Evaluation of the Effectiveness of an Innovative Polycomponent Formulation on Adult and Aged Human Dermal Fibroblasts.

Skin aging is a dynamic process that determines structural alterations in ECM and reduction in dermal fibroblasts. The recent availability on the market of an innovative polycomponent formulation (KARISMA Rh Collagen® FACE, K) containing noncrosslinked high-molecular-weight hyaluronic acid (HMW-HA), a human recombinant polypeptide of collagen-1 alpha chain, and carboxymethyl cellulose (CMC), attracted our scientific interest in evaluating its biomolecular effects on human dermal adult and aged fibroblasts. After treatment with increasing K concentrations, cell proliferation, collagen I, prolyl 4-hydroxylase (P4HA1), an essential protein in collagen biosynthesis, and α-SMA levels were assessed. The fibroblast contractility, TGF-ß1 levels, and oxidative stress markers were also evaluated. K formulation exposure led to a significant and dose-dependent increase in the proliferation and migration of adult fibroblasts. Of note, the K exposure counteracted the H2O2-induced aging by promoting cell proliferation, reducing ß-galactosidase activity, and neutralizing the aging-associated oxidative damage. Moreover, an increase in collagen I, P4HA1, α-SMA, TGF-ß1 levels, and improved contractility of adult and aged fibroblasts were observed after treatment. Overall, our results show evidence that the K treatment is efficacious in improving biological functions in adult fibroblasts and suppressing the biomolecular events associated with H2O2-induced cellular aging, thus supporting the regenerative and bio-revitalizing action of the K formulation helpful in preventing or treating skin aging.

Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-ß1-induced fibrotic response in normal human dermal fibroblasts.

BACKGROUND: Abnormal and deregulated skin wound healing associated with prolonged inflammation may result in dermal fibrosis. Since the current therapeutic strategies revealed unsatisfactory, the investigation of alternative approaches such as those based on the use of specific probiotic strains could provide promising therapeutic options. In this study, we aimed to evaluate whether the lysate from S. thermophilus could antagonize the fibrogenic effects of TGF-ß1 in normal human dermal fibroblasts (NHDF). METHODS: NHDF were exposed to TGF-ß1 to establish a fibrotic phenotype. Proliferation rate and cell number were measured using the IncuCyte® Live Cell Imager system and the trypan blue dye exclusion test. Phenoconversion markers (α-SMA and fibronectin) and collagen I levels were assessed by western blot and immunofluorescence. The mRNA levels of TGF-ß1 were evaluated by RT-PCR. The Smad2/3 phosphorylation level as well as ß-catenin and PPARγ expression, were assessed by western blot. The cell contractility function and migration of NHDF were studied using collagen gel retraction assay, and scratch wound healing assay, respectively. The effects of S. thermophilus lysate, alone or combined with TGF-ß1, were evaluated on all of the above-listed parameters and markers associated with TGF-ß1-induced fibrotic phenotype. RESULTS: Exposure to the S. thermophilus lysate significantly reduced the key mediators and events involved in the abnormal activation of myofibroblasts by TGF-ß1 within the fibrotic profile. The S. thermophilus treatment significantly reduced cell proliferation, migration, and myo-differentiation. In addition, the treatment with probiotic lysate reduced the α-SMA, fibronectin, collagen-I expression levels, and affected the collagen contraction ability of activated dermal fibroblasts. Moreover, the probiotic targeted the TGF-ß1 signaling, reducing Smad2/3 activation, TGF-ß1 mRNA level, and ß-catenin expression through the upregulation of PPARγ. CONCLUSION: This is the first report showing that S. thermophilus lysate had a remarkable anti-fibrotic effect in TGF-ß1-activated NHDF by inhibiting Smad signaling. Notably, the probiotic was able to reduce ß-catenin and increase PPARγ levels. The findings support our point that S. thermophilus may help prevent or treat hypertrophic scarring and keloids.

Soluble Fraction from Lysate of a High Concentration Multi-Strain Probiotic Formulation Inhibits TGF-ß1-Induced Intestinal Fibrosis on CCD-18Co Cells.

