Probabilistic spatial analysis in quantitative microscopy with uncertainty-aware cell detection using deep Bayesian regression

The investigation of biological systems with three-dimensional microscopy demands automatic cell identification methods that not only are accurate but also can imply the uncertainty in their predictions. The use of deep learning to regress density maps is a popular successful approach for extracting cell coordinates from local peaks in a postprocessing step, which then, however, hinders any meaningful probabilistic output. We propose a framework that can operate on large microscopy images and output probabilistic predictions (i) by integrating deep Bayesian learning for the regression of uncertainty-aware density maps, where peak detection algorithms generate cell proposals, and (ii) by learning a mapping from prediction proposals to a probabilistic space that accurately represents the chances of a successful prediction. Using these calibrated predictions, we propose a probabilistic spatial analysis with Monte Carlo sampling. We demonstrate this in a bone marrow dataset, where our proposed methods reveal spatial patterns that are otherwise undetectable

Characterization of tissue properties on the sub-micron scale in human bone by means of synchrotron radiation CT

Gesunder humaner Knochen unterliegt einem permanenten Umbau, um sich den mechanischen Anforderungen anzupassen, Mikrofrakturen zu reparieren und das Mineraliengleichgewicht zu erhalten. Dieser Umbauprozess wird durch Osteoblasten- und Osteoklastenaktivität realisiert, den knochenbildenden bzw. knochenresorbierenden Zellen. Gesteuert wird dieser Prozess durch Osteozyten, dessen Netzwerk mechanosensorische Fähigkeiten zugesprochen werden. Bisphosphonate (BP), hemmen die Osteoklastenaktivität und erhöhen somit die Knochenumsatzzeit. Im ersten Teil dieser Arbeit wurden morphologische Eigenschaften der Osteozyten-Lakunen (OL) in humanem Knochen mittels Synchrotron-µCT untersucht. Dabei wurden sowohl gesunde als auch mit BP behandelte Spender verglichen. Anschließend haben wir Synchrotron-Nano-CT in Kombination mit Phasenkontrast angewandt, um unsere Untersuchungen auf die Morphologie des lakuno-kanalikulären Netzwerkes (LKN) und die Gewebeeigenschaften in der Umgebung des LKN auszuweiten. Wir nahmen an, dass der sekundäre Mineralisierungsprozess mittels eines Diffusionsprozesses durch die Grenzfläche der extrazellulären Flüssigkeit im LKN stattfindet, was zu Gradienten der Massendichte in der Umgebung des LKN führen sollte. Unsere Untersuchungen haben gezeigt, dass sowohl in der Umgebung der OL als auch der Kanäle Massendichtegradienten existieren. Daraus schließen wir, dass der Mineralienaustausch zwischen der extrazellulären Flüssigkeit und der mineralisierten Matrix an der gesamten Oberfläche des LKN stattfindet. Wir schätzten, dass die Kapazität, unter Berücksichtigung des gesamten LKN, Mineralien auszutauschen etwa eine Größenordnung höher ist, gegenüber der Annahme, dass der Austausch lediglich an den Grenzflächen der OL stattfindet. Zukünftige Studien sollten nicht nur die peri-LKN Gewebeeigenschaften während der sekundären Mineralisierung untersuchen, sondern auch Schwankungen der Mineralienkonzentration bei hohen Kalziumanforderungen des Körpers berücksichtigen.Under healthy conditions human bone undergoes permanent remodeling to adjust to mechanical demands, to repair micro-cracks and to maintain mineral homeostasis. This process of remodeling is performed by osteoblasts and osteoclasts: bone-forming and bone-resorbing cells. The activity of osteoclasts and osteoblasts is triggered by osteocytes, the most frequently occurring type of bone cell, via mechanosensation processes. Bisphosphonates (BP) prescribed during treatment for osteoporosis or bone metastasis inhibit osteoclast activity and thus decrease the bone turnover. In this work, the distribution and morphology of osteocyte lacunae of human cortical jaw bone was investigated in 3D, and a comparison between healthy and BP-treated donors was performed using synchrotron radiation (SR) µCT. In a second approach, we used SR nano-CT with phase contrast to investigate the morphology of the canalicular network and the bone tissue properties in the vicinity of the lacuna-canalicular network of human jaw bone, originating from both healthy subjects and patients treated with BPs. We hypothesized that secondary mineralization takes place via a diffusion process through the fluid-matrix interface at both the lacunar and the canalicular surfaces. This should result in mass density gradients with respect to the distance to the pore boundary. Such mass density gradients were indeed observed at both lacunar and canalicular interfaces. We concluded that mineral exchange between extracellular fluid and mineralized matrix occurs at all bone surfaces, including the canaliculi. Our data suggested that the capacity of the pore network to exchange mineral with the bone matrix would increase by one order of magnitude if the canalicular surface is taken into account. However, more studies should be performed, targeting not only the changes of tissue properties during secondary mineralization, but also during fluctuations of mineral concentration in periods of high mineral demand

