One of the main effects of ageing on skin is the reduction on its ability to selfregenerate and create new extracellular matrix (ECM). This has an impact on the production of key structural proteins, such as collagen, but also on proteoglicans, which play a role on cellular signalling.
This paper presents the results on the activity of a combination of synthetic peptides whose structures have been specifically designed to reorganise into the skin to build up a three-dimensional polymeric net that is completely biocompatible. This net not only provides a structural support to the skin helping the anchoring of cells, but also activates cellular regeneration processes. Moreover, it is able to activate intracellular signals that stimulate the proliferation and differentiation of skin cells, activating synthesis of extracellular proteins. Therefore, this specific combination of peptides constitutes the scaffold to create a structure that behaves like the extracellular matrix itself.
Skin ageing is caused by intrinsic biological factors combined with external effects (environmental impacts like UV or pollutants). Most of the skin ageing macroscopic signs (such as wrinkles, skin thinning or loss of firmness) are related to a reduction either of the quantity or quality of the extracellular matrix (ECM). Hence, the improvement of the structure of ECM is a key goal to minimise the effects of ageing and also for regeneration processes. Wound repair is an essential physiological process that plays a key role for tissue homeostasis. Any regeneration of injured or damaged skin, including damage by environmental factors, requires restoration of ECM. A large number of growth factors, such as the Fibroblast Growth Factor (FGF) or Transforming Growth Factor-??(TGF?), are known to promote various aspects of the repair process, including angiogenesis, inflammation, and migration of both keratinocytes and fibroblasts. Interactions between ECM cells also play a crucial role during the whole wound healing process. The aim of this work has been to study a combination of Tissue Regenerative Peptides (named Renaissance and here described as TRP), specially designed to construct a scaffold for tissue regeneration and ECM formation and able to be linked to fibroblasts and to enhance their proliferation. This approach is a step ahead of cosmetic regeneration strategies due to an action at two different levels: constructing itself a 3D regenerative matrix, which improves ECM properties, and also contributing to cellular signalling to enhance cellular proliferation, differentiation and multiplication.
Technology description
Tissue lesions represent a clinical challenge, and consequently, the repair and functional recovery of the damaged and degenerated tissue is likewise a great goal for researchers of several scientific disciplines (biomaterials, dermatology etc.). New and unprecedented solutions for tissue lesions have become possible by means of cellular and tissue engineering and, in particular, by using bioactive materials in order to provide structure and shape for tissue assembly. Tissue regeneration involves the development of living tissues such as blood vessels, cartilaginous tissue, bones, tendons and nerves. In skin, it essentially plays a role in the recovery from damage and ageing. The latest generation of scaffold biomaterials used for tissue engineering includes self-assembling peptides and non-biological amphiphiles, able to selfassemble and exhibit the physical and chemical properties of natural ECM. These materials have the potential for extensive modification by incorporation of peptide domains that influence cell adhesion, differentiation, and proliferation. Outstanding examples of the application of this technology are bioactive peptidic 3D fibril scaffolds capable of inducing remineralisation of damaged or diseased dental enamel or self-assembling peptidic structures that are ECM-like, with potential application in myocardial tissue regeneration. TRP has been designed to incorporate the latest advances in this field of research, to provide cosmetic science with a new approach to skin regeneration. The skin has a considerable potential for self-repair. However the cutaneous full repair is a regenerative process only in the fetus. Inflammation and scar markings prevail in the final stage of gestation and during life, consequently they alter the repair result. In adults, skin is regenerated forming a scar in the place of the wound. The aim of the TRP technology is to regenerate skin through a complete restoration of its structure, functions, and physiology. The process begins with the spontaneous formation of 3D scaffolding of peptides, which serves to organise and to reverse the damage by means of several functional activities. The initial activation of TRP shoots up the activation of growth factor and other morphogens that are deposited inside the 3D scaffolding of natural peptides. This scaffolding contains peptides of cellular adherence that show a specific ligament in a variety of integrin receptors found in the fibroblasts’ surface. These interactions, coupled with the accommodative activity of proteases, stimulate cellular migration in the scaffolding. The eventual activation and increase of the matrix by the same cells inside the initial scaffolding of this new connective tissue results in the formation of a new and renovated skin.
