Multimodal analysis and comparison of stoichiometric and structural characteristics of parosteal and conventional osteosarcoma with massive sclerosis in human bone.

Osteosarcoma (OS) is the most common malignant primary bone tumor in humans and occurs in various subtypes. Tumor formation happens through malignant osteoblasts producing immature bone. In the present paper we studied two different subtypes of osteosarcoma, from one individual with conventional OS with massive sclerosis and one individual with parosteal OS, based on a multimodal approach including small angle x-ray scattering (SAXS), wide angle x-ray diffraction (WAXS), backscattered electron imaging (BEI) and Raman spectroscopy. It was found that both tumors showed reduced mineral particle sizes and degree of orientation of the collagen-mineral composite in the affected areas, alongside with a decreased crystallinity. Distinct differences between the tumor material from the two individuals were found in the degree of mineralization. Further differences were observed in the carbonate to phosphate ratio, which is related to the degree of carbonate substitution in bone mineral and indicative of the turnover rate. The contraction of the c-axis of the bone mineral crystals proved to be a further, very sensitive parameter, potentially indicative of malignancy.

Antibody-ligand interactions on a high-capacity staphylococcal protein A resin.

Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.

Ultrasonic fatigue of unfilled and carbon nanotube (CNT) reinforced polyetheretherketone (PEEK).

Fatigue properties of polyetheretherketone (PEEK) and multiwall carbon nanotube (CNT) reinforced PEEK were investigated with the ultrasonic fatigue testing method. Lifetimes were measured in the high and very high cycle fatigue regime at resonance frequency 19 kHz and load ratio R = -1. Pulse-pause loading served to avoid specimen self-heating and led to effective cycling frequencies in the range from several hundred Hz to about two kHz. Stress amplitude for 50 % fracture probability at 109 cycles is 21.2 ± 4.3 MPa for unreinforced PEEK (22 % of its tensile strength) and 33.5 ± 3.5 MPa for CNT reinforced PEEK (33 % of its tensile strength). Servohydraulic fatigue tests at 22 Hz with CNT reinforced PEEK delivered fatigue lifetimes comparable to ultrasonic tests, i.e. no frequency effect and no influence of load versus displacement control was observed. Keeping specimen temperature far below the glass transition temperature, ultrasonic fatigue testing of a high temperature resistant plastic was successfully implemented.

Comparative Analysis of Various Spider Silks in Regard to Nerve Regeneration: Material Properties and Schwann Cell Response.

Peripheral nerve reconstruction through the employment of nerve guidance conduits with Trichonephila dragline silk as a luminal filling has emerged as an outstanding preclinical alternative to avoid nerve autografts. Yet, it remains unknown whether the outcome is similar for silk fibers harvested from other spider species. This study compares the regenerative potential of dragline silk from two orb-weaving spiders, Trichonephila inaurata and Nuctenea umbratica, as well as the silk of the jumping spider Phidippus regius. Proliferation, migration, and transcriptomic state of Schwann cells seeded on these silks are investigated. In addition, fiber morphology, primary protein structure, and mechanical properties are studied. The results demonstrate that the increased velocity of Schwann cells on Phidippus regius fibers can be primarily attributed to the interplay between the silk's primary protein structure and its mechanical properties. Furthermore, the capacity of silk fibers to trigger cells toward a gene expression profile of a myelinating Schwann cell phenotype is shown. The findings for the first time allow an in-depth comparison of the specific cellular response to various native spider silks and a correlation with the fibers' material properties. This knowledge is essential to open up possibilities for targeted manufacturing of synthetic nervous tissue replacement.

Does early post-operative exercise influence bone healing kinetics? Preclinical evaluation of non-critical sized femur defect healing.