DEVELOPMENT OF NANOPARTICLE RATE-MODULATING AND SYNCHROTRON PHASE CONTRAST-BASED ASSESSMENT TECHNIQUES FOR CARDIAC TISSUE ENGINEERING

Myocardial infarction (MI) is the most common cause of heart failure. Despite advancements in cardiovascular treatments and interventions, current therapies can only slow down the progression of heart failure, but not tackle the progressive loss of cardiomyocytes after MI. One aim of cardiac tissue engineering is to develop implantable constructs (e.g. cardiac patches) that provide physical and biochemical cues for myocardium regeneration. To this end, vascularization in these constructs is of great importance and one key issue involved is the spatiotemporal control of growth-factor (GF)-release profiles. The other key issue is to non-invasively quantitatively monitor the success of these constructs in-situ, which will be essential for longitudinal assessments as studies are advanced from ex-vivo to animal models and human patients. To address these issues, the present research aims to develop nanoparticles to modulate the temporal control of GF release in cardiac patches, and to develop synchrotron X-ray phase contrast tomography for visualization and quantitative assessment of 3D-printed cardiac patch implanted in a rat MI model, with four specific objectives presented below. The first research objective is to optimize nanoparticle-fabrication process in terms of particle size, polydispersity, loading capacity, zeta potential and morphology. To achieve this objective, a comprehensive experimental study was performed to examine various process parameters used in the fabrication of poly(lactide-co-glycolide) (PLGA) nanoparticles, along with the development of a novel computational approach for the nanoparticle-fabrication optimization. Results show that among various process parameters examined, the polymer and the external aqueous phase concentrations are the most significant ones to affect the nanoparticle physical and release characteristics. Also, the limitations of PLGA nanoparticles such as initial burst effect and the lack of time-delayed release patterns are identified. The second research objective is to develop bi-layer nanoparticles to achieve the controllable release of GFs, meanwhile overcoming the above identified limitations of PLGA nanoparticles. The bi-layer nanoparticle is composed of protein-encapsulating PLGA core and poly(L-lactide) (PLLA)-rate regulating shell, thus allowing for low burst effect, protein structural integrity and time-delayed release patterns. The bi-layer nanoparticles, along with PLGA ones, were successfully fabricated and then used to regulate simultaneous and/or sequential release of multiple angiogenic factors with the results demonstrating that they are effective to promote angiogenesis in fibrin matrix. The third objective is to develop novel mathematical models to represent the controlled-release of bioactive agents from nanoparticles. For this, two models, namely the mechanistic model and geno-mechanistic model, were developed based on the local and global volume averaging approaches, respectively, and then validated with experiments on both single- and bi-layer nanoparticles, by which the ovalbumin was used as a protein model for the release examination. The results illustrates the developed models are able to provide insight on the release mechanism and to predict nanoparticle transport and degradation properties of nanoparticles, thus providing a means to regulate and control the release of bioactive agents from the nanoparticles for tissue engineering applications. The fourth objective of this research is to develop a synchrotron-based phase contrast non-invasive imaging technique for visualization and quantitative assessment of cardiac patch implanted in a rat MI model. To this end, the patches were created from alginate strands using the three-dimensional (3D) printing technique and then surgically implanted on rat hearts for the assessment based on phase contrast tomography. The imaging of samples was performed at various sample-to-detector distances, CT-scan time, and areas of the region of interest (ROI) to examine their effects on imaging quality. Phase-retrieved images depict visible and quantifiable structural details of the patch at low radiation dose, which, however, are not seen from the images by means of dual absorption-phase and a 3T clinical magnetic resonance imaging. Taken together, this research represents a significant advance in cardiac tissue engineering by developing novel nano-guided approaches for vascularization in myocardium regeneration as well as non-invasive and quantitative monitoring techniques for longitudinal studies on the cardiac patch implanted in animal model and eventually in human patients