Methodology
Microarray (DNA Chip)
RNA was harvested from 3D skin model (Skinethic) after 36 hours of TRP treatment. TRP solution (2% of 50 ppm solution). Briefly, total RNA was extracted using Trizol (Gibco BRL Life Technologies) and purified on RNeasy Micro kit columns (Qiagen). Extracted RNA was used to synthesise double stranded cDNA using the One Cycle cDNA Synthesis Kit (Affymetrix, Inc.) CEL files were imported into the affy package in Bioconductor and pre-processed using the RMA (robust multiarray analysis) algorithm with the default parameters. Genes were filtered according to the following criteria: Signal ?log (100), Mean AbsFC (absolute fold-change) ?1.25.
Characterisation of formed structures. Scanning Electron Microscopy (SEM)
0.02% TRP solution was incubated (37°C, eight days) and analysed on a Jeol JSM-6310 scanning electron microscope. Transmission Electron Microscopy (TEM): JEOL JEM 1010 (Japan) with a 80 kV acceleration voltage. Images were captured with a CCD Megaview III (SIS) camera (Münster, Germany), after four days of incubation.
Cell proliferation studies
The study was undertaken with HDF (1 x 10 cells/cm3) and keratinocytes (2.5 x 10 cells/cm3) using cellular count by Trypan Blue. Three concentrations were tested (0.1%, 0.5%, 1.0%) of a TRP solution of 100 ppm. Counting was done on days two and four, and cell morphology was qualitatively evaluated by optical microscopy.
Affinity assay for fibroblasts
Human Dermal Fibroblasts (HDF) at low confluence were treated with a range of concentrations of TRP (0 ?g/mL, 5 ?g/mL, 10 ?g/mL, 20 ?g/mL and 40 ?g/mL) and fluoresceine-labelled during one hour at room temperature in the growth medium. Then HDF were washed several times on type V plates at 1500 rpm during two minutes each time, to remove non-binded peptides. Treated/non-treated cells were analysed by flow citometry (FACS).
Fibroblasts binding studies by SEM
HDF were seeded at low confluence on top of glass. Twenty-four hours later they were treated with 0.3% of a solution of 100 ppm of TRP. Samples were fixed 24 hours later and finally treated/nontreated cells were compared using SEM (Microscope NOVA NANOSEM2).
Increase of collagen I and VI formation by fluorescence microscopy
HDF were seeded at low confluence on top of glass. Twenty-four hours later they were treated with 1.0% of a solution of 100 ppm of TRP. Samples were incubated with the corresponding antibodies against collagen I and VI. Finally treated/non treated cells were compared using Spectral Confocal Microscopy (FV1000 Olympus).
Increase of collagen I biosynthesis
HDF were incubated with the matrix forming peptides (TRP, 72 hours). Human antibody of collagen type I (marked with biotin) was used for quantification.
Evaluation of the anti-wrinkle and of the re-densifying effect of TRP an in vivo test
The panel consisted of 20 women between 40-60 years old. The product was applied twice a day on the face during 56 days and formulated at 3% of solution (2.5 g/L of TRP). Elasticity and skin compactness were evaluated using a Cutometer MPA 580. Measurements of fold thickness were done with a Plicometer Fat Track Pro on the cutaneous fold between eye contour and temple. Wrinkle analysis was done with a VisioFace Quick camera to record images of the face (fixed at the same position) and image analysis was performed with Skin Surface Analyzer (SSA) software to measure wrinkle volume variation. Ecography measurements were carried out with DUB 22 Taberna Pro Medicum. Expert evaluation in the medical studio and volunteers’ self-evaluation was performed according to VAS scale where 0 is the minimum and 10 is the maximum value.