Physical exercise is a well-known modality for maintaining healthy locomotor mechanism. A detailed preclinical research on physical exercise effect on bone healing kinetics could help to improve the rehabilitation process after fracture treatment and bone remodeling. Our aim was to evaluate the effect of early post-operative exercise effect on bone microstructural changes in a rat model. Twenty Sprague Dawley male rats underwent bi-cortical 1.6 mm hole drilling in both femur diaphysis, after which (n = 10) underwent continuous treadmill training (TR) over two weeks, while the other group of rats (n = 10) was assigned to non-training (NT) control group. New bone formation labeling was performed by subcutaneous fluorochrome injections at day 5, 14 and 31. In vivo micro-computed tomography (µCT) scans were performed once a week during the 6-week post-operative period. Ten animals (five from each group) were euthanized at 3rd week while remaining animals were euthanized at 6th week. Femur samples were extracted and underwent ex vivo µCT and histological evaluation, while serum was used for evaluating alkaline phosphatase (ALP). µCT data demonstrated increased volume and surface of newly formed bone in defect area of TR group. Bone volume/Tissue volume (BV/TV) ratio and number of osteocytes showed an increase in TR group after 3-week period. Fluorochrome distances were increased between day 5 and 14 within the training group. Serum ALP level increased in both groups over 3- and 6-weeks. Post-operative exercise increases the bone healing kinetics and stimulates the new bone formation during and after the training protocol has ended.

Insights into the material properties of dragline spider silk affecting Schwann cell migration.

Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood. In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.

Mechanics of a Biomimetic Moisture Sensitive Actuator Based on Compression Wood.

Various mechanisms of plant organ movements have been reported, including the close association of two layers with expressed differences in hygroscopic properties. Following this principle, actuator beams composed of thin veneers out of normal and compression wood cut from Scots pine (Pinus sylvestris L.) were prepared by using two types of adhesives. The mismatch of the swelling properties of the two layers in tight combination resulted in an expressed bending deflection in response to set humidity changes. The resulting curvatures were measured and analyzed by the Timoshenko bi-metal-model, as well as with an enhanced three-layer model, with the latter also considering the mechanical influence of the glueline on the actuator bending. The thermally induced strain in the original model was replaced by another strain due to moisture changes. The strain was modelled as a function of wood density, along with changes in wood moisture. Experiments with free movement of the bilayer to measure curvature, and with constraints to determine forces, were performed as well. Deformation and magnitude of actuators movements were in close agreement with the enhanced bilayer-model for the phenol-resorcinol-formaldehyde adhesive, which deviated substantially from the casein adhesive glued actuators. The obtained results are seen as critical for wood-based actuator systems that are potentially used in buildings or other applications.

3D nanoscale analysis of bone healing around degrading Mg implants evaluated by X-ray scattering tensor tomography.

The nanostructural adaptation of bone is crucial for its biocompatibility with orthopedic implants. The bone nanostructure also determines its mechanical properties and performance. However, the bone's temporal and spatial nanoadaptation around degrading implants remains largely unknown. Here, we present insights into this important bone adaptation by applying scanning electron microscopy, elemental analysis, and small-angle X-ray scattering tensor tomography (SASTT). We extend the novel SASTT reconstruction method and provide a 3D scattering reciprocal space map per voxel of the sample's volume. From this reconstruction, parameters such as the thickness of the bone mineral particles are quantified, which provide additional information on nanostructural adaptation of bone during healing. We selected a rat femoral bone and a degrading ZX10 magnesium implant as model system, and investigated it over the course of 18 months, using a sham as control. We observe that the bone's nanostructural adaptation starts with an initially fast interfacial bone growth close to the implant, which spreads by a re-orientation of the nanostructure in the bone volume around the implant, and is consolidated in the later degradation stages. These observations reveal the complex bulk bone-implant interactions and enable future research on the related biomechanical bone responses. STATEMENT OF SIGNIFICANCE: Traumatic bone injuries are among the most frequent causes of surgical treatment, and often require the placement of an implant. The ideal implant supports and induces bone formation, while being mechanically and chemically adapted to the bone structure, ensuring a gradual load transfer. While magnesium implants fulfill these requirements, the nanostructural changes during bone healing and implant degradation remain not completely elucidated. Here, we unveil these processes in rat femoral bones with ZX10 magnesium implants and show different stages of bone healing in such a model system.

Pore Development during the Carbonization Process of Lignin Microparticles Investigated by Small Angle X-ray Scattering.

