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This small-scale design study describes disciplinary learning in mathematical modeling and science from an authentic engineeringthemed module. Current research in tissue engineering served as source material for the module, including science content for readings and a mathematical modeling activity in which students work in small teams to design a model in response to a problem from a client. The design of the module was guided by well-established principles of model-eliciting activities (a special class of problem-solving activities deeply studied in mathematics education) and recently published implementation design principles, which emphasize the portability of model-eliciting activities to many classroom settings. Two mathematical modeling research questions were addressed: 1. What mathematical approaches did student-teams take when they designed mathematical models to evaluate the quality of blood vessel networks? and 2. What attributes of mature mathematical models were captured in the mathematical models that the student-teams designed? One science content research question was addressed: 1. Before and after the module, what aspects of angiogenesis did students describe when they were asked what they knew about the process of blood vessel growth from existing vessels? Participants who field-tested the module included high school students in a summer enrichment program and early college students enrolled in four general-studies mathematics courses. Data collected from participants included mathematical models produced by small teams of students, as well as students’ individual responses before and after the module to a prompt asking them what they knew about the process of new blood vessel growth from existing vessels. The data were analyzed for mathematical model type and science content by adopting methods of grounded theory, in which researchers suspend expectations about what should be in the data and, instead, allow for the emergence of patterns and trends. The mathematical models were further analyzed for mathematical maturity using an a priori coding scheme of attributes of a mathematical model. Analyses showed that student-teams created mathematical models of varying maturity using four different mathematical approaches, and comparisons of students’ responses to the science prompt showed students knew essentially nothing about angiogenesis before the module but described important aspects of angiogenesis after the module. These findings were used to set up an agenda for future research about the design of the module and the relationship between disciplinary learning and authentic engineering problems.

With metastasis accounting for approximately 90% of breast cancer deaths and an alarming number of over 300,000 new breast cancer cases to be diagnosed by the end of 2018, there is growing need to understand the process of breast cancer. Changes in the extracellular matrix (ECM) of the tumor microenvironment play an essential role in this deadly tumor progression. Specifically, the glycoprotein fibronectin (FN), has been identified to be up-regulated in patients with worse clinical outcomes. During tumor progression fibronectin undergoes conformational changes that aid in metastatic dissemination. In order to analyze the dynamic changes in FN expression and evaluate architectural changes within the metastatic niche, electrophoretic tissue clearing was used to image changes in whole murine lungs that have undergone breast cancer metastasis. The X-CLARITY tissue clearing system utilizes electrophoresis which removes the lipid compounds from tissues to make them optically transparent. Coupling this with the c-PRESTO immuno-labelling technique and confocal light microscopy allows imaging of FN expression and structure in whole murine organs. In this study, a tissue clearing, and staining protocol was developed and used to visualize the 3D FN structure in whole murine lungs. Future studies will evaluate changes in FN expression and structure over time during cancer progression. This would contribute to on-going research on the development of more effective breast cancer drugs that aim to combat metastasis.

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) sensor able to detect reactive oxygen species (peroxide, hydroxyl, superoxide, etc.) inside of neurons, giving one the ability to enhance their understanding of the role these species play in the onset of the disease. This sensor relies on Förster Resonance Energy Transfer (FRET) between a green fluorescent protein and a red fluorescent protein to facilitate red-shifting of the sensor’s emission spectrum. Linked via a short polypeptide chain, the energy transfer efficiency of these combined FPs can vary greatly. Various linker lengths and FP combinations were experimentally tested to draw conclusions about their performance. The current trajectory of the research currently implies that those combinations with the shortest linker lengths will yield the highest-performing sensors. This sensor is another vital piece in the library of tools which can be used to help us begin answering the many questions we have about PD and its pathology.

