Nonlinear Microscopy Techniques: Principles and Biomedical Applications

Nonlinear optical microscopy techniques have emerged as a set of successful tools within the biomedical research field. These techniques have been successfully used to study autofluorescence signals in living tissues, structural protein arrays, and to reveal the presence of lipid bodies inside the tissular volume. In the first section, the nonlinear contrast technique foundations is described, and also, a practical approach about how to build and combine this setup on a single confocal system platform shall be provided. In the next section, examples of the usefulness of these approaches to detect early changes associated with the progression of different epithelial and connective tissular diseases are presented

Scanning Electron Microscopy in Bone Pathology: Review of Methods, Potential and Applications

This article reviews the applications of SEM methods to human bone pathologies referring to studies made at UCL. We consider the methods which may be most suitable; these prove to be not routine in the context of most bio-medical applications of SEM. Valuable information can be obtained from a bone sample if its edges are ground flat, before making either (a) a matrix surface preparation by washing away all the cells or (b) a mineralising front preparation, by also dissolving the osteoid -for which hydrogen peroxide is recommended to produce a robust specimen. BSE contrast from a cut block surface can be used to measure bone phase volume. SE contrasts from natural surfaces (trabeculae, canals and lacunae) can be used to study forming, resting and resorbing* surfaces both qualitatively and quantitatively (*except in the case of histological osteomalacia, where the existence of osteoid will go undetected and reversal lines will be difficult to distinguish from recently resorbed surfaces). We also recommend the use of PMMA embedded bone blocks, which can be used as obtained from the pathologist, but are better embedded by a more rigorous procedure. BSE image analysis can be used to quantitate bone density fractions opening up a completely new investigative method for the future. Osteoid can be measured automatically using CL if the bone sample is block stained with brilliant sulphaflavine before embedding or if a scintillant is added to the embeddant. We give examples of observations made from a number of bone diseases: vitamin D resistant rickets, ostegenesis imperfecta; osteomalacia; osteoporosis; hyperparathyroidism; fluorosis; Paget \u27s disease; tumour metastasis to bone

Diagnosis of osteoporosis from dental panoramic radiographs using the support vector machine method in a computer-aided system

<p>Abstract</p> <p>Background</p> <p>Early diagnosis of osteoporosis can potentially decrease the risk of fractures and improve the quality of life. Detection of thin inferior cortices of the mandible on dental panoramic radiographs could be useful for identifying postmenopausal women with low bone mineral density (BMD) or osteoporosis. The aim of our study was to assess the diagnostic efficacy of using kernel-based support vector machine (SVM) learning regarding the cortical width of the mandible on dental panoramic radiographs to identify postmenopausal women with low BMD.</p> <p>Methods</p> <p>We employed our newly adopted SVM method for continuous measurement of the cortical width of the mandible on dental panoramic radiographs to identify women with low BMD or osteoporosis. The original X-ray image was enhanced, cortical boundaries were determined, distances among the upper and lower boundaries were evaluated and discrimination was performed by a radial basis function. We evaluated the diagnostic efficacy of this newly developed method for identifying women with low BMD (BMD T-score of -1.0 or less) at the lumbar spine and femoral neck in 100 postmenopausal women (≥50 years old) with no previous diagnosis of osteoporosis. Sixty women were used for system training, and 40 were used in testing.</p> <p>Results</p> <p>The sensitivity and specificity using RBF kernel-SVM method for identifying women with low BMD were 90.9% [95% confidence interval (CI), 85.3-96.5] and 83.8% (95% CI, 76.6-91.0), respectively at the lumbar spine and 90.0% (95% CI, 84.1-95.9) and 69.1% (95% CI, 60.1-78.6), respectively at the femoral neck. The sensitivity and specificity for identifying women with low BMD at either the lumbar spine or femoral neck were 90.6% (95% CI, 92.0-100) and 80.9% (95% CI, 71.0-86.9), respectively.</p> <p>Conclusion</p> <p>Our results suggest that the newly developed system with the SVM method would be useful for identifying postmenopausal women with low skeletal BMD.</p

Familial adenomatous polyposis : new insights into the craniofacial radiograph features

