In vivo bio-integration of three hyaluronic acid fillers in human skin: a histological study.

BACKGROUND: Hyaluronic acid (HA) formulations are used for aesthetic applications. Different cross-linking technologies result in HA dermal fillers with specific characteristic visco-elastic properties. OBJECTIVE: Bio-integration of three CE-marked HA dermal fillers, a cohesive (monophasic) polydensified, a cohesive (monophasic) monodensified and a non-cohesive (biphasic) filler, was analysed with a follow-up of 114 days after injection. Our aim was to study the tolerability and inflammatory response of these fillers, their patterns of distribution in the dermis, and influence on tissue integrity. METHODS: Three HA formulations were injected intradermally into the iliac crest region in 15 subjects. Tissue samples were analysed after 8 and 114 days by histology and immunohistochemistry, and visualized using optical and transmission electron microscopy. RESULTS: Histological results demonstrated that the tested HA fillers showed specific characteristic bio-integration patterns in the reticular dermis. Observations under the optical and electron microscopes revealed morphological conservation of cutaneous structures. Immunohistochemical results confirmed absence of inflammation, immune response and granuloma. CONCLUSION: The three tested dermal fillers show an excellent tolerability and preservation of the dermal cells and matrix components. Their tissue integration was dependent on their visco-elastic properties. The cohesive polydensified filler showed the most homogeneous integration with an optimal spreading within the reticular dermis, which is achieved by filling even the smallest spaces between collagen bundles and elastin fibrils, while preserving the structural integrity of the latter. Absence of adverse reactions confirms safety of the tested HA dermal fillers.

The cutaneous lesions of dioxin exposure: lessons from the poisoning of Victor Yushchenko.

Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called "chloracne" are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics.

A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.

Three major difficulties must be overcome to establish a quantitative method for melanosomal transfer analysis: (i) establishing a three-dimensional co-culture reassuring direct melanocyte to keratinocyte transfer, (ii) separation of melanocytes and keratinocytes following co-culture and (iii) melanosome quantification in each cell population. Melanocytes and keratinocytes are cultured on the opposite sides of the porous membrane of hanging cell inserts (1µm pores, 2×10(6) pores/cm(2) ). Cell separation is performed after 3days of co-culture by simple trypsinisation. Melanosome quantification in separated cell populations was accomplished by an ELISA-like method using gp-100 as the antigen. Melanocytes and keratinocytes come into 'direct' contact through the pores, and melanosomal transfer is accomplished without cell passage through the membrane. Cell separation by simple trypsinisation results in pure melanocyte and keratinocyte populations. Melanosome quantification by the ELISA-like method proved to be sensitive and specific to distinguish the known inhibitors and inducers of melanosomal transfer.

Synergistic effect of hyaluronate fragments in retinaldehyde-induced skin hyperplasia which is a Cd44-dependent phenomenon.

BACKGROUND: CD44 is a polymorphic proteoglycan and functions as the principal cell-surface receptor for hyaluronate (HA). Heparin-binding epidermal growth factor (HB-EGF) activation of keratinocyte erbB receptors has been proposed to mediate retinoid-induced epidermal hyperplasia. We have recently shown that intermediate size HA fragments (HAFi) reverse skin atrophy by a CD44-dependent mechanism. METHODOLOGY AND PRINCIPAL FINDINGS: Treatment of primary mouse keratinocyte cultures with retinaldehyde (RAL) resulted in the most significant increase in keratinocyte proliferation when compared with other retinoids, retinoic acid, retinol or retinoyl palmitate. RAL and HAFi showed a more significant increase in keratinocyte proliferation than RAL or HAFi alone. No proliferation with RAL was observed in CD44-/- keratinocytes. HA synthesis inhibitor, 4-methylumbelliferone inhibited the proliferative effect of RAL. HB-EGF, erbB1, and tissue inhibitor of MMP-3 blocking antibodies abrogated the RAL- or RAL- and HAFi-induced keratinocyte proliferation. Topical application of RAL or RAL and HAFi for 3 days caused a significant epidermal hyperplasia in the back skin of wild-type mice but not in CD44-/- mice. Topical RAL and HAFi increased epidermal CD44 expression, and the epidermal and dermal HA. RAL induced the expression of active HB-EGF and erbB1. However, treatment with RAL and HAFi showed a more significant increase in pro-HB-EGF when compared to RAL or HAFi treatments alone. We then topically applied RAL and HAFi twice a day to the forearm skin of elderly dermatoporosis patients. After 1 month of treatment, we observed a significant clinical improvement. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that (i) RAL-induced in vitro and in vivo keratinocyte proliferation is a CD44-dependent phenomenon and requires the presence of HA, HB-EGF, erbB1 and MMPs, (ii) RAL and HAFi show a synergy in vitro and in vivo in mouse skin, and (iii) the combination of RAL and HAFi seems to have an important therapeutic effect in dermatoporosis.