Stereology as the 3D tool to quantitate lung architecture

Stereology is the method of choice for the quantitative assessment of biological objects in microscopy. It takes into account the fact that, in traditional microscopy such as conventional light and transmission electron microscopy, although one has to rely on measurements on nearly two-dimensional sections from fixed and embedded tissue samples, the quantitative data obtained by these measurements should characterize the real three-dimensional properties of the biological objects and not just their "flatland" appearance on the sections. Thus, three-dimensionality is a built-in property of stereological sampling and measurement tools. Stereology is, therefore, perfectly suited to be combined with 3D imaging techniques which cover a wide range of complementary sample sizes and resolutions, e.g. micro-computed tomography, confocal microscopy and volume electron microscopy. Here, we review those stereological principles that are of particular relevance for 3D imaging and provide an overview of applications of 3D imaging-based stereology to the lung in health and disease. The symbiosis of stereology and 3D imaging thus provides the unique opportunity for unbiased and comprehensive quantitative characterization of the three-dimensional architecture of the lung from macro to nano scale

Mikrokeskkonna mõju naharakkudele

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Peamised rakud inimese nahakoes on keratinotsüüdid, melanotsüüdid ja fibroblastid. Kõigil kolmel rakutüübil on oma spetsiifiline ülesanne nahakoes: tihedalt mitmekihilise struktuurina paiknevad keratinotsüüdid tagavad organismile esmase kaitse väliste keskkonnamõjude, sealhulgas patogeenide eest; pigmendisünteesi eest vastutavad melanotsüüdid; ning fibroblastid, mis on peamised rakuväliste valkude tootjateks. Kuid ülesandeid, mida naharakud peavad täitma, on kordades rohkem ning paljud neist pole veel teada või vajavad täiendavaid uuringuid. Selleks, et rakud oma spetsiifilisi funktsioone täita saaksid, on vajalik koele omase võrkja tugisüsteemi – rakuvälise maatriksi olemasolu. Väga oluline on rakkude õige funktsioneerimine tagada tehiskude arendamisel. Vajadus nahakoe siirdamiste järele on maailmas üha suurenemas. Biotehnoloogilisel teel saadud nahakoest soovitakse leida abi nii suurte põletuste, krooniliste haavandite kui ka kaasasündinud ja omandatud nahadefektide ravis. Käesoleva uurimistöö laiemaks eesmärgiks oli töötada välja nahale iseloomulike keemiliste ja füüsikaliste omadustega struktuurne materjal, mis võimalikult hästi imiteeriks füsioloogilist rakuvälist maatriksit. Selleks, et eelnevalt teada saada rakkude võimalikud ülesanded, tegime kindlaks rakkudes ekspresseeruvad geenid kogu transkriptoomi tasemel. Arendasime välja biosobivad materjalid ning hindasime neil kasvatatud naharakkude bioloogilisi omadusi – elulemust, paljunemist, morfoloogiat. Kokkuvõtteks, rakuvälise keskkonna omadused mõjutavad oluliselt rakkude kasvu, paljunemist ning morfoloogiat. Selleks, et saavutada füsioloogilisele nahale võimalikult sarnaselt funktsioneeriv nahaanaloog on lisaks keemilistele omadustele ülioluline saavutada ka koele iseloomulik struktuursus ja mehaanilised omadused.In skin tissue the predominant cell types are keratinocytes, melanocytes and fibroblasts. All these cells have specific roles to play: keratinocytes form a dense multilayer structure that acts as the primary defense mechanism of the organism against environmental influences, including pathogens; melanocytes are responsible for pigment synthesis; and fibroblasts are the main producers of extracellular proteins. However, skin cells have a plethora of functions and many of these are still unknown or require additional studies. For the cells to perform their specific functions, a net-like support structure called extracellular matrix, typical for tissues, is needed. Guaranteeing the right functioning of cells is very important in the development of artificial tissues. The need for skin transplants is steadily increasing all over the world. It is hoped that skin tissues produced by tissue engineering may help in the treatment of extensive burns, chronic ulcers and both congenital and acquired skin defects. The general aim of this study was to develop a structural material with chemical and physical properties characteristic to natural skin that would mimic physiological extracellular matrix as exactly as possible. To determine the possible functions of the cells, we performed a gene expression analysis at the whole transcriptome level. After that we developed biocompatible materials and evaluated the biological properties – survival, proliferation and morphology – of the skin cells grown on these materials. In conclusion, the properties of the extracellular environment have major impact on the growth and morphology of the cells. For a skin analogue to have a functionality very similar to physiological skin, not only its chemical properties, but also its structurality and mechanical properties should mimic those of natural tissue

