J. van Marle

Confocal Microscopy Study of Undisturbed and Chlorhexidine-treated Dental Biofilm

Culturing of dispersed plaque samples and vitality staining of plaque smears are the most commonly used methods for evaluating the effects of antimicrobials on dental plaque. The visualization of the antimicrobial action on oral biofilm present on the substrate surface (in situ) would add valuable information to the existing knowledge about the treatment effects. This study aimed at combining the advantage of confocal laser scanning microscopy (CLSM) to visualize plaque non-destructively with a vitality staining technique to assess the immediate bactericidal effect of chlorhexidine (CHX) on biofilm. Three 200-μmwide grooves were cut into bovine dentin discs for plaque accumulation. The discs were worn by six subjects for 6, 24, and 48 hrs, then broken into halves, one of which received a one-minute extra-oral 0.2% CHX treatment, while the other served as control. Both halves were stained for vital fluorescence measurements and visualized by CLSM. Plaque vitality (in %) was quantified by image analysis in three plaque layers-outer, middle, and inner. The CHX effect was significant in six-hour samples (p < 0.001) and only in the outer layer of the 48-hour plaque (p < 0.001), demonstrating a resistant nature of dental biofilm to a single CHX treatment. With the present approach, we have shown that it is possible to visualize and quantitate the antimicrobial treatment effect on biofilm still present on the substrate on which it was grown.

Reduced wound contraction and scar formation in punch biopsy wounds. Native collagen dermal substitutes. A clinical study

In full‐thickness skin wounds dermal regeneration usually fails, resulting in scar formation and wound contraction. We studied dermal regeneration by implantation of collagenous matrices in a human punch biopsy wound model.

Lichen planus is associated with human herpesvirus type 7 replication and infiltration of plasmacytoid dendritic cells

Background  Lichen planus (LP) is a common inflammatory skin disease of unknown aetiology. Viral causes have been suggested.

Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes

SummaryRheumatoid arthritis, a disease of unknown aetiology, is characterized by joint inflammation and, in its later stages, cartilage destruction. Inflammatory mediators may exert not only suppression of matrix synthesis but also cartilage degradation, which eventually leads to severe cartilage depletion. Systemically and locally produced growth factors and hormones regulate cartilage metabolism. Alterations in levels of these factors or in their activity can influence the pathogenesis of articular cartilage destruction in arthritic joints. The main topic of the present review is the role of the anabolic factor insulin-like growth factor-1 in the regulation of chondrocyte metabolic functions in normal and in diseased cartilage. This is the most important growth factor that balances chondrocyte proteoglycan synthesis and catabolism to maintain a functional cartilage matrix. A brief overview of how chondrocytes keep the cartilage matrix intact, and how catabolic and anabolic vactors are thought to be involved in pathological cartilage destruction precedes the review of the role of this growth factor in proteoglycan metabolism in cartilage.

Otoconial alterations after embryonic development in hypergravity

The relation between prolonged hypergravity and structural adaptation of otoconia was studied in hamsters (n = 56). Three groups of hamsters (n = 27), were conceived and born in a centrifuge: group 1 (n = 10) 1 month under 2.5 G, group 2 (n = 9) 5 months under 2.5 G and 4 months under 1 G, group 3 (n = 8) 1 month under 2.5 G and 8 months under 1 G. Control hamsters (n = 29) were conceived and born under 1 G (1 month old, n = 7; 9 months old, n = 22). Histological study of the otoconial layers (energy dispersive x-ray element analysis and scanning electron microscopy) showed similar calcium content, size, and shape in utricular and saccular otoconia in all groups. Different were the utricular otoconial size classes, large, medium-sized, and small. The area with small otoconia increased in group 1 (p = 0.002). In group 2, the large otoconial area decreased (p = 0.001) and the medium-sized one increased (p < 0.001). In group 3, the large otoconial area decreased (p = 0.003) and the medium-sized one increased (p = 0.007). For age-related effects we found group 1 with an increased area of large otoconia (p = 0.001) and a decreased medium-sized one compared to groups 2 (p < 0.001) and 3 (p = 0.02). Hypergravity during formation of otoconia does not affect calcium content, size, or shape, but changes relative size of the areas with large, medium-sized, or small otoconia and the development of these areas. This resulted in a structural adaptation to hypergravity.

