Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 59-60). / Similar to biological human ankle, today's commercially available powered ankle-foot prostheses can vary impedance and deliver net positive ankle work. These commercially available prostheses are intrinsically controlled. Users cannot intuitively change ankle controller's behavior to perform movements that are not part of the repetitive walking gait cycle. For example, when transition from level ground walking to descending stairs, user cannot intuitively initiate or control the amount of ankle angle deflexion for a more normative stair descent gait pattern. This paper presents a hybrid controller that adds myoelectric control functionality to an existing intrinsic controller. The system employs input from both mechanical sensors on the ankle as well as myoelectric signals from gastrocnemius muscle of the user. This control scheme lets the user to modulate the gain of command ankle torque upon push off during level ground walking and stair ascent. It also allows the user to interrupt level ground walking control cycle and initiate ankle plantar flexion during stair descent. As a preliminary study, ankle characteristics such as ankle angle and torque were measured and compared to biological ankle characteristics. Results show that the proposed hybrid controller can maintain existing controller's biomimetic characteristics. In addition, it can also recognize to a qualitative extent the intended command torque for ankle push off and user's desire to switch between control modalities for different terrains. The study shows that it is possible and desirable to use neural signals as control signals for prosthetic leg controllers. Keyword: Myoelectric control, powered prosthesis, proportional torque control / by Jing Wang. / M.Eng.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Vita. Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / The inability of articular cartilage to repair itself after acute injury has been implicated in the development of osteoarthritis. The objective of this work was to develop methods for delivering growth factors to cartilage and to test the ability of a self-assembling peptide scaffold, (KLDL)3, with or without growth factors to augment repair. Delivery methods included growth factor adsorption, scaffold-tethering, and modification of growth factor structure. (KLDL)3 was modified to deliver IGF-1 and TGF-[beta]1 to chondrocytes and bone marrow- derived stromal cells (BMSCs), respectively, by adsorption and by biotin-streptavidin tethering. This study showed that while TGF-[beta]1 can be effectively delivered by adsorption, IGF-1 can not. Additionally, while tethering these factors provided longterm sequestration, tethering did not stimulate proteoglycan production in vitro. A full-thickness, critically sized, rabbit cartilage defect model was used to test the ability of (KLDL)3 with or without chondrogenic factors (TGF-[beta]1, dexamethasone, and IGF-1) and BMSCs to stimulate cartilage regeneration in vivo. (KLDL)3 alone showed the greatest repair after 12 weeks with significantly higher Safranin-O, collagen II immunostaining, and cumulative histology scores compared to untreated contralateral controls. Ongoing studies include the evaluation of (KLDL)3 in a clinically relevant sized equine defect co-treated with micro-fracture and subjected to strenuous exercise. A fusion protein was created by adding a heparin-binding domain to IGF-1 (HBIGF- 1), converting IGF-1 from a short-acting growth factor to one that can be retained and locally delivered in articular cartilage in vivo. It was shown that HB-IGF-1 is retained in cartilage through binding to negatively charged glycosaminoglycan chains, with chondroitin sulfate the most prevalent type in cartilage. HB-IGF-1 was shown to bind adult human cartilage and to be preferentially delivered and retained in rat articular cartilage after intra-articular injection. In contrast, unmodified IGF-1 was not detectable after intra-articular injection. These results suggest that modification of growth factors with heparin-binding domains may be a clinically relevant strategy for local delivery to cartilage. Taken together, these results show that (KLDL)3 self-assembling peptide hydrogels are customizable for growth factor delivery and can promote cartilage repair in vivo. In addition, the fusion protein HB-IGF-1 is preferentially retained in cartilage tissue compared to un-modified IGF-1. / by Rachel E. Miller. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Inflammation is a major risk factor for many types of cancer, and the physiological processes involved in inflammation can contribute to many aspects of cancer development. Inflammation entails reprogramming of cell behaviors that resemble cancer, such as increased proliferation and signals for survival and migration, and it also entails production of reactive chemical species, which can damage DNA to promote genetic instability, another hallmark of cancer. While much research has been dedicated to studying the relationships between inflammation and cancer, it has been difficult to distinguish the relative contributions of modified cell behavior and de novo mutagenesis to the development of cancer. Furthermore, few studies have addressed the role(s) inflammation plays in cancer initiation versus promotion. Here, we utilized a transgenic mouse for detecting mutations in a variety of models of inflammation to parse the mechanisms by which inflammation contributes to mutations and cancer. The RaDR mouse, developed in the Engelward lab, contains a ubiquitously expressed transgene that enables detection of sequence rearrangement mutations following aberrant homologous recombination (HR). These mice also contain the Gpt-[delta] transgene for detecting point mutations and deletions, enabling unprecedented breadth and depth of possible mutation analyses in a single tissue. Our studies began by querying whether elements that regulate inflammation protect against mutagenesis in RaDR animals. We then studied RaDR mutagenesis in several models of intestinal inflammation and cancer. Together, these experiments showed that inflammation does not significantly induce de novo sequence rearrangement mutations, but it greatly increases the overall burden of mutant cells in a tissue as a result of heightened proliferation and clonal expansion. We also used the RaDR mouse model to expand upon studies of DNA repair pathway balance. DNA damage is addressed by a network of pathways, each designed to identify and repair specific types of lesions. One of the most important repair pathways for DNA damage caused by inflammation is the Base Excision Repair (BER) pathway, and we have previously found that BER intermediates can increase the frequency of mutagenic HR. Here, we expand upon that information, showing that acceleration of the BER pathway by increased expression of an initiating enzyme does not increase sequence rearrangement mutations, provided the downstream pathway can be resolved efficiently. Together, the studies described herein demonstrate that inflammation is unlikely to initiate cancer via sequence rearrangement mutations, but inflammation is a strong promoter of cancer in part through increased clonal expansion of mutant cells. / by Jennifer Elizabeth Kay. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Tissue engineering is a potentially valuable tool for clinical treatment of diseases where host tissues or organs need to be replaced. Progression of engineering metabolically complex organs and tissues has been severely limited by the lack of established, functional vasculature. The thesis work described herein focused on methods of establishing and studying specific endothelial cell types in vitro for potential applications in establishing functional microvascular architecture. To achieve these objectives, a model system of primary liver sinusoidal endothelial cells (LSEC) was initially studied due to the high metabolic requirements of the liver, as well as the unique phenotype that they possess. We were able to demonstrate that free fatty acids were able to rescue LSEC in culture, promote proliferation, and maintain their differentiated phenotype. Our work with lipid supplementation in serum-free conditions provides flexibility in engineering liver tissue with a functional vasculature comprised with relevant endothelial types encountered in vivo. Following up our work with LSEC, we explored the human dermal microvascular endothelial cell (HDMVEC) system to understand the signaling mechanisms involved in sprouting angiogenesis. Engineered tissues that are implanted will require integration with host vasculature. We established a method to collect large signaling data sets from a physiologically relevant in vitro culture system of HDMVEC that permitted angiogenic sprouting. We were able to find statistically significant data regarding how angiostatic cues like Platelet Factor 4 can modulate angiogenesis signaling pathways. Our results from working with both types of endothelial cell systems provide insight into potential methods for establishing specialized microvasculature for engineered tissues, both in propagation of differentiated endothelial cells in vitro and promotion of tissue/organ survival following their implantation. / by Ta-Chun Hang. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / The DNA in every cell accrues nearly 100,000 lesions daily from both endogenous and exogenous sources. The accumulated damage, e.g. strand breaks and base lesions, can lead to mutations, cell death, and cancer if not repaired efficiently. To protect genome integrity, organisms have evolved multiple DNA repair processes. A deeper comprehension of DNA damage and repair during disease pathogenesis can aid the development of novel therapeutics to reduce the damage and ameliorate the disease. Here, we studied DNA damage and repair in two inflammatory contexts. First, we investigated the role of DNA damage and repair during influenza infection, a common viral respiratory disease with an active inflammatory response. Second, we examined the effects of S-nitrosation, a post-translational modification that is common in inflammatory regions, on repair of alkylation damage. Influenza induces an excessive inflammatory response in the host and a reduction in inflammation reduces morbidity. While inflammation can cause DNA damage and induce DNA repair in other inflammatory contexts, there has been minimal analysis on the existence and function of DNA damage and repair during influenza infection. Utilizing immuno-fluorescent analysis of double strand break markers, we observed an increase in strand breaks both in vitro and in vivo. Influenza infected mice also displayed a significant increase in homologous recombination (HR) gene and protein expression during the recovery phase of infection in multiple virus and mouse backgrounds. Moreover, influenza infected mice deficient in DNA repair proteins AAG, ALKBH2, and ALKBH3, displayed increased morbidity and HR protein expression when compared to wild type. Together, these results raise the possibility of a role for DNA repair and more specifically HR during influenza infection. To study the effects of inflammation on DNA repair protein function, we analyzed the capacity of cells treated with S-nitrosoglutathione (GSNO), a nitrosating agent, to repair alkylation damage. GSNO-exposed cells displayed dysregulation in the activities base excision repair (BER) proteins. Following challenge with an alkylating agent, GSNO-exposed cells had an increase in repair intermediates and reduced viability, suggesting that GSNO exposure inhibits BER completion. The knowledge gained from these studies lays the groundwork for new prevention strategies and novel therapeutics. / by Marcus Curtis Parrish. / Ph. D.

Thesis: M. Eng., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages. 49-52). / Post-traumatic osteoarthritis (PTOA) is a subtype of OA associated with cartilage defects caused by traumatic joint injury. Because articular cartilage has a limited innate healing response, due to its avascular, aneural, and alymphatic nature, these defects lead to chronic degenerative joint disease if left untreated. Current treatments to repair articular cartilage generally result in fibrocartilage that is mechanically and biochemically inferior to native hyaline tissue. This has motivated the development of tissue engineering strategies for cartilage defect repair. Hydrogel approaches have shown promising results in their ability to induce chondrogenesis, proliferation, and cartilage-like matrix production, but are often very soft at early time points and at risk of damage from joint articulation. Solid scaffolds solve this mechanical problem, but often sacrifice bioactivity and integration with native tissue. In order to avoid the drawbacks of each of these approaches, we proposed a composite scaffold approach using a synthetic solid scaffold, made of bioabsorbable polyglycolic acid:trimethylene carbonate (PGA:TMC) or expanded polytetrafluoroethylene (ePTFE), loaded with a chondrocyte-seeded self-assembling peptide hydrogel, [KLDL]₃. We hypothesized that these composite scaffolds would benefit from the mechanical protection of the solid scaffolds as well as the pro-chondrogenic and proliferative effects of the KLD hydrogel, allowing chondrocytes to produce cartilage-like extracellular matrix in a protected mechanical environment. To test the potential of these composite scaffolds for use in cartilage repair, we measured cell distribution, viability, matrix production and accumulation, and static and dynamic mechanical properties. We found that cells could be evenly distributed through at least one of the solid scaffolds tested, with all showing proliferation and maintenance of viability over four-week culture. Per-cell matrix production was an order of magnitude higher than in KLD hydrogels alone. Mechanical properties of composite scaffolds appeared to be dominated by the solid scaffolds, showing that they offered mechanical protection to the soft hydrogel within. Use in a cartilage defect model showed potential for integration with native tissue given optimization of gel-casting methods. Overall, our results show that these composite scaffolds are a viable tissue engineering strategy for articular cartilage repair. / "Funded by the National Science Foundation and W.L.Gore & Associates, Inc."--Page 3. / by Jennifer R. Ibañez. / M. Eng.