Metastasis is one of the primary reasons for the high mortality rates in female patients diagnosed with breast cancer. It involves the migration of cancer cells into the circulatory system allowing for the dissemination of cancer cells in distal tissues. Understanding the major processes that occur in cells and tissues during metastasis can help improve currently existing therapeutic methods. In order to understand such mechanisms, developing physiologically relevant tissue models is crucial. Advancements in microfluidics have led to the fabrication of 3D culture models with shear stress gradients and flow control that can recapitulate aspects of the tumor microenvironment in vitro. However, most of these 3D culture models were fabricated using photolithography techniques performed in a clean room, which requires extensive training and can be cost prohibitive. Other studies have used more accessible techniques such as mold casting to construct a vessel-like microchannel. The main drawback of this method is that it limits the usage of high magnification objective lenses. Here, we propose a simple, high-fidelity and cost-effective approach in fabricating in vitro tissue platforms using a 3D printer that is optimal for live-cell imaging. To demonstrate proof-of-concept, we imaged endothelial and fibroblast cells cultured inside the 3D microfluidic collagen hydrogel, which was compared with the control group cultured in the 2D platform. The integrity of the microchannel fabricated inside the collagen hydrogel was imaged by using confocal reflectance microscopy. This feature has broader implications to bioengineered tissue fabrication and should be further explored in depth in the future.

Fluorescence microscopy, which allows multiple-color imaging, plays an important role in observing structures inside cells with high specificity. The advent of super-resolution fluorescence microscopy, or nanoscopy techniques such as single-molecule switching nanoscopy (SMSN), has extended the application range of fluorescence microscopy beyond the diffraction limit, achieving up to 10-fold improvement in spatial resolution. At the same time, the recent development of expansion microscopy (ExM) allows samples to be physically expanded by 4-fold in the lateral dimensions providing another independent method to resolve structures beyond the diffraction limit. When combined, ExM-SMSN makes it possible to achieve another significant leap in resolution of light microscopy. However, the need for specialized protein labels prevents the efficient combination of these two techniques, especially for multiple-color imaging. Here, we demonstrate our work in progress to effectively combine these two super-resolution techniques to provide another 2-4-fold improvement from the current achievable resolution for multi-color imaging. The developed technique will further push the boundary of achievable resolution of fluorescence microscopy and pave the way towards resolving protein-specific ultra-structures in the cellular context.

In excitatory neurons, the ability of a synaptic connection to strengthen or weaken is known as synaptic plasticity and is thought to be the cellular basis for learning and memory. Understanding the mechanism of synaptic plasticity is an important step towards understanding and developing treatment methods for learning and memory disorders. A key molecular process in synaptic plasticity for mammalian glutamatergic neurons is the exocytosis (delivery to the synapse) of AMPA-type glutamate receptors (AMPARs). While the protein signaling pathways responsible for exocytosis have long been investigated with experimental methods, it remains unreasonable to study the system in its full complexity via only in vitro and in vivo studies. A large number of protein interaction states are observed, creating a system both difficult to monitor and limited in spatiotemporal resolution in an experimental setting. Thus, a computational modeling approach could be employed to help elucidate the underlying protein interaction mechanisms. Here we develop a systematic model to investigate the spatiotemporal patterning of AMPARs. We replicate in silico two distinct mechanisms of AMPAR trafficking related to variation in AMPAR subunit functionality. This model is validated against current knowledge of AMPAR trafficking and used to explore spatial localization of AMPARs to specific synaptic sites, as well as to describe the differences in the spatiotemporal dynamics between the two interacting pathways. These findings help to explain how AMPAR trafficking occurs and can serve as a step towards understanding the role it plays in synaptic plasticity.

Type 1 diabetes, an autoimmune disease in which the body’s immune system destroys the insulin-producing beta cells necessary for managing a person’s blood glucose levels, affects 1.25 million Americans. A potential treatment for this disease is islet cell transplantation where Islets of Langerhans, containing the beta cells, are transplanted from a normal donor to a diabetic recipient to regulate blood glucose levels and provide insulin independence. Similar to whole organ transplantation, immune modulation through immunosuppression therapy is necessary for successful transplantation of islets without rejection. However, long-term systemic immunosuppression therapy can be toxic to the patient and the islets. Because of this, there is a need for alternative strategies where immune protection is provided locally through islet encapsulation or localized delivery of immunomodulatory agents. In this study, we investigated the toxicity of the inactive and active forms of an FDA approved immunosuppressant drug by assessing the morphology, viability, and protein expression of human islets embedded in a novel collagen encapsulation material. Overall, there was decreased islet cell death and improved morphology with i) decreasing drug concentration from 1.0 mg/mL to 0.01 mg/mL; ii) use of the inactive form of the drug vs the active form of the drug; and iii) incorporation of the drug into the collagen encapsulation material rather than the culture medium. Islet encapsulation strategies incorporating localized immunosuppression has the potential to be a successful solution for improving islet longevity and function following transplant without impacting the patient systemically.

