Werner Baschong

Dynamic Elastic Modulus of Porcine Articular Cartilage Determined at Two Different Levels of Tissue Organization by Indentation-Type Atomic Force Microscopy

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, |E(*)|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = approximately 2.5 microm) and sharp pyramidal tips (radius = approximately 20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. |E(*)| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was approximately 2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on |E(*)| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Enhanced Cartilage Tissue Engineering by Sequential Exposure of Chondrocytes to FGF-2 During 2D Expansion and BMP-2 During 3D Cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor‐2 (FGF‐2), were cultured on three‐dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein‐2 (BMP‐2). Chondrocytes expanded without FGF‐2 exhibited high intensity immunostaining for smooth muscle α‐actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF‐2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7‐fold higher cell number, 4.2‐fold higher wet weight, and 2.8‐fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF‐2. Chondrocytes expanded with FGF‐2 and cultured on PGA scaffolds in the presence of BMP‐2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5‐fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP‐2. The presence of BMP‐2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF‐2. In summary, the presence of FGF‐2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP‐2 during 3D cultivation on PGA scaffolds.

Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy

The introduction of ultrasmall (∼1–3 nm) colloidal gold markers in immunoelectron microscopy (IEM) in 1989 has considerably improved the sensitivity of this marker system. Ultrasmall gold markers have opened the field of pre‐embedding labeling studies to gold markers without the need of harsh permeabilizing steps. They are recommended for the detection of scarce antigens in ultrathin cryosections which may otherwise escape immunodetection. However, reports concerning the preparation of ultrasmall gold colloids, their conjugation to proteins, and their use in high‐resolution studies (without an additional enlargement step) are very limited. Also, the available enlargement techniques necessary for the use of this marker in conventional electron microscopy require detailed discussion to clarify the large number of contradictory observations. The present review summarizes and discusses the findings accumulated within the last 10 years on the application of ultrasmall gold markers in IEM with regard to their merits, limitations, detection sensitivity, and suitability for different labeling techniques. It should provide practical hints for the use of ultrasmall gold colloids and discusses problems arising with enlargement techniques such as silver enhancement and gold toning procedures. Microsc. Res. Tech. 42:66–79, 1998.

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or Nycodenz-adjusted density of the hydrated detergent micelle

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.

Head structure of bacteriophages T2 and T4

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Triclosan is minimally effective in rodent malaria models

To the Editor: The discovery several years ago that Plasmodium parasites use a fatty acid synthesis type II pathway (FAS-II), shared by plants and bacteria, raised hopes for the discovery of new antimalarial targets1 and stimulated the search for FAS-II inhibitors. This pathway is harbored in the Plasmodium apicoplast, a nonphotosynthetic plastid of cyanobacterial origin, and is composed of four enzymes. The rate-limiting step is mediated by enoyl acyl carrier protein reductase (FabI)2. In mammals, all four enzymatic steps reside in the single multifunctional fatty acid synthesis type I (FAS-I) protein. The finding that the microbicide triclosan, a chlorinated hydroxyldiphenyl ether, targets lipid synthesis in Escherichia coli via inhibition of FabI3 led to the investigation of its effect on Plasmodium. An article published in 2001 in Nature Medicine4 reported that triclosan inhibited in vitro propagation of asexual blood stage forms of the human pathogen Plasmodium falciparum and cured mice infected with the rodent malarial species Plasmodium berghei. This article and a related patent5 have stimulated a wave of investigations and substantial investments in this agent as a potential new antimalarial drug. Our groups subsequently confirmed triclosan’s in vitro activity, yielding half-maximal inhibitory concentration values with P. falciparum strains that were comparable to the initial report in the remodeled human IPF lung, both in vascular and in alveolarinterstitial compartments, where they appear to be closely associated with myofibroblasts. This supports the possibility that strategies targeting NOX-4 may be beneficial, not only for treatment of alveolarinterstitial fibrosis but also possibly for the vascular remodeling and pulmonary hypertension that are associated with advanced IPF.

Confocal laser scanning microscopy and scanning electron microscopy of tissue Ti-implant interfaces

Microscopic inspection of heterogenous three-dimensional (3D) objects such as oral implants, or implants in general, is conventionally performed either on ground sections of methyl-metacrylate-embedded material, at the cellular level by histologic analysis of the peri-implant tissue by light microscopy (LM), or at the supramolecular level by transmission electron microscopy (TEM). Alternatively, the architecture of the tissue/implant interface is visualized by scanning electron microscopy (SEM). The two approaches exclude each other because of the sample preparation.We elaborate conditions for the non-invasive analysis of tissue/implant interfaces by confocal laser scanning microscopy (CLSM) in buffer, hoping to obtain a 3D view of fluorescently labeled tissue constituents at the tissue implant interface and, through subsequent SEM, of the metal surface. The use of water-immersion objectives, originally developed for high LM under physiological conditions is essential. In an exploratory approach, the tissue/Ti-interfaces of two retrieved dental implants were analyzed. One was a step-cylinder used for orthodontic anchoring and the other was an endosseous step-screw implant retrieved after infection-related loosening prior to load. The adhering tissue fragments were fluorescently triple-labeled for actin, fibronectin, and sm-alpha-actin. Optical sections for fluorescent images and for the laser reflection map were registered concomitantly. This approach allowed the labeled structures to be located on the metal surface. Subsequently, the same implants were prepared for SEM of the tissue/implant interface, and upon removal of the adhering structures, of the underlying metal surface. Thus, specific proteins can be identified and their spatial architecture as well as that of the underlying metal surface can be visualized for one and the same implant. The immediate visualization after fluorescence labeling in buffer by means of water immersion objective lenses proved most critical.

[11] Three-dimensional visualization of cytoskeleton by confocal laser scanning microscopy

Publisher Summary The chapter presents a study on three-dimensional (3-D) visualization of cytoskeleton by confocal laser scanning microscopy (CLSM). Imaging of the 3-D architecture in the cytoskeleton of cells in culture and in tissue by CLSM depends on two features, they are: proper sample preparation and adequate resolution. Functional studies of the cytoskeleton require CLSM for the 3-D documentation of the cytoskeletal constituents at optimal resolution. Practical suggestions for specific cell cultivation techniques and tissue excision are presented. The chapter addresses key problems with permeabilization and fixation of cells and tissue. Fixation, especially cross-linking by aldehydes, may lead to substantial background signals, either by the binding of fluorescent labels to free aldehyde groups or by the formation of fluorescent aldehyde polymers. Moreover, tissue constituents, especially the extracellular matrix, may display substantial autofluorescence. The removal of autofluorescence, therefore, is critical for achieving adequate resolution of the cytoskeleton in multilayer cultures or in tissue biopsies. Proper treatment with NaBH 4 can minimize aldehyde-related background labeling and auto fluorescence. Immunolabeling techniques and sample mounting are discussed. Accordingly, hardware requirements, data acquisition, and image processing will relate to analyzing the 3-D nature of the cytoskeleton.