Application of low-cost carbon black from lignin highly depends on the materials properties, which might by determined by raw material and processing conditions. Four different technical lignins were subjected to thermostabilization followed by stepwise heat treatment up to a temperature of 2000 °C in order to obtain micro-sized carbon particles. The development of the pore structure, graphitization and inner surfaces were investigated by X-ray scattering complemented by scanning electron microscopy and FTIR spectroscopy. Lignosulfonate-based carbons exhibit a complex pore structure with nanopores and mesopores that evolve by heat treatment. Organosolv, kraft and soda lignin-based samples exhibit distinct pores growing steadily with heat treatment temperature. All carbons exhibit increasing pore size of about 0.5-2 nm and increasing inner surface, with a strong increase between 1200 °C and 1600 °C. The chemistry and bonding nature shifts from basic organic material towards pure graphite. The crystallite size was found to increase with the increasing degree of graphitization. Heat treatment of just 1600 °C might be sufficient for many applications, allowing to reduce production energy while maintaining materials properties.

Metal-Insulator Transition of Ultrathin Sputtered Metals on Phenolic Resin Thin Films: Growth Morphology and Relations to Surface Free Energy and Reactivity.

Nanostructured metal assemblies on thin and ultrathin polymeric films enable state of the art technologies and have further potential in diverse fields. Rational design of the structure-function relationship is of critical importance but aggravated by the scarcity of systematic studies. Here, we studied the influence of the interplay between metal and polymer surface free energy and reactivity on the evolution of electric conductivity and the resulting morphologies. In situ resistance measurements during sputter deposition of Ag, Au, Cu and Ni films on ultrathin reticulated polymer films collectively reveal metal-insulator transitions characteristic for Volmer-Weber growth. The different onsets of percolation correlate with interfacial energy and energy of adhesion weakly but as expected from ordinary wetting theory. A more pronounced trend of lower percolation thickness for more reactive metals falls in line with reported correlations. Ex situ grazing incidence small angle X-ray scattering experiments were performed at various thicknesses to gain an insight into cluster and film morphology evolution. A novel approach to interpret the scattering data is used where simulated pair distance distributions of arbitrary shapes and arrangements can be fitted to experiments. Detailed approximations of cluster structures could be inferred and are discussed in view of the established parameters describing film growth behavior.

Solvent-Free Ultrasonic Dispersion of Nanofillers in Epoxy Matrix.

Dispersion of carbon nanotubes and carbon nanofibers is a crucial processing step in the production of polymer-based nanocomposites and poses a great challenge due to the tendency of nanofillers to agglomerate. One of the most effective methods for dispersion is the use of a three-roll mill, which is a well-established method and results in agglomerates below 5 µm. Nevertheless, this process is time-consuming and thus a limiting factor for industrial applications. Our aim was to establish an easy and efficient ultrasonic dispersion process, characterize the dispersion parameters, and compare both methods, ultrasonication and the three-roll mill. We applied rheological tests and analyzed the agglomerate sizes by an image fit of the microscopy images. All these analyses combined deliver a valuable set of information about the dispersion's quality and, therefore, allows the improvement and further adaptation of the dispersion process.

Cobalt Chromium Molybdenum Surface Modifications Alter the Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells.

Surface roughness on orthopedic implant materials has been shown to be highly influential on the behavior of osteogenic cells. Mesenchymal stem and progenitor cells (MSPCs) migrate to the interface, adhere, proliferate, and differentiate into osteoblasts, which subsequently form bone matrix. Modifications of the implant surfaces should accelerate this process and improve biocompatibility. In this study, five surface topographies on cobalt chromium molybdenum (CoCrMo) were engineered to examine the influence on MSPCs. Scanning electron microscopy revealed significant differences in the morphology of untreated CoCrMo discs in comparison with CoCrMo with a titanium nitride (TiN) coating, polished and porous coated CoCrMo surfaces, and CoCrMo with a pure titanium (cpTi) coating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDX). Human primary MSPCs were expanded from tissue samples of spongiosa bone and characterized according to the criteria of the International Society for Cellular Therapy. The characteristic phenotype of MSPC was confirmed by flow cytometry and multilineage differentiation. Alcaline phosphatase and osteopontin expression increased significantly in all groups about 5-fold and 10-fold, respectively, in comparison to the undifferentiated controls. The porous coated surface showed a reduced expression of osteogenic markers. Due to the osteogenic differentiation, the expression of integrin α5ß1, which is particularly important for cell-material contact, increased 4-7-fold. In the dynamic process of bone biology, MSPCs cultured and differentiated on cpTi, showed significant upregulation of IL6 and leptin.