Benzene, a toxin and carcinogen found in air polluted by cigarette smoke, car exhaust, and industrial processes, is associated with the development of leukemia and lymphoma. Other than avoiding exposure, there is no current method to deter the effects of benzene. One potential strategy to prevent these effects is to engineer the bacteria of the human lung microbiome to degrade benzene. To evaluate this novel approach, we must verify that the bacteria remain viable within the lung microenvironment. To do so, lungs were harvested from rats and swabbed to determine the contents of the original lung microbiome. Then green fluorescent protein (GFP)-transformed E. coli were introduced to the lungs and the lungs were ventilated for five minutes before being swabbed again. The lungs were sliced with a vibratome and cultured for three days. They were analyzed under a microscope and swabbed daily to determine how the bacteria disperse upon delivery and detect changes within the lung microbiome. If results show that introduction of a new bacterial species does not significantly change the lung microbiome over time, the project can move forward to test the engineered bacteria’s viability in the lung environment and effectiveness in rescuing lung cells from benzene’s toxicity.

Over 240 million people missing teeth worldwide experience lingering problems such as difficulty speaking and eating, undesirable aesthetics, and resorption of bone supporting neighboring teeth. The gold standard of treatment utilizes grafts to attach a function-restoring implant to supporting bone. Current graft materials suffer from problems including autologous donor site morbidity, long resorption time, incomplete integration with the maxillae or mandible, and structural weakness. Patient-specific, cellularized bone grafts may be a solution to these issues by accelerating and improving the quality of regenerated bone. Recently, encapsulation of mesenchymal stem cells within self-assembling type I collagen oligomer matrices has been shown to support rapid mineralization of small-scale bone constructs (cylinders with diameter and height of 6mm and 1mm, respectively) in vitro. However, this method’s volume and geometric constraints for nutrient transport and cell viability are still unknown. In this study, the effects of construct size and medium formulation on mineralization were investigated using conventional static culture methods. To create constructs, human adipose stem cells (hASCs) were embedded in oligomer matrices, allowed to polymerize, and compressed to final cell and fibril densities of 3x107 cells/mL and 50 mg/mL, respectively. Varying construct sizes (maximum diameter and thickness of 11 mm and 0.81 mm) were cultured for 1 week in growth medium or osteogenic medium with varying calcium concentrations. Alizarin red staining was used to detect calcium deposits indicative of cell-induced mineralization. Preliminary data suggests that culture in osteogenic medium supplemented with both 8 mM and 16 mM calcium may induce rapid, uniform mineralization across all sizes tested, and 16 mM calcium supplementation induces greater mineralization. However, additional validation by direct measurement of cell viability and osteogenic differentiation will be needed to better compare bone regeneration as a function of scale.

Neonatal sepsis, resulting from a bloodstream infection within the first few weeks of life, is the leading cause of newborn deaths worldwide. The gold standard of neonatal sepsis diagnosis requires a blood culture to identify the infecting bacteria, however require days of incubation, expensive equipment, and expertise. Any delay in diagnosis is critical, as the condition can be treated easily if appropriate antibiotics are administered promptly. A low-cost, rapid, and sensitive diagnostic test would enable more timely treatment and lead to better patient outcomes with fewer required resources. Point-of-care, nucleic acid amplification assays are a promising alternative to blood culture that quickly deliver sensitive results with minimal sample volume. However, these require isolated and purified template DNA from the pathogenic bacteria, a task that is difficult to achieve in a field setting. This study sought to develop a simple one-step lysis and amplification protocol for three common bacterial causes of neonatal sepsis, Streptococcus agalacitae, Klebsiella pnemoniae, and Staphylococcus aureus. The combined efficacy of enzymes, proteinase K and achromopeptidase (ACP), and heat to lyse each of these bacteria for direct DNA amplification was examined. Results showed that all three strains could be effectively lysed by applying 1 U of ACP and incubating for 10 minutes at 37° C. No adverse effects were seen in amplification reactions containing ACP if the compound was inactivated prior to amplification at 85° C for 2 minutes. The demonstrated effectiveness of ACP in rapid bacterial lysis validates its usefulness in a point-of-care device for neonatal sepsis.

