Small diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties. To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture� duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion). In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue. Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies.

Polymer-Based membranes play a critical role in several industrially important gas separation processes, e.g., carbon dioxide removal from natural gas. However, an intrinsic trade-off between membrane flux (characterized by its permeability) and selectivity to one gas over the other has limited their effectiveness in practical environments. While some incremental success has been obtained by empirically developing new polymer chemistries, the best hopes for transformative improvements may require novel architectures employing predictive structure/property relationships. In this work, we develop a novel hybrid membrane construct comprised of inorganic nanoparticles grafted with polymer chains to form grafted nanoparticles. We find that the grafting architecture almost exclusively results in enhanced gas transport properties, in contrast with those expected from conventional predictions. These enhancements, found to be a result of elevated diffusion constants, are broadly tunable with the grafted chain length and leads to order of magnitude increases in gas permeability. We conjecture that the grafted polymer chains serve to impart added free volume to the composite material, which manifests itself as enhanced gas diffusion relative to the pure polymer. Indeed, multiple experimental and simulation probes verify this picture, and indicate that the free volume increases are a result of the grafted chains adopting anisotropic conformations to fill space. Building off of this finding, we systematically study the effects of the nanoparticle core size and chain grafting density, and find that both the chain length where the maximum permeability occurs, as well as the extent of the enhancement, varies depending on the relative sizes of the chains and the nanoparticle. A thorough structural analysis of the grafted nanoparticles in dilute solution as well as bulk samples indicate that the relation between the measured polymer brush height and the chain length undergoes a transition at intermediate chain lengths, similar to the observed gas permeability enhancements. Using a simple scaling approach, we show that this transition is related to the crossover from a concentrated polymer brush with higher order scaling to a semi-dilute brush where the chains are more ideal. We hypothesize that this impenetrable concentrated brush phase is the source of the added free volume, and that this effect is diminished when the grafted chains are longer than the transition point and the penetrable, semi-dilute polymer brush begins to dominate gas diffusion. When cast in the framework of free volume theories, this prediction accurately captures the trends in gas diffusion; the result is a unique structure/property relation that can be used to design optimal membrane materials. We expand on these constructs to probe other grafted nanoparticle-based architectures incorporating free polymer chains and advanced chemistries to further manipulate the gas transport properties of these mixed-matrix materials. The addition of free chains with judiciously chosen molecular weights and loadings gives a nearly independent means to tune membrane selectivity, which when combined with the intrinsic permeability increases in the matrix-free grafted nanoparticles results in superior materials that can exceed the current performance Upper Bound. We relate this result to the spacial distribution of the free chains throughout the grafted polymer corona, and how this affects the distribution of the free volume in the material as it selectively cuts off larger gas molecules. We further leverage this universal grafting platform by grafting polymer chains with novel chemistries to design membranes with record-setting selectivities while also increasing permeability by nearly two orders of magnitude. We conclude that grafted nanoparticle constructs allow for precise and predictive control of gas transport properties through a new structure/property relation, and serve as a novel material design platform with the potential to function as high performance gas separation technologies.

