Stefan Schwan

Micromechanical measurements on P-protein aggregates (forisomes) from Vicia faba plants

Forisomes are chemomechanically active P-protein aggregates found in the phloem of legumes. They can convert chemical energy into mechanical work when induced by divalent metal ions or changes in pH, which control the folding state of individual forisome proteins. We investigated the changing geometric parameters of individual forisomes and the strength and dynamics of the forces generated during this process. Three different divalent ions were tested (Ca2+, Sr2+ and Ba2+) and were shown to induce similar changes to the normalized length and diameter. In the concentration range from 0.1 to 4 M, K+ and Cl(-) ions had no influence on the contraction behaviour of the forisomes induced by 10 mM Ca2+. In the absence of dissolved oxygen, these changes were independent of the radius of the metal ion, water uptake and the strength of binding between the selected metal ions and those protein molecules responsible for forisome conformational transformation. In the absence of any load, bound Ca2+, Sr2+ and Ba2+ ions showed apparent and averaged dissociation constants of 14, 62 and 1070 microM, respectively. Various forisomes generated bending on a quartz glass fibre with a diameter of 9 microm. The fibre bending was measured microscopically also by correlation between the digital patterns of a predefined window of observation before and after bending. Similar bending forces of approximately 90 nN were measured for a single forisome sequentially exposed to 10 mM Ca2+, Sr2+ and Ba2+. In the absence of dissolved oxygen, the same conditions resulted in averaged bending forces of (93+/-40) nN, (58+/-20) nN, and (91+/-20) nN after contacting different forisomes with 10 mM Ca2+, 10 mM Sr2+, and 10 mM Ba2+ respectively, demonstrating that the force generated was independent on ion concentrations above a certain threshold value.

Microstructure analysis method for evaluating degenerated intervertebral disc tissue.

Degeneration of intervertebral disc (IVD) tissue is characterized by several structural changes that result in variations in disc physiology and loss of biomechanical function. The complex process of degeneration exhibits highly intercorrelated biomechanical, biochemical, and cellular interactions. There is currently some understanding of the cellular changes in degenerated intervertebral disc tissue, but microstructural changes and deterioration of the tissue matrix has previously been rarely explored. In this work, sequestered IVD tissue was successfully characterized using histology, light microscopy, and scanning electron microscopy (SEM) to quantitatively evaluate parameters of interest for intervertebral disc degeneration (IDD) such as delamination of the collagenous matrix, cell density, cell size, and extra cellular matrix (ECM) thickness. Additional qualitative parameters investigated included matrix fibration and irregularity, neovascularization of the IVD, granular inclusions in the matrix, and cell cluster formation. The results of this study corroborated several previously published findings, including those positively correlating female gender and IVD cell density, age and cell size, and female gender and ECM thickness. Additionally, an array of quantitative and qualitative investigations of IVD degeneration could be successfully evaluated using the given methodology, resin-embedded SEM in particular. SEM is especially practical for studying micromorphological changes in tissue, as other microscopy methods can cause artificial tissue damage due to the preparation method. Investigation of the microstructural changes occurring in degenerated tissue provides a greater understanding of the complex process of disc degeneration as a whole. Developing a more complete picture of the degenerative changes taking place in the intervertebral disc is crucial for the advancement and application of regenerative therapies based on the pathology of intervertebral disc degeneration.

Micro-computed tomography, scanning electron microscopy and energy X-ray spectroscopy studies of facet joint degeneration: A comparison to clinical imaging

Segmental degeneration in the human lumbar spine affects both the intervertebral discs and facet joints. Facet joint degeneration not only affects the cartilage surface, but also alters the cellular properties of the cartilage tissue and the structure of the subchondral bone. The primary focus of this study is the investigation of these microstructural changes that are caused by facet joint degeneration. Microstructural analyses of degenerated facet joint samples, obtained from patients following operative lumbar interbody fusion, have not previously been extensively investigated. This study analyzes human facet joint samples from the inferior articular process using scanning electron microscopy, micro-computed tomography, and energy dispersive X-ray spectroscopy to evaluate parameters of interest in facet joint degeneration such as elemental composition, cartilage layer thickness and cell density, calcification zone thickness, subchondral bone portion, and trabecular bone porosity. These microstructural analyses demonstrate fragmentation, cracking, and destruction of the cartilage layer, a thickened calcification zone, localized calcification areas, and cell cluster formation as pathological manifestations of facet joint degeneration. The detailed description of these microstructural changes is critical for a comprehensive understanding of the pathology of facet joint degeneration, as well as the subsequent development and efficacy analysis of regenerative treatment strategies.