Fibrosis is a severe complication of chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Current strategies are not fully effective in treating fibrosis; therefore, innovative anti-fibrotic approaches are urgently needed. TGF-ß1 plays a central role in the fibrotic process by inducing myofibroblast differentiation and excessive extracellular matrix (ECM) protein deposition. Here, we explored the potential anti-fibrotic impact of two high concentration multi-strain probiotic formulations on TGF-ß1-activated human intestinal colonic myofibroblast CCD-18Co. Human colonic fibroblast CCD-18Co cells were cultured in the presence of TGF-ß1 to develop a fibrotic phenotype. Cell viability and growth were measured using the Trypan Blue dye exclusion test. The collagen-I, α-SMA, and pSmad2/3 expression levels were evaluated by Western blot analysis. Fibrosis markers were also analyzed by immunofluorescence and microscopy. The levels of TGF-ß1 in the culture medium were assessed by ELISA. The effects of commercially available probiotic products VSL#3® and Vivomixx® were evaluated as the soluble fraction of bacterial lysates. The results suggested that the soluble fraction of Vivomixx® formulation, but not VSL#3®, was able to antagonize the pro-fibrotic effects of TGF-ß1 on CCD-18Co cells, being able to prevent all of the cellular and molecular parameters that are related to the fibrotic phenotype. The mechanism underlying the observed effect appeared to be associated with inhibition of the TGF-ß1/Smad signaling pathway. To our knowledge, this study provides the first experimental evidence that Vivomixx® could be considered to be a promising candidate against intestinal fibrosis, being able to antagonize TGF-ß1 pro-fibrotic effects. The differences that were observed in our fibrosis model between the two probiotics used could be attributable to the different number of strains in different proportions.

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro.

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

Soluble Fraction from Lysates of Selected Probiotic Strains Differently Influences Re-Epithelialization of HaCaT Scratched Monolayer through a Mechanism Involving Nitric Oxide Synthase 2.

A growing body of evidence supports the use of probiotics in the treatment of several skin conditions, including wounds. Even if in vitro and in vivo studies have highlighted the pro-healing effects of some probiotic bacteria, the underlying mechanisms are still not fully defined. The current investigation aimed to determine the re-epithelialization potential of the soluble fraction from lysate of seven different probiotic strains belonging to different genera (i.e., Streptococcus, Lactobacillus, and Bifidobacterium) on in vitro physically wounded HaCaT monolayer model. The results suggested that the soluble fraction of S. thermophilus,L. plantarum, and L. acidophilus promoted the re-epithelialization of scratched HaCaT monolayers, whereas those from B. longum,B. infantis, and B. breve significantly inhibited the process. On the other hand, L. bulgaricus showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of S. thermophilus, L. plantarum, and L. acidophilus.

Secretome of Adipose Tissue-Derived Stem Cells (ASCs) as a Novel Trend in Chronic Non-Healing Wounds: An Overview of Experimental In Vitro and In Vivo Studies and Methodological Variables.

Wound healing is a complex process with a linear development that involves many actors in a multistep timeline commonly divided into four stages: Hemostasis, inflammation, proliferation, and remodeling. Chronic non-healing wounds fail to progress beyond the inflammatory phase, thus precluding the next steps and, ultimately, wound repair. Many intrinsic or extrinsic factors may contribute to such an occurrence, including patient health conditions, age-related diseases, metabolic deficiencies, advanced age, mechanical pressure, and infections. Great interest is being focused on the adipose tissue-derived stem cell's (ASC) paracrine activity for its potential therapeutic impact on chronic non-healing wounds. In this review, we summarize the results of in vitro and in vivo experimental studies on the pro-wound healing effects of ASC-secretome and/or extracellular vesicles (EVs). To define an overall picture of the available literature data, experimental conditions and applied methodologies are described as well as the in vitro and in vivo models chosen in the reported studies. Even if a comparative analysis of the results obtained by the different groups is challenging due to the large variability of experimental conditions, the available findings are undoubtedly encouraging and fully support the use of cell-free therapies for the treatment of chronic non-healing wounds.

Reducing complications in post-bariatric plastic surgery: our experience and literature review.

BACKGROUND: With the rise in obesity, there has been a similar increase in bariatric surgery. This resulted in numerous patients losing significant weight with accompanying circumferential body contouring issues. This has led to an amazing increase in the number of body contouring procedures performed. METHODS: The aim of this work is to revise the cases of body contouring in 78 ex-obese patients who underwent body contouring surgery in the Department of Health Life and Environmental Sciences - Plastic Reconstructive and Aesthetic Plastic Surgery Section, from 2007 to 2016. RESULTS: The authors have noticed a deep relationship between adverse events and cigarette smoking and with pre-operative BMI. Regardless of these variables, the authors focused on the protocol for the management of patients, which required a collaboration between medical and nursing staff. CONCLUSIONS: Ex-obese patients have an important risk for complications, but the comparison of our personal data with those of the international literature confirms the efficacy of our management protocol with regard to the prevention of complications. (www.actabiomedica.it).

