Browsing by Subject "Biomaterial"

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

Many implanted devices fail due to the formation of an avascular capsule. Fat is known to promote healing and vascularization. It is possible that isolating and attaching ASCs (adipose stem cells) to an implanted device improves the healing in the adjacent tissue.

Various attachment methods were studied, and the fibronectin treatment was found comparable to or better than other treatments. Next, bare and ASC coated polyurethane were implanted into rats. The fibrous capsule surrounding the bare polyurethane was thicker and contained more collagen at 8 weeks. Additionally, the microvessel density in the tissue surrounding the ASC coated polyurethane was significantly higher at 4 and 8 weeks. Quantification of glucose sensor response following ASC attachment for 1 week found no measurable significant differences in function.

The bioluminescence technique, which quantifies the tissue glucose concentration around the implant at the moment of freezing, was used to determine if ASC attachment to biomaterials impacts the tissue glucose concentration profile. ASC attachment to polyurethane and to glucose sensors did not significantly change the glucose profiles in the tissue. However, a quantifiable glucose concentration profile was observed around all glucose sensors.

The final experiments were performed to identify a possible mechanism that adipose tissue uses to alter the foreign body response. In vitro experiments showed that VEGF (VEGF-A specifically) secretion following ASC attachment to polyurethane was 10-20 times higher than with fibroblast attachment after three days and 40-70 times higher after six days. This high secretion of VEGF would likely have in vivo physiological affects on microvasculature.

In conclusion, the attachment of ASCs to polyurethane reduced the thickness and collagen content of the fibrous capsule surrounding ASC coated implants and increased the microvessel density in adjacent tissue. In addition, ASC attachment did not enhance glucose sensor function, nor did it decrease the glucose concentration in the adjacent tissue. Finally, ASCs were found to secrete high amounts of pro-vascular cytokines, which likely plays a key role in the observed improvement of the foreign body response.

The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction.

This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.

When designing biomaterials for clinical applications, the performance of these platforms hinges on their interaction with the host immune system. A failure in engaging and incorporating the correct immune response would lead to foreign body response and subsequent rejection of the materials. To improve the biocompatibility of biomaterials and avoid undesired immune reactions, the key immunomodulatory cell type macrophage needs to be engaged and its phenotype modulated properly and timely. Therefore, the design parameters of biomaterials should be carefully considered in the context of macrophage modulation. Microporous annealed particle scaffolds (MAPS) are a new class of immunomodulatory granular materials generated through the interlinking of microgels. The modular nature of MAPS offers enormous tunability in not only the individual microgel design but also the homogenous or heterogenous microgel assembly into the bulk scaffold. We leveraged the plug-and-play feature of MAPS to study the effect of two design parameters, microgel crosslinking peptide (comprised of L- or D-amino acids) and spatial confinement (achieved through varying microgel size), on macrophage modulation and host responses. We uncovered that a fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes in MAPS with D-amino acid-based crosslinker was an indicator for regenerative scaffolds in a subcutaneous implantation model. We also discovered that scaffolds comprised of large microgels with pore size that can accommodate ~40 µm diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced inflammation level. The role of spatial confinement on macrophage response was further explored in vitro, where we demonstrated that size-dependent macrophage response to M1/M2 cytokine stimulations was tied to the change in cell morphology and motility. This work offers valuable insights into the dynamic immune response to synthetic porous scaffolds with a specific focus on macrophages, and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes for would healing and biomaterial implants.

Soft biomaterials are used in a variety of applications such as scaffolds for cell growth and coatings for implants or transplants. We aim to create a protein hydrogel that will self-assemble upon the mixing of two different protein constructs. This is accomplished using Streptavidin, a protein that tetramerizes, and SpyTag-SpyCatcher, a protein-peptide that spontaneously forms covalent bonds, as the crosslinking mechanisms. Further, using protein building blocks whose viscoelastic properties are known from single-molecule force spectroscopy (SMFS), we aim to create a hydrogel whose physical properties are tailorable by altering the building blocks incorporated in the constructs. This thesis focuses on using an atomic force microscope for force spectroscopy and imaging to analyze the formation of networks upon mixing various protein constructs. We find that small scale networks form using both Streptavidin and SpyTag-SpyCatcher as crosslinkers and that SpyCatcher can be used to detect molecular crosslinking and polyprotein polarization through SMFS.

Extending the functional lifetime of acrylic poly(methyl methacrylate) (PMMA) bone cement may reduce the number of revision total joint replacement (TJR) surgeries performed each year. We developed a system utilizing an encapsulated water-reactive, FDA-approved tissue adhesive, 2-octyl cyanoacrylate (OCA), as a healing agent to repair microcracks within a bone cement matrix. The proposed research tested the following hypotheses: (1) reactive OCA can be successfully encapsulated and the resulting capsules thoroughly characterized; (2) the static mechanical properties of the PMMA composite can be improved or maintained through inclusion of an optimal wt% of OCA-containing capsules; (3) PMMA containing encapsulated OCA has a prolonged lifetime when compared with a capsule-free PMMA control as measured by the number of cycles to failure; and (4) the addition of capsules to the PMMA does not significantly alter the biocompatibility of the material. Based on the experiments reported herein, the primary conclusions of this dissertation are as follows: (1) functional OCA can be encapsulated within polyurethane spheres and successfully incorporated into PMMA bone cement; (2) lower wt% of capsules maintained the tensile, compressive, fracture toughness, and bending properties of the PMMA; (3) inclusion of 5 wt% of OCA-containing capsules in the matrix increased the number of cycles to failure when compared to unfilled specimens and those filled with OCA-free capsules; and (4) MG63 human osteosarcoma cell proliferation and viability were unchanged following exposure to OCA-containing PMMA when compared with a capsule-free control.