Results and discussion
The pattern of the affected genes studied though the DNA chip showed an induction of genes involved in the expression of different proteins, in which more than 20% of them are located and play their function in the cytoplasm, more than 5% in the extracellular space and more than 15% in the plasma membrane. Several pathways related to the regeneration and proliferation are modulated, such as cellular assembly and organisation mechanisms, cell growth, cell cycle, lipid metabolism and other interesting ones, like cell-to-cell signalling and interaction and cell mediated immune response. It is worth highlighting the effects on proteins such as actin and mucin 12, collagen XI, defensins and perforins, cytochrome P450, family 4, subfamily A, polypeptide 11 and glycerol-3- phosphate acylransferase 1, mitochondrial and stomatin, and formin-1. This activity profile is in agreement with the expected mechanism of action of TRP and its properties as unique scaffold for skin regeneration. The ability to create 3D structures was confirmed by electron microscopy (SEM and TEM, Figures 2 and 3). Fibre characterisation demonstrates that TRP self-assembles to promote threedimensional structures, able to act as a support structure ECM-like. The images show at different augmentations how peptides self-assemble to promote fibre structures up to 100 micrometers as measured by SEM. The affinity and binding of TRP on fibroblasts was confirmed at in vitro level. A dose-dependent effect was observed (Fig. 4). Hence, as the concentration increases, more fibroblasts bind to TRP and to the matrix that they create together with other ECM polymers. SEM images showed how treated fibroblasts exhibited a higher density of extracellular matrix components at the surface, demonstrating that the affinity of TRP by the fibroblast enhances interaction and production of ECM components (Fig. 5). Therefore, the 3D scaffold structure created by the peptides behaves like the natural ECM, linking cell and matrix. It should be noted that the new created 3D matrix has a specific and cellular structure difficult to distinguish it from the natural one. Cell proliferation studies showed that TRP enhanced fibroblast and keratinocyte proliferation in a dose-dependent manner. After four days of treatment with 0.1%, 0.5% and 1% of a 100 ppm solution of TRP, the increase in cell proliferation was: 51%, 62% and 82%, respectively in fibroblasts, and 54%, 71% and 83%, respectively, in keratinocytes. Hence, the studied combination of peptides provides biological signals for the cells. Moreover, the product exhibited the ability to promote formation of proteins such as collagen I and VI. After fibroblasts treatment, fluorescence microscopy images showed an increase in the density of fibroblasts and collagen (type I and VI). This confirms that TRP enhances fibroblast proliferation and extracellular matrix components production (Figs. 6 and 7). Increase in collagen I biosynthesis by fibroblasts was also quantified showing a dose-dependent effect 110% at 5 mg/mL, 122% at 10 mg/mL, 135% at 20 mg/mL, 160% at 40 mg/mL and 178% at 80 mg/mL. In vivo the combination of peptides TRP has confirmed its ability to improve biomechanical properties of skin, related to ECM quality. The statistical analysis showed at D28 an improvement of skin compactness of 30% and skin elasticity of 18%, being increased up to 37% and 24% at D56, respectively. Compactness of skin was also assessed by plicometry, showing an increase of skin thickness of 12% at D28 and 15% at D56. An increase in density and compactness of skin was also confirmed by ecography (Fig. 8). The images showed that global dermal profiles at D56 were more regular, indicating the redensifying power of the tested product. Data taken from volunteers showed an increase in dermal thickness (up to 7% in the best case). Wrinkle analyses demonstrated the efficacy of the combination of peptides to reduce the appearance of wrinkles (Fig. 9). The statistical analysis showed a significant improvement of wrinkle volume of 18%, reaching 27% in the best case. The objectively measured data were in agreement with the evaluation of clinical experts and volunteers’ self-assessment (Table 1), who evaluated the whole improvement of macroscopic signs of ageing.
Conclusions
Renaissance (TRP) has proved that this specific combination of peptides provides a 3D regenerative matrix that completes the natural renovation of skin with a completely new approach. It provides structural support to cells and adequate signalling to fibroblasts, visibly improving biomechanical properties of skin and repairing damage caused by ageing.
References
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