A Practical Approach for the In Vitro Safety and Efficacy Assessment of an Anti-Ageing Cosmetic Cream Enriched with Functional Compounds.

(1) Background: Cosmeceuticals are topical products applied to human skin to prevent skin ageing and maintain a healthy skin appearance. Their effectiveness is closely linked to the compounds present in a final formulation. In this article, we propose a panel of in vitro tests to support the efficacy assessment of an anti-ageing cream enriched with functional compounds. (2) Methods: biocompatibility and the irritant effect were evaluated on reconstructed human epidermis (RHE) and corneal epithelium (HCE) 3D models. After a preliminary MTT assay, normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) were used to evaluate the extracellular matrix (ECM) protein synthesis, and interleukin-6 (IL-6) and metalloproteinase-1 (MMP-1) production. (3) Results: data collected showed good biocompatibility and demonstrated the absence of the irritant effect in both 3D models. Therefore, we demonstrated a statistical increase in collagen and elastin productions in NHDF cells. In HaCaT cells, we highlighted an anti-inflammatory effect through a reduction in IL-6 levels in inflammatory stimulated conditions. Moreover, the reduction of MMP-1 production after UV-B radiation was demonstrated, showing significant photo-protection. (4) Conclusion: a multiple in vitro assays approach is proposed for the valid and practical assessment of the anti-ageing protection, anti-inflammatory and biocompatible claims that can be assigned to a cosmetic product containing functional compounds.

Red grape (Vitis vinifera L.) flavonoids down-regulate collagen type III expression after UV-A in primary human dermal blood endothelial cells.

Red grape (Vitis vinifera L.) flavonoids including flavan-3-ols (eg, catechin and epicatechin), flavonols (eg, quercetin) and anthocyanins (eg, malvidin) exert anti-inflammatory and antioxidant activities. In the skin they also have a photoprotective action, and their effects have been extensively investigated in keratinocytes, melanocytes and fibroblasts. Despite their known effects also on blood vasculature, little is known on their activities on human dermal blood endothelial cells (HDBECs), which are critically involved in skin homeostasis as well as in the pathogenesis of neoplastic and inflammatory skin diseases. We sought to study the biological effects of selected red grape flavonoids in preventing the consequences of ultraviolet (UV)-A irradiation in vitro. Our results show that red grape flavonoids prevent UV-A-induced sICAM-1 release in HDBECs, suggesting that this cell type could represent an additional target of the anti-inflammatory activity of flavonoids. In addition, flavonoids effectively inhibited UV-A-induced synthesis of collagen type III at both RNA and protein level, indicating that dermal blood microvasculature could be actively involved in ECM remodelling as a consequence of skin photo-ageing, and that this can be prevented by red grape flavonoids.

Investigation on the Biological Safety and Activity of a Gluconolactone-Based Lotion for Dermocosmetic Application.

(1) Background: Cosmeceuticals are formulas enriched with active ingredients that exert efficacy on different skin molecular targets. (2) Methods: Cell viability and the absence of potential irritant risk were evaluated on keratinocytes (HaCaT), fibroblasts (NHDF), adipocytes (3T3-L1), sebocytes (PCi-SEB_CAU) and reconstructed human epidermis (RHE), respectively. Several treatments were performed to evaluate the ability of the lotion to stimulate the production of collagen and elastin, stimulate the differentiation of keratinocytes and reduce the number of senescent cells following UVB stimulation. In addition, the modulation of genes involved in the production, storage and accumulation of sebum were investigated. (3) Results: The results obtained demonstrated the biosafety of the formula in all cell lines tested. The 24-h treatment with non-cytotoxic concentrations determined an increase in the expression of the collagen (COL1A1), elastin (ELN) and involucrin (IVL) genes, while a reduction of peroxisome proliferator-activated receptor-gamma (PPARγ) gene expression and a reduction of SA-ßgal-positive cells were found. Moreover, the treatment did not interfere with normal steroid 5-alpha reductase (5RDA3) gene expression levels. (4) Conclusions: Data collected demonstrated the biosafety of the lotion, the non-comedogenic property and a multi targets anti-aging effect. In particular, data collected on the booster lotion make it a valid way to counteract the pore dilatation aging related.

