Biology subjects

Grandfield, K.

Publications and source records attributed to Grandfield, K..

10 recordsLinked to original sources

bioRxiv2026

Functionalization of Gold Surfaces with Dithiobis(succinimidyl propionate) for Immobilization of Fetuin-A and Assessment of the Attachment and Proliferation of Osteoblast-like Cells

Surface functionalization of biomaterials enables the immobilization of proteins and other molecules and can be utilized to direct the biological response to devices and implants. Fetuin-A is a blood plasma protein involved in numerous physiological processes, including the regulation of mineralization. Notably, many investigations of fetuin-A have explored its cellular interaction when in solution, but limited studies report the role of fetuin-A when used as a surface modifier. The present investigation explores the response elicited by fetuin-A on Saos-2 cells when it is immobilized on a model gold surface through the covalent reaction with dithiobis(succinimdyl propionate) (DSP). Comparative surface characterization using x-ray photoelectron spectroscopy (XPS), atomic force microscopy - infrared spectroscopy (AFM-IR) and surface plasmon resonance (SPR) confirmed the surface modifications but indicate partial inhomogeneity in the functionalizer surface coverage. The interaction of albumin and fetuin-A with the surface was quantified by radiolabeling, quartz crystal microbalance with dissipation (QCM-D) and SPR, demonstrating a higher mass of fetuin-A bound to the surface in comparison to serum albumin. Over 7 days, cells bound to the surfaces with immobilized fetuin-A showed significantly hindered proliferation of osteoblast-like cells compared to the positive control (fibronectin), presumably due to a decrease in cell metabolism. This study provides new insights into the role of fetuin-A in regulating Saos2 cell response and elucidates its potential use in combination with chemical functionalizers for biomedical applications requiring surface modification.

bioengineering↗

bioRxiv2025

The Role of Fetuin-A on the Attachment and Proliferation of Osteoblast-like Cells on Model Gold Surfaces

Fetuin-A is a plasma protein of interest for bone-interfacing applications due to its role in mineralization processes through calcium/phosphate ion-binding capabilities. However, the role of fetuin-A in the initial stages of cellular interaction with biomaterials and the mechanisms involved are not fully clear. This work investigated the response of osteoblast-like Saos-2 cells to model gold substrates presenting pre-adsorbed fetuin-A as a surface modification, to determine the role of the protein in cell attachment and proliferation. Correlative quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance, and radiolabeling confirmed fetuin-A adsorbed on model surfaces in similar quantities compared to serum albumin but formed a less packed layer with increased water entrapment. Surfaces presenting pre-adsorbed fetuin-A enhanced cellular adhesion, similar to fibronectin, but attached cells displayed morphological characteristics more similar to those with pre-adsorbed albumin, with lower average surface area and maximum axis. Over 3 days, fetuin-A exhibited lower cellular proliferation compared to the fibronectin control, likely correlated to the decrease in cellular metabolism observed at the same time-point, and persisted over 7 days. These results provide insight into the role of adsorbed fetuin-A for bone-interfacing implant applications, suggesting the pre-adsorption of the protein alone aids cellular attachment, but is not sufficient to promote early stages of osseointegration.

bioengineering↗

bioRxiv2025

Real-Time Visualization of Calcium Phosphate Formation on Titanium Dioxide Nanoparticles Using Liquid Transmission Electron Microscopy

The integration of titanium dioxide (TiO2) with calcium phosphate (CaP)-based hydroxyapatite (HAP) is a promising strategy for enhancing the bioactivity of bone implants. However, a fundamental understanding of the interfacial reactions governing CaP mineralization on TiO2 remains limited due to lack of characterization techniques with sufficient spatial and temporal resolution in hydrated state. In this study, we combined in situ liquid TEM imaging and correlative ex situ TEM analyses to investigate the nucleation, aggregation, and crystallization of the CaP layer on TiO2 nanoparticle surfaces. Our findings reveal a three-step mineralization process: (1) initial aggregation of TiO2 nanoparticles in solution, (2) formation of an amorphous calcium phosphate-like (ACP-like) layer on the TiO2 surface, leading to an ACP-like coated TiO2 nanoparticle structure, and (3) progressive crystallization of ACP into HAP, forming a HAP-like coated TiO2 nanoparticle structure. Liquid-TEM imaging captured dynamic transformations, including nanoparticle aggregation, structural evolution, and phase transitions, providing unprecedented insights into the physicochemical interactions underlying mineralization. Additionally, we evaluated the effects of electron beam exposure on TiO2 nanoparticles, demonstrating that high electron flux densities can induce morphological instability. This study advances our understanding of CaP-TiO2 interfacial mineralization and offers valuable guidance for optimizing bioactive coatings to improve osseointegration and the long-term stability of bone implants.

