Impact of collagen crosslinking on the second harmonic generation signal and the fluorescence lifetime of collagen autofluorescence.

BACKGROUND/PURPOSE: Collagen is the major structural protein of the skin and its crosslinks are essential for its mechanical stability. In photodamaged skin, a decrease of the mature collagen crosslink histidinohydroxylysino-norleucine was reported. In this study, we investigated the consequences and measurability of the reduced crosslinking. METHODS: In order to determine the consequences of reduced collagen crosslinking, in vitro models of reduced collagen crosslinking were established. The collagen synthesis and structure was analyzed using the signals second harmonic generation (SHG) and the fluorescence lifetime of the collagen autofluorescence by a multiphoton laser scanning microscope. RESULTS: Reduced collagen crosslinking results in a posttranscriptionally diminished collagen synthesis, a modified structure of the collagen fibers and fibrils and a higher intensity of the SHG signal. The SHG signal might be influenced by the interspaces of the collagen molecules within one collagen fibril. Because of these findings, it can be speculated that reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril, resulting in an enhanced SHG signal. Alternative explanations are discussed. Furthermore, the fluorescence lifetime was reduced in the in vitro models of reduced collagen crosslinking. In the crosslink sites of the collagen molecules, the main ratio of fluorescence is found. As the fluorescence lifetime is determined not only by the fluorescent molecule itself but also by its microenvironment, the change in the fluorescence lifetime might be explained by reduced crosslinking at the crosslink site. CONCLUSION: A reduction of collagen crosslinking (as seen in photodamaged skin) results in an increase of the SHG signal and a decrease of the fluorescence lifetime in vitro. In vivo measurements of the two parameters might reveal the status of collagen crosslinking and therefore help to identify the status of dermal photodamage or pathogenesis using collagen crosslinking determination.

Folic acid and creatine improve the firmness of human skin in vivo.

BACKGROUND: The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. AIMS: Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. PATIENTS/METHODS: Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. RESULTS: The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. CONCLUSIONS: Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.

Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.

BACKGROUND: Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. METHODS: Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. RESULTS: The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-ss had an inhibitory effect on microcarrier expanded chondrocytes. CONCLUSIONS: Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.