Lower back pain from intervertebral disc injury affects around 84% of the population at some point in their life, which at its worst may cause total immobilization. This pain can only be temporarily relieved by spinal fusion or intervertebral disc replacement; however, both of these cause loss of natural motion in patients by removing damaged fibrocartilage discs. While these techniques help mitigate pain briefly, no permanent solution exists currently to both relieve pain and preserve natural motion. My work may be a solution by eventually providing patient-specific implants that resemble native tissue in the regeneration process that could be absorbed and remodeled by the body. The purpose of this study is to use tunable type I oligomeric collagen matrices for culturing of patient-derived stem cells to optimize chondrogenic media. Human adipose stem cells (hASCs) were passaged and used in conjunction with oligomer collagen, which was polymerized as cell/oligomer mixtures and plastically compressed to a density of 24.5mg/mL, with 4.5x105 cells per sample. These cell-matrix constructs were cultured with different media and supplements (namely TGF-β (3) and L-ascorbic-acid-2-phosphate) for 1 week. Safranin-o staining was used to detect sulfated glycosaminoglycans, a direct measure of chondrogenesis. Preliminary results show that supplemented DMEM media has the most chondrogenic potential, but further study is required. These results will be used to further improve the process of chondrogenesis in vitro in order to develop fibrocartilage constructs for use in vivo, eventually allowing for implantable constructs that both preserve natural disc height and relieve pain more permanently.

Bone fracture has recently become prevalent, especially with an increasingly aging population. Current bone grafts procedures, including autografts and allografts, are hindered by multiple factors, such as limited supplies and inconsistent bone healing. Scaffold-based bone tissue engineering emerges as a prospective strategy to aid in bone regeneration through delivery of growth factors such as bone morphogenic proteins (BMPs). However, the use of BMPs suffers from several drawbacks such as protein instability and immunogenicity. Therefore, there exists a great need for the development of novel therapies to promote bone healing. Notch signaling, a pathway critical for cell-fate determination has been shown to regulate osteogenesis, which suggests the potential of targeting Notch to enhance bone repair. The long-term goal of this work is to develop biomaterial-based regenerative technologies to induce bone regeneration by fine-tuning Notch signaling. In this study, a three-dimensional (3D) porous scaffold system was fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) to mimic structural and mechanical properties of native bone using a microsphere sintering technology. In vitro studies were conducted to evaluate the effects of notch inhibition via a γ-secretase inhibitor DAPT on osteoblast responses. When the DAPT was added during the 2D culture on tissue culture polystyrene (TCPS), the osteoblast mineral deposition was significantly enhanced. Intriguingly, the enhancement was more pronounced on the 3D PLGA scaffolds, which may be attributed to the increased cell-cell contact in the 3D culture environment. Current efforts are focused on scaffold-based modulation of Notch signaling with both quantitative and temporal precision for enhanced osteogenesis.

Actomyosin cortex, a thin network underlying cell membrane, is known to generate a large portion of tensile forces required for various cellular processes. Recently, theoretical studies predicted that buckling of actin filaments breaks symmetry between tensile and compressive forces developed by myosin motors, resulting in tensile stress at a network level. However, the significance of the filament buckling of the cortex has yet to be demonstrated either computationally or experimentally. Here, buckling-dependent stress generation of the cortex-like actomyosin network was investigated using an agent-based computational model consisting of actin filaments, actin cross-linking proteins (ACPs), and molecular motors. First, a wide parametric space of filament length and the densities of ACP and motors was explored. It was found that the importance of the buckling varies depending on conditions; with higher ACP/motor densities and longer actin filaments, symmetry breaking induced by buckling is more severe. Using the data collected from the model, it was explained why the buckling is more likely to occur under such conditions. In addition, by analyzing temporal evolutions of filament behaviors, it was showed that some of the buckled filaments become subjected to tensile forces in a later stage, which was not predicted by the theoretical models.