Metabolism and biological activities of topical 4-oxoretinoids in mouse skin.

Retinoic acid mediates most of the biological actions of vitamin A. It is oxidized by CYP26A1 to 4-oxoretinoic acid, considered as an inactive catabolite of retinoic acid. However, in the light of studies reporting the presence of 4-oxoretinal or 4-oxoretinol as the predominant retinoids during morphogenesis, we analyzed the retinoid-like biological activity of these oxoretinoids in mouse skin in vivo. Topical 4-oxoretinal and 4-oxoretinol promoted significant epidermal hyperplasia and metaplasia in mouse tail. They induced a moderate response for epidermal inflammation, compared with retinal, whereas neither 4-oxoretinal nor 4-oxoretinol prevented menadione-induced epidermal lipid peroxidation, unlike retinal and retinol. As analyzed by quantitative PCR, 4-oxoretinal and 4-oxoretinol did not reproduce the significant increased expression of genes coding for keratin 4, amphiregulin, heparin-EGF and CYP26A1, that did induce retinal and retinol. However, both retinal and 4-oxoretinal significantly inhibited the lipopolysaccharide-induced maturation of human dendritic cells in vitro. As analyzed in vivo and in vitro, 4-oxoretinal and 4-oxoretinol were not converted into retinoic acid. We conclude that 4-oxoretinal and 4-oxoretinol exert a moderate direct retinoid-like activity in vivo, thus confirming previous in vitro studies in amphibians showing 4-oxometabolites of vitamin A as bioactive agents rather than inactive catabolites.

Effect of intense pulsed-light exposure on lipid peroxides and thymine dimers in human skin in vivo.

BACKGROUND: Intense pulsed light (IPL) generates high-intensity short flashes of visible light and has been used for about 10 years to improve dermatological conditions such as telangiectasia, pigmented lesions, and skin aging. Although these systems deliver a moderate dose (10-30 J/cm(2)) of visible light, this dose is delivered during a short pulse (2-5 milliseconds), which implies a very high fluence rate (approximately 4000 W/cm(2)). For this reason, we speculated whether the Bunsen-Roscoe law of reciprocity could still be valid in these conditions. OBSERVATIONS: Nine healthy volunteers were exposed to IPL or UV-A or simulated solar UV radiation, and then thymine dimer and lipid peroxide concentrations were determined in skin biopsy specimens of the exposed sites. Only exposure to solar UV radiation (7-J/cm(2) UV-A + 80-mJ/cm(2) UV-B) produced measurable amounts of thymine dimers in DNA from skin biopsy specimens, whereas UV-A radiation (40 J/cm(2)) and IPL (9 J/cm(2)) induced 3-fold and 6-fold increases of cutaneous lipid peroxides, respectively. CONCLUSIONS: These preliminary results indicate that IPL, although filtered for wavelengths shorter than 500 nm, can generate oxidative stress, a typical hallmark of UV-A, but does not induce thymine dimers. This emphasizes the need for long-term studies involving IPL before using this technique in a recurrent manner.

Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism.

BACKGROUND: Skin atrophy is a common manifestation of aging and is frequently accompanied by ulceration and delayed wound healing. With an increasingly aging patient population, management of skin atrophy is becoming a major challenge in the clinic, particularly in light of the fact that there are no effective therapeutic options at present. METHODS AND FINDINGS: Atrophic skin displays a decreased hyaluronate (HA) content and expression of the major cell-surface hyaluronate receptor, CD44. In an effort to develop a therapeutic strategy for skin atrophy, we addressed the effect of topical administration of defined-size HA fragments (HAF) on skin trophicity. Treatment of primary keratinocyte cultures with intermediate-size HAF (HAFi; 50,000-400,000 Da) but not with small-size HAF (HAFs; <50,000 Da) or large-size HAF (HAFl; >400,000 Da) induced wild-type (wt) but not CD44-deficient (CD44-/-) keratinocyte proliferation. Topical application of HAFi caused marked epidermal hyperplasia in wt but not in CD44-/- mice, and significant skin thickening in patients with age- or corticosteroid-related skin atrophy. The effect of HAFi on keratinocyte proliferation was abrogated by antibodies against heparin-binding epidermal growth factor (HB-EGF) and its receptor, erbB1, which form a complex with a particular isoform of CD44 (CD44v3), and by tissue inhibitor of metalloproteinase-3 (TIMP-3). CONCLUSIONS: Our observations provide a novel CD44-dependent mechanism for HA oligosaccharide-induced keratinocyte proliferation and suggest that topical HAFi application may provide an attractive therapeutic option in human skin atrophy.

Topical calcineurin inhibitors decrease the production of UVB-induced thymine dimers from hairless mouse epidermis.