Homing- und Chemokin-Rezeptoren können wichtige Informationen liefern über Aktivierungsstand und Organspezifität der einzelnen Zelle, aber auch über Homöostase und Gleichgewicht von T-Zellen untereinander. Mittels FACS-Analyse konnte in murinen Transplantationsmodellen durch die Hochregulation einzelner Homing-Rezeptoren auf T-Zellen die Entwicklung einer aGvHD vorhergesagt werden. Ob sich ein solches diagnostisches Fenster auch beim Menschen darstellen könnte, sollte in einer klinischen Pilotstudie untersucht werden. Zu diesem Zweck wurde der Expressionsverlauf von 19 verschiedenen Aktivierungsmarkern, Chemokin- und Homingrezeptoren auf T-Zell-Subpopulationen bei allogen transplantierten Patienten charakterisiert. Anschließend wurde versucht, eine Assoziation zwischen der Höhe der jeweiligen Rezeptorexpression und der späteren Entwicklung einer akuten Graft-versus-Host-Disease herzustellen. Die Expression der meisten untersuchten Rezeptoren nahm im Zeitverlauf ab und war insgesamt nur schwach ausgeprägt. Dabei zeigte sich insbesondere im frühen Verlauf nach SZT eine hohe relative Expression dieser Marker bei Patienten ohne aGvHD verglichen mit Patienten mit aGvHD im Verlauf. Insofern scheint eine hohe relative Expression dieser Rezeptoren kein Hinweis auf eine gesteigerte Alloreaktivität der jeweiligen T-Zellen zu sein. Gleichzeitig schien die absolute Anzahl an rezeptorpositiven Zellen eher positiv mit einer aGvHD korreliert zu sein. Die erhöhte Anzahl rezeptorpositiver Zellen errechnete sich allerdings aus der höheren absoluten Anzahl aller T-Zellen bei Patienten mit aGvHD. Damit war diese nicht rezeptorspezifisch, so dass sich Rezeptoren vom Expressionstyp 1 nicht eignen, um in der klinischen Routine eine aGvHD vorherzusagen. Einige Rezeptoren waren auf T-Helfer-Zellen auf mittlerem Niveau konstant exprimiert. Für manche von diesen zeigten sich unterschiedlich hohe relative Expressionsniveaus vor GvHD-Beginn bei Grad 2-4 verglichen mit Grad 1. Die absolute Anzahl rezeptorpositiver Zellen war bei allen Rezeptoren bei aGvHD Grad 2-4 höher als bei Grad 1. Die Expression einiger Marker war auf T-Zellen ausgeprägt, aber im Zeitverlauf fluktuierend. Für die Expression von beta 7 integrin allein und auch für dessen Koexpression zusammen mit CD 49d alpha 4 deuteten sich bei Patienten mit aGvHD Grad 2-4 höhere relative und absolute Werte an als für Patienten mit aGvHD Grad 1. Die Expression der übrigen hoch exprimierten Rezeptoren schien nicht vom Ausmaß der später einsetzenden aGvHD beeinflusst zu werden. Ob durch FACS-Analyse der Rezeptoren CLA, CCR 4, CD 45RA und/oder alpha 4 beta 7 allein oder in Kombination tatsächlich die alloreaktiven T-Zellen der aGvHD charakterisiert und quantifiziert werden können, muss an einem größeren Patientenkollektiv untersucht werden. Da zum jetzigen Zeitpunkt der prädiktive Wert dieser Marker weder postuliert noch ausgeschlossen werden kann, erscheint eine weitergehende Untersuchung sinnvoll. / Homing and chemokine receptors can provide important information about the activation and organ specifity of the cell itself as well as about homeostasis and equilibrium among t cells in general. In murine transplantion models, FACS analysis of t cells could detect the upregulation of homing receptors on t cells prior to the clinical onset of aGvHD. We tried to invesigate if such a diagnostic window could also be found in a clinical setting. Therefore we characterized the expression of 19 homing and chemokine receptors on t cells subsets in allogeneicly transplantated patients. We then looked for any association between the amount of the very receptor expression and the later development of aGvHD. The expression of most studied receptors was decreasing over the course of time and overall low. Especially in the first weeks after alloHCT, a high percentage of receptor expressing cells could be seen in patients without aGvHD compared to patients who developed aGvHD though. Therefore a relatively high expression of these receptors during lymphopenia did not seem to be associated with alloreactivity of the t cells. At the same time, an increased amount of receptor positive cells seemed to correlate positively with aGvHD. Yet, this was because patients with aGvHD showed overall higher rates of leukocytes and lymphocytes than the controls. The effect did not seem to be receptor specific so that receptors of the expression type 1 do not qualify for predicting aGvHD in clinical routine. Some receptors were constantely expressed on a medium level on helper t cells. A part of those showed higher expression levels when patients developed aGvHD grade 2-4 compared to patients with milder grade 1. The absolute amount of receptor positive cells was higher in severe aGvHD in all markers classified as expression type 2. Type 3 is characterized by an overall high but fluctuating homing receptor expression on t-cells. The receptors beta 7 integrin and CD 49 d alpha 4 were upregulated before patients developed severe aGvHD grade 2-4. The expression of the other type 3 receptors didn't seen to be influenced by later aGvHD. Further investigation in a larger cohort is needed to test the value of CLA, CCR4, CD 45 RA, beta 7 integrin and CD 49 d alpha 4 as predictive aGvHD biomarkers.