Determination of the scattering anisotropy with optical coherence tomography

In this work we demonstrate measurements with optical coherence tomography (OCT) of the scattering phase function in the backward direction and the scattering anisotropy parameter g. Measurements of the OCT attenuation coefficient and the backscattering amplitude are performed on calibrated polystyrene microspheres with a time-domain OCT system. From these measurements the phase function in the backward direction is determined. The measurements are described by the single scattering model and match Mie calculations very well. Measurements on Intralipid demonstrate the ability to determine the g of polydisperse samples and, for Intralipid, g = 0.35 ± 0.03 is measured, which is well in agreement with g from literature. These measurements are validated using the Intralipid particle size distribution determined from TEM measurements. Measurements of g and the scattering phase function in the backward direction can be used to monitor changes in backscattering, which can indicate morphological changes of the sample or act as contrast enhancement mechanism.

Chondrocyte IGF-1 receptor expression and responsiveness to IGF-1 stimulation in mouse articular cartilage during various phases of experimentally-induced arthritis.

OBJECTIVE--To examine the distribution of insulin like growth factor-1 (IGF-1) receptors and the biological response to IGF-1 stimulation in articular cartilage of normal mouse knee joints and arthritic joints taken at various stages of experimentally induced arthritis. METHODS--In situ IGF-1 receptor expression and responsiveness to IGF-1 stimulation were examined in murine articular cartilage at different phases in two models of experimentally induced arthritis. IGF-1 receptor expression was visualised in joint sections with the use of anti-IGF-1 receptor antibodies and quantified by confocal laser scanning microscopy. Chondrocyte proteoglycan (PG) synthesis was measured by incorporation of 35S-sulphate. RESULTS--In control cartilage, the majority of IGF-1 receptors were found on chondrocytes localised in the middle and deeper zones of the cartilage, whereas receptor expression in surface zone chondrocytes was very low. During culture of normal articular cartilage, IGF-1 was able to maintain chondrocyte PG synthesis at the in vivo level. Concurrently with the development of arthritis, cartilage lost its capacity to react to IGF-1, but IGF-1 stimulation recovered when the inflammatory response waned. Shortly after induction of arthritis, IGF-1 receptor expression initially declined, but it had returned to normal levels by day 1 and remained increased thereafter. CONCLUSION--The distribution of IGF-1 receptor expression in the different zones of normal articular cartilage reflects IGF-1 stimulation and metabolic activity of chondrocytes in these layers. This correlation is disturbed in arthritic cartilage, suggesting inadequate or overruled signalling.

Histatin 5 and derivatives: Their localization and effects on the ultra-structural level

Histatins, a family of cationic peptides present in saliva, are active against the opportunistic yeast Candida albicans. The mechanism of action is still unclear. Histatin 5 and more potent synthetic variants, dhvar4 and dhvar5, were used to study localization and effects on morphology on the ultra-structural level. Although all peptides induced leakage, no association with the plasma membrane, indicative for permanent pores, was observed with immuno-gold-labeling. Freeze-fracturing showed severe changes of the plasma membrane. Together with, for the dhvars, the loss of intracellular integrity, this suggests that leakage may be a secondary effect rather than an effect of formation of permanent pores.

Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans.

Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17-30 and LFampin265-284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17-30 and LFampin265-284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity.

Effects of Sustained Acceleration on the Morphological Properties of Otoconia in Hamsters

We investigated the effect of prolonged hypergravity on the otoconial layer of the maculae utriculi and the maculae sacculi in hamsters. The animals were placed in a centrifuge under conditions of 2.5 G, and remained there for 6 months. We then determined the calcium contents of the otoconia with energy dispersive X-ray element analysis, and recorded the size, shape and distribution of the otoconia. Scanning electron microscopy was used to make photos to determine the effects of hypergravity on the shape and size of the otoconia, and on the distribution of smaller and larger otoconia. No differences were found in the calcium content, shape, size or distribution of otoconia between centrifuged hamsters and control animals. Our findings indicate that structural adaptation to hypergravity does not take place at the otoconial level, at least not in animals subjected to hypergravity after the vestibular system was fully matured.