<p>To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype. </p><p>In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.</p><p>In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated. </p><p>Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.</p> / Dissertation

The synthetic biodegradable polymer poly(lactide-co-glycolide) (PLGA) has been widely explored as substrate biomaterials for controlled drug delivery and tissue engineering. ECM component heparin and bone mineral hydroxyapatite (HA) are attractive biomaterials which can functionalize the PLGA surface to improve cell cell response and to bring in the dual growth factor delivery, because heparin and HA both can improve cell responses and bind with various proteins. To combine the osteoconductivity of HA and the controlled drug release of PLGA microspheres, HA coated PLGA microspheres were developed by a 3 hour rapid HA precipitation on the PLGA microsphere surface. Effects of various fabrication parameters on microsphere and HA coating morphology were evaluated. This core-shell composite worked as a dual drug delivery device and demonstrated better cell cell response than PLGA microspheres without HA coating. Three different methods, including osmogen, extractable porogen and gas-foaming porogen, were evaluated to fabricate porous microspheres as injectable cell scaffolds in the tissue engineering. The gas-foaming method produced covered porous PLGA microspheres, on which a skin layer covered all the surface pores. The skin layer was hydrolysed by NaOH to control the surface porosity. The modified open porous microspheres have large continued surface areas between pores, which provided more continued areas for cell adhesion. The porous microspheres with controllable surface porosity and large surface continuity between pores could be novel injectable cell scaffolds. Heparin was immobilized on the open porous PLGA microspheres by a facile layer-by-layer assemble to combine the advantages of porous structure and the protein binding from heparin. The heparin-coated porous microspheres promoted cell adhesion, spreading, proliferation and osteogenic differentiation. Growth factor-like protein lactoferrin was immobilized on the heparin coated porous microspheres, which further enhanced MG-63 proliferation and osteogenic differentiation. The heparin-coated porous microspheres are promising multi-functional devices for controlled drug delivery and injectable cell delivery.

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.

Cartilage tissue engineered constructs using mesenchymal stem cells were stimulated with 3 different stimulation algorithms to achieve characteristics mimicking the superficial tangential zone of articular cartilage. The stimulation algorithm of both compression and tension without an offset had the best properties out of all the evaluated groups.

Electrospun materials have potential use in many biomedical applications such as soft tissue replacements or as scaffolds to target drug delivery to local sites. Electrospinning is a polymer processing technique that can be used to create materials composed of fibers with diameters ranging from the micron to the nanoscale. We investigated the effects of microfibrous and nanofibrous electrospun polycaprolactone (EPCL) on innate, cell-mediated, and humoral components of the immune system. Results demonstrated that in both young (12 week) and old (6 month) mice, EPCL had no effect on various immune parameters. With its lack of immunotoxicity, EPCL presents an excellent polymer scaffold for use in delivering drugs to local sites. Drug delivery studies focused on using EPCL nanofiber scaffolds with the known immunosuppressive compound dexamethasone (DEX) incorporated within the matrix. The ability of the EPCL-DEX scaffold to suppress cell-mediated immunity (CMI) was evaluated using the delayed-type hypersensitivity (DTH) response to Candida albicans. Preliminary studies were conducted following subcutaneous implantation of a single disk (6-mm or 3-mm diameter) with 3, 10, 30, or 100 % w/w DEX in EPCL in the thigh region. Based on footpad swelling, dose -responsive suppression of the DTH was observed based on DEX equivalent units (DEU) at all but the lowest dose. The animals that received the high dose (100% in 6-mm) had decreased spleen weights, however no change in spleen weight was observed at the lower doses. Thymus weights were only affected at the four highest doses. These preliminary results suggest that implantation of a drug-containing electrospun scaffold may achieve local immunosuppression without systemic toxicity. Finally, we evaluated the EPCL-DEX scaffold in an acute inflammatory model (keyhole limpet hemocyanin) and a mouse model of rheumatoid arthritis (collagen induced arthritis). While similar trends were observed in the other models, the EPCL-DEX system achieved greatest success in the DTH model.