Examination of intestinal ultrastructure, bowel wall apoptosis and tight junctions in the early phase of sepsis.

Gut hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. This project aims to elucidate these mechanisms in the early phase of sepsis. Eighteen male wild type mice were randomized to two groups. All mice received one single gavage of fluorescein isothiocyanate (FITC) dextran 30 min before intervention. One group (n = 10) underwent cecal ligation and puncture to induce sepsis. The other group (n = 8) was sham operated. Septic animals exhibited significantly increased permeability for FITC 8 h post-operatively. Significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin-2 and decreased expressions of claudin-4, tight-junction-protein-1 and occludin-1 resembling increased permeability of tight junctions. However, these alterations could not be confirmed at the protein level. Light microscopy revealed significant dilatation of intercellular spaces at the basal sections of intestinal epithelial cells (IEC) in septic animals confirmed by increased intercellular spaces at the level of tight junctions and adherens junctions in electron microscopy (TEM). In small angle X-ray scattering no increase in number or size of nanopores could be shown in the bowel wall. HOECHST staining and PCR of ileum samples for apoptosis markers proofed no relevant differences in intestinal epithelial cell apoptosis between the groups. Intestinal hyperpermeability in septic animals was most likely caused by alterations of the intercellular contacts and not by apoptosis or increased size/number of nanopores of intestinal epithelial cells in this murine model of early sepsis.

CoCrMo surface modifications affect biocompatibility, adhesion, and inflammation in human osteoblasts.

In this study, different surface modifications were performed on a Cobalt-Chrome-Molybdenum (CoCrMo) alloy and the effects on cell viability and cytotoxicity as well as the adhesion potential of human osteoblasts (hFOB) and their inflammation reaction were investigated in vitro. CoCrMo discs were coated with TiN, with polished and porous coated surfaces, or with pure titanum (cpTi) surfaces and examined by Scanning Electron Microscopy to evaluate surface modifications. In vitro cell viability, adhesion behaviour, and expression of inflammation markers of hFOB human osteoblasts were measured via CellTiter-Glo, CytoTox, ELISA, and RT-PCR respectively. All results were compared to CoCrMo without surface modifications. The biocompatibility data showed high compatibility for the TiN hard coatings. Likewise, the porous surface coating increased cell viability significantly, compared to an untreated CoCrMo alloy. None of the investigated materials influenced cytotoxicity. Different surface modifications did not influence expression of fibronectin, although TiN, porous surface coatings and polished surfaces showed highly significant reductions in integrin subunit expression. In addition to the regulation of adhesion potential these three surfaces stimulated an anti-inflammatory response by osteocytes. Improved biocompatibility and adhesion properties may contribute to better osteointegration of prosthetics.

Insight into the nanostructure of anisotropic cellulose aerogels upon compression.

Cellulose II aerogels are a highly porous class of biobased ultra-light-weight materials. They consist of interlinked networks of loosely aggregated cellulose fibrils. The latter typically have random orientation due to spontaneous phase separation triggered by addition of antisolvent to moleculardisperse cellulose solutions. Deceleration of phase separation has been recently proposed as a novel approach towards aerogels featuring preferred cellulose orientation. Here, we investigate the mechanical response of such oriented cellulose aerogels towards load up to 80% compression. Stress-strain curves were recorded and in situ small angle X-ray scattering (SAXS) was performed during compression test to obtain information about the structural alterations of the aerogel fibril networks on the nano-scale related to deformation. Using SAXS in addition, structural changes can be followed in much more detail than by recording stress-strain curves alone. Buckling and coalescence of fibers and a change in fibril orientation can be related to certain regimes in the stress-strain curve. If the loading axis is oriented parallel to the network orientation the aerogels show higher resilience towards the compression.

Antibody Binding Heterogeneity of Protein A Resins.

