Chemical & Biomolecular Engineering
Permanent URI for this communityhttp://hdl.handle.net/1903/2219
Formerly known as the Department of Chemical Engineering.
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Item CO-CULTURE OF BONE MARROW STROMAL CELLS AND CHONDROCYTES FOR BONE TISSUE ENGINEERING: MICROARRAY STUDY OF CHONDROCYTE SECRETED FACTORS(2011) Janardhanan, Sathyanarayana; Fisher, John P; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue engineering refers to the assembly of biomaterials, cells and signaling molecules to develop functional tissues based on strategies derived from developmental processes. Cells play a crucial role, in that they can secrete a library of molecules, not entirely characterized in the laboratory, and yet provide repeatable results during in vitro experiments. Under conditions of co-culture with mesenchymal stem cells, the underlying biology of chondrocytes can elucidate the signal expression during the early bone development process called endochondral ossification. This interaction is tightly regulated in chondrocytes and results in the recruitment and differentiation of mesenchymal stem cells (MSCs) into osteoblasts. We executed a co-culture system, to observe the potential of alginate encapsulated bovine articular cartilage chondrocytes to induce osteogenic differentiation of bovine bone marrow stromal cells and to observe the interaction on a global scale by making use of the microarray platform. We identified certain genes expressed by chondrocytes that show substantial activity in co-culture systems such as versican (VCAN), secreted frizzled related protein 1 (SFRP1), matrix metallopeptidase 13 (MMP13), extracellular matrix protein 1 ( ECM1) and collagen type 1 ( Col1A1, Col1A2).Item Desing of Click Hydrogels for Cell Encapsulation(2011) Breger, Joyce; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The long-term stability of ionically crosslinked alginate hinders the development of a bioartificial pancreas for the treatment of Type I Diabetes. Ionically crosslinked alginate with divalent cations is traditionally utilized to encapsulate islets of Langerhans serving as a protective barrier between the host's immune system and the donor islets of Langerhans. However, due to ion exchange with monovalent ions from the surrounding serum, alginate degrades exposing donor tissue to the host's immune system. The overall goal of this dissertation was to explore the possibility of utilizing `click' chemistry to introduce covalent crosslinking in alginate for therapeutic cell encapsulation. `Click' chemistry is customarily defined as the Cu (I) catalyzed reaction between an azide and alkyne to form a 1,2,3 triazole ring. To achieve the goal of covalently crosslinked polysaccharides, the following aims were determined: (1) synthesis and characterization of functionalized polysaccharides (alginate and/or hyaluronic acid) with alkyne or azide end groups; (2) measurement and comparison of the stability and transport properties of covalently crosslinked alginate hydrogels to that of ionically crosslinked alginate hydrogels; (3) determination of the inflammatory potential and cytotoxicity of these functionalized polysaccharides and `click' reagents by employing RAW264.7, a murine macrophage cell line under various simulated inflammatory states (with or without endotoxin, with or with out the inflammatory cytokine gamma-interferon); (4) optimization of the `click' reaction for therapeutic cell encapsulation utilizing RIN-5F, a rat insulinoma cell line, while minimizing cytotoxicity and maintaining insulin production; (5) encapsulation of primary porcine islets of Langerhans in either ionically and/or covalently crosslinked alginate capsulation and comparing insulin response to a glucose challenge. The results of these experiments demonstrate the utility of employing `click' chemistry to increase the overall stability of alginate hydrogels while maintaining therapeutic cell function.Item Proteomic Analysis of Human Urinary Exosomes(2009) Gonzales, Patricia Amalia; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells. These small vesicles (40-100 nm) have been shown to be secreted by most cell types throughout the body. In the kidney, urinary exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane of renal tubular epithelia. Exosomes contain apical membrane and cytosolic proteins and can be isolated using differential centrifugation. The analysis of urinary exosomes provides a non-invasive means of acquiring information about the physiological or pathophysiological state of renal cells. The overall objective of this research was to develop methods and knowledge infrastructure for urinary proteomics. We proposed to conduct a proteomic analysis of human urinary exosomes. The first objective was to profile the proteome of human urinary exosomes using liquid chromatography-tandem spectrometry (LC-MS/MS) and specialized software for identification of peptide sequences from fragmentation spectra. We unambiguously identified 1132 proteins. In addition, the phosphoproteome of human urinary exosomes was profiled using the neutral loss scanning acquisition mode of LC-MS/MS. The phosphoproteomic profiling identified 19 phosphorylation sites corresponding to 14 phosphoproteins. The second objective was to analyze urinary exosomes samples isolated from patients with genetic mutations. Polyclonal antibodies were generated to recognize epitopes on the gene products of these genetic mutations, NKCC2 and MRP4. The potential usefulness of urinary exosome analysis was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I and using the single nucleotide polymorphism in the ABCC4 gene. The third objective was to study the normal variability between proteomes of female and male urinary exosomes, and to implement a normalization method to analyze urinary exosome samples. Only 19 proteins had a 2-fold change representing 4.9% of the total number of proteins identified which shows that there is high concordance between proteomes of urinary exosomes isolated from males and females. The normalization method, timed urine collection did not correlate as expected with the intensity signal of MVB markers, TSG101 and Alix. This research shows that the proteomic analysis of human urinary exosomes can be the basis for future biomarker studies as well as physiological studies.