Optimisation and characterisation of human corneal stromal models

The native corneal structure is highly organised and unified in architecture with structural and functional integration which mediates its transparency and mechanical strength. Two of the most demanding challenges in corneal tissue engineering are the replication of the native corneal stromal architecture and the preservation of stromal cell phenotype which prevents scar-like tissue formation. A concerted effort in this thesis has been devoted to the generation of a functional human corneal stromal model by the manipulation of chemical, topographical and cellular cues. To achieve this, previously built non-destructive, online, real-time monitoring techniques, micro-indentation and optical coherence tomography (OCT), which allow for the mechanical and contraction properties of corneal equivalents to be monitored, have been improved. These macroscopic parameters have been cross-validated by histological, imunohistochemical, morphological and genetic expression analysis

Mechanical properties of collagen-based scaffolds for tissue regeneration

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 219-231).Collagen-glycosaminoglycan (CG) scaffolds for the regeneration of skin and nerve have previously been fabricated by freeze-drying a slurry containing a co-precipitate of collagen and glycosaminoglycan. Recently, mineralized collagen-glycosaminoglycan (MCG) scaffolds for bone regeneration have been developed by freeze-drying a slurry containing a co-precipitate of calcium phosphate, collagen and glycosaminoglycan. Bi-layer scaffolds with CG and MCG layers have been developed for cartilage-bone joint regeneration. The mechanical properties (Young's modulus and strength) of scaffolds are critical for handling during surgery as well as for cell differentiation. The mechanical properties of the MCG scaffolds are low in the dry state (e.g. they can be crushed under hard thumb pressure) as well as in the hydrated state (e.g. they do not have the optimal modulus for mesenchymal stem cells (MSC) to differentiate into bone cells). In addition, there is interest in extending the application of CG scaffolds to tendon and ligament, which carry significant mechanical loads. This thesis aims to improve the mechanical properties of the both CG and MCG scaffolds and to characterize their microstructure and mechanical properties. Models for cellular solids suggest that the overall mechanical properties of the scaffold can be increased by either increasing the mechanical properties of the solid from which the scaffold is made or by increasing the relative density of the scaffold. In an attempt to increase the solid properties, the MCG scaffolds with increasing mineral content were fabricated.(cont.) The mechanical properties were lower for the more highly mineralized scaffolds as a result of an increase in the number of defects such as cracked and disconnected walls. Next, we attempted to increase the mechanical properties by increasing the relative density of the MCG scaffolds. The volume fraction of solids in the slurry was increased by a vacuum-filtration technique. The slurry was then freeze-dried in the conventional manner to produce scaffolds with increased relative densities. Increasing the relative density by a factor of 3 increased the dry Young's modulus and crushing strength roughly by 9 and 7 times, respectively, allowing the dry scaffolds to withstand hard thumb pressure. The Young's modulus for the densest scaffold in the hydrated state was in the optimum range for MSC to differentiate into bone cells. Further, we attempted to improve the mechanical properties of the CG scaffold using the same technique. We were able to achieve an increase in its tensile Young's modulus in the dry state by a factor of aboutl0 times. Finally, the fraction of MC3T3 cells attaching to the CG scaffolds was found to increase linearly with the specific surface area of the scaffold, or with the number of binding sites available for cell attachment.by Biraja P. Kanungo.Ph.D

Engineering patterned and dynamic surfaces for the spatio-temporal control of cell behaviour

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Recent advancements in microfabrication technologies in combination with the use of light-responsive materials, allow to manipulate the shape of living cells in real-time in a non-invasive Spatio-temporal controlled way to introduce additional geometrically defined adhesion sites and to study relative cell behaviour. Here, the confocal laser technique is exploited for dynamically evaluate the variation over time of the tensional and morphological cell state. This method allows the precise control of specific actin structures that regulate cell architecture. Actin filament bundles, initially randomly organized in circular-shaped cells, are induced to align and distribute to form a rectangular-shaped cell in response to specific dynamic changes in the cell adhesion pattern. The changes in morphology also reflect dramatic changes in FAs distribution, cell mechanics, nuclear morphology, and chromatin conformation. The reported strategy is convenient to explore the cell-substrate interface and the mechanisms through which cell geometry regulates cell signalling in a facile and cost-effective manner and it open new routes to understand how the field of dynamic platforms should potentially contribute to unveil complex biological events such as the modulation of cell shape

Mechanics of Biomaterials

The mechanical behavior of biomedical materials and biological tissues are important for their proper function. This holds true, not only for biomaterials and tissues whose main function is structural such as skeletal tissues and their synthetic substitutes, but also for other tissues and biomaterials. Moreover, there is an intimate relationship between mechanics and biology at different spatial and temporal scales. It is therefore important to study the mechanical behavior of both synthetic and livingbiomaterials. This Special Issue aims to serve as a forum for communicating the latest findings and trends in the study of the mechanical behavior of biomedical materials