Tese (doutorado) — Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2020.A Polipose Adenomatosa Familial (FAP) é uma doença com padrão de herança autossômico dominante predisponente ao câncer colorretal. No Brasil, o câncer colorretal está entre as quatro neoplasias malignas mais frequentes e é o terceiro em mortalidade em ambos os sexos. Os pacientes com FAP, além de apresentarem manifestações intestinais, apresentam alterações dento-ósseas. Dentre essas, são relatados presença de osteomas, odontomas, dentes supranumerários, escleroses ósseas no complexo maxilomandibular que podem se manifestar precocemente - antes do aparecimento dos pólipos intestinais. Uma revisão sistemática da literatura demonstrou a importância da investigação de doenças sistêmicas por meio de alterações ósseas presentes em radiografias panorâmicas - rotineiramente requisitadas por cirurgiões-dentistas. Baseando em artigos incluídos na revisão, regiões de interesse foram mapeadas como pontos de referência para uma futura área de análise de índices radiomorfométricos. Alterações ósseas foram detectadas quando condiçōes sistêmicas acometiam os pacientes. Um segundo projeto demonstrou que o trabeculado ósseo mandibular de pacientes FAP, quando comparados com controles pareados, apresentou alterações micro estruturais no osso trabecular mandibular quando submetidos a análise de dimensão fractal. Numa tentativa de englobar pacientes pediátricos e adultos em países diferentes, um estudo multicêntrico foi elaborado em parceria com a Universidade de Brasília e o Mercy’s Children Hospital nos Estados Unidos. Pacientes pediátricos FAP mostraram alterações ósseas similares aos adultos. Quando esses pacientes foram comparados aos controles, os pacientes FAP apresentaram alterações no padrão trabeculado ósseo, além de alterações dentais. Esse último estudo têm como objetivo recomendar o acompanhamento odontológico periódico através de radiografia panorâmica convencional anual em pacientes FAP e nas famílias em risco. Além de enfatizar a necessidade de participação do dentista nas equipes médicas multiprofissionais que acompanham essas famílias. Assim, esse trabalho alerta e conscientiza de forma crítica, baseada em evidências, nas equipes de saúde bucal sobre a importância de investigar doenças sistêmicas, alterações ósseas e FAP nos exames radiográficos rotineiros.Familial Adenomatous Polyposis (FAP) is an autosomal dominant disorder caused by mutations in the Adenomatous Polyposis Coli gene (APC). Worldwide, colorectal cancer (CRC) is within the third most frequent malignant neoplasm. CRC ranked as the third modality associated-death with females and males. The FAP patients, in addition to present extraintestinal manifestations, also show dento-osseous alterations. These alterations are mostly associated with odontomas, osteomas, supernumerary teeth, and idiopathic osteosclerosis. These last could precede the clinical evidence of intestinal polyps. A systemic review of the literature demonstrated the importance of the systemic disease investigation through mandibular trabecular bone alterations using conventional panoramic radiographs – which are routinely prescribed by dentists in general practice. Based on the articles included in this systematic review, regions of interest were mapped and used as reference-points to investigate radiomorphometric indexes. Besides, trabecular and cortical bone alterations were possibly associated with systemic conditions. A second project demonstrated that the mandibular trabecular bone pattern in FAP patients when compared to healthy individuals, showed texture discrepancies and narrow bone alterations via the fractal dimension analysis. In an attempt to radiographically assess FAP children and adults in different locations, we developed a multicentric study in partnership with the Children’s Mercy Hospital in Kansas City, United States. Pediatric FAP demonstrated osseous alterations that were similar to the adults affected by the same disease. Compared to the healthy controls, the FAP patients, presented alterations in the trabecular bone texture of the mandible. These studies aim to recommend the annual dental follow-up on the FAP patients and families at risk using the panoramic radiograph. In addition to emphasizing the importance of a dentist collaborating in the FAP multispecialty team. Thus, our objective is to alert and create critical thinking, based on scientific evidence, in the dental health teams about the importance of the opportunistic surveillance and screening of systemic diseases and FAP extraintestinal manifestations on the routinely taken dental radiographs

Osteogenesis Imperfecta: Ultrastructural and Microanalytical Changes in Bone

Osteogenesis Imperfecta (OI) is an hereditary disease of connective tissue, characterised clinically by frequent fracture following minimal trauma. Advances in molecular biology have demonstrated type I procollagen gene mutations in OI, this has not been matched by studies in the transmission electron microscope. The aim of this study was to investigate histological and ultrastructural changes of bone collagen and microanalytical changes of bone mineral in OI. Ultrastructurally, cellular changes were observed, possibly due to the poor secretion of abnormal type I procollagen chains. Fibrils were observed associated with type I collagen, possibly representing degraded collagen or abnormal collagen formed de novo. Histomorphometry of bone osteoid type I collagen fibre diameters, demonstrated larger diameter fibrils in OI compared to normal bone, possibly indicating an altered packing of collagen molecules. An X-ray microanalysis technique was developed, and determined the molar calcium to phosphorus (Ca/P) ratio of OI cortical bone to be lower than normal. Analytical studies on bone mineral suggested an apatite lattice was maintained, despite the possible ionic substitutions resulting in a low Ca/P ratio. A transgenic mouse with a mutated type I procollagen was investigated using the methodologies applied to human OI bone. Radiography and alizarin red staining demonstrated multiple fractures. Fibrils associated with type I collagen were observed. Analytical studies correlated well with OI data, a lower Ca/P ratio was observed in transgenic mouse bone. Molecular biological results have indicated that some mutations cause substitutions for glycine, this study has shown that these appear to alter the quarternary structure of the type I collagen. Alteration in collagen architecture may change the stereochemistry and hence the nucleation and growth environment for apatite. Also, an increased opportunity for ionic substitution and adsorption of ions may result. The findings of this study point to certain abnormalities in type I collagen and mineral formation and may be associated with the increased ease and frequency of bone fractures in OI

Macro and Microfluidic Flows for Skeletal Regenerative Medicine

Fluid flow has a great potential as a cell stimulatory tool for skeletal regenerative medicine, because fluid flow-induced bone cell mechanotransduction in vivo plays a critical role in maintaining healthy bone homeostasis. Applications of fluid flow for skeletal regenerative medicine are reviewed at macro and microscale. Macroflow in two dimensions (2D), in which flow velocity varies along the normal direction to the flow, has explored molecular mechanisms of bone forming cell mechanotransduction responsible for flow-regulated differentiation, mineralized matrix deposition, and stem cell osteogenesis. Though 2D flow set-ups are useful for mechanistic studies due to easiness in in situ and post-flow assays, engineering skeletal tissue constructs should involve three dimensional (3D) flows, e.g., flow through porous scaffolds. Skeletal tissue engineering using 3D flows has produced promising outcomes, but 3D flow conditions (e.g., shear stress vs. chemotransport) and scaffold characteristics should further be tailored. Ideally, data gained from 2D flows may be utilized to engineer improved 3D bone tissue constructs. Recent microfluidics approaches suggest a strong potential to mimic in vivo microscale interstitial flows in bone. Though there have been few microfluidics studies on bone cells, it was demonstrated that microfluidic platform can be used to conduct high throughput screening of bone cell mechanotransduction behavior under biomimicking flow conditions

Novel Models to Image and Quantify Bone Drug Efficacy and Disease Progression In Vivo: Addressing the Fragility Phenotype

Bone is a composite biomaterial of mineral crystals, organic matrix, and water. Each contributes to bone quality and strength and may change independently, or together, with disease progression and treatment. Even so, there is a near ubiquitous reliance on ionizing x-ray-based approaches to characterize bone mineral density (BMD) which only accounts for ~60% of bone strength and may not adequately predict fracture risk. In a rare and severe bone disease such as osteogenesis imperfecta (OI), the hallmark genotypic and phenotypic variability makes clinical management particularly challenging. Treatment strategies rely on anti-resorptive bisphosphonates which address osteoclastic, but not osteoblastic deficiencies. Radiographic characterization of efficacy identifies structural, but not biomaterial-level alterations. Together, there is an unmet need for improved treatment strategies and means to longitudinally monitor treatment outcomes at the biomaterial-level to improve clinical management of bone disease. This thesis will describe a novel model to understand and predict individual patient treatment response to an emerging therapeutic, sclerostin antibody (SclAb) prior to clinical exposure. We then challenge the current bone imaging gold-standard with the characterization of a novel zero echo time (ZTE) magnetic resonance imaging (MRI) technique that may hold promise in identifying matrix-level and biochemical changes characteristic of OI and other diseases. SclAb has gained interest as a promising bone-forming therapeutic suggesting a novel treatment strategy through inhibition of endogenous sclerostin but effects in human pediatric OI bone remains unknown. We treated bone samples retrieved from pediatric OI patients during surgery with SclAb in vitro and quantified transcriptional response of Wnt-related genes. Results demonstrated a bone-forming response in a manner paralleling pre-clinical experience. Factors inherent to the unique phenotypic/genotypic patient profile such as the patient’s baseline cellular phenotype appear to govern response magnitude; OI patients with low untreated expression of osteoblast-related genes demonstrated the greatest magnitude of upregulation during treatment. To expand findings in vivo, we developed a novel OI xenograft model where bone was implanted into a host-derived microenvironment. The model was efficacious; bone was bioaccessible by the host and retained patient-derived bone cells throughout implantation. Treatment increased bone density and volume with a variable outcome between cortical and trabecular bone. Patients with low baseline osterix demonstrated robust human-derived osterix-expression with treatment supporting in vitro findings. The validated xenograft model can be used to establish patient-specific factors influencing treatment response suggesting a personalized medicine approach to managing OI. Characterization of treatment efficacy for OI, as well as other metabolic bone diseases, is complicated by the lack of imaging modality able to safely monitor material-level and biochemical changes in vivo. To improve upon BMD, we tested the efficacy of a 3D ZTE-MRI approach in an estrogen-deficient (OVX) model of osteoporosis during growth. ZTE-MRI-derived BMD correlated significantly with BMD measured using the gold standard, µCT, which significantly increased longitudinally over the duration of the study. Growth appeared to overcome estrogen-deficient changes in bone mass yet ZTE-MRI detected significant changes consistent with estrogen deficiency by ten weeks in cortical water, cortical matrix organization (T1) and marrow fat. Findings point to ZTE-MRI’s ability to quantify BMD in good agreement with the gold standard and detect biochemical alterations consistent with disease independent of the mineral phase suggesting its value for bone imaging. Together, results from this thesis indicate a new treatment design and non-ionizing imaging strategy to improve management of bone diseases such as OI.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149889/1/rachelks_1.pd

Phenotypic variability of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA): clinical, molecular and biochemical delineation

BACKGROUND: The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA) (OMIM 225400) is a rare inheritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1; EC 1.14.11.4) due to mutations in PLOD1. Biochemically this results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) crosslinks excreted in the urine. Clinically the disorder is characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Severe hypotonia usually leads to delay in gross motor development, whereas cognitive development is reported to be normal. METHODS: We describe the clinical, biochemical and molecular characterisation, as well as electron microscopy findings of skin, in 15 patients newly diagnosed with this rare type of Ehlers-Danlos syndrome. RESULTS: Age at diagnosis ranged from 5 months to 27 years, with only 1/3 of the patients been diagnosed correctly in the first year of life. A similar disease frequency was found in females and males, however a broad disease severity spectrum (intra- and interfamilial), independent of molecular background or biochemical phenotype, was observed. Kyphoscoliosis, one of the main clinical features was not present at birth in 4 patients. Importantly we also noted the occurrence of vascular rupture antenatally and postnatally, as well as developmental delay in 5 patients. CONCLUSION: In view of these findings we propose that EDS VIA is a highly variable clinical entity, presenting with a broad clinical spectrum, which may also be associated with cognitive delay and an increased risk for vascular events. Genotype/phenotype association studies and additional molecular investigations in more extended EDS VIA populations will be necessary to further elucidate the cause of the variability of the disease severity

Electron microscopy characterisation of in vivo collagen and mineral ultra-structures, their development and pathologies

The investigation of the nanostructure of mineralised tissues is challenging due to the intrinsic complexity of the hierarchical biocomposites and heterogeneity of biological samples. Despite numerous transmission electron microscopy (TEM) studies investigating a variety of mineralised tissues, details of the collagen-mineral ultrastructure and the origins of collagen and mineral formation are not fully understood. Analytical scanning transmission electron microscopy (STEM), combined with electron energy-loss spectroscopy (EELS), has the potential of identifying bone composition at a very high spatial resolution. We used these techniques to investigate the in vivo mineralisation process, and to probe the structural/compositional origins of bone-related pathologies at high spatial resolution. High-pressure freezing/freeze substitution (HPS/FS) methods were employed to preserve features of mineralisation process, specifically the structure and chemistry of the mineral and collagen phases and the distribution of diffusible mineral ions. The application of STEM-EELS in the biomineralisation studies is, however, limited, partially due to the absence of an EELS library of mineral and collagen standards. We developed an EELS spectra collection of biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite) so that biominerals can be identified by composition and coordination environment. For the first time, an extensive collection of all major elemental edges (phosphorus, carbon, calcium, oxygen) is presented and compared. We then used this library to characterise in vivo mineralisation processes. We examined turkey tendon, which calcifies with age, in order to understand the mineralisation process. We identified chemical and structural signatures representative of the non-, poorly and well mineralised tissues. In particular, a chemical signature of pyridine-based compounds was identified and a protocol was developed to assess changes in the nano-scale chemistry of the collagen-mineral matrix in disrupted tissues. We observed a change in the oxidation state of pyridine-based compounds in the collagen fibrils, which most likely occurs pror to nucleation. Mineral ions (calcium, phosphate) were delivered into the collagen matrix, either in the form of amorphous calcium phosphate vesicles or by diffusion from the body fluids. We are first to show in vivo that the mineral nucleated in the gap region of collagen fibrils in the form of ellipsoidal grains of amorphous calcium phosphate, which transformed into crystalline apatite with time. Inside the collagen fibril, pyridine-based compounds changed their oxidation stage prior, or during, the mineralisation process. We also compared healthy and abnormal (osteogenesis imperfecta or OI) mice tissues to reveal defects in the fibril architecture and mineral chemistry in the OI model. Abnormal tissues were capable of producing collagen fibrils with a characteristic banding pattern, typical for the normal collagen. However, the diameter of the abnormal fibrils was lower. Moreover, in OI-affected tissues, large regions of disorganised fibrils were seen. Defects in fibril formation have previously been predicted by bulk chromatographic and modelling studies. The morphology and crystallinity of mineral in healthy and abnormal tissues were similar. In contrast, a much stronger signal, characteristic of carbonate ion presence, was observed in the EELS spectra taken from the mineral in the OI tissue. More generally, the library of biominerals, identification of early in vivo mineralisation patterns, and identification of alterations in disease, demonstrate that STEM-EELS provides a method to identify chemical and structural features present within mineralising with unprecedented spatial resolution. Understanding the mechanisms of bone mineralisation and the nature of the collagen-mineral interaction will help in determining the source of bone’s toughness at the molecular level.Open Acces

Electron microscopy characterisation of in vivo collagen and mineral ultra-structures, their development and pathologies

The investigation of the nanostructure of mineralised tissues is challenging due to the intrinsic complexity of the hierarchical biocomposites and heterogeneity of biological samples. Despite numerous transmission electron microscopy (TEM) studies investigating a variety of mineralised tissues, details of the collagen-mineral ultrastructure and the origins of collagen and mineral formation are not fully understood. Analytical scanning transmission electron microscopy (STEM), combined with electron energy-loss spectroscopy (EELS), has the potential of identifying bone composition at a very high spatial resolution. We used these techniques to investigate the in vivo mineralisation process, and to probe the structural/compositional origins of bone-related pathologies at high spatial resolution. High-pressure freezing/freeze substitution (HPS/FS) methods were employed to preserve features of mineralisation process, specifically the structure and chemistry of the mineral and collagen phases and the distribution of diffusible mineral ions. The application of STEM-EELS in the biomineralisation studies is, however, limited, partially due to the absence of an EELS library of mineral and collagen standards. We developed an EELS spectra collection of biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite) so that biominerals can be identified by composition and coordination environment. For the first time, an extensive collection of all major elemental edges (phosphorus, carbon, calcium, oxygen) is presented and compared. We then used this library to characterise in vivo mineralisation processes. We examined turkey tendon, which calcifies with age, in order to understand the mineralisation process. We identified chemical and structural signatures representative of the non-, poorly and well mineralised tissues. In particular, a chemical signature of pyridine-based compounds was identified and a protocol was developed to assess changes in the nano-scale chemistry of the collagen-mineral matrix in disrupted tissues. We observed a change in the oxidation state of pyridine-based compounds in the collagen fibrils, which most likely occurs pror to nucleation. Mineral ions (calcium, phosphate) were delivered into the collagen matrix, either in the form of amorphous calcium phosphate vesicles or by diffusion from the body fluids. We are first to show in vivo that the mineral nucleated in the gap region of collagen fibrils in the form of ellipsoidal grains of amorphous calcium phosphate, which transformed into crystalline apatite with time. Inside the collagen fibril, pyridine-based compounds changed their oxidation stage prior, or during, the mineralisation process. We also compared healthy and abnormal (osteogenesis imperfecta or OI) mice tissues to reveal defects in the fibril architecture and mineral chemistry in the OI model. Abnormal tissues were capable of producing collagen fibrils with a characteristic banding pattern, typical for the normal collagen. However, the diameter of the abnormal fibrils was lower. Moreover, in OI-affected tissues, large regions of disorganised fibrils were seen. Defects in fibril formation have previously been predicted by bulk chromatographic and modelling studies. The morphology and crystallinity of mineral in healthy and abnormal tissues were similar. In contrast, a much stronger signal, characteristic of carbonate ion presence, was observed in the EELS spectra taken from the mineral in the OI tissue. More generally, the library of biominerals, identification of early in vivo mineralisation patterns, and identification of alterations in disease, demonstrate that STEM-EELS provides a method to identify chemical and structural features present within mineralising with unprecedented spatial resolution. Understanding the mechanisms of bone mineralisation and the nature of the collagen-mineral interaction will help in determining the source of bone’s toughness at the molecular level.Open Acces