Protein A affinity chromatography is a core unit operation in antibody manufacturing. Nevertheless, there is not enough understanding of in-column antibody adsorption in the Protein A capture step. This work aims to investigate in situ the establishment of an antibody (trastuzumab) layer during Protein A chromatography both in terms of energetic contributions and uptake kinetics. Flow microcalorimetry is employed as a technique with an in situ operating detector, which provides an understanding of the thermodynamics of the adsorption process. In addition, the antibody uptake rate is also investigated in order to establish a correlation between its diffusion on the stationary phase and the associated thermodynamics. Two resins with different particle size, intraparticle porosity, and a Protein A ligand structure are studied: the synthetically engineered B-domain tetrameric MabSelect SuRe and the synthetically engineered C-domain hexameric TOYOPEARL AF-rProtein A HC. The uptake rate follows a pore diffusion model at low equilibrium time, showing a slower diffusivity after a certain time because of the heterogeneous binding nature of these two resins. In addition, the microcalorimetric studies show that adsorption enthalpy is highly favourable at low isotherm concentrations and evolves toward an equilibrium with increasing surface concentration. These data suggest that the relationship between adsorption enthalpy and the establishment of the antibody layer in the Protein A chain is consistent with heterogeneous adsorption.

High-resolution large-area imaging of nanoscale structure and mineralization of a sclerosing osteosarcoma in human bone.

Osteosarcoma is the most common primary bone cancer type in humans. It is predominantly found in young individuals, with a second peak later in life. The tumour is formed by malignant osteoblasts and consists of collagenous, sometimes also mineralized, bone matrix. While the morphology of osteosarcoma has been well studied, there is virtually no information about the nanostructure of the tumour and changes in mineralization on the nanoscale level. In the present paper, human bone tissue inside, next to and remote from a sclerosing osteosarcoma was studied with small angle x-ray scattering, x-ray diffraction and electron microscopy. Quantitative evaluation of nanostructure parameters was combined with high resolution, large area mapping to obtain microscopic images with nanostructure parameter contrast. It was found that the tumour regions were characterized by a notable reduction in mineral particle size, while the mineral content was even higher than that in normal bone. Furthermore, the normal preferential orientation of mineral particles along the longitudinal direction of corticalis or trabeculae was largely suppressed. Also the bone mineral crystal structure was affected: severe crystal lattice distortions were detected in mineralized tumour tissue pointing to a different ion substitution of hydroxyl apatite in tumorous tissue than in healthy tissue.

Vacuum Casting and Mechanical Characterization of Nanocomposites from Epoxy and Oxidized Multi-Walled Carbon Nanotubes.

Sample preparation is an important step when testing the mechanical properties of materials. Especially, when carbon nanotubes (CNT) are added to epoxy resin, the increase in viscosity complicates the casting of testing specimens. We present a vacuum casting approach for different geometries in order to produce specimens from functional nanocomposites that consist of epoxy matrix and oxidized multi-walled carbon nanotubes (MWCNTs). The nanocomposites were characterized with various mechanical tests that showed improved fracture toughness, bending and tensile properties performance by addition of oxidized MWCNTs. Strengthening mechanisms were analyzed by SEM images of fracture surfaces and in-situ imaging by digital image correlation (DIC).

Enhanced Cu and Cd sorption after soil aging of woodchip-derived biochar: What were the driving factors?

Biochar (BC) is increasingly tested as a soil amendment for immobilization of heavy metals (HMs) and other pollutants. In our study, an acidic soil amended with wood chip-derived BC showed strongly enhanced Cu and Cd sorption after 15 months of aging under greenhouse conditions. X-ray absorption near edge structure suggested formation of Cu(OH)2 and CuCO3 and upon aging increasingly Cu sorption to the BC organic phase (from 9.2% to 40.7%) as main binding mechanisms of Cu on the BCs. In contrast, Cd was predominantly bound as CdCO3 on the BCs even after 15 months (82.7%). We found indications by mid-infrared spectroscopy that the formation of organic functional groups plays a role for increased HM sorption on aged BCs. Yet, our data suggest that the accessibility of BC's pore network and reactive surfaces is likely to be the overriding factor responsible for aging-related changes in HM sorption capacity, rather than direct interactions of HMs with oxidized functional groups. We observed highly weathered BC surface structures with scanning electron microscopy along with strongly increased wettability of the BCs after 15 months of soil aging as indicated by a decrease of water contact angles (from 62.4° to 4.2°).

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface.

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.