The cost of new cultivar development is high due to long juvenile periods and large tree size in tree fruit breeding programs. For apples, sour cherries, and saskatoon berries, grafting seedling scions onto the tips of branches of mature plants was hypothesized to shorten the juvenile period and reduce land use under the Canadian prairie conditions. For apples, a tip grafting system (tip grafting onto mature crabapple rootstocks) was compared with the traditional grafting system (grafting onto young Ottawa 3 rootstocks). Apple scions of Golden Delicious, McIntosh, and SK Prairie Sun which exhibit a range of inherent cold hardiness, were grafted in the spring of 2001. Over a two year period, winter survival of the scions was improved by 37% by the tip grafting system as compared to the traditional grafting system making it not feasible for evaluation of cold hardiness of scions. Vegetative growth of scions approximated the rootstocks on which the scions were grafted. Winter survival was highly correlated with shoot growth cessation (r = +0.83) and terminal bud stage (r = +0.85) observed around the time of first frost. Juvenile seedlings of saskatoon berry and sour cherry hybrids were tip grafted onto mature plants of their own species in the spring of 2000. After two growing seasons, the tip grafting system in sour cherries had reduced flowering by 69.7%, shoot length by 84%, and shoot diameter by 76% compared with the juvenile seedlings on their own roots (scion donors). Tip grafting saskatoon berry seedlings increased flowering by 68%, shoot length by 257%, and shoot diameter by 42% compared with scion donors. For sour cherries, the tip grafting system reduced winter dieback by 99.6%, hastened terminal bud development and leaf drop compared with the scion donors. Tip grafting of saskatoon berry seedlings had little effect on terminal bud development and cold hardiness of scions perhaps due to the cold hardy character of this species. For apples and sour cherries, the tip grafting system tested in this study enhanced cold hardiness of scions when combined with the appropriated rootstocks and may be useful for maintaining germplasm that otherwise would not be hardy in northern locations.

The cost of new cultivar development is high due to long juvenile periods and large tree size in tree fruit breeding programs. For apples, sour cherries, and saskatoon berries, grafting seedling scions onto the tips of branches of mature plants was hypothesized to shorten the juvenile period and reduce land use under the Canadian prairie conditions. For apples, a tip grafting system (tip grafting onto mature crabapple rootstocks) was compared with the traditional grafting system (grafting onto young Ottawa 3 rootstocks). Apple scions of Golden Delicious, McIntosh, and SK Prairie Sun which exhibit a range of inherent cold hardiness, were grafted in the spring of 2001. Over a two year period, winter survival of the scions was improved by 37% by the tip grafting system as compared to the traditional grafting system making it not feasible for evaluation of cold hardiness of scions. Vegetative growth of scions approximated the rootstocks on which the scions were grafted. Winter survival was highly correlated with shoot growth cessation (r = +0.83) and terminal bud stage (r = +0.85) observed around the time of first frost. Juvenile seedlings of saskatoon berry and sour cherry hybrids were tip grafted onto mature plants of their own species in the spring of 2000. After two growing seasons, the tip grafting system in sour cherries had reduced flowering by 69.7%, shoot length by 84%, and shoot diameter by 76% compared with the juvenile seedlings on their own roots (scion donors). Tip grafting saskatoon berry seedlings increased flowering by 68%, shoot length by 257%, and shoot diameter by 42% compared with scion donors. For sour cherries, the tip grafting system reduced winter dieback by 99.6%, hastened terminal bud development and leaf drop compared with the scion donors. Tip grafting of saskatoon berry seedlings had little effect on terminal bud development and cold hardiness of scions perhaps due to the cold hardy character of this species. For apples and sour cherries, the tip grafting system tested in this study enhanced cold hardiness of scions when combined with the appropriated rootstocks and may be useful for maintaining germplasm that otherwise would not be hardy in northern locations.

No description available.

No description available.