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview.

Considering the increasing interest in adipose-derived stem cells (ASCs) in regenerative medicine, optimization of methods aimed at isolation, characterization, expansion and evaluation of differentiation potential is critical to ensure (a) the quality of stem cells also in terms of genetic stability; (b) the reproducibility of beneficial effects; and (c) the safety of their use. Numerous studies have been conducted to understand the mechanisms that regulate ASC proliferation, growth and differentiation, however standard protocols about harvesting and processing techniques are not yet defined. It is also important to note that some steps in the procedures of harvesting and/or processing have been reported to affect recovery and/or the physiology of ASCs. Even considering the great opportunity that the ASCs provide for the identification of novel molecular targets for new or old drugs, the definition of homogeneous preparation methods that ensure adequate quality assurance and control, in accordance with current GMPs (good manufacturing practices), is required. Here, we summarize the literature reports to provide a detailed overview of the methodological issues underlying human ASCs isolation, processing, characterization, expansion, differentiation techniques, recalling at the same time their basilar principles, advantages and limits, in particular focusing on how these procedures could affect the ASC quality, functionality and plasticity.

Improving the outcome of fractional CO2 laser resurfacing using a probiotic skin cream: Preliminary clinical evaluation.

As known, fractional CO2 resurfacing treatments are more effective than non-ablative ones against aging signs, but post-operative redness and swelling prolong the overall downtime requiring up to steroid administration in order to reduce these local systems. In the last years, an increasing interest has been focused on the possible use of probiotics for treating inflammatory and allergic conditions suggesting that they can exert profound beneficial effects on skin homeostasis. In this work, the Authors report their experience on fractional CO2 laser resurfacing and provide the results of a new post-operative topical treatment with an experimental cream containing probiotic-derived active principles potentially able to modulate the inflammatory reaction associated to laser-treatment. The cream containing DermaACB (CERABEST™) was administered post-operatively to 42 consecutive patients who were treated with fractional CO2 laser. All patients adopted the cream twice a day for 2 weeks. Grades were given according to outcome scale. The efficacy of the cream containing DermaACB was evaluated comparing the rate of post-operative signs vanishing with a control group of 20 patients topically treated with an antibiotic cream and a hyaluronic acid based cream. Results registered with the experimental treatment were good in 22 patients, moderate in 17, and poor in 3 cases. Patients using the study cream took an average time of 14.3 days for erythema resolution and 9.3 days for swelling vanishing. The post-operative administration of the cream containing DermaACB induces a quicker reduction of post-operative erythema and swelling when compared to a standard treatment.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process.

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-ß antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc.

In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

Cystic chondromalacia of the auricle treated with dual-plane excision with intracartilaginous dissection.

Cystic chondromalacia of the auricle is an uncommon condition in which a degenerative process occurs within the cartilage. The disorder affects young and middle-aged people. Clinically, it manifests as a painless, fluctuant swelling that frequently relapses despite various therapeutic approaches. In this article we report a typical case of cystic chondromalacia of the auricle that was successfully treated by surgery-specifically, dual-plane dissection-and we briefly review the literature.

Basal cell carcinoma in childhood: a case report.

Basal cell carcinoma is the most common tumor of skin. Its incidence increases with age, although it is relatively infrequent under the age of 50 years, and its occurrence in childhood is exceedingly rare. This article describes an otherwise typical basal cell carcinoma arising in young patient, with emphasis on histologic diagnosis.

Chemotherapeutically induced cutaneous tuberculosis after BCG injection in a patient with pelvic osteosarcoma.

BACKGROUND: Tuberculosis (TB) is a serious infection afflicting a multitude of people worldwide. Recently, its prevalence has increased. The incidence of skin involvement generally is low. Bacillus Calmette-Guérin (BCG) is a live attenuated strain of Mycobacterium bovis that typically is administered as a vaccine to stimulate the immune system when treating some early neoplasms or to guard against tuberculosis. METHODS: Case report and literature review. CASE REPORT: The authors describe a young man with osteosarcoma of the left hemipelvis who received intradermal BCG injection for immune stimulation prior to surgery. In the course of neoadjuvant chemotherapy, he developed cutaneous tuberculosis. CONCLUSION: It is our opinion that BCG injection should be avoided in all patients requiring surgery, especially in oncologic patients, where the immunodeficiency brought on by chemotherapy predisposes to active opportunistic infection.