BACKGROUND: An increased incidence of ultraviolet-light-related skin tumours is a well-known problem in patients undergoing posttransplantation immunosuppression with systemic calcineurin inhibitors such as cyclosporine A or tacrolimus. UV-related carcinogenesis as a consequence of long-term treatment of sun-exposed sites with topical calcineurin inhibitors is therefore of theoretical concern. RESULTS: In this study, we show that tacrolimus acts as a UVB filter when incorporated into liposome membranes. In hairless mice pretreated with 1% pimecrolimus cream, 0.1% tacrolimus ointment or vehicle, the amount of epidermal thymine dimers, measured 1 h after 1 J/cm2 of UVB irradiation, was decreased by 89, 84 and 47%, respectively, as compared to untreated mice. Forty-eight hours after UVB irradiation, 97, 89 and 93% of epidermal thymine dimer levels were removed in pimecrolimus-, tacrolimus- or vehicle-treated mice, respectively. In contrast, 69% of thymine dimers, originally present in much higher amounts than in treated mice, were removed from untreated controls. UVB-induced apoptosis was less pronounced in treated mice. CONCLUSION: Taken together, these results suggest that topical calcineurin inhibitors prevent DNA photodamage due to a filter effect of both vehicle and active components, whereas they do not affect the clearance of DNA photoproducts.

Pharmacology of RALGA, a mixture of retinaldehyde and glycolic acid.

BACKGROUND: Retinoids and alpha-hydroxy acids (AHAs) are major compounds in topical therapy. They exert distinct but potentially complementary activities. However, their association is limited by their respective irritating potential. Recently, the first association between a retinoid and an AHA has been achieved; this formulation (RALGA) associates retinaldehyde (RAL)--a precursor of retinoic acid (RA)--and glycolic acid (GA)--an AHA. OBJECTIVE: To study the pharmacological properties of RALGA. METHODS: The bioavailability of RAL into the skin after topical RALGA was studied by HPLC, and its bioconversion to RA was analysed by measuring the enzyme activity of retinaldehyde dehydrogenase and the RA content in the epidermis and dermis. The retinoid activity of RALGA was studied on the modulation of Hhb4 keratin mRNA on the tail of C57BL/6 mice, and its comedolytic properties on the size and density of dermal cysts and the morphology of sebaceous glands in hairless mice. RESULTS: Epidermal and dermal concentrations of RAL and RA were higher after RALGA treatment, as compared to both RAL 0.1% alone and RA 0.05% alone; this indicates that the presence of GA favours the bioavailability and biotransformation of RAL into RA. The retinoid activity of RALGA (suppression of Hhb4 mRNA keratin) was similar to that of RAL alone, indicating that the presence of GA does not interfere with specific retinoid activity; GA alone had no effect in this test, which confirms the specificity of Hhb4 mRNA keratin modulation for retinoid activity. The diameter and the density of dermal cysts as well as the size of sebaceous glands were significantly decreased by RALGA. CONCLUSION: These observations indicate that the addition of an AHA such as GA to a retinoid such as RAL results in a better bioavailability of the retinoid, thus a higher delivery of RA, which potentiates the biological activities of the retinoid. This combination allows a delivery of high amounts of RA in the skin while preventing the side-effects usually observed with high concentrations of topical RA.

Vitamin A exerts a photoprotective action in skin by absorbing ultraviolet B radiation.

Retinyl esters, a storage form of vitamin A, concentrate in the epidermis, and absorb ultraviolet radiation with a maximum at 325 nm. We wondered whether these absorbing properties of retinyl esters might have a biologically relevant filter activity. We first used an in vitro model to assess the photoprotective properties of retinyl palmitate. We then applied topical retinyl palmitate on the back of hairless mice before exposing them to 1 J per cm2 ultraviolet B, and assayed the levels of thymine dimers produced in epidermal DNA 2 h following ultraviolet B exposure. Finally, we applied topical retinyl palmitate or a sunscreen on the buttocks of human volunteers before exposing them to four minimal erythema doses of ultraviolet B; we assayed the levels of thymine dimers produced 2 h following ultraviolet B exposure, and determined the intensity of erythema 24 h after ultraviolet B. In vitro, retinyl palmitate was shown to be as efficient as the commercial filter octylmethoxycinnamate in preventing ultraviolet-induced fluorescence or photobleaching of fluorescent markers. The formation of thymine dimers in mouse epidermis was significantly inhibited by topical retinyl palmitate. In human subjects, topical retinyl palmitate was as efficient as a sun protection factor 20 sunscreen in preventing sunburn erythema as well as the formation of thymine dimers. These results demonstrate that epidermal retinyl esters have a biologically relevant filter activity and suggest, besides their pleomorphic biologic actions, a new role for vitamin A that concentrates in the epidermis.