The field of regenerative medicine seeks to create replacement tissues and organs, both to repair deficiencies in biological function and to treat structural damage caused by injury. Scaffoldings mimicking extracellular matrix (ECM), the structure to which cells attach to form tissues, have been developed from synthetic polymers and also been prepared by decellularizing adult tissue. However, the structure of ECM undergoes significant remodeling during natural tissue repair, suggesting that ECM-replacement constructs that mirror developing tissues may promote better regeneration than those modeled on adult tissues. This work investigated the effectiveness of a method of viewing the extracellular matrix of developing embryos. Embryos were perfused with acrylamide, polymerized, and decellularized with sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS). Cell nuclei were stained with a fluorescent DNA-binding compound to verify decellularization. Whole-mount embryos were stained and used to verify that the overall structure of the ECM was not altered. Antibody staining was combined with fluorescent confocal microscopy to determine the presence of fibrillin-2 (FBN2), fibronectin (FN), tenascin-C (TNC), and collagen-VI (COL6). This study demonstrated that while the results of SDS decellularization depend on SDS concentration, embryonic stage, the protein under consideration, and the region of the embryo examined, FBN2 and FN tended to maintain their natural structure and to become more visible with treatment, while TNC and COL6 tended to be disrupted and removed. Future efforts to develop tissue-replacement constructs may benefit by using this method to examine, quantify, and mimic the structure and composition of specific proteins of developing ECM.

Patients with kidney failure usually undergo hemodialysis, a process by which toxins produced by the body are filtered from the blood, in order to survive. The preferred form for vascular access is called an arteriovenousfistula (AVF), a surgically created connection between an artery and vein that is utilized to undergo dialysis. However, AVFs have a failure rate of 50-60%. One of the contributions to AVF failure is endothelial cell dysfunction and loss of glycocalyx, which allows neutrophils and other native cells into the media of the vessel, which causes an inflammatory response. Our lab addresses endothelial dysfunction by mimicking the function of the glycocalyx to prevent transmigration of inflammatory cells and ultimately create a healthier vessel for hemodialysis. We have synthesized several glycocalyx mimics consisting of a dermatan sulfate backbone with multiple selectin and ICAM-binding peptides attached. Initial testing involved determining the ability of the variants to bind to inflamed endothelial cells. We also cultured human promyelocytic leukemia cells (HL60) and used retinoic acid to differentiate them into neutrophils. These cells would then test the glycocalyx mimics ability to prevent migration of neutrophils. Thus far, we have seen that the glycocalyx mimics binding to endothelial cells and that this binding is dependent upon the type of selectin and/or ICAM-binding peptides as well as how many peptides are present per dermatan sulfate backbone. We have also shown that proliferation occurs 10 days after seeding, and that rentinoic acid (RA) differentiates HL60 cells into neutrophils. We have developed a protocol for differentiation of HL60 cells to neutrophils, a promising set of glycocalyx mimics, and culturing method for HL60 cells.

