Figures
Figure 1
Macroscopic appearance (A-E) , histological visualization (F-J) and HA detection (K-O) of the reconstructed skin samples at the end of the culturing period (day 21). Dermal equivalents were prepared without exogenous material (A, F, K) , with 100 μl free HA (B, D, G, I, L, N) or with 100 μL cross-linked HA (C, E, H, J, M, O) . In both the mixed and the inclusion model (C, E) , cross-linked HA incorporation resulted in an increase in the transparency of the dermis. Free HA had no or a reduced macroscopic impact on the mixed and inclusion models, respectively (B, D) . Arrows show localization of exogenous HA inclusion. In histological staining (G-J) , HA is revealed using alcian blue staining, in contrast to the collagen stained with Sirius red. Specific detection of endogenous and exogenous HA was seen in the epidermis and dermis via immunostaining (K-O) . In the dermis, exogenous free HA could not be detected (L, N) whereas exogenous cross-linked HA was observed with a strong intensity (M, O) . Top scale bar: 1 cm, middle scale bar: 200 μm, bottom scale bar: 100 μm.
Figure 1
Figure 2
Control of HA-specific immunodetection in the reconstructed skin with cross-linked HA . In the inclusion reconstructed skin model, endogenous HA is detected in both the epidermis and dermis (A) ; the exogenous HA inclusion is detected in the middle part of the reconstructed skin with a strong green intensity (B) . Specificity of the detection was confirmed by an absence of signal when the samples were treated with hyaluronidase prior to staining (C, D ). Counterstaining of nuclei by propidium iodide in red. Scale bar: 100 μm.
Figure 2
Figure 3
MMP-1 protein secretion in culture media of dermal equivalents at day 4 (A) and full skin equivalents at day 21 (B). Dermal equivalents were prepared with 100 μL or 400μl free, cross-linked HA+ or cross-linked HA++ (low and high cross-linked HA, respectively). Total MMP-1 protein expression was measured in supernatants at day 4 or day 21. Note that the protein secretion level is modulated by HA volume, the incorporation method and the degree of cross-linking. MMP-1 secretion is higher with 400 μl of HA versus 100μl from day 4 in both mixed and inclusion dermal equivalent models (A). MMP-1 modulation with 100μl was seen later in skin equivalents, after 21 days (B). A higher increase in MMP-1 expression with exogenous HA was observed in the inclusion model as compared to the mixed model. Cross-linking of HA also seems to be modulating MMP-1 secretion (A, B). *Significant difference (p<0.05); # significant difference between the inclusion model and the mixed model for the same type of HA (p<0.05).
Figure 3
Figure 4
Collagen neosynthesis by fibroblasts with cross-linked HA in the mixed reconstructed skin model and the inclusion reconstructed skin model (day 21). Skin equivalents were prepared with 100μl free, cross-linked HA+, or cross-linked HA++ in the dermal compartment and stained for human N-terminal type I procollagen (A) and human N-terminal type III procollagen (B) after 21 days of culture. Counterstaining of nuclei by propidium iodide (red) or bis-benzimide (blue). Scale bars: 50 μm. Type I procollagen C-terminal peptide (PCI-PIP) was detected in supernatants of the cultures (C) . Significant differences between the inclusion model and the mixed model (n = 4 per condition) for the same type of HA are reported (p<0.05). Note that in the inclusion model, 100 μL cross-linked HA increased type I and III procollagen expression of dermal fibroblasts, as shown by immunostainings (A, B) , and cleaved peptide C-terminal of procollagen secretion, as compared to the mixed model (C) .
Figure 4
Figure 5
Impact of cross-linked HA on surrounding fibroblasts in the inclusion model. A-C) Double labeling of reconstructed skin samples (inclusion model with 100μl free or cross-linked HA) for C-terminal of type I procollagen (green) and HA (red). D-F) Double labeling of reconstructed skin for vimentin (green) and HA (red). G-I) Double labeling of reconstructed skin for α-SMA (green) and HA (red). Note that immunostainings illustrate an increase of collagen synthesis for fibroblasts close to the exogenous cross-linked HA+ (C ). Visualization of vimentin cytoskeleton expression revealed an elongated shape of fibroblasts localized close to cross-linked HA+ (F) together with an increase of α-SMA expression (I ). Dotted lines on pictures mark the border between inclusion of exogenous HA and the dermis. Counterstaining of nuclei by bis-benzimide (blue). Scale bar: 25 μm, inset: 8 μm.
Immunofluorescence around each cell in several sections of samples was quantified in dermal area 1 and dermal area 2 in the inclusion model. Positive cells were counted according to their fluorescent level emission (level.μm2 ), high: >5000; intermediary: 5000-1000; low: 0-1000 (J, K ) and mean expression per cell in each area was compared.
Figure 5
Figure 6
CD44 receptor expression of fibroblasts in inclusion skin equivalent models with HA. Pictures are representative of standard CD44 pattern expression (green) in fibroblasts in the control skin equivalent (A), close to free HA inclusion (B), and close to cross-linked HA+ inclusion (C). Higher expression of CD44 was seen with exogenous HA as compared to control skin equivalents.Counterstaining of nuclei by propidium iodide (red). Scale bar: 25 μm, inset: 8 μm. Immunofluorescence in several sections of samples was quantified around each cell (visible nucleus) in the dermal part of the reconstructed skin. Results are expressed as mean ± SEM (D). Integrated emission of CD44 receptor was detected significantly higher with exogenous HAs (p<0.001).
Figure 6
Authors
L’OREAL Research and Innovation,
1, avenue Eugène Schueller,
93600 Aulnay-sous-bois, France
Facial aging is mainly characterized by wrinkles, folds, volume loss and sagging, which reflect gradual and permanent changes occurring in the deeper compartments of the skin. Reducing the clinical signs of aging through soft-tissue augmentation using materials referred to as ‘fillers’ is an expanding field of aesthetic medicine and a prevalent anti-skin aging strategy. A wide range of fillers which differ in their origin (natural or synthetic) and their formulation can be found on the market, in [...]