MARINE INGREDIENTS To study the effects of the
exopolysaccharide on dermal fibroblasts, we compared retracting collagen lattices, in which collagen fibres are remodelled to a floating disc and the tension is very low and distributed isotropically, to stressed lattices that cannot be retracted due to adherence of the collagen lattice to a nylon thread placed at the inner perimeter of the dish.6 Within collagen lattices, fibroblasts
utilise α2β1 integrin receptors to mediate cell-collagen contact.7,8
By their
transmembrane nature, α2β1 receptors physically connect external collagen fibres with the actin filament network and thereby transmit forces and information required for contraction into the cells.9 In relaxed lattices, fibroblasts appear stellar with short processes; in stretched lattices, fibroblasts are elongated, bipolar, and oriented along the lines of tension according to observations made by Lambert and Bellows.6,10
Effect on lattice contraction Bell reported that the incorporation of fibroblasts in a collagen gel induces a progressive contraction of the gel, resulting in the formation of a dense collagen disc, called retracted lattice. Contraction is determined by measurements of gel diameter over time. Retraction of the dermis equivalent is proportional to the number of fibroblasts incorporated and to the collagen concentration.5 In our experiment, the relaxed lattices are made with normal human dermal fibroblasts (NHDF) in passage 6 and collagen I containing or not 0.02% or 0.2% of EPS Seafill (now referred to as ‘the marine exopolysaccharide’). Then the obtained lattices were deposited on multi- well plates. They were seeded and placed in an incubator for 52 hours. After 52 hours, the lattices were photographed and the area occupied by the lattice in the culture well was measured. The treatment with 0.02% and 0.2% of the marine exopolysaccharide improves the contraction of the lattice in the Petri dishes (see Fig. 1). The marine exopolysaccharide generates a tightening effect on the collagen fibre network of the relaxed lattice. This macroscopic contraction is related to the force induced by fibroblasts on collagen fibrils. Models have been developed to associate with this macroscopic phenomenon, microscopic phenomena taking place within the collagen gel.11
Effect on stretched lattice The stretched lattices were made with NHDF and collagen I containing or not 0.2% of the marine exopolysaccharide. Then the obtained lattices were deposited
48 PERSONAL CARE April 2012
Figure 3: 3D observation of collagen lattices which are a) untreated or b) treated with 0.2% of EPS Seafill. The arrows point to fibroblasts in the collagen lattice.
a b a b
Figure 4: Immunostaining of elastin fibres produced by human fibroblasts a) untreated or b) treated with 0.2% of EPS Seafill.
on multi-well plates, at the bottom of each is deposited a nylon ring. They were seeded and placed in an incubator for five days. After five days the lattices were photographed, the medium was then vacuumed out, the lattices rinsed and then fixed.
Microscopic observation was performed with a Nikon TE300 Eclipse microscope equipped with a Nikon DS DS-2Mv camera head. One photograph in three dimensions per well was taken.
The control lattice shows fibroblasts with a relaxed form and a flexible collagen
25 20 15 10 5 2% 0
Non-treated fibroblasts
Fibroblasts
treated with 0.2% of EPS Seafill
Figure 5: Quantification of the elastin staining intensity in human fibroblasts untreated or treated with EPS Seafill.
23%
matrix (see arrow on the control lattice in Figure 3).
In the presence of 0.2% of the marine exopolysaccharide, the shape of the fibroblasts is even more stretched, the collagen network is under tension, and a tensile force is generated over the entire lattice.
The marine exopolysaccharide induces the contraction of fibroblasts which consequently stretch the collagen network for a resulting tensing effect. The marine exopolysaccharide generates a tightening effect on the collagen fibre network.
Effect on elastin synthesis NHDF were used in passage 4 for elastin synthesis in stretched lattices. The microscopic observation allows analysing fibroblast morphology in a stretched lattice. As described by Lambert and Bellows6,10
are elongated, bipolar, and oriented along the lines of tension.
In the absence of product, the staining of elastin fibres is very low. In the presence of 0.2% the marine exopolysaccharide, an important increase in elastin staining is also observed (see Fig. 4). This effect was confirmed by an image analysis. The treatment with 0.2% of the marine exopolysaccharide increases by 23% the synthesis of elastin by human fibroblasts. This result suggests that the marine
we observed that fibroblasts
Elastin staining (%)
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