Development and Applications of Advanced Ultrasound Techniques for Characterization and Stimulation of Engineered Tissues

Mechanobiology is central in the development, pathology, and regeneration of musculoskeletal tissues, in which mechanical factors play important roles. Therefore, there is a need for methods to characterize the composition and mechanical properties of developing musculoskeletal tissues over time. Ultrasound elastographic techniques have been developed for noninvasive imaging of spatial heterogeneity in tissue stiffness. However, their application for quantitative assessment of tissue mechanical properties, especially viscoelastic properties, has not been exploited. Additionally, ultrasound energy may be used to apply mechanical stimulation to engineered constructs at the microscale, and thereby to enhance tissue regeneration. We have developed a multimode ultrasound viscoelastography (MUVE) system for assessing microscale mechanical properties of engineered hydrogels. MUVE uses focused ultrasound pulses to apply acoustic radiation force (ARF) to deform samples, while concurrently measuring sample dimensions using coaxial high frequency ultrasound imaging. We used MUVE to perform creep tests on agarose, collagen, and fibrin hydrogels of defined concentrations, as well as to monitor the mechanical properties of cell-seeded constructs over time. Local and bulk viscoelastic properties were extracted from strain-time curves through fitting of relevant constitutive models, showing clear differences between concentrations and materials. In particular, we showed that MUVE is capable of mapping heterogeneity of samples in 3D. Using inclusion of dense agarose microbeads within agarose, collagen and fibrin hydrogels, we determined the spatial resolution of MUVE to be approximately 200 μm in both the lateral and axial directions. Comparison of MUVE to nanoindentation and shear rheometry showed that our ultrasound-based technique was superior in generating consistent, microscale data, particularly for very soft materials. We have also adapted MUVE to generate localized cyclic compression, as a means to mechanically stimulate engineered tissue constructs at the microscale. Selected treatment protocols were shown to enhance the osteogenic differentiation of human mesenchymal stem cells in collagen-fibrin hydrogels. Constructs treated at 1 Hz at an acoustic pressure of 0.7 MPa for 30 minutes per day showed accelerated osteogenesis and increased mineralization by 10 to 30 percent, relative to unstimulated controls. In separate experiments, the ultrasound pulse intensity was increased over time to compensate for changes in matrix properties over time, and a 35 percent increase in mineralization was achieved. We also extended the application of a previously-developed spectral ultrasound imaging (SUSI) technique to an animal model for early detection of heterotopic ossification (HO). The quantitative information on acoustic scatterer size and concentration derived from SUSI was used to differentiate tissue composition in a burn/tenotomy mice model from the control model. Importantly, HO foci were detected as early as one week after injury using SUSI, which is 3-5 weeks earlier than when using conventional micro-computed tomography. Taken together, these results demonstrate that ultrasound-based techniques can non-invasively and quantitatively characterize viscoelastic properties of soft materials in 3D, as well as their composition over time. Ultrasound pulses can also be used to stimulate engineered constructs to promote musculoskeletal tissue formation. MUVE, SUSI, and ultrasound stimulation can be combined into an integrated system to investigate the roles of matrix composition, static mechanical environment, and dynamic mechanical stimuli in tissue regeneration, as well as the interactions of these factors and their evolution over time. Ultrasound-based techniques therefore have promising potential in noninvasively characterizing the composition and biomechanics, as well as providing mechanical intervention in native and engineered tissues as they develop over time.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144116/1/xho_1.pd

