La MedicinaEsteticaYear 39 N. 3
July· September 2015
Biorivolumetria® seen undera microscope. In vitro tests toevaluate regenerative effect ofBiorivolumetria® products
Andrea Alessandrini
Marco Zazzetta
La MedicinaEsteticaYear 39 N. 3
July· September 2015
ABSTRACT
There is a new method that has been developed in aesthetic medicine:"Biorivolumetria®". It is an anti-aging treatment that combines avolumising effect with an effect of cellular regeneration. The innovationis based on the use of a special chemical formula that implements a highlypure cross-linked Hyaluronic Acid (HA) together with a native intercalatedhyaluronic acid. When injected, this formulation,besides an immediatevolume restoration, enables as well a long-lasting receptor stimulationwhich therefore, performs a regenerative action on the derma and thehypoderm. In vitro tests have mainly been performed to evaluate theregenerative action. The tests were performed on human endothelial cellsand fibroblasts, and the results revealed an increase in the endothelialmigration process and proliferation of fibroblasts. To evaluate theeffects of the treatment on angiogenesis markers immunofluorescenceanalyses were performed that have shown an increase in trans-cellularpermeability. Tests on the presence of proteins related to the cell-to-cellcontact have demonstrated an inhibition of occludin, a junction proteinthat impedes cellular migration. The biomaterial Regenyal Idea Bioexpander® favours the expression of CD40 (a receptor that participatesin the formation of new vessels) and the expression of fibronectin (thekey element in the regeneration processes of tissues), while the level ofFGF2 - a growth factor that takes part in the proliferation process of fibroblasts-remains constant.
Introduction
In Aesthetic Medicine and was developed an innovative method, which is called "Biorivolumetria®": it is an anti-aging treatment which produces volume and cell regenerationat the same time.
The innovation is based on the use of special formulations of pure hyaluronic acid (HA), devoid of protein residues and solvents; the production process is protected by patents, because thehyaluronic acid, cross-linked with BDDE, traps native hyaluronic acid inside, intercalated in different quantities according to the formulations. The cross-linked hyaluronic acid may be likened to a sort of scaffold that protects and encloses the bio-interactive HA. This native HA is released more slowly than the same acid injected into the skin and not protected; in this way it exerts a prolonged action of receptor stimulation. Moreover the presence of native HA gives greater elasticity[1].
Numerous studies have confirmed the regenerativepropertiesof native hyaluronic acid[2],[3],[4],[5],[6]. Our aim was to evaluate the biological effects of biorivolumetria products on cells, confirming the clinical data[7],[8] that have amply demonstrated improved tissue. In this regard we have carried out in vitro studies of proliferation and migration of endothelial cells and human fibroblasts. The study is run by Noxamet, a spin-off of the University of Siena and Pavia.
Materials and methods
Cell cultures
endothelial cells from human umbilical vein (HUVEC) and endothelial cells of human blood (HDBEC) were purchased from Promocell (Heidelberg, Germany). Both cell lines were grown in complete endothelial growth medium (EGM-2) (Lonza, Basel, Switzerland), it integrates with 10% fetal bovine serum (FBS).
The human dermal fibroblasts (HDF) are from Lonza (Verviers, Belgium). The cells were cultured at 37 ° C with a concentration of 5% C02. The cells were split 1:3 twice a week, and used up to passage 10.
Cell survival and proliferation
The proliferation of endothelial cells and fibroblasts, was evaluated according to the same protocol. 1000 cells per well (96 well plate) were left to adhere in culture medium containing 10% serum for 3-4 hours, then the test substances were added to the medium with 1% serum and 2%. All experiments were performed in triplicate. After 2 days, the cells were fixed, stained and counted randomly to 20x magnification originated, in 5 fields[9]. The data are reported as the number of cellscounted/well.
Migration of cells in vitro by means of scratch test
Endothelial cells and fibroblasts were seeded into 24-well plates (1 x 105 cells/well) and, after confluence, were scratched by the use of a sterile tip. Once gently washed to remove debris, the cells were treated with the test substances for 18 h. The results are expressed as percentage of the wound area with respect to time 0, for measurement by image analysis software[10].
Immunofluorescence analysis
Occludin (cell-cell adhesion marker), fibronectin (extracellular matrix protein), fibroblast growth factor-2 (FGF-2) (autocrine growth factor) and CD40 (a marker of angiogenesis and inflammation) expressed on the cell surface or intracellular, were visualised by confocal analysis. 5 x 104 cells were seeded on circular glass slides by 1cm. After 18 h, the cells were washed and treated with the biomaterial Regenyal Idea Bio-expander® to 1 mg / ml in medium containing serum to 2%. The analysis and immunofluorescence was performed as previously reported10,[11],[12].
