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.

Although researchers have been able to print small, simple, and avascular tissues, they have been unsuccessful in creating large, complex and vascularized organs. Printing large and complex three-dimensional tissues or organs involves utilizing a large quantity of cellular spheroids and layer-by-layer addition of spheroids. In this study, an in-house cell spheroid fabrication system was developed to produce cell spheroids with human liver cells (hepG2), human endothelial cells (hEC), human neural stem cells (hNSC), and induced pluripotent stem cells (iPSC). It offers the ability of fabricating uniform-sized spheroids repeatedly, which is essential when large and complex structures need to be produced. In order to test the spheroids’ ability to fuse, hEC spheroids were placed in line with one another and revealed successful fusion. Overall, the results indicate the in- house developed cell spheroid fabrication system can play a major role in bioprinting by providing researchers with uniform-sized spheroids in large quantities, consistently.

Traumatic defects in articular cartilage can lead to joint disease and disability including osteoarthritis. Because cartilage is unable to regenerate when injured, the field of tissue engineering holds promise in restoring functional tissue. In this research, type II collagen was electrospun, cross-linked, and tested as scaffolds for supporting chondrocyte growth. The mechanical, biochemical, and histological characteristics of the engineered tissue were assessed as a function of the electrospinning solution concentration (i.e. scaffold fiber diameter and pore properties) and as a function of the time in culture (evaluated at 2, 4, and 6 weeks). Fiber diameter had a linear relationship with concentration: mean diameter increased from 107 to 446 nm and from 289 to 618 nm, measured with SEM and permeability meter, respectively, with increasing concentration, from 60 mg/mL to 120 mg/mL. Pore size revealed no relationship with concentration but mean values ranged in size from 1.76 to 3.17 μ2 or from 0.00055 to 0.0028 μ2, depending on the measurement technique. Average porosity ranged from 84.1 to 89.1%, and average permeability was between 6.82x10-4 and 35.0 x10-4 D. Histological analysis revealed a relatively high number of spherical cells, possibly indicating the expression of the chondrocyte phenotype. However, there were very little glycosaminoglycans and type II collagen synthesized by the cells despite an increase in the cell density over time for the 60, 80, and 100 mg/mL concentrations. The mean values for peak stress (between 0.17 and 0.35 MPa) and tangential modulus (between 0.32 and 0.64 MPa) for the mats are at least an order of magnitude less than that for native cartilage, while the mean values for strain at break (between 93 and 150%) for the mats are at least an order of magnitude greater than that for native cartilage. The equilibrium stiffness for all concentrations decreased from week 2 to week 6 of tissue culture (which may correlate with increasing cell density); the 100 mg/mL concentration had the highest mean value (0.084 MPa at week 2) and the lowest mean value (0.010 MPa at week 6). This research did not indicate any significant findings regarding the influence of concentration or culture time on chondrogenesis. Because the cells appeared to grow on the surface of the scaffold but there was a lack of cell migration into the scaffold, the scaffold material may be sufficient to support chondrocyte growth but the scaffold physical design must be reconsidered.

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.

The purpose of this work was to develop an automated method of measuring fiber diameters of electrospun scaffolds from scanning electron microscopy images of these scaffolds. Several automated methods were developed and evaluated by comparison to known values and data obtained via the standard manual method. Simulated images with known diameters were used as test images to evaluate the accuracy of each measurement technique. Eight scanning electron microscopy images were also used for the evaluation of the automated methods compared to the standard manual method. All diameter measurements were made in pixels. Five new automated methods coded in MATLAB were developed. The five methods varied the approach of identifying edges of fibers as well as assigning edges to single fibers and calculating the distance between edges assigned to the same fiber. One-way analysis of variance and the Tukey-Kramer tests were performed for comparison of all methods per image. The Custom Canny Slopes automated method was shown to accurately approximate the mean diameters in ten simulated images as well as microscopy image of real scaffolds (p<0.05).

The purpose of this study was to construct a flowmeter that could accurately measure the hydraulic permeability of electrospun fibrinogen scaffolds, providing insight into the transport properties of electrospun scaffolds while making the measurement of their topographical features (fiber and pore size) more accurate. Three different concentrations of fibrinogen were used (100, 120, and 150mg/ml) to create scaffolds with three different fiber diameters and pore sizes. The fiber diameters and pore sizes of the electrospun scaffolds were analyzed through scanning electron microscopy and image analysis software. The permeability of each scaffold was measured and used to calculate permeability-based fiber diameters and pore sizes, which were compared to values obtained through image analysis. Permeability measurement revealed scaffold permeability to increase linearly with fibrinogen concentration, much like average fiber diameter and pore size. Comparison between the two measurement methods proved the efficacy of the flowmeter as a way to measure scaffold features.