The Anti-Ageing and Whitening Potential of a Cosmetic Serum Containing 3-O-ethyl-l-ascorbic Acid.

Skin ageing has many manifestations such as wrinkles, dryness, hyperpigmentation, and uneven skin tone. Extrinsic and intrinsic factors, especially solar ultraviolet light (UVB), contribute to skin ageing; its main features are brown spots, alterations in melanin pigmentation, and a decrease in collagen and hyaluronic acid linked to oxidative stress. Several studies showed that topical products containing ingredients with antioxidant activity can reduce oxidative damage; to provide a maximum anti-ageing effect to the skin, topical products can combine various ingredients. C-SHOT SERUM contains a combination of two molecules with a proven anti-ageing activity: a high percentage (30%) of a more stable vitamin C derivative, 3-O-ethyl-l-ascorbic acid, and lactic acid (1%). The product showed a high biocompatibility, assessed through an MTT assay on keratinocytes and on Reconstructed Human Epidermis (RHE, SkinEthic); the anti-ageing activity was demonstrated on human dermal fibroblasts and keratinocytes by a statistically significant increase in collagen production and a reduction of a UVB-induced DNA damage marker (γ-H2AX histone), indicating DNA protection. Moreover, a depigmenting activity, shown by a highly significant decrease in melanin content on treated Reconstructed Human Pigmented Epidermis (RHPE), was assessed. According to the data of our study, the tested product contrasts the effect of skin ageing and irregular pigmentation due to the physiological decline of the skin.

In Vitro Hair Growth Promoting Effect of a Noncrosslinked Hyaluronic Acid in Human Dermal Papilla Cells.

Dermal papilla cells (DPCs) are a source of nutrients and growth factors, which support the proliferation and growth of keratinocytes as well as promoting the induction of new hair follicles and maintenance of hair growth. The protection from reactive oxygen species (ROS) and the promotion of angiogenesis are considered two of the basal mechanisms to preserve the growth of the hair follicle. In this study, a noncrosslinked hyaluronic acid (HA) filler (HYDRO DELUXE BIO, Matex Lab S.p.A.) containing several amino acids was tested with in vitro assays on human follicle dermal papilla cells (HFDPCs). The experiments were carried out to investigate the possible protection against oxidative stress and the ability to increase the vascular endothelial growth factor (VEGF) release. The results demonstrated the restoration of cell viability against UVB-induced cytotoxicity and an increase in the VEGF secretion. These data demonstrate the capability of the product to modulate human dermal papilla cells, suggesting a future use in mesotherapy, a minimally invasive local intradermal therapy (LIT), after further clinical investigations.

In Vitro Evaluation of the Effect of a Not Cross-Linked Hyaluronic Acid Hydrogel on Human Keratinocytes for Mesotherapy.

The multicomponent preparations for mesotherapy are based on the principle that skin and hair aging can be prevented by supplying the fundamental substrates for correct cellular functioning, such as nucleotides, vitamins, amino acids, and biomolecules including hyaluronic acid (HA) that promote skin hydration and several biological activities. The study provides evidence for the application of HYDRO DELUXE BIO (Matex Lab S.p.A), a biocompatible hydrogel containing not cross-linked HA, for the treatment of the scalp's skin by mesotherapy. Using an in vitro model of immortalized human keratinocytes, we studied markers involved in hair aging prevention and growth, such as inflammatory markers, angiogenesis, and oxidative damage. HYDRO DELUXE BIO showed high biocompatibility and the ability to significantly reduce the expression of the inflammation marker interleukin (IL)-8 in Tumor Necrosis Factor (TNF)-stimulated cells. Then, we evaluated angiogenesis, a pivotal event during hair growth, measuring the Vascular Endothelial Growth Factor (VEGF) expression that resulted to be significantly increased in treated cells, suggesting a pro-angiogenetic capability. A protective activity against the oxidative stress agent was showed, increasing the survival rate in treated cells. Concluding, HYDRO DELUXE BIO is suitable for treatment by mesotherapy of the scalp's skin as it modulates the expression levels of markers involved in the biorevitalization of the hair follicle.