Skeletal muscles can self-repair minor strains, lacerations, and contusions; however, in cases of volumetric muscle lossand muscle degenerative diseases, tissue fails to regenerate. Current cell-based therapies, such as myoblast transplantation, have significant drawbacks of low survival rates and engraftment efficacy, mainly due to the absence of supportive cell microenvironment. Scaffolds that mimic the natural cell microenvironment provide a robust platform to support cell adhesion, migration, proliferation, and differentiation. Electrospinning is a versatile technology platform used for fabricating the fiber scaffold that mimics the extracellular matrix. Thus, we aim to reconstitute the cell microenvironment through development of aligned fiber scaffolds by electrospinning as oriented muscle fibers create natural microenvironment of myogenic cells. In particular, aligned fiber scaffolds will be optimized in term of mechanical properties and fiber diameters as fiber curvature and mechanical stiffness provide significant physical cues for myogenic cell behaviors. Here, we fabricated and characterized electrospun polyester fiber scaffolds with different diameters from micro-scale to nano-scale. The mechanical properties of the fabricated nanofibers were found to be in the range of contractile muscles as evidenced from atomic force microscopy measurements. With these scaffolds, C2C12 myoblasts were seeded and analyzed for the initial attachment. It was shown that aligned fibers with varying diameters resulted in different responses in cell attachment, indicating the role of cell topography sensing in cell-biomaterial interactions. Current ongoing studies focus on long-term in vitro culture of scaffolds in a custom-made muscle bioreactor emulating the contraction/relaxation of skeletal muscle tissue.

Protein post-translational modifications serve to give proteins new cellular function, spatial localization, or enzymatic activity. Myristoylation is a common post-translational modification where the enzyme N-myristoyltransferase adds myristic acid onto the N-terminus of a variety of proteins. In this work we use a myristic acid analog, 12-azidododecanoic acid (12ADA) to facilitate the implementation of azide-alkyne cycloaddition reactions on myristoylated proteins. Selective protein labeling methods such as these are useful in research because they can be used to help determine the biological function of this protein lipid modification and can be extended to study disregulated protein myristoylation in disease states. To validate 12ADA incorporation onto proteins, C2C12 myoblast cell lysates were reacted with an alkyne functionalized fluorophore and analyzed via SDS-PAGE. In order to visualize 12ADA tagged proteins in vivo, fixed C2C12 cells were reacted with an alkyne functionalized fluorophore and were imaged with a fluorescent microscope. The results clearly indicate selective protein tagging in in vitro lysates and in vivo. There is a distinct difference in the patterning of 12ADA protein tagging between differentiated and non-differentiated cells. The purpose of this research is to develop a selective protein labeling method. In our research, this selective protein labeling method is used to studying cellular differentiation in the context of developmental biology. Currently, there is not a clear understanding of the proteins associated with cellular differentiation related to development. Understanding this can allow scientists to track development progress and understand unique proteins associated with differentiating cells.

Balloon angioplasty, the most prevalent non-surgical treatment for Atherosclerosis, damages the endothelial layer of the artery, baring an underlying collagenous layer, which causes platelet adhesion and activation and eventual thrombosis and intimal hyperplasia. Previous work in our lab has used a collagen-binding peptidoglycan, dermatan-sulfate-SILY (DS-SILY), that has been shown to bind to type I collagen and prevent platelet adhesion and activation. Our goal is to fabricate nanoparticle-SILY by cross-linking SILY to a poly(N-isopropylacrylamide) (NIPAm) nanoparticle instead of a DS backbone, while retaining the SILY’s high collagen binding affinity and platelet inhibition capacity observed in DS-SILY. Using a biotin-streptavidin assay, we showed that nanoparticle-SILY has a high binding affinity when applied to a collagen-coated surface, starting at a concentration of 1 mg/mL and maximized at concentrations at or above 4 mg/mL. Using dynamic light scattering, we showed that the nanoparticle-SILY maintains the thermodynamic properties characteristic of NIPAm. The ability of the particles to inhibit platelet binding and activation will be tested by using an ELISA to measure NAP-2 and PF-4 expression in platelet rich plasma applied to a collagen surface treated with nanoparticle-SILY. We expect to find that nanoparticle-SILY is able to inhibit platelet activation, as evidenced by lower levels of NAP-2 and PF-4 released. If it can be shown that nanoparticle-SILY has the capability to prevent platelet adhesion and activation, future work could explore the potential of the nanoparticles to be loaded with anti-inflammatory peptides and used for the dual purpose of targeted drug delivery and collagen shielding.