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 60-64). / Oceanic cyanobacteria are amongst the most populous species on the planet and have been found in every ocean around the world. These photosynthetic organisms play a major role in the global carbon cycle. They have adapted to a number of different temperature, light, and nutrient niches. However, as important primary producers in the oceans, these organisms play a vital role which may be threatened by global climate change and pollution. As research on cyanobacterial species progresses, these organisms have been found to show promise as potential sources of biofuel, renewable energy, and agents for bioremediation. In order to utilize these organisms for future engineering applications and basic scientific research, it is important to be able to grow the organism in a stable and reproducible manner. This research characterizes the growth of Synechococcus WH8109 in the laboratory. In the laboratory, cell culture densities of greater than 109 cells/mL with a doubling time of approximately 24 hours were achieved when grown at 28'C with a 24 hour light cycle in sea water and artificial salt water media. Not only did cyanobacteria evolve long before their distant enteric cousins, but they harness nearly all of their energy through photosynthesis. The photosystem is constantly subjected to photo-oxidative damage and degradation. Interesting insight may be gained by studying this complex repair process in the bacterial counterpart to plants, prior to applying these concepts to higher order plant species. Chaperones have been implicated in this repair process. In order to better characterize the stress response of WH8109, I have also isolated the Synechococcus homologue of GroEL using anion exchange and gel filtration chromatography and sucrose gradient centrifugation. The expression levels of this chaperone were analyzed under normal and stress conditions and they have been shown to respond to heat shock and infection. / by Erika M. Erickson. / M.Eng.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. Vita. / Includes bibliographical references (pages 132-153). / The Plcsmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult due to a lack of human liver models that robustly recapitulate host-pathogen interactions in a physiologically relevant cell type. Through the application of hepatic tissue engineering concepts and techniques, this thesis sought to develop advanced models of liver-stage malaria that will allow the facile interrogation of potential antimalarial drugs in primary human hepatocytes. In the first part of this work, we established liver-stage Plasmodium infection in an engineered microscale human liver platform based on micropatterned cocultures of primary human hepatocytes and supportive stromal cells, enabling medium-throughput phenotypic screens for potential antimalarial drugs in a more authentic host cell, and demonstrated the utility of this model for malaria vaccine testing. We further hypothesized and showed that recapitulation of a more physiologically relevant oxygen tension that is experienced by hepatocytes in vivo improved infection rates and parasite growth in vitro. Next, we demonstrated the feasibility of establishing liver-stage malaria infections in human induced pluripotent stem cell-derived hepatocyte-like cells (iHLCs), thus enabling the study of host genetic variation on liver-stage malaria infection and antimalarial drug responses. We also applied recently discovered small molecules to induce further hepatic maturation, thus increasing the utility of using iHLCs for antimalarial drug development. Finally, we designed and provided a proof-of-concept for a humanized mouse model of liver-stage malaria that involves the fabrication and ectopic implantation of PEG-cryogel-based engineered human artificial livers, and can be generated in a facile, rapid and scalable fashion for future preclinical antimalarial drug testing in vivo. The results of this research represent a three-pronged approach towards engineering scalable human liver models that recapitulate liver-stage malaria infection which may ultimately facilitate antimalarial drug discovery at various stages of the drug development pipeline. / by Shengyong Ng. