In Vitro Grown Sheep Preantral Follicles Yield Oocytes with Normal Nuclear-Epigenetic Maturation

BACKGROUND: Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes. CONCLUSIONS/SIGNIFICANCE: In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation

Influence of a biomimetic gelatin porous scaffold in chondrogenic and osteogenic differentiation of mesenchymal stem cells

Recently, tissue engineering has merged with stem cell technology to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes (1). Gelatin based scaffolds are considered to be a highly suitable 3D material for tissue regeneration, due to high biocompatibility and adaptation to native tissues. In the present study, the chondrogenic and osteogenic potential of a porous gelatin based scaffold (2), alone or in combination with hydroxyapatite crystals, was investigated in human mesenchymal stem cells. Cells were culture up to 4 weeks on the scaffold and on monolayer. MTT assay was performed to evaluate cell viability, light and transmission electron microscopy were carried out to demonstrate cell colonization inside the porous architecture of the biomaterial and scaffold adhesion. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan and versican and osteogenic markers such as Collagen type I, Runx -2, osteopontin and bone matrix protein, were investigated by Real Time PCR. Results showed an high cell viability, adhesion and colonization of the scaffold. Real Time PCR data demonstrated an up-regulation of all the chondrogenic and osteogenic markers analyzed. In conclusion, gelatin porous scaffold provides an improved environment for chondrogenic and osteogenic differentiation of stem cells compared to cell monolayer culture system

Comparative study between autogenous graft and muscular graft covered with autogenous vein tube in wistar rats' tibial nerves using the fluoro-gold® as a neuronal marker

The purpose of this work was to study nervous regeneration through neurons counts by comparing two surgical techniques for addressing nervous gaps on 15 rats' lower limbs. Initially, a 12-mm long vein tube from the left outer jugular was obtained, and then both lower limbs are operated, exposing the tibial nerve at each side and performing a resection of an 8-mm nerve segment, at the same time simulating a gap and an autogenous nerve graft. Left gap repair consisted of a usual conventional graft for nervous injury repair by means of microsurgical suture. The gap repair on right lower limbs was made through quadriceps muscle, treated with liquid nitrogen, covered with an 8-mm tube of jugular vein. After four months, the animals were submitted to a new surgery for exposing tibial nerves to the Fluoro-Gold® neuronal marker. After 48 hours, the rats were perfused and medullar segment between L3 and S1 was removed and subsequently cut into 40µm sections. Neurons on all sections were counted, and no statistical differences were found between both surgical techniques.Este trabalho teve como objetivo o estudo da regeneração nervosa através da contagem de neurônios comparando duas técnicas cirúrgicas no tratamento da perda de substância nervosa nos membros inferiores em 15 ratos. Inicialmente obteve-se tubo de veia de 12mm de comprimento retirado da jugular externa esquerda. A seguir, opera-se os dois membros inferiores, expondo o nervo tibial de cada lado e ressecando um segmento de 8 mm do nervo, simulando, ao mesmo tempo, a perda de substância e a obtenção do enxerto nervoso autógeno. A reparação da perda de substância do lado esquerdo consistiu numa enxertia convencional simples para a reparação de lesão nervosa por meio de sutura microcirúrgica. A do membro inferior direito foi pela tubulização com 8 mm de enxerto de músculo quadríceps denaturado com nitrogênio líquido coberto com veia jugular. Após quatro meses, os animais foram submetidos à nova cirurgia para exposição dos nervos tibiais ao marcador neuronal Fluoro Gold®. Após 48 horas, foram perfundidos e o segmento medular entre L3 e S1 foi removido e posteriormente cortado em secções de 40 µm. Houve contagem neuronal de todos os cortes e não foram verificadas diferenças estatísticas entre as duas técnicas cirúrgicas.UNIFESP Departamento de Ortopedia e TraumatologiaUNIFESP Departamento de Neurologia e NeurocirurgiaUNIFESP Departamento de BioquímicaUNIFESP, Depto. de Ortopedia e TraumatologiaUNIFESP, Depto. de Neurologia e NeurocirurgiaUNIFESP, Depto. de BioquímicaSciEL

matricellular protein expression and cell ultrastructure as parameters to test in vitro cytotoxicity of a biomimetic scaffold