Ductal Carcinoma In Situ (DCIS) is a non-invasive cancer that forms around breast milk ducts that can potentially progress into invasive breast cancer if untreated. Lack of models to study its diverse pathophysiology and differential response to treatments poses a challenge to develop standard treatment modalities with improved therapeutic outcomes. The traditional in vitro models such as cell monolayer are convenient but insufficient to represent the physiological characteristics of DCIS tumor microenvironment and often fail to predict clinical outcomes. The animal models effectively simulate the in vivo environment but also lack the ability to control the environmental parameters to match specific conditions making it difficult to address the heterogeneities in disease state and patient-to-patient variations. It is critical to develop a new DCIS model system that offers physiologically relevant features with high degree of control. In order to address this need, a novel microfluidic in vitro model was developed. A lumen structure to represent the milk duct in breast was generated along the microfluidic channel using a fluid dynamic phenomenon called viscous finger patterning in which as the less viscous fluid passes through, it leaves a continuous trail that makes a hollow tubular structure in the collagen hydrogel. Consequently, MCF-7 breast cancer cell lines were cultured along the lumen surface with BR5 stromal fibroblast in collagen hydrogel. A relatively straight, smooth lumen was achieved at a higher concentration of collagen gel by viscous finger patterning with an optimal flow rate. The interaction between a non-invasive breast cancer cell line, MCF-7 and stromal fibroblast most likely remain unchanged, thus mimicking the DCIS. This new model system is a potential tool to study DCIS progression and treatment response by offering physiologically relevant features that can be tailored to match disease state and patient specific conditions.

Satellite cells are anatomically localized along the surface of muscle fibers and have been regarded as a population of muscle-specific progenitors that are responsible for muscle regeneration. In response to muscle injuries, satellite cells are activated to enter the cell cycle, then proliferate and differentiate into mature muscle cells to regenerate damaged myofibers. Unfortunately, this natural repair mechanism is interrupted in conditions such as muscle degenerative diseases or volumetric muscle loss. The function of stem cells is regulated by signals from their local microenvironment which is called the stem cell niche. Current satellite cell-based strategies such as direct cell transplantation are hindered by the low survival and poor distribution of transplanted cells due to lack of the cell niche. Thus, the objective of this project is to develop a cell/biomaterial combination strategy for muscle regeneration based on understanding of the characteristics of satellite cells and their cell niche. First, satellite cells were isolated from the skeletal muscles of a wild-type mouse and expanded in culture. The scaffolds were synthesized to mimic the anisotropic organization of myofibers. Cells were then seeded on scaffolds and cultured. Cellular responses of satellite cells to the scaffolds were measured and analyzed for adhesion, self-renewal, and proliferation. These experiments provided critical insight on the interactions between synthetic biomaterials and satellite cells. In the future, this synthetic scaffold system will optimized with appropriate structural and mechanical properties to facilitate transplantation of satellite cells for muscle regeneration.

Due to the high prevalence of arthritis and cartilage-related injuries, tissue engineers are studying ways to grow cartilage tissue replacements. Resilin, an elastomeric protein found in insect cuticles, is known for its extraordinary resilience and elasticity. In previous studies, recombinant resilin-based hydrogels, or cross-linked protein networks, exhibited potential for use in cartilage tissue scaffolds. Our lab successfully developed resilin-based proteins with a sequence based on the mosquito gene and showed that resilin-based hydrogels possess mechanical properties of the same order of magnitude as native articular cartilage. In addition, these mechanical properties can be controlled by changing the protein concentration. To understand how these resilin-based hydrogels will behave in physiological environments, it is necessary to characterize their physical properties, such as swelling ratio and water content. In this study, we developed a protocol to fabricate hydrogels and characterize their swelling ratio and water content using bovine serum albumin (BSA) as a model protein and a cross-linker, tri(hydroxymethyl)phosphine (THP). Hydrogels of varying diameters (4mm and 6mm) and protein concentrations (8 – 14 wt%) were fabricated at 37oC in silicone molds. To mimic a physiological environment, hydrogels were stored in phosphate buffered saline (PBS). Each hydrogel was weighed until fully swollen, indicated by a constant weight for 3 time points. Freeze-drying was performed to obtain dry hydrogels. The swelling ratio and water content were calculated using the final swollen weight and the dry weight. Results showed that size did not significantly affect swelling ratio or water content of 8, 12, and 14 wt% BSA gels but did affect 10 wt% hydrogels. Furthermore, an increase in protein concentration resulted in a decrease in swelling ratio and water content. Because this procedure allowed successful characterization of BSA hydrogels, this protocol will be adapted to characterize our own resilin-based protein hydrogels in future studies.