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 198-215). / Focal cartilage defects caused by joint injury have a limited capacity to self-repair, and if left untreated, can lead to the early onset of osteoarthritis. The current gold standard of care, microfracture surgery, induces an endogenous repair response, but typically results in poorly integrated fibrocartilage, rather than native hyaline cartilage. The objective of this thesis was to test the hypothesis that a self-assembling peptide hydrogel functionalized with chemotactic and pro-anabolic growth factors and placed into the defect during surgery could induce migration of endogenous progenitor cells into the repair tissue. Since these progenitors are naturally accessed during microfracture surgery, clinical translation of this approach could ultimately steer repair to a more hyaline-like response. Poor cartilage repair is believed to be the result of an insufficient number of progenitor cells at the defect site. We hypothesized that the addition of a single dose combination of chemotactic growth factors (such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-P 1 (TGF-[beta]1), and heparin-binding IGF-l (HB-IGF-1)) premixed into a hydrogel scaffold could stimulate bone-marrow progenitor cell migration into the hydrogel. A novel 3D gel-to-gel migration assay, using (KLDL)₃ self-assembling peptide gels, demonstrated that the combination of PDGF-BB and TGF-[beta]1 induced significant migration into the gel compared to growth-factor free controls. Importantly, these growth factors were retained in the hydrogel and exhibited a slow release over 1-2 weeks. We also hypothesized that a brief enzymatic pre-treatment of the defect site could release proteoglycans from the walls of the surrounding native cartilage in a controlled manner, and thereby create space for newly synthesized repair tissue to anchor and integrate with this adjacent host cartilage. We used an in vitro model in which a cylindrical annulus of native cartilage was pre-treated with trypsin over a 2-minute period and then filled with a chondrocyte-seeded (KLDL) ³ hydrogel ftnctionalized with pro-anabolic HB-IGF-I that had been premixed into the gel. (This procedure was deemed to be clinically tractable by collaborating equine surgeons now using this approach in parallel animal studies.) Trypsin pre-treatment depleted proteoglycan content of adjacent cartilage in a controlled manner, and HB-IGF-l was found to be delivered to the surrounding cartilage from the peptide gel. HB-IGF-I was found to stimulate matrix biosynthesis both in the surrounding cartilage and the chondrocyte-seeded KLD scaffold, and to enhance mechanical integration. We further explored the uptake and diffusive transport properties of HB-IGF-l into cartilage motivated by the need to understand the pharmacokinetics of delivery from the repair construct to surrounding cartilage. The positively charged heparin-binding domain of HB-IGF-l results in high uptake into cartilage, making it an effective method of delivering the pro-anabolic attributes of IGF-1, which in its native form would be rapidly cleared from the joint. The observed high and rapid uptake of HB-IGF-l into cartilage will enable characterization of dosing for HB-IGF-l delivery to cartilage by either intra-articular injection or from implanted hydrogel scaffolds. In summary our results show that of (KLDL)₃ peptide hydrogel scaffolds can foster growth-factor induced progenitor cell migration to increase progenitor cell recruitment into a cartilage defect. Thus, the use of a peptide gel premixed with PDGF-BB and HB-IGF-l to enhance progenitor migration into the scaffold, combined with a trypsin pre-treatment to help promote subsequent integration, is a promising strategy towards improving integrative repair. The combination of these approaches is currently being tested in an in vivo rabbit model. / by Paul Hancock Liebesny. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Recent evidence demonstrates a correlative relationship between metabolic disorders and cancer prevalence. In addition, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and killing cancer cell lines in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders still respond to the compound. Given the metabolic implications of these data and the role of metabolism in prostate cancer incidence, we evaluated resveratrol in an in vitro disease progression model of prostate cancer and found that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. Inhibiting downstream pro-survival signaling with the MEK inhibitor U0126 rescued the C4-2 cells from resveratrol-induced death, however other MEK inhibitors did not recapitulate this response. In fact, U0126 acted independently of MEK, inhibiting mitochondrial function and shifting cells to aerobic glycolysis. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we investigated the role of mitochondrial metabolism and focused on how glutamine supplementation of citric acid cycle intermediate a-ketoglutarate may be involved. Suppression of the conversion of glutamate to a-ketoglutarate with the transaminase inhibitors cycloserine and amino oxyacetate rescued C4-2 cells from resveratrol-induced death. In addition, reducing extracellular glutamine concentration in the culture medium also inhibited apoptosis. These results imply resveratrolinduced death is dependent on glutamine metabolism, a pathway dysregulated in a variety of cancers linked to oncogenic signaling. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications. / by Amy Marie Nichols. / Ph.D.