bioengineering↗

bioRxiv2025

3D Focused Ion Beam Microscopy of Fossilized Albertosaurus sarcophagus reveals Nano to Microscale Structures

Osteohistological investigations of fossilized bone can reveal details about the specimens biological, geological and environmental conditions. Micro-to-nanoscale imaging provides insight into the structural organization of bone and can also reveal indicators of the fossilization process. We examined a petrographic thin section of the left fibula of a [~]71.5 million-year-old Albertosaurus sarcophagus (Canadian Museum of Nature [CMN] catalogue number FV 11315) using nanoscale scanning electron microscopy (SEM) and focused ion beam (FIB)-SEM tomographic imaging to study the arrangement of mineral and organic components of fossil bone in multidimensions. Here, we present evidence of permineralization in Haversian canals by energy dispersive X-ray spectroscopy. Nanoscale 3D FIB-SEM imaging revealed that the characteristic 67 nm banding periodicity of collagen fibrils was remarkably well preserved over 70M years, and 3D imaging allowed for the detection of collagen fibril bundles in parallel fibered and lamellar bone arrangements. A newly discovered structure in modern bone, the ellipsoidal mineral cluster, was tiled throughout the 3D space of fibrolamellar fossil bone. These observations, afforded by the high-resolution and site-specific nature of FIB-SEM, link key fossilized features with the micro-nanoscale structure of modern-day bone. This investigation highlights the persistence of bone formation and organization persisting for over millions of years.

paleontology↗

bioRxiv2023

Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization

Collagen biomineralization is foundational to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood as the majority of theories are derived from electron microscopy (EM) in static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs dynamically. Herein, novel liquid transmission EM (TEM) strategies are presented, where collagen mineralization was explored in liquid conditions for the first time. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium-phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points, precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates this liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

bioengineering↗

bioRxiv2023

Shedding light (... electrons) on human bone ultrastructure with correlative on-axis electron tomography and energy dispersive X-ray spectroscopy tomography

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with a diameter up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: i) low-contrast nanosized circular spaces ("holes") observed in sections of bone oriented perpendicular to the long axis of a long bone; and ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most "holes" are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signature (Ca and P) within the gap zones, but the signal appears weaker than that associated to the extra-fibrillar mineral, pointing towards the existence of dissimilarities between the two types of mineralization.

bioengineering↗

bioRxiv2023

Characterizing Mineral Ellipsoids in New Bone Formation at the Interface of Ti6Al4V Porous Implants

The hierarchy of newly formed bone contains elements of disorder within an ordered multiscale structure, spanning from the macroscale to below the nanoscale. With mineralized structures presenting in the shape of ellipsoids in mature and mineralizing tissue, this work characterizes the heterogeneity in mineral ellipsoid packing at the interface of porous titanium implants. The characterization of mineral at the bone-implant interface offers insight into the osseointegration of titanium implants and the mechanical properties of the interfacial bone tissue. Using scanning transmission electron microscopy and plasma focused ion beam - scanning electron microscopy, mineral ellipsoids are characterized at the implant interface in both 2D and 3D. Heterogeneous in their size and shape within the newly formed bone tissue, ellipsoids are observed with alternating orientations corresponding to unique lamellar packets within 2-3 m of the titanium implant interface - although this motif is not universal, and a mineral-dense zone can also appear at the implant interface. Short-order ellipsoid orientation shifts are also present in the 3D probe of the implant interface, where an approximate 90{degrees} misorientation angle between neighbouring packets of mineral ellipsoid resolves with increasing distance from the titanium, possibly providing a strengthening mechanism to prevent crack propagation in the peri-implant bone. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/524810v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@3c34a1org.highwire.dtl.DTLVardef@a3437borg.highwire.dtl.DTLVardef@a374e1org.highwire.dtl.DTLVardef@14e9572_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Osseointegration of functionally-graded Ti6Al4V porous implants: Histology of the pore network