In Vitro Embryo Production in Buffalo Species (Bubalus bubalis).

In the last years there has been a growing attention in the world regarding buffalo (Bubalus bubalis) breeding. In tropical countries, climatic and environmental conditions make buffalo an irreplaceable milk producer. Since cattle cannot express their own productive potential in these conditions, buffaloes are best suited to satisfy animal protein demand, as they are easy to maintain, long-lived, and parasite resistant. Here we describe how to produce buffalo embryos from slaughterhouse oocytes, with protocols for media formulation, oocyte selection, semen capacitation, and in the end embryo evaluation.

Global transcriptome profiles of Italian Mediterranean buffalo embryos with normal and retarded growth.

The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW<2.7 mm were classed as retarded embryos. Three buffaloes with embryos of the largest EW (3.7, 3.7 and 3.9 mm) and three buffaloes with embryos of the smallest EW (1.5, 1.6 and 1.9 mm) were slaughtered on Day 27 to recover embryos for transcriptome analysis using a bovine custom designed oligo array. A total of 1,047 transcripts were differentially expressed between embryos with normal growth and embryos with retarded growth. Retarded embryos showed 773/1,047 (74%) transcripts that were down-regulated and 274/1,047 (26%) transcripts that were up-regulated relative to normal embryos; in silico analyses focused on 680/1,047 (65%) of the differentially expressed transcripts. The most altered transcripts observed in retarded embryos were associated with membrane structure and function and with metabolic and homeostasis maintenance functions. Other notable functions altered in retarded embryos were developmental processes and in particular nervous system differentiation and function. Specific biochemical pathways such as the complement cascade and coagulation were also altered in retarded embryos. It was concluded from the findings that buffalo embryos with retarded growth on Day 25 after mating show altered gene expression compared with normal embryos, and some de-regulated functions are associated with attachment to the uterine endometrium.

Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis).

The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.

Ovum pick-up and in vitro embryo production (OPU-IVEP) in Mediterranean Italian buffalo performed in different seasons.

This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%, respectively, in spring-summer and in the transitional period; P<0.01). The results of this trial demonstrated that the morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes.

The effect of season on oocyte quality and developmental competence in Italian Mediterranean buffaloes (Bubalus bubalis).

At Italian latitudes, buffalo (Bubalus bubalis) is a seasonally polyestrous species, showing an improved reproductive efficiency when daylight decreases (autumn). The aim of the present study was to evaluate the influence of the season on buffalo oocyte recovery rate, on oocyte quality, assessed on morphological basis, and developmental competence after in vitro fertilization. For this purpose, buffalo ovaries were collected from a local abattoir and the oocytes obtained by aspirating the follicles were evaluated, classified and, if considered of good quality, devolved to the different procedures of IVEP. In general, no differences were found in terms of oocyte recovery per ovary among seasons, but interestingly, the percentage of small oocytes was higher (P<0.05) during spring and summer (0.9±0.1 and 0.9±0.2) compared to autumn and winter (0.3±0.1 and 0.2±0.1). Both cleavage and embryo rate increased during the period from October to December (71.7±3.1 and 26.5±2.1, respectively) compared to the period from April to June (58.0±2.4 and 18.8±1.6, respectively), thus reflecting the in vivo reproductive behavior. Nevertheless, it is worth emphasizing that transferrable embryos were produced in vitro, even during the unfavorable season, but with decreased efficiency. In conclusion, these results suggest to avoid the oocyte collection during spring when planning OPU trials in order to save resources and improve the benefits/costs ratio.