Multifrequency ultrasound-assisted liposuction: 5 years of experience.

BACKGROUND: The use of ultrasound in liposuction was first introduced by Kloehn. The ultrasound frequencies used in this technology have a high degree of selectivity for fat cells, thus reducing blood loss, postoperative edema, and ecchymosis and avoiding contour irregularities. The authors report their 5 years of experience with a multifrequency ultrasound generator and describe their series with the aim to focus attention on the problems linked with ultrasound use. METHODS: A total of 797 patients were selected to receive ultrasound assisted liposuction (UAL). Accurate preoperative planning was conducted for all the patients before admission to the surgical quarter. Liposuction was performed using general anesthesia for 538 patients (63 %), peripheral anesthesia for 220 patients (25.76 %), and local anesthesia for 96 patients (11.24 %). The ultrasound generator device was set by choosing the better working protocol (continuous or pulsed mode) according to the tissue characteristics, and the frequency test was performed. A photo of the patient was taken before surgery and at every follow-up visit to estimate the results and to have an objective evaluation of the outcome. The patient's opinion of the results also was recorded using a graduated scale. RESULTS: The results were excellent in 204 cases (25.4 %), good in 356 cases (44.6 %), moderate in 174 cases (22 %), and poor in 63 cases (8 %). CONCLUSIONS: For body contour improvement, UAL is an essential procedure. The introduction of multifrequency UAL, or rather the possibility of using the better working frequency during the treatment, allows better results, thus improving patient outcome. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article.

Melasma treated with intense pulsed light.

BACKGROUND: Hypermelanosis includes a diverse group of genetic and acquired skin anomalies that appear as darker, hyperpigmented areas. Melasma, in particular, is a hypermelanotic condition that affects sun-exposed skin in females. Whether this condition is acquired or genetic is still controversial. However, it clearly correlates with exposure to UV light, a genetic predisposition, and hormonal variations (from pregnancy or oral contraceptives). METHODS: Between October 2006 and March 2008, 38 patients with melasma were treated with intense pulsed light (IPL) at the LASER Center of the Department of Health Science, Plastic and Reconstructive Surgery Session, University of L'Aquila. Diagnosis was based on medical history, physical examination, and video microscopy. RESULTS: Results were graded as excellent, good, moderate, or poor. Grades were given according to outcome scale and reported complications. All 38 patients had follow-up checks at 30 days, 3 months, and 6 months and someone at more than 1 year. Results were excellent in 18 patients (47.37%), good in 11 (28.95%), moderate in 5 (13.16%), and poor in 4 cases (10.52%). CONCLUSION: From a careful review of the scientific literature and according to our personal clinical experience, IPL stands out as an effective tool in the treatment and healing of a high percentage of hypermelanosis and melasma, with a very low risk of complications and an excellent satisfaction rate among patients.

[Body image disorders and other psychiatric symptoms in aesthetic plastic surgery]. / Disturbi dell'immagine corporea e sintomatologia psichiatrica in pazienti candidati ad interventi di chirurgia estetica.

OBJECT: The aim of our study has been to investigate the presence of alterations of body image and psychiatric symptoms in a sample of patients candidates to participate in cosmetic surgery. MATERIAL: It has been recruited 56 outpatients (M=12; F=44) of mean age of 37, 55 years (ds 13.31) consecutively afferent to the Plastic Surgery Ward at the University of L'Aquila. All patients were Plastic Surgery candidates. They have been evaluated by administration of the following instruments: Self-Report Symptom Inventory-Revised (SCL-90) and the Body Uneasiness Test (BUT). RESULTS: The sample was divided into two groups in order to BUT positivity (33.92% positive versus 66.02% negative). Both groups have showed statistically significant differences (p < 0.01) in all SCL-90 dimensions moreover the BUT positive patients have showed greater scores in all SCL-90 dimensions than BUT negative patients. DISCUSSION: Our data indicate the high prevalence of body image disorders and the presence of other psychiatric symptoms in Plastic Surgery patients. CONCLUSIONS: Our study confirm the importance of psychiatric evaluation in those patients. Therefore, a liaison between Psychiatric Service and Plastic Surgery is needed and the consequent psychiatric evaluation results essential to give indications for complete surgical cosmetic procedures.