Quantitative volumetric Raman imaging of three dimensional cell cultures

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in 3D cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy

Quantitative volumetric Raman imaging of three dimensional cell cultures

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy

Modulating Temporal and Spatial Oxygenation over Adherent Cellular Cultures

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1α. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab

Tuning Hydrogel Degradation for Cartilage Tissue Engineering

Cartilage tissue engineering using biodegradable scaffolds as carriers for cartilage cells (chondrocytes) presents a promising strategy to regenerate cartilage damaged by age, injury, or disease. State-of-the-art clinical therapies implement chondrocytes harvested from the patient, however these treatments suffer from patient-to-patient variability and ineffectiveness due to aging. Photopolymerizable poly(ethylene glycol) (PEG) hydrogel scaffolds that can be modified to permit tunable degradation present an opportunity to tailor scaffolds to the patient\u27s cells. Scaffold degradation is crucial to encourage cartilaginous matrix deposition by entrapped chondrocytes, however the rate of degradation must be matched to matrix deposition, which is a significant design challenge further complicated by the effects of age. The goal of this thesis was to characterize degradable PEG hydrogels towards developing a suitable cartilage tissue engineering platform that can be applied to a wide range of patients regardless of age. Initial work focused on bulk hydrolytically degradable scaffolds with poly(lactic acid) in the PEG crosslinks. Chondrocytes were isolated from skeletally mature and immature (adult and juvenile) bovine cartilage, and encapsulated in hydrolytically degradable scaffolds, revealing lower anabolic and higher catabolic activity of adult cells, further motivating the need to tailor scaffolds to donor age. Bulk degrading scaffolds swelled with time, increasing the network mesh size and permitting diffusive matrix loss. To improve matrix production and retention, degradable scaffolds were bioactively modified by entrapping a native matrix molecule involved in tissue assembly, and matching the culture medium to physiological osmolarity. Both strategies stimulated matrix deposition short-term but could not overcome long-term matrix loss due to bulk degradation. A new cartilage-specific enzymatically degradable hydrogel was created to encourage cell-localized degradation, by incorporating a peptide sequence into the PEG crosslinks that is degraded by chondrocyte-secreted enzymes. Enzymatically degradable scaffolds encouraged cartilaginous matrix deposition and interconnectivity. Experimental control over diffusion- and reaction dominated degradation (bulk vs. localized), both possible in enzymatically degradable hydrogels, was demonstrated using a model system where enzyme-laden microparticles simulated enzyme secretion from cells. This thesis demonstrated that enzymatically degradable hydrogels and bioactive modification are valuable tools to create a tunable degradable platform to promote tissue regeneration using cells from any patient

Demographics, Growth, and Survivorship of Spiranthes parksii Correll and Spiranthes cernua (L.) Rich are Influenced by Vertebrate and Invertebrate Herbivory and Supplemental Water During Summer Dormancy

Spiranthes parksii Correll is a rare terrestrial orchid endemic to only thirteen Texas counties, and is a federally listed endangered species in the United States due to habitat loss and fragmentation. Individual plants of S. parksii show irregular patterns of appearance aboveground and may exhibit vegetative dormancy for one or more seasons, even several years. Their transient behavior poses research difficulties and to determine effective conservation practices, the biology and ecology of the species must be assessed further. This thesis presents data for a population of S. parksii and its sympatric congener, Spiranthes cernua (L.) Rich, in eastern Grimes County that was monitored from 2014 to 2018 to determine if (1) population survival is affected by annual weather and consistency in aboveground presence as rosettes and flower stalks, (2) growth and survival are affected by the type and timing of herbivory, and (3) scheduled summertime watering events will decrease the prevalence of summer rosette dormancy and increase survival and inflorescence growth in the fall. A detailed analysis of demographics on these two species was conducted and results suggest the sample population presence is declining over time. Summer temperatures appeared to be negatively correlated with reproductive presence proportions and S. parksii flower stalk size, which indicated potential threats by climate change. Precipitation in previous and current years largely accounted for variations in rosette and reproductive proportions, and high and low precipitation thresholds possibly dictated stalk height and number of flowers. Contrary to previous research and regardless of the overall sample population decline, over 50% of the sample population flowered in three or more years during the study period, and most plants that flowered returned as rosettes each spring. Variability in seedling presence was perhaps also caused by variability of weather conditions and its effects on germination and soil moisture. Experimentally mesh-protected plants that allowed only minute invertebrate access had the greatest presence proportions at all life stages and frequently exhibited the lowest herbivory rates while plants exposed to both vertebrates and invertebrates consistently sustained the greatest herbivory. Rosette herbivory did not affect flower stalk growth except in 2015 when the study site received unusually low precipitation, which indicated negative interactive effects of weather and herbivory on plant vigor. In general, greater rosette herbivory led to a greater probability of reproductive absence. Minimum fall season herbivory rates by treatment did not coincide with maximum flower stalk growth, therefore, the timing rather than type of herbivory appeared to have a greater impact on growth. Historically small and large plants that received supplemental water in 2017 both showed reduced summer dormancy when compared to controls but reproductive growth was not affected. Results suggested that water-treated individuals were also less likely to forgo reproductive season dormancy and instead return aboveground as a fall rosette. Reproduction is considered a costly process that can diminish subsequent growth, but data here indicated that large flower stalks generally became large rosettes. Counter to expectations, there were no differences in dormancy by species yet soil around S. parksii presented lower volumetric water content, deeper claypans, and less slope than that of S. cernua. It is expected that microhabitat parameters have some influence on dormancy as large plants in this study resided on steeper slopes and exhibited a reduced tendency toward summer and fall dormancy than small plants, but results will benefit from more detailed soil analyses and the inclusion of genetic factors