Micro-CT evaluation of asymmetrical ovine intervertebral disc height loss from surgical approach

PurposeThe primary goal of this study is to clearly define and evaluate new intervertebral disc height parameters in analysing the morphological pathology of disc degeneration for application in damage model and regeneration therapy development, as well as applying traditional variables to 3-D characterization methods.MethodsA posterolateral surgical approach was used to induce disc degeneration in an ovine model. At 12-months post-operation, sheep vertebral segments were removed and characterized using micro-CT to evaluate disc height parameters in regard to injury localization.ResultsStatistically significant differences between the disc height loss of the left and right side of the disc, consistent with the lateral surgical approach used were seen using the modified average disc height method by Dabbs et al. However, convexity index and the newly proposed Cross Tilt Index did not conclusively demonstrate a difference.ConclusionTwo-dimensional morphological evaluations can be applied in 3-D to provide a more complete picture of disc height loss for injury models. New 3-D parameters that are tailored to the type of surgical approach used should be investigated, with the 9-point system described herein providing a useful basis for derived values. Additionally, the surgical approach chosen when artificially injuring the disc can result in asymmetrical degeneration, as indicated by uneven disc height loss.

Morphological Characterization of the Self‐Assembly of Virus Movement Proteins into Nanotubes in the Absence of Virus Particles

One infection mechanism of plant viruses is the generation of nanotubes by viral movement proteins, allowing cell‐to‐cell virus particle transport. Previously, it was assumed that viral nanotubes extend directly from the host‐cell plasma membrane. In virus‐infected plants, these nanotubes reach an extraordinary diameter:length ratio (≈100 nm:µm or mm range). Here, viral nanotubes are produced in a transient protoplast system; the coding sequence for alfalfa mosaic virus movement protein is translationally fused to green fluorescent protein. The maximum extension of viral nanotubes into the culture medium is achieved 24–48 h posttransfection, with lengths in the micro‐ and millimeter ranges. Scanning electron microscopy and transmission electron microscopy show that strong inhomogeneous viral nanotubes are formed compared to particle‐filled systems. The nanotubes have similar length, but fluctuating wall thickness and diameter and are susceptible to entanglement and recombination. Indirect methods demonstrate that movement proteins assemble independently at the top of the nanotube. These viral nanotubes grow distinctly from previously known natural particle‐filled systems and are a unique biological tubular nanomaterial that has the potential for micro‐ or nanoapplications as a mechanically stable structural component.

The influence of injecting an epidural contrast agent into the sacral canal on the fluoroscopic visibility of bony landmarks for sacroiliac screw fixation: a feasibility study

OBJECT In sacroiliac screw fixation of unstable pelvic injuries in geriatric patients, poor bone quality often obscures important bony landmarks in fluoroscopic images. The authors analyzed the feasibility of injecting a transhiatal contrast agent (CA) into the sacral canal to improve fluoroscopic visualization in the sacral epidural space. METHODS Eight fresh cadaveric whole-body specimens from human donors whose mean age at the time of death was 78 years (range 69-87 years) were used. First, to identify bony landmarks without CA enhancement, the authors acquired fluoroscopy images of the native sacral canal, using lateral, inlet, and outlet projections. Through puncture of the sacral hiatus, 8-10 ml of CA was injected into the epidural space. Fluoroscopy images were then acquired in the standard pelvic views to identify the bony landmarks. To assess the effect of the CA enhancement, visibility of the landmarks was assessed before and after CA injection. Each identified landmark was scored as 1, and summative landmark scores of up to 10 were determined for each specimen. RESULTS The cadaveric specimens were representative of bone structures in the geriatric population. In all specimens, epidural CA injection enhanced the fluoroscopic visualization of the sacral canal and of the S-1 foramina. The enhancement increased the total bony landmark score from 5.9 (range 4-8) without CA injection to 8.1 (range 6-10) after CA injection. Considering only intrasacral landmarks, the score was increased from 1.5 to 3. CONCLUSIONS Injection of a transhiatal epidural CA improves fluoroscopic imaging of the sacral canal and of the neural foramina. Hence, this technique could be applied to help the surgeon identify anatomical landmarks during sacroiliac screw fixation in geriatric patients.