3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60mm of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications

short term evaluation of the effects of orthodontic clear aligners on sleep bruxism activity

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Ultrastructural features of human sperm cells cryopreserved by different methods

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied

Recommendations for Face Coverings While Exercising During the COVID-19 Pandemic

In an effort to reduce transmission and number of infections of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19) virus, governments and official bodies around the world have produced guidelines on the use of face masks and face coverings. While there is a growing body of recommendations for healthcare professionals and the wider population to use facial protection in “enclosed spaces” where minimal distancing from other individuals is not possible, there is a dearth of clear guidelines for individuals undertaking exercise and sporting activity. The present viewpoint aims to propose recommendations for face coverings while exercising during the COVID-19 pandemic that consider physical distancing, the environment, the density of active cases associated with the specific sports activity, and the practical use of face coverings in order to reduce potential viral transmission. Recommendations are provided on the basis of very limited available evidence in conjunction with the extensive collective clinical experience of the authors and acknowledging the need to consider the likelihood of the presence of the SARS-CoV-2 in the general population. We recommend that face coverings should be used in any environment considered to be of a high or moderate transmission risk, where tolerated and after individual risk assessment. In addition, as national caseloads fluctuate, individual sporting bodies should consider up to date guidance on the use of face coverings during sport and exercise, alongside other preventative measures

Growth and stratification of epithelial cells in minimal culture conditions.

Biological risk management is required in modern tissue engineering. Particular attention should be paid to the culture medium and the scaffold used. In this perspective, it is important to define minimal culture conditions which allow proper growth and differentiation of epithelial cells in vitro. We propose a simple experimental system which permits the generation of three-dimensional epidermal constructs using a collagen layer as a scaffold mimicking the entire dermal tissue and without the need of any feeder layer. Although showing significant differences compared to natural epidermis, these epidermal constructs appear useful to study keratinocyte differentiation and epidermis histogenesis

Expression of p63 transcription factor in ectoderm-derived oral tissues.

The p63 gene encodes six splice variants expressed with transactivating or dominant-negative activities. Animal studies with p63 -/- mutants have suggested that p63 is important for proper development of several organs, including tooth and salivary gland. Moreover, mutations of p63 have been detected in patients affected by ectrodactyly, ectodermal dysplasia and facial clefts. To clarify the role of p63 in craniofacial development, we have studied the localization of p63 protein in human and rat oral tissues using immunohistochemistry. p63 immunostaining was identified in the enamel organ, oral epithelium and developing salivary glands. All compartments of the enamel organ were immunolabelled, whereas only basal and some suprabasal cells of the oral epithelium were stained. Ectomesenchyme-derived cells, including pulp cells, odontoblasts, bone cells and chondrocytes, were negative. The staining pattern was identical in human and rat tissues. These data lend support to the hypothesis that p63 is involved in growth and differentation of ectoderm-derived oral tissues and may be useful to clarify molecular and developmental aspects observed in animal knockout experiments and human syndromes related to p63 gene alteration

Models of epithelial histogenesis

Epithelial tissues emerge from coordinated sequences of cell renewal, specialization and assembly. Like corresponding immature tissues, adult epithelial tissues are provided by stem cells which are responsible for tissue homeostasis. Advances in epithelial histogenesis has permitted to clarify several aspects related to stem cell identification and dynamics and to understand how stem cells interact with their environment, the socalled stem cell niche. The development and maintenance of epithelial tissues involves epithelial-mesenchymal signalling pathways and cell-matrix interactions which control target nuclear factors and genes. The tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for the study of genetic pathways during development. Clonogenic epithelial cells isolated from developing as well mature epithelial tissues has been used to engineer epithelial tissue- equivalents, e.g. epidermal constructs, that are used in clinical practise and biomedical research. Information on molecular mechanisms which regulate epithelial histogenesis, including the role of specific growth/differentiation factors and cognate receptors, is essential to improve epithelial tissue engineering

Cell kinetic analysis in artificial skin using immunochemical methods.

Cell kinetic studies provide important information on histogenesis in vivo and in vitro. In this regard, specific antibodies to cell cycle-related antigens have been raised and characterized, thus permitting the study of cell kinetics using immunochemical methods. Recent advances in culture technology permitted the generation of human skin equivalents in vitro. We here provide detailed practical procedures for the study of epidermal cell kinetics in a model of artificial skin using immunohistochemistry and flow cytometry. The combined application of both techniques allows a precise detection of tissue growth sites and a quantitative assessment of cell growth. Moreover, simultaneous analysis of differentiation markers and proliferation antigens may be useful to understand molecular mechanisms that regulate tissue growth and development

Immunomagnetic separation and biological characterization of a putative mesenchymal stem cells subpopulation

Our previous data demonstrated the presence of MSCs isolated from ovarian follicular liquid. A novel immunomagnetic procedure to isolate rere cells in suspension was used, using Dynal microbeads and a dedicated multiwells magnetic device. It was possible to isolate CD44+ cells and recovere them onto coverslips for the next steps of immunostaining and/or biological characterization. Further experiments have been designed in order to verify the stemness of these cells, seeding them in culture and inducing differentiation into other cell lineages to assess in vitro their plasticity

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60m of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications