Biology subjects

Sommerdijk, N.

Publications and source records attributed to Sommerdijk, N..

9 recordsLinked to original sources

bioRxiv2025

Overglycosylation introduces local changes in triple helix alignment in collagen type I fibril structure

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.

biochemistry↗

bioRxiv2025

Combined Raman Microscopy and Transmission Electron Microscopy shows the co-existence of whitlockite crystals and carbonated hydroxyapatite-mineralized collagen fibrils in human calcified aortic valves

Aortic valve stenosis (AS), the leading valvular disease in aging populations, is driven by complex extracellular matrix (ECM) modifications and progressive calcification. Despite its clinical significance, the molecular mechanisms underlying AS remain poorly understood, limiting therapeutic options to invasive valve replacement. This study employs Raman microscopy, a label-free imaging technique, and its integration with transmission electron microscopy (TEM) to elucidate the biochemical and ultrastructural changes in stenotic aortic valves. High-resolution spectroscopic imaging revealed distinct extracellular matrix modification across different regions, involving elastin degradation, cholesterol deposition, and mineral formation, comprising not only hydroxyapatite (cHAp) but also whitlockite. Elastin-rich domains associated with cHAp deposition exhibited cross-link degradation, while collagen matrices supported mineralized phases with varying mineral-to-matrix ratios that, in heavily mineralized regions, went far above those of mature human bone. For the first time we demonstrated whitlockite as a mineral deposit in calcified aortic valves, in areas with different degrees of calcification. This implies that this Mg containing mineral, which has been considered a precursor to cHAp in pathological calcification, forms independently, challenging prevailing models of calcification. The combination of Raman and TEM showed how bone-like cHAp mineralized collagen matrix in later stages engulfs the initial non-physiological whitlockite deposits. This correlative multimodal approach advances our understanding of AS by capturing spatially resolved chemical and structural dynamics at the nanoscale. The findings highlight Raman microscopys potential for probing calcification mechanisms across diverse tissue types and suggest its role in identifying novel therapeutic targets. This study underscores the value of integrative imaging methodologies in unraveling complex pathological processes and advancing patient care.

biochemistry↗

bioRxiv2025

Two Transport Routes for Fetuin-A-based Mineral Complexes in Osteogenic Matrix Mineralization

Living organisms must transport calcium and phosphate at high concentrations to enable bone formation without triggering uncontrolled mineral precipitation. Fetuin-A binds calcium phosphate to form soluble calciprotein complexes, but how these complexes contribute to physiological mineralization has remained unclear. Here we show that Fetuin-A-based mineral complexes exist in functionally distinct states that determine mineral bioavailability. Using cryogenic and liquid-phase electron microscopy, biochemical analysis, and osteoblast cell cultures, we demonstrate that small, chemically labile calciprotein monomers directly mineralize collagen fibrils, whereas larger, chemically matured primary calciprotein particles cannot. Instead, these particles require cellular uptake and lysosomal processing to release mineral for matrix deposition. This functional divergence arises from a irreversible chemical transformation of the mineral phase that drives the primary CPP assembly. Together, our findings establish nanoscale chemical maturation as a key control point that separates mineral transport from mineralization, reframing our understanding both physiological bone formation and pathological calcification.

biochemistry↗

bioRxiv2024

Development of an Organ-on-a-Chip for Correlative Microscopy: Visualizing Early Osteogenesis in 3D with High Resolution

Correlative microscopy approaches offer powerful means to study tissue development across spatial scales, but combining 3D light and electron imaging remains technically challenging. Here, we present a practical workflow that integrates organ-on-a-chip culture with longitudinal fluorescence imaging and volume electron microscopy. By modifying an existing chip platform designed for aligned tissue growth, we demonstrate the feasibility of extended 3D live imaging and subsequent high-pressure freezing of intact microtissues. Fluorescence-guided targeting enables focused ion beam/scanning electron microscopy (FIB/SEM) of selected regions, revealing ultrastructural features such as cellular organization, collagen alignment, and matrix mineralization. While not aimed at new biological discoveries, this study highlights the compatibility and potential of this pipeline for future high-resolution, multiscale studies of tissue morphogenesis and pathology in controlled microenvironments.

bioengineering↗

bioRxiv2023

Osteoblast-induced collagen alignment in a 3D in vitro bone model

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized by hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we developed a 3D cell culture system in which osteogenic cells deposit an oriented collagen matrix, that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenic cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration towards the periphery of the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenesis and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

cell biology↗

bioRxiv2023

Unravelling the relationship between isolated bone matrix vesicles and forming mineral at the nanometer scale

Matrix vesicles (MVs) are involved in the initial deposition of hydroxyapatite (HAp) during bone mineralization, but their mechanism of action is not yet fully understood. In vitro studies propose two pathways by which MVs can trigger HAp precipitation: the first is mediated by their enhanced phosphatase activity, and the second suggested to depend on structural components present in MVs to mediate nucleation directly from soluble ions without the requirement of phosphatase activity. However, the relevance of these two pathways for bone mineralization and the relationship between MVs and forming mineral in such in vitro experiments remains unclear. Here, we used near-native cryoTEM nanoscale imaging in combination with bulk characterizations to disentangle the content and action of MVs during in vitro mineralization. We show that MVs isolation by conventional ultracentrifugation results in heterogeneous dispersions containing non-vesicular particles, including collagens and proteoglycans, in addition to bilayered vesicles. The separation of phosphatase-enriched MVs from non-vesicular particles and comparative mineralization experiments demonstrated that the ability of MVs to induce fast mineralization, independently of phosphatase activity, depends on the presence of non-vesicular particles. Therefore, we conclude that the primary pathway by which MVs trigger mineralization is through the action of their resident phosphatase enzymes, with the direct mineral nucleation to be a secondary event consequential of their membrane components. Lastly, we observed mineral formation restricted to the extravesicular space or in close proximity to the membrane interface, suggesting that the relationship between MVs and forming mineral is more intricate than previously understood.

biophysics↗

bioRxiv2023

A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

Liquid phase electron microscopy (LP-EM) has emerged as a powerful technique for in-situ observation of material formation in liquid. However, monitoring these processes requires the repeated interaction of the electron beam with the aqueous environment leading to the decomposition of water molecules (radiolysis), which affects the formation processes under investigation. Graphenes ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. Using the strategy, the process of interest is initiated when the graphene liquid cells (GLCs) are sealed. This means that the process cannot be imaged at early time points since microscope preparation and initiation of image acquisition at the region of interest are time-consuming. Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the most significant limitations of the graphene liquid cells, while combining the advantages of fluorescence and electron microscopy. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at a selected time point. We demonstrate the workflow first by observing multiple day crystallization processes and highlight its potential by observing a biological process: the complexation of calciprotein particles. With this observation, we show the exciting possibilities for LP-EM in biology.

biochemistry↗

bioRxiv2022

A 3D CELL-FREE BONE MODEL SHOWS COLLAGEN MINERALIZATION IS A PHYSICOCHEMICAL PROCESS DRIVEN AND CONTROLLED BY THE MATRIX

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization and different degrees of mineralization where the intrafibrillar and extrafibrillar mineral is intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization, and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, we demonstrate the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement. We then explore this cell-free in vitro system as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicates that glycosaminoglycans play a more prominent role than generally assumed in the matrix-mineral interactions. Our experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.

biochemistry↗

bioRxiv2020

An Organoid for woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in vivo osteocytes the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under influence of external triggers (mechanical stimulation, drug administration).

bioengineering↗