The knowledge of how cells interact with and sense their surroundings is missing the key components of time dependency and how substrate stiffness affects amount and rate of strain. This new knowledge of cell-substrate interaction can be applied further to research regarding chromatin spatiotemporal dynamics to better understand gene accessibility for transcription. Studying how the cell functions on a deeper level will provide understanding of cellular morphological changes and proliferation. This study uses the methods of optical microscopy and traction force microscopy (TFM) to image substrate deformation as well as analyze its strain profile to find where forces are interacting with the substrate the most. A 60X objective on a confocal microscope was used to image the cell membrane, nucleus, and fluorescent beads in PDMS gels of varying stiffness on which the cells were placed. Based on how the nucleus deforms as well as how the beads move due to cell-substrate interaction, a strain profile of the gel along with traction force analysis can be determined to quantify how the cell is interacting with its substrate. The results are that as the cell is trypsinized after spreading along the substrate, the focal adhesions made by the cell will disconnect, causing the beads to spread out locally around the cell. It was also found that as substrate stiffness increases, the rate of cell spreading increases. From these findings, it can be concluded that the cell responds more positively in environments of higher stiffness and spreads at a faster rate.

Motor proteins such as kinesin move along microtubules in order to transport cellular cargos throughout the cell by obtaining energy from RNA hydrolysis which allows the cell to complete the tasks needed to stay alive. In this work, we developed synthetic molecular motors using DNA enzymes (DNAzyme) and fluorescent nanomaterials which mimic the functions and structures of motor proteins. A DNAzyme-capped CdS nanoparticle and a RNA-functionalized single-walled carbon nanotube (SWCNT) were used as a walker and a track in the motor platform, respectively. As a walking mechanism, the DNAzyme cleaved the RNA substrates in the presence of metal cations. The RNA molecules were functionalized with SWCNTs using pi-pi stacking. Due to their fluorescent properties under specific light excitations, they were visualized to track the position of our motor. In addition, we studied the kinetics of molecular motors in different environments. As a result, the fastest translocation velocity was found to be 1nm min-1 and the maximum displacement was 3µm. A turnover rate of 0.025s-1 was determined by making a kinetic model based on the density of the single motor reactions. We demonstrated that the cation concentration, type of metal cation, pH, and temperature all modify the kinetics of the molecular motor. In conclusion, we developed the bio-inspired synthetic motors using DNA nanotechnology and showed how to control their movements using design of structures and modification of chemical environments. In the future, we will develop the kinetic model to analyze their kinetics and design the optimized molecular motors on purpose.

The motivation is to applying engineering knowledge to develop 3D bio-printing in inkjet printer (first stage--monolayer). To achieve the goal, there are three problems to solve. First, we have to figure out regulation of growth of target cells; inability to regulate the location and pattern of growing cells make us even unable to build 3D printer in the direct way. Second problem is how to protect of yeast cells from high temperature and viscous force when printing. The third issue is how to modify the inkjet printer especially the feeding system in order to implement printing on other materials rather than paper. We obtain inspiration that building a synthetic polarization system that include positive feedback and mutual inhibition can generate artificial PIP3 and CDC42 poles in living cells. We program the proper genetic circuit inside yeast that leading polarization in specific spatial position. After building the exogenous polarization network, we found we cannot solve the upward/downward overlap of yeast cells for any ways that difficult to form single layer. The attempt solution of protecting cells is adding certain amount of sugars and salts into medium; as a result viscosity of medium, heat conductivity will decrease meanwhile the medium would not be toxic to yeast cells. The experimental results demonstrate glucose tends to decrease the surface tension of cells that help distribute cells monolayer in the physical way. Result shows yeast cells are growing well in the pattern that being set before printing, meanwhile, they are distributed in monolayer at a high possibility just as expected, the validity supports polarization could be a feasible way in 3D tissue printing.