Materials and Reagents
The cell culture reagents are sourced from Sigma-Aldrich (St. Louis, MO, USA). the foetal bovine serum is from Hyclone (Euroclone, Milan, Italy). 40 kDa FITC-dextran supplied by Life Technologies (Carlsbad, CA, USA). The antibodies anti-CD40 and anti-FGF-2 from Santa Cruz Biotechnology (Dallas, Texas). The Anti-occludin is supplied by Zymed - Life Technologies (Carlsbad, CA, USA), the Anti-Fibronectin from Sigma (Saint Louis, MO, USA).
Data analysis and statistical procedures
Results are representative or averages of at least three independent experiments done in triplicate. Statistical analysis was performed using ANOVA followed by Bonferroni test, and when appropriate by the Student t-test (GraphPad). P <0.05 was considered statistically significant.
Results
RegenyalIdeaBio-expander® promotes the proliferation of fibroblasts but not the proliferation of endothelial cells.
The endothelial cells, exposed to increasing concentrations of biomaterial-hyaluronic acid dissolved in the culture medium containing two different percentages of serum (2% FBS and 1, v: v), have not increased their proliferative potential (Figure 1). Fibroblasts exposed to increasing concentrations of hyaluronic acid-biomaterial did not show any impairment of cell survival. The number of cells in the presence of the biomaterial Regenyal Idea Bio-expander® was kept as in the control condition (medium, 2% FBS), with a considerable increase in the number of cells in 1 mg / ml of hyaluronic acid (Figure 2 ).
Figure 1 - The endothelial cells have not increased their proliferative potential.
Figure 2 - The number of fibroblasts in the presence of the biomaterial has undergone a considerable increase in the number of cells at a concentration of 1mg / ml of hyaluronic acid
Regenyal Idea Bio-expander® promotes the migration of endothelial cells, but not migration of fibroblasts in the wound healing assay
The scratch test was made on confluent cell cultures, in order to evaluate the cellular motility in the presence of the biomaterial to mimic wound healing. A clear increase of cell migration was measured in endothelial cells exposed to 1 mg / ml of hyaluronic acid (Figure 3a). The spontaneous migration of fibroblasts had not been further stimulated by the biomaterial at any concentration tested (0.1 to 10 mg) (Figure 3b).
Figure 3a - Increased migration of endothelial cells
in the scratch test
Figure 3b - The spontaneous migration of fibroblasts not been further Stimulated by biomaterial Regenyal Idea Bio-expander® at any concentration tested (0.1 to 10 mg)
Hyaluronic acid and markers of angiogenesis
Cultured endothelial cells, once confluent, were studied in terms of the transcellular permeability, a parameter for the acquisition of an inflammatory phenotype. The incubation of the monolayers HDBEC with hyaluronic acid for 18 h has madethe cell capable of increasing the transcellular permeability. An increase of 60% of the permeability was found compared to untreated cells, the value is maintained over time (the trend of the spontaneous permeability is increased 1.10, 1.62 and 1.63 fold compared to basal conditions, respectively after 15, 30 and 45 min of incubation with the tracer).
The endothelial cells were then studied for the presence / localisation of proteins related to cell-cell contact such as occludin. In the control condition the cells show the presence of labeling of occludin especially in the cell periphery. Treatment with hyaluronic acid, in agreement with the migration characteristic of the cells and with the increase of the permeability, reduces the cytoplasmic expression of occludin and its periplasmic localisation (Figure 4). The labeling of the cells for a marker of inflammatory cells such as CD-40, an activation marker of endothelial cells against both inflammatory phenotype toangiogenic[13], is greater after incubation with hyaluronic acid (Figure 4).
Figure 4 - Regenyal Idea Bio-expander reduced the expression of occludin and cell labeling for CD-40 and result increased after incubation
Markers related to the functions of fibroblasts were then assessed in response to biomaterial Regenyal Idea Bio-expander®. In cells exposed to 1 mg / ml of biomaterial, the expression of extracellular matrix proteins, such as fibronectin and the growth factor FGF-2 fibroblasts, were maintained (Figure 5). Ie documents that the biomaterial can support the proper function of the connective once implanted in vivo.
Figure 5 - The expression of extracellular matrix proteins,
such as Fibronectin and the fibroblast growth factor
FGF-2, have been maintained
Discussions and conclusions
From our in vitro models we can confirm that Regenyal Bio-expander® Idea has excellent biocompatibility with fibroblasts whose functions and whose markers are maintained at an optimum level. Endothelial cells, being more sensitive to physical stimuli, indicate a positive response to the biomaterial Regenyal Idea Bio-expander® in a range of rigid concentration (0.1-1mg / ml) with an angiogenic phenotype activated. In vitro data reinforce the clinical data in our possession giving us indications on improving tissue probably due to the proliferation of fibroblasts and the improvement of microcirculation. Furtherin vivo studies are underway in animal models to refute the data obtained so far on biorivolumetria products.
MD, Lecturer at theInternational School ofAesthetic Medicine of theFatebenefratelliFoundation inRome; Head of the AngiologyDepartment of the ItalianMinistry of Health - Rome
Biologist, Technical Manager at Regenyal Laboratories srl
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