Aortic valve disease is a complex process characterized by valve interstitial cell activation, disruption of the extracellular matrix culminating in valve mineralization occurring over many years. We ...explored the function of the retinoblastoma protein (pRb) in aortic valve disease, given its critical role in mesenchymal cell differentiation including bone development and mineralization.
We generated a mouse model of conditional pRb knockout (cKO) in the aortic valve regulated by Tie2-Cre-mediated excision of floxed RB1 alleles. Aged pRb cKO animals showed significantly more aortic valve regurgitation by echocardiography compared to pRb het control animals. The pRb cKO aortic valves had increased leaflet thickness without increased cellular proliferation. Histologic studies demonstrated intense α-SMA expression in pRb cKO leaflets associated with disorganized extracellular matrix and increased leaflet stiffness. The pRb cKO mice also showed increased circulating cytokine levels.
Our studies demonstrate that pRb loss in the Tie2-lineage that includes aortic valve interstitial cells is sufficient to cause age-dependent aortic valve dysfunction.
Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells, but the ...persistence of white patches in stable disease is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine noninvasive multiphoton microscopy (MPM) imaging and single-cell RNA-Seq (scRNA-Seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, compared with nonlesional skin, some keratinocyte subpopulations are enriched in lesional vitiligo skin and shift their energy utilization toward oxidative phosphorylation. Systematic investigation of cell-to-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in nonresponders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-to-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.
Optical spectroscopy and imaging approaches offer the potential to noninvasively assess different aspects of the cellular, extracellular matrix, and scaffold components of engineered tissues. In ...addition, the combination of multiple imaging modalities within a single instrument is highly feasible, allowing acquisition of complementary information related to the structure, organization, biochemistry, and physiology of the sample. The ability to characterize and monitor the dynamic interactions that take place as engineered tissues develop promises to enhance our understanding of the interdependence of processes that ultimately leads to functional tissue outcomes. It is expected that this information will impact significantly upon our abilities to optimize the design of biomaterial scaffolds, bioreactors, and cell systems. Here, we review the principles and performance characteristics of the main methodologies that have been exploited thus far, and we present examples of corresponding tissue engineering studies.
Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers ...because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.
In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.
A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients.
Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix ...changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.
Three-dimensional (3D) in vitro cultures recapitulate key features of the brain including morphology, cell-cell and cell-extracellular matrix interactions, gradients of factors, and mechanical ...properties. However, there remains a need for experimental and computational tools to investigate network functions in these 3D models. To address this need, we present an experimental system based on 3D scaffold-based cortical neuron cultures in which we expressed the genetically encoded calcium indicator GCaMP6f to record neuronal activity at the millimeter-scale. Functional neural network descriptors were computed with graph-theory-based network analysis methods, showing the formation of functional networks at 3 weeks of culture. Changes to the functional network properties upon perturbations to glutamatergic neurotransmission or GABAergic neurotransmission were quantitatively characterized. The results illustrate the applicability of our 3D experimental system for the study of brain network development, function, and disruption in a biomimetic microenvironment.
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•3D biomimetic in vitro cultures engineered to study functional neural networks•Millimeter-scale network analysis using GCaMP6f expression and widefield microscopy•Connected, unspecialized networks form in the developing 3D neural culture•Network properties change upon pharmacological perturbations of neurotransmission
Optical Imaging; Techniques in Neuroscience; Biomedical Engineering
Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow‐derived mesenchymal stem cells (hMSCs) seeded on aqueous‐derived porous silk scaffolds. ...Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes (≈900–1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone‐like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous‐derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone‐like outcomes during dynamic cultivation.
Abstract Designing biomaterial scaffolds remains a major challenge in tissue engineering. Key to this challenge is improved understanding of the relationships between the scaffold properties and its ...degradation kinetics, as well as the cell interactions and the promotion of new matrix deposition. Here we present the use of non-linear spectroscopic imaging as a non-invasive method to characterize not only morphological, but also structural aspects of silkworm silk fibroin-based biomaterials, relying entirely on endogenous optical contrast. We demonstrate that two photon excited fluorescence and second harmonic generation are sensitive to the hydration, overall β sheet content and molecular orientation of the sample. Thus, the functional content and high resolution afforded by these non-invasive approaches offer promise for identifying important connections between biomaterial design and functional engineered tissue development. The strategies described also have broader implications for understanding and tracking the remodeling of degradable biomaterials under dynamic conditions both in vitro and in vivo.
Calcifications occur during the development of healthy bone, and at the onset of calcific aortic-valve disease (CAVD) and many other pathologies. Although the mechanisms regulating early calcium ...deposition are not fully understood, they may provide targets for new treatments and for early interventions. Here, we show that two-photon excited fluorescence (TPEF) can provide quantitative and sensitive readouts of calcific nodule formation, in particular in the context of CAVD. Specifically, by means of the decomposition of TPEF spectral images from excised human CAVD valves and from rat bone prior to and following demineralization, as well as from calcific nodules formed within engineered gels, we identified an endogenous fluorophore that correlates with the level of mineralization in the samples. We then developed a ratiometric imaging approach that provides a quantitative readout of the presence of mineral deposits in early calcifications. TPEF should enable non-destructive, high-resolution imaging of three-dimensional tissue specimens for the assessment of the presence of calcification.
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