Following scaffold implantation, cell sufferance, in-vivo encapsulation, foreign body reaction and inflammatory response has been reported and the up- regulation of matricellular proteins is often connected with this condition. Cytotoxicity of biomaterials is generally tested according to ISO standard 10993-5 based mainly on viability tests. Additional assays, based on improved cytotoxicity knowledge, are suggested in order to better analyze the biocompatibility of implant materials. The purpose of the study was to evaluate the matricellular protein expression as biomarker for in vitro-testing the biocompatibility of implant materials. Tenascin-C, osteocalcin and osteopontin belong to the matricellular protein family and were chosen as cytotoxicity markers. Mesenchymal stem cells were seeded on collagen/hydroxyapatite scaffold and on carboxymethyl cellulose based hydrogel in order to evaluate gene/protein expression by cell viability test, Real Time PCR and western blot. Electron microscopy was carried out to evaluate the morphological changes induced by cell/scaffold interactions. A low expression of tenascin-c, osteonectin and osteopontin was demonstrated in collagen/hydroxyapatite scaffold compared to the cells cultured on tissue flasks and on hydrogel scaffold. Based on our results, we propose matricellular protein expression as parameter for testing in vitro biocompatibility of implant materials

A case report of vibration-induced hand comorbidities in a postwoman

<p>Abstract</p> <p>Background</p> <p>Prolonged exposure to hand-transmitted vibration is associated with an increased occurrence of symptoms and signs of disorders in the vascular, neurological and osteoarticular systems of the upper limbs. However, the available epidemiological evidence is derived from studies on high vibration levels caused by vibratory tools, whereas little is known about possible upper limb disorders caused by chronic exposure to low vibration levels emitted by fixed sources.</p> <p>Case presentation</p> <p>We present the case of a postwoman who delivered mail for 15 years using a low-powered motorcycle. The woman was in good health until 2002, when she was diagnosed with bilateral Raynaud's phenomenon. In March 2003 a bilateral carpal tunnel syndrome was electromyographically diagnosed; surgical treatment was ineffective. Further examinations in 2005 highlighted the presence of chronic tendonitis (right middle finger flexor).</p> <p>Risk assessment</p> <p>From 1987, for 15 years, our patient rode her motorcycle for 4 h/day, carrying a load of 20-30 kg. For about a quarter of the time she drove over country roads. Using the information collected about the tasks carried out every day by the postwoman and some measurements performed on both handles of the motorcycle, as well as on both iron parts of the handlebars, we reconstructed the woman's previous exposure to hand-arm vibration. 8-hour energy-equivalent frequency weighted acceleration was about 2.4 m/s². The lifetime dose was 1.5 × 10⁹(m²/s⁴)hd.</p> <p>Conclusions</p> <p>The particular set of comorbidities presented by our patient suggests a common pathophysiological basis for all the diseases. Considering the level of exposure to vibrations and the lack of specific knowledge on the effects of vibration in women, we hypothesize an association between the work exposure and the onset of the diseases.</p

Impact of the Covid-19 pandemic on perinatal mental health (Riseup-PPD-COVID-19): protocol for an international prospective cohort study

Corona Virus Disease 19 (COVID-19) is a new pandemic, declared a public health emergency by the World Health Organization, which could have negative consequences for pregnant and postpartum women. The scarce evidence published to date suggests that perinatal mental health has deteriorated since the COVID-19 outbreak. However, the few studies published so far have some limitations, such as a cross-sectional design and the omission of important factors for the understanding of perinatal mental health, including governmental restriction measures and healthcare practices implemented at the maternity hospitals. Within the Riseup-PPD COST Action, a study is underway to assess the impact of COVID-19 in perinatal mental health. The primary objectives are to (1) evaluate changes in perinatal mental health outcomes; and (2) determine the risk and protective factors for perinatal mental health during the COVID-19 pandemic. Additionally, we will compare the results between the countries participating in the study