Sost deficiency does not alter bone’s lacunar or vascular porosity in mice

SCLEROSTIN (Sost) is expressed predominantly in osteocytes acting as a negative regulator of bone formation. In humans, mutations in the SOST gene lead to skeletal overgrowth and increased bone mineral density, suggesting that SCLEROSTIN is a key regulator of bone mass. The function of SCLEROSTIN as an inhibitor of bone formation is further supported by Sost knockout (KO) mice which display a high bone mass with elevated bone formation. Previous studies have indicated that Sost exerts its effect on bone formation through Wnt-mediated regulation of osteoblast differentiation, proliferation and activity. Recent in vitro studies have also suggested that SCLEROSTIN regulates angiogenesis and osteoblast-to-osteocyte transition. Despite this wealth of knowledge of the mechanisms responsible for SCLEROSTIN action, no previous studies have examined whether SCLEROSTIN regulates osteocyte and vascular configuration in cortices of mouse tibia. Herein, we image tibiae from Sost KO mice and their wild-type (WT) counterparts with high resolution CT to examine whether lack of SCLEROSTIN influences the morphometric properties of lacunae and vascular canal porosity relating to osteocytes and vessels within cortical bone. Male Sost KO and WT mice (n = 6 /group) were sacrificed at 12 weeks of age. Fixed tibiae were analysed using microCT to examine cortical bone mass and architecture. Then, samples were imaged by using benchtop and synchrotron nanoCT at the tibiofibular junction. Our data, consistent with previous studies show that, Sost deficiency leads to significant enhancement of bone mass by cortical thickening and bigger cross-sectional area and we find that this occurs without modifications of tibial ellipticity, a measure of bone shape. In addition, our data show that there are no significant differences in any lacunar or vascular morphometric or geometric parameters between Sost KO mouse tibia and WT counterparts. We therefore conclude that the significant increases in bone mass induced by Sost deficiency are not accompanied by any significant modification in the density, organisation or shape of osteocyte lacunae or vascular content within the cortical bone. These data may imply that SCLEROSTIN does not modify the frequency of osteocytogenic recruitment of osteoblasts to initiate terminal osteocytic differentiation in mice

A high-throughput analysis of high-resolution X-ray CT images of stems of olive and citrus plants resistant and susceptible to Xylella fastidiosa

The bacterial plant pathogen Xylella fastidiosa causes disease in several globally important crops. However, some cultivars harbour reduced bacterial loads and express few symptoms. Evidence considering plant species in isolation suggests xylem structure influences cultivar susceptibility to X. fastidiosa. We test this theory more broadly by analysing high-resolution synchrotron X-ray computed tomography of healthy and infected plant vasculature from two taxonomic groups containing susceptible and resistant varieties: two citrus cultivars (sweet orange cv. Pera, tangor cv. Murcott) and two olive cultivars (Koroneiki, Leccino). Results found the susceptible plants had more vessels than resistant ones, which could promote within-host pathogen spread. However, features associated with resistance were not shared by citrus and olive. While xylem vessels in resistant citrus stems had comparable diameters to those in susceptible plants, resistant olives had narrower vessels that could limit biofilm spread. And while differences among olive cultivars were not detected, results suggest greater vascular connectivity in resistant compared to susceptible citrus plants. We hypothesize that this provides alternate flow paths for sustaining hydraulic functionality under infection. In summary, this work elucidates different physiological resistance mechanisms between two taxonomic groups, while supporting the existence of an intertaxonomical metric that could speed up the identification of candidate-resistant plants.</p