Bicomponent composite fibers have been widely used in the textile industry and are gaining increasing attention on biomedical applications. In this research, polycaprolactone/poly (lactic acid) side-by-side bicomponent fibers were created for the application of a biodegradable scaffold. The side-by-side structure endowed the fiber with self-crimps when it was processed under certain conditions. This material was produced by electrospinning and collected on a high speed rotating mandrel to get highly oriented fibers. A mechanical stretch at the same direction was done followed by a wet heat treatment for polymer retraction. Crimped fibers were demonstrated by scanning electron microscopy. The quantitative porosity and uniaxial tensile strength was not affected by the post-treatments, but the cell ingrowth and proliferation after seeding the scaffold were significantly improved. In conclusion, the side-by-side crimped material serves as a better extracellular matrix analogue without sacrificing mechanical properties.

The field of tissue engineering aims to develop viable substitutes with the ability to repair and regenerate the functions of damaged tissue. Common practices to supplement bone regeneration in larger defects include bone graft biomaterials such as autografts, allografts, xenografts, and synthetic biomaterials. Autologous bone grafting is the current gold-standard procedure used to replace missing or damaged bone. However, these grafts have disadvantages such as donor site morbidity, limited availability, and the need for a secondary surgery. The focus of this study is to tissue engineer a lyophilized gelatin composite sponge composed of hydroxyapatite (HA), chitin whiskers (CW), and preparations rich in growth factors (PRGF) to provide sufficient structural support to the defect site while enhancing the body’s own reparative capacity, ultimately eliminating the need for autologous tissue harvesting or repeat operations. The present study investigates several in vitro evaluations on multiple compositions of modified gelatin sponge scaffolds for use in bone graft applications. Gelatin sponges were fabricated via freeze-drying, enhanced with PRGF, HA, and/or CW, and cross-linked with 50 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) either during or post gelation. Initial evaluation of all scaffold combinations indicated that scaffolds released contents up to 90 days, EDC cross-linking during gelation allowed for more protein release, and had the ability to swell. Since the incorporation of PRGF, HA, and CW increased cell infiltration, and production of cell-created mineral matrix over 90 days in culture, these scaffolds were further characterized. Freeze-dried gelatin sponges enhanced with PRGF, HA, and CW and cross-linked during gelation with EDC (PHCE) were mineralized (M) in 5x revised simulated body fluid (r-SBF) for 1 hour to create a bone-like mineral surface. Gelatin EDC scaffold controls (GE), GE-M, PHCE, and PHCE-M scaffolds were characterized for their ability to swell, mineralizing potential, surface morphology, growth factor incorporation and release, uniaxial compression properties, and cell attachment, proliferation, infiltration, and protein/cytokine secretion.. After mineralization, scanning electron microscopy showed sparse clusters of mineral deposition for GE-M scaffolds while PHCE-M scaffolds exhibited a more uniform mineral deposition. Both GE and PHCE scaffolds were porous structures that swelled up to 50% of their original volume upon hydration. Over 21 days incubation, PHCE-M scaffolds cumulatively released about 30% of their original protein content, significantly more than all other scaffolds. Multiplex Luminex assays confirmed the successful incorporation of PRGF growth factors within PRGF sponges. For acellular uniaxial compression testing, PHCE-M scaffolds reported lower Young’s modulus values (1.3 - 1.6 MPa) when compared to GE and GE-M scaffolds (1.6 – 3.2 MPa). These low modulus values were comparable to values of tissue found in early stages of bone healing. DAPI (4',6-diamidino-2-phenylindole) staining and imaging showed an increase in initial cell attachment and infiltration of PHCE and PHCE-M scaffolds on day 1. GE-M scaffolds also appeared to attach more cells than the GE control. MTS cell proliferation assay results indicated that on days 4 and 7, PHCE scaffolds increased cell proliferation (compared to GE controls). MTS also illustrated that the addition of a mineral coating increases and decreases cell proliferation on GE-M and PHCE-M scaffolds, respectively. Multiplexer analysis of MG-63 protein/cytokine secretion suggests that cells are responding in a bone regenerative fashion on all scaffolds, as evidence of osteocalcin secretion. Little to no secretion of osteopontin, IL-1β, and TNF-α demonstrates that scaffolds are not influencing cells to secrete factors associated with bone resorption. The compressive mechanical properties of cellularized scaffolds did not differ much from acellular scaffolds. The collective results indicated increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion by cells on GE-M scaffolds, which support the addition of a bone-like mineral surface on GE scaffolds. Cellularized PHCE and PHCE-M scaffolds report similar advantages as well as Young’s modulus values in the range of native tissues present in the early stages of bone healing. The results of this study propose that the developed PHCE and PHCE-M scaffolds exhibit good cellular responses and mechanical properties for use in early bone healing applications.

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.