Development of high-speed two-photon microscopy for biological and medical applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (p. 135-144).Two-photon microscopy (TPM) is one of the most powerful microscopic technologies for in-vivo 3D tissue imaging up to a few hundred micrometers. It has been finding important applications in neuronal imaging, tumor physiology study, and optical biopsy. A practical limitation of TPM is its slow imaging speed (0.3 1 frames/s). We designed high-speed two-photon microscopes (HSTPMs) whose imaging speed is more than 10 times faster than traditional TPM, while their imaging depths, image contrast are comparable to those of TPM. The first high speed system is HSTPM based on polygonal mirror scanner. The scanning speed reaches 13 frames/s for typical tissues using a polygonal mirror scanner. This system is based on single-focus scanning and single-pixel signal collection. The usage of higher input power is required to compensate for the signal reduction due to higher scanning speed. However, since fluorescence signal is ultimately limited by the saturation of fluorophores due to their finite lifetimes, is the signal to noise ratio (SNR) of single focus scanning systems are also ultimately limited at high speed. This problem is circumvented in a second system based on parallelization by scanning specimens with multiple foci of excitation light and collecting signals with spatially resolved detectors. The imaging speed is increased proportional to the number of foci and similar excitation laser power per focus circumventing the problem of fluorophore saturation. However, it has been recognized that this method is severely limited for deep tissue imaging due to photon scattering.(cont.) We quantitatively measured the photon scattering effect and demonstrated that its image resolution is the same as conventional TPM but its image contrast is degraded to the faster signal decay with the increase of imaging depth. We designed a new MMM based on multi-anode photomultiplier tube (MAPMT) which utilizes the advantage of MMM in terms of parallelization but overcomes the emission photon scattering problem by optimizing the design detector geometry. This method achieved equivalent SNR as conventional TPM with imaging speed more than 10 times higher than TPM. We applied these HSTPMs to a number of novel biomedical applications focusing on studying biological problems that needs to resolve the high speed kinetics processes or or the imaging of large tissue sections with subcellular resolution to achieve the requisite statistical accuracy. In the study of transdermal drug delivery mechanisms with chemical enhancers,, large section imaging enables microscopic transport properties to be measured even in skin which is highly topographical heterogeneous. This methodology allowed us to identify the novel transport pathways through the stratum corneum of skin. In the study of tumor physiology, microvasculature in tumor tissue deep below the surface was characterized to be densely distributed and tortuous compared to that of normal tissue. The interaction of leukocyte and endothelium in tumor tissue was measured by imaging the kinetics of leukocyte interaction with blood vessel wall in tumor tissues using HSTPM. The capability of large section imaging was further applied to develop a 3D tissue cytometer with the advantages that cell-cell and cell- extracellular matrix interaction can be quantified in tissues.(cont.) The statistical accuracy of this instrument was verified by quantitatively measuring cell population ratios in engineered tissue constructs composed of a mixture of two cell subpopulations. Further, this 3D tissue cytometer was applied to screen and to identify rare recombination events in transgenic mice that carry novel fluorescent genetic reporters.by Ki Hean Kim.Ph.D

Microscopy Conference 2021 (MC 2021) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2021"

Development and applications of high speed and hyperspectral nonlinear microscopy

Nonlinear microscopy refers to a range of laser scanning microscopy techniques that are based on nonlinear optical processes such as two-photon excited fluorescence and second harmonic generation. Nonlinear microscopy techniques are powerful because they enable the visualization of highly scattering biological samples with subcellular resolution. This capability is especially valuable for in vivo and live tissue imaging since it can provide both structural and functional information about tissues in their native environment. With the use of a range of exogenous dyes and intrinsic contrast, in vivo nonlinear microscopy can be used to characterize and measure dynamic processes of tissues in their normal environment. These advances have been particularly relevant in neuroscience, where truly understanding the function of the brain requires that its neural and vascular networks be observed while undisturbed. Despite these advantages, in vivo nonlinear microscopy still faces several major challenges. First, observing dynamics that occur in large areas over short time scales, such as neuronal signaling and blood flow, is challenging because nonlinear microscopy generally requires scanning to create an image. This limits the study of dynamic behavior to either a single plane or to a small subset of regions within a volume. Second, applications that rely on the use of exogenous dyes can be limited by the need to stain tissues before imaging, the availability of dyes, and specificity that can be achieved. Usually considered a nuisance, endogenous tissue contrast from autofluorescence or structures exhibiting second harmonic generation can produce stunning images for visualizing subcellular morphology. Imaging endogenous contrast can also provide valuable information about the chemical makeup and metabolic state of the tissue. Few methods have been developed to carefully and quantitatively examine endogenous fluorescence in living tissues. In this thesis, these two challenges in nonlinear microscopy are addressed. The development of a novel hyperspectral two-photon microscopy method to acquire spectroscopic data from tissues and increase the information available from endogenous contrast is presented. This system was applied to visualize and identify sources of endogenous contrast in gastrointestinal tissues, providing robust references for the assessment of normal and diseased tissues. Secondly, three methods for high speed volumetric imaging using laser scanning nonlinear microscopy were developed to address the need for improved high-speed imaging in living tissues. A spectrally-encoded high-speed imaging method that can provide simultaneous imaging of multiple regions of the living brain in parallel is presented and used to study spontaneous changes in vascular tone in the brain. This technique is then extended for use with second harmonic generation microscopy, which has the potential to greatly increase the degree of multiplexing. Finally, a complete system design capable of volumetric scan rates >1Hz is shown, offering improved performance and versatility to image brain activity