Demographics, Growth, and Survivorship of Spiranthes parksii Correll and Spiranthes cernua (L.) Rich are Influenced by Vertebrate and Invertebrate Herbivory and Supplemental Water During Summer Dormancy

Spiranthes parksii Correll is a rare terrestrial orchid endemic to only thirteen Texas counties, and is a federally listed endangered species in the United States due to habitat loss and fragmentation. Individual plants of S. parksii show irregular patterns of appearance aboveground and may exhibit vegetative dormancy for one or more seasons, even several years. Their transient behavior poses research difficulties and to determine effective conservation practices, the biology and ecology of the species must be assessed further. This thesis presents data for a population of S. parksii and its sympatric congener, Spiranthes cernua (L.) Rich, in eastern Grimes County that was monitored from 2014 to 2018 to determine if (1) population survival is affected by annual weather and consistency in aboveground presence as rosettes and flower stalks, (2) growth and survival are affected by the type and timing of herbivory, and (3) scheduled summertime watering events will decrease the prevalence of summer rosette dormancy and increase survival and inflorescence growth in the fall. A detailed analysis of demographics on these two species was conducted and results suggest the sample population presence is declining over time. Summer temperatures appeared to be negatively correlated with reproductive presence proportions and S. parksii flower stalk size, which indicated potential threats by climate change. Precipitation in previous and current years largely accounted for variations in rosette and reproductive proportions, and high and low precipitation thresholds possibly dictated stalk height and number of flowers. Contrary to previous research and regardless of the overall sample population decline, over 50% of the sample population flowered in three or more years during the study period, and most plants that flowered returned as rosettes each spring. Variability in seedling presence was perhaps also caused by variability of weather conditions and its effects on germination and soil moisture. Experimentally mesh-protected plants that allowed only minute invertebrate access had the greatest presence proportions at all life stages and frequently exhibited the lowest herbivory rates while plants exposed to both vertebrates and invertebrates consistently sustained the greatest herbivory. Rosette herbivory did not affect flower stalk growth except in 2015 when the study site received unusually low precipitation, which indicated negative interactive effects of weather and herbivory on plant vigor. In general, greater rosette herbivory led to a greater probability of reproductive absence. Minimum fall season herbivory rates by treatment did not coincide with maximum flower stalk growth, therefore, the timing rather than type of herbivory appeared to have a greater impact on growth. Historically small and large plants that received supplemental water in 2017 both showed reduced summer dormancy when compared to controls but reproductive growth was not affected. Results suggested that water-treated individuals were also less likely to forgo reproductive season dormancy and instead return aboveground as a fall rosette. Reproduction is considered a costly process that can diminish subsequent growth, but data here indicated that large flower stalks generally became large rosettes. Counter to expectations, there were no differences in dormancy by species yet soil around S. parksii presented lower volumetric water content, deeper claypans, and less slope than that of S. cernua. It is expected that microhabitat parameters have some influence on dormancy as large plants in this study resided on steeper slopes and exhibited a reduced tendency toward summer and fall dormancy than small plants, but results will benefit from more detailed soil analyses and the inclusion of genetic factors

Temporally degradable collagen–mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications

Activatable cell-biomaterial interfacing with photo-caged peptides

Spatio-temporally tailoring cell–material interactions is essential for developing smart delivery systems and intelligent biointerfaces. Here we report new photo-activatable cell–material interfacing systems that trigger cellular uptake of various cargoes and cell adhesion towards surfaces. To achieve this, we designed a novel photo-caged peptide which undergoes a structural transition from an antifouling ligand to a cell-penetrating peptide upon photo-irradiation. When the peptide is conjugated to ligands of interest, we demonstrate the photo-activated cellular uptake of a wide range of cargoes, including small fluorophores, proteins, inorganic (e.g., quantum dots and gold nanostars) and organic nanomaterials (e.g., polymeric particles), and liposomes. Using this system, we can remotely regulate drug administration into cancer cells by functionalizing camptothecin-loaded polymeric nanoparticles with our synthetic peptide ligands. Furthermore, we show light-controlled cell adhesion on a peptide-modified surface and 3D spatiotemporal control over cellular uptake of nanoparticles using two-photon excitation. We anticipate that the innovative approach proposed in this work will help to establish new stimuli-responsive delivery systems and biomaterials