Barbara Zavan

High glucose induces adipogenic differentiation of muscle-derived stem cells.

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Active Silver Nanoparticles for Wound Healing

In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, Acticoat™ Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment.

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkins lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL. Methodology/Principal Findings The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkins lymphomas with an indolent (EBV− Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV+ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures. Conclusions/Significance Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.

Redox control of protein kinase C: cell- and disease-specific aspects.

Hormones, growth factors, electrical stimulation, and cell-cell interactions regulate numerous cellular processes by altering the levels of second messengers, thus influencing biochemical reactions inside the cells. The Protein Kinase C family (PKCs) is a group of serine/threonine kinases that are dependent on calcium (Ca(2+)), diacylglycerol, and phospholipids. Signaling pathways that induce variations on the levels of PKC activators have been implicated in the regulation of diverse cellular functions and, in turn, PKCs are key regulators of a plethora of cellular processes, including proliferation, differentiation, and tumorigenesis. Importantly, PKCs contain regions, both in the N-terminal regulatory domain and in the C-terminal catalytic domain, that are susceptible to redox modifications. In several pathophysiological conditions when the balance between oxidants, antioxidants, and alkylants is compromised, cells undergo redox stress. PKCs are cell-signaling proteins that are particularly sensitive to redox stress because modification of their redox-sensitive regions interferes with their activity and, thus, with their biological effects. In this review, we summarize the involvement of PKCs in health and disease and the importance of redox signaling in the regulation of this family of kinases.

Characteristics of repair tissue in second-look and third-look biopsies from patients treated with engineered cartilage: relationship to symptomatology and time after implantation

IntroductionThe present study established characteristics of tissue regrowth in patients suffering knee lesions treated with grafts of autologous chondrocytes grown on three-dimensional hyaluronic acid biomaterials.MethodsThis multicentred study involved a second-look arthroscopy/biopsy, 5 to 33 months post implant (n = 63). Seven patients allowed a third-look biopsy, three of which were performed 18 months post implant. Characteristics of tissues were histologically and histochemically evaluated. The remaining bone stubs were evaluated for cartilage/bone integration. For data analysis, biopsies were further divided into those obtained from postoperative symptomatic patients (n = 41) or from asymptomatic patients (n = 22).ResultsThe percentage of hyaline regenerated tissues was significantly greater in biopsies obtained after, versus within, 18 months of implantation. Differences were also observed between symptomatic and asymptomatic patients: reparative tissues taken from symptomatic patients 18 months after grafting were mainly fibrocartilage or mixed (hyaline–fibrocartilage) tissue, while tissues taken from asymptomatic patients were hyaline cartilage in 83% of biopsies. In a small group of asymptomatic patients (n = 3), second-look and third-look biopsies taken 18 months after surgery confirmed maturation of the newly formed tissue over time. Cartilage maturation occurred from the inner regions of the graft, in contact with subchondral bone, towards the periphery of the implant.ConclusionsThe study indicates that, in asymptomatic patients after chondrocyte implantation, regenerated tissue undergoes a process of maturation that in the majority of cases takes longer than 18 months for completion and leads to hyaline tissue and not fibrous cartilage. Persistence of symptoms might reflect the presence of a nonhyaline cartilage repair tissue.

Graphene based scaffolds effects on stem cells commitment

Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal structure, with extraordinary electrical, thermal, and physical properties. Moreover, the molecular structure of graphene can be chemically modified with molecules of interest to promote the development of high-performance devices. Although carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase. Study prove that graphene is highly biocompatible, has low toxicity and a large dosage loading capacity. This review describes the ability of graphene and its related materials to induce stem cells differentiation into osteogenic, neuronal, and adipogenic lineages.

In vitro reconstruction of human dermal equivalent enriched with endothelial cells

Experiences coming from many cell-culture studies has brought about the concept that tissue and organ reconstruction should be performed in a three-dimensional environment as it normally occurs in vivo. As far as endothelial cell culture is concerned, it has been shown that angiogenesis can be successfully achieved only when cells are cultured in the presence of collagen-based matrices or basal membrane substrates. The aim of the present investigation is to demonstrate that human umbilical vein endothelial cells (HUVEC) can be grown and differentiated on an artificial dermis obtained by fibroblasts cultured on hyaluronic acid-based scaffolds. For this purpose, we have cultured HUVEC, retrieved by collagenase digestion of perfused human umbilical vein either alone and with fibroblast at 1/1 ratio into HYAFF-11 non-woven mesh. Cultures were maintained for up to 3 weeks. Samples were taken at different time points within this period for the MTT proliferation test and for immunohistochemical analysis. Our results demonstrate that hyaluronan-based biomaterials (HYAFF-11 NW mesh) represent a suitable substrate for HUVEC adhesion, proliferation and reorganization in microcapillary network.

Silver Nanoparticles and Mitochondrial Interaction

Nanotechnology has gone through a period of rapid growth, thus leading to the constant increase in the application of engineered nanomaterials in daily life. Several different types of nanoparticles have been engineered to be employed in a wide array of applications due to their high surface to volume ratio that leads to unique physical and chemical properties. So far, silver nanoparticles (AgNps) have been used in many more different medical devices than any other nanomaterial, mainly due to their antimicrobial properties. Despite the promising advantages posed by using AgNps in medical applications, the possible health effects associated with the inevitable human exposure to AgNps have raised concerns as to their use since a clear understanding of their specific interaction with biological systems has not been attained yet. In light of such consideration, aim of the present work is the morphological analysis of the intracellular behavior of AgNps with a diameter of 10 nm, with a special attention to their interaction with mitochondria.

Hyaluronan benzyl ester as a scaffold for tissue engineering.

Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian tissues. In order to allow a similar three-dimensional organization of in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense momentum for research on “smart scaffolds” for use in cell culture. One of the most promising materials for tissue engineering and regenerative medicine is a hyaluronan derivative: a benzyl ester of hyaluronan (HYAFF®). HYAFF® can be processed to obtain several types of devices such as tubes, membranes, non-woven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are resorbed by the host tissues. Human hepatocytes, dermal fibroblasts and keratinocytes, chondrocytes, Schwann cells, bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells have been successfully cultured in these meshes. The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue.

In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds

Successful in vitro reconstruction of skin requires the inclusion of several cell types that give rise in coculture to the specific elements present in native skin, and the appropriate scaffolding structure to house and support these cells. In addition to the two main structural components, epidermis and dermis, one critical apparatus of the skin is a capillary network that guarantees adequate perfusion of nutrients and oxygen. The aim of the present study was to develop an in vitro coculture system that assumed the human dermal‐epidermal architecture and included a microcapillary network in a three‐dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full‐thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro, engineered skin obtained with this composite culture developed into a well‐differentiated upper layer of stratified keratinocytes lining a dermal‐like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the perioperative period.