To study the pathogenesis and preventive strategies of acute and chronic graft-versus-host disease (aGVHD, cGVHD) after hematopoietic stem cell transplantation (HSCT), murine models of aGVHD and cGVHD were constructed. In addition, the role of CD4+CD25+ regulatory T cells (Tregs) in GVHD was also investigated in these models. My project consisted of three parts, including MHCI,II mismatched (part 1) and haploidentical BMT(part2) based aGVHD, and MHC matched, minor histocompatibility antigen (miHA)mismatched cGVHD(part 3). In the first model, aGVHD resulting from an MHC I, II mismatched aGVHD (B6(H-2b)→BALB/c(H-2d)) HSCT was studied, particularly with respect to the role that CD4+CD25+Tregs played. The results showed that CD4+CD25-T-cells induced more severe aGVHD than CD4+ T-cells, resulting in more extensive target organ lesions, especially in colon. The possible mechanism might be due to the enhanced proliferation and differentiation towards pathogenic Th1 cells. In the second model, haploidentical (B6(H-2b)→[C57BL/6×CBA/Ca]F1(H-2b×k)) HSCT was used to studied the role of CD4+or CD8+T-cells in aGVHD.Both high dose of donor CD4+-and CD8+-T-cells have the ability to induce lethal aGVHD in the hosts. However, the clinical and histological features of aGVHD induced by CD4+T-cells were significantly different from that induced by CD8+T-cells. Both donor CD4+-and CD8+-T-cells showed marked proliferation and differentiation towards CD4+IFN-γ+Th1and CD8+IFN-γ+cells, respectively. Polyclonalexpanded freshly isolatednTregs (exp-nTregs) showed obvious proliferation, increased apoptosis, and rapid loss of Foxp3 expression with impaired suppressive function. Exp-nTregs were further investigatedfor their preventive function in aGVHD in this haploidentical HSCT model. The results showed that exp-nTregs were capable of attenuating either CD4+-or CD8+-T-cell-induced aGVHD with significantly prolonged survival rate. In the third model, cGVHD was investigatedin DBA/2 (H-2d)→BALB/c (H-2d) HSCT, where the biologic readout was proteinuria and skin fibrosis. The results showed that donor CD4+T-and B220+B-cells were the main effectors in the pathogenesis of cGVHD. Notably, a more active germinal center (GC) reaction existed in the cGVHD cohorts compared with the control syngeneic cohort. Furthermore, Tfh and GC B-cells were shown to have originated from donor CD4+T-and B220+B-cells, respectively. Importantly, Tfh and GC B cells were mutually stimulatory and inter-dependent. In conclusion, three murine models were used to investigate aGVHD and cGVHD. The results showed that Tregs played a significant suppressive role in aGVHD complicating haploidentical HSCT. Furthermore, a hyperactive germinal center reaction might be the main cause of cGVHD. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The aim of this study was to test the application of an in situ–formed synthetic polyethylene glycol (PEG) as a biodegradable membrane with a variety of graft materials and hyperbaric oxygen (HBO) for enhanced bone regeneration. Critical-sized rabbit calvarial defects were created in bilateral parietal bones. Group 1 served as a control with unfilled defects, Group 2 had defects filled with morcelized autogenous bone, and Group 3 had defects filled with biphasic calcium phosphate. One defect was protected PEG membrane and half the animals were subjected to HBOT treatment. The unsupported membrane didn’t produce the desired bone regeneration in the unfilled and bone grafted groups. HBO didn't ameliorate the bone grafted or ceramic filled defects over the 6-week time period. Caution is recommended with the membrane over unsupported defects. Future assessments with HBO should be completed at the 12-week time point.

Objective: The purpose of this study was to compare the accuracy of cone beam CT (CBCT) imaging with digital orthopantomograms for determining bone quantity in the mandibular ramus. Methods: Twenty-nine cadaveric mandibles marked bilaterally with three fiducial markers were imaged using both CBCT and digital orthopantomography. After sectioning, four cross sectional measurements were made on the specimens and on the CBCT images. Two corresponding linear measurements were made on the orthopantomograms. Statistical analysis was used to compare the CBCT and orthopantomogram measurements with measurements from the anatomic specimens. Results: CBCT measurements were found to significantly differ from those made on the anatomic specimens (P<0.05). Linear measurements from the orthopantomograms varied by 15.9 percent compared to the anatomic specimens. Conclusion: CBCT and orthopantomogram measurements were significantly different from those of the anatomic specimens suggesting inaccuracies in the radiographic technology or a lack of precision in landmark identification.