The additive manufacturing of titanium into porous geometries offers a means to generate low-stiffness endosseous implants with a greater surface area to improve osseointegration. In order to optimize pore size in the scaffolds, it is important to first understand the timeline of osseointegration in pre-clinical models. In this work, selective laser melting was used to produce gyroid-based scaffolds with a uniform pore size of 300 m or functionally-graded pore size from 600 m to 300 m before implantation in New Zealand white rabbit tibiae for 4 and 12 weeks. Initial in vitro assessment with Saos-2 cells showed favourable cell proliferation at pore sizes of 300 and 600 m. At four weeks, histological observations indicated some residual inflammation alongside neovessel infiltration into the scaffold interior and some early apposition of mineralized bone tissue. At twelve weeks, both scaffolds were filled with a mixture of adipocyte-rich marrow, micro-capillaries, and mineralized bone tissue. X-ray microcomputed tomography showed a higher bone volume fraction (BV/TV) and percentage of bone-implant contact (BIC) in the implants with 300 m pores than in the functionally-graded specimens, indicating that these smaller pore sizes may be favourable for osseointegration in leporine bone. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/521963v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@2e319aorg.highwire.dtl.DTLVardef@baa9deorg.highwire.dtl.DTLVardef@e66075org.highwire.dtl.DTLVardef@c6c8c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

A Facile Method for Simultaneous Visualization of Wet Cells and Nanostructured Biomaterials in SEM using Ionic Liquids

This work presents a successful methodology to image mammalian cells adhered to nanostructured biomaterials using scanning electron microscopy (SEM) operating in low-vacuum mode following ionic liquid treatment. Human osteoblast-like Saos-2 cells were treated with a room-temperature ionic liquid, 1-Ethyl-3-methylimidazolium tetrafluoroborate, and subsequently imaged on titanium utilizing SEM. Titanium substrates were modified to create laser-induced periodic surface structures (LIPSS) for visualizing at the sub-micron scale. Using a combination of fluorescence-based cell metabolism along with light microscopy and SEM image analysis, the shape and location of irradiated cells were confirmed to be unchanged after multiple irradiation sessions while the viability of minimally irradiated cells was unaltered. The wet imaging conditions combined with a rapid facile protocol using ionic liquid allows this technique to fulfill a niche in examining cellular behavior on biomaterials with sub-micron surface features. The demonstrated method to track observed cell adhesion to sub-micron surface features with SEM has great implications for the understanding of cell migration on nanostructured surfaces as well as on the exploration of simpler SEM preparation methods for cellular imaging.

cell biology↗

bioRxiv2020

Human bone mesoscale 3D structure revisited by plasma focused ion beam serial sectioning

Visualizing bone mineralization and collagen microfibril organization at intermediate scales between the nanometer and the 100s of microns range, the mesoscale, is still an important challenge. Similarly, visualizing cellular components which locally affect the tissue structure requires a precision of a few tens of nanometers at maximum while spanning several tens of micrometers. To address this issue, we employed a plasma focused ion beam (PFIB) equipped with a scanning electron microscope (SEM) to sequentially section nanometer-scale layers of demineralized and mineralized human femoral lamellar bone over volumes of approximately 46 x 40 x 9 m3, and 29 x 26 x 9 m3, respectively. This large scale view retained high enough resolution to visualize the collagen microfibrils while partly visualizing the lacuno-canalicular network (LCN) in three-dimensions (3D). We showed that serial sectioning can be performed on mineralized sections, and does not require demineralization. Moreover, this method revealed ellipsoidal mineral clusters, noted by others in high resolution studies, as a ubiquitous motif in lamellar bone over tens of microns, suggesting a heterogeneous and yet regular pattern of mineral deposition past the single collagen fibril level. These findings are strong evidence for the need to revisit bone mineralization over multi-length scales. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/180729v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1fe58a0org.highwire.dtl.DTLVardef@16c726corg.highwire.dtl.DTLVardef@cc28c6org.highwire.dtl.DTLVardef@1891d1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