effect of SF molecular weight on the sol-gel kinetics is determined using the low-pressure CO2 method. The results from these studies demonstrate that low-pressure CO2 processing proves to be a facile method for synthesizing 3D SF hydrogels. We also determined the effect of SF solution concentration on the morphology and textural properties of SF aerogels. Changing the solution concentration from 2 wt% to 6 wt% yielded a higher surface area (260 to 308 m2/g) and different macro structure, but similar mesopore pore volume and size, and micro structure. Furthermore, we determined the effect of drying method on the morphology and textural properties of SF hydrogels gelled via CO2 acidification. Drying with supercritical carbon dioxide (scCO2) yielded an aerogel surface area five times larger than aerogels that were freeze dried. Moreover, a freeze dried hydrogel initially frozen at -20 °C had pores approximately 10 µm larger than a hydrogel initially frozen at -196 °C. The results presented here also demonstrate the potential of SF aerogels as drug delivery devices for the extended release of ibuprofen, a model drug compound. SF aerogels are loaded with ~21 wt% of ibuprofen using scCO2 at 40 °C and 100 bar. Differential scanning calorimetry of the ibuprofen-loaded SF aerogels indicates that the ibuprofen is amorphous. Scanning electron microscopy and nitrogen adsorption/desorption analysis are used to investigate the morphology and textural properties. Phosphate buffer solution (PBS) soaking studies at 37 °C and pH 7.4 reveal that the SF aerogels do not swell or degrade for up to six hours. In vitro ibuprofen release in PBS at 37 °C and pH 7.4 occurs over a six-hour period when the ibuprofen is loaded in SF aerogel discs with an aspect ratio of ~1.65 (diameter/thickness), whereas the dissolution of the same amount of pure ibuprofen occurs in 15 minutes. Furthermore, the release of ibuprofen from these SF aerogel discs are modeled using the Fu model which indicates that ibuprofen release follows Fickian diffusion for the first 65 wt% of ibuprofen release, and non-Fickian diffusion for the next 25 wt% of ibuprofen release. We also showed that SF aerogel scaffolds support in vitro human foreskin fibroblast cell attachment, proliferation, propagation, and cell seeding of different densities (10x103, 30x103, and 60x103). In summary, we created and characterized a tunable 3D SF aerogel scaffold with potential for applications in drug delivery and tissue engineering applications.

The resurgence, and eventual rise to prominence in the field of tissue engineering, that electrospinning has experienced over the last decade speaks to the simplicity and adaptability of the process. Electrospinning has been used for the fabrication of tissue engineering scaffolds intended for use in nearly every part of the human body: blood vessel, cartilage, bone, skin, nerve, connective tissue, etc. Diverse as the aforementioned tissues are in both form and function, electrospinning has found a niche in the repair of each due to its capacity to consistently create non-woven structures of fibers ranging from nano-to-micron size in diameter. These structures have had success in tissue engineering applications because of their ability to mimic the body’s natural structural framework, the extracellular matrix. In this study we examine a number of different techniques for altering scaffold properties (i.e. mechanical strength, degradation rate, permeability, and bioactivity) to create electrospun structures tailored to unique tissue specific applications; the end goal being the creation of a cellularized tissue engineering ligament analogue. To alter the mechanical properties of electrospun structures while maintaining high levels of bioactivity, synthetic polymers such as polydioxanone were blended in solution with naturally occurring proteins like elastin and fibrinogen prior to electrospinning. Cross-linking of electrospun structures, using glutaraldehyde, carbodiimide hydrochloride, and genipin, was also investigated as a means to both improve the mechanical stability and slow the rate of degradation of the structures. Fiber orientation and scaffold anisotropy were controlled through varying fabrication parameters, and proved effective in altering the mechanical properties of the structures. Finally, major changes in the structure of electrospun scaffolds were achieved through the implementation of air-gap electrospinning. Scaffolds created through air-gap electrospinning exhibited higher porosity’s than their traditionally fabricated counterparts, allowing for greater cell penetration into the scaffold. Overall, this collection of results provides insight into the diversity of electrospinning and reveals innumerous options, both pre and post fabrication, for the tissue engineer to create site-specific engineering scaffolds capable of mimicking both the form and function of native tissue.