The process of electrospinning has given the field of tissue engineering insight into many aspects of tissue engineered scaffolds, including how factors such as fiber diameter and porosity are affected by polymer concentration. However, the affects of fiber alignment upon the material properties of electrospun scaffolds remains unclear. The purpose of this study is to determine how the material properties of electrospun gelatin scaffolds are affected by changes in fiber alignment and starting gelatin concentration. Gelatin scaffolds, with starting concentrations of 80, 100, and 130mg/m1, were electrospun onto a target mandrel rotating at various speeds. Samples of each scaffold were taken parallel and perpendicular to the axis of mandrel rotation. Fast Fourier Transform (FFT) analysis was performed on these samples, to determine how fiber alignment is affected by starting polymer concentration and the rotational speed of the target mandrel. Mechanical tests were aiso performed on these samples. Results were analyzed by Three-way ANOVA. It was determined that starting gelatin concentration, mandrel speed, and direction of fiber alignment interact together to produce effects on the mechanical properties of electrospun gelatin scaffolds.

The goal of this research was to develop a system of injectable hydrogels to deliver stem cells to musculoskeletal defects, thereby allowing cells to remain at the treatment site and secrete soluble factors that will facilitate tissue regeneration. First, production parameters for encapsulating cells in microbeads were determined. This involved investigating the effects of osmolytes on alginate microbead properties, and the effects of alginate microbead cell density, alginate microbead density, and effects of osteogenic media on microencapsulated cells. Although cells remained viable in the microbeads, alginate does not readily degrade in vivo for six months. Therefore, a method to incorporate alginate lyase in microbeads was developed and optimized to achieve controlled release of viable cells. Effectiveness of this strategy was determined through cell release studies and measuring proteins and expression of genes that are characteristic of the cell’s phenotype. Lastly, in vivo studies were done to assess the ability of alginate microbeads to localize microencapsulated cells and support chondrogenesis and osteogenesis. This project will provide insight to the tissue engineering field regarding cell-based therapies and healing musculoskeletal defects.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. A hallmark of epithelial cells is strong cell-cell junctions. These junctions include E-Cadherin, a type of adherens junction that is critical for both barrier function and contact inhibition. Prior experiments by other groups have shown that adherens junctions are subject to mechanical tension. Externally applied forces (e.g. stretch) results in changes in E-Cadherin forces that coordinate proliferation. My current work tests the hypothesis that E-Cadherin forces mediate the spatial regulation of cell proliferation even in the absence of externally applied forces.

Diseases involving fibrosis cause tens of thousands of deaths per year in the US alone. These diseases are characterized by a large amount of extracellular matrix, causing stiff abnormal tissues that may not function correctly. To take steps towards curing these diseases, a fundamental understanding of how cells interact with their substrate and how mechanical forces alter signaling pathways is vital. Studying the mechanobiology of cells and the interaction between a cell and its extracellular matrix can help explain the mechanisms behind stem cell differentiation, cell migration, and metastasis. Due to the correlation between force, extracellular matrix assembly, and substrate stiffness, it is vital to make in vitro models that more accurately simulate biological stiffness as well as measure the amount of force and extracellular matrix assembly. To accomplish this, blends of two types of poly(dimethylsiloxane) (PDMS) were made and the material properties of these polymer blends were characterized. A field of 5µm or 7µm microscopic pillars (referred to as posts) with a diameter of 2.2µm were fabricated from these blends. Each combination of PDMS blend and post height were calibrated and the stiffness was recorded. Additionally, polymer attachment experiments were run to ensure cells survived and had a normal phenotype on the different blends of PDMS when compared to pure PDMS. Finally, cells were placed onto a field of posts and their forces were calculated using the new stiffness found for each blend of post. Varying the PDMS material stiffness using blends allow posts to be much more physiologically relevant and help to create more accurate in vitro models while still allowing easy and accurate force measurement. More biologically relevant in vitro models can help us acquire more accurate results